Note: Descriptions are shown in the official language in which they were submitted.
WO 2022/204581
PCT/1JS2022/022063
TGF-BETA INHIBITORS AND USE THEREOF
RELATED APPLICATIONS
[1] This Application claims the benefit of and priority to US Provisional
Applications 63/166,824 filed March 26,
2021; 63/202,260 filed June 3, 2021; 63/302,999 filed January 25, 2022; and
63/313,386 filed February 24, 2022,
each entitled "TGF-BETA INHIBITORS AND USE THEREOF," the contents of which are
expressly incorporated
herein by reference in their entirety.
FIELD
[2] The instant application relates generally to TGFp inhibitors and
therapeutic use thereof, as well as related
assays for diagnosing, monitoring, prognosticating, and treating disorders,
including cancer.
BACKGROUND
[3] Transforming growth factor beta 1 (TGF[31) is a member of the TGFp
superfamily of growth factors, along
with two other structurally related isoforms, namely, TGFp2 and TG933, each of
which is encoded by a separate
gene. These TGFp isoforms function as pleiotropic cytokines that regulate cell
proliferation, differentiation,
immunomodulation (e.g., adaptive immune response), and other diverse
biological processes both in homeostasis
and in disease contexts. The three TGFp isoforms signal through the same cell-
surface receptors and trigger
similar canonical downstream signal transduction events that include the
SMAD2/3 pathway.
[4] TGFp has been implicated in the pathogenesis and progression of a
number of disease conditions, such as
cancer, fibrosis, and immune disorders. In many cases, such conditions are
associated with dysregulation of the
extracellular matrix (ECM). For these and other reasons, TGFP has been an
attractive therapeutic target for the
treatment of immune disorders, various proliferative disorders, and fibrotic
conditions. However, observations from
preclinical studies, including in rats and dogs, have revealed serious
toxicities associated with systemic inhibition
of TGFr3s in vivo, and to date, there are no TGF13 therapeutics available in
the market which are deemed both safe
and efficacious.
[51 Dose-limiting toxicities noted with inhibition of the TGFP
pathway have remained a major concern in the
development of anti-TGFp therapies. These include cardiovascular
abnormalities, skin lesions, epithelial oral
hyperplasia, and gingival bleeding (Vitsky 2009; Lonning 2011; Stauber 2014;
Mitra 2020). Although many of these
toxicities are either reversible or manageable, the cardiovascular lesions
such as inflammation, hemorrhage or
hyperplasia in the valves, aortic arch and associated arteries of the heart,
are not reversible and therefore continue
to be key safety issues when developing TGFp inhibitors (Stauber 2014;
Anderton 2011; Mitra 2020).
[6] Previously, Applicant described a class of monoclonal antibodies
that have a novel mechanism of action to
modulate growth factor signaling (see, for example, WO 2014/182676, the
contents of which are herein
incorporated by reference in their entirety). These antibodies were designed
to exploit the fact that TGFI31 is
expressed as latent pro-protein complex comprised of prodomain and growth
factor, which requires an activation
step that releases the growth factor from the latent complex. Rather than
taking the traditional approach of directly
targeting the mature growth factor itself post-activation (such as
neutralizing antibodies), the novel class of
inhibitory antibodies specifically targeted the inactive pro-proprotein
complex itself so as to preemptively block the
activation step, upstream of ligand-receptor interaction. Without being bound
by theory, it was reasoned that this
unique mechanism of action should provide advantages for achieving both
spatial and temporal benefits in that
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they act at the source, that is, by targeting the latent proTGF01 complex
within a disease microenvironment before
activation takes place.
[7] Using this approach, further monoclonal antibodies that specifically
bind and inhibit the activation step of
TGF131 (that is, release of mature growth factor from the latent complex) in
an isoform-selective manner have been
generated (see, WO 2017/156500, the contents of which are herein incorporated
by reference in their entirety).
Data presented for those antibodies support the notion that isoform-specific
inhibition (as opposed to pan-inhibition)
of TGFp may render improved safety profiles of antagonizing TGFp in vivo.
Taking this into consideration, the
instant inventors have sought to develop TGFp1 inhibitors that are both i)
isoform-specific; and, ii) capable of
broadly targeting multiple TGFI31 signaling complexes that are associated with
different presenting molecules, as
therapeutic agents for conditions driven by multifaceted TGF31 effects and
dysregulation thereof. A non-limiting
example of such an isoform-specific inhibitor is a TGF(31-selective antibody,
e.g., Ab4, Ab5, Ab6, Ab21, Ab22,
Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, or Ab34
disclosed herein.
[8] Examples of such antibodies were subsequently described in WO
2018/129329 and PCT/US2019/041373,
the contents of each of which are herein incorporated by reference in their
entirety. These isoform-specific
inhibitory agents demonstrated both efficacy and safety in vivo.
[9] For example, PCT/US2019/041373 discloses that isoform-selective, high
affinity antibodies capable of
targeting large latent complexes (LLCs) of TGFP1 may be effective to treat
TGFP1-related indications, such as
diseases involving abnormal gene expression (e.g., TGFB1, Acta2, Col1a1,
Col3a1, Fn1, Itga11, Lox, LoxI2, CCL2
and Mmp2), diseases involving ECM dysregulation (e.g., fibrosis, myelofibrosis
and solid tumor), diseases
characterized by increased immunosuppressive cells (e.g., Tregs, MDSCs and/or
M2 macrophages), diseases
involving mesenchymal transition, diseases involving proteases, diseases
related to abnormal stem cell
proliferation and/or differentiation.
[10] In multiple preclinical tumor models, such TGF31 inhibitors were shown
to overcome tumor primary
resistance (i.e., present before treatment initiation) to an immunotherapy
(e.g., checkpoint inhibitors), where the
tumor is infiltrated with immunosuppressive cell types, such as regulatory T
cells, M2-type macrophages, and/or
myeloid-derived suppressive cells (tumor-associated MDSCs). Upon treatment, a
reduction in the number of tumor-
associated immunosuppressive cells (e.g., MDSCs) and a corresponding increase
in the number of anti-tumor
effector T cells were observed. In multiple preclinical models, (including
tumors co-expressing TGFp1/3 isoforms),
significant and durable antitumor effects were achieved, coupled with survival
benefits, when used in conjunction
with a checkpoint blockade therapy, suggesting that inhibition of TGFpl alone
was sufficient to sensitize
immunosuppressive tumors to cancer immunotherapy such as checkpoint
inhibitors. See, Martin et al. Science
Translational Medicine (2020), 12(536): eaay8456.
[11] As of the filing date of this application, the prevailing view of the
field as a whole appears to be that it is
necessary or advantageous to inhibit multiple isoforms of TGFp to achieve
therapeutic effects, while managing
toxicities by careful dosing regimen. Consistent with this premise, numerous
groups are developing TGFp inhibitors
that target more than one isoform. These include low molecular weight
antagonists of TGFp receptors, e.g., ALK5
antagonists, such as Galunisertib (LY2157299 monohydrate); monoclonal
antibodies (such as neutralizing
antibodies) that inhibit all three isoforms ("pan-inhibitor" antibodies) (see,
for example, WO 2018/134681);
monoclonal antibodies that preferentially inhibit two of the three isoforms
(e.g., antibodies against TGFp1/2 (for
example WO 2016/161410) and TGFp1/3 (for example \NO 2006/116002 and WO
2020/051333); integrin inhibitors
such as antibodies that bind to c6/133, aVP5, aVP6, aVP8, a51, allbP3, or a8P1
integrins and inhibit downstream
activation of TGFp. e.g., selective inhibition of TGFp1 and/or TGFp3 (e.g.,
PLN-74809), and engineered molecules
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(e.g., fusion proteins) such as ligand traps (for example, WO 2018/029367; WO
2018/129331 and WO
2018/158727).
[12] Whilst immune checkpoint inhibitors have become one of the most
remarkable success stories of cancer
therapy in recent years, these therapies are effective in only a small portion
of patient populations (Hedge et al.,
Immunity. 2020 Jan 14;52(1):17-35). As single agents, many immune checkpoint
inhibitors typically have response
rates of only about 10-35%. An unmet need in cancer immunotherapy has been the
limited availability of reliable
predictable biomarkers (see, for example, Zhang et al., Front. Med. 2019,
13(1): 32-44, "Monitoring checkpoint
inhibitors: predictive biomarkers in immunotherapy" and Arora et al., Adv.
Ther. 2019, 36: 2638-2678, "Existing and
emerging biomarkers for immune checkpoint immunotherapy in solid tumors).
Although traditional tumor biopsy
offers valuable information on the disease, possible limitations with biopsy
include being invasive, not always
feasible for sample collection/access, and potentially not being
representative of the whole tumoral landscape.
Alternatives to biopsies are being actively explored, including gene
expression profiling and noninvasive imaging
techniques. Certain serum markers may be useful for diagnostic purposes, but
less so for prognostic purposes
(see, for example, Zhang et.al.). This has led to the suggestion that blood-
based evaluation is likely a poor
surrogate of what happens in the tumor microenvironment (TME) (Galon & Bruni,
Nature Reviews Drug Discovery,
2019 Mar;18(3):197-218 "Approaches to treat immune hot, altered and cold
tumors with combination
immunotherapies"). There remains a need for better guidance as to both
selection of suitable=TGFp inhibitors
tailored to certain patient populations and related therapeutic regimen which
may provide improved cancer therapy.
SUMMARY
[13] The present disclosure relates to compositions comprising TGFp
inhibitors and methods for selecting
suitable subjects to treat with TGFI3 inhibitors, as well as related methods
of treatments and monitoring treatment
parameters such as efficacy and target engagement. The disclosure provides
better and more targeted
therapeutics and treatment modalities, including improved ways of identifying
candidates for treatment and/or
monitoring treatment efficacy, e.g., patients or patient populations who are
likely to benefit from the TGFp inhibitor
therapy. Related methods, including therapeutic regimens, and methods for
manufacturing such inhibitors are
encompassed herein. The selection of particular subjects and TGFP inhibitors
for therapeutic use is aimed to
achieve in vivo efficacy.
[14] More specifically, the present disclosure provides, inter alia, i)
enhanced methods for analysis aimed to
provide better characterization of the cellular architecture within and
surrounding a tumor; ii) improved methods for
determining circulatory TGFp levels aimed to achieve greater accuracy; iii)
improved methods for assessing
circulating MDSC levels, including identification of LRRC33 as a novel marker
for circulating MDSC cells and
improved surface markers for identification of mMDSC and gMDSC sub-
populations; and iv) additional biomarkers,
including p-Smad2, which are useful for predicting and monitoring therapeutic
efficacy, as well as determining
target engagement. Thus, one or more of these features may be employed as part
of diagnostic and/or therapeutic
regimen for subjects (e.g., patients) with a condition associated with TGFp1
dysregulation, such as cancer.
[15] In some embodiments, the pharmaceutical composition and/or treatment
regimen disclosed herein
comprises a TGFp inhibitor and one or more additional therapies such as a
genotoxic therapy and/or a checkpoint
inhibitor therapy. In some embodiments, the one or more additional therapies
may be administered in conjunction
with the TGFp inhibitor, either as a separate molecular entity administered
separately, as a single formulation (e.g.,
an admixture), or as part of a single molecular entity, e.g., an engineered
multifunctional construct that functions
as both a checkpoint inhibitor and a TGFp inhibitor. In some embodiments, the
checkpoint inhibitor therapy is an
antibody or an antigen-binding fragment that targets PD-1, PD-L1, CTLA-4, or
LAG3. In some embodiments, the
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genotoxic therapy is a chemotherapy or a radiation therapy. In some
embodiments, the TGFI3 inhibitor and the one
or more additional therapies are administered concurrently, separately, or
sequentially. In some embodiments, the
TGFI3 inhibitor is a TGF(31 inhibitor, such as apitegromab or Ab6. In some
embodiments, the TGFp inhibitor is any
one of the antibodies or antigen-binding fragments disclosed in
PCT/JP2015/006323, the content of which is hereby
incorporated by reference in its entirety. In some embodiments, the TGFP
inhibitor is GYM329. In some
embodiments, the TGFp inhibitor is an inhibitor of TGFp1 and TGFp2, such as
NIS793/X0MA-089 or GC1008. In
some embodiments, the TGFI3 inhibitor is an inhibitor of TGFpl and TGFp3, such
as M7824 (bintrafusp alpha) or
AVID200. In some embodiments, the TGFI3 inhibitor is a pan TGFp inhibitor
(i.e., an agent that inhibits TGFp1,
TGFP2, and TGFI33), including GC1008 or derivative thereof, SAR439459,
LY3022859, or an agent that blocks a
ligand-binding domain of a TGFI3 receptor. In some embodiments, the TGFp
inhibitor is an agent that binds the
RGD motif present in latent TGFpl and/or TGFp3, e.g., a low molecular weight
(small molecule) compound or an
antibody or antigen-binding fragment. In some embodiments, the TGFp inhibitor
is an RNA-based inhibitor of
TGFI31 expression. In some embodiments, the TGFI3 inhibitor is a soluble
ligand trap such as M7824 (bintrafusp
alpha) or AVID200.
[16] In various embodiments, the disclosure provides a method of treating,
predicting, and/or monitoring
therapeutic efficacy of a TGFp inhibitor treatment in a subject by measuring
or monitoring circulating TGFI3 levels
(e.g., circulating TG931 levels, e.g., circulating latent TGFp1 levels).
Without being bound by theory, the instant
inventors have discovered that administering a TGFp inhibitor to a subject
increases TGFI3 levels in the subject's
blood, possibly due to an accumulation of latent TGFI3 as a result of
inhibiting the TGFI3 activation pathway. Thus,
the disclosure contemplates the use of circulating TGFI3, e.g., from a blood
sample obtained from a subject, as a
biomarker to determine and monitor therapeutic efficacy and/or target
engagement, and to guide decisions on
treatment. The terms circulating and circulatory (as in "circulating TGFp" and
"circulatory TGFp") may be used
interchangeably.
[17] In some embodiments, the disclosure provides a method of treating,
predicting, and/or monitoring
therapeutic efficacy of a TGFI3 inhibitor treatment in a subject, the method
comprising (i) determining a level of
circulating TGFp in the subject prior to administering a TGFI3 inhibitor; (ii)
administering to the subject a
therapeutically effective amount of the TGFI3 inhibitor; and (iii) determining
a level of circulating TGFI3 in the subject
after administration, wherein an increase in circulating TGFI3 after the
administration as compared to before the
administration indicates therapeutic efficacy. In some embodiments, the
increase is at least 1.5-fold, at least 2-fold,
at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more.
In some embodiments, the treatment alters
the level of circulating TGFp. In some embodiments, continued treatment is
contingent on an observed increase in
circulating TGFp.
[18] In some embodiments, the disclosure encompasses a method of
determining target engagement and/or
therapeutic efficacy of a TGFp inhibitor treatment in a subject, wherein the
treatment comprises (i) determining the
circulating TGFP level in a sample obtained from the subject prior to
administering the TGFP inhibitor; (ii)
administering a first dose of the TGFp inhibitor to the subject; and (iii)
determining the circulating TGFI3 level in a
sample obtained from the subject after the administration, wherein an increase
in the circulating TGFp level after
the administration as compared to before the administration is indicative of
target engagement and/or therapeutic
efficacy. In some embodiments, the method comprises administering to the
subject a second dose of the TGFI3
inhibitor if an increase in the circulating TGFI3 level after the
administration as compared to before the administration
is observed. In some embodiments, the treatment is continued if an increase in
the circulating TGFI3 level after the
administration as compared to before the administration is observed. In some
embodiments, the increase is at least
1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-
fold, at least 5-fold, or more.
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[19] In any one of the previous embodiments, the treatment may further
comprise administering to the subject
one or more additional therapies, e.g., a genotoxic therapy and/or
immunotherapy, wherein the one or more
additional therapies are administered concurrently (e.g., simultaneously),
separately, or sequentially. For instance,
the additional therapy may comprise a checkpoint inhibitor therapy.
[20] In some embodiments, the disclosure provides an improved method for
measuring a circulating TGFI3 level
from a blood sample or a sample derived from blood, the method comprising
processing the sample at a
temperature of 2-8 DC in a sample tube comprising an anticoagulant. In some
embodiments, the anticoagulant is
citrate-theophylline-adenosine-dipyridamole (CTAD). In some embodiments, the
tube is coated with a citrate-
theophylline-adenosine-dipyridamole (CTAD) solution, a 0.11 M buffered
trisodium citrate solution, 15 M
theophylline, 3.7 M adenosine, and 0.198 M dipyridamole, wherein the solution
has a pH of 5Ø In some
embodiments, the sample processing comprises one or more centrifugation steps
at a speed of greater than 100xg
and/or one or more centrifugation steps at a speed of below 15000xg. In some
embodiments, the sample
processing comprises a centrifugation protocol comprising: i) a first step of
10 minutes at 150xg and a second step
of 20 minutes at 2500xg; or ii) a first step of 10 minutes at 2500xg and a
second step of 20 minutes at 2500xg; or
iii) a first step of 10 minutes at 1500xg and a second step of 5 minutes at
12000xg. In some embodiments, the
method comprises analyzing a level of plasma factor 4 (PF4) in the same sample
from which the circulating TGF13
level is determined, such that the PF4 level provides quality control for the
sample. In some embodiments, a sample
is only used to determine a circulating TGFp level if the PF4 level in the
same sample is below a concentration
indicative of plasma activation. In some embodiments, a PF4 level of greater
than 500 ng/ml is indicative of plasma
activation. Thus, in some embodiments, a sample is used to assess a
circulating TGF13 level only if the sample has
a PF4 level of 500 ng/ml or less.
[21] In some embodiments, the disclosure provides a method of treating,
predicting, and/or monitoring
therapeutic efficacy of a TGFp inhibitor treatment in a subject, the method
comprising (i) determining a level of
phosphorylated Smad2 (P-Smad2) nuclear translocation in a tumor sample
obtained from the subject prior to
administering a TGFI3 inhibitor (pre-treatment tumor sample); (ii)
administering to the subject one or more doses of
the TGFp inhibitor; and (iii) determining a level of P-Smad2 nuclear
translocation in a tumor sample obtained from
the subject after the administration (post-treatment tumor sample); wherein a
decrease in P-8mad2 nuclear
translocation after the administration as compared to before the
administration indicates therapeutic efficacy. In
some embodiments, the treatment is continued if a decrease in P-Smad2 nuclear
translocation is observed in the
post-treatment tumor sample. In some embodiments, the decrease is by at least
1.3-fold, at least 1.5-fold, at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold. In some
embodiments, the level of P-Smad2 nuclear
translocation is determined by nuclear masking.
[22] In various embodiments, the disclosure provided herein involves the
use of circulating MDSC levels as a
predictive biomarker to improve the diagnosis, monitoring, patient selection,
prognosis, and/or continued treatment
of a subject being administered a TGFP inhibitor (e.g., a TGFI31-selective
inhibitor such as Ab6) by monitoring
circulating MDSC levels. In some embodiments, the disclosure also encompasses
methods of determining
therapeutic efficacy and therapeutic agents (e.g., compositions) or regiments
for use in subjects with cancer by
measuring levels of circulating MDSCs. Wthout being bound by theory, the
instant inventors have discovered that
reversal of or overcoming an immunosuppressive phenotype, e.g., in a cancer or
related condition that manifests
dysregulation of the ECM, such as by administration of a TGFI3 inhibitor, can
be indicated by analyzing circulating
MDSC levels, e.g., in a sample obtained from a subject, e.g., in blood or a
blood component, e.g., prior to the time
point when a reduction in tumor volume or other biomarkers might be used to
confirm treatment efficacy. In some
embodiments, circulatory MDSCs are characterized by cell-surface expression of
LRRC33. In some embodiments,
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sub-populations of circulatory MDSCs are measured, such as m-MDSCs and/or g-
MDSCs. The terms circulating
and circulatory (as in "circulating MDSCs" and "circulatory MDSCs") may be
used interchangeably.
[23] Tumor-associated MDSC cells may contribute to TGF31-mediated
immunosuppression in the tumor
microenvironment. Previously, Applicant showed that MDSCs were indeed enriched
in solid tumors and that
inhibition of TGF31 in conjunction with a checkpoint inhibitor treatment
significantly reduced intratumoral MDSCs,
which correlated with slowed tumor growth and, in some cases, achieved
complete regression in multiple preclinical
tumor models (PCT/US2019/041373). In these efficacy studies, effectiveness of
such combination therapy was
observed over the course of weeks to months (for example, 6-12 weeks) by
monitoring tumor growth. Tumor biopsy
may reveal an immune profile of a tumor microenvironment (TME); however, in
addition to being invasive, biopsy-
based information may be inaccurate or skewed because tumor-infiltrating
lymphocytes (TILs) may not be uniformly
present within the whole tumor, and therefore, depending on which portion of
the tumor is sampled by biopsy,
results may vary. To overcome the limitations of biopsy-based analyses, data
presented herein now establish the
correlation between tumor-associated (e.g., intratumoral) MDSC levels arid
circulatory MDSC levels, raising the
possibility that MDSCs measured in blood samples (e.g., whole blood or a blood
component, e.g., PBMCs) may
serve as a surrogate to more accurately predict patient populations that are
likely to benefit from certain therapeutic
regimens. Furthermore, evidence suggests the degree of tumor burden (e.g., the
size of tumor) correlates with the
relative level of circulating MDSCs in the subject bearing the tumor.
Therefore, by monitoring circulating MDSC
levels in a subject after receiving the therapy, response to the therapy
(e.g., therapeutic effects) may be evaluated
without the need for invasive biopsies, and results may be obtained sooner
than conventional methods.
Additionally, more recent findings presented herein identify, inter alia,
LRRC33 as a novel cell-surface marker for
MDSCs in circulation (e.g., blood samples). This observation raises the
possibility that surface LRRC33 expression
may be used as a blood-based predictive biomarker.
[24] In various embodiments, the methods disclosed herein employ
circulating MDSCs as an early biomarker to
predict the efficacy of combination therapy comprising a TGF3 inhibitor. Data
disclosed herein show that after
TGF31 inhibitor treatment, there is a marked reduction in circulating MDSC
levels relative to baseline, which can
be a reduction in mMDSC levels and/or gMDSC levels. Such circulating MDSC
levels can be measured in blood
or a blood component, which can be detected well before antitumor efficacy
outcome can readily be obtained, in
some cases shortening the timeline by weeks. Thus, the disclosure provides the
use of circulating MDSCs as a
predictive biomarker for the patient's responsiveness to a cancer therapy,
e.g., a combination therapy. In related
aspects of the disclosure provided herein, the level of circulating MDSC cells
may be determined within 1-10 weeks,
e.g., 3-6 weeks, following administration of a dose of a TGF3 inhibitor,
optionally within 3 weeks or at about 3
weeks following administration of the dose of TGF3 inhibitor. In some
embodiments, the level of circulating MDSC
cells may be determined within 2 weeks following administration of the dose of
TGF3 inhibitor. In some
embodiments, the level of circulating MDSC cells may be determined at about 10
days following administration of
the dose of TGF3 inhibitor.
[25] Cancer immunotherapy may harness or enhance the body's immunity to combat
cancer. Without being
bound by theory, it is contemplated that low levels of circulating MDSCs in
subjects with cancer indicate that the
body has retained or restored disease-fighting immunity (e.g., antitumor
activity), more specifically, lymphocytes
such as CD8+ T cells, which can be mobilized to attack malignant cells. Thus,
reduced levels of circulating MDSCs
upon TGF3 inhibitor treatment may indicate pharmacodynamic effects of TGF3
inhibition (e.g., TGF31 inhibition)
and serve as an early predictive biomarker for therapeutic efficacy when
treated with a cancer therapy such as
checkpoint inhibitors.
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[26] Where cancer patients receive a combination therapy comprising a cancer
therapy (such as checkpoint
inhibitor) and a TGFI3 inhibitor that is not selective for TGFI31 (non-
selective TGFI3 inhibitor), there may be a greater
risk of toxicity. To mitigate or manage such risk, non-selective TGFI3
inhibitor may be administered infrequently or
intermittently, for example on an "as-needed" basis. For example, circulating
MDSC levels may be monitored
periodically in order to determine that the effects of overcoming
immunosuppression are sufficiently maintained, so
as to ensure antitumor effects of the cancer therapy. During the course of
cancertreatment, if MDSC levels become
elevated, this may indicate that the patient may benefit from additional
dose(s) of a TGFI3 inhibitor. Such approach
may help reduce unnecessary risk and adverse events associated with over-
exposure to a TGFI3 inhibitor,
particularly a non-TG931 selective inhibitor. In some embodiments, the TGFI3
inhibitor targets TGFI31/2 signaling.
In some embodiments, the TGFI3 inhibitor targets TGFp1/3 signaling. In some
embodiments, the TGFI3 inhibitor
targets TGF[31/2/3 signaling.
[27] In some embodiments, disclosed herein are methods of treating cancer
(also described herein in the context
of compositions for use in treating cancer or cancer treatments). Also
disclosed are methods of predicting,
determining, or monitoring therapeutic efficacy in subjects with cancer, e.g.,
monitoring a patient's responsiveness
to treatment and/or making continued treatment decisions based on the
monitored parameters. In some
embodiments, the cancer is an immune excluded cancer and/or a
myeloproliferative disorder, wherein the
myeloproliferative disorder may be myelofibrosis. In some embodiments, the
cancer is a TGF[31-positive cancer.
The TGF131-positive cancer may co-express TGFI31, TGFI32, and/or TGFI33. The
TG931-positive cancer may be a
TGF131-dominant tumor. The TGF[31-positive cancer may be a TGF[31-dominant
tumor and may co-express
TGFp1, TGF[32, and/or TGF[33. For instance, the TGF(31-positive cancer may be
a TGFp1-dominant tumor and
may co-express TGFp1 and TGF[32. As another example, The TGFp1-positive cancer
may be a TGFp1-dominant
tumor and may co-express TGF[31 and TGF133. Such cancer includes advanced
cancer, e.g., metastatic cancer
(e.g., metastatic solid tumors) and cancer with a locally advanced tumor
(e.g., locally advanced solid tumors). In
some embodiments, the treatment comprises administering to the subject a TGFI3
inhibitor in an amount sufficient
to reduce circulating MDSC levels.
[28] In some embodiments, the disclosure encompasses a method of predicting
or determining therapeutic
efficacy in a subject having cancer comprising the steps of determining
circulating MDSC levels in the subject prior
to administering a TGFI3 inhibitor (alone or in conjunction with a cancer
therapy), administering to the subject a
therapeutically effective amount of the TGFI3 inhibitor (alone or in
conjunction with a cancer therapy), and
determining circulating MDSC levels in the subject after the administration,
wherein a reduction in circulating MDSC
levels after administration, as compared to circulating MDSC levels before
administration, predicts therapeutic
efficacy.
[29] In some embodiments, the disclosure encompasses a method of
determining therapeutic efficacy of a TGF13
inhibitor treatment in a subject, wherein the treatment comprises (i)
determining the circulating MDSC level in a
sample obtained from the subject prior to administering the TGFP inhibitor;
(ii) administering a first dose of the
TGFI3 inhibitor to the subject; and (iii) determining the circulating MDSC
level in a sample obtained from the subject
after the administration, wherein a reduction in the circulating MDSC level
after the administration as compared to
before the administration is indicative of therapeutic efficacy. In some
embodiments, the method comprises
administering to the subject a second dose of the TGFI3 inhibitor if a
reduction in the circulating MDSC level after
the administration as compared to before the administration is observed. In
some embodiments, the treatment is
continued if a reduction in the circulating MDSC level after the
administration as compared to before the
administration is observed.
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[30] In some embodiments, the disclosure encompasses a combination therapy
comprising a dose of a TGFp
inhibitor and a checkpoint inhibitor therapy and/or a genotoxic therapy for
use in the treatment of cancer, wherein
the treatment comprises concurrent (e.g., simultaneous), separate, or
sequential administration of a dose of the
TGFp inhibitor, wherein a reduction in circulating MDSC level after the
administration as compared to before the
administration has been determined.
[31] In some embodiments, the disclosure encompasses a TGFp inhibitor for
use in the treatment of cancer in
a subject, wherein the subject is administered a dose of the TGFp inhibitor,
and wherein the TGFp inhibitor reduces
or reverses immune suppression in the cancer, wherein said reduced or reversed
immune suppression has been
determined by a reduction in the circulating MDSC level in the subject
measured after the administration of the
TGFp inhibitor as compared to the circulating MDSC level measured in the
subject prior to administering the dose
of the TGFp inhibitor.
[32] In some embodiments, the disclosure encompasses a method of treating
advanced cancer in a human
subject comprising the steps of selecting a subject with advanced cancer and
administering a combination therapy
comprising a TGFP inhibitor and a checkpoint inhibitor therapy, wherein the
advanced cancer comprises a locally
advanced tumor and/or metastatic cancer with primary resistance to a
checkpoint inhibitor therapy, wherein the
subject has elevated circulating MDSC levels. In some embodiments, the
combination therapy reduces the
circulating MDSC level in the subject. In some embodiments, continued
treatment is contingent on an observed
reduction in the subject's circulating MDSC level.
[33] In various embodiments, the circulating MDSC level may be a level of
mMDSC and/or gMDSC. In some
embodiments, mMDSCs are identified by cell surface markers of CD11b+, HLA-DR-
/low, CD14+, CD15-,
CD33+/high, and CD66b-. In some embodiments, gMDSCs are identified by cell
surface markers of CD11 b+, HLA-
DR-, CD14-, CD15+, CD33+/low, and CD66b+. In some embodiments, the reduction
in a circulating MDSC level
(e.g., circulating mMDSC and/or circulating gmMDSC) may be a reduction of at
least 10%.
[34] In some embodiments, the disclosure encompasses a method of treating,
predicting, determining, and/or
monitoring therapeutic efficacy of a cancer treatment in a subject
administered a TGFp inhibitor alone or in
combination with one or more additional cancer therapies (e.g., a checkpoint
inhibitor therapy and/or a genotoxic
therapy), the method comprising the steps of: (i) obtaining a pre-treatment
biopsy sample from the subject, (ii)
determining a level of tumor-associated CD8+ cells in the pre-treatment biopsy
sample, (iii) administering the
treatment to the subject, (iv) obtaining a post-treatment biopsy sample from
the subject, and (v) determining a level
of tumor-associated CD8+ cells in the post-treatment biopsy sample, wherein
the levels of tumor-associated CD8+
cells in the biopsy samples are determined by immunohistochemical analysis of
individual tumor nests within the
tumor.
[35] In some embodiments, a subject having an immune inflamed tumor
characterized by a pre-treatment biopsy
sample having greater than 5% CD8+ cells in individual tumor nests is selected
for the treatment. In some
embodiments, the immune inflamed tumor is characterized by having greater than
5% CD8+ cells in greater than
50% of the individual tumor nests detected.
[36] In some embodiments, a subject having an immune excluded tumor
characterized by a pre-treatment biopsy
sample having less than 5% intratumor CD8+ cells and greater than 5% margin
CD8+ cells is selected for
treatment. In some embodiments, the immune excluded tumor is characterized by
having less than 5% CD8+ cells
in individual tumor nests. In some embodiments, the immune excluded tumor is
characterized by having less than
5% CD8+ cells in greater than 50% of the individual tumor nests detected.
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[37] In some embodiments, therapeutic efficacy can be monitored by
comparing the levels of CD8+ cells in the
pre-treatment biopsy sample and the post-treatment biopsy sample such that a
change in CD8+ level in the post-
treatment biopsy sample as compared to the CD8+ level in the pre-treatment
indicates therapeutic efficacy. In
some embodiments, an increase of intratumor CD8+ cells (e.g., an increase of
CD8+ cells inside tumor nests) in
the post-treatment biopsy sample as compared to the pre-treatment biopsy
sample indicates therapeutic efficacy.
[38] In some embodiments, any one of the biomarkers disclosed herein may be
used in conjunction with one or
more of the other biomarkers provided herein. For example, circulating TGF8
may be monitored in combination
with one or more biomarkers disclosed herein, e.g., circulating MDSC, tumor-
associated CD8+ cell, intratumor or
circulating cytokines, and/or p-Smad2 nuclear translocation. In some
embodiments, treatment efficacy and/or
continued treatment may be contingent on observed changes in two or more sets
of biomarkers.
[39] In various embodiments, the methods and compositions for use of the
present disclosure comprise treating
or selecting for treatment a subject having a cancer, wherein the cancer may
be a highly metastatic cancer and/or
a solid cancer. In some embodiments, the subject has melanoma, triple-negative
breast cancer, HER2-positive
breast cancer colorectal cancer (e.g., microsatellite stable-colorectal
cancer, lung cancer (e.g., non-small cell lung
cancer or small cell lung cancer), pancreatic cancer, bladder cancer, kidney
cancer (e.g., transitional cell
carcinoma, renal sarcoma, and renal cell carcinoma (RCC), including clear cell
RCC, papillary RCC, chromophobe
RCC, collecting duct RCC, or unclassified RCC, uterine cancer, prostate
cancer, stomach cancer (e.g., gastric
cancer), or thyroid cancer.
[40] In some embodiments, the methods and compositions for use of the
present disclosure comprise treating
or selecting for treatment a subject having a cancer that is resistant to
immunotherapy. The subject may be
treatment-naïve (e.g., has not previously received a cancer therapy), may have
primary resistance to an
immunotherapy (i.e., resistance is present before treatment initiation), or
may have acquired resistance to an
immunotherapy (i.e., resistance as a result of at least one dose of
treatment). In some embodiments, the
immunotherapy is a checkpoint inhibitor therapy, e.g., an anti-PD-1 or anti-PD-
L1 antibody.
[41] In some embodiments, the methods and compositions for use according to
the present disclosure
encompass providing treatment to a treatment-naïve subject. In some
embodiments, the methods and
compositions for use according to the present disclosure encompass providing
treatment to a subject who has
previously received a cancer therapy or who is currently receiving cancer
therapy. A previous cancer therapy may
be the same cancer therapy to be administered according to the invention. The
cancer therapy may be checkpoint
inhibitor (CPI) therapy. In some embodiments, the methods and compositions for
use according to the present
disclosure encompass providing treatment to a cancer subject wherein the
cancer is or is suspected of being
immune suppressive (e.g., having a tumor with an immune excluded or
immunosuppressive phenotype).
[42] In some embodiments, the methods and compositions for use according to
the present disclosure
encompass providing treatment to a subject having a cancer with a high
response rate to checkpoint inhibitor
therapy (e.g., overall response rate of greater than 30%, greater 40%, greater
than 50%, or greater). Examples of
cancer with high response rates to checkpoint inhibitor therapy include, but
are not limited to, microsatellite
instability-colorectal cancer (MSI-CRC), renal cell carcinoma (RCC), melanoma
(e.g., metastatic melanoma),
Hodgkin's lymphoma, NSCLC, cancer with high microsatellite instability (MSI-
H), cancer with mismatch repair
deficiency (dMMR), primary mediastinal large B-cell lymphoma (PMBCL), and
Merkel cell carcinoma (e.g., as
reported in Haslam et al., JAMA Network Open. 2019;2(5): e192535).
[43] In some embodiments, the methods and compositions for use according to
the present disclosure
encompass providing treatment to a subject having a cancer with a low response
rate to checkpoint inhibitor therapy
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(e.g., overall response rate of 30% or less, 20% or less, or 10%, or less). In
some embodiments, the subject may
be treatment-naïve. In some embodiments, the subject may be resistant to
checkpoint inhibitor therapy. Examples
of cancer with low response rates to checkpoint inhibitor therapy include, but
are not limited to, ovarian cancer,
gastric cancer, and triple-negative breast cancer.
[44] In various embodiments, the methods and compositions for use according
to the present disclosure
encompass providing treatment to a subject having a solid cancer. In some
embodiments, the solid cancer is
selected from melanoma (e.g., metastatic melanoma), renal cell carcinoma,
breast cancer, e.g., triple-negative
breast cancer, HER2-positive breast cancer, colorectal cancer, e.g.,
microsatellite stable-colorectal cancer and
colon adenocarcinoma, lung cancer (e.g., metastatic non-small cell lung
cancer, small cell lung cancer),
esophageal cancer, pancreatic cancer, bladder cancer, kidney cancer, e.g.,
transitional cell carcinoma, renal
sarcoma, and renal cell carcinoma (RCC), including clear cell RCC, papillary
RCC, chromophobe RCC, collecting
duct RCC, or unclassified RCC, uterine cancer, e.g., uterine corpus
endometrial carcinoma, prostate cancer,
stomach cancer (e.g., gastric cancer), head and neck cancer, e.g., head and
neck squamous cell cancer, urothelial
carcinoma, hepatocellular carcinoma, thyroid cancer, or tenosynovial giant
cell tumor (TGCT).
[45] In some embodiments, a TGFp inhibitor of the present disclosure may be
used to treat, including to improve
rates or ratios of complete verses partial responses among the responders of a
cancer therapy. Typically, even in
cancer types where response rates to a cancer therapy (e.g., a checkpoint
inhibitor therapy) are relatively high
(e.g., nO% responders), complete response rates are low. The TGFP inhibitors
of the present disclosure may
therefore be used to increase the fraction of complete responders within the
responder population. In preferred
embodiments, the TGFI3 inhibitor is Ab6.
[46] In various embodiments, the TGFp inhibitor of the present disclosure
does not inhibit TGF[32 signaling at a
therapeutically effective dose. In some embodiments, the TGFp inhibitor does
not inhibit TGFp3 signaling at a
therapeutically effective dose. In some embodiments, the TGFp inhibitor does
not inhibit 1GF132 signaling and
TGFI33 signaling at a therapeutically effective dose.
[47] In various embodiments, the TGFp inhibitor is a TGFpl -selective
inhibitor. In some embodiments, the TGFp
inhibitor may bind TGFp1 with an affinity of 0.5 nM or greater (KD < 0 5 nM)
with a dissociation rate of no more
than 10.0E-4 (1/s) as measured by SPR. More preferably, the TGFP inhibitor is
an activation inhibitor of TGFP1.
For example, the activation inhibitor may be a monoclonal antibody or an
antigen-binding fragment thereof that
binds the latent lasso region of a latent TGFp1 complex. In some embodiments,
the TGFp inhibitor is Ab4, Ab5,
Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33,
or Ab34. Most preferably, the
TGFp inhibitor is Ab6 or a variant thereof (e.g., a variant of Ab6 as used
herein is one that retains at least 80%,
90%, 95% or greater sequence similarity to Ab6 and/or retains one or more
binding and/or therapeutic properties
of Ab6, so as to achieve a desired therapeutic effect).
[48] In various embodiments, the methods and compositions for use disclosed
herein comprise use of a TGFp
inhibitor disclosed herein in conjunction (e.g., in combination) with a
checkpoint inhibitor and/or a genotoxic therapy,
wherein the checkpoint inhibitor is an anti-PD-1 antibody, anti-PD-L1
antibody, anti-CTLA-4-antibody, anti-LAG3
antibody, or an antigen-binding fragment thereof; and/or the genotoxic therapy
is a chemotherapy or a radiation
therapy, wherein optionally, the chemotherapy is a PARP inhibitor therapy.
[49] In various embodiments, the methods and compositions for use disclosed
herein comprise use of a TGFp
inhibitor disclosed herein in conjunction with at least one additional
therapy. In some embodiments, the at least
one additional therapy is a cancer therapy, such as an immunotherapy, a
genotoxic therapy, including
chemotherapy and radiation therapy (including radiotherapeutic agents), an
engineered immune cell therapy (e.g.,
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CAR-T therapy), a cancer vaccine therapy, and/or an oncolytic viral therapy.
In some embodiments, the at least
one additional therapy is chemotherapy or radiation therapy (including
radiotherapeutic agents). In some
embodiments, the at least one additional cancer therapy is a checkpoint
inhibitor therapy. In some embodiments,
the checkpoint inhibitor may comprise an agent targeting programmed cell death
protein 1 (PD-1) or programmed
cell death protein 1 ligand (PD-L1). For instance, the checkpoint inhibitor
may comprise an anti-PD-1 or anti-PD-
L1 antibody. In some embodiments, the TGF8 inhibitors disclosed herein may be
used in conjunction with at least
one additional therapy selected from: a PD-1 antagonist (e.g., a PD-1
antibody), a PDL1 antagonist (e.g., a PDL1
antibody), a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist (e.g., a CTLA4
antibody), a GITR agonist e.g., a
GITR antibody), an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3
antibody, an anti-B7H4 antibody,
an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-0X40 antibody (0X40
agonist), an anti-CD27 antibody, an
anti-CD70 antibody, an anti-CD47 antibody, an anti-41 BB antibody, an anti-PD-
1 antibody, an anti-CD20 antibody,
an anti-CD3 antibody, an anti-PD-1/anti-PDL1 bispecific or multispecific
antibody, an anti-CD3/anti-CD20 bispecific
or multispecific antibody, an anti-HER2 antibody, an anti-CD79b antibody, an
anti-CD47 antibody, an antibody that
binds T cell immunoglobulin and ITIM domain protein (TIGIT), an anti-ST2
antibody, an anti-beta7 integrin (e.g., an
anti-alpha4-beta7 integrin and/or alphaE beta7 integrin), a CDK inhibitor, an
oncolytic virus, an indoleamine 2,3-
dioxygen2se (IDO) inhibitor, and/or a PARP inhibitor.
BRIEF DESCRIPTION OF THE FIGURES
[50] FIG. 1 shows tumor MDSC levels measured in MBT-2 tumors.
[51] FIG. 2 shows tumor volume and circulating G-MDSC and M-MDSC levels in
MBT-2 mice.
[52] FIG. 3 shows tumor volume in MBT-2 mice across treatment groups.
[53] FIG. 4 shows baseline level of circulating MDSCs in non-tumor bearing
mice.
[54] FIG. 5 shows levels of circulating MDSCs in tumor-bearing mice.
[55] FIG. 6 shows a comparison of circulating MDSC levels in non-tumor
bearing mice and tumor-bearing mice.
[56] FIG. 7A shows a comparison of circulating M-MDSC and G-MDSC levels on
days 3-10; FIG. 7B shows
time-course of changes in circulating M-MDSC and G-MDSC levels from days 3-10.
[57] FIG. 8 is a plot of circulating MDSC level and tumor volume on day 10
across treatment groups.
[58] FIG. 9A shows tumor MDSC levels in different treatment groups; FIG. 9B
shows a comparison of circulating
G-MDSC levels and tumor MDSC levels on day 10 across treatment groups.
[59] FIG. 10 shows correlation of tumor MDSC levels to circulating MDSC
levels.
[60] FIG. 11 shows tumor G-MDSC and tumor 008+ cells across all treatment
groups.
[61] FIG. 12A shows circulating gMDSC and mMDSC levels in whole blood of
mice bearing MBT2 tumors; FIG.
12B shows intratumoral gMDSC and mMDSC levels in mice bearing MBT2 tumors.
[62] FIG. 13A shows circulating TGFI31 levels (pg/mL) in MBT-2 mice; FIG.
13B shows plasma levels of Ab6
(pg/mL, left) and TGF31 (pg/mL, right); FIG. 13C shows correlation of plasma
levels of Ab6 (pg/mL) and TGF31
(pg/mL) in MBT-2 mice treated with AB6 alone or in combination with an anti-
PD1 antibody.
[63] FIG. 14 shows plasma platelet factor 4 levels (ng/mL) in MBT-2 mice
(right) and sample outliers as
determined by interguartile range (left).
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[64] FIG. 15 shows identified sample outliers (left) and outlier-corrected
levels (pg/mL) of circulatory TGFpl
(right).
[65] FIG. 16 shows circulatory TGF13 levels in NHP following a single dose
of Ab6.
[66] FIG. 17 shows circulatory TGFp levels in rats following a single dose
of Ab6.
[67] FIG. 18 shows an exemplary sample collection and processing method for
evaluating circulating TGF31
levels in blood.
[68] FIG. 19A shows circulating TGF(31 levels in blood samples as evaluated
under various sample processing
conditions.
[69] FIG. 19B shows platelet factor 4 (PF4) levels in blood samples as
evaluated under various sample
processing conditions.
[70] FIG. 20 shows correlation of circulating TGF(31 levels and PF4 levels
in blood samples as evaluated under
various sample processing conditions.
[71] FIG. 21A shows PF4 levels in blood samples as evaluated under various
sample processing conditions;
FIG. 21B and FIG. 21C show exemplary outlier analysis based on measurement of
PF4 levels.
[72] FIG. 22 shows PF4 vs. TGFI31 levels pre-dose and 1 hour post-dose.
[73] FIG. 23A shows fold change in TGFp levels over time in subjects
treated with 80-240 mg of Ab6; FIG. 23B
shows fold change in TGFp levels over time in subjects treated with 800 mg of
Ab6; FIG. 23C shows fold change
in TGFp levels over time in subjects treated with 1600 mg of Ab6.
[74] FIG. 24 shows a P-Smad2 IHC analysis of melanoma samples.
[75] FIG. 25 shows pSmad-2 signaling in MBT2 tumors following treat with
Ab6-mIgG1.
[76] FIG. 26A shows tissue compartment data of bladder cancer samples; FIG.
26B shows tissue compartment
data of melanoma samples.
[77] FIG. 27 shows density of CD8+ cells in bladder cancer samples as
analyzed based on tumor nest.
[78] FIG. 28 shows immune phenotype analysis of a single bladder cancer
sample based on density of CD8+
cells measured in tumor nests.
[79] FIG. 29A shows average percentages of CD8+ cells and immune
phenotyping in bladder cancer and
melanoma samples, as analyzed by tumor compartments (left) and tumor nests
(right); underlined phenotype
reflects differences between analyses; FIG. 29B shows average percentages of
CD8+ cells and immune
phenotyping in bladder cancer and melanoma samples, as analyzed by tumor
compartments (left) and tumor nests
(right); underlined phenotype reflects differences between analyses; FIG. 29C
shows tumor nest data and immune
phenotyping for individual tumor nests identified from bladder cancer samples;
FIG. 29D shows tumor nest CD8+
data and immune phenotyping for bladder cancer and melanoma samples; FIG. 29E
shows percent CD8+ cells in
tumor, tumor margin, and stroma compartments of commercially available bladder
cancer samples.
[80] FIG. 30A shows representative CD8+ staining in bladder cancer samples;
FIG. 30B shows subdivision of
CD8+ staining in the tumor margin compartment; FIG. 30C shows subdivision of
CD8+ staining in the tumor margin
compartment of a bladder sample.
[81] FIG. 31 shows comparison of compartment CD8+ ratio and absolute
percent CD8 positivity.
[82] FIG. 32 shows comparison of CD8+ cell density and absolute percent CD8
positivity.
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[83] FIG. 33 shows tumor depth of bladder samples.
[84] FIG. 34 shows CD8 density in a melanoma sample.
[85] FIGs. 35A-c show exemplary analysis of MDSC by signal filtering.
[86] FIGs. 36A-C shows identification of tumor MDSC populations in various
solid cancer samples.
[87] FIGs. 37A-C shows analysis of gMDSC and mMDSC populations in various
solid cancer samples.
[88] FIG. 38 shows a schematic of an exemplary pathology analysis of tumor
tissue sample.
[89] FIG. 39 shows a schematic of an exemplary pathology analysis of tumor
tissue sample.
[90] FIG. 40 shows a schematic of an exemplary TGFp inhibitor treatment
regimen.
[91] FIG. 41 illustrates identification of three binding regions (Region 1,
Region 2, and Region 3) following
statistical analyses. Region 1 overlaps with a region called "Latency Lasso"
within the prodomain of proTGFI31,
while Regions 2 and 3 are within the growth factor domain.
[92] FIG. 42 depicts various domains and motifs of proTGF131, relative to
the three binding regions involved in
Ab6 binding. Sequence alignment among the three isoforms is also provided.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Definitions
[93] In order that the disclosure may be more readily understood, certain
terms are first defined. These
definitions should be read in light of the remainder of the disclosure and as
understood by a person of ordinary skill
in the art. Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as
commonly understood by a person of ordinary skill in the art. Additional
definitions are set forth throughout the
detailed description.
[94] Advanced cancer, advanced malignancy: The term "advanced cancer" or
"advanced malignancy" as used
herein has the meaning understood in the pertinent art, e.g., as understood by
oncologists in the context of
diagnosing or treating subjects/patients with cancer. Advanced malignancy with
a solid tumor can be locally
advanced or metastatic. The term "locally advanced cancer" is used to describe
a cancer (e.g., tumor) that has
grown outside the organ it started in but has not yet spread to distant parts
of the body. Thus, the term includes
cancer that has spread from where it started to nearby tissue or lymph nodes.
By contrast, "metastatic cancer" is
a cancer that has spread from the part of the body where it started (the
primary site) to other parts (e.g., distant
parts) of the body.
[95] Affinity: Affinity is the strength of binding of a molecule (such as
an antibody) to its ligand (such as an
antigen). It is typically measured and reported by the equilibrium
dissociation constant (KO. In the context of
antibody-antigen interactions, Ko is the ratio of the antibody dissociation
rate ("off rate" or Koff), how quickly it
dissociates from its antigen, to the antibody association rate ("on rate" or
K.) of the antibody, how quickly it binds
to its antigen. For example, an antibody with an affinity of s 5 nM has a Ko
value that is 5 nM or lower (i.e., 5 nM
or higher affinity) determined by a suitable in vitro binding assay. Suitable
in vitro assays can be used to measure
KD values of an antibody for its antigen, such as Biolayer lnterferometry
(BLI) and Solution Equilibrium Titration
(e.g., MSD-SET). In a preferred embodiment, affinity is measured by surface
plasmon resonance (e.g., Biacoree).
An antibody with a suitable affinity in a surface plasmon resonance assay may
have, e.g., a Ko of at most about 1
nM, e.g., at most about 0.5 nM, e.g., at most about 0.5, 0.4, 0.3, 0.2, 0.15
nM, or less.
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[96] Antibody: The term "antibody" encompasses any naturally-occurring,
recombinant, modified or engineered
immunoglobulin or immunoglobulin-like structure or antigen-binding fragment or
portion thereof, or derivative
thereof, as further described elsewhere herein. Thus, the term refers to an
immunoglobulin molecule that
specifically binds to a target antigen, and includes, for instance, chimeric,
humanized, fully human, and mu ltispecific
antibodies (including bispecific antibodies). An intact antibody will
generally comprise at least two full-length heavy
chains and two full-length light chains, but in some instances can include
fewer chains such as antibodies naturally
occurring in camelids which can comprise only heavy chains. Antibodies can be
derived solely from a single
source, or can be "chimeric," that is, different portions of the antibody can
be derived from two different antibodies.
Antibodies, or antigen binding portions thereof, can be produced in
hybridomas, by recombinant DNA techniques,
or by enzymatic or chemical cleavage of intact antibodies. The term
antibodies, as used herein, includes
monoclonal antibodies, multispecific antibodies such as bispecific antibodies,
minibodies, domain antibodies,
synthetic antibodies (sometimes referred to herein as "antibody mimetics"),
chimeric antibodies, humanized
antibodies, human antibodies, antibody fusions (sometimes referred to herein
as "antibody conjugates"),
respectively. In some embodiments, the term also encompasses peptibodies.
[97] Antigen: The term "antigen" broadly includes any molecules comprising
an antigenic determinant within a
binding region(s) to which an antibody or a fragment specifically binds. An
antigen can be a single-unit molecule
(such as a protein monomer or a fragment) or a complex comprised of multiple
components. An antigen provides
an epitope, e.g., a molecule or a portion of a molecule, or a complex of
molecules or portions of molecules, capable
of being bound by a selective binding agent, such as an antigen binding
protein (including, e.g., an antibody). Thus,
a selective binding agent may specifically bind to an antigen that is formed
by two or more components in a
complex. In some embodiments, the antigen is capable of being used in an
animal to produce antibodies capable
of binding to that antigen. An antigen can possess one or more epitopes that
are capable of interacting with
different antigen binding proteins, e.g., antibodies. In the context of the
present disclosure, a suitable antigen is a
complex (e.g., multimeric complex comprised of multiple components in
association) containing a proTGF dimer in
association with a presenting molecule. Each monomer of the proTGF dimer
comprises a prodomain and a growth
factor domain, separated by a furin cleavage sequence. Two such monomers form
the proTGF dimer complex.
This in turn is covalently associated with a presenting molecule via disulfide
bonds, which involve a cysteine residue
present near the N-terminus of each of the proTGF monomer. This multi-complex
formed by a proTGF dimer
bound to a presenting molecule is generally referred to as a large latent
complex. An antigen complex suitable for
screening antibodies or antigen-binding fragments, for example, includes a
presenting molecule component of a
large latent complex. Such presenting molecule component may be a full-length
presenting molecule or a
fragment(s) thereof. Minimum required portions of the presenting molecule
typically contain at least 50 amino
acids, but more preferably at least 100 amino acids of the presenting molecule
polypeptide, which comprises two
cysteine residues capable of forming covalent bonds with the proTGFI31 dimer.
[98] Antigen-binding portion/fragment: The terms "antigen-binding portion" or
"antigen-binding fragment" of an
antibody, as used herein, refers to one or more fragments of an antibody that
retain the ability to specifically bind
to an antigen (e.g., TGFI31). Antigen binding portions include, but are not
limited to, any naturally occurring,
enzymatically obtainable, synthetic, or genetically engineered polypeptide or
glycoprotein that specifically binds an
antigen to form a complex. In some embodiments, an antigen-binding portion of
an antibody may be derived, e.g.,
from full antibody molecules using any suitable standard techniques such as
proteolytic digestion or recombinant
genetic engineering techniques involving the manipulation and expression of
DNA encoding antibody variable and
optionally constant domains. Non-limiting examples of antigen-binding portions
include: (i) Fab fragments, a
monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2
fragments, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the hinge region;
(iii) Fd fragments consisting of the
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VH and CHI domains;; (iv) Fv fragments consisting of the VL and VH domains of
a single arm of an antibody; (v)
single-chain Fv (scFv) molecules (see, e.g., Bird et al., (1988) Science
242:423-426; and Huston et al., (1988)
Proc. Nat'l. Acad. Sci. USA 85:5879-5883); (vi) dAb fragments (see, e.g., Ward
et al., (1989) Nature 341: 544-546);
and (vii) minimal recognition units consisting of the amino acid residues that
mimic the hypervariable region of an
antibody (e.g., an isolated complementarity determining region (CDR)). Other
forms of single chain antibodies,
such as diabodies are also encompassed. The term antigen binding portion of an
antibody includes a "single chain
Fab fragment" otherwise known as an "scFab," comprising an antibody heavy
chain variable domain (VH), an
antibody constant domain 1 (CH1), an antibody light chain variable domain
(VL), an antibody light chain constant
domain (CL) and a linker, wherein said antibody domains and said linker have
one of the following orders in N-
terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-
CH1, c) VH-CL-linker-VL-CH1 or d)
VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide of at least 30
amino acids, preferably between 32
and 50 amino acids.
[99] Bias: In the context of the present disclosure, the term "bias" (as in
"biased binding") refers to skewed or
uneven affinity towards or against a subset of antigens to which an antibody
is capable of specifically binding.
For example, an antibody is said to have bias when the affinity for one
antigen complex and the affinity for
another antigen complex are not equivalent. Context-independent antibodies
according to the present disclosure
have equivalent affinities towards such antigen complexes (i.e., unbiased or
uniform).
[100] Binding region: As used herein, a "binding region" is a portion of an
antigen that, when bound to an
antibody or a fragment thereof, can form an interface of the antibody-antigen
interaction. Upon antibody binding,
a binding region becomes protected from surface exposure, which can be
detected by suitable techniques, such
as HDX-MS. Antibody-antigen interaction may be mediated via multiple (e_g ,
two or more) binding regions. A
binding region can comprise an antigenic determinant, or epitope.
[101] Biolayer Interferometry (BLI): BLI is a label-free technology for
optically measuring biomolecular
interactions, e.g., between a ligand immobilized on the biosensor tip surface
and an analyte in solution. BLI
provides the ability to monitor binding specificity, rates of association and
dissociation, or concentration, with
precision and accuracy. BLI platform instruments are commercially available,
for example, from ForteBio and are
commonly referred to as the Octet System.
[102] Cancer: The term "cancer" as used herein refers to the physiological
condition in multicellular eukaryotes
that is typically characterized by unregulated cell proliferation and
malignancy. The term broadly encompasses,
solid and liquid malignancies, including tumors, blood cancers (e.g.,
leukemias, lymphomas and myelomas), as
well as myelofibrosis.
[103] Cell-associated proTGR31: The term refers to TGF61 or its signaling
complex (e.g., pro/latent TGF61) that
is membrane-bound (e.g., tethered to cell surface). Typically, such cell is an
immune cell. TGF61 that is presented
by GARP or LRRC33 is a cell-associated TGF(31. GARP and LRRC33 are
transmembrane presenting molecules
that are expressed on cell surface of certain cells. GARP-proTGF61 and LRRC33-
may be collectively referred to
as "cell-associated" (or "cell-surface") proTGF61 complexes, that mediate cell
proTGF61-associated (e.g., immune
cell-associated) TGF61 activation/signaling. The term also includes
recombinant, purified GARP-proTGF61 and
LRRC33-proTG931 complexes in solution (e.g., in vitro assays) which are not
physically attached to cell
membranes. Average KD values of an antibody (or its fragment) to a GARP-
proTGF61 complex and an LRRC33-
proTGF61 complex may be calculated to collectively represent affinities for
cell-associated (e.g., immune cell-
associated) proTGF61 complexes. See, for example, Table 5, column (G). Human
counterpart of a presenting
molecule or presenting molecule complex may be indicated by an "h" preceding
the protein or protein complex,
e.g., "hGARP," "hGARP-proTGF61," hLRRC33" and "hLRRC33-proTGF61." In addition
to blocking release of
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active TG931 growth factor from cell-tethered complexes, cell-associated
proTGFI31 may be a target for
internalization (e.g., endocytosis) and/or cell killing such as ADCC, ADCP, or
ADC-mediated depletion of the target
cells expressing such cell surface complexes.
[104] Checkpoint inhibitor: In the context of this disclosure, checkpoint
inhibitors refer to immune checkpoint
inhibitors and carries the meaning as understood in the art. A "checkpoint
inhibitor therapy" or "checkpoint blockade
therapy" is one that targets a checkpoint molecule to partially or fully alter
its function. Typically, a checkpoint is a
receptor molecule on a T cell or NK cell, or a corresponding cell surface
ligand on an antigen-presenting cell (APC)
or tumor cell. Without being bound by theory, immune checkpoints are activated
in immune cells to prevent
inflammatory immunity developing against the "self". Therefore, changing the
balance of the immune system via
checkpoint inhibition may allow it to be fully activated to detect and
eliminate the cancer. The best known inhibitory
receptors implicated in control of the immune response are cytotoxic T-
lymphocyte antigen-4 (CTLA-4),
programmed cell death protein 1 (PD-1), programmed cell death receptor ligand
1 (PD-L1), T-cell immunoglobulin
domain and mucin domain-3 (TIM3), lymphocyte-activation gene 3 (LAG3), killer
cell immunoglobulin-like receptor
(KIR), glucocorticoid-induced tumor necrosis factor receptor (GITR) and V-
domain immunoglobulin (Ig)-containing
suppressor of T-cell activation (VISTA). Non-limiting examples of checkpoint
inhibitors include: Nivolumab,
Pembrolizumab, cemiplimab, BMS-936559, Atezolizumab, Avelumab, Durvalumab,
1pilimumab, Tremelimumab,
IMP-321 (Eftilagimod alpha or ImmuFacte), BMS-986316 (Relatlimab), budigalimab
(ABBV-181), and Lirilumab.
Keytrudae is one example of anti-PD-1 antibodies, while Opdivo0 is one example
of an anti-PD-L1 antibody.
Therapies that employ one or more of immune checkpoint inhibitors may be
referred to as checkpoint blockade
therapy (CBT) or checkpoint inhibitor therapy (CPI).
[105] Clinical benefit: As used herein, the term "clinical benefits" is
intended to include both efficacy and safety
of a therapy. Thus, therapeutic treatment that achieves a desirable clinical
benefit is both efficacious (e.g., achieves
therapeutically beneficial effects) and safe (e.g., with tolerable or
acceptable levels of toxicities or adverse events).
[106] Combination therapy: "Combination therapy" refers to treatment regimens
for a clinical indication that
comprise two or more therapeutic agents. Thus, the term refers to a
therapeutic regimen in which a first therapy
comprising a first composition (e.g., active ingredient) is administered in
conjunction with at least a second therapy
comprising a second composition (active ingredient) to a patient, intended to
treat the same or overlapping disease
or clinical condition. The term may further encompass a therapeutic regimen in
which a first therapy comprising a
first composition (e.g., active ingredient) is administered in conjunction
with a second therapy comprising a second
composition (e.g., active ingredient such as a checkpoint inhibitor), a third
therapy comprising a third composition
(e.g., active ingredient such as a chemotherapy), or more (e.g., additional
distinct active ingredients). The first,
second, and (optionally additional) compositions may act on the same cellular
target, or discrete cellular targets.
The phrase "in conjunction with," in the context of combination therapies,
means that therapeutic effects of a first
therapy overlaps temporally and/or spatially with therapeutic effects of a
second and additional therapy in the
subject receiving the combination therapy. The first, second, and/or
additional compositions may be administered
concurrently (e.g., simultaneously), separately, or sequentially. Thus, the
combination therapies may be formulated
as a single formulation for concurrent administration, or as separate
formulations, for sequential, concurrent, or
simultaneous administration of the therapies. When a subject who has been
treated with a first therapy to treat a
disease is administered with a second and additional therapies to treat the
same disease, the second and additional
therapies may be referred to as an add-on therapy or adjunct therapy.
[107] Combinatory or combinatorial epitope: A combinatorial epitope is an
epitope that is recognized and bound
by a combinatorial antibody at a site (i.e., antigenic determinant) formed by
non-contiguous portions of a component
or components of an antigen, which, in a three-dimensional structure, come
together in close proximity to form the
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epitope. Thus, antibodies of the disclosure may bind an epitope formed by two
or more components (e.g., portions
or segments) of a pro/latent TG931 complex. A combinatory epitope may comprise
amino acid residue(s) from a
first component of the complex, and amino acid residue(s) from a second
component of the complex, and so on.
Each component may be of a single protein or of two or more proteins of an
antigenic complex. A combinatory
epitope is formed with structural contributions from two or more components
(e.g., portions or segments, such as
amino acid residues) of an antigen or antigen complex.
[108] Compete or cross-compete; cross-block: The term "compete" when used in
the context of antigen binding
proteins (e.g., an antibody or antigen binding portion thereof) that compete
for the same epitope means competition
between antigen binding proteins as determined by an assay in which the
antigen binding protein being tested
prevents or inhibits (e.g., reduces) specific binding of a reference antigen
binding protein to a common antigen
(e.g., TGFI31 or a fragment thereof). Numerous types of competitive binding
assays can be used to determine if
one antigen binding protein competes with another, for example: solid phase
direct or indirect radioimmunoassay
(RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich
competition assay; solid phase direct
biotin-avidin EIA; solid phase direct labeled assay, and solid phase direct
labeled sandwich assay. Usually, when
a competing antigen binding protein is present in excess, it will inhibit
(e.g., reduce) specific binding of a reference
antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-
55%, 55-60%, 60-65%, 65-70%, 70-
75% or 75% or more. In some instances, binding is inhibited by at least 80-
85%, 85-90%, 90-95%, 95-97%, or
97% or more when the competing antibody is present in excess. In some
embodiments, an SPR (e.g., Biacore)
assay is used to determine competition. In some embodiments, a BLI (e.g.,
Octet ) assay is used to determine
competition
[109] In some embodiments, a first antibody or antigen-binding portion thereof
and a second antibody or antigen-
binding portion thereof "cross-block" with each other with respect to the same
antigen, for example, as assayed by
Biolayer lnterferometry (such as Octets) or by surface plasmon resonance (such
as Biacore System), using
standard test conditions, e.g., according to the manufacturer's instructions
(e.g., binding assayed at room
temperature, ¨20-25 C). In some embodiments, the first antibody or fragment
thereof and the second antibody or
fragment thereof may have the same epitope. In other embodiments, the first
antibody or fragment thereof and the
second antibody or fragment thereof may have non-identical but overlapping
epitopes. In yet further embodiments,
the first antibody or fragment thereof and the second antibody or fragment
thereof may have separate (different)
epitopes which are in close proximity in a three-dimensional space, such that
antibody binding is cross-blocked via
steric hindrance. "Cross-block" means that binding of the first antibody to an
antigen prevents binding of the second
antibody to the same antigen, and similarly, binding of the second antibody to
an antigen prevents binding of the
first antibody to the same antigen.
[110] Antibody binning (sometimes referred to as epitope binning or epitope
mapping) may be carried out to
characterize and sort a set (e.g., "a library") of monoclonal antibodies made
against a target protein or protein
complex (i.e., antigen). Such antibodies against the same target are tested
against all other antibodies in the library
in a pairwise fashion to evaluate if antibodies block one another's binding to
the antigen. Closely related binning
profiles indicate that the antibodies have the same or closely related (e.g.,
overlapping) epitope and are "binned"
together. Binning provides useful structure-function profiles of antibodies
that share similar binding regions within
the same antigen because biological activities (e.g., intervention; potency)
effectuated by binding of an antibody to
its target is likely to be carried over to another antibody in the same bin.
Thus, among antibodies within the same
epitope bin, those with higher affinities (lower KD) typically have greater
potency.
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[111] In some embodiments, an antibody that binds the same epitope as Ab6
binds a proTGF61 complex such
that the epitope of the antibody includes one or more amino acid residues of
Region 1, Region 2 and Region 3,
identified as the binding region of Ab6.
[112] Complementary determining region: As used herein, the term "CDR" refers
to the complementarity
determining region within antibody variable sequences. There are three CDRs in
each of the variable regions of
the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3,
for each of the variable regions.
The term "CDR set" as used herein refers to a group of three CDRs that occur
in a single variable region that can
bind the antigen. The exact boundaries of these CDRs have been defined
differently according to different systems.
The system described by Kabat (Kabat et al., (1987; 1991) Sequences of
Proteins of Immunological Interest
(National Institutes of Health, Bethesda, Md.) not only provides an
unambiguous residue numbering system
applicable to any variable region of an antibody, but also provides precise
residue boundaries defining the three
CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers
(Chothia & Lesk (1987) J. MoL
Biol. 196: 901-917; and Chothia et al., (1989) Nature 342: 877-883) found that
certain sub-portions within Kabat
CDRs adopt nearly identical peptide backbone conformations, despite having
great diversity at the level of amino
acid sequence. These sub-portions were designated as Li, L2 and L3 or H1, H2
and H3, or L-CDR1, L-CDR2 and
L-CDR3 or H-CDR1, H-CDR2 and H-CDR3, where the "L" and the "H" designate the
light chain and the heavy
chain regions, respectively. These regions may be referred to as Chothia CDRs,
which have boundaries that
overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the
Kabat CDRs have been described
by Padlan (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. MoL Biol. 262(5):
732-45. Still other CDR
boundary definitions may not strictly follow one of the herein systems, but
will nonetheless overlap with the Kabat
CDRs, although they may be shortened or lengthened in light of prediction or
experimental findings that particular
residues or groups of residues or even entire CDRs do not significantly impact
antigen binding (see, for example:
Lu X et al., MAbs. 2019 Jan;11(1):45-57). The methods used herein may utilize
CDRs defined according to any of
these systems, although certain embodiments use Kabat or Chothia defined CDRs.
[113] Conformational epitope: A conformational epitope is an epitope that is
recognized and bound by a
conformational antibody in a three-dimensional conformation, but not in an
unfolded peptide of the same amino
acid sequence. A conformational epitope may be referred to as a conformation-
specific epitope, conformation-
dependent epitope, or conformation-sensitive epitope. A corresponding antibody
or fragment thereof that
specifically binds such an epitope may be referred to as conformation-specific
antibody, conformation-selective
antibody, or conformation-dependent antibody. Binding of an antigen to a
conformational epitope depends on the
three-dimensional structure (conformation) of the antigen or antigen complex.
[114] Constant region: An immunoglobulin constant domain refers to a heavy or
light chain constant domain.
Human IgG heavy chain and light chain constant domain amino acid sequences are
known in the art.
[115] Context-biased: As used herein, "context-biased antibodies" refer to a
type of conformational antibodies
that binds an antigen with differential affinities when the antigen is
associated with (i.e.., bound to or attached to)
an interacting protein or a fragment thereof. Thus, a context-biased antibody
that specifically binds an epitope
within proTGF61 may bind LTBP1-proTGF61, LTBP3-proTGF61, GARP-proTGF61 and
LRRC33-proTGF61 with
different affinities. For example, an antibody is said to be "matrix-
biased" if it has higher affinities for matrix-
associated broTGF61 complexes (e.g., LTBP1-proTGF61 and LTBP3-proTGF61) than
for cell-associated
proTGFpl complexes (e.g., GARP-proTGF61 and LRRC33-proTGF61). Relative
affinities of [matrix-associated
complexes] : [cell-associated complexes] may be obtained by taking average KID
values of the former, taking
average Ko values of the latter, and calculating the ratio of the two, as
exemplified herein.
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[116] Context-independent: According to the present disclosure, "a context-
independent antibody that binds
proTGF131 has equivalent affinities across the four known presenting molecule-
proTGF61 complexes, namely,
LTBP1-proTGF61, LTBP3-proTGF61, GARP-proTGF61 and LRRC33-proTGF131. Context-
independent
antibodies disclosed in the present application may also be characterized as
unbiased. Typically, context-
independent antibodies show equivalent (i.e., no more than five-fold bias in)
affinities, such that relative ratios of
measured KD values between matrix-associated complexes and cell-associated
complexes are no greater than 5
as measured by a suitable in vitro binding assay, such as surface plasmon
resonance, Biolayer Interferometry
(BLI), and/or solution equilibrium titration (e.g., MSD-SET). In a preferred
embodiment, surface plasmon resonance
is used.
[117] ECM-associated TGFI31/proTGF131: The term refers to TGF61 or its
signaling complex (e.g., pro/latent
TGF61) that is a component of (e.g., deposited into) the extracellular matrix.
TGF61 that is presented by LTBP1
or LTBP3 is an ECM-associated TGF131, namely, LTBP1-proTGF61 and LTBP3-
proTGF61, respectively. LTBPs
are critical for correct deposition and subsequent bioavailability of TGF6 in
the ECM, where fibrillin (Fbn) and
fibronectin (FN) are believed to be the main matrix proteins responsible for
the association of LTBPs with the ECM.
Such matrix-associated latent complexes are enriched in connective tissues, as
well as certain disease-associated
tissues, such as tumor stroma and fibrotic tissues. Human counterpart of a
presenting molecule or presenting
molecule complex may be indicated by an "h" preceding the protein or protein
complex, e.g., "hLTBP1," "hLTBP1-
proTGF131," hLTBP3" and "hLTBP3-proTGF61."
[118] Effective amount: The terms "effective" and "therapeutically effective"
refer to the ability or an amount to
sufficiently produce a detectable change in a parameter of a disease, e.g., a
slowing, pausing, reversing,
diminution, or amelioration in a symptom or downstream effect of the disease.
The term encompasses but does
not require the use of an amount that completely cures a disease. An
"effective amount" (or therapeutically effective
amount, or therapeutic dose) may be a dosage or dosing regimen that achieves a
statistically significant clinical
benefit (e.g., efficacy) in a patient population. For example, Ab6 has been
shown to be efficacious at doses as low
as 3 mg/kg and as high as 30 mg/kg in preclinical models. The term "minimum
effective dose" or "minimum effective
amount" refers to the lowest amount, dosage, or dosing regimen that achieves a
detectable change in a parameter
of a disease, e.g., a statistically significant clinical benefit. References
herein to a dose of an agent (e.g., a dose of
a TGF61 inhibitor) may be a therapeutically effective dose, as described
herein. In a clinical setting, such as human
clinical trials, the term "pharmacological active dose (PAD)" may be used to
refer to effective dosage. Effective
amounts may be expressed in terms of doses being administered or in terms of
exposure levels achieved as a
result of administration (e.g., serum concentrations).
[119] Effective tumor control: The term "effective tumor control" may be used
to refer to a degree of tumor
regression achieved in response to treatment, where, for example, the tumor is
regressed by a defined fraction
(such as <25%) of an endpoint tumor volume. For instance, in a particular
model, if the endpoint tumor volume is
set at 2,000 mm3, effective tumor control is achieved if the tumor is reduced
to less than 500 mm3 assuming the
threshold of <25%. Therefore, effective tumor control encompasses complete
regression. Clinically, effective
tumor control can be measured by objective response, which includes partial
response (PR) and complete
response (CR) as determined by art-recognized criteria, such as RECIST v1.1
and corresponding iRECIST
(iRECIST v1.1). In some embodiments, effective tumor control in clinical
settings also includes stable disease,
where tumors that are typically expected to grow at certain rates are
prevented from such growth by the treatment,
even though shrinkage is not achieved.
[120] Effector T cells: Effector T cells, as used herein, are T lymphocytes
that actively respond immediately to a
stimulus, such as co-stimulation and include, but are not limited to, C04+ T
cells (also referred to as T helper or
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Th cells) and CD8+ T cells (also referred to as cytotoxic T cells). Th cells
assist other white blood cells in
immunologic processes, including maturation of B cells into plasma cells and
memory B cells, and activation of
cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells
because they express the CD4
glycoprotein on their surfaces. Helper T cells become activated when they are
presented with peptide antigens by
MHC class II molecules, which are expressed on the surface of antigen-
presenting cells (APCs). Once activated,
they divide rapidly and secrete small proteins called cytokines that regulate
or assist in the active immune response.
These cells can differentiate into one of several subtypes, including Th1,
Th2, Th3, Th17, Th9, or TFh, which
secrete different cytokines to facilitate different types of immune responses.
Signaling from the APC directs T cells
into particular subtypes. Cytotoxic (Killer). Cytotoxic T cells (TO cells,
CTLs, 1-killer cells, killer T cells), on the
other hand, destroy virus-infected cells and cancer cells, and are also
implicated in transplant rejection. These cells
are also known as CD8+ T cells since they express the CD8 glycoprotein at
their surfaces. These cells recognize
their targets by binding to antigen associated with MHC class I molecules,
which are present on the surface of all
nucleated cells. Cytotoxic effector cell (e.g., CD8+ cells) markers include,
e.g., perforin and granzyme B.
[121] Endpoint: In studies aimed to assess effectiveness (e.g., clinical
benefit or improvements) of a therapy,
such as in clinical trials for a cancer therapy, endpoints represent the
measures of predetermined parameters
indicative of treatment effects. In oncology, suitable endpoints may include
overall survival, disease-free survival
(DFS), event-free survival (EFS), progression-free survival (PFS), objective
response rate (ORR), complete
response (CR), partial response (PR), time to progression (TTP), as well as
patient-reported outcomes (e.g.,
symptom assessment) and biomarker assessment such as blood or body fluid-based
assessments.
[122] Epithelial hyperplasia: The term "epithelial hyperplasia" refers to an
increase in tissue growth resulting
from proliferation of epithelial cells. As used herein, epithelial hyperplasia
refers to the undesired toxicity resulting
from TGFP inhibition which may include, but is not limited to, abnormal growth
of epithelial cells in the oral cavity,
esophagus, breast, and ovary.
[123] Epitope: The term "epitope" may be also referred to as an antigenic
determinant, is a molecular determinant
(e.g., polypeptide determinant) that can be specifically bound by a binding
agent, immunoglobulin, or T-cell
receptor. Epitope determinants include chemically active surface groupings of
molecules, such as amino acids,
sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may
have specific three- dimensional
structural characteristics, and/or specific charge characteristics. An epitope
recognized by an antibody or an
antigen-binding fragment of an antibody is a structural element of an antigen
that interacts with CDRs (e.g., the
complementary site) of the antibody or the fragment. An epitope may be formed
by contributions from several
amino acid residues, which interact with the CDRs of the antibody to produce
specificity. An antigenic fragment
can contain more than one epitope. In certain embodiments, an antibody may
specifically bind an antigen when it
recognizes its target antigen in a complex mixture of proteins and/or
macromolecules. For example, antibodies
are said to "bind to the same epitope" if the antibodies cross-compete (one
prevents the binding or modulating
effect of the other).
[124] Extended Latency Lasso: The term "Extended Latency Lasso" as used herein
refers to a portion of the
prodomain that comprises Latency Lasso and Alpha-2 Helix, e.g.,
LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ
ID NO: 127). In some embodiments, Extended Latency Lasso further comprises a
portion of Alpha-1 Helix, e.g.,
LVKRKRIEA (SEQ ID NO: 132) or a portion thereof.
[125] Fibrosis: The term "fibrosis" or "fibrotic condition/disorder" refers
to the process or manifestation
characterized by the pathological accumulation of extracellular matrix (ECM)
components, such as collagens,
within a tissue or organ.
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[126] Finger-1 (of TGF/31 Growth Factor): As used herein, "Finger-1" is a
domain within the TGF31 growth factor
domain. In its unmutated form, Finger-1 of human proTGF131 contains the
following amino acid sequence:
CVRQLYIDFRKDLGVVKWIHEPKGYHANFC (SEQ ID NO: 124). In the 3D structure, the
Finger-1 domain comes
in close proximity to Latency Lasso.
[127] Finger-2 (of TGFI31 Growth Factor): As used herein, "Finger-2" is a
domain within the TGFp1 growth factor
domain. In its unmutated form, Finger-2 of human proTGF61 contains the
following amino acid sequence:
CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 125). Finger-2 includes the
"binding region 6",
which spatially lies in close proximity to Latency Lasso.
[128] GARP-proTGFAI complex: As used herein, the term "GARP-TGF61 complex"
refers to a protein complex
comprising a pro-protein form or latent form of a transforming growth factor-
61 (TGF61) protein and a glycoprotein-
A repetitions predominant protein (GARP) or fragment or variant thereof. In
some embodiments, a pro-protein form
or latent form of TGF61 protein may be referred to as "pro/latent TGF61
protein". In some embodiments, a GARP-
TGF61 complex comprises GARP covalently linked with pro/latent TGF31 via one
or more disulfide bonds. In
nature, such covalent bonds are formed with cysteine residues present near the
N-terminus (e.g., amino acid
position 4) of a proTGF61 dimer complex. In other embodiments, a GARP-TGF61
complex comprises GARP non-
covalently linked with pro/latent TGF61. In some embodiments, a GARP-TGF(31
complex is a naturally-occurring
complex, for example a GARP-TGF61 complex in a cell. The term "hGARP" denotes
human GARP.
[129] High-affinity: As used herein, the term "high-affinity" as in "a high-
affinity proTGF61 antibody" refers to in
vitro binding activities having a Ko value of
5 nM, more preferably .. 1 nM. Thus, a high-affinity, context-
independent proTGF61 antibody encompassed by the disclosure herein has a KB
value of 5 nM, more preferably
1 nM, towards each of the following antigen complexes: LTBP1-proTGF61, LTBP3-
proTGF61, GARP-proTGF61
and LRRC33-proTGF61.
[130] Human antibody: The term "human antibody," as used herein, is intended
to include antibodies having
variable and constant regions derived from human germline immunoglobulin
sequences. The human antibodies
of the present disclosure may include amino acid residues not encoded by human
germline immunoglobulin
sequences (e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in
vivo), for example in the CDRs and in particular CDR3. However, the term
"human antibody," as used herein, is
not intended to include antibodies in which CDR sequences derived from the
germline of another mammalian
species, such as a mouse, have been grafted onto human framework sequences.
[131] Humanized antibody: The term "humanized antibody" refers to antibodies,
which comprise heavy and light
chain variable region sequences from a non-human species (e.g., a mouse) but
in which at least a portion of the
VH and/or VL sequence has been altered to be more "human-like," i.e., more
similar to human germline variable
sequences. One type of humanized antibody is a CDR-grafted antibody, in which
human CDR sequences are
introduced into non-human VH and VL sequences to replace the corresponding
nonhuman CDR sequences. Also
"humanized antibody" is an antibody, or a variant, derivative, analog or
fragment thereof, which immunospecifically
binds to an antigen of interest and which comprises an FR region having
substantially the amino acid sequence of
a human antibody and a CDR region having substantially the amino acid sequence
of a non-human antibody. As
used herein, the term "substantially" in the context of a CDR refers to a CDR
having an amino acid sequence at
least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least
99% identical to the amino acid
sequence of a non-human antibody CDR. A humanized antibody comprises
substantially all of at least one, and
typically two, variable domains (Fab, Fab, F(ab')2, FabC, Fv) in which all or
substantially all of the CDR regions
correspond to those of a non-human immunoglobulin (i.e., donor antibody) and
all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence. In an
embodiment a humanized antibody
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also comprises at least a portion of an immunoglobulin Fc region, typically
that of a human immunoglobulin. In
some embodiments a humanized antibody contains the light chain as well as at
least the variable domain of a
heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4
regions of the heavy chain. In
some embodiments a humanized antibody only contains a humanized light chain.
In some embodiments a
humanized antibody only contains a humanized heavy chain. In specific
embodiments a humanized antibody only
contains a humanized variable domain of a light chain and/or humanized heavy
chain.
[132] Immune-excluded or immuno-excluded tumor: As used herein, tumors
characterized as "immune excluded"
are devoid of or substantially devoid of intratumoral anti-tumor lymphocytes.
For example, tumors with poorly
infiltrated T cells may have T cells that surround the tumor, e.g., the
external perimeters of a tumor mass and/or
near the vicinity of vasculatures ('perivascular") of a tumor, which
nevertheless fail to effectively swarm into the
tumor to exert cytotoxic function against cancer cells. In other situations,
tumors fail to provoke a strong immune
response (so-called "immune desert" tumors) such that few T cells are present
near and in the tumor environment.
In contrast to immune-excluded tumors, tumors that are infiltrated with anti-
tumor lymphocytes are sometimes
characterized as 'hot" or "inflamed" tumors; such tumors tend to be more
responsive to and therefore are the target
of immune checkpoint blockade therapies ("CBTs"). Typically, however, only a
fraction of patients responds to a
CBT due to immune exclusion that renders the tumor resistant to the CBT.
[133] Immune safety (assessment): As used herein, the term refers to safety
assessment related to immune
responses (immune activation), Acceptable immune safety criteria include no
significant cytokine release as
determined by in vitro or in vivo cytokine release testing (e.g., assays); and
no significant platelet aggregation,
activation as determined with human platelets. Statistical significance in
these studies may be determined against
a suitable control as reference. For example, fora test molecule which is a
human monoclonal antibody, a suitable
control may be an immunoglobulin of the same subtype, e.g., an antibody of the
same subtype known to have a
good safety profile in a human.
[134] Immunosuppression, immune suppression, immunosuppressive: The terms
refer to the ability to suppress
immune cells, such as T cells, NK cells and B cells. The gold standard for
evaluating immunosuppressive function
is the inhibition of T cell activity, which may include antigen-specific
suppression and non-specific suppression.
Regulatory T cells (Tregs) and MDSCs may be considered immunosuppressive
cells. M2-polarized macrophages
(e.g., disease-localized macrophages such as TAMs and FAMs) may also be
characterized as
immunosuppressive.
[135] Immunological memory: Immunological memory refers to the ability of the
immune system to quickly and
specifically recognize an antigen that the body has previously encountered and
initiate a corresponding immune
response. Generally, these are secondary, tertiary, and other subsequent
immune responses to the same antigen.
Immunological memory is responsible for the adaptive component of the immune
system, special T and B cells ¨
the so-called memory T and B cells. Antigen-naïve T cells expand and
differentiate into memory and effector T
cells after they encounter their cognate antigen within the context of an MHC
molecule on the surface of a
professional antigen presenting cell (e.g., a dendritic cell). The single
unifying theme for all memory T cell subtypes
is that they are long-lived and can quickly expand to large numbers of
effector T cells upon re-exposure to their
cognate antigen. By this mechanism they provide the immune system with
"memory" against previously
encountered pathogens. Memory T cells may be either CD4+ or CD8+ and usually
express C045R0. In a
preclinical setting, immunological memory may be tested in a tumor rechallenge
paradigm.
[136] Inhibit or inhibition of: The term "inhibit" or "inhibition of," as used
herein, means to reduce by a measurable
amount, and can include but does not require complete prevention or
inhibition.
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[137] lsoform-non-specific: The term "isoform non-specific" refers to an
agent's ability to bind to more than one
structurally related isoforms. An isoform-non-specific TGFp inhibitor exerts
its inhibitory activity toward more than
one isoform of TGF13, such as TGFp1/3, TGF(31/2, TGFp2/3, and TGF131/2/3.
[138] lsoform-specific: The term "isoform specificity" refers to an agent's
ability to discriminate one isoform over
other structurally related isoforms (i.e., isoform selectivity). An isoform-
specific TGFp inhibitor exerts its inhibitory
activity towards one isoform of TG93 but not the other isoforms of TGFp at a
given concentration. For example,
an isoform-specific TGFp1 antibody selectively binds TGFp1. A TGF(31-specific
inhibitor (antibody) preferentially
targets (binds thereby inhibits) the TGF(31 isoform over TGFp2 or TGFp3 with
substantially greater affinity. For
example, the selectivity in this context may refer to at least a 10-fold, 100-
fold, 500-fold, 1000-fold, or greater
difference in respective affinities as measured by an in vitro binding assay
such as BLI (Octet ) or preferably SPR
(Biacore0). In some embodiments, the selectivity is such that the inhibitor
when used at a dosage effective to
inhibit TGFP1 in vivo does not inhibit TGFP2 and TGFP3. For such an inhibitor
to be useful as a therapeutic,
dosage to achieve desirable effects (e.g., therapeutically effective amounts)
must fall within the window within
which the inhibitor can effectively inhibit the TGFp1 isoform without
inhibiting TGFp2 or 1G933. In some
embodiments, a TGFp1-selective inhibitor is a pharmacological agent that
interferes with the function or activities
of TGFp1, but not of TGFp2 and/or TGFp3, irrespective of the mechanism of
action.
[139] Isolated: An "isolated" antibody as used herein, refers to an antibody
that is substantially free of other
antibodies having different antigenic specificities. In some embodiments, an
isolated antibody is substantially free
of other unintended cellular material and/or chemicals.
[140] Large Latent Complex: The term "large latent complex" ("LLC") in the
context of the present disclosure
refers to a complex comprised of a proTGFp1 dimer bound to so-called a
presenting molecule. Thus, a large latent
complex is a presenting molecule-proTGFp1 complex, such as LTBP1-proTGF(31,
LTBP3-proTGFp1, GARP-
proTGFpl and LRRC33-proTGFp1 . Such complexes may be formed in vitro using
recombinant, purified
components capable of forming the complex. For screening purposes, presenting
molecules used for forming such
LLCs need not be full length polypeptides; however, the portion of the protein
capable of forming disulfide bonds
with the proTGFp1 dimer complex via the cysteine residues near its N-terminal
regions is typically required.
[141] Latency associated peptide (LAP): LAP is so-called the "prodomain" of
proTGFP1. As described in more
detail herein, LAP is comprised of the "Straight Jacket" domain and the "Arm"
domain. Straight Jacket itself is
further divided into the Alpha-1 Helix and Latency Lasso domains.
[142] Latency Lasso: As used herein, "Latency Lasso," sometimes also referred
to as Latency Loop, is a domain
flanked by Alpha-1 Helix and the Arm within the prodomain of proTGFb1. In its
unmutated form, Latency Lasso of
human proTGF131 comprises the amino acid sequence: LASPPSQGEVPPGPL (SEQ ID NO:
126) which is spanned
by Region 1 identified in FIG. 41. As used herein, the term Extended Latency
Lasso region" refers to the Latency
Lasso together with its immediate C-terminal motif referred to as Alpha-2
Helix (a2-Helix) of the prodomain. The
proline residue that is at the C-terminus of the Latency Lasso provides the
perpendicular "turn" like an "elbow" that
connects the lasso loop to the a2-Helix. Certain high affinity TGFp1
activation inhibitors bind at least in part to
Latency Lasso or a portion thereof to confer the inhibitory potency (e.g., the
ability to block activation), wherein
optionally the portion of the Latency Lasso is ASPPSQGEVPPGPL (SEQ ID NO:
170). In some embodiments, the
antibodies of the present disclosure bind a proTGFp1 complex at ASPPSQGEVPPGPL
(SEQ ID NO: 170) or a
portion thereof. Certain high affinity TGFp1 activation inhibitors bind at
least in part to Extended Latency Lasso or
a portion thereof to confer the inhibitory potency (e.g., the ability to block
activation), wherein optionally the portion
of the Extended Latency Lasso is KLRLASPPSQGEVPPGPLPEAVL (SEQ ID NO: 142).
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[143] Localized: In the context of the present disclosure, the term
"localized" (as in "localized tumor", "disease-
localized" etc.) refers to anatomically isolated or isolatable abnormalities,
such as solid malignancies, as opposed
to systemic disease. Certain leukemia, for example, may have both a localized
component (for instance the bone
marrow) and a systemic component (for instance circulating blood cells) to the
disease.
[144] LRRC33-proTGFp1 complex: As used herein, the term "LRRC33-TGFI31
complex" refers to a complex
between a pro-protein form or latent form of transforming growth factor-131
(TG931) protein and a Leucine-Rich
Repeat-Containing Protein 33 (LRRC33; also known as Negative Regulator of
Reactive Oxygen Species or
NRROS) or fragment or variant thereof. In some embodiments, a LRRC33-TGFI31
complex comprises LRRC33
covalently linked with pro/latent TGFI31 via one or more disulfide bonds. In
nature, such covalent bonds are formed
with cysteine residues present near the N-terminus (e.g., amino acid position
4) of a proTGF131 dimer complex. In
other embodiments, a LRRC33-TGFI31 complex comprises LRRC33 non-covalently
linked with pro/latent TGFI31.
In some embodiments, a LRRC33-TGFP1 complex is a naturally-occurring complex,
for example a LRRC33-TGFP1
complex in a cell. The term "hLRRC33" denotes human LRRC33. In vivo, LRRC33
and LRRC33-containing
complexes on cell surface may be internalized. LRRC33 is expressed on a subset
of myeloid cells, including M2-
polarized macrophages (such as TAMs) and MDSCs. MDSCs that express LRRC33 on
cell surface include tumor-
associated MDSCs and circulatory MDSCs. LRRC33-expressing tumor-associated
MDSCs may include gMDSCs.
LRRC33-expressing MDSCs in circulation may include g-MDSCs.
[145] LTBP1-proTGF(31 complex: As used herein, the term "LTBP1-TGFI31 complex"
refers to a protein complex
comprising a pro-protein form or latent form of transforming growth factor-131
(TGFp1) protein and a latent TGF-
beta binding protein 1 (LTBP1) or fragment or variant thereof. In some
embodiments, a LTBP1-TG931 complex
comprises LTBP1 covalently linked with pro/latent TGFI31 via one or more
disulfide bonds. In nature, such covalent
bonds are formed with cysteine residues present near the N-terminus (e.g.,
amino acid position 4) of a proTGFP1
dimer complex. In other embodiments, a LTBP1-TGF131 complex comprises LTBP1
non-covalently linked with
pro/latent TGF131. In some embodiments, a LTBP1-TGF131 complex is a naturally-
occurring complex, for example
a LTBP1-TGFI31 complex in a cell. The term "hLTBP1" denotes human LTBP1.
[146] LTBP3-proTGFp1 complex: As used herein, the term "LTBP3-TG931 complex"
refers to a protein complex
comprising a pro-protein form or latent form of transforming growth factor-I31
(TGFp1) protein and a latent TGF-
beta binding protein 3 (LTBP3) or fragment or variant thereof. In some
embodiments, a LTBP3-TGFI31 complex
comprises LTBP3 covalently linked with pro/latent TGFp1 via one or more
disulfide bonds. In nature, such covalent
bonds are formed with cysteine residues present near the N-terminus (e.g.,
amino acid position 4) of a proTG931
dimer complex. In other embodiments, a LTBP3-TGF[31 complex comprises LTBP1
non-covalently linked with
pro/latent TGF131. In some embodiments, a LTBP3-TGFp1 complex is a naturally-
occurring complex, for example
a LT3P3-TGF131 complex in a cell. The term "hLTBP3" denotes human LTBP3.
[147] M2 or M2-like macrophage: M2 macrophages represent a subset of activated
or polarized macrophages
and include disease-associated macrophages in both fibrotic and tumor
microenvironments. Cell-surface markers
for M2-polarized macrophages typically include CD206 and CD163 (i.e.,
0D206+/0D163+). M2-polarized
macrophages may also express cell-surface LRRC33. Activation of M2 macrophages
is promoted mainly by IL-4,
IL-13, IL-10 and TG93; they secrete the same cytokines that activate them (IL-
4, IL-13, IL-10 and TGF13). These
cells have high phagocytic capacity and produce ECM components, angiogenic and
chemotactic factors. The
release of TGF13 by macrophages may perpetuate the myofibroblast activation,
EMT and EndMT induction in the
disease tissues, such as fibrotic tissue and tumor stroma. For example, M2
macrophages play a role in TGF13-
driven lung fibrosis and are also enriched in a number of tumors.
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[148] Matrix-associated proTGF)31: LTBP1 and LTBP3 are presenting molecules
that are components of the
extracellular matrix (ECM). LTBP1-proTGF131 and LTBP3-proTGF81 may be
collectively referred to as "ECM-
associated" (or "matrix-associated") proTGF31 complexes, that mediate ECM-
associated 1GF31
activation/signaling. The term also includes recombinant, purified LTBP1-
proTGF31 and LTBP3-proTG931
complexes in solution (e.g., in vitro assays) which are not physically
attached to a matrix or substrate.
[149] Maximally tolerated dose (MTD): The term MTD generally refers to, in the
context of safety/toxicology
considerations, the highest amount of a test article (such as a TGF81
inhibitor) evaluated with no-observed-
adverse-effect level (NOAEL). For example, the NOAEL for Ab6 in rats was the
highest dose evaluated (100
mg/kg), suggesting that the MTD for Ab6 is >100 mg/kg, based on a four-week
toxicology study. The NOAEL for
Ab6 in non-human primates was the highest dose evaluated (300 mg/kg),
suggesting that the MTD for Ab6 in the
non-human primates is >300 mg/kg, based on a four-week toxicology study.
[150] Meso-Scale Discovery: "Meso-Scale Discovery" or "MSD" is a type of
immunoassays that employs
electrochemiluminescence (ECL) as a detection technique. Typically, high
binding carbon electrodes are used to
capture proteins (e.g., antibodies). The antibodies can be incubated with
particular antigens, which binding can
be detected with secondary antibodies that are conjugated to
electrochemiluminescent labels. Upon an electrical
signal, light intensity can be measured to quantify analytes in the sample.
[151] Myelofibrosis: "Myelofibrosis," also known as osteomyelofibrosis, is a
relatively rare bone marrow
proliferative disorder (e.g., cancer), Myelofibrosis is generally
characterized by the proliferation of an abnormal
clone of hematopoietic stem cells in the bone marrow and other sites results
in fibrosis, or the replacement of the
marrow with scar tissue. The term myelofibrosis encompasses primary
myelofibrosis (PMF), also be referred to as
chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary mean
that in these cases the disease is
of unknown or spontaneous origin), as well as secondary types of
myelofibrosis, such as myelofibrosis that
develops secondary to polycythemia vera (PV) or essential thrombocythaemia
(ET). Myelofibrosis is a form of
myeloid metaplasia, which refers to a change in cell type in the blood-forming
tissue of the bone marrow, and often
the two terms are used synonymously. The terms agnogenic myeloid metaplasia
and myelofibrosis with myeloid
metaplasia (MMM) are also used to refer to primary myelofibrosis.
Myelofibrosis is characterized by mutations that
cause upregulation or overactivation of the downstream JAK pathway.
[152] Myeloid cells: In hematopoiesis, myeloid cells are blood cells that
arise from a progenitor cell for
granulocytes, monocytes, erythrocytes, or platelets (the common myeloid
progenitor, that is, CMP or CFU-GEMM),
or in a narrower sense also often used, specifically from the lineage of the
myeloblast (the myelocytes, monocytes,
and their daughter types), as distinguished from lymphoid cells, that is,
lymphocytes, which come from common
lymphoid progenitor cells that give rise to B cells and T cells. Certain
myeloid cell types, their general morphology,
typical cell surface markers, and their immune-suppressive ability in both
mouse and human, are summarized
below. In some embodiments, a human neutrophil can be identified by at least
one (e.g., all) of the cell surface
markers CDi1b, CD14-, CD15*, and CD66b*. In some embodiments, a human
neutrophil is LOX-1-. In some
embodiments, a human neutrophil is I¨ILA-DR-Med. In some embodiments, a
classical human monocyte can be
identified by at least one (e.g., all) of the cell surface markers CD14+ CD15-
CD16- HLA-DR4. In some
embodiments, a classical human monocyte is CD33* and/or CD111p*. In some
embodiments, a classical human
monocyte is CD16-. In some embodiments, an intermediate human monocyte can be
identified by at least one
(e.g., all) of the cell surface markers CD14+ CD15- CD16+ HLA-DIR+. In some
embodiments, a non-classical human
monocyte can be identified by at least one (e.g., all) of the cell surface
markers CD14- CD15- CD16. HLA-DIR*. In
some embodiments, a human M1 macrophage can be identified by at least one
(e.g., all) of the cell surface markers
CD15- CD16+ CD80+ HLA-DR-'1"'gh CD33. In some embodiments, a human M1
macrophage is CD66b-. In some
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embodiments, a human M1 macrophage is CD11 b+. In some embodiments, a human M1
macrophage is CD14-. In
some embodiments, a human M2 macrophage can be identified by at least one
(e.g., all) of the cell surface markers
CD1113 and CD15-. In some embodiments, a human M2 macrophage is CD206'. In
some embodiments, a human
M2 macrophage is CD163'. In some embodiments, a human M2 macrophage is HLA-
DR+. In some embodiments,
a human M2 macrophage is CD14-. In some embodiments, a human M2 macrophage is
CD33+. In some
embodiments, a human M2 macrophage is CD66b-.
Myeloid cells Typical Morphology Select surface phenotype
Immune
suppression
Mouse
Neutrophils Round shape with a CD11 Ly6G' Ly6C1
segmented nucleus
Monocytes Round shape with an CD11b+ Ly6G- Ly6Ch'
indented nucleus
Macrophages Round shape with CD11b+ F4/80h' Ly6G- Ly6CI CD80+
pseudopodia (M1)
F4/80+ CD206+ CD163+
LRRC33+ (M2)
Dendritic cells Dendritic shape with CD11b+ CD11e Ly6G- Ly6C-il0
polypodia (classical)
CD11b- CD11c+ Ly6G- Ly6C-
(classical)
CD11 b- CD11ci Ly6G- Ly6C+ PDCA-1+
(plasmacytoid)
Fibrocytes Spindle shape CD11b. Coll* Ly6G- Ly6C+
G-MDSCs Round shape with a CD11b+ Ly6G + Ly6CI
(PMN-MDSCs) banded nucleus LRRC33+
M-MDSCs Round shape with an CD1113+ Ly6G- Ly6Ch1
indented nucleus LRRC33+
Human
Neutrophils Round shape with a CD11b+ CD14- CD15+ CD66b+ LOX-1-
segmented nucleus
Monocytes Round shape with an CD14+ CD15- CD16- HLA-DR+
indented nucleus (classical)
CD14+ CD15- CD16+ HLA-DR+
(intermediate)
CD14- CD15- CD16+ HLA-DR+
(non-classical)
Macrophages Round shape with CD15- CD16+ CD80+ HLA-DR+ CD33+
pseudopodia (M1)
CD11b+ CD15- CD206+ CD163+ HLA-DR+
+/-
(M2)
Dendritic cells Dendritic shape with CD14- CD16- CD1C+ C083+
polypodia (classical)
CD14- CD16- CD141+ CD83+
(classical)
CD14- C016- CD303+ CD83+
(plasmacytoid)
Fibrocytes Spindle shape CD11b+ Coll* CD13+ C034+ CD45R0+ HLA-
DR+
G-MDSCs Round shape with an CD11b+ CD33+ C014- CD15+ CD66b+
LOX-1+
(PMN-MDSCs) annular nucleus HLA-DR-il
M-MDSCs Round shape with an CD11b+ CD33+ CD14+ CD15- HLA-DR-
il
indented nucleus
[153] Myeloid-derived suppressor cell: Myeloid-derived suppressor cells
(MDSCs) are a heterogeneous
population of cells generated during various pathologic conditions. MDSCs
include at least two categories of cells
termed i) "granulocytic" (G-MDSC) or polymorphonuclear (PMN-MDSC), which are
phenotypically and
morphologically similar to neutrophils; and ii) monocytic (M-MDSC) which are
phenotypically and morphologically
similar to monocytes. MDSCs are characterized by a distinct set of genomic and
biochemical features, and can be
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distinguished by specific surface molecules. In certain embodiments, suitable
cell surface markers for identifying
MDSCs may include one or more of CD11 b, CD33, CD14, C015, HLA-DR and CD66b.
For example, human G-
MDSCs/PMN-MDSCs typically express the cell-surface markers CD11b, CD33, CD15
and CD66b. In some
embodiments, human G-MDSCs may express low levels of the CD33 cell surface
marker. In some embodiments,
human G-MDSCs/PMN-MDSCs may express LOX-1 and/or Arginase. By comparison,
human M-MDSCs typically
express the cell surface markers CD11b, 0D33 and CD14. Additionally, both
human G-MDSCs/PMN-MDSCs and
M-MDSCs may also exhibit low levels or undetectable levels of HLA-DR. In some
embodiments, human G-MDSCs
may be HLA-DR-. In certain embodiments, G-MDSCs may be differentiated from M-
MDSCs based on the presence
or absence of certain cell surface marker (e.g., CD14, CD15, and/or CD66b). In
some embodiments, G-MDSCs
may be identified by the presence or elevated expression of surface markers
CD11b, CD33, CD15, CD66b, and/or
LOX-1, and the absence of CD14, whereas M-MDSCs may be identified by the
presence or elevated expression
of surface markers CD11b, CD33, and/or 0014, and the absence of 0015. In some
embodiments, M-MDSCs may
be CD66b-. In addition to such cell-surface markers, MDSCs may be
characterized by the ability to suppress
immune cells, such as T cells, NK cells and B cells. Immune suppressive
functions of MDSCs may include inhibition
of antigen-non-specific function and inhibition of antigen-specific function.
MDSCs, including tumor-associated
MDSCs and MDSCs in circulation, can express cell surface LRRC33 and/or LRRC33-
proTGF131. In some
embodiments, a signal intensity of a cell surface marker may be categorized,
or binned, as "low", "medium", or
"high" based on normalization of signal intensity to reduce background and
bleed through signals. In some
embodiments, a signal intensity of a cell surface marker may be categorized
based on cutoff thresholds provided
in Table 38A. In some embodiments, a signal intensity of a cell surface marker
may be determined by binary
intensity selection. In some embodiments, the binary intensity selection
comprises categorizing a signal intensity
measured for a particular cell surface marker as "positive" or "negative." In
some embodiments, a signal intensity
of a cell surface marker may be categorized based on the cutoff thresholds
provided in Table 38B. In some
embodiments, signal intensities of a set of surface markers may be determined
by sequential application of signal
filtering, where the signal intensity threshold for one or more surface
markers is determined before the threshold is
determined for one or more additional surface markers.
[154] Myofibroblast: Myofibroblasts are cells with certain phenotypes of
fibroblasts and smooth muscle cells and
generally express vimentin, alpha-smooth muscle actin (a-SMA; human gene
ACTA2) and paladin. In many
disease conditions involving extracellular matrix dysregulations (such as
increased matrix stiffness), normal
fibroblast cells become de-differentiated into myofibroblasts in a TGFP-
dependent manner. Aberrant
overexpression of TGFp is common among myofibroblast-driven pathologies. TGFp
is known to promote
myofibroblast differentiation, cell proliferation, and matrix production.
Myofibroblasts or myofibroblast-like cells
within the fibrotic microenvironment may be referred to as fibrosis-associated
fibroblasts (or "FAFs"), and
myofibroblasts or myofibroblast-like cells within the tumor microenvironment
may be referred to as cancer-
associated fibroblasts (or "CAFs").
[155] Pan-TGFO inhibitor/pan-inhibition of TGFI3: The term "pan-TGFp
inhibitor" refers to any agent that is
capable of inhibiting or antagonizing all three isoforms of TGFP. Such an
inhibitor may be a small molecule inhibitor
of TGFp isoforms, such as those known in the art. The term includes pan-TGFp
antibody which refers to any
antibody capable of binding to each of TGFp isoforms, i.e., TGFpl , TGFp2, and
TGFp3. In some embodiments, a
pan-TGFp antibody binds and neutralizes activities of all three isoforms,
i.e., TGF(31, TGF(32, and TGF03. The
antibody 1011 (or the human analog fresolimumab (GC1008)) is a well-known
example of a pan-TGFp antibody
that neutralizes all three isoforms of TGFp. Examples of small molecule pan-
TGFp inhibitors include galunisertib
(LY2157299 monohydrate), which is an antagonist for the TGFp receptor I
kinase/ALK5 that mediates signaling of
all three TGFI3 isoforms.
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[156] Perivascular (infiltration): The prefix "pen-" means "around"
"surrounding" or "near," hence "perivascular"
literally translates to around the blood vessels. As used herein in the
context of tumor cell infiltrates, the term
"perivascular infiltration" refers to a mode of entry for tumor-infiltrating
immune cells (e.g., lymphocytes) via the
vasculature of a solid tumor.
[157] Potency: The term "potency' as used herein refers to activity of a drug,
such as an inhibitory antibody (or
fragment) having inhibitory activity, with respect to concentration or amount
of the drug to produce a defined effect.
For example, an antibody capable of producing certain effects at a given
dosage is more potent than another
antibody that requires twice the amount (dosage) to produce equivalent
effects. Potency may be measured in cell-
based assays, such as TGF6 activation/inhibition assays, whereby the degree of
TGF3 activation, such as
activation triggered by integrin binding, can be measured in the presence or
absence of test article (e.g., inhibitory
antibodies) in a cell-based system. Typically, among those capable of binding
to the same or overlapping binding
regions of an antigen (e.g., cross-blocking antibodies), antibodies with
higher affinities (lower Ko values) tend to
show higher potency than antibodies with lower affinities (greater KD values).
[158] Preclinical model: The term "preclinical model" refers to a cell line or
an animal that exhibits certain
characteristics of a human disease which is used to study the mechanism of
action, efficacy, pharmacology, and
toxicology of a drug, procedure, or treatment before it is tested on humans.
Typically, cell-based preclinical studies
are referred to as "in vitro" studies, whereas animal-based preclinical
studies are referred to as "in vivo" studies.
For example, in vivo mouse preclinical models encompassed by the current
disclosure include the MBT2 bladder
cancer model, the Cloudman S91 melanoma model, and the EMT6 breast cancer
model.
[159] Predictive biomarker Predictive biomarkers provide information on the
probability or likelihood of response
to a particular therapy. Typically, a predictive biomarker is measured before
and after treatment, and the changes
or relative levels of the marker in samples collected from the subject
indicates or predicts therapeutic benefit.
[160] Presenting molecule: Presenting molecules in the context of the present
disclosure refer to proteins that
form covalent bonds with latent pro-proteins (e.g., proTGF61) and tether
("present") the inactive complex to an
extracellular niche (such as ECM or immune cell surface) thereby maintaining
its latency until an activation event
occurs Known presenting molecules for proTGF31 include: LTBP1, LTBP3, GARP and
LRRC33, each of which
can form a presenting molecule-proTGF31 complex (i.e., LLC), namely, LTBP1-
proTGF61, LTBP3-proTGF31,
GARP-proTGF31 and LRRC33-proTGF61, respectively. In nature, LTBP1 and LTBP3
are components of the
extracellular matrix (ECM); therefore, LTBP1-proTGF61 and LTBP3-proTGF31 may
be collectively referred to as
"ECM-associated" (or "matrix-associated") proTGF31 complexes, that mediate ECM-
associated TGF31
signaling/activities. GARP and LRRC33, on the other hand, are transmembrane
proteins expressed on cell surface
of certain cells; therefore, GARP-proTGF61 and LRRC33-proTGF31 may be
collectively referred to as "cell-
associated" (or "cell-surface") proTGF61 complexes, that mediate cell-
associated (e.g., immune cell-associated)
TGF31 signaling/activities.
[161] ProTGFpl: The term "proTGF61" as used herein is intended to encompass
precursor forms of inactive
TGF61 complex that comprises a prodomain sequence of TGF31 within the complex.
Thus, the term can include
the pro-, as well as the latent- forms of TGF31. The expression "pro/latent
TGF31" may be used interchangeably.
The 'pro" form of TGF31 exists prior to proteolytic cleavage at the furin
site. Once cleaved, the resulting form is
said to be the "latent" form of TGF31. The "latent" complex remains non-
covalently associated until further
activation trigger, such as integrin-driven activation event. The proTGF31
complex is comprised of dimeric TGF61
pro-protein polypeptides, linked with disulfide bonds. The latent dimer
complex is covalently linked to a single
presenting molecule via the cysteine residue at position 4 (Cys4) of each of
the proTGF61 polypeptides. The
adjective "latent" may be used generally/broadly to describe the "inactive"
state of TGF31, prior to integrin-mediated
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or other activation events. The proTGF31 polypeptide contains a prodomain
(LAP) and a growth factor domain
(SEQ ID NO: 119).
[162] Regression (tumor regression): Regression of tumor or tumor growth can
be used as an in vivo efficacy
measure. For example, in preclinical settings, median tumor volume (MTV) and
Criteria for Regression Responses
Treatment efficacy may be determined from the tumor volumes of animals
remaining in the study on the last day.
Treatment efficacy may also be determined from the incidence and magnitude of
regression responses observed
during the study. Treatment may cause partial regression (PR) or complete
regression (CR) of the tumor in an
animal. Complete regression achieved in response to therapy (e.g.,
administration of a drug) may be referred to as
"complete response" and the subject that achieves complete response may be
referred to as a "complete
responder". Thus, complete response excludes spontaneous complete regression.
In some embodiments of
preclinical tumor models, a PR response is defined as the tumor volume that is
50% or less of its Day 1 volume for
three consecutive measurements during the course of the study, and equal to or
greater than 13.5 mm3 for one or
more of these three measurements. In some embodiments, a CR response is
defined as the tumor volume that is
less than 13.5 mm3 for three consecutive measurements during the course of the
study. In preclinical model, an
animal with a CR response at the termination of a study may be additionally
classified as a tumor-free survivor
(TFS). The term "effective tumor control" may be used to refer to a degree of
tumor regression achieved in
response to treatment, where, for example, the tumor volume is reduced to <25%
of the endpoint tumor volume in
response to treatment. For instance, in a particular model, if the endpoint
tumor volume is 2,000 mm3, effective
tumor control is achieved if the tumor is reduced to less than 500 mm3.
Therefore, effective tumor control
encompasses complete regression, as well as partial regression that reaches
the threshold reduction.
[163] Resistance (to therapy): Resistance to a particular therapy (such as
CBT) may be due to the innate
characteristics of the disease such as cancer ("primary resistance", i.e.,
present before treatment initiation), or due
to acquired phenotypes that develop over time following the treatment
("acquired resistance"). Patients who do
not show therapeutic response to a therapy (e.g., those who are non-responders
or poorly responsive to the
therapy) are said to have primary resistance or acquired resistance to the
therapy. Patients who have never
previously received a treatment and do not show a therapeutic response to the
treatment are said to have primary
resistance. Patients who initially show therapeutic response to a therapy but
later lose effects (e.g., progression or
recurrence despite continued therapy) are said to have acquired resistance to
the therapy. In the context of
immunotherapy, such resistance can indicate immune escape.
[164] Response Evaluation Criteria in Solid Tumors (RECIST) and iRECIST:
RECIST is a set of published rules
that define when tumors in cancer patients improve ("respond"), stay the same
("stabilize"), or worsen (''progress")
during treatment. The criteria were published in February 2000 by an
international collaboration including the
European Organisation for Research and Treatment of Cancer (EORTC), National
Cancer Institute of the United
States, and the National Cancer Institute of Canada Clinical Trials Group.
Subsequently, a revised version of the
RECIST guideline (RECIST v 1.1) has been widely adapted (see: Eisenhauera et
al., (2009), "New response
evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)"
Eur J Cancer 45: 228-247,
incorporated herein).
[165] Response criteria are as follows: Complete response (CR): Disappearance
of all target lesions; Partial
response (PR): At least a 30% decrease in the sum of the LD of target lesions,
taking as reference the baseline
sum LD; Stable disease (SD): Neither sufficient shrinkage to qualify for PR
nor sufficient increase to qualify for PD,
taking as reference the smallest sum LD since the treatment started;
Progressive disease (PD): At least a 20%
increase in the sum of the LD of target lesions, taking as reference the
smallest sum LD recorded since the
treatment started or the appearance of one or more new lesions.
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[166] On the other hand, iRECIST provides a modified set of criteria that
takes into account immune-related
response (see: www.ncbi.nlm.nih.gov/pmc/articles/PMC5648544/ contents of which
are incorporated herein by
reference). The RECIST and iRECIST criteria are standardized, may be revised
from time to time as more data
become available, and are well understood in the art.
[167] Response rate: The term response rate (as in "low response rates") as
used herein carries the ordinary
meaning as understood by the skilled person in medicine, such as oncologists.
A response rate is the proportion
(e.g., fraction or percentage) of subjects in a patient population who shows
clinical improvement upon receiving a
treatment (e.g., pharmacological intervention) and may include complete
response and partial response. In
oncology, clinical improvement may include tumor shrinkage (e.g., partial
response) or disappearance (e.g.,
complete response). When used as a clinical endpoint for clinical trials of
cancer treatments, this is typically
expressed as the objective response rate (ORR). The FDA defines ORR as the
proportion of patietns with tumor
size reduction of a predefined amount and for a minimum time period. See:
"Clinical Trial Endpoints for the
Approval of Cander Drugs and Biologics ¨ Guidance for Industry" published by
U.S. Department of Health and
Human Services, Food and Drug Administration, Oncology Center of Excellence,
Center for Drug Evaluation and
Research (CDER), Center for Biologics Evaluation and Research (CBER), the
contents of which is incorporated
herein by reference.
[168] Solid tumor: The term "solid tumor" refers to proliferative disorders
resulting in an abnormal growth or mass
of tissue that usually does not contain cysts or liquid areas. Solid tumors
may be benign (non-cancerous), or
malignant (cancerous). Solid tumors include tumors of advanced malignancies,
such as locally advanced solid
tumors and metastatic cancer. Solid tumors are typically comprised of multiple
cell types, including, without
limitation, cancerous (malignant) cells, stromal cells such as CAFs, and
infiltrating leukocytes, such as
macrophages, MDSCs and lymphocytes. Solid tumors to be treated with an isoform-
selective inhibitor of TGF[31,
such as those described herein, are typically TGF61-positive (TGFI31+) tumors,
which may include multiple cell
types that produce TGF131. In certain embodiments, the TGF81+ tumor may also
co-express TGFI33 (i.e., TGFI33-
positive). For example, certain tumors are TGFI31/3-co-dominant. In some
embodiments, such tumors are caused
by cancer of epithelial cells, e.g., carcinoma. In certain embodiments, such
tumors include ovarian cancer, breast
cancer, bladder cancer, pancreatic cancer, e.g., pancreatic adenocarcinoma,
prostate cancer, e.g., prostate
adenocarcinoma, melanoma, e.g., skin cutaneous melanoma, lung cancer, e.g.,
lung squamous cell carcinoma
and lung adenocarcinoma, liver cancer (e.g., liver hepatocellular carcinoma),
uterine cancer, e.g., uterine corpus
endometrial carcinoma, kidney cancer, e.g., renal clear cell carcinoma, head
and neck cancer, e.g., head and neck
squamous cell carcinoma, colon cancer, e.g., colon adenocarcinoma, esophageal
carcinoma, and tenosynovial
giant cell tumor (TGCT). In some embodiments, a solid tumor treated herein,
such as one or more of those listed
above, exhibits elevated TG931 expression as compared to other tumor types and
exhibits a reduced
responsiveness to mainline therapy, e.g., genotoxic therapy. Accordingly,
TGFI3 inhibitors (e.g., Ab6) may be used
in conjunction with one or more genotoxic therapies (e.g., chemotherapy and/or
radiation therapy, including
radiotherapeutic agents) to treat such cancer in a subject.
[169] Specific binding: As used herein, the term "specific binding" or
"specifically binds" means that an antibody,
or antigen binding portion thereof, exhibits a particular affinity for a
particular structure (e.g., an antigenic
determinant or epitope) in an antigen (e.g., a Ko measured by Biacoree). In
some embodiments, an antibody, or
antigen binding portion thereof, specifically binds to a target, e.g., TGFI31,
if the antibody has a Ko for the target of
at least about 10-8 M, 10-9 M, 1010 M, 10-11 M, 10-12 M, or less. In some
embodiments, the term "specific binding
to an epitope of proTGF81", "specifically binds to an epitope of proTGFf31",
"specific binding to proTGFf31", or
"specifically binds to proTG931" as used herein, refers to an antibody, or
antigen binding portion thereof, that binds
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to proTGFp1 and has a dissociation constant (Ko) of 1.0 x 10-8 M or less, as
determined by suitable in vitro binding
assays, such as surface plasmon resonance and Biolayer lnterferometry (BLI).
In preferred embodiments, kinetic
rate constants (e.g., Ko) are determined by surface plasmon resonance (e.g., a
Biacore system). In one
embodiment, an antibody, or antigen binding portion thereof, can specifically
bind to both human and a non-human
(e.g., mouse) orthologues of proTGFP1. In some embodiments, an antibody may
also "selectively" (i.e.,
"preferentially") bind a target antigen if it binds that target with a
comparatively greater strength than the strength
of binding shown to other antigens, e.g., a 10-fold, 100-fold, 1000-fold, or
greater comparative affinity for a target
antigen (e.g., TGFp1) than for a non-target antigen (e.g., TGFp2 and/or
TGFp3). In preferred embodiments, an
isoform-selective inhibitor exhibits no detectable binding or potency towards
other isoforms or counterparts. In
some embodiments, an antibody that binds specifically to a set of antigens may
have high affinity toward said
antigens but may not distinguish said antigens from one another (i.e., the
antibody is specific but not selective). In
some embodiments, an antibody that binds to an antigen with a particularly
high affinity as compared to other
antigens may be considered selective for said antigen. For instance, an
antibody that binds to antigen X with 1000-
fold higher affinity as compared to antigen Y may be considered an antibody
that is selective for antigen X over
antigen Y. In the context of the present disclosure, "an antibody that
specifically binds an antigen with high affinity"
generally refers to a KD of 1.0 x 10-8 M or less.
[170] Subject: The term "subject" in the context of therapeutic applications
refers to an individual who receives
or is in need of clinical care or intervention, such as treatment, diagnosis,
etc. Suitable subjects include vertebrates,
including but not limited to mammals (e.g., human and non-human mammals).
Where the subject is a human
subject, the term "patient" may be used interchangeably. In a clinical
context, the term "a patient population" or
"patient subpopulation" is used to refer to a group of individuals that falls
within a set of criteria, such as clinical
criteria (e.g., disease presentations, disease stages, susceptibility to
certain conditions, responsiveness to therapy,
etc.), medical history, health status, gender, age group, genetic criteria
(e.g., carrier of certain mutation,
polymorphism, gene duplications, DNA sequence repeats, etc.) and lifestyle
factors (e.g., smoking, alcohol
consumption, exercise, etc.).
[171] Target engagement: As used herein, the term target engagement refers to
the ability of a molecule (e.g.,
TGFp inhibitor) to bind to its intended target in vivo (e.g., endogenous
TGFp). In case of activation inhibitors, the
intended target can be a large latent complex.
[172] TGFgl-related indication: A "TGFp1-related indication" is a TGFpl-
associated disorder and means any
disease or disorder, and/or condition, in which at least part of the
pathogenesis and/or progression is attributable
to TGFp1 signaling or dysregulation thereof. Certain TGFp1 -associated
disorders are driven predominantly by the
TGFp1 isoform. Subjects having a TGFp1-related indication may benefit from
inhibition of the activity and/or levels
TGFp1. Certain TGFp1-related indications are driven predominantly by the TGFp1
isoform. TGFp1-related
indications include, but are not limited to: fibrotic conditions (such as
organ fibrosis, and fibrosis of tissues involving
chronic inflammation), proliferative disorders (such as cancer, e.g., solid
tumors and myelofibrosis), disease
associated with ECM dysregulation (such as conditions involving matrix
stiffening and remodeling), disease
involving mesenchymal transition (e.g., EndMT and/or EMT), disease involving
proteases, disease with aberrant
gene expression of certain markers described herein. These disease categories
are not intended to be mutually
exclusive.
[173] TGFp inhibitor: The term "TG93 inhibitor" refers to any agent capable of
antagonizing biological activities,
signaling or function of TGFp growth factor (e.g., TGFp1, TGFp2 and/or TGFp3).
The term is not intended to limit
its mechanism of action and includes, for example, neutralizing inhibitors,
receptor antagonists, soluble ligand
traps, TGFI3 activation inhibitors, and integrin inhibitors (e.g., antibodies
that bind to aVp1, aVp3, aVp5, aN/136,
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aN/38, 0531, al 103, or a831 integrins, and inhibit downstream activation of
TG93. e.g., selective inhibition of
TGF31 and/or TGF133). The term encompasses TGFI3 inhibitors that are isoform-
selective and non-selective
inhibitors. The latter include, for example, small molecule receptor kinase
inhibitors (e.g., ALK5 inhibitors),
antibodies (such as neutralizing antibodies) that preferentially bind two or
more isoforms, and engineered
constructs (e.g., fusion proteins) comprising a ligand-binding moiety. In
certain embodiments, these antibodies
may include or may be engineered to include a mutation or modification that
causes an extended half-life of the
antibody. In some embodiments, such mutations or modifications may be within
the Fc domain of the antibodies
(e.g., Fc-modified antibodies). In some embodiments, the mutation is so-called
YTE mutation.TGFp inhibitors also
include antibodies that are capable of reducing the availability of latent
proTGFP which can be activated in the
niche, for example, by inducing antibody-dependent cell mediated cytotoxicity
(ADCC), and/or antibody-dependent
cellular phagocytosis (ADPC), as well as antibodies that result in
internalization of cell-surface complex comprising
latent proTGF3, thereby removing the precursor from the plasma membrane
without depleting the cells themselves.
Internalization may be a suitable mechanism of action for LRRC33-containing
protein complexes (such as human
LRRC33-proTGF[31) which results in reduced levels of cells expressing LRRC33-
containing protein complexes on
cell surface.
[174] The "TGFp family' is a class within the TGF[3 superfamily and in human
contains three members: TGF131 ,
TGF32, and TGF[33, which are structurally similar. The three growth factors
are known to signal via the same
receptors.
[175] TGFgl-positive cancer/tumor: The term, as used herein, refers to a
cancer/tumor with aberrant TGF31
expression (overexpression). Many human cancer/tumor types show predominant
expression of the TGF131 (note
that "TGFB" is sometimes used to refer to the gene as opposed to protein)
isoform. In some cases, such
cancer/tumor may show co-dominant expression of another isoform, such as
TGF[33. A number of epithelial
cancers (e.g., carcinoma) may co-express TGF[31 and TGF[33. Within the tumor
environment of TGF[31-positive
tumors, TGF131 may arise from multiple sources, including, for example, cancer
cells, tumor-associated
macrophages (TAMs), cancer-associated fibroblasts (CAFs), regulatory T cells
(Tregs), myeloid-derived
suppressor cells (MDSCs), and the surrounding extracellular matrix (ECM). In
the context of the present disclosure,
preclinical cancer/tumor models that recapitulate human conditions are TGF[31-
positive cancer/tumor.
[176] Therapeutic window: The term "therapeutic window" refers to a dosage
range that produces therapeutic
response without causing significant/observable/unacceptable adverse effect
(e.g., within adverse effects that are
acceptable or tolerable) in subjects. Therapeutic window may be calculated as
a ratio between minimum effective
concentrations (MEC) to the minimum toxic concentrations (MTC). To illustrate,
a TGF131 inhibitor that achieves in
vivo efficacy at 10 mg/kg dosage and shows tolerability or acceptable
toxicities at 100 mg/kg provides at least a
10-fold (e.g., 10x) therapeutic window. By contrast, a pan-inhibitor of TGFI3
that is efficacious at 10 mg/kg but
causes adverse effects at less than the effective dose is said to have "dose-
limiting toxicities." Generally, the
maximally tolerated dose (MTD) may set the upper limit of the therapeutic
window. For example, Ab6 was shown
to be efficacious at dosage ranging between about 3-30 mg/kg/week and was also
shown to be free of observable
toxicities associated with pan-inhibition of TGF3 at dosage of at least 100 or
300 mg/kg/week for 4 weeks in rats
or non-human primates. Based on this, Ab6 shows at minimum a 3.3-fold and up
to 100-fold therapeutic window.
In some embodiments, the concept of therapeutic window may be expressed in
terms of safety factors.
[177] Toxicity: As used herein, the term "toxicity" or "toxicities" refers to
unwanted in vivo effects in subjects (e.g.,
patients) associated with a therapy administered to the subjects (e.g.,
patients), such as undesirable side effects
and adverse events. "Tolerability" refers to a level of toxicities associated
with a therapy or therapeutic regimen,
which can be reasonably tolerated by patients, without discontinuing the
therapy due to the toxicities. Typically,
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toxicity/toxicology studies are carried out in one or more preclinical models
prior to clinical development to assess
safety profiles of a drug candidate (e.g., monoclonal antibody therapy).
Toxicity/toxicology studies may help
determine the 'no-observed-adverse-effect level (NOAEL)" and the "maximally
tolerated dose (MTD)" of a test
article, based on which a therapeutic window may be deduced. Preferably, a
species that is shown to be sensitive
to the particular intervention should be chosen as a preclinical animal model
in which safety/toxicity study is to be
carried out. In case of TGF6 inhibition, suitable species include rats, dogs,
and cynos. Mice are reported to be
less sensitive to pharmacological inhibition of TGF3 and may not reveal
toxicities that are potentially dangerous in
other species, including human, although certain studies report toxicities
observed with pan-inhibition of TGF3 in
mice. To illustrate in the context of the present disclosure, the NOAEL for
Ab6 in rats was the highest dose
evaluated (100 mg/kg), suggesting that the MTD is >100 mg/kg, based on a four-
week toxicology study. The MTD
of Ab6 in non-human primates is >300 mg/kg based on a four-week toxicology
study.
[178] For determining NOAELs and MTDs, preferably, a species that is shown to
be sensitive to the particular
intervention should be chosen as a preclinical animal model in which
safety/toxicology study is to be carried out.
In case of TGF3 inhibition, suitable species include, but are not limited to,
rats, dogs, and cynos. Mice are reported
to be less sensitive to pharmacological inhibition of TGF6 and may not reveal
toxicities that are potentially serious
or dangerous in other species, including human.
[179] Treat/treatment: The term "treat" or "treatment" includes therapeutic
treatments, prophylactic treatments,
and applications in which one reduces the risk that a subject will develop a
disorder or other risk factor. Thus the
term is intended to broadly mean: causing therapeutic benefits in a patient
by, for example, enhancing or boosting
the body's immunity; reducing or reversing immune suppression; reducing,
removing or eradicating harmful cells
or substances from the body; reducing disease burden (e.g., tumor burden);
preventing recurrence or relapse;
prolonging a refractory period, and/or otherwise improving survival. The term
includes therapeutic treatments,
prophylactic treatments, and applications in which one reduces the risk that a
subject will develop a disorder or
other risk factor. Treatment does not require the complete curing of a
disorder and encompasses embodiments in
which one reduces symptoms or underlying risk factors. In the context of
combination therapy, the term may also
refer to: i) the ability of a second therapeutic to reduce the effective
dosage of a first therapeutic so as to reduce
side effects and increase tolerability; ii) the ability of a second therapy to
render the patient more responsive to a
first therapy; and/or iii) the ability to effectuate additive or synergistic
clinical benefits.
[180] Tumor-associated macrophage (TAM): TAMs are polarized/activated
macrophages with pro-tumor
phenotypes (M2-like macrophages). TAMs can be either marrow-originated
monocytes/macrophages recruited to
the tumor site or tissue-resident macrophages which are derived from erythro-
myeloid progenitors. Differentiation
of monocytes/macrophages into TAMs is influenced by a number of factors,
including local chemical signals such
as cytokines, chemokines, growth factors and other molecules that act as
ligands, as well as cell-cell interactions
between the monocytes/macrophages that are present in the niche (tumor
microenvironment). Generally,
monocytes/macrophages can be polarized into so-called "Ml" or "M2" subtypes,
the latter being associated with
more pro-tumor phenotype. In a solid tumor, up to 50% of the tumor mass may
correspond to macrophages, which
are preferentially M2-polarized. Among tumor-associated monocytes and myeloid
cell populations, M1
macrophages typically express cell surface HLA-DR, CD68 and CD86, while M2
macrophages typically express
cell surface HLA-DR, CD68, CD163 and CD206. Tumor-associated, M2-like
macrophages (such as M2c and M2d
subtypes) can express cell surface LRRC33 and/or LRRC33-proTGF61.
[181] Tumor microenvironment: The term "tumor microenvironment (TME)" refers
to a local disease niche, in
which a tumor (e.g., solid tumor) resides in vivo. The TME may comprise
disease-associated molecular signature
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(a set of chemokines, cytokines, etc.), disease-associated cell populations
(such as TAMs, CAFs, MDSCs, etc.) as
well as disease-associated ECM environments (alterations in ECM components
and/or structure).
[182] Variable region: The term "variable region" or "variable domain" refers
to a portion of the light and/or heavy
chains of an antibody, typically including approximately the amino-terminal
120 to 130 amino acids in the heavy
chain and about 100 to 110 amino terminal amino acids in the light chain. In
certain embodiments, variable regions
of different antibodies differ extensively in amino acid sequence even among
antibodies of the same species. The
variable region of an antibody typically determines specificity of a
particular antibody for its target.
[183] Other than in the operating examples, or where otherwise indicated, all
numbers expressing quantities of
ingredients or reaction conditions used herein should be understood as
modified in all instances by the term
"about." The term "about" means 10% of the recited value.
[184] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly
indicated to the contrary, should be understood to mean "at least one."
[185] The phrase "and/or," as used herein in the specification and in the
claims, should be understood to mean
"either or both" of the elements so conjoined, i.e., elements that are
conjunctively present in some cases and
disjunctively present in other cases. Other elements may optionally be present
other than the elements specifically
identified by the "and/or" clause, whether related or unrelated to those
elements specifically identified unless clearly
indicated to the contrary. Thus, as a non-limiting example, a reference to "A
and/or B," when used in conjunction
with open-ended language such as "comprising" can refer, in one embodiment, to
A without B (optionally including
elements other than B); in another embodiment, to B without A (optionally
including elements other than A); in yet
another embodiment, to both A and B (optionally including other elements);
etc.
[186] As used herein in the specification and in the claims, the phrase "at
least one," in reference to a list of one
or more elements, should be understood to mean at least one element selected
from any one or more of the
elements in the list of elements, but not necessarily including at least one
of each and every element specifically
listed within the list of elements and not excluding any combinations of
elements in the list of elements. This
definition also allows that elements may optionally be present other than the
elements specifically identified within
the list of elements to which the phrase "at least one" refers, whether
related or unrelated to those elements
specifically identified. Thus, as a non-limiting example, "at least one of A
and B" (or, equivalently, "at least one of
A or B," or, equivalently "at least one of A and/or B") can refer, in one
embodiment, to at least one, optionally
including more than one, A, with no B present (and optionally including
elements other than B); in another
embodiment, to at least one, optionally including more than one, B, with no A
present (and optionally including
elements other than A); in yet another embodiment, to at least one, optionally
including more than one, A, and at
least one, optionally including more than one, B (and optionally including
other elements); etc.
[187] Use of ordinal terms such as "first," "second," "third," etc., in the
claims to modify a claim element does not
by itself connote any priority, precedence, or order of one claim element over
another or the temporal order in
which acts of a method are performed, but are used merely as labels to
distinguish one claim element having a
certain name from another element having a same name (but for use of the
ordinal term) to distinguish the claim
elements.
[188] Ranges provided herein are understood to be shorthand for all of the
values within the range. For example,
a range of 1 to 50 is understood to include any number, combination of
numbers, or sub-range from the group
consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or
50, e.g., 10-20, 1-10, 30-40, etc.
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Transforming Growth Factor-beta (TGF13)
[189] The Transforming Growth Factor-beta (TGFp) activities and subsequent
partial purification of the soluble
growth factors were first described in the late 1970's to early 1980's, with
which the TGFP field began some 40
years ago. To date, 33 gene products have been identified that make up the
large TGFp superfamily. The TGFp
superfamily can be categorized into at least three subclasses by structural
similarities: TGFps, Growth-
Differentiation Factors (GDFs) and Bone-Morphogenetic Proteins (BMPs). The
TGFp subclass is comprised of
three highly conserved isoforms, namely, TGF[31, TGF[32 and TGF[33, which are
encoded by three separate genes
in human.
[190] The TGFPs are thought to play key roles in diverse processes, such as
inhibition of cell proliferation,
extracellular matrix (ECM) remodeling, and immune homeostasis. The importance
of TGF(31 for T cell homeostasis
is demonstrated by the observation that TGFp1 -/- mice survive only 3-4 weeks,
succumbing to multi-organ failure
due to massive immune activation (Kulkarni, A.B., et al., Proc Natl Acad Sci U
S A, 1993. 90(2): p. 770-4; Shull,
M.M., et al., Nature, 1992. 359(6397): p. 693-9). The roles of TGFp2 and TGFp3
are less clear. Whilst the three
TGFp isoforms have distinct temporal and spatial expression patterns, they
signal through the same receptors,
TGFpRI and TGFpRII, although in some cases, for example for TGFp2 signaling,
type III receptors such as
betaglycan are also required (Feng, X.H. and R. Derynck, Annu Rev Cell Dev
Biol, 2005.21: p. 659-93; Massague,
J., Annu Rev Biochem, 1998. 67: p. 753-91). Ligand-induced oligomerization of
TGFpRI/II triggers the
phosphorylation of SMAD transcription factors, resulting in the transcription
of target genes, such as Coll al,
Col3a1, ACTA2, and SERPINE1 (Massague, J., .J. Seoane, and D. VVotton, Genes
Dev, 2005. 19(23): p. 2783-
810). SMAD-independent TGFp signaling pathways have also been described, for
example in cancer or in the
aortic lesions of Marfan mice (Derynck, R. and Y.E. Zhang, Nature, 2003.
425(6958): p. 577-84; Holm, T.M., et al.,
Science, 2011. 332(6027): p. 358-61).
[191] The biological importance of the TGFp pathway in humans has been
validated by genetic diseases.
Camurati-Engelman disease results in bone dysplasia due to an autosomal
dominant mutation in the TGFB1 gene,
leading to constitutive activation of TGFp1 signaling (Janssens, K., et al., J
Med Genet, 2006. 43(1): p. 1-11).
Patients with Loeys/Dietz syndrome carry autosomal dominant mutations in
components of the TGFP signaling
pathway, which cause aortic aneurism, hypertelorism, and bifid uvula (Van
Laer, L., H. Dietz, and B. Loeys, Adv
Exp Med Blot, 2014. 802: p. 95-105). As TGFp pathway dysregulation has been
implicated in multiple diseases,
several drugs that target the TGFp pathway have been developed and tested in
patients, but with limited success.
[192] Dysregulation of the TGFp signaling has been associated with a wide
range of human diseases. Indeed,
in a number of disease conditions, such dysregulation may involve multiple
facets of TGFp function. Diseased
tissue, such as fibrotic and/or inflamed tissues and tumors, may create a
local environment in which TGFp
activation can cause exacerbation or progression of the disease, which may be
at least in part mediated by
interactions between multiple TGFp-responsive cells, which are activated in an
autocrine and/or paracrine fashion,
together with a number of other cytokines, chemokines and growth factors that
play a role in a particular disease
setting.
[193] For example, a tumor microenvironment (TME) contains multiple cell types
expressing TGFpl , such as
activated myofibroblast-like fibroblasts, stromal cells, infiltrating
macrophages, MDSCs and other immune cells, in
addition to cancer (i.e., malignant) cells. Thus, the TME represents a
heterogeneous population of cells expressing
and/or responsive to TGFpl but in association with more than one types of
presenting molecules, e.g., LTBP1,
LTBP3, LRRC33 and GARP, within the niche.
[194] Advances in immunotherapy have transformed the effective treatment
landscape for a growing number of
cancer patients. Most prominent are the checkpoint blockade therapies (CBT),
which have now become part of
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standard of care regimens for an increasing number of cancers. While profound
and durable responses to CBT
have been observed across a growing number of cancer types, it is now clear
that a significant fraction of tumors
appear to be refractory to CBT even at the outset of treatment, hence pointing
to primary resistance as a major
challenge to enabling many patients' immune systems to target and eliminate
tumor cells. Efforts to understand
and address the underlying mechanisms conferring primary resistance to CBT
have been undertaken in order to
broaden treatment efficacy for a greater number of patients. However, this
enthusiasm has been curbed by
lackluster clinical trial results and failures when combining CBTs with agents
known to affect the same tumor type
or to modulate seemingly relevant components of the immune system. A likely
reason is that a clear mechanistic
rationale for the given combination is often not rooted in clinically-derived
data, and has thus led to uncertain and
confounding outcomes in trials intended to enhance approved single-agent
therapies. It has become clear that the
design of combination immunotherapy should be rooted in scientific evidence of
relevance to underlying tumor and
immune system biology.
[195] Recently, a phenomenon referred to as "immune exclusion" was coined to
describe a tumor environment
from which anti-tumor effector T cells (e.g., CD8+ T cells) are kept away
(hence "excluded") by immunosuppressive
local cues. More recently, a number of retrospective analyses of clinically-
derived tumors have implicated TGF8
pathway activation in mediating primary resistance to CBT. For example,
transcriptional profiling and analysis of
pretreatment melanoma biopsies revealed an enrichment of TG93-associated
pathways and biological processes
in tumors that are non-responsive to anti-PD-1 CBT. In an immune-excluded
tumor, effector cells, which would
otherwise be capable of attacking cancer cells by recognizing cell-surface
tumor antigens, are prevented from
gaining access to the site of cancer cells. In this way, cancer cells evade
host immunity and immuno-oncologic
therapeutics, such as checkpoint inhibitors, that exploit and rely on such
immunity. Indeed, such tumors show
resistance to checkpoint inhibition, such as anti-PD-1 and anti-PD-L1
antibodies, presumably because target T
cells are blocked from entering the tumor hence failing to exert anti-cancer
effects.
[196] A number of retrospective analyses of clinically-derived tumors points
to TGF8 pathway activation in
mediating primary resistance to CBT. For example, transcriptional profiling
and analysis of pretreatment melanoma
biopsies revealed an enrichment of TGF8-associated pathways and biological
processes in tumors that are non-
responsive to anti-PD-1 CBT. More recently, similar analyses of tumors from
metastatic urothelial cancer patients
revealed that lack of response to PD-L1 blockade with atezolizumab was
associated with transcriptional signatures
of TGF8 signaling, particularly in tumors wherein CD8+ T cells appear to be
excluded from entry into the tumor.
The critical role of TGFI3 signaling in mediating immune exclusion resulting
in anti-PD-(L)1 resistance has been
verified in the EMT-6 syngeneic mouse model of breast cancer. While the EMT-6
tumors are weakly responsive
to treatment with an anti-PD-L1 antibody, combining this checkpoint inhibitor
with 1D11, an antibody that blocks
the activity of all TGFI3 isoforms, resulted in a profound increase in the
frequency of complete responses when
compared to treatment with individual inhibitors. The synergistic antitumor
activity is proposed to be due to a
change in cancer-associated fibroblast (CAF) phenotype and a breakdown of the
immune excluded phenotype,
resulting in infiltration of activated CD8+ T cells into the tumors. Similar
results were found in a murine model of
colorectal cancer and metastasis using a combination of an anti-PD-L1 antibody
with galunisertib, a small molecule
inhibitor of the type I TGFI3 receptor ALK5 kinase. Collectively, these
findings suggest that inhibiting the TGFI3
pathway in CBT-resistant tumors could be a promising approach to improve or
increase the number of clinical
responses to CBT. While recent work has implicated a relationship between
TGFI3 pathway activation and primary
CBT resistance, TGF8 signaling has long been linked to features of cancer
pathogenesis. As a potent
immunosuppressive factor, TGFI3 prevents antitumor T cell activity and
promotes immunosuppressive
macrophages. Malignant cells often become resistant to TGF8 signaling as a
mechanism to evade its growth and
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tumor-suppressive effects. TGF13 activates CAFs, inducing extracellular matrix
production and promotion of tumor
progression. Finally, TGFI3 induces EMT, thus supporting tissue invasion and
tumor metastases.
[197] Mammals have distinct genes that encode and express the three TG93
growth factors, TGFp1, TG932, and
TGFp3, all of which signal through the same heteromeric TGFp receptor complex.
Despite the common signaling
pathway, each TGFI3 isoform appears to have distinct biological functions, as
evidenced by the non-overlapping
TGFp knockout mouse phenotypes. All three TG93 isoforms are expressed as
inactive prodomain-growth factor
complexes, in which the TGFI3 prodomain, also called latency-associated
peptide (LAP), wraps around its growth
factor and holds it in a latent, non-signaling state. Furthermore, latent TGFp
is co-expressed with latent TGFp-
binding proteins and forms large latent complexes (LLCs) through disulfide
linkage. Association of latent TGFp
with Latent TGF13 Binding Protein-1 (LTBP1) or LTBP3 enables tethering to
extracellular matrix, whereas
association to the transmembrane proteins GARP or LRRC33 enables elaboration
on the surface of Tregs or
macrophages, respectively. In vivo, latent TG931 and latent TG933 are
activated by a subset of aV integrins,
which bind a consensus RGD sequence on LAP, triggering a conformational change
to release the growth factor.
The mechanism by which latent TGF(32 is activated is less clear as it lacks a
consensus RGD motif. TGFp1 release
by proteolytic cleavage of LAP has also been implicated as an activation
mechanism, but its biological relevance
is less clear.
[198] Although the pathogenic role of TGFp activation is clear in several
disease states, it is equally clear that
therapeutic targeting of the TGFP pathway has been challenging due to the
pleiotropic effects that result from broad
and sustained pathway inhibition. For example, a number of studies have shown
that small molecule-mediated
inhibition of the TGFp type I receptor kinase ALK5 (TGFBR1) or blockade of all
three highly related TGFp growth
factors with a high-affinity antibody resulted in severe cardiac
valvulopathies in mice, rats and dogs. These "pan"-
TGFP approaches that block all TGFP signaling therefore have a very narrow
therapeutic window, which has proven
to be an impediment to the treatment of a number of disease-relevant processes
with very high unmet medical
need. No TGFp-targeting therapy has been approved to date and clinical trial
results with such modalities have
largely been disappointing, likely due to the use of what proved to be
inefficacious dosing regimens that were
required in order to accommodate safety concerns.
[199] All references cited herein are incorporated by reference for any
purpose. Where a reference and the
specification conflict, the specification will control. It is to be
appreciated that certain features of the disclosed
compositions and methods, which are, for clarity, described herein in the
context of separate embodiments, may
also be provided in combination in a single embodiment. Conversely, various
features of the disclosed compositions
and methods that are, for brevity, described in the context of a single
embodiment, may also be provided separately
or in any subcombination.
Methods of Treatment and Biomarkers of Therapeutic Efficacy
[200] The subject matter of the present disclosure generally relates to the
disclosure of PCT/2021/012969 filed
January 11,2021, the entire content of which is incorporated by reference
herein.
Circulating/circulatory MDSCs as a biomarker
[201] MDSCs are a heterogeneous population of cells named for their myeloid
origin and their main immune
suppressive function (Gabrilovich. Cancer Immunol Res. 2017 Jan; 5(1): 3-8).
MDSCs generally exhibit high
plasticity and strong capacity to reduce cytotoxic functions of T cells and
natural killer (NK) cells, including their
ability to promote T regulatory cell (Treg) expansion and in turn suppress T
effector cell function (Gabrilovich et al.,
Nat Rev Immunol. (2012) 12:253-68). MDSCs are typically classified into two
subsets, monocytic (m-MDSCs) and
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granulocytic (G-MDSCs or PMN-MDSCs), based on their expression of surface
markers (Consonni et al., Front
Immunol. 2019 May 3; 10:949). Suppressive G-MDSCs can be characterized by
their production of reactive oxygen
species (ROS) as the major mechanism of immune suppression. In contrast, M-
MDSCs mediate immune
suppression primarily by upregulating the inducible nitric oxide synthase gene
(iNOS) and produce nitric oxide (NO)
as well as an array of immune suppressive cytokines (Youn and Garilovich, EurJ
Immunol. 2010 Nov; 40(11):
2969-2975).
[202] MDSCs have been implicated in various diseases, such as chronic
inflammation, infection, autoimmune
diseases, and graft-versus-host diseases. In recent years, MDSCs have become
an immune population of interest
in cancer due to their role in inducing T cell tolerance through checkpoint
blockade molecules such as the
programmed death-ligand 1 (PD-L1) and the cytotoxic T-lymphocyte antigen 4
(CTLA4) (Trovato et al., J
Immunother Cancer. 2019 Sep 18;7(1):255). Furthermore, MDSCs have generally
been characterized as favoring
tumor progression by mechanisms in addition to immune suppression, including
promoting tumor angiogenesis.
Studies to date have focused on MDSCs present in tumor biopsies, given their
propensity to enrich around inflamed
tissue. (Passro et al., Clin Trans/ Oncol. 2019 Jun 28.; Al et al., BMC
Cancer. 2018 Dec 5;18(1):1220; Nakamura.
Front Med (Lausanne). 2019; 6: 119). However, such studies had not been
reported in the literature to have
elucidated a clear relationship between MDSC levels and therapeutic response.
For instance, low baseline
monocytic MDSC frequency was shown to correlate poorly with treatment benefits
(Pico de Coafia et al.,
Oncotarget. 2017 Mar 28; 8(13): 21539-21553).
[203] Many human cancers (e.g., solid tumors) are known to show elevated
levels of MDSCs in biopsies from
patients, as compared to healthy controls (reviewed, for example, in Elliott
et al., (2017) Frontiers in Immunology,
Vol. 8, Article 86). These human cancers include but are not limited to
bladder cancer, colorectal cancer, prostate
cancer, breast cancer, glioblastoma, hepatocellular carcinoma, head and neck
squamous cell carcinoma, lung
cancer, melanoma, NSCLC, ovarian cancer, pancreatic cancer, and renal cell
carcinoma. The compositions and
methods according to the present disclosure may be applied to one or more of
these cancers.
[204] Previously, it was demonstrated by Applicant that immunosuppressive
tumors contain elevated levels of
tumor-infiltrating or intratumoral MDSCs, also referred to as tumor-associated
MDSCs, and evidence indicated that
this was inversely correlated with anti-tumor immunity in a TGF61-dependent
manner. Data provided in
PCT/US2019/041373, the content of which is hereby incorporated in its
entirety. These data suggested that
probing tumor-associated immune cells, by, for example, biopsies, can be
useful for characterizing anti-tumor
effects in cancer patients. Further, Applicant made a surprising finding that
relatively simple and noninvasive blood
tests may provide equivalent information, leading to the recognition that
pharmacological effects of TG931 inhibition
on overcoming an immunosuppressive phenotype can be determined by measuring
circulating MDSC levels.
[205] The present disclosure includes the finding that circulating MDSC levels
(including gMDSCs and/or
mMDSCs) may be determined by detecting or measuring LRRC33-positive cells in a
blood sample, identifying
LRRC33 as a novel blood-based biomarker for circulating MDSCs. For example,
LRRC33-positive cells in a blood
sample collected from a patient (such as cancer patient) may be detected or
measured by a FACS-based assay
using an antibody that binds cell-surface LRRC33. In some embodiments, the
LRRC33-expressing cells in a blood
sample collected from a subject having cancer are G-MDSCs. While MDSCs are
derived from bone marrow-
originated monocytes, cell-surface expression of LRRC33 appears to be narrowly
restricted to MDSCs, and not
monocytes, in circulation. This recognition raises a new possibility of using
LRRC33 as a blood-based marker for
circulating MDSCs. In some embodiments, LRRC33 expression may be determined by
any of the antibodies
disclosed in WO/2018/208888 and WO/2018/081287, the contents of which are
incorporated herein in their entirety.
Applicant has now established a correlation between circulatory MDSC levels
and tumor-associated MDSC levels.
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Together with the finding that circulatory MDSCs appear to show robust and
uniform cell-surface LRRC33
expression, determination of LRRC33 levels measured in blood samples may serve
as an effective surrogate to
assess tumor immune-phenotype, such as immunosuppression, without the need for
more invasive procedures
such as tumor biopsy.
[206] In various embodiments, the present disclosure provides methods of
treating cancer, predicting, or
determining efficacy, and/or confirming pharmacological response by monitoring
the levels of circulating MDSCs
in a sample obtained from a patient (e.g., in the blood or a blood component
of a patient) receiving a TGF3 inhibitor,
e.g., a TGFp1-selective inhibitor (such as a selective pro- or latent-TGF31
inhibitor, e.g., Ab6), isoform-non-
selective TGFI3 inhibitors (such as low molecular weight ALK5 antagonists,
neutralizing antibodies that bind two or
more of TGF31/2/3, e.g., GC1008 and variants, antibodies that bind TGFp1 /3,
ligand traps, e.g., TGF31/3
inhibitors), and/or an integrin inhibitor (and integrin inhibitors (e.g.,
antibodies that bind to aV31, aV133, aV35, aVp6,
aVP8, a5P1, 011bI33, or a8]1 integrins, and inhibit downstream activation of
TGFP. e.g., selective inhibition of
TGF31 and/or TGFp3). Exemplary integrin inhibitors include the anti-aVp8
integrin antibodies provided in
W02020051333, the disclosure of which is incorporated by reference. In various
embodiments disclosed herein,
the circulating MDSCs may be measured within 1, 2, 3, 4, 5, 6, or 7 days, or
within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
weeks (e.g., preferably less than 6 weeks) following administration of a
treatment to a subject, e.g., administration
of a therapeutic dose of a TGFp inhibitor.
[207] In certain embodiments, the TGFP treatment may be administered alone or
in conjunction with an additional
cancer therapy. The treatment may be administered to subjects with an
immunosuppressive cancer or a
myeloproliferative disorder. In some embodiments, the TGFp inhibitor is a
TGF31-selective antibody or antigen-
binding fragment thereof encompassed in the current disclosure (e.g., Ab6). In
some embodiments, the TG931-
selective antibody or antigen-binding fragment does not inhibit TGFI32 and
TGFP3 at a therapeutically effective
dose. In some embodiments, the TGF13 inhibitor is an isoform-non-selective
TGFp inhibitor (such as low molecular
weight ALK5 antagonists, neutralizing antibodies that bind two or more of
TGFp1 /2/3, e.g., GC1 008 and variants,
antibodies that bind TGF31 /3, and ligand traps, e.g., TGFp1 /3 inhibitors).
In some embodiments, the TGF13 inhibitor
is an integrin inhibitor (e.g., an antibody that binds to aV31, aVI33, aVp5,
aV36, aN/38, a5131, a11b33, or a831
integrins, and inhibits downstream activation of TGFp. e.g., selective
inhibition of TGF31 and/or TGF33).
Exemplary integrin inhibitors include the anti-aVp8 integrin antibodies
provided in W02020051333, the disclosure
of which is incorporated by reference. In some embodiments, the additional
cancer therapy may include
chemotherapy, radiation therapy (including radiotherapeutic agents), cancer
vaccine or immunotherapy including
checkpoint inhibitor therapies such as anti-PD-1, anti-PD-L1, and anti-CTLA-4
antibodies. In some embodiments,
the checkpoint inhibitor therapy is selected from the group consisting of
ipilimumab (e.g., Yervoy0); nivolumab
(e.g., Opdivoil0); pembrolizumab (e.g., Keytrudae); avelumab (e.g.,
Bavencioe); cemiplimab (e.g., Libtayo410);
atezolizumab (e.g., Tecentrige); budigalimab (e.g., ABBV-181); and durvalumab
(e.g., Imfinzie). In preferred
embodiments, a combination cancer therapy comprises Ab6 and at least one
checkpoint inhibitor (such as those
listed above). Thus, in some embodiments, a combination of Ab6 and a
checkpoint inhibitor is used for the
treatment of cancer in a human patient in amounts effective to treat the
cancer. In some embodiments, the TGFP
treatment may further or alternatively include a second checkpoint inhibitor.
In some embodiments, the TGFp
treatment may further or alternatively include a chemotherapy (e.g., a
genotoxic therapy or radiation therapy).
[208] For example, without being bound by theory, evidence suggests that
overactive TGF3 pathways may
correlate with unresponsiveness of a tumor to genotoxic therapies, such as
chemotherapy and radiation therapy
(Liu et al., Sci Trans/Med. 2021 Feb 10;13(580):eabc4465). This is observed
across multiple cancer types, e.g.,
cancers of the epithelia, e.g., carcinoma. In certain embodiments, such cancer
types include ovarian cancer, breast
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cancer, bladder cancer, pancreatic cancer, e.g., pancreatic adenocarcinoma,
prostate cancer, e.g., prostate
adenocarcinoma, melanoma, e.g., skin cutaneous melanoma, lung cancer, e.g.,
lung squamous cell carcinoma
and lung adenocarcinoma, liver cancer (e.g., liver hepatocellular carcinoma),
uterine cancer, e.g., uterine corpus
endometrial carcinoma, kidney cancer, e.g., renal clear cell carcinoma, head
and neck cancer, e.g., head and neck
squamous cell carcinoma, colon cancer, e.g., colon adenocarcinoma, esophageal
carcinoma, and tenosynovial
giant cell tumor (TGCT). In some embodiments, the cancer is a cancer having
elevated TGP[31 levels associated
with ROS (e.g., elevated ROS). Without being bound by theory, ROS may induce
an increase in TGFI3 levels (e.g.,
TGF61 levels) which may be reduced by a TGFI3 inhibitor (e.g., a TGF[31
inhibitor) disclosed herein.
[209] Accordingly, TGFI3 inhibitors (e.g., Ab6) may be used in conjunction
with one or more genotoxic therapies
(e.g., chemotherapy and/or radiation therapy, including radiotherapeutic
agents) to treat such a cancer in a subject.
In certain embodiments, such a cancer may have elevated TGFI3 levels, e.g.,
elevated TGFI3 activity, as indicated
by direct measurement and/or one or more changes in downstream gene regulation
(e.g., in one or more genes
involved in DNA repair). For instance, a cancer, such as one of the cancers
listed above, may have elevated TGFI3
signaling as indicated by upregulation of one or more genes associated with
non-homologous end joining (NHEJ),
e.g., Cyclin Dependent Kinase Inhibitor 1A (CDKN1A), or downregulation of one
or more genes relating to
alternative end joining, e.g., LIG1 (DNA ligase 1), PARP1, and/or POLO.
[210] The present disclosure also provides methods of using measurements of
circulating MDSCs in treating
cancer in subjects administered a TGFI3 inhibitor alone or in conjunction with
an immunotherapy. Furthermore, the
descriptions presented herein provide support for the circulating MDSC
population as an early predictive marker of
efficacy, particularly in cancer subjects treated with a TGFp inhibitor and
checkpoint inhibitor combination therapy,
e.g., at a time point before other markers of treatment efficacy, such as a
reduction in tumor volume, can be
detected.
[211] In certain embodiments, a TGF13 inhibitor, e.g., a TGF[31-selective
inhibitor such as Ab6, an isoform-non-
selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing
antibodies that bind two or more of
TGF131/2/3, e.g., GC1008 and variants, antibodies that bind TGFI31/3, ligand
traps, e.g., TGF131/3 inhibitors, and/or
an integrin inhibitor (e.g., an antibody that binds to aV131, aV133, aV[35,
aV136, aV[38, 05131, al lb[33, or a861 integrins,
and inhibits downstream activation of TGFI3. e.g., selective inhibition of
TGFI31 and/or TGFp3) is administered
concurrently (e.g., simultaneously), separately, or sequentially to a
checkpoint inhibitor therapy such that the
amount (e.g., dose) of TGF61 inhibition administered is sufficient to reduce
circulating MDSC levels by at least
10%, at least 15%, at least 20%, at least 25%, or more, as compared to
baseline MDSC levels. Circulating MDSC
levels may be measured prior to or after each treatment or each dose of the
TGF[3 inhibitor such that a decrease
of at least 10%, at least 15%, at least 20%, at least 25%, or more in
circulating MDSC levels may be indicative or
predictive of treatment efficacy. In some embodiments, the level of
circulating MDSCs may be used to determine
disease burden (e.g., as measured by a change in relative tumor volume before
and after a treatment regimen). In
some embodiments, the level of circulating mMDSCs may be used to determine
disease burden (e.g., as measured
by a change in relative tumor volume before and after a treatment regimen).
[212] In certain embodiments, a decrease in circulating MDSC levels (e.g.,
mMDSC levels) may be indicative of
a decrease in disease burden (e.g., a decrease in relative tumor volume). For
instance, circulating MDSC levels
(e.g., mMDSC levels) may be measured prior to and after the administration of
a dose of TGF inhibitor (such as
isoform-selective inhibitors, e.g., Ab6, isoform-non-selective TGFI3
inhibitors, e.g., low molecular weight ALK5
antagonists, neutralizing antibodies that bind two or more of TGFI31/2/3,
e.g., GC1008 and variants, antibodies that
bind TGF[31/3, ligand traps, e.g., TGF[31/3 inhibitors, and/or an integrin
inhibitor (e.g., an antibody that binds to
aV[31, aV[33, aV[35, aV[36, aV[38, a5[31, al lb[33, or a8[31 integrins, and
inhibits downstream activation of TGF[3, e.g.,
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selective inhibition of TG931 and/or TG933) and a reduction in the circulating
MDSC levels may be indicative or
predictive of pharmacological effects, e.g., of a reduction in disease burden
(e.g., a reduction in relative tumor size).
[213] In certain embodiments, circulating MDSC levels (e.g., circulating mMDSC
levels) may be measured prior
to and following administration of a first dose of a TGFp inhibitor, such as a
TGF131-selective inhibitor, e.g., Ab6,
an isoform-non-selective inhibitor, e.g., low molecular weight ALK5
antagonists, neutralizing antibodies that bind
two or more of TGF(31/2/3, e.g., GC1008 and variants, antibodies that bind
TGF131/3, ligand traps, e.g., TGF(31/3
inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to
0VI31, aVp3, aVp5, aVp6, aVp8, a5(31, al Ibp3,
0r a8131 integrins, and inhibits downstream activation of TGFp. e.g.,
selective inhibition of TGF131 and/or TGF133).
In some embodiments, administration of a first dose of TGFI3 inhibitor e.g.,
Ab6, isoform-non-selective TGFp
inhibitors, e.g., low molecular weight ALK5 antagonists, neutralizing
antibodies that bind two or more of TG931/2/3,
e.g., GC1008 and variants, antibodies that bind TGF131/3, ligand traps, e.g.,
TGF01 /3 inhibitors, and/or an integrin
inhibitor, e.g., an antibody that binds to aVP1, aVP3, aVP5, aVP6, aVP8,
05131, allb(33, or a8P1 integrins, and
inhibits downstream activation of TGFp. e.g., selective inhibition of TGF131
and/or TGF133 may be used to reduce
tumor volume, such that administration of the TG93 inhibitor reduces
circulating MDSC levels by at least 10%, at
least 20%, at least 25%, or more, as compared to circulating MDSC levels
(e.g., circulating mMDSC levels) prior
to administration.
[214] In some embodiments, reduction in circulating MDSC levels (e.g.,
circulating mMDSC levels) is indicative
or predictive of pharmacological effects and further warrants administration
of a second or more dose(s) of the
TGFp inhibitor. In some embodiments, reduction in circulating mMDSC levels is
indicative or predictive of
pharmacological effects and further warrants administration of a second or
more dose(s) of the TGFp inhibitor. In
some embodiments, the first dose of the TGF13 inhibitor is the very first dose
of TGFp inhibitor received by the
patient. In some embodiments, the first dose of the TGFP inhibitor is the
first dose of a given treatment regimen
comprising more than one dose of TGFI3 inhibitor. In another embodiment,
circulating MDSC levels (e.g., circulating
mMDSC levels) may be measured prior to and after combination treatment
comprising a TGFp inhibitor (e.g., Ab6)
and a checkpoint inhibitor therapy, administered concurrently (e.g.,
simultaneously), separately, or sequentially,
and a reduction in the circulating MDSC levels is indicative or predictive of
therapeutic efficacy. In some
embodiments, the reduction of circulating MDSC levels following the
combination treatment of a TGFp inhibitor,
such as a TGF131 inhibitor, such as a TG931-selective inhibitor, e.g., Ab6, an
isoform-non-selective inhibitor, e.g.,
low molecular weight ALK5 antagonists, neutralizing antibodies that bind two
or more of TGF131/2/3, e.g., GC1008
and variants, antibodies that bind TGFI31/3, ligand traps, e.g., TGF131/3
inhibitors, and/or an integrin inhibitor (e.g.,
an antibody that binds to aVp1, aV(33, aV135, aVp6, aVp8, a5131, all bp3, 0r
a8131 integrins, and inhibits downstream
activation of TGFp. e.g., selective inhibition of TGF(31 and/or TGFI33), and a
checkpoint inhibitor therapy, may
warrant continuation of treatment. In some embodiments, the reduction of
circulating mMDSC levels following the
combination treatment of a checkpoint inhibitor therapy and a TGFp inhibitor,
such as a TGFI31 inhibitor, such as
a TGFp1-selective inhibitor, e.g., Ab6, or an isoform-non-selective inhibitor,
e.g., a low molecular weight ALK5
antagonist, a neutralizing antibody that bind two or more of TGF131/2/3, e.g.,
GC1008 and variants, an antibody
that bind TGFP1/3, ligand traps, e.g., TGFP1/3 inhibitors, and/or an integrin
inhibitor (e.g., an antibody that binds
to aV131, aV133, aV35, aVp6, aVI38, a5131, a11 b133, or a8p1 integrins, and
inhibits downstream activation of TGFp.
e.g., selective inhibition of TGFp1 and/or TGFp3), may warrant continuation of
treatment. In some embodiments,
the reduction of circulating gMDSC levels following the combination treatment
of a checkpoint inhibitor therapy and
a TGFp inhibitor, such as a TGFp1 inhibitor, such as a TGF131 -selective
inhibitor, e.g., Ab6, or an isoform-non-
selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a
neutralizing antibody that bind two or more of
TGF131/2/3, e.g., GC1008 and variants, an antibody that bind TGF131/3, ligand
traps, e.g., TG931/3 inhibitors,
and/or an integrin inhibitor (e.g., an antibody that binds to aVp1, aVp3,
aV135, aVp6, aVp8, a5131, al Ibp3, or a8131
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integrins, and inhibits downstream activation of TGFp. e.g., selective
inhibition of TGF131 and/or TGFp3), may
warrant continuation of treatment.
[215] In certain embodiments of the present disclosure, levels of circulating
MDSCs may be used to predict,
determine, and monitor pharmacological effects of treatment comprising a dose
of TGFp inhibitor, such as a
TGF131-selective inhibitor, e.g., Ab6, an isoform-non-selective inhibitor,
e.g., low molecular weight ALK5
antagonists, neutralizing antibodies that bind two or more of TGFp1 /2/3,
e.g., GC1008 and variants, antibodies that
bind TGFp1/3, ligand traps, e.g., TGFp1/3 inhibitors, and/or an integrin
inhibitor (e.g., an antibody that binds to
aV131, aV133, aV135, aV136, aV138, a5131, a1 1b133, or a8131 integrins, and
inhibits downstream activation of TGFp. e.g.,
selective inhibition of TGFI31 and/or TGFI33) administered alone or in
conjunction with another cancer therapy such
as a checkpoint inhibitor. In certain embodiments, a baseline circulating MDSC
level may be measured before
administering an initial treatment (e.g., a first dose of a TGF13 inhibitor).
In certain embodiments, a low baseline
circulating MDSC level is predictive of better response to the TGFP inhibitor
treatment. In certain embodiments, a
low baseline circulating mMDSC level is predictive of better response to the
TGFp inhibitor treatment. In certain
embodiments, a low baseline circulating mMDSC level is predictive of better
response to the TGFp inhibitor
treatment. In certain embodiments, a patient administered the TGFp inhibitor
treatment has a low baseline
circulating mMDSC level. In certain embodiments, circulating MDSCs may be
measured within six weeks following
administration of the initial treatment. In certain embodiments, circulating
MDSC levels may be measured within
thirty days following administration of the initial dose of TGFp inhibitor. In
some embodiments, MDSC levels may
be measured within or at about three weeks following administration of the
initial dose of TGFp inhibitor. In some
embodiments, MDSC levels may be measured within or at about two weeks
following administration of the initial
dose of TGFp inhibitor. In some embodiments, MDSC levels may be measured
within or at about ten days following
administration of the initial dose of TGFp inhibitor. In certain embodiments,
the MDSCs are circulating mMDSCs.
In certain embodiments, the MDSCs are circulating gMDSCs.
[216] In certain embodiments, circulating MDSC levels may be used to select,
inform treatment, and/or predict
response in patients who have not received a checkpoint inhibitor treatment
previously. Patients diagnosed with a
cancer type with reported high response rates to checkpoint inhibitor therapy
(e.g., overall response rate of greater
than 30%, greater 40%, greater than 50%, or greater, as reported in the art)
who have not received a checkpoint
inhibitor therapy previously may be tested to first determine whether their
tumors exhibit an immune-excluded or
immunosuppressive phenotype. In some embodiments, circulating MDSCs may be
used in conjunction with
immunohistochemistry, flow cytometry, and/or in vivo imaging methods known in
the art to determine the immune
phenotype of the tumor. Patients with cancers exhibiting an immune-excluded or
immunosuppressive phenotype
may be selected to receive a TGFp inhibitor, such as a TGFp1-selective
inhibitor, e.g., Ab6, an isoform-non-
selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing
antibodies that bind two or more of
TGFp1/2/3, e.g., GC1008 and variants, antibodies that bind TGFp1/3, ligand
traps, e.g., TGFp1/3 inhibitors, and/or
an integrin inhibitor (e.g., an antibody that binds to aVp1, aVp3, aVp5,
aV(36, aV138, 05131, a1 1b133, 0r a8]31 integrins,
and inhibits downstream activation of TGFp. e.g., selective inhibition of
TGFI31 and/or TGF133) and checkpoint
inhibitor combination therapy (e.g., an anti-PD1 or anti-PD-L1 antibody). In
some embodiments, the circulating
MDSCs are circulating mMDSCs. In some embodiments, the circulating MDSCs are
circulating gMDSCs. In some
embodiments, patients exhibiting an immune-excluded or immunosuppressive
phenotype are treated with a TGFp
inhibitor.
[217] In some embodiments, circulating MDSC levels (e.g., circulating mMDSC
levels) may be further monitored
as an early predictor of treatment response. In certain embodiments, patients
diagnosed with a cancer type with
reported low response rates to checkpoint inhibitor therapy (e.g., overall
response rate of 30% or less, 20% or less,
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or 10%, or less, as reported in the art) who have not received a checkpoint
inhibitor therapy previously may be
treated with a combination of a TGFI3 inhibitor, such as a TGFI31-selective
inhibitor, e.g., Ab6, an isoform-non-
selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing
antibodies that bind two or more of
TGF131/2/3, e.g., GC1008 and variants, antibodies that bind TGF[31/3, ligand
traps, e.g., TGF131/3 inhibitors, and/or
an integrin inhibitor (e.g., an antibody that binds to aV131, aV133, aV135,
aV[36, aVP8, a5131, allb133, or a8131 integrins,
and inhibits downstream activation of TGFI3. e.g., selective inhibition of
TGF[31 and/or TGF[33) and a checkpoint
inhibitor therapy. In some embodiments, treatment response in these patients
may be predicted by monitoring
circulating MDSC levels. In some embodiments, treatment response in these
patients may be predicted by
monitoring circulating mMDSC levels. In some embodiments, treatment response
in these patients may be
predicted by monitoring circulating gMDSC levels. In some embodiments,
treatment is continued based on the
circulating MDSC levels.
[218] In certain embodiments, circulating MDSC levels may be used for
selecting, informing treatment in, and
predicting response in patients who are resistant to checkpoint inhibitor
therapy or who do not tolerate checkpoint
inhibitor therapy (e.g., due to adverse effects). These patients may have
primary resistance (i.e., have never shown
response to checkpoint inhibitor therapy) or have acquired resistance (i.e.,
have responded checkpoint inhibitor
therapy initially and developed resistance over time). In some embodiments,
resistance to checkpoint inhibitor
therapy in patients is indicative of immune suppression or exclusion, thus
these patients may be selected as
candidates for receiving a TGFI3 inhibitor therapy, such as a TGFI31-selective
inhibitor, e.g., Ab6, an isoform-non-
selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing
antibodies that bind two or more of
TGFp1/2/3, e.g., GC1008 and variants, antibodies that bind TGF[31/3, and
ligand traps, e.g., TGF[31/3 inhibitors,
and/or an integrin inhibitor (e.g., an antibody that binds to aVp1, aVp3,
aVp5, aVp6, aV138, a5p1, a1 1b133, or a8p1
integrins, and inhibits downstream activation of TGF[3. e.g., selective
inhibition of TGF[31 and/or TGFp3). In certain
embodiments, patients with either primary resistance or acquired resistance to
checkpoint inhibitor may be
administered a TGFI3 inhibitor, such as a TGFI31-selective inhibitor, e.g.,
Ab6, an isoform-non-selective inhibitor,
e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind
two or more of TGF[31/2/3, e.g.,
GC1008 and variants, antibodies that bind TG931/3, ligand traps, e.g., TG931/3
inhibitors, and/or an integrin
inhibitor (e.g., an antibody that binds to aV[31, aV[33, aV[35, aV[36, aV[38,
a5[31, a11b133, or a8p1 integrins, and
inhibits downstream activation of TGF[3. e.g., selective inhibition of TGFpl
and/or TGF[33), and their response to
treatment may be monitored and/or predicted by circulating MDSC levels. In
some embodiments, a reduction of at
least 10%, at least 15%, at least 20%, at least 25%, or more in circulating
MDSC levels may be indicative of
response to the TGFI3 inhibitor therapy. In some embodiments, a reduction of
at least 10%, at least 15%, at least
20%, at least 25%, or more in circulating MDSC levels may indicate
pharmacological effects of a treatment, e.g.,
with a TGF[3 inhibitor. In certain embodiments, a decrease in circulating MDSC
levels may be indicative of a
decrease in tumor size. In certain embodiments, a decrease in circulating
mMDSC levels may be indicative of a
decrease in tumor size. In certain embodiments, a decrease in circulating
gMDSC levels may be indicative of a
decrease in tumor size. A chart summarizing exemplary treatment regimens is
provided in FIG. 40.
[219] Most TGFI3 inhibitors currently in development are not isoform-
selective. These include pan-inhibitors of
TGFI3, and inhibitors that target TGF[31/2 and TGF[31/3. Approaches taken to
manage possible toxicities
associated with such inhibitors include careful dosing regimens to hit a
narrow window in which both efficacy and
acceptable safety profiles may be achieved. This may include sparing of an
isoform non-selective inhibitor, which
may include infrequent dosing and/or reducing dosage per administration. For
instance, in lieu of weekly dosing
of a biologic TGFI3 inhibitor, monthly dosing may be considered. Another
example is to dose only in an initial phase
of a combination immunotherapy so as to avoid or minimize toxicities
associated with TGFp inhibition.
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[220] Because a combination therapy comprising a cancer therapy (such as
checkpoint inhibitor therapy) and an
isoform-non-selective TGFI3 inhibitor may result in a greater risk of toxicity
as compared to a TGFI31-selective
inhibitor (e.g. Ab6), in order to mitigate or manage such risk, the isoform-
non-selective TGFp inhibitor may be
administered infrequently or intermittently, for example on an "as-needed"
basis. In such treatment paradigm,
circulating MDSC levels may be monitored periodically in order to determine
that the effects of overcoming
immunosuppression are sufficiently maintained, so as to ensure antitumor
effects of the cancer therapy. During
the course of cancer treatment, if MDSCs become elevated, it indicates that
the patient benefits from additional
doses of a TGFI3 inhibitor. Such approach may help reduce unnecessary risk and
adverse events associated with
TGFI3 inhibition, non-isoform-selective inhibitors in particular. In some
embodiments, the TGFI3 inhibitor targets
TGF131/2. In some embodiments, the TGFI3 inhibitor targets TGFI31/3. In some
embodiments, the TGFI3 inhibitor
targets TG931/2/3. In some embodiments, the TGFI3 inhibitor selectively
targets TGFI31.
[221] Accordingly, the present disclosure provides a TGFI3 inhibitor for use
in an intermittent dosing regimen for
cancer immunotherapy in a patient, wherein the intermittent dosing regimen
comprises the following steps:
measuring circulating MDSCs (e.g., circulating mMDSCs and/or circulating
gMDSCs) in a first sample collected
from the patient prior to a TGFI3 inhibitor treatment; administering a TGFI3
inhibitor to the patient treated with a
cancer therapy, wherein the cancer therapy is optionally a checkpoint
inhibitor therapy and/or a chemotherapy;
measuring circulating MDSCs (e.g., circulating mMDSCs and/or circulating
gMDSCs) in a second sample collected
from the patient after the TGFI3 inhibitor treatment; continuing with the
cancer therapy if the second sample shows
reduced levels of circulating MDSCs as compared to the first sample. In some
embodiments, the intermittent dosing
regimen further comprises measuring circulating MDSCs (e.g., circulating
mMDSCs and/or circulating gMDSCs) in
a third sample; and, administering to the patient an additional dose of a
TGFI3 inhibitor, if the third sample shows
elevated levels of circulating MDSC levels as compared to the second sample.
In some embodiments, the TGF13
inhibitor is an isoform-non-selective inhibitor. In some embodiments, the
sample is blood or a blood component
sample. In some embodiments, the isoform-non-selective inhibitor inhibits
TGF(31/2/3, TGFI31/2 or TGFp1/3.
Baseline circulating MDSC levels are likely to be elevated in cancer patients
as compared to healthy individuals,
and subjects with immunosuppressive cancers may have even more elevated
circulating MDSC levels. As such,
decreases in circulating MDSC levels (e.g., decreases in circulating mMDSC
levels) in patients treated with a TG93
inhibitor therapy such as a TGF131-selective inhibitor (e.g., Ab6), an isoform-
non-selective inhibitor (e.g., low
molecular weight ALK5 antagonists), neutralizing antibodies that bind two or
more of TGF[31/2/3 (e.g., GC1008 and
variants), antibodies that bind TGFI31/3, ligand traps (e.g., TGFP1/3
inhibitors), and/or an integrin inhibitor (e.g., an
antibody that binds to aVp1, aVp3, 0V135, aV[36, aVI38, a5[31, a1 1b133, or
a831 integrins, and inhibits downstream
activation of TGFI3. e.g., selective inhibition of TGFI31 and/or TGFI33),
either alone or in combination with a
checkpoint inhibitor therapy, may be indicative of a reduction or reversal of
immune suppression in the cancer.
[222] In certain embodiments, a TGFI3 inhibitor, such as a TGFI31-selective
inhibitor (e.g., Ab6), an isoform-non-
selective inhibitor (e.g., low molecular weight ALK5 antagonists),
neutralizing antibodies that bind two or more of
TGFI31/2/3 (e.g., GC1008 and variants), antibodies that bind TGFI31/3, ligand
traps (e.g., TGFI31/3 inhibitors),
and/or an integrin inhibitor (e.g., an antibody that binds to aVP1, aVP3,
aVI35, aVP6, aVP8, a5P1, al IbP3, or a8P1
integrins, and inhibits downstream activation of TGFp. e.g., selective
inhibition of TGFI31 and/or TGF133) is
administered to a subject with cancer such that the dose of the TGFI3
inhibitor is sufficient to reduce or reverse
immune suppression in the cancer as indicated by a reduction of circulating
MDSC levels (e.g., a reduction of
circulating mMDSC levels) and/or a change in the levels of tumor-associated
immune cells measured after
administering the TGFI3 inhibitor treatment as compared to levels measured
before administration. In some
embodiments, levels of circulating MDSC and/or tumor-associated immune cells
are measured before and after
administration of a TGFp inhibitor treatment such as a TGFI31-selective
inhibitor (e.g., Ab6), an isoform-non-
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selective inhibitor (e.g., low molecular weight ALK5 antagonists),
neutralizing antibodies that bind two or more of
TGFI31/2/3 (e.g., GC1008 and variants), antibodies that bind TGFI31/3, ligand
traps (e.g., TGFp1/3 inhibitors),
and/or an integrin inhibitor (e.g., an antibody that binds to aV[31, aV[33,
aVp5, aV[36, aVp8, a5[31, al Ibp3, or a8[31
integrins, and inhibits downstream activation of TGF[3. e.g., selective
inhibition of TGF[31 and/or TGF[33) in
combination with a checkpoint inhibitor therapy, and a reduction of
circulating MDSC levels and/or change(s) in the
levels of tumor-associated immune cells measured after treatment as compared
to levels measure before treatment
indicates reduction or reversal of immune suppression in the cancer. In some
embodiments, a reduction of
circulating mMDSC levels and/or change(s) in the levels of tumor-associated
immune cells measured after
treatment as compared to levels measure before treatment indicates reduction
or reversal of immune suppression
in the cancer. In some embodiments, a reduction of circulating gMDSC levels
and/or change(s) in the levels of
tumor-associated immune cells measured after treatment as compared to levels
measure before treatment
indicates reduction or reversal of immune suppression in the cancer. In some
embodiments, treatment is continued
in a patient exhibiting a reversal of immune suppression.
[223] Circulating MDSC levels may be determined in a sample such as a whole
blood sample or a blood
component (e.g., PBMCs). In some embodiments, the sample is fresh whole blood
or a blood component of a
sample that has not been previously frozen. In certain embodiments,
circulating MDSCs may be collected by
drawing peripheral blood into heparinized tubes. From peripheral blood,
peripheral blood mononuclear cells may
be isolated using, e.g., elutriation, magnetic beads separation, or density
gradient centrifugation methods (e.g.,
Ficoll-Paguee) known in the art. In some embodiments, MDSCs may be separated
from peripheral blood
mononuclear cells by surface marker selection (e.g., using immunofluorescence,
e.g., flow cytometry/FACS
analysis or immunohistochemistry). G-MDSCs and M-MDSCs may be further
distinguished using surface markers
provided herein.
Tumor-associated immune cell markers
[224] Immune cell markers may be used to determine whether a cancer has an
immune-excluded phenotype,
and/or may be used in determining treatment efficacy or treatment regimen,
alone or in combination with other
circulating biomarkers such as circulating MDSCs. If the tumor is determined
to have an immune-excluded
phenotype, cancer therapy (such as CBT) alone may not be efficacious. Without
being bound by theory, the tumor
may lack sufficient cytotoxic cells within the tumor environment for effective
CBT treatment alone. Thus, an
alternative and/or add-on therapy with a TGF[3 inhibitor (such as those
described herein) may reduce immuno-
suppression, thereby providing an improved treatment alone or rendering the
resistant tumor more responsive to
a cancer therapy. In some embodiments, immune cell markers are measured in
biopsies (e.g., core needle
biopsies). In some embodiments, patients having an immune-excluded tumor are
administered a treatment
comprising one or more TGF[3 inhibitor (e.g., TGFI31 inhibitor, e.g., Ab6). In
some embodiments, patients having
an immune-excluded tumor are administered a treatment comprising one or more
TGF[3 inhibitor (e.g., TGF[31
inhibitor, e.g., Ab6) inhibitor and monitored for improvement in condition
(e.g., increased immune cell penetration
into a tumor, reduced tumor volume, etc.). In some embodiments, a patient
exhibiting an improvement in condition
after a first round of treatment is administered one or more additional rounds
of treatment. In some embodiments,
subjects are administered one or more additional treatment in combination with
the one or more TGFI3 inhibitor
(e.g., TGFP1 inhibitor, e.g., Ab6).
[225] Tumor-associated immune cells that may be used to indicate the immune
contexture of a tumor/cancer
microenvironment include, but are not limited to, cytotoxic T cells and tumor-
associated macrophages (TAMs), as
well as tumor-associated MDSCs. Biomarkers to detect cytotoxic T cell levels
may include, but are not limited to,
the C08 glycoprotein, granzyme B, perforin, and IFNy, of which the latter
three markers may also be indicative of
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activated cytotoxic T cells. To measure the level of TAMs, protein markers
such as HLA-DR, CD68, C0163, CO206,
and other biomarkers, any method known in the art may be used. In certain
embodiments, increased levels of
cytotoxic T cells, e.g., activated cytotoxic T cells, detected within the
tumor microenvironment may be indicative of
reduction or reversal of immune suppression. For example, an increase in CD8
expression and perforin, granzyme
B, and/or IFNy expression by tumor-associated immune cells may be indicative
of reduction or reversal of immune
suppression in the cancer. In certain embodiments, decreased levels of TAMs or
tumor-associated MDSCs
detected within the tumor microenvironment may be indicative of reduced or
reversal of immune suppression. For
example, a decrease of HLA-DR, CD68, CD163, and CD206 expression by tumor-
associated immune cells may
indicate reduced or reversal of immune suppression in the cancer. In certain
embodiments, tumor-associated
immune cells, e.g., CD8+ T cells, may be used in combination with one or more
additional biomarkers to indicate
immune contexture of a tumor/cancer microenvironment. In certain embodiments,
the immune contexture of a
tumor may be characterized by the density, location, organization, and/or
functional orientation of tumor-infiltrating
immune cells. In certain embodiments, such markers may be used to determine
the immune phenotype of a tumor,
e.g., to determine if a tumor is immune excluded, inflamed, or desert.
[226] In various embodiments, cytotoxic T cells, e.g., in a patient sample,
may be used to determine whether a
cancer has an immune-excluded phenotype, and/or may be used in determining
treatment efficacy or treatment
regimen, alone or in combination with other biomarkers such as circulating
MDSCs. For example, C08 expression
and/or the distribution of CD8 expression in a tumor sample may be used. For
instance, CD8 expression may be
examined in a sample to determine distribution in the tumor (i.e., tumor
compartment), stroma (i.e., stroma
compartment), and margin (i.e., margin compartment; identified, e.g., by
assessing the region approximately 10-
100 pm, or 25-75 pm, or 30-60 pm, e.g., 50 pm, between tumor and stroma). In
certain embodiments, tumor,
stroma, and/or margin compartments within the tumor may be identified using
histological methods (e.g.,
pathologist assessment, pathologist-trained machine learning algorithms,
and/or immunohistochemistry). In certain
embodiments, CD8+ T cells in a tumor compartment may be referred to as "tumor-
associated CD8+ cells". In
certain embodiments, CD8+ T cells in a stroma compartment may be referred to
as "stroma-associated CD8+
cells". In certain embodiments, CD8+ T cells in a margin compartment may be
referred to as "margin-associated
CD8+ cells". In some embodiments, CD8 distribution may be determined in a
tumor nest (e.g., a mass of cells
extending from a common center seen in a cancerous growth), the stroma
surrounding the tumor nest, and the
margin between the tumor nest and its surrounding stroma (identified, e.g., by
assessing the region approximately
10-100 pm, or 25-75 pm, or 30-60 pm, e.g., 50 pm, between the tumor nest and
the surrounding stroma).
[227] In certain embodiments, tumor nests may be identified using histological
methods (e.g., pathologist
assessment, pathologist-trained machine learning algorithms, and/or
immunohistochemistry). In certain
embodiments, one or more tumor nests may be found within a tumor compartment.
In certain embodiments, a
tumor may comprise multiple (e.g., at least 5, at least 10, at least 20, at
least 25, at least 50, or more) tumor nests.
[228] By default, unless otherwise indicated by context, the term "stroma" or
"stroma compartment" refers to the
stroma surrounding the tumor, and the term "margin" or "margin compartment"
refers to the margin between the
tumor and the stroma surround the tumor. In some embodiments, the structural
interface between the tumor/tumor
nest and the surrounding stroma is determined by imaging analysis. A margin
can then be defined as the region
surrounding the interface in either direction by a predetermined distance, for
example, 10-100 pm. In some
embodiments, this distribution may be used prior to administering a TGFI3
inhibitor, such as a TGFI31 inhibitor (e.g.,
Ab6) to select a patient for treatment and/or predict and/or determine the
likelihood of a therapeutic response (e.g.,
an anti-tumor response) to an anti-cancer therapy comprising an anti-TGFr3
inhibitor. For instance, if no or few
cytotoxic T cells (e.g., less than 5% CD8+ T cells) are seen in a tumor
sample, including in stroma and margin, this
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may indicate a patient who would not benefit from TGF inhibitor therapy
(without being bound by theory, this may
be because there are few immune cells to recruit to the tumor). Similarly, if
a high density of cytotoxic T cells (e.g.,
greater than 5% CD8+ T cells) is observed in tumor as well as stroma and
margin, this patient may also have
limited benefit from TGF inhibitor therapy (without being bound by theory,
this may be because immune cells have
already infiltrated the tumor).
[229] In contrast, in certain embodiments, the subject's cancer may exhibit an
immune-excluded phenotype, in
which cytotoxic T cells (e.g., CD8+ T cells) are observed clustered primarily
in or near the margin, e.g., at the
border between the margin and the tumor, and not significantly infiltrated
into the tumor itself (e.g., less than 5%
CD8+ T cells in the tumor compartment and greater than 10% CD8+ T cells in the
margin and/or stroma
compartment). In certain embodiments, the subject's cancer may exhibit an
immune-excluded phenotype, in which
cytotoxic T cells (e.g., CD8+ T cells) are observed clustered primarily in or
near the margin, e.g., at the border
between the margin and the tumor (or peri-vasculature), and not significantly
infiltrated into the tumor core itself
(e.g., less than 5% CD8+ T cells in the tumor compartment and greater than 5%
CD8+ T cells in the margin and/or
stroma compartment). In certain embodiments, the subject's cancer may exhibit
an immune-excluded phenotype,
in which cytotoxic T cells (e.g., CD8+ T cells) are observed clustered
primarily in or near the margin, e.g., at the
border between the margin and the tumor, and not significantly infiltrated
into the tumor itself (e.g., less than 5%,
less than 10%, less than 15%, or fewer CD8+ T cells in the tumor compartment
and greater than 5%, greater than
10%, greater than 15%, or more CD8+ T cells in the margin and/or stroma
compartment). In some embodiments,
CD8+ content in tumor compartments may be based on any of the methods
described in Ziai et al. (PLoS One.
2018; 13(1): e0190158), Massi et al. (J I mmunother Cancer. 2019 Nov
15;7(1):308), Sharma et al. (Proc Natl Acad
Sci U S A. 2007 Mar 6;104(10):3967-72), or Echarti et al. (Cancers (Basel).
2019 Sep; 11(9): 1398), the contents
of which are hereby incorporated in their entirety. Any of these methods may
be used to determine the immune
phenotype of the tumor. Tumor samples with this pattern from a patient may
indicate a patient likely to benefit from
TGF inhibitor therapy (without being bound by theory, this may be because the
tumor is actively suppressing the
immune response, preventing sufficient ingress of cytotoxic T cells, which
could be partially or completely reversed
by the TGF inhibitor).
[230] In some embodiments, an immune-excluded phenotype is characterized by
determining a cluster score of
cytotoxic T cells (e.g., CD8+ T cells) within a tumor-associated compartment,
e.g., in the tumor, in the margin near
the external perimeters of a tumor mass, and/or in the vicinity of tumor
vasculatures. In some embodiments, the
cluster score of cytotoxic T cells (e.g., CD8+ T cells) can be determined
based on the homogeneity of immune cells
in a particular tumor-associated compartment, such that a compartment
containing highly uniform distribution of
cytotoxic T cells (e.g., CD8+ T cells) yields a high cluster score. In certain
embodiments, tumors exhibiting an
immune-excluded phenotype may be characterized by lower densities of cytotoxic
T cells (e.g., CD8+ T cells)
inside the tumor as compared to densities outside of the tumor (e.g., the
external perimeters of a tumor mass
and/or near the vicinity of vasculatures of a tumor). In some embodiments, the
immune-excluded phenotype is
characterized by cytotoxic T cells (e.g., CD8+ T cells) in the tumor stroma
that are located in close vicinity (e.g.,
less than 100 pm) to the tumor. In some embodiments, the immune-excluded
phenotype is characterized by
cytotoxic T cells (e.g., CD8+ T cells) capable of infiltrating the tumor nest
and locating at a close distance (e.g.,
less than 100 pm) to the tumor. In some embodiments, CD8+ T cells can be
observed in clusters within a tumor
near intratumoral blood vessels as determined for example by endothelial
markers. By comparison, upon
overcoming immunosuppression by TGF beta inhibitors, more uniform distribution
of CD8+ T cells within the tumor
can be observed, presumably as a result of the CD8+ cells being able to
infiltrate from the perivascular regions
and possibly proliferate in the tumor.
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[231] In certain embodiments, levels of tumor-infiltrating cytotoxic T cells
(e.g., CD8+ T cells) and their activation
status may be determined from a tumor biopsy sample obtained from the subject.
In some embodiments, tumor
biopsy samples, e.g., core needle biopsies, may be obtained at least 28 days
prior to and at least 100 days following
treatment administration. In some embodiments, tumor biopsy samples, e.g.,
core needle biopsies, may be
obtained about 21 days to about 45 days following treatment administration. In
some embodiments, tumor biopsy
samples may be obtained via core needle biopsy. In some embodiments, treatment
is continued if an increase is
detected.
[232] In certain embodiments, the immune phenotype of a subject's cancer may
be determined by measuring the
cell densities of cytotoxic T cells (e.g., percent of CD8+ T cells per square
millimeter or other defined square
distance) in a tumor biopsy sample. In certain embodiments, the immune
phenotype of a subject's cancer may be
determined by comparing the densities of cytotoxic T cells (e.g., CD8+ T
cells) inside the tumor to that outside the
tumor (e.g., to cells in the margin, e.g., at the external perimeters of a
tumor mass and/or near the vicinity of
vasculatures of a tumor). In some embodiments, the immune phenotype of a
subject's cancer may be determined
by comparing the percentage of CD8+ lymphocytes inside the tumor to that
outside the tumor. In certain
embodiments, the immune phenotype of a subject's cancer may be determined by
comparing the cluster or
dispersion of cytotoxic T cells (e.g., average number of CD8+ T cells
surrounding other CD8+ T cells) in the tumor,
stroma, or margin. In certain embodiments, the immune phenotype of a subject's
cancer may be determined by
measuring the average distance from cytotoxic T cells (e.g., CD8+ T cells) in
the stroma to the tumor. In certain
embodiments, the immune phenotype of a subject's cancer may be determined by
measuring the average depth
of cytotoxic T cell (e.g., CD8+ T cell) penetration into the tumor nest. Cell
counts and density may be determined
using immunostaining and computerized or manual measurement protocols. In
certain embodiments, levels of
cytotoxic T cells (e.g., CD8+ T cells) may be measured using
immunohistochemical analysis of tumor biopsy
samples. In certain embodiments, levels of cytotoxic T cells (e.g., CD8+ T
cells) may be determined at least 28
days prior to and/or at least 100 days following administering a TGFp therapy.
In certain embodiments, levels of
cytotoxic T cells (e.g., CD8+ T cells) may be determined up to about 45 days
(e.g., about 21 days to about 45 days)
following administering a TGFp therapy. In some embodiments, levels of
cytotoxic T cells (e.g., CD8+ T cells) are
determined 5, 10, 15, 20, 25, 30, or more days prior to and/or at least 50,
60, 70, 80, 90, 100, 110, 120, 130, 140,
or 150 days following administering a TGF6 therapy (or at any time point in
between).
[233] In some embodiments, a tumor with lower levels of cytotoxic T cells
(e.g., CD8+ T cells) inside the tumor as
compared to cytotoxic T cell levels (e.g., CD8+ T cells) outside the tumor
(e.g., the external perimeters of a tumor
and/or near the vicinity of vasculatures of a tumor) may be identified as an
immune-excluded tumor. In some
embodiments, immune-excluded tumors may also have higher levels of cytotoxic T
cells (e.g., CD8+ T cells) in the
tumor stroma as compared to inside the tumor. In certain embodiments, immune-
excluded tumors may be identified
by determining the ratio of cytotoxic T cell density (e.g., CDS+ T cells)
inside the tumor to outside of the tumor,
wherein the ratio is less than 1. In certain embodiments, immune-excluded
tumors may be identified by determining
the cytotoxic T cell density ratio inside the tumor to density in the tumor
margin, wherein the ratio is less than 1. In
certain embodiments, immune-excluded tumors may be identified by determining
the cell density ratio inside the
tumor to density in the tumor stoma, wherein the ratio is less than 1. In
certain embodiments, immune-excluded
tumors may be identified by comparing the absolute number, percentage, and/or
density of cytotoxic T cells (e.g.,
CD8+ T cells) inside the tumor to outside the tumor (e.g., margin and/or
stroma). In some embodiments, the
absolute number, percentage, and/or density of cytotoxic T cells (e.g., CD8+ T
cells) outside the tumor is at least
2-fold, 3-fold, 4-fold, 5-fold, 7-fold, or 10-fold greater than inside the
tumor in an immune-excluded tumor. In some
embodiments, an immune-excluded tumor comprises less than 5% CD8+ T cells
inside the tumor and greater than
10% CD8+ T cells in the tumor margin and/or stroma. In some embodiments,
immune-excluded tumors may be
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identified by comparing a ratio of compartmentalized cytotoxic T cell density
(e.g., density of CD8+ cells inside the
tumor to density in the tumor margin and/or stroma) and the ratio of whole
tissue cytotoxic T cell density (e.g.,
CD8+ cells inside the tumor to CD8+ cells in the entire tumor tissue or
biopsy), wherein the compartmentalized
ratio is greater than the whole tissue ratio. In some embodiments, a tumor
with increased cell density of cytotoxic
T cells (e.g., CD8+ T cells) at an average distance of about 100 pm or less
outside of the tumor may be identified
as an immune-excluded tumor. In some embodiments, cytotoxic T cell density
(e.g., CD8+ T cells) may be used in
conjunction with one or more parameters, such as average CD8+ cluster score.
In some embodiments, an average
CD8+ clustering score of 50% or less in the tumor indicates immune exclusion.
[234] In some embodiments, a tumor with lower levels of CD8+ T cells inside
(e.g., core of) the tumor as compared
to CD8+ T cells outside the tumor (e.g., peripheries of the tumor, e.g., the
external perimeters of a tumor and/or
near the vicinity of vasculatures of a tumor, e.g., in the tumor margin and/or
stroma) may be identified as an
immune-excluded tumor. In some embodiments, an immune-excluded tumor comprises
less than 5%, less than
10%, or less than 15% CD8+ T cells inside the tumor and/or inside one or more
tumor nests and greater than 5%,
greater than 10%, or greater than 15% CD8+ T cells outside the tumor and/or
outside one or more tumor nests. In
some embodiments, an immune-excluded tumor comprises less than 5% CD8+ T cells
inside the tumor and/or
inside one or more tumor nests and greater than 5% CD8+ T cells outside of the
tumor and/or outside one or more
tumor nests. In some embodiments, an immune-excluded tumor comprises less than
10% CD8+ T cells inside the
tumor and/or inside one or more tumor nests and greater than 10% CD8+ T cells
outside of the tumor and/or
outside one or more tumor nests. In some embodiments, an immune-excluded tumor
comprises less than 15%
CD8+ T cells inside the tumor and/or inside one or more tumor nests and
greater than 15% CD8+ T cells outside
of the tumor and/or outside one or more tumor nests.
[235] In some embodiments, a tumor with higher levels of CD8+ T cells inside
the tumor as compared to CD8+ T
cells outside the tumor (e.g., the external perimeters of a tumor and/or near
the vicinity of vasculatures of a tumor,
e.g., in the tumor margin and/or stroma) may be identified as an immune-
inflamed tumor. In some embodiments,
an immune-inflamed tumor comprises greater than 5% CD8+ T cells inside the
tumor. In some embodiments, an
immune-inflamed tumor comprises greater than 10% CD8+ T cells inside the tumor
and/or inside one or more
tumor nests. In some embodiments, an immune-inflamed tumor comprises greater
than 15% CD8+ T cells inside
the tumor and/or inside one or more tumor nests.
[236] In some embodiments, a tumor with low levels of CD8+ T cells both inside
and outside the tumor may be
identified as an immune desert tumor. In some embodiments, an immune desert
tumor comprises less than 5%
CD8+ T cells inside the tumor and less than 10% CD8+ T cells in the tumor
margin and/or stroma. In some
embodiments, an immune desert tumor comprises less than 5% CD8+ T cells inside
the tumor (and/or inside one
or more tumor nests) and less than 5% CD8+ T cells in the tumor margin and/or
stroma.
[237] In some embodiments, CD8+ content in tumor compartments may be
determined based on any of the
methods described in Ziai et al. (PLoS One. 2018; 13(1): e0190158), Massi et
al. (J Immunother Cancer. 2019 Nov
15;7(1):308), Sharma et al. (Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3967-
72), or Echarti et al. (Cancers
(Basel). 2019 Sep; 11(9): 1398), the contents of which are hereby incorporated
in their entirety. In some
embodimehnts, any of these methods may be used to determine the immune
phenotype of the tumor.
[238] In certain embodiments, the immune phenotype of a subject's cancer may
be determined by average
percent CD8 positivity (i.e., percentage of CD8+ lymphocytes) as measured over
multiple (e.g., at least 5, at least
15, at least 25, at least 50, or more) tumor nests of a tumor (e.g., in one or
more tumor biopsy samples). In certain
embodiments, the immune phenotype of a given tumor nest may be determined by
comparing the CD8 positivity
inside the tumor nest to the CD8 positivity outside the tumor nest (e.g., in
the tumor nest margin and/or the tumor
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nest stroma). In certain embodiments, a tumor nest may be identified as immune
inflamed if the C08 positivity
inside the tumor nest is greater than 5%. In certain embodiments, a tumor nest
may be identified as immune
excluded if the CD8 positivity inside the tumor nest is less than 5% and the
CD8 positivity in the tumor nest margin
is greater than 5%. In certain embodiments, a tumor nest may be identified as
an immune desert if the CD8 positivity
inside the tumor nest is less than 5% and CD8 positivity in the tumor nest
margin is less than 5%. In certain
embodiments, a subject's cancer may be identified immune inflamed if greater
than 50% of the total tumor area
analyzed comprises tumor nests exhibiting immune inflamed phenotype. In
certain embodiments, a subject's
cancer may be identified as immune excluded if greater than 50% of the total
tumor area analyzed comprises tumor
nests exhibiting immune excluded phenotype. In certain embodiments, a
subject's cancer may be identified as an
immune desert if greater than 50% of the total tumor area analyzed comprises
tumor nests exhibiting immune
desert phenotype. In certain embodiments, a subject's cancer may be identified
based on determination of CD8
positivity from more than one sample (e.g., at least three samples, e.g., four
samples) taken from the same tumor.
[239] In certain embodiments, tumor biopsy samples may be obtained by core
needle biopsy. In certain
embodiments, three to five samples (e.g., four samples) may be taken from the
same tumor. In certain
embodiments, the needle may be inserted along a single trajectory, wherein
multiple samples (e.g., three to five
samples, e.g., four samples) may be taken at different tumors depths along the
same needle trajectory. In certain
embodiments, samples taken at different tumor depths may be used to analyze
combined CD8 positivity over
multiple tumor nests. In certain embodiments, the combined CD8 positivity
determined in these samples may be
representative of CD8 positivity in the rest of the tumor. In certain
embodiments, the combined CD8 positivity
determined in these samples may be used to identify immune phenotype of a
subject's cancer.
[240] In certain embodiments, the immune phenotype of a subject's tumor may be
determined by combined
analysis of the absolute number, percentage, ratio, and/or density of CD8+
cells in the tumor and the combined
CD8 positivity (i.e., percentage of CD8+ lymphocytes) across tumor nests
throughout the tumor.
[241] In certain embodiments, tumor compartments may be identified,
determined, and/or analyzed for markers
such as CD8 content manually, e.g., by a pathologist inspection of tumor
samples. In some embodiments, tumor
compartments may be identified, determined, and/or analyzed for markers such
as CD8 content by digital analysis,
e.g., by using a software or computer program for automated identification. In
certain embodiments, a skilled artisan
may use such a software or computer program for automated identification of
tumor nests and the boundaries
between a tumor nest, stroma compartment, and/or tumor margin compartment. In
certain embodiments, a
software or computer program may be used to evaluate the distribution of
suitable markers such as CD8+ T cells
in the identified tumor nest, stromal compartment, and/or tumor margin
compartment. In certain embodiments, the
software or computer program may be based on one or more machine learning
algorithms. In certain embodiments,
the one or more machine learning algorithms may be based initially on manual
classification of reference samples,
e.g., by a trained pathologist. In some embodiments, the software or computer
program may use a neural network
approach with machine learning based on reference samples categorized
manually, e.g., by a pathologist.
Exemplary softwares or computer programs include any software or computer
program that has the capability of
intaking an image (e.g., microscope images of a tumor sample comprising immune
staining), processing and
analyzing the image, and segmenting the tumor compartments in the image based
on specific parameters (e.g.,
nuclear staining, fibroblast staining, 0D8+ staining, other biomarkers). In
certain embodiments, the softwares or
computer program may be any of those provided by Visiopharm, HALO (Indica
Labs), CellProfiler Analyst, Aperio
Image Analysis, Zeiss ZEN Intellesis, or ImageJ. Such programs may
advantageously achieve sufficient resolution
for visualizing certain characteristics of individual tumor nests within a
solid tumor (e.g., boundaries for tumor nest,
stroma, and/or margin compartmenrs), as opposed to analyzing substantially the
entire tumor as a whole.
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[242] In certain embodiments, a subject whose cancer exhibits an immune-
excluded phenotype may be more
responsive to a therapy comprising administration of a TGFI3 inhibitor (e.g.,
Ab6). In some embodiments, such a
subject is identified for treatment. In some embodiments, such a subject is
administered a treatment comprising a
TGF inhibitor, such as a TGF[31-selective inhibitor (e.g., Ab6), an isoform-
non-selective inhibitor (e.g., low
molecular weight ALK5 antagonists), neutralizing antibodies that bind two or
more of TGFP1/2/3 (e.g., GC1008 and
variants), antibodies that bind TGFI31/3, ligand traps (e.g., TGFI31/3
inhibitors), and/or an integrin inhibitor (e.g., an
antibodies that bind to aV131, aV133, aV135, aV136, aVp8, a5131, all b[33, or
a8p1 integrins, and inhibit downstream
activation of TGF(3. e.g., selective inhibition of TGF131 and/or TGF133).
[243] In certain embodiments, a subject whose cancer exhibits an immune-
excluded phenotype may be more
responsive to a combination therapy comprising a TGFI3 inhibitor, such as a
TGFp1-selective inhibitor (e.g., Ab6),
an isoform-non-selective inhibitor (e.g., low molecular weight ALK5
antagonists), neutralizing antibodies that bind
two or more of TGFP1/2/3 (e.g., GC1008 and variants), antibodies that bind
TGFI31/3, ligand traps (e.g., TGFP1/3
inhibitors), and/or an integrin inhibitor (e.g., an antibody that binds to
(11/(31, aV[33, aVp5, aV[36, aV[38, 05131, al I b[33,
or a8131 integrins, and inhibits downstream activation of TGFI3. e.g.,
selective inhibition of TGF131 and/or TGF133),
and an additional cancer therapy, e.g., a checkpoint inhibitor. In some
embodiments, the additional cancer therapy
may comprise chemotherapy, radiation therapy (including radiotherapeutic
agents), a cancer vaccine, or an
immunotherapy comprising a checkpoint inhibitor such as an anti-PD-1, anti-PD-
L1, or anti-CTLA-4 antibody. In
some embodiments, the checkpoint inhibitor therapy is selected from the group
consisting of ipilimumab (e.g.,
Yervoy0); nivolumab (e.g., Opdivo0); pembrolizumab (e.g., Keytruda0); avelumab
(e.g., Bavencio0); cemiplimab
(e.g., Libtayo0); atezolizumab (e.g., Tecentrig0); budigalimab (e.g., A8BV-
181); and durvalumab (e.g., Imfinzie).
In certain embodiments, a subject whose cancer exhibits an immune-excluded
phenotype is administered a
combination therapy comprising a TGFI3 inhibitor, such as a TGFp1-selective
inhibitor (e.g., Ab6), and an additional
cancer therapy, e.g., a checkpoint inhibitor.
[244] In certain embodiments, a subject whose cancer exhibits an immune-
excluded phenotype may be more
responsive to a combination therapy comprising a TGFI3 inhibitor, such as a
TGFI31-selective inhibitor (e.g., Ab6),
and a checkpoint inhibitor therapy (e.g., a P01 or PDL1 antibody). In some
embodiments, such a subject is
identified for receiving the combination therapy. In some embodiments, such a
subject is identified for receiving the
combination therapy prior to receiving the checkpoint inhibitor therapy alone.
In some embodiments, such a subject
is identified for receiving the combination therapy prior to receiving either
the checkpoint inhibitor therapy or the
TGFp inhibitor alone. In some embodiments, such a subject is treatment-naïve.
In some embodiments, such a
subject has previously received a checkpoint inhibitor therapy and is non-
responsive to the checkpoint inhibitor
therapy. In some embodiments, such a subject has cancer that exhibits an
immune-excluded phenotype. In some
embodiments, such a subject has previously received a checkpoint inhibitor
therapy and is directly given a
combination therapy (e.g., bypassing the need to first try treatment with a
checkpoint inhibitor alone). In some
embodiments, such a subject is administered a combination therapy comprising a
TGFp inhibitor, such as a TGF131-
selective inhibitor (e.g., Ab6), and an additional cancer therapy, e.g., a PD1
or PDL1 antibody.
[245] In some embodiments, a subject whose cancer exhibits an immune-excluded
phenotype may be selected
for treatment and/or monitored during and/or after administration of the
therapy comprising a TGFI3 inhibitor, such
as a TGFP1-selective inhibitor (e.g., Ab6). In some embodiments, patient
selection and/or treatment efficacy is
determined by measuring the level of cytotoxic T cells (e.g., CD8+ T cells)
inside the tumor as compared to the
level of cytotoxic T cells (e.g., CD8+ T cells) outside the tumor (e.g., in
the margin). In certain embodiments, an
increase in the levels of tumor-infiltrating cytotoxic T cells (e.g., C08+ T
cells) inside the tumor relative to outside
the tumor (e.g., margin and/or stroma) following administration of a TGFI3
inhibitor therapy (e.g., Ab6), alone or in
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combination with an additional therapy (e.g., a checkpoint inhibitor therapy),
may indicate a therapeutic response
(e.g., anti-tumor response). For instance, an increase of at least 10%, 15%,
20%, 25%, or more in tumor-infiltrating
cytotoxic T cell levels following TGF8 inhibitor treatment (e.g., Ab6) as
compared to tumor-infiltrating cytotoxic T
cell levels before the treatment may be indicative of therapeutic response
(e.g., anti-tumor response). In some
embodiments, an increase of at least 10%, 15%, 20%, 25%, or more in total
tumor area comprising immune
inflamed tumor nests may be indicative of therapeutic response. In some
embodiments, levels of cytolytic proteins
such as perforin or granzyme B or proinflammatory cytokines such as IFNy
expressed by the tumor-infiltrating
cytotoxic T cells may also be measured to determine the activation status of
the tumor-infiltrating cytotoxic T cells.
In some embodiments, an increase of at least 1.5-fold, or 2-fold, or 5-fold,
or more in cytolytic protein levels may
be indicative of therapeutic response (e.g., anti-tumor response). In some
embodiments, a change of at least a
1.5-fold, 2-fold, 5-fold, or 10-fold, or more increase in IFNy levels may be
indicative of a therapeutic response (e.g.,
anti-tumor response). In some embodiments, treatment is continued if an
increase in tumor-infiltrating cytotoxic T
cells (e.g., CD8+ T cells) is detected.
[246] In certain embodiments, a subject whose cancer exhibits an immune-
inflamed phenotype may be more
responsive to a therapy comprising a checkpoint inhibitor without a TGF8
inhibitor than would a subject having an
immune-excluded phenotype. In some embodiments, the checkpoint inhibitor
therapy is selected from the group
consisting of ipilimumab (e.g., Yervoy0); nivolumab (e.g., Opdivo0);
pembrolizumab (e.g., Keytruda0); avelumab
(e.g., Bavencioe); cemiplimab (e.g., LibtayoS); atezolizumab (e.g.,
Tecentriqe); budigalimab (e.g., ABBV-181);
and durvalumab (e.g., Imfinzi8). In certain embodiments, a subject whose
cancer exhibits an immune-inflamed
phenotype is administered a checkpoint inhibitor.
[247] In certain embodiments, immune phenotyping of a subject's tumor may be
determined from a tumor biopsy
sample (e.g., core needle biopsy sample), for example histologically, using
one or more parameters such as, but
not limited to, distribution of cytotoxic T cells (e.g., 0D8+ T cells),
percentage of cytotoxic T cells (e.g., CD8+ T
cells) in the tumor versus stromal compartment, and percentage of cytotoxic T
cells (e.g., CD8+ T cells) in the
tumor margin.
[248] Recognizing that samples collected by a traditional needle biopsy
protocol risk inadvertent bias, depending
on where within the tumor the needle was inserted, the present disclosure also
provides improved methods, where
needle biopsy is employed for tumor analysis. According to the present
disclosure, the risk of bias inherent to
needle biopsy may be significantly reduced by collecting adjacent tumor
samples, for example, at least three, but
preferably four samples collected from adjacent tumor tissue (e.g., from the
same tumor). This may be carried out
from a single needle insertion point, by, for example, altering the angle
and/or the depth of insertion. Taking into
account that some tissue sections prepared from needle biopsy samples may not
remain intact during sample
processing, and the possibility that a needle may be inserted in the portion
of the tumor tissue that does not
accurately represent the tumor phenotype, collecting four samples may help
mitigate such limitations and provides
more representative tumor phenotyping for improved accuracy.
[249] In certain embodiments, a sample may be analyzed for its distribution of
cytotoxic T cells (e.g., CD8+ T
cells) using a method such as CD8 immunostaining. In certain embodiments, the
distribution of cytotoxic T cells
(e.g., CD8+ T cells) may be relatively uniform (e.g., distribution is
homogeneous throughout the sample, e.g., CD8
density across tumor nests have a variance of 10% or lower). In some
embodiments, a tumor nest (or cancer nest)
refers to a mass of cells extending from a common center of a cancerous
growth. In some embodiments, a tumor
nest may comprise cells interspersed in stroma. In certain embodiments, a
sample, such as a sample with an even
distribution of cytotoxic T cells (e.g., 008 T cells), may be analyzed to
determine the percentages of cytotoxic T
cells (e.g., CD8+ T cells) in the tumor and in the stoma. In certain
embodiments, a high percentage (e.g., greater
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than 5%) of cytotoxic T cells (e.g., CD8+ T cells) in the tumor and a low
percentage (e.g., less than 5%) of cytotoxic
T cells (e.g., CD8+ T cells) in the stroma may be indicative of an inflamed
tumor phenotype. In certain embodiments,
a low percentage of cytotoxic T cells (e.g., CD8+ T cells) in both the tumor
and the stroma (e.g., combined tumor
and stroma C08 percentage of less than 5%) may be indicative of a poorly
immunogenic tumor phenotype (e.g.,
an immune desert phenotype). In certain embodiments, a low percentage (e.g.,
less than 5%) of cytotoxic T cells
(e.g., CD8+ T cell cells) in the tumor and a high percentage (e.g., greater
than 5%) of cytotoxic T cells (e.g., CD8+
T cell cells) in the stroma may be indicative of an immune-excluded tumor
phenotype. In certain embodiments, a
tumor-to-stroma CD8 ratio may be determined by dividing C08 percentage in the
tumor over the percentage in the
stroma. In certain embodiments, a tumor-to-stroma CD8 ratio of greater than 1
may be indicative of an inflamed
tumor phenotype. In certain embodiments, a tumor-to-stroma CD8 ratio of less
than 1 may be indicative of an
immune-excluded tumor. In certain embodiments, percentages of cytotoxic T
cells may be determined by
immunohistochemical analysis of CD8 immunostaining.
[250] In certain embodiments, a sample, such as a sample with uneven
distribution of cytotoxic T cells (e.g., CD8
density across tumor nests have a variance of greater than 10%), may be
analyzed to determine the marg in-to-
stroma CD8 ratio. In certain embodiments, such ratio may be calculated by
dividing CD8 density in the tumor
margin over CD8 density in the tumor stroma. In certain embodiments, an immune
excluded tumor exhibits a
margin-to-stroma CD8 ratio of greater than 0.5 and less than 1.5.
[251] In certain embodiments, a sample having a margin-to-stroma CD8 ratio of
greater than 1.5 may be further
analyzed to determine and/or confirm immune phenotyping (e.g., to determine
and/or confirm whether the tumor
has an immune-excluded phenotype) by evaluating tumor depth. In certain
embodiments, tumor depth may be
measured in increments of 20 pm-200 pm (e.g., 100 pm). In certain embodiments,
tumor depth may be determined
by pathological analysis and/or digital image analysis. In certain
embodiments, a significant tumor depth may be
indicated by a distance of about 2-fold or greater than the depth of the tumor
margin. In certain embodiments, a
tumor sample may have a tumor margin depth of 100 pm and a tumor depth
measurement of greater than 200 pm,
such sample would have a tumor depth score of greater than 2, and would
therefore have significant tumor depth.
In certain embodiments, significant tumor depth may be indicated by a ratio of
2 or greater as determined by dividing
tumor depth by the depth of the tumor margin. In certain embodiments, tumor
depth may be measured in
increments corresponding to the depth of the tumor margin. For instance, the
tumor depth of a tumor nest having
a tumor margin of 100 pm may be measured in increments of 100 pm. In certain
embodiments, a tumor sample
with significant tumor depth may exhibit shallow penetration by cytotoxic T
cells (e.g., the tumor sample having
greater than 5% CD8 T cells but does not exhibit tumor penetration beyond one
tumor depth increment). In certain
embodiments, a tumor sample with significant tumor depth that exhibits shallow
CD8 penetration may be indicative
of an immune excluded tumor.
[252] In certain embodiments, a tumor phenotype analysis may be conducted
according to any part of the
exemplary flow chart shown in FIG. 38, e.g., using all the steps in that
figure.
[253] In certain embodiments, a subject whose cancer exhibits an immune
excluded phenotype may be selected
for TG93 inhibitor therapy (e.g., a TGF[31 inhibitor such as Ab6). In certain
embodiments, a subject whose cancer
exhibits an immune excluded phenotype may be more responsive to a TGFI3
inhibitor therapy (e.g., a TG931
inhibitor such as Ab6). In certain embodiments, a subject whose cancer
exhibits an immune-excluded phenotype
may be more responsive to a combination therapy comprising a TGFr3 inhibitor,
such as a TGF[31-selective inhibitor
(e.g., Ab6), and a second cancer therapy, e.g., a checkpoint inhibitor therapy
(e.g., a P01 or PDL1 antibody).
[254] In certain embodiments, a response to TGFr3 inhibitor therapy (e.g., a
TGF[31 inhibitor such as Ab6) may
be monitored and/or determined using parameters such as any of the ones
described above. In certain
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embodiments, a change in a distribution of cytotoxic T cells (e.g., CD8+ T
cells) in a pre-treatment tumor sample
as compared to a corresponding post-treatment sample from the corresponding
tumor may be indicative of a
therapeutic response to treatment. In certain embodiments, a change (e.g.,
increase) of at least 1-fold (e.g., 1.1-
fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,
1.9-fold, 2-fold, or greater) in the tumor-to-
stroma CD8 density ratio between the pre-treatment and post-treatment tumor
samples may be indicative of a
therapeutic response. In certain embodiments, a change (e.g., increase) of 1.5-
fold or greater in the tumor-to-
stroma CD8 density ratio between the pre-treatment and post-treatment tumor
samples may be indicative of a
therapeutic response. In certain embodiments, the tumor-to-stroma CD8 density
ratio may be determined by
dividing CD8 cell density in the tumor nest over CD8 cell density in the tumor
stroma. In certain embodiments, a
change (e.g., increase) of 1.5-fold or greater in the density of cytotoxic T
cells (e.g., CD8+ T cells) in the tumor
margin between the pre-treatment and post-treatment tumor samples may be
indicative of a therapeutic response.
In certain embodiments, a change (e.g., increase) of 1.5-fold or greater in
the tumor depth score of pre-treatment
and post-treatment tumor samples may be indicative of a therapeutic response.
In some embodiments, the TGF13
inhibitor therapy (e.g., a TGF31 inhibitor such as Ab6) achieves at least a 2-
fold, e.g., 3-fold, 4-fold, 5-fold, 6-fold,
7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or a greater degree of
increase in the number of intratumoral T cells,
e.g., when used in conjunction with a checkpoint inhibitor such as a PD-(L)1
antibody, relative to pre-treatment. In
certain embodiments, treatment with a TGF3 inhibitor therapy (e.g., a TGF131
inhibitor such as Ab6), e.g., alone or
in combination with one or more additional cancer therapies, may be continued
if a therapeutic response is
observed.
[255] In certain embodiments, the pre-treatment and post-treatment samples
have comparable tumor depth
scores (e.g., variance of less than 0.25 in tumor depth scores of pre-
treatment and post-treatment tumor samples)
and the samples may be analyzed to determine therapeutic response according to
one or more of the parameters
described above. In certain embodiments, the pre-treatment and post-treatment
samples have comparable total
and compartmental areas (e.g., variance of less than 0.25 in analyzable total
and compartmental area of pre-
treatment and post-treatment tumor samples) and the samples may be analyzed to
determine therapeutic response
according to one or more of the parameters described above.
[256] In some embodiments, percent necrosis in a tumor sample may be assessed
by histological and/or digital
image analysis, which may reflect the presence or activities of cytotoxic
cells in the tumor. In some embodiments,
percent necrosis in tumor samples may be compared in pre-treatment and post-
treatment tumor samples collected
from a subject administered a TGF3 inhibitor (e.g., Ab6). In some embodiments,
increase of greater than 10% in
percent necrosis (e.g., the proportion of necrotic area to total tissue area
in a tumor sample) between pre-treatment
and post-treatment samples may be indicative of a therapeutic response to TGF6
inhibitor therapy, e.g., TGF61
inhibitor such as Ab6. In some embodiments, an increase of 10% or greater in
percent necrosis in or near the
center of the tumor (e.g., the proportion of necrotic area inside the tumor
margin) may be indicative of a therapeutic
response.
[257] In certain embodiments, a therapeutic response may be determined
according to any part of the exemplary
flow chart shown in FIG. 39.
[258] In some embodiments, an increased level of tumor-infiltrating cytotoxic
T cells (e.g., CD8+ T cells),
especially activated cytotoxic T cells, following TGF3 inhibitor therapy
(e.g., a TGF31 inhibitor such as Ab6) may
indicate conversion of an immune-excluded tumor microenvironment toward an
immune-infiltrated or "inflamed"
microenvironment. For instance, an increase of at least 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 15%, 20%,
25%, or more in tumor-associated cytotoxic T cell levels following TGF6
inhibitor treatment (e.g., Ab6) as compared
to tumor-associated cytotoxic T cell levels before the treatment may be
indicative of a reduction or reversal of
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immune suppression in the cancer. In some embodiments, an increase of at least
1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10%, 15%, 20%, 25%, or more in tumor area comprising immune inflamed
tumor nests may be indicative
of a reduction or reversal of immune suppression in the cancer. In some
embodiments, levels of cytolytic proteins
such as perforin or granzyme B or proinflammatory cytokines such as IFNy
expressed by the tumor-associated
cytotoxic T cells may be measured to determine the activation status of the
tumor-associated cytotoxic T cells. In
some embodiments, an increase of at least 1-fold, 1.1-fold, 1.2-fold, 1.3-
fold, 1.4-fold, 1.5-fold, or 2-fold, or 5-fold,
or more in cytolytic protein levels may be indicative of reduction or reversal
of immune suppression in the cancer.
In some embodiments, a change of at least a 1.5-fold, 2-fold, 5-fold, or 10-
fold, or more increase in IFNy levels
may be indicative of a reduction or reversal of immune suppression in the
cancer. In some embodiments, treatment
with the TGFp inhibitor therapy (e.g., a TGFp1 inhibitor such as Ab6) is
continued if such a reduction or reversal of
immune suppression in the cancer is detected.
[259] lmmunosuppressive lymphocytes associated with TMEs include TAMs and
MDSCs. A significant fraction
of tumor-associated macrophages is of so-called "M2" type, which has an
immunosuppressive phenotype. Most
of these cells are monocyte-derived cells that originate in the bone marrow.
Intratumoral (e.g., tumor-associated)
levels of immunosuppressive cells such as TAMs and MDSCs may also be measured
to determine the status of
immune suppression in a cancer. In some embodiments, a decrease of at least
10%, 15%, 20%, 25%, or more in
the level of TAMs may be indicative of reduced or reversal of immune
suppression. In certain embodiments, tumor-
associated immune cells may be measured from a biopsy sample from the subject
prior to and following TGFp
inhibitor treatment (e.g., Ab6). In certain embodiments, biopsy samples may be
obtained between 28 days and 130
days following treatment administration.
[260] The concept of "immune contexture" examines the TME from the perspective
of tumor-infiltrating
lymphocytes (i.e., tumor immune microenvironment or TIME). Tumor immune
contexture refers to the localization
(e.g., spatial organization) and/or density of the immune infiltrate in the
TME. TIME is usually associated with the
clinical outcome of cancer patients and has been used for estimating cancer
prognosis (see, for example, Fridman
et al., (2017) Nat Rev Clin Oncol. 14(12): 717-734) "The immune contexture in
cancer prognosis and treatment").
Typically, tissue samples from tumors are collected (e.g., biopsy such as core
needle biopsy) for TIL analyses. In
some embodiments, TILs are analyzed by FACS-based methods. In some
embodiments, TILs are analyzed by
immunohistochemical (INC) methods. In some embodiments, TILs are analyzed by
so-called digital pathology (see,
for example, Saltz et al., (2018) Cell Reports 23, 181-193. "Spatial
organization and molecular correlation of tumor-
infiltrating lymphocytes using deep learning on pathology images.");
(Scientific Reports 9:13341 (2019) "A novel
digital score for abundance of tumor infiltrating lymphocytes predicts disease
free survival in oral squamous cell
carcinoma"). In some embodiments, tumor biopsy samples may be used in various
DNA- and/or RNA-based assays
(e.g. RNAseq or Nanostring) to evaluate the tumor immune contexture. Without
wishing to be bound by theory, it
is possible that a reduction or reversal of immune suppression in a
cancer/tumor, as indicated by increased
cytotoxic T cells and decreased TAMs, may be predictive of therapeutic
efficacy in subjects administered with
TGFp inhibitor alone (e.g., Ab6) or in conjunction with a checkpoint inhibitor
therapy.
Circulating/circulatory latent-TGFp
[261] According to the present disclosure, circulating latent TGFP may serve
as a target engagement biomarker.
Where an activation inhibitor is selected as a therapeutic candidate, for
example, such biomarker may be employed
to evaluate or confirm in vivo target engagement by monitoring the levels of
circulating TGF beta (circulating TGFp)
before and after administration. In some embodiments, a target engagement
marker comprising circulating latent
TGFP (e.g., circulating latent TGFP1) is measured in a sample. In some
embodiments, circulating TGFP1 in a blood
sample (e.g., plasma and/or serum) comprises both latent and mature forms, the
former of which representing vast
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majority of circulatory TGF01. In some embodiments, total circulating TGFI3
(e.g., total circulating TG931) may be
measured, i.e., comprising both latent and mature TGFI3, for example by using
an acid treatment step to liberate
the mature growth factor (e.g., TGFp1) from its latent complex and detecting
with an enzyme-linked immunosorbent
assay (ELISA) assay. In some embodiments, reagents such as antibodies that
specifically bind the latent form of
TGFI3 (e.g., TGFP1) may be employed to specifically measure circulatory latent
TGF131. In some embodiments, a
majority of the measured circulating TGFI3 (e.g., circulating TGFp1) is
released from a latent complex. In some
embodiments, the total circulating TGFI3 (e.g., circulating TGF131) measured
is equivalent to dissociated latent
TGFI3 (e.g., latent TGF(31) in addition to any free TGFp (e.g., TGFp1) present
prior to acid treatment, which is
known to be only a small fraction of circulating TGFP1. In some embodiments,
only circulating latent TGFI3 (e.g.,
circulating latent TGFI31) is detectable. In some embodiments, circulating
latent TGFI3 (e.g., circulating latent
circulating TG931) is measured.
[262] In some embodiments, circulating TGFI3 (e.g., circulating latent TGFP1)
can be measured from a blood
sample by any of the methods described in or adapted from Mussbacher et al.,
PLos One. 2017 Dec 8;
12(12):e0188921 and Mancini et al. Trans/ Res. 2018 Feb; 192: 15-29, the
contents of which are hereby
incorporated by reference in their entirety.
[263] Challenges associated with determining blood/serum TGFp levels with
accuracy have been well recognized.
Platelets are a main source of TGFp1 in circulation, and even moderate
handling of blood samples, such as blood
collection, pipetting of blood samples, mechanical agitation, etc., is known
to cause the release of TGFP1 from
platelets in the sample, resulting in skewed readout.
[264] Aspects of the present disclosure include improved assays for measuring
circulatory TGFP levels. Such
assays comprise a sample collection step, sample processing step and measuring
step.
[265] Sample collection comprises placing a blood sample obtained from a
subject (e.g., cancer patient) into a
container (e.g., collection tube). Preferably, the collection tube is a
sterile, evacuated glass or plastic tube
containing anticoagulant. In some embodiments, such tube is about 13 mm times
75 mm in size and has a capacity
of about 2.7 mL. In some embodiments, the collection tube contains an
anticoagulant solution which includes a
form of sodium citrate. In preferred embodiments, the anticoagulant solution
is so-called CTAD. The CTAD
contains buffered trisodium citrate solution, theophylline, adenosine and
dipyrudamole. For example, the CTAD
may contain 0.11M buffered trisodium citrate solution (pH about 5.0), 15M
theophylline, 3.7M adenosine and
0.198M dipyridamole. Such collection tubes may contain an internal silicone
coating to minimize contact activation.
Such tubes may be equipped with a closing means (e.g., cap or stopper) aimed
to protect users from blood which
might splatter when the tube is opened. Such closure may be a rubber stopper,
which may be recessed inside the
plastic shield, preventing exposure to blood present on the stopper. Examples
of commercially available collection
tubes include BD VacutainerT" CTAD Blood Collection Tubes, which is equipped
with a Hemogard TM closure. The
manufacture's product description suggests that upon collection of blood into
the tube, the samples be centrifuged
at 1500g for 15 minutes at room/ambient temperature (18-25-C). Surprisingly,
however, Applicant has found that,
contrary to the manufacturer's recommendation, sample collection and
processing carried out at 2-8-C (e.g., about
4-C) produces superior results for purposes of measuring circulatory TGF(31
levels.
[266] Accordingly, in some embodiments, circulating TGFP (e.g., circulating
latent TGF131) in a blood sample is
measured by collecting the blood sample in a collection tube that comprises
(containing or coated with) an
anticoagulant. In some embodiments, the collection tube comprises a citrate
coating. In some embodiments, the
collection tube is coated with a solution comprising 0.1-0.5 M buffered
trisodium citrate. In some embodiments, the
collection tube is coated with a solution comprising 10-20 M theophylline. In
some embodiments, the collection
tube is coated with a solution comprising 2-5 M adenosine. In some
embodiments, the collection tube is coated
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with a solution comprising 0.1-0.25 M dipyridamole. In some embodiments, the
collection tube is coated with a
solution having a pH of 4.0-6Ø In some embodiments, the collection tube is
coated with an anticoagulant selected
from citrate-theophylline-adenosine-dipyridamole (CTAD), citrate (e.g., sodium
citrate), acid-citrate-dextrose
(ACD), ethylenediaminetetraacetic acid (EDTA), and heparin. In some
embodiments, the collection tube is coated
with CTAD. In some embodiments, the collection tube is coated with a CTAD
solution comprising about 0.11M
buffered trisodium citrate solution, about 15 M theophylline, about 3.7 M
adenosine, and about 0.198 M
dipyridamole. In some embodiments, the CTAD solution has a pH of about 5Ø In
some embodiments, the
collection tube is glass. In some embodiments, the collection tube has a
silicone coating. In some embodiments,
the collection tube has a HemogardTM closure. In some embodiments, the
collection tube has a volume capacity of
2-3 mL (e.g., 2.7 mL). In some embodiments, the collection tube is sterile. In
some embodiments, the collection
tube is a BD VacutainerTM CTAD blood collection tube (Macey et al. Clin Chem.
2002 Jun;48(6 Pt 1):891-9).
[267] Sample processing refers to any handling or processing of a biological
sample (e.g., blood sample) following
the sample collection step discussed above. The sample processing step may
include, for example, centrifugation,
fractionation or separation of sample, pipetting, mechanical agitation (e.g.,
shaking or mixing), etc. In some
embodiments, sample processing is carried out to prepare platelet-poor plasma
(PPP). A PPP fraction may be
prepared from a blood sample for the measurement of circulatory TGF[31 levels.
The term PPP typically refers to
blood plasma that contains less than 10,000 platelets per microliter (i.e., <
10 x 103/pL).
[268] In some embodiments, processing the blood sample comprises incubation
and/or centrifugation at a
temperature that is lower than room temperature. In some embodiments,
processing the blood sample comprises
incubation and/or centrifugation at a temperature that is lower than 20 C,
lower than 15 C, lower than 10 C, lower
than 5 00, or lower. In some embodiments, processing the blood sample
comprises incubation and/or centrifugation
at 2-8 'C. In some embodiments, processing the blood sample comprises
incubation and/or centrifugation at about
4 'C. In some embodiments, processing the blood sample comprises one or more
incubation steps as described
in Example 4.
[269] In some embodiments, processing the blood sample comprises one or more
centrifugation steps. In some
embodiments, processing the blood sample comprises one or more centrifugation
steps carried out at about 4 'C.
In some embodiments, processing the blood sample comprises a centrifugation
step at a speed of below 1500xg,
below 1000xg, below 800xg, below 400xg, below 250xg, below 200xg, or lower. In
some embodiments, processing
the blood sample comprises a centrifugation step at a speed of about 150xg. In
some embodiments, processing
the blood sample comprises a centrifugation step at a speed of above 1500xg,
above 2000xg, above 2500xg,
above 5000xg, above 7500xg, above 10000xg, above 12000xg, or higher. In some
embodiments, processing the
blood sample comprises a centrifugation step at a speed of about 2500xg. In
some embodiments, processing the
blood sample comprises a centrifugation step at a speed of about 12000xg.
[270] In some embodiments, processing the blood sample comprises a first
centrifugation step at a speed below
1000xg, and a second centrifugation step at a speed above 2000xg, optionally
with one or both steps at about 4
C. In some embodiments, processing the blood sample comprises a first
centrifugation step at a speed of about
150xg, and a second centrifugation step at a speed of about 2000xg. In some
embodiments, processing the blood
sample comprises a first centrifugation step at a speed below 2500xg, and a
second centrifugation step at a speed
above 10000xg. In some embodiments, processing the blood sample comprises a
first centrifugation step at a
speed of about 1500xg, and a second centrifugation step at a speed of about
12000xg. In some embodiments,
processing the blood sample comprises a first centrifugation step at a speed
of between 1000xg to 5000xg, and a
second centrifugation step at a speed of between 1000xg to 5000xg. In some
embodiments, processing the blood
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sample comprises a first step and a second centrifugation step, wherein the
two centrifugation steps are carried
out at the same speed. In some embodiments, processing the blood sample
comprises a first centrifugation step
at a speed of about 2500xg, and a second centrifugation step at a speed of
about 2500xg.
[271] In some embodiments, processing the blood sample comprises one or more
centrifugation steps carried
out for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least
20 minutes, at least 25 minutes, at least
30 minutes, or longer. In some embodiments, the blood sample is processed by a
first centrifugation step for at
least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20
minutes, at least 25 minutes, at least 30
minutes, or longer, followed by a second centrifugation step for at least 5
minutes, at least 10 minutes, at least 15
minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or
longer. In some embodiments, processing
the blood sample comprises a first centrifugation step for about 10 minutes,
and a second centrifugation step for
about 20 minutes. In some embodiments, processing the blood sample comprises a
first centrifugation step for
about 10 minutes, and a second centrifugation step for about 5 minutes. In
some embodiments, processing the
blood sample comprises transferring the supernatant portion of the sample to a
separate tube after the first
centrifugation step, and further processing the supernatant in a second
centrifugation step. In some embodiments,
the supernatant portion of the sample following the second centrifugation step
is used for measuring circulating
TGFp (e.g., circulating latent TGFp1) levels. In some embodiments, TGFp (e.g.,
circulating latent TGFp1) levels
may be determined using Bio-Plex Pro TM TGF-p Assays (Strauss et al. Olin
Cancer Res. 2018 Mar 15;24(6):1287-
1295).
[272] In some embodiments, collection, processing, and/or determination of
circulating TGFI3 (e.g., circulating
latent TGFp1) levels are conducted at about 4 C.
[273] In some embodiments, processing the blood sample comprises a first
centrifugation step of 100-500xg for
5-25 minutes, and a second centrifugation step of 1000-3000xg for 10-40
minutes, each step is optionally carried
out at about 4 C. In some embodiments, processing the blood sample comprises a
first centrifugation step of 1000-
3000xg for 5-25 minutes, and a second centrifugation step of 1000-3000xg for
10-40 minutes, each step is
optionally carried out at about 4cC. In some embodiments, processing the blood
sample comprises a first
centrifugation step of 1000-3000xg for 5-25 minutes, and a second
centrifugation step of 5000-15000xg for 2-10
minutes, each step is optionally carried out at about 4 C.
[274] In some embodiments, processing the blood sample comprises a first
centrifugation step of 1500xg for 10
minutes, and a second centrifugation step of 12000xg for 5 minutes, optionally
with one or both steps carried out
at about 4 C. In some embodiments, the blood sample is processed by a first
centrifugation step of 2500xg for 10
minutes, followed by a second centrifugation step of 2500xg for 10 minutes,
optionally with one or both steps at
about 4 C. In some embodiments, one or more additional centrifugation step is
applied.
[275] In various embodiments, the present disclosure provides methods of
determining and monitoring the level
of circulating latent TGFp in a sample obtained from a patient, such that
unwanted or inadvertent TGFp activation
associated with sample processing and preparation is reduced. In certain
embodiments, the methods disclosed
herein may be used to determine or monitor the level of circulating latent
TGFP1, e.g., by using sample collection
methods disclosed herein and/or by normalzing to control markers of platelet
activation during collection, e.g., PF4
levels.
[276] Following the sample processing step described above, the resulting
samples (e.g., PPP et al.) may be
used to carry out one or more measuring steps for circulatory TGF13.
Accordingly, the present disclosure provides,
in various embodiments, a method for measuring circuating TGFp levels in a
blood sample, wherein the method
comprises a collection step and a processing step, each of which is carried
out at 2-8 C using a CTAD collection
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tube. The processing step may comprise two centrifugation steps as described
above, to generate a PPP fraction
from the blood sample. The resulting PPP is used to measure TGFI3 levels. In
some embodiments, total TGFp
levels, which include both the active and latent TGFp forms, are measured. In
some embodiments, active TGFp
(mature growth factor) levels are measured. In some embodiments, latent TGFp
levels are measured. In some
embodiments, a majority of the TGFP measured in an acidified sample is from
circulating latent TGFP. In some
embodiments, the level of the TGFp1 isoform is selectively measured. In some
embodiments, the measuring step
may include acidification of the sample to release TGFp (i.e., mature growth
factor) from the latent complex (i.e.,
proTGFp, such as proTGFp1). ELISA-based methods may be employed to then
capture and detect/quantitate
TGFP present in the sample.
[277] Such assay steps may be incorporated in a treatment regimen for a
patient. For example, such assays may
be used for providing information to aid prognosis, diagnosis, target
engagement, monitoring therapeutic response,
etc.
[278] In some embodiments, circulatory TGFp levels may serve as a predictive
biomarker.
[279] In some embodiments, circulatory TGFp levels may serve as a predictive
biomarker for therapeutic
response to a checkpoint inhibitor therapy. In some embodiments, high baseline
levels of circulatory TGFp levels
(e.g., in the plasma) may be predictive of poor therapeutic response to a
checkpoint inhibitor therapy (e.g.,
pembrolizumab) (Feun et al. Cancer. 2019 Oct 15;125(20):3603-3614).
[280] Accordingly, in various embodiments, the treatment regimen may include
administration of a therapy that
includes a TGFp inhibitor, such as TGFp1 inhibitor. The TGFp inhibitors
include, for example, monoclonal
antibodies that bind the latent form of TGFp (i.e., proTGFp, such as proTGFp1)
thereby preventing the release of
the growth factor, such as Ab6 and other anitbodies that work by the same
mechanism of action (see, for example,
WO 2000/014460, WO 2000/041390, PCT/2021/012930, WO 2018/013939, WO
2020/160291). The TGFp
inhibitors include neutralizing antibodies and engineered constructs that
incorporate an antigen-binding fragment
thereof. Examples of neutralizing antibodies include GC1008 and its variants,
and NIS-793 (X0MA089). The
TGFp inhibitors also include so-called ligand traps, which comprise the ligand
binding fragment(s) of the TGFp
receptor(s). Examples of ligand traps include M7824 (bintrafusp alpha) and
AVID200. The TGFp inhibitors also
include low molecular weight receptor kinase inhibitors, such as ALK5
inhibitors.
[281] In various embodiments, the patient being administered the treatment
regimen is diagnosed with, at risk of
developing, or suspected to have a TGFP-related disease, such as cancer,
myeloproliferative disorders (such as
myelofibrosis), fibrosis and immune disorders. Thus, in some embodiments, the
present disclosure provides a
TGFp inhibitor for use in the treatment of a TGFp-related disease in a
subject, wherein the treatment comprises
administration of a composition comprising a TGFp inhibitor in an amount
sufficient to treat the disease, wherein
the treatment further comprises determination of circulatory TGFP levels in
accordance with the disclosure herein.
In some embodiments, the treatment further comprises determination of
circulatory MDSCs. In some
embodiments, circulatory MDSC levels are determined by measuring cell-surface
marker(s). In some
embodiments, the cell-surface marker is LRRC33. In some embodiments, the
patient is a cancer patient, wherein
optionally the cancer comprises a solid tumor, such as locally advanced or
metastatic tumor. In some
embodiments, the patient previously received a cancer therapy, wherein the
cancer therapy is checkpoint inhibitor,
radiation therapy and/or chemotherapy. In some embodiments, the subject was
unresponsive or refractory to the
cancer therapy, wherein optionally the cancer therapy comprises a checkpoint
inhibitor (e.g., chechpoint inhibitor-
resistant). In some embodiments, the tumor is refractory to the cancer
therapy. In some embodiments, the patient
is naïve to a cancer therapy, e.g., a checkpoint inhibitor (i.e., a checipoint
inhibitor-naïve patient). In some
embodiments, the checkpoint inhibitor-naive patient is diagnosed with a type
of cancer that has statistically shown
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to have low response rates (e.g., below 30%, below 25%, below 20%, below 15%,
etc.) to checkpoint inhibitors,
such as anti-PD-(L)1. In some embodiments, the solid tumor has an immune
excluded phenotype. In some
embodiments, the solid tumor has low expression of PD-L1.
[282] In various embodiments, the present disclosure provides methods of
treating a TGFp-related disorder,
comprising monitoring the level of circulating TGFI3, e.g., circulating latent
TGFp (e.g., TGF131) in a sample obtained
from a patient (e.g., in the blood, e.g., plasma and/or serum, of a patient)
receiving a TGFp inhibitor. In certain
embodiments, circulating TGFp, e.g., circulating latent TGF13 (e.g., TGF(31)
may be measured in plasma samples
collected from the subject. In certain embodiments, measuring TGFp, e.g.,
circulating latent TGFp (e.g., TGF31)
from the plasma may reduce the risk of inadvertently activating TGFp, such as
that observed during serum
preparations and/or processing. Accordingly, the present disclosure includes a
TGFp inhibitor for use in the
treatment of diseases such as cancer, myelofibrosis, and fibrosis, in a
subject, wherein the treatment comprises a
step of measuring circulating TGFP levels from a plasma sample collected from
the subject. Such samples may be
collected before and/or after administration of a TGFp inhibitor to treat such
diseases.
[283] The level of circulating TGFP (e.g., circulating latent TGFI31) may be
monitored alone or in conjunction with
one or more of the biomarkers disclosed herein (e.g., MDSCs). In certain
embodiments, circulating TGFp (e.g.,
circulating latent TGF131) may be monitored alone or in conjunction with one
or more of total platelet count,
phosphorylated Smad2 level, and/or treatment duration. In certain embodiments,
the TG93 inhibitor may be
administered alone or in conjunction with an additional cancer therapy. In
some embodiments, the treatment may
be administered to a subject afflicted with a TGFp-related cancer or
myeloproliferative disorder. In some
embodiments, the TGFp inhibitor is a TGFI31-selective antibody or antigen-
binding fragment thereof encompassed
in the current disclosure (e.g., Ab6). In some embodiments, the TGFp inhibitor
is an isoform-non-selective TGFp
inhibitor (such as low molecular weight ALK5 antagonists, neutralizing
antibodies that bind two or more of
TGFI31/2/3, e.g., GC1008 and variants, antibodies that bind TGF(31/3, and
ligand traps, e.g., TGFp1/3 inhibitors).
In some embodiments, the TGF13 inhibitor is an integrin inhibitor (e.g., an
antibody that binds to aVE1 , aVp3, aV35,
c6/136, aVf38, a5]31, a11b133, or a8131 integrins, and inhibits downstream
activation of TGFp. e.g., selective inhibition
of TGFp1 and/or TGFp3). In some embodiments, the additional cancer therapy may
comprise chemotherapy,
radiation therapy (including radiotherapeutic agents), a cancer vaccine, or an
immunotherapy, such as a checkpoint
inhibitor therapy, e.g., an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody. In
some embodiments, the checkpoint
inhibitor therapy is selected from the group consisting of ipilimumab (e.g.,
Yervoy0); nivolumab (e.g., Opdivo0);
pembrolizumab (e.g., Keytruda0); avelumab (e.g., Bavencio0); cemiplimab (e.g.,
Libtayo0); atezolizumab (e.g.,
Tecentrige); budigalimab (ABBV-181); and durvalumab (e.g., Imfinzie).
[284] In various embodiments, circulating TGFp (e.g., circulating latent
TGF131) may be measured in a sample
obtained from a subject (e.g., whole blood or a blood component). In various
embodiments, the circulating latent
TGFp levels (e.g., latent TGF31) may be measured within 1, 2, 3, 4, 5, 6, 7,
8, 10, 12, 14, 16, 18, 21, 22, 25, 28,
30, 35, 40, 45, 48, 50, or 56 days following administration of the TGFP
inhibitor to a subject, e.g., up to 56 days
after administration of a therapeutic dose of a TG93 inhibitor. In various
embodiments, the circulating latent TGFp
levels (e.g., circulating latent TGF(31) may be measured about 8 to about 672
hours following administration of a
therapeutic dose of a TGFp inhibitor. In various embodiments, the circulating
latent TGF(3 levels (e.g., circulating
latent TGFP1) may be measured about 72 to about 240 hours (e.g., about 72 to
about 168 hours, about 84 to about
156 hours, about 96 to about 144 hours, about 108 to about 132 hours)
following administration of a therapeutic
dose of a TGFp inhibitor. In various embodiments, the circulating latent TGFp
levels (e.g., circulating latent TGFp1)
may be measured about 120 hours following administration of a therapeutic dose
of a TGFp inhibitor. In some
embodiments, the circulating latent TGFP levels (e.g., circulating latent
TGFP1) may be measured by any method
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known in the art (e.g., ELISA). In preferred embodiments, circulating TGFI3
levels are measured from a blood
sample (e.g., a plasma sample).
[285] In various embodiments, the present disclosure encompasses a method of
treating cancer in a subject,
wherein the treatment comprises determining a level of circulating TGFp in the
subject prior to administering a
TGFI3 inhibitor, administering to the subject a therapeutically effective
amount of the TGFI3 inhibitor, and
determining a level of circulating TGFp in the subject after administration.
In some embodiments, the circulating
TGFI3 level is determined or has been determined by processing a blood sample
from the subject below room
temperature in a sample tube coated with an anticoagulant.
[286] In various embodiments, a method of treating a cancer or other TGF-
related disorder comprises
administering a TGFP inhibitor (e.g., an anti-TGFP1 antibody) to a patient in
need thereof and confirming the level
of target engagement by the inhibitor. In some embodiments, determining the
level of target engagement comprises
determining the levels of circulating TGFI3 (e.g., circulating latent TGF131)
in a sample obtained from a patient (e.g.,
in the blood or a blood component of a patient) receiving the TGFI3 inhibitor.
In some embodiments, an increase in
circulating TGFP (e.g., circulating latent TGFI31) after administration of the
TGF inhibitor indicates target
engagement. In some embodiments, the present disclosure provides a method of
determining targeting
engagement in a subject having cancer, comprising determining a level of
circulating TGFI3 (e.g., circulating latent
TGFI31) in the subject prior to administering a TGFp inhibitor, administering
to the subject a therapeutically effective
amount of the TGFI3 inhibitor, and determining a level of circulating TGFP in
the subject after administration. In
some embodiments, an increase in circulating TGFI3 levels (e.g., circulating
latent TGFI31 levels) after
administration as compared to before administration indicates target
engagement of the TGFp inhibitor. In some
embodiments, an increase in circulating TGFp (e g., circulating latent TGF(31)
after administration of the TGF13
inhibitor indicates target engagement, wherein the increase is at least 1.5-
fold, at least 2-fold, at least 2.5-fold, at
least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-
fold, at least 8-fold, at least 9-fold, at least 10-
fold, or more over baseline level. In some embodiments, the circulating TGFI3
levels are determined or have been
determined by processing a blood sample from the subject below room
temperature in a sample tube coated with
an anticoagulant. In some embodiments, further therapeutically effective
amount of the TGFI3 inhibitor are
administered if target engagement is detected.
[287] In various embodiments, the present disclosure also provides methods of
using circulating TGFI3 levels
(e.g., circulating latent TGFI31 levels) to predict therapeutic response, as
well as for informing further treatment
decisions (e.g., by continuing treatment if an increase is observed). In some
embodiments, an additional dose of
the TGFp inhibitor (e.g., an anti-TGF[31 antibody) is administered if target
engagement is detected. In some
embodiments, the method of determining therapeutic efficacy comprises
determining a level of circulating TGFI3 in
the subject prior to administering a TGFp inhibitor, administering to the
subject a therapeutically effective amount
of the TGFI3 inhibitor, and determining a level of circulating TGFI3 in the
subject after administration. In preferred
embodiments, circulating TGFP levels are measured from a blood sample. In some
embodiments, the circulating
TGFI3 levels are determined or have been determined by processing the blood
sample from the subject below room
temperature in a sample tube coated with an anticoagulant. In some
embodiments, further therapeutically effective
amount of the TGFI3 inhibitor are administered if efficacy is detected.
[288] In one aspect of the current disclosure, levels of circulating TGFI3
(e.g., circulating latent TGF(31) are
determined to inform treatment and predict therapeutic efficacy in subjects
administered a TGFp inhibitor such as
a TG931-selective inhibitor described herein. In certain embodiments, a TGFI3
inhibitor (e.g., Ab6) is administered
alone or concurrently (e.g., simultaneously), separately, or sequentially with
an additional cancer therapy, e.g., a
checkpoint inhibitor therapy, such that the amount of TGFI31 inhibition
administered is sufficient to increase the
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levels of circulating TGFp (e.g., circulating latent TGF(31) as compared to
baseline levels. Circulating TGFp levels
(e.g., circulating latent TGF131) may be measured prior to or after each
treatment such that an increase in
circulating latent-TGFp levels (e.g., latent TGFp1) following the treatment
indicates therapeutic efficacy. For
instance, circulating TGFp levels (e.g., circulating latent TGFp1) may be
measured prior to and after the
administration of a TGFP inhibitor (e.g., Ab6) and an increase in circulating
TGFP levels (e.g., latent TGFP1)
following the treatment predicts therapeutic efficacy. In some embodiments,
treatment is continued if an increase
in circulating TGFp is detected. In certain embodiments, circulating TGFp
levels (e.g., circulating latent TGF131)
may be measured prior to and following administration of a first dose of a
TGFp inhibitor such as a TGFpl inhibitor
described herein (e.g., Ab6), and an increase in circulating TGFP levels
(e.g., circulating latent TGFP1) following
the administration predicts therapeutic efficacy and further warrants
administration of a second or more dose(s) of
the TGFp inhibitor. In some embodiments, circulating TGFp levels (e.g.,
circulating latent TGFp1) may be
measured prior to and after a combination treatment of TGFp inhibitor such as
a TGFpl -selective inhibitor (e.g.,
Ab6), and an additional therapy (e.g., a checkpoint inhibitor therapy),
administered concurrently (e.g.,
simultaneously), separately, or sequentially, and a change in circulating
latent-TGFp levels following the treatment
predicts therapeutic efficacy. In some embodiments, treatment is continued if
an increase is detected. In some
embodiments, an increase in circulating TGFp (e.g., circulating latent TGF131)
after administration of the TGFp
inhibitor indicates therapeutic efficacy, wherein the increase is at least 1.5-
fold, at least 2-fold, at least 2.5-fold, at
least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-
fold, at least 8-fold, at least 9-fold, at least 10-
fold, or more, as compared to baseline. In some embodiments, the increase in
circulating TGFp levels (e.g.,
circulating latent TGFp1) following a combination treatment may warrant
continuation of treatment. In preferred
embodiments, circulating TGFp levels are measured from a blood sample, wherein
the blood sample is optionally
processed below room temperature in a sample tube coated with an
anticoagulant.
[289] In certain embodiments, the current disclosure provides a method of
treating a cancer in a subject,
comprising administering a second dose of a TGFp inhibitor to a subject having
an elevated level of circulating
TGFp after receiving a first dose the TGFp inhibitor, wherein the level of
TGFp has been measured by processing
a blood sample from the subject below room temperature in a sample tube coated
with an anticoagulant.
[290] In certain embodiments, the current disclosure provides a method of
treating a cancer in a subject
comprising determining a level of circulating TGFp in the subject prior to
administering a TGFp inhibitor,
administering to the subject a first dose of TGFp inhibitor, determining a
level of circulating TGFp in the subject
after administration, and administering a second dose of the TGFp inhibitor to
the subject if the level of circulating
TGFp is elevated. In some embodiments, measuring the level of circulating TGFp
comprises processing a blood
sample from the subject below room temperature in a sample tube coated with an
anticoagulant. In some
embodiments, the level of circulating TGFP after the first dose of the TGFP
inhibitor is elevated by at least 1.5-fold,
at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least
5-fold, or more as compared to baseline (e.g.,
the level of circulating TGFp before the first dose of the TGFp inhibitor). In
some embodiments, the circulating
TGFp is latent TGFp. In some embodiments, the circulating TGFp is circulating
TGFp1.
[291] In some embodiments, the cancer comprises a solid tumor, wherein
optionally the solid tumor is selected
from: melanoma (e.g., metastatic melanoma), triple-negative breast cancer,
HER2-positive breast cancer,
colorectal cancer (e.g., microsatellite stable-colorectal cancer), lung cancer
(e.g., metastatic non-small cell lung
cancer, small cell lung cancer), esophageal cancer, pancreatic cancer, bladder
cancer, kidney cancer (e.g.,
transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC),
including clear cell RCC, papillary RCC,
chromophobe RCC, collecting duct RCC, or unclassified RCC, uterine cancer,
prostate cancer, stomach cancer
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(e.g., gastric cancer), head and neck squamous cell cancer, urothelial
carcinoma (e.g., metastatic urothelial
carcinoma), hepatocellular carcinoma, or thyroid cancer.
[292] In various embodiments, the current disclosure encompasses a method of
treating a TGFp-related disorder
comprising administering a therapeutically effective amount of a TGFI3
inhibitor to a subject having a TGFp-related
disorder, wherein the therapeutically effective amount is an amount sufficient
to increase the level of circulating
TGFI3 (e.g., circulating latent TGF131). In certain embodiments, the TGFI3
inhibitor is a TGFp activation inhibitor. In
certain embodiments, the TGFI3 inhibitor is a TGFI31 inhibitor (e.g., Ab6). In
certain embodiments, the circulating
TGFI3 is latent TGFpi . In some embodiments, the therapeutically effective
amount of the TGFp inhibitor (e.g., Ab6)
is between 0.1-30 mg/kg per dose. In some embodiments, therapeutically
effective amount of the TGFI3 inhibitor
(e.g., Ab6) is between 1-30 mg/kg per close. In some embodiments, the
therapeutically effective amount of the
TGFI3 inhibitor (e.g., Ab6) is between 5-20 mg/kg per dose. In some
embodiments, the therapeutically effective
amount of the TGFI3 inhibitor (e.g., Ab6) is between 3-10 mg/kg per dose. In
some embodiments, the therapeutically
effective amount of the TGFp inhibitor (e.g., Ab6) is between 1-10 mg/kg per
dose. In some embodiments, the
therapeutically effective amount of the TGFp inhibitor (e.g., Ab6) is between
2-7 mg/kg per dose. In some
embodiments, the therapeutically effective amount of the TGFI3 inhibitor
(e.g., Ab6) is about 2-6 mg/kg per dose.
In some embodiments, the therapeutically effective amount of the TGFI3
inhibitor (e.g., Ab6) is about 1 mg/kg per
dose. In some embodiments, doses are administered about every three weeks. In
some embodiments, the TGF13
inhibitor (e.g., Ab6) is dosed weekly, every 2 weeks, every 3 weeks, every 4
weeks, monthly, every 6 weeks, every
8 weeks, bi-monthly, every 10 weeks, every 12 weeks, every 3 months, every 4
months, every 6 months, every 8
months, every 10 months, or once a year. In preferred embodiments, circulating
TGFI3 levels are measured from
a blood sample (e.g., a plasma sample, serum sample, etc.).
[293] In various embodiments, total circulatory TGFP1 (e.g., circulating
latent TGFP1) in blood samples collected
from patients may range between about 2-200 ng/mL at baseline, although the
measured amounts vary depending
on the individuals, health status, and the exact assays being employed. In
certain embodiments, total circulatory
TGF131 (e.g., circulating latent TGFI31) in blood samples collected from
patients may range between about 1 ng/mL
to about 10 ng (e.g., about 1000 pg/mL to about 7000 pg/mL). In certain
embodiments, the level of circulating latent
TGFI3 (e.g., latent TGF31) following administration of a TGFI3 inhibitor
(e.g., Ab6) is increased by at least 1.5-fold
(e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold,
at least 5-fold, at least 6-fold, at least 7-fold, at
least 8-fold, at least 9-fold, at least 10-fold, or more) as compared to
circulating latent TGFp levels prior to the
administration (e.g., baseline). In preferred embodiments, circulating TGFp
levels are measured from a blood
sample (e.g., a plasma sample, serum sample, etc.).
[294] In certain embodiments, circulating TGFI3 levels (e.g., circulating
latent TGFI31) may be used to monitor
target engagement and pharmacological activity of a TGFp inhibitor in a
subject receiving a TGFp inhibitor therapy
(e.g., a TGFp activation inhibitor, e.g., Ab6). In certain embodiments,
circulating TGFI3 levels (e.g., circulating latent
TGFP1 levels) may be measured prior to and after administration of a first
dose of TGFI3 inhibitor (e.g., Ab6) such
that an increase of at least 1.5-fold (e.g. at least 1.5-fold, at least 2-
fold, at least 3-fold, at least 4-fold, at least 5-
fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at
least 10-fold, or more) over baseline in circulating
latent TGFI3 levels following the administration indicates target engagement
(e.g., binding of the TGFI3 inhibitor to
human large latent proTGFP1 complex). In certain embodiments, circulating TGFP
levels (e.g., circulating latent
TGFp1) may be measured prior to and after administration of a first dose of
TGFI3 inhibitor (e.g., Ab6) such that an
increase in circulating TGFp levels (e.g., circulating latent TGFp1) following
the administration indicates therapeutic
efficacy. In certain embodiments, treatment is continued if an increase in
circulating TGFp levels (e.g., circulating
latent TGF131) following administration of a TGFP inhibitor (e.g., Ab6) is
detected. In preferred embodiments,
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circulating TGFI3 levels (e.g., circulating latent TG931) are measured from a
blood sample (e.g., a plasma sample,
serum sample, etc.).
[295] In some embodiments, circulating TG93 levels (e.g., circulating latent
TGFp1) may be measured prior to
and after administration of a first dose of a TGFp inhibitor (e.g., Ab6), and
an increase in circulating TGFp levels
(e.g., circulating latent TGFI31) after the administration indicates target
engagement, treatment response, and/or
further warrants administration of a second or more dose(s) of the TGFp
inhibitor. In another embodiment,
circulating TGFp levels (e.g., circulating latent TGF(31) may be measured
prior to and after administration of a first
dose of a combination treatment comprising a checkpoint inhibitor therapy and
a TGFp inhibitor such as a TGF31-
selective inhibitor (e.g., Ab6), and an increase in circulating TGFI3 levels
(e.g., circulating latent TGFI31) after the
administration indicates target engagement, treatment response, and/or further
warrants continuation of treatment.
In various embodiments, the combination therapy comprises a checkpoint
inhibitor therapy and a TGF3 inhibitor
such as a TGFP1-selective inhibitor (e.g., Ab6), an isoform-non-selective
inhibitor (e.g., low molecular weight ALK5
antagonists), neutralizing antibodies that bind two or more of TGFp1/2/3
(e.g., GC1008 and variants), antibodies
that bind TGFp1/3, and/or an integrin inhibitor (e.g., an antibody that binds
to aVp1 , aVp3, aVp5, aVp6, aV38,
a5p1, allbp3, or a8131 integrins, and inhibits downstream activation of TGFO
e.g., selective inhibition of TGFpl
and/or TGF[33). In preferred embodiments, circulating TGFp levels are measured
from a blood (e.g., plasma
sample, serum sample, etc.).
[296] In any of the preceding embodiments, circulating TGFI3 can be
circulating TGFP1 or circulating latent
TGFpl . In various embodiments, the circulating TGFI31 or circulating latent
TGF131 is measured from a blood
sample collected from the subject. In various embodiments, the blood sample is
processed below room temperature
in a sample tube containing or coated with an anticoagulant.
Smad2 phosphorylation
[297] According to the present disclosure, activation of Smad2 may serve as a
marker for target engagement
and/or therapeutic efficacy. In certain embodiments, Smad2 activation is
detected by measuring a level of Smad2
phosphorylation (p-Smad2) and/or p-Smad2 nuclear translocation. In certain
embodiments, p-Smad2 levels and/or
p-Smad2 nuclear translocation is measured by immunohistochemistry. In certain
embodiments, p-Smad2 nuclear
translocation is determined by nuclear masking analysis.
[298] In certain embodiments, a method for determining therapeutic efficacy in
a subject being treated for cancer
comprises determining a level of p-Smad2 nuclear translocation in a tumor
sample obtained from the subject prior
to administering a therapy comprising a TGFp inhibitor; administering to the
subject one or more doses of the TGFp
inhibitor; and determining a level of p-Smad2 nuclear translocation in a tumor
sample obtained from the subject
after the administration; wherein a decrease in p-Smad2 nuclear translocation
after the administration as compared
to before the administration indicates therapeutic efficacy. In certain
embodiments, the p-Smad2 nuclear
translocation after the administration is decreased by at least 1.3-fold, at
least 1.5-fold, at least 2-fold, at least 3-
fold, at least 4-fold, at least 5-fold, or more as compared to the p-Smad2
nuclear translocation before the
administration. In certain embodiments, one or more additional doses of the
treatment is administered if a decrease
in p-Smad2 nuclear translocation is observed.
[299] In certain embodiments, a method for determining target engagement in a
subject having cancer comprises
determining a level of p-Smad2 nuclear translocation in a tumor sample
obtained from the subject prior to
administering a therapy comprising a TGFp inhibitor; administering to the
subject one or more doses of the TGFp
inhibitor; determining a level of p-Smad2 nuclear translocation in a tumor
sample obtained from the subject after
the administration; and wherein a decrease in p-Smad2 nuclear translocation
after the administration as compared
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to before the administration indicates target engagement of the TGF6
inhibitor. In certain embodiments, the p-
Smad2 nuclear translocation after the administration is decreased by at least
1.3-fold, at least 1.5-fold, at least 2-
fold, at least 3-fold, at least 4-fold, at least 5-fold, or more as compared
to the p-Smad2 nuclear translocation before
the administration. In certain embodiments, one or more additional doses of
the treatment is administered if a
decrease in p-Smad2 nuclear translocation is observed.
[300] In certain embodiments, a method for treating cancer in a subject or a
TGF(3 inhibitor for use in treating
cancer, wherein the method or the TGF6 inhibitor for use comprises determining
a level of p-Smad2 nuclear
translocation in a tumor sample obtained from the subject prior to
administering a therapy comprising a TGFp
inhibitor; administering to the subject a first dose of the TGF6 inhibitor;
determining a level of p-Smad2 nuclear
translocation in a tumor sample obtained from the subject after the
administration; and administering to the subject
one or more additional doses of the TGFI3 inhibitor if the p-Smad2 nuclear
translocation after the administration of
the first dose is decreased as compared to the p-Smad2 nuclear translocation
before the administration of the first
dose. In certain embodiments, the p-Smad2 nuclear translocation after the
administration is decreased by at least
1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-
fold, at least 5-fold, or more as compared to the
p-Smad2 nuclear translocation before the administration. In certain
embodiments, the treatment comprises
administering one or more additional doses of the treatment if a decrease in p-
Smad2 nuclear translocation is
observed.
[301] In any of the preceding embodiments, the TGF6 inhibitor is a TGF61
inhibitor, e.g., Ab6.
Immune Safety
[302] Cytokines play an important role in normal immune responses, but when
the immune system is triggered to
become hyperactive, the positive feedback loop of cytokine production can lead
to a "cytokine storm" or
hypercytokinemia, a situation in which excessive cytokine production causes an
immune response that can
damage organs, especially the lungs and kidneys, and even lead to death. Such
condition is characterized by
markedly elevated proinflammatory cytokines in the serum. Historically, a
Phase 1 Trial of the anti-0D28
monoclonal antibody TGN1412 in healthy volunteers led to a life-threatening
"cytokine storm" response resulted
from an unexpected systemic and rapid induction of proinflammatory cytokines
(Suntharalingam G et al., N Engl J
Med. 2006 Sep 7;355(10):1018-28). This incident prompted heightened awareness
of the potential danger
associated with pharmacologic stimulation of T cells.
[303] Whilst TGFp-directed therapies do not target a specific T cell receptor
or its ligand, Applicant of the present
disclosure reasoned that it was prudent to carry out immune safety assessment,
including, for example, in vitro
cytokine release assays, in vivo cytokine measurements from plasma samples of
non-human primate treated with
a TGF6 inhibitor, and platelet assays using human platelets. Exemplary assays
were previously described, for
instance in PCT/U52021/012969 at Example 23.
[304] In some embodiments, one or more of the cytokines IL-2, TNFa, IFNy, IL-
16, CCL2 (MCP-1), and IL-6 may
be assayed, e.g., by exposure to peripheral blood mononuclear cell (PBMC)
constituents from heathy donors.
Cytokine response after exposure to an antibody disclosed herein, e.g., Ab6,
may be compared to release after
exposure to a control, e.g., an IgG isotype negative control antibody.
Cytokine activation may be assessed in plate-
bound and/or soluble assay formats. Levels of IFNy, IL-2, IL-16, TNFa, IL-6,
and CCL2 (MCP-1) should not exceed
10-fold, e.g., 8-, 6-, 4-, or 2-fold the activation in the negative control.
In some embodiments, a positive control may
also be used to confirm cytokine activation in the sample, e.g., in the PBMCs.
In some embodiments, these in vitro
cytokine release results may be further confirmed in vivo, e.g., in an animal
model such as a monkey toxicology
study, e.g., a 4-week GLP repeat-dose monkey study as described in
PCT/US2021/012969 at Example 24.
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[305] Human platelets have been reported to express CARP, which can form
TGFI31 LLCs (Iran et al., 2009.
Proc Nat! Acad Sci U S A. 106(32): 13445-13450). In some embodiments, an
antibody disclosed herein, e.g., Ab6,
does not significantly bind to and/or activate platelets. In some embodiments,
platelet activation is evaluated in
vitro, as described in Example 23. In some embodiments, platelet aggregation,
binding, and activation may be
assessed in human whole blood or platelet-rich plasma from healthy donors.
Platelet aggregation and binding after
exposure to an antibody disclosed herein, e.g., Ab6 may be compared to
exposure to a negative control, e.g.,
saline solution, or a reference sample, e.g., a buffered solution. In certain
embodiments, platelet aggregation and
binding do not exceed 10% above the aggregation in the negative control. In
some embodiments, platelet activation
following exposure to an antibody disclosed herein, e.g., Ab6, may be compared
to exposure to a positive control,
e.g., adenosine di phosphate (ADP). The activation status of platelets may be
determined by surface expression of
activation markers e.g., CD62P (P-Selectin) and GARP detectable by flow
cytometry. Platelet activation should not
exceed 10% above the activation in the negative control. In some embodiments,
in vitro platelet response results
may be further confirmed in vivo, e.g., in an animal model such as a monkey
toxicology study, e.g., a 4-week GLP
repeat-dose monkey study.
[306] In some embodiments, selection of an antibody or an antigen-binding
fragment thereof for therapeutic use
may include: identifying an antibody or antigen-binding fragment that meets
the criteria of one or more of those
described herein; carrying out an in vivo efficacy study in a suitable
preclinical model to determine an effective
amount of the antibody or the fragment; carrying out an in vivo
safety/toxicology study in a suitable model to
determine an amount of the antibody that is safe or toxic (e.g., MTD, NOAEL,
or any art-recognized parameters for
evaluating safety/toxicity); and, selecting the antibody or the fragment that
provides at least a three-fold therapeutic
window (preferably 6-fold, more preferably a 10-fold therapeutic window, even
more preferably a 15-fold
therapeutic window). In certain embodiments, the in vivo efficacy study is
carried out in two or more suitable
preclinical models that recapitulate human conditions. In some embodiments,
such preclinical models comprise a
TGFpl-positive cancer, which may optionally comprise an immunosuppressive
tumor. The immunosuppressive
tumor may be resistant to a cancer therapy such as CBT, chemotherapy and
radiation therapy (including a
radiotherapeutic agent). In some embodiments, the preclinical models are
selected from MBT-2, Cloudman S91
and EMT6 tumor models.
[307] Identification of an antibody or antigen-binding fragment thereof for
therapeutic use may further include
carrying out an immune safety assay, which may include, but is not limited to,
measuring cytokine release and/or
determining the impact of the antibody or antigen-binding fragment on platelet
binding, activation, and/or
aggregation. In certain embodiments, cytokine release may be measured in vitro
using PBMCs or in vivo using a
preclinical model such as non-human primates. In certain embodiments, the
antibody or antigen-binding fragment
thereof does not induce a greater than 10-fold release in IL-6, IFNy, and/or
TNFa levels as compared to levels in
an IgG control sample in the immune safety assessment. In certain embodiments,
assessment of platelet binding,
activation, and aggregation may be carried out in vitro using PBMCs. In some
embodiments, the antibody or
antigen-binding fragment thereof does not induce a more than 10% increase in
platelet binding, activation, and/or
aggregation as compared to buffer or isotype control in the immune safety
assessment.
[308] The selected antibody or the fragment may be used in the manufacture of
a pharmaceutical composition
comprising the antibody or the fragment. Such pharmaceutical composition may
be used in the treatment of a
TGFp indication in a subject as described herein. For example, the TGFI3
indication may be a proliferative disorder,
e.g., a TGF31-positive cancer. Thus, the invention includes a method for
manufacturing a pharmaceutical
composition comprising a TGFr3 inhibitor, wherein the method includes the step
of selecting a TGFr3 inhibitor which
is tested for immune safety as assessed by immune safety assessment comprising
cytokine release assays and
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optionally further comprising a platelet assay. The TGFI3 inhibitor selected
by the method does not trigger
unacceptable levels of cytokine release (e.g., no more than 10-fold, but more
preferably within 2.5-fold as compared
to control such as IgG control). Similarly, the TGFr3 inhibitor selected by
the method does not cause unacceptable
levels of platelet aggregation, platelet activation and/or platelet binding.
Such TGFr3 inhibitor is then manufactured
at large-scale, for example 250L or greater, e.g., 1000L, 2000L, 3000L, 4000L
or greater, for commercial production
of the pharmaceutical composition comprising the TGFI3 inhibitor.
Cancer! malignancies
[309] Various cancers involve TG93 activities, e.g., TG931 activities, and may
be treated with the antibodies,
compositions, and methods of the present disclosure. As used herein, the term
"cancer" comprises any of various
malignant neoplasms, optionally associated with TGFI31-positive cells.
Such malignant neoplasms are
characterized by the proliferation of anaplastic cells that tend to invade
surrounding tissue and metastasize to new
body sites and also refers to the pathological condition characterized by such
malignant neoplastic growths. The
source of TGF[31 may vary and may include the malignant (cancer) cells
themselves, as well as their surrounding
or support cells/tissues, including, for example, the extracellular matrix,
various immune cells, and any
combinations thereof.
[310] Examples of cancer which may be treated in accordance with the present
disclosure include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid
malignancies. More particular
examples of such cancers include, but are not limited to, anal carcinoma; bile
duct cancer; brain tumor (including
glioblastoma); breast cancer, e.g., HER2+ breast cancer and triple-negative
breast cancer (TNBC), ductal
carcinoma in situ (DCIS); cervical cancer; colorectal cancer; endometrial or
uterine carcinoma; esophageal cancer;
gastric or gastrointestinal cancer; gastrointestinal carcinoid tumor;
gastrointestinal stromal tumors (GIST); head
and neck cancer, e.g. head and neck squamous cell cancer (HNSCC); liver
cancer, e.g., hepatocellular carcinoma
(HCC); lung cancer, including small-cell lung cancer (SCLC), non-small cell
lung cancer (NSCLC), metastatic
NSCLC, adenocarcinoma of the lung, and squamous carcinoma of the lung;
melanoma; ovarian cancer; pancreatic
cancer (e.g., pancreatic ductal adenocarcinoma (PDAC);penile carcinoma;
prostate cancer, e.g., castration-
resistant prostate cancer (CRPC); renal cell carcinoma (RCC), e.g., clear cell
RCC; cancer of the peritoneum;
salivary gland carcinoma; thyroid cancer; urothelial carcinoma (UC) of the
bladder and urinary tract, including
metastatic UC (mUC); urothelial bladder cancer, muscle-invasive bladder cancer
(MIBC), and non-muscle-invasive
bladder cancer (NMIBC); myeloproliferative neoplasms (MPN), including chronic
myeloid leukemia (CML),
polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia
(ET), chronic neutrophilic
leukemia (CNL); myelodysplastic syndromes (MDS); myeloproliferative neoplasms
(MDS/MPN); acute
lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); chronic
lymphocytic leukemia (CLL); multiple
myeloma; Hodgkin's lymphoma; non-Hodgkin's lymphoma (NHL), including diffuse
large B cell lymphoma (DLBCL),
follicular lymphoma, hairy cell leukemia; mantle cell lymphoma; monoclonal
gammopathy of undetermined
significance (MGUS); plasma cell myeloma; waldenstrOm macroglobulinemia; and
mature T and NK neoplasms.
In certain embodiments, a cancer which may be treated in accordance with the
present disclosure includes one
having high tumor mutational burden.
[311] Affirmative identification of cancer as "TGFI31-positive" is not
required for carrying out the therapeutic
methods described herein but is encompassed in some embodiments. Typically,
certain cancer types are known
to be or suspected, based on credible evidence, to be associated with TG931
signaling.
[312] Cancers may be localized (e.g., solid tumors) or systemic. In the
context of the present disclosure, the term
"localized" (as in "localized tumor") refers to anatomically isolated or
isolatable abnormalities/lesions, such as solid
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malignancies, as opposed to systemic disease (e.g., so-called liquid tumors or
blood cancers). Certain cancers,
such as certain types of leukemia (e.g., myelofibrosis) and multiple myeloma,
for example, may have both a
localized component (for instance the bone marrow) and a systemic component
(for instance circulating blood
cells) to the disease. In some embodiments, cancers may be systemic, such as
hematological malignancies.
Cancers that may be treated according to the present disclosure are TGFI31-
positive and include but are not limited
to, all types of lymphomas/leukemias, carcinomas arid sarcomas, such as those
cancers or tumors found in the
anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum,
endometrium, esophagus, eye, gallbladder, head
and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries,
pancreas, penis, prostate, skin, small
intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus. In
some embodiments, the cancer may
be an advanced cancer, such as a locally advanced solid tumor and metastatic
cancer.
[313] In some embodiments, the cancer may be a cancer having elevated 1GF81
levels associated with reactive
oxygen species (ROS). In some embodiments, the cancer may be a cancer having
elevated ROS levels and
expressing high levels of TGF81.
[314] Antibodies or antigen-binding fragments thereof encompassed by the
present disclosure may be used in
the treatment of cancer, including, without limitation: myelofibrosis,
melanoma, adjuvant melanoma, renal cell
carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC,
collecting duct RCC, or unclassified
RCC, bladder cancer, colorectal cancer (CRC) (e.g., microsatellite-stable CRC,
mismatch repair deficient colorectal
cancer), colon cancer, rectal cancer, anal cancer, breast cancer, triple-
negative breast cancer (TNBC), HER2-
negative breast cancer, HER2-positive breast cancer, BRCA-mutated breast
cancer, hematologic malignancies,
non-small cell carcinoma, non-small cell lung cancer/carcinoma (NSCLC), small
cell lung cancer/carcinoma
(SCLC), extensive-stage small cell lung cancer (ES-SCLC), lymphoma (classical
Hodgkin's and non-Hodgkin's),
primary mediastinal large B-cell lymphoma (PMBCL), 1-cell lymphoma, diffuse
large B-cell lymphoma, histiocytic
sarcoma, follicular dendritic cell sarcoma, interdigitating dendritic cell
sarcoma, myeloma, chronic lymphocytic
leukemia (CLL), acute myeloid leukemia (AML), small lymphocytic lymphoma
(SLL), head and neck cancer (e.g.,
head and neck squamous cell cancer), urothelial cancer e.g., metastatic
urothelial carcinoma), merkel cell
carcinoma (e.g., metastatic merkel cell carcinoma), merkel cell skin cancer,
cancer with high microsatellite
instability (MSI-H), cancer with mismatch repair deficiency (dMMR), tumor
mutation burden high cancer,
mesothelioma (e.g., malignant pleural mesothelioma), gastric cancer,
gastroesophageal junction cancer (GEJ),
gastric adenocarcinoma, neuroendocrine tumors, gastrointestinal stromal tumors
(GIST), gastric cardia
adenocarcinoma, renal cancer, biliary cancer, cholangiocarcinoma, pancreatic
cancer, prostate cancer,
adenocarcinoma, squamous cell carcinoma, non-squamous cell carcinoma,
cutaneous squamous cell carcinoma
(CSCC), ovarian cancer, endometrial cancer, fallopian tube cancer, cervical
cancer, peritoneal cancer, stomach
cancer, brain cancers, malignant glioma, glioblastoma, gliosarcoma,
neuroblastoma, thyroid cancer, adrenocortical
carcinoma, oral intra-epithelial neoplasia, esophageal cancer, nasal cavity
and paranasal sinus squamous cell
carcinoma, nasopharynx carcinoma, salivary gland cancer, liver cancer, basal
cell carcinoma; and hepatocellular
cancer (HCC). However, any cancer (e.g., patients with such cancer) in which
TGF81 is overexpressed or is at
least a predominant isoform, as determined by, for example biopsy, may be
treated with an isoform-selective
inhibitor of TGF61 in accordance with the present disclosure.
[315] In cancer, TGFI3 (e.g., TGFI31) may be either growth promoting or growth
inhibitory. As an example, in
pancreatic cancers, SMAD4 wild type tumors may experience inhibited growth in
response to TGF6, but as the
disease progresses, constitutively activated type ll receptor is typically
present. Additionally, there are SMAD4-
null pancreatic cancers. In some embodiments, antibodies, antigen binding
portions thereof, and/or compositions
of the present disclosure are designed to selectively target components of
TGF5 signaling pathways that function
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uniquely in one or more forms of cancer. Leukemias, or cancers of the blood or
bone marrow that are characterized
by an abnormal proliferation of white blood cells, i.e., leukocytes, can be
divided into four major classifications
including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia
(CLL), acute myelogenous leukemia
or acute myeloid leukemia (AML) (AML with translocations between chromosome 10
and 11 [t(10, 11)],
chromosome 8 and 21 [t(8;21)], chromosome 15 and 17 [t(15;17)1, and inversions
in chromosome 16 [inv(16)];
AML with multilineage dysplasia, which includes patients who have had a prior
myelodysplastic syndrome (MDS)
or myeloproliferative disease that transforms into AML; AML and
myelodysplastic syndrome (MDS), therapy-
related, which category includes patients who have had prior chemotherapy
and/or radiation and subsequently
develop AML or MDS; d) AML not otherwise categorized, which includes subtypes
of AML that do not fall into the
above categories; and e) acute leukemias of ambiguous lineage, which occur
when the leukemic cells cannot be
classified as either myeloid or lymphoid cells, or where both types of cells
are present); and chronic myelogenous
leukemia (CML).
[316] In some embodiments, any one of the above referenced TGFp1-positive
cancer may also be TGFp3-
positive. In some embodiments, tumors that are both TGFpl-positive and TGFp3-
positive may be TGFp1/TG933
co-dominant. In some embodiments, such cancer is carcinoma comprising a solid
tumor. In some embodiments,
such tumors are breast carcinoma. In some embodiments, the breast carcinoma
may be of triple-negative
genotype (triple-negative breast cancer). In some embodiments, subjects with
TGFp1-positive cancer have
elevated levels of MDSCs. For example, such tumors may comprise MDSCs
recruited to the tumor site resulting
in an increased number of MDSC infiltrates. In some embodiments, elevated
levels of MDSCs may be detected in
the blood (i.e., circulating MDSCs). In some embodiments, subjects with breast
cancer show elevated levels of C-
Reactive Protein (CRP), an inflammatory marker associated with recurrence and
poor prognosis. In some
embodiments, subjects with breast cancer show elevated levels of IL-6.
[317] The TGFp inhibitors of the disclosure may be used to treat patients
suffering from chronic myeloid leukemia,
which is a stem cell disease, in which the BCR/ABL oncoprotein is considered
essential for abnormal growth and
accumulation of neoplastic cells. lmatinib is an approved therapy to treat
this condition; however, a significant
fraction of myeloid leukemia patients show Imatinib-resistance. TGFp
inhibition achieved by the inhibitor such as
those described herein may potentiate repopulation/expansion to counter
BCR/ABL-driven abnormal growth and
accumulation of neoplastic cells, thereby providing clinical benefit.
[318] TGFp inhibitors such as those described herein may be used to treat
multiple myeloma. Multiple myeloma
is a cancer of B lymphocytes (e.g., plasma cells, plasmablasts, memory B
cells) that develops and expands in the
bone marrow, causing destructive bone lesions (i.e., osteolytic lesion).
Typically, the disease manifests enhanced
osteoclastic bone resorption, suppressed osteoblast differentiation (e.g.,
differentiation arrest) and impaired bone
formation, characterized in part, by osteolytic lesions, osteopenia,
osteoporosis, hypercalcemia, as well as
plasmacytoma, thrombocytopenia, neutropenia and neuropathy. The TGFp inhibitor
therapy described herein may
be effective to ameliorate one or more such clinical manifestations or
symptoms in patients. The TGFP1 inhibitor
may be administered to patients who receive additional therapy or therapies to
treat multiple myeloma, including
those listed elsewhere herein. In some embodiments, multiple myeloma may be
treated with a TGFp inhibitor such
as an isoform-specific context-independent inhibitor, e.g., Ab6, in
combination with a myostatin inhibitor (such as
an antibody disclosed in WO 2017/049011, e.g., apitegromab, also known as SRK-
015) or an IL-6 inhibitor. In
some embodiments, the TGFp inhibitor may be used in conjunction with
traditional multiple myeloma therapies,
such as bortezomib, lenalidomide, carfilzomib, pomalidomide, thalidomide,
doxorubicin, corticosteroids (e.g.,
dexamethasone and prednisone), chemotherapy (e.g., melphalan), radiation
therapy (including radiotherapeutic
agents), stem cell transplantation, plitidepsin, elotuzumab, lxazomib,
masitinib, and/or panobinostat.
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[319] The types of carcinomas which may be treated by the methods of the
present disclosure include, but are
not limited to, papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor,
teratoma,
adenoma/adenocarcinoma, melanoma, fibroma, liporna, leiomyoma, rhabdomyoma,
mesothelioma, angioma,
osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small
cell carcinoma, large cell
undifferentiated carcinomas, basal cell carcinoma and sinonasal
undifferentiated carcinoma.
[320] The types of sarcomas include, but are not limited to, soft tissue
sarcoma such as alveolar soft part sarcoma,
angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round
cell tumor, extraskeletal
chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma,
hemangiosarcoma, Kaposi's
sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma,
malignant fibrous histiocytoma,
neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor,
Ewing's sarcoma (primitive
neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma,
osteosarcoma, and
chondrosarcoma.
[321] TGF8 inhibitors such as those described herein may be suited for
treating malignancies involving cells of
neural crest origin. Cancers of the neural crest lineage (i.e., neural crest-
derived tumors) include, but are not limited
to: melanoma (cancer of melanocytes), neuroblastoma (cancer of sympathoadrenal
precursors), ganglioneuroma
(cancer of peripheral nervous system ganglia), medullary thyroid carcinoma
(cancer of thyroid C cells),
pheochromocytoma (cancer of chromaffin cells of the adrenal medulla), and
MPNST (cancer of Schwann cells). In
some embodiments, antibodies and methods of the disclosure may be used to
treat one or more types of cancer
or cancer-related conditions that may include, but are not limited to, colon
cancer, renal cancer, breast cancer,
malignant melanoma, urothelial carcinoma, and glioblastoma (Schlingensiepen et
al., 2008. Cancer Res. 177: 137-
50; Ouhtit et al., 2013. J Cancer. 4 (7). 566-572.
Immunological Characteristics
[322] Under normal conditions, regulatory T cells (Tregs) represent a small
subset of the overall CD4-positive
lymphocyte population and play key roles for maintaining immune system in
homeostasis. In nearly all cancers,
however, the number of Tregs is markedly increased. While Tregs play an
important role in dampening immune
responses in healthy individuals, an elevated number of Tregs in cancer has
been associated with poor prognosis.
Elevated Tregs in cancer may dampen the host's anti-cancer immunity and may
contribute to tumor progression,
metastasis, tumor recurrence and/or treatment resistance. For example, human
ovarian cancer ascites are
infiltrated with Foxp3+ GARP+ Tregs (Downs-Canner et al., Nat Commun. 2017, 8:
14649). Similarly, Tregs
positively correlated with a more immunosuppressive and more aggressive
phenotype in advanced hepatocellular
carcinoma (Kalathil et al., Cancer Res. 2013, 73(8): 2435-44). Tregs can
suppress the proliferation of effector T
cells. In addition, Tregs exert contact-dependent inhibition of immune cells
(e.g., naïve CD4+ T cells) through the
production of TGF61. To combat a tumor, therefore, it is advantageous to
inhibit Tregs so sufficient effector T cells
can be available to exert anti-tumor effects.
[323] Increasing lines of evidence suggest the role of macrophages in
tumor/cancer progression. The present
disclosure encompasses the notion that this is in part mediated by TGFI3
activation, especially TGF61 activation,
in the tumor microenvironment. Bone marrow-derived monocytes (e.g., CD11b+)
are recruited to tumor sites in
response to tumor-derived cytokines/chemokines (such as CCL2, CCL3 and CCL4),
where monocytes undergo
differentiation and polarization to acquire pro-cancer phenotype (e.g., M2-
biased or M2-like macrophages, TAMs).
As previously demonstrated (WO 2018/129329), monocytes isolated from human
PBMCs can be induced to
polarize into different subtypes of macrophages, e.g., M1 (pro-fibrotic, anti-
cancer) and M2 (pro-cancer). A majority
of TAMs in many tumors are M2-biased. Among the M2-like macrophages, M2c and
M2d subtypes, but not Ml,
are found to express elevated LRRC33 on the cell surface. Moreover,
macrophages can be further skewed or
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activated by certain cytokine exposure, such as M-CSF, resulting in a marked
increase in LRRC33 expression,
which coincides with TGFp1 expression. Increased levels of circulating M-CSF
(i.e., serum M-CSF concentrations)
in patients with myeloproliferative disease (e.g., myelofibrosis) have also
been observed. Generally, tumors with
high macrophage (TAM) and/or MDSC infiltrate are associated with poor
prognosis. Similarly, elevated levels of
M-CSF are also indicative of poor prognosis. Thus, in some embodiments, the
TGFI3 inhibitors such as those
encompassed herein can be used in the treatment of cancer that is
characterized by elevated levels of pro-cancer
macrophages and/or MDSCs. In some embodiments, the TGF6 inhibitors such as
those encompassed herein can
be used in the treatment of cancer that is characterized by elevated levels of
MDSCs regardless of levels of other
macrophages. The LRRC33-arm of the inhibitors may at least in part mediate its
inhibitory effects against disease-
associated immunosuppressive myeloid cells, e.g., M2-macrophages and MDSCs.
[324] High prevalence of tumor-associated M2-like macrophages is recapitulated
in murine syngeneic tumor
models, such as those described previously by the Applicant, see data
disclosed in PCT/US2019/041373. In MBT-
2 tumors, for example, nearly 40% of CD45-positive cells isolated from an
established tumor are M2 macrophages.
This is reduced by half in animals treated with a combination of an isoform-
selective TGF131 and anti-PD-1. By
comparison, no significant change in the number of tumor-associated M1
macrophages is observed in the same
animals. Like M2 macrophages, tumor-associated MDSCs are also elevated in
established tumors (about 10-12%
of CD45+ cells) and are markedly reduced (to negligible levels) by inhibiting
both PD-1 and TGF[31 in the treated
animals. As Applicant previously disclosed, a majority of tumor-infiltrating
M2 macrophages and MDSCs express
cell-surface LRRC33 and/or LRRC33-proTGF[31 complex. Interestingly, cell-
surface expression of LRRC33 (or
LRRC33-proTGF[31 complex) appears to be highly regulated. The TGF6 inhibitors
described herein, e.g., Ab6, are
capable of becoming rapidly internalized in cells expressing LRRC33 and
proTGF[31 , and the rate of internalization
achieved with the TGFp inhibitor is significantly higher than that with a
reference antibody that recognizes cell-
surface LRRC33. Similar results are obtained from primary human macrophages.
These observations show that
Ab6 can promote internalization upon binding to its target, LRRC33-proTGFI31,
thereby removing the LRRC33-
containing complexes from the cell surface. Thus, target engagement by a TGF[3
inhibitor of the present disclosure,
e.g., Ab6 may induce antibody-dependent downregulation of the target protein
(e.g., cell-associated proTG931
complexes). At the disease loci, this may reduce the availability of
activatable latent LRRC33-proTGF[31 levels.
Therefore, the TGFI3 inhibitors of the disclosure may inhibit the LRRC33 arm
of TGF[31 via dual mechanisms of
action: i) blocking the release of mature growth factor from the latent
complex; and, ii) removing LRRC33-proTGF[31
complexes from cell-surface via internalization. In the tumor
microenvironment, the antibodies may target cell-
associated latent proTGF[31 complexes, augmenting the inhibitory effects on
the target cells, such as M2
macrophages (e.g., TAMs), MDSCs, and Tregs. Phenotypically, these are
immunosuppressive cells, contributing
to the immunosuppressive tumor microenvironment, which is at least in part
mediated by the TGFp1 pathway.
Given that many tumors are enriched with these cells, the antibodies that are
capable of targeting multiple arms of
TGFp1 function, such as those described herein, should provide a particular
functional advantage.
[325] Many human cancers are known to cause elevated levels of MDSCs in
patients, as compared to healthy
control (reviewed, for example, in Elliott et al., (2017) "Human tumor-
infiltrating myeloid cells: phenotypic and
functional diversity" Frontiers in Immunology, Vol. 8, Article 86). These
human cancers include but are not limited
to: bladder cancer, colorectal cancer, prostate cancer, breast cancer,
glioblastoma, hepatocellular carcinoma, head
and neck squamous cell carcinoma, lung cancer, melanoma, NSCL, ovarian cancer,
pancreatic cancer, and renal
cell carcinoma. Elevated levels of MDSCs may be detected in biological samples
such as peripheral blood
mononuclear cell (PBMC) and tissue samples (e.g., tumor biopsy). For example,
frequency of or changes in the
number of MDSCs may be measured as: percent (%) of total PBMCs, percent (%) of
CD14+ cells, percent (%) of
CD45+ cells; percent (%) of mononuclear cells, percent (%) of total cells,
percent (%) of CD11b+ cells, percent (%)
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of monocytes, percent (%) of non-lymphocytic MNCs, percent (%) of KLA-DR
cells, using suitable cell surface
markers (phenotype).
[326] On the other hand, macrophage infiltration into a tumor may also signify
effectiveness of a therapy. As
exemplified herein, tumors effectively penetrated by effector T cells (e.g.,
CD8+ T cells) following the treatment
with a combination of a checkpoint inhibitor and a context-independent TGFp1
inhibitor. Intratumoral effector T
cells may lead to recruitment of phagocytic monocytes/macrophages that clean
up cell debris.
[327] It was observed that the combination of anti-PD-1 and a TGFp inhibitor
resulted in robust CD8 T cell
influx/expansion throughout the tumor, as compared to anti-PD-1 treatment
alone. Correspondingly, robust
increase in CD8 effector genes may be achieved by the combination treatment.
Thus, the TG931 inhibitors of the
present disclosure may be used to promote effector 1-cell infiltration into
tumors.
[328] In addition, extensive infiltration/expansion of the tumor by F4/80-
positive macrophages is observed. This
may be indicative of M1 (anti-tumor) macrophages clearing cancer cell debris
generated by cytotoxic cells and is
presumably a direct consequence of TGFp1 inhibition.
As previously described in further detail in
PCT/US2019/041373, these tumor-infiltrating macrophages were identified
predominantly as non-M2
macrophages for their lack of CD163 expression, indicating that circulating
monocytes are recruited to the tumor
site upon checkpoint inhibitor and TGF31 inhibitor treatment and differentiate
into M1 macrophages, and this
observation is accompanied by a marked influx of CD8+ T cells into the tumor
site. Thus, the TGF31 inhibitors of
the present disclosure may be used to increase non-M2 macrophages associated
with tumor.
[329] Recently, checkpoint blockade therapy (CBT) has become a standard of
care for treating a number of cancer
types. Despite the profound advances in cancer immunotherapy, primary
resistance to CBT remains a major unmet
need for patients; a majority of patients' cancers still fail to respond to PD-
(L)1 inhibition. Retrospective analysis
of urothelial cancer and melanoma tumors has recently implicated TGFp
activation as a potential driver of primary
resistance, very likely via multiple mechanisms including exclusion of
cytotoxic T cells from the tumor as well as
their expansion within the tumor microenvironment (immune exclusion). These
observations and subsequent
preclinical validation have pointed to TGFp pathway inhibition as a promising
avenue for overcoming primary
resistance to CBT. However, therapeutic targeting of the TGFp pathway has been
hindered by dose-limiting
preclinical cardiotoxicities, most likely due to inhibition of signaling from
one or more TGFP isoforms.
[330] Many tumors lack of primary response to CBT. In this scenario, CD8+ T
cells are commonly excluded from
the tumor parenchyma, suggesting that tumors may co-opt the immunomodulatory
functions of TGFP signaling to
generate an immunosuppressive microenvironment. These insights from
retrospective clinical tumor sample
analyses provided the rationale for investigating the role of TGFp signaling
in primary resistance to CBT.
[331] With respect to TGFP and responses to CBT, herein we observe the
prevalent expression of 1GFP1 in many
human tumors, suggesting that this family member may be the key driver of this
pathway's contribution to primary
resistance.
[332] Increasing evidence suggests that TGFp may be a primary player in
creating and/or maintaining
immunosuppression in disease tissues, including the immune-excluded tumor
environment. Therefore, TGFp
inhibition may unblock the immunosuppression and enable effector T cells
(particularly cytotoxic CD8+ T cells) to
access and kill target cancer cells. In addition to tumor infiltration, TGFP
inhibition may also promote CD8+ T cell
expansion. Such expansion may occur in the lymph nodes and/or in the tumor
(intratumorally). While the exact
mechanism underlining this process has yet to be elucidated, it is
contemplated that immunosuppression is at least
in part mediated by immune cell-associated TGFf31 activation involving
regulatory T cells and activated
macrophages. It has been reported that TGFP directly promotes Foxp3 expression
in CD4+ T cells, thereby
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converting them into a regulatory (immunosuppressive) phenotype (i.e., Treg).
Moreover, Tregs suppress effector
T cell proliferation, thereby reducing immune responses. This process is shown
to be TGF131-dependent and likely
involves GARP-associated TGF(31 signaling. Observations in both humans and
animal models have indicated that
an increase in Tregs in TME is associated with poor prognosis in multiple
types of cancer. In addition, Applicant
has previously shown that M2-polarized macrophages exposed to tumor-derived
factors such as M-CSF
dramatically upregulate cell-surface expression of LRRC33, which is a
presenting molecule for TGF31 (see, for
example: PCT/U32018/031759). These so-called tumor-associated macrophages (or
TAMs) are thought to
contribute to the observed TGFp1-dependent immunosuppression in TMEs and
promote tumor growth.
[333] A number of solid tumors are characterized by having tumor stroma
enriched with myofibroblasts or
myofibroblast-like cells. These cells produce collagenous matrix that
surrounds or encases the tumor (such as
desmoplasia), which at least in part may be caused by overactive TGFI31
signaling. It is contemplated that the
TGF131 activation is mediated via ECM-associated presenting molecules, e.g.,
LTBP1 and LTBP3 in the tumor
stroma.
[334] Selective inhibition of TGFP activation, such as TGFP1 inhibition, may
be sufficient to overcome primary
resistance to CBT. By targeting the prodomain of latent TGFp1, an isoform-
selective inhibitor of TGFp1 may
achieve isoform specificity and inhibit latent TGF131 activation.
[335] Selective inhibition of the TGFp pathway, such as the TGF(31 pathway,
may result in significantly improved
preclinical safety versus broad inhibition of all isoform activity.
Pleiotropic effects associated with broad TGFp
pathway inhibition have hindered therapeutic targeting of the TGFp pathway.
Most experimental therapeutics to
date (e.g., galunisertib, LY3200882, fresolimumab) lack selectivity for a
single TGFP isoform, potentially
contributing to the dose-limiting toxicities observed in nonclinical and
clinical studies. Genetic data from knockout
mice and human loss-of-function mutations in the TGF(32 or TGFp3 genes suggest
that the cardiac toxicities
observed with nonspecific TGFp inhibitors may be due to inhibition of TGFp2 or
1G933. The present disclosure
teaches that selective inhibition of TGF131 activation with such an antibody
has an improved safety profile and is
sufficient to elicit robust antitumor responses when combined with PD-1
blockade, enabling the evaluation of the
TGF131 inhibitor efficacy at clinically tractable dose levels.
[336] The preclinical studies and results presented herein and in
PCT/US2019/041373 demonstrate that
combination treatment with a TGF131 inhibitor (e.g., Ab6) and a checkpoint
inhibitor may have profound effects on
the intratumoral immune contexture (e.g., increased levels of tumor-associated
CD8+ T cells). These may include
an unexpected enrichment of Treg cells by the combination treatment with anti-
PD-1/TG931 inhibitor.
[337] In addition to the expected and observed impact on the disposition of
cytotoxic T cells within tumors, the
TGFp inhibitor/anti-PD-1 combination treatment may also beneficially impact
the immunosuppressive myeloid
compartment. Therefore, a therapeutic strategy that includes targeting of
these important immunosuppressive cell
types may have a greater effect than targeting a single immunosuppressive cell
type (i.e., only Treg cells) in the
tumor microenvironment. Thus, the TG931 inhibitors of the present disclosure
may be used to reduce tumor-
associated immunosuppressive cells, such as M2 macrophages and MDSCs.
[338] Applicant's data also demonstrate that highly specific inhibition of
TGFp1 activation may enable the host
immune system to overcome a key mechanism of primary resistance to checkpoint
blockade therapy, while
avoiding the previously recognized toxicities of broader TGFp inhibition that
have been a key limitation for clinical
application.
[339] Accordingly, TGFr3 inhibitors such as selective TGFpl inhibitors may be
used to counter primary resistance
to CBT, thereby rendering the tumor/cancer more susceptible to the CBT. Such
effects may be applicable to
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treating a wide spectrum of malignancy types, where the cancer/tumor is TG931-
positive. In some embodiments,
such tumor/cancer may further express additional isoform, such as TGFI33. Non-
limiting examples of the latter
may include certain types of carcinoma, such as breast cancer.
[340] Accordingly, the disclosure provides, in some embodiments, selection
criteria for identifying or selecting a
patient or patient populations/sub-populations for which the TGFI31 inhibitors
are likely to achieve clinical benefit.
In some embodiments, suitable phenotypes of human tumors include: i) a
subset(s) are shown to be responsive to
CBT (e.g., PD-(L)1 axis blockade); ii) evidence of immune exclusion; and/or,
iii) evidence of TGFB1 expression
and/or TGFp signaling. Various cancer types fit the profile, including, for
example, melanoma and bladder cancer.
[341] As mentioned above, TGF8 inhibitors such as those described herein may
be used in the treatment of
melanoma. The types of melanoma that may be treated with such inhibitors
include, but are not limited to, Lentigo
maligna, Lentigo maligna melanoma, Superficial spreading melanoma, Acral
lentiginous melanoma, Mucosa!
melanoma, Nodular melanoma, Polypoid melanoma, and Desmoplastic melanoma. In
some embodiments, the
melanoma is a metastatic melanoma. In some embodiments, the melanoma is a
cutaneous melanoma.
[342] More recently, immune checkpoint inhibitors have been used to
effectively treat advanced melanoma
patients. In particular, anti-programmed death (PD)-1 antibodies (e.g.,
nivolumab and pembrolizumab) have now
become the standard of care for certain types of cancer such as advanced
melanoma, which have demonstrated
significant activity and durable response with a manageable toxicity profile.
However, effective clinical application
of PD-1 antagonists is encumbered by a high rate of innate resistance (-60-
70%) (see Hugo et al., (2016) Cell 165:
35-44), illustrating that ongoing challenges continue to include the questions
of patient selection and predictors of
response and resistance as well as optimizing combination strategies (Perrot
et al., (2013) Ann Dermatol 25(2):
135-144). Moreover, studies have suggested that approximately 25% of melanoma
patients who initially responded
to an anti-PD-1 therapy eventually developed acquired resistance (Ribas et
al., (2016) JAMA 315: 1600-9).
[343] The number of tumor-infiltrating CD8+ T cells expressing PD-1 and/or
CTLA-4 appears to be a key indicator
of success with checkpoint inhibition, and both PD-1 and CTLA-4 blockade may
increase the infiltrating T cells. In
patients with higher presence of tumor-associated macrophages, however, anti-
cancer effects of the CD8 cells
may be suppressed.
[344] It is contemplated that LRRC33-expressing cells, such as myeloid cells,
including myeloid precursors,
MDSCs and TAMs, may create or support an immunosuppressive environment (such
as TME and myelofibrotic
bone marrow) by inhibiting T cells (e.g., T cell depletion), such as CD4
and/or CD8 T cells, which may at least in
part underline the observed anti-PD-1 resistance in certain patient
populations. Indeed, evidence suggests that
resistance to anti-PD-1 monotherapy was marked by failure to accumulate CD8+
cytotoxic T cells and reduced
Teff/Treg ratio. Notably, the present inventors have recognized that there is
a bifurcation among certain cancer
patients, such as a melanoma patient population, with respect to LRRC33
expression levels: one group exhibits
high LRRC33 expression (LRRC33"), while the other group exhibits relatively
low LRRC33 expression
(LRRC33I0w). Thus, the disclosure includes the notion that the LRRC33huh
patient population may represent those
who are poorly responsive to or resistant to immune checkpoint inhibitor
therapy. Accordingly, agents that inhibit
LRRC33, such as those described herein, may be particularly beneficial for the
treatment of cancer, such as
melanoma, lymphoma, and myeloproliferative disorders, that is resistant to
checkpoint inhibitor therapy (e.g., anti-
PD-1).
[345] In some embodiments, a cancer/tumor is intrinsically resistant to or
unresponsive to an immune checkpoint
inhibitor (e.g., primary resistance). Without intending to be bound by
particular theory, the inventors of the present
disclosure contemplate that this may be at least partly due to upregulation of
TGF81 signaling pathways, which
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may create an immunosuppressive microenvironment where checkpoint inhibitors
fail to exert their effects. TGFpl
inhibition may render such cancer more responsive to checkpoint inhibitor
therapy. Non-limiting examples of
cancer types which may benefit from a combination of an immune checkpoint
inhibitor and a TGFp1 inhibitor
include: myelofibrosis, melanoma, renal cell carcinoma, bladder cancer, colon
cancer, hematologic malignancies,
non-small cell carcinoma, non-small cell lung cancer/carcinoma (NSCLC),
lymphoma (classical Hodgkin's and non-
Hodgkin's), head and neck cancer, urothelial cancer e.g., metastatic
urothelial carcinoma), cancer with high
microsatellite instability, cancer with mismatch repair deficiency, gastric
cancer, renal cancer, and hepatocellular
cancer. However, any cancer (e.g., patients with such cancer) in which TGFp1
is overexpressed, is co-expressed
with TGFP3, or is the dominant isoform over TGFP2/3, as determined by, for
example biopsy, may be treated with
a TGFI3 inhibitor in accordance with the present disclosure.
[346] In some embodiments, a cancer/tumor becomes resistant over time. This
phenomenon is referred to as
acquired resistance. Like primary resistance, in some embodiments, acquired
resistance is at least in part
mediated by TGFp1-dependent pathways. TGFp inhibitors described herein may be
effective in restoring anti-
cancer immunity in these cases. The TGFp inhibitors of the present disclosure
may be used to reduce recurrence
of tumor. The TGFp inhibitors of the present disclosure may be used to enhance
durability of cancer therapy such
as CBT. The term "durability" used in the context of therapies refers to the
time between clinical effects (e.g., tumor
control) and tumor re-growth (e.g., recurrence). Presumably, durability and
recurrence may correlate with
secondary or acquired resistance, where the therapy to which the patient
initially responded stops working. Thus,
the TGFp inhibitors of the present disclosure may be used to increase the
duration of time the cancer therapy
remains effective. The TGFp inhibitors of the present disclosure may be used
to reduce the probability of
developing acquired resistance among the responders of the therapy.
The TGFp inhibitors of the present
disclosure may be used to enhance progression-free survival in patients. In
some embodiments, the TGFp
inhibitors described herein may be used to improve disease-free survival time
in patients. In some embodiments,
the TGFp inhibitors of the present disclosure may be effective for improving
patient-reported outcomes, reduced
complications, faster time to treatment completion, more durable treatment,
longer time between retreatment, etc.
In some embodiments, the TGFp inhibitors of the present disclosure may be used
to improve overall survival in
patients.
[347] In some embodiments, the TGFp inhibitors of the present disclosure may
be used to improve rates or ratios
of complete verses partial responses among the responders of a cancer therapy.
Typically, even in cancer types
where response rates to a cancer therapy (such as CBT) are relatively high
(e.g., a 35%), CR rates are quite low.
The TGFp inhibitors of the present disclosure are therefore used to increase
the fraction of complete responders
within the responder population.
[348] In addition, the TGFp inhibitor may be also effective to enhance or
augment the degree of partial response
among partial responders.
[349] In some embodiments, clinical endpoints for the TGFp inhibitors
described herein include those described
in the 2018 Food and Drug Administration Guidelines for Clinical Trial
Endpoints for the Approval of Cancer Drugs
and Biologics, the content of which is incorporated herein in its entirety.
[350] In some embodiments, combination therapy comprising an immune checkpoint
inhibitor and an LRRC33
inhibitor (such as those described herein) may be used with the methods
disclosed herein and may be effective to
treat such cancer. In addition, high LRRC33-positive cell infiltrate in
tumors, or otherwise sites/tissues with
abnormal cell proliferation, may serve as a biomarker for host
immunosuppression and immune checkpoint
resistance. Similarly, effector T cells may be precluded from the
imrnunosuppressive niche which limits the body's
ability to combat cancer.
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[351] As previously disclosed in PCT/US2019/041373, Tregs that express CARP-
presented TGF61 suppress
effector T cell proliferation. Together, TGFI31 is likely a key driver in the
generation and maintenance of an immune
inhibitory disease microenvironrnent (such as TME), and multiple TGF61
presentation contexts are relevant for
tumors. In some embodiments, the combination therapy may achieve more
favorable Teff/Treg ratios.
[352] In some embodiments, the antibodies, or antigen binding portions
thereof, that specifically bind a GARP-
TGF61 complex, a LTBP1-TGF61 complex, a LTBP3-TGFp1 complex, and/or a LRRC33-
TGF61 complex, as
described herein, may be used in methods for treating cancer in a subject in
need thereof, said method comprising
administering the antibody, or antigen binding portion thereof, to the subject
such that the cancer is treated. In
certain embodiments, the cancer is colon cancer. In certain embodiments, the
cancer is melanoma. In certain
embodiments, the cancer is bladder cancer. In certain embodiments, the cancer
is head and neck cancer. In
certain embodiments, the cancer is lung cancer.
[353] In some embodiments, the antibodies, or antigen binding portions
thereof, that specifically bind a GARP-
TGF61 complex, a LTBP1-TGF61 complex, a LTBP3-TGF61 complex, and/or a LRRC33-
TGFI31 complex, as
described herein, may be used in methods for treating solid tumors. In some
embodiments, solid tumors may be
desmoplastic tumors, which are typically dense and hard for therapeutic
molecules to penetrate. By targeting the
ECM component of such tumors, such antibodies may "loosen" the dense tumor
tissue to disintegrate, facilitating
therapeutic access to exert its anti-cancer effects. Thus, additional
therapeutics, such as any known anti-tumor
drugs, may be used in combination.
[354] Additionally or alternatively, isoform-specific, context-independent
antibodies for fragments thereof that are
capable of inhibiting TGF61 activation, such as those disclosed herein, may be
used in conjunction with the
chimeric antigen receptor 1-cell ("CAR-T") technology as cell-based
immunotherapy, such as cancer
immunotherapy for combatting cancer.
[355] In some embodiments, the antibodies, or antigen binding portions
thereof, that specifically bind a GARP-
TGF61 complex, a LTBP1-TGF61 complex, a LTBP3-TGF61 complex, and/or a LRRC33-
TGF61 complex, as
described herein, may be used in methods for inhibiting or decreasing solid
tumor growth in a subject having a
solid tumor, said method comprising administering the antibody, or antigen
binding portion thereof, to the subject
such that the solid tumor growth is inhibited or decreased. In certain
embodiments, the solid tumor is a colon
carcinoma tumor. In some embodiments, the antibodies, or antigen binding
portions thereof useful for treating a
cancer is an isoform-specific, context-independent inhibitor of TGF61
activation. In some embodiments, such
antibodies target a GARP-TGF61 complex, a LTBP1-TGF61 complex, a LTBP3-TGF61
complex, and a LRRC33-
TGF61 complex. In some embodiments, such antibodies target a GARP-TGF61
complex, a LTBP1-TGF61
complex, and a LTBP3-TGF61 complex. In some embodiments, such antibodies
target a LTBP1-TGF61 complex,
a LTBP3-TGF61 complex, and a LRRC33-TGF61 complex. In some embodiments, such
antibodies target a
GARP-TGF61 complex and a LRRC33-TGF61 complex.
[356] The disclosure includes the use of TGF6 inhibitors, such as context-
independent, isoform-specific inhibitors
of TGF61, in the treatment of cancer comprising a solid tumor in a subject. In
some embodiments, such TGFp
inhibitors may inhibit the activation of TGF61. In some embodiments, such TGF6
inhibitors comprise an antibody
or antigen-binding portion thereof that binds a proTGF61 complex. The binding
can occur when the complex is
associated with any one of the presenting molecules, e.g., LTBP1, LTBP3, CARP
or LRRC33, thereby inhibiting
release of mature TGF61 growth factor from the complex. In some embodiments,
the solid tumor is characterized
by having stroma enriched with CD8+ T cells making direct contact with CAFs
and collagen fibers. Such a tumor
may create an immuno-suppressive environment that prevents anti-tumor immune
cells (e.g., effector T cells) from
effectively infiltrating the tumor, limiting the body's ability to fight
cancer. Instead, such cells may accumulate within
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or near the tumor stroma. These features may render such tumors poorly
responsive to an immune checkpoint
inhibitor therapy. As discussed in more detail below, TGFI31 inhibitors
disclosed herein may unblock the
suppression so as to allow effector cells to reach and kill cancer cells, for
example, used in conjunction with an
immune checkpoint inhibitor.
[357] TGFI3, especially TGFI31, is contemplated to play multifaceted roles in
a tumor microenvironment, including
tumor growth, host immune suppression, malignant cell proliferation,
vascularity, angiogenesis, migration, invasion,
metastasis, and chemo-resistance. Each "context" of TGF(31 presentation in the
environment may therefore
participate in the regulation (or dysregulation) of disease progression. For
example, the GARP axis is particularly
important in Treg response that regulates effector T cell response for
mediating host immune response to combat
cancer cells. The LTBP1/3 axis may regulate the ECM, including the stroma,
where cancer-associated fibroblasts
(CAFs) play a role in the pathogenesis and progression of cancer. The LRRC33
axis may play a crucial role in
recruitment of circulating monocytes to the tumor microenvironment, subsequent
differentiation into tumor-
associated macrophages (TAMs), infiltration into the tumor tissue and
exacerbation of the disease.
[358] In some embodiments, TGFP1-expressing cells infiltrate the tumor,
creating or contributing to an
immunosuppressive local environment. The degree by which such infiltration is
observed may correlate with worse
prognosis. In some embodiments, higher infiltration is indicative of poorer
treatment response to another cancer
therapy, such as immune checkpoint inhibitors. In some embodiments, TGF(31-
expressing cells in the tumor
microenvironment comprise immunosuppressive immune cells such as Tregs and/or
myeloid cells. In some
embodiments, the myeloid cells include, but are not limited to, macrophages,
monocytes (tissue resident or bone
marrow-derived), and MDSCs.
[359] In some embodiments, LRRC33-expressing cells in the TME are myeloid-
derived suppressor cells
(MDSCs). MDSC infiltration (e.g., solid tumor infiltrate) may underline at
least one mechanism of immune escape,
by creating an immunosuppressive niche from which host's anti-tumor immune
cells become excluded. Evidence
suggest that MDSCs are mobilized by inflammation-associated signals, such as
tumor-associated inflammatory
factors, Opon mobilization, MDSCs can influence immunosuppressive effects by
impairing disease-combating
cells, such as CD8+ T cells and NK cells. In addition, MDSCs may induce
differentiation of Tregs by secreting
TGFp and IL-10, further adding to the immunosuppressive effects. Thus, TGFI3
inhibitor such as those described
herein may be administered to patients with immune evasion (e.g., compromised
immune surveillance) to restore
or boost the body's ability to fight the disease (such as a cancer or tumor).
As described in more detail herein, this
may further enhance (e.g., restore or potentiate) the body's responsiveness or
sensitivity to another therapy, such
as cancer therapy.
[360] In some embodiments, elevated frequencies (e.g., number) of circulating
MDSCs in patients are predictive
of poor responsiveness to checkpoint blockade therapies, such as PD-1
antagonists and PD-L1 antagonists. For
example, biomarker studies showed that circulating pre-treatment HLA-
DRI0/CD14+/CD11b+ myeloid-derived
suppressor cells (MDSC) were associated with progression and worse OS (p =
0.0001 and 0.0009). In addition,
resistance to PD-1 checkpoint blockade in inflamed head and neck carcinoma
(HNC) associates with expression
of GM-CSF and Myeloid Derived Suppressor Cell (MDSC) markers. This observation
suggested that strategies to
deplete MDSCs, such as chemotherapy, should be considered in combination
(e.g., administered concurrently
(e.g., simultaneously), separately, or sequentially) with anti-PD-1. LRRC33 or
LRRC33-TGFp complexes represent
a novel target for cancer immunotherapy due to selective expression on
immunosuppressive myeloid cells.
Therefore, without intending to be bound by particular theory, targeting this
complex may enhance the effectiveness
of standard-of-care checkpoint inhibitor therapies in the patient population.
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[361] The disclosure therefore provides the use of TGFI3 inhibitors, such as
the isoform-specific TGFI31 inhibitor
described herein, for the treatment of cancer that comprises a solid tumor.
Such treatment comprises
administration of a TGFp inhibitor encompassed by the disclosure, e.g., Ab6,
to a subject diagnosed with cancer
that includes at least one localized tumor (solid tumor) in an amount
effective to treat the cancer. Preferably, the
subject is further treated with a cancer therapy, such as CBT, chemotherapy,
and/or radiation therapy (such as a
radiotherapeutic agent). In some embodiments, the TGFp inhibitor increases the
rate/fraction of a primary
responder patient population to the cancer therapy. In some embodiments, the
TGFp inhibitor increases the degree
of responsiveness of primary responders to the cancer therapy. In some
embodiments, the TGF1 inhibitor
increases the ratio of complete responders to partial responders to the cancer
therapy. In some embodiments, the
TGFp inhibitor increases the durability of the cancer therapy such that the
duration before recurrence and/or before
the cancer therapy becomes ineffective is prolonged. In some embodiments, the
TGFp inhibitor reduces
occurrences or probability of acquired resistance to the cancer therapy among
primary responders.
[362] In some embodiments, cancer progression (e.g., tumor
proliferation/growth, invasion, angiogenesis and
metastasis) may be at least in part driven by tumor-stroma interaction. In
particular, CAFs may contribute to this
process by secretion of various cytokines and growth factors and ECM
remodeling. Factors involved in the process
include but are not limited to stromal-cell-derived factor 1 (SCD-1), MMP2,
MMP9, MMP3, MMP-13, INF-a, TGF[31,
VEGF, IL-6, M-CSF. In addition, CAFs may recruit TAMs by secreting factors
such as CCL2/MCP-1 and SDF-
1/CXCL12 to a tumor site; subsequently, a pro-TAM niche (e.g., hyaluronan-
enriched stromal areas) is created
where TAMs preferentially attach. Since TGFpl has been suggested to promote
activation of normal fibroblasts
into myofibroblast-like CAFs, administration of an isoform-specific, context-
independent TGFpl inhibitor such as
those described herein may be effective to counter cancer-promoting activities
of CAFs. Data previously disclosed
in PCT/US2019/041373 suggest that an isoform-specific context-independent
antibody that blocks activation of
TGFpl can inhibit UUO-induced upregulation of maker genes such as CCL2/MCP-1,
a-SMA. FN1 and Coil, which
are also implicated in many cancers.
[363] In certain embodiments, the antibodies, or antigen binding portions
thereof, that specifically bind a GARP-
TGFpl complex, a LTBP1-TGFp1 complex, a 1:1131P3-TGFp1 complex, and/or a
LRRC33-TGFI31 complex, as
described herein, are administered to a subject having cancer or a tumor,
either alone or in combination with an
additional agent, e.g., an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist).
Other combination therapies which
are included in the disclosure are the administration of an antibody, or
antigen binding portion thereof, described
herein, with radiation (radiation therapy, including radiotherapeutic agents),
or a chemotherapeutic agent
(chemotherapy). Exemplary additional agents to use with an anti-TGFp inhibitor
include, but are not limited to, a
PD-1 antagonist (e.g., a PD-1 antibody), a PDL1 antagonist (e.g., a PDL1
antibody), a PD-Ll or PDL2 fusion
protein, a CTLA4 antagonist (e.g., a CTLA4 antibody), a GITR agonist e.g., a
GITR antibody), an anti-ICOS
antibody, an anti-ICOSL antibody, an anti-57H3 antibody, an anti-1371-14
antibody, an anti-TIM3 antibody, an anti-
LAG3 antibody, an anti-OX40 antibody (0X40 agonist), an anti-CD27 antibody, an
anti-0070 antibody, an anti-
CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an anti-CD20
antibody, an anti-CD3 antibody, an
anti-CD3/anti-CD20 bispecific or multispecific antibody, an anti-HER2
antibody, an anti-CD79b antibody, an anti-
CD47 antibody, an antibody that binds T cell immunoglobulin and ITIM domain
protein (TIGIT), an anti-ST2
antibody, an anti-beta7 integrin (e.g., an anti-a1pha4-beta7 integrin and/or
alphaE beta7 integrin), a CDK inhibitor,
an oncolytic virus, an indoleamine 2,3-dioxygenase (IDO) inhibitor, and/or a
PARP inhibitor. Examples of useful
oncolytic viruses include, adenovirus, reovirus, measles, herpes simplex,
Newcastle disease virus, senecavirus,
enterovirus and vaccinia. In certain embodiments, the oncolytic virus is
engineered for tumor selectivity.
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[364] In some embodiments, determination or selection of therapeutic approach
for combination therapy that suits
particular cancer types or patient population may involve the following: a)
considerations regarding cancer types
for which a standard-of-care therapy is available (e.g., immunotherapy-
approved indications); b) considerations
regarding treatment-resistant subpopulations (e.g., immune excluded); and c)
considerations regarding
cancers/tumors that are or generally suspected to be "TGFP1 pathway-active" or
otherwise at least in part TGFP1-
dependent (e.g., TGFp1 inhibition-sensitive). For example, many cancer samples
show that TGFp1 is the
predominant isoform by, for instance, TCGA RNAseq. In some embodiments, DNA-
and/or RNA-based assays
(e.g. RNAseq or Nanostring) may be used to evaluate the level of TGFp
signaling (e.g. TGFp1 signaling) in tumor
samples. In some embodiments, over 50% (e.g., over 50%, 60%, 70%, 80% and 90%)
of samples from each tumor
type are positive for TGFp1 isoform expression. In some embodiments, the
cancers/tumors that are "TGF131
pathway-active" or otherwise at least in part TGFp1-dependent (e.g., TGFp1
inhibition-sensitive) contain at least
one Ras mutation, such as mutations in K-ras, N-ras and/or H-ras. In some
embodiments, the cancer/tumor
comprises at least one K-ras mutation.
[365] Confirmation of TGF(31 expression in clinical samples collected from
patients (such as biopsy samples) is
not prerequisite to TGF(31 inhibition therapy, where the particular condition
has been generally known or suspected
to involve the TGFp pathway.
[366] In some embodiments, a TGFp inhibitor such as those described herein is
administered in conjunction with
checkpoint inhibitory therapy to patients diagnosed with cancer for which one
or more checkpoint inhibitor therapies
are approved or shown effective. These include, but are not limited to:
bladder urothelial carcinoma (such as
metastatic urothelial carcinoma), squamous cell carcinoma (such as head &
neck), kidney clear cell carcinoma,
kidney papillary cell carcinoma, liver hepatocellular carcinoma, lung
adenocarcinoma, skin cutaneous melanoma,
and stomach adenocarcinoma. In certain embodiments, such patients are poorly
responsive or non-responsive to
the checkpoint inhibitor therapy. In some embodiments, the poor responsiveness
is due to primary resistance. In
some embodiments, the cancer that is resistant to checkpoint blockade shows
downregulation of TCF7 expression.
In some embodiments, TCF7 downregulation in checkpoint inhibition-resistant
tumor may be correlated with a low
number of intratumoral CD8+ T cells.
[367] A TGFP inhibitor such as those described herein may be used in the
treatment of chemotherapy- or
radiotherapy-resistant cancers. Thus, in some embodiments, a TGFp1 inhibitor,
e.g., Ab6, may be administered
to patients diagnosed with cancer for which they receive or have received
chemotherapy and/or radiation therapy
(such as a radiotherapeutic agent). In particular, the use of the TG931
inhibitor is advantageous where the cancer
(patient) is resistant to such therapy. In some embodiments, such cancer
comprises quiescent tumor propagating
cancer cells (TPCs), in which TGFp signaling controls their reversible entry
into a growth arrested state, which
protects TPCs from chemotherapy or radiation therapy (such as a
radiotherapeutic agent). It is contemplated that
upon pharmacological inhibition of TG931, TPCs with compromised fail to enter
quiescence and thus rendered
susceptible to chemotherapy and/or radiation therapy (such as a
radiotherapeutic agent). Such cancer includes
various carcinomas, e.g., squamous cell carcinomas. See, for example, Brown et
al., (2017) "TGF-p-lnduced
Quiescence Mediates Chemoresistance of Tumor-Propagating Cells in Squamous
Cell Carcinoma." Cell Stem Cell.
21 (5):650-664.
[368] In some embodiments, a TGFp inhibitor such as an isoform-selective TG931
inhibitor (e.g., Ab6) may be
used to treat (e.g., reduce) anemia in a subject, e.g., in a cancer patient.
In some embodiments, a TGFp inhibitor
such as an isoform-selective TGF(31 inhibitor (e.g., Ab6) may be used in
combination with a BMP inhibitor (e.g., a
BMP6 inhibitor, e.g., a RGMc inhibitor, e.g., any of the RGMc inhibitor
disclosed in WO/2020/086736, the content
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of which is hereby incorporated in its entirety) to treat (e.g., reduce)
anemia, e.g., in the subject. In some
embodiments, the anemia results from reduced or impaired red blood cell
production (e.g., as a result of
myelofibrosis or cancer), iron restriction (e.g., as a result of cancer or
treatment-induced anemia, such as
chemotherapy-induced anemia), or both. In some embodiments, the combination of
a TGFp inhibitor and a BMP
inhibitor (antagonist) may be administered at a therapeutically effective
amount or amounts that is/are sufficient to
relieve one or more anemia-related symptom and/or complication in the subject,
e.g., a cancer patient. In some
embodiments, the combination of a TGFp inhibitor and a BMP inhibitor
(antagonist) may be administered at a
therapeutically effective amount that is sufficient to increase or normalize
red blood cell production and/or reduce
iron restriction. Without wishing to be bound by theory, it is contemplated
that TGFP1 inhibitors (e.g., Ab6) may
alleviate symptoms and/or complications related to anemia through their
hematopoiesis-promoting effects and that
BMP inhibitors (antagonists) (e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor)
may improve iron-deficiency anemia
(e.g., chemotherapy-induced anemia). In some embodiments, the treatment for
anemia further comprises
administering one or more JAK inhibitor (e.g., Jak1/2 inhibitor, Jak1
inhibitor, and/or Jak2 inhibitor).
[369] In some embodiments, the BMP inhibitor is an antagonist of the kinase
associated with the BMP receptor
(e.g., type I receptor and/or type ll receptor).
[370] In some embodiments, the BMP inhibitor is a "ligand trap" that binds (or
sequesters) the BMP growth
factor(s), including BMP6.
[371] In some embodiments, the BMP inhibitor is an antibody that neutralizes
the BMP growth factor(s), including
BMP6. Examples include anti-BMP6 antibodies (e.g., WO 2016/098079, Novartis;
and, KY-1070, KyMab).
[372] In some embodiments, the BMP inhibitor is an inhibitor of a BMP6 co-
receptor, such as RGMc. For example,
such inhibitor may include an antibody that binds RGMa/c. (BOser et al. AAPS
J. 2015 Jul;17(4): 930-938). More
preferably, such inhibitor is an antibody that selectively binds RGMc (see,
for example, WO 2020/086736).
Therapeutic Indications and/or Subjects Likely to Benefit from a Therapy
Comprising a TGFp-Inhibitor
[373] The current disclosure encompasses methods of treating cancer and
predicting or monitoring therapeutic
efficacy using a TGFp inhibitor, e.g., Ab6. In some embodiments, the
identification/screening/selection of suitable
indications and/or patient populations for which TGFp inhibitors, such as
those described herein, are likely to have
advantageous therapeutic benefits comprise: i) whether the disease is driven
by or dependent predominantly on
the TGFp1 isoform over the other isoforms in human (or at least co-dominant);
ii) whether the condition (or affected
tissue) is associated with an immunosuppressive phenotype (e.g., an immune-
excluded tumor); and, iii) whether
the disease involves both matrix-associated and cell-associated TGFp1
function.
[374] Differential expression of the three known TGFp isoforms, namely, TGFp1,
TGFp2, and TGFp3, has been
observed under normal (healthy; homeostatic) as well as disease conditions in
various tissues. Nevertheless, the
concept of isoform selectivity has neither been fully exploited nor robustly
achieved with conventional approaches
that favor pan-inhibition of TGFp across multiple isoforms. Moreover,
expression patterns of the isoforms may be
differentially regulated, not only in normal (homeostatic) vs abnormal
(pathologic) conditions, but also in different
subpopulations of patients. Because most preclinical studies are conducted in
a limited number of animal models,
which may or may not recapitulate human conditions, data obtained with the use
of such models may be biased,
resulting in misinterpretations of data or misleading conclusions as to the
translatability for purposes of developing
therapeutics.
[375] Previous analyses of human tumor samples implicated TGFp signaling as an
important contributor to
primary resistance to disease progression and treatment response, including
checkpoint blockade therapy ("CBT'')
for various types of malignancies. Studies reported in literature reveal that
the TGFB gene expression may be
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particularly relevant to treatment resistance, suggesting that activity of
this isoform may be driving TGFp signaling
in these diseases. As detailed in PCT/US2019/041373, across the majority of
human tumor types profiled at The
Cancer Genome Atlas (TCGA), TGFB1 expression appears to be the most prevalent,
suggesting that selection of
preclinical models that more closely recapitulate human disease expression
patterns of TGFp isoforms may be
beneficial.
[376] Without being bound by theory, TGF131 and TGFp3 are often co-dominant
(co-expressed at similar levels)
in certain murine syngeneic cancer models (e.g., EMT-6 and 411) that are
widely used in preclinical studies (e.g.,
see PCT/US2019/041373 at FIG. 21B). By contrast, numerous other cancer models
(e.g., S91, B16 and MBT-2)
express almost exclusively TGFI31, similar to that observed in many human
tumors, in which TGFI31 appears to be
more frequently the dominant isoform over TGF132/3 (see PCT/US2019/041373 at
FIGs. 20 and 21A).
Furthermore, the TGFp isoform(s) predominantly expressed under homeostatic
conditions may not be the disease-
associated isoform(s). For example, in normal lung tissues in healthy rats,
tonic TGFP signaling appears to be
mediated mainly by TGFp3. However, TGFp1 appears to become markedly
upregulated in disease conditions,
such as lung fibrosis. Taken together, while not prerequisite, it may be
beneficial to test or confirm relative
expression of TGFp isoforms in clinical samples so as to select suitable
therapeutics to which the patient is likely
to respond. In some embodiments, determination of relative isoform expression
may be made post-treatment. In
such circumstances, patients' responsiveness (e.g., clinical response/benefit)
in response to TGFp1 inhibition
therapy may be correlated with relative expression levels of TGFI3 isoforms.
In some embodiments, overexpression
of the TGF(31 isoform shown ex post facto correlates with greater
responsiveness to the treatment.
[377] Whilst inhibition of TGF131 alone appears to be sufficient to overcome
primary resistance to cancer
immunotherapy as demonstrated in a tumor model expressing both TGFp1 and
TGF133, findings disclosed herein
suggests that inhibition of TGFP3 may in fact be harmful. Surprisingly, in a
murine liver fibrosis model, mice treated
with an isoform-selective inhibitor of TGFp3 manifest exacerbation of
fibrosis. A significant increase of collagen
deposits in liver sections of these animals suggest that inhibition of TGF33
in fact may result in greater
dysregulation of the ECM. Without being bound by theory, this suggests that
TGFp3 inhibition may promote a pro-
fibrotic phenotype.
[378] A hallmark of pro-fibrotic phenotypes is increased deposition and/or
accumulation of collagens in the ECM,
which is associated with increased stiffness of tissue ECMs. This has been
observed during pathological
progression of cancer, fibrosis and cardiovascular disease. Consistent with
this, Applicant previously demonstrated
the role of matrix stiffness on integrin-dependent activation of TG93, using
primary fibroblasts grown on silicon-
based substrates with defined stiffness (e.g., 5 kPa, 15 kPa or 100 kPa) (see
WO 2018/129329). Matrices with
greater stiffness enhanced TGF131 activation, and this was suppressed by
isoform-specific inhibitors of TGFp1.
These observations suggest that the pharmacologic inhibition of TGF(33 may
exert opposing effects to TGF(31
inhibition by creating a pro-tumor microenvironment, where greater stiffness
of the tissue matrix may support cancer
progression.
[379] Given the common pathways involved in fibrotic phenotypes and many
aspects of cancer progression such
as increased invasiveness and metastasis (see, for example: Chakravarthy et
al., Nat Corn (2018)9:4692. "TGF-
p-associated extracellular matrix genes link cancer-associated fibroblasts to
immune evasion and immunotherapy
failure"), pro-fibrotic effects of TGF133 inhibition observed in a fibrosis
model may be applicable to cancer contexts.
[380] The finding mentioned above therefore raises the possibility that TGFp
inhibitors with inhibitory potency
against TGFp3 may not only be ineffective in treating cancer but may in fact
be detrimental. In some embodiments,
TGFp3 inhibition is avoided in patients suffering from a cancer type that is
statistically highly metastatic. Cancer
types that are typically considered highly metastatic include, but are not
limited to, colorectal cancer, lung cancer,
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bladder cancer, kidney cancer (e.g., transitional cell carcinoma, renal
sarcoma, and renal cell carcinoma (RCC),
including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC,
or unclassified RCC, uterine
cancer, prostate cancer, stomach cancer, and thyroid cancer. Moreover, TGF(33
inhibition may be best avoided in
patients having or are at risk of developing a fibrotic condition and/or
cardiovascular disease. Such patients at risk
of developing a fibrotic condition and/or cardiovascular disease include, but
are not limited to, those with metabolic
disorders, such as NAFLD and NASH, obesity, and type 2 diabetes. Similarly,
TGFp3 inhibition may be best
avoided in patients diagnosed with or at risk of developing myelofibrosis.
Those at risk of developing myelofibrosis
include those with one or more genetic mutations implicated in the
pathogenesis of myelofibrosis.
[381] In addition to the possible concerns of inhibiting TGFI33 addressed
above, Takahashi et al. (Nat Metab.
2019,1(2): 291-303) recently reported a beneficial role of TGFp2 in regulating
metabolism. The authors identified
TGFp2 as an exercise-induced adipokine, which stimulated glucose and fatty
acid uptake in vitro, as well as tissue
glucose uptake in vivo; which improved metabolism in obese mice; and, which
reduced high fat diet-induced
inflammation. Moreover, the authors observed that lactate, a metabolite
released from muscle during exercise,
stimulated TGF132 expression in human adipocytes and that a lactate-lowering
agent reduced circulating 1G932
levels and reduced exercise-stimulated improvements in glucose tolerance.
Thus, in some embodiments, a TGFp
inhibitor may be used in treating a subject that does not have inhibitory
activity towards the TGFp2 isoform, e.g.,
to avoid a potentially harmful impact on one or more metabolic functions of a
treated subject.
[382] More recently, a potential link between cancer and various metabolic
conditions has been recognized. For
example, as reviewed by Braun et al., an enhanced risk of cancer mortality is
associated with metabolic syndrome
among men (Braun et al. Int J Biol Sci. 2011; 7(7): 1003-1015). Similarly, the
authors noted "metabolic
dysregulation may play an important role in the etiology and progression of
certain cancer types and worse
outcome for some cancers. Obesity and diabetes, individually, have been
associated with breast, endometrial,
colorectal, pancreatic, hepatic and renal cancer" (Braun et al. Int J Biol
Sci. 2011; 7(7): 1003-1015).
[383] Accordingly, in various embodiments, a TGF13 inhibitor may be used in
the treatment of a TGFp-related
indication (e.g., cancer) in a subject, wherein, the TGFp inhibitor inhibits
TGFp1 but does not inhibit TGFp2 at the
therapeutically effective dose administered. In some embodiments, the subject
benefits from improved metabolism
after such treatment, wherein optionally, the subject has or is at risk of
developing a metabolic disease, such as
obesity, high fat diet-induced inflammation, and glucose dysregulation (e.g.,
diabetes). In some embodiments, the
TGFp-related indication is cancer, wherein optionally the cancer comprises a
solid tumor, such as locally advanced
cancer and metastatic cancer.
[384] In some embodiments, the TGFp inhibitor is TGFpl-selective (e.g., it
does not inhibit TGFp2 and/or TGFp3
signaling at a therapeutically effective dose). In certain embodiments, a
TGF(31-selective inhibitor is selected for
use in treating a cancer patient. In some embodiments, such a treatment: i)
avoids TGFp3 inhibition to reduce the
risk of exacerbating ECM dysregulation (which may contribute to tumor growth
and invasiveness) and ii) avoids
TGFp2 inhibition to reduce the risk of increasing metabolic burden in the
patients. Related methods for selecting a
TGFp inhibitor for therapeutic use are also encompassed herein.
[385] The disclosure includes methods for selecting a TGFp inhibitor for use
in the treatment of cancer, wherein
the TGFp inhibitor has no or little inhibitory potency against TGFp3 (e.g.,
the TGFp inhibitor does not target TGF(33).
In certain embodiments, the TGFp inhibitor is a TG931-selective inhibitor
(e.g., antibodies or antigen binding
fragments that do not inhibit TGFp2 and/or TGF33 signaling at therapeutically
effective doses). It is contemplated
that this selection strategy may reduce the risk of exacerbating ECM
dysregulation in cancer patients and still
provide benefits of TGFp1 inhibition to treat cancer. In some embodiments, the
cancer patients are also treated
with a cancer therapy, such as immune checkpoint inhibitors. In some
embodiments, the cancer patient is at risk
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of developing a metabolic disease, such as fatty liver, obesity, high fat diet-
induced inflammation, and glucose or
insulin dysregulation (e.g., diabetes).
[386] The present disclosure also includes related methods for selecting
and/or treating suitable patient
populations who may be candidates for receiving a TGFI3 inhibitor capable of
inhibiting TGF33. Such methods
include use of a TGFI3 inhibitor capable of inhibiting TGFp3 for the treatment
of cancer in subjects who are not
diagnosed with a fibrotic disorder (such as organ fibrosis), who are not
diagnosed with myelofibrosis, who are not
diagnosed with a cardiovascular disease and/or those who are not at risk of
developing such conditions. Similarly,
such methods include use of a TGFI3 inhibitor capable of inhibiting TGF33 for
the treatment of cancer in subjects,
wherein the cancer is not considered to be highly metastatic. The TGFI3
inhibitor capable of inhibiting TGFp3 may
include pan-inhibitors of TGFI3 (such as low molecular weight antagonists of
TG93 receptors, e.g., ALK5 inhibitors,
and neutralizing antibodies that bind TGF131 /2/3), isoform-non-selective
inhibitors such as antibodies that bind
TGF131/3 and engineered fusion proteins capable of binding TG931/3, e.g.,
ligand traps, and integrin inhibitors
(e.g., an antibody that binds to aVp1, aVp3, aVp5, aVp6, aVp8, a5]31, a11bp3,
or a8131 integrins, and inhibits
downstream activation of TGFI3. e.g., selective inhibition of TGF131 and/or
TGFp3).
[387] The surprising notion that TGFp3 inhibition may in fact be disease-
promoting suggests that patients who
have been previously treated with or currently undergoing treatment with a
TGFp inhibitor with inhibitory activity
towards TG933 may benefit from additional treatment with a TG931-selective
inhibitor to counter the possible pro-
fibrotic effects of the TGFI33 inhibitor. Accordingly, the disclosure includes
a TGF31-selective inhibitor for use in
the treatment of cancer in a subject, wherein the subject has been treated
with a TGFI3 inhibitor that inhibits TGFI33
in conjunction with a checkpoint inhibitor, comprising the step of:
administering to the subject a TGFp1-selective
inhibitor, wherein optionally the cancer is a metastatic cancer, a
desmoplastic tumor, myelofibrosis, and/or, wherein
the subject has a fibrotic disorder or is at risk of developing a fibrotic
disorder and/or cardiovascular disease,
wherein optionally the subject at risk of developing a fibrotic disorder or
cardiovascular disease suffers from a
metabolic condition, wherein optionally the metabolic condition is NAFLD,
NASH, obesity or diabetes.
[388] As described herein, the isoform-selective TGFp1 inhibitors are
particularly advantageous for the treatment
of diseases in which the TGF(31 isoform is predominantly expressed relative to
the other isoforms (e.g., referred to
as TG931-dominant). As an example, a non-limiting list of human cancer
clinical samples with relative expression
levels of TGFB1 (left), TGFB2 (center) and TGFB3 (right) were previously
described in PCT/US2019/041 373, e.g.,
at FIGs. 20 and 21A. Each horizontal line across the three isoforms represents
a single patient. As can be seen,
overall TG931 expression (TGFB1) is significantly higher in most of these
human tumors/cancers than the other
two isoforms across many tumor/cancer types, suggesting that TG931-selective
inhibition may be beneficial in
these disease types. Taken together, these lines of evidence support the
notion that selective inhibition of 1GF131
activity may overcome primary resistance to CBT. Generation of highly
selective TGFp1 inhibitors will also enable
evaluation of whether such an approach will address key safety issues observed
with pan-TGFI3 inhibition, which
will be important for assessment of their therapeutic utility.
[389] It was previously considered that TGFp1 inhibitors may not be
efficacious, particularly in cancer types in
which TG931 is co-dominant with another isoform or in which TG932 and/or TG933
expression is significantly
greater than TGF131. However more recently, the inventors of the present
application have made an unexpected
finding that TGFp inhibitors, e.g., TGF131 inhibitors, such as a TGFpl-
selective inhibitor (e.g., Ab6), used in
conjunction with a checkpoint inhibitor (e.g., anti-PD-1 antibody), is capable
of causing significant tumor regression
in the EMT-6 model, which is known to express both TGFp1 and TGFp3 at similar
levels. The co-dominance has
been confirmed by both RNA measurements and ELISA assays (see
PCT/US2019/041373 at FIG. 35). This
observation was surprising because it had been previously hypothesized that in
order to achieve material efficacy
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in tumors co-expressing TGF31 and TGF33 in a checkpoint blockade context, both
of the co-dominant isoforms
would have to be specifically inhibited. Accordingly, methods of treatment
disclosed herein include the use of
TGF61 inhibitor for promoting tumor regression, where the tumor is
TGF31+/TGF33+. Such tumor may include,
for example, cancers of epithelial origin, i.e., carcinoma (e.g., basal cell
carcinoma, squamous cell carcinoma, renal
cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma,
and adenocarcinoma). In some
embodiments, TGE131 is predominantly the disease-associated isoform, whilst
TGFp3 supports homeostatic
function in the tissue, such as epithelia.
[390] Aberrant activity of the TGFp signaling pathway has been reported to
impact gene expressions involved in
both fibrotic and cancer processes. For instance, dysregulation of the TGF131
signal transduction pathway has been
observed to alter genes such as SNA11, MMP2, MMP9, and TIMP1, all of which are
important for cellular processes
like adhesion and extracellular matrix remodeling and have been implicated in
fibrosis and the epithelial
mesenchymal transition (EMT) process in cancer. Accordingly, in some
embodiments, the methods of treatment
herein, e.g., of fibrosis-related cancer indications, comprise the
administration of a TGF3 inhibitor that does not
inhibit TGF(33, e.g., using a TGFpl-selective antibody, e.g., Ab6. Certain
tumors, such as various carcinomas, may
be characterized as low mutational burden tumors (MBTs). Such tumors are often
poorly immunogenic and fail to
elicit sufficient T cell response. Cancer therapies that include chemotherapy,
radiation therapy (such as a
radiotherapeutic agent), cancer vaccines and/or oncolytic virus, may be
helpful to elicit T cell immunity in such
tumors. Therefore, TGF131 inhibition therapy detailed herein can be used in
conjunction with one or more of these
cancer therapies to increase anti-tumor effects. Essentially, such combination
therapy is aimed at converting "cold"
tumors (e.g., poorly immunogenic tumors) into "hot" tumors by promoting neo-
antigens and facilitating effector cells
to attack the tumor. Examples of such tumors include breast cancer, ovarian
cancer, and pancreatic cancer, e.g.,
pancreatic ductal adenocarcinoma (PDAC). Accordingly, any one or more of the
antibodies or fragments thereof
described herein may be used to treat poorly immunogenic tumor (e.g., an
"immune-excluded" tumor) sensitized
with a cancer therapy aimed to promote T cell immunity.
[391] In immune-excluded tumors where effector T cells are kept away from the
site of tumor (hence "excluded"),
the immunosuppressive tumor environment may be mediated in a TGFI31-dependent
fashion. These are tumors
that are typically immunogenic; however, T cells cannot sufficiently
infiltrate, proliferate, and elicit their cytotoxic
effects due to the immune-suppressed environment. Typically, such tumors are
poorly responsive to cancer
therapies such as CBTs. As suggested by data provided herein and previously in
PCT/US2019/041373, adjunct
therapy comprising a TGF31 inhibitor may overcome the immunosuppressive
phenotype, allowing T cell infiltration,
proliferation, and anti-tumor function, thereby rendering such tumor more
responsive to cancer therapy such as
CBT.
[392] Thus, the second inquiry is drawn to identification or selection of
patients who have immunosuppressive
tumor(s), who are likely to benefit from a TGF3 inhibitor therapy, e.g., a
TGF[31 inhibitor such as Ab6. The presence
or the degree of frequencies of effector T cells in a tumor is indicative of
anti-tumor immunity. Therefore, detecting
anti-tumor cells such as CD8+ cells in a tumor provides useful information for
assessing whether the patient may
benefit from a CET and/or TGF[31 inhibitor therapy.
[393] Detection may be carried out by known methods such as
immunohistochemical analysis of tumor biopsy
samples, including digital pathology methods. More recently, non-invasive
imaging methods are being developed
which will allow the detection of cells of interest (e.g., cytotoxic T cells)
in vivo. See for example,
http://www.imaginab.com/technology/; Tavare et al., (2014) PNAS, 111(3): 1108-
1113; Tavare et al., (2015)J Nucl
Med 56(8): 1258-1264; Rashidian et al., (2017) J Exp Med 214(8): 2243-2255;
Beckford Vera et al., (2018) PLoS
ONE 13(3): e0193832; and Tavare et al., (2015) Cancer Res 76(1): 73-82, each
of which is incorporated herein by
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reference. Typically, antibodies or antibody-like molecules engineered with a
detection moiety (e.g., radiolabel)
can be infused into a patient, which then will distribute and localize to
sites of the particular marker (for instance
CD8+). In this way, it is possible to determine whether the tumor has an
immune-excluded phenotype. If the tumor
is determined to have an immune-excluded phenotype, cancer therapy (such as
CBT) alone may not be efficacious
because the tumor lacks sufficient cytotoxic cells within the tumor
environment. Add-on therapy with a TGFP
inhibitor such as those described herein may reduce immuno-suppression thereby
rendering the cancer therapy-
resistant tumor more responsive to a cancer therapy.
[394] Non-invasive in vivo imaging techniques may be applied in a variety of
suitable methods for purposes of
diagnosing patients; selecting or identifying patients who are likely to
benefit from TGFI3 inhibitor therapy, e.g., a
TGFp inhibitor therapy; and/or, monitoring patients for therapeutic response
upon treatment. Any cells with a
known cell-surface marker may be detected/localized by virtue of employing an
antibody or similar molecules that
specifically bind to the cell marker. Typically, cells to be detected by the
use of such techniques are immune cells,
such as cytotoxic T lymphocytes, regulatory T cells, MDSCs, tumor-associated
macrophages, NK cells, dendritic
cells, and neutrophils. Antibodies or engineered antibody-like molecules that
recognize such markers can be
coupled to a detection moiety.
[395] Non-limiting examples of suitable immune cell markers include monocyte
markers, macrophage markers
(e.g., M1 and/or M2 macrophage markers), CTL markers, suppressive immune cell
markers, MDSC markers (e.g.,
markers for G- and/or M-MDSCs), including but are not limited to: 008, CD3,
004, CD11b, CD33, C0163, 00206,
CD68, 0014, CD15, CD66b, 0D34, CD25, and CD47. In some embodiments, the in
vivo imaging comprises T cell
tracking, such as cytotoxic CD8-positive T cells. Accordingly, any one of the
TGFp inhibitors of the present
disclosure may be used in the treatment of cancer in a subject with a solid
tumor, wherein the treatment comprises:
i) carrying out an in vivo imaging analysis to detect T cells in the subject,
wherein optionally the T cells are CD8+
T cells, and if the solid tumor is determined to be an immune-excluded solid
tumor based on the in vivo imaging
analysis of step (i), then, administering to the subject a therapeutically
effective amount of a TGF6 inhibitor, e.g.,
Ab6. In some embodiments, the subject has received a CBT, wherein optionally
the solid tumor is resistant to the
CBT. In some embodiments, the subject is administered with a CBT in
conjunction with the TGFp1 inhibitor, as a
combination therapy. The combination may comprise administration of a single
formulation that comprises both a
checkpoint inhibitor and a TGFp inhibitor. The TGF13 inhibitor may be a TGF61
inhibitor, such as a TG931-selective
inhibitor, e.g., Ab6, an isoform-non-selective inhibitor, e.g., low molecular
weight ALK5 antagonists, neutralizing
antibodies that bind two or more of TGFp1/2/3, e.g., GC1008 and variants,
antibodies that bind TGFI31/3, ligand
traps, e.g., TGF(31/3 inhibitors, and/or an integrin inhibitor (e.g., an
antibody that binds to aVp1, aVp3, aVp5, aV66,
aVp8, a5p1, 011b133, or a861 integrins, and inhibits downstream activation of
TG93. e.g., selective inhibition of
TGF131 and/or TG933). Alternatively, the combination therapy may comprise
administration of a first formulation
comprising a checkpoint inhibitor and a second formulation comprising a TGFI3
inhibitor, wherein the TGFp inhibitor
may be a TGF(31 inhibitor, such as a TGFp1-selective inhibitor, e.g., Ab6, an
isoform-non-selective inhibitor, e.g.,
a low molecular weight ALK5 antagonist, a neutralizing antibody that bind two
or more of TGFp1/2/3, e.g., GC1008
or variants, an antibody that bind TG931/3, a ligand trap, e.g., a TG931/3
inhibitor, and/or an integrin inhibitor (e.g.,
an antibody that binds to aVp1, aV133, aVI35, aVp6, aV38, (1561, all bp3, or
a8131 integrins, and inhibits downstream
activation of TGF(3. e.g., selective inhibition of TGF131 and/or TGFp3).
[396] In some embodiments, the in vivo imaging comprises MDSC tracking, such
as G-MDSCs and M-MDSCs.
For example, MDSCs may be enriched at a disease site (such as fibrotic tissues
and solid tumors) at the baseline.
Upon therapy (e.g., TGFf31 inhibitor therapy), fewer MDSCs may be observed, as
measured by reduced intensity
of the label (such as radioisotope and fluorescence), indicative of
therapeutic effects.
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[397] In some embodiments, the in vivo imaging comprises tracking or
localization of LRRC33-positive cells.
LRRC33-positive cells include, for example, MDSCs and activated M2-like
macrophages (e.g., TAMs and activated
macrophages associated with fibrotic tissues). For example, LRRC33-positive
cells may be enriched at a disease
site (such as fibrotic tissues and solid tumors) at the baseline. Upon therapy
(e.g., TGF31 inhibitor therapy), fewer
cells expressing cell surface LRRC33 may be observed, as measured by reduced
intensity of the label (such as
radioisotope and fluorescence), indicative of therapeutic effects.
[398] In some embodiments, the in vivo imaging comprises the use of PET-SPECT,
MRI and/or optical
fluorescence/bioluminescence in order to detect target of interest (e.g.,
molecules or entities which can be bound
by the labeled reagent, such as cells and tissues expressing appropriate
marker(s)).
[399] In some embodiments, labeling of antibodies or antibody-like molecules
with a detection moiety may
comprise direct labeling or indirect labeling.
[400] In some embodiments, the detection moiety may be a tracer. In some
embodiments, the tracer may be a
radioisotope, wherein optionally the radioisotope may be a positron-emitting
isotope. In some embodiments, the
radioisotope is selected from the group consisting of, 18F. 11C, 13N, 150,
68Ga, 177Lu, 18F and 86Zr.
[401] Thus, such methods may be employed to carry out in vivo imaging with the
use of labeled antibodies in
immune-PET.
[402] In some embodiments, such in vivo imaging is performed for monitoring a
therapeutic response to the
TGF31 inhibition therapy in the subject. For example, the therapeutic response
may comprise conversion of an
immune excluded tumor into an inflamed tumor, which correlates with increased
immune cell infiltration into a
tumor. This may be visualized by increased intratumoral immune cell frequency
or degree of detection signals,
such as radiolabeling and fluorescence.
[403] Accordingly, the disclosure includes a method for treating cancer which
may comprise the following steps:
i) selecting a patient diagnosed with cancer comprising a solid tumor, wherein
the solid tumor is or is suspected to
be an immune excluded tumor; and, ii) administering to the patient an antibody
or the fragment encompassed
herein in an amount effective to treat the cancer. In some embodiments, the
patient has received, or is a candidate
for receiving a cancer therapy such as immune checkpoint inhibition therapies
(e.g., PD-(L)1 antibodies),
chemotherapies, radiation therapies, engineered immune cell therapies, and
cancer vaccine therapies. In some
embodiments, the selection step (i) comprises detection of immune cells or one
or more markers thereof, wherein
optionally the detection comprises a tumor biopsy analysis, serum marker
analysis, and/or in vivo imaging.
[404] In some embodiments, the patient is diagnosed with cancer for which a
CBT has been approved, wherein
optionally, statistically a similar patient population with the particular
cancer shows relatively low response rates to
the approved CBT, e.g., under 25%. For example, the response rates for the CBT
may be between about 10-25%,
for example about 10-15%. Such cancer may include, for example, ovarian
cancer, gastric cancer, and triple-
negative breast cancer. The TGFI3 inhibitors of the present disclosure may be
used in the treatment of such cancer,
where the subject has not yet received a CBT. The TGF31 inhibitor may be
administered to the subject in
combination with a CBT. In some embodiments, the subject may receive or may
have received additional cancer
therapy, such as chemotherapy and radiation therapy (including a
radiotherapeutic agent).
[405] In vivo imaging techniques described above may be employed to detect,
localize, and/or track certain
MDSCs in a patient diagnosed with a TGF3-associated disease, such as cancer.
Healthy individuals have no or
low frequency of MDSCs in circulation. With the onset of or progression of
such a disease, elevated levels of
circulating and/or disease-localized MDSCs may be detected. For example, CCR2-
positive M-MDSCs have been
reported to accumulate to tissues with inflammation and may cause progression
of fibrosis in the tissue (such as
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pulmonary fibrosis), and this is shown to correlate with TG931 expression.
Similarly, MDSCs are enriched in a
number of solid tumors (including triple-negative breast cancer) and in part
contribute to the immunosuppressive
phenotype of the TME. Therefore, treatment response to TGFI3 inhibition, such
as TGF61 inhibition, according to
the present disclosure may be monitored by localizing or tracking circulating
MDSCs. Reduction of or low frequency
of circulating MDSC levels is typically indicative of therapeutic benefits or
better prognosis. Accordingly, the current
disclosure provides methods of predicting and monitoring therapeutic efficacy
of TGFI3 inhibitor therapy, e.g.,
combination therapy of a TGFI31 inhibitor and a checkpoint inhibitor, by
measuring circulating MDSCs in the blood
or a blood component of the subject. The current disclosure also provides
methods of selecting patients, e.g.,
patients with immunosuppressive cancers and determining treatment regimens
based on levels of circulating
MDSCs measured. The TGFI3 inhibitor may be a TG931 inhibitor, such as a TGFI31-
selective inhibitor, e.g., Ab6,
an isoform-non-selective inhibitor, e.g., a low molecular weight ALK5
antagonist, a neutralizing antibody that bind
two or more of TGF131/2/3, e.g., GC1008 or variants, an antibody that bind
TG931/3, a ligand trap, e.g., a TGFI31/3
inhibitor, and/or an integrin inhibitor (e.g., an antibody that binds to
aVf31, aV133, aVf35, aV66, aVf38, a5131, a1 1b133,
or a8p1 integrins, and inhibits downstream activation of TGFI3. e.g.,
selective inhibition of TGFpl and/or TGFp3).
[406] The TGFI3 inhibitors of the present disclosure may be used in the
treatment of cancer in a subject, wherein
the cancer is characterized by immune suppression, wherein the cancer
optionally comprises a solid tumor that is
TGF131-positive and TGF(33-positive. Such subject may be diagnosed with
carcinoma. In some embodiments, the
carcinoma is breast carcinoma, wherein optionally the breast carcinoma is
triple-negative breast cancer (TNBC).
Such treatment can further comprise a cancer therapy, including, without
limitation, chemotherapies, radiation
therapies, cancer vaccines, engineered immune cell therapies (such as CAR-T),
and immune checkpoint blockade
therapies, such as anti-PD(L)-1 antibodies. The TGFI3 inhibitor may be a TGFp1
inhibitor, such as a TGFp1-
selective inhibitor, e.g., Ab6, or an isoform-non-selective inhibitor, e.g., a
low molecular weight ALK5 antagonist, a
neutralizing antibody that bind two or more of TGFI31/2/3, e.g., GC1008 or
variants, an antibody that bind TGF131/3,
a ligand trap, e.g., a TGFI31/3 inhibitor, and/or an integrin inhibitor (e.g.,
an antibody that binds to aVf31, aVf33,
aV135, aV136, aVf38, a531, a11bp3, or a8p1 integrins, and inhibits downstream
activation of TGFI3. e.g., selective
inhibition of TG931 and/or TGFp3).
[407] In some embodiments, a cold tumor is identified, in which few effector
cells are present both inside and
outside the tumor or is known to be a type of cancer characterized as poorly
immunogenic (e.g., a tumor
characterized as an immune desert). A subject/patient with such a tumor is
treated with an immune-sensitizing
cancer therapy, such as chemotherapy, radiation therapy (such as a
radiotherapeutic agent), oncolytic viral
therapy, and cancer vaccine, in order to elicit stronger T cell response to
tumor antigens (e.g., neo-antigens). This
step may convert the cold tumor into an "immune excluded" tumor. The subject
optionally further receives a CBT,
such as anti-PD-(L)1. The subject is further treated with a TGFP1 inhibitor,
such as the antibodies disclosed herein.
This may convert the cold or immune excluded tumor into an "inflamed" or "hot"
tumor, which confers
responsiveness to immunotherapy. Non-limiting examples of poorly immunogenic
cancers include breast cancer
(such as TNBC), prostate cancer (such as Castration resistant prostate cancer
(CRPC)) and pancreatic cancer
(such as pancreatic adenocarcinoma (PDAC)).
[408] As Applicant has previously shown, high affinity, isoform-selective
inhibitors of TGF(31 of the present
disclosure, such as Ab6, can inhibit Plasmin-induced activation of TGF61. The
plasmin-plasminogen axis has
been implicated in certain tumorigenesis, invasion and/or metastasis, of
various cancer types, carcinoma in
particular, such as breast cancer. Therefore, it is possible that the TGFI3
inhibitors such as those described herein
may exert the inhibitory effects via this mechanism in tumors or tumor models,
such as EMT6, involving the
epithelia. Indeed, Plasmin-dependent destruction or remodeling of epithelia
may contribute to the pathogenesis of
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conditions involving epithelial injuries and invasion/dissemination of
carcinoma. The latter may be triggered by
epithelial to mesenchymal transition ("EMT"). It has been reported that
plasminogen activation and plasminogen-
dependent invasion were more prominent in epithelial-like cells and were
partly dictated by the expression of
S100A10 and PAI-1 (Bydoun et al., (2018) Scientific Reports, 8:14091).
[409] The TGFp inhibitors of the present disclosure (e.g., a TGFp1 inhibitor,
e.g., Ab6) may be used in the
treatment of anemia in a subject in need thereof. In some embodiments, the
subject is diagnosed with cancer. In
some embodiments, the subject is diagnosed with a myeloproliferative disorder
(e.g., myelofibrosis). In some
embodiments, a TGFp inhibitor (e.g., Ab6) is used alone to treat anemia. In
some embodiments, the TGFp inhibitor
is used in combination with an additional agent, e.g., a BMP antagonist (e.g.,
a BMP6 inhibitor, e.g., a RGMc
inhibitor). In some embodiments, a combination comprising a TGFp1 inhibitor
(e.g., Ab6) and a BMP antagonist
(e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor) is used to improve anemia
resulting from insufficient erythrocyte
production, iron deficiency, and/or chemotherapy. In some embodiments, the
treatment for anemia further
comprises administering one or more JAK inhibitor (e.g., Jak1/2 inhibitor,
Jak1 inhibitor, and/or Jak2 inhibitor).
[410] The disclosure includes a method for selecting a patient population or a
subject who is likely to respond to
a therapy comprising a TGFp inhibitor such as those described herein. Subjects
selected according to such
methods may be the subjects treated according to the various aspects of the
present disclosure. Such method may
comprise the steps of: providing a biological sample (e.g., clinical sample)
collected from a subject, determining
(e.g., measuring or assaying) relative levels of TGFI31, TGFP2 and TGFP3 in
the sample, and, administering to the
subject a composition comprising a TGFp inhibitor, such as a TGFI31 inhibitor
described herein, if TG931 is the
dominant isoform over TGFp2 and TGFp3; and/or, if TGFp1 is significantly
overexpressed or upregulated as
compared to control. In some embodiments, such method comprises the steps of
obtaining information on the
relative expression levels of TGFP1, TGFP2 and TGFP3 which was previously
determined; identifying a subject to
have TGF131-positive, preferably TGFpl-dominant, disease; and administering to
the subject a composition
comprising a TGFp inhibitor disclosed herein. In some embodiments, such
subject has a disease (such as cancer)
that is resistant to a therapy (such as cancer therapy). In some embodiments,
such subject shows intolerance to
the therapy and therefore has or is likely to discontinue the therapy.
Addition of the TGFp inhibitor to the therapeutic
regimen may enable reducing the dosage of the first therapy and still achieve
clinical benefits in combination. In
some embodiments, the TG93 inhibitor may delay or reduce the need for
surgeries. In some embodiments, the
TGFp inhibitor is a 1GF131 inhibitor described herein, e.g., Ab6.
[411] Relative levels of the isoforms may be determined by RNA-based assays
and/or protein-based assays,
which are well-known in the art. In some embodiments, the step of
administration may also include another therapy,
such as immune checkpoint inhibitors, or other agents provided elsewhere
herein. Such methods may optionally
include a step of evaluating a therapeutic response by monitoring changes in
relative levels of TGFp1, TGFp2 and
TGFp3 at two or more time points. In some embodiments, clinical samples (such
as biopsies) are collected both
prior to and following administration. In some embodiments, clinical samples
(such as biopsies) are collected
multiple times following treatment to assess in vivo effects overtime.
[412] In addition to the above inquiries, the third inquiry interrogates the
breadth of TGFp function, such as TGFpl
function, involved in a particular disease. In particular, this may be
represented by the number of TGFp1 contexts,
namely, which presenting molecule(s) mediate disease-associated TGFI31
function. TGFpl -specific, broad-
context inhibitors, such as context-independent inhibitors, are advantageous
for the treatment of diseases that
involve both an ECM component and an immune component of TGFp1 function. Such
disease may be associated
with dysregulation in the ECM as well as perturbation in immune cell function
or immune response. Thus, the
TGFp1 inhibitors described herein are capable of targeting ECM-associated
TGFp1 (e.g., presented by LTBP1 or
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LTBP3) as well as immune cell-associated TG931 (e.g., presented by GARP or
LRRC33). Such inhibitors inhibit
all four of the therapeutic targets (e.g., "context-independent" inhibitors):
GARP-associated pro/latent TGF131;
LRRC33-associated pro/latent TGFp1; LTBP1-associated pro/latent TGFp1; and,
LTBP3-associated pro/latent
TGF131, so as to broadly inhibit TG931 function in these contexts.
[413] Whether or not a particular condition of a patient involves or is driven
by multiple aspects of TGFp1 function
may be assessed by evaluating expression profiles of the presenting molecules,
in a clinical sample collected from
the patient. Various assays are known in the art, including RNA-based assays
and protein-based assays, which
may be performed to obtain expression profiles. Relative expression levels
(and/or changes/alterations thereof) of
LTBP1, LTBP3, GARP, and LRRC33 in the sample(s) may indicate the source and/or
context of TGFp1 activities
associated with the condition. For instance, a biopsy sample taken from a
solid tumor may exhibit high expression
of all four presenting molecules. For example, LTBP1 and LTBP3 may be highly
expressed in CAFs within the
tumor stroma, while GARP and LRRC33 may be highly expressed by tumor-
associated immune cells, such as
Tregs and leukocyte infiltrate, respectively.
[414] Accordingly, the disclosure includes a method for determining (e.g.,
testing or confirming) the involvement
of TGFp1 in the disease, relative to TGFp2 and TGFp3. In some embodiments, the
method further comprises a
step of: identifying a source (or context) of disease-associated TGFp1. In
some embodiments, the source/context
is assessed by determining the expression of TGFp presenting molecules, e.g.,
LTBP1, LTBP3, GARP and
LRRC33 in a clinical sample taken from patients. In some embodiments, such
methods are performed ex post
facto.
[415] With respect to LRRC33-positive cells, Applicant of the present
disclosure has recognized that there can be
a significant discrepancy between RNA expression and protein expression of
LRRC33. In particular, while a select
cell type appears to express LRRC33 at the RNA level, only a subset of such
cells express the LRRC33 protein on
the cell-surface. It is contemplated that LRRC33 expression may be
highly regulated via protein
trafficking/localization, for example, in terms of plasma membrane insertion
and rapid internalization. Therefore, in
certain embodiments, LRRC33 protein expression may be used as a marker
associated with a diseased tissue
(such as tumor tissues) enriched with, for example, activated/M2-like
macrophages and MDSCs.
[416] In a related aspect, the present disclosure provides therapeutic use and
related treatment methods
comprising an immune checkpoint inhibitor, e.g., a PD-(L)1 antibody. Non-
limiting examples of useful checkpoint
inhibitors include: ipilimumab (Yervoy0); nivolumab (Opdivo0); pembrolizumab
(Keytruda0); avelumab
(Bavencio0); cemiplimab (Libtayog; atezolizumab (Tecentriq0); budigalimab
(ABBV-181); durvalumab (Imfinzie),
etc.
[417] According to the present disclosure, a cancer treatment method may
include a checkpoint inhibitor for use
in the treatment of cancer in a subject, wherein the treatment comprises
administration of a checkpoint inhibitor to
the subject who is treated with a TGFp inhibitor or vice versa (i.e.,
administration of a TGFI3 inhibitor to a subject
who is treated with a checkpoint inhibitor), wherein, upon treatment of the
TGFp inhibitor, circulating MDSC levels
in a sample collected from the subject are reduced, as compared to prior to
the treatment. The sample may be a
blood sample or a sample of blood component. The checkpoint inhibitor may be a
PD-1 antibody. The checkpoint
inhibitor may be a PD-L1 antibody. The checkpoint inhibitor may be a CTLA4
antibody. In some embodiments, the
checkpoint inhibitor is selected from the group consisting of ipilimumab
(e.g., Yervoye); nivolumab (e.g., Opdivo0);
pembrolizumab (e.g., Keytruda0); avelumab (e.g., Bavencio0); cemiplimab (e.g.,
Libtayo0); atezolizumab (e.g.,
Tecentriqe); budigalimab (ABBV-181); and durvalumab (e.g., Imfinzie).
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[418] According to the present disclosure, a cancer treatment method may
include a checkpoint inhibitor for use
in the treatment of cancer in a subject who is poorly responsive to the
checkpoint inhibitor, or wherein the subject
has a cancer with primary resistant to the checkpoint inhibitor, wherein the
treatment comprises administering to
the subject a TGFp inhibitor, measuring circulating MDSC levels before and
after the administration of the TGF13
inhibitor, and if circulating MDSCs are reduced after the TGFP inhibitor
administration, further administering a
checkpoint inhibitor to the subject in an amount sufficient to treat cancer.
The checkpoint inhibitor may be a PD-1
antibody. The checkpoint inhibitor may be a PD-L1 antibody. The checkpoint
inhibitor may be a CTLA4 antibody.
In some embodiments, the checkpoint inhibitor is selected from the group
consisting of ipilimumab (e.g., Yervoye);
nivolumab (e.g., Opdivo0); pembrolizumab (e.g., KeytrudaS); avelumab (e.g.,
Bavencio0); cemiplimab (e.g.,
Libtayoe); atezolizumab (e.g., Tecentrig0); budigalimab (ABBV-181); and
durvalumab (e.g., Imfinzie). Optionally,
the TGFp inhibitor is an isoform-selective inhibitor of TGF(31, wherein
optionally the inhibitor is an activation inhibitor
of TG931 or neutralizing antibody that selectively binds TGFI31; or an isoform-
non-selective inhibitor (e.g., inhibitors
of TGFp1/2/3, TGF51/3, TGFp1/2).
Combination Therapy
[419] Disclosed herein are pharmaceutical compositions of a TGFp inhibitor,
e.g., an antibody or antigen-binding
portion thereof, described herein, and related methods used as, or referring
to, combination therapies for treating
subjects who may benefit from TGFP inhibition in vivo. In any of these
embodiments, such subjects may receive
combination therapies that include a first composition comprising at least one
TGFp inhibitor, e.g., Ab6, in
conjunction with at least a second composition comprising at least one
additional therapeutic intended to treat the
same or overlapping disease or clinical condition. In some embodiments, such
subjects may receive an additional
third composition comprising at least one additional therapeutic intended to
treat the same or overlapping disease
or clinical condition. The TGFp inhibitor may be a TGFp1 inhibitor, such as a
TGFp1-selective inhibitor (e.g., one
which does not inhibit TGFp2 and/or TGFp3 signaling at a therapeutically
effective dose), e.g., Ab6, or an isoform-
non-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a
neutralizing antibody that bind two or more
of TGFp1/2/3, e.g., GC1008 or variants, an antibody that bind TGF31/3, ligand
trap, e.g., a TGFp1/3 inhibitor,
and/or an integrin inhibitor (e.g., an antibody that binds to aVp1, aVp3,
aVI35, aVp6, aV138, a5(31, a11b133, or a8p1
integrins, and inhibits downstream activation of TGFp. e.g., selective
inhibition of TGFp1 and/or TGFp3). The first,
second, and third compositions may both act on the same cellular target, or
discrete cellular targets. In some
embodiments, the first, second, and third compositions may treat or alleviate
the same or overlapping set of
symptoms or aspects of a disease or clinical condition. In some embodiments,
the first, second, and third
compositions may treat or alleviate a separate set of symptoms or aspects of a
disease or clinical condition. In
some embodiments, the combination therapy may comprise more than three
compositions, which may act on the
same target or discrete cellular targets, and which may treat or alleviate the
same or overlapping set of symptoms
or aspects of a disease or clinical condition. To give but one example, the
first composition may treat a disease or
condition associated with TGF(3 signaling, while the second composition may
treat inflammation or fibrosis
associated with the same disease, etc. As another example, the first
composition may treat a disease or condition
associated with TGFp signaling, while the second and third compositions may
have anti-neoplastic effects and/or
help reverse immune suppression. In certain embodiments, the first composition
may be a TGFp inhibitor (e.g., a
TGFP1 inhibitor described herein), the second composition may be a checkpoint
inhibitor, and the third composition
may be a checkpoint inhibitor distinct from the second composition. In certain
embodiments, a first composition
comprising a TGFp inhibitor (e.g., a TGFp1 inhibitor described herein) is
combined with a checkpoint inhibitor and
a chemotherapeutic agent. In certain embodiments, a first composition
comprising a TGFp inhibitor (e.g., a TGF(31
inhibitor described herein) is combined with two distinct checkpoint
inhibitors and a chemotherapeutic agent. Such
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combination therapies may be administered in conjunction with each other. As
noted above, the phrase "in
conjunction with," in the context of combination therapies, means that
therapeutic effects of a first therapy overlap
temporally andlor spatially with therapeutic effects of a second therapy in
the subject receiving the combination
therapy. The first, second, and/or additional compositions may be administered
concurrently (e.g., simultaneously),
separately, or sequentially. Thus, the combination therapies may be formulated
as a single formulation for
concurrent or simultaneous administration, or as separate formulations for
concurrent (e.g., simultaneous),
separate, or sequential administration of the therapies. As used herein, a
combination therapy may comprise two
or more therapies (e.g., compositions) given in a single bolus or
administration, or in a single patient visit (e.g., to
or with a medical professional) but in two or more separate boluses or
administrations, or in separate patient visits
(and, e.g., in two or more separate boluses or administrations). For instance,
the therapies may be given less than
about 5 minutes apart, or 1 minute apart. The therapies may be given less than
about 30 minutes or 1 hour apart
(e.g., in a single patient visit). In some embodiments, the therapies may be
given more than about 1 minute, about
2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 30
minutes, about 45 minutes, about 1
hour, about 2 hour, about 4 hours, about 6 hours, about 8 hours, about 10
hours, about 1 day, about 2 day, about
3 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 1
month, or more, apart. In some
embodiments, the therapies may be given more than about 1 day apart (e.g., in
separate visits). The therapies
may be given within 3 months (e.g., within 1 month) of one another. In some
embodiments, a therapy may be given
according to the dosing schedule of one or more approved therapeutics for
treating the condition (e.g., administered
at the same frequency as for an approved checkpoint inhibitor or other
chemotherapeutic agent).
[420] In certain embodiments, the TGFp inhibitor (e.g., a TGFp1 inhibitor
described herein) may be administered
in an amount of about 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80
mg, or less. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered in an amount
of about 3000 mg, 2400 mg, 2000
mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less, e.g., at a frequency of once
every two weeks, three weeks, or any
multiples of two weeks or three weeks (e.g., once every four weeks, once every
six weeks), wherein the TGFp
inhibitor (e.g., Ab6) is administered alone or in combination with a
checkpoint inhibitor therapy, (e.g., any approved
checkpoint inhibitor therapy, including, but not limited to, antibodies or
other agents against cytotoxic 1-lymphocyte
antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell
death receptor ligand 1 (PD-L1), T-
cell immunoglobulin domain and mucin domain-3 (TIM3), lymphocyte-activation
gene 3 (LAG3), killer cell
immunoglobulin-like receptor (KIR), glucocorticoid-induced tumor necrosis
factor receptor (GITR), or V-domain
immunoglobulin (Ig)-containing suppressor of 1-cell activation (VISTA)).
[421] In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at 3000 mg once every
six weeks. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered at 3000 mg once every six
weeks, wherein the TGFP inhibitor (e.g., Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered alone
at 2400 mg once every six weeks.
In certain embodiments, the TG93 inhibitor (e.g., Ab6) may be administered at
2400 mg once every six weeks,
wherein the TGFp inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFP inhibitor (e.g., Ab6) may be administered alone at 2000
mg once every six weeks. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at
2000 mg once every six weeks,
wherein the TGFp inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered alone at 1600
mg once every six weeks. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at
1600 mg once every six weeks,
wherein the TGFp inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered alone at 800
mg once every six weeks. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at 800 mg once
every six weeks, wherein the
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TGFI3 inhibitor (e.g., Ab6) is administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments,
the TGFI3 inhibitor (e.g., Ab6) may be administered alone at 240 mg once every
six weeks. In certain embodiments,
the TGF[3 inhibitor (e.g., Ab6) may be administered at 240 mg once every six
weeks, wherein the TGFp inhibitor
(e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGF[3 inhibitor
(e.g., Ab6) may be administered alone at 80 mg once every six weeks. In
certain embodiments, the TGFP inhibitor
(e.g., Ab6) may be administered at 80 mg once every six weeks, wherein the
TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6)
may be administered alone at an amount of less than 80 mg once every six
weeks. In certain embodiments, the
TGFI3 inhibitor (e.g., Ab6) may be administered at an amount of less than 80
mg once every six weeks, wherein
the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an anti-PD-
(L)1 therapy. In some embodiments,
the TGFI3 inhibitor (e.g., Ab6) may be administered every six weeks at a dose
of 50-3000 mg, e.g., 200-3000 mg,
200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg,
1000-3000 mg, 1500-2500
mg, 2000-3000 mg.
[422] In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered alone at 3000 mg once every
four weeks. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 3000 mg once every
four weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered alone
at 2400 mg once every four weeks.
In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
2400 mg once every four weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGF[3 inhibitor (e.g., Ab6) may be administered alone at 2000
mg once every four weeks. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
2000 mg once every four weeks,
wherein the TGFp inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered alone at 1600
mg once every four weeks. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
1600 mg once every four weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered alone at 800
mg once every four weeks. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at 800
mg once every four weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGF13 inhibitor (e.g., Ab6) may be administered alone at 240
mg once every four weeks. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
240 mg once every four weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered alone at 80
mg once every four weeks. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at 80 mg once
every four weeks, wherein the
TGFI3 inhibitor (e.g., Ab6) is administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the
TGFI3 inhibitor (e.g., Ab6) may be administered alone at an amount of less
than 80 mg once every four weeks. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at an
amount of less than 80 mg once
every four weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1
therapy. In some embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered every four weeks at a dose of
50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000
mg, 1000-2000 mg, 250-
2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-3000 mg.
[423] In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at 3000 mg once every
three weeks. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 3000 mg once every
three weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy.
In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
3000 mg at a frequency of any
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multiples of three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered alone at 2400 mg once
every three weeks. In certain embodiments, the TGF[3 inhibitor (e.g., Ab6) may
be administered at 2400 mg once
every three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFP inhibitor (e.g., Ab6) may be
administered at 2400 mg at a frequency of
any multiples of three weeks, wherein the TGFp inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may
be administered alone at 2000 mg
once every three weeks. In certain embodiments, the TGFI3 inhibitor (e.g.,
Ab6) may be administered at 2000 mg
once every three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 2000 mg at a frequency
of any multiples of three weeks, wherein the TG93 inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may
be administered alone at 1600 mg
once every three weeks. In certain embodiments, the TGFI3 inhibitor (e.g.,
Ab6) may be administered at 1600 mg
once every three weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered
in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered at 1600 mg at a frequency
of any multiples of three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may
be administered alone at 800 mg
once every three weeks. In certain embodiments, the TGFI3 inhibitor (e.g.,
Ab6) may be administered at 800 mg
once every three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered at 800 mg at a frequency
of any multiples of three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at 240 mg
once every three weeks. In certain embodiments, the TGFI3 inhibitor (e.g.,
Ab6) may be administered at 240 mg
once every three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered at 240 mg at a frequency
of any multiples of three weeks, wherein the TGF13 inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may
be administered alone at 80 mg once
every three weeks. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may
be administered at 80 mg once
every three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 80 mg at a frequency of
any multiples of three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may
be administered alone at an amount
of less than 80 mg once every three weeks. In certain embodiments, the TGFI3
inhibitor (e.g., Ab6) may be
administered at an amount of less than 80 mg once every three weeks, wherein
the TGFp inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6)
may be administered at less than 80 mg at a frequency of any multiples of
three weeks, wherein the TGFI3 inhibitor
(e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In
some embodiments, the TGFI3 inhibitor
(e.g., Ab6) may be administered every three weeks at a dose of 50-3000 mg,
e.g., 200-3000 mg, 200-1000 mg,
250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg,
1500-2500 mg, 2000-
3000 mg.
[424] In certain embodiments, the TGF[3 inhibitor (e.g., Ab6) may be
administered alone at 3000 mg once every
two weeks. In certain embodiments, the TGF[3 inhibitor (e.g., Ab6) may be
administered at 3000 mg once every
two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
3000 mg at a frequency of any
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multiples of two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered alone at 2400 mg once every
two weeks. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered at 2400 mg once every
two weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFP inhibitor (e.g., Ab6) may be administered at
2400 mg at a frequency of any
multiples of two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at 2000 mg once every
two weeks. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered at 2000 mg once every
two weeks, wherein the TGFP inhibitor (e.g. Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
2000 mg at a frequency of any
multiples of two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at 1600 mg once every
two weeks. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 1600 mg once every
two weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at
1600 mg at a frequency of any
multiples of two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at 800 mg once every
two weeks. In certain embodiments, the TGF[3 inhibitor (e.g., Ab6) may be
administered at 800 mg once every two
weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at BOO
mg at a frequency of any multiples
of two weeks, wherein the TGFp inhibitor (e.g., Ab5) is administered in
combination with an anti-PD-(L)1 therapy.
In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered
alone at 240 mg once every two weeks.
In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
240 mg once every two weeks,
wherein the TGFP inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at 240 mg at a
frequency of any multiples of two
weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered alone
at 80 mg once every two weeks.
In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
80 mg once every two weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at 80 mg at a
frequency of any multiples of two
weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered alone
at an amount of less than 80 mg
once every two weeks. In certain embodiments, the TGFp inhibitor (e.g., Ab6)
may be administered at an amount
of less than 80 mg once every two weeks, wherein the TGFI3 inhibitor (e.g.,
Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor
(e.g., Ab6) may be administered at less
than 80 mg at a frequency of any multiples of two weeks, wherein the TGFI3
inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In some embodiments, the TGFp
inhibitor (e.g., Ab6) may be
administered every two weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-
1000 mg, 250-750 mg, 500-2000
mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-
3000 mg.
[425] In certain embodiments, a TGFI3 inhibitor (e.g., a TGF01 inhibitor
described herein, e.g, Ab6) may be
administered in combination or in conjunction with a genotoxic therapy.
Without wishing to be bound by theory, in
some embodiments, a subject receiving a genotoxic therapy may have elevated
levels of TGF(31, for example
where administration of the genotoxic therapy may cause an increase in TGF(31
levels in the subject. Thus, in some
embodiments, administration of a TGFI3 inhibitor (e.g., a TGFp1 inhibitor
described herein, e.g, Ab6) in combination
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or in conjunction with a genotoxic therapy may provide added therapeutic
benefit by binding to this increased
TGF131 following treatment with the genotoxic therapy. In some embodiments,
the genotoxic therapy may be a
chemotherapy and/or a radiation therapy.
[426] In certain embodiments, a TGFp inhibitor (e.g., a TGFp1 inhibitor
described herein, e.g, Ab6) may be
administered in combination or in conjunction with checkpoint inhibitor
therapy, e.g., an anti-PD-(L)1 therapy), to a
subject who is a non-responder to checkpoint inhibitor therapy, e.g., an anti-
PD-(L)1 therapy). In certain
embodiments, the subject has not previously received a checkpoint inhibitor
therapy. Exemplary checkpoint
inhibitors include, but are not limited to, nivolumab (Opdivo , anti-PD-1
antibody), pembrolizumab (Keytruda ,
anti-PD-1 antibody), cemiplimab (Libtayo , anti-PD-1 antibody), budigalimab
(ABBV-181, anti-PD-1 antibody);
BMS-936559 (anti-PD-L1 antibody), atezolizumab (Tecentriq , anti-PD-L1
antibody), avelumab (Bavencio , anti-
PD-L1 antibody), durvalumab (Imfinzi , anti-PD-L1 antibody), ipilimumab
(Yervoy , anti-CTLA4 antibody),
tremelimumab (anti-CTLA4 antibody), IMP-321 (eftilgimod alpha or "ImmuFact0",
anti-LAG3 large molecule), BMS-
986016 (Relatlimab, anti-LAG3 antibody), and lirilumab (anti-KIR20L-1, -2, -3
antibody).
[427] In certain embodiments, the TGFP inhibitor (e.g. Ab6) is administered
alone to a subject having advanced
solid cancer. In certain embodiments, the TGFp inhibitor (e.g., Ab6) is
administered in combination with a
checkpoint inhibitor therapy to a subject having advanced solid cancer. In
certain embodiments, the TGFp inhibitor
(e.g., Ab6) is administered in combination with a checkpoint inhibitor therapy
to a subject having advanced solid
cancer, wherein the subject is a non-responder to prior checkpoint inhibitor
therapy. In some embodiments, the
subject has non-small cell lung cancer (NSCLC), melanoma (MEL), or urothelial
carcinoma (UC), including
metastatic urothelial carcinoma (mUC). In some embodiments, the subject has
ovarian cancer, colorectal cancer
(CRC), bladder cancer, renal cell carcinoma (RCC), including clear cell RCC,
papillary RCC, chromophobe RCC,
collecting duct RCC, or unclassified RCC, or head and neck cancer (e.g., head
and neck squamous cell carcinoma
(HNSCC) or oropharynx cancer)). In some embodiments, the subject has
esophageal cancer, gastric cancer,
hepatocellular carcinoma (HCC), triple-negative breast cancer (TNBC), cervical
cancer, endometrial cancer, basal
cell carcinoma (BCC), cutaneous squamous cell carcinoma (CSCC), merkel cell
carcinoma (MCC), small-cell lung
cancer (SCLC), primary mediastinal large B-cell lymphoma (PMBCL), Hodgkin's
lymphoma, microsatellite
instability high cancer (MSI-H) (e.g., MSI-H CRC), mismatch repair deficient
cancer (dMMR)(e.g., dMMR CRC),
tumor mutational burden-high (TMB-H) cancer, or malignant pleural mesothelioma
(MPM). In certain embodiments,
the TGFp inhibitor (e.g., Ab6) is administered in combination with a
checkpoint inhibitor therapy to a subject having
urothelial carcinoma (UC), including metastatic urothelial carcinoma (mUC),
melanoma (MEL), or non-small cell
lung cancer NSCLC. In certain embodiments, the subject is a non-responder to
checkpoint inhibitor therapy. In
certain embodiments, the checkpoint inhibitor therapy is pembrolizumab (e.g.,
Keytruda0). In certain embodiments,
the checkpoint inhibitor therapy is nivolumab (e.g., Opdivo ). In certain
embodiments, the checkpoint inhibitor
therapy is cemiplimab (e.g., Libtayo0). In certain embodiments, the checkpoint
inhibitor therapy is atezolizumab
(e.g., Tecentrige). In certain embodiments, the checkpoint inhibitor therapy
is avelumab (e.g., BavencioG). In
certain embodiments, the checkpoint inhibitor therapy is durvalumab (e.g.,
Imfinzi0). In certain embodiments, the
checkpoint inhibitor therapy is budigalimab (e.g., ABBV-181).
[428] In certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered
at 3000 mg, 2400 mg, 2000 mg,
1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with pembrolizumab at a
frequency of once every two
weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once
every four weeks, once every six
weeks). In certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered
in combination with pembrolizumab
to a subject having NSCLC, UC, MEL, esophageal cancer, gastric cancer, HNSCC,
HCC, cervical cancer, SCLC,
PMBCL, Hodgkin's lymphoma, MSI-H or dMMR cancer, or TMB-H cancer. In certain
embodiments, the subject is
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a non-responder to pembrolizumab. In certain embodiments, the subject has not
received pembrolizumab
previously. In certain embodiments, the TGFp inhibitor (e.g., Ab6) is
administered in combination with
pembrolizumab to a subject having NSCLC who is a non-responder to
pembrolizumab treatment. In certain
embodiments, the subject having NSCLC has not received pembrolizumab
previously. In certain embodiments, the
TGFP inhibitor (e.g., Ab6) is administered in combination with pembrolizumab
to a subject having MEL who is a
non-responder to pembrolizumab treatment. In certain embodiments, the subject
having MEL has not received
pembrolizumab previously. In certain embodiments, the TGFp inhibitor (e.g.,
Ab6) is administered in combination
with pembrolizumab to a subject having UC or mUC who is a non-responder to
pembrolizumab treatment. In certain
embodiments, the subject having UC or mUC has not received pembrolizumab
previously.
[429] In certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered
at 3000 mg, 2400 mg, 2000 mg,
1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with nivolumab at a
frequency of once every two weeks,
three weeks, or any multiples of two weeks or three weeks (e.g., once every
four weeks, once every six weeks). In
certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered in
combination with nivolumab to a subject
having NSCLC, UC, MEL, esophageal cancer, HNSCC, HCC, RCC, Hodgkin's lymphoma,
MSI-H or dMMR CRC,
or MPM. In certain embodiments, the subject is a non-responder to nivolumab.
In certain embodiments, the subject
has not received nivolumab previously.
[430] In certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered
at 3000 mg, 2400 mg, 2000 mg,
1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with cemiplimab at a
frequency of once every two weeks,
three weeks, or any multiples of two weeks or three weeks (e.g., once every
four weeks, once every six weeks). In
certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered in
combination with cemiplimab to a subject
having BCC or CSCC. In certain embodiments, the subject is a non-responder to
cemiplimab. In certain
embodiments, the subject has not received cemiplimab previously.
[431] In certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered
at 3000 mg, 2400 mg, 2000 mg,
1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with atezolizumab at a
frequency of once every two
weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once
every four weeks, once every six
weeks). In certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered
in combination with atezolizumab
to a subject having NSCLC, MEL, HOC, TNBC, or SOLO. In certain embodiments,
the subject is a non-responder
to atezolizumab. In certain embodiments, the subject has not received
atezolizumab previously.
[432] In certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered
at 3000 mg, 2400 mg, 2000 mg,
1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with avelumab at a
frequency of once every two weeks,
three weeks, or any multiples of two weeks or three weeks (e.g., once every
four weeks, once every six weeks).
In embodiments, the TGFp inhibitor (e.g., Ab6) is administered in combination
with avelumab to a subject having
UC or MCC. In certain embodiments, the subject is a non-responder to avelumab.
In certain embodiments, the
subject has not received avelumab previously.
[433] In certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered
at 3000 mg, 2400 mg, 2000 mg,
1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with durvalumab at a
frequency of once every two weeks,
three weeks, or any multiples of two weeks or three weeks (e.g., once every
four weeks, once every six weeks). In
embodiments, the TGFp inhibitor (e.g., Ab6) is administered in combination
with durvalumab to a subject having
NSCLC or SCLC. In certain embodiments, the subject is a non-responder to
durvalumab. In certain embodiments,
the subject has not received durvalumab previously.
[434] In certain embodiments, the TGFp inhibitor (e.g., Ab6) is administered
at 3000 mg, 2400 mg, 2000 mg,
1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with budigalimab (e.g.,
ABBV-181), e.g., at a dose of
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250 mg, 375 mg, or 500 mg at a frequency of once every two weeks, three weeks,
or any multiples of two weeks
or three weeks (e.g., once every four weeks, once every six weeks). In certain
embodiments, budigalimab (e.g.,
ABBV-181) is administered at 250 mg once every two weeks. In certain
embodiments, budigalimab (e.g., ABBV-
181) is administered at 375 mg once every three weeks. In certain embodiments,
budigalimab (e.g., ABBV-181) is
administered at 500 mg once every four weeks. In certain embodiments, the TGFP
inhibitor (e.g., Ab6) is
administered in combination with budigalimab to a subject having a locally
advanced or metastatic solid tumor.
See, e.g., NCT 03821 935 (Study to determine the safety, tolerability,
pharmacokinetics and recommended phase
2 dose (RP2D) of ABBV-151 as a single agent and in combination with ABBV-181
in participants with locally
advanced or metastatic solid tumors.
https://clinicaltrials.govict2/show/NCT03821935). In certain embodiments,
budigalimab is administered once every four weeks. In certain embodiments, the
TGFI3 inhibitor (e.g., Ab6) is
administered in combination with budigalimab to a subject having triple-
negative breast cancer (TN BC), pancreatic
adenocarcinoma, urothelial cancer, or Hepatocellular carcinoma (HCC). In
certain embodiments, the TGFp inhibitor
(e.g., Ab6) is administered in combination with budigalimab to a subject
having non-small cell lung cancer or head
and neck squamous cell carcinoma. Italian et al. Cancer Immunol lmmunother.
2022 Feb; 71(2):417-431. In
certain embodiments, the subject is a non-responder to budigalimab. In certain
embodiments, the subject has not
received budigalimab previously.
[435] In certain embodiments, combination therapies produce synergistic
effects in the treatment of a disease.
The term "synergistic" refers to effects that are greater than additive
effects (e.g., greater efficacy) of each
monotherapy in aggregate.
[436] In some embodiments, combination therapies comprising a pharmaceutical
composition described herein
produce efficacy that is overall equivalent to that produced by another
therapy (such as monotherapy of a second
agent) but are associated with fewer unwanted adverse effect or less severe
toxicity associated with the second
agent, as compared to the monotherapy of the second agent. In some
embodiments, such combination therapies
allow lower dosage of the second agent but maintain overall efficacy. Such
combination therapies may be
particularly suitable for patient populations where a long-term treatment is
warranted and/or involving pediatric
patients.
[437] The disclosure provides pharmaceutical compositions and methods for use
in, and as, combination
therapies for the reduction of TGFp1 protein activation and the treatment or
prevention of diseases or conditions
associated with TGFI31 signaling, as described herein. Accordingly, the
methods or the pharmaceutical
compositions may further comprise a second therapy. In some embodiments, the
methods or pharmaceutical
compositions disclosed herein may further comprise a third therapy. In some
embodiments, the second therapy
and/or the third therapy may be useful in treating or preventing diseases or
conditions associated with TGFp1
signaling. The second therapy and/or the third therapy may diminish or treat
at least one symptom(s) associated
with the targeted disease. The first, second, and third therapies may exert
their biological effects by similar or
unrelated mechanisms of action; or either one or both of the first and second
therapies may exert their biological
effects by a multiplicity of mechanisms of action. In some embodiments, the
second therapy and a TGFI3 inhibitor
disclosed herein (e.g., a TGF(31-selective inhibitor disclosed herein) are
present in a single formulation or in
separate formulations contained within in a single package or kit. In some
embodiments, the second therapy, the
third therapy, and a TGFP inhibitor disclosed herein (e.g., a TGFP1-selective
inhibitor disclosed herein) are present
in a single formulation or in separate formulations contained within in a
single package or kit. In some embodiments,
the second therapy, and a TGFp inhibitor disclosed herein (e.g., a TGFp1-
selective inhibitor disclosed herein) are
comprised in a single molecule, e.g., in a bispecific antibody or other
multispecific construct or, wherein the
checkpoint inhibitor is a small molecule, in an antibody-drug conjugate. In
some embodiments, the second therapy,
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the third therapy, and a TGFp inhibitor disclosed herein (e.g., a TGF(31-
selective inhibitor disclosed herein) are
comprised in a single molecule, e.g., in a bispecific antibody or other
multispecific construct or, wherein the
checkpoint inhibitor is a small molecule, in an antibody-drug conjugate.
Examples of engineered constructs with
TGFp inhibitory activities include M7824 (Bintrafusp alfa) and AVID200. M7824
is a bifunctional fusion protein
composed of 2 extracellular domains of TGF-]3R11 (a TGF-P "trap") fused to a
human IgG1 monoclonal antibody
against PD-L1. AVID200 is an engineered TGE-p ligand trap comprised of TGF- p
receptor ectodomains fused to
a human Fc domain.
[438] It should be understood that the pharmaceutical compositions described
herein may have the first and
second therapies in the same pharmaceutically acceptable carrier or in a
different pharmaceutically acceptable
carrier for each described embodiment. It further should be understood that
the first and second therapies may be
administered concurrently (e.g., simultaneously), separately, or sequentially
within described embodiments.
[439] The one or more anti-TGFp antibodies, or antigen binding portions
thereof, of the disclosure may be used
in conjunction with one or more of additional therapeutic agents. Examples of
the additional therapeutic agents
which can be used with an anti-TGFP antibody of the disclosure include, but
are not limited to: cancer vaccines,
engineered immune cell therapies, chemotherapies, radiation therapies (e.g.,
radiotherapeutic agents), a modulator
of a member of the TGFp superfamily, such as a myostatin inhibitor (e.g., a
myostatin inhibitor disclosed in
W02016/073853 and W02017/049011, the contents of which are hereby incorporated
in their entirety), and a
GDF11 inhibitor; a VEGF agonist; a VEGF inhibitor (such as bevacizumab); an
IGF1 agonist; an FXR agonist; a
CCR2 inhibitor; a CCR5 inhibitor; a dual CCR2/CCR5 inhibitor; CCR4 inhibitor,
a lysyl oxidase-like-2 inhibitor; an
ASK1 inhibitor; an Acetyl-CoA Carboxylase (ACC) inhibitor; a p38 kinase
inhibitor; pirfenidone; nintedanib; an M-
CSF inhibitor (e.g., M-CSF receptor antagonist and M-CSF neutralizing agents);
a MAPK inhibitor (e.g., Erk
inhibitor), an immune checkpoint agonist or antagonist; an IL-11 antagonist;
and IL-6 antagonist, and the like. Other
examples of the additional therapeutic agents which can be used with the TGFp
inhibitors include, but are not
limited to, an indoleamine 2,3-dioxygenase (IDO) inhibitor, an arginase
inhibitor, a tyrosine kinase inhibitor, Ser/Thr
kinase inhibitor, a dual-specific kinase inhibitor. In some embodiments, such
an agent may be a PI3K inhibitor, a
PKC inhibitor, or a JAK inhibitor.
[440] While checkpoint inhibitor (CPI) therapies have transformed the
treatment of solid tumors, less than half of
cancer patients are eligible fortreatment with an approved CPI and of those, <
13% respond to CPI therapy (Haslam
2019). Given these data, there remains a significant unmet need across solid
tumor indications with approved and
unapproved therapies.
[441] Recent data suggest that the effectiveness of immunomodulatory
strategies require the presence of a
baseline immune response. Tumors lacking a pre-existing immune response or
tumors with low numbers of T cells
in the tumor core and an enrichment of T cells in the invasive margin or
stroma (e.g., in an immune-excluded tumor)
have been associated with poor response to CPI (Galon and Bruni 2019. Nat Rev
Drug Discov. 18(3): 197-218).
The TGFp pathway has been implicated in mediating primary resistance to CPI
therapies, and as such, combination
therapy with an anti-latent TGFp monoclonal antibody may increase efficacy in
patients with an inadequate
response to CPI monotherapy.
[442] The current disclosure includes use of a TGFp inhibitor, e.g., Ab6, as a
potential anti-cancer therapy alone
or in combination with other therapies for the treatment of solid tumors and
rare hematological malignancies for
which TGFp signaling dysregulation has been implicated as a mediator of the
disease process. In some
embodiments, combination therapy comprising a TGFp inhibitor, e.g., Ab6, and
at least one additional agent may
be efficacious in patients with advanced solid tumors such as cutaneous
melanoma, urothelial carcinoma (UC),
non-small cell lung cancer (NSCLC), and head and neck cancer. In some
embodiments, combination therapy
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comprising a TGFp inhibitor, e.g., Ab6, and at least one additional agent may
be efficacious in patients with
immune-excluded tumors such as non-small cell lung cancer, melanoma, renal
cell carcinoma, triple-negative
breast cancer, gastric cancer, microsatellite stable-colorectal cancer,
pancreatic cancer, small cell lung cancer,
HER2-positive breast cancer, or prostate cancer.
[443] In some embodiments, the at least one additional agent (e.g., cancer
therapy agent) used in a method or
composition disclosed herein is a checkpoint inhibitor. In some embodiments,
the at least one additional agent is
selected from the group consisting of a PD-1 antagonist, a PD-L1 antagonist, a
PD-L1 or PD-L2 fusion protein, a
CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody, an anti-ICOSL
antibody, an anti-B7H3 antibody, an
anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-0X40
antibody (0X40 agonist), an anti-
CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41 BB
antibody, an anti-PD-1 antibody, an
oncolytic virus, and a PARP inhibitor. Exemplary checkpoint inhibitors
include, but are not limited to, nivolumab
(Oodivoe, anti-PD-1 antibody), pembrolizumab (Keytruda , anti-PD-1 antibody),
cemiplimab (Libtayo , anti-PD-1
antibody), budigalimab (ABBV-181, anti-PD-1 antibody), BMS-935559 (anti-PD-L1
antibody), atezolizumab
(Tecentriqe, anti-PD-L1 antibody), avelumab (Bavencioe, anti-PD-L1 antibody),
durvalumab (Imfinzi , anti-PD-L1
antibody), ipilimumab (Yervoy , anti-CTLA4 antibody), tremelimumab (anti-CTLA4
antibody), IMP-321 (eftilgimod
alpha or "ImmuFacte", anti-LAG3 large molecule), BMS-986016 (Relatlimab, anti-
LAG3 antibody), and lirilumab
(anti-KIR2DL-1, -2, -3 antibody). In some embodiments, the TGFp inhibitors
disclosed herein is used in the
treatment of cancer in a subject who is a poor responder or non-responder of a
checkpoint inhibition therapy, such
as those listed herein. In some embodiments, the checkpoint inhibitor and a
TGF6 inhibitor (e.g., a TGF131-
selective inhibitor disclosed herein) are comprised in a single molecule,
e.g., in a bispecific antibody or other
multispecific construct or, wherein the checkpoint inhibitor is a small
molecule, in an antibody-drug conjugate.
[444] In some embodiments, the disclosure encompasses use of a TGFP inhibitor,
e.g., Ab6, in combination with
at least one checkpoint inhibitor therapy for the treatment of solid tumors
and/or hematological malignancies for
which TGFp signaling dysregulation has been implicated as a mediator of the
disease process. In certain
embodiments, the combination therapy may be administered to patients who are
not responsive to checkpoint
inhibitor therapy (e.g., anti-PD-1 or anti-PD-L1 therapy). Such patients may
include, but are not limited to, those
diagnosed with non-small cell lung cancer, urothelial bladder carcinoma,
melanoma, triple-negative breast cancer,
or other advance solid cancers. In certain embodiments, the combination
therapy may comprise a TGFp inhibitor,
e.g., Ab6, and a checkpoint inhibitor therapy (e.g., pembrolizumab). In
certain embodiments, the combination
therapy may be administered to immunotherapy-naive patients (e.g., patients
who have riot previously received a
checkpoint inhibitor therapy) diagnosed with a cancer that has received FDA
approval for treatment with a
checkpoint inhibitor therapy. Such cancer may be gastric cancer (e.g.,
metastatic gastric cancer), urothelial bladder
carcinoma, lung cancer, triple-negative breast cancer, renal cell carcinoma,
including clear cell RCC or papillary
RCC, cervical cancer, or head and neck squamous cell carcinoma. In certain
embodiments, the combination
therapy may comprise a TGFp inhibitor, e.g., Ab6, and a checkpoint inhibitor
therapy (e.g., pembrolizumab). certain
embodiments, the combination therapy may further comprise an additional agent,
e.g., an additional checkpoint
inhibitory and/or another chemotherapeutic agent. In certain embodiments, the
combination therapy may be
administered to immunotherapy-naïve patients (e.g., patients who have not
previously received a checkpoint
inhibitor therapy) diagnosed with a cancer that has not received FDA approval
for treatment with a checkpoint
inhibitor therapy. Such cancer may be a microsatellite-stable colorectal
cancer or pancreatic cancer. In certain
embodiments, the combination therapy may comprise a TGFI3 inhibitor, e.g.,
Ab6, a checkpoint inhibitor therapy
(e.g., pembrolizumab), and at least one chemotherapeutic agent (e.g.,
axitinib, paclitaxel, cisplatin, and/or 5-
fluorouracil). In certain embodiments, the checkpoint inhibitor therapy may be
pembrolizumab, nivolumab, and/or
atezolizumab. In certain embodiments, the combination therapy is administered
to patients who have cancers
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characterized as exhibiting an immune-excluded phenotype. In certain
embodiments, additional analyses of a
patient's cancer may be carried out to further inform treatment, and such
analyses may use known cancer-specific
markers including microsatellite instability levels, PD-1 and/or PD-L1
expression level, and/or the presence of
mutations in known cancer driver genes such as EGFR, ALK, ROS1, BRAF. In
certain embodiments, tumor PD-L1
expression may be used as a biomarker of therapeutic response.
[445] In certain embodiments, the TGFp inhibitor, e.g., Ab6, may be
administered in an amount of about 3000
mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less. In certain
embodiments, the TGFp inhibitor
(e.g., Ab6) may be administered in an amount of about 3000 mg, 2400 mg, 2000
mg, 1600 mg, 800 mg, 240 mg,
80 mg, or less, e.g., at a frequency of once every six weeks, once every four
weeks, once every three weeks, once
every two weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered alone
or in combination with a checkpoint
inhibitor therapy, e.g., an anti-PD-(L)1 therapy. In certain embodiments, the
TGFp inhibitor (e.g., Ab6) may be
administered alone at 3000 mg once every three weeks.
[446] In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at 3000 mg once every
six weeks. In certain embodiments, the TGFP inhibitor (e.g., Ab6) may be
administered at 3000 mg once every six
weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered alone
at 2400 mg once every six weeks.
In certain embodiments, the TG93 inhibitor (e.g., Ab6) may be administered at
2400 mg once every six weeks,
wherein the TGFP inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered alone at 2000
mg once every six weeks. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at
2000 mg once every six weeks,
wherein the TGFp inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy In certain
embodiments, the TGFP inhibitor (e.g., Ab6) may be administered alone at 1600
mg once every six weeks. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at
1600 mg once every six weeks,
wherein the TGFp inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered alone at 800
mg once every six weeks. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at 800 mg once
every six weeks, wherein the
TGFp inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1
therapy. In certain embodiments,
the TGFp inhibitor (e.g., Ab6) may be administered alone at 240 mg once every
six weeks. In certain embodiments,
the TGFp inhibitor (e.g., Ab6) may be administered at 240 mg once every six
weeks, wherein the TGFp inhibitor
(e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFI3 inhibitor
(e.g., Ab6) may be administered alone at 80 mg once every six weeks. In
certain embodiments, the TGFp inhibitor
(e.g., Ab6) may be administered at 80 mg once every six weeks, wherein the
TGFp inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1 therapy. In certain
embodiments, the TGFP inhibitor (e.g., Ab6)
may be administered alone at an amount of less than 80 mg once every six
weeks. In certain embodiments, the
TGFp inhibitor (e.g., Ab6) may be administered at an amount of less than 80 mg
once every six weeks, wherein
the TGFp inhibitor (e.g., Ab6) is administered in combination with an anti-PD-
(L)1 therapy. In some embodiments,
the TGFP inhibitor (e.g., Ab6) may be administered every six weeks at a dose
of 50-3000 mg, e.g., 200-3000 mg,
200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg,
1000-3000 mg, 1500-2500
mg, 2000-3000 mg.
[447] In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at 3000 mg once every
four weeks. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered at 3000 mg once every
four weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFP inhibitor (e.g., Ab6) may be administered alone
at 2400 mg once every four weeks.
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In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
2400 mg once every four weeks,
wherein the TGFp inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered alone at 2000
mg once every four weeks. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
2000 mg once every four weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered alone at 1600
mg once every four weeks. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
1600 mg once every four weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered alone at 800
mg once every four weeks. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
800 mg once every four weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGF13 inhibitor (e.g., Ab6) may be administered alone at 240
mg once every four weeks. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
240 mg once every four weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGF[3 inhibitor (e.g., Ab6) may be administered alone at 80
mg once every four weeks. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at 80 mg once
every four weeks, wherein the
TGFI3 inhibitor (e.g., Ab6) is administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the
TGFI3 inhibitor (e.g., Ab6) may be administered alone at an amount of less
than 80 mg once every four weeks. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at an
amount of less than 80 mg once
every four weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1
therapy. In some embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered every four weeks at a dose of
50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000
mg, 1000-2000 mg, 250-
2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-3000 mg.
[448] In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered alone at 3000 mg once every
three weeks. In certain embodiments, the TGF[3 inhibitor (e.g., Ab6) may be
administered at 3000 mg once every
three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy.
In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
3000 mg at a frequency of any
multiples of three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TGF[3 inhibitor (e.g., Ab6) may be
administered alone at 2400 mg once
every three weeks. In certain embodiments, the TGFP inhibitor (e.g., Ab6) may
be administered at 2400 mg once
every three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 2400 mg at a frequency of
any multiples of three weeks, wherein the TGFr3 inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFP inhibitor (e.g., Ab6) may be
administered alone at 2000 mg
once every three weeks. In certain embodiments, the TGFI3 inhibitor (e.g.,
Ab6) may be administered at 2000 mg
once every three weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered
in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TG93 inhibitor (e.g., Ab6) may be
administered at 2000 mg at a frequency
of any multiples of three weeks, wherein the TGF13 inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may
be administered alone at 1600 mg
once every three weeks. In certain embodiments, the TGFI3 inhibitor (e.g.,
Ab6) may be administered at 1600 mg
once every three weeks, wherein the TGF[3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TGF[3 inhibitor (e.g., Ab6) may be
administered at 1600 mg at a frequency
of any multiples of three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may
be administered alone at 800 mg
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once every three weeks. In certain embodiments, the TGFI3 inhibitor (e.g.,
Ab6) may be administered at 800 mg
once every three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 800 mg at a frequency
of any multiples of three weeks, wherein the TGFp inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFP inhibitor (e.g., Ab6) may be
administered alone at 240 mg
once every three weeks. In certain embodiments, the TGFp inhibitor (e.g., Ab6)
may be administered at 240 mg
once every three weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered
in combination with an anti-PD-
(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 240 mg at a frequency
of any multiples of three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may
be administered alone at 80 mg once
every three weeks. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may
be administered at 80 mg once
every three weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 80 mg at a frequency of
any multiples of three weeks, wherein the TGFp inhibitor (e.g., Ab6) is
administered in combination with an anti-
PD-(L)1 therapy. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at an amount
of less than 80 mg once every three weeks. In certain embodiments, the TGFI3
inhibitor (e.g., Ab6) may be
administered at an amount of less than 80 mg once every three weeks, wherein
the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6)
may be administered at less than 80 mg at a frequency of any multiples of
three weeks, wherein the TGFI3 inhibitor
(e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In
some embodiments, the TGFp inhibitor
(e.g., Ab6) may be administered every three weeks at a dose of 50-3000 mg,
e.g., 200-3000 mg, 200-1000 mg,
250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg,
1500-2500 mg, 2000-
3000 mg.
[449] In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered alone at 3000 mg once every
two weeks. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 3000 mg once every
two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
3000 mg at a frequency of any
multiples of two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be
administered alone at 2400 mg once every
two weeks. In certain embodiments, the TGFP inhibitor (e.g., Ab6) may be
administered at 2400 mg once every
two weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at
2400 mg at a frequency of any
multiples of two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFP inhibitor (e.g., Ab6) may be
administered alone at 2000 mg once every
two weeks. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 2000 mg once every
two weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at
2000 mg at a frequency of any
multiples of two weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered
in combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered alone at 1600 mg once every
two weeks. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 1600 mg once every
two weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at
1600 mg at a frequency of any
multiples of two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with an anti-PD-(L)1
therapy. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered alone at 800 mg once every
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two weeks. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be
administered at 800 mg once every two
weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at 800
mg at a frequency of any multiples
of two weeks, wherein the TGFI3 inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy.
In certain embodiments, the TGFP inhibitor (e.g., Ab6) may be administered
alone at 240 mg once every two weeks.
In certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at
240 mg once every two weeks,
wherein the TGFp inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFp inhibitor (e.g., Ab6) may be administered at 240 mg at a
frequency of any multiples of two
weeks, wherein the TGFP inhibitor (e.g., Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered alone
at 80 mg once every two weeks.
In certain embodiments, the TG93 inhibitor (e.g., Ab6) may be administered at
80 mg once every two weeks,
wherein the TGFI3 inhibitor (e.g., Ab6) is administered in combination with an
anti-PD-(L)1 therapy. In certain
embodiments, the TGFI3 inhibitor (e.g., Ab6) may be administered at 80 mg at a
frequency of any multiples of two
weeks, wherein the TGFp inhibitor (e.g., Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In
certain embodiments, the TGFp inhibitor (e.g., Ab6) may be administered alone
at an amount of less than 80 mg
once every two weeks. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6)
may be administered at an amount
of less than 80 mg once every two weeks, wherein the TGFI3 inhibitor (e.g.,
Ab6) is administered in combination
with an anti-PD-(L)1 therapy. In certain embodiments, the TGFI3 inhibitor
(e.g., Ab6) may be administered at less
than 80 mg at a frequency of any multiples of two weeks, wherein the TGFI3
inhibitor (e.g., Ab6) is administered in
combination with an anti-PD-(L)1 therapy. In some embodiments, the TGFp
inhibitor (e.g., Ab6) may be
administered every two weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-
1000 mg, 250-750 mg, 500-2000
mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-
3000 mg.
[450] In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) is administered
in combination with pembrolizumab
to a subject having NSCLC, UC, MEL, esophageal cancer, gastric cancer, HNSCC,
HCC, cervical cancer, SCLC,
PMBCL, Hodgkin's lymphoma, MSI-H or dMMR cancer, or TMB-1-1 cancer. In certain
embodiments, the subject is
a non-responder to pembrolizumab. In certain embodiments, the subject has not
received pembrolizumab
previously. In certain embodiments, the TGFI3 inhibitor (e.g., Ab6) is
administered in combination with
pembrolizumab to a subject having NSCLC who is a non-responder to
pembrolizumab treatment. In certain
embodiments, the subject having NSCLC has not received pembrolizumab
previously. In certain embodiments, the
TGFI3 inhibitor (e.g., Ab6) is administered in combination with pembrolizumab
to a subject having MEL who is a
non-responder to pembrolizumab treatment. In certain embodiments, the subject
having MEL has not received
pembrolizumab previously. In certain embodiments, the TGFI3 inhibitor (e.g.,
Ab6) is administered in combination
with pembrolizumab to a subject having UC or mUC who is a non-responder to
pembrolizumab treatment. In certain
embodiments, the subject having UC or mUC has not received pembrolizumab
previously.
[451] In certain embodiments, the TGF13 inhibitor (e.g., Ab6) is administered
in combination with nivolumab to a
subject having NSCLC, UC, MEL, esophageal cancer, HNSCC, HCC, RCC, Hodgkin's
lymphoma, MSI-H or dMMR
CRC, or MPM. In certain embodiments, the subject is a non-responder to
nivolumab. In certain embodiments, the
subject has not received nivolumab previously.
[452] In certain embodiments, the TGFP inhibitor (e.g., Ab6) is administered
in combination with cemiplimab to a
subject having BCC or CSCC. In certain embodiments, the subject is a non-
responder to cemiplimab. In certain
embodiments, the subject has not received cemiplimab previously.
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[453] In certain embodiments, the TGF8 inhibitor (e.g., Ab6) is administered
in combination with atezolizumab to
a subject having NSCLC, MEL, HCC, TNBC, or SCLC. In certain embodiments, the
subject is a non-responder to
atezolizumab. In certain embodiments, the subject has not received
atezolizumab previously.
[454] In certain embodiments, the TGF8 inhibitor (e.g., Ab6) is administered
in combination with avelumab to a
subject having UC or MCC. In certain embodiments, the subject is a non-
responder to avelumab. In certain
embodiments, the subject has not received avelumab previously.
[455] In certain embodiments, the 1GF8 inhibitor (e.g., Ab6) is administered
in combination with durvalumab to a
subject having NSCLC or SCLC. In certain embodiments, the subject is a non-
responder to durvalumab. In certain
embodiments, the subject has not received durvalumab previously.
[456] In certain embodiments, the TGF8 inhibitor (e.g., Ab6) is administered
in combination with a checkpoint
inhibitor therapy to a subject having a solid tumor for which a checkpoint
inhibitor therapy has been approved. In
certain embodiments, the subject has a tumor type that has been approved for
treatment with a combination of a
checkpoint inhibitor therapy and a chemotherapy. In certain embodiments, the
subject has a tumor type that
typically exhibits immune exclusion in more than 50% of the tumor area (e.g.,
tumor nests). In certain embodiments,
the immune excluded tumor types include triple-negative breast cancer or renal
cell carcinoma.
[457] In certain embodiments, the TGF8 inhibitor (e.g., Ab6) is administered
in combination with a checkpoint
inhibitor therapy to a subject having a solid tumor for which a checkpoint
inhibitor monotherapy has been approved.
In certain embodiments, the subject has a tumor type that typically exhibits
immune exclusion in more than 50% of
the tumor area (e.g., tumor nests), such as non-small cell lung cancer,
urothelial carcinoma, gastric cancer, and
renal cell carcinoma. In certain embodiments, the subject has a tumor type
that typically exhibits immune exclusion
in less than 50% of the tumor area (e.g., tumor nests), such as small-cell
lung cancer or melanoma.
[458] In certain embodiments, the TGF8 inhibitor (e.g., Ab6) is administered
in combination with a checkpoint
inhibitor therapy to a subject having a solid tumor for which a checkpoint
inhibitor has not been approved. In certain
embodiments, the subject has a tumor type that typically exhibits immune
exclusion in more than 50% of the tumor
area (e.g., tumor nests), such as microsatellite stable colorectal cancer,
pancreatic cancer, and prostate cancer.
VVithout wishing to be bound by theory, it is possible that tumor types that
do not have an approved checkpoint
inhibitor therapy respond poorly to checkpoint inhibitor therapy with or
without a conventional chemotherapy. Thus,
it is contemplated that such tumor types, particularly tumors exhibiting
immune exclusion, may have better
response to checkpoint inhibitor therapy when combined with a TGFI3 inhibitor
therapy (e.g., Ab6).
[459] Thus, TGF8 inhibitors (e.g., Ab6) may be used in conjunction (e.g., in
combination) with a checkpoint
inhibitor therapy for the treatment of cancer in a subject, wherein the cancer
comprises an immunosuppressive
tumor, and wherein the immunosuppressive tumor is resistant to checkpoint
inhibitor therapy. Non-limiting
examples of immunosuppressive tumors include acute myeoid leukemia,
adrenocortical cancer, brain lower grade
glioma, cholangiocarcinoma, colon adenocarcinoma, diffuse large B-cell
lymphoma, esophageal carcinoma,
glioblastoma multiforme, kidney chromophobe, lung squamous cell carcinoma,
mesothelioma, ovarian serous
cystadenocarcinoma, pheochromocytoma and paraganglioma, prostate
adenocarcinoma, rectum
adenocarcinoma, sarcoma, testicular germ cell tumor, thymoma, thyroid
carcinoma, uterine carcinosarcoma,
uterine corpus endometrioid carcinoma, or uveal melanoma.
[460] In some embodiments, the at least one additional agent binds a T-cell
costimulation molecule, such as
inhibitory costimulation molecules and activating costimulation molecules. In
some embodiments, the at least one
additional agent is selected from the group consisting of an anti-CD40
antibody, an anti-CD38 antibody, an anti-
KIR antibody, an anti-CD33 antibody, an anti-00137 antibody, and an anti-0D74
antibody.
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[461] In some embodiments, the at least one additional therapy is radiation.
In some embodiments, the at least
one additional agent is a radiotherapeutic agent. In some embodiments, the at
least one additional agent is a
chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is
Taxol. In some embodiments,
the at least one additional agent is an anti-inflammatory agent. In some
embodiments, the at least one additional
agent inhibits the process of monocyte/macrophage recruitment and/or tissue
infiltration. In some embodiments,
the at least one additional agent is an inhibitor of hepatic stellate cell
activation. In some embodiments, the at least
one additional agent is a chemokine receptor antagonist, e.g., CCR2
antagonists and CCR5 antagonists. In some
embodiments, such chemokine receptor antagonist is a dual specific antagonist,
such as a CCR2/CCR5
antagonist. In some embodiments, the at least one additional agent to be
administered as combination therapy is
or comprises a member of the TGFp superfamily of growth factors or regulators
thereof. In some embodiments,
such agent is selected from modulators (e.g., inhibitors and activators) of
GDF8/myostatin and GDF11. In some
embodiments, such agent is an inhibitor of GDF8/myostatin signaling. In some
embodiments, such agent is a
monoclonal antibody that specifically binds a pro/latent myostatin complex and
blocks activation of myostatin. In
some embodiments, the monoclonal antibody that specifically binds a pro/latent
myostatin complex and blocks
activation of myostatin does not bind free, mature myostatin; see, for
example, WO 2017/049011.
[462] In some embodiments, an additional therapy comprises cell therapy, such
as CAR-T therapy and CAR-NK
therapy.
[463] In some embodiments, an additional therapy comprises administering an
anti-VEGF therapy, such as a
VEGF inhibitor, e.g., bevacizumab. In some embodiments, inhibitors of TGFp
contemplated herein may be used in
conjunction with (e.g., combination therapy, add-on therapy, etc.) a VEGF
inhibitor (e.g., bevacizumab) for the
treatment of solid cancer (e.g., ovarian cancer). In some embodiments,
inhibitors of TGFp contemplated herein
may be used in conjunction with (e.g., combination therapy, add-on therapy,
etc.) a VEGF inhibitor (e.g.,
bevacizumab) for the treatment of hematopoietic cancers.
[464] In some embodiments, an additional therapy is a cancer vaccine. Numerous
clinical trials that tested
peptide-based cancer vaccines have targeted hematological malignancies
(cancers of the blood), melanoma (skin
cancer), breast cancer, head and neck cancer, gastroesophageal cancer, lung
cancer, pancreatic cancer, prostate
cancer, ovarian cancer, and colorectal cancers. The antigens included peptides
from HER2, telomerase (TERT),
survivin (BIRC5), and Wilms' tumor 1 (VVT1). Several trials also used
"personalized" mixtures of 12-15 distinct
peptides. That is, they contain a mixture of peptides from the patient's tumor
that the patient exhibits an immune
response against. Some trials are targeting solid tumors, glioma,
glioblastoma, melanoma, and breast, cervical,
ovarian, colorectal, and non-small lung cell cancers and include antigens from
MUC1, ID01 (Indoleamine 2,3-
dioxygenase), CTAG1B, and two VEGF receptors, FLT1 and KDR. Notably, the IDO1
vaccine is tested in patients
with melanoma in combination with the immune checkpoint inhibitor ipilimumab
and the BRAF (gene) inhibitor
vemurafenib.
[465] Non-limiting examples of tumor antigens useful as cancer vaccines
include: NY-ESO-1, HER2, HPV16 E7
(Papillomaviridae#E7), CEA (Carcinoembryonic antigen), VVT1, MART-1, gp100,
tyrosinase, URLC10, VEGFR1,
VEGFR2, surviving, MUC1 and MUC2.
[466] Activated immune cells primed by such cancer vaccine may, however, be
excluded from the TME in part
through TGFp1-dependent mechanisms. To overcome the immunosuppression, use of
TGFp1 inhibitors of the
present disclosure may be considered so as to unleash the potential of the
vaccine.
[467] Combination therapies contemplated herein may advantageously utilize
lower dosages of the administered
therapeutic agents, thus avoiding possible toxicities or complications
associated with the various monotherapies.
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In some embodiments, use of an isoform-specific inhibitor of TGF(31 described
herein may render those who are
poorly responsive or not responsive to a therapy (e.g., standard of care) more
responsive. In some embodiments,
use of an isoform-specific inhibitor of TGFp1 described herein may allow
reduced dosage of the therapy (e.g.,
standard of care) which still produces equivalent clinical efficacy in
patients but fewer or lesser degrees of drug-
related toxicities or adverse events.
[468] In some embodiments, inhibitors of TGFp contemplated herein may be used
in conjunction with (e.g.,
combination therapy, add-on therapy, etc.) a selective inhibitor of myostatin
(GDF8). In some embodiments, the
selective inhibitor of myostatin is an inhibitor of pro/latent myostatin
activation. See, for example, the antibodies
disclosed in WO 2017/049011, such as apitegromab.
Advantages of TGF131 Inhibitors as a Therapeutic
[469] It has been recognized that various diseases involve heterogeneous
populations of cells as sources of
TGF131 that collectively contribute to the pathogenesis and/or progression of
the disease. More than one types of
TGF131-containing complexes ("contexts") likely coexist within the same
disease microenvironment. In particular,
such diseases may involve both an ECM (or "matrix") component of TG931
signaling (e.g., ECM dysregulation)
and an immune component of TGF31 signaling. In such situations, selectively
targeting only a single TGFpl
context (e.g., TGFp1 associated with one particular type of presenting
molecule) may provide limited relief. Thus,
broadly inhibitory TGFp1 antagonists are desirable for therapeutic use.
Previously described inhibitory antibodies
that broadly targeted multiple latent complexes of TGFp1 exhibited skewed
binding profiles among the target
complexes (see, for example, WO 2018/129329 and \NO 2019/075090). The
inventors therefore set out to identify
more uniformly inhibitory antibodies that selectively inhibit TGFp1
activation, irrespective of particular presenting
molecule linked thereto. It was reasoned that particularly for immune-oncology
applications, it is advantageous to
potently inhibit both matrix-associated TGFp1 and immune cell-associated
TGF31.
[470] In various embodiments, context-independent inhibitors of TGFpl are used
in the treatments and methods
disclosed herein to target the pro/latent forms of TGFp1. More specifically,
in one modality, the inhibitor targets
ECM-associated TGF131 (LTBP1/3-TGFp1 complexes). In another modality, the
inhibitor targets immune cell-
associated TGFP1. This includes GARP-presented TGF131, such as GARP-TG931
complexes expressed on Treg
cells and LRRC33-TGFI31 complexes expressed on macrophages and other
myeloid/lymphoid cells, as well as
certain cancer cells.
[471] Such antibodies may include isoform-specific inhibitors of TGFp1 that
bind and prevent activation (or
release) of mature TGF(31 growth factor from a pro/latent TGFp1 complex in a
context-independent manner, such
that the antibodies can inhibit activation (or release) of TGFp1 associated
with multiple types of presenting
molecules. In particular, the present disclosure provides antibodies capable
of blocking ECM-associated TGF31
(LTBP-presented and LTBP3-presented complexes) and cell-associated TGFpl (GARP-
presented and LRRC33-
presented complexes).
[472] Various disease conditions have been suggested to involve dysregulation
of TGFp signaling as a
contributing factor. Indeed, the pathogenesis and/or progression of certain
human conditions appear to be
predominantly driven by or dependent on TGFp1 activities. In particular, many
such diseases and disorders involve
both an ECM component and an immune component of TGF(31 function, suggesting
that TGFP1 activation in
multiple contexts (e.g., mediated by more than one type of presenting
molecules) is involved. Moreover, it is
contemplated that there is crosstalk among TGFp1-responsive cells. In some
cases, interplays between
multifaceted activities of the TGFp1 axis may trigger a cascade of events that
lead to disease progression,
aggravation, and/or suppression of the host's ability to combat disease.
For example, certain disease
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microenvironments, such as tumor microenvironment (TME) and fibrotic
microenvironment (FME), may be
associated with TGF61 presented by multiple different presenting molecules,
e.g., LTBP1-proTGF131, LTBP3-
proTGF61, GARP-proTGF61, LRRC33-proTGF61 , and any combinations thereof. TGF61
activities of one context
may in turn regulate or influence TGF61 activities of another context, raising
the possibility that when dysregulated,
this may result in exacerbation of disease conditions. Therefore, it is
desirable to broadly inhibit across multiple
modes of TGF61 function (i.e., multiple contexts) while selectively limiting
such inhibitory effects to the TGF61
isoform. The aim is not to perturb homeostatic TGF6 signaling mediated by the
other isoforms, including TGF63,
which plays an important role in would healing.
[473] Immune components of TGFp1 activities are largely mediated by cell-
associated TGF61 (e.g., GARP-
proTGF61 and LRRC33-proTGF61). Both the GARP- and LRRC33-arms of TGF61
function are associated with
immunosuppressive features that contribute to the progression of many
diseases. Thus, TGF6 inhibitors such as
the TGF61 inhibitors described herein, may be used to inhibit TGF61 associated
with immunosuppressive cells.
The immunosuppressive cells include regulatory T-cells (Tregs), M2
macrophages/tumor-associated
macrophages, arid MDSCs. The TGF6 inhibitors of the current disclosure may
inhibit, reduce, or reverse
immunosuppressive phenotype at a disease site such as the tumor
microenvironment.
[474] In some embodiments, the TGF61 inhibitor inhibits TGF61 associated with
a cell expressing the GARP-
TGF61 complex or the LRRC33-TGF61 complex, wherein optionally the cell may be
a 1-cell, a fibroblast, a
myofibroblast, a macrophage, a monocyte, a dendritic cell, an antigen
presenting cell, a neutrophil, a myeloid-
derived suppressor cell (MDSC), a lymphocyte, a mast cell, or a microglial
cell. The T-cell may be a regulatory T
cell (e.g., immunosuppressive T cell). The neutrophil may be an activated
neutrophil. The macrophage may be
an activated (e.g., polarized) macrophage, including profibrotic and/or tumor-
associated macrophages (TAM), e.g.,
M2c subtype and M2d subtype macrophages. In some embodiments, macrophages are
exposed to tumor-derived
factors (e.g., cytokines, growth factors, etc.) which may further induce pro-
cancer phenotypes in macrophages. In
some embodiments, such tumor-derived factor is CSF-1/M-CSF.
[475] In some embodiments, the cell expressing the GARP-TGFI31 complex or the
LRRC33-TGF61 complex is a
cancer cell, e.g., circulating cancer cells and tumor cells.
TG93 Inhibitors Useful for Carrying Out the Compositions and Methods of the
Disclosure
[476] TGF6 inhibitors suitable for the therapeutic use and related methods
disclosed herein include small
molecule (i.e., low molecular weight) antagonists and biologics. Such
inhibitors include isoform-selective inhibitors
and isoform-non-selective inhibitors. Biologics inhibitors include antibodies,
antigen-binding fragments thereof,
antibody-based or immunoglobulin-like molecules, as well as other engineered
constructs, typically fusion proteins,
such as ligand traps. Ligand traps typically include a ligand-binding moiety
that is derived from ligand-binding
portion or portions of TGF6 receptor(s). Such biologics may be multifunctional
constructs, such as bi-functional
fusion proteins and bispecific antibodies.
[477] In some embodiments, methods disclosed herein may employ one or more of
the following: low molecular
weight antagonists of TGF6 receptors, e.g., ALK5 antagonists, such as
Galunisertib (LY2157299 monohydrate);
monoclonal antibodies (such as neutralizing antibodies) that inhibit all three
isoforms ("pan-inhibitor" antibodies)
(see, for example, WO 2018/134681); monoclonal antibodies that preferentially
inhibit two of the three isoforms
(e.g., antibodies against TGF61/2 (for example WO 2016/161410) and TGF131/3
(for example WO 2006/116002);
and engineered molecules (e.g., fusion proteins) such as ligand traps (for
example, WO 2018/029367; WO
2018/129331 and WO 2018/158727). In some embodiments, methods disclosed herein
may employ one or more
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of the TGFI3 inhibitors disclosed in Bathe and Massague (Immunity, 2019. Apr
16;50(4):924-940), the content of
which is incorporated herein in its entirety.
[478] In some embodiments, the TGFp inhibitor is a TG931-selective inhibitor,
such as a TG931-selective inhibitor
disclosed herein, e.g., Ab6. In some embodiments, the TGFp1-selective
inhibitor is one disclosed in
PCTIUS2019/041390, PCT/US2019/041373, or PCT/U52021/012930, the contents of
each of which are hereby
incorporated by reference in their entirety.
[479] In some embodiments, the low molecular weight antagonists of TGFp
receptors may include Vactosertib
(TEW-7197, EW-7197), LY3200882, PF-06952229, AZ 12601011, and/or AZ 12799734.
[480] In some embodiments, the neutralizing pan-TGFp antibody is GC 1008, also
known as fresolimumab, or a
derivative thereof. In some embodiments, such antibody comprises the sequence
in accordance with the disclosure
of WO/2018/134681. In some embodiments, the pan-TG93 antibody is SAR439459 or
a derivative thereof.
[481] In some embodiments, the TGFp1/2 antibodies include XPA-42-089 or a
derivative thereof.
[482] In some embodiments, the antibody is a neutralizing antibody that
specifically binds both TGFp1 and
TGF[33. In some embodiments such antibody preferentially binds TGF(31 over
TGF(33. For example, the antibody
comprises the sequence in accordance with the disclosure of WO/2006/116002. In
some embodiments, the
antibody is 21D1.
[483] In some embodiments, the antibody is a neutralizing antibody that
specifically binds both TGFP1 and
TGFp2. In some embodiments, the antibody comprises the sequence in accordance
with the disclosure of
WO/2016/161410. In some embodiments, the antibody is XOMA-089, or NIS-793.
[484] In some embodiments, the antibody is an activation inhibitor antibody
that is selective for TGF31. In some
embodiments, the antibody comprises the sequence in accordance with the
disclosure of WO/2015/015003,
WO/2019/075090 or WO/2016/115345.
[485] In some embodiments, the antibody is a neutralizing antibody that is
selective for TGF131. In some
embodiments, the antibody comprises the sequence in accordance with the
disclosure of WO/2013/134365 or
WO/2018/043734.
[486] In some embodiments, the TGFp inhibitor is a ligand trap. In some
embodiments, the ligand trap comprises
the structure in accordance with the disclosure of WO/2018/158727. In some
embodiments, the ligand trap
comprises the structure in accordance with the disclosure of WO 2018/029367;
WO 2018/129331. In some
embodiments, the ligand trap is a construct known as CTLA4- TGFbRII. In some
embodiments, the ligand trap is
a bi-functional fusion protein comprising a checkpoint inhibitor function and
a TGFp inhibitor function. In some
embodiments, the bi-functional fusion protein is a construct known as M7824 or
PDL1-TGFbRII. In some
embodiments, the TGFp inhibitor is a receptor based TGFp trap, e.g., AVID200.
[487] In some embodiments, the TGFp inhibitor is an integrin inhibitor. In
some embodiments, the TGFp inhibitor
is an inhibitor of an integrin such as aV31, aVp3, aVp5, aV36, aVp8, a5p1,
allbp3, and/or a8p1. lntegrin inhibitors
include small molecule inhibitors and antibodies that bind to an integrin
and/or inhibit the binding of an integrin to
the RGD motif of proTGFp1 and/or proTGF(33.
[488] In some embodiments, the TGFI3 inhibitor is an inhibitor of latent TGFP
(e.g., latent TGFP1 or latent TGFP3).
In some embodiments, the TG93 inhibitor is an inhibitor that binds the RGD
motif of proTG931 and/or proTGF(33.
[489] In some embodiments, the TGFI3 inhibitor is an LTBP-selective inhibitor,
such as an LTBP-selective inhibitor
disclosed in PCT/US2020/015915. In some embodiments, the TGFp inhibitor is an
LTBP1-selective inhibitor. In
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some embodiments, the TGF6 inhibitor is an LTBP3-selective inhibitor. In some
embodiments, the TGF6 inhibitor
is an LTBP-1 and LTBP3-selective inhibitor.
Isoform-Selective Antibodies of proTG931
[490] Preferably, the therapeutic use and related methods in accordance with
the present disclosure are carried
out with an isoform-selective inhibitor of TGF61, e.g., Ab6 (the sequence of
which is as disclosed in
PCT/US2019/041373, the contents of which are herein incorporated by reference
in its entirety).
[491] Applicant previously disclosed improved antibodies which embody all or
most of the following features: 1)
selectivity towards TGFI31 is maintained to minimize unwanted toxicities
associated with pan-inhibition ("isoform-
selectivity') (see, for example, PCT/U32017/021972); 2) exhibit broad binding
activities across various biological
contexts, or, both matrix-associated and cell-associated categories ("context-
independent") (see, for example, WO
2018/129329); 3) achieve more even or unbiased affinities across multiple
antigen complexes (uniformity'); 4)
show strong binding activities for each ofthe antigen complexes, ("high-
affinity') and have robust inhibitory activities
for each context ("potency') (see, for example, PCT/US2019/041373); and, 5)
the preferred mechanism of action
is to inhibit the activation step so the inhibitor can target a tissue-
tethered, latent TG931 complex, so as to
preemptively prevent downstream activation events to achieve durable effects,
rather than to directly target
soluble/free growth factors ("durability'). As disclosed in PCT/US2019/041373,
such TGF61 inhibitors are highly
potent and highly selective inhibitor of latent TGF61 activation. Data
presented therein demonstrated, inter alia,
that this mechanism of isoform-selective inhibition is sufficient to overcome
primary resistance to anti-PD-1 in
syngeneic mouse models that closely recapitulate some of the features of
primary resistance to CBT found in
human cancers. In addition, 6) such inhibitors have an improved safety profile
as compared to pan-inhibitors or
other isoform-non-selective inhibitors of TGFp, Together, these efficacy and
safety data provide a rationale for
exploring the therapeutic use of selective TGF61 inhibition to broaden and
enhance clinical responses to
checkpoint blockade in cancer immunotherapy, as well as to treat a number of
additional TGF61-related indications.
General features of certain TGF81 inhibitors
[492] Exemplary antibodies that may be used for carrying out the present
disclosure are disclosed in
W02020014460, the content of which is incorporated herein by reference in its
entirety.
[493] Preferred antibodies and corresponding nucleic acid sequences that
encode such antibodies useful for
carrying out the present disclosure include one or more of the CDR amino acid
sequences shown in Tables 1 and
2. Each set of the H-CDRs (H-CDR1, H-CDR2 and H-CDR3) listed in Table 1 can be
combined with the L-CDRs
(L-CDR1, L-CDR2 and L-CDR3) provided in Table 2.
[494] Thus, the disclosure provides an antibody or antigen binding fragment
thereof, e.g., an isolated antibody or
antigen-binding fragment thereof, comprising six CDRs (e.g., an H-CDR1, an H-
CDR2, an H-CDR3, an L-CDR1,
an L-CDR2 and an L-CDR3) as listed in Table 1, and wherein the L-CDR1
comprises QASQDITNYLN (SEQ ID
NO: 78), the L-CDR2 comprises DASNLET (SEQ ID NO: 79), and the L-CDR3
comprises QQADNHPPVVT (SEQ
ID NO: 6), wherein optionally, the H-CDR1 may comprise FTFSSFSMD (SEQ ID NO:
80); the H-CDR-2 may
comprise YISPSADTIYYADSVKG (SEQ ID NO: 76); and/or, the H-CDR3 may comprise
ARGVLDYGDMLMP (SEQ
ID NO: 3). In some embodiments, the antibody or the fragment comprises H-CDR1
having the amino acid
sequence FTFSSFSMD (SEQ ID NO: 80), H-CDR2 having the amino acid sequence
YISPSADTIYYADSVKG
(SEQ ID NO: 76), and H-CDR-3 having the amino acid sequence ARGVLDYGDMLMP (SEQ
ID NO: 3); L-CDR1
having the amino acid sequence QASQDITNYLN (SEQ ID NO: 78), L-CDR2 having the
amino acid sequence
DASNLET (SEQ ID NO: 79), and L-CDR3 having the amino acid sequence QQADNHPPVVT
(SEQ ID NO: 6).
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Additional examples of antibodies or antigen-binding fragments thereof
encompassed by the present disclosure
are provided in PCT/US2019/041390, PCT/US2019/041373, PCT/US2021/012930, the
contents of each of which
are herein incorporated by reference in their entirety.
Table 1. Complementary determining regions of the heavy chain of exemplary
antibodies, as
determined using the numbering scheme described in Lu et al.
Ab I-I-CDR1 H-CDR2 H-CDR3
FTFSSYSMN YISSSSSTIYYADSVKG ARGVLDYGDMLDP
Ab4 (SEQ ID NO: 81) (SEQ ID NO: 82) (SEQ ID NO:
83)
FTFSSFSMD YISPDASTIYYADSVKG ARGVLDYGDMLDP
Ab5 (SEQ ID NO: 80) (SEQ ID NO: 84) (SEQ ID NO:
83)
FTFSSFSMD YISPSADTIYYADSVKG ARGVLDYGDMLMP
Ab6 (SEQ ID NO: 80) (SEQ ID NO: 76) (SEQ ID NO: 3)
FTFSSFSMD YISPDASTIYYADSVKG ARGVLDYGDMLDP
Ab21 (SEQ ID NO: 80) (SEQ ID NO: 84) (SEQ ID NO:
83)
FTFGSFSMN YII-ISDASTIYYADSVKG ARGVLDYGDMLDP
Ab22 (SEQ ID NO: 88) (SEQ ID NO: 86) (SEQ ID NO:
83)
FTFSSFSMN YISPSADTIYYADSVKG ARGVLDYGDMLDP
Ab23 (SEQ ID NO: 87) (SEQ ID NO: 76) (SEQ ID NO:
83)
FTFSSFAMY YISPDASTIYYADSVKG ARGVLDYGDMLDP
Ab24 (SEQ ID NO: 88) (SEQ ID NO: 84) (SEQ ID NO:
83)
FTFGSFSMD YISPDASTIYYADSVKG ARGVLDYGDMLDP
Ab25 (SEQ ID NO: 88) (SEQ ID NO: 84) (SEQ ID NO:
83)
FTFSSFSMD YISPDASTIYYADSVKG ARGVLDYGDMLDP
Ab26 (SEQ ID NO: 80) (SEQ ID NO: 84) (SEQ ID NO:
83)
FTFSFYAMN YISPDASTIYYADSVKG ARGVLDYGDMLDP
Ab27 (SEQ ID NO: 90) (SEQ ID NO: 84) (SEQ ID NO:
83)
FTFSSFSMD YISPDASTIYYADSVKG VRGVLDYGDMLDP
Ab28 (SEQ ID NO: 80) (SEQ ID NO: 84) (SEQ ID NO:
91)
FTFSSFAMN YISPDASTIYYAGSVKG VRAVLDYGDM LDP
Ab29 (SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ID NO:
94)
FTFSSFSMD YISPDASTIYYADSVKG ARGTLDYGDMLDP
Ab30 (SEQ ID NO: 80) (SEQ ID NO: 84) (SEQ ID NO:
95)
FTFSSFSMD YISPDASTIYYADSVKG ARAVLDYGDM LDP
Ab31 (SEQ ID NO: 80) (SEQ ID NO: 84) (SEQ ID NO:
96)
FTFSSFSMN YISPSADTIYYADSVKG ARGVVVDMGDMLDP
Ab32 (SEQ ID NO: 87) (SEQ ID NO: 76) (SEQ ID NO:
97)
FTFSSFSMN YISPSADTIYYADSVKG AHGVLDYGDMLDP
Ab33 (SEQ ID NO: 87) (SEQ ID NO: 76) (SEQ ID NO:
98)
FTFAFYSMN YISPDASTIYYADSVKG ARGVLDYGDMLDP
Ab34 (SEQ ID NO: 99) (SEQ ID NO: 84) (SEQ ID NO:
83)
Table 2. Complementary determining regions of the light chain of exemplary
antibodies, as
determined using the Kabat numbering scheme or the numbering system of Lu et
al.
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L-CDR1 L-CDR2 L-CDR3
QASQDITNYLN DASNLET QQADNHPPVVT
(SEQ ID NO: 78) (SEQ ID NO: 79) (SEQ ID NO: 6)
[495] Determination of CDR sequences within an antibody depends on the
particular numbering scheme being
employed. Commonly used systems include but are not limited to: Kabat
numbering system, IMTG numbering
system, Chothia numbering system, and others such as the numbering scheme
described by Lu et al., (Lu X et
al., MAbs. 2019 Jan;11(1):45-57). To illustrate, 6 CDR sequences of Ab6 as
defined by four different numbering
systems are exemplified below. Any art-recognized CDR numbering systems may be
used to define CDR
sequences of the antibodies of the present disclosure.
Table 3. Six CDRs of an exemplary antibody (Ab6) based on four numbering
schemes
IMTG numbering Kabat numbering Chothia numbering
System of Lu et al.
H-CDR1 GFTFSSFS SFSMD GFTFSSF FTFSSFSMD
(SEQ ID NO: 1) (SEQ ID NO: 75) (SEQ ID NO: 168) (SEQ
ID NO: 80)
H-CDR2 ISPSADTI YISPSADTIYYADSVKG SPSADT
YISPSADTIYYADSVKG
(SEQ ID NO: 2) (SEQ ID NO: 76) (SEQ ID NO: 169) (SEQ
ID NO: 76)
H-CDR3 ARGVLDYGDMLMP GVLDYGDMLMP GVLDYGDMLMP ARGVLDYGDMLMP
(SEQ ID NO: 3) (SEQ ID NO: 77) (SEQ ID NO: 77) (SEQ ID
NO: 3)
L-CDR1 QDITNY QASQDITNYLN QASQDITNYLN QASQDITNYLN
(SEQ ID NO: 4) (SEQ ID NO: 78) (SEQ ID NO: 78) (SEQ ID
NO: 78)
L-CDR2 DAS DASNLET DASNLET DASNLET
(SEQ ID NO: 5) (SEQ ID NO: 79) (SEQ ID NO: 79) (SEQ ID
NO: 79)
L-CDR3 QQADNHPPWT QQADNHPPVVT QQADNHPPVVT QQADNHPPVVT
(SEQ ID NO: 6) (SEQ ID NO: 6) (SEQ ID NO: 6) (SEQ ID
NO: 6)
[496] Amino acid sequences of the heavy chain variable domain and the light
chain variable domain of exemplary
antibodies of the present disclosure are provided in Table 4. Thus, in some
embodiments, the isoform-selective
TGF31 inhibitor of the present disclosure may be an antibody or an antigen-
binding fragment thereof comprising a
heavy chain variable domain (VH) and a light chain variable domain (VL),
wherein the VH and the VL sequences are
selected from any one of the sets of VH and VL sequences listed in Table 4
below.
Table 4. Heavy chain variable domains and light chain variable domains of
exemplary antibodies
Heavy Chain Variable Domain (VH) Light Chain Variable
Domain (VL)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
YSMNVVVRQAPGKGLEWVSYISSSSSTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab4 SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNHPPVVTFGG
YCARGVLDYGDMLDPWGQGTLVTVSS GTKVEIK
(SEQ ID NO: 100) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMDVVVRQAPGKGLEWVSYISPDASTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab5 SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNHPPVVTFGG
YCARGVLDYGDMLDPWGQGTLVTVSS GTKVEIK
(SEQ ID NO: 101) (SEQ ID NO: 8)
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EVQLVESGGGLVQPGGSLRLSCTASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMDWVRQAPGKG LEWVSYISPSADTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab6 SVKGRFT IS RDNAKNTLYLQMNSLRAE DTAVY
GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGVLDYGDMLMPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 7) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMDWVRQAPGKG LEWVSYISPDASTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab21 SVKGRFT IS RDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGVLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 102) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFGS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMNWVRQAPGKG LEWVSYIHSDASTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab22 SVKGRFT IS RDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGVLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 103) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMNWVRQAPGKG LEWVSYISPSADTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab23 SVKGRFT IS RDNAKNTLYLQMNSLRAE DTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGVLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 104) (SEQ ID NO: 8)
EVQLVESGGGLVQPGRSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FAMYVVVRQAPGKGLEWVSYISPDASTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab24 SVKGRFT IS RDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGVLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 105) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFGS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMDWVRQAPGKG LEWVSYISPDASTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab25 SVKGRFT IS RDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGVLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 106) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMDWVRQAPGKG LEWVSYISPDASTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab26 SVKGRFT IS RDNAKNTLYLQMNSLRAE DTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGVLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 107) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSF DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
YAMNVVVRQAPGKGLEWVSYISPDASTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab27 SVKGRFT IS RDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGVLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 108) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMDWVRQAPGKG LEVVVSYISPDASTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab28 SVKGRFT IS RDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCVRGVLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 109) (SEQ ID NO: 8)
EVQLVESGGGLVQPGRSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FAMNWVRQAPGKG LEWVSYISPDASTIYYAG VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab29 SVKGRFT IS RDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCVRAVLDYGDMLDPWGQGTLVTVSS GTKVEIK
(SEQ ID NO: 110) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMDWVRQAPGKG LEWVSYISPDASTIYYAD WYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab30 SVKGRFT IS RDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGTLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 111) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMDWVRQAPGKG LEWVSYISPDASTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab31 SVKGRFT IS RDNAKNTLYLQMNSLRAE DTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARAVLDYGDMLDPWGQGTLVTVSS GTKVE I K
(SEQ ID NO: 112) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
FSMNWVRQAPGKG LEVVVSYISPSADTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab32 SVKGRFT IS RDNAKNTLYLQMNSLRAE DTAVY GTDFTFTISSLQPEDIATYYCQQADNH PPVVTFGG
YCARGVWDMGDMLDPVVGQGTLVTVSS GTKVE I K
(SEQ ID NO: 113) (SEQ ID NO: 8)
Ab33 EVQLVESGGGLVQPGGSLRLSCAASGFTFSS DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
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FSMNVVVRQAPGKGLEVVVSYISPSADTIYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
SVKGRFTISRDNAKNTLYLQMNSLRAEDTAVY GTDFTFTISSLCIPEDIATYYCQQADNHPPVVTFGG
YCAHGVLDYGDMLDPWGQGTLVTVSS GTKVEIK
(SEQ ID NO: 114) (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFAF DIQMTQSPSSLSASVGDRVTITCQASQDITNYLN
YSMNVVVRQAPGKGLEWVSYISPDAST IYYAD VVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
Ab34 SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY GTDFTFTISSLOPEDIATYYCQQADNHPPVVTFGG
YCARGVLDYGDMLDPWGQGTLVT VSS GTKVEIK
(SEQ ID NO: 115) (SEQ ID NO: 8)
[497] In some embodiments, an antibody or an antigen-binding fragment thereof
is disclosed that comprises a
heavy chain variable domain and a light chain variable domain, wherein, the
heavy chain variable domain has at
least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 k 99% and
100%) sequence identity
with any one of the sequences selected from the group consisting of: Ab4, Ab5,
Ab6, Ab21, Ab22, Ab23, Ab24,
Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34; and, wherein
the light chain variable
domain has at least 90% identity with any one of the sequences selected from
Ab4, Ab5, Ab6, Ab21, Ab22, Ab23,
Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34, wherein,
optionally, the heavy chain
variable domain may optionally have at least 95% sequence identity, and/or,
the light chain variable domain may
have at least 95% (e.g., at least 95%, 96%, 97%, 98% 99% and 100%) sequence
identity. In some
embodiments, the heavy chain variable domain of the antibody or the fragment
has at least 90% sequence
identity with SEQ ID NO: 7, and wherein optionally, the light chain variable
domain of the antibody or the
fragment has at least 90% sequence identity with SEQ ID NO: 8. In some
embodiments, the heavy chain
variable domain of the antibody or the fragment has at least 95% sequence
identity with SEQ ID NO: 7, and
wherein optionally, the light chain variable domain of the antibody or the
fragment has at least 95% sequence
identity with SEQ ID NO: 8. In some embodiments, the heavy chain variable
domain of the antibody or the
fragment has at least 98% sequence identity with SEQ ID NO: 7, and wherein
optionally, the light chain variable
domain of the antibody or the fragment has at least 98% sequence identity with
SEQ ID NO: 8. In some
embodiments, the heavy chain variable domain of the antibody or the fragment
has 100% sequence identity with
SEQ ID NO: 7, and wherein optionally, the light chain variable domain of the
antibody or the fragment has 100%
sequence identity with SEQ ID NO: 8.
[498] In various embodiments, an antibody or an antigen-binding fragment
thereof disclosed herein comprises 6
CDRs from, or the full sequences of, the heavy and light chain variable
domains of SEQ ID Nos: 7 and 8,
respectively. In some embodiments, the antibody or an antigen-binding fragment
thereof comprises heavy and
light chain variable domain sequences with at least 90% sequence identity
(e.g., at least 95% identity) to SEQ ID
NOs: 7 and 8, respectively. For instance, the antibody or an antigen-binding
fragment thereof may comprise a
set of 6 respective H- and L- CDRs selected from those set out in Tables 1 and
2 above. In some certain
embodiments, the antibody or antigen-binding fragment thereof comprises a set
of 6 respective H- and L- CDRs
as set out in Table 3 (e.g., using the system of Lu et al.).
[499] Alternatively, or in addition, the antibody or an antigen-binding
fragment thereof used in the context of the
present disclosure may comprise heavy and light chain variable domains with at
least 90% sequence identity
(e.g., at least 95% identity) to SEQ ID Nos: 7 and 8, respectively, and
specifically binds a proTGF31 complex at
(i) a first binding region comprising at least a portion of Latency Lasso (SEQ
ID NO: 126); and ii) a second
binding region comprising at least a portion of Finger-1 (SEQ ID NO: 124);
characterized in that when bound to
the proTGF31 complex in a solution, the antibody or the fragment protects the
binding regions from solvent
exposure as determined by hydrogen-deuterium exchange mass spectrometry (HDX-
MS). The first binding
region may comprise PGPLPEAV (SEQ ID NO: 134) or a portion thereof and the
second binding region may
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comprise RKDLGVVKVV (SEQ ID NO: 143) or a portion thereof. As used herein,
protection of the binding region
refers to protein-protein interactions, such as antibody-antigen binding, the
degree by which a protein (e.g., a
region of a protein containing an epitope) is exposed to a solvent as assessed
by an HDX-MS-based assay of
protein-protein interactions. Protection of binding may be determined by the
level of proton exchange occurring at
a binding site, which is inversely correlates with the degree of
binding/interaction. Therefore, when an antibody
described herein binds to a region of an antigen, the binding region is
"protected" from being exposed to the
solvent because the protein-protein interaction precludes the binding region
from being accessible by the
surrounding solvent. The protected region is thus indicative of a site of
interaction. The antibody or the fragment
may further bind the proTGF31 complex at one or more of the following binding
regions or a portion thereof:
LVKRKRIEA (SEQ ID NO: 132); LASPPSQGEVPPGPL (SEQ ID NO: 126); LALYNSTR (SEQ ID
NO: 135);
REAVPEPVL (SEQ ID NO: 136); YQKYSNNSVVR (SEQ ID NO: 137); RKDLGWKVVIHE (SEQ ID
NO: 144);
HEPKGYHANF (SEQ ID NO: 145); LGPCPYIVVS (SEQ ID NO: 139); ALEPLPIV (SEQ ID NO:
140); and,
VGRKPKVEQL (SEQ ID NO: 141).
[500] In some embodiments, the antibody or antigen-binding fragments may
further be characterized in that it
cross-blocks (cross-competes) for binding to TGF31 (e.g., to pro- and/or
latent- TGF31) with an antibody having
the heavy chain variable domain of SEQ ID NO: 7, and the light chain variable
domain of SEQ ID NO: 8. In some
embodiments, the antibody that cross-blocks or cross-competes comprises heavy
and light chain variable
domains that are at least about 90% (e.g., 95% or 99%) identical to those of
SEQ ID NOs 7 and 8, respectively.
[501] In some embodiments, the antibody or antigen binding portion thereof,
that specifically binds to a GARP-
TGF31 complex, a LTBP1-TGF31 complex, a LTBP3-TGF31 complex, and/or a LRRC33-
TGF31 complex
comprises a heavy chain variable domain amino acid sequence encoded by a
nucleic acid sequence having at
least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the
nucleic acid sequence set forth in
SEQ ID NO: 7, and a light chain variable domain amino acid sequence encoded by
a nucleic acid sequence having
at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the
nucleic acid sequence set forth
in SEQ ID NO: 8. In some embodiments, the antibody or antigen binding portion
thereof, comprises a heavy chain
variable domain amino acid sequence encoded by the nucleic acid sequence set
forth in SEQ ID NO: 7, and a light
chain variable domain amino acid sequence encoded by the nucleic acid sequence
set forth in SEQ ID NO: 8.
[502] In some examples, any of the antibodies of the disclosure that
specifically bind to a GARP-TGF31 complex,
a LTBP1-TGF31 complex, a LTBP3-TGF31 complex, and/or a LRRC33-TGF31 complex
include any antibody
(including antigen binding portions thereof) having one or more CDR (e.g.,
CDRH or CDRL) sequences
substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3. For
example, the antibodies
may include one or more CDR sequences as shown in Table 1 containing up to 5,
4, 3, 2, or 1 amino acid residue
variations as compared to the corresponding CDR region in any one of SEQ ID
NOs: 3, 6, 76, 78, 79, 80, 81, 82,
83 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99. In some
embodiments, one or more of the six
CDR sequences contain up to three (3) amino acid changes as compared to the
sequences provided in Table I.
Such antibody variants comprising up to 3 amino acid changes per CDR are
encompassed by the present
disclosure. In some embodiments, such variant antibodies are generated by the
process of optimization, such as
affinity maturation. The complete amino acid sequences for the heavy chain
variable region and light chain variable
region of the antibodies listed in Table 4 (e.g., Ab6), as well as nucleic
acid sequences encoding the heavy chain
variable region and light chain variable region of certain antibodies are
provided below:
Ab6 - Heavy chain variable region amino acid sequence
EVQLVESGGGLVQPGGSLRLSCTASGFTFSSFSMDVVVRQAPGKGLEWVSYISPSADTIYYADSVKGRFTISRDN
AKNTLYLQMNSLRAEDTAVYYCARGVLDYGDMLMPVVGQGTLVTVSS (SEQ ID NO: 7)
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Ab6 ¨ Light chain variable region amino acid sequence
DIQMTQSPSSLSASVGDRVT ITCQASQDITNYLNINYQQKPG
KAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTIS
SLQPEDIATYYCQQADNHPPVVTFGGGTKVEIK (SEQ ID NO: 8)
Ab6 ¨ Heavy chain amino acid sequence
EVQLVESGGGLVQPGGSLRLSCTASGFTFSSFSMDVVVRQAPGKGLEVVVSYISPSADTIYYADSVKGRFTISRDN
AKNTLYLQMNSLRAEDTAVYYCARGVLDYGDMLMPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK
YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM IS RTPEVTCVVVDVSQE DPEVQFN VVYVDGVEVH
NAKTKPREEQ
FNSTYRVVSVLTVLHQDVVLNG KEYKCKVSN KG LPSS I
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSD IAVEVVESNGQP EN NYKTTP PVLDSDGSFF LYS RLTVDKS RVVQEG NVFSCSVMH EALH N
HYTQKSLSL
SLG (SEQ ID NO: 9)
Ab6 ¨ Heavy chain nucleic acid sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAG
CCTCTGGATTCACCTTCAGTAGCTTCAGCATGGACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG
GGTTTCATACATTAGTCCCAGTGCAGACACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTC
CAGAGACAATGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACT
ACTGCGCCAGAGGGGTGCTCGACTACGGAGACATGTTAATGCCATGGGGCCAGGGAACCCTGGTCACCGT
CTCCTCAGCGTCGACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCCA
CCGCCGCTCTGGGCTGTCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGAGCTGGAACTCTGGCGC
CCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGG
TGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACC
AAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCCTGCCCTGCCCCTGAGTTCCTGG
GCGGACCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTG
ACCTGCGTGGTGGTGGACGTGTCCCAGGAAGATCCTGAGGTCCAGTTCAATTGGTACGTGGATGGCGTGG
AGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTACCGGGTGGTGTCTGTGCT
GACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCAGCAACAAGGGCCTGCCC
TCCTCCATCGAGAAAACCATCTCCAAGGCCAAGG GCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCC
TAGCCAGGAAGAGATGACCAAGAATCAGGTGTCCCTGACATGCCTGGTGAAGGGCTTCTACCCTTCCGATA
TCGCCGTGGAGTGGGAGAGCAACGGCCAGCCAGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTC
CGACGGCTCCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGGTGGCAGGAAGGCAACGTCTTTT
CCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGC
(SEQ ID NO: 10)
Ab6 ¨ Light chain amino acid sequence
DIQMTQSPSSLSASVGDRVT ITCQASQDITNYLNVVYQQKPG
KAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTIS
SLQP ED IATYYCQQAD NH P PWTFGGGTKVEI KRTVAAPSVF I FP PSDEQL KSGTASVVCLLN NFYP
REAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:
11)
Ab6 ¨ Light chain nucleic acid sequence (human kappa)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAG
GCGAGTCAGGACATTACCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATC
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TACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACT
TTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAGCAGGCCGACAATCACCCTCCT
TGGACTTTTGGCGGAGGGACCAAGGTTGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC
GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA
GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG
GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAA
AGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG
TGT (SEQ ID NO: 12)
[503] In some embodiments, the "percent identity" of two amino acid sequences
is determined using the algorithm
of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified
as in Karlin and Altschul Proc. Natl.
Acad. Sal. USA 90:5873-77, 1993. Such an algorithm is incorporated into the
NBLAST and XBLAST programs
(version 2.0) of Altschul, et al., J. MoL Biol. 215:403-10, 1990. BLAST
protein searches can be performed with the
XBLAST program, score=50, word length=3 to obtain amino acid sequences
homologous to the protein molecules
of interest. Where gaps exist between two sequences, Gapped BLAST can be
utilized as described in Altschul et
al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and
Gapped BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
[504] In any of the antibodies or antigen-binding fragments described herein,
one or more conservative mutations
can be introduced into the CDRs or framework sequences at positions where the
residues are not likely to be
involved in an antibody-antigen interaction. In some embodiments, such
conservative mutation(s) can be
introduced into the CDRs or framework sequences at position(s) where the
residues are not likely to be involved
in interacting with a GARP-TGF31 complex, a LTBP1-TGF81 complex, a LTBP3-TGF61
complex, and a LRRC33-
TGF31 complex as determined based on the crystal structure. In some
embodiments, likely interface (e.g.,
residues involved in an antigen-antibody interaction) may be deduced from
known structural information on another
antigen sharing structural similarities.
[505] As used herein, a "conservative amino acid substitution" refers to an
amino acid substitution that does not
alter the relative charge or size characteristics of the protein in which the
amino acid substitution is made. Variants
can be prepared according to methods for altering polypeptide sequence known
to one of ordinary skill in the art
such as are found in references which compile such methods, e.g., Molecular
Cloning: A Laboratory Manual, J.
Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York, 1989,
or Current Protocols in Molecular Biology, P.M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York.
Conservative substitutions of amino acids include substitutions made amongst
amino acids within the following
groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q,
N; and (g) E, D.
[506] In some embodiments, the antibodies provided herein comprise mutations
that confer desirable properties
to the antibodies. For example, to avoid potential complications due to Fab-
arm exchange, which is known to occur
with native IgG4 mAbs, the antibodies provided herein may comprise a
stabilizing 'Adair' mutation (Angal et al 'A
single amino acid substitution abolishes the heterogeneity of chimeric
mouse/human (IgG4) antibody," Mol
Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat
numbering) is converted to
proline resulting in an IgG1-like (CPPCP (SEQ ID NO: 43)) hinge sequence.
Accordingly, any of the antibodies
may include a stabilizing 'Adair' mutation or the amino acid sequence CPPCP
(SEQ ID NO: 43).
[507] Isoform-specific, context-independent inhibitors of TGF31 of the present
disclosure may optionally comprise
antibody constant regions or parts thereof. For example, a VL domain may be
attached at its C-terminal end to a
light chain constant domain like CK or CA. Similarly, a VH domain or portion
thereof may be attached to all or part
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of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any isotype subclass.
Antibodies may include suitable
constant regions (see, for example, Kabat et al., Sequences of Proteins of
Immunological Interest, No. 91-3242,
National Institutes of Health Publications, Bethesda, Md. (1991)). Therefore,
antibodies within the scope of this
may disclosure include VH and VL domains, or an antigen binding portion
thereof, combined with any suitable
constant regions.
[508] Additionally or alternatively, such antibodies may or may not include
the framework region of the antibodies
of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, and 8. In some
embodiments, antibodies that specifically bind to a GARP-TGF31 complex, a
LTBP1-TGF31 complex, a LTBP3-
TGF31 complex, and a LRRC33-TGF131 complex are murine antibodies and include
murine framework region
sequences.
[509] In some embodiments, such antibodies bind to a GARR-TGF61 complex, a
LTBR1-TGF31 complex, a
LTBP3-TGF61 complex, and a LRRC33-TGF61 complex with relatively high affinity,
e.g., with a KD less than 1O-
M, 10-10 M, 10-11 M or lower. For example, such antibodies may bind a GARP-
TGF31 complex, a LTBP1-TGF31
complex, a LTBP3-TGF61 complex, and/or a LRRC33-TGF61 complex with an affinity
between 5 pM and 1 nM,
e.g., between 10 pM and 1 nM, e.g., between 10 pM and 500 pM. The disclosure
also includes antibodies or
antigen binding fragments that compete with any of the antibodies described
herein for binding to a GARP-TGF31
complex, a LTBP1-TGF31 complex, a LTBP3-TGFI31 complex, and/or a LRRC33-TGF61
complex and that have
a KD value of 1 nM or lower (e.g., 1 nM or lower, 500 pM or lower, 100 pM or
lower). The affinity and binding
kinetics of the antibodies that specifically bind to a GARP-TGF31 complex, a
LTBP1-TGF31 complex, a LTBP3-
TGF31 complex, and/or a LRRC33-TGF61 complex can be tested using any suitable
method including but not
limited to biosensor-based technology (e.g., OCTET or Biacoree) and solution
equilibrium titration-based
technology (e.g., MSD-SET). In some embodiments, affinity and binding kinetics
are measured by SPR, such as
Biacore systems. In preferred embodiments, such antibodies dissociate from
each of the aforementioned large
latent complex with an OFF rate of 10e-4 or less.
[510] In some embodiments, inhibitors of cell-associated TGF31 (e.g., GARP-
presented TGF31 and LRRC33-
presented TGF131) according to the disclosure include antibodies or fragments
thereof that specifically bind such
complex (e.g., GARP-pro/latent TGF131 and LRRC33-pro/latent TGFI31) and
trigger internalization of the
complex. This mode of action causes removal or depletion of the inactive TGF61
complexes (e.g., GARP-
proTGF31 and LRRC33-proTGF31) from the cell surface (e.g., Treg, macrophages,
etc.), hence reducing TGF31
available for activation. In some embodiments, such antibodies or fragments
thereof bind the target complex in a
pH-dependent manner such that binding occurs at a neutral or physiological pH,
but the antibody dissociates
from its antigen at an acidic pH; or, dissociation rates are higher at acidic
pH than at neutral pH. Such antibodies
or fragments thereof may function as recycling antibodies.
Antibodies Competing with the Preferred Antibodies of TG931
[511] Aspects of the disclosure relate to antibodies that compete or cross-
compete with any of the antibodies
provided herein. The term "compete", as used herein with regard to an
antibody, means that a first antibody binds
to an epitope (e.g., an epitope of a GARP-proTGF31 complex, a LTBP1-proTGF131
complex, a LTBP3-proTGF31
complex, and a LRRC33-proTGF61 complex) in a manner sufficiently similar to or
overlapping with the binding of
a second antibody, such that the result of binding of the first antibody with
its epitope is detectably decreased in
the presence of the second antibody compared to the binding of the first
antibody in the absence of the second
antibody. The alternative, where the binding of the second antibody to its
epitope is also detectably decreased in
the presence of the first antibody, can, but need not be the case. That is, a
first antibody can inhibit the binding of
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a second antibody to its epitope without that second antibody inhibiting the
binding of the first antibody to its
respective epitope. However, where each antibody detectably inhibits the
binding of the other antibody with its
epitope or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with
each other for binding of their respective epitope(s). Both competing and
cross-competing antibodies are within
the scope of this disclosure. Regardless of the mechanism by which such
competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a common
epitope, or portion thereof), the skilled
artisan would appreciate that such competing and/or cross-competing antibodies
are encompassed and can be
useful for the methods and/or compositions provided herein. The term "cross-
blocking" and "cross-competing" may
be used interchangeably.
[512] Two different monoclonal antibodies (or antigen-binding fragments) that
bind the same antigen may be able
to simultaneously bind to the antigen if the binding sites are sufficiently
further apart in the three-dimensional space
such that each binding does not interfere with the other binding. By contrast,
two different monoclonal antibodies
may have binding regions of an antigen that are the same or overlapping, in
which case, binding of the first antibody
may prevent the second antibody from being able to bind the antigen, or vice
versa. In the latter case, the two
antibodies are said to "cross-block" with each other with respect to the same
antigen.
[513] Antibody "binning" experiments are useful for classifying multiple
antibodies that are made against the same
antigen into various "bins" based on the relative cross-blocking activities.
Each "bin" therefore represents a discrete
binding region(s) of the antigen. Antibodies in the same bin by definition
cross-block each other. Binning can be
examined by standard in vitro binding assays, such as Biacore or Octet , using
standard test conditions, e.g.,
according to the manufacturer's instructions (e.g., binding assayed at room
temperature, ¨20-25 C).
[514] Aspects of the disclosure relate to antibodies that compete or cross-
compete with any of the specific
antibodies, or antigen binding portions thereof, as provided herein. In some
embodiments, an antibody, or antigen
binding portion thereof, binds at or near the same epitope as any of the
antibodies provided herein. In some
embodiments, an antibody, or antigen binding portion thereof, binds near an
epitope if it binds within 15 or fewer
amino acid residues of the epitope. In some embodiments, any of the antibody,
or antigen binding portion thereof,
as provided herein, binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14
or 15 amino acid residues of an epitope
that is bound by any of the antibodies provided herein.
[515] In another embodiment, provided herein is an antibody, or antigen
binding portion thereof, competes or
cross-competes for binding to any of the antigens provided herein (e.g., a
GARP-TGF61 complex, a LTBP1-TGF61
complex, a LTBP3-TGF131 complex, and/or a LRRC33-TGF61 complex) with an
equilibrium dissociation constant,
KD, between the antibody and the protein of less than 10-8 M. In other
embodiments, an antibody competes or
cross-competes for binding to any of the antigens provided herein with a Ko in
a range from 10-12 M to 10-9 M. In
some embodiments, provided herein is an anti-TGF[31 antibody, or antigen
binding portion thereof that competes
for binding with an antibody, or antigen binding portion thereof, described
herein. In some embodiments, provided
herein is an anti-TGF61 antibody, or antigen binding portion thereof, that
binds to the same epitope as an antibody,
or antigen binding portion thereof, described herein.
[516] Any of the antibodies provided herein can be characterized using any
suitable methods. For example, one
method is to identify the epitope to which the antigen binds, or "epitope
mapping." There are many suitable methods
for mapping and characterizing the location of epitopes on proteins, including
solving the crystal structure of an
antibody-antigen complex, competition assays, gene fragment expression assays,
and synthetic peptide-based
assays, as described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an
additional example, epitope mapping
can be used to determine the sequence to which an antibody binds. The epitope
can be a linear epitope, i.e.,
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contained in a single stretch of amino acids, or a conformational epitope
formed by a three-dimensional interaction
of amino acids that may not necessarily be contained in a single stretch
(primary structure linear sequence). In
some embodiments, the epitope is a TGF(31 epitope that is only available for
binding by the antibody, or antigen
binding portion thereof, described herein, when the TGFp1 is in a GARP-
proTGF131 complex, a LTBP1-proTG931
complex, a LTBP3-proTGFP1 complex, or a LRRC33-proTGFP1 complex. Peptides of
varying lengths (e.g., at
least 4-6 amino acids long) can be isolated or synthesized (e.g.,
recombinantly) and used for binding assays with
an antibody. In another example, the epitope to which the antibody binds can
be determined in a systematic screen
by using overlapping peptides derived from the target antigen sequence and
determining binding by the antibody.
According to the gene fragment expression assays, the open reading frame
encoding the target antigen is
fragmented either randomly or by specific genetic constructions and the
reactivity of the expressed fragments of
the antigen with the antibody to be tested is determined. The gene fragments
may, for example, be produced by
PCR and then transcribed and translated into protein in vitro, in the presence
of radioactive amino acids. The
binding of the antibody to the radioactively labeled antigen fragments is then
determined by immunoprecipitation
and gel electrophoresis. Certain epitopes can also be identified by using
large libraries of random peptide
sequences displayed on the surface of phage particles (phage libraries).
Alternatively, a defined library of
overlapping peptide fragments can be tested for binding to the test antibody
in simple binding assays. In an
additional example, mutagenesis of an antigen binding domain, domain swapping
experiments and alanine
scanning mutagenesis can be performed to identify residues required,
sufficient, and/or necessary for epitope
binding. For example, domain swapping experiments can be performed using a
mutant of a target antigen in which
various fragments of the GARP-proTGFp1 complex, a LTBP1-proTGF31 complex, a
LTBP3-proTGF(31 complex,
and/or a proLRRC33-TGFp1 complex have been replaced (swapped) with sequences
from a closely related, but
antigenically distinct protein, such as another member of the TGFp protein
family (e.g., GDF11).
[517] Alternatively, competition assays can be performed using other
antibodies known to bind to the same
antigen to determine whether an antibody binds to the same epitope as the
other antibodies. Competition assays
are well known to those of skill in the art.
[518] In some embodiments, a pharmaceutical composition may be made by a
process comprising a step of:
selecting an antibody or antigen-binding fragment thereof, which cross-
competes with an antibody having a heavy
chain variable domain of SEQ ID NO: 7 and a light chain variable domain of SEQ
ID NO: 8 for binding to TGFI31
(e.g., to pro-TGFI31 and/or latent TGFp1).
[519] In some embodiments, a pharmaceutical composition may be made by the
process comprising a step of:
selecting an antibody or antigen-binding fragment thereof, which cross-
competes with the antibody selected from
the group consisting of Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,
Ab27, Ab28, Ab29, Ab30, Ab31,
Ab32, Ab33 and Ab34; and, formulating into a pharmaceutical composition.
[520] Preferably, the antibody selected by the process is a high-affinity
binder characterized in that the antibody
or the antigen-binding fragment is capable of binding to each of human LLCs
(e.g., hLTBP1-proTGF(31, hLTBP3-
proTGFP1, hGARP-proTGFP1 and hLRRC33-proTGFP1) with a Ko of 1 nM, as measured
by solution equilibrium
titration. Such cross-competing antibodies may be used in the treatment of
TGFI31-related indications a subject in
accordance with the present disclosure.
Various Modifications and Variations of Antibodies
[521] Non-limiting variations, modifications, and features of any of the
antibodies or antigen-binding fragments
thereof encompassed by the present disclosure are briefly discussed below.
Embodiments of related analytical
methods are also provided.
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[522] Naturally-occurring antibody structural units typically comprise a
tetramer. Each such tetramer typically is
composed of two identical pairs of polypeptide chains, each pair having one
full-length "light" (in certain
embodiments, about 25 kDa) and one full-length "heavy" chain (in certain
embodiments, about 50-70 kDa). The
amino-terminal portion of each chain typically includes a variable region of
about 100 to 110 or more amino acids
that typically is responsible for antigen recognition. The carboxy-terminal
portion of each chain typically defines a
constant region that can be responsible for effector function. Human antibody
light chains are typically classified
as kappa and lambda light chains. Heavy chains are typically classified as mu,
delta, gamma, alpha, or epsilon,
and define the isotype of the antibody. An antibody can be of any type (e.g.,
IgM, IgD, IgG, IgA, IgY, and IgE) and
class (e.g., IgG-1, IgG2, lgG3, lgG4, IgMi, IgM2, IgAt and IgA2). Within full-
length light and heavy chains, typically,
the variable and constant regions are joined by a "J" region of about 12 or
more amino acids, with the heavy chain
also including a "D" region of about 10 more amino acids (see, e.g.,
Fundamental Immunology, Ch. 7 (Paul, W.,
ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its
entirety)). The variable regions of each
light/heavy chain pair typically form the antigen binding site.
[523] The variable regions typically exhibit the same general structure of
relatively conserved framework regions
(FR) joined by three hyper variable regions, also called complementarity
determining regions or CDRs. The CDRs
from the two chains of each pair typically are aligned by the framework
regions, which can enable binding to a
specific epitope. From N-terminal to C-terminal, both light and heavy chain
variable regions typically comprise the
domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids
to each domain is typically
in accordance with the definitions of Kabat Sequences of Proteins of
Immunological Interest (National Institutes of
Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol.
196: 901-917; Chothia et al., (1989)
Nature 342: 878-883. The CDRs of a light chain can also be referred to as CDR-
L1, CDR-L2, and CDR-L3, and
the CDRs of a heavy chain can also be referred to as CDR-H1, CDR-H2, and CDR-
H3. In some embodiments, an
antibody can comprise a small number of amino acid deletions from the carboxy
end of the heavy chain(s). In
some embodiments, an antibody comprises a heavy chain having 1-5 amino acid
deletions in the carboxy end of
the heavy chain. In certain embodiments, definitive delineation of a CDR and
identification of residues comprising
the binding site of an antibody is accomplished by solving the structure of
the antibody and/or solving the structure
of the antibody-ligand complex. In certain embodiments, that can be
accomplished by any of a variety of techniques
known to those skilled in the art, such as X-ray crystallography. In some
embodiments, various methods of analysis
can be employed to identify or approximate the CDR regions. Examples of such
methods include, but are not
limited to, the Kabat definition, the Chothia definition, the AbM definition,
the definition described by Lu et al (see
above), and the contact definition.
[524] An "affinity matured" antibody is an antibody with one or more
alterations in one or more CDRs thereof,
which result in an improvement in the affinity of the antibody for antigen
compared to a parent antibody, which does
not possess those alteration(s). Exemplary affinity matured antibodies will
have nanomolar or even picomolar
affinities (e.g., Ko of ¨10-9 M-10-12 M range) for the target antigen.
Affinity matured antibodies are produced by
procedures known in the art. Marks et al., (1992) Bio/Technology 10: 779-783
describes affinity maturation by VH
and VL domain shuffling. Random mutagenesis of CDR and/or framework residues
is described by Barbas, et al.,
(1994) Proc Nat. Acad. Sci. USA 91: 3809-3813; Schier et al., (1995) Gene 169:
147- 155; Yelton et al., (1995) J.
lmmunol. 155: 1994-2004; Jackson et al., (1995) J. lmmunol. 154(7): 3310-9;
and Hawkins et al., (1992) J. Mol.
Biol. 226: 889-896; and selective mutation at selective mutagenesis positions,
contact or hypermutation positions
with an activity enhancing amino acid residue is described in U.S. Patent No.
6,914,128. Typically, a parent
antibody and its affinity-matured progeny (e.g., derivatives) retain the same
binding region within an antigen,
although certain interactions at the molecular level may be altered due to
amino acid residue alternation(s)
introduced by affinity maturation.
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[525] The term "CDR-grafted antibody" refers to antibodies, which comprise
heavy and light chain variable region
sequences from one species but in which the sequences of one or more of the
CDR regions of VH and/or VL are
replaced with CDR sequences of another species, such as antibodies having
murine heavy and light chain variable
regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced
with human CDR sequences.
[526] The term "chimeric antibody" refers to antibodies, which comprise heavy
and light chain variable region
sequences from one species and constant region sequences from another species,
such as antibodies having
murine heavy and light chain variable regions linked to human constant
regions.
[527] As used herein, the term "framework" or "framework sequence" refers to
the remaining sequences of a
variable region minus the CDRs. Because the exact definition of a CDR sequence
can be determined by different
systems, the meaning of a framework sequence is subject to correspondingly
different interpretations. The six
CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -I-12, and -H3 of heavy
chain) also divide the framework
regions on the light chain and the heavy chain into four sub-regions (FR1,
FR2, FR3 and FR4) on each chain, in
which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and
CDR3 between FR3 and
FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a
framework region, as referred by
others, represents the combined FR's within the variable region of a single,
naturally occurring immunoglobulin
chain. As used herein, a FR represents one of the four sub-regions, and FRs
represents two or more of the four
sub-regions constituting a framework region.
[528] In some embodiments, the antibody or antigen-binding fragment thereof
comprises a heavy chain
framework region 1 (H-FR1) having the following amino acid sequence with
optionally 1, 2 or 3 amino acid changes:
EVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 147). For example, the Gly residue at
position 16 may be
replaced with an Arg (R); and/or, the Ala residue at position 23 may be
replaced with a Thr (T).
[529] In some embodiments, the antibody or antigen-binding fragment thereof
comprises a heavy chain
framework region 2 (H-FR2) having the following amino acid sequence with
optionally 1, 2 or 3 amino acid changes:
VVVRQAPGKGLEVVVS (SEQ ID NO: 148).
[530] In some embodiments, the antibody or antigen-binding fragment thereof
comprises a heavy chain
framework region 3 (H-FR3) having the following amino acid sequence with
optionally 1, 2 or 3 amino acid changes:
RFTISRDNAKNSLYLQMNSLRAEDTAVYYC (SEQ ID NO: 149). For example, the Ser residue
at position 12 may
be replaced with a Thr (T).
[531] In some embodiments, the antibody or antigen-binding fragment thereof
comprises a heavy chain
framework region 4 (H-FR4) having the following amino acid sequence with
optionally 1, 2 or 3 amino acid changes:
WGQGTLVTVSS (SEQ ID NO: 150).
[532] In some embodiments, the antibody or antigen-binding fragment thereof
comprises a light chain framework
region 1 (L-FR1) having the following amino acid sequence with optionally 1, 2
or 3 amino acid changes:
DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 151).
[533] In some embodiments, the antibody or antigen-binding fragment thereof
comprises a light chain framework
region 2 (L-FR2) having the following amino acid sequence with optionally 1, 2
or 3 amino acid changes:
VVYQQKPGKAPKLLIY (SEQ ID NO: 152).
[534] In some embodiments, the antibody or antigen-binding fragment thereof
comprises a light chain framework
region 3 (L-FR3) having the following amino acid sequence with optionally 1, 2
or 3 amino acid changes:
GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 153).
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[535] In some embodiments, the antibody or antigen-binding fragment thereof
comprises a light chain framework
region 4 (L-FR4) having the following amino acid sequence with optionally 1, 2
or 3 amino acid changes:
FGGGTKVEIK (SEQ ID NO: 154).
[536] In some embodiments, the antibody, or antigen binding portion thereof,
comprises a heavy chain
immunoglobulin constant domain of a human IgM constant domain, a human IgG
constant domain, a human IgG1
constant domain, a human IgG2 constant domain, a human IgG2A constant domain,
a human IgG2B constant
domain, a human IgG2 constant domain, a human IgG3 constant domain, a human
IgG3 constant domain, a human
IgG4 constant domain, a human IgA constant domain, a human IgA1 constant
domain, a human IgA2 constant
domain, a human IgD constant domain, or a human IgE constant domain. In some
embodiments, the antibody, or
antigen binding portion thereof, comprises a heavy chain immunoglobulin
constant domain of a human IgG1
constant domain or a human IgG4 constant domain. In some embodiments, the
antibody, or antigen binding portion
thereof, comprises a heavy chain immunoglobulin constant domain of a human
IgG4 constant domain. In some
embodiments, the antibody, or antigen binding portion thereof, comprises a
heavy chain immunoglobulin constant
domain of a human IgG4 constant domain having a backbone substitution of Ser
to Pro that produces an IgGl-like
hinge and permits formation of inter-chain disulfide bonds.
[537] In some embodiments, the antibody or antigen binding portion thereof,
further comprises a light chain
immunoglobulin constant domain comprising a human Ig lambda constant domain or
a human Ig kappa constant
domain.
[538] In some embodiments, the antibody is an IgG having four polypeptide
chains which are two heavy chains
and two light chains.
[539] In some embodiments, wherein the antibody is a humanized antibody, a
diabody, or a chimeric antibody.
In some embodiments, the antibody is a humanized antibody. In some
embodiments, the antibody is a human
antibody. In some embodiments, the antibody comprises a framework having a
human germline amino acid
sequence.
[540] In some embodiments, the antigen binding portion is a Fab fragment, a
F(ab')2 fragment, a scFab fragment,
or an scFv fragment.
[541] In some embodiments, the antibody contains one or more amino acid
modifications in the Fc region. In
some embodiments, modifications in the Fc region may provide altered
properties, such as altered half life in
circulation, e.g., by altering affinity for Fc receptors such as FcRn (Front
lmmunol. 2019 Jun 7;10:1296). In some
embodiments, modifications to the Fc region of the antibody provides increased
FcyR binding. In some
embodiments, modifications in the Fc region provide improved antibody effector
function. In some embodiments,
modifications in the Fc region provide increased in vivo half-life for the
antibody. In some embodiments, the half
life of the antibody may be increased by increasing its affinity for binding
to FcRn at low pH (e.g., pH <6.5). In some
embodiments, such Fc modifications may include Met252Tyr, Ser254Thr, and
Thr256Glu substitutions (see, e.g.,
US7083784).
[542] As used herein, the term "germline antibody gene" or "gene fragment"
refers to an immunoglobulin
sequence encoded by non-lymphoid cells that have not undergone the maturation
process that leads to genetic
rearrangement and mutation for expression of a particular immunoglobulin (see,
e.g., Shapiro et al., (2002) Crit.
Rev. lmmunol. 22(3): 183-200; Marchalonis et al., (2001) Adv. Exp. Med. Biol.
484: 13-30). One of the advantages
provided by various embodiments of the present disclosure stems from the
recognition that germline antibody
genes are more likely than mature antibody genes to conserve essential amino
acid sequence structures
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characteristic of individuals in the species, hence less likely to be
recognized as from a foreign source when used
therapeutically in that species.
[543] As used herein, the term "neutralizing" refers to counteracting the
biological activity of an antigen (e.g.,
target protein) when a binding protein specifically binds to the antigen. In
an embodiment, the neutralizing binding
protein binds to the antigen/ target, e.g., cytokine, kinase, growth factor,
cell surface protein, soluble protein,
phosphatase, or receptor ligand, and reduces its biologically activity by at
least about 20%, 40%, 60%, 80%, 85%,
90%, 95%. 96%, 97%. 98%, 99% or more. In some embodiments, a neutralizing
antibody to a growth factor
specifically binds a mature, soluble growth factor that has been released from
a latent complex, thereby preventing
its ability to bind its receptor to elicit downstream signaling. In some
embodiments, the mature growth factor is
TGFp1 or TGFp3. The term "binding protein" as used herein includes any
polypeptide that specifically binds to an
antigen (e.g., TGF(31), including, but not limited to, an antibody, or antigen
binding portions thereof, and a bispecific
or multispecific construct that comprises an antigen binding region (e.g., a
region capable of binding TGFP1) and
a region capable of binding one or more additional antigens or additional
epitopes on a single antigen. Examples
include a DVD-IgTM, a TVD-Ig, a RAb-Ig, a bispecific antibody, and a dual
specific antibody. A binding protein may
also comprise an antibody-drug conjugate, e.g., wherein a second agent (e.g.,
a small molecule checkpoint
inhibitor) is linked to an antibody or antigen-binding fragment thereof
capable of binding TGFI31 (e.g., capable of
binding pro- and/or latent-TGFp1)
[544] The term "monoclonal antibody" or "mAb" when used in a context of a
composition comprising the same
may refer to an antibody preparation obtained from a population of
substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical except for
possible naturally occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly specific,
being directed against a single
antigen. Furthermore, in contrast to polyclonal antibody preparations that
typically include different antibodies
directed against different determinants (epitopes), each mAb is directed
against a single determinant on the
antigen. The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular
method.
[545] The term ''recombinant human antibody," as used herein, is intended to
include all human antibodies that
are prepared, expressed, created or isolated by recombinant means, such as
antibodies expressed using a
recombinant expression vector transfected into a host cell (described further
in Section II C, below), antibodies
isolated from a recombinant, combinatorial human antibody library (Hoogenboom,
H.R. (1997) TIE Tech. 15: 62-
70; Azzazy, H. and Highsmith, W.E. (2002) Clin. Biochem. 35: 425-445;
Gavilondo, J.V. and Larrick, J.W. (2002)
BioTechniques 29: 128-145; Hoogenboom, H. and Chames, P. (2000) Immunot Today
21: 371-378, incorporated
herein by reference), antibodies isolated from an animal (e.g., a mouse) that
is transgenic for human
immunoglobulin genes (see, Taylor, L. D. et al., (1992) Nucl. Acids Res. 20:
6287-6295; Kellermann, S-A. and
Green, L.L. (2002) Cur. Opin. in Biotechnol. 13: 593-597; Little, M. et al.,
(2000) Immunol. Today 21: 364-370) or
antibodies prepared, expressed, created or isolated by any other means that
involves splicing of human
immunoglobulin gene sequences to other DNA sequences. Such recombinant human
antibodies have variable
and constant regions derived from human germline immunoglobulin sequences. In
certain embodiments, however,
such recombinant human antibodies are subjected to in vitro mutagenesis (or,
when an animal transgenic for
human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino
acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while derived from
and related to human germline VH
and VL sequences, may not naturally exist within the human antibody germline
repertoire in vivo.
[546] As used herein, "Dual Variable Domain lmmunoglobulin" or "DVD-IgTM" and
the like include binding proteins
comprising a paired heavy chain DVD polypeptide and a light chain DVD
polypeptide with each paired heavy and
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light chain providing two antigen binding sites. Each binding site includes a
total of 6 CDRs involved in antigen
binding per antigen binding site. A DVD-IgTM is typically has two arms bound
to each other at least in part by
dimerization of the CH3 domains, with each arm of the DVD being bispecific,
providing an immunoglobulin with
four binding sites. DVD-IgTM are provided in US Patent Publication Nos.
2010/0260668 and 2009/0304693, each
of which are incorporated herein by reference including sequence listings.
[547] As used herein, "Triple Variable Domain Immunoglobulin" or "TVD-Ig" and
the like are binding proteins
comprising a paired heavy chain TVD binding protein polypeptide and a light
chain TVD binding protein polypeptide
with each paired heavy and light chain providing three antigen binding sites.
Each binding site includes a total of
6 CDRs involved in antigen binding per antigen binding site. A TVD binding
protein may have two arms bound to
each other at least in part by dimerization of the CH3 domains, with each arm
of the TVD binding protein being
trispecific, providing a binding protein with six binding sites.
[548] As used herein, "Receptor-Antibody lmmunoglobulin" or "RAb-Ig" and the
like are binding proteins
comprising a heavy chain RAb polypeptide, and a light chain RAb polypeptide,
which together form three antigen
binding sites in total. One antigen binding site is formed by the pairing of
the heavy and light antibody variable
domains present in each of the heavy chain RAb polypeptide and the light chain
RAb polypeptide to form a single
binding site with a total of 6 CDRs providing a first antigen binding site.
Each the heavy chain RAb polypeptide
and the light chain RAb polypeptide include a receptor sequence that
independently binds a ligand providing the
second and third "antigen" binding sites. A RAb-Ig typically has two arms
bound to each other at least in part by
dimerization of the CH3 domains, with each arm of the RAb-Ig being
trispecific, providing an immunoglobulin with
six binding sites. RAb-Igs are described in US Patent Application Publication
No. 2002/0127231, the entire
contents of which including sequence listings are incorporated herein by
reference).
[549] In various embodiments, the present disclosure provides, in part, novel
antibodies and antigen-binding
fragments that may be used alone, linked to one or more additional agents
(e.g., as ADCs), or as part of a larger
macromolecule (e.g., a bispecific antibody, dual-specific antibody, or as a
multispecific antibody, or as part of a
construct further comprising a ligand trap, e.g., in combination with a TGFB
ligand trap such as M7824 (Merck) and
AVID200 (Forbius)), or as part of a bifunctional or multifunctional engineered
construct (e.g., fusion proteins and
ligand traps) and may be administered as part of pharmaceutical compositions
or combination therapies.
[550] The term "bispecific antibody," as used herein, and as differentiated
from a "bispecific half-Ig binding protein"
or "bispecific (half-Ig) binding protein", refers to full-length antibodies
that are generated by quadroma technology
(see Milstein, C. and Cuello, A.G. (1983) Nature 305(5934): p. 537-540), by
chemical conjugation of two different
monoclonal antibodies (see Staerz, U.D. et al., (1985) Nature 314(6012): 628-
631), or by knob-into-hole or similar
approaches, which introduce mutations in the Fc region that do not inhibit CH3-
CH3 dimerization (see Holliger, P.
et al., (1993) Proc. Natl. Acad. Sci USA 90(14): 6444-6448), resulting in
multiple different immunoglobulin species
of which only one is the functional bispecific antibody. By molecular
function, a bispecific antibody binds one
antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and
binds a different antigen (or epitope)
on its second arm (a different pair of HC/LC). By this definition, a
bispecific antibody has two distinct antigen
binding arms (in both specificity and CDR sequences), and is monovalent for
each antigen it binds to. For example,
a bispecific antibody comprising two binding arms directed toward TGF131 and
PD-1 may be used to combine a
TGF31 inhibitor (Ab6 or Ab6-derived binding moiety) and a checkpoint inhibitor
(e.g., an anti-PD1 antibody or
moiety). Such a bispecific antibody may be used as an exemplary form of
treatment for patients selected to receive
a TGF31 inhibitor and checkpoint inhibitor combination therapy.
[551] The term "dual-specific antibody," as used herein, and as differentiated
from a bispecific half-Ig binding
protein or bispecific binding protein, refers to full-length antibodies that
can bind two different antigens (or epitopes)
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in each of its two binding arms (a pair of HC/LC) (see PCT Publication No. WO
02/02773). Accordingly, a dual-
specific binding protein has two identical antigen binding arms, with
identical specificity and identical CDR
sequences, and is bivalent for each antigen to which it binds.
[552] The term "multispecific antibody" refers to an antibody or antigen
binding fragment that displays binding
specificity for two or more epitopes, where each binding site differs and
recognizes a different epitope (on the same
or different antigens). A bispecific antibody is an exemplary type of
multispecific antibody. Higher order
multispecifics (i.e., antibodies exhibiting more than two specificities)
include but are not limited to trispecific
antibodies in TriMAb, triple body, and tribody formats. For exemplary types of
multispecific antibodies and/or
methods of generating the same, see, e.g., Castoldi et al., Protein Eng Des
Se/2012;25:551-9; Schubert et al.,
MAbs 20113:21-30; Kugler et al., Br J Haematol 2010;150:574-86; Schoonjans et
al., J Immunol 2000;165:7050-
7; and Egan et al., MAbs 2017;9(1):68-84, which are all incorporated herein by
reference for such types and
methods.
[553] The term "Kon," as used herein, is intended to refer to the on rate
constant for association of a binding
protein (e.g., an antibody) to the antigen to form the, e.g., antibody/antigen
complex as is known in the art. The
"Kon" also is known by the terms "association rate constant," or "ka," as used
interchangeably herein. This value
indicating the binding rate of an antibody to its target antigen or the rate
of complex formation between an antibody
and antigen also is shown by the equation: Antibody ("Ab") + Antigen
("Ag")¨>Ab-Ag.
[554] The term "Koff," as used herein, is intended to refer to the off rate
constant for dissociation of a binding
protein (e.g., an antibody) from the, e.g., antibody/antigen complex as is
known in the art. The "Koff" also is known
by the terms "dissociation rate constant" or "kd" as used interchangeably
herein. This value indicates the
dissociation rate of an antibody from its target antigen or separation of Ab-
Ag complex over time into free antibody
and antigen as shown by the equation: Ab + Ag<¨Ab-Ag.
[555] The terms "equilibrium dissociation constant" or "Ko," as used
interchangeably herein, refer to the value
obtained in a titration measurement at equilibrium, or by dividing the
dissociation rate constant (koff) by the
association rate constant (kon). The association rate constant, the
dissociation rate constant, and the equilibrium
dissociation constant are used to represent the binding affinity of a binding
protein, e.g., antibody, to an antigen.
Methods for determining association and dissociation rate constants are well
known in the art. Using fluorescence¨
based techniques offers high sensitivity and the ability to examine samples in
physiological buffers at equilibrium.
Other experimental approaches and instruments, such as a Biacore
(biomolecular interaction analysis) assay,
can be used (e.g., instrument available from Biacore International AB, a GE
Healthcare company, Uppsala,
Sweden). Additionally, a KinExA (Kinetic Exclusion Assay) assay, available
from Sapidyne Instruments (Boise,
Idaho), can also be used.
[556] The terms "crystal" and "crystallized" as used herein, refer to a
binding protein (e.g., an antibody), or antigen
binding portion thereof, that exists in the form of a crystal. Crystals are
one form of the solid state of matter, which
is distinct from other forms such as the amorphous solid state or the liquid
crystalline state. Crystals are composed
of regular, repeating, three-dimensional arrays of atoms, ions, molecules
(e.g., proteins such as antibodies), or
molecular assemblies (e.g., antigen/antibody complexes). These three-
dimensional arrays are arranged according
to specific mathematical relationships that are well-understood in the field.
The fundamental unit, or building block,
that is repeated in a crystal is called the asymmetric unit. Repetition of the
asymmetric unit in an arrangement that
conforms to a given, well-defined crystallographic symmetry provides the "unit
cell" of the crystal. Repetition of the
unit cell by regular translations in all three dimensions provides the
crystal. See Giege, R. and Ducruix, A. Barrett,
Corstailization of Nucleic Acids and Proteins, a Practical Approach, 2nd ed.,
pp. 201-16, Oxford University Press,
New York, New York, (1999). The term "linker" is used to denote polypeptides
comprising two or more amino acid
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residues joined by peptide bonds and are used to link one or more antigen
binding portions. Such linker
polypeptides are well known in the art (see, e.g., Holliger, P. et al., (1993)
Proc. Natl. Acad. Sci. USA 90: 6444-
6448; Pollak, R.J. et al., (1994) Structure 2:1121-1123). Exemplary linkers
include, but are not limited to,
ASTKGPSVFPLAP (SEQ ID NO: 44), ASTKGP (SEQ ID NO: 45); TVAAPSVFIFPP (SEQ ID
NO: 46); TVAAP (SEQ
ID NO: 47); AKTTPKLEEGEFSEAR (SEQ ID NO: 48); AKTTPKLEEGEFSEARV (SEQ ID NO:
49); AKTTPKLGG
(SEQ ID NO: 50); SAKTTPKLGG (SEQ ID NO: 51); SAKTTP (SEQ ID NO: 52); RADAAP
(SEQ ID NO: 53);
RADAAPTVS (SEQ ID NO: 54); RADAAAAGGPGS (SEQ ID NO: 55); RADAAAA(G4S)4 (SEQ ID
NO: 56);
SAKTTPKLEEGEFSEARV (SEQ ID NO: 57); ADAAP (SEQ ID NO: 58); ADAAPTVSIFPP (SEQ
ID NO: 59);
QPKAAP (SEQ ID NO: 60); QPKAAPSVTLFPP (SEQ ID NO: 61); AKTTPP (SEQ ID NO: 62);
AKTTPPSVTPLAP
(SEQ ID NO: 63); AKTTAP (SEQ ID NO: 64); AKTTAPSVYPLAP (SEQ ID NO: 6576);
GGGGSGGGGSGGGGS
(SEQ ID NO: 66); GENKVEYAPALMALS (SEQ ID NO: 67); GPAKELTPLKEAKVS (SEQ ID NO:
68);
GHEAAAVMQVQYPAS (SEQ ID NO: 69); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO: 70); and
ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 71).
[557] "Label" and "detectable label" or "detectable moiety" mean a moiety
attached to a specific binding partner,
such as an antibody or an analyte, e.g., to render the reaction between
members of a specific binding pair, such
as an antibody and an analyte, detectable, and the specific binding partner,
e.g., antibody or analyte, so labeled is
referred to as "detectably labeled." Thus, the term "labeled binding protein"
as used herein, refers to a protein with
a label incorporated that provides for the identification of the binding
protein. In an embodiment, the label is a
detectable marker that can produce a signal that is detectable by visual or
instrumental means, e.g., incorporation
of a radiolabeled amino acid or attachment to a polypeptide of biotinyl
moieties that can be detected by marked
avidin (e.g., streptavidin containing a fluorescent marker or enzymatic
activity that can be detected by optical or
colorimetric methods). Examples of labels for polypeptides include, but are
not limited to, the following:
radioisotopes or radionuclides (e.g., 18F. 11C, 13N, 150, 88Ga, 18F, 89Zr, 3H,
14C, 35s, 90y, 99-rc, 111in, 1251, 1311, 177Lu,
1661-10, and 15351n); chromogens; fluorescent labels (e.g., FITC, rhodamine,
and lanthanide phosphors); enzymatic
labels (e.g., horseradish peroxidase, luciferase, and alkaline phosphatase);
chemiluminescent markers; biotinyl
groups; predetermined polypeptide epitopes recognized by a secondary reporter
(e.g., leucine zipper pair
sequences, binding sites for secondary antibodies, metal binding domains, and
epitope tags); and magnetic agents,
such as gadolinium chelates. Representative examples of labels commonly
employed for immunoassays include
moieties that produce light, e.g., acridinium compounds, and moieties that
produce fluorescence, e.g., fluorescein.
Other labels are described herein. In this regard, the moiety itself may not
be detectably labeled but may become
detectable upon reaction with yet another moiety. Use of "detectably labeled"
is intended to encompass the latter
type of detectable labeling.
[558] In some embodiments, the binding affinity of an antibody, or antigen
binding portion thereof, to an antigen
(e.g., protein complex), such as presenting molecule-proTGF131 complexes, is
determined using ELI (e.g., an
Octet assay). A BLI (e.g., Octet ) assay is an assay that determines one or
more a kinetic parameters indicative
of binding between an antibody and antigen. In some embodiments, an Octet
system (ForteBio , Menlo Park,
CA) is used to determine the binding affinity of an antibody, or antigen
binding portion thereof, to presenting
molecule-proTGF31 complexes. For example, binding affinities of antibodies may
be determined using the
FortelBio Octet QKe dip and read label free assay system utilizing bio-layer
interferometry. In some embodiments,
antigens are immobilized to biosensors (e.g., streptavidin-coated biosensors)
and the antibodies and complexes
(e.g., biotinylated presenting molecule-proTGF131 complexes) are presented in
solution at high concentration (50
pg/mL) to measure binding interactions. In some embodiments, the binding
affinity of an antibody, or antigen
binding portion thereof, to a presenting molecule-proTGF31 complex is
determined using the protocol outlined
herein.
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Characterization of Exemplary Antibodies Against proTGF131
Binding profiles
[559] Exemplary antibodies according to the present disclosure include those
having enhanced binding activities
(e.g., subnanomolar KD). Included are a class of high-affinity, context-
independent antibodies capable of
selectively inhibiting TGF[31 activation. Note that the term "context
independent" is used herein with a greater
degree of stringency as compared to previous more general usage. According to
the present disclosure, the term
confers a level of uniformity in relative affinities (i.e., unbias) that the
antibody can exert towards different antigen
complexes. Thus, the context-independent antibody of the present disclosure is
capable of targeting multiple types
of TGF[31 precursor complexes (e.g., presenting molecule-proTGF[31 complexes)
and of binding to each such
complex with equivalent affinities (i.e., no greater than three-fold
differences in relative affinities across the
complexes) with KID values lower than 10 nM, preferably lower than 5 nM, more
preferably lower than 1 nM, even
more preferably lower than 100 pM, as measured by, for example, MSD-SET. As
presented below, many
antibodies encompassed by the disclosure have KD values in a sub-nanomolar
range.
[560] Thus, the antibodies are capable of specifically binding to each of the
human presenting molecule-
proTGFI31 complexes (sometimes referred to as "Large Latency Complex" which is
a ternary complex comprised
of a proTGFI31 dimer coupled to a single presenting molecule), namely, LT6P1-
proTGFI31, LTBP3-proTGF131,
GARP-proTGFp1 and LRRC33-proTGFp1. Typically, recombinantly produced, purified
protein complexes are
used as antigens (e.g., antigen complexes) to evaluate or confirm the ability
of an antibody to bind the antigen
complexes in suitable in vitro binding assays. Such assays are well known in
the art and include, but are not limited
to Bio-Layer Interferometry (BLI)-based assays (such as Octet ) and solution
equilibrium titration-based assays
(such as MSD-SET).
[561] BLI-based binding assays are widely used in the art for measuring
affinities and kinetics of antibodies to
antigens. It is a label-free technology in which biomolecular interactions are
analyzed on the basis of optical
interference. One of the proteins, for example, an antibody being tested, can
be immobilized on the biosensor tip.
VVhen the other protein in solution, for example, an antigen, becomes bound to
the immobilized antibody, it causes
a shift in the interference pattern, which can be measured in real-time. This
allows the monitoring of binding
specificity, rates of association and dissociation, as well as concentration
dependency. Thus, BLI is a kinetic
measure that reveals the dynamics of the system. Due to its ease of use and
fast results, BLI-based assays such
as the Octet system (available from ForteBio /Molecular Devices , Fremont
California), are particularly
convenient when used as an initial screening method to identify and separate a
pool of "binders" from a pool of
"non-binders" or "weak binders" in the screening process.
[562] BLI-based binding assays revealed that the novel antibodies are
characterized as "context-
balanced/context-independent" antibodies when binding affinity is measured by
Octet . As can be seen in Table
summarizing BLI-based binding profiles of non-limiting examples of antibodies,
these antibodies show relatively
uniform KD values in a sub-nanomolar range across the four target complexes,
with relatively low matrix-to-cell
differentials (no greater than five-fold bias) (see column (H)). This can be
contrasted against the previously
identified antibody Ab3, provided as a reference antibody, which shows
significantly higher relative affinities
towards matrix-associated complexes (27+ fold bias) over cell-associated
complexes.
[563] Table 5 below provides non-limiting examples of context-independent
proTGF[31 antibodies encompassed
by the present disclosure. The table provides representative results from in
vitro binding assays, as measured by
Octet . Similar results are also obtained by an SPR-based technique (Biacore
System).
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[564] Column (A) of the table lists monoclonal antibodies with discrete amino
acid sequences. Ab3 (shown in
bold) is a reference antibody identified previously, which was shown to be
potent in cell-based assays; efficacious
in various animal models; and, with a clean toxicology profile (disclosed in:
WO 2018/129329). Columns (B), (D),
(E) and (F) provide affinities of each of the listed antibodies, measured in
KD. Column (B) shows the affinity to a
recombinant human LTBP1-proTGF131 complex; column (C) shows the affinity to a
recombinant human LTBP3-
proTGF31 complex; (E) shows the affinity to a recombinant human GARP-proTGF31
complex; and (F) shows the
affinity to a recombinant human LRRC33-proTGF31 complex, of each of the
antibodies. Average KD values of (B)
and (C) are shown in the corresponding column (D), which collectively
represents affinities of the antibodies to
ECM- or matrix-associated proTGF31 complexes. Similarly, Average KD values of
(E) and (F) are shown in the
corresponding column (G), which collectively represents affinities of the
antibodies to cell-surface or cell-associated
proTGF31 complexes. Finally, relative ratios between the average KD values
from columns (D) and (G) are
expressed as "fold bias" in column (H). Thus, the greater the number of column
(H) is, the greater bias exists for
the particular antibody, when comparing binding preferences of the antibody
for matrix-associated complexes and
cell-surface complexes. This is one way of quantitatively representing and
comparing inherent bias of antibodies
to their target complexes. Such analyses may be useful in guiding the
selection process for a candidate antibody
for particular therapeutic use.
Table 5. Non-limiting examples of context-independent TGF131 antibodies and KD
values measured
by BLI
(A) Matrix-associated proTGFb1 Cell-associated proTGFb1 (H)
Ab (B) (C) (D) (E) (F) (G) G/D
Ref hLTBP1 hLTBP3 ECM AVRG hGARP hLRRC33 Cell AVRG
(fold
(nM)
(nM) bias)
Ab3 4.70E-10 4.59E-10 0.4645 1.73E-08
8.52E-09 12.91 27.79
Ab21 2.25E-10 2.68E-10 0.2465 8.33E-10
4.55E-10 0.644 2.613
Ab22 3.18E-10 3.29E-10 0.3235 9.74E-10
4.15E-10 0.6945 2.147
Ab23 4.17E-10 4.68E-10 0.4425 1.34E-09
4.55E-10 0.8975 2.028
Ab24 2.46E-10 1.98E-10 0.222 6.65E-10
4.10E-10 0.5375 2.421
Ab25 2.17E-10 1.52E-10 0.1845 4.88E-10
4.09E-10 0.4485 2.431
Ab26 2.21E-10 1.73E-10 0.197 6.25E-10
3.60E-10 0.4925 2.500
Ab27 1.78E-10 2.38E-10 0.208 4.24E-10
2.99E-10 0.3615 1.738
Ab28 3.40E-10 3.16E-10 0.328 7.97E-10
4.09E-10 0.603 1.838
Ab29 1.89E-10 1.21E-10 0.155 3.07E-10
3.02E-10 0.3045 1.965
AB30 3.32E-10 2.61E-10 0.2965 8.33E-10
5.35E-10 0.684 2.307
Ab31 2.36E-10 1.81E-10 0.2085 5.81E-10
4.10E-10 0.4955 2.376
Ab6 2.07E-10 1.23E-10 0.165 4.04E-10
3.36E-10 0.37 2.242
Ab32 2.69E-10 2.15E-10 0.242 4.96E-10
6.98E-10 0.597 2.467
Ab33 1.79E-10 1.11E-10 0.145 2.65E-10
3.39E-10 0.302 2.083
[565] The disclosure provides a class of high-affinity, context-independent
antibodies, each of which is capable
of binding with equivalent affinities to each of the four known presenting
molecule-proTGF31 complexes, namely,
LTBP1-proTGF31, LTBP3-proTGF31 , GARP-proTGF31, and LRRC33-proTGF31. In some
embodiments, the
antibody binds each of the presenting molecule-proTGF31 complexes with
equivalent or higher affinities, as
compared to the previously described reference antibody, Ab3. According to the
disclosure, such antibody
specifically binds each of the aforementioned complexes with an affinity
(determined by KD) of 5 5 nM as measured
by a suitable in vitro binding assay, such as Biolayer Interferometry and
surface plasmon resonance. In some
embodiments, the antibody or the fragment binds a human LTBP1-proTGF31 complex
with an affinity of 5 5 nM, 5
4 nM, 5 3 nM, 5 2 nM, 5 1 nM, 5 5 nM or 5 0.5 nM. In some embodiments, the
antibody or the fragment binds a
human LTBP3-proTGF31 complex with an affinity of 5 5 nM, 5 4 nM, 5 3 nM, 5 2
nM, 5 1 nM, 5 5 nM or 5 0.5 nM.
In some embodiments, the antibody or the fragment binds a human GARP-proTGF31
complex with an affinity of 5
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nM, 5 4 nM, 5 3 nM, 5 2 nM, 1 nM, 5 5 nM or 5 0.5 nM. In some embodiments, the
antibody or the fragment
binds a human LRRC33-proTGFI31 complex with an affinity of 5 5 nM, 54 nM, 5 3
nM, 5 2 nM, 5 1 nM or 5 0.5 nM.
[566] In certain embodiments, such antibody is human- and murine-cross-
reactive. Thus, in some embodiments,
the antibody or the fragment binds a murine LTBP1-proTGF31 complex with an
affinity of 5 5 nM, 5 4 nM, 5 3 nM,
5 2 nM, 5 1 nM, 5 5 nM or 5 0.5 nM. In some embodiments, the antibody or the
fragment binds a murine LTBP3-
proTGF31 complex with an affinity of 5 5 nM, 5 4 nM, 5 3 nM, 5 2 nM, 1 nM or 5
0.5 nM. In some embodiments,
the antibody or the fragment binds a murine GARP-proTGFI31 complex with an
affinity of 5 5 nM, 5 4 nM, 5 3 nM,
5 2 nM, 1 nM or 5 0.5 nM. In some embodiments, the antibody or the fragment
binds a murine LRRC33-proTGFp1
complex with an affinity of 5 5 nM, 5 4 nM, 5 3 nM, 5 2 nM, 5 1 nM or 5 0.5
nM.
[567] As shown, the proTGF31 antibodies of the present disclosure have
particularly high affinities for matrix-
associated proTGE31 complexes. In some embodiments, the average KD value of
the matrix-associated
complexes (i.e., LTBP1-proTGF31 and LTBP3-proTGF31) is 5 1 nM or 5 0.5 nM.
[568] As shown, the proTGF31 antibodies of the present disclosure have high
affinities for cell-associated
proTGF31 complexes. In some embodiments, the average KD value of the cell-
associated complexes (i.e., GARP-
proTGF31 and LRRC33-proTGF31) is 5 2 nM or 5 1 nM.
[569] The high-affinity proTGFpl antibodies of the present disclosure are
characterized by their uniform
(unbiased) affinities towards the all four antigen complexes (compare, for
example, to Ab3). No single antigen
complex among the four known presenting molecule-proTGF31 complexes described
herein deviates significantly
in KD. In other words, more uniform binding activities have been achieved by
the present disclosure relative to
previously described proTGF31 antibodies (including Ab3) in that each such
antibody shows equivalent affinities
across the four antigen complexes. In some embodiments, the antibody orthe
fragment shows unbiased or uniform
binding profiles, characterized in that the difference (or range) of
affinities of the antibody or the fragments across
the four proTGF31 antigen complexes is no more than five-fold between the
lowest and the highest KD values. In
some embodiments, the relative difference (or range) of affinities is no more
than three-fold.
[570] The concept of "uniformity" or lack of bias is further illustrated in
Table 5. Average KD values between the
two matrix-associated and cell-associated complexes are calculated,
respectively (see columns (D) and (G)).
These average KD values can then be used to ask whether bias in binding
activities exists between complexes
associated with matrix vs. complexes associated with cell surface (e.g.,
immune cells). Bias may be expressed
as "fold-difference" in the average KD values, as illustrated in Table 5. As
compared to the previously described
antibody, Ab3, the high-affinity, context-independent proTGF31 antibodies
encompassed by the present
disclosure are remarkably unbiased in that many show no more than three-fold
difference in average KD values
between matrix- and cell-associated complexes (compare this to 25+ fold bias
in Ab3).
[571] Accordingly, a class of context-independent monoclonal antibodies or
fragments is provided, each of
which is capable of binding with equivalent affinities to each of the
following presenting molecule-proTGF31
complexes with an affinity of 5 1 nM as measured by Biolayer Interferometry or
surface plasmon resonance:
LTBP1-proTGF31, LTBP3-proTGF31, GARP-proTGF31, and LRRC33-proTGF31. Such
antibody specifically
binds each of the aforementioned complexes with an affinity of 5 5 nM as
measured by Biolayer lnterferometry or
surface plasmon resonance, wherein the monoclonal antibody or the fragment
shows no more than a three-fold
bias in affinity towards any one of the above complexes relative to the other
complexes, and wherein the
monoclonal antibody or the fragment inhibits release of mature TGF31 growth
factor from each of the proTGF31
complexes but not from proTGF32 or proTGF33 complexes.
[572] Whilst the kinetics of binding profiles (e.g
and and "off" rates) obtainable from BLI-based assays provide
useful information, Applicant of the present disclosure contemplated that,
based on the mechanism of action of
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the activation inhibitors disclosed herein, that is, antibodies that work by
binding to a tethered (e.g., tissue-
localized) inactive (e.g., latent) target thereby preventing it from getting
activated, binding properties measured at
equilibrium might more accurately reflect their in vivo behavior and potency.
To put this in perspective, as an
example, antibodies with fast "on" rate ("K.") which would be reflected in
binding measurements obtained by BLI,
may provide relevant parameters for evaluating neutralizing antibodies (e.g.,
antibodies that directly target and
must rapidly sequester the active, soluble growth factor itself for them to
function as effective inhibitors).
However, the same may not necessarily apply for antibodies that function as
activation inhibitors, such as those
disclosed herein. As described, the mechanism of action of the novel TGF131
inhibitors of the present disclosure
is via the inhibition of the activation step, which is achieved by targeting
the tissue/cell-tethered latent complex,
as opposed to sequestration of soluble, post-activation growth factor. This is
because an activation inhibitor of
TGF31 targets the inactive precursor localized to respective tissues (e.g.,
within the ECM, immune cell surface,
etc.) thereby preemptively prevent the mature growth factor from being
released from the complex. This
mechanism of action is thought to allow the inhibitor to achieve target
saturation (e.g., equilibrium) in vivo, without
the need for rapidly competing for transient growth factor molecules against
endogenous receptors as required
by conventional neutralizing inhibitors.
[573] Taking this difference in the mechanism of action into consideration,
further evaluation of binding
properties was carried out by the use of another mode of in vitro binding
assays that allows the determination of
affinity at equilibrium.
[574] In view of this, it is contemplated that assays that measure binding
affinities of such antibodies at
equilibrium may more accurately represent the mode of target engagement in
vivo. Thus, MSD-SET-based binding
assays (or other suitable assays) may be performed, as exemplified in Table 6
below.
[575] Solution equilibrium titration ("SET") is an assay whereby binding
between two molecules (such as an
antigen and an antibody that binds the antigen) can be measured at equilibrium
in a solution. For example, Meso-
Scale Discovery ("MSD")-based SET, or MSD-SET, is a useful mode of determining
dissociation constants for
particularly high-affinity protein-protein interactions at equilibrium (see,
for example: Ducata et al., (2015) J
Biomolecular Screening 20(10): 1256-1267). The SET-based assays are
particularly useful for determining KD
values of antibodies with sub-nanomolar (e.g., picomolar) affinities.
Table 6. Non-limiting examples of high-affinity context-independent TG931
antibodies (hIgG4) and
KD values measured by MSD-SET ("h" denotes human complex)
(A) Matrix-associated proTGFI31 Cell-associated proTGFI31
(H)
Ab Ref (B) (C) (D) (E) (F) (G)
G/D (fold
hLTBP1 hLTBP3 ECM AVRG hGARP hLRRC33 Cell AVRG
bias)
(nM) (nM)
Cl 3.30E-08 1.40E-08 23.2 5.10E-09 2.20E-09
3.65 0.16
C2 2.10E-08 1.20E-08 16.5 8.80E-09 6.10E-09
7.45 0.48
Ab3 1.30E-08 1.62E-08 14.6 2.80E-08 3.50E-08
31.5 2.16
Ab6 1.8E-11 2.9E-11 0.024 2.7E-11 6.3E-11
0.045 1.88
Ab22 5.00E-11 3.30E-11 0.042 2.70E-11 2.00E-10
0.114 2.71
Ab24 2.40E-11 2.10E-11 0.023 1.90E-11 1.80E-10
0.100 4.35
Ab26 2.80E-11 2.30E-11 0.026 1.40E-1 1 1.30E-
10 0.072 2.77
Ab29 1.20E-11 1.10E-11 0.012 5.50E-12 4.30E-11
0.024 2.00
Ab30 3.10E-11 2.60E-11 0.029 2.20E-11 1.40E-10
0.081 2.80
Ab31 1.90E-11 1.40E-11 0.017 1.90E-11 9.60E-11
0.058 3.41
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Ab32 3.70E-11 2.60E-11 0.032 1.50E-11 8.70E-11
0.051 1.60
Ab33 1.10E-11 7.00E-12 0.009 7.80E-12 4.60E-11
0.027 3.00
Ab4 4.6E-9 5.5E-9 5.05 2.5E-9 2.1E-9 2.3
0.42
[576] Table 6 also includes three previously described TGFP1-selective
antibodies (Cl, 02 and Ab3) as reference
antibodies. Cl and C2 were first disclosed in PCT/US2017/021972 published as
WO 2017/156500 (corresponding
to "Abl" and "Ab2" therein), and Ab3 was described in PCT/1J52018/012601
published as WO 2018/129329
(corresponding to "Ab3" therein).
[577] As can be seen from the affinity data provide in Table 6, binding
activities of the novel antibodies according
to the present disclosure are significantly higher than the previously
identified reference antibodies. Moreover, the
novel TGFP1 antibodies are "context-independent" in that they bind to each of
the human LLC complexes with
equivalent affinities (e.g., sub-nanomolar range, e.g., with Ko of < 1 nM).
The high-affinity, context-independent
binding profiles suggest that these antibodies may be advantageous for use in
the treatment of TGFp1 -related
indications that involve dysregulation of both the ECM-related and immune
components, such as cancer.
[578] For solution equilibrium titration-based binding assays, protein
complexes that comprise one of the
presenting molecules such as those shown above may be employed as antigen
(presenting molecule-TGFpl
complex, or an LLC). Test antibodies are allowed to form antigen-antibody
complex in solution. Antigen-
antibody reaction mixtures are incubated to allow an equilibrium to be
reached; the amount of the antigen-
antibody complex present in the assay reactions can be measured by suitable
means well known in the art. As
compared to BLI-based assays, SET-based assays are less affected by on/off
rates of the antigen-antibody
complex, allowing sensitive detection of very high affinity interactions. As
shown in Table 6, in the present
disclosure, certain high-affinity inhibitors of TGFp1 show a sub-nanomolar
(e.g., picomolar) range of affinities
across all large latent complexes tested, as determined by SET-based assays.
[579] Accordingly, a class of context-independent monoclonal antibodies or
fragments is provided, each of
which is capable of binding with equivalent affinities to each of the
following human presenting molecule-
proTGFI31 complexes with a Ko of 5 1 nM as measured by a solution equilibrium
titration assay, such as MSD-
SET: hLTBP1-proTGFp1, hLTBP3-proTGFp1, hGARP-proTGFp1, and hLRRC33-proTGFp1.
Such antibody
specifically binds each of the aforementioned complexes with a Ko of 5 1 nM as
measured by MSD-SET, and
wherein the monoclonal antibody or the fragment inhibits release of mature
TGFp1 growth factor from each of the
proTGFp1 complexes but not from proTGFp2 or proTGFp3 complexes. In certain
embodiments, such antibody
or the fragment binds each of the aforementioned complexes with a KD of 500 pM
or less (i.e., 5 500 pM), 250 pM
or less (i.e., 5 250 pM), or 200 pM or less (i.e., 5 200 pM). Even more
preferably, such antibody or the fragment
binds each of the aforementioned complexes with a Ko of 100 pM or less (i.e.,
5 100 pM). In some embodiments,
the antibody or the fragment does not bind to free TGFp1 growth factor which
is not associated with the
prodomain complex. In some embodiments, the antibody or the fragment does not
bind to LTBP1/TGFp2 or
LTBP3/TGFP3 LLCs. This can be tested or confirmed by suitable in vitro binding
assays known in the art, such
as biolayer interferometry.
[580] In further embodiments, such antibodies or the fragments are also cross-
reactive with murine (e.g., rat
and/or mouse) and/or non-human primate (e.g., cyno) counterparts. To give but
one example, Ab6 is capable of
binding with high affinity to each of the large latent complexes of multiple
species, including: human, murine, rat,
and cynomolgus monkey, as exemplified in Table 7 below.
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Table 7. Non-limiting example of a TG931 antibody with cross-species
reactivities as measured by
MSD-SET ("h" denotes human; "m" denotes murine)
Ag hLTBP1- hLTBP3- hGARP- hLRRC33mLTBP1- mLTBP3- mGARP- mLRRC33
comple proTG93 proTG93 proTG93 proTGFI3 proTG93 proTG93
proTG93
1 1 1 1 1 1
proTG931
1
Ab6 1.80E-11 2.90E-11 2.70E-11 6.30E-11 2.40E-11
2.80E-11 2.10E-11 4.80E-11
[581] Surface plasmon resonance (SPR) provides useful binding kinetics
information with good resolution and
sensitivity, which enables detection of unlabeled biomolecular interactants
(such as antibody-antigen interactions)
in real time. The SPR-based biosensors (such as Biacore systems) can be used
in determination of active
concentration as well as characterization of molecular interactions in terms
of both affinity and chemical kinetics.
With respect to antibodies that target latent prodomain complex and inhibit
the activation step of growth factor from
the latent complex, in addition to having high overall affinities (typically
expressed as the equilibrium dissociation
constant or KD), it may be particularly advantageous to have slow off rates,
or KoFF, As previously exemplified in
Example 1 of PCT/US2019/041373, Ab6, which is an activation inhibitor of.TGF31
, binds each LLC with a KID of
less than 0.5 nM with KoFF of less than 10.0E-4 (1/s), as measured by SPR. The
off rate of an antibody may
therefore be an important binding kinetics criterion for selection
consideration for a therapeutic antibody to be
manufactured and for use in human therapy described herein.
[582] Accordingly, the invention includes a TGFI3 inhibitor which is an
antibody or antigen-binding fragment
thereof, for use in the treatment of cancer in a subject (according to the
present disclosure), wherein the antibody
or the fragment with a KOFF of less than 10.0E-4 (1/s) is selected, wherein
optionally the selected antibody has a
KD of less than 0.5 nM as measured by SPR.
[583] The invention further includes a method for manufacturing a
pharmaceutical composition comprising a
TGF3 inhibitor which is an antibody or antigen-binding fragment thereof, for
use in the treatment of cancer in a
subject (according to the present disclosure), the method comprising the step
of selecting an antibody or antigen-
binding fragment which has a KoFF of less than 10.0E-4 (1/s) and optionally
has a KD of less than 0.5 nM as
measured by SPR.
Potency
[584] Antibodies disclosed herein may be broadly characterized as "functional
antibodies" for their ability to inhibit
TGF31 signaling. As used herein, "a functional antibody" confers one or more
biological activities by virtue of its
ability to bind a target protein (e.g., antigen), in such a way as to modulate
its function. Functional antibodies
therefore broadly include those capable of modulating the activity/function of
target molecules (i.e., antigen). Such
modulating antibodies include inhibiting antibodies (or inhibitory antibodies)
and activating antibodies. The present
disclosure is drawn to antibodies which can inhibit a biological process
mediated by TGF3 signaling associated
with multiple contexts of TGF31. Inhibitory agents used to carry out the
present disclosure, such as the antibodies
described herein, are intended to be TGF31-selective and not to target or
interfere with TGF32 and TGF33 when
administered at a therapeutically effective dose (dose at which sufficient
efficacy is achieved within acceptable
toxicity levels). The novel antibodies of the present disclosure have enhanced
inhibitory activities (potency) as
compared to previously identified activation inhibitors of TGF31.
[585] In some embodiments, potency of an inhibitory antibody may be measured
in suitable cell-based assays,
such as CAGA reporter cell assays described herein. Generally, cultured cells,
such as heterologous cells and
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primary cells, may be used for carrying out cell-based potency assays. Cells
that express endogenous TGFpl
and/or a presenting molecule of interest, such as LTBP1, LTBP3, GARP and
LRRC33, may be used. Alternatively,
exogenous nucleic acids encoding protein(s) of interest, such as TGF(31 and/or
a presenting molecule of interest,
such as LTBP1, LTBP3, GARP and LRRC33, may be introduced into such cells for
expression, for example by
transfection (e.g., stable transfection or transient transfection) or by viral
vector-based infection. In some
embodiments, LN229 cells are employed for such assays. The cells expressing
TGFp1 and a presenting molecule
of interest (e.g., LTBP1, LTBP3, GARP or LRRC33) are grown in culture, which
"present" the large latent complex
either on cell surface (when associated with GARP or LRRC33) or deposit into
the ECM (when associated with an
LTBP). Activation of TGFI31 may be triggered by integrin, expressed on another
cell surface. The integrin-
expressing cells may be the same cells co-expressing the large latent complex
or a separate cell type. Reporter
cells are added to the assay system, which incorporates a TGFp-responsive
element. In this way, the degree of
TGFp activation may be measured by detecting the signal from the reporter
cells (e.g., TGFp-responsive reporter
genes, such as luciferase coupled to a TGF[3-responsive promoter element) upon
TGF13 activation. Using such
cell-based assay systems, inhibitory activities of the antibodies can be
determined by measuring the change
(reduction) or difference in the reporter signal (e.g., luciferase activities
as measured by fluorescence readouts)
either in the presence or absence of test antibodies. Such assays are
exemplified in PCT/US2019/041373 at
Example 2.
[586] Thus, in some embodiments, the inhibitory potency (IC50) of the novel
antibodies of the present disclosure
calculated based on cell-based reporter assays for measuring TGFI31 activation
(such as LN229 cell assays
described elsewhere herein) may be 5 nM or less, measured against each of the
hLTBP1-proTGF31, hLTBP3-
proTGFp1, hGARP-proTGFp1 and hLRRC33-proTGFp1 complexes. In some embodiments,
the antibodies have
an IC50 of 2 nM or less (i.e., 2 nM) measured against each of the LLCs. In
certain embodiments, the IC50 of the
antibody measured against each of the LLC complexes is 1nM or less. In some
embodiments, the antibody has
an IC50 of less than 1 nM against each of the hLTBP1-proTGFI31, hLTBP3-
proTGFI31, hGARP-proTGFpl and
hLRRC33-proTGFp1 complexes.
Table 8. Inhibitory potencies (in IC50) of select antibodies as measured by
reporter cell assays
Ab Ref. IC50 (nM)
hLTBP1-proTGF[31 hLTBP3-proTGF[31 hGARP-proTGF[31
hLRRC33-proTGF131
Ab4 5.2 5.6 0.8
3.5
Ab5 1.3 1.0 0.1
0.6
Ab6 1.0-2.7 0.8-2.7 0.3-1.6 0.5-
1.9
Ab21 1.6 0.8 0.4
0.6
Ab23 0.8 0.9 0.3
0.6
Ab25 6.1 0.51 0.4
0.7
Ab26 0.7 0.7 0.3
0.3
Ab29 0.5 0.8 0.3
0.5
Ab33 1.6 1.1 0.2
0.7
[587] Activation of TGFI31 may be triggered by an integrin-dependent mechanism
or protease-dependent
mechanism. The inhibitory activities (e.g., potency) of the antibodies
according to the present disclosure may be
evaluated for the ability to block TGF(31 activation induced by one or both of
the modes of activation. The reporter
cell assays described above are designed to measure the ability of the
antibodies to block or inhibit integrin-
dependent activation of TGFI31 activation. Inhibitory potency may also be
assessed by measuring the ability of the
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antibodies to block protease-induced activation of TGF81 . Non-limiting
embodiments of such assays were
previously disclosed. See, e.g., PCT/U52019/041373 at Example 3. Accordingly,
in some embodiments of the
disclosure, the isoform-selective inhibitor according to the present
disclosure is capable of inhibiting integrin-
dependent activation of TGF61 and protease-dependent activation of TGF61. Such
inhibitor may be used to treat
a TGF61-related indication characterized by EDM dysregulation involving
protease activities. For example, such
TGF31-related indication may be associated with elevated myofibroblasts,
increased stiffness of the ECM, excess
or abnormal collagen deposition, or any combination thereof. Such conditions
include, for example, fibrotic
disorders and cancer comprising a solid tumor (such as metastatic carcinoma)
or myelofibrosis.
[588] In some embodiments, potency may be evaluated in suitable in vivo models
as a measure of efficacy and/or
pharmacodynamics effects. For example, if the first antibody is efficacious in
an in vivo model at a certain
concentration, and the second antibody is equally efficacious at a lower
concentration than the first in the same in
vivo model, then, the second antibody can be said to me more potent than the
first antibody. Any suitable disease
models known in the art may be used to assess relative potencies of TGF81
inhibitors, depending on the particular
indication of interest, e.g., cancer models and fibrosis models. Preferably,
multiple doses or concentrations of each
test antibody are included in such studies.
[589] Similarly, pharmacodynamics (PD) effects may be measured to determine
relative potencies of inhibitory
antibodies. Commonly used PD measures for the TGFp signaling pathway include,
without limitation,
phosphorylation of SMAD2/3 and expression of downstream effector genes, the
transcription of which is sensitive
to TGF6 activation, such as those with a TGF6-responsive promoter element
(e.g., Smad-binding elements). In
some embodiments, the antibodies of the present disclosure are capable of
completely blocking disease-induced
SMAD2/3 phosphorylation in preclinical fibrosis models when the animals are
administered at a dose of 3 mg/kg
or less. In some embodiments, the antibodies of the present disclosure are
capable of reducing and/or completely
blocking disease-induced SMAD2/3 phosphorylation. In some embodiments, the
antibodies of the present
disclosure are capable of reducing and/or completely blocking disease-induced
SMAD2 phosphorylation (e.g.,
regardless of any change in SMAD3) , e.g., in the nucleus. In some
embodiments, reduction is measured as a ratio
of phosphorylated SMAD2/3 over total SMAD2/3. In some embodiments, reduction
is measured as a ratio of
phosphorylated SMAD2 over total SMAD2. In some embodiments, the antibodies of
the present disclosure are
capable of reducing nuclear localization of phosphorylated SMAD2, as measured,
for example, by IHC.
[590] In some embodiments, measurement of nuclear localization of
phosphorylated Smad2 (P-Smad2) can use
one or more digital image analysis parameters to identify degrees of P-Smad2
nucleus staining intensities (e.g., a
digital image analysis parameter that allows visualization and measurement of
INC signal intensity of an individual
nucleus, e.g., a nucleus mask). P-Smad2 positive nuclei can be sorted into
categories (e.g., 1+, 2+, 3+) based on
chromogenic intensity relative to normalized scanning parameters. The analysis
can allow for data to be considered
in multiple contexts (e.g., % Nuclear Positivity, H-Score, Density), and can
be combined with additional analysis
parameters (e.g., compartment area, total number of quantified cells, number
of P-Smad2 positive cells, and/or
number or percent of 0 P-Smad2, 1+ P-Smad2, 2+ P-Smad2, or 3+ P-Smad2 cells).
Pseudocolor image masks
can be applied to each staining category for visualization and QC purposes.
Exemplary tools for analyzing nuclear
masking include, but are not limited to, software developed by Visiopharm,
Indica Labs (e.g., HALO imaging
analysis platform), and Flagship Biosciences. VVithout being bound by theory,
in some embodiments, measuring
SMAD2 phosphorylation e.g., nuclear SMAD2 phosphorylation (without measuring
SMAD3) may improve the
accurate detection of a treatment-related effect (e.g., therapeutic efficacy
and/or target engagement). Denis et al.,
Development 143: 3481-90 (2016); Liu et al., J. Biol. Chem. 278: 11721-8
(2003); David et al., Oncoimmunology
6: e1349589 (2017).
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[591] In some embodiments, P-Smad2 nuclear translocation may be used in a
method of determining therapeutic
efficacy in a subject administered one or more doses of a TGFI3 inhibitor
(e.g., Ab6). In some embodiments, a level
of P-Smad2 nuclear translocation is determined in a tumor sample obtained from
the subject before and after
administering one or more doses of a TGFp inhibitor (e.g., Ab6), wherein a
decrease in P-Smad2 nuclear
translocation after the administration as compared to before the
administration indicates therapeutic efficacy. In
some embodiments, the P-Smad2 nuclear translocation may be determined using
immunohistochemistry. In some
embodiments, the P-Smad2 nuclear translocation may be determined using nuclear
masking. In some
embodiments, the P-Smad2 nuclear translocation may be determined using a
digital image analysis tool, such as
one developed by Flagship Biosciences, Visiopharm, or Indica Labs. In some
embodiments, a decrease of at least
1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-
fold, 1.9-fold, 2-fold, 2.2-fold, 2.4-fold, 2.6-fold,
2.8-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or more, in P-Smad2
nuclear translocation levels after the
administration as compared to before the administration indicates therapeutic
efficacy. In some embodiments, one
or more additional doses of the TGFp inhibitor (e.g., Ab6) is administered if
a decrease is detected.
[592] In some embodiments, P-Smad2 nuclear translocation may be used in a
method of determining target
engagement in a subject administered one or more doses of a TGFI3 inhibitor
(e.g., Ab6). In some embodiments,
a level of P-Smad2 nuclear translocation is determined in a tumor sample
obtained from the subject before and
after administering one or more doses of a TGFp inhibitor (e.g., Ab6), wherein
a decrease in P-Smad2 nuclear
translocation after the administration as compared to before the
administration indicates target engagement. In
some embodiments, the P-Smad2 nuclear translocation may be determined using
immunohistochemistry. In some
embodiments, the P-Smad2 nuclear translocation may be determined using nuclear
masking. In some
embodiments, the P-Smad2 nuclear translocation may be determined using a
digital image analysis tool, such as
one developed by Flagship Biosciences, Visiopharm, or Indica Labs. In some
embodiments, a decrease of at least
1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-
fold, 1.9-fold, 2-fold, 2.2-fold, 2.4-fold, 2.6-fold,
2.8-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or more, in P-Smad2
nuclear translocation levels after the
administration as compared to before the administration indicates target
engagement. In some embodiments, one
or more additional doses of the TGFI3 inhibitor (e.g., Ab6) is administered if
a decrease is detected.
[593] In some embodiments, P-3mad2 nuclear translocation may be used in
conjunction with other biomarkers,
such as tumor CD8+ cells (e.g., percent CD8+ cells in various tumor
compartments, e.g., tumor nests, stroma,
margin), to determine therapeutic efficacy and/or target engagement in a
subject administered one or more doses
of a TGFp inhibitor (e.g., Ab6). In some embodiments, therapeutic efficacy
and/or target engagement may be
indicated by, inter alia, a decrease in P-Smad2 nuclear translocation in a
tumor sample obtained from the subject
after the administration of a TGF8 inhibitor (e.g., Ab6) as compared to before
the administration. In some
embodiments, therapeutic efficacy and/or target engagement may be indicated by
a decrease in P-Smad2 nuclear
translocation and an increase in CD8+ cells in a tumor sample obtained from
the subject after the administration
as compared to before the administration. In some embodiments, therapeutic
efficacy and/or target engagement
may be indicated by a decrease in P-Smad2 nuclear translocation and an
increase in total tumor area comprising
immune-inflamed tumor nests in a tumor sample obtained from the subject after
the administration as compared to
before the administration. In some embodiments, more than one additional
marker is used to assess therapeutic
efficacy and/or target engagement in conjunction with P-Smad2 nuclear
translocation. In some embodiments, the
TGFp inhibitor (e.g., Ab6) is administered in conjunction with a checkpoint
inhibitor therapy. In some embodiments,
additional doses of the TGFp inhibitor (e.g., Ab6) may be administered to
patients whose tumors show therapeutic
efficacy and/or target engagement.
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[594] In some embodiments, the antibodies of the present disclosure are
capable of significantly suppressing
fibrosis-induced expression of a panel of marker genes including Acta2, Coll
al, Col3a1 , Fnl , ltgal 1, Lox, LoxI2,
when the animals are administered at a dose of 10 mg/kg or less in the UUO
model of kidney fibrosis.
[595] In some embodiments, the selection process of an antibody or antigen-
binding fragment thereof for
therapeutic use may therefore include identifying an antibody or fragment that
shows sufficient inhibitory potency.
For example, the selection process may include a step of carrying out a cell-
based TGF(31 activation assay to
measure potency (e.g., IC50) of one or more test antibodies or fragments
thereof, and, selecting a candidate
antibody or fragment thereof that shows desirable potency. In some
embodiments, IC50 for each of the human
LLCs 5 nM or less. The selected antibody or the fragment may then be used in
the treatment of a TGF61-related
indication described herein.
Binding regions
[596] In the context of the present disclosure, "binding region(s)" of an
antigen provides a structural basis for the
antibody-antigen interaction. As used herein, a "binding region" refers to the
areas of interface between the
antibody and the antigen, such that, when bound to the proTGF61 complex
("antigen") in a physiological solution,
the antibody or the fragment protects the binding region from solvent
exposure, as determined by suitable
techniques, such as hydrogen-deuterium exchange mass spectrometry (HDX-MS).
Identification of binding regions
is useful in gaining insight into the antigen-antibody interaction and the
mechanism of action for the particular
antibody. Identification of additional antibodies with similar or overlapping
binding regions may be facilitated by
cross-blocking experiments that enable epitope binning. Optionally, X-ray
crystallography may be employed to
identify the exact amino acid residues of the epitope that mediate antigen-
antibody interactions.
[597] The art is familiar with HDX-MS, which is a widely used technique for
exploring protein conformation or
protein-protein interactions in solution. This method relies on the exchange
of hydrogens in the protein backbone
amide with deuterium present in the solution. By measuring hydrogen-deuterium
exchange rates, one can obtain
information on protein dynamics and conformation (reviewed in: Wei et al.,
(2014) "Hydrogen/deuterium exchange
mass spectrometry for probing higher order structure of protein therapeutics:
methodology and applications." Drug
Discov Today. 19(1): 95-102; incorporated by reference). The application of
this technique is based on the premise
that when an antibody-antigen complex forms, the interface between the binding
partners may occlude solvent,
thereby reducing or preventing the exchange rate due to steric exclusion of
solvent.
[598] The present disclosure includes antibodies or antigen-binding fragments
thereof that bind a human LLC at
a region (binding region") comprising Latency Lasso or a portion thereof.
Latency Lasso is a protein module within
the prodomain. It is contemplated that many potent activation inhibitors may
bind this region of a proTGFpl
complex in such a way that the antibody binding would "lock in" the growth
factor thereby preventing its release.
Interestingly, this is the section of the complex where the butterfly-like
elongated regions of the growth factor (e.g.,
corresponding to, for example, Finger-1 and Finger-2) closely interact with
the cage-like structure of the prodomain.
Based on the data previously disclosed in PCT/US2019/041373, it is envisaged
that an antibody that tightly wraps
around the binding regions identified may effectively prevent the proTGF81
complex from disengaging (i.e.,
releasing the growth factor), thereby blocking activation.
[599] Using the HDX-MS technique, binding regions of proTGF81 can be
determined. In some embodiments, a
portion on proTGF81 identified to be important in binding an antibody or
fragment includes at least a portion of the
prodomain and at least a portion of the growth factor domain. Antibodies or
fragments that bind a first binding
region ("Region 1" in FIG. 41) comprising at least a portion of Latency Lasso
are preferable. More preferably, such
antibodies or fragments further bind a second binding region ("Region 2" in
FIG. 41) comprising at least a portion
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of the growth factor domain at Finger-1 of the growth factor domain. Such
antibodies or fragments may further
bind a third binding region (Region 3" in FIG. 41) comprising at least a
portion of Finger-2 of the growth factor
domain.
[600] Additional regions within the proTGFI31 may also contribute, directly or
indirectly, to the high-affinity
interaction of these antibodies disclosed herein. Regions that are considered
important for mediating the high-
affinity binding of the antibody to the proTGF[31 complex may include, but are
not limited to: LVKRKRIEA (SEQ ID
NO: 132); LASPPSQGEVP (SEQ ID NO: 133); PGPLPEAV (SEQ ID NO: 134); LALYNSTR
(SEQ ID NO: 135);
REAVPEPVL (SEQ ID NO: 136); YQKYSNNS1NR (SEQ ID NO: 137); RKDLGWKWIHEPKGYHANF
(SEQ ID NO:
138); LGPCPYIWS (SEQ ID NO: 139); ALEPLPIV (SEQ ID NO: 140); and, VGRKPKVEQL
(SEQ ID NO: 141)
(based on the native sequence of human proTGF31).
[601] Among regions that may contribute to the antibody-antigen interaction,
in some embodiments, the high-
affinity antibody of the present disclosure may bind an epitope that comprises
at least one residue of the amino
acid sequence KLRLASPPSQGEVPPGPLPEAVL ("Region 1") (SEQ ID NO: 142).
[602] In some embodiments, the high-affinity antibody of the present
disclosure may bind an epitope that
comprises at least one residue of the amino acid sequence RKDLGWKWIHEPKGYHANF
("Region 2") (SEQ ID
NO: 138).
[603] In some embodiments, the high-affinity antibody of the present
disclosure may bind an epitope that
comprises at least one residue of the amino acid sequence VGRKPKVEQL ("Region
3") (SEQ ID NO: 141).
[604] In some embodiments, the high-affinity antibody of the present
disclosure may bind an epitope that
comprises at least one residue of the amino acid sequence
KLRLASPPSQGEVPPGPLPEAVL ("Region 1") (SEQ
ID NO: 142) and at least one residue of the amino acid sequence
RKDLGWKWIHEPKGYHANF ("Region 2") (SEQ
ID NO: 138).
[605] In some embodiments, the high-affinity antibody of the present
disclosure may bind an epitope that
comprises at least one residue of the amino acid sequence
KLRLASPPSQGEVPPGPLPEAVL (Region 1") (SEQ
ID NO: 142) and at least one residue of the amino acid sequence VGRKPKVEQL
("Region 3") (SEQ ID NO: 141).
[606] In some embodiments, the high-affinity antibody of the present
disclosure may bind an epitope that
comprises at least one residue of the amino acid sequence
KLRLASPPSQGEVPPGPLPEAVL ("Region 1") (SEQ
ID NO: 142), at least one residue of the amino acid sequence
RKDLGWKWIHEPKGYHANF ("Region 2") (SEQ ID
NO: 138), and, at least one residue of the amino acid sequence VGRKPKVEQL
("Region 3") (SEQ ID NO: 141).
[607] In addition to contributions from Regions 1,2 and/or 3, such epitope may
further include at least one amino
acid residues from a sequence selected from the group consisting of: LVKRKRIEA
(SEQ ID NO: 132);
LASPPSQGEVP (SEQ ID NO: 133); PGPLPEAV (SEQ ID NO: 134); LALYNSTR (SEQ ID NO:
135); REAVPEPVL
(SEQ ID NO: 136); YQKYSNNSWR (SEQ ID NO: 137); RKDLGWKWIHEPKGYHANF (SEQ ID NO:
138);
LGPCPYIWS (SEQ ID NO: 139); ALEPLPIV (SEQ ID NO: 140); and, VGRKPKVEQL (SEQ ID
NO: 141).
[608] Notably, many of the binding regions identified in structural studies
using four representative isoform-
selective TGFI31 antibodies are found to be overlapping, pointing to certain
regions within the proTGFI31 complex
that may be particularly important in maintaining latency of the proTGFI31
complex. Thus, advantageously,
antibodies or fragments thereof may be selected at least in part on the basis
of their binding region(s) that include
the overlapping portions identified across multiple inhibitors described
herein. These overlapping portions of
binding regions include, for example, SPPSQGEVPPGPLPEAVL (SEQ ID NO: 165),
VVKWIHEPKGYHANF (SEQ
ID NO: 166), and PGPLPEAVL (SEQ ID NO: 167). Thus, the high-affinity, isoform-
selective TGF[31 inhibitor
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according to the present disclosure may bind a proTG931 complex (e.g., human
LLCs) at an epitope that comprises
one or more amino acid residues of SPPSQGEVPPGPLPEAVL (SEQ ID NO: 165),
VVKWIHEPKGYHANF (SEQ
ID NO: 166), arid/or PGPLPEAVL (SEQ ID NO: 167).
[609] Thus, any of the antibody or antigen-binding fragment encompassed by the
present disclosure, such as
antibodies or fragments of Categories 1 through 5 disclosed herein, may bind
one or more of the binding regions
identified herein. Such antibodies may be used in the treatment of a TGF(31
indication in a subject as described
herein. Accordingly, selection of an antibody or antigen-binding fragment
thereof suitable for therapeutic use in
accordance with the present disclosure may include identifying or selecting an
antibody or a fragment thereof that
binds SPPSQGEVPPGPLPEAVL (SEQ ID NO: 165), VVKINIHEPKGYHANF (SEQ ID NO: 166),
PGPLPEAVL (SEQ
ID NO: 167), or any portion(s) thereof.
[610] Non-limiting examples of protein domains or motifs of human proTGFI31 as
previously described (WO
2014/182676) are provided in Table 9.
Table 9. Select protein domains/motifs of human TGFI31-related polypeptides
Human TGFI31 Amino Acid Sequence SEQ
ID NO
domain/module
Latency Associated LSTCKTIDMELVKRKR lEAIRGQILSKLRLASPPSOGEVPPGPLPEAVLA
119
Peptide (LAP) LYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQS
(prodomain) THSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSN
NSVVRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHC
SCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHL
QSSRHRR
("First binding region" is underlined)
Straight Jacket LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLP 120
(Latency Lasso" is underlined)
Growth Factor ALDTNYCFSSTEKNCCVRQLYIDFRKDLGVVKWIHEPKGYHANFCLGP 121
Domain CPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRK
PKVEQLSNMIVRSCKCS
("Finger-1" and 'Finger-2" are underlined, respectively)
Fastener residues 74-76, YYA n/a
Furin cleavage site RHRR 122
Arm EAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYD 123
KFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELY
QKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFR
LSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLE
RAQHLQSSRHRR
Finger-1 CVRQLYIDFRKDLGVVKVVIHEPKGYHANFC 124
("Second binding region" is underlined)
Finger-2 CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 125
("Third binding region" is underlined)
Residue for Cys 4 n/a
presenting molecule
association
Latency Lasso LASPPSQGEVPPGPL 126
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(Portion of the binding regions shared across 4 different iso form-
selective proTGFpl antibodies is underlined)
Extended Latency LASPPSQGEVPPGPLPEAVLALYNSTR 127
Lasso (Portion of the binding regions shared across 4
different iso form-
selective proTGF01 antibodies is underlined)
Alpha-1 Helix LSTCKTIDMELVKRKRIEAIRGQILSKLR 128
Alpha-2 Helix AVLALYNSTR 129
Trigger Loop NGFTTGRRGDLATIHGMNRP 130
I nteg rin binding residue 215-217, ROD n/a
Bowtie CSCDSRDNTLQVD 131
Safety/toxicology
[611] The development of TGFI3 inhibitors remains challenging due to the need
to identify a therapy with the
desired pharmacological effects and sufficient therapeutic window, which also
eliminates on-target toxicities. The
majority of TG93 inhibitors, including monoclonal antibodies and small
molecule kinase inhibitors (SMIs), non-
selectively target either multiple TGF3 isoforms or the TGF3 receptor, which
mediates signaling from all three TGF13
isoforms. Unfortunately, these inhibitors have not demonstrated promising
clinical data in cancer patients mainly
due to a lack of efficacy (Akhurst 2017; Cohn 2014; Voelker 2017), an
unfavorable safety profile, or both (Tolcher
2017; Volker 2017; Cohn 2014). The toxicities associated with these molecules
include cardiovascular
abnormalities, epithelial hyperplasia, gastrointestinal abnormalities, and
skin lesions. Each of these toxicities have
been characterized in multiple animal species (e.g., rodents, dogs, and
cynomolgus monkeys) in studies ranging
in duration from 1-2 weeks up to 6-months (Lonning 2011; Stauber 2014; Mitra
2020). Amongst these toxicities,
the irreversible cardiovascular inflammatory lesions, hemorrhage and
hyperplasia in heart valves, and arterial
lesions that include the aorta and coronary arteries, are of major concern.
[612] Conventional pan-inhibitors of TGF3 capable of antagonizing multiple
isoforms have been known to cause
a number of toxicities, including, for example, cardiovascular toxicities
(cardiac lesions, most notably valvulopathy)
reported across multiple species including dogs and rats. These include,
hyperplasia in aortic valve, right AV valve,
and left AV valve; inflammation in aortic valve, left AV valve, and ascending
aorta; hemorrhage in ascending aorta,
aortic valve and left AV valve; connective tissue degeneration in ascending
aorta (see for example, Strauber et al.,
(2014) "Nonclinical safety evaluation of a Transforming Growth Factor 13
receptor I kinase inhibitor in Fischer 344
rats and beagle dogs" J. Olin. Pract 4(3): 1000196).
[613] In addition, neutralizing antibodies that bind all three TGFI3 isoforms
have been associated with certain
epithelial toxicities observed across multiple species, some of which are
summarized below.
Table 10. Epithelial toxicities associated with pan-inhibitors of TGF0
Mice Cyno Human
Toxicities = Hyperplasia and = Hyperplasia of
gingiva, . Gigival bleeding
inflammation of tongue, nasal epithelium, and =
Epistaxis
gingiva, and esophagus. bladder = Headache
= Findings not reversible =
Anemia lead to = Fatigue
(12wk recovery) cessation of treatment .
Various skin disorders,
= Changes
were including
reversible (except
keratoacanthornas (KA),
bladder)
hyperkeratosis,
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cutaneous SCC, and
basal cell carcinoma
Drug / Dose / 1011 GC1008 GC1008
D Dosing: 50 mg/kg (3x/week) Dosing: 10
arid 50 mg/kg Dose: 0.1,0.3, 1, 3, 10, 15
uration
Duration: 9-12 weeks Duration: 6 months mg/kg
Duration: 4 monthly doses
Exposure Serum conc. = 1-2 mg/mL Not disclosed
Half life: 21.7d
(over 4-12 weeks) ON Cmax ¨(350
ng/mL)mg
*Vitsky et. al., Am. J Pathology vol. 174, 2009; and Lonning et. al., Current
Pharmaceutical Biotech 12, 2176-
2189, 2011
[614] Building upon the earlier recognition by the applicant of the present
disclosure (see PCT/US2017/021972)
that lack of isoform-specificity of conventional TGFI3 antagonists may
underlie the source of toxicities associated
with TGF3 inhibition, the present inventors sought to further achieve broad-
spectrum TGF81 inhibition for treating
various diseases that manifest multifaceted TGFI31 dysregulation, while
maintaining the safety/tolerability aspect
of isoform-selective inhibitors.
[615] In clinical setting, therapeutic benefit is achieved only when the
minimum effective concentrations (MEC) of
a drug (e.g., monoclonal antibody) are below the minimum toxic concentrations
(MTC) of the drug. This was not
achieved with most, if not all, conventional pan-inhibitors of TGF3, which in
fact appeared to cause dose-limiting
toxicities. Applicant's previous work described isoform-selective inhibitors
of TGF(31 that showed markedly
improved safety profile, as compared to conventional pan-inhibitors, such as
small molecule receptor antagonists
and neutralizing antibodies. WO 2017/156500 disclosed an isoform-selective
inhibitor of TGFI31 activation, which,
when administered at a dose of up to 100 mg/kg per week for 4 weeks in rats,
no test article-related toxicities were
observed, establishing the NOAEL for the antibody as the highest dose tested,
i.e., 100 mg/kg. Applicant's
subsequent work also showed that an antibody with enhanced function also
showed the equivalent safety profiles.
Here, one of the objectives was to identify antibodies with even higher
affinities and potencies, but with at least the
same or equivalent levels of safety.
[616] Results from four-week rat toxicology studies were previously disclosed
in PCT/US2019/041373, see, e.g.,
data provided in FIG. 24B. Two isoform-selective TGF[31 inhibitors (Ab3 and
Ab6) were tested in separate studies,
together with a small molecule ALK5 inhibitor and a monoclonal neutralizing
antibody as control. No test article-
related toxicities were noted with either of the isoform-selective antibodies,
while the non-selective inhibitors as
expected caused a variety of adverse events consistent with published studies.
In contrast to treatments that
broadly block TGF13 signaling, Ab6 showed no cardiac toxicities in a 4-week,
non-GLP pilot toxicology study in rats,
suggesting that selective inhibition of the 1GFI31 isoform may have an
improved safety profile compared to pan-
TGF3 inhibitors. Moreover, Ab6 was shown to be safe (e.g., no observed adverse
events) at a dose level as high
as 300 mg/kg in cynomolgus monkeys when dosed weekly for 4 weeks. Since Ab6
has been shown to be
efficacious in a number of in vivo models at a dose as low as 3 mg/kg, this
offers an up to 100-fold of a therapeutic
window. Importantly, this demonstrates that high potency does not have to mean
greater risk of toxicity. Without
wishing to be bound by a particular theory, it is contemplated that the highly
selective nature of the antibodies
disclosed herein likely account for the lack of observed toxicities.
[617] Thus, in some embodiments, the novel antibody according to the present
disclosure has the maximally
tolerated dose (MTD) of >100 mg/kg when dosed weekly for at least 4 weeks. In
some embodiments, the novel
antibody according to the present disclosure has the no-observed-adverse-
effect level (NOAEL) of up to 100 mg/kg
when dosed weekly for at least 4 weeks Suitable animal models to be used for
conducting safety/toxicology
studies for TGFI3 inhibitors and TGFI31 inhibitors include, but are not
limited to: rats, dogs, cynos, and mice. In
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certain embodiments, the minimum effective amount of the antibody based on a
suitable preclinical efficacy study
is below the NOAEL. More preferably, the minimum effective amount of the
antibody is about one-third or less of
the NOAEL. In certain embodiments, the minimum effective amount of the
antibody is about one-sixth or less of
the NOAEL. In some embodiments, the minimum effective amount of the antibody
is about one-tenth or less of
the NOAEL.
[618] In some embodiments, the disclosure encompasses an isoform-selective
antibody capable of inhibiting
TGF131 signaling, which, when administered to a subject, does not cause
cardiovascular or known epithelial
toxicities at a dose effective to treat a TGFpl-related indication. In some
embodiments, the antibody has a
minimum effective amount of about 3-10 mg/kg administered weekly, biweekly or
monthly. Preferably, the antibody
causes no to minimum toxicities at a dose that is at least six-times the
minimum effective amount (e.g., a six-fold
therapeutic window). More preferably, the antibody causes no to minimum
toxicities at a dose that is at least ten-
times the minimum effective amount (e.g., a ten-fold therapeutic window). Even
more preferably, the antibody
causes no to minimum toxicities at a dose that is at least fifteen-times the
minimum effective amount (e.g., a fifteen-
fold therapeutic window).
[619] Therapeutic agents that engage immune cells pose the potential risk of
activating immune cells when
administered to patients. In selecting a TGFp inhibitor for therapeutic use,
it is therefore important to determine or
confirm that a candidate inhibitor does not trigger a proinflammatory cytokine
response (e.g., cytokine release) in
human peripheral blood mononuclear cells (PBMCs). Proinflammatory cytokines
include, for example, IFNy, IL-2,
IL-l]3, INFa, CCL2 and IL-6. In some embodiments, acceptable levels of
cytokine release triggered by a test agent
(candidate inhibitor) are within 2.5-fold of the response as compared to
vehicle control (e.g., IgG).
[620] Accordingly, the present disclosure provides a TGFI3 inhibitor for use
in the treatment of a TGFp-related
condition (e.g., cancer, myelofibrosis, fibrosis, etc.) in a human patient,
which includes i) selection of a TGFp
inhibitor, which has been shown not to trigger unsafe levels of
proinflammatory cytokine release in human PBMCs;
and, ii) administration of a composition comprising a therapeutically
effective amount of the TGFI3 inhibitor to the
patient, to treat the condition, In some embodiments, the TGFI3 inhibitor does
not trigger unsafe levels of cytokine
release from human PBMCs at an amount that is at least three times the
therapeutically effective amount.
Preferably, at least five times the therapeutically effective amount of the
TGFI3 inhibitor does not cause unsafe
levels of cytokine release in human PBMCs.
[621] Human platelets have been reported to express latent TGF(31.
Pharmacological intervention that targets
platelets may cause unwanted effects on platelet function, such as platelet
aggregation and activation, which could
result in blood coagulation dysregulation. Therefore, it is important to
determine or confirm that a candidate inhibitor
does not cause unwanted platelet activation or interfere with the normal
function of platelets.
[622] Accordingly, the present disclosure provides a TGFI3 inhibitor for use
in the treatment of a TGFP-related
condition (e.g., cancer, myelofibrosis, fibrosis, etc.) in a human patient,
which includes i) selection of a TGF13
inhibitor, which has been shown not to cause platelet aggregation or
activation; and, ii) administration of a
composition comprising a therapeutically effective amount of the TGFp
inhibitor to the patient, to treat the condition,
In some embodiments, the TGFI3 inhibitor does not cause spontaneous or ADP-
induced platelet activation in a
dose-dependent manner at an amount that is at least three times the
therapeutically effective amount. Preferably,
at least five times the therapeutically effective amount of the TGFI3
inhibitor does not cause platelet activation. In
certain embodiments, the TGFI3 inhibitor does not inhibit ADP-induced platelet
activation in a dose-dependent
manner at an amount that is at least three times the therapeutically effective
amount. Preferably, at least five times
the therapeutically effective amount of the TGFp inhibitor does not inhibit
platelet activation.
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[623] The present disclosure includes a TGFI3 inhibitor for use in the
treatment of cancer in a human patient,
wherein the treatment comprises: i) selecting a TGFI3 inhibitor shown to be
both efficacious and safe in a preclinical
model(s), and, ii) administering to the human patient an effective dose of the
TGFI3 inhibitor, wherein optionally the
TGFI3 inhibitor is effective to reduce tumor burden when used in conjunction
with a checkpoint inhibitor, wherein
further optionally the TGFI3 inhibitor does not trigger platelet activation in
human blood samples and does not cause
inflammatory cytokine release in PBMCs at doses greater than a minimum
efficacious dose; and, further optionally
the TGFI3 inhibitor does not cause unacceptable adverse events as evaluated in
a standard toxicology study in one
or more preclinical models in which NOAEL is at least 10 times the minimum
efficacious dose.
[624] Thus, selection of an antibody or an antigen-binding fragment thereof
for therapeutic use may include:
selecting an antibody or antigen-binding fragment that meets the criteria of
one or more of Categories 1-5 described
herein; carrying out an in vivo efficacy study in a suitable preclinical model
to determine an effective amount of the
antibody or the fragment; carrying out an in vivo safety/toxicology study in a
suitable model to determine an amount
of the antibody that is safe or toxic (e.g., MTD, NOAEL, cytokine release,
effects on platelets, or any art-recognized
parameters for evaluating safety/toxicity); and, selecting the antibody or the
fragment that provides at least a three-
fold therapeutic window (preferably 6-fold, more preferably a 10-fold
therapeutic window, even more preferably a
15-fold therapeutic window). In preferred embodiments, the in vivo efficacy
study is carried out in two or more
suitable preclinical models that recapitulate human conditions. In some
embodiments, such preclinical models
comprise TGF131-positive cancer, which may optionally comprise an
immunosuppressive tumor. The
immunosuppressive tumor may be resistant to a cancer therapy such as CBT,
chemotherapy and radiation therapy
(such as a radiotherapeutic agent). In some embodiments, the preclinical
models are selected from MBT-2,
Cloudman S91 and EMT6 tumor models.
[625] The selected antibody or the fragment may be used in the manufacture of
a pharmaceutical composition
comprising the antibody or the fragment. Such pharmaceutical composition may
be used in the treatment of a
TGF131 indication in a subject as described herein. For example, the TGFp1
indication may be a proliferative
disorder and/or a fibrotic disorder.
Mechanism of action
[626] Antibodies of the present disclosure that are useful as therapeutics are
inhibitory antibodies of TGFP1.
Further, the antibodies are activation inhibitors, that is, the antibodies
block the activation step of TGF(31, rather
than directly chasing after already activated growth factor.
[627] In a broad sense, the term "inhibiting antibody' refers to an antibody
that antagonizes or neutralizes the
target function, e.g., growth factor activity. Advantageously, certain
inhibitory antibodies of the present disclosure
are capable of inhibiting mature growth factor release from a latent complex,
thereby reducing growth factor
signaling. Inhibiting antibodies include antibodies targeting any epitope that
reduces growth factor release or
activity when associated with such antibodies. Such epitopes may lie on the
prodomains of TGFI3 proteins (e.g.,
TGF131), growth factors or other epitopes that lead to reduced growth factor
activity when bound by antibody.
Inhibiting antibodies of the present disclosure include, but are not limited
to, TG931-inhibiting antibodies. In some
embodiments, inhibitory antibodies of the present disclosure specifically bind
a combinatory epitope, i.e., an epitope
formed by two or more comporients/portions of an antigen or antigen complex.
For example, a combinatorial
epitope may be formed by contributions from multiple portions of a single
protein, i.e., amino acid residues from
more than one non-contiguous segments of the same protein. Alternatively, a
combinatorial epitope may be formed
by contributions from multiple protein components of an antigen complex. In
some embodiments, inhibitory
antibodies of the present disclosure specifically bind a conformational
epitope (or conformation-specific epitope),
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e.g., an epitope that is sensitive to the three-dimensional structure (i.e.,
conformation) of an antigen or antigen
complex.
[628] Traditional approaches to antagonizing TGF6 signaling have been to i)
directly neutralize the mature growth
factor after it has already become active so as to deplete free ligands (e.g.,
released from its latent precursor
complex) that are available for receptor binding; ii) employ soluble receptor
fragments capable of sequestering free
ligands (e.g., so-called ligand traps); or, iii) target its cell-surface
receptor(s) to block ligand-receptor interactions.
Each of these conventional approaches requires the antagonist to compete
against endogenous counterparts.
Moreover, the first two approaches (i and ii) above target the active ligand,
which is a transient species. Therefore,
such antagonist must be capable of kinetically outcompeting the endogenous
receptor during the brief temporal
window. The third approach may provide a more durable effect in comparison but
inadvertently results in unwanted
inhibitory effects (hence possible toxicities) because many growth factors
(e.g., up to ¨20) signal via the same
receptor(s).
[629] To provide solutions to these drawbacks, and to further enable greater
selectivity and localized action, the
mechanism of action underlining the inhibitory antibodies such as those
described herein acts upstream of TGF61
activation and ligand-receptor interaction. Thus, it is contemplated that high-
affinity, isoform-specific, context-
independent inhibitors of TGF131 suitable for carrying out the present
disclosure should preferably target the inactive
(e.g., latent) precursor TGF61 complex (e.g., a complex comprising pro/latent
TGF61) prior to its activation, in order
to block the activation step at its source (such as in a disease
microenvironment, e.g., TME). According to certain
embodiments of the disclosure, such inhibitors target with equivalent
affinities both ECM-associated and cell
surface-tethered pro/latent TGF61 complexes, rather than free ligands that are
transiently available for receptor
binding.
[630] Advantages of locally targeting tissue/cell-tethered complex at the
source, as opposed to soluble active
species (i.e., mature growth factors after being released from the source),
are further supported by a recent study.
lshihara et al., (Sci. Transl. Med. 11, eaau3259 (2019) "Targeted antibody and
cytokine cancer immunotherapies
through collagen affinity") reported that when systemically administered drugs
are targeted to the tumor sites by
conjugating with a collagen-binding moiety, they were able to enhance anti-
tumor immunity and reduce treatment-
related toxicities, as compared to non-targeted counterparts.
[631] The mechanism of action achieved by the antibodies of the present
disclosure may further contribute to
enhanced durability of effect, as well as overall greater potency and safety.
[632] Interestingly, these antibodies may exert additional inhibitory
activities toward cell-associated TGFI31
(LRRC33-proTGF61 and GARP-proTGF61). Applicant has found that LRRC33-binding
antibodies tend to become
internalized upon binding to cell-surface LRRC33. Whether the internalization
is actively induced by antibody
binding, or alternatively, whether this phenomenon results from natural (e.g.,
passive) endocytic activities of
macrophages is unclear. However, the high-affinity, isoform-selective TGF61
inhibitor, Ab6, is capable of becoming
rapidly internalized in cells transfected with LRRC33 and proTGF61 , and the
rate of internalization achieved with
Ab6 is significantly higher than that with a reference antibody that
recognizes cell-surface LRRC33. Similar results
are obtained from primary human macrophages. These observations raise the
possibility that Ab6 can induce
internalization upon binding to its target, LRRC33-proTGF61, thereby removing
the LRRC33-containing complexes
from the cell surface. At the disease loci, this may reduce the availability
of activatable latent LRRC33-proTGF61
levels. Therefore, the isoform-selective TGF61 inhibitors may inhibit the
LRRC33 arm of TGF61 via two parallel
mechanisms of action: i) blocking the release of mature growth factor from the
latent complex; and, ii) removing
LRRC33-proTGF61 complexes from cell-surface via internalization.
It is possible that similar inhibitory
mechanisms of action may apply to GARP-proTGF61.
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[633] In some embodiments, the antibody is a pH-sensitive antibody that binds
its antigen with higher affinity at a
neutral pH (such as pH of around 7) than at an acidic pH (such as pH of around
5). Such antibodies may have
higher dissociation rates at acidic conditions than neutral or physiological
conditions. For example, the ratio
between dissociation rates measured at an acidic pH and dissociation rates
measured at neutral pH (e.g., Koff at
pH5 over Koff at pH 7) may be at least 1.2. Optionally, the ratio is at least
1.5. In some embodiments, the ratio is
at least 2. Such pH-sensitive antibodies may be useful as recycling
antibodies. Upon target engagement on cell
surface, the antibody may trigger antibody-dependent internalization of (hence
removal of) membrane-bound
proTGFp1 complexes (associated with LRRC33 or GARP). Subsequently, in an
acidic intracellular compartment
such as lysosome, the antibody-antigen complex dissociates, and the free
antibody may be transported back to
the extracellular domain.
[634] Thus, in some embodiments, selection of an antibody or an antigen-
binding fragment for therapeutic use
may be in part based on the ability to induce antibody-dependent
internalization and/or pH-dependency of the
antibody.
Antigen Complexes and Components Thereof
[635] The novel antibodies of the present disclosure specifically bind each of
the four known human large latency
complexes (e.g., hLTBP1-proTGFp1, hLTBP3-proTGFp1, hGARP-proTGF131 and hLRRC33-
proTGFp1),
selectively inhibits TGFp1 activation.
[636] Screening (e.g., identification and selection) of such antibodies
involves the use of suitable antigen
complexes, which are typically recombinantly produced. Useful protein
components that may comprise such
antigen complexes are provided, including TG93 isoforms and related
polypeptides, fragments and variants,
presenting molecules (e.g., LTBPs, GARP, LRRC33) and related polypeptides,
fragments and variants. These
components may be expressed, purified, and allowed to form a protein complex
(such as large latent complexes),
which can be used in the process of antibody screening. The screening may
include positive selection, in which
desirable binders are selected from a pool or library of binders and non-
binders, and negative selection, in which
undesirable binders are removed from the pool. Typically, at least one matrix-
associated complex (e.g., LTBP1-
proTG931 and/or LTBP1-proTGF131) and at least one cell-associated complex
(e.g., GARP-proTG931 and/or
LRRC33-proTGFI31) are included for positive screening to ensure that binders
being selected have affinities for
both such biological contexts.
[637] In some embodiments, the TGF131 comprises a naturally occurring
mammalian amino acid sequence. In
some embodiment, the TGF(31 comprises a naturally occurring human amino acid
sequence. In some
embodiments, the TGF(31 comprises a human, a monkey, a rat or a mouse amino
acid sequence. In some
embodiments, an antibody, or antigen binding portion thereof, described herein
does not specifically bind to TGFP2.
In some embodiments, an antibody, or antigen binding portion thereof,
described herein does not specifically bind
to TGF133. In some embodiments, an antibody, or antigen binding portion
thereof, described herein does not
specifically bind to TGF(32 or TGF(33. In some embodiments, an antibody, or
antigen binding portion thereof,
described herein specifically binds to a TGFP1 comprising the amino acid
sequence set forth in SEQ ID NO: 23.
The amino acid sequences of TGFp2, and TGFp3 amino acid sequence are set forth
in SEQ ID NOs: 27 and 21,
respectively. In some embodiments, an antibody, or antigen binding portion
thereof, described herein specifically
binds to a TGFI31 comprising a non-naturally-occurring amino acid sequence
(otherwise referred to herein as a
non-naturally-occurring TGFp1). For example, a non-naturally-occurring TGFp1
may comprise one or more
recombinantly generated mutations relative to a naturally-occurring TGFp1
amino acid sequence. In some
embodiments, a TGFp1, TGFp2, or TGFp3 amino acid sequence comprises the amino
acid sequence as set forth
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in SEQ ID NOs: 13-24, as shown in Table 11. In some embodiments, a TGF131,
TGF32, or TGF33 amino acid
sequence comprises the amino acid sequence as set forth in SEQ ID NOs: 25-32,
as shown in Table 12.
[638] TGF31 (prodomain + growth factor domain)
LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYA
KEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRY
LSNRLLAPSDSPEVVLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNR
PFLLLMATPLERAQHLOSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYI
WSLDTQYSKVLALYNQH NPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 13)
[639] TGF32 (prodomain + growth factor domain)
SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDE
EYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVREDVSAMEKNASNLVKAEFRVERLQNPKARVPEQRIELYQILK
SKDLTSPTQRYIDSKVVKTRAEGEVVLSFDVTDAVHEVVLHHKDRNLGFKISLHCPCCTFVPSNNYI
IPNKSEELEAR
FAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYI
DFKRDLGVVKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKI
EQLSNMIVKSCKCS (SEQ ID NO: 17)
[640] TGF33 (prodomain + growth factor domain)
SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENT
ESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQI
LRPDEHIAKQRYIGGKNLPTRGTAEVVLSFDVTDTVREVVLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKF
KGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYID
FRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPK
VEQLSNMVVKSCKCS (SEQ ID NO: 21)
Table 11. Exemplary TGFI31, TGFI32, and TGFI33 amino acid sequences
Protein Sequence
SEQ ID
NO
proTGF31 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLAL
13
YNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKOST
HSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNS
WRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSC
DSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATP LERAQHLQSS
RHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFC
LGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVG
RKPKVEQLSNMIVRSCKCS
proTGF31 C4S LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSOGEVPPGPLPEAVLAL
14
YNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQST
HSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNS
WRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSC
DSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATP LERAQHLQSS
RHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFC
LGPCPYIWSLDTQYSKVLALYNOHNPGASAAPCCVPQALEPLPIVYYVG
RKPKVEQLSNMIVRSCKCS
proTGF31 D2G LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLAL
15
YNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQST
HSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNS
WRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSC
DSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATP LERAQHLQSS
RHGALDTNYCFSSTEKNCCVRQLYIDFRKDLGVVKWIHEPKGYHANFCL
GPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR
KPKVEQLSNMIVRSCKCS
proTGF31 C4S D2G LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLAL
16
YNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKOST
HSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNS
WRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSC
145
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DSRDNTLQVDI NGFTTGRRGDLATIHGMNRPFLLLMATP LERAQHLQSS
RHGALDTNYCFSSTEKNCCVROLYIDFRKDLGVVKWIHEPKGYHANFCL
GPCPYIVVSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR
KPKVEQLSNMIVRSCKCS
proTGF32 SLSTCSTLDMDQFMRKRIEAIRGOILSKLKLTSPPEDYPEPEEVPPEVISI
17
YNSTRDLLQEKASRRAAAC ERERSDEEYYAKEVYKI DMPP FFPSENA IP
PTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIEL
YQI LKSKDLTSPTQRYIDSKVVKTRAEGEVVLSFDVTDAVHEINLHHKDRN
LGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIK
STRKKNSGKTPH LLLMLLPSYRLESQQTN RRKKRALDAAYCFRNVQDN
CCLRPLYIDFKRDLGVVKVVIHEPKGYNANFCAGACPYLWSSDTQHSRVL
SLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS
proTGF32 C5S SLSTSSTLDMDQFMRKRI
EAIRGOILSKLKLTSPPEDYPEPEEVPPEVISI 18
YNSTRDLLQEKASRRAAAC ERERSDEEYYAKEVYKI DMPP FFPSENA IP
PTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIEL
YQI LKSKDLTSPTQRYIDSKVVKTRAEGEVVLSFDVTDAVH EVVLH HKDRN
LGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIK
STRKKNSGKTPH LLLMLLPSYRLESQQTN RRKKRALDAAYCFRNVQDN
CCLRPLYIDFKRDLGVVKWIHEPKGYNANFCAGACPYLWSSDTQHSRVL
SLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS
proTGF32 C5S D2G SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISI
19
YNSTRDLLQEKASRRAAAC ERERSDEEYYAKEVYKI DMPP FFPSENA IP
PTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIEL
YQI LKSKDLTSPTQRYIDSKVVKTRAEGEVVLSFDVTDAVH EVVLH HKDRN
LGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIK
STRKKNSGKTPH LLLMLLPSYRLESQQTN RRKGALDAAYCFRNVQDNC
CLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLVVSSDTQHSRVLS
LYNT IN PEASASPCCVSQDLEPLT ILYYIGKTPKIEQLSNM IVKSCKCS
proTGF32 D2G SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISI
20
YNSTRDLLQEKASRRAAAC ERERSDEEYYAKEVYKI DMPP FFPSENA IP
PTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIEL
YQI LKSKDLTSPTQRYIDSKVVKTRAEGEVVLSFDVTDAVHEINLHHKDRN
LGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDOKTIK
STRKKNSGKTPH LLLMLLPSYRLESQQTN RRKGALDAAYCFRNVQDNC
C LRPLYIDFKRDLGWKWI IHEPKGYNANFCAGACPYLWSSDTQHSRVLS
LYNT IN PEASASPCCVSQDLEPLT ILYYIGKTPKIEQLSNM IVKSCKCS
proTGF33 SLSLSTCTTLDFGH
IKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLA 21
LYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNEL
AVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIE
LFQILRPDEH IAKQRYIGGKNLPTRGTAEVVLSFDVTDTVREVVLLRRESN
LGLE IS I HCPCHTFQP NGDI LEN I HEVMEI KFKGVDNEDDHGRGDLGRLK
KQKDHHNPH LI LMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCC
VRPLYIDFRQDLGWKVVVHEPKGYYANFCSGPCPYLRSADTTHSTVLGL
YNTLNPEASASPCCVPQDLEPLTI LYYVGRTPKVEQLSNMVVKSCKCS
proTGF33 C7S SLSLSTSTTLDFGH IKKKRVEAIRGQ I
LSKLRLTSPPEPTVMTHVPYQVLA 22
LYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNEL
AVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIE
LFQILRPDEH IAKQRYIGGKNLPTRGTAEVVLSFDVTDTVREVVLLRRESN
LGLE IS I HCPCHTFQP NGDI LEN I HEVMEI KFKGVDNEDDHGRGDLGRLK
KQKDHHNPH LI LMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCC
VRPLYI DFRQDLGWKVVVHEP KGYYANFCSGPCPYLRSADTTH STVLGL
YNTLNPEASASPCCVPQDLEPLTI LYYVGRTPKVEQLSNMVVKSCKCS
proTGF33 C7S D2G SLSLSTSTTLDFGH IKKKRVEAIRGQ I
LSKLRLTSPPEPTVMTHVPYQVLA 23
LYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNEL
AVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIE
LFQILRPDEH IAKQRYIGGKNLPTRGTAEVVLSFDVTDTVREVVLLRRESN
LGLE IS I HCPCHTFQP NGDI LEN I HEVMEI KFKGVDNEDDHGRGDLGRLK
KQKDHHNPH LI LMMIPPHRLDNPGQGGQRKGALDTNYCFRNLEENCCV
RPLYIDFRQDLGVVKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLY
NTLN PEASASPCCVPQDLEPLT I LYYVGRTP KVEQLSN MVVKSCKCS
proTGF33 D2G SLSLSTCTTLDFGH IKKKRVEAI RGQ I
LSKLRLTSPPEPTVMTHVPYQVLA 24
LYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNEL
AVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIE
LFQILRPDEH IAKQRYIGGKNLPTRGTAEVVLSFDVTDTVREVVLLRRESN
LGLE IS I HCPCHTFQP NGDI LEN I HEVMEI KFKGVDNEDDHGRGDLGRLK
KQKDHHNPH LI LMMIPPHRLDNPGQGGQRKGALDTNYCFRNLEENCCV
146
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RPLYIDFRQDLGVVKVVVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLY
NTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS
Table 12. Exemplary non-human amino acid sequences
Protein Species Sequence
SEQ ID
NO
proTGF61 Mouse
LSTCKTIDMELVKRKRIEAIRGOILSKLRLASPPSOGEVPPGPLPEAVL 25
ALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKT
KDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQ
KYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGF
RFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATP
LERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWK
VVIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPC
CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS
proTGF(31 Cyno
LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVL 26
ALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFK
QSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQK
YSNNSWRYLSNRLLAPSDSPEVVLSFDVTGVVRQWLSRGGEIEGFR
LSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPL
ERAQHLQSSRHRRALDTNYCFSSTEKNCCVROLYIDFRKDLGVVKWI
HEPKGYHANFCLGPCPYIVVSLDTQYSKVLALYNQHNPGASAAPCCV
PQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS
TGF61 LAP Mouse
LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVL 27
C4S ALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKT
KDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQ
KYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGF
RFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATP
LERAQHLHSSRHRR
TGF61 LAP Cyno
LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVL 28
C4S ALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFK
QSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQK
YSNNSWRYLSNRLLAPSDSPEVVLSFDVTGVVRQVVLSRGGEIEGFR
LSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPL
ERAQHLQSSRHRR
proTGF61 Mouse
LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVL 29
C4S D2G ALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKT
KDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQ
KYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGF
RFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATP
LERAQHLHSSRHGALDTNYCFSSTEKNCCVROLYIDFRKDLGWKWI
HEPKGYHANFCLGPCPYIVVSLDTQYSKVLALYNQHNPGASASPCCV
PQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS
proTGF61 Mouse
LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVL 30
C4S ALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKT
KDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQ
KYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGF
RFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATP
LERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWK
WIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPC
CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS
proTGF61 Cyno
LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVL 31
C4S ALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFK
QSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQK
YSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQVVLSRGGEIEGFR
LSAHCSCDSKDNTLQVD I NGFTTGRRGDLATI HGMNRPFLLLMATPL
ERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWI
HEPKGYHANFCLGPCPYIVVSLDTCYSKVLALYNOHNPGASAAPCCV
PQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS
proTGF(31 Cyno LSTSKTI
DMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVL 32
C4S D2G ALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFK
QSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQK
YSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQVVLSRGGEIEGFR
LSAHCSCDSKDNTLQVD I NGFTTGRRGDLATI HGMNRPFLLLMATPL
ERAQHLQSSRHGALDTNYCFSSTEKNCCVRQLYIDFRKDLG \A/KWIH
147
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EP KGYHANFC LG PC PYIWSLDTQYSKVLALYNQH N PGASAAPCCVP
QALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS
LTBP3 CYN 0 GPAGERGAGGGGALARERFKVVFAPVICKRTCLKGQCRDSCQQGS
33
NMTLIGENGHSTDTLTGSGFRVVVCPLPCMNGGQCSSRNQCLCPP
DFTGRFCQVPAGGAGGGTGGSGPGLSRAGALSTGALPP LAP EGDS
VASKHA IYAVQVIADPPGPGEGPPAQHAAFLVP LGPGQ ISAEVQAPP
PVVNVRVH HP PEASVQVHRI ESSNAEGAAPSOFILLPHPKPSHPRPP
TQKPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWGQSKC
HKCPQ LQYTGVQKPGPVRG EVGADCPQGYKR LNSTHCQ D IN ECAM
PGVCRHGDCLNNPGSYRCVCPPGHSLGPSRTQCIADKPEEKSLCF
RLVSPEHQCQHPLTTRLTRQLCCCSVGKAWGARCQRCPADGTAAF
KE ICPAGKGYH I LTSHQTLTIQGESDFSLFLHPDGPPKPQQLPESPS
QAPPPEDTEEERGVTTDSPVSEERSVQQSHPTATTSPARPYPELIS
RPSPPTMRVVFLPDLPPSRSAVEIAPTQVTETDECRLNQNICGHGEC
VPGPPDYSCHCNPGYRSHPQHRYCVDVNECEAEPCGPGRGICMN
TGGSYNCHCNRGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINF
PGHYKCNCYPGYRLKASRPPVCEDIDECRDPSSCPDGKCENKPGS
FKCIACQPGYRSQGGGACRDVNECAEGSPCSPGVVCENLPGSFRC
TCAQGYAPAPDGRSCVDVDECEAGDVCDNGICTNTPGSFQCQCLS
GYHLSRDRSHCEDIDECDFPAACIGGDCI NTNGSYRCLCPQGHRLV
GGRKCQDIDECTQDPGLCLPHGACKNLQGSYVCVCDEGFTPTQDQ
HGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAG
VVGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNN IVNYGIPAHRDIDE
CMLFGAEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECL
DESNCRNGVCENTRGGYRCACTPPAEYSPAQRQCLSPEEMDVDE
CODPAACRPGRCVNLPGSYRCECRPPVVVPGPSGRDCOLPESPAE
RAPERRDVCVVSQRGEDGMCAGPQAGPALTFDDCCCRQGRGVVGA
QCRPCPPRGAGSQCPTSQSESNSFWDTSPLLLGKPRRDEDSSEED
SDECRCVSGRCVPRPGGAVCECPGGFQLDASRARCVDIDECRELN
QRGLLCKSERCVNTSGSFRCVCKAGFARSRPHGACVPQRRR
LTBP3 Mouse GPAGERGTGGGGALARERFKVVFAPVICKRTCLKGQCRDSCQQGS
34
NMTLIGENGHSTDTLTGSAFRVVVCPLPCMNGGQCSSRNOCLCPP
DFTGRFCQVPAAGTGAGTGSSG PG LARTGAMSTG PLP PLAPEGES
VASKHA IYAVQVIADPPGPGEGPPAQHAAFLVP LGPGQ ISAEVQAPP
PVVNVRVH HP PEASVQVHRI EGPNAEGPASSQHLLPHPKPPHPRPP
TQKPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWGQSKC
HKCPQLQYTGVQKPVPVRGEVGADCPQGYKRLNSTHCQDINECAM
PGNVCHGDCLNNPGSYRCVCPPGHSLGPLAAQCIADKPEEKSLCFR
LVSTEHQCQHPLTTRLTRQLCCCSVGKAWGARCQRCPADGTAAFK
EICPGKGYH I LTSHQTLTIQGESDFSLFLH PDGP PKPQQLPESPSRAP
PLEDTEEERGVTMDPPVSEERSVQQSHPTTTTSPPRPYPELISRPSP
PTFHRFLPDLPPSRSAVEIAPTQVTETDECRLNQNICGHGQCVPGPS
DYSCHCNAGYRSHPQHRYCVDVNECEAEPCGPGKGICMNTGGSY
NCHCNRGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINFPGHYK
CNCYPGYRLKASRPPICEDIDECRDPSTCPDGKCENKPGSFKCIAC
QPGYRSQGGGAC RDVN ECSEGTPCSPGVVC EN LPGSYRCTCAQYE
PAQDGLSCIDVDECEAGKVCQDGICTNTPGSFQCQCLSGYHLSRDR
SRCEDIDECDFPAACIGGDCI NTNGSYRCLCPLGHRLVGGRKCKKDI
DECSQDPGLCLPHACENLQGSYVCVCDEGFTLTQDQHGCEEVEQP
HHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYP
CPVYSSAEFHSLVPDGKRLHSGQQHCELCIPAHRDIDECILFGAEICK
EGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVC
ENTRGGYRCACTPPAEYSPAQAQCLIPERWSTPQRDVKCAGASEE
RTACVVVGPWAGPALTFDDCCCRQPRLGTQCRPCPPRGTGSQCPT
SQSESNSFVVDTSPLLLGKSPRDEDSSEEDSDECRCVSGRCVPRPG
GAVCECPGGFQLDASRARCVDIDECRELNQRGLLCKSERCVNTSG
SFRCVCKAGFTRSRPHGPACLSAAADDAAIAHTSVIDHRGYFH
LTBP1S Cyno NHTGRIKVVFTPSICKVTCTKGSCQNSCEKGNTTTLISENGHAADTLT
35
ATNFRVVLCHLPCMNGGQCSSRDKCQCPPNFTGKLCQIPVHGASV
PKLYQHSQQPGKALGTHVI HSTHTLPLTVTSQQGVKVKFPPN IVN I H
VKH PPEASVQ I H QVSRI DG PTGQ KTKEAQPGQSQVSYQG LPVQ KTQ
TIHSTYSHQQVIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSKQE
DCCGTVGTSWGFNKCQKCPKKPSYHGYNQMMECLPGYKRVNNTF
COD IN ECOLOGVCPNGECLNTMGSYRCTCKIGFGPDPTFSSCVPDP
PVISEEKGPCYRLVSSGRQC MHPLSVHLTKQLCCCSVGKAWGPHC
EKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIHHHVGKGPVFVKPK
148
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NTQPVAKSTHPPPLPAKEEPVEALTFSREH GPGVAEPEVATAPPEK
EIPSLDQEKTKLEPGQPQLSPGISTIHLHPQFPVVI EKTSPPVPVEVAP
EASTSSASQVIAPTQVTE I N ECTVN PDICGAG HCI NLPVRYTC I CYEG
YKFSEQQRKCVDIDECTQVQH LCSQGRCENTEGSFLC I CPAGFMAS
EEGTNC I DVDECLRPDVCGEGHCVNTVGAFRC EYCDSGYRMTQRG
RCEDI DECLNPSTCPDEQCVNSPGSYQCVPCTEGFRGVVNGQCLDV
DECLEPNVCTNGDCSNLEGSYMCSCHKGYTRTPDHKHCKDIDECQ
QGNLCVNGQCKNTEGSFRCTCGQGYQLSAAKDQCEDI DECQHHHL
CAHGQCRNTEGSFQCVCDQGYRASGLGDHCED IN ECLEDKSVCQR
GDC I NTAGSYDCTCPDGFQLDDNKTCQDINECEHPGLCGPQGECL
NTEGSFHCVCQQGFSISADGRTCED IDECVNNTVCDSHGFCDNTAG
SFRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFCENVEGSFL
CVCADENQEYSPMTGQCRSRTSTDLDVEQPKEEKKECYYNLNDAS
LC DNVLAPNVTKQ ECCCTSGAGWGDNC El FPCPVLGTAEFTEMCPK
GKG FVPAGESSSEAGGENYKDADEC LLFG QEICKNG FCLNTRPGYE
CYCKQGTYYDPVKLQCFDMDECQDPSSCIDGQCVNTEGSYNCFCT
H PMVLDASE KRCIRPAESN EQ I EETDVYQD LCVVE H LSD EYVCSRPL
VG KQTTYTECCCLYGEAWGMQCALCPM KDSD DYAQLC N I PVTGRR
QPYGRDALVDFSEQYAPEADPYFIQDRFLNSFEELQAEECGILNGCE
NGRCVRVQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNRMSLCK
NAKCINTEGSYKCLCLPGYVPSDKPNYCTPLNTALNLEKDSDLE
LTDP 1 S mouse NHTGRIKVVFTPSICKVTCTKGNCQNSCQKGNTTTLISENGHAADTL
36
TATNFRVVICHLPCMNGGQCSSRDKCQCPPNFTGKLCQIPVLGASM
PKLYQHAQQQGKALGSHVIHSTHTLPLTMTSQQGVKVKFPPN IVNIH
VKH PPEASVQ I H QVSRI DSPGGQKVKEAQPGQSQVSYQGLPVQKT
QTVHSTYSHQQLIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSK
QEDCCGTVGTSWGFNKCQKCPKKQSYHGYTQMMECLQGYKRVN
NTFCQD I NECQLQGVCP NGEC LNTMGSYRCSCKMGFGP DPTFSSC
VP OPPVIS EEKG PCYRLVS PGRH CM HPLSVH LTKQ ICCCSVG KAVVG
PHCEKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIHQHIGKEAVYV
KPKNTQPVAKSTHPPPLPAKEEPVEALTSSWEHGPRGAEPEVVTAP
PEKEIPSLDQEKTRLEPGQPQLSPGVSTIHLHPQFPVVVEKTSPPVP
VEVAPEASTSSASQVIAPTQVTE I NECTVNPD ICGAGHC I N LPVRYTC
ICYEGYKFSEQLRKCVDI DECAQVRHLCSQGRCENTEGSFLCVCPA
GFMASEEGTNC IDVDEC LRPDMCRDGRCINTAGAFRCEYCDSGYR
MSRRGYCEDIDECLKPSTCP EEQCVNTPGSYQCVPCTEGFRGVVNG
QCLDVDECLQPKVCTNGSCTNLEGSYMCSCHRGYSPTPDHRHCQ
DIDECQQGNLCMNGQCRNTDGSFRCTCGQGYQLSAAKDQCEDIDE
CEHHHLCSHGQCRNTEGSFQCVCNQGYRASVLGDHCEDINECLED
SSVCQGGDCINTAGSYDCTCPDGFQLNDNKGCQD IN ECAQPGLCG
SHGECLNTQGSFHCVCEQGFSISADGRTCEDIDECVNNTVCDSHGF
CDNTAGSFRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFCE
NVEGSFLCVCADENQEYSPMTGQCRSRVTEDSGVDRQPREEKKEC
YYNLNDASLCDNVLAPNVTKQECCCTSGAGWGDNCEIFPCPVQGT
AEFTEMCPRGKGLVPAGESSYDTGGENYKDADECLLFGEE ICKNGY
CLNTQPGYECYCKQGTYYDPVKLQCFDMDECQDPNSCIDGQCVNT
EGSYNCFCTHPMVLDASEKRCVQPTESNEQ1 EETDVYQDLCWEHLS
EEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALCPMKDSDDYAQL
CN I PVTGRRRPYG RDALVDFSEQYGPETDPYFI QDRFLNSFEELQAE
ECG ILNGCENGRCVRVQEGYTCDCFDGYHLDMAKMTCVDVNECSE
LNNRMSLCKNAKCINTEGSYKCLCLPGYI PSDKPNYCTPLNSALNLD
KESDLE
GARP mouse ISQRREQVPCRTVNKEALCH
GLGLLQVPSVLSLDIQALYLSGNQLQSI 37
LVSPLGFYTALRHLDLSDNQISFLQAGVFQALPYLEHLNLAHNRLAT
GMALNSGGLGRLPLLVSLDLSGNSLHGN LVERLLGETPRLRTLSLAE
NSLTRLARHTFVVGMPAVEQLDLHSNVLMD I EDGAFEALPHLTHLN LS
RNSLTC ISDFSLQQLQVLDLSC NSIEAFQTAPEPQAQFQLAVVLDLRE
NKLLHFPDLAVFPRLIYLNVSNNLIQLPAGLPRGSEDLHAPSEGVVSA
SP LSNPSRNASTH PLSQLLNLDLSYN E I ELVPASFLEHLTSLRFLNLS
RNCLRSFEARQVDSLPCLVLLDLSHNVLEALELGTKVLGSLQTLLLQ
DNALQELPPYTFASLASLQRLNLQGNQVSPCGGPAEPGPPGCVDFS
GIPTLHVLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATGALV
GLEASLEVLELQGNGLTVLRVDLPCFLRLKRLNLAENQLSHLPAWTR
AVSLEVLDLRNNSFSLLPGNAMGGLETSLRRLYLQGNPLSCCGNGVV
LAAQLHQGRVDVDATQDLICRFGSQEELSLSLVRPEDCEKGGLKNV
N L I LLLSFTLVSAIVLTTLATI CFLRRQKLSQQYKA
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sGARP mouse ISQRREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQALYLSGNQLQS1
38
LVSPLGFYTALRHLDLSDNQISFLQAGVFQALPYLEHLNLAHNRLAT
GMALNSGGLGRLPLLVSLDLSGNSLHGN LVERLLGETPRLRTLSLAE
NSLTRLARHTFINGMPAVEQLDLHSNVLMDIEDGAFEALPHLTHLNLS
RNSLTCISDFSLQQLQVLDLSCNSIEAFQTAPEPQAQFQLAVVLDLRE
NKLLHFPDLAVFPRLIYLNVSNNLIQLPAGLPRGSEDLHAPSEGINSA
SP LSNPSRNASTH PLSQLLNLDLSYN El ELVPASFLEHLTSLRFLNLS
RNCLRSFEARQVDSLPCLVLLDLSHNVLEALELGTKVLGSLQTLLLQ
DNALQELPPYTFASLASLQRLNLQGNQVSPCGGPAEPGPPGCVDFS
GIPTLHVLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATGALV
GLEASLEVLELQGNGLTVLRVDLPCFLRLKRLNLAENQLSHLPAVVTR
AVSLEVLDLRNNSFSLLPGNAMGGLETSLRRLYLQGNPLSCCGNGVV
LAAQLHQGRVDVDATQDLICRFGSQEELSLSLVRPEDCEKGGLKNV
[641] In some embodiments, antigenic protein complexes (e.g., a LTBP-TGF31
complex) may comprise one or
more presenting molecules, such as LTBP proteins (e.g., LTBP1, LTBP2, LTBP3,
and LTBP4), GARP proteins,
LRRC33 proteins, or fragment(s) thereof. Typically, a minimum required
fragment suitable for carrying out the
embodiments disclosed herein includes at least 50 amino acids, preferably at
least 100 amino acids, of a presenting
molecule protein, comprising at least two cysteine residues capable of forming
disulfide bonds with a proTGF131
complex. Specifically, these Cys residues form covalent bonds with Cysteine
resides present near the N-terminus
of each monomer of the proTGF31 complex. In the three-dimensional structure of
a proTGF31 dimer complex, the
N-terminal so-called "Alpha-1 Helix" of each monomer comes in close proximity
to each other, setting the distance
between the two cysteine residues (one from each helix) required to form
productive covalent bonds with a
corresponding pair of cysteines present in a presenting molecule (see, for
example, Cuende et al., (2015) Sci
Trans. Med. 7: 284ra56). Therefore, when a fragment of a presenting molecule
is used to form an LLC in the
screening process (e.g., immunization, library screening, identification, and
selection), such fragment should
include the cysteine residues separated by the right distance, which will
allow proper disulfide bond formation with
a proTGF31 complex in order to preserve correct conformation of the resulting
LLC. LTBPs (e.g., LTBP1, LTBP3
and LTBP4), for example, may contain "cysteine-rich domains" to mediate
covalent interactions with proTGF31.
[642] An antibody, or antigen binding portion thereof, as described herein, is
capable of binding to a LTBP1-
TGF31 complex. In some embodiments, the LTBP1 protein is a naturally-occurring
protein or fragment thereof. In
some embodiments, the LTBP1 protein is a non-naturally occurring protein or
fragment thereof. In some
embodiments, the LTBP1 protein is a recombinant protein. Such recombinant
LTBP1 protein may comprise
LTBP1, alternatively spliced variants thereof and/or fragments thereof.
Recombinant LTBP1 proteins may also be
modified to comprise one or more detectable labels. In some embodiments, the
LTBP1 protein comprises a leader
sequence (e.g., a native or non-native leader sequence). In some embodiments,
the LTBP1 protein does not
comprise a leader sequence (i.e., the leader sequence has been processed or
cleaved). Such detectable labels
may include, but are not limited to biotin labels, polyhistidine tags, myc
tags, HA tags and/or fluorescent tags. In
some embodiments, the LTBP1 protein is a mammalian LTBP1 protein. In some
embodiments, the LTBP1 protein
is a human, a monkey, a mouse, or a rat LTBP1 protein. In some embodiments,
the LTBP1 protein comprises an
amino acid sequence as set forth in SEQ ID NOs: 35 and 36 in Table 12. In some
embodiments, the LTBP1
protein comprises an amino acid sequence as set forth in SEQ ID NO: 39 in
Table 14.
[643] An antibody, or antigen binding portion thereof, as described herein, is
capable of binding to a LTBP3-
TGF31 complex. In some embodiments, the LTBP3 protein is a naturally-occurring
protein or fragment thereof. In
some embodiments, the LTBP3 protein is a non-naturally occurring protein or
fragment thereof. In some
embodiments, the LTBP3 protein is a recombinant protein. Such recombinant
LTBP3 protein may comprise
LTBP3, alternatively spliced variants thereof and/or fragments thereof. In
some embodiments, the LTBP3 protein
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comprises a leader sequence (e.g., a native or non-native leader sequence). In
some embodiments, the LTBP3
protein does not comprise a leader sequence (i.e., the leader sequence has
been processed or cleaved).
Recombinant LTBP3 proteins may also be modified to comprise one or more
detectable labels. Such detectable
labels may include, but are not limited to biotin labels, polyhistidine tags,
myc tags, HA tags and/or fluorescent
tags. In some embodiments, the LTBP3 protein is a mammalian LTBP3 protein. In
some embodiments, the LTBP3
protein is a human, a monkey, a mouse, or a rat LTBP3 protein. In some
embodiments, the LTBP3 protein
comprises an amino acid sequence as set forth in SEQ ID NOs: 33 and 34 in
Table 12. In some embodiments,
the LTBP1 protein comprises an amino acid sequence as set forth in SEQ ID NO:
40 in Table 14.
[644] An antibody, or antigen binding portion thereof, as described herein, is
capable of binding to a GARP-TGF131
complex. In some embodiments, the GARP protein is a naturally-occurring
protein or fragment thereof. In some
embodiments, the GARP protein is a non-naturally occurring protein or fragment
thereof. In some embodiments,
the GARP protein is a recombinant protein. Such a GARP may be recombinant,
referred to herein as recombinant
GARP. Some recombinant GARPs may comprise one or more modifications,
truncations and/or mutations as
compared to wild type GARP. Recombinant GARPs may be modified to be soluble.
In some embodiments, the
GARP protein comprises a leader sequence (e.g., a native or non-native leader
sequence). In some embodiments,
the GARP protein does not comprise a leader sequence (i.e., the leader
sequence has been processed or cleaved).
In other embodiments, recombinant GARPs are modified to comprise one or more
detectable labels. In further
embodiments, such detectable labels may include, but are not limited to biotin
labels, polyhistidine tags, flag tags,
myc tags, HA tags and/or fluorescent tags. In some embodiments, the GARP
protein is a mammalian GARP
protein. In some embodiments, the GARP protein is a human, a monkey, a mouse,
or a rat GARP protein. In
some embodiments, the GARP protein comprises an amino acid sequence as set
forth in CEO ID NOs: 37-38 in
Table 12. In some embodiments, the GARP protein comprises an amino acid
sequence as set forth in SEQ ID
NOs: 41 and 42 in Table 14. In some embodiments, the antibodies, or antigen
binding portions thereof, described
herein do not bind to TGF31 in a context-dependent manner, for example binding
to TGF31 would only occur when
the TGF31 molecule was complexed with a specific presenting molecule, such as
GARP. Instead, the antibodies,
and antigen-binding portions thereof, bind to TGF31 in a context-independent
manner. In other words, the
antibodies, or antigen-binding portions thereof, bind to TGF31 when bound to
any presenting molecule: GARP,
LTBP1, LTBP3, and/or LRRC33.
[645] An antibody, or antigen binding portion thereof, as described herein, is
capable of binding to a LRRC33-
TGF31 complex. In some embodiments, the LRRC33 protein is a naturally-
occurring protein or fragment thereof.
In some embodiments, the LRRC33 protein is a non-naturally occurring protein
or fragment thereof. In some
embodiments, the LRRC33 protein is a recombinant protein. Such a LRRC33 may be
recombinant, referred to
herein as recombinant LRRC33. Some recombinant LRRC33 proteins may comprise
one or more modifications,
truncations and/or mutations as compared to wild type LRRC33. Recombinant
LRRC33 proteins may be modified
to be soluble. For example, in some embodiments, the ectodomain of LRRC33 may
be expressed with a C-terminal
His-tag in order to express soluble LRRC33 protein (sLRRC33; see, e.g., SEQ ID
NO: 73). In some embodiments,
the LRRC33 protein comprises a leader sequence (e.g., a native or non-native
leader sequence). In some
embodiments, the LRRC33 protein does not comprise a leader sequence (i.e., the
leader sequence has been
processed or cleaved). In other embodiments, recombinant LRRC33 proteins are
modified to comprise one or
more detectable labels. In further embodiments, such detectable labels may
include, but are not limited to biotin
labels, polyhistidine tags, flag tags, myc tags, HA tags and/or fluorescent
tags. In some embodiments, the LRRC33
protein is a mammalian LRRC33 protein. In some embodiments, the LRRC33 protein
is a human, a monkey, a
mouse, or a rat LRRC33 protein. In some embodiments, the LRRC33 protein
comprises an amino acid sequence
as set forth in SEQ ID NOs: 72, 73, and 74 in Table 14.
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Table 13. Exemplary LTBP amino acid sequences
Protein Sequence
SEQ
ID NO
LTBP1S NHTGRIKVVFTPSICKVTCTKGSCQNSCEKGNTTTLISENGHAADTLT 39
ATNFRVVICHLPCMNGGQCSSRDKCQCPPNFTGKLCQI PVHGASVP
KLYQHSQQPGKALGTHVI HSTHTLPLTVTSQQGVKVKFPPN IVN I HVK
HP PEASVQ IHQVSRI DGPTGQKTKEAQPGQSQVSYQG LPVQKTQTI H
STYSHQQVIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSKQEDCC
GTVGTSWGFN KCQKC PKKPSYH GYNQMM ECLPGYKRVNNTFCQD I
NECQLQGVCPNGECLNTMGSYRCTCKIGFGPDPTFSSCVPDPPVI SE
EKGPCYRLVSSGRQCMHPLSVHLTKQLCCCSVGKAWGPHCEKCPL
PGTAAFKEICPGGMGYTVSGVHRRRPIHHHVGKGPVFVKPKNTQPV
AKSTHPPPLPAKEEPVEALTFSREHGPGVAEPEVATAPPEKEIPSLDQ
EKTKLEPGQ PQLSPGISTI HLHPQFPVVIEKTSPPVPVEVAP EASTSSA
SQVIAPTQVTEINECTVNPDICGAGHCINLPVRYTCICYEGYRFSEQQ
RKCVDIDECTOVQHLCSOGRCENTEGSFLCICPAGFMASEEGTNCID
VDECLRPDVCGEGHCVNTVGAFRCEYCDSGYRMTQRGRCEDIDECL
NPSTCPDEQCVNSPGSYQCVPCTEGFRGWNGQCLDVDECLEPNVC
ANGDCSNLEGSYMCSCHKGYTRTPDHKHCRDIDECQQGNLCVNGQ
CKNTEGSFRCTCGQGYQLSAAKDQCEDI DECQHRHLCAHGQCRNT
EGSFQCVCDQGYRASGLGDHCED IN ECLEDKSVCQRG DCINTAGSY
DCTCPDGFQLDDN KTCQD IN ECEH PG LCGPQG ECLNTEGS FHCVCQ
QGFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYQGFQ
APQDGQGCVDVNECELLSGVCGEAFCENVEGSFLCVCADENQEYSP
MTGQCRSRTSTDLDVDVDQPKEEKKECYYNLNDASLCDNVLAPNVT
KQECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAGESS
SEAGGENYKDADECLLFGQEICKNGFCLNTRPGYECYCKQGTYYDP
VKLQCFDM DECQDPSSC I DGQCVNTEGSYNCFCTHP MVLDASEKRC
IRPAESNEQI EETDVYQDLCVVEHLSDEYVCSRPLVGKQTTYTECCCL
YG EA1NGMQCALCPLKDSD DYAQLCN I PVTG RRQPYGRDALVDFS EQ
YTPEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCD
CFDGYHLDTAKMTCVDVNECDELNNRMSLCKNAKCINTDGSYKCLCL
PGYVPSDKPNYCTPLNTALNLEKDSDLE
LTBP3 GPAGERGAGGGGALARERFKVVFAPVICKRTCLKGQCRDSCQQGS 40
NMTLIGENGHSTDTLTGSGFRVVVCPLPC MNGGQCSSRNQCLCPPD
FTGRFCQVPAGGAGGGTGGSGPGLSRTGALSTGALPP LAP EGOS VA
SKHAIYAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISAEVQAPPPVV
NVRVHH PP EASVQVHRI ESSNAESAAPSQHLLPH PKPSHPRPPTQKP
LGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWGQSKCHKCPQ
LQYTGVQKPGPVRGEVGADCPQGYKRLNSTHCQDINECAMPGVCR
HGDCLNNPGSYRCVCPPGHSLGPSRTQCIADKPEEKSLCFRLVSPEH
QCQHPLTTRLTRQLCCCSVGKAWGARCQRCPTDGTAAFKEICPAGK
GYH ILTSHQTLTIQGESDFSLFLHPDGPPKPQQLP ESPSQAPPPEDTE
EERGVTTDSPVSEERSVQQSH PTATTTPARPYPELISRPSPPTM RVVF
LPDLPPSRSAVEIAPTQVTETDECRLNQN ICGHGECVPGPPDYSCHC
NPGYRSHPQHRYCVDVNECEAEPCGPGRGICMNTGGSYNCHCNRG
YRLHVGAGGRSCVDLNECAKPHLCGDGGFC IN FPGHYKCNCYPGYR
LKASRPPVCEDIDECRDPSSCPDGKCENKPGSFKC IACQPGYRSQG
GGACRDVNECAEGSPCSPGWCENLPGSFRCTCAQGYAPAPDGRSC
LDVDECEAGDVCDNG ICSNTPGSFQCQCLSGYHLSRDRSHCEDI DE
CDFPAACIGGDCINTNGSYRCLCPQGHRLVGGRKCQDI DECSQDPSL
CLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVEQPHHKKECYL
NFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEF
HSLCP DGKGYTODN N I VNYGIPAHRDIDECMLFGSEICKEGKCVNTQ
PGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVCENTRGGYR
CACTPPAEYSPAQRQCLSPEEMDVDECQDPAACRPGRCVNLPGSY
RCECRPPVVVPGPSGRDCQLPESPAERAPERRDVCVVSQRGEDGMC
AG PLAGPALTFDDCCCRQGRGWGAQCRPCPP RGAGSHCPTSQSES
NSFVVDTSPLLLGKPPRDEDSSEEDSDECRCVSGRCVPRPGGAVCEC
PGGFQLDASRARCVDIDECRELNQ RGLLCKSERCVNTSGSFRCVCK
AG FARSRPHGACVPQRRR
Table 14. Exemplary GARP and LRRC33 amino acid sequences
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Protein Sequence
SEQ
ID NO
GARP AQHQDKVPCKMVDKKVSCQVLG LLQVPSVLPP DTETLD LSGN QLRS
ILA 41
SPLGFYTALRHLDLSTNE IS FLQPGAFQALTH LE H LS LAH NRLAMATALS
AGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLS LAE NSLTRLTR
HTFRDM PAL EQLD LHS NVLM D I EDGAFEGLPRLTHLN LSRNSLTCISDFS
LQQLRVLDLSCN S I EAFQTASQPQAEFQLTWLD LRENKLLH FP DLAALP
RLIYLNLSNN LI RLPTGP PQDSKG I HAPSEGWSALPLSAPSGNASGRPLS
QLLN LDLSYN E I ELI PDSFLEH LTSLCFLNLSRNCLRTFEARRLGSLPCLM
LLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPYTFANLASLQRLN
LQGNRVSPCGGPDEPGPSGCVAFSG ITS LRS LSLVDN E I ELLRAGAFLH
TPLTELDLSSN PGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFIC
LKRLNLAENRLSHLPAVVTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLR
RLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSH
VRPEDCEKGG LKN I N LI I ILTFILVSAI LLTTLAACCCVRRQKFNQQYKA
sGARP AQHQDKVPCKMVDKKVSCQVLG LLQVPSVLPP DTETLD LSGN QLRS
ILA 42
SPLGFYTALRHLDLSTNE IS FLQPGAFQALTH LE H LS LAH NRLAMATALS
AGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLS LAE NSLTRLTR
HTFRDM PAL EQLD LHS NVLM D I EDGAFEGLPRLTHLN LSRNSLTCISDFS
LQQLRVLDLSCN S I EAFQTASQPQAEFQLTWLD LRENKLLH FP DLAALP
RLIYLNLSNN LI RLPTGP PQDSKG I HAPSEGWSALPLSAPSGNASGRPLS
QLLN LDLSYN E I ELI PDSFLEH LTSLCFLNLSRNCLRTFEARRLGSLPCLM
LLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPYTFANLASLQRLN
LQGNRVSPCGGPDEPGPSGCVAFSG ITS LRS LSLVDN E I ELLRAGAFLH
TPLTELDLSSN PGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFIC
LKRLNLAENRLSHLPAVVTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLR
RLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSH
VRPEDCEKGG LKN IN
LRRC33 (also known as MELLPLWLCLGFHFLTVGWRNRSGTATAASQGVCKLVGGAADCRGQ
72
NRROS; Uniprot SLASVPSS LP PHARMLTLDAN PLKTLWNHSLQPYPLLESLSLHSCH
LE RI
Accession No. Q86YC3) SRGAFQEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLSGNAL
TEDMAALMLQNLSSLRSVSLAGNTI MRLDDSVFEGLERLRELDLQRNYI
FE IEGGAFDGLAELRHLN LAFNNLPCIVDFGLTRLRVLNVSYNVLEWFLA
TGGEAAFELETLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDLYN
TSSPREMVAQFLLVDGNVTN ITTVSLWEEFSSSDLADLRFLDMSQNQF
QYLPDGFLRKMPSLSHLNLHQNCLMTLHIREHEPPGALTELDLSHNQLS
ELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQIS
LCPLPAAS DRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGTSL
TYLDLSSNWGVLNGSLAPLQDVAPMLQVLSLRNMGLHSSFMALDFSGF
GN LRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSR
GLRTIYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNLSSKI IRVTE
LPGGVPRDCKVVERLDLGLLYLVL I LPSCLILLVACTVIVLTEKKPLLQVIK
SRCHVVSSVY
* Native signal peptide is depicted in bold font.
soluble LRRC33 MDMRVPAQLLGLLLLWFSGVLGWRNRSGTATAASQGVCKLVGCAAD
73
(sLRRC33) CRGQSLASVPSSLPPHARMLTLDANPLKTLWNHSLQPYPLLESLSLHSC
HLERISRGAFQEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLS
GNALTEDMAALMLQNLSSLRSVSLAGNTI MRLDDSVFEGLERLRELDLQ
RNYI FE IEGGAFDG LAELRH LNLAFN N LPCIVDFGLTRLRVLNVSYNVLE
VVFLATGGEAAFELETLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYR
DLYNTSSPREMVAQFLLVDGNVTN ITTVSLWEEFSSSDLADLRFLDMSQ
NQFQYLPDGFLRKMPSLSH LN LH QNCLMTLH I REH EP PGALTELDLSH N
QLSELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFANARN ITTLDMSH
NQIS LCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQ
GTSLTYLDLSSNWGVLNGSLAPLQDVAPMLQVLSLRNMGLHSSFMALD
FSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSE
QLSRGLRTIYLSQN PYDCCGVDGWGALQHGQTVADWAMVTCN LSSKI I
RVTELPGGVPRDCKWERLDLGLHHHHHH
* Modified human kappa light chain signal peptide is depicted in bold font.
Histidine tag is underlined.
Human LRRC33-GARP MDMRVPAQLLGLLLLVVFSGVLGWRNRSGTATAASQGVCKLVGGAAD
74
chimera CRGQSLASVPSSLPPHARMLTLDANPLKTLWNHSLQPYPLLESLSLHSC
HLERISRGAFQEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLS
GNALTEDMAALMLQNLSSLRSVSLAGNTI MRLDDSVFEGLERLRELDLQ
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RNYIFEIEGGAFDGLAELRHLNLAFNN LPCIVDFGLTRLRVLNVSYNVLE
VVFLATGGEAAFELETLDLSHNOLLFFPLLPQYSKLRTLLLRDNNMGFYR
DLYNTSSPREMVAQFLLVDGNVINITTVSLVVEEFSSSDLADLRFLDMSQ
NQFQYLPDGFLRKMPSLSHLNLHQNCLMTLH IREHEPPGALTELDLSHN
QLSELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSH
NQISLCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQ
GTSLTYLDLSSNWGVLNGSLAPLQDVAPMLQVLSLRNMGLHSSFMALD
FSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSE
QLSRGLRTIYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNLSSKI I
RVTELPGGVPRDCKWERLDLGLLIIILTF/LVSAILLTTLAACCCVRROKFN
QQYKA
* Modified human kappa light chain signal peptide is depicted in bold font.
** LRRC33 ectodomain is underlined.
# GARP transmembrane domain is italicized.
414# GARP intracellular tail is double underlined.
Pharmaceutical Compositions and Formulations
[646] The disclosure further provides pharmaceutical compositions used as a
medicament suitable for
administration in human and non-human subjects. One or more high-affinity,
context-independent antibodies
encompassed by the disclosure can be formulated or admixed with a
pharmaceutically acceptable carrier
(excipient), including, for example, a buffer, to form a pharmaceutical
composition. Such formulations may be used
for the treatment of a disease or disorder that involves TGFp signaling. In
certain embodiments, such formulations
may be used for immuno-oncology applications.
[647] The pharmaceutical compositions of the disclosure may be administered to
patients for alleviating a TGFp-
related indication (e.g., fibrosis, immune disorders, and/or cancer).
"Acceptable" means that the carrier is
compatible with the active ingredient of the composition (and preferably,
capable of stabilizing the active ingredient)
and not deleterious to the subject to be treated. Examples of pharmaceutically
acceptable excipients (carriers),
including buffers, would be apparent to the skilled artisan and have been
described previously. See, e.g.,
Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott
Williams and Wilkins, Ed. K. E.
Hoover. In one example, a pharmaceutical composition described herein contains
more than one antibody that
specifically binds a GARP-proTGF31 complex, a LTBP1-proTGF31 complex, a LTBP3-
proTGF31 complex, and a
LRRC33-proTGF131 complex where the antibodies recognize different
epitopes/residues of the complex.
[648] The pharmaceutical compositions to be used in the present methods can
comprise pharmaceutically
acceptable carriers, excipients, or stabilizers in the form of lyophilized
formulations or aqueous solutions
(Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott
VVilliams and Wilkins, Ed. K. E.
Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations
used, and may comprise buffers such as phosphate, citrate, and other organic
acids; antioxidants including
ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl
ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides; proteins, such as
serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides,
and other carbohydrates including
glucose, man nose, or dextrans; chelating agents such as EDTA; sugars such as
sucrose, man nitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-
protein complexes); and/or non-ionic
surfactants such as TWEENT", PLURONIC or polyethylene glycol (PEG).
Pharmaceutically acceptable
excipients are further described herein.
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[649] The disclosure also includes pharmaceutical compositions that comprise
an antibody or fragment thereof
according to the present disclosure, and a pharmaceutically acceptable
excipient.
[650] Thus, the antibody or a molecule comprising an antigen-binding fragment
of such antibody can be
formulated into a pharmaceutical composition suitable for human
administration.
[651] The pharmaceutical formulation may include one or more excipients. In
some embodiments, excipient(s)
may be selected from the list provided in
the following:
https://www.accessdata.fda.gov/scripts/cder/iig/index.Cfm?event=browseByLetter.
page&Letter=A
[652] The pharmaceutical composition is typically formulated to a final
concentration of the active biologic (e.g.,
monoclonal antibody, engineered binding molecule comprising an antigen-binding
fragment, etc.) to be between
about 20 mg/mL and about 200 mg/mL. For example, the final concentration
(wt/vol) of the formulations may range
between about 20-200, 20-180, 20-160, 20-150, 20-120, 20-100, 20-80, 20-70, 20-
60, 20-50, 20-40, 30-200, 30-
180, 30-160, 30-150, 30-120, 30-100, 30-80, 30-70, 30-60, 30-50, 30-40, 40-
200, 40-180, 40-160, 40-150, 40-120,
40-100, 40-80, 40-70, 40-60, 40-50, 50-200, 50-180, 50-160, 50-150, 50-120, 50-
100, 50-80, 50-70, 50-60, 60-
200, 60-180, 60-160, 60-150, 60-120, 60-100, 60-80, 60-70, 70-200, 70-180, 70-
160, 70-150, 70-120, 70-100, 70-
80 mg/mL. In some embodiments, the final concentration of the biologic in the
formulation is about 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, or 200
mg/mL.
[653] The pharmaceutical compositions of the present disclosure are preferably
formulated with suitable buffers.
Suitable buffers include but are not limited to: phosphate buffer, citric
buffer, and histidine buffer.
[654] The final pH of the formulation is typically between pH 5.0 and 8Ø For
example, the pH of the
pharmaceutical composition may be about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,
5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,
6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8.
[655] The pharmaceutical composition of the present disclosure may comprise a
surfactant, such as nonionic
detergent, approved for the use in pharmaceutical formulations. Such
surfactants include, for example,
polysorbates, such as Polysorbate 20 (TweenTm-20), Polysorbate 80 (Tween-80)
and NP-40.
[656] The pharmaceutical composition of the present disclosure may comprise a
stabilizer. For liquid-protein
preparations, stability can be enhanced by selection of pH-buffering salts,
and often amino acids can also be used.
It is often interactions at the liquid/air interface or liquid/solid interface
(with the packaging) that lead to aggregation
following adsorption and unfolding of the protein. Suitable stabilizers
include but are not limited to: sucrose,
maltose, sorbitol, as well as certain amino acids such as histidine, glycine,
methionine and arginine.
[657] The pharmaceutical composition of the present disclosure may contain one
or any combinations of the
following excipients: Sodium Phosphate, Arginine, Sucrose, Sodium Chloride,
Tromethamine, Mannitol, Benzyl
Alcohol, Histidine, Sucrose, Polysorbate 80, Sodium Citrate, Glycine,
Polysorbate 20, Trehalose, Poloxamer 188,
Methionine, Trehalose, Hyaluronidase, Sodium Succinate, Potassium Phosphate,
Disodium Edetate, Sodium
Chloride, Potassium Chloride, Maltose, Histidine Acetate, Sorbitol, Pentetic
Acid, Human Serum Albumin, Pentetic
Acid.
[658] In some embodiments, the pharmaceutical composition of the present
disclosure may contain a
preservative.
[659] The pharmaceutical composition of the present disclosure is typically
presented as a liquid or a lyophilized
form. Typically, the products can be presented in vial (e.g., glass vial).
Products available in syringes, pens, or
autoinjectors may be presented as pre-filled liquids in these
container/closure systems.
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[660] In some examples, the pharmaceutical composition described herein
comprises liposomes containing an
antibody that specifically binds a GARP-proTGF131 complex, a LTBP1-proTGF131
complex, a LTBP3-proTGP131
complex, and a LRRC33-proTGF[31 complex, which can be prepared by any suitable
method, such as described
in Epstein et al., Proc. NatL Acad. Sci. USA 82:3688 (1985); Hwang et al.,
Proc. Natl. Acad. Sal. USA 77:4030
(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced
circulation time are disclosed in
U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the
reverse phase evaporation method
with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-
derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of
defined pore size to yield
liposomes with the desired diameter.
[661] In some embodiments, liposomes with targeting properties are selected to
preferentially deliver or localize
the pharmaceutical composition to certain tissues or cell types. For example,
certain nanoparticle-based carriers
with bone marrow-targeting properties may be employed, e.g., lipid-based
nanoparticles or liposomes. See, for
example, Sou (2012) "Advanced drug carriers targeting bone marrow",
ResearchGate publication 232725109.
[662] In some embodiments, pharmaceutical compositions of the disclosure may
comprise or may be used in
conjunction with an adjuvant. It is contemplated that certain adjuvant can
boost the subjects immune responses
to, for example, tumor antigens, and facilitate T effector function, DC
differentiation from monocytes, enhanced
antigen uptake and presentation by APCs, etc. Suitable adjuvants include but
are not limited to retinoic acid-based
adjuvants and derivatives thereof, oil-in-water emulsion-based adjuvants, such
as MF59 and other squalene-
contain ing adjuvants, Toll-like receptor (TRL) ligands (e.g., CpGs), a-
tocopherol (vitamin E) and derivatives thereof.
[663] The antibodies described herein may also be entrapped in microcapsules
prepared, for example, by
coacervation techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-
microcapsules and poly-(methylmethacylate) microcapsules, respectively, in
colloidal drug delivery systems (for
example, liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in
macroemulsions. Exemplary techniques have been described previously, see,
e.g., Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing (2000).
[664] In other examples, the pharmaceutical composition described herein can
be formulated in sustained-release
format. Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic
polymers containing the antibody, which matrices are in the form of shaped
articles, e.g., films, or microcapsules.
Examples of sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-
methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),
copolymers of L-glutamic acid and 7
ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic acid copolymers such as
the LUPRON DEPOTTm (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide
acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[665] The pharmaceutical compositions to be used for in vivo administration
must be sterile. This is readily
accomplished by, for example, filtration through sterile filtration membranes.
Therapeutic antibody compositions
are generally placed into a container having a sterile access port, for
example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[666] The pharmaceutical compositions described herein can be in unit dosage
forms such as tablets, pills,
capsules, powders, granules, solutions or suspensions, or suppositories, for
oral, parenteral or rectal
administration, or administration by inhalation or insufflation.
[667] Suitable surface-active agents include, in particular, non-ionic agents,
such as polyoxyethylenesorbitans
(e.g., TweenTm 20, 40, 60, 80 or 85) and other sorbitans (e.g., SpanTM 20, 40,
60, 80 or 85). Compositions with a
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surface-active agent will conveniently comprise between 0.05 and 5% surface-
active agent, and can be between
0.1 and 2.5%. It will be appreciated that other ingredients may be added, for
example mannitol or other
pharmaceutically acceptable vehicles, if necessary.
[668] Suitable emulsions may be prepared using commercially available fat
emulsions, such as lntralipidTM,
LiposynTM, lntonutrolTM, LipofundiriTM and LipiphysanTM. The active ingredient
may be either dissolved in a pre-
mixed emulsion composition or alternatively it may be dissolved in an oil
(e.g., soybean oil, safflower oil, cottonseed
oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing
with a phospholipid (e.g., egg
phospholipids, soybean phospholipids or soybean lecithin) and water. It will
be appreciated that other ingredients
may be added, for example glycerol or glucose, to adjust the tonicity of the
emulsion. Suitable emulsions will
typically contain up to 20% oil, for example, between 5 and 20%.
[669] The emulsion compositions can be those prepared by mixing an antibody of
the disclosure with lntralipidTM
or the components thereof (soybean oil, egg phospholipids, glycerol and
water).
Kits for Use in Detecting, Monitoring or Alleviating a 1G93-Related Indication
[670] The present disclosure also provides kits for use in treating,
diagnosing, or predicting response in a subject
having a diseases/disorders associated with a TGF8-related indication. Such
kits can include one or more
containers comprising an antibody, or antigen binding portion thereof, that
specifically binds to TGF3 (e.g., TGF31
or latent TG931), phosphorylated SMAD (e.g., phosphorylated SMAD2), or any one
of the surface markers
described herein for identifying immune cells such as MDSCs (e.g., mMDSCs
and/or gMDSCs) or CD8+ T cells.
[671] In some embodiments, the kit can comprise instructions for use in
accordance with any of the methods
described herein. The included instructions can comprise a description of
using the antibody, or antigen binding
portion thereof, that specifically binds TGF8 (e.g., TGF31 or latent TGF31),
phosphorylated SMAD (e.g.,
phosphorylated SMAD2), or any one of the surface markers described herein for
identifying immune cells such as
MDSCs (e.g., mMDSCs and/or gMDSCs) or CD8+ T cells to treat, diagnose, or
predict response in a subject having
a target disease described herein. In some embodiments, the kit may comprise a
description of selecting an
individual suitable for treatment based on identifying whether that individual
has the target disease or is likely to
achieve clinical efficacy following treatment with a particular regimen, e.g.,
a treatment comprising a TGF31
inhibitor, e.g., Ab6, alone or in conjunction with a checkpoint inhibitor
therapy or a chemotherapy. In still other
embodiments, the instructions comprise a description for determining target
engagement or therapeutic efficacy in
an individual treated with a TGFI31 inhibitor, e.g., Ab6.
[672] In some embodiments, the instructions may include information such as
use of the antibodies or antigen-
binding fragments binding to biomarekrs described herein, e.g., TGF3 (e.g.,
TGF31 or latent TGF31),
phosphorylated SMAD (e.g., phosphorylated SMAD2), or any one of the surface
markers described herein for
identifying immune cells such as MDSCs (e.g., mMDSCs and/or gMDSCs) or CD8+ T
cells, as part of a treatment
regimen. Instructions supplied in the kits of the disclosure are typically
written instructions on a label or package
insert (e.g., a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a
magnetic or optical storage disk) are also acceptable.
[673] The label or package insert indicates that the composition is used for
treating, diagnosing, or predicting
response for a disease or disorder associated with a TGFp-related indication.
Instructions may be provided for
practicing any of the methods described herein.
[674] The kits of this disclosure are in suitable packaging. Suitable
packaging includes, but is not limited to, vials,
bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages
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for use in combination with a specific device, such as an inhaler, nasal
administration device (e.g., an atomizer) or
an infusion device such as a minipump. A kit may have a sterile access port
(for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a hypodermic
injection needle). The container
may also have a sterile access port (for example the container may be an
intravenous solution bag or a vial having
a stopper pierceable by a hypodermic injection needle).
[675] Kits may optionally provide additional components such as buffers and
interpretive information. Normally,
the kit comprises a container and a label or package insert(s) on or
associated with the container. In some
embodiments, the disclosure provides articles of manufacture comprising
contents of the kits described above.
Process of Screening, Identification and Manufacture of Preferred Isoform-
specific Inhibitors of TGFpi
[676] The disclosure encompasses screening/selection methods, production
methods and manufacture
processes of antibodies or fragments thereof capable of binding each of: a
GARP-proTGFB1 complex, a LTBP1-
proTGF[31 complex, a LTBP3-proTGF[31 complex, and a LRRC33-proTGF[31 complex
with equivalent affinities,
and pharmaceutical compositions and related kits comprising the same. In some
embodiments, for screening
purposes, at least one of the LTBP1-proTGF[31 and LTBP3-proTGF[31 complexes
and at least one of the GARP-
proTG931 and LRRC33-proTGF[31 complexes are included. Antibodies or fragments
thereof identified in the
screening process are preferably further tested to confirm its ability to bind
each of the LLCs of interest with high
affinity.
[677] Numerous methods may be used for obtaining antibodies, or antigen
binding fragments thereof, of the
disclosure. For example, antibodies can be produced using recombinant DNA
methods. Monoclonal antibodies
may also be produced by generation of hybridomas (see e.g., Kohler and
Milstein (1975) Nature, 256: 495-499) in
accordance with known methods. Hybridomas formed in this manner are then
screened using standard methods,
such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon
resonance (e.g., OCTET or
BIACORE) analysis, to identify one or more hybridomas that produce an antibody
that specifically binds to a
specified antigen. Any form of the specified antigen may be used as the
immunogen, e.g., recombinant antigen,
naturally occurring forms, any variants or fragments thereof, as well as
antigenic peptide thereof (e.g., any of the
epitopes described herein as a linear epitope or within a scaffold as a
conformational epitope). One exemplary
method of making antibodies includes screening protein expression libraries
that express antibodies or fragments
thereof (e.g., scFv), e.g., phage or ribosome display libraries. Phage display
is described, for example, in Ladner
et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson
et al., (1991) Nature, 352: 624-
628; Marks et al., (1991) J. Mol. Biol., 222: 581-597; WO 92/18619; WO
91/17271; WO 92/20791; WO 92/15679;
WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.
[678] In addition to the use of display libraries, the specified antigen
(e.g., presenting molecule-TGFI31 complexes)
can be used to immunize a non-human host, e.g., rabbit, guinea pig, rat,
mouse, hamster, sheep, goat, chicken,
camelid, as well as non-mammalian hosts such as shark. In one embodiment, the
non-human animal is a mouse.
[679] Immunization of a non-human host may be carried out with the use of a
purified recombinant protein
complex as an immunogen, such as proTGFI31 with or without a presenting
molecule (or fragment thereof)
associated thereto. These include, but are not limited to: LTBP1-proTGFp1 ,
LTBP3-proTG931, GARP-proTGF131
and LRRC33-proTGFI31. The associated presenting molecule need not be full
length counterpart but preferably
includes the two cysteine residues that form covalent bonds with the proTGFI31
dimer complex.
[680] Alternatively, immunization of a non-human host may be carried out with
the use of a cell-based antigen.
The term cell-based antigen refers to cells (e.g., heterologous cells)
expressing the proTGFI31 protein complex.
This may be achieved by overexpression of proTGF[31, optionally with co-
expression of a presenting molecule. In
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some embodiments, endogenous counterpart(s) may be utilized as cell-based
antigen. Cell-surface expression of
the proteins that form the proTGF81-containing protein complex may be
confirmed by well-known methods such
as FAGS. Upon immunization of the host with such cells (a cell-based antigen),
immune responses to the antigen
are elicited in the host, allowing antibody production and subsequent
screening. In some embodiments, suitable
knockout animals are used to facilitate stronger immune responses to the
antigen. Alternatively, structural
differences among different species may be sufficient to trigger antibody
production in the host.
[681] In another embodiment, a monoclonal antibody is obtained from the non-
human animal, and then modified,
e.g., chimeric, using suitable recombinant DNA techniques. A variety of
approaches for making chimeric antibodies
have been described. See e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A.
81:6851, 1985; Takeda et al., Nature
314:452, 1985, Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat.
No. 4,816,397; Tanaguchi et al.,
European Patent Publication EP171496; European Patent Publication 0173494,
United Kingdom Patent GB
2177096B.
[682] For additional antibody production techniques, see Antibodies: A
Laboratory Manual, eds. Harlow et al.,
Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily
limited to any particular source,
method of production, or other special characteristics of an antibody.
[683] Some aspects of the present disclosure relate to host cells transformed
with a polynucleotide or vector.
Host cells may be a prokaryotic or eukaryotic cell. The polynucleotide or
vector which is present in the host cell
may either be integrated into the genome of the host cell or it may be
maintained extrachromosomally. The host
cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect,
fungal, plant, animal or human cell. In
some embodiments, fungal cells are, for example, those of the genus
Saccharomyces, in particular those of the
species S. cerevisiae. The term "prokaryotic" includes all bacteria which can
be transformed or transfected with
a DNA or RNA molecules for the expression of an antibody or the corresponding
immunoglobulin chains.
Prokaryotic hosts may include gram negative as well as gram positive bacteria
such as, for example, E. coli, S.
typhimurium, Serratia marcescens and Bacillus subtilis. The term ''eu ka
ryotic" includes yeast, higher plants, insects
and vertebrate cells, e.g., mammalian cells, such as NSO and CHO cells.
Depending upon the host employed in
a recombinant production procedure, the antibodies or immunoglobulin chains
encoded by the polynucleotide may
be glycosylated or may be non-glycosylated. Antibodies or the corresponding
immunoglobulin chains may also
include an initial methionine amino acid residue.
[684] In some embodiments, once a vector has been incorporated into an
appropriate host, the host may be
maintained under conditions suitable for high level expression of the
nucleotide sequences, and, as desired, the
collection and purification of the immunoglobulin light chains, heavy chains,
light/heavy chain dimers or intact
antibodies, antigen binding fragments or other immunoglobulin forms may
follow; see, Beychok, Cells of
Immunoglobulin Synthesis, Academic Press, N.Y., (1979). Thus, polynucleotides
or vectors are introduced into
the cells which in turn produce the antibody or antigen binding fragments.
Large-scale production of the antibody
or antibody fragments (for example, about 250 L or greater, e.g., 1000L,
2000L, 3000L, 4000L or greater) is suitable
for commercial-scale manufacture of pharmaceutical compositions comprising the
antibody and is typically carried
out in a culture system, such as a suspension cell culture. Such culture may
be a eukaryotic cell culture, wherein
optionally the eukaryotic cell culture is a mammalian cell culture, plant cell
culture, or an insect cell culture. In some
embodiments, the mammalian cell culture comprises a CHO cell, MDCK cell, NSO
cell, Sp2/0 cell, BHK cell, M urine
C127 cell, Vero cell, HEK293 cell, HT-1080 cell, or PER.C6 cell.
[685] The transformed host cells can be grown in fermenters and cultured using
any suitable techniques to
achieve optimal cell growth. Once expressed, the whole antibodies, their
dimers, individual light and heavy chains,
other immunoglobulin forms, or antigen binding fragments, can be purified
according to standard procedures of the
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art, including ammonium sulfate precipitation, affinity columns, column
chromatography, gel electrophoresis and
the like; see, Scopes, Protein Purification, Springer Verlag, N.Y. (1982). The
antibody or antigen binding fragments
can then be isolated from the growth medium, cellular lysates, or cellular
membrane fractions. The isolation and
purification of the, e.g., microbially expressed antibodies or antigen binding
fragments may be by any conventional
means such as, for example, preparative chromatographic separations and
immunological separations such as
those involving the use of monoclonal or polyclonal antibodies directed, e.g.,
against the constant region of the
antibody.
[686] Aspects of the disclosure relate to a hybridoma, which provides an
indefinitely prolonged source of
monoclonal antibodies. As an alternative to obtaining immunoglobulins directly
from the culture of hybridomas,
immortalized hybridoma cells can be used as a source of rearranged heavy chain
and light chain loci for subsequent
expression and/or genetic manipulation. Rearranged antibody genes can be
reverse transcribed from appropriate
mRNAs to produce cDNA. In some embodiments, heavy chain constant region can be
exchanged for that of a
different isotype or eliminated altogether. The variable regions can be linked
to encode single chain Fv regions.
Multiple Fv regions can be linked to confer binding ability to more than one
target or chimeric heavy and light chain
combinations can be employed. Any appropriate method may be used for cloning
of antibody variable regions and
generation of recombinant antibodies.
[687] In some embodiments, an appropriate nucleic acid that encodes variable
regions of a heavy and/or light
chain is obtained and inserted into an expression vectors which can be
transfected into standard recombinant host
cells. A variety of such host cells may be used. In some embodiments,
mammalian host cells may be
advantageous for efficient processing and production. Typical mammalian cell
lines useful for this purpose include
CHO cells, 293 cells, or NSO cells. The production of the antibody or antigen
binding fragment may be undertaken
by culturing a modified recombinant host under culture conditions appropriate
for the growth of the host cells and
the expression of the coding sequences. The antibodies or antigen binding
fragments may be recovered by
isolating them from the culture. The expression systems may be designed to
include signal peptides so that the
resulting antibodies are secreted into the medium; however, intracellular
production is also possible.
[688] The disclosure also includes a polynucleotide encoding at least a
variable region of an immunoglobulin
chain of the antibodies described herein. In some embodiments, the variable
region encoded by the polynucleotide
comprises at least one complementarity determining region (CDR) of the VH
and/or VL of the variable region of the
antibody produced by any one of the above described hybridomas.
[689] Polynucleotides encoding antibody or antigen binding fragments may be,
e.g., DNA, cDNA, RNA or
synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic
acid molecule comprising any
of those polynucleotides either alone or in combination. In some embodiments,
a polynucleotide is part of a vector.
Such vectors may comprise further genes such as marker genes which allow for
the selection of the vector in a
suitable host cell and under suitable conditions.
[690] In some embodiments, a polynucleotide is operatively linked to
expression control sequences allowing
expression in prokaryotic or eukaryotic cells. Expression of the
polynucleotide comprises transcription of the
polynucleotide into a translatable mRNA. Regulatory elements ensuring
expression in eukaryotic cells, preferably
mammalian cells, are well known to those skilled in the art. They may include
regulatory sequences that facilitate
initiation of transcription and optionally poly-A signals that facilitate
termination of transcription and stabilization of
the transcript. Additional regulatory elements may include transcriptional as
well as translational enhancers, and/or
naturally associated or heterologous promoter regions. Possible regulatory
elements permitting expression in
prokaryotic host cells include, e.g., the PL, Lac, Trp or Tac promoter in E.
coli, and examples of regulatory elements
permitting expression in eukaryotic host cells are the A0X1 or GAL1 promoter
in yeast or the CMV-promoter, SV40-
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promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a
globin intron in mammalian
and other animal cells.
[691] Beside elements which are responsible for the initiation of
transcription such regulatory elements may also
include transcription termination signals, such as the SV40-poly-A site or the
tk-poly-A site, downstream of the
polynucleotide. Furthermore, depending on the expression system employed,
leader sequences capable of
directing the polypeptide to a cellular compartment or secreting it into the
medium may be added to the coding
sequence of the polynucleotide and have been described previously. The leader
sequence(s) is (are) assembled
in appropriate phase with translation, initiation and termination sequences,
and preferably, a leader sequence
capable of directing secretion of translated protein, or a portion thereof,
into, for example, the extracellular medium.
Optionally, a heterologous polynucleotide sequence can be used that encode a
fusion protein including a C- or N-
terminal identification peptide imparting desired characteristics, e.g.,
stabilization or simplified purification of
expressed recombinant product.
[692] In some embodiments, polynucleotides encoding at least the variable
domain of the light and/or heavy chain
may encode the variable domains of both immunoglobulin chains or only one.
Likewise, polynucleotides may be
under the control of the same promoter or may be separately controlled for
expression. Furthermore, some aspects
relate to vectors, particularly plasmids, cosmids, viruses and bacteriophages
used conventionally in genetic
engineering that comprise a polynucleotide encoding a variable domain of an
immunoglobulin chain of an antibody
or antigen binding fragment; optionally in combination with a polynucleotide
that encodes the variable domain of
the other immunoglobulin chain of the antibody.
[693] In some embodiments, expression control sequences are provided as
eukaryotic promoter systems in
vectors capable of transforming or transfecting eukaryotic host cells, but
control sequences for prokaryotic hosts
may also be used. Expression vectors derived from viruses such as
retroviruses, vaccinia virus, adeno-associated
virus, herpes viruses, or bovine papilloma virus, may be used for delivery of
the polynucleotides or vector into
targeted cell population (e.g., to engineer a cell to express an antibody or
antigen binding fragment). A variety of
appropriate methods can be used to construct recombinant viral vectors. In
some embodiments, polynucleotides
and vectors can be reconstituted into liposomes for delivery to target cells.
The vectors containing the
polynucleotides (e.g., the heavy and/or light variable domain (s) of the
immunoglobulin chains encoding sequences
and expression control sequences) can be transferred into the host cell by
suitable methods, which vary depending
on the type of cellular host.
[694] The screening methods may include a step of evaluating or confirming
desired activities of the antibody or
fragment thereof. In some embodiments, the step comprises selecting for the
ability to inhibit target function, e.g.,
inhibition of release of mature/soluble growth factor (e.g., TGF61) from a
latent complex. In certain embodiments,
such step comprises a cell-based potency assay, in which inhibitory activities
of test antibody or antibodies are
assayed by measuring the level of growth factor released in the medium (e.g.,
assay solution) upon activation,
when proTGFp complex is expressed on cell surface. The level of growth factor
released into the medium/solution
can be assayed by, for example, measuring TGF3 activities. Non-limiting
examples of useful cell-based potency
assays are described in PCT/US2019/041373, e.g., at Example 2.
[695] In some embodiments, the step of screening desirable antibodies or
fragments comprises selecting for
antibodies or fragments thereof that promote internalization and subsequent
removal of antibody-antigen
complexes from the cell surface. In some embodiments, the step comprises
selecting for antibodies or fragments
thereof that induce ADCC. In some embodiments, the step comprises selecting
for antibodies or fragments thereof
that accumulate to a desired site(s) in vivo (e.g., cell type, tissue or
organ). In some embodiments, the step
comprises selecting for antibodies or fragments thereof with the ability to
cross the blood brain barrier. The
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methods may optionally include a step of optimizing one or more antibodies or
fragments thereof to provide variant
counterparts that possess desirable profiles, as determined by criteria such
as stability, binding affinity, functionality
(e.g., inhibitory activities, Fc function, etc.), immunogenicity, pH
sensitivity and developability (e.g., high solubility,
low self-association, etc.).
[696] The process for making a composition comprising an antibody or a
fragment according to the disclosure
may include optimization of an antibody or antibodies that are identified to
possess desirable binding and functional
(e.g., inhibitory) properties. Optimization may comprise affinity maturation
of an antibody or fragment thereof.
Further optimization steps may be carried out to provide physicochemical
properties that are advantageous for
therapeutic compositions. Such steps may include, but are not limited to,
mutagenesis or engineering to provide
improved solubility, lack of self-aggregation, stability, pH sensitivity, Fc
function, and so on. The resulting optimized
antibody is preferably a fully human antibody or humanized antibody suitable
for human administration.
[697] Manufacture process for a pharmaceutical composition comprising such an
antibody or fragment thereof
may comprise the steps of purification, formulation, sterile filtration,
packaging, etc. Certain steps such as sterile
filtration, for example, are performed in accordance with the guidelines set
forth by relevant regulatory agencies,
such as the FDA. Such compositions may be made available in a form of single-
use containers, such as pre-filled
syringes, or multi-dosage containers, such as vials.
Modifications
[698] Antibodies, or antigen binding portions thereof, of the disclosure may
be modified with a detectable label or
detectable moiety, including, but not limited to, an enzyme, prosthetic group,
fluorescent material, luminescent
material, bioluminescent material, radioactive material, positron emitting
metal, nonradioactive paramagnetic metal
ion, and affinity label for detection and isolation of a GARP-proTGF61
complex, a LTBP1-proTG931 complex, a
LTBP3-proTGF[31 complex, and/or a LRRC33-proTGF[31 complex. The detectable
substance or moiety may be
coupled or conjugated either directly to the polypeptides of the disclosure or
indirectly, through an intermediate
(such as, for example, a linker (e.g., a cleavable linker)) using suitable
techniques. Non-limiting examples of
suitable enzymes include horseradish peroxidase, alkaline phosphatase, 6-
galactosidase, glucose oxidase, or
acetylcholinesterase; non-limiting examples of suitable prosthetic group
complexes include streptavidin/biotin and
avidin/biotin; non-limiting examples of suitable fluorescent materials include
biotin, umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride, or phycoerythrin; an
example of a luminescent material includes luminol; non-limiting examples of
bioluminescent materials include
luciferase, luciferin, and aequorin; and examples of suitable radioactive
material include a radioactive metal ion,
e.g., alpha-emitters or other radioisotopes such as, for example, iodine
(1311, 1251, 1231, 1211), carbon (14C), sulfur
(35S), tritium (3H), indium (115mln, 113mln, 1121n, 111In), and technetium
(99Tc, 99mTc), thallium (201Ti), gallium
(68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine
(18F), 153Sm, Lu (177Lu),
159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 86R, 188Re, 142Pr, 105Rh, 97Ru,
68Ge, 57Co, 65Zn, 85Sr,
32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, Zirconium (89Zr) and tin (113Sn, 117Sn).
In some embodiments, the radio
label may be selected from the group consisting of:
13N, 150, 58Ga, 177Lu, 18F and 89Zr. In some embodiments,
useful labels are positron-emitting isotopes, which may be detected by
positron-emission tomography. The
detectable substance may be coupled or conjugated either directly to the
antibodies of the disclosure that bind
specifically to a GARP-proTGF61 complex, a LTBP1-proTGF131 complex, a LTBP3-
proTGF61 complex, and/or a
LRRC33-proTG931 complex, or indirectly, through an intermediate (such as, for
example, a linker) using suitable
techniques. Any of the antibodies provided herein that are conjugated to a
detectable substance may be used for
any suitable diagnostic assays, such as those described herein.
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[699] In addition, antibodies, or antigen binding portions thereof, of the
disclosure may also be modified with a
drug. The drug may be coupled or conjugated either directly to the
polypeptides of the disclosure, or indirectly,
through an intermediate (such as, for example, a linker (e.g., a cleavable
linker)) using suitable techniques.
Targeting Agents
[700] In some embodiments methods of the present disclosure comprise the use
of one or more targeting agents
to target an antibody, or antigen binding portion thereof, as disclosed
herein, to a particular site in a subject for
purposes of modulating mature TGFp release from a GARP-proTGF131 complex, a
LTBP1-proTGF(31 complex, a
LTBP3-proTGFp1 complex, and/or a LRRC33-proTG931 complex. For example, LTBP1-
proTGFp1 and LTBP3-
proTGF131 complexes are typically localized to extracellular matrix. Thus, in
some embodiments, antibodies
disclosed herein can be conjugated to extracellular matrix targeting agents
for purposes of localizing the antibodies
to sites where LTBP-associated TGFp1 complexes reside. In such embodiments,
selective targeting of antibodies
leads to selective modulation of LTBP1-proTGFp1 and LTBP3-proTGFp1 complexes.
In some embodiments,
extracellular matrix targeting agents include heparin binding agents, matrix
metalloproteinase binding agents, lysyl
oxidase binding domains, fibrillin-binding agents, hyaluronic acid binding
agents, and others.
[701] Similarly, GARP-proTG931 and LRRC33-proTGFp1 complexes are typically
localized and anchored to the
surface of cells. The former is expressed on activated FOXP3+ regulatory T
cells (Tregs), while the latter is
expressed on certain myeloid cells and some cancer cells such as AML. Thus, in
some embodiments, antibodies
disclosed herein can be conjugated to immune cell (e.g., Treg cell, activated
macrophages, etc.) binding agents
for purposes of localizing antibodies to sites where these cell-associated
proTGFP1 complexes reside. In such
embodiments, selective targeting of antibodies leads to selective inhibition
of cell associated-proTGFp1 complexes
(e.g., selective inhibition of the release of mature TGFpl for purposes of
immune modulation, e.g., in the treatment
of cancer). In such embodiments, immune cell targeting agents may include, for
example, CCL22 and CXCL12
proteins or fragments thereof.
[702] In some embodiments, bispecific antibodies may be used having a first
portion that selectively binds a
proTGF01 complex and a second portion that selectively binds a component of a
target site, e.g., a component of
the ECM (e.g., fibrillin) or a component of a Treg cell (e.g., CTLA-4).
[703] As further detailed herein, the present disclosure contemplates that
isoform-selective TG931 inhibitors, such
as those described herein, may be used for promoting or restoring
hematopoiesis in the bone marrow. Accordingly,
in some embodiments, a composition comprising such an inhibitor (e.g., high-
affinity, isoform-selective inhibitor of
TGFp1) may be targeted to the bone marrow. One mode of achieving bone marrow
targeting is the use of certain
carriers that preferentially target the bone marrow localization or
accumulation. For example, certain nanoparticle-
based carriers with bone marrow-targeting properties may be employed, e.g.,
lipid-based nanoparticles or
liposomes. See, for example, Sou (2012) "Advanced drug carriers targeting bone
marrow", ResearchGate
publication 232725109.
[704] In some embodiments, targeting agents include immune-potentiators, such
as adjuvants comprising
squalene and/or a-tocopherol and adjuvants comprising a TLR ligand/agonist
(such as TLR3 ligands/agonists).
For example, squalene-containing adjuvant may preferentially target certain
immune cells such as monocytes,
macrophages and antigen-presenting cells to potentiate priming, antigen
processing and/or immune cell
differentiation to boost host immunity. In some embodiments, such adjuvant may
stimulate host immune responses
to neo-epitopes for T cell activation.
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Therapeutic Targets and in vivo Mechanisms of Action
[705] Accordingly, the TGFI3 inhibitors (e.g., high-affinity, isoform-
selective TGFI31 inhibitors) disclosed herein
may be used to inhibit TGF31 in any suitable biological systems, such as in
vitro, ex vivo and/or in vivo systems.
Related methods may comprise contacting a biological system with the TGF31
inhibitor. The biological system
may be an assay system, a biological sample, a cell culture, arid so on. In
some cases, these methods include
modifying the level of free growth factor in the biological system.
[706] Accordingly, such pharmaceutical compositions and formulations may be
used to target TGFI3-containing
latent complexes accessible by the inhibitors in vivo. Thus, the antibody of
the disclosure is aimed to target the
following complexes in a disease site (e.g., TME) where it preemptively binds
the latent complex thereby preventing
the growth factor from being released: i) proTGFI31 presented by GARP; ii)
proTGF31 presented by LRRC33; iii)
proTGF[31 presented by LTBP1; and iv) proTGF[31 presented by LTBP3. Typically,
complexes (i) and (ii) above
are present on cell surface because both GARP and LRRC33 are transmembrane
proteins capable of anchoring
or tethering latent proTGFI31 on the extracellular face of the cell expressing
LRRC33, whilst complexes (iii) and (iv)
are components of the extracellular matrix. In this way, the inhibitors
embodied herein do away with having to
complete binding with endogenous high affinity receptors for exerting
inhibitory effects. Moreover, targeting
upstream of the ligand/receptor interaction may enable more durable effects
since the window of target accessibility
is longer and more localized to relevant tissues than conventional inhibitors
that target transient, soluble growth
factors only after it has been released from the latent complex. Thus,
targeting the latent complex tethered to
certain niches may facilitate improved target engagement in vivo, as compared
to conventional neutralizing
antibodies that must compete binding with endogenous receptors during its
short half-life as a soluble (free) growth
factor, e.g., ¨two minutes, once it is released from the latent complex.
[707] A number of studies have shed light on the mechanisms of TGFI31
activation. Three integrins, c6/131 , aV36,
aVI38, and 0VI31 have been demonstrated to be key activators of latent TGFI31
(Reed, NI., et al., Sci Transl Med,
2015. 7(288): p. 288ra79; Travis, M.A. and D. Sheppard, Aririu Rev Immuriol,
2014. 32: p. 51-82; Munger, J.S., et
al., Cell, 1999. 96(3): p.319-28; Sheppard. Cancer Metastasis Rev, 2005.
24(3): 395-402). aV integrins bind the
RGD sequence present in TGF31 and TG931 LAPs with high affinity (Dong, X., et
al., Nat Struct Mol Biol, 2014.
21(12): p. 1091-6). Transgenic mice with a mutation in the TGF[31 RGD site
that prevents integrin binding, but not
secretion, phenocopy the TGF131-/- mouse (Yang, Z., et al., J Cell Biol, 2007.
176(6): p. 787-93). Mice that lack
both 36 and pa integrins recapitulate all essential phenotypes of TGF31 and
TGF133 knockout mice, including
multiorgan inflammation and cleft palate, confirming the essential role of
these two integrins for TGF31 activation
in development and homeostasis (Aluwihare, P., et al., J Cell Sci, 2009.
122(Pt 2): p. 227-32). Key for integrin-
dependent activation of latent TGF31 is the covalent tether to presenting
molecules; disruption of the disulfide
bonds between GARP and TGF31 LAP by mutagenesis does not impair complex
formation, but completely
abolishes TGF31 activation by aV36 (Wang, R., et al., Mol Biol Cell, 2012.
23(6): p. 1129-39). The recent structure
study of latent TGFI31 illuminates how integrins enable release of active
TGFI31 from the latent complex: the
covalent link of latent TGF31 to its presenting molecule anchors latent TGF31,
either to the ECM through LTBPs,
or to the cytoskeleton through GARP or LRRC33. lntegrin binding to the RGD
sequence results in a force-
dependent change in the structure of LAP, allowing active TGF31 to be released
and bind nearby receptors (Shi,
M., et al., Nature, 2011. 474(7351): p. 343-9). The importance of integrin-
dependent TGF31 activation in disease
has also been well validated. A small molecule inhibitor of 01/31 protects
against bleomycin-induced lung fibrosis
and carbon tetrachloride-induced liver fibrosis (Reed, NJ., et al., Sci Trans!
Med, 2015. 7(288): p. 288ra79), and
aV36 blockade with an antibody or loss of integrin 36 expression suppresses
bleomycin-induced lung fibrosis and
radiation-induced fibrosis (Munger, J.S., et al., Cell, 1999. 96(3): p. 319-
28); Horan, G.S., et al., Am J Resph- Crit
Care Med, 2008. 177(1): p. 56-65).
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[708] In addition to integrins, other mechanisms of TGF31 activation have been
implicated, including
thrombospondin-1 and activation by proteases such as Plasmin, matrix
metalloproteinases (MMPs, e.g., MMP2,
MMP9 and MMP12), cathepsin D, kallikrein, thrombin, and the ADAMs family of
zinc proteases (e.g., ADAM10,
ADAM12 and ADAM17). Knockout of thrombospondin-1 recapitulates some aspects of
the TGFI31-/- phenotype
in some tissues, but is not protective in bleomycin-induced lung fibrosis,
known to be TGFP-dependent (Ezzie,
ME., et al., Am J Respir Cell Mol Biol, 2011. 44(4): p. 556-61). Additionally,
knockout of candidate proteases did
not result in a TGFp1 phenotype (Worthington, J.J., J.E. Klementowicz, and
M.A. Travis, Trends Biochem Sci,
2011. 36(1): p. 47-54). This could be explained by redundancies or by these
mechanisms being critical in specific
diseases rather than development and homeostasis.
[709] Thus, the TGF13 inhibitors (e.g., high-affinity, isoform-specific
inhibitors of TGFp1) described herein include
inhibitors that work by preventing the step of TGF(31 activation. In some
embodiments, such inhibitors can inhibit
integrin-dependent (e.g., mechanical or force-driven) activation of TGFP1. In
some embodiments, such inhibitors
can inhibit protease-dependent or protease-induced activation of TGF31. The
latter includes inhibitors that inhibit
the TGFpl activation step in an integrin-independent manner. In some
embodiments, such inhibitors can inhibit
TGF31 activation irrespective of the mode of activation, e.g., inhibit both
integrin-dependent activation and
protease-dependent activation of TGF31. Non-limiting examples of proteases
which may activate TGF31 include
serine proteases, such as Kallikreins, Chemotrypsin, Trypsin, Elastases,
Plasmin, thrombin, as well as zinc
metalloproteases (MMP family) such as MMP-2, MMP-9 and MMP-13. Kallikreins
include plasma-Kallikreins and
tissue Kallikreins, such as KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8,
KLK9, KLK10, KLK11, KLK12,
KLK13, KLK14 and KLK15. Data previously presented in PCT/U62019/041373
demonstrate examples of an
isoform-specific TGF31 inhibitors, capable of inhibiting Kallikrein-dependent
activation of TGFp1 in vitro. In some
embodiments, inhibitors of the present disclosure prevent release or
dissociation of active (mature) TG931 growth
factor from the latent complex. In some embodiments, such inhibitors may work
by stabilizing the inactive (e.g.,
latent) conformation of the complex. Applicant's previous data further
demonstrated that a high-affinity, context-
independent TGF(31 inhibitor (e,g, Ab6) can also inhibit Plasmin-dependent
TGFI31 activation. Surprisingly,
however, a context-biased TG931 inhibitor (Ab3) was less effective to inhibit
this process. Both Ab3 and Ab6 have
similar affinities for matrix-associated proTGF31 complexes. However, Ab3 has
a significantly weaker binding
affinity for cell-associated proTGF31 complexes. The relative difference
between the two categories is more than
20-fold ("bias"). By comparison, Ab6 shows equivalent high affinities towards
both categories of the antigen
complexes. One possible explanation is that the observed functional difference
may stem from the bias feature of
Ab3. Another possible explanation is that it is mediated by differences in
epitopes or binding regions.
[710] The disclosure is particularly useful for therapeutic use for certain
diseases that are associated with multiple
biological roles of TGFP signaling that are not limited to a single context of
TGFP function. In such situations, it
may be beneficial to inhibit TGFp1 effects across multiple contexts. Thus, the
present disclosure provides methods
for targeting and inhibiting TGF31 in an isoform-specific manner, rather than
in a context-specific manner. Such
agents may be referred to as "isoform-specific, context-independent" TGF31
modulators.
[711] A body of evidence supports the notion that many diseases manifest
complex perturbations of TGFp
signaling, which likely involve participation of heterogeneous cell types that
confer different effects of TGFp
function, which are mediated by its interactions with so-called presenting
molecules. At least four such presenting
molecules have been identified, which can "present" TGFI3 in various
extracellular niches to enable its activation in
response to local stimuli. In one category, TGF3 is deposited into the ECM in
association with ECM-associated
presenting molecules, such as LTBP1 and LTBP3, which mediate ECM-associated
TGFp activities. In another
category, TGFP is tethered onto the surface of immune cells, via presenting
molecules such as GARP and LRRC33,
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which mediate certain immune function. These presenting molecules show
differential expression, localization
and/or function in different tissues and cell types, indicating that
triggering events and outcome of TGF6 activation
will vary, depending on the microenvironment. Based on the notion that many
TGF3 effects may interact and
contribute to disease progression, therapeutic agents that can antagonize
multiple facets of TGF3 function may
provide greater efficacy.
LRRC33-proTGFI31 as target
[712] LRRC33 is expressed in selective cell types, in particular those of
myeloid lineage, including monocytes
and macrophages. Monocytes originated from progenitors in the bone marrow and
circulate in the bloodstream
and reach peripheral tissues. Circulating monocytes can then migrate into
tissues where they become exposed to
the local environment (e.g., tissue-specific, disease-associated, etc.) that
includes a panel of various factors, such
as cytokines and chemokines, triggering differentiation of monocytes into
macrophages, dendritic cells, etc. These
include, for example, alveolar macrophages in the lung, osteoclasts in bone
marrow, microglia in the CNS,
histiocytes in connective tissues, Kupffer cells in the liver, and brown
adipose tissue macrophages in brown adipose
tissues. In a solid tumor, infiltrated macrophages may be tumor-associated
macrophages (TAMs), tumor-
associated neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs),
etc. Such macrophages may
activate and/or be associated with activated fibroblasts, such as carcinoma-
associated (or cancer-associated)
fibroblasts (OAFS) and/or the stroma. Thus, inhibitors of TGF31 activation
described herein which inhibits release
of mature TGF61 from LRRC33-containing complexes can target any of these cells
expressing LRRC33-proTGF31
on cell surface. At a fibrotic microenvironment, LRRC33-expressing cells may
include M2 macropahges, tissue
resident macrophages, and/or MDSCs.
[713] In some embodiments, the LRRC33-TGF31 complex is present at the outer
surface of profibrotic (M2-like)
macrophages. In some embodiments, the profibrotic (M2-like)
macrophages are present in the fibrotic
microenvironment. In some embodiments, targeting of the LRRC33-TGF61 complex
at the outer surface of
profibrotic (M2-like) macrophages provides a superior effect as compared to
solely targeting LTBP1-TGF61 and/or
LTBP1-TGF131 complexes. In some embodiments, M2-like macrophages, are further
polarized into multiple
subtypes with differential phenotypes, such as M2c and M2d TAM-like
macrophages. In some embodiments,
macrophages may become activated by various factors (e.g., growth factors,
chemokines, cytokines and ECM-
remodeling molecules) present in the tumor microenvironment, including but are
not limited to TGF31, CCL2 (MCP-
1), CCL22, SDF-1/0X0L12, M-CSF (CSF-1), IL-6, IL-8, IL-10, IL-11, CXCR4, VEGF,
PDGF, prostaglandin-
regulating agents such as arachidonic acid and cyclooxygenase-2 (COX-2),
parathyroid hormone-related protein
(PTHrP), RUNX2, HIF1a, and metalloproteinases. Exposures to one or more of
such factors may further drive
monocytes/macrophages into pro-tumor phenotypes. To give but one example, CCL2
and VEGF co-expression
in tumors has been shown to be correlated with increased TAM and poor
diagnosis. In turn, activated tumor-
associated cells may also facilitate recruitment and/or differentiation of
other cells into pro-tumor cells, e.g., CAFs,
TANs, MDSCs, and the like. Stromal cells may also respond to macrophage
activation and affect ECM remodeling,
and ultimately vascularization, invasion, and metastasis. For example, 00L2
not only functions as a monocyte
attractant but also promotes cell adhesion by upregulating MAC-1, which is a
receptor for ICAM-1, expressed in
activated endothelium. This may lead to CCL2-dependent arteriogenesis and
cancer progression. Thus, TGF31
inhibitors described herein may be used in a method for inhibiting
arteriogenesis by interfering with the CCL2
signaling axis.
[714] A subset of myeloid cells express cell surface LRRC33, including M2-
polarized macrophages and myeloid-
derived suppressor cells (MDSCs), both of which have immunosuppressive
phenotypes and are enriched at
disease environments (e.g., TME and FME). Bone marrow-derived circulating
monocytes do not appear to express
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cell surface LRRC33. The restrictive expression of LRRC33 makes this a
particularly appealing therapeutic target.
While a majority of studies available in the literature have focused on
effector T cell biology (e.g., CD8+ cytotoxic
cells) in cancer, increasing evidence (such as data presented in
PCT/US2019/041373) points to important roles of
suppressive myeloid cell populations in diseases. Importantly, the highly
selective TGF131 inhibitory antibodies
disclosed herein, are capable of targeting this arm of TG931 function in vivo.
More specifically, tumor-associated
M2 macrophages and MDSCs express cell-surface LRRC33, with a strong
correlation to disease progression. The
high-affinity, TGF31-selective antibodies disclosed herein are capable of
overcoming primary resistance to
checkpoint blockade therapy (CBT) of tumors in multiple pharmacological
models. Indeed, anti-tumor efficacy
coincides with a significant decrease in tumor-associated macrophages and MDSC
levels, suggesting that targeting
this facet of TGFI31 function may contribute to therapeutically beneficial
effects. This is likely applicable to other
disease where these immunosuppressive cells are enriched. A number of fibrotic
conditions are also associated
with elevated local frequencies of these cell populations. Thus, the high-
affinity, TGF31-selective antibodies are
expected to exert similar in vivo effects in such indications. Notably,the new
findings provided herein identify
LRRC33 as a novel cell surface marker for MDSCs in circulation, this, coupled
with Applicant's previous findings
that i) the degree of tumor burden correlates with tumor associated MDSCs, and
that ii) tumor-associated MDSCs
correlate with circulatory MDSCs, points to a new use for LRRC33 as a blood-
based biomarker, e.g., indicative of
immune suppression. As described herein, an immune-suppressed tumor may be
particularly responsive to
treatment comprising a TGFp inhibitor (e.g., TGF31-selective inhibitor, e.g.,
Ab6) and optionally in combination
with a checkpoint inhibitor therapy.
TGFI31-Related Indications
General features of TGF/31-related indications
[715] TGFf31 inhibitors, such as isoform-selective inhibitors described
herein, may be used to treat a wide variety
of diseases, disorders and/or conditions that are associated with TGF31
dysregulation (i.e., "TGR31-related
indications") in human subjects. As used herein, "disease (disorder or
condition) associated with TGF131
dysregulation" or "TGF[31-related indication" means any disease, disorder
and/or condition related to expression,
activity and/or metabolism of a TGF131 or any disease, disorder and/or
condition that may benefit from inhibition of
the activity and/or levels TGFI31. A plethora of evidence exists in literature
pointing to the dysregulation of the
TGF13 signaling pathway in pathologies such as cancer and fibrosis.
[716] Based on the inventors' recognition that TGF131 appears to be the
predominant disease-associated
isoform, the present disclosure includes the use of an isoform-selective,
context-independent TGFI31 inhibitor in a
method for treating a TGF31-related indication in a human subject. Such
inhibitor is typically formulated into a
pharmaceutical composition that further comprises a pharmaceutically
acceptable excipient. Advantageously,
the inhibitor targets both ECM-associated TGF31 and immune cell-associated
TGFI31 but does not target TGFI32
or TGFI33 in vivo. In some embodiments, the inhibitor inhibits the activation
step of TGF131. The disease may be
characterized by dysregulation or impairment in at least two of the following
attributes: a) regulatory T cells
(Treg); b) effector T cell (left) proliferation or function; c) myeloid cell
proliferation or differentiation; d) monocyte
recruitment or differentiation; e) macrophage function; f) epithelial-to-
mesenchymal transition (EMT) and/or
endothelial-to-mesenchymal transition (EndMT); g) gene expression in one or
more of marker genes selected
from the group consisting of: PAI-1, ACTA2, CCL2, Coll al, Col3a1, FN-1, CTGF,
and TGFB1; h) ECM
components or function; i) fibroblast differentiation. A therapeutically
effective amount of such inhibitor is
administered to the subject suffering from or diagnosed with the disease.
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[717] In some embodiments, such therapeutic use incorporates the step of
diagnosing and/or monitoring
treatment response as detailed herein. For example, circulating MDSCs and/or
circulating latent=TGF131 may be
used as biomarker, in accordance with the present disclosure. Such therapeutic
use may further include a step
of selecting a suitable TGFr3 inhibitor as therapy and/or selecting a patient
or patient population likely to benefit
from such therapy.
[718] In some embodiments, a disease treated herein may involve dysregulation
or impairment of ECM
components or function comprises that show increased collagen I deposition. In
some embodiments, the
dysregulation of the ECM includes increased stiffness of the matrix. In some
embodiments, the dysregulation of
the ECM involves fibronectin and/or fibrillin.
[719] In some embodiments, the dysregulation or impairment of fibroblast
differentiation comprises increased
myofibroblasts or myofibroblast-like cells. In some embodiments, the
myofibroblasts or myofibroblast-like cells
are cancer-associated fibroblasts (CAFs). In some embodiments, the CAFs are
associated with a tumor stroma
and may produce CCL2/MCP-1 and/or CXCL12/SDF-1. In some embodiments, the
myofibroblasts or
myofibroblast-like cells are localized to a fibrotic tissue.
[720] In some embodiments, the dysregulation or impairment of regulatory T
cells comprises increased Treg
activity.
[721] In some embodiments, the dysregulation or impairment of effector T cell
(Teff) proliferation or function
comprises suppressed CD4+/CD8+ cell proliferation.
[722] In some embodiments, the dysregulation or impairment of myeloid cell
proliferation or differentiation
comprises increased proliferation of myeloid progenitor cells. The increased
proliferation of myeloid cells may
occur in a bone marrow,
[723] In some embodiments, the dysregulation or impairment of monocyte
differentiation comprises increased
differentiation of bone marrow-derived and/or tissue resident monocytes into
macrophages at a disease site,
such as a fibrotic tissue and/or a solid tumor.
[724] In some embodiments, the dysregulation or impairment of monocyte
recruitment comprises increased
bone marrow-derived monocyte recruitment into a disease site such as TME,
leading to increased macrophage
differentiation and M2 polarization, followed by increased TAMs.
[725] In some embodiments, the dysregulation or impairment of macrophage
function comprises increased
polarization of the macrophages into M2 phenotypes.
[726] In some embodiments, the dysregulation or impairment of myeloid cell
proliferation or differentiation
comprises an increased number of Tregs, MDSCs and/or TANs.
[727] TGFI3-related indications may include conditions comprising an immune-
excluded disease
microenvironment, such as tumor or cancerous tissue that suppresses the body's
normal defense
mechanism/immunity in part by excluding effector immune cells (e.g., CD4+
and/or CD8+ T cells). In some
embodiments, such immune-excluding conditions are associated with poor
responsiveness to treatment (e.g.,
cancer therapy). Non-limiting examples of the cancer therapies, to which
patients are poorly responsive, include
but are not limited to: checkpoint inhibitor therapy, cancer vaccines,
chemotherapy, and radiation therapy (such as
a radiotherapeutic agent). Without intending to be bound by particular theory,
it is contemplated that TGFI3
inhibitors, such as those described herein, may help counter the tumor's
ability to evade or exclude anti-cancer
immunity by restoring immune cell access, e.g., T cell (e.g., CD8+ cells) and
macrophage (e.g., F4/80+ cells, Ml-
polarized macrophages) access by promoting T cell expansion and/or
infiltration into tumor.
[728] Thus, TGFI3 inhibition may overcome treatment resistance (e.g., immune
checkpoint resistance, cancer
vaccine resistance, CAR-T resistance, chemotherapy resistance, radiation
therapy resistance (such as resistance
to a radiotherapeutic agent), etc.) in immune-excluded disease environment
(such as TME) by unblocking and
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restoring effector T cell access and cytotoxic effector functions. Such
effects of TGFp inhibition may further provide
long-lasting immunological memory mediated, for example, by CD8+ T cells.
[729] In some embodiments, tumor is poorly immunogenic (e.g., "desert" or
"cold" tumors). Patients may benefit
from cancer therapy that triggers neo-antigens or promote immune responses.
Such therapies include, but are not
limited to, chemotherapy, radiation therapy (such as a radiotherapeutic
agent), oncolytic viral therapy, oncolytic
peptides, tyrosine kinase inhibitors, neo-epitope vaccines, anti-CTLA4,
instability inducers, DDR agents, NK cell
activators, and various adjuvants such as TLR ligands/agonists. TGFp1
inhibitors, such as those described herein,
can be used in conjunction to boost the effects of cancer therapies. One mode
of action for TGFp1 inhibitors may
be to normalize or restore MHC expression, thereby promoting T cell immunity.
[730] Non-limiting examples of TGFP-related indications include: fibrosis,
including organ fibrosis (e.g., kidney
fibrosis, liver fibrosis, cardiac/cardiovascular fibrosis, muscle fibrosis,
skin fibrosis, uterine fibrosis/endometriosis
and lung fibrosis), scleroderma, Alport syndrome, cancer (including, but not
limited to: blood cancers such as
leukemia, myelofibrosis, multiple myeloma, colon cancer, renal cancer, breast
cancer, malignant melanoma,
glioblastoma), fibrosis associated with solid tumors (e.g., cancer
desmoplasia, such as desmoplastic melanoma,
pancreatic cancer-associated desmoplasia and breast carcinoma desmoplasia),
stromal fibrosis (e.g., stromal
fibrosis of the breast), radiation-induced fibrosis (e.g., radiation fibrosis
syndrome), facilitation of rapid
hematopoiesis following chemotherapy, bone healing, wound healing, dementia,
myelofibrosis, myelodysplasia
(e.g., myelodysplasic syndrome or MDS), a renal disease (e.g., end-stage renal
disease or ESRD), unilateral
ureteral obstruction (UUO), tooth loss and/or degeneration, endothelial
proliferation syndromes, asthma and
allergy, gastrointestinal disorders, anemia of the aging, aortic aneurysm,
orphan indications (such as Marfan's
syndrome and Camurati-Engelmann disease), obesity, diabetes, arthritis,
multiple sclerosis, muscular dystrophy,
bone disorders, amyotrophic lateral sclerosis (ALS), Parkinson's disease,
osteoporosis, osteoarthritis, osteopenia,
metabolic syndromes, nutritional disorders, organ atrophy, chronic obstructive
pulmonary disease (COPD), and
anorexia.
[731] Evidence suggests that the ectonucleotidases CD39 and CD73 may at least
in part contribute to elevated
levels of adenosine in disease conditions. Notably, the 0039/0D73-TGFp axis
may play a role in modulating
immune cells implicated in the TGFp signaling, including Tregs and MDSCs. Both
regulatory T cells (Tregs) and
myeloid-derived suppressive cells (MDSCs) generally exhibit immunosuppressive
phonotypes. In many pathologic
conditions (e.g., cancer, fibrosis), these cells are enriched at disease sites
and may contribute to creating and/or
maintaining an immunosuppressive environment. This may be at least in part
mediated by the ectonucleotidases
CD39 and C073 which together participates in the breakdown of ATP into
nucleoside adenosine, leading to
elevated local concentrations of adenosine in the disease environment, such as
tumor microenvironment and
fibrotic environment. Adenosine can bind to its receptors expressed on target
cells such as T cells and NK cell,
which in turn suppress anti-tumor function of these target cells.
Diseases with aberrant gene expression; biomarkers
[732] It has been observed that abnormal activation of the TGFp signal
transduction pathway in various disease
conditions is associated with altered gene expression of a number of markers.
These gene expression markers
(e.g., as measured by rriRNA) include, but are riot limited to: Serpine 1
(encoding PAI-1), MCP-1 (also known as
CCL2), Col1a1, Col3a1, FN1, TGFB1, CTGF, ACTA2 (encoding a-SMA), SNAll (drives
EMT in fibrosis and
metastasis by downregulating E-cadherin (Cdh1), MMP2 (matrix metalloprotease
associated with EMT), MMP9
(matrix metalloprotease associated with EMT), TIMP1 (matrix metalloprotease
associated with EMT), FOXP3
(marker of Treg induction), CDH1 (E cadherin (marker of epithelial cells)
which is downregulated by TGFp), and,
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CDH2 (N cadherin (marker of mesenchymal cells) which is upregulated by TG93).
Interestingly, many of these
genes are implicated to play a role in a diverse set of disease conditions,
including various types of organ fibrosis,
as well as in many cancers, which include myelofibrosis. Indeed,
pathophysiological link between fibrotic conditions
and abnormal cell proliferation, tumorigenesis and metastasis has been
suggested. See for example, Cox and
Erler (2014) Clinical Cancer Research 20(14): 3637-43 "Molecular pathways:
connecting fibrosis and solid tumor
metastasis"; Shiga et al., (2015) Cancers 7:2443-2458 "Cancer-associated
fibroblasts: their characteristics and
their roles in tumor growth"; Wynn and Barron (2010) Semin. Liver Dis. 30(3):
245-257 "Macrophages: master
regulators of inflammation and fibrosis", contents of which are incorporated
herein by reference. Without wishing
to be bound by a particular theory, the inventors of the present disclosure
contemplate that the TGF81 signaling
pathway may in fact be a key link between these broad pathologies.
[733] The ability of chemotactic cytokines (or chemokines) to mediate
leukocyte recruitment (e.g.,
monocytes/macrophages) to injured or disease tissues has crucial consequences
in disease progression.
Members of the C-C chemokine family, such as monocyte chemoattractant protein
1 (MCP-1), also known as
CCL2, macrophage inflammatory protein 1-alpha (MIP-1a), also known as CCL3,
and MIP-113, also known as
CCL4, and M IP-2a, also known as CXCL2, have been implicated in this process.
[734] For example, MCP-1/CCL2 is thought to play a role in both fibrosis and
cancer. MCP-1/CCL2 is
characterized as a profibrotic chemokine and is a monocyte chemoattractant,
arid evidence suggests that it may
be involved in both initiation and progression of cancer. In fibrosis, MCP-
1/CCL2 has been shown to play an
important role in the inflammatory phase of fibrosis. For example,
neutralization of MCP-1 resulted in a dramatic
decrease in glomerular crescent formation and deposition of type I collagen.
Similarly, passive immunotherapy
with either anti-MCP-1 or anti-MIP-1 alpha antibodies is shown to
significantly reduce mononuclear phagocyte
accumulation in bleomycin-challenged mice, suggesting that MIP-1 alpha and MCP-
1 contribute to the recruitment
of leukocytes during the pulmonary inflammatory response (Smith, Biol Signals.
1996 Jul-Aug;5(4):223-31,
"Chemotactic cytokines mediate leukocyte recruitment in fibrotic lung
disease"). Elevated levels of MIP-1alpha in
patients with cystic fibrosis and multiple myeloma have been reported (see,
for example: Mrugacz et al., J Interferon
Cytokine Res. 2007 Jun;27(6):491-5), supporting the notion that MIP-la is
associated with localized or systemic
inflammatory responses.
[735] Lines of evidence point to the involvement of C-C chemokines in tumor
progression/metastasis. For
example, tumor-derived MCP-1/CCL2 can promote "pro-cancer" phenotypes in
macrophages. For example, in
lung cancer, MCP-1/CCL2 has been shown to be produced by stromal cells and
promote metastasis. In human
pancreatic cancer, tumors secrete CCL2, and immunosuppressive CCR2-positive
macrophages infiltrate these
tumors. Patients with tumors that exhibit high CCL2 expression/low CD8 T-cell
infiltrate have significantly
decreased survival. Without wishing to be bound by particular theory, it is
contemplated that monocytes that are
recruited to an injured or diseased tissue environment may subsequently become
polarized in response to local
cues (such as in response to tumor-derived cytokines), thereby further
contributing to disease progression. These
M2-like macrophages are likely to contribute to immune evasion by suppressing
effector cells, such as CD4+ and
CD8+ T cells. In some embodiments, this process is in part mediated by LRRC33-
TGF[31 expressed by activated
macrophages. In some embodiments, the process is in part mediated by GARP-
TGF[31 expressed by Tregs.
[736] Similarly, in certain carcinomas, such as breast cancer (e.g., triple
negative breast cancer), CXCL2/CCL22-
mediated recruitment of MDSCs has been shown to promote angiogenesis and
metastasis (see, for example,
Kumar et al., (2018) J Clin Invest 128(11): 5095-5109). It is therefore
contemplated that this process is at least in
part mediated by TGF81, such as LRRC33-TGF81. Moreover, because proteases such
as MMP9 are implicated
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in the process of matrix remodeling that contributes to tumor invasion and
metastasis, the same or overlapping
signaling pathways may also play a role in fibrosis.
[737] Involvement of PAI-1/Serpine1 has been implicated in a variety of
fibrotic conditions, cancers, angiogenesis,
inflammation, as well as neurodegenerative diseases (e.g., Alzheimer's
Disease). Elevated expression of PAI-1 in
tumor and/or serum is correlated with poor prognosis (e.g., shorter survival,
increased metastasis) in various
cancers, such as breast cancer and bladder cancer (e.g., transitional cell
carcinoma) as well as myelofibrosis. In
the context of fibrotic conditions, PAI-1 has been recognized as an important
downstream effector of TGF131-
induced fibrosis, and increased PAI-1 expression has been observed in various
forms of tissue fibrosis, including
lung fibrosis (such as IPF), kidney fibrosis, liver fibrosis and scleroderma.
In some embodiments, the process is in
part mediated by ECM-associated TGF31, e.g., via LTBP1-proTGF31 and/or LTBP3-
proTGF31.
[738] In some embodiments, in vivo effects of the TGFI31 inhibitor therapy may
be assessed by measuring
changes in expression levels of suitable gene markers. Suitable markers
include TGF3 (e.g., TGFB1, TGFB2, and
TGFB3). Suitable markers may also include one or more presenting molecules for
TGF3 (e.g., TGF31, TGF32,
and TGF33), such as LTBP1, LTBP3, GARP (or LRRC32) and LRRC33. In some
embodiments, suitable markers
include mesenchymal transition genes (e.g., AXL, ROR2, WNT5A, LOXL2, TVVIST2,
TAGLN, and/or FAP),
immunosuppressive genes (e.g., IL10, VEGFA, VEGFC), monocyte and macrophage
chemotactic genes (e.g.,
CCL2, CCL3, CCL4, CCL7, CCL8, CCL13 and CCL22), and/or various fibrotic
markers discussed herein.
Exemplary markers are plasma/serum markers.
[739] As previously described in PCT/US2019/041373, isoform-specific, context-
independent inhibitors of TGF31
can be used to reduce expression levels of many of these markers in suitable
preclinical models, including
mechanistic animal models, such as UUO, which has been shown to be TGF31-
dependent. Therefore, such
inhibitors may be used to treat a disease or disorder characterized by
abnormal expression (e.g.,
overexpression/upregulation or underexpression/downregulation) of one or more
of the gene expression markers
of the disease.
[740] Thus, in some embodiments, an isoform-specific, context-independent
inhibitor of TGF31 is used in the
treatment of a disease associated with overexpression of one or more of the
following: PAI-1 (encoded by
Serpine1), MCP-1 (also known as CCL2), Coll a1, Col3a1, FN1, TGFB1, CTGF, a-
SMA, ITGA11, and ACTA2,
wherein the treatment comprises administration of the inhibitor to a subject
suffering from the disease in an amount
effective to treat the disease. In some embodiments, the inhibitor is used to
treat a disease associated with
overexpression of PAI-1, MCP-1/CCL2, CTGF, and/or a-SMA. In some embodiments,
the disease is myelofibrosis.
In some embodiments, the disease is cancer, for example, cancer comprising a
solid tumor.
[741] Involvement of the TGF31 pathway in controlling key facets of both the
ECM and immune components may
explain the observations that a remarkable number of dysregulated genes are
shared across a wide range of
pathologies such as proliferative disorders and fibrotic disorders. This
supports the notion that the aberrant pattern
of expression in the genes involving TGF31 signaling is likely a generalizable
phenomenon. These marker genes
may be classified into several categories such as: genes involved in
mesenchymal transition (e.g., EndMT and
EMT); genes involved in angiogenesis; genes involved in hypoxia; genes
involved in wound healing; and genes
involved in tissue injury-triggered inflammatory response.
[742] A comprehensive study carried out by Hugo et al., (Cell, 165(1): 35-44)
elegantly demonstrated the
correlation between differential gene expression patterns of these classes of
markers and the responsiveness to
checkpoint blockade therapy (CBT) in metastatic melanoma. The authors found co-
enrichment of the set of genes
coined "IPRES signatures" defined a transcriptomic subset within not only
melanoma, but also all major common
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human malignancies analyzed. Indeed, the work links tumor cell phenotypic
plasticity (i.e., mesenchymal
transition) and the resultant impacts on the microenvironment (e.g., ECM
remodeling, cell adhesion, and
angiogenesis features of immune suppressive wound healing) to CBT resistance.
In addition to !PRES, other gene
signatures such as TIDE (Jing et al., Nat Med. 2018 Oct;24(10):1550-1558), TIS
(Danaher et al., J lmmunother
Cancer. 2018 Jun 22;6(1):63), F-TBRS (Mariathasan et al., Nature. 2018 Feb 22;
554(7693): 544-548), IMPRES
(Auslander et al., Nat Med. 2018 Oct; 24(10): 1545-1549), and xCell (Aran et
al. Genome Biol. 2017 Nov
15;18(1):220) may also be used to evaluate the tumor immune microenvironment.
[743] Recognizing that each of these IPRES gene categories has been implicated
in disease involving TGF8
dysregulation, Applicant previously contemplated that the TGF81 isoform in
particular may mediate these
processes in disease conditions (see, for example, WO 2017/156500). Work
disclosed previously further
supported this notion (e.g., PCT/US2019/041373 at Example 11; FIG. 37A),
further confirming that therapies that
selectively target TGF81 (as opposed to non-selective alternatives) may offer
an advantage both with respect to
efficacy and safety.
[744] Accordingly, the present disclosure includes a method/process of
selecting or identifying a candidate patient
or patient population likely to respond to a TGF81 inhibition therapy, and
administering to the patient(s) an effective
amount of a high-affinity isoform-selective inhibitor of TGF81. Observation of
a patient's lack of responsiveness to
a CBT (e.g., resistance) may indicate that the patient is a candidate for the
TGF81 inhibition therapy described
herein. Thus, an isoform-selective inhibitor of TGFI31 such as Ab6 may be used
in the treatment of cancer in a
subject, wherein the subject is poorly responsive to a CBT. The subject may
have advanced cancer, such as a
locally advanced solid tumor or metastatic cancer. A patient is said to be
"poorly responsive" when there is no or
little meaningful therapeutic effects achieved (e.g., do not meet the criteria
of partial response or compete response
based on standard guidelines, such as RECIST and iRECIST) following a duration
of time which is expected to be
sufficient to show meaningful therapeutic effects of the particular therapy.
Typically, such duration of time for CBTs
is at least about 3 months of treatment, either with or without additional
therapies such as chemotherapy. Such
patients may be referred to as "refractory" or "non-responders." Where such
patients are poorly responsive to the
initial CBT, the patients may be referred to as "primary non-responders."
Cancer (or patients with such cancer) in
this category may be characterized as having "primary resistance" to the CBT.
In some embodiments, the subject
is a primary non-responder after receiving at least about 3 months of the CBT
treatment, wherein optionally, after
at least about 4 months of the CBT treatment. In some embodiments, the subject
also received additional therapy
in combination with the CBT, such as chemotherapy.
[745] Upon identification of the subject as a non-responder of a CBT, the high-
affinity, isoform-selective inhibitor
of TGF131 may be administered to the subject in conjunction with a CBT, which
may or may not comprise the same
checkpoint inhibitor as the first CBT to which the subject failed to respond.
Any suitable immune checkpoint
inhibitors may be used, e.g., approved checkpoint inhibitors. In some
embodiments, the high-affinity, isoform-
selective inhibitor of TGFI31 is administered to the subject in conjunction
with a CBT comprising an anti-PD-1
antibody or anti-PD-L1 antibody. The high-affinity, isoform-selective
inhibitor of TGF81 is aimed to overcome the
resistance by rendering the cancer more susceptible to the CBT.
[746] The process of selecting or identifying a candidate patient or patient
population likely to respond to, or
otherwise likely to benefit from, a TGF81 inhibition therapy may comprise a
step of testing a biological sample
collected from the patient (or patient population), such as biopsy samples,
for the expression of one or more of the
markers discussed herein. Similarly, such genetic marker(s) may be used for
purposes of monitoring the patient's
responsiveness to a therapy. Monitoring may include testing two or more
biological samples collected from the
patient, for example, before and after administration of a therapy, and during
the course of a therapeutic regimen
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over time, to evaluate changes in gene expression levels of one or more of the
markers, indicative of therapeutic
response or effectiveness. In some embodiments, a liquid biopsy may be used.
[747] In some embodiments, a method of selecting a candidate patient or
patient population likely to respond to
a TGFI31 inhibition therapy may comprise a step of identifying a patient or
patient population previously tested for
the genetic marker(s), such as those described herein, which showed aberrant
expression thereof. These same
methods are also applicable to later confirming or correlating with the
patients' response to the therapy.
[748] In some embodiments, the aberrant marker expression includes elevated
levels of at least one of the
following: TGFI31, LRRC33, GARP, LTBP1, LTBP3, CCL2, CCL3, PAI-1/Serpinet In
some embodiments, the
patient or patient population (e.g., biological samples collected therefrom)
shows elevated TGFp1 activation,
phospho-Smad2, phospho-Smad2/3, or combination thereof. In some embodiments,
the patient or patient
population (e.g., biological samples collected therefrom) shows elevated
MDSCs. In some embodiments, such
patient or patient population has cancer, which may comprise a solid tumor
that is TGF131-positive. The solid tumor
may be a TGF131-dominant tumor, in which TGF(31 is the predominant isoform
expressed in the tumor, relative to
the other isoforms. In some embodiments, the solid tumor may be a TGFP1-co-
dominant tumor, in which TG931
is the co-dominant isoform expressed in the tumor, e.g., TGFp1+/ TGF133+. In
some embodiments, such patient
or patient population exhibits resistance to a cancer therapy, such as
chemotherapy, radiation therapy (such as a
radiotherapeutic agent) and/or immune checkpoint therapy, e.g., anti-PD-1
(e.g., pembrolizumab, nivolumab,
spartalizumab), anti-PD-L1 (e.g., atezolizumab), anti-CTLA4 (e.g.,
ipilimumab), engineered immune cell therapy
(e.g., CAR-T), and cancer vaccines, etc. According to the disclosure, TGFI31
inhibitors provided herein, such as
Ab6, overcome the resistance by unblocking immunosuppression so as to allow
effector cells to gain access to
cancer cells thereby achieving anti-tumor effects. TG931 inhibitor therapy may
therefore promote effector cell
infiltration and/or expansion in the tumor. Additionally, TGFP1 inhibitor
therapy may reduce the frequency of
immunosuppressive immune cells, such as Tregs and MDSCs, in the tumor.
[749] In some embodiments, the aberrant marker expression includes one or more
panels of genes: mesenchymal
transition markers (e.g., AXL, ROR2, VVNT5A, LOXL2, TWIST2, TAGLN, FAP);
immunosuppressive genes (e.g.,
IL10, VEGFA, VEGFC); monocyte and macrophage chemotactic genes (e.g., CCL2,
CCL7, CCL8, CCL13); genes
involved in angiogenesis and wound healing (e.g., T cell suppressive); cell
adhesion markers; ECM remodeling;
skeletal system and bone development markers; and genes involved in tissue
injury-triggered inflammatory
response.
[750] In some embodiments, lack or downregulation of MHC expression (such as
MHC class 1) may serve as a
biomarker for TGF31-associated conditions for which the antibodies or antigen-
binding fragments encompassed
by the present disclosure may be used as therapy. Reduced MHC levels may
signal immune escape, which may
correlate with poor responsiveness of the patients to immune therapies, such
as CBT. Selective inhibition of TG931
therefore may at least in part restore effector cell function.
[751] The present disclosure provides a TGFp inhibitor (e.g., TGFp1-selective
inhibitor such as Ab6) for use in
the treatment of a TGFp-related disorder with aberrant gene expression (e.g.,
as described herein) in a patient,
wherein the treatment comprises administration of a composition comprising the
TGFp inhibitor (e.g., TGF(31
inhibitor) which has been selected, at least in part, on the basis of its
immune safety profile. A suitable immune
safety profile of the TGFp inhibitor is characterized in that i) it does not
trigger unacceptable levels of cytokine
release (e.g., within 2.5-fold of control); ii) it does not promote
unacceptable levels of platelet aggregation; or both
in field-accepted cell-based assay(s) and/or in in vivo assay(s) (such as
those described herein).
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Diseases involving mesenchymal transition
[752] Mesenchymal transition is a process of phenotypic shift of cells, such
as epithelial cells and endothelial
cells, towards a mesenchymal phenotype (such as myofibroblasts). Examples of
genetic markers indicative of
mesenchymal transition include AXL, ROR2, WNT5, LOXL2, TWIST2, TAGLN and FAP.
In cancer, for example,
mesenchymal transition (e.g., increased EndMT and EMT signatures) indicates
tumor cell phenotypic plasticity.
Thus, inhibitors of TGFp, e.g., TGFpl inhibitors, such as Ab6, may be used to
treat a disease that is initiated or
driven by mesenchymal transition, such as EMT and EndMT.
[753] EMT (epithelial-to-mesenchymal transition) is the process by which
epithelial cells with tight junctions switch
to mesenchymal properties (phenotypes) such as loose cell-cell contacts. The
process is observed in a number of
normal biological processes as well as pathological situations, including
embryogenesis, wound healing, cancer
metastasis and fibrosis (reviewed in, for example, Shiga et al., (2015)
"Cancer-Associated Fibroblasts: Their
Characteristics and Their Roles in Tumor Growth." Cancers, 7: 2443-2458).
Generally, it is believed that EMT
signals are induced mainly by TGFP. Many types of cancer, for example, appear
to involve transdifferentiation of
cells towards mesenchymal phenotype (such as myofibroblasts and CAFs) which
correlate with poorer prognosis.
Thus, isoform-specific, context-independent inhibitors of TGF(31 , such as
those described herein, may be used to
treat a disease that is initiated or driven by EMT. Indeed, data exemplified
in PCT/US2019/041373 (e.g., FIGs. 4-
6) show that such inhibitors have the ability to suppress expression of
myofibroblastJCAF markers in vivo, such as
a-SMA, LOXL2, Coll (Type I collagen), and FN (fibronectin). Thus, TGFp
inhibitors, e.g., TGFpl inhibitors, such
as Ab6, may be used for the treatment of a disease characterized by EMT. A
therapeutically effective amount of
the inhibitor may be an amount sufficient to reduce expression of markers such
as a-SMA/ACTA2, LOXL2Col1
(Type I collagen), and FN (fibronectin). In some embodiments, the disease is a
proliferative disorder, such as
cancer.
[754] Similarly, TGFI3 is also a key regulator of the endothelial-to-
mesenchymal transition (EndMT) observed in
normal development, such as heart formation. However, the same or similar
phenomenon is also seen in many
disease-associated tissues, such as cancer stroma and fibrotic sites. In some
disease processes, endothelial
markers such as CD31 become downregulated upon TGFpl exposure and instead the
expression of mesenchymal
markers such as FSP-1, a-SMA/ACTA2 and fibronectin becomes induced. Indeed,
stromal CAFs may be derived
from vascular endothelial cells. Thus, TGFp inhibitors, e.g.,TGFpl inhibitors,
such as Ab6, may be used for the
treatment of a disease characterized by EndMT. A therapeutically effective
amount of the inhibitor may be an
amount sufficient to reduce expression of markers such as FSP-1, a-SMA/ACTA2
and fibronectin. In some
embodiments, the disease is a proliferative disorder, such as cancer.
[755] The present disclosure provides a TG93 inhibitor (e.g., TGFpl -selective
inhibitor such as Ab6) for use in
the treatment of a TGFp-related disorder involving mesenchymal transition
(e.g., as described herein) in a patient,
wherein the treatment comprises administration of a composition comprising the
TGFp inhibitor (e.g., TGFpl
inhibitor) which has been selected, at least in part, on the basis of its
immune safety profile. A suitable immune
safety profile of the TGFp inhibitor is characterized in that i) it does not
trigger unacceptable levels of cytokine
release (e.g., within 2.5-fold of control); ii) it does not promote
unacceptable levels of platelet aggregation; or both
in field-accepted cell-based assay(s) and/or in in vivo assay(s) (such as
those described herein).
Diseases involving matrix stiffening and remodeling
[756] Progression of various TGFpl -related indications, such as fibrotic
conditions and cancer (e.g., tumor growth
and metastasis), involves increased levels of matrix components deposited into
the ECM and/or
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maintenance/remodeling of the ECM. It has been reported that increased
deposition of ECM components such as
collagens can alter the mechanophysical properties of the ECM (e.g., the
stiffness of the matrix/substrate) and this
phenomenon is associated with TGF[31 signaling. Applicant previously
demonstrated the role of matrix stiffness
on integrin-dependent activation of TGF13, using primary fibroblasts
transfected with proTGF[31 and LTBP1 and
grown on silicon-based substrates with defined stiffness (e.g., 5 kPa, 15 kPa
01 100 kPa). As disclosed in WO
2018/129329, matrices with greater stiffness enhance TGF[31 activation, and
this can be suppressed by isoform-
specific inhibitors of TGF[31. These observations suggest that TGF[31
influences ECM properties (such as
stiffness), which in turn can further induce TG931 activation, reflective of
disease progression.
[757] Thus, TGF[31 inhibitors, such as Ab6, may be used to block this process
to counter disease progression
involving ECM alterations, such as fibrosis, tumor growth, invasion,
metastasis and desmoplasia. The LTBP-arm
of such inhibitors can directly target ECM-associated pro/latent TGF131
complexes which are presented by LTBP1
and/or LTBP3, thereby preventing activation/release of the growth factor from
the complex in the disease niche. In
some embodiments, the TGF[31 inhibitors may normalize ECM stiffness to treat a
disease that involves integrin-
dependent signaling. In some embodiments, the integrin comprises an oil chain,
[31 chain, or both. The
architecture of the ECM, e.g., ECM components and organization, can also be
altered by matrix-associated
proteases. Thus, in some embodiments, the TGF[31 inhibitors may normalize ECM
stiffness to treat a disease that
involves protease-dependent signaling associated with disease-associated ECM,
e.g., in tumor and fibrotic tissues.
[758] As reviewed in Lampi and Reinhart-King (Science Translational Medicine,
10(422): eaao0475, "Targeting
extracellular matrix stiffness to attenuate disease: From molecular mechanisms
to clinical trials"), increased
stiffness of tissue ECMs occurs during pathological progression of cancer,
fibrosis and cardiovascular disease.
The mechanical properties associated with the process involve phenotypically
converted myofibroblasts, TGF[3 and
matrix cross-linking. A major cause of increased ECM stiffness during cancer
and fibrotic diseases is dysregulated
matrix synthesis and remodeling by activated fibroblasts that have de-
differentiated into myofibroblasts (e.g., CAFs
and FAFs). Remodeling of the tumor stroma and organ fibrosis exhibit striking
similarities to the wound healing
response, except that in the pathological state the response is sustained.
Myofibroblasts are a heterogeneous
cell population with pathology-specific precursor cells originating from
multiple cell sources, such as bone marrow-
derived and tissue resident cells. Commonly used myofibroblast markers include
alpha-smooth muscle actin (a-
SMA). As previously shown in PCT/US2019/041373, high-affinity, isoform-
specific TGF[31 inhibitors are able to
reduce ACTA2 expression (which encodes a-SMA), collagens, as well as FN
(fibronectin) in in vivo studies.
Fibronectin is important in the anchoring of LTBP-associated proTGF31
complexes onto the matrix structure.
[759] The importance of the TGF[3 pathway in ECM regulation is well-
established. Because TGF[31 (and TG933)
can be mechanically activated by certain integrins (e.g., av integrins), the
integrin-TGF31 interaction has become
a therapeutic target. For example, a monoclonal antibody to av[36 has been
investigated for idiopathic lung fibrosis.
However, such approach is expected to also interfere with TGF[33 signaling
which shares the same integrin-binding
motif, RGD, and furthermore, such antibody will not be effective in blocking
TGF31 activated via other modes, such
as protease-induced activation. In comparison, high-affinity, isoform-specific
TGF[31 inhibitors, such as Ab6, can
also block protease-dependent activation of 1GF[31, as well as integrin-
dependent activation of TGF[31. Therefore,
such TGF[31 inhibitors may provide superior attributes. Based on Applicant's
previous work, high-affinity isoform-
selective inhibitors of TGF31 may be effective in treating disease associated
with ECM stiffening.
[760] Thus, the disclosure includes therapeutic use of isoform-selective
inhibitors of TGF[31 in the treatment of a
disease associated with matrix stiffening, or in a method for reducing matrix
stiffness, in a subject. Such use
comprises administration of a therapeutically effective amount of the isoform-
selective inhibitor of TGF[31, such as
Ab6.
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[761] The present disclosure provides a TGF13 inhibitor (e.g., TGF01-selective
inhibitor such as Ab6) for use in
the treatment of a TGFI3-related disorder involving matrix stiffening and
remodeling (e.g., as described herein) in a
patient, wherein the treatment comprises administration of a composition
comprising the TGF3 inhibitor (e.g.,
TGF31 inhibitor) which has been selected, at least in part, on the basis of
its immune safety profile. A suitable
immune safety profile of the TGF3 inhibitor is characterized in that i) it
does not trigger unacceptable levels of
cytokine release (e.g., within 2.5-fold of control); ii) it does not promote
unacceptable levels of platelet aggregation;
or both in field-accepted cell-based assay(s) and/or in in vivo assay(s) (such
as those described herein).
Diseases involving proteases
[762] Activation of TGFp from its latent complex may be triggered mechanically
by integrin in a force-dependent
manner, and/or by proteases. Evidence suggests that certain classes of
proteases may be involved in the process,
including but are not limited to Ser/Thr proteases such as Kallikreins,
chemotrypsin, elastases, plasmin, thrombin,
as well as zinc metalloproteases of MMP family, such as MMP-2, MMP-9 and MMP-
13, and the Adam family of
proteases, such as Adam10 and Adam17. MMP-2 degrades the most abundant
component of the basement
membrane, Collagen IV, raising the possibility that it may play a role in ECM-
associated TGF31 regulation. MMP-
9 has been implicated to play a central role in tumor progression,
angiogenesis, stromal remodeling and metastasis,
including in carcinoma, such as breast cancer. Thus, protease-dependent
activation of TGFp1 in the ECM may be
important for treating ECM-associated diseases such as fibrosis and cancer.
[763] Kallikreins (KLKs) are trypsin- or chymotrypsin-like serine proteases
that include plasma Kallikreins and
tissue Kallikreins. The ECM plays a role in tissue homeostasis acting as a
structural and signaling scaffold and
barrier to suppress malignant outgrowth. KLKs may play a role in degrading ECM
proteins and other components
which may facilitate tumor expansion and invasion. For example, KLK1 is highly
upregulated in certain breast
cancers and can activate pro-MMP-2 and pro-MMP-9. KLK2 activates latent
TGFI31, rendering prostate cancer
adjacent to fibroblasts permissive to cancer growth. KLK3 has been widely
studied as a diagnostic marker for
prostate cancer (PSA). KLK3 may directly activate TGFp1 by processing
plasminogen into plasmin, which
proteolytically cleaves LAP, thereby causing the TGFI31 growth factor to be
released from the latent complex. KLK6
may be a potential marker for Alzheimer's disease.
[764] Moreover, data previously provided by Applicant indicated that such
proteases may be a Kallikrein. Thus,
the disclosure encompasses the use of an isoform-specific, context-independent
inhibitor of TGF31 in a method
for treating a disease associated with Kallikrein or a Kallikrein-like
protease. In some embodiments, the TGFpl
inhibitor is Ab6, or derivatives thereof.
[765] Known activators of TGF31, such as plasmin, TSP-1 and aV36 integrin, all
interact directly with LAP. It is
postulated that proteolytic cleavage of LAP may destabilize the LAP-TGFp
interaction, thereby releasing active
TGF31 (the growth factor domain) from the latent complex. It has been
suggested that the region containing the
amino acid stretch 54-LSKLRL-59 is important for maintaining TGFI31 latency.
Thus, agents (e.g., antibodies) that
stabilize the interaction, or block the proteolytic cleavage of LAP may
prevent TCF31 activation.
[766] Many of these proteases associated with pathological conditions (e.g.,
cancer) function through distinct
mechanisms of action. Thus, targeted inhibition of particular proteases, or
combinations of proteases, may provide
therapeutic benefits for the treatment of conditions involving the protease-
TGFp axis. Accordingly, it is
contemplated that inhibitors (e.g., TGFI31 antibodies) that selectively
inhibit protease-induced activation of TGFI31
may be advantageous in the treatment of such diseases (e.g., cancer).
Similarly, selective inhibition of TGFpl
activation by one protease over another protease may also provide therapeutic
benefit, depending on the condition
being treated.
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[767] Plasmin is a serine protease produced as a precursor form called
Plasminogen. Upon release, Plasmin
enters circulation and therefore is detected in serum. Elevated levels of
serum Plasmin appear to correlate with
cancer progression, possibly through mechanisms involving disruption of the
extracellular matrix (e.g., basement
membrane and stromal barriers) which facilitates tumor cell motility,
invasion, and metastasis. Plasmin may also
affect adhesion, proliferation, apoptosis, cancer nutrition, oxygen supply,
formation of blood vessels, and activation
of VEGF (Didiasova etal., Int. J. Mol. Sc!, 2014, 15,21229-21252). In
addition, Plasmin may promote the migration
of macrophages into the tumor microenvironment (Philips et al., Cancer Res.
2011 Nov 1;71(21):6676-83 and
Choong et al., Clin. Orthop. Re/at. Res. 2003, 415S, 546-S58). Indeed, tumor-
associated macrophages (TAMs)
are well characterized drivers of tumorigenesis through their ability to
promote tumor growth, invasion, metastasis,
and angiogenesis.
[768] Plasmin activities have been primarily tied to the disruption of the
ECM. However, there is mounting
evidence that Plasmin also regulates downstream MMP and TGFI3 activation.
Specifically, Plasmin has been
suggested to cause activation of TGF3 through proteolytic cleavage of the
Latency Associated Peptide (LAP),
which is derived from the N-terminal region of the TGF3 gene product
(Horiguchi et al., J Biochern. 2012 Oct;
152(4):321-9), resulting in the release of active growth factor. Since TGF31
may promote cancer progression, this
raises the possibility that plasmin-induced activation of TGF3 may at least in
part mediate this process.
[769] TGF31 has also been shown to regulate expression of uPA, which is a
critical player in the conversion of
Plasminogen into Plasmin (Santibanez, Juan F., /SRN Dermatology, 2013:
597927). uPA has independently been
shown to promote cancer progression (e.g., adhesion, proliferation, and
migration) by binding to its cell surface
receptor (uPAR) and promoting conversion of Plasminogen into Plasmin.
Moreover, studies have shown that
expression of uPA and/or plasminogen activator inhibitor-1 (PAI-1) are
predictors of poor prognosis in colorectal
cancer (D. Q. Seetoo, etal., Journal of Surgical Oncology, vol. 82, no. 3, pp.
184-193, 2003), breast cancer (N.
Harbeck et al., Clinical Breast Cancer, vol. 5, no. 5, pp. 348-352, 2004), and
skin cancer (Santibanez, Juan F.,
ISRN Dermatology, 2013: 597927). Thus, without wishing to be bound by a
particular theory, the interplay between
Plasmin, TGF31, and uPA may create a positive feedback loop towards promoting
cancer progression.
Accordingly, inhibitors that selectively inhibit Plasmin-dependent 1GF31
activation may be particularly suitable for
the treatment of cancers reliant on the Plasmin/TGF31 signaling axis.
[770] In one aspect of the disclosure, TGF3 inhibitors such as the isoform-
specific inhibitors of TGF31 described
herein can inhibit protease-dependent activation of TGF31. In some
embodiments, the inhibitors can inhibit
protease-dependent TG931 activation in an integrin-independent manner. In some
embodiments, such inhibitors
can inhibit TGF31 activation irrespective of the mode of activation, e.g.,
inhibit both integrin-dependent activation
and protease-dependent activation of TGF31. In some embodiments, the protease
is selected from the group
consisting of: serine proteases, such as Kallikreins, Chemotrypsin, Trypsin,
Elastases, Plasmin, as well as zinc
metalloproteases (MMP family) such as MMP-2, MMP-9 and MMP-13.
[771] In some embodiments, the TGF3 inhibitors (e.g., TGF31 antibody) can
inhibit Plasmin-induced activation of
TGF31.
In some embodiments, the inhibitors can inhibit Plasmin- and integrin-
induced TGF31 activation. In
some embodiments, the antibody is a monoclonal antibody that specifically
binds proTGF31. In some
embodiments, the antibody binds latent proTGF31 thereby inhibiting release of
mature growth factor from the latent
complex. In some embodiments, the high-affinity, context-independent inhibitor
of TGF31 activation suitable for
use in the method of inhibiting Plasmin-dependent activation of TGF31 is Ab6
or a derivative or variant thereof.
[772] In some embodiments, the TGF3 inhibitor (e.g., TGF31 antibody) inhibits
cancer cell migration. In some
embodiments, the inhibitor inhibits macrophage migration.
In some embodiments, the inhibitor inhibits
accumulation of TAMs.
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[773] In another aspect, provided herein is a method for treating cancer in a
subject in need thereof, the method
comprising administering to the subject an effective amount of an TGFp
inhibitor (e.g., TGFp1 antibody), wherein
the inhibitor inhibits protease-induced activation of TGFp1 (e.g., Plasmin),
thereby treating cancer in the subject.
[774] In another aspect, provided herein is a method of reducing tumor growth
in a subject in need thereof, the
method comprising administering to the subject an effective amount of an TGFp
inhibitor (e.g., TGFp1 antibody),
wherein the inhibitor inhibits protease-induced activation of TGFP1 (e.g.,
Plasmin), thereby reducing tumor growth
in the subject.
[775] The present disclosure provides a TGF13 inhibitor (e.g., TGFI31-
selective inhibitor such as Ab6) for use in
the treatment of a TGFp-related disorder involving protease(s) (e.g., as
described herein) in a patient, wherein the
treatment comprises administration of a composition comprising the TGFp
inhibitor (e.g., TGFp1 inhibitor) which
has been selected, at least in part, on the basis of its immune safety
profile. A suitable immune safety profile of
the TGFp inhibitor is characterized in that i) it does not trigger
unacceptable levels of cytokine release (e.g., within
2.5-fold of control); ii) it does not promote unacceptable levels of platelet
aggregation; or both in field-accepted cell-
based assay(s) and/or in in vivo assay(s) (such as those described herein).
Myeloproliferative disorders / myelofibrosis
[776] The present disclosure provides therapeutic use of TGFpl inhibitors,
such as Ab6, in the treatment of
myeloproliferative disorders. These include, for example, myelodysplastic
syndrome (MDS) and myelofibrosis
(e.g., primary myelofibrosis and secondary myelofibrosis).
[777] Myelofibrosis, also known as osteomyelofibrosis, is a relatively rare
bone marrow proliferative disorder
(cancer), which belongs to a group of diseases called myeloproliferative
disorders. Myelofibrosis is classified into
the Philadelphia chromosome-negative (-) branch of myeloproliferative
neoplasms. Myelofibrosis is characterized
by clonal myeloproliferation, aberrant cytokine production, extramedullary
hematopoiesis, and bone marrow
fibrosis. The proliferation of an abnormal clone of hematopoietic stem cells
in the bone marrow and other sites
results in fibrosis, or the replacement of the marrow with scar tissue. The
term myelofibrosis, unless otherwise
specified, refers to primary myelofibrosis (PMF). This may also be referred to
as chronic idiopathic myelofibrosis
(cIMF) (the terms idiopathic and primary mean that in these cases the disease
is of unknown or spontaneous
origin). This is in contrast with myelofibrosis that develops secondary to
polycythemia vera or essential
thrombocythaemia. Myelofibrosis is a form of myeloid metaplasia, which refers
to a change in cell type in the blood-
forming tissue of the bone marrow, and often the two terms are used
synonymously. The terms agnogenic myeloid
metaplasia and myelofibrosis with myeloid metaplasia (MMM) are also used to
refer to primary myelofibrosis. In
some embodiments, the hematologic proliferative disorders which may be treated
in accordance with the present
disclosure include myeloproliferative disorders, such as myelofibrosis. So-
called "classical" group of BCR-ABL
(Ph) negative chronic myeloproliferative disorders includes essential
thrombocythemia (ET), polycythemia vera
(PV) and primary myelofibrosis (PMF).
[778] Myelofibrosis disrupts the body's normal production of blood cells. The
result is extensive scarring in the
bone marrow, leading to severe anemia, weakness, fatigue and often an enlarged
spleen. Production of cytokines
such as fibroblast growth factor by the abnormal hematopoietic cell clone
(particularly by megakaryocytes) leads
to replacement of the hematopoietic tissue of the bone marrow by connective
tissue via collagen fibrosis. The
decrease in hematopoietic tissue impairs the patient's ability to generate new
blood cells, resulting in progressive
pancytopenia, a shortage of all blood cell types. However, the proliferation
of fibroblasts and deposition of collagen
is thought to be a secondary phenomenon, and the fibroblasts themselves may
not be part of the abnormal cell
clone.
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[779] Myelofibrosis may be caused by abnormal blood stem cells in the bone
marrow. The abnormal stem cells
produce mature and poorly differentiated cells that grow quickly and take over
the bone marrow, causing both
fibrosis (scar tissue formation) and chronic inflammation.
[780] Primary myelofibrosis is associated with mutations in Janus kinase 2
(JAK2), thrombopoietin receptor (MPL)
and calreticulin (CALR), which can lead to constitutive activation of the JAK-
STAT pathway, progressive scarring,
or fibrosis, of the bone marrow occurs. Patients may develop extramedullary
hematopoiesis, i.e., blood cell
formation occurring in sites other than the bone marrow, as the haemopoetic
cells are forced to migrate to other
areas, particularly the liver and spleen. This causes an enlargement of these
organs. In the liver, the abnormal size
is called hepatomegaly. Enlargement of the spleen is called splenomegaly,
which also contributes to causing
pancytopenia, particularly thrombocytopenia and anemia. Another complication
of extramedullary hematopoiesis
is poikilocytosis, or the presence of abnormally shaped red blood cells.
[781] The principal site of extramedullary hematopoiesis in myelofibrosis is
the spleen, which is usually markedly
enlarged in patients suffering from myelofibrosis. As a result of massive
enlargement of the spleen, multiple
subcapsular infarcts often occur in the spleen, meaning that due to
interrupted oxygen supply to the spleen partial
or complete tissue death happens. On the cellular level, the spleen contains
red blood cell precursors, granulocyte
precursors and megakaryocytes, with the megakaryocytes prominent in their
number and in their abnormal shapes.
Megakaryocytes may be involved in causing the secondary fibrosis seen in this
condition.
[782] It has been suggested that TGF6 may be involved in the fibrotic aspect
of the pathogenesis of myelofibrosis
(see, for example, Agarwal et al., "Bone marrow fibrosis in primary
myelofibrosis: pathogenic mechanisms and the
role of TGF6" (2016) Stem Cell lnvestig 3:5). Bone marrow pathology in primary
myelofibrosis is characterized by
fibrosis, neoangeogenesis and osteosclerosis, and the fibrosis is associated
with an increase in production of
collagens deposited in the ECM.
[783] A number of biomarkers have been described, alternations of which are
indicative of or correlate with the
disease. In some embodiments, the biomarkers are cellular markers. Such
disease-associated biomarkers are
useful for the diagnosis and/or monitoring of the disease progression as well
as effectiveness of therapy (e.g.,
patients' responsiveness to the therapy). These biomarkers include a number of
fibrotic markers, as well as cellular
markers. In lung cancer, for example, TGF61 concentrations in the
bronchoalveolar lavages (BAL) fluid are
reported to be significantly higher in patients with lung cancer compared with
patients with benign diseases (-2+
fold increase), which may also serve as a biomarker for diagnosing and/or
monitoring the progression or treatment
effects of lung cancer.
[784] Because myelofibrosis is associated with abnormal megakaryocyte
development, certain cellular markers
of megakaryocytes as well as their progenitors of the stem cell lineage may
serve as markers to diagnose and/or
monitor the disease progression as well as effectiveness of therapy. In some
embodiments, useful markers include,
but are not limited to: cellular markers of differentiated megakaryocytes
(e.g., CD41, CD42 and Tpo R), cellular
markers of megakaryocyte-erythroid progenitor cells (e.g., CD34, CD38, and
CD45RA-), cellular markers of
common myeloid progenitor cells (e.g., IL-3a/CD127, C034, SCF R/c-kit and Flt-
3/Flk-2), and cellular markers of
hematopoietic stem cells (e.g., CD34, CD38-, Flt-3/Flk-2). In some
embodiments, useful biomarkers include fibrotic
markers. These include, without limitation: TGF61/TGFB1, PAI-1 (also known as
Serpinel), MCP-1 (also known
as CCL2), Coll al, Col3a1, FN1, CTGF, a-SMA, ACTA2, Timpl , Mmp8, and Mmp9. In
some embodiments, useful
biomarkers are serum markers (e.g., proteins or fragments found and detected
in serum samples).
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[785] Based on the finding that TGFp is a component of the leukemic bone
marrow niche, it is contemplated that
targeting the bone marrow microenvironment with TGFI3 inhibitors may be a
promising approach to reduce
leukemic cells expressing presenting molecules that regulate local TGFp
availability in the effected tissue.
[786] Indeed, due to the multifaceted nature of the pathology which manifests
TGFp-dependent dysregulation in
both myelo-proliferative and fibrotic aspects (as the term "myelofibrosis"
itself suggests), isoform-specific, TGFp
inhibitors such as those described herein may provide particularly
advantageous therapeutic effects for patients
suffering from myelofibrosis. It is contemplated that the LTBP-arm of such
inhibitor can target ECM-associated
TGF131 complex in the bone marrow, whilst the LRRC33-arm of the inhibitor can
block myeloid cell-associated
TGF131. In addition, abnormal megakaryocyte biology associated with
myelofibrosis may involve both GARP- and
LTBP-mediated TGFp1 activities. Thus, TGFp inhibitors such as the isoform-
specific, context-independent inhibitor
of TGF(31 disclosed herein, may be capable of targeting such complexes and
thereby inhibiting release of active
TGF131 in the niche.
[787] TGFp inhibitors such as the TGFp1-selective inhibitors described herein
are useful for treatment of patients
with primary and secondary myelofibrosis, who have had an inadequate response
to or are intolerant of other (or
standard-of-care) treatments, such as hydroxyurea and JAK inhibitors. Such
inhibitors are also useful for treatment
of patients with intermediate or high-risk myelofibrosis (MF), including
primary MF, post-polycythemia vera MF and
post-essential thrombocythemia MF. In some embodiments, such TGFp inhibitors
may be used in combination with
a checkpoint inhibitor therapy.
[788] Accordingly, one aspect of the disclosure relates to methods for
treating primary myelofibrosis. The method
comprises administering to a patient suffering from primary myelofibrosis a
therapeutically effective amount of a
composition comprising a TGFp inhibitor that causes reduced TGFp availability.
In some embodiments, an isoform-
specific, context- context-independent monoclonal antibody inhibitor of TGFp1
activation is administered to patients
with myelofibrosis. Such antibody may be administered at dosages ranging
between 0.1 and 100 mg/kg, such as
between 1 and 30 mg, e.g., 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20
mg/kg, 30 mg/kg, etc. For example,
suitable dosing regimens include between 1-30 mg/kg administered weekly. In
some embodiments, the TGFI31
inhibitor is dosed at about 10 mg/kg per week. Optionally, the frequency of
administration may be adjusted after
the initial phase, for example, from about once a week (during an initial
phase) to once a month (during a
maintenance phase). In some embodiments, the TGFI3 inhibitor (e.g., a TGF31
inhibitor) may be administered in
combination with a checkpoint inhibitor therapy.
[789] Exemplary routes of administration of a pharmaceutical composition
comprising the antibody is intravenous
or subcutaneous administration. When the composition is administered
intravenously, the patient may be given
the therapeutic over a suitable duration of time, e.g., approximately 30-120
minutes (e.g., 30 min, 60 min, 75 min,
90 min, and 120 min), per treatment, and then repeated every several weeks,
e.g., 3 weeks, 4 weeks, 6 weeks,
etc., for a total of several cycles, e.g., 4 cycles, 6, cycles, 8 cycles, 10
cycles, 12 cycles, etc. In some embodiments,
patients are treated with a composition comprising the inhibitory antibody at
dose level of 1-10 mg/kg (e.g., 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 mg/kg per dosing) via intravenous administration every
28 days (4 weeks) for 6 cycles or 12
cycles. In some embodiments, such treatment is administered as a chronic (long-
term) therapy (e.g., to be
continued indefinitely, as long as deemed beneficial) in lieu of discontinuing
following a set number of cycles of
administration.
[790] While myelofibrosis is considered a type of leukemia, it is also
characterized by the manifestation of fibrosis.
Because TGFp is known to regulate aspects of ECM homeostasis, the
dysregulation of which can lead to tissue
fibrosis, it is desirable to inhibit TGFp activities associated with the ECM.
Accordingly, antibodies or fragments
thereof that bind and inhibit proTGFp presented by LTBPs (such as LTBP1 and
LTBP3) are encompassed by this
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disclosure. In some embodiments, antibodies or fragments thereof suitable for
treating myelofibrosis are "context-
independent" in that they can bind multiple contexts of proTGFp complex, such
as those associated with LRRC33,
GARP, LTBP1, LTBP3, or any combination thereof. In some embodiments, such
antibody is a context-independent
inhibitor of TGFp activation, characterized in that the antibody can bind and
inhibit any of the following latent
complexes: LTBP1-proTGFP, LTBP3-proTGFP, GARP-proTGF3 and LRRC33-proTG93. In
some embodiments,
such an antibody is an isoform-specific antibody that binds and inhibits such
latent complexes that comprise one
but not the other isoforms of TGF3. These include, for example, LTBP1-
proTGFp1, LTBP3-proTGFp1, GARP-
proTGFp1 and LRRC33-proTGFp1 . In some embodiments, such antibody is an
isoform-selective antibody that
preferentially binds with high affinity and inhibits TGF31 signaling.
[791] Early in vivo data indicate that TGF3 inhibitors such as an isoform-
selective context-independent inhibitor
of TGFpl described herein, can be used to treat myelofibrosis in a
translatable murine model of primary
myelofibrosis. Unlike the current standard of care JAK2 inhibitor, which only
provides symptomic relief but does
riot provide clinical or survival benefits, the TGFp inhibitor (e.g., an
isoform-selective context-independent inhibitor
of TGFpl described herein) achieves significant anti-fibrotic effects in the
bone marrow of the diseased mice and
may also prolong survival, supporting the notion that the TGF(31 inhibitor may
be effective to treat myeloproliferative
disorders in human patients.
[792] Suitable patient populations of myeloproliferative neoplasms who may be
treated with the compositions and
methods described herein may include, but are not limited to: a) a patient
population that is Philadelphia (+); b) a
patient population that is Philadelphia (-); c) a patient population that is
categorized "classical" (PV, ET and PMF);
d) a patient population carrying the mutation JAK2V617F(+); e) a patient
population carrying JAK2V617F(-); f) a
patient population with JAK2 exon 12(+); g) a patient population with MPL(+);
arid h) a patient population with
CALR(+).
[793] In some embodiments, the patient population includes patients with
intermediate-2 or high-risk
myelofibrosis. In some embodiments, the patient population comprises subjects
with myelofibrosis who are
refractory to or not candidates for available therapy. In some embodiments,
the subject has platelet counts between
100-200 x 109/L. In some embodiments, the subject has platelet counts > 200 x
109/L prior to receiving the
treatment.
[794] In some embodiments, a subject to receive (and who may benefit from
receiving) an isoform-specific,
context-independent TGF31 inhibitor therapy is diagnosed with intermediate-1
or higher primary myelofibrosis
(PMF), or post-polycythemmia vera/essential thrombocythemia myelofibrosis
(post-PV/ET MF). In some
embodiments, the subject has documented bone marrow fibrosis prior to the
treatment. In some embodiments,
the subject has MF-2 or higher as assessed by the European consensus grading
score and grade 3 or higher by
modified Bauermeister scale prior to the treatment. In some embodiments, the
subject has the ECOG performance
status of 1 prior to the treatment. In some embodiments, the subject has white
blood cell count (109/L) ranging
between 5 and 120 prior to the treatment. In some embodiments, the subject has
the JAK2V617F allele burden
that ranges between 10-100%.
[795] In some embodiments, a subject to receive (and who may benefit from
receiving) an isoform-specific,
context-independent TGF31 inhibitor therapy is transfusion-dependent (prior to
the treatment) characterized in that
the subject has a history of at least two units of red blood cell transfusions
in the last month for a hemoglobin level
of less than 8.5 g/dL that is not associated with clinically overt bleeding.
[796] In some embodiments, a subject to receive (and who may benefit from
receiving) an isoform-specific,
context-independent TGF31 inhibitor therapy previously received a therapy to
treat myelofibrosis. In some
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embodiments, the subject has been treated with one or more of therapies,
including but are not limited to: AZD1480,
panobinostat, EPO, IFNa, hydroxyurea, pegylated interferon, thalidomide,
prednisone, and JAK2 inhibitor (e.g.,
Lestaurtinib, CEP-701).
[797] In some embodiments, the patient has extramedullary hematopoiesis. In
some embodiments, the
extramedullary hematopoiesis is in the liver, lung, spleen, and/or lymph
nodes. In some embodiments, the
pharmaceutical composition of the present disclosure is administered locally
to one or more of the localized sites
of disease manifestation.
[798] In some embodiments, a TGF6 inhibitor such as an isoform-specific,
context-independent TGF61 inhibitor
described herein is administered alone or in combination with a checkpoint
inhibitortherapy to patients in an amount
effective to treat myelofibrosis. The therapeutically effective amount is an
amount sufficient to relieve one or more
symptoms and/or complications of myelofibrosis in patients, including but are
not limited to: excessive deposition
of ECM in bone marrow stroma (fibrosis of the bone marrow), neoangiogenesis,
osteosclerosis, splenomegaly,
hematomegaly, anemia, bleeding, bone pain and other bone-related morbidity,
extramedullary hematopoiesis,
thrombocytosis, leukopenia, cachexia, infections, thrombosis and death. Thus,
TGF13 inhibition therapies
comprising the antibodies or antigen-binding fragments of the disclosure may
achieve clinical benefits, which
include, inter alia, anti-fibrotic effects and/or normalization of blood cell
counts. Such therapy may prolong survival
and/or reduce the need for bone marrow transplantation.
[799] In some embodiments, the amount of TGF6 inhibitor is effective to reduce
TGF61 expression and/or
secretion (such as of megakaryocytic cells) in patients. Such inhibitor may
therefore reduce TGF61 mRNA levels
in treated patients. In some embodiments, such inhibitor reduces TGF61 mRNA
levels in bone marrow, such as
in mononuclear cells. PMF patients typically show elevated plasma TGF61 levels
of above ¨2,500 pg/mL, e.g.,
above 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000,9,000, and 10,000
pg/mL (contrast to normal ranges
of ¨600-2,000 pg/mL as measured by ELISA) (see, for example, Mascaremhas et
al., (Leukemia & Lymphoma,
2014, 55(2): 450-452)). Zingariello (Blood, 2013, 121(17): 3345-3363)
quantified bioactive and total TGF61
contents in the plasma of PMF patients and control individuals. According to
this reference, the median bioactive
TGF61 in PMF patients was 43 ng/mL (ranging between 4-218 ng/mL) and total
TGF61 was 153 ng/mL (32-1000
ng/mL), while in control counterparts, the values were 18(0.05-144) and 52 (8-
860), respectively. Thus, based on
these reports, plasma TGF61 contents in PMF patients are elevated by several
fold, e.g., 2-fold, 3-fold, 4-fold, 5-
fold, etc., as compared to control or healthy plasma samples. Treatment with
the inhibitor, e.g., following 4-12
cycles of administration (e.g., 2, 4, 6, 8, 10, 12 cycles) or chronic or long-
term treatment, for example every 4
weeks, at dosage of 0.1-100 mg/kg, for example, 1-30 mg/kg monoclonal
antibody) described herein may reduce
the plasma TGF61 levels by at least 10% relative to the corresponding baseline
(pre-treatment), e.g., at least 15%,
20%, 25%, 30%, 35%, 40%, 45%, and 50%.
[800] Some of the therapeutic effects may be observed relatively rapidly
following the commencement of the
treatment, for example, after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6
weeks. For example, the inhibitor
may effectively increase the number of stem cells and/or precursor cells
within the bone marrow of patients treated
with the inhibitor within 1-8 weeks. These include hematopoietic stem cells
and blood precursor cells. A bone
marrow biopsy may be performed to assess changes in the frequencies/number of
marrow cells. Correspondingly,
the patient may show improved symptoms such as bone pain and fatigue.
[801] Subjects suffering from a myeloproliferative disorder (e.g.,
myelofibrosis) may manifest an elevated level of
white blood cell counts (e.g., leukemic). In some embodiments, the
therapeutically effective amount of the TGFp
inhibitor (e.g., TGF61 inhibitor) is an amount that is effective to normalize
blood cell counts. In some embodiments,
the amount is effective to reduce total white cell counts in the subject, as
compared to pre-treatment. In some
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embodiments, the amount is effective to reduce total platelet counts in the
subject, as compared to pre-treatment.
In some embodiments, the amount is effective to increase (e.g., normalize or
restore) hemoglobin levels in the
subject, as compared to pre-treatment. In some embodiments, the amount is
effective to increase (e.g., normalize
or restore) hematocrit levels in the subject, as compared to pre-treatment.
[802] One of the morphological hallmarks of myelofibrosis is fibrosis in the
bone marrow (e.g., marrow stroma),
characterized in part by aberrant ECM. In some embodiments, the amount of TGF8
inhibitor (e.g., TGF131 inhibitor)
is effective to reduce fibrosis, characterized by excessive collagen
deposition, e.g., by mesenchymal stromal cells.
In some embodiments, the TGF[3 inhibitor is effective to reduce the number of
CD41-positive cells, e.g.,
megakaryocytes, in treated subjects, as compared to control subjects that do
not receive the treatment. In some
embodiments, baseline frequencies of megakaryocytes in PMF bone marrow may
range between 200-700 cells
per square millimeters (mm2), and between 40-300 megakaryocites per square-
millimeters (mm2) in PMF spleen,
as determined with randomly chosen sections. In contrast, megakaryocyte
frequencies in bone marrow and spleen
of normal donors are fewer than 140 and fewer than 10, respectively. Treatment
with the TGF[3 inhibitor (e.g.,
TGF131 inhibitor) may reduce the number (e.g., frequencies) of megakaryocytes
in bone marrow and/or spleen. In
some embodiments, treatments with the inhibitor may reduce or inhibit
autocrine TGFI31 signaling in
megakaryocytes. In some embodiments, treatments with the inhibitor may cause
reduced levels of downstream
effector signaling, such as phosphorylation of SMAD2/3, e.g., phosphorylation
of SMAD2. In some embodiments,
the TGFI3 inhibitor (e.g., TGFI31 inhibitor) is effective to reduce expression
levels of fibrotic markers, such as those
described herein. Patients with myelofibrosis may suffer from enlarged spleen.
Thus, clinical effects of a
therapeutic may be evaluated by monitoring changes in spleen size. Spleen size
may be examined by known
techniques, such as assessment of the spleen length by palpation and/or
assessment of the spleen volume by
ultrasound. In some embodiments, the subject to be treated with an isoform-
specific, context-independent inhibitor
of TGFI31 has a baseline spleen length (prior to the treatment) of 5 cm or
greater, e.g., ranging between 5 and 30
cm as assessed by palpation. In some embodiments, the subject to be treated
with an isoform-specific, context-
independent inhibitor of TGF[31 has a baseline spleen volume (prior to the
treatment) of 300 mL or greater, e.g.,
ranging between 300-1500 mL, as assessed by ultrasound. Treatment with the
inhibitor, e.g., following 4-12 cycles
of administration (e.g., 2, 4, 6, 8, 10,12 cycles), for example every 4 weeks,
at dosage of 0.1-30 mg/kg monoclonal
antibody) described herein may reduce spleen size in the subject. In some
embodiments, the effective amount of
the inhibitor is sufficient to reduce spleen size in a patient population that
receives the inhibitor treatment by at least
10%, 20%, 30%, 35%, 40%, 50%, and 60%, relative to corresponding baseline
values. For example, the treatment
is effective to achieve a n5% reduction in spleen volume from baseline in 12-
24 weeks as measured by MRI or
CT scan, as compared to placebo control. In some embodiments, the treatment is
effective to achieve a a35%
reduction in spleen volume from baseline in 24-48 weeks as measured by MRI or
CT scan, as compare to best
available therapy control. Best available therapy may include hydroxyurea,
glucocorticoids, as well as no
medication, anagrelide, epoetin alfa, thalidomide, lenalidomide,
mercaptopurine, thioguanine, danazol,
peginterferon alfa-2a, interferon-a, melphalan, acetylsalicylic acid,
cytarabine, and colchicine.
[803] In some embodiments, a patient population treated with a TGFP inhibitor
such as an isoform-specific,
context-independent TGFI31 inhibitor described herein shows a statistically
improved treatment response as
assessed by, for example, International Working Group for Myelofibrosis
Research and Treatment (IWG-MRT)
criteria, degree of change in bone marrow fibrosis grade measured by the
modified Bauermeister scale and
European consensus grading system after treatment (e.g., 4, 6, 8, or 12
cycles), symptom response using the
Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF).
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[804] In some embodiments, the treatment with an isoform-specific, context-
independent TGF31 inhibitor such
as those described herein, achieves a statistically improved treatment
response as assessed by, for example,
modified Myelofibrosis Symptom Assessment Form (MFSAF), in which symptoms are
measured by the MFSAF
tool (such as v2.0), a daukt diary capturing the debilitating symptoms of
myelofibros is (abdominal discomfort, early
satiety, pain under left ribs, pruritus, night sweats, and bone/muscle pain)
using a scale of 0 to 10, where 0 is
absent and 10 is the worst imaginable. In some embodiments, the treatment is
effective to achieve a 50 /0
reduction in total MFSAF score from the baseline in, for example, 12-24 weeks.
In some embodiments, a significant
fraction of patients who receive the therapy achieves a 50% improvement in
Total Symptom Score, as compared
to patients taking placebo. For example, the fraction of the patient pool to
achieve 501D/o improvement may be
over 40%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.
[805] In some embodiments, the therapeutically effective amount of the
inhibitor is an amount sufficient to attain
clinical improvement as assessed by an anemia response. For example, an
improved anemia response may
include longer durations of transfusion-independence, e.g., 8 weeks or longer,
following the treatment of 4-12
cycles, e.g., 6 cycles.
[806] In some embodiments, the therapeutically effective amount of the
inhibitor is an amount sufficient to
maintain stable disease for a duration of time, e.g., 6 weeks, 8 weeks, 12
weeks, six months, etc. In some
embodiments, progression of the disease may be evaluated by changes in overall
bone marrow cellularity, the
degree of reticulin or collagen fibrosis, and/or a change in JAK2V617F allele
burden.
[807] In some embodiments, a patient population treated with an isoform-
specific, context-independent TGF31
inhibitor such as those described herein, shows statistically improved
(prolonged) survival, as compared to a control
population that does not receive the treatment. For example, in control
groups, median survival of PMF patients is
approximately six years (approximately 16 months in high-risk patients), and
fewer than 20% of the patients are
expected to survive 10 years or longer post-diagnosis. Treatment with the
isoform-specific, context-independent
TGF31 inhibitor such as those described herein, may prolong the survival time
by, at least 6 months, 12 months,
18 months, 24 months, 30 months, 36 months, or 48 months. In some embodiments,
the treatment is effective to
achieve improved overall survival at 26 weeks, 52 weeks, 78 weeks, 104 weeks,
130 weeks, 144 weeks, or 156
weeks, as compared to patients who receive placebo.
[808] Clinical benefits of the therapy, such as those exemplified above, may
be seen in patients with or without
new onset anemia.
[809] One of the advantageous features of the isoform-specific, context-
independent TGF31 inhibitors is that they
maintain improved safety profiles enabled by isoform selectivity, as compared
to conventional TGFI3 antagonists
that lack the selectivity. Therefore, it is anticipated that treatment with an
isoform-specific, context-independent
inhibitor, such as those described herein, may reduce adverse events in a
patient population, in comparison to
equivalent patient populations treated with conventional TGFI3 antagonists,
with respect to the frequency and/or
severity of such events. Thus, the isoform-specific, context-independent TG931
inhibitors may provide a greater
therapeutic window as to dosage and/or duration of treatment.
[810] Adverse events may be graded by art-recognized suitable methods, such as
Common Terminology Criteria
for Adverse Events (CTCAE) version 4. Previously reported adverse events in
human patients who received TG93
antagonists, such as GC1008, include: leukocytosis (grade 3), fatigue (grade
3), hypoxia (grade 3), asystole (grade
5), leukopenia (grade 1), recurrent, transient, tender erythematous, nodular
skin lesions, suppurative dermatitis,
and herpes zoster.
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[811] The TG931 inhibitor therapy may cause less frequent and/or less severe
adverse events (side effects) as
compared to JAK inhibitor therapy in myelofibrosis patients, with respect to,
for example, anemia,
thrombocytopenia, neutropenia, hypercholesterolemia, elevated alanine
transaminase (ALT), elevated aspartate
transaminase (AST), bruising, dizziness, and headache, thus offering a safer
treatment option.
[812] It is contemplated that inhibitors of TGFp signaling may be used in
conjunction with one or more therapeutic
agents to treat myelofibrosis as a combination (e.g., "add-on") therapy. In
some embodiments, the TGFp inhibitor
is an inhibitor of TGFp activation, e.g., TGFp1 activation, e.g., Ab6, which
is administered in combination with one
or more checkpoint inhibitors disclosed herein to a patient suffering from
myelofibrosis. In some embodiments, the
TGFp inhibitor such as Ab6 is administered to a patient suffering from
myelofibrosis who has received or is a
candidate for receiving a JAK1 inhibitor, JAK2 inhibitor or JAK1/JAK2
inhibitor. In some embodiments, such
patients are responsive to the JAK1 inhibitor, JAK2 inhibitor or JAK1/JAK2
inhibitor therapy, while in other
embodiments such patients are poorly responsive or not responsive to the JAK1
inhibitor, JAK2 inhibitor or
JAK1/JAK2 inhibitor therapy. In some embodiments, use of a TGFI3 inhibitor
such as an isoform-specific inhibitor
of TGF(31 described herein may render those who are poorly responsive or not
responsive to the JAK1 inhibitor,
JAK2 inhibitor or JAK1/JAK2 inhibitor therapy more responsive. In some
embodiments, use of a TGFI3 inhibitor
such as an isoform-specific inhibitor of TGFp1 described herein may allow
reduced dosage of the JAK1 inhibitor,
JAK2 inhibitor or JAK1/JAK2 inhibitor which still produces equivalent or
meaningful clinical efficacy or benefits in
patients but with fewer or lesser degrees of drug-related toxicities or
adverse events (such as those listed above).
In some embodiments, treatment with the inhibitor of TGFp activation described
herein used in conjunction with
JAK1 inhibitor, JAK2 inhibitor or JAK1/JAK2 inhibitor therapy may produce
synergistic or additive therapeutic
effects in patients. In some embodiments, treatment with the inhibitor of TGFp
activation described herein may
boost the benefits of JAK1 inhibitor, JAK2 inhibitor or JAK1/JAK2 inhibitor or
other therapy given to treat
myelofibrosis. In some embodiments, patients may additionally receive a
therapeutic to address anemia
associated with myelofibrosis.
[813] In some embodiments, a TGFIP inhibitor described herein, such as a TGFp1-
selective inhibitor described
herein (e.g., Ab6), may be used to provide therapeutic benefit in conjunction
with a checkpoint inhibitor therapy for
the treatment of myelofibrosis. Primary cells isolated from patients with JAK2
mutation exhibit higher PD-L1
expression as compared to primary cells from healthy donors. This indicates
that constitutive activation of the
JAK2/STAT pathway in megakaryocytes and platelets may contribute to immune
escape via PD-L1-mediated
reduction of T cell activation, metabolic activity, and cell cycle progression
of T cells (Prestipino et al., Sci Transl
Med 2018; 10(429)). Additionally, activation of the TGF(3 signaling pathway
has also been shown to increase PD-
1 expression on cytotoxic T cells and decrease sensitivity to PD-1/PD-L1-
mediated checkpoint blockade (Chen et
al., Int J Cancer 2018;143:2561). Wthout being bound by theory, these
findings, along with low response rates to
checkpoint inhibitor therapy (e.g., anti-PD-1 therapy) observed in
myelofibrosis patients, provide support for the
potential importance of TGFI3 signaling in mediating clinical resistance to
checkpoint inhibitor therapy.
[814] In some embodiments, a TGFP inhibitor such as a TGFP1 inhibitor (e.g.,
Ab6) may be used in conjunction
with a BMP antagonist (e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor) for
treating anemia in a patient with a
myeloproliferative disorder such as myelofibrosis. Without wishing to be bound
by theory, it is contemplated that
TGFp1 inhibitors (e.g., Ab6) may be helpful for promoting hematopoiesis, while
BMP antagonists (e.g., BMP6
inhibitors, e.g., RGMc inhibitors) may reduce iron deficiency (such as a
deficiency arising from a cancer and/or
chemotherapy). In some embodiments, a treatment comprising a TGF(31 inhibitor
(e.g., Ab6) and a BMP antagonist
(e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor) may be administered at a
therapeutically effective amount that is
sufficient to relieve one or more anemia-related symptom and/or complication.
In some embodiments, a treatment
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comprising a TG931 inhibitor (e.g., Ab6) and a BMP antagonist (e.g., a BMP6
inhibitor, e.g., a RGMc inhibitor) may
be administered at a therapeutically effective amount to increase red blood
cell production and/or reduce iron
restriction, in a patient with a myeloproliferative disorder (e.g.,
myelofibrosis). In some embodiments, the treatment
for anemia further comprises administering one or more JAK inhibitor (e.g.,
Jak1/2 inhibitor, Jak1 inhibitor, and/or
Jak2 inhibitor). In some embodiments, an improved anemia response may include
a longer duration of transfusion-
independence, e.g., 8 weeks or longer, e.g., following treatment for 4-12
cycles, e.g., 6 cycles. In some
embodiments, the treatment further includes one or more checkpoint inhibitors
such as anti-PD1 antibodies, anti-
PD-L1 antibodies, and/or anti-CTLA-4 antibodies.
[815] Accordingly, the present disclosure provides a TGFp inhibitor (e.g.,
TGF131-selective inhibitor such as Ab6)
for use in the treatment of a myeloproliferative disorder such as primary
myelofibrosis in a patient, wherein the
treatment comprises administration of a composition comprising the TGFp
inhibitor (e.g., TGF(31 inhibitor) which
has been selected, at least in part, on the basis of its immune safety
profile. A suitable immune safety profile of
the TGFp inhibitor is characterized in that i) it does not trigger
unacceptable levels of cytokine release (e.g., within
2.5-fold of control); ii) it does not promote unacceptable levels of platelet
aggregation; or both in field-accepted cell-
based assay(s) and/or in in vivo assay(s) (such as those described herein).
Conditions involving MHC downregulation or mutation
[816] TGFP-related indications may also include conditions in which major
histocompatibility complex (MHC) class
I is deleted or deficient (e.g., downregulated). Such conditions include
genetic disorders in which one or more
components of the MHC-mediated signaling is impaired, as well as conditions in
which MHC expression is altered
by other factors, such as cancer, infections, fibrosis, and medications.
[817] For example, MHC I downregulation in tumor is associated with tumor
escape from immune surveillance.
Indeed, immune escape strategies aimed to avoid T-cell recognition, including
the loss of tumor MHC class I
expression, are commonly found in malignant cells. Tumor immune escape has
been observed to have a negative
effect on the clinical outcome of cancer immunotherapy, including treatment
with antibodies blocking immune
checkpoint molecules (reviewed in, for example: Garrido et al., (2017) Curr
Opin Immunol 39: 44-51. "The urgent
need to recover MHC class I in cancers for effective immunotherapy",
incorporated by reference herein). Thus,
the isoform-selective, context-independent TGFP1 inhibitors encompassed by the
present disclosure may be
administered either as a monotherapy or in conjunction with another therapy
(such as checkpoint inhibitor,
chemotherapy, radiation therapy (such as a radiotherapeutic agent), etc.) to
unleash or boost anti-cancer immunity
and/or enhance responsiveness to or effectiveness of another therapy.
[818] In some embodiments, MHC downregulation is associated with acquired
resistance to a cancer therapy,
such as CBT. It is contemplated that the isoform-selective inhibitors of
TGFI31 may be used to treat patients who
are primary responders of a cancer therapy such as CBT, to reduce the
probability of developing acquired
resistance. Thus, among those treated with the TGFpl inhibitor, who are
primary responders of cancer therapy,
occurrence of secondary or acquired resistance to the cancer therapy over time
may be reduced.
[819] Downregulation of MHC class I proteins are also associated with certain
infectious diseases, including viral
infections such as HIV. See for example, Cohen et al., (1999) Immunity 10(6):
661-671. "The selective
downregulation of class I major histocompatibility complex proteins by HIV-1
protects HIV-infected cells from NK
Cells", incorporated herein by reference. Thus, the isoform-selective, context-
independent TGFpl inhibitors
encompassed by the present disclosure may be administered either as a
monotherapy or in conjunction with
another therapy (such as anti-viral therapy, protease inhibitor therapy, etc.)
to unleash or boost host immunity
and/or enhance responsiveness to or effectiveness of another therapy.
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[820] The present disclosure provides a TGF13 inhibitor (e.g., TG931-selective
inhibitor such as Ab6) for use in
the treatment of a TGF13-related disorder involving MHC downregulation or
mutation (e.g., as described herein) in
a patient, wherein the treatment comprises administration of a composition
comprising the TGFp inhibitor (e.g.,
TGF131 inhibitor) which has been selected, at least in part, on the basis of
its immune safety profile. A suitable
immune safety profile of the TGFP inhibitor is characterized in that i) it
does not trigger unacceptable levels of
cytokine release (e.g., within 2.5-fold of control); ii) it does not promote
unacceptable levels of platelet aggregation;
or both in field-accepted cell-based assay(s) and/or in in vivo assay(s) (such
as those described herein).
Conditions involving stem cell self-renewal, tissue regeneration and stem cell
repopulation
[821] Evidence suggests that the TGFp pathway plays a role in regulating the
homeostasis of various stem cell
populations and their differentiation/repopulation within a tissue. During
homeostasis, tissue-specific stem cells
are held predominantly quiescent but are triggered to enter cell cycle upon
certain stress. TGFp1 is thought to
function as a "break" during the process that tightly regulates stem cell
differentiation and reconstitution, and the
stress that triggers cell cycle entry coincides with TGFp1 inhibition that
removes the "break." Thus, it is
contemplated that isoform-selective inhibitors of TGFI31, such as those
described herein, may be used to skew or
correct cell cycle and GO entry decision of stem cells/progenitor cells within
a particular tissue.
[822] Accordingly, the inventors of the present disclosure contemplate the use
of isoform-selective TGFpl
inhibitors in conditions in which: i) stem cell/progenitor cell
differentiation/reconstitution is halted or perturbed due
to a disease or induced as a side effect of a therapy/mediation; ii) patients
are on a therapy or mediation that
causes healthy cells to be killed or depleted; iii) patients may benefit from
increased stem cell/progenitor cell
differentiation/reconstitution; iv) disease is associated with abnormal stem
cell differentiation or reconstitution.
[823] In self-renewing tissues, such as bone marrow (blood cell production)
and the epidermis, the balance
between proliferation and differentiation processes is tightly regulated to
ensure the maintenance of the stem cell
population during lifetime. Reviewed by D'Arcangel et al., (2017) Int. J Mol
Sci. 18(7): 1591. TGFP1 acts as a
potent negative regulator of the cell cycle and tumor suppressor in part
through induction of cyclin-dependent
kinase inhibitors, p15/INK4b, p21 and p57. Evidence suggests that TGFp1
contributes to the induction of
p16/INK4a and p19/ARF to mediate growth arrest and senescence in certain cell
types. Accordingly, in some
embodiments, an isoform-selective inhibitor of TGFP1 activation, such as those
described herein, is used to
regulate p16/INK4a-dependent cellular senescence and stem cell dynamics in
various stem cell populations.
[824] For example, mesenchymal stromal/stem cells (MSCs) are a small
population of stromal cells present in
most adult connective tissues, such as bone marrow, fat tissue, and umbilical
cord blood. MSCs are maintained in
a relative state of quiescence in vivo but, in response to a variety of
physiological and pathological stimuli, are
capable of proliferating then differentiating into osteoblasts, chondrocytes,
adipocytes, or other mesoderm-type
lineages like smooth muscle cells (SMCs) and cardiomyocytes. Multiple
signaling networks orchestrate MSCs
differentiating into functional mesenchymal lineages, among which TGF-131 has
emerged as a key player (reviewed
for example by Zhao & Hantash (2011. Vitam Horm 87:127-41).
[825] Similarly, hematopoietic stem cells are required for lifelong blood cell
production; to prevent exhaustion, the
majority of hematopoietic stem cells remain quiescent during steady-state
hematopoiesis. During hematologic
stress, however, these cells are rapidly recruited into cell cycle and undergo
extensive self-renewal and
differentiation to meet increased hematopoietic demands. TGF131 may work as
the "switch" to control the
quiescence-repopulation transition/balance.
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[826] Thus, the isoform-selective inhibitors of TG931 can be used in the
treatment of conditions involving
hematopoietic stem cell defects and bone marrow failure. In some embodiments,
depletion or impairment of the
hematopoietic stem cell reservoir leads to hematopoietic failure or
hematologic malignancies. In some
embodiments, such conditions are DNA repair disorder characterized by
progressive bone marrow failure. In some
embodiments, such condition is caused by stem and progenitor cell attrition.
In some embodiments, such
conditions are associated with anemia. In some embodiments, such condition is
Fanconi Anemia (FA). In some
embodiments, such conditions are characterized by hyperactive TGFI3 pathway
that suppresses the survival of
certain cell types upon DNA damage. Thus, it is contemplated that the isoform-
selective inhibitors of TGF[31 can
be used for rescuing proliferation defects of FA hematopoietic stem cells
and/or bone marrow failure in subjects
with FA. See, for example, Zhang et al., (2016), Cell Stem Cell, 18: 668-681,
"TGF-I3 inhibition rescues
hematopoietic stem cell defects and bone marrow failure in Fanconi Anemia."
[827] The present disclosure provides a TGFI3 inhibitor (e.g., TGF131-
selective inhibitor such as Ab6) for use in
the treatment of a TGFI3-related disorder involving stem cell self-renewal,
tissue regeneration and/or stem cell
repopulation (e.g., as described herein) in a patient, wherein the treatment
comprises administration of a
composition comprising the TGFI3 inhibitor (e.g., TGF131 inhibitor) which has
been selected, at least in part, on the
basis of its immune safety profile. A suitable immune safety profile of the
TGF[3 inhibitor is characterized in that i)
it does not trigger unacceptable levels of cytokine release (e.g., within 2.5-
fold of control); ii) it does not promote
unacceptable levels of platelet aggregation; or both in field-accepted cell-
based assay(s) and/or in in vivo assay(s)
(such as those described herein).
Conditions involving treatment-induced hematopoietic dys regulation
[828] It is recognized that certain drugs which are designed to treat various
disease conditions, often induce or
exacerbate anemia in the patient being treated (e.g., treatment- or drug-
induced anemia, such as chemotherapy-
induced anemia and radiation therapy-induced anemia). In some embodiments, the
patient is treated with a
myelosuppressive drug that may cause side effects that include anemia. Such
patient may benefit from
pharmacological TGFI31 inhibition in order to boost hematopoiesis. In some
embodiments, the TGFI31 inhibitor
may promote hematopoiesis in patients by preventing entry into a quiescent
state. In some embodiments, the
patient may receive a G-CSF therapy (e.g., Filgrastim).
[829] Accordingly, the disclosure includes the use of an isoform-selective
inhibitor of TGF61, such as those
disclosed herein, to be administered to patients who receive myelosuppressive
therapy (e.g., therapy with side
effects including myelosuppressive effects). Examples of myelosuppressive
therapies include but are not limited
to: peginterferon alfa-2a, interferon alfa-n3, peginterferon alfa-2b,
aldesleukin, gemtuzumab ozogamicin,
interferon alfacon-1, rituximab, ibritumomab tiuxetan, tositumomab,
alemtuzumab, bevacizumab, L-
Phenylalanine, bortezomib, cladribine, carmustine, amsacrine, chlorambucil,
raltitrexed, mitomycin, bexarotene,
vindesine, floxuridine, tioguanine, vinorelbine, dexrazoxane, sorafenib,
streptozocin, gemcitabine, teniposide,
epirubicin, chloramphenicol, lenalidomide, altretamine, zidovudine, cisplatin,
oxaliplatin, cyclophosphamide,
fluorouracil, propylthiouracil, pentostatin, methotrexate, carbamazepine,
vinblastine, linezolid, imatinib,
clofarabine, pemetrexed, daunorubicin, irinotecan, meth imazole, etoposide,
dacarbazine, temozolomide,
tacrolimus, sirolimus, mechlorethamine, azacitidine, carboplatin,
dactinomycin, cytarabine, doxorubicin,
hydroxyurea, busulfan, topotecan, mercaptopurine, thalidomide, melphalan,
fludarabine, flucytosine,
capecitabine, procarbazine, arsenic trioxide, idarubicin, ifosfamide,
mitoxantrone, lomustine, paclitaxel,
docetaxel, dasatinib, decitabine, nelarabine, everolimus, vorinostat,
thiotepa, ixabepilone, nilotinib, belinostat,
trabectedin, trastuzumab emtansine, temsirolimus, bosutinib, bendamustine,
cabazitaxel, eribulin, ruxolitinib,
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carfilzomib, tofacitinib, ponatinib, pomalidomide, obinutuzumab, tedizolid
phosphate, blinatumomab, ibrutinib,
palbociclib, olaparib, dinutuximab, and colchicine.
[830] Additional TG93-related indications may include any of those disclosed
in US Pub. No. 2013/0122007, US
Pat. No. 8,415,459 or International Pub. No. WO 2011/151432, the contents of
each of which are herein
incorporated by reference in their entirety.
[831] In certain embodiments, antibodies, antigen binding portions thereof,
and compositions of the disclosure
may be used to treat a wide variety of diseases, disorders and/or conditions
associated with TGFp1 signaling. In
some embodiment, target tissues/cells preferentially express the TGF(31
isoform over the other isoforms. Thus,
the disclosure includes methods for treating such a condition associated with
TGFp1 expression (e.g.,
dysregulation of TGF(31 signaling and/or upregulation of TGF131 expression)
using a pharmaceutical composition
that comprises an antibody or antigen-binding portion thereof described
herein.
[832] In some embodiments, the disease involves TGF(31 associated with (e.g.,
presented on or deposited from)
multiple cellular sources. In some embodiments, such disease involves both an
immune component and an ECM
component of TGFp1 function. In some embodiments, such disease involves: i)
dysregulation of the ECM (e.g.,
overproduction/deposition of ECM components such as collagens and proteases;
altered stiffness of the ECM
substrate; abnormal or pathological activation or differentiation of
fibroblasts, such as myofibroblasts, fibrocytes
and OAFS); ii) immune suppression due to increased Tregs and/or suppressed
effector T cells (Teff), e.g., elevated
ratios of Treg/Teff; increased leukocyte infiltrate (e.g., macrophage and
MDSCs) that causes suppression of 004
and/or COB T cells; and/or iii) abnormal or pathological activation,
differentiation, and/or recruitment of myeloid
cells, such as macrophages (e.g., bone marrow-derived monocytic/macrophages
and tissue resident
macrophages), monocytes, myeloid-derived suppresser cells (MDSCs),
neutrophils, dendritic cells, and NK cells.
[833] In some embodiments, the condition involves TGFpl presented by more than
one types of presenting
molecules (e.g., two or more of: GARP, LRRC33, LTBP1 and/or LTBP3). In some
embodiments, an affected
tissues/organs/cells that include TGFp1 from multiple cellular sources. To
give but one example, a solid tumor
(which may also include a proliferative disease involving the bone marrow,
e.g., myelofibrosis and multiple
myeloma) may include TGF(31 from multiple sources, such as the cancer cells,
stromal cells, surrounding healthy
cells, and/or infiltrating immune cells (e.g., 0045+ leukocytes), involving
different types of presenting molecules.
Relevant immune cells include but are not limited to myeloid cells and
lymphoid cells, for example, neutrophils,
eosinophils, basophils, lymphocytes (e.g., B cells, T cells, and NK cells),
and monocytes. Context-independent
inhibitors of TGFp1 may be useful for treating such conditions.
[834] In some embodiments, hematopoietic dysregulation associated with cancer
may cause anemia. The TGFp
therapy may further include a BMP6 inhibitor, such as a RGMc inhibitor. In
some embodiments, cancer-associated
anemia may be caused by the disease itself; while in some embodiments the
anemia may be caused by cancer
therapy (such as chemotherapy).
[835] The present disclosure provides a TGFI3 inhibitor (e.g., TGFI31-
selective inhibitor such as Ab6) for use in
the treatment of a TGFp-related disorder involving treatment-induced
hematopoietic dysregulation (e.g., as
described herein) in a patient, wherein the treatment comprises administration
of a composition comprising the
TGFp inhibitor (e.g., TGFp1 inhibitor) which has been selected, at least in
part, on the basis of its immune safety
profile. A suitable immune safety profile of the TGFp inhibitor is
characterized in that i) it does not trigger
unacceptable levels of cytokine release (e.g., within 2.5-fold of control);
ii) it does not promote unacceptable levels
of platelet aggregation; or both in field-accepted cell-based assay(s) and/or
in in vivo assay(s) (such as those
described herein).
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[836] Non-limiting examples of conditions or disorders that may be treated
with isoform-specific context-
independent inhibitors of TGF131, such as antibodies or fragments thereof
described herein, are provided below.
Treatment Regimen, Administration
[837] To practice the method disclosed herein, an effective amount of the
pharmaceutical composition described
above can be administered to a subject (e.g., a human) in need of the
treatment via a suitable route, such as
intravenous administration, e.g., as a bolus or by continuous infusion over a
period of time, by intramuscular,
intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, inhalation or topical
routes. Commercially available nebulizers for liquid formulations, including
jet nebulizers and ultrasonic nebulizers
are useful for administration. Liquid formulations can be directly nebulized
and lyophilized powder can be nebulized
after reconstitution. Alternatively, antibodies, or antigen binding portions
thereof, that specifically bind a GARP-
TGF61 complex, a LTBP1-TGF61 complex, a LTBP3-TGF61 complex, and/or a LRRC33-
TGF(31 complex can be
aerosolized using a fluorocarbon formulation and a metered dose inhaler, or
inhaled as a lyophilized and milled
powder.
[838] The subject to be treated by the methods described herein can be a
mammal, more preferably a human.
Mammals include, but are not limited to, farm animals, sport animals, pets,
primates, horses, dogs, cats, mice and
rats. A human subject who needs the treatment may be a human patient having,
at risk for, or suspected of having
a TGF6-related indication, such as those noted above. A subject having a TGF6-
related indication can be identified
by routine medical examination, e.g., laboratory tests, organ functional
tests, CT scans, or ultrasounds. A subject
suspected of having any of such indication might show one or more symptoms of
the indication. A subject at risk
for the indication can be a subject having one or more of the risk factors for
that indication.
[839] As used herein, the terms "effective amount" and "effective dose" refer
to any amount or dose of a
compound or composition that is sufficient to fulfill its intended purpose(s),
i.e., a desired biological or medicinal
response in a tissue or subject at an acceptable benefit/risk ratio. For
example, in certain embodiments of the
present invention, the intended purpose may be to inhibit TG93-1 activation in
vivo, to achieve clinically meaningful
outcome associated with the TG93-1 inhibition. Effective amounts vary, as
recognized by those skilled in the art,
depending on the particular condition being treated, the severity of the
condition, the individual patient parameters
including age, physical condition, size, gender and weight, the duration of
the treatment, the nature of concurrent
therapy (if any), the specific route of administration and like factors within
the knowledge and expertise of the health
practitioner. These factors are well known to those of ordinary skill in the
art and can be addressed with no more
than routine experimentation. It is generally preferred that a maximum dose of
the individual components or
combinations thereof be used, that is, the highest safe dose according to
sound medical judgment. It will be
understood by those of ordinary skill in the art, however, that a patient may
insist upon a lower dose or tolerable
dose for medical reasons, psychological reasons or for virtually any other
reasons.
[840] Empirical considerations, such as the half-life, generally will
contribute to the determination of the dosage.
For example, antibodies that are compatible with the human immune system, such
as humanized antibodies or
fully human antibodies, may be used to prolong half-life of the antibody and
to prevent the antibody being attacked
by the host's immune system. Frequency of administration may be determined and
adjusted over the course of
therapy, and is generally, but not necessarily, based on treatment and/or
suppression and/or amelioration and/or
delay of a TGF6-related indication. Alternatively, sustained continuous
release formulations of an antibody that
specifically binds a GARP-TGF61 complex, a LTBP1-TGF131 complex, a LTBP3-TGF61
complex, and/or a
LRRC33-TGF(31 complex may be appropriate. Various formulations and devices for
achieving sustained release
would be apparent to the skilled artisan and are within the scope of this
disclosure.
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[841] In one example, dosages for an antibody as described herein may be
determined empirically in individuals
who have been given one or more administration(s) of the antibody. Individuals
are given incremental dosages of
the antagonist. To assess efficacy, an indicator of the TGF6-related
indication can be followed. For example,
methods for measuring for myofiber damage, myofiber repair, inflammation
levels in muscle, and/or fibrosis levels
in muscle are well known to one of ordinary skill in the art.
[842] The present disclosure encompasses the recognition that agents capable
of modulating the activation step
of TGF6s in an isoform-specific manner may provide improved safety profiles
when used as a medicament.
Accordingly, the disclosure includes antibodies and antigen-binding fragments
thereof that specifically bind and
inhibit activation of TGF61, but not TGF62 or TGF63, thereby conferring
specific inhibition of the TGFI31 signaling
in vivo while minimizing unwanted side effects from affecting TGF132 and/or
TGF133 signaling.
[843] In some embodiments, the antibodies, or antigen binding portions
thereof, as described herein, are not toxic
when administered to a subject. In some embodiments, the antibodies, or
antigen binding portions thereof, as
described herein, exhibit reduced toxicity when administered to a subject as
compared to an antibody that
specifically binds to both TGF61 and TGF62. In some embodiments, the
antibodies, or antigen binding portions
thereof, as described herein, exhibit reduced toxicity when administered to a
subject as compared to an antibody
that specifically binds to both TGF61 and TGF63. In some embodiments, the
antibodies, or antigen binding
portions thereof, as described herein, exhibit reduced toxicity when
administered to a subject as compared to an
antibody that specifically binds to TGF61, TGF62 and TGF63.
[844] Generally, for administration of any of the antibodies described herein,
an initial candidate dosage can be
about 1-20 mg/kg per administration, with dosing frequency of, e.g., once
weekly (QM, once every 2 weeks (Q2W),
once every 3 weeks (Q3VV), once every 4 weeks (Q4VV), monthly, etc. For
example, patients may receive an
injection of about 1-10 mg/kg per 1 week, per 2 weeks, per 3 weeks, or per 4
weeks, etc., in an amount effective
to treat a disease (e.g., cancer) wherein the amount is well-tolerated (within
acceptable toxicities or adverse
events).
[845] For the purpose of the present disclosure, a typical dosage (per
administration, such as an injection and
infusion) might range from about 0.1 mg/kg to 30 mg/kg, depending on the
factors mentioned above. For repeated
administrations over several days or longer, depending on the condition, the
treatment is sustained until a desired
suppression of symptoms occurs or until sufficient therapeutic levels are
achieved to alleviate a TGF6-related
indication, or a symptom thereof. For example, suitable effective dosage for
Ab6 may be between 1 mg/kg and 30
mg/kg, (e.g., 1-10 mg/kg, 1-15 mg/kg, 3-20 mg/kg, 5-30 mg/kg, etc.) dosed
twice a week, once a week, every two
weeks, every 4 weeks or once a month. Suitable effective dose for Ab6
includes, about 1 mg/kg, about 3 mg/kg,
about 5 mg/kg, about 10 mg/kg, for example, dosed weekly.
[846] An exemplary dosing regimen comprises administering an initial dose,
followed by one or more of
maintenance doses. For example, an initial dose may be between about 1 and 30
mg/kg, for instance, once a
week or twice a week. Thereafter, maintenance dose(s) may follow, for example,
between about 0.1 and 20 mg/kg,
for instance, once a week, every other week, once a month, etc. However, other
dosage regimens may be useful,
depending on the pattern of pharmacokinetic decay that the practitioner wishes
to achieve. Pharmacokinetics
experiments have shown that the serum concentration of an antibody disclosed
herein (e.g., Ab3) remains stable
for at least 7 days after administration to a preclinical animal model (e.g.,
a mouse model). Without wishing to be
bound by any particular theory, this stability post-administration may be
advantageous since the antibody may be
administered less frequently while maintaining a clinically effective serum
concentration in the subject to whom the
antibody is administered (e.g., a human subject). In some embodiments, dosing
frequency is once every week,
every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks,
every 7 weeks, every 8 weeks, every
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9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3
months, or longer. The progress of
this therapy is easily monitored by conventional techniques and assays. The
dosing regimen (including the
antibody used) can vary over time.
[847] In some embodiments, for an adult patient of normal weight, doses
ranging from about 1 mg/kg to 30 mg/kg,
or from 80 mg to 3000 mg, e.g., 30, 50, 100, 500, 1000, 2000, or 3000 mg, may
be administered. The particular
dosage regimen, e.g.., dose, timing, and repetition, will depend on the
particular individual and that individual's
medical history, as well as the properties of the individual agents (such as
the half-life of the agent, and other
relevant considerations).
[848] Serum concentrations of the TGF6 inhibitor antibody that are
therapeutically effective to treat a TGF31-
related indication in accordance with the present disclosure may be at least
about 10 pg/mL, e.g., between about
pg/mL and 1.0 mg/mL. In some embodiments, effective amounts of the antibody as
measured by serum
concentrations are about 20-400 ug/mL. In some embodiments, effective amounts
of the antibody as measured
by serum concentrations are about 100-800 pg/mL. In some embodiments,
effective amounts of the antibody as
measured by serum concentrations are at least about 20 pg/mL, e.g., at least
about 50 pg/mL, 100 pg/mL, 150
pg/mL, or 200 pg/mL. As shown previously, a single dose of Ab6 administered
intravenously at 1, 3, 10, 30, or 37.5
mg/kg resulted in Cma. values of about 25 ug/mL to about 900 ug/mL.
Furthermore, in non-human primates, there
were no observed toxicities (for example: no cardiotoxicities, hyperplasia and
inflammation, dental and gingival
findings) associated with Ab6 after maintaining serum concentration levels of
about 2,000-3,000 pg/mL for at least
8 weeks (data provided in PCT/US2019/041373 at Example 12). Therefore, about
10-100 fold therapeutic window
may be achieved.
[849] For the purpose of the present disclosure, the appropriate dosage of an
antibody that specifically binds a
GARP-TGF31 complex, a LTBP1-TGF31 complex, a LTBP3-TGF131 complex, and/or a
LRRC33-TGF31 complex
will depend on the specific antibody (or compositions thereof) employed, the
type and severity of the indication,
whether the antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical
history and response to the antagonist, and the discretion of the attending
physician. In some embodiments, a
clinician will administer an antibody that specifically binds a GARP-TGF31
complex, a LTBP1-TGF31 complex, a
LTBP3-TGF31 complex, and/or a LRRC33-TG931 complex, until a dosage is reached
that achieves the desired
result. Administration of an antibody that specifically binds a GARP-TGF131
complex, a LTBP1-TGF131 complex, a
LTBP3-TGF31 complex, and/or a LRRC33-TGF31 complex can be continuous or
intermittent, depending, for
example, upon the recipient's physiological condition, whether the purpose of
the administration is therapeutic or
prophylactic, and other factors known to skilled practitioners. The
administration of antibody that specifically binds
a GARP-TGF31 complex, a LTBP1-TGF31 complex, a LTBP3-TGF31 complex, and/or a
LRRC33-TGF31 complex
may be essentially continuous over a preselected period of time or may be in a
series of spaced dose, e.g., either
before, during, or after developing a TGF3-related indication.
[850] In order to minimize potential risk and adverse events associated with
TGF3 inhibition, a TGF3 inhibitor
such as any one of the antibodies disclosed herein may be administered
intermittently. For instance, the TGF3
inhibitor may be administered on an "as needed" basis in a therapeutically
effective amount sufficient to achieve
and maintain clinical benefit (e.g., reduction of tumor volume and/or reversal
or reduction of immunosuppression).
In some embodiments, administration of a TGF3 inhibitor such as any one of the
antibodies disclosed herein (e.g.,
a TGF31 inhibitor, e.g., Ab6) may be used in combination with a method of
determining or monitoring therapeutic
efficacy (e.g., measuring of circulating MDSCs). In some embodiments, the TGF3
inhibitor is administered in
patients only when clinical benefit from additional doses of the TGF13
inhibitor is determined, e.g., when an elevation
in circulating MDSCs is detected.
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[851] As used herein, the term "treating" refers to the application or
administration of a composition including one
or more active agents to a subject, who has a TGFI3-related indication, a
symptom of the indication, or a
predisposition toward the indication, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate,
improve, or affect the indication, the symptom of the indication, or the
predisposition toward the indication.
[852] Alleviating a TGFI3-related indication with an antibody that
specifically binds a GARP-TGF131 complex, a
LTBP1-TGF[31 complex, a LTBP3-TGF131 complex, and/or a LRRC33-TGF31 complex
includes delaying the
development or progression of the indication, or reducing indication's
severity. Alleviating the indication does not
necessarily require curative results. As used therein, "delaying" the
development of an indication associated with
a TGF3-related indication means to defer, hinder, slow, retard, stabilize,
and/or postpone progression of the
indication. This delay can be of varying lengths of time, depending on the
history of the indication and/or individuals
being treated. A method that "delays" or alleviates the development of an
indication, or delays the onset of the
indication, is a method that reduces probability of developing one or more
symptoms of the indication in a given
time frame and/or reduces extent of the symptoms in a given time frame, when
compared to not using the method.
Such comparisons are typically based on clinical studies, using a number of
subjects sufficient to give a statistically
significant result.
Diagnostics, Patient Selection, Monitoring
[853] Therapeutic methods that include TGF3 inhibition therapy may comprise
diagnosis of a TGF3 indication
and/or selection of patients likely to respond to such therapy. Additionally,
patients who receive the TGF3 inhibitor
may be monitored for therapeutic effects of the treatment, which typically
involves measuring one or more suitable
parameters, which are indicative of the condition, and which can be measured
(e.g., assayed) before and after the
treatment, and evaluating treatment-related changes in the parameters. For
example, such parameters may
include levels of biomarkers present in biological samples collected from the
patients. Biomarkers may be RNA-
based, protein-based, cell-based, and/or tissue-based. For example, genes that
are overexpressed in certain
disease conditions may serve as the biomarkers to diagnose and/or monitor the
disease or response to the therapy.
Cell-surface proteins of disease-associated cell populations may serve as
biomarkers. Such methods may include
the direct measurements of disease parameters indicative of the extent of the
particular disease, such as tumor
size/volume. Any suitable sampling methods may be employed, such as
serum/blood samples, biopsies, and
imaging. The biopsy may include tissue biopsies (such as tumor biopsy, e.g.,
core needle biopsy) and liquid
biopsies.
[854] While biopsies have traditionally been the standard for diagnosing and
monitoring various diseases, such
as proliferative disorders (e.g., cancer), less invasive alternatives may be
preferred. For example, many non-
invasive in vivo imaging techniques may be used to diagnose, monitor, and
select patients for treatment. Thus,
the disclosure includes the use of in vivo imaging techniques to diagnose
and/or monitor disease in a patient or
subject. In some embodiments, the patient or subject is receiving an isoform-
specific TGFI31 inhibitor as described
herein. In other embodiments, an in vivo imaging technique may be used to
select patients for treatment with an
isoform-specific TGF31 inhibitor. In some embodiments, such techniques may be
used to determine if or how
patients respond to a therapy, e.g., TGF31 inhibition therapy.
[855] Exemplary in vivo imaging techniques used for the methods include, but
are not limited to X-ray radiography,
magnetic resonance imaging (MRI), medical ultrasonography or ultrasound,
endoscopy, elastography, tactile
imaging, thermography, medical photography. Other imaging techniques include
nuclear medicine functional
imaging, e.g., positron emission tomography (PET) and Single-photon emission
computed tomography (SPEC).
Methods for conducting these techniques and analyzing the results are known in
the art.
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[856] Non-invasive imaging techniques commonly used to diagnose and monitor
cancer include, but are not
limited to: magnetic resonance imaging (MRI), computed tomography (CT),
ultrasound, positron emission
tomography (PET), single-photon emission computed tomography (SPECT),
fluorescence reflectance imaging
(FRI), and fluorescence mediated tomography (FMT). Hybrid imaging platforms
may also be used to diagnose and
monitor cancer. For example, hybrid techniques include, but are not limited
to: PET-CT, FMT-CT, FMT-MRI, and
PET-MRI. Dynamic contrast enhanced MRI (DCE-MRI) is another imaging technique
commonly used to detect
breast cancers. Methods for conducting these techniques and analyzing the
results are known in the art.
[857] More recently, non-invasive imaging methods are being developed which
will allow the detection of cells of
interest (e.g., cytotoxic T cells, macrophages, and cancer cells) in vivo.
See, for example,
www.imaginab.com/technology/; Tavare et al., (2014) PNAS, 111(3): 1108-1113;
Tavare et al., (2015) J Nucl Med
56(8): 1258-1264; Rashidian et al., (2017) J Exp Med 214(8): 2243-2255;
Beckford Vera et al., (2018) PLoS ONE
13(3): e0193832; and Tavare et al., (2015) Cancer Res 76(1): 73-82, each of
which is incorporated herein by
reference. So-called "T-cell tracking" is aimed to detect and localize anti-
tumor effector T-cells in vivo. This may
provide useful insights into understanding the immunosuppressive phenotype of
solid tumors. Tumors that are
well-infiltrated with cytotoxic T cells ("inflamed" or "hot" tumors) are
likely to respond to cancer therapies such as
checkpoint blockade therapy (CBT). On the other hand, tumors with
immunosuppressive phenotypes tend to have
poor T-cell infiltration even when there is an anti-tumor immune response.
These so-called "immune excluded"
tumors likely fail to respond to cancer therapies such as CBT. T-cell tracking
techniques may reveal these different
phenotypes and provide information to guide in therapeutic approach that would
likely benefit the patients. For
example, patients with an "immune excluded" tumor are likely to benefit from a
TGFp inhibitor therapy such as a
TGF131 inhibitor therapy to help reverse the immunosuppressive phenotype. It
is contemplated that similar
techniques may be used to diagnose and monitor other diseases, for example,
fibrosis. Typically, antibodies or
antibody-like molecules engineered with a detection moiety (e.g., radiolabel,
fluorescence, etc.) can be infused into
a patient, which then will distribute and localize to sites of the particular
marker (for instance CD8+ and M2
macrophages).
[858] Non-invasive in vivo imaging techniques may be applied in a variety of
suitable methods for purposes of
diagnosing patients; selecting or identifying patients who are likely to
benefit from TGF(3 inhibitor therapy such as
TGF131 inhibitor therapy; and/or, monitoring patients for therapeutic response
upon treatment. Any cells with a
known cell-surface marker may be detected/localized by virtue of employing an
antibody or similar molecules that
specifically bind to the cell marker. Typically, cells to be detected by the
use of such techniques are immune cells,
such as cytotoxic T lymphocytes, regulatory T cells, MDSCs, disease-associated
macrophages (M2 macropahges
such as TAMs and FAMs), NK cells, dendritic cells, and neutrophils.
[859] Non-limiting examples of suitable immune cell markers include monocyte
markers, macrophage markers
(e.g., M1 and/or M2 macrophage markers), CTL markers, suppressive immune cell
markers, MDSC markers (e.g.,
markers for G- and/or M-MDSCs), including but are not limited to: CD8, CD3,
CD4, CD11b, CD33, CD163, CO206,
CD68, CD14, CD15, CD66b, CD34, CD25, and CD47. In some embodiments, levels of
circulating MDSCs (e.g.,
circulating mMDSCs and/or gMDSCs) may be used to predict, determine, and
monitor pharmacological effects of
treatment comprising a dose of TGFp inhibitor (e.g., a TG931 -selective
inhibitor, e.g., Ab6). In some embodiments,
circulating mMDSCs may be identified by surface markers CD11b+, HLADR-/low,
CD14+, CD15-, CD33+/high,
and CD66b-. In some embodiments, gMDSCs may be identified by surface markers
CD11 b+, HLADR-, CD14-,
CD15+, CD33+/low, and CD66b-'-. In some embodiments, LRRC33 is used as a
marker for MDSCs. In some
embodiments, LRRC33 is used as a marker for MDSCs in circulation. In some
embodiments, a reduction in
circulating MDSC levels following treatment with a TGFP inhibitor (e.g., a
TGFP1-selective inhibitor, e.g., Ab6) may
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be indicative of therapeutic efficacy. In some embodiments, levels of
circulating MDSCs may be used to predict,
determine, arid monitor pharmacological effects of treatment comprising a dose
of TGF3 inhibitor (e.g., a TGF31-
selective inhibitor, e.g., Ab6). Thus, LRRC33 levels in blood samples may
serve as a predictive biomarker for
assessing therapeutic response in conditions associated with immune
suppression.
[860] In vivo imaging techniques described above may be employed to detect,
localize and/or track certain
MDSCs in a patient diagnosed with a TGF31-associated disease, such as cancer
and fibrosis. Healthy individuals
have no or low frequency of MDSCs in circulation. With the onset of or
progression of such a disease, elevated
levels of circulating and/or disease-associated MDSCs may be detected. For
example, CCR2-positive M-MDSCs
have been reported to accumulate to tissues with inflammation and may cause
progression of fibrosis in the tissue
(such as pulmonary fibrosis), and this is shown to correlate with TGF31
expression. Similarly, MDSCs are enriched
in a number of solid tumors (including triple-negative breast cancer) and in
part contribute to the
immunosuppressive phenotype of the TME. Therefore, treatment response to TGF3
inhibition therapy such as a
TGF31 inhibitor therapy according to the present disclosure may be monitored
by localizing or tracking MDSCs.
Reduction of or low frequency of detectable MDSCs is typically indicative of
therapeutic benefits or better prognosis.
[861] Accordingly, the disclosure also includes a method for treating a TGF31-
related disease or condition which
may comprise the following steps: i) selecting a patient diagnosed with a
TGF31-related disease or condition; and,
ii) administering to the patient an antibody or the fragment encompassed
herein in an amount effective to treat the
disease or condition. In some embodiments, the selection step (i) comprises
detection of disease markers (e.g.,
fibrosis or cancer markers as described herein), wherein optionally the
detection comprises a biopsy analysis,
serum marker analysis, and/or in vivo imaging. In some embodiments, the
selection step (i) comprises an in vivo
imaging technique as described herein.
[862] The disclosure also includes a method for treating cancer which may
comprise the following steps: i)
selecting a patient diagnosed with cancer comprising a solid tumor, wherein
the solid tumor is or is suspected to
be an immune excluded tumor; and, ii) administering to the patient an antibody
or the fragment encompassed
herein in an amount effective to treat the cancer. Preferably, the patient has
received, is receiving, or is a candidate
for receiving a cancer therapy such as an immune checkpoint inhibition therapy
(e.g., a PD-(L)1 antibody), a
chemotherapy, a radiation therapy, an engineered immune cell therapy, or a
cancer vaccine therapy. In some
embodiments, the selection step (i) comprises detection of immune cells or one
or more markers thereof, wherein
optionally the detection comprises a tumor biopsy analysis, serum marker
analysis, and/or in vivo imaging. In some
embodiments, the selection step (i) comprises an in vivo imaging technique as
described here. In some
embodiments, the method further comprises monitoring for a therapeutic
response as described herein. In certain
embodiments, circulating MDSCs, such as G-MDSCs and M-MDSCs, are measured
before and after (e.g., 1-7
days or 1-10 weeks before or after) administering a therapeutically effective
dose of a TGF3 inhibitor such as a
TGF31 inhibitor described herein as an indicator of therapeutic efficacy
and/or a predictor of response.
[863] In some embodiments, in vivo imaging is performed for monitoring a
therapeutic response to the TGF31
inhibition therapy in the subject. The in vivo imaging can comprise any one of
the imaging techniques described
herein and measure any one of the markers and/or parameters described herein.
In the case of cancer, the
therapeutic response may comprise conversion of an immune excluded tumor into
an inflamed tumor (which
correlates with increased immune cell infiltration into a tumor), reduced
tumor size, and/or reduced disease
progression. Increased immune cell infiltration may be visualized by increased
intratumoral immune cell frequency
or degree of detection signals, such as radiolabeling and fluorescence.
[864] In some embodiments, the in vivo imaging used for diagnosing, selecting,
treating, or monitoring patients,
comprises MDSC tracking, such as tracking G-MDSCs (also known as PMN-MDSCs)
and M-MDSCs. For
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example, MDSCs (e.g., mMDSCs and/or gMDSCs) may be enriched at a disease site
(such as fibrotic tissues and
solid tumors) at the baseline. Upon administering a therapy (e.g., a TGFpl
inhibitor therapy), MDSCs levels may
decrease, as measured by reduced intensity of the label (such as radioisotope
and fluorescence), indicative of
therapeutic effects. In some embodiments, circulating MDSCs, including
circulating G-MDSCs and M-MDSCs, may
be detected in the blood or a blood component of the subject receiving a TGFP
inhibitor, e.g., a TGFP1 inhibitor,
e.g., Ab6. In some embodiments, MDSCs are detected, measured, or quantified by
the use of an antibody that
binds LRRC33. In some embodiments, mMDSCs and/or gMDSCs are detected,
measured, or quantified by
detecting a set of cell surface markers such as those provided herein.
[865] In certain embodiments, assays useful in determining the efficacy and/or
therapeutic response in a subject
treated with a TGFp inhibitor (e.g., Ab6) include, but are not limited to,
immunohistochemical or
immunofluorescence analyses of certain immune cell markers (e.g., flow
cytometry or immunohistochemistry)
known in the art for measuring levels of circulating MDSCs (e.g., G-MDSCs and
M-MDSCs). In some embodiments,
human G-MDSCs may be identified by the expression of the surface markers CD1
1b+, CD14-, C033+/low, C015+,
HLADR-, and CD66b+. In some embodiments, human G-MDSCs may also express HLA-
DR, LOX-1, and/or
Arginase. In some embodiments, M-MDSCs may be identified by the expression of
surface markers CD11b+,
CD33+/high, CD15-, HLADR-/low, CD66-, and CD14+. In some embodiments, the TGFp
inhibitors such as those
encompassed herein may be used to detect reduction in circulatory MDSCs, but
not levels of other circulatory
monocytes, after administration to a patient in need thereof. In some
embodiments, circulatory MDSCs are
measured by the expression of cell surface expressed LRRC33.
[866] In some embodiments, the in vivo imaging comprises tracking or
localization of LRRC33-positive cells.
LRRC33-positive cells include, for example, MDSCs and activated M2-like
macrophages (e.g., TAMs and activated
macrophages associated with fibrotic tissues). For example, LRRC33-positive
cells may be enriched at a disease
site (such as solid tumors) at the baseline. Upon therapy (e.g., TGFpl
inhibitor therapy), fewer cells expressing
cell surface LRRC33 may be observed, as measured by reduced intensity of the
label (such as radioisotope and
fluorescence), indicative of therapeutic effects.
[867] In some embodiments, the in vivo imaging techniques described herein may
comprise the use of PET-
SPECT, MRI and/or optical fluorescence/bioluminescence in order to detect
cells of interest.
[868] In some embodiments, labeling of antibodies or antibody-like molecules
with a detection moiety may
comprise direct labeling or indirect labeling.
[869] In some embodiments, the detection moiety may be a tracer. In some
embodiments, the tracer may be a
radioisotope, wherein optionally the radioisotope may be a positron-emitting
isotope. In some embodiments, the
radioisotope is selected from the group consisting of: 18F, 11C, 13N, 150,
68Ga, 177Lu, 18F and 89Zr.
[870] Thus, such methods may be employed to carry out in vivo imaging with the
use of labeled antibodies in
immune-PET.
[871] Accordingly, the disclosure also includes a method for treating a TGFpl
indication in a subject, which
method comprises a step of diagnosis, patient selection, and/or monitoring
therapeutic effects using an imaging
technique. In some embodiments, a TGFp inhibitor such as an isoform-selective
TGFpl inhibitor according to the
present disclosure is used in the treatment of a TGFP1 indication, wherein the
treatment comprises administration
of an effective amount of the TGFp inhibitor (e.g., Ab6) to treat the
indication, and further comprising a step of
monitoring therapeutic effects in the subject by in vivo imaging. Optionally,
the subject may be selected as a
candidate for receiving the TGF3 inhibitor therapy (e.g., a TGFpl inhibitor
therapy), using a diagnostic or selection
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step that comprises in vivo imaging. The TG931 indication may be a
proliferative disorder (such as cancer with a
solid tumor and myelofibrosis).
[872] In some embodiments, the subject has cancer, wherein the method
comprises the following steps: i)
selecting a patient diagnosed with cancer comprising a solid tumor, wherein
the solid tumor is or is suspected to
be an immune excluded tumor; and, ii) administering to the patient an antibody
or the fragment encompassed
herein in an amount effective to treat the cancer. Preferably, the patient has
received, or is a candidate for receiving
a cancer therapy such as immune checkpoint inhibition therapies (e.g., PD-(L)1
antibodies), chemotherapies,
radiation therapies, engineered immune cell therapies, and cancer vaccine
therapies. In some embodiments, the
selection step (i) comprises detection of immune cells or one or more markers
thereof, wherein optionally the
detection comprises a tumor biopsy analysis, serum marker analysis, and/or in
vivo imaging. In some
embodiments, the selection step (i) comprises an in vivo imaging technique as
described here. In some
embodiments, the method further comprises monitoring for a therapeutic
response as described herein.
Cell-Based Assays for Measuring TG93 Activation
[873] Activation of TGFI3 (and inhibition thereof by a TGFI3 test inhibitor,
such as an antibody) may be measured
by any suitable method known in the art. For example, integrin-mediated
activation of TGF[3 can be utilized in a
cell-based potency assay, such as the "CAGA12" reporter (e.g., luciferase)
assay, described in more detail herein.
As shown, such an assay system may comprise the following components: i) a
source of TGF13 (recombinant,
endogenous or transfected); ii) a source of activator such as integrin
(recombinant, endogenous, or transfected);
and iii) a reporter system that responds to TGFP activation, such as cells
expressing TGFP receptors capable of
responding to TGFp and translating the signal into a readable output (e.g.,
luciferase activity in CAGA12 cells or
other reporter cell lines). In some embodiments, the reporter cell line
comprises a reporter gene (e.g., a luciferase
gene) under the control of a TGFp-responsive promoter (e.g., a PAI-1
promoter). In some embodiments, certain
promoter elements that confer sensitivity may be incorporated into the
reporter system. In some embodiments,
such promoter element is the CAGA12 element. Reporter cell lines that may be
used in the assay have been
described, for example, in Abe et al., (1994) Anal Biochem. 216(2): 276-84,
incorporated herein by reference. In
some embodiments, each of the aforementioned assay components are provided
from the same source (e.g., the
same cell). In some embodiments, two of the aforementioned assay components
are provided from the same
source, and a third assay component is provided from a different source. In
some embodiments, all three assay
components are provided from different sources. For example, in some
embodiments, the integrin and the latent
TGFI3 complex (proTGFI3 and a presenting molecule) are provided for the assay
from the same source (e.g., the
same transfected cell line). In some embodiments, the integrin and the TGF are
provided for the assay from
separate sources (e.g., two different cell lines, a combination of purified
integrin and a transfected cell). When cells
are used as the source of one or more of the assay components, such components
of the assay may be
endogenous to the cell, stably expressed in the cell, transiently transfected,
or any combination thereof.
[874] A skilled artisan could readily adapt such assays to various suitable
configurations. For instance, a variety
of sources of TGFp may be considered. In some embodiments, the source of TGF[3
is a cell that expresses and
deposits TGFI3 (e.g., a primary cell, a propagated cell, an immortalized cell
or cell line, etc.). In some embodiments,
the source of TGFI3 is purified and/or recombinant TGFI3 immobilized in the
assay system using suitable means.
In some embodiments, TGFI3 immobilized in the assay system is presented within
an extracellular matrix (ECM)
composition on the assay plate, with or without de-cellularization, which
mimics fibroblast-originated TGF[3. In
some embodiments, TGFI3 is presented on the cell surface of a cell used in the
assay. Additionally, a presenting
molecule of choice may be included in the assay system to provide suitable
latent-TGFp complex. One of ordinary
skill in the art can readily determine which presenting molecule(s) may be
present or expressed in certain cells or
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cell types. Using such assay systems, relative changes in TGFp activation in
the presence or absence of a test
agent (such as an antibody) may be readily measured to evaluate the effects of
the test agent on TGFp activation
in vitro. Data from exemplary cell-based assays are provided in the Example
section below.
[875] Such cell-based assays may be modified or tailored in a number of ways
depending on the TGFp isoform
being studied, the type of latent complex (e.g., presenting molecule), and the
like. In some embodiments, a cell
known to express integrin capable of activating TGFp may be used as the source
of integrin in the assay. Such
cells include SW480/(36 cells (e.g., clone 1E7). In some embodiments, integrin-
expressing cells may be co-
transfected with a plasmid encoding a presenting molecule of interest (such as
GARP, LRRC33, LTBP (e.g., LTBP1
or LTBP3), etc.) and a plasmid encoding a pro-form of the TGFp isoform of
interest (such as proTGFI31). After
transfection, the cells are incubated for sufficient time to allow for the
expression of the transfected genes (e.g.,
about 24 hours), cells are washed, and incubated with serial dilutions of a
test agent (e.g., an antibody). Then, a
reporter cell line (e.g., CAGA12 cells) is added to the assay system, followed
by appropriate incubation time to
allow TGFp signaling. After an incubation period (e.g., about 18-20 hours)
following the addition of the test agent,
signal/read-out (e.g., luciferase activity) is detected using suitable means
(e.g., for luciferase-expressing reporter
cell lines, the Bright-Glo reagent (Promega) can be used). In some
embodiments, Luciferase fluorescence may be
detected using a BioTek (Synergy H1) plate reader, with autogain settings.
[876] Data demonstrating that exemplary antibodies of the disclosure which are
capable of selectively inhibiting
the activation of 1GFI31 in a context-independent manner can be found, e.g.,
in PCT/US2019/041390,
PCT/US2019/041373, and PCT/US2021/012930.
Nucleic Acids
[877] In some embodiments, antibodies, antigen binding portions thereof,
and/or compositions of the present
disclosure may be encoded by nucleic acid molecules. Such nucleic acid
molecules include, without limitation,
DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNA
molecules, vectors, plasmids and the
like. In some embodiments, the present disclosure may comprise cells
programmed or generated to express
nucleic acid molecules encoding compounds and/or compositions of the present
disclosure. In some cases, nucleic
acids of the disclosure include codon-optimized nucleic acids. Methods of
generating codon-optimized nucleic
acids are known in the art and may include, but are not limited to, those
described in US Patent Nos. 5,786,464
and 6,114,148, the contents of each of which are herein incorporated by
reference in their entirety.
List of Certain Embodiments
1. A method of treating cancer in a subject, wherein the treatment
comprises administering to the subject a
TGFp inhibitor in an amount sufficient to reduce circulating MDSC levels.
2. The method of embodiment 1, wherein the reduced circulating MDSCs are G-
MDSCs.
3. The method of embodiment 1 or 2, wherein the G-MDSCs express one or more
of LOX-1+, CD11b+,
HLADR-, CD14-, CD15+, CD33+/low, and CD66+.
4. The method of any one of embodiments 1-3, wherein the treatment further
comprises administering a
cancer therapy.
5. The method of any one of embodiments 1-4, wherein the TGFp inhibitor and
the cancer therapy are
administered concurrently (e.g., simultaneously), separately, or sequentially.
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6. The method of embodiment 4 or 5, comprising determining whether
a subject has a reduction in
circulating MDSC levels following administration of the TGFp inhibitor, and
administering the cancer therapy if
MDSC levels have been reduced.
7. The method of any one of embodiments 1-6, wherein the cancer
therapy comprises a checkpoint
inhibitor therapy, optionally an agent targeting PD-1 or PD-L1, optionally an
anti-PD-1 or anti-PD-L1 antibody.
8. A method of predicting therapeutic efficacy in a subject having
cancer, comprising:
(i) determining circulating MDSC levels in the subject prior to administering
a TGFp inhibitor (alone or in
combination with a cancer therapy);
(ii) administering to the subject a therapeutically effective amount of the
TGFp inhibitor (alone or in
combination with a cancer therapy); and
iii) determining circulating MDSC levels in the subject after the
administration, wherein a reduction in
circulating MDSC levels after administration, as compared to circulating MDSC
levels before administration,
predicts pharmacological effects.
9. A method of treating cancer in a subject, comprising the steps
of:
(i) determining circulating MDSC levels in the subject prior to administering
a TGFP inhibitor;
(ii) administering to the subject a first therapeutically effective dose of
the TGFp inhibitor;
(iii) determining circulating MDSC levels in the subject after administering
the TGFI3 inhibitor;
(iv) administering to the subject a second therapeutically effective dose of
the TGFp inhibitor if the
circulating MDSC levels measured after administering the first therapeutically
effective dose of the TGFp inhibitor
are reduced as compared to the circulating MDSC levels measured prior to
administering the first therapeutically
effective dose of the TGFp1 inhibitor.
10. The method of embodiment 8 or embodiment 9, comprising
administering a checkpoint inhibitor therapy
concurrently (e.g., simultaneously), separately, or sequentially with the TGFp
inhibitor.
11. A method of treating cancer in a subject, comprising the steps
of:
(i) determining circulating MDSC levels in the subject prior to administering
a combination therapy
comprising a therapeutically effective amount of a TGFp inhibitor and a
therapeutically effective amount of a
checkpoint inhibitor therapy;
(ii) administering to the subject the combination therapy;
(iii) determining circulating MDSC levels in the subject after administering
the combination therapy;
(iv) continuing the combination therapy if the circulating MDSC levels
measured after administering the
first therapeutically effective dose of the combination therapy are reduced as
compared to the circulating MDSC
levels measured prior to administering the first therapeutically effective
dose.
12. The method of embodiment 11, wherein the combination therapy
comprises administering the TGFp
inhibitor concurrently (e.g., simultaneously), separately, or sequentially
with the checkpoint inhibitor therapy.
13. A method of treating advanced cancer in a human subject, the
method comprising the steps of
i) selecting a subject with advanced cancer comprising a locally advanced
tumor and/or metastatic
cancer with primary resistance to a checkpoint inhibitor therapy,
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ii) administering a TGFp inhibitor; and,
ii) administering to the subject a checkpoint inhibitor therapy.
14 The method of embodiment 13, wherein the checkpoint inhibitor
therapy is administered concurrently
(e.g., simultaneously), separately, or sequentially with the TGFp inhibitor.
15. A method for treating advanced cancer in a human subject, the
method comprising the steps of
i) selecting a subject with advanced cancer comprising a locally advanced
tumor and/or metastatic
cancer with primary resistance to a CPI therapy, wherein the subject has
received a TGFr3 inhibitor which is a
TGFP1-selective inhibitor or a TGFP inhibitor that does not inhibit TGFI33;
and,
ii) administering to the subject a CPI therapy, optionally in conjunction with
the TG93 inhibitor.
16 The method of any one of embodiments 13-15, further comprising
measuring the levels of circulating
MDSC levels before and after administering the treatment, wherein a reduction
in circulating MDSC levels is
indicative of a treatment response.
17. The method of embodiment 16, further comprising continuing the
treatment if a reduction in circulating
MDSC levels is determined.
18. The method of any one of embodiments 8-17, wherein the reduced
circulating MDSCs are G-MDSCs.
19. The method of embodiment 18, wherein the G-MDSCs express one or more of
LOX-1+, CD11b+,
HLADR-, CD14-, CD15+, CD33+/low, and CD66+.
20. The method of any one of embodiments 1-19, wherein the circulating MDSC
levels are determined from
whole blood or a blood component collected from the subject.
21 The method of any one of embodiments 1-20, wherein the
treatment reduces circulating MDSC levels
by at least 10%, optionally by at least 15%, 20%, 25%, or more.
22. The method of any one of embodiments 1-21, wherein the TGFI3
inhibitor is a TGFp1 inhibitor, optionally
a TGFp1-specific inhibitor.
23 The method of any one of embodiments 1-21, wherein the subject
has circulating MDSC levels at least
2-fold above circulating MDSC levels in a healthy subject prior to a
treatment.
24 A method of selecting a subject for treatment, wherein the
subject has circulating MDSC levels at least
2-fold above circulating MDSC levels in a healthy subject prior to treatment.
25 The method of 24, wherein the subject has or is suspected of
having cancer.
26 A method of treating a subject for cancer, wherein the subject
has circulating MDSC levels at least 2-
fold above circulating MDSC levels in a healthy subject prior to treatment,
comprising administering to the subject
a TG93 inhibitor in an amount sufficient to reduce circulating MDSC levels.
27 The method of any one of embodiments 1-26, wherein the level of
circulating MDSC cells is determined
within 3-6 weeks, e.g., within or at about 3 weeks, following administration
of a TGFp inhibitor.
28 The method of any one of embodiments 1-27, wherein the level of
circulating MDSC cells is determined
within 2 weeks, e.g., 10 days, following administration of the TGFp inhibitor.
29. The method of any one of embodiments 1-28, further comprising
the steps of:
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(i) determining the levels of tumor-associated immune cells in the subject
prior to administering a
treatment;
(ii) administering the treatment to the subject; and
(iii) determining the levels of tumor-associated immune cells in the subject
after administering the
treatment;
wherein a change in the level of one or more tumor-associated immune cell
populations after inhibitor
administration, as compared to the levels of tumor-associated immune cells
before administration, indicates
therapeutic efficacy.
30. The method of embodiment 29, wherein a change in levels of
tumor-associated immune cells in step (iii)
indicates reduction or reversal of immune suppression in the cancer.
31. The method of embodiment 29 or 30, wherein the tumor-associated
immune cells comprise CD8+ T
cells and/or tumor-associated macrophages (TAMs).
32. The method of embodiments 29-31, wherein the change in the
levels of tumor-associated immune cells
comprises at least a 10%, optionally at least a 15%, 20%, 25%, or more,
increase in CD8+ T cell levels.
33. The method of embodiments 29-32, wherein the change in the
levels of tumor-associated immune cells
comprises at least a 10%, optionally at least a 15%, 20%, 25%, or more,
increase in the level of TAMs.
34. The method of any one of embodiments 29-33, wherein the levels
of tumor-associated immune cells are
determined in a sample collected from the subject by immunohistochemistry
analysis.
35. The method of any one of embodiments 29-34, wherein the levels
of tumor-associated immune cells are
determined by in vivo imaging.
36. The method of any one of embodiments 29-34, wherein the sample
is a tumor biopsy sample.
37. The method of any one of embodiments 29-36, further comprising
continuing to administer the treatment
if a change in the level of one or more tumor-associated immune cell
populations is detected.
38. The method of any one of embodiments 1-37, further comprising
the steps of:
(i) determining the levels of circulating latent TGFp in the subject prior to
administering a treatment;
(ii) administering the treatment to the subject; and
(iii) determining the levels of circulating latent TGFp in the subject after
administering the treatment; and
wherein an increase in circulating latent TGF13 after inhibitor
administration, as compared to circulating
latent TGFp before administration, indicates therapeutic efficacy.
39. The method of embodiment 38, further comprising continuing to
administer the treatment if a change in
the level of circulating latent TGFp is detected.
40 The method of 38 or 39, wherein the level of circulating latent
TGFp is determined in a sample obtained
from the subject.
41 The method of 40, wherein the sample is a whole blood sample or
a blood component.
42 The method of any one of embodiments 39-41, wherein the
circulating latent TGFp is circulating latent
TGFpl.
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43. A method of treating cancer, comprising administering to a subject a
TGFp inhibitor in a therapeutically
effective amount that does not cause a significant release of one or more
cytokines selected from interferon
gamma (IFNy), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis
factor alpha (TNFa), interleukin 1 beta (IL-
113), and chemokine C-C motif ligand 2 (CCL2) / monocyte chemoattractant
protein 1 (MCP-1).
44. A method for identifying whether a TGFI3 inhibitor will be tolerated in
a patient, comprising contacting a
cell culture or fluid sample with the TGFp inhibitor and determining whether
it causes a significant release of one
or more cytokines selected from interferon gamma (IFNy), interleukin 2 (IL-2),
interleukin 6 (IL-6), tumor necrosis
factor alpha (TNFa), interleukin 1 beta (IL-1p) and chemokine C-C motif ligand
2 (CCL2) / monocyte
chemoattractant protein 1 (MCP-1), wherein a significant release indicates the
TGFp inhibitor will not be well
tolerated.
45. The method of embodiment 43 or 44, wherein cytokine release is assessed
in an in vitro cytokine
release assay, optionally an assay in peripheral blood mononuclear cells
(PBMCs) or whole blood, optionally
wherein the PBMCs or whole blood are obtained from the subject prior to
administering a TGFp inhibitor therapy.
46. The method of any one of embodiments 43-45, wherein cytokine release is
assessed in an in vitro
cytokine release assay of peripheral blood mononuclear cells (PBMCs) or whole
blood obtained from a healthy
subject.
47. The method of embodiment 45 or 46, wherein the cytokine release assay
comprises a soluble phase
and/or a solid phase assay format.
48. The method of any one of embodiments 45-47, wherein the cytokine
release assay comprises: i) a solid
phase assay, ii) a high-density PBMC pre-culture assay, and/or iii) a PBL-1-
11.JVEC co-culture assay.
49. The method of any one of embodiments 45-48, wherein the cytokine
release assay comprises a
multiplex array, e.g., a Luminexa array system.
50. The method of any one of embodiments 45-49, wherein the cytokine
release assay comprises
comparing cytokine release from a TGFp inhibitor to release from one or more
control antibodies selected from
an anti-CD3 antibody and an anti-CD28 antibody, optionally wherein the CO28
antibody optionally comprises
TGN1412.
51. The method of any one of embodiments 43-50, wherein the TGFp inhibitor
does not induce more than a
10-fold increase in IL-6 levels, optionally less than a 2-fold, 4-fold, 6-
fold, or 8-fold increase in IL-6 levels, as
compared to levels in the absence of the inhibitor or in the presence of a
control antibody.
52. The method of any one of embodiments 43-51, wherein the TGFp inhibitor
does not induce more than a
10-fold increase in IFNy levels, optionally less than a 2-fold, 4-fold, 6-
fold, or 8-fold increase in IFNy levels, as
compared to levels in the absence of the inhibitor or in the presence of a
control antibody.
53. The method of embodiments 43-52, wherein the TGFp inhibitor does not
induce more than a 10-fold
increase in TNFa levels, optionally less than a 2-fold, 4-fold, 6-fold, or 8-
fold increase in TNFa levels, as
compared to levels in the absence of the inhibitor or in the presence of a
control antibody.
54. The method of embodiment any one of embodiments 43-53, wherein the TGFp
inhibitor is administered
in a therapeutically effective amount that does not cause a significant
release of one or more cytokines in an
animal model comprising a non-human primate.
55. The method of embodiments 43-54, wherein the therapeutically effective
amount of the TGFP inhibitor is
an amount sufficient to reduce circulating MDSC levels.
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56. The method of embodiment 55, wherein the reduced MDSCs are G-MDSCs.
57. The method of embodiment 56, wherein the G-MDSCs express one or more of
LOX-1+, CD11b+,
HLADR-, CD14-, CD15+, CD33+/low, and CD66+.
58. The method of any one of embodiments 55-57, wherein the circulating
MDSC levels are determined
from whole blood or a blood component collected from the subject.
59. The method of any one of embodiments 43-58, wherein the TGFp inhibitor
is a TGFp1 inhibitor,
optionally a TGF(31-specific inhibitor, e.g., Ab6.
60. A TGFI3 inhibitor for use in the treatment of cancer in a subject,
wherein the treatment comprises
administration of a dose of said TGFI3 inhibitor to the subject having cancer,
wherein said TGFp inhibitor does not
cause a significant release of one or more cytokines selected from interferon
gamma (IFNy), interleukin 2 (IL-2),
interleukin 6 (IL-6), tumor necrosis factor alpha (TNFa), interleukin 1 beta
(IL-1p) and chemokine C-C motif ligand
2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1).
61. A combination therapy comprising a dose of a TGFI3 inhibitor and a
cancer therapy agent for use in the
treatment of cancer, wherein the treatment comprises simultaneous, separate or
sequential administration to a
subject of a dose of the TGFI3 inhibitor and the cancer therapy agent, wherein
said TGFI3 inhibitor does not cause
a significant release of one or more cytokines selected from interferon gamma
(IFNy), interleukin 2 (IL-2),
interleukin 6 (IL-6), tumor necrosis factor alpha (TNFa), interleukin 1 beta
(IL-1p) and chemokine C-C motif ligand
2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1).
62. The TGFp inhibitor for use according to embodiment 60 or the
combination therapy for use according to
embodiment 61, wherein the TGFI3 inhibitor is administered in a
therapeutically effective amount that does not
cause a significant release of one or more cytokines in an animal model
comprising a non-human primate.
63. The TGFI3 inhibitor for use according to embodiment 60 or 62, or the
combination therapy for use
according to embodiment 61, wherein the TGF13 inhibitor is administered in a
therapeutically effective amount
that is sufficient to reduce circulating MDSC levels.
64 The TGFp inhibitor for use or the combination therapy for use
according to embodiment 63, wherein the
reduced MDSCs are G-MDSCs.
65 The TGFI3 inhibitor for use or the combination therapy for use
according to embodiment 64, wherein the
G-MDSCs express one or more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+,
CD33+/low, and CD66+.
66 The TGFI3 inhibitor for use or the combination therapy for use
according to any one of embodiments 63-
65, wherein the circulating MDSC levels are determined from whole blood or a
blood component collected from
the subject.
67. The method of embodiment 44 or any embodiment dependent thereon, the
TGFI3 inhibitor for use
according to embodiment 60 or any embodiment dependent thereon, or the
combination therapy for use
according to embodiment 61 or any embodiment dependent thereon, wherein the
TGFp inhibitor is a TGFp1
inhibitor, optionally a TGF131-specific inhibitor.
68. The TGFp inhibitor for use according to embodiment 60 or any embodiment
dependent thereon, or the
combination therapy for use according to embodiment 61 or any embodiment
dependent thereon, wherein the
TGFI3 inhibitor does not cause significant release of one or more cytokines as
determined by the method of
embodiment 44 or any embodiment dependent thereon.
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69. A method of treating cancer, comprising administering to a subject a
TGF13 inhibitor in a therapeutically
effective amount that does not induce a significant increase in platelet
binding, activation, and/or aggregation.
70. The method of embodiment 69, wherein platelet binding, activation,
and/or aggregation is measured in a
sample of plasma or whole blood.
71. A method for determining whether a TGFp inhibitor causes a significant
increase in platelet binding,
activation and/or aggregation following exposure of the sample to said TGFp
inhibitor, which method comprises
measuring platelet binding, activation and/or aggregation in a blood sample.
72 The method of any one of embodiments 70 or 71, wherein the
sample is obtained from the subject prior
to administering a TGFp inhibitor therapy.
73. The method of any one of embodiment 69-72, wherein the sample is
obtained from a healthy subject.
74. The method of any one of embodiments 69-73, wherein administering the
TGFp inhibitor does not
increase platelet binding by more than 10% as compared to binding in the
absence of the TGFp inhibitor and/or
in the presence of a buffer or isotype control.
75. The method of any one of embodiments 69-74, wherein administering the
TGFp inhibitor does not
increase platelet activation by more than 10%, as compared to activation in
the absence of the inhibitor.
76. The method of any one of embodiments 69-75, wherein administering the
TGFp inhibitor does not
increase platelet aggregation in vitro by more than 10% of the activation
induced by a known platelet aggregation
agonist, e.g., adenosine diphosphate (ADP).
77. The method of any one of embodiments 69-76, wherein administering the
TGFp inhibitor does not
increase platelet aggregation by more than 10%, as compared to aggregation
caused by a negative control.
78. The method of embodiments any one of 69-77, wherein the therapeutically
effective amount of the
TGFp inhibitor is an amount sufficient to reduce levels of circulating MDSCs.
79. The method of embodiment 78, wherein the reduced MDSCs are G-MDSCs.
80. The method of embodiment 79, wherein the G-MDSCs express one or more of
LOX-1+, CD11b+,
HLADR-, CD14-, CD15+, CD33+/low, and C066+.
81. The method of any one of embodiments 78-80, wherein the circulating
MDSC levels are determined
from whole blood or a blood component collected from the subject.
82. The method of any one of embodiments 69-81, wherein the TGFp inhibitor
is a TGF(31 inhibitor,
optionally a TGFp1-specific inhibitor.
83. A TGFp inhibitor for use in the treatment of cancer by administering to
a subject a dose of said TGFp
inhibitor, wherein said TGFp inhibitor does not cause a significant increase
in platelet binding, activation and/or
aggregation.
84. A combination therapy comprising a dose of a TGFp inhibitor and a
cancer therapy agent for the
treatment of cancer, wherein the treatment comprises simultaneous, separate,
or sequential administration to a
subject of a dose of the TGFp inhibitor and the cancer therapy agent, wherein
said TGFp inhibitor does not cause
a significant increase in platelet binding, activation and/or aggregation.
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85. The TGFp inhibitor for use according to embodiment 83 or the
combination therapy for use according to
embodiment 84, wherein the TGFp inhibitor is administered in a therapeutically
effective amount that is sufficient
to reduce circulating MDSC levels.
86 The TGFp inhibitor for use or the combination therapy for use
according to embodiment 85, wherein the
reduced MDSCs are G-MDSCs.
87 The TGFp inhibitor for use or the combination therapy for use
according to embodiment 85, wherein the
M-MDSCs express one or more of LOX-1+, CD11 b+, HLADR-, CD14-, CD15+,
CD33+/low, and CD66+.
88 The TGFp inhibitor for use or the combination therapy for use
according to embodiment 85, wherein the
reduced MDSCs are M-MDSCs that express one or more of CD11b+, HLADR-/low,
CD14+, CD15-, CD33+/high,
and CD66b-.
89. The method of embodiment 71 or any embodiment dependent thereon, the
TGF13 inhibitor for use
according to embodiment 83 or any embodiment dependent thereon, or the
combination therapy for use
according to embodiment 84 or any embodiment dependent thereon, wherein the
TGFp inhibitor is a TGF[31
inhibitor, optionally a TGFpl-specific inhibitor.
90. The TGFI3 inhibitor for use according to embodiment 83 or any
embodiment dependent thereon, or the
combination therapy for use according to embodiment 84 or any embodiment
dependent thereon, wherein the
TGFp inhibitor has been determined not to cause a significant increase in
platelet binding, activation and/or
aggregation by the method of embodiment 71 or any embodiment dependent
thereon.
91. The TGFp inhibitor for use according to embodiment 83 or any embodiment
dependent thereon, or the
combination therapy for use according to embodiment 84 or any embodiment
dependent thereon, wherein the
TGFp inhibitor is a TGFI3 inhibitor according to any one of embodiments 60-68.
92. A method of making a TGFp inhibitor for treating cancer in a subject,
comprising the steps of selecting a
TGFI3 inhibitor which satisfies one or more, e.g., all of, the following
criteria:
a) the TGFp inhibitor is efficacious in one or more preclinical models;
b) the TGFp inhibitor does not cause valvulopathies or epithelial hyperplasia
in toxicology studies in one
or more animal species at a dose at least greater than a minimum efficacious
dose; and
C) the TGFp inhibitor does not induce significant cytokine release from human
PBMCs or whole blood in
an in vitro cytokine release assay at the minimum efficacious dose as
determined in the one or more preclinical
models of (a);
93 A method of making a TGFI3 inhibitor for treating cancer in a
subject, comprising the steps of selecting a
TGFp inhibitor which satisfies one or more, e.g., all of, the following
criteria:
a) the TGFp inhibitor is efficacious in one or more preclinical models;
b) the TGFp inhibitor does not cause valvulopathies or epithelial hyperplasia
in toxicology studies in one
or more animal species at a dose at least greater than a minimum efficacious
dose;
C) the TGFp inhibitor does not induce significant cytokine release from human
PBMCs or whole blood in
an in vitro cytokine release assay at the minimum efficacious dose as
determined in the one or more preclinical
models of (a);
d) the TGFp inhibitor does not induce a significant increase in platelet
binding, activation, and/or
aggregation at the minimum efficacious close as determined in the one or more
preclinical models of (a); and
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e) the TGF3 inhibitor reduces circulating MDSCs at the minimum efficacious
dose as determined in the
one or more preclinical models of (a),
wherein the method further comprises manufacturing a pharmaceutical
composition comprising the TGF3
inhibitor and a pharmaceutically acceptable excipient.
94. The method of embodiment 92 or 93, wherein the TGF13 inhibitor is a
TGF31 inhibitor, optionally a
TGF31-specific inhibitor.
95. A method of treating cancer in a subject, comprising administering a
therapeutically effective amount of
the TGF3 inhibitor manufactured according to the method of any one of
embodiments 92-94.
96. A TG93 inhibitor for use in an intermittent dosing regimen for cancer
immunotherapy in a patient,
wherein the intermittent dosing regimen comprises:
(i) measuring circulating MDSCs in a first sample, e.g., a blood sample,
collected from the patient prior
to a TGF3 inhibitor treatment,
(ii) administering a TGF3 inhibitor to the patient treated with a cancer
therapy, wherein the cancer
therapy is optionally a checkpoint inhibitor therapy,
(iii) measuring circulating MDSCs in a second sample collected from the
patient after the TGF3 inhibitor
treatment,
(iv) continuing with the cancer therapy if the second sample shows reduced
levels of circulating MDSCs
as compared to the first sample; and
(v) repeating the process as needed after a further blood sample from a
patient shows elevated levels of
circulating MDSC levels.
97 The method of embodiment 96, further comprising measuring
circulating MDSCs in a third sample, and
administering to the patient an additional dose of a TGF3 inhibitor if the
third sample shows elevated levels of
circulating MDSC levels as compared to the second sample.
98. The method of embodiment 96 or 97, wherein the TGF13 inhibitor inhibits
TG931 signaling.
99. The method of embodiment 96 or 97, wherein the TGF3 inhibitor inhibits
TGF31 signaling but does not
inhibit TGFI32 signaling and/or TGF33 signaling at a therapeutically effective
dose.
100. The method of embodiment 96 or 97, wherein the TGF13 inhibitor is
TGF31-selective.
101. The method of embodiment 96 or 97, wherein the TGF13 inhibitor is an
integrin inhibitor.
102. The method of 101, wherein the integrin inhibitor inhibits integrin
0V31, 0V33, aV35, aN/36, c6/138, a531,
a11b33, and/or a8(31.
103. The method of 101 or 102, wherein the integrin inhibitor inhibits
downstream TGF31/3 activation.
104. A TGF31-selective inhibitor for use in the treatment of cancer in a
subject, wherein the subject has been
treated with a TGF3 inhibitor that inhibits TGF33 in conjunction with a
checkpoint inhibitor.
105. The method of 104, wherein the cancer is a metastatic cancer, a
desmoplastic tumor, or myelofibrosis.
106. The method of 104 or 105, wherein the subject has a disorder involving
dysregulated ex-tracellular matrix
(ECM) or is at risk of developing such a disorder.
107. The method of 106, wherein the disorder involving dysregulated ECM is
NASH,
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108. The TGFp1-selective inhibitor for use according to any one of
embodiments 104-107, wherein the prior
TGFp inhibitor inhibits TGF131/2/3 or TGF131/3.
109. A non-isoform-selective TGFp inhibitor for use in the treatment
of cancer in a subject, comprising the
steps of:
(i) selecting a subject who is not diagnosed with a fibrotic disorder or who
is not at high risk of
developing a fibrotic disorder; and,
(ii) administering to the subject the non-isoform-selective TGFp inhibitor in
an amount effective to treat
the cancer.
110. An isoform-non-selective TGFp inhibitor for use in the
treatment of cancer in a subject, wherein the
treatment comprises the steps of selecting a subject whose cancer is not a
highly metastatic cancer and
administering to the subject the isoform-non-selective TGFp inhibitor.
111. The method of 109 or 110, wherein the isoform-non-selective
TGFp inhibitor is an antibody that inhibits
TGF131/2/3 or TGF131/3.
112 The method of any one of embodiments 109-111, wherein the
isoform-non-selective TGFp inhibitor is an
integrin inhibitor binding to integrins aVp1, aVp6, aV138, and/or aVp3.
113 The method of 112, wherein the integrin inhibitor is an
inhibitor of TGF131/3 activation.
114. The method of 110, wherein the isoform-non-selective TGFp
inhibitor is an engineered construct
comprising a TGFP receptor ligand-binding moiety.
115. The isoform-non-selective TG93 inhibitor for use according to
embodiment 110, wherein the highly
metastatic cancer is colorectal cancer, lung cancer (e.g., NSCLC), bladder
cancer, kidney cancer (e.g.,
transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC),
including clear cell RCC, papillary
RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC, uterine
cancer, prostate cancer, stomach
cancer, or thyroid cancer.
116. A TGFpl-selective inhibitor for use in the treatment of cancer
in a subject wherein the treatment
comprises the steps of
(i) selecting a subject whose cancer is a highly metastatic cancer, and
(ii) administering to the subject an isoform-selective TGF(31 inhibitor;
wherein the highly metastatic cancer comprises colorectal cancer, lung cancer,
bladder cancer, kidney cancer
(e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma
(RCC), including clear cell RCC,
papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC,
uterine cancer, prostate cancer,
stomach cancer, or thyroid cancer.
117. A TG931-selective inhibitor for use in the treatment of cancer
in a subject wherein the treatment
comprises the steps of:
(i) selecting a subject having myelofibrosis, a fibrotic disorder or is at
risk of developing a fibrotic
disorder, and,
(ii) administering to the subject an isoform-selective TGFp1 inhibitor in an
amount effective to treat the
cancer.
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118. The TGFp1-selective inhibitor for use according to embodiment 117,
wherein the subject is further
treated with a cancer therapy, wherein optionally the cancer therapy comprises
a checkpoint inhibitor.
119. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of the preceding embodiments, wherein the subject is a patient who
has not received cancer therapy.
120. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of the preceding embodiments, wherein the subject is receiving
cancer therapy.
121. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of the preceding embodiments, wherein the subject has previously
received cancer therapy.
122. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of the preceding embodiments, wherein the subject is or will be
receiving cancer therapy.
123. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of the preceding embodiments, wherein the subject has cancer that
is resistant to a cancer therapy
that does not comprise a TGFI3 inhibitor.
124. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of the preceding embodiments, wherein the subject is poorly
responsive to a cancer therapy that does
not comprise a TGFp inhibitor.
125. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of the preceding embodiments, wherein the subject is currently
receiving or previously received a
cancer therapy that does not comprise a TGFP inhibitor.
126. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of embodiments 121-125, wherein the cancer therapy does not
comprise a TGFp inhibitor.
127. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of embodiments 121-126, wherein the cancer therapy comprises a
chemotherapy, radiation therapy
(e.g., a radiotherapeutic agent), engineered immune cell therapy (e.g., CAR-T
therapy), oncolytic viral therapy,
and/or cancer vaccine therapy.
128. The method, the medical use, the TGFp inhibitor for use, or the
combination therapy for use according
to any one of embodiments 121-127, wherein the cancer therapy comprises
immunotherapy comprising a
checkpoint inhibitor therapy.
129. A method of treating a subject having a solid cancer, comprising
determining the level of cytotoxic T
cells (e.g., CD8+ T cells) in a sample obtained from the subject prior to
administering a TGFp inhibitor, wherein
the level of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor is lower
than the level of cytotoxic T cells (e.g.,
CD8+ T cells) outside the tumor prior to treatment, and administering to the
subject a therapeutically effective
amount of a TGFP inhibitor, wherein the therapeutically effective amount is an
amount sufficient to increase the
level of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor relative to
the level outside the tumor.
130. A method of treating a subject having a solid cancer, comprising
determining in a sample obtained from
the subject the cytotoxic T cell (e.g., CD8+ T cell) levels inside and outside
the tumor, selecting a subject having
a ratio of cytotoxic T cell (e.g., CD8+ T cell) levels inside the tumor to
outside the tumor of less than 1, and
administering to the subject a therapeutically effective amount of a TGFp
inhibitor.
1 31 . The method of embodiment 129 or 130, wherein the level of
cytotoxic T cells (e.g., CD8+ T cells)
outside the tumor is determined from the tumor margin and/or stroma.
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132. The method of any one of embodiments 129-131, wherein the level of
cytotoxic T cells (e.g., CD8+ T
cells) outside of the tumor is determined from the margin.
133. The method of embodiment 131 or embodiment 132, wherein the margin is
approximately 10-100 pm in
width (e.g., 50 pm in width).
134. The method of any one of embodiments 129-133, wherein the level of the
cytotoxic T cells (e.g., CD8+ T
cells) in the margin and/or the stroma is at least 2-fold, 3-fold, 4-fold, 5-
fold, 7-fold, or 10-fold greater than the
level inside the tumor.
135. A method of treating a subject having a solid cancer, comprising
measuring levels of CD8+ cells in one
or more tumor nests from at least one tumor tissue sample obtained from the
subject, and administering to the
subject a therapeutically effective amount of a TGF6 inhibitor if greater than
50% of the area of the sample
measured comprises tumor nests comprising lower levels of CD8-positive cells
inside the tumor nest relative to
levels of CD8-positive cells outside of the tumor nest (e.g., less than 5%
CD8+ cells inside the tumor nest and
greater than 5% CD8+ cells outside the tumor nest).
136. A method of treating a subject having a solid cancer, comprising:
(i) determining the cytotoxic T cell (e.g., CDS+ T cell) levels inside and
outside the tumor in a first
sample and selecting a subject having a ratio of cytotoxic T cell (e.g., CD8+
T cell) density inside the tumor to
density outside the tumor of less than 1;
(ii) administering to the subject a first dose of a TGF6 inhibitor; and
(iii) determining the level of cytotoxic T cells (e.g., CD8+ T cells) inside
the tumor in a second sample;
and
(iv) administering to the subject a second dose of the TGF6 inhibitor if the
level of cytotoxic T cells (e.g.,
CD8+ T cells) inside the tumor determined in step (iii) is increased as
compared to the level of cytotoxic T cells
(e.g., CD8+ T cells) inside the tumor determined in step (i).
137. A method of determining therapeutic efficacy of a cancer treatment in
a subject comprising:
(i) determining the level of cytotoxic T cells (e.g., CD8+ T cells) inside the
tumor in a first sample;
(ii) administering to the subject a dose of a TGF6 inhibitor;
(iii) determining the level of cytotoxic T cells (e.g., CD8+ T cells) inside
the tumor in a second sample;
and
(iv) determining whether the level of cytotoxic T cells (e.g., CD8+ T cells)
determined in step (iii) is
increased as compared to step (i), such increase being indicative of
therapeutic efficacy of the cancer treatment.
138. The method of embodiment 136 or embodiment 137, wherein step (ii)
further comprises administering to
the subject an additional cancer therapy simultaneously, separately, or
sequentially to the TGF13 inhibitor.
139. The method of embodiment 138, wherein the cancer therapy comprises a
checkpoint inhibitor therapy
(e.g., an agent targeting PD-1 or PD-L1, or an anti-PD-1 or anti-PD-L1
antibody).
140. The method of any one of embodiments 136-139, wherein the level of
cytotoxic T cells (e.g., CD8+ T
cells) in the tumor is increased by at least 10%, 15%, 20%, 25%, or more.
141. The method of any one of embodiments 129-140, wherein the TGF6
inhibitor is a TG961-selective
inhibitor, e.g., Ab6.
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142. The method of any one of embodiments 129-141, wherein the sample is a
tumor biopsy sample.
143. The method of embodiment 142, wherein the tumor biopsy sample is a
core needle biopsy sample of
the tumor.
144. The method of any of embodiments 129-143, wherein the level of
cytotoxic T cells (e.g., CD8+ T cells)
are determined by immunohistochemical analysis.
145. The method of embodiment 136, further comprising determining the level
of circulating MDSCs before
and after administration of the first dose of the TGFp inhibitor, wherein a
second dose of the TG93 inhibitor is
administered if a reduction of MDSC levels is determined after the
administration of the first dose of the TGFp
inhibitor and the level of cytotoxic T cells (e.g., CD8+ T cells) inside the
tumor determined in step (iii) is increased
as compared to the level of cytotoxic T cells (e.g., CD8+ T cells) inside the
tumor determined in step (i).
146. The method of embodiment 137, further comprising determining the level
of circulating MDSCs before
and after administration of the TGFp inhibitor, wherein a reduction of MDSC
levels and/or an increase in cytotoxic
T cells (e.g., CD8+ T cells) levels inside the tumor after the administration
indicates therapeutic efficacy.
147 The method of embodiment 145 or 146, wherein the circulating
MDSCs are G-MDSCs.
148. The method of embodiment 147, wherein the G-MDSCs express one or more
of LOX-1+, CD11b+,
HLADR-, CD14-, CD15+, CD33+/low, and CD66+.
149. The method of any one of embodiments 145-148, wherein the circulating
MDSC levels are determined
from whole blood or a blood component collected from the subject.
150. The method of any one of embodiments 145-149, wherein the level of
circulating MDSC levels is
reduced by at least 10%, optionally by at least 15%, 20%, 25%, or more.
151. The method of any one of embodiments 145-150, wherein the level of
cytotoxic T cells (e.g., CD8+ T cells)
inside the tumor is increased by at least 10%, 15%, 20%, 25%, or more, and the
level of circulating MDSCs is
decreased by at least 15%, 20%, 25%, or more.
152. The method of any one of embodiments 129-151, wherein the level of
cytotoxic T cells (e.g., CD8+ T cells)
is the percentage of CD8+ T cells or the CD8+ cell density (e.g., number of
CD8+ T cells per millimeter squared).
153. The method of any one of embodiments 129-152, wherein the
therapeutically effective amount of the
TGFp inhibitor is between 0.1 mg/kg to 30 mg/kg per dose.
154. The method of any one of embodiments 129-153, wherein the
therapeutically effective amount of the
TGFp inhibitor is between 1 mg/kg and 10 mg/kg per dose.
155. The method of any one of embodiments 129-154, wherein the
therapeutically effective amount of the
TGFp inhibitor is between 2 mg/kg and 7 mg/kg per dose.
156. The method of any one of embodiments 129-155, wherein the TGFI3
inhibitor is dosed weekly, every 2
weeks, every 3 weeks, every 4 weeks, monthly, every 6 weeks, every 8 weeks,
bimonthly, every 10 weeks, every
12 weeks, every 3 months, every 4 months, every 6 months, every 8 months,
every 10 months, or once a year.
157. The method of any one of embodiments 129-156, wherein the TGFp
inhibitor is dosed about every 3
weeks.
158. The method of any one of embodiments 129-157, wherein the TGFp
inhibitor is administered
intravenously or subcutaneously.
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159. The method of any one of embodiments 129-158, wherein the cancer is
non-small cell lung cancer,
melanoma, renal cell carcinoma, triple-negative breast cancer, gastric cancer,
microsatellite stable-colorectal
cancer, pancreatic cancer, small cell lung cancer, HER2-positive breast
cancer, or prostate cancer.
160. A method of determining therapeutic efficacy of a cancer treatment in
a subject, wherein the treatment
comprises administering to the subject a combination therapy for simultaneous,
separate or sequential
administration comprising a dose of a TGF8 inhibitor and a cancer therapy,
which method comprises:
(i) determining the circulating myeloid-derived suppressor cell (MDSC)
level in a sample obtained from the
subject prior to administering the TGF8 inhibitor, wherein optionally the MDSC
level is determined by the
use of an antibody that binds LRRC33;
(ii) determining the circulating MDSC level in a sample obtained from the
subject after the administration of
the TGFI3 inhibitor, wherein optionally the MDSC level is determined by the
use of an antibody that binds
LRRC33; and
(iii) determining whether the level determined in step (ii) is reduced
compared to the level determined in
step (i), such reduction being indicative of therapeutic efficacy of the
cancer treatment.
161. The method of embodiment 160, wherein the level of circulating MDSC
cells is determined within 3-6
weeks following administration of the dose of TGF8 inhibitor, optionally
within 3 weeks or at about 3 weeks
following administration of the dose of TGF8 inhibitor.
162. The method of embodiment 160, wherein the level of circulating MDSC
cells is determined within 2
weeks following administration of the dose of TGF[3 inhibitor, optionally at
about 10 days following administration
of the dose of TGFI3 inhibitor.
163. The method of any one of embodiments 160-162, wherein the subject in
step (i) or (ii) has not received
previous cancer therapy, optionally wherein the subject in steps (i) and (ii)
has not received previous cancer
therapy.
164. The method of any one of embodiments 160-163, wherein the subject is
to receive the cancer therapy if
circulating MDSC levels are determined to be reduced.
165. The method of any one of embodiments 160-164, wherein the subject in
step (i) or (ii) has received
previous cancer therapy or is receiving cancer therapy, optionally wherein the
subject in step (i) and (ii) has
received previous cancer therapy or is receiving cancer therapy.
166. The method of embodiment 165, wherein the subject is to receive
further cancer therapy if circulating
MDSC levels are determined to be reduced.
167. The method of any one of embodiments 160-166, wherein the subject
receives more than one dose of
the TGF8 inhibitor prior to step (ii).
168. The method of any one of embodiments 160-167, wherein the sample is a
whole blood sample or a
blood component.
169. A cancer therapy agent for use in the treatment of cancer in a
subject, wherein the subject has received
a dose of a TGFp inhibitor and wherein the circulating MDSC level in the
subject measured after the
administration of the TGF8 inhibitor has been determined to be reduced as
compared to the circulating MDSC
level measured in the subject prior to administering the dose of the TGF13
inhibitor, wherein optionally the
circulating MDSC level is measured using an antibody that binds LRRC33.
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170. A combination therapy comprising a dose of a TGFI3 inhibitor and a
cancer therapy agent for use in the
treatment of cancer, wherein the treatment comprises simultaneous, separate,
or sequential administration to a
subject of a dose of the TGFp inhibitor and the cancer therapy agent, and
wherein the circulating MDSC level in
the subject measured after the administration of the TGFp inhibitor has been
determined to be reduced as
compared to the circulating MDSC level measured in the subject prior to
administering the dose of the TGFI3
inhibitor, wherein optionally the circulating MDSC level is measured using an
antibody that binds LRRC33.
171. The combination therapy for use according to embodiment 170, wherein
the subject has not received
previous cancer therapy and wherein the circulating MDSC level in the subject
has been determined to be
reduced prior to administration of the cancer therapy agent.
172. The combination therapy for use according to embodiment 170 or
embodiment 171, wherein the subject
has not received previous cancer therapy, wherein the subject receives the
TGFp inhibitor prior to the cancer
therapy agent.
173. The combination therapy for use according to embodiment 170, wherein
the subject receives the cancer
therapy agent prior to the TGFp inhibitor.
174. A TGFp inhibitor for use in the treatment of cancer in a subject,
wherein the subject has received at
least a first dose of the TGFI3 inhibitor, and wherein the treatment comprises
administering a further dose of the
TGFI3 inhibitor, provided that: the circulating MDSC level in the subject
measured after the administration of the
at least first dose of the TGFI3 inhibitor is reduced as compared to the
circulating MDSC level measured in the
subject prior to administering a dose of the TGFp inhibitor, wherein
optionally the circulating MDSC level is
measured using an antibody that binds LRRC33.
175. A TGFI3 inhibitor for use in the treatment of cancer in a subject,
wherein the subject is administered a
dose of the TGFI3 inhibitor, and wherein the TGFI3 inhibitor reduces or
reverses immune suppression in the
cancer, wherein said reduced or reversed immune suppression has been
determined by a reduction in the
circulating MDSC level in the subject measured after the administration of the
TGFI3 inhibitor as compared to the
circulating MDSC level measured in the subject prior to administering the dose
of the TGFI3 inhibitor, wherein
optionally the circulating MDSC level is measured using an antibody that binds
LRRC33.
176. The cancer therapy agent for use according to embodiment 169, the
combination therapy for use
according to embodiment 170 or any embodiment dependent thereon, or the TGFp
inhibitor for use according to
embodiment 174 or embodiment 175, wherein the subject has received more than
one dose of the TGFI3 inhibitor
prior to the determination that the circulating MDSC levels are reduced.
177. The cancer therapy agent for use according to embodiment 169 or any
embodiment dependent thereon,
the combination therapy for use according to embodiment 170 or any embodiment
dependent thereon, or the
TGFI3 inhibitor for use according to embodiment 174 or embodiment 175 or any
embodiment dependent thereon,
wherein the level of circulating MDSC cells is determined within 3-6 weeks
following administration of the dose of
TGF3 inhibitor, optionally within 3 weeks or at about 3 weeks, optionally
within 2 weeks or at about 10 days,
following administration of the dose of TGFI3 inhibitor, optionally wherein
said dose of TGFI3 inhibitor is the first
dose of the TGFI3 inhibitor that the subject has received.
178. The cancer therapy agent for use according to embodiment 169 or any
embodiment dependent thereon,
the combination therapy for use according to embodiment 170 or any embodiment
dependent thereon, or the
TGFI3 inhibitor for use according to embodiment 174 or embodiment 175 or any
embodiment dependent thereon,
wherein the subject has not received previous cancer therapy.
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179. The cancer therapy agent for use according to embodiment 169 or any
embodiment dependent thereon,
the combination therapy for use according to embodiment 170 or any embodiment
dependent thereon, or the
TGFp inhibitor for use according to embodiment 174 or embodiment 175 or any
embodiment dependent thereon,
wherein the subject is receiving cancer therapy.
180. The cancer therapy agent for use according to embodiment 169 or any
embodiment dependent thereon,
the combination therapy for use according to embodiment 170 or any embodiment
dependent thereon, or the
TGF3 inhibitor for use according to embodiment 174 or embodiment 175 or any
embodiment dependent thereon,
wherein the subject will be receiving cancer therapy.
181. The cancer therapy agent for use according to embodiment 169 or any
embodiment dependent thereon,
the combination therapy for use according to embodiment 170 or any embodiment
dependent thereon, or the
TGFp inhibitor for use according to any one of embodiments 178-180, wherein
the cancer therapy comprises
immunotherapy, chemotherapy, radiation therapy, engineered immune cell therapy
(e.g., CAR-T therapy), cancer
vaccine therapy and/or oncolytic viral therapy.
182. The cancer therapy agent for use according to embodiment 169 or any
embodiment dependent thereon,
the combination therapy for use according to embodiment 170 or any embodiment
dependent thereon, or the
TGF3 inhibitor for use according to any one of embodiments 178-180, wherein
the cancer therapy is
immunotherapy comprising checkpoint inhibitor therapy, optionally wherein the
checkpoint inhibitor comprises an
agent targeting programmed cell death protein 1 (PD-1) or programmed cell
death protein 1 ligand (PD-L1),
optionally wherein the checkpoint inhibitor comprises an anti-PD-(L)1
antibody.
183. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
16901 any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
dependent thereon, or the TGFI3 inhibitor for use according to embodiment 174
or embodiment 175 or any
embodiment dependent thereon, wherein the circulating MDSCs are G-MDSCs.
184. The method of determining therapeutic efficacy, the cancer therapy
agent for use, the combination
therapy for use, or TGFp inhibitor for use according to embodiment 183,
wherein the G-MDSCs express one or
more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+, CD33+/low, and CD66+.
185. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
169 or any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
dependent thereon, or the TGFI3 inhibitor for use according to embodiment 174
or embodiment 175 or any
embodiment dependent thereon, wherein the circulating MDSC levels are reduced
by at least 10%, optionally by
at least 15%, 20%, 25%, or more.
186. The cancer therapy agent for use according to embodiment 169 or any
embodiment dependent thereon,
the combination therapy for use according to embodiment 170 or any embodiment
dependent thereon, or the
TGFp inhibitor for use according to embodiment 174 or embodiment 175 or any
embodiment dependent thereon,
wherein the circulating MDSC levels have been determined from a whole blood
sample or a blood component
obtained from the subject.
187. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
169 or any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
dependent thereon, or the TGF8 inhibitor for use according to embodiment 174
or embodiment 175 or any
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embodiment dependent thereon, wherein the TGF13 inhibitor is a TG931
inhibitor, optionally wherein the TGFp
inhibitor is a TGFI31-specific inhibitor.
188. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
169 or any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
dependent thereon, or the TGFp inhibitor for use according to embodiment 174
or embodiment 175 or any
embodiment dependent thereon, wherein the subject has circulating MDSC levels
at least 2-fold above circulating
MDSC levels in a healthy subject prior to a treatment.
189. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
169 or any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
dependent thereon, or the TGFp inhibitor for use according to embodiment 174
or embodiment 175 or any
embodiment dependent thereon, wherein the subject has or is suspected of
having cancer.
190. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
169 or any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
dependent thereon, or the TGFp inhibitor for use according to embodiment 174
or embodiment 175 or any
embodiment dependent thereon, wherein the level of a tumor-associated immune
cell in the subject measured
after the administration of the first dose of the TGFp inhibitor is changed as
compared to the level of said tumor-
associated immune cell in the subject measured prior to the administration of
the first dose of the TGFP inhibitor.
191. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, which further comprises:
(iv) determining the level of one or more tumor-associated immune cells in
a sample obtained from the
subject prior to administering the TGFI3 inhibitor;
(v) determining the level of one or more tumor-associated immune cells in a
sample obtained from the
subject after the administration of the TGFp inhibitor; and
(vi) determining whether the level determined in step (v) is changed
compared to the level determined in
step (iv), such change being indicative of therapeutic efficacy of the cancer
treatment.
192. The method according to embodiment 191, wherein a change in level of
one or more tumor-associated
immune cells indicates reduction or reversal of immune suppression in the
cancer.
193. The method according to embodiment 191 or embodiment 192, wherein the
tumor-associated immune
cells comprise CD8+ T cells and/or tumor-associated macrophages (TAMs).
194. The method according to any one of embodiments 191-193, wherein the
change in the levels of one or
more tumor-associated immune cells comprises at least a 10%, optionally at
least a 15%, 20%, 25%, or more,
increase in CD8+ T cell levels.
195. The method according to any one of embodiments 191-194, wherein the
change in the levels of one or
more tumor-associated immune cells comprises at least a 10%, optionally at
least a 15%, 20%, 25%, or more,
increase in the level of TAMs.
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196. The method according to any one of embodiments 191-195, wherein the
level of one or more tumor-
associated immune cells is determined, in a sample obtained from the subject,
by immunohistochemistry
analysis.
197. The method according to any one of embodiments 191-196, wherein the
level of one or more tumor-
associated immune cells is determined by in vivo imaging.
198. The method according to any one of embodiments 191-197, wherein the
sample is a tumor biopsy
sample, wherein the tumor biopsy sample is optionally a core needle biopsy
sample of the tumor.
199. The cancer therapy agent for use according to embodiment 169 or any
embodiment dependent thereon,
the combination therapy for use according to embodiment 170 or any embodiment
dependent thereon, or the
TGFp inhibitor for use according to embodiment 174 or embodiment 175 or any
embodiment dependent thereon,
wherein the level of circulating latent TGFp (e.g., circulating latent TGFp1)
in the subject measured after the
administration of the first dose of the TGFp inhibitor is changed as compared
to the level of said circulating latent
TGFp in the subject measured prior to the administration of the first dose of
the TGFp inhibitor.
200. The method of determining therapeutic efficacy according to embodiment
160 or embodiment 191 or
any embodiment dependent thereon, which further comprises:
(vii) determining the level of circulating latent TGFp in a sample obtained
from the subject prior to
administering the TGFp inhibitor;
(viii) determining the level of circulating latent TGFp in a sample
obtained from the subject after the
administration of the TGFp inhibitor; and
(ix) determining whether the level determined in step (viii) is increased
compared to the level determined in
step (vii), such increase being indicative of therapeutic efficacy of the
cancer treatment.
201. The method according to embodiment 200, wherein the level of
circulating latent TGFP is determined in
a sample obtained from the subject and wherein the sample is a whole blood
sample or a blood component.
202. The method according to embodiment 200, wherein the circulating latent
TGFP is circulating latent
TGFp1.
203. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
169 or any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
dependent thereon, or the TGFp inhibitor for use according to embodiment 174
or embodiment 175 or any
embodiment dependent thereon, wherein the subject has cancer that is resistant
to a cancer therapy that does
not comprise a TGFP inhibitor.
204. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
169 or any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
dependent thereon, or the TGFp inhibitor for use according to embodiment 174
or embodiment 175 or any
embodiment dependent thereon, wherein the subject is poorly responsive to a
cancer therapy that does not
comprise a TGFP inhibitor.
205. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
169 or any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
215
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dependent thereon, or the TGFp inhibitor for use according to embodiment 174
or embodiment 175 or any
embodiment dependent thereon, wherein the subject is currently receiving or
previously received a cancer
therapy that does not comprise a TGFp inhibitor.
206. The method of determining therapeutic efficacy according to embodiment
160 or any embodiment
dependent thereon, the cancer therapy agent for use according to embodiment
169 or any embodiment
dependent thereon, the combination therapy for use according to embodiment 170
or any embodiment
dependent thereon, or the TGF3 inhibitor for use according to embodiment 174
or embodiment 175 or any
embodiment dependent thereon, wherein the subject has cancer that is resistant
to a cancer therapy that does
not comprise a TGFp inhibitor.
207. The method, the medical use, the cancer therapy agent for use, the
TGFP inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor is
administered to the subject intravenously.
208. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor is
administered at a concentration of about 37.5 mg/kg, 30 mg/kg, 20 mg/kg, 10
mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg,
4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, or less.
209. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor is
administered in an amount of about 3000 mg, 2400 mg, 1600 mg, 800 mg, 240 mg,
80 mg, or less.
210. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to embodiment 204 or embodiment 205,
wherein the TGFp inhibitor is
administered about every three weeks.
211. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the cancer comprises
an immune-excluded tumor.
212. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the cancer is a
myeloproliferative disorder, wherein optionally the myeloproliferative
disorder is myelofibrosis.
213. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the cancer is a highly
metastatic cancer.
214. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the cancer is
colorectal cancer, lung cancer, bladder cancer, kidney cancer (e.g.,
transitional cell carcinoma, renal sarcoma,
and renal cell carcinoma (RCC), including clear cell RCC, papillary RCC,
chromophobe RCC, collecting duct
RCC, or unclassified RCC, uterine cancer, prostate cancer, stomach cancer, or
thyroid cancer.
215. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the subject is at risk of
developing aortic stenosis.
216
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216. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the cancer is TGFpl-
positive.
217. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the cancer co-
expresses TGF(31 and TG933.
218. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the tumor is a TGF(31-
dominant tumor.
219. A method for manufacturing a pharmaceutical composition comprising a
TGFI3 inhibitor, the method
comprising the steps of:
i) providing a TGFI3 inhibitor that meets the following criteria:
a) the TGFp inhibitor is a monoclonal antibody, an antigen-binding fragment
thereof, or a
multispecific construct that is capable of binding a TGFp;
b) the TGFp inhibitor binds the TGFp with a Ko of < 1.0 nM, preferably Ko <
500 pM, as
measured by a SPR-based assay (e.g., Biacore) and inhibits TGFpl ;
c) the TGFp inhibitor is effective in vivo in a preclinical model at a dose
that does not cause a
toxicity associated with pan-inhibition of TGFp when dosed with at least 10
times the minimum efficacious
amount for at least 4 weeks in an animal model;
ii) carrying out an immune safety assessment comprising:
a) a cytokine release assay (in vitro and/or in vivo); and/or,
b) a platelet assay
iii) producing the TGFp inhibitor at a scale of 250L or larger; and,
iv) formulating the TGFp inhibitor into a pharmaceutical composition with one
or more excipients.
220. The method of embodiment 219, wherein the TGFp is TGFpl
221. The method of embodiment 219, wherein the TGFp is a proTGFp complex,
mature TGFp growth factor,
or a ligand-binding domain of a TGFp receptor.
222. The method of embodiment 219, wherein the TGFP inhibitor is effective
in causing tumor growth
regression, prolonged survival, and/or normalized gene expression of PAI-1,
CCL2, FN-1, ACTA2, Coll al,
Col3a1, FN-1, CTGF, and/or TGF131.
223. The method of embodiment 222, wherein the tumor is a TGFpl -dominant
tumor, wherein optionally the
tumor further expresses TGFp3.
224. The method of embodiment 219 wherein the toxicity associated with pan-
inhibition of TGFp comprises
one or more of a cardiovascular toxicity (e.g., a valvulopathy), epithelial
hyperplasia, bleeding, and skin lesion.
225. The method of embodiment 219, wherein the immune safety assessment
comprises an in vitro cytokine
release assay.
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226. The method of embodiment 219, wherein the scale of the production is
at least 500L, at least 1000L, at
least 2000L.
227. The method of any one of embodiments 219 to 226, wherein the
production comprises a eukaryotic cell
culture, wherein optionally the eukaryotic cell culture is a mammalian cell
culture, plant cell culture, or an insect
cell culture.
228. The method of embodiment 227, wherein the mammalian cell culture
comprises a CHO cell, MOCK cell,
NSO cell, Sp2/0 cell, BHK cell, Murine C127 cell, Vero cell, HEK293 cell, HT-
1080 cell, or PER.C6 cell.
229. A method of treating a TGFp-related disorder in a subject, the method
comprising administering to the
subject a therapeutically effective amount of a TGFI3 inhibitor to treat the
disorder, wherein the therapeutically
effective amount is an amount sufficient to increase the level of circulating
latent TGFp after the administration.
230. A method of treating a TGFp-related disorder in a subject, the method
comprising administering a TGFp
inhibitor and monitoring levels of circulating latent TGFp after
administration.
231. The method of embodiments 229 or 230, wherein the TGF13-related
disorder is a TG931-related
disorder.
232. The method of embodiment 231, wherein the TGFI31-related disorder is a
cancer.
233. The method of embodiment 231, wherein the TGFp1-related disorder is an
immune disorder
234. The method of any one of embodiments 229-233, wherein if the level of
circulating latent TGFp after the
administration of the TGFP inhibitor is increased, e.g., by at least 2-fold, 3-
fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-
fold, 9-fold, 10-fold, or more, relative to the level prior to the
administration, an additional dose of the TGFp
inhibitor is administered.
235. The method of any one of embodiments 229-234, wherein the level of
circulating latent TGFp after the
administration of the TGFp inhibitor is increased to a maximum of at least
1000 pg/ml.
236. The method of any one of embodiments 229-235, wherein the level of
circulating latent TGFp after the
administration of the TGFp inhibitor is increased to a maximum of about 1000
pg/ml to about 8000 pg/ml.
237. The method of any one of embodiments 229-236, wherein the level of
circulating latent TGFp after the
administration of the TGFp inhibitor is increased to a maximum about 2000
pg/ml to about 6500 pg/ml.
238. The method of any one of 229-237, wherein the level of circulating
latent TGFp after the administration
of the TGFp inhibitor is increased by a minimum of about 1.5-fold.
239. The method of any one of embodiments 229-238, wherein the level of
circulating latent TGFp is
measured about 8 to about 672 hours following administration of the TGF13
inhibitor.
240. The method of any one of embodiments 229-239, wherein the level of
circulating latent TGFp is
measured about 24 hours to about 336 hours following administration of the
TGFp inhibitor.
241. The method of any one of embodiments 229-240, wherein the level of
circulating latent TGFp is
measured about 72 hours to about 240 hours following administration of the
TGFp inhibitor.
242. The method of any one of embodiments 229-241, wherein the TGFp
inhibitor is administered at a dose
of about 1 mg/kg to about 30 mg/kg.
243. The method of any one of embodiments 229-242, wherein the TGFP
inhibitor is administered at a dose
of about 5 mg/kg to about 20 mg/kg.
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244. The method of any one of embodiments 229-243, wherein the TGFp
inhibitor is administered at a dose
of about 2 mg/kg to about 7 mg/kg.
245. The method of any one of embodiments 229-245, wherein the TG93
inhibitor is administered about
every three weeks.
246. A method of determining the efficacy of a cancer treatment in a
subject, comprising determining the
level of circulating latent TGFp1 in a first sample from the subject,
administering a dose of a TGFp1 inhibitor to
the subject, and determining the level of circulating latent TGFp in a second
sample from the subject after
administration, wherein an increase of at least 2-fold, 3-fold, 4-fold, 5-
fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or
more in circulating latent TGFp levels between the first sample and the second
sample indicates treatment
efficacy.
247. A method of treating a subject with a solid cancer, comprising
determining the level of circulating latent
TGF131 in a first sample from the subject, administering to the subject a dose
of a TGFp1 inhibitor, and
determining the level of circulating latent TGFp in a second sample from the
subject after administration.
248. The method of embodiment 247, wherein if the subject has a ratio of
circulating latent TG93 after the
administration to before the administration of at least 1.2, an additional
dose of the TGFp inhibitor is
administered.
249. The method of any one of embodiments 246-248, wherein the second
sample is collected from the
subject 24 hours to 56 days after the administration.
250. The method of any one of embodiments 229-249, wherein the TG93
inhibitor is a TGFp activation
inhibitor, e.g., a TGF31-selective inhibitor.
251. The method of embodiment 2250, wherein the TGFp inhibitor is Ab6.
252. The method of any one of embodiments 229-251, wherein the
therapeutically effective amount of the
TGFp inhibitor is between 0.1 mg/kg to 30 mg/kg per dose.
253. The method of any one of embodiments 229-252, wherein the
therapeutically effective amount of the
TGFp inhibitor is between 1 mg/kg and 10 mg/kg per dose.
254. The method of any one of embodiments 229-253, wherein the
therapeutically effective amount of the
TGFp inhibitor is between 2 mg/kg and 7 mg/kg per dose.
255. The method of any one of embodiments 229-254, wherein the TG93
inhibitor is dosed weekly, every 2
weeks, every 3 weeks, every 4 weeks, monthly, every 6 weeks, every 8 weeks,
bimonthly, every 10 weeks, every
12 weeks, every 3 months, every 4 months, every 6 months, every 8 months,
every 10 months, or once a year.
256. The method of any one of embodiments 229-255, wherein the TGFp
inhibitor is dosed about every 3
weeks.
257. The method of any one of embodiments 229-256, wherein the TG93
inhibitor is administered
intravenously or subcutaneously.
258. The method of any one of embodiments 229-257, wherein the latent TGFp
is latent TGFp1.
259. The method of any one of embodiments 229-258, wherein the level of
circulating latent TGFP is
measured in a blood sample.
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260. The method of any one of embodiments 229-259, wherein the blood sample
is a serum sample or a
plasma sample.
261. The method of any one of embodiments 229-260, wherein the circulating
latent TG93 levels are
measured by ELISA.
262. The method of any one of embodiments 229-261, further comprising
determining the levels of circulating
MDSCs in the subject prior to and after administration of the TGFp inhibitor,
wherein optionally the circulating
MDSC levels are determined with the use of an antibody that binds LRRC33.
263. The method of embodiment 262, wherein a reduction in the levels of
circulating MDSCs after the
administration as compared to before the administration indicates therapeutic
efficacy and, optionally, one or
more additional treatments comprising the TGFp inhibitor is administered.
264. The method of embodiment 262, wherein the circulating MDSCs are G-
MDSCs.
265. The method of embodiment 264, wherein the G-MDSCs express one or more
of CD11 b, CD33, CD15,
LOX-1, CD66b, and HLA-DRI /-.
266. The method of any one of embodiments 243-246, wherein the circulating
MDSC levels are determined
from whole blood or a blood component collected from the subject.
267. The method of any one of embodiments 262-266, wherein administration
of the TGFp inhibitor reduces
circulating MDSC levels by at least 10%, optionally by at least 15%, 20%, 25%,
or more.
268. The method of any one of embodiments 262-267, wherein circulating latent
TGFP levels are increased by at
least 50% and circulating MDSC levels are decreased by at least 15%, 20%, 25%,
or more.
269. The method, the medical use, the cancer therapy agent for use, the
TGFI3 inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor
inhibits TGFpl signaling.
270. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor
Inhibits TGF131 signaling but does not inhibit TGF(33 signaling at a
therapeutically effective dose.
271. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor
does not inhibit TGFp2 signaling and TGFp3 signaling at a therapeutically
effective dose.
272. The method, the medical use, the cancer therapy agent for use, the
TGFI3 inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor
does not bind to free TGFI3 growth hormones.
273. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TG93 inhibitor
binds to pro- and/or -latent TGFpl.
274. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor
binds to at least a portion of a Latency Lasso in TGFpl.
220
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275. The method, the medical use, the cancer therapy agent for use, the
TGF3 inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGF3 inhibitor
binds to at least a portion of a Finger-1 domain in TGF31.
276. The method, the medical use, the cancer therapy agent for use, the
TGF3 inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGF3 inhibitor is a
neutralizing antibody or a ligand trap.
277. The method, the medical use, the cancer therapy agent for use, the
1GF3 inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGF3 inhibitor
binds selectively to TGF31, optionally selectively to a pro- and/or latent-
TGF31.
278. The method, the medical use, the cancer therapy agent for use, the
TGF3 inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGF3 inhibitor is
an isolated antibody or antigen-binding fragment thereof which is capable of
specifically binding a proTGF31
complex at (i) a first binding region comprising at least a portion of Latency
Lasso (SEQ ID NO: 126); and ii) a
second binding region comprising at least a portion of Finger-1 (SEQ ID NO:
124).
279. The method, the medical use, the cancer therapy agent for use, the
TGF3 inhibitor for use, or the
combination therapy for use according to embodiment 278, wherein the first
binding region further comprises an
amino acid sequence of SEQ ID NO: 134 or a portion thereof.
280. The method, the medical use, the cancer therapy agent for use, the
TGF3 inhibitor for use, or the
combination therapy for use according to embodiment 278, wherein the second
binding region further comprises
an amino acid sequence of SEQ ID NO: 143 or a portion thereof.
281. The method, the medical use, the cancer therapy agent for use, the
TGF3 inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGF3 inhibitor
comprises an isolated antibody or antigen-binding fragment thereof, comprising
three heavy chain
complementarity determining regions comprising amino acid sequences of SEQ ID
NO: 1 (H-CDR1), SEQ ID NO:
2 (H-CDR2), and SEQ ID NO: 3 (H-CDR3), and three light chain complementarity
determining regions comprising
amino acid sequences of SEQ ID NO: 4 (L-CDR1), SEQ ID NO: 5 (L-CDR2), and SEQ
ID NO: 6 (L-CDR3), as
defined by the IMTG numbering system.
282. The method, the medical use, the cancer therapy agent for use, the
TGF3 inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGF3 inhibitor
comprises an isolated antibody or antigen-binding fragment thereof, comprising
three heavy chain
complementarity determining regions (H-CDR1, H-CDR2, and H-CDR3) from a heavy
chain variable region
comprising an amino acid sequence of SEQ ID NO: 7, and three light chain
complementarity determining regions
(L-CDR1, L-CDR2, and L-CDR3) from a light chain variable region comprising an
amino acid sequence of SEQ
ID NO: 8.
283. The method, the medical use, the cancer therapy agent for use, the
TGF3 inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the TGF3 inhibitor
comprises an isolated antibody or antigen-binding fragment thereof, comprising
three heavy chain
complementarity determining regions (H-CDR1, H-CDR2, and H-CDR3) from a heavy
chain variable region that
is at least 90% identical to an amino acid sequence of SEQ ID NO: 7, and three
light chain complementarity
determining regions (L-CDR1, L-CDR2, and L-CDR3) from a light chain variable
region that is at least 90%
identical to an amino acid sequence of SEQ ID NO: 8.
221
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284. The method, the medical use, the cancer therapy agent for use, the
TGFI3 inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the TGFI3 inhibitor
comprises an isolated antibody or antigen-binding fragment thereof, comprising
a heavy chain variable region
that is at least 90% identical to an amino acid sequence of SEQ ID NO: 7 and a
light chain variable region that is
at least 90% identical to an amino acid sequence of SEQ ID NO: 8.
285. The method, the medical use, the cancer therapy agent for use, the
TGFI3 inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the TGFI3 inhibitor
comprises an isolated antibody or antigen-binding fragment thereof, comprising
a heavy chain variable region
comprising an amino acid sequence of SEQ ID NO: 7 and a light chain variable
region comprising an amino acid
sequence of SEQ ID NO: 8.
286. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the TGFI3 inhibitor
comprises an isolated antibody or antigen-binding fragment thereof, comprising
a heavy chain comprising an
amino acid sequence of SEQ ID NO: 9 and a light chain comprising an amino acid
sequence of SEQ ID NO: 11.
287. The method, the medical use, the cancer therapy agent for use, the
TGFI3 inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the TGFI3 inhibitor cross-
blocks and/or competes for binding to TGFp1 with an antibody or antigen-
binding fragment comprising a heavy
chain variable domain of SEQ ID NO: 7, and a light chain variable domain of
SEQ ID NO: 8.
288. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor is a
monoclonal antibody, optionally a fully human or humanized antibody, or an
antigen binding fragment thereof.
289. The method, the medical use, the cancer therapy agent for use, the
TGFI3 inhibitor for use, or the
combination therapy for use according to any one of the preceding embodiments,
wherein the TGFp inhibitor is
present in a multispecific or bispecific construct.
290. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to embodiment 289, wherein the
multispecific or bispecific construct is
also capable of binding to an immune cell-surface antigen,
291. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to embodiment 290, wherein the immune
cell-surface antigen is PD-1, PD-
L1, CTLA4, or LAG3.
292. The method, the medical use, the cancer therapy agent for use, the
TGFI3 inhibitor for use, or the
combination therapy for use according to embodiment 290 or 291, wherein the
immune cell-surface antigen is
PD-1 or PD-L1, optionally comprising an anti-PD-1 or anti-PD-L1 antibody or
antigen binding fragment thereof.
293. The method, the medical use, the cancer therapy agent for use, the
TGFI3 inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the TGFp inhibitor
comprises a human IgG4 or IgGi constant region.
294. The method, the medical use, the cancer therapy agent for use, the
TGFI3 inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the subject is a human
patient and wherein the patient has a historically documented solid tumor that
is metastatic or locally advanced,
for which standard-of-care therapy does not exist, has failed in the patient,
or is not tolerated by the patient, or for
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which the patient has been assessed as not suitable candidate or otherwise
ineligible for the standard-of-care
therapy.
295. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the subject is a human
patient and wherein the patient has a history of primary anti-PD-(L)1 antibody
nonresponse presenting either as
progressive disease or stable disease (e.g., not improving, but also not
worsening, clinically or radiographically)
after at least 3 cycles of treatment with an anti-PD-(L)1 antibody therapy
(optionally alone or in combination with
chemotherapy) approved for that tumor type.
296. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the subject is a human
patient and wherein the patient has received the most recent dose of anti-PD-
(L)1 antibody therapy within 6
months of the administration of the TGFp inhibitor.
297. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the subject is a human
patient and wherein the patient has NSCLC and has genomic tumor aberrations
for which a targeted therapy is
available (wherein optionally the targeted therapy targets anaplastic lymphoma
kinase and/or EGFR), wherein
further optionally the patient has progressed on an approved therapy for these
aberrations or did not tolerate an
approved therapy for these aberrations, or was not considered suitable
candidates or was otherwise ineligible for
an approved therapy for these aberrations.
298. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the subject is a human
patient and wherein the patient has measurable disease as determined by
Response Evaluation Criteria in Solid
Tumor (RECIST) v1.1.
299. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the subject is a human
patient and wherein the patient has an Eastern Cooperative Oncology Group
performance status (PS) 0-1.
300. The method, the medical use, the cancer therapy agent for use, the
TGFp inhibitor for use, or the
combination therapy for use according to any of the preceding embodiments,
wherein the subject is a human
patient and wherein the patient has a predicted life expectancy of a 3 months.
301. The method of embodiment 1, wherein the reduced circulating MDSCs are
M-MDSCs.
302. The method of embodiment 1 or embodiment 2, wherein the M-MDSCs
express one or more of
CD11b+, HLADR-/low, CD14+, CD15-, CD33+/high, and CD66b-.
303. The composition, composition for use, or method of any one of the
preceding embodiments, wherein the
TGFp inhibitor is shown to cause no significant adverse events (e.g., dose-
limiting toxicities) in a preclinical
animal model when dosed at up to 100, 200, or 300 pg/kg weekly for 4 weeks, 8
weeks or up to12 weeks, as
assessed by standard toxicology analyses or according to the present
disclosure.
304. The composition for use, the TGFp inhibitor for use, or the method
according to any one of the
preceding embodiments, wherein selection of the composition or the TGFp
inhibitor comprises in vivo efficacy
and safety criteria, wherein the safety criteria includes: i) lack of platelet
aggregation, activation and/or binding
when assessed under the condition according to the present disclosure, and, in
lack of significant (e.g., within
2.5-fold of control) cytokine release, when assessed under the condition
according to the present disclosure.
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305. The composition for use, the TGFp inhibitor for use, or the
method according to any one of the
preceding embodiments, wherein the pharmacodynamics of the TGFP inhibitor are
assessed by measuring
circulatory latent TGFp1 levels before and after the administration of the
TGFp1 inhibitor in blood (serum)
samples collected from the subject.
List of Certain Other Embodiments
1. A TGFI3 inhibitor for use in the treatment of a cancer in a
subject, wherein the treatment comprises
determining a level of circulating TGFI3 in a blood sample collected from the
subject and administering the TGFI3
inhibitor to the subject, wherein the circulating TGFp level is determined or
has been determined by processing
the blood sample below room temperature in a sample tube comprising an
anticoagulant.
2. A method of treating a cancer in a subject, comprising:
(i) determining a level of circulating TGFp in the subject prior to
administering a TGFp inhibitor;
(ii) administering to the subject a therapeutically effective amount of the
TGFI3 inhibitor;
(iii) determining a level of circulating TGFp in the subject after
administration; and
wherein the circulating TGFp level is determined or has been determined by
processing a blood sample
from the subject below room temperature in a sample tube comprising an
anticoagulant .
3. A method of determining therapeutic efficacy in a subject being
treated for a cancer, comprising:
(i) determining a level of circulating TGFp in the subject prior to
administering a TGFp inhibitor;
(ii) administering to the subject a therapeutically effective amount of the
TGFI3 inhibitor; and
(iii) determining a level of circulating TGFI3 in the subject after
administration;
wherein an increase in circulating TGFI3 levels after administration as
compared to before administration
indicates therapeutic efficacy, and wherein the circulating TGFI3 level is
determined or has been determined by
processing a blood sample from the subject below room temperature in a sample
tube comprising an
anticoagulant.
4. The method of embodiment 3, wherein an increase of at least 1.5-
fold, at least 2-fold, at least 2.5-fold, at
least 3-fold, at least 4-fold, at least 5-fold, or more in circulating TGFI3
levels after administration as compared to
before administration indicates therapeutic efficacy.
5. A method of determining targeting engagement in a subject
having cancer, comprising:
(i) determining a level of circulating TGFI3 in the subject prior to
administering a TGFI3 inhibitor;
(ii) administering to the subject a therapeutically effective amount of the
TGFI3 inhibitor;
(iii) determining a level of circulating TGFI3 in the subject after
administration; and
wherein an increase in circulating TGFI3 levels after administration as
compared to before administration
indicates target engagement of the TGFp inhibitor, and wherein a circulating
TGFI3 level is determined or has
been determined by processing a blood sample from the subject below room
temperature in a sample tube
comprising an anticoagulant.
6. The method of embodiment 4, wherein an increase of at least 1.5-
fold, at least 2-fold, at least 2.5-fold, at
least 3-fold, at least 4-fold, at least 5-fold, or more in circulating TGFp
levels after administration as compared to
before administration indicates target engagement of the TGFI3 inhibitor.
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7. The method of any one of embodiments 2-6, wherein the
administration of the TGFp inhibitor is
continued if the level of circulating TGFp after the administration is
increased by at least 1.5-fold, at least 2-fold,
at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more
as compared to the level of circulating
TGFp before the administration.
8. A method of treating a cancer in a subject, comprising
administering a second dose of a TGFp inhibitor
to a subject having an elevated level of circulating TGFp after receiving a
first dose the TGF6 inhibitor, wherein
the level of TGFp has been measured by processing a blood sample from the
subject below room temperature in
a sample tube comprising an anticoagulant.
9. A method of treating a cancer in a subject, comprising:
(i) determining a level of circulating TGFI3 in the subject prior to
administering a TGFp inhibitor;
(ii) administering to the subject a first dose of TGF13 inhibitor;
(iii) determining a level of circulating TGFp in the subject after
administration; and
(iv) administering a second dose of the TGFP inhibitor to the subject if the
level of circulating TGFP is elevated,
wherein determining the level of TGFp comprises processing a blood sample from
the subject below room
temperature in a sample tube comprising an anticoagulant.
10. The method of embodiment 8 or 9, wherein the level of
circulating TGFp after the first dose of the TGFp
inhibitor is elevated by at least 1.5-fold, at least 2-fold, at least 2.5-
fold, at least 3-fold, at least 4-fold, at least 5-
fold, or more as compared the level of circulating TGFP before the first dose
of the TGFP inhibitor.
11. The TGFp inhibitor or method of any one of embodiments 1-10,
wherein the cancer comprises a solid
tumor, wherein optionally the solid tumor is selected from: melanoma (e.g.,
metastatic melanoma), renal cell
carcinoma, triple-negative breast cancer, HER2-positive breast cancer,
colorectal cancer (e.g., microsatellite
stable-colorectal cancer), lung cancer (e.g., metastatic non-small cell lung
cancer, small cell lung cancer),
esophageal cancer, pancreatic cancer, bladder cancer, kidney cancer (e.g.,
transitional cell carcinoma, renal
sarcoma, and renal cell carcinoma (RCC), including clear cell RCC, papillary
RCC, chromophobe RCC, collecting
duct RCC, or unclassified RCC, uterine cancer, prostate cancer, stomach cancer
(e.g., gastric cancer), head and
neck squamous cell cancer, urothelial carcinoma, hepatocellular carcinoma, or
thyroid cancer.
12. The TGFp inhibitor or method of any one of embodiments 1-11,
comprising administering a checkpoint
inhibitor therapy concurrently (e.g., simultaneously), separately, or
sequentially with the TGFp inhibitor, wherein
the checkpoint inhibitor is optionally an anti-PD-1 antibody, anti-PD-L1
antibody, anti-CTLA-4-antibody, anti-
LAG3 antibody, or an antigen-binding fragment thereof.
13. The TGFp inhibitor or method of embodiment 12, wherein the
cancer has an immune excluded
phenotype, e.g., characterized by containing less than 5% intratumor CD8+
cells and greater than 5% margin
CD8+ cells as assessed by an immunohistochemistry analysis capable of
detecting individual tumor nest(s)
within the tumor.
14. The TGFp inhibitor or method of embodiment 13, wherein the
cancer is characterized by having greater
than 50% tumor area comprising tumor nest(s) comprising lower levels of CD8+
cells inside the tumor nest(s)
relative to levels of CD8+ cells outside of the tumor nest, e.g., less than 5%
CD8+ cells inside the tumor nest(s)
and greater than 5% CD8+ cells outside the tumor nest(s), e.g., in the margin.
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15. The TGF6 inhibitor or method of any one of embodiments 1-14, wherein
processing the blood samples
comprises one or more centrifugation steps at a speed of greater than 100xg
and/or one or more centrifugation
steps at a speed of below 15000xg.
16. The TGF6 inhibitor or method of any one of embodiments 1-15, wherein
processing the blood samples
comprises a centrifugation protocol comprising:
i) a first step of 5-25 minutes at 100-500xg and a second step of 10-40
minutes at 1000-5000xg; or
ii) a first step of 5-25 minutes at 1000-5000xg and a second step of 10-40
minutes at 1000-5000xg; or
iii) a first step of 5-25 minutes at 500-3000xg and a second step of 2-10
minutes at 7500-15000xg.
17. The TGF6 inhibitor or method of any one of embodiments 1-16, wherein
processing the blood samples
comprises a centrifugation protocol comprising:
i) a first step of 10 minutes at 150xg and a second step of 20 minutes at
2500xg; or
ii) a first step of 10 minutes at 2500xg and a second step of 20 minutes at
2500xg; or
iii) a first step of 10 minutes at 1500xg and a second step of 5 minutes at
12000xg.
18. The TGF6 inhibitor or method of any one of embodiments 1-17, wherein
processing the blood samples
comprises a centrifugation protocol comprising a first step of 10 minutes at
2500xg and a second step of 20
minutes at 2500xg.
19. The TGF6 inhibitor or method of any one of embodiments 1-18, wherein
the anticoagulant comprises
0.1-0.5 M buffered trisodium citrate.
20. The TGF6 inhibitor or method of any one of embodiments 1-19, wherein
the anticoagulant comprises
10-20 M theophylline.
21. The TGF6 inhibitor or method of any one of embodiments 1-20, wherein
the anticoagulant comprises 2-
M adenosine.
22. The TGF6 inhibitor or method of any one of embodiments 1-21, wherein
the anticoagulant comprises
0.1-0.25 M dipyridamole.
23. The TGF6 inhibitor or method of any one of embodiments 1-22, wherein
the anticoagulant comprises a
citrate-theophylline-adenosine-dipyridamole (CTAD) solution, optionally
comprising about 0.11M buffered
trisodium citrate solution, about 15 M theophylline, about 3.7 M adenosine,
and about 0.198 M dipyridamole.
24. The TGF6 inhibitor or method of any one of embodiments 1-23, wherein
the collection tube is coated
with a solution that has a pH of 4.0-6.0, e.g., about 5Ø
25. The TGF6 inhibitor or method of any one of embodiments 1-24, wherein
the collection tube is glass or
plastic.
26. The TGF6 inhibitor or method of any one of embodiments 1-25, wherein
the collection tube comprises a
silicone coating.
27. The TGFp inhibitor or method of any one of embodiments 1-26, wherein
the collection tube has a
HemogardTM closure.
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28. The TGFp inhibitor or method of any one of embodiments 1-27, wherein
the collection tube has a
volume capacity of 2-3 mL, e.g., 2.7 mL.
29. The TGFp inhibitor or method of any one of embodiments 1-28, wherein
the collection tube is sterile.
30. The TGFp inhibitor or method of any one of embodiments 1-29, wherein
the collection tube is a BD
VacutainerTM CTAD blood collection tube.
31. The TGF6 inhibitor or method of any one of embodiments 1-30, wherein
the blood sample is processed
at 2-8 C.
32. The TGFP inhibitor or method of any one of embodiments 1-31, wherein
the blood sample is processed
at 4 C.
33. The TGFp inhibitor or method of any one of embodiments 1-32, wherein
processing the blood sample
comprises determining a level of platelet factor 4 (PF4) in the same sample
from which the circulating TGFp is
determined, wherein the sample is used for measuring optionally the level of
circulating TGFp in a sample is
normalized based on the PF4 levels determined from the same sampleif the PF4
level is 500 ng/ml or less.
34. The TGFp inhibitor or method of any one of embodiments 1-33, wherein
the circulating TGFp is
circulating TGFp1.
35. The TGF6 inhibitor or method of any one of embodiments 1-34, wherein
the circulating TGF6 is
circulating latent TGF61.
36. The TGFP inhibitor or method of any one of embodiments 1-35, wherein
the TGFP inhibitor is
administered at a dose of 2000 mg or 3000 mg and a frequency of once every two
weeks, three weeks, or any
multiples of two weeks or three weeks.
37. A method of treating a cancer in a human subject comprising
administering to the subject a 2000 mg or
3000 mg dose of a TGFp inhibitor at a frequency of once every two weeks, three
weeks, or any multiples of two
weeks or three weeks, wherein the subject is not receiving a checkpoint
inhibitor therapy.
38. A TGFp inhibitor for use in the treatment of a cancer in a human
subject, wherein the treatment
comprises administering to the subject a 2000 mg or 3000 mg dose of a TGFp
inhibitor at a frequency of once
every two weeks, three weeks, or any multiples of two weeks or three weeks,
wherein the subject is not receiving
a checkpoint inhibitor therapy.
39. A method of treating a cancer in a subject comprising administering to
the subject a TGFp inhibitor and
a checkpoint inhibitor therapy, where the TGFP inhibitor is administered at a
dose of 2000 mg or 3000 mg and a
frequency of once every two weeks, three weeks, or any multiples of two weeks
or three weeks.
40. A TGFp inhibitor for use in the treatment of a cancer in a subject,
wherein the treatment comprises
administering to the subject a TGF6 inhibitor and a checkpoint inhibitor
therapy, where the TGF6 inhibitor is
administered at a dose of 2000 mg or 3000 mg and a frequency of once every two
weeks, three weeks, or any
multiples of two weeks or three weeks.
41. The method or TGFp inhibitor for use of any one of embodiments 37-40,
wherein the subject is a non-
responder to checkpoint inhibitor therapy.
42. The method or TGFp inhibitor for use of any one of embodiments 37-41,
wherein the subject has not
received a checkpoint inhibitor therapy previously.
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43. The method or TGF3 inhibitor for use of any one of embodiments 37-42,
wherein the checkpoint
inhibitor therapy is pembrolizumab, nivolumab, cemiplimab, atezolizumab,
avelumab, budigalimab, or
durvalumab.
44. The method or TGF8 inhibitor for use of any one of embodiments 37-43,
wherein the TGF3 inhibitor is
administered at a frequency of once every two weeks.
45. The method or TGF3 inhibitor for use of any one of embodiments 37-43,
wherein the TGF3 inhibitor is
administered at a frequency of once every three weeks.
46. The method or TGF8 inhibitor for use of any one of embodiments 37-43,
wherein the TGF3 inhibitor is
administered at a frequency of once every four weeks.
47. The method or TGF8 inhibitor for use of any one of embodiments 37-43,
wherein the TGF3 inhibitor is
administered at a frequency of once every six weeks.
48. The method or TGF3 inhibitor for use of any one of embodiments 37-47,
wherein the TGF3 inhibitor is
administered at a dose of 2000 mg.
49. The method or TGF3 inhibitor for use of any one of embodiments 37-47,
wherein the TGF3 inhibitor is
administered at a dose of 3000 mg.
50. The method or TGF3 inhibitor for use of any one of embodiments 37-49,
wherein the TGF3 inhibitor is
administered consecutively or concurrently with a checkpoint inhibitor,
wherein the checkpoint inhibitor is
optionally an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4-antibody,
anti-LAG3 antibody, or an antigen-
binding fragment thereof.
51. The method or TGF8 inhibitor for use of embodiment 50, wherein the TGF8
inhibitor is administered at
the same frequency as the checkpoint inhibitor therapy.
52. A method of determining therapeutic efficacy in a subject being treated
for a cancer, comprising:
(i) determining a level of P-Smad2 nuclear translocation in a tumor sample
obtained from the subject
prior to administering a TGF3 inhibitor;
(ii) administering to the subject one or more doses of the TGF3 inhibitor; and
(iii) determining a level of P-Smad2 nuclear translocation in a tumor sample
obtained from the subject
after the administration;
wherein a decrease in P-Smad2 nuclear translocation after the administration
as compared to before the
administration indicates therapeutic efficacy.
53 The method of embodiment 52, wherein a decrease of at least 1 3-
fold, at least 1.5-fold, at least 2-fold,
at least 3-fold, at least 4-fold, at least 5-fold, or more in P-Smad2 nuclear
translocation levels after the
administration as compared to before the administration indicates therapeutic
efficacy.
54. A method of determining targeting engagement in a subject
having cancer, comprising:
(i) determining a level of P-Smad2 nuclear translocation in a tumor sample
obtained from the subject
prior to administering a TGF3 inhibitor;
(ii) administering to the subject one or more doses of the TGF8 inhibitor;
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(iii) determining a level of P-Smad2 nuclear translocation in a tumor sample
obtained from the subject
after the administration; and
wherein a decrease in P-Smad2 nuclear translocation after the administration
as compared to before the
administration indicates target engagement of the TGF6 inhibitor.
55. The method of embodiment 54, wherein a decrease of at least 1.3-
fold, at least 1.5-fold, at least 2-fold,
at least 3-fold, at least 4-fold, at least 5-fold, or more in P-Smad2 nuclear
translocation levels after the
administration as compared to before the administration indicates target
engagement.
56. A method of treating cancer in a subject, comprising:
(i) determining a level of P-Smad2 nuclear translocation in a tumor sample
obtained from the subject
prior to administering a TGFp inhibitor;
(ii) administering to the subject a first dose of the TGFp inhibitor;
(iii) determining a level of P-Smad2 nuclear translocation in a tumor sample
obtained from the subject
after the administration; and
(iv) administering to the subject one or more additional doses of the TGF6
inhibitor if the P-Smad2
nuclear translocation after the administration of the first dose is decreased
as compared to the P-Smad2 nuclear
translocation before the administration of the first dose.
57. The method of embodiment 56, wherein P-Smad2 nuclear
translocation is decreased by at least 1.3-
fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at
least 5-fold, or more.
58. The method or TGFp inhibitor for use according to any one of
embodiments 54-57, wherein the
determination of nuclear translocation of p-Smad2 comprises
immunohistochemistry.
59. The method or TGFp inhibitor for use according to any one of
embodiments 54-58, wherein the
determination of nuclear translocation of p-Smad2 comprises nuclear masking.
60. The TGF6 inhibitor or method of any one of embodiments 1-59,
wherein the TGF6 inhibitor inhibits
TGF31 signaling.
61. The TGF6 inhibitor or method of any one of embodiments 1-60,
wherein the TGF6 inhibitor binds
selectively to pro/latent-TGF6.
62. The TGF6 inhibitor or method of embodiment 60, wherein the TGF6
inhibitor binds selectively to
pro/latent-TGF61.
63. The TGF6 inhibitor or method of embodiment 61 or embodiment 62,
wherein the TGF6 inhibitor does
not bind to mature TGF[31.
64 The TGF6 inhibitor or method of any one of embodiments 1-63,
wherein the TGF6 inhibitor is a TGF61-
selective inhibitor, wherein optionally the TGF31-selective inhibitor is a
neutralizing antibody that binds mature
TGF61 or an activation inhibitor that binds proTGF61.
65. The TGF6 inhibitor or method of embodiment 64, wherein the
TGF61-selective inhibitor is:
a) a monoclonal antibody designated as Ab6 herein, a TGFp1-selective variant
thereof, or an antigen-
binding fragment thereof or an antibody comprising the CDRs and/or variable
domains from Ab6; or
b) an antibody or an antigen-binding fragment thereof that competes for
binding and/on binds the same
epitope as Ab6.
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66. The TG93 inhibitor or method of any one of embodiments 1-65, wherein
the TG93 inhibitor comprises
an isolated antibody or antigen-binding fragment thereof comprising three
heavy chain complementarity
determining regions comprising amino acid sequences of SEQ ID NO: 1 (H-CDR1),
SEQ ID NO: 2 (H-CDR2),
and SEQ ID NO: 3 (H-CDR3) and three light chain complementarity determining
regions comprising amino acid
sequences of SEQ ID NO: 4 (L-CDR1), SEQ ID NO: 5 (L-CDR2), and SEQ ID NO: 6 (L-
CDR3), as defined by the
IMTG numbering system, wherein optionally the TGFp inhibitor comprises an
isolated antibody or antigen-binding
fragment thereof comprising a heavy chain variable region comprising an amino
acid sequence of SEQ ID NO: 7
and a light chain variable region comprising an amino acid sequence of SEQ ID
NO: 8 or amino acid sequences
90% identical thereto.
67. The TG93 inhibitor or method of any one of embodiments 1-66 wherein the
TG93 inhibitor is a TG931/2
inhibitor, wherein optionally the inhibitor is NIS793/X0MA-089 or GC1008.
68. The TG93 inhibitor or method of any one of embodiments 1-66, wherein
the TG93 inhibitor is a
TG931/3 inhibitor wherein optionally the inhibitor is M7824 (bintrafusp alpha)
or AVID200.
69. The TGF13 inhibitor or method of any one of embodiments 1-68, wherein
the TGF13 inhibitor is a low
molecular weight antagonist of the TG93 receptor kinase, wherein optionally
the antagonist is an ALK5 inhibitor.
70. A method for treating cancer a human subject having a solid tumor, the
method comprising
administering to the subject a checkpoint inhibitor in an amount effective to
reduce tumor growth, wherein the
solid tumor has an inflamed phenotype characterized by containing greater than
5% intratumor CD8+ cells as
assessed by an immunohistochemistry analysis capable of detecting individual
tumor nest(s) within the tumor.
71. The method of embodiment 70, wherein the tumor is characterized by
having greater than 50% tumor
area comprising tumor nest(s) comprising greater than 5% CD8+ cells inside the
tumor nest(s).
72. A TG93 inhibitor for use in the treatment of cancer in a subject,
wherein the treatment comprises
administration of a TG93 inhibitor, a cancer therapy, or a combination
thereof, wherein optionally the cancer
therapy is a checkpoint inhibitor therapy or genotoxic therapy, and wherein
the treatment comprises
determination of a level of MDSCs in a blood sample obtained from the subject,
wherein optionally, the MDSC
levels are measured with the use of an antibody that binds LRRC33, wherein
further optionally, the antibody that
binds LRRC33 is used in a FACS-based assay or ELISA-based assay.
73. The TG93 inhibitor for use according to embodiment 72, wherein the
treatment further comprises
determination of CD8+ cells in a tumor biopsy sample obtained from the
subject, optionally using an image
analysis method that allows resolution of individual tumor nests in accordance
with the disclosure herein.
74. The TG93 inhibitor for use according to embodiment 72 or 73, wherein
the treatment further comprises
determination of circulatory TG93 levels in a blood sample obtained from the
subject, wherein the blood sample
is collected and/or processed below room temperature, optionally in accordance
with the disclosure herein,
wherein optionally, the blood sample is processed to prepare a platelet-poor
plasma (PPP) sample.
75. The TG93 inhibitor for use according to any one of embodiments 72-74,
wherein the TG93 inhibitor is
an isoform-selective inhibitor of TG931, wherein optionally the isoform-
selective inhibitor is a neutralizing
antibody that selectively binds TG931, or an antibody that binds a latent
TG931 complex; or wherein the TG93
inhibitor is an inhibitor of TGF131 and TG932, wherein optionally the
inhibitor is NI5793/X0MA-089 or GC1008.
76. The TGF3 inhibitor for use according to any one of embodiments 48-50,
wherein the TGF13 inhibitor is
an inhibitor of TGF31 and TGFp3, wherein optionally the inhibitor is M7824
(bintrafusp alpha) or AVID200.
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77. The TGFI3 inhibitor for use or method according to any one of
embodiments 1-66 and 69-74, wherein
the TGFI3 inhibitor is a pan inhibitor that inhibits TGFI31, TGFI32 and
TGFI33, wherein optionally the pan inhibitor
is GC1008 or derivative thereof, SAR439459, LY3022859, or an agent that blocks
ligand-binding domain of a
TGFI3 receptor.
78. The TGFp inhibitor for use or method according to any one of
embodiments 1-66 and 69-74, wherein
the TGFp inhibitor is an activation inhibitor of TGFp pathway that interferes
with integrin-TGFp interaction,
wherein optionally the inhibitor is an agent that binds an integrin, or an
agent that binds the RGD motif present in
latent TGF131 and/or TG933, wherein further optionally the agent is a low
molecular weignt weight (small
molecule) compound or an antibody or antigen-binding fragment thereof.
79. The TGFI3 inhibitor for use or method according to any one of
embodiments 1-66 and 69-74, wherein
the TGFp inhibitor is a low molecular weight (small molecule) antagonist of
ALK5.
80. The TGFI3 inhibitor for use or method according to any one of
embodiments 1-66 and 69-74, wherein
the TGFp inhibitor is an RNA-based inhibitor of TGF131 expression.
81. The TGFI3 inhibitor for use or method according to any one of
embodiments 1-66 and 69-74, wherein
the TGFp inhibitor is a soluble ligand trap that comprises a ligand-binding
fragment of a TGFp receptor, capable
of sequestering mature growth factor, wherein the mature growth factor is
TGFpl , TGFI32, TGF(33, or
combination thereof, wherein further optionally, the ligand trap is M7824
(bintrafusp alpha) or AVID200.
82. The TGFI3 inhibitor for use or method according to any one of
embodiments 70-81, wherein the
checkpoint inhibitor is anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4-
antibody, anti-LAG3 antibody, or an
antigen-binding fragment thereof.
83. The TGFp inhibitor for use of embodiment 72, wherein the cancer therapy
is a genotoxic therapy.
84. The method or TGFp inhibitor for use of any one of embodiments 1-35, 52-
69, wherein the TGFI3
inhibitor is administered concurrently (e.g., simultaneously or sequentially)
with a genotoxic therapy.
85. A method of treating solid cancer, comprising:
i) selecting a subject having a cancer with an elevated TGFp level and/or TGFp
activity; and
ii) administering to the subject a therapeutically effective amount of a TGFI3
inhibitor;
wherein the TGFp inhibitor is administered concurrently (e.g., simultaneously
or sequentially) with a
genotoxic therapy.
86. A TGFp inhibitor for use in treating solid cancer, comprising:
i) selecting a subject having a cancer with an elevated TGFp level and/or
signaling; and
ii) administering to the subject a therapeutically effective amount of a TGFp
inhibitor;
wherein the TGFI3 inhibitor is administered concurrently (e.g., simultaneously
or sequentially) with a
genotoxic therapy.
87. The method or TGFI3 inhibitor for use of embodiment 85 or embodiment
86, wherein the subject has
intrinsic or acquired resistance to the genotoxic therapy.
88. The method or TGFp inhibitor for use of embodiment 85 or embodiment 86,
wherein TGFI3 inhibitor
produces synergistic effects with the genotoxic therapy.
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89. The method or TGFI3 inhibitor for use of any one of embodiments 83-88,
wherein the genotoxic therapy
is a chemotherapy or a radiation therapy.
90. The method or TGFI3 inhibitor for use of any one of embodiments 83-89,
wherein subject has ovarian
cancer, breast cancer, bladder cancer, pancreatic cancer, e.g., pancreatic
adenocarcinoma, prostate cancer,
e.g., prostate adenocarcinoma, melanoma, e.g., skin cutaneous melanoma, lung
cancer, e.g., lung squamous
cell carcinoma and lung adenocarcinoma, liver cancer (e.g., liver
hepatocellular carcinoma), uterine cancer, e.g.,
uterine corpus endometrial carcinoma, kidney cancer, e.g., renal clear cell
carcinoma, head and neck cancer,
e.g., head and neck squamous cell carcinoma, colon cancer, e.g., colon
adenocarcinoma, esophageal
carcinoma, or tenosynovial giant cell tumor (TGCT).
91. The method or TGFI3 inhibitor for use of any one of embodiments 83-90,
wherein the TGFI3 inhibitor
comprises an isolated antibody or antigen-binding fragment thereof comprising
three heavy chain
complementarity determining regions comprising amino acid sequences of SEQ ID
NO: 1 (H-CDR1), SEQ ID NO:
2 (H-CDR2), and SEQ ID NO: 3 (H-CDR3) and three light chain complementarity
determining regions comprising
amino acid sequences of SEQ ID NO: 4 (L-CDR1), SEQ ID NO: 5 (L-CDR2), and SEQ
ID NO: 6 (L-CDR3), as
defined by the IMTG numbering system, wherein optionally the TGFI3 inhibitor
comprises an isolated antibody or
antigen-binding fragment thereof comprising a heavy chain variable region
comprising an amino acid sequence
of SEQ ID NO: 7 and a light chain variable region comprising an amino acid
sequence of SEQ ID NO: 8 or amino
acid sequences 90% identical thereto.
92. A method of distinguishing MDSC populations from a biological sample
obtained from a patient, wherein
gMDSC populations are identified by cell surface markers of CD11b+, HLA-DR-,
CD14-, CD15+, CD33+/low, and
CD66b+; and wherein mMDSC populations are identified by cell surface markers
of CD11 b+, HLA-DR-/low,
CD14+, CD15-, CD33+/high, and CD66b-.
93. The method of embodiment 92, wherein the identification comprises
binary intensity selection.
94. The method of embodiment 93, wherein the binary intensity selection
comprises binning signal
intensities according to one or more of the cutoff points shown in Table 38B.
95. The method of any one of embodiments 92-94, wherein the identification
comprises sequential
application of signal filtering.
96. The method of any one of embodiments 92-95, wherein the identification
comprises signal binning
according to HLD-DR signal intensity, wherein a signal intensity of <0.105 is
binned as negative signal, a signal
intensity of 0.105-0.125 is binned as low signal, a signal intensity of 0.125-
0.155 is binned as medium signal, and
a signal intensity of greater than 0.155 is binned as high signal.
97. The method of any one of embodiments 92-96, wherein the identification
comprises signal binning
according to CD33 signal intensity, wherein a signal intensity of <0.17 is
binned as negative signal, a signal
intensity of 0.17-0.18 is binned as low signal, a signal intensity of 0.18-
0.19 is binned as medium signal, and a
signal intensity of greater than 0.19 is binned as high signal.
EXAMPLES
[878] It will be readily apparent to those skilled in the art that other
suitable modifications and adaptations of the
composition and methods described herein may be made using suitable
equivalents without departing from the
scope of the disclosure or the embodiments disclosed herein. This disclosure
is further illustrated by the following
examples which should not be construed as limiting.
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[879] We previously observed that tumor-associated MDSCs express cell-surface
LRRC33 in tumor-bearing
mice. Here, we investigated whether circulating MDSCs might also express cell-
surface LRRC33. Previously, we
and others found that presence of LRRC33 rriRNA transcripts does not always
correlate with protein expression of
LRRC33, particularly cell-surface LRRC33. Therefore, FAGS experiments were
carried out to assess whether
circulating MDSCs in tumor-bearing mice express cell-surface LRRC33. A
monoclonal antibody that binds cell-
surface LRRC33, and an IgG control, were used to isolate/enrich MDSCs,
including the G-MDSC and M-MDSC
pools from blood samples collected from MBT-2 mice. Data show rubust and
substantially uniform expression of
LRRC33 in circulating MDSCs. In fact, nearly all MDSCs, including G-MDSCs and
M-MDSCs, in circulation appear
to express cell-surface LRRC33 at high levels. Taken together, these data show
that LRRC33 is expressed on cell
surface by circulating MDSCs, as well as in tumor (i.e., tumor-associated
MDSCs), that circulating monocytes do
not express LRRC33, and that LRRC33 is identrified as a novel blood-based
marker for MDSCs. This raises the
possibility that LRRC33 may serve as a surrogate blood-based biomarker for
immunosuppression, particularly in
cancer.
Example 1: Detection of Circulating MDSCs
[880] The effects of Ab6 treatment on circulating immune cell subsets in vivo
were determined using an MBT-2
mouse model. Tumor-bearing mice were dosed with 10 mg/kg of Ab6 alone on days
1 and 8 or in combination with
an anti-PD-1 antibody dosed on days 1, 4, and 8 at 10 mg/kg.
[881] Whole blood was collected on day 10 and processed for flow cytometry
analysis. Levels of circulating G-
MDSCs and M-MDSCs were determined based on the expression of surface protein
markers for G-MDSCs (CD45+
CD11b+ Ly6G+ Ly6CI0w) and M-MDSCs (CD45+ CD11b+ Ly6G- Ly6Chi9h). Values were
expressed as percentages
of total CD45+ cells detected in the blood. Circulating G-MDSC levels were
decreased in groups treated with both
Ab6 alone and combination treatments (i.e. Ab6 in combination with anti-PD-1),
whereas circulating M-MDSC levels
in Ab6-treated groups did not differ as compared to groups treated with IgG
control or anti-PD-1 treatment alone.
Tumor and circulating MDSC levels as assessed at day 10 following treatment
initiation are shown in FIG. 1 and
FIG. 2.
[882] Flow cytometry analysis of T-cells was also performed at day 10
following treatment initiation from whole
blood. Circulating T-cell levels were determined based on the expression of T
cell surface protein markers. CD8+
and CD3+ T cell levels and values were normalized to total circulating CD45+
cells detected in whole blood. Groups
treated with Ab6 alone exhibited a slight increase in both CD8+ and CD3+
circulating T-cell levels compared IgG
control. Ab6 and anti-PD-1 combination treatment did not lead to significant
changes in either CD8+ or CD3+
circulating T cell levels.
[883] A second in vivo study was carried out to further evaluate the effects
of Ab6 treatment on circulating and
intratumoral MDSC populations. MBT-2 mice were treated with IgG control, Ab6
alone (10 mg/kg), an anti-PD-1
antibody (10 mg/kg), or a combination of Ab6 (1 mg/kg, 3 mg/kg, or 10 mg/kg)
with an anti-PD-1 antibody (10
mg/kg). Treatments were administered on day 1 and 8. Whole blood was collected
on day 17 prior to administering
the first dose of treatment, and days 3, 6, and 10. Tumor volume was monitored
throughout the study, and
intratumoral MDSC analysis was carried out at day 10.
[884] Measurement of tumor volume on days 1, 4, 7, and 10 showed a
statistically significant treatment response
in animals treated with anti-PD-1 antibody alone and in all animals treated
with the combination of anti-PD-1
antibody and Ab6, but not in animals treated with Ab6 alone before day 10
(FIG. 3) . The lack of treatment response
observed in animals treated with Ab6 alone before day 10 was unlikely the
result of incorrect dosing, as
pharmacokinetic results confirmed that all animals were administered the
correct Ab6 dosage. These results
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indicate that, in the case of MBT-2 tumors, concurrent inhibition of PD-1 and
TGF31 pathways can reduce (e.g.,
delay or regress) tumor growth to a greater extent than inhibition of the PD-1
or TGFI31 pathway alone.
[885] Levels of circulating immune cell populations were determined from whole
blood samples via FAGS
analysis. Total CD11b+ myeloid cells were identified from whole blood, from
which M-MDSC populations were then
identified by the expression of cell surface markers Ly6C (Ly6ChI9h), and G-
MDSC populations were identified by
the expression of cell surface marker Ly6G (Ly6G+). Baseline circulating MDSC
levels were determined from whole
blood samples collected from non-tumor bearing mice and consisted of 17.8%
myeloid cells, which comprised 30%
G-MDSCs and 29.1% M-MDSCs (percentages of total myeloid population) (FIG. 4).
Levels of circulating immune
cells were assessed on day 10 from tumor-bearing mice. Compared to baseline
levels of non-tumor bearing mice,
tumor-bearing mice exhibited markedly increased levels of total myeloid
population and circulating G-MDSCs, but
not M-MDSC cells (FIG. 6). Blood samples from tumor-bearing mice were found to
consist of 64.9% myeloid cells,
which comprised 70% G-MDSCs and 6.95 M-MDSCs. Furthermore, G-MDSCs were also
found to make up 45.4%
of the total CD45+ immune cell population in the blood of tumor-bearing mice,
as compared to 5.45% of total CD45+
cells in the blood of non-tumor bearing mice (FIG. 5).
[886] Circulating MDSC populations were evaluated in tumor-bearing animals
throughout treatment. As shown in
FIG. 6, levels of M-MDSCs remained low throughout treatment, whereas levels of
G-MDSCs exhibited a decreasing
trend, with statistically significant decreases in G-MDSC levels detected in
all groups by day 10. A decrease in
circulating G-MDSC levels in animals treated with Ab6 alone was not observed
until day 10 (FIG. 7A and FIG. 7B).
This suggests that Ab6 treatment alone may be sufficient to reduce circulating
MDSC levels albeit a a delayed rate
as compared to a combination of Ab6 and anti-PD-1 treatment.
[887] The association of circulating G-MDSC levels to tumor volume was also
assessed at day 10. FIG. 8 shows
a linear correlation between circulating G-MDSC levels and tumor volume in all
groups.
[888] Levels of circulating and intratumorial MDSCs were compared to tumor
volume measurements at day 10.
Intratumoral M-MDSC levels in treated animals were similar across all
treatment groups and did not decrease as
compared to control animals. In contrast, intratumoral G-MDSC levels in
animals treated with anti-PD-1 antibody
alone or combination of anti-PD-1 antibody and Ab6 were reduced as compared to
control animals (FIG. 9A). While
Ab6 treatment alone resulted in a decrease in circulating G-MDSC levels,
intratumoral MDSC levels were not
affected by Ab6 treatment alone (FIG. 9B). A correlation of relative MDSC
levels in tumor and in circulation is
shown in FIG. 10. Additionally, reduced intratumoral G-MDSC levels at day 10
were found to correlate with elevated
tumor CD8+ cells across all treatment groups (FIG. 11), suggesting a decrease
in overall tumor immune
suppression.
Example 2: In vivo assessment of LRRC33 expression on circulating MDSC in MBT2
Syngeneic Bladder
Cancer Mouse Model
[889] LRRC33 expression levels on circulating MDSCs were determined using FACS
analysis using whole blood
samples collected from mice bearing MBT2 tumors. M-MDSC and G-MDSC populations
were identified on the
basis of Ly6C and Ly6G expression, respectively, and then further
characterized using a monoclonal antibody that
binds cell-surface LRRC33, and an IgG control. Nearly all circulating MDSCs,
including both G-MDSCs and M-
MDSCs, appeared to express LRRC33. As shown in FIG. 12A, LRRC33 expression was
detected on 89.1% of G-
MDSCs and 100% of the M-MDSCs from whole blood of the tumor bearing mice.
Consistent with previous
observations, LRRC33 was also detected on intratumoral MDSCs, including G-
MDSCs and M-MDSCs, albeit at
varying levels (FIG. 12B). In contrast, LRRC33 expression was not detected on
monocytes.
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Example 3: Ab6 treatment modulates circulatory TGFI31 levels
[890] Circulating TGFp1 levels were assessed before arid after treatment with
Ab6 arid/or an anti-PD-1 antibody
in an MBT-2 mouse model. Tumor-bearing mice were dosed on days 1 and 8 with
Ab6 alone at 10 mg/kg, a PD-1
antibody alone at 10 mg/kg, or 1 mg/kg, 3 mg/kg, or 10 mg/kg of Ab6 in
combination with 10 mg/kg of the anti-P D1
antibody. Control animals were dosed with IgG control.
[891] Blood samples were collected 14 before tumor implantation (day -14), 1
day before treatment, and on days
3, 6, and 10 following treatment. Samples were processed, including an acid
treatment step, and circulatory TG931
levels (pg/mL) were determined using an enzyme-linked immunosorbent assay
(ELISA, e.g., R&D Systems
Quantikine assay). The acid treatment step liberates TG931 from its latent
complex and, without being bound by
theory, it is believed that most of the TGF(31 in circulation is present in
the latent complex. Blood samples were
also analyzed for platelet activation as a quality control measure. Samples
with significant platelet activation were
not further analyzed for circulating TGFp levels because such samples were
likely to contain TGFp released from
the activated platelets, thus likely rendering the TGFp readout inaccurate.
Platelet factor 4 (PF4) concentration was
used as a surrogate marker for identifying samples containing significant
platelet activation. Samples with high PF4
levels (greater than 500 ng/ml) were identified as having significant platelet
activation and thus eliminated from
further analysis for TGFp concentration. Pharmacodynamic readouts such as
assessment of tumor size, target
engagement, and immune infiltration are carried out using tumor samples.
[892] Results showed an increasing trend of circulating TGF31 levels in
animals treated with Ab6 (alone or in
combination with the anti-PD-1 antibody) as compared to circulating TGF131
levels pre-implantation and in IgG
control-treated animals In contrast, animals treated with the anti-PD1
antibody alone did not exhibit increased
circulating TGFP1 levels compared to controls (FIG. 13A). A statistically
significant correlation was found between
circulating TGFI31 levels and plasma levels of Ab6 for each treatment group
(R2 = 0.714) (FIG. 13B and FIG. 13C).
[893] In order to avoid measuring additional circulatory TGFP1 released as an
artifact of sample collection and
processing, plasma platelet factor 4 (PF4) levels were determined in each
sample by ELISA and resulting PF4
levels were used to normalize circulatory TGF(31 release. PF4 levels may be
used as an indicator of platelet
activation induced during sample collection that may contribute to TGFp1
release. PF4 levels (ng/mL) were found
to be low across drug-treated and IgG control samples. In comparison, pre-
implant samples exhibited higher PF4
levels (FIG. 14, left panel). Sample outliers were identified using
interquartile range, with an upper bound PF4 level
of 60.45 ng/mL and a lower bound of 42.41 ng/mL (FIG. 14, right panel).
Results corrected for PF4 outliers showed
a statistically significant, dose-dependent increase in circulating TGF31
levels following Ab6 treatment (alone or in
combination with the anti-PD-1 antibody). Furthermore, outlier-correct results
also revealed elevated circulating
TGF131 levels in tumor-bearing animals as compared to non-tumor-bearing
controls (pre-implantation) (FIG. 15).
As shown in FIG. 15, outlier-corrected total circulatory TGFp1 levels were
about 2000 pg/mL pre-implantation,
about 3000 pg/mL in mice treated with IgG control, about 2500 pg/mL in mice
treated with anti-PD-1 alone, about
9000 pg/mL in mice treated with 10 mg/kg of Ab6 alone, and above 6000 pg/mL,
7200 pg/mL, and 9000 pg/mL in
mice treated with combination therapy comprising 1 mg/kg, 3 mg/kg, and 10
mg/kg of Ab6, respectively.
[894] Without wishing to be bound by theory, PF4 levels may be useful for
identifying and eliminating samples
contaminated by platelet activation during sample collection and processing.
In some embodiments, a sample with
a PF4 concentration of greater than 500 ng/mg may indicate that the sample
contains platelet activation. In some
embodiments, the sample with a PF4 concentration of greater than 500 ng/mg may
be treated as an outlier sample.
[895] Similar trends of dose-dependent increase in circulatory TGF131 levels
were also observed in non-human
primates and rats treated with a single dose of Ab6. In non-human primates
treated with 1 mg/kg, 3 mg/kg, 10
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mg/kg, or 30 mg/kg of Ab6, the extent and duration of increases in circulatory
TGF31 levels were dose-dependent.
Circulatory TGF131 levels were measured around 72-240 hrs following Ab6
administration, and peak circulatory
TGF31 levels were between about 2000 pg/ml to about 4000 pg/ml (FIG. 16).
Similarly, rats that were treated with
0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, or 30 mg/kg of Ab6 also exhibited dose-
dependent increase in circulatory
TGF31 levels. Circulatory TGF31 levels were detected around 24-360 hrs
following Ab6 administration, with peak
circulatory TGF31 levels detected between about 5000 pg/ml to about 7500
pg/ml. Duration of circulatory TGF31
elevation for each treatment group appeared to be dose-dependent (FIG. 17).
Example 4: Sample collection and processing method for assessment of
circulating latent TGFpl in
human blood samples
[896] A pilot study was conducted to identify improved collection and
processing conditions for assessing
circulation latent TGF31 in human plasma samples. Whole blood samples were
collected from 6 healthy donors
and processed under various conditions to generate platelet poor plasma (PPP),
including variations in
temperature, types of collection tubes, time prior to processing, and
centrifugation conditions (FIG. 18, Table 15).
PPP processing was assessed using tubes coated with anticoagulant citrate-
theophylline-adenosine-dipyridamole
(CTAD) or sodium citrate (TSC). Samples were processed after 2 hours or 4
hours of incubation following sample
collection. Samples were centrifuged according to one of three centrifugation
protocols: a first step of 150xg for 10
minutes followed by a second step of 2500xg for 20 minutes (P1), a first step
of 2500xg for 10 minutes followed by
a second step of 2500xg for 20 minutes (P2), and a first step of 1500xg for 10
minutes followed by a second step
of 12000xg for 5 minutes (P3). Acceleration and deceleration of the
centrifugation steps were set at 20% of
centrifuge maximum. Following the first centrifugation step, the supernatant
portion of each tube was transferred
to a separate tube, removing approximately 70% of the volume without
disturbing buffy coat and red cell layer, and
the supernatant is further processed the second centrifugation step. The
resulting supernatant was used to
measure circulating TGF31 levels. Sample incubation and processing were
carried out under 4 C or room
temperature (RT).
Table 15: Sample processing conditions
Assay Component Parameters
CTAD
Anticoagulant
TSC
4 C
Temperature
RT
2 hours
Incubation time
4 hours
P1:10 min at 150xg, 20 min at 2500xg
Centrifugation protocol P2: 10 min at 2500xg, 20 min at
2500xg
P3: 10 min at 1500xg, 5 min at 12000xg
[897] Circulatory TGF131 was evaluated by ELISA after acidification of PPP,
using the using R&D System
Quantikine assay. The average concentration of TCF31 ranged from 500 pg/mIto
1500 pg/ml (FIG. 19A). Samples
processed at 4 C showed significantly lower level of TGF31 as compared to
samples processed at room
temperature. Platelet Factor 4 (PF4) levels were evaluated as control for
platelet-induced TGF31 activation.
Significantly higher level of TGFpi and PF4 were detected in samples processed
at room temperature as compared
to samples processed at 4 C (FIG. 19B). Lowest level of PF4 was observed in
samples collected in CTAD tubes
and processed under centrifugation condition P2 (FIG. 19B). Correlation of PF4
and TGF31 was observed in
samples processed at room temperature (FIG. 20). Exemplary analyses of outlier
removal based on high PF4
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levels are shown in FIGs. 21A-21C. Regression analysis used in FIG. 21B is
described in Motu!sky and Brown.
BMC Bioinformatics. 2006 Mar 9;7:123.
Example 5: In vivo assessment of circulating latent TGFpl in human plasma
samples
[898] Circulating latent TGF3 levels were assessed in human platelet-poor
plasma samples both before Ab6
administration and one-hour following Ab6 administration. PF4 levels were
analyzed either at baseline or 1 hour
after administration as quality control for platelet activation in each sample
(FIG. 22). General methods for
determining circulating latent TGF3 levels are provided in Example 3, above.
Circulating TGF3 levels were
measured at additional time points following Ab6 administration and results
are shown in FIGs. 23A-C. Tables 16A-
C below show exemplary baseline TGF3 and PF4 concentrations from plasma
samples collected from patients
treated with Ab6.
Table 16A
Circulatory TGFb1 level at pre-dose(pg/m1)
80mg to 240 mg Ab6 Patient 101-001 Patient 104-
003
Baseline TGF[31 (pg/ml) 3920 6060 (1hr)
PF4 (pg/ml) 266 451
Table 16B
Circulatory TGFb1 level at predose(pg/m1)
800mg Ab6 Patient 101-002 Patient 104-001
Patient 105-002
Baseline TGFI31 (pg/ml) 2700 3780 5480
PF4 (pg/m1) 82.3 277 258
Table 16C
Circulatory TGFb1 level at predose(pg/m1)
1600mg Patient 101-003 Patient 101-004
Patient 103-004 Patient 104-002 Patient 105-003
Baseline TGFI31 4690 5790 7160 (1hr) 2850 3220
(pg/ml)
PF4 (pg/ml) 145 223 442 192 113
Example 6: Measurement of latent TGFp activation
[899] Inhibitory activity of Ab6 was measured as previously described in
Martin et al., 2020. Briefly, LN229 cells
(ATCC) were transfected with a plasmid encoding either human, rat, or
cynomolgus macaque proTGF31. About
24 hours after cell transfection, Ab6 was added to the transfectants together
with CAGA12 reporter cells (Promega,
Madison, WI). Approximately 16-20 hours after setting up the co-culture, the
assay was developed and
luminescence read out on a plate reader. Dose-response activities were
nonlinearly fit to a three-parameter log
inhibitor vs. response model using Prism 8 and best-fit I050 values
calculated.
[900] Modulation of TGF3 signaling in Ab6-treated tumors will be detected
using a P-Smad 2 IHC assay, where
decreased expression in P-Smad2 is indicative of TGF3 inhibition. As shown in
FIG. 24, a P-Smad2 IHC assay
was developed using commercially available melanoma samples and a digital
nuclear masking analysis. Nucleus
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mask is a digital image analysis parameter that enables visualization and
measurement of IHC signal intensity of
an individually marked cell or nucleus. The assay was established using the
commercial melanoma samples to
enable identification of a range of P-Smad2 nucleus staining intensity ranging
from high (red), medium (orange),
low (yellow) to negative (blue). Cells were sorted into categories (e.g., 0,
1+, 2+, 3+) based on chromogenic
intensity relative to scanning parameters. Variables analyzed during assay
development are shown in Table 17
below. Analysis was carried out using software developed by Flagship
Biosciences.
Table 17. Primary Data Variables for P-Smad 2 IHC Assay
Variable Name Description
Total Number of Quantified Cells Total number of cells quantified by
IA in compartment
Percent Total Cells Percent of Cells in compartment
out of All Cells
Number of p-Smad2 positive Cells Number of p-Smad2 positive cells
in compartment
Number of p-Smad2 negative Cells Number of p-Smad2 negative cells
in compartment
Percent Positive Cells Percent of p-Smad2 in the
compartment identified
Compartment Area (mm2) Area of compartment in mm2
Compartment Area (Percent)
Percent of the total tissue area occupied by compartmental area
% 0 p-Smad2 cells Percent of cells at 0
intensity
% 1+ p-Smad2 cells Percent of cells at 1+
intensity
% 2+ p-Smad2 cells Percent of cells at 2+
intensity
% 3+ p-Smad2 cells Percent of cells at 3+
intensity
Number of 0 p-Smad2 cells Number of cells at 0
intensity
Number of 1+ p-Smad2 cells Number of cells at 1+
intensity
Number of 2+ p-Smad2 cells Number of cells at 2+
intensity
Number of 3+ p-Smad2 cells Number of cells at 3+
intensity
H-Score Standard scoring paradigm representing both the prevalence and
intensity of biomarker expression. H-Score scale ranges from 0-300.
[901] pSmad2 phosphorylation was measured in MBT2 mice, which further
supported the rationale for measuring
tumor phosphorylated smad 2 (p-Smad2) as a biomarker for SRK-181-mediated
TGF81 inhibition. As shown in
FIG. 25, p-Smad2 levels were analyzed in MBT-2 mice (bladder cancer) at day 10
following weekly dosing of Ab6-
mIgG1 at 1 mg/kg, 3 mg/kg, 10 mg/kg, or 30 mg/kg. Ab6-mIgG1 treatment reduced
tumor p-3mad2
phosphorylation, thereby supporting p-Smad2 as a pharmacodynamic biomarker.
[902] Briefly, samples from MBT-2 mice were fixed, processed, embedded, and
sectioned at 4um thickness.
Slides were deparaffinized and stained using a Leica Bond Rx autostainer.
Sections were blocked using a lx
diluted Animal Free Blocking solution from Cell Signaling Technologies
(#15019). Staining was performed using a
phospho-Smad2 rabbit monoclonal antibody from Cell Signaling Technologies
(#3108, 138D4) at a final
concentration of 0.174 ug/mL. Heat induced epitope retrieval was performed
using a citrate-based solution for 20
minutes at 95 C. Polymer-Rabbit HRP was incubated as a post-primary for 25
minutes, procured from Cell
Signaling Technologies (#8114S). Secondary fluorophores, spectral DAPI, and
Tyramide Signal Amplification
reagents were procured from Akoya Biosciences (NEL741001KT, FP1490). Sections
were dried and cover-slipped
with antifade mounting media (ThermoFisher, P36961), and imaged on a Zeiss
AxioScan z1 with an additional Cy5
band to further separate autofluorescence signal.
[903] Image quantification was performed using a Visiopharm analysis platform.
Nuclei were segmented first on
the basis of DAPI staining intensity and resulting objects were reclassified
as pSmad2 strong, medium, or weakly
positive. These classifications were thresholded by FITC intensity, with a
background threshold of 15000 ABU and
requiring at least 30% nuclear coverage. Segmented objects with area less than
23 um2 or greater than 230 um2
were discarded before pSmad2 classifications were applied. Strong, medium, and
weak classifications were used
for QC purposes, and total % pSmad2 positivity is reported as the sum of these
groups, divided by the total number
of cells, and multiplied by 100.
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Example 7: Analysis of CD8-positive cells in Ab6-treated tumors
[904] Investigation of CD8 T cell status in biopsied tissues typically
describes each tissue as one of three main
phenotypes: immune desert, immune-excluded, or inflamed. Immune desert
phenotypes do not express
appreciable levels of CD8 throughout the tissue. Immune-excluded tissues
exhibit CD8 expression, but the
expression is mostly localized to the stroma or the stromal margin surrounding
tumor nests. Inflamed tissues show
appreciable levels of CD8 expression or CD8-positive cells within the tumor
nests of the tissues. While this
phenotypic categorization is beneficial, these percentages of expression are
often calculated as a mean of
expression through the tissue and does not take in to account the
heterogeneous nature of tumor biology. This
may result in a tumor containing one highly inflamed tumor nest being averaged
out with multiple deserted tumor
nests to categorize the tissue as excluded or deserted even though
inflammation is present.
[905] To better represent the heterogeneity of inflammation present within
tumor tissues, an image analysis-based
algorithm was developed which not only separated out the tumor, stroma, and
tumor/stroma margin, but identified
each tumor nest within the tissue as its own discrete object. This analysis
allowed for the enumeration of number
and size of all tumor nests within the tissue, and further quantified the
percentage of CD8 expression within and
outside of each tumor nest. Each tumor nest was given its own phenotypic
classification of inflamed, excluded, or
deserted, and the percentage of tumor nests displaying each phenotype was
calculated to represent the
heterogenic inflammation within tumors.
[906] Core needle biopsy was used to obtain tumor samples from 36 subjects
diagnosed with bladder cancer or
melanoma. Three to four core biopsy samples were collected from each tumor
using a 16- to 18-gauge needle.
Samples were fixed at room temperature in a formalin container for 24 to 48
hours. Once fixation was completed,
biopsies were transferred to a histocassette between sponges pre-soaked with
PBS. The histocassette was then
submerged in cold PBS and stored for no more than 3-4 days prior to analysis.
[907] The percentage of CD8+ cells was determined by immunohistochemical
analysis in 23 whole-tissue tumor
resection samples of bladder cancer and 13 samples of melanoma. Data from the
whole tissue, as well as tumor,
stroma, and margin (25 pm in each direction from the tumor/stromal interface)
compartments of the whole tissue,
were evaluated. Margins between tumor nests and the surrounding stroma and
margin compartments were
identified by digital analysis of the stained samples using pathologist
assessment and automated machine learning
software developed by Flaship Biosciences (Westminster, CO). Percentage of
CD8+ cells was also evaluated for
individual tumor nests throughout samples that contained tumor nests with
potentially mixed immunophenotypes.
For samples which were poorly-defined or distinctly lacked analyzable tumor,
only whole tissue was analyzed.
Results from compartment analysis demonstrated variation in the percentage of
CD8+ cells among different
compartments in bladder cancer (FIG. 26A) and melanoma (FIG. 26B) samples.
Cell counts, compartmental area,
CD8+ cell density (average number of CD8+ cells / mm2), and CD8+ cell
clustering were measured. Results from
tumor nest analysis are shown in FIGs. 27-29E. Cell count based on FIG. 29C is
provided below.
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Table 18
Sample Name Tumor Nest Tumor Nest Total Cell Tumor Nest CD8
Interface CD8 Tumor Nest
Number Count % Positive %
Positive Status
BladderCA 26 1 281 0 2.303
Excluded
BladderCA 26 2 535 0,51-3 2.399
Excluded
BladderCA 26 3 703 2,418 2.883
Excluded
BladderCA 26 4 256 5,078 4.281
inflamed
.õ .õ .., ,.. ...
.õ
immune
BladderCA 26 84 405 0 0
Desert
BladderCA 26 85 420 0.23,5 0.716
Excluded
immune
BlaaderC.,A 26 86 250 0 0
Desert
BledcierCA 26 87 780 0 0.703
Excluded
BiadderCA 25 88 1196 0.0838 0.536
'Excluded
BledcierCA 26 89 452 0 0 287
Excluded
BladiderCA 26 90 445 4.045 2.953
Excluded
BladderCA 26 91 527 0 0.623
Exc.luded
BladclerCA 26 92 1068 0 0.109
'Excluded
Bladde:CA 26 93 910 0.33 0,631
Excluded
BladderCA 25 94 761 0,657 2.111
Excluded
BladderCe-126 95 293 0,341 1.636
Excluded
BladderCA 25 % 336 2,063 5.398
Excluded
BladderCA 26 97 305 0.759 2 143
Excluded
BladderCA 25 98 852 0.284 1.674
Excluded
[908] To determine the immune phenotype, the percentage of CD8+ cells in the
tumor compartment was
compared to that of the stromal and margin compartment. The ratio of CD8+
cells in the tumor compartment to that
of the stromal or margin compartments varied across immune phenotypes. As an
example, FIG. 30A shows that
bladder cancer samples #26, #30, and #9 exhibited different percentages of
CD8+ cells across compartments,
which indicated that these tumors likely had different immune phenotypes.
Bladder sample #26 (FIG. 30A, left)
exhibited an immune desert phenotype, as demonstrated by low CD8+ staining
across all three compartments
(0.8% CD8+ staining in the tumor, 1.9% in the stroma, and 1.3% in the margin).
Bladder sample #9 (FIG. 30A,
right), which exhibited an immune inflamed phenotype, showed similarly high
percentages of CD8+ staining across
all three compartments (11% CD8+ staining in the tumor, 8.7% in the stroma,
and 12% in the margin). In contrast,
bladder sample #30 (FIG. 30A, middle), which exhibited an immune excluded
phenotype, showed greater
percentages of CD8+ cells in the stroma and margin as compared to the tumor
(5.2% CD8+ staining in the tumor,
compared to 39% in the stroma and 24% in the margin). In some cases, further
analysis of CD8+ expressing in the
stroma and margin, by subdividing the margin compartment, may provide even
more information for immune
phenotyping. For instance, as shown in FIG. 30B, 18.3% and 4.8% of CD8+ cells
outside the tumor are located in
the stroma and margin compartments, respectively, and subdividing the margin
component further reveals that
nearly all of the CD8+ cells in the margin lie on the stromal-facing side of
the margin with almost no CD8+ cells
found in the tumor-facing side. Similarly, FIG. 30C shows strong CD8 staining
in the tumor margin of bladder
sample #30, and subdividing the margin component further demonstrates that
nearly all of the CD8 positivity is
localized on the stromal side of the margin compartment and nearly no CD8
positivity is localized on the tumor
side. These observations indicate that the tumor is likely immune excluded.
[909] Compartment ratios of CD8+ expression for the tumor, stroma, and margin
were compared to absolute
percent C08 positivity in whole tissue. As demonstrated in FIG. 31, tumors
that exhibit similar percentages of CD8+
cells display distinct CD8+ expression profiles in each tumor compartment,
suggesting that compartment ratios of
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CD8+ expression may provide more information for immune phenotyping as
compared to absolute percent CD8
positivity data of the whole tumor alone. Likewise, CD8+ cell density in each
tumor compartment, as determined
by the number of CD8-F cells per millimeter squared, was compared to percent
CD8+ expression of whole tissue.
IHC staining data in FIG. 32 show two different tumor stroma sections have an
approximately 10-fold difference in
CD8+ cell densities despite exhibiting similar overall percentages of CD8+
expression (FIG. 32). These findings
suggest that cell density may be used as an additional or alternative
measurement to absolute CD8 positivity, and
that in some cases, cell density data may better represent tumor immune
populations for immune phenotyping.
[910] Tumor depth was determined for two bladder samples by determining the
distance available for CD8+ T cell
penetration in the tumor nest of a given sample. Bladder sample #22 had a
tumor depth of greater than 8
(measurement continued toward opposite side of the tumor nest), whereas
bladder sample #30 had a tumor depth
of less than 2 (FIG. 33). Given that the percent CD8 expression in these
samples were found to be similar, these
results suggest that tumor depth may be a useful parameter for
determining/confirming tumor immunophenotyping
when used in combination with other parameters such as percent CD8 expression.
[911] Localized CD8 expression was further analyzed for melanoma sample #30,
which showed low overall 0D8
percentages. Localized areas of CD8 expression showed a concentration of CD8
cells near necrotic regions, which
may be indicative of potential treatment effects (FIG. 34).
[912] CD8 positivity (e.g., percentage of CD8+ cells) was determined for
individual tumor nests and compared to
CD8 positivity as measured in tumor compartments. As shown in Fig. 27, further
breakdown of tumor compartments
into tumor nests reveals varying degrees of CD8 positivity in different parts
of the tumor and provides additional
insight into the immune phenotype of the tumor. For instance, the immune
phenotype of a bladder tumor sample
was determined by first measuring the CDS positivity of 74 tumor nests, then
assigning each individual tumor nest
a relative phenotype (e.g., immune inflamed, immune excluded, or immune
desert) based on CD8 positivity, and
finally calculating the combined tumor areas exhibiting each immune phenotype
(FIG. 28). In some cases, immune
phenotypes as determined based on tumor nest analysis differed from the immune
phenotypes determined based
on analysis of tumor compartments alone (FIGS. 29A-E).
Example 7: Prevalence of tumor MDSC in various solid tumors
[913] A signal intensity filter for detecting cell surface markers of human
MDSCs was established. Multiple
markers were selected and developed to distinguish MDSC subtypes from other
monocytes. Cell surface markers
for distinguishing human MDSCs include CD11 b, CD33, CD66b, 0014, CD15, and
HLA-DR. A chromogenic assay
was performed to define the immunofluorescence dynamic range of each antibody
used to detect the cell surface
markers. Two types of signal intensity filters were used to identify MDSCs: 1)
binary intensity selection, i.e., based
on positive vs. negative signal, e.g., distinguishing CD14+ mMDSC from CD15+
gMDSC; and 2) categorical binning
of signal intensities to define a range of signal intensities, e.g., to
distinguish HLA-DRI w-ncgmMDSC from HLA-
DRneggMDSC. Signal intensity filters were applied sequentially, and
categorization was reviewed and confirmed by
pathologists. Exemplary thresholds for detecting cell surface markers after
application of signal intensity filters are
shown in Tables 19A-C below.
Table 19A
HLADR CD33
Negative <0.105 <0.17
Low 0.105 - 0.125 0.17 - 0.18
Med 0.125 - 0.155 0.18 - 0.19
High >0.155 >0.19
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Table 19B
CD11 b C014 CD15
CD66b
Negative <0.145 <0.24 <0.19 <0.19
Positive >0.145 >0.24 >0.19 >0.19
Table 19C
CD11 b HLADR CD14 CD15 C033
CD66b
gMDSC +/low
mMDSC -/low +/high
classical monocyte
neutrophil -/med +/med
ml macrophage +/hig h
m2 macrophage
[914] An exemplary analysis of signal intensity filtering of MDSC markers in
bladder cancer is shown in FIG. 35A.
An example of HLA-DR detection using threshold cutoffs is shown in FIG. 35B.
All cells were plotted to illustrate
threshold cuttoffs, where the x-axis shows staining intensity (binned columns)
and the y-axis shows the number of
cells. Visualization of signal intensities were normalized by channel in order
to reduce background and bleed
through (spectral unmixing). Because of this, threshold cutoffs varied from
channel to channel. Examples of signal
normalization are shown in the panels of FIG. 35C.
[915] gMDSC populations in tumor samples were identified by first filtering
for CD15 and CD66b positivity (FIG.
36A). This filtering distinguished gMDSCs from mMDSCs, which are CD15 and
CD66b negative. Sequential
application of signal filters was then applied to specify gMDSCs according to
the markers and thresholds described
above (FIG. 36B). A comparison of the original tumor image from a bladder
cancer sample (right panel), without
signal filtering, the image after applying CD15+/CD66bhgh filtering (top left
panel), and the image after applying
CD15-VCD661Pgh/CD14-/CD3Vw/HLADR-/CD111D+ filtering (bottom left panel) is
shown in FIG. 36C. A large portion
of the gMDSC population appeared to be at the tumor edge, near the stromal
compartment.
[916] Using the same signal filtering described above, gMDSC and mMDSC
populations were analyzed in
samples from bladder cancer, melanoma, NSCLC, and ovarian cancer (FIGs. 37A-
C). The samples had much
higher gMDSC populations as compared to mMDSC populations. Ovarian cancer
exhibited the lowest gMDSC
levels and the highest mMDSC levels among the various indications examined.
mMDSC levels were the lowest in
melanoma samples.
Example 8: Phase 1 clinical study
[917] A multi-center, open-label, Phase 1, first-in-human, dose escalation and
dose expansion study (DRAGON;
NCT04291079) is ongoing to evaluate the safety, tolerability,
pharmacokinetics, pharmacodynamics, and efficacy
of Ab6 administered alone and in combination with anti-PD-(L)1 therapy in
adult patients with locally advanced or
metastatic solid tumors. The study is divided into three treatment parts, Part
Al, Part A2, and Part B, and a Long-
Term Extension Phase (LTEP). Part Al (dose escalation) determines the maximum
tolerated dose (MTD) or
maximum administered dose (MAD) of Ab6 as a single agent and the recommended
Phase 2 dose (RP2D) of Ab6
as a single-agent. Part A2 (dose escalation) determines the MTD or MAD of Ab6
in combination with anti-PD-(L)1
antibody therapy and the RP2D of Ab6 in combination with anti-PD-(L)1 antibody
therapy for use in Part B. In Part
B (dose expansion), parallel cohorts of patients with Non-small cell lung
cancer (NSCLC), Urothelial Carcinoma
(UC), Cutaneous melanoma (MEL), renal cell carcinoma, or another advanced or
metastatic solid tumor type that
is not NSCLC, UC, or MEL, are enrolled to confirm the tolerability of the RP2D
of Ab6 (determined in Part A2) and
to evaluate the anti-tumor activity of Ab6 in combination with an anti-PD-(L)1
antibody therapy. Patients in Part Al
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may continue treatment with Ab6 as a single agent at the RP2D in the LTEP
following 3 cycles of treatment with
Ab6 as a single agent in Part Al. Patients in Part A2 may continue treatment
with Ab6 at the RP2D in combination
with anti-PD-(L)1 antibody therapy in the LTEP following 3 cycles of treatment
with Ab6 in combination with anti-
PD-(L)1 antibody therapy in Part A2. Patients in Part B may continue treatment
with Ab6 in combination with anti-
PD-(L)1 antibody therapy in the LTEP following 9 cycles of treatment with Ab6
in combination with anti-PD-(L)1
antibody therapy in Part B.
[918] In Part Al, patients with advanced solid tumors are administered 80 mg,
240 mg, 800 mg, 1600 mg, 2400
mg, or 3000 mg of Ab6 via intravenous infusion once every three weeks. In Part
A2, patients who are non-
responders to prior anti-PD-(L)1 therapy are administered a combination of an
approved anti-PD-(L)1 therapy
based on tumor type and Ab6, where Ab6 is administered intravenously at 240
mg, 800 mg, 1600 mg, or 2400 mg
once every 3 weeks. In Part B, patients who are non-responders to prior anti-
PD-(L)1 therapy are administered a
combination of an anti-PD-(L)1 therapy and Ab6 via intravenous infusion.
Cohort A of Part B consists of patients
with non-small cell lung cancer (NSCLC) who are administered Ab6 in
combination with pembrolizumab. Cohort B
of Part B consists of patients with urothelial carcinoma (UC) who are
administered Ab6 in combination with
pembrolizumab. Cohort C of Part B consists of patients with melanoma (MEL) who
are administered Ab6 in
combination with pembrolizumab. Cohort D of Part B consists of patients with a
cancer that is not NSCLC, UC, or
MEL, and the patients are administered Ab6 in combination with an approved
anti-PD-(L)1 therapy based on tumor
type. Blood chemistry assays are performed as a safety assessment in Part B,
including measuring blood levels of
cytokines interleukin-1 beta (IL-1(3), tumor necrosis factor alpha (TNBa),
interleukin-6 (IL-6), interleukin-8 (IL-8),
interferon gamma (IFNy), and interleukin 10 (IL-10).
[919] Primary outcome measures for the study include 1) evaluating the safety
and tolerability of Ab6 (Part Al)
or in combination with an anti-PD-(L)1 therapy (Part A2) and 2) determining
the MID or MAD, RP2D, and dose
limiting toxicities (DLTs). DLTs response as assessed in the first 21 days of
the study. MTD, MAD, RP2D, and
DLTs are assessed using RECIST v1.1 and do not include toxicities clearly
related to disease progression and
intercurrent illness. Safety endpoints include adverse events, clinical
observations (e.g., vital signs, physical
examination), laboratory tests, electrocardiogram, and echocardiogram.
[920] Secondary outcome measures of the study include evaluating the
pharmacokinetics of Ab6 alone or in
combination with an anti-PD-(L)1 therapy. Specific pharmacokinetic parameters
include maximum drug
concentration (C.), time to C. (Tr.), last validated plasma concentration
(Ci.st), time to Clast (Ti.st), and half-
life (T1/2). Time frame for evaluating pharmacokinetic parameters are cycles 1
and 3, where each cycle is 21 days.
The anti-tumor activity of Ab6 alone or in combination with an anti-PD-(L)1
therapy is also evaluated as a secondary
outcome. Specific clinical response parameters include objective response,
defined as a complete response (CR)
or partial response (PR), as determined by RECIST v1.1 or iRECIST v1.1. Other
secondary outcome measures
include evaluating anti-drug antibodies and biomarkers. Biomarker assessment
includes evaluation of the immune
lanscape (e.g., identify immune-inflamed, immune-excluded, and immune desert
tumors) and TGFI3 pathway status
within the tumor. Orthogonal biomarker strategies are also applied to assess
multiple biologically related pathways,
such assessments include as tumor-based multiplex immunohistochemistry (INC),
next-generation sequencing,
and evaluation of blood-based biomarkers.
[921] Key inclusion criteria for the study include patients who are L18 years
of age, with a predicted life expectancy
of L 3 months and patients who have measurable disease per RECIST v1.1 at
screening. Additionally, patients
must have an Eastern Cooperative Oncology Group performance status (PS) 0-1.
To meet eligibility for part Al,
patients must have a histologically documented solid tumor that is metastatic
or locally advanced, for which
standard of care therapy does not exist, has failed in the patient, or is not
tolerated by the patient, or for which the
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patient has been assessed by the Investigator as not being a suitable
candidate or otherwise ineligible for the
standard of care therapy. To meet eligibility for Part A2 and Part B, patients
must have a history of primary anti-
PD-(L)1 antibody nonresponse presenting (based upon the Investigator's
assessment) either as progressive
disease or stable disease (e.g., not improving, but also not worsening,
clinically or radiographically) after at least 3
cycles of treatment with an anti-PD-(L)1 antibody therapy (alone or in
combination with chemotherapy) approved
for that tumor type. Additionally, patients must also have received their most
recent dose of anti-PD-(L)1 antibody
therapy within 6 months of enrollment (or 9 months for UC cohort) in order to
meet eligibility for Part B.
[922] Key exclusion criteria include patients who have ECOG performance status
concurrent anti-cancer
treatment, a history of active metastatic CNS disease, an active or prior
history of autoimmune disease,
hypersensitive or presence of anti-drug antibodies to anti-PD-(L)1 antibody
therapy, and/or concurrent second
malignancy. Additional exclusion criteria include patients who have second
malignancy at other sites with the
exceptions of adequately treated in situ carcinoma, e.g., cervical carcinoma,
non-melanoma skin cancer, bilateral
synchronous discordance breast cancer, or indolent prostate cancer under
observation. A past history of other
malignancies are allowed as long as the patient has been free of recurrence
for years, or if the patient has been
treated with curative intent within the past 2 years, and in the opinion of
the Investigator, is unlikely to have a
recurrence. Women who are pregnant or breastfeeding are also excluded.
Exclusion criteria for Part A2 and Part
B include patients who are receiving concurrent anticancer treatment, with the
exception of an anti-PD-(L)1
antibody therapy for Part A2 or Part B, either approved or investigational,
within 28 days prior to administration of
Ab6, patients who have received biologic therapy (except for anti-PD-(01
antibody therapy for Part A2 or Part B),
<28 days prior to administration of Ab6, pateints who have received systemic
cytotoxic chemotherapy (except for
in combination with anti-PD-(L)1 antibody therapy) <28 days prior to
administration of Ab6, patients who have
received targeted small molecule therapy within 5 half-lives of the compound
prior to administration of Ab6, or
pateints who have a history of intolerance or treatment discontinuation due to
severe irAE or other adverse reaction
from prior anti-PD-(L)1 antibody therapy.
[923] Twenty patients have been dosed (14 patients in Part Al and 6 patients
in Part A2). Dose escalation phase
of the study is on-going. Early results from Part Al showed that escalation of
Ab6 from 80 mg to 2400 mg resulted
in no DLTs. Similarly, escalation of Ab6 from 240 mg to 800 mg, when dosed in
combination with an anti-PD-(L)1
therapy, did not result in DLTs in Part A2. A dose of 3000 mg of Ab6 is
currently under evaluation in Part A2.
Example 9: Effects of TGFI31 inhibition on MK differentiation in cells
isolated from myelofibrosis patients
[924] TGF6 is capable of promoting the proliferation of marrow stromal cells
and collagen deposition as well as
endothelial cell proliferation thereby promoting microenvironmental changes
that resemble those observed in MF
bone marrow. Furthermore, increased levels of TGF6 in myelofibrosis patients
have been implicated in both the
development of anemia and thrombocytopenia as well as disease development in
patients with myelofibrosis. Thus,
it is hypothesized that treatment with a TGF6 inhibitor may be able to reverse
the undesired effects resulting from
increased TGFp levels in myelofibrosis patients and myelofibrotic cancers.
Since megakaryocytes (MK) and
platelets are the major sources of TGF61 (Blood 2007;110:986-993), the
therapeutic potential of the TGF61
inhibitor Ab6 is explored by culturing mononuclear cells or CD34+ cells from
myelofibrosis patients under conditions
that generate megakaryocyte-enriched populations.
[925] Culture conditions that generate MK enriched populations are as
described according to Mosoyan et al.,
(Leukemia. 2017 November; 31(11): 2458-2467, the contents of which are herein
incorporated by reference to
their entirety). Briefly, cells are cultured using a two-step liquid culture
system of either mononuclear cells or CD34+
cells from healthy donors or myelofibrosis patients (MF-MNCs). Cells are
suspended in IMDM medium (Invitrogen,
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Grand Island, NY) supplemented with 1% penicillin/streptomycin, 1% L-Glutamine
(lnvitrogen, Grand Island, NY),
20mM 13-mercaptoethanol, 1% bovine serum albumin (BSA) Fraction V (Sigma, St.
Louis, MO), 30% serum
substitute BIT 9500, 100 rig/ml recombinant human stern cell factor (hSCF), 50
nM/m1 recombinant human
thrombopoietin (hTP0) (R&D Systems, Minneapolis, MN, USA) (lancu-Rubin et al.,
Exp Hematol. 2012
Jul;40(7):564-74, the contents of which are herein incorporated by reference
to their entirety). MK colony forming
unit (CFU-MK) assays are performed by using the MegaCult System and Detection
Kit according to the
manufacturer's instructions (Stern Cell Technologies, Vancouver, BC, Canada).
Isolation of CD61+ MK is
performed using an Immunomagnetic Selection Kit as per manufacturer's
recommendations (Miltenyi Biotech Inc.,
Auburn, CA, USA).
[926] MF-MNC or CD34+ cells from healthy individuals or myelofibrosis patients
are cultured with SCF and TPO
for 7 days, after which the cells were cultured for 2-8 more days with TPO
alone. Ab6, a negative control antibody
(e.g., isotype control), or the TGFI3R1 kinase inhibitor galunisertib is added
for 24-72 hours during conditioning
periods of cell cultures as described. Conditioning media is collected from
each of the cultures and assayed for
levels of TGF131, TG932, and TG933 by ELISA. The effects of the conditioned
media treated with a negative control
antibody, Ab6, or galunisertib on normal fibroblasts and endothelial cell
proliferation and on collagen deposition are
evaluated. Effects of the conditioned media on normal and myelofibrotic CD34+
colony formation in the presence
of cytokine combination are also assessed.
[927] To determine the effects of conditioned media treated with negative
control, Ab6, or galunisertib on
malignant hematopoiesis, hematopoietic colonies cloned from myelofibrotic
CD34+ cells are genotyped for
myelofibrosis driver mutations. To determine whether TGF81 inhibition
eliminates downstream effects of excessive
TGF3 signaling, target cells such as fibroblasts, endothelial cells and
hematopoietic cells are analyzed for SMAD
activation.
[928] MK cultures are analyzed using flow cytometry and MK cells are
identified based on expression of CD41
and CD42 protein markers. Treatment of MK cultures with up to 100 nM of Ab6
inhibits autocrine TGF31 signaling
in MKs from MF-MNCs but does not inhibit pSMAD2 activation by recombinant
TGFI31. In patient cell cultures
where Smad phosphorylation is demonstrated, TGF3 activation occurs in a cell-
autonomous manner. Suppression
of phosphorylation by Ab6 confirms that phosphorylation is induced by TGFb1.
Addition of exogenous TGF131
growth factor to cell cultures is used to demonstrate Smad signaling
competence in cultures.
Example 10: Exacerbation of ECM dys regulation in mice treated with TGFp3
inhibitor
[929] From a safety standpoint, there has been a wide recognition that pan
inhibition of TGFI3 can cause toxicities,
which underscores the fact that no TGF-13 inhibitors have been successfully
developed to this day. To circumvent
potentially dangerous adverse effects, a number of groups have recently turned
to identifying inhibitors that target
a subset but not all - of the isoforms and still retain efficacy.
[930] Pro-fibrotic phenotypes (e.g., increased collagen deposit into the ECM)
are associated not only with fibrosis,
but also with aspects of cancer progression, such as invasion and metastasis.
See, for example, Chakravarthy et
al., (Nature Communications, (2018) 9:4692. "TGF-P-associated extracellular
matrix genes link cancer-associated
fibroblasts to immune evasion and immunotherapy failure"). Diseased tissues
with dysregulated ECM, including
stroma of various cancer types, can express both TG931 and TGF83. Indeed, as
recently as in 2019, multiple
groups are making effort to develop TGFI3 inhibitors that target both of these
isoforms, such as ligand traps and
integrin inhibitors.
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[931] Previously, we have shown that inhibition of TGF61 alone is sufficient
to overcome primary resistant to
checkpoint blockade therapy in tumor models. To further examine in vivo role
of TGF63 in the regulation of ECM,
a TGF61-selective inhibitor and TGF63-selective inhibitor were tested in a
diet-induced murine liver fibrosis model.
[932] In control animals that received regular diet, the baseline fibrosis
score as measured by percentage of PSR-
positive area by histology was less than 2%. After 12 weeks of fibrosis-
causing diet (antibody treatment in the last
8 weeks, with continued diet), control animals treated with IgG alone showed
approximately 6.5% of PSR-positive
area by histology. Animals treated with the TGF61-selective inhibitor reduced
that to approximately 4% of PSR-
positive area (p < 0.001 vs IgG control group). Animals treated with the TGF63-
selective inhibitor were found to
develop significantly worse fibrosis with approximately 12.5% PSR-positive
area (p < 0.001 vs IgG control group),
while animals treated with a combination of the TGF63-selective inhibitor and
the TGF61 -selective inhibitor showed
milder fibrosis with approximately 8% PSR-positive area (p < 0.001 vs IgG
control group).
[933] These results suggest that inhibition of TGF63 exacerbated ECM
dysregulation as indicated by increased
collagen accumulation. Data also show that concurrent inhibition of the 1/3
isoforrns in fact attenuates the efficacy
of TGF61 inhibition in vivo, raising the possibility that TGF83 inhibition may
be detrimental to ECM regulation.
Equivalents
[934] The various features and embodiments of the present disclosure, referred
to in individual sections above
apply, as appropriate, to other sections, mutatis mutandis. Consequently,
features specified in one section may be
combined with features specified in other sections, as appropriate.
[935] Those skilled in the art will recognize, or be able to ascertain using
no more than routine experimentation,
many equivalents to the specific embodiments of the disclosure described
herein. Such equivalents are intended
to be encompassed by the following claims.
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