Note: Descriptions are shown in the official language in which they were submitted.
TLR9 MODULATORS FOR TREATING CANCER
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
The contents of the text file submitted electronically herewith are
incorporated
herein by reference in their entirety: A computer readable format copy of the
Sequence
Listing (filename: 105968-5180 PR Sequence Listing).
FIELD OF THE INVENTION
The invention relates to the field of oncology, and use of immunotherapy in
the
treatment of cancer.
BACKGROUND OF THE INVENTION
Toll-like receptors (TLRs) are present on many cells of the immune system and
are involved in the innate immune response. In vertebrates, this family
consists of eleven
proteins called TLR1 to TLR11 that recognize pathogen associated molecular
patterns
from bacteria, fungi, parasites, and viruses. TLRs are a key mechanism by
which
vertebrates recognize and mount immune responses to foreign molecules and also
provide a link between the innate and adaptive immune responses. Some TLRs are
located on the cell surface to detect and initiate a response to extracellular
pathogens and
other TLRs are located inside the cell to detect and initiate a response to
intracellular
pathogens.
TLR9 recognizes unmethylated CpG motifs in bacterial DNA and in synthetic
oligonucleotides. While agonists of TLR9, and other TLR agonists, can initiate
anti-
tumor immune responses, TLR agonists can also induce immune suppressive
factors that
may be counterproductive for effective tumor responses.
There is a need for cancer immunotherapies that induce antitumor responses,
and
keep the immune system productively engaged to improve the overall response.
Additionally, there is a need to identify patients who may best benefit from
such cancer
immunotherapies and be more likely to respond to treatment.
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Date Recue/Date Received 2022-02-10
SUMMARY OF THE INVENTION
In various aspects, the present invention provides a method for treating a
tumor,
including, without limitation, metastatic melanoma, comprising intratumorally
administering an oligonucleotide TLR9 agonist (e.g., IMO-2125 or other
immunostimulatory oligonucleotides described herein) to a cancer patient. The
method
further comprises administering an immune checkpoint inhibitor therapy, such
as a
therapy targeting CTLA-4, PD-1/PD-Li/PD-L2, TIM3, LAG3, and/or IDO. The TLR9
agonist upon intratumoral injection induces global increases in expression of
checkpoint
genes, including ID01, PDL1, PD1, ID02, CEACAM1, 0X40, TIM3, LAG3, CTLA4,
and OX4OL. By altering immune signaling in the tumor microenvironment, such
changes
in gene expression provide opportunities to improve responsiveness to
checkpoint
inhibitor therapy, including in some embodiments, a complete response. The
invention
further provides the opportunity to balance anti-tumor responses with
inhibitory signals,
thereby also minimizing immune-related adverse events (irAEs) of checkpoint
inhibitor
therapy. The invention further provides the opportunity to select patients
with metastatic
disease whose tumor is more likely to respond to therapy.
In various embodiments, the patient has a cancer that was previously
unresponsive to, or had become resistant to, a checkpoint inhibitor therapy,
such as anti-
CTLA-4, anti-PD-1, or anti-PD-Li and/or anti-PD-L2 agent. The invention finds
use for
treating primary cancer or a metastatic cancer, including cancers that
originate from skin,
colon, breast, or prostate, among other tissues. In some embodiments, the
cancer is
progressive, locally advanced, or metastatic carcinoma. In some embodiments,
the cancer
is metastatic melanoma.
In accordance with embodiments of the invention, the immunostimulatory
oligonucleotide (e.g., IMO-2125) is administered intratumorally. Intratumoral
administration alters immune signaling in the tumor microenvironment, priming
the
immune system for an effective anti-tumor response, while inducing changes
that are
compatible with more effective checkpoint inhibitor therapy. For example, the
TLR9
agonist (e.g., IMO-2125) may be administered intratumorally at from about 4 mg
to about
64 mg per dose, with from about 3 to about 12 doses being administered over 10
to 12
weeks. For example, therapy may be initiated with 3 to 5 weekly doses of IM0-
2125,
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Date Recue/Date Received 2022-02-10
optionally followed by 3 to 8 maintenance doses, which are administered about
every
three weeks.
During the regimen of IMO-2125 (or other TLR9 agonist), one or more
checkpoint inhibitor therapies are administered to take advantage of the
changes in
immune signaling. In some embodiments, the patient receives an anti-CTLA-4
agent
(e.g., ipilimumab or tremelimumab) and/or an anti-PD-1 agent (e.g., nivolumab
or
pembrolizumab). The immune checkpoint inhibitor can be administered
parenterally,
such as, in some embodiments, subcutaneously, intratumorally, intravenously.
For
example, in various embodiments the immune checkpoint inhibitor is
administered at a
dose of from about 1 mg/kg to about 5 mg/kg intravenously. The initial dose of
the
immune checkpoint inhibitor can be administered at least one week after the
initial TLR9
agonist dose, for example in about weeks 2, 3 or 4. In some embodiments, the
immunotherapy agent is administered from about 2 to about 6 times (e.g., about
4 times,
preferably every three weeks).
In some embodiments, IMO-2125 is administered intratumorally to a metastatic
melanoma patient previously found to be unresponsive or only partially
responsive to
PD-1 blockade therapy. For example, IMO-2125 is administered at a dose of from
4 to
32 mg per dose in weeks 1, 2, 3, 5, 8, and 11, with ipilimumab i.v. at 3
mg/kg. Ipilimumab
can be administered every three weeks, beginning in week 2. Alternatively,
pembrolizumab can be administered i.v. at 2 mg/kg every three weeks beginning
on week
2.
In some embodiments, IMO-2125 is administered intratumorally to a metastatic
melanoma patient exhibiting low expression of MHC Class I genes, e.g., in a
tumor
biopsy. For example, IMO-2125 is administered at a dose of from 4 to 32 mg per
dose
in weeks 1, 2, 3, 5, 8, and 11, with ipilimumab i.v. at 3 mg/kg. Ipilimumab
can be
administered every three weeks, beginning in week 2. Alternatively,
pembrolizumab can
be administered i.v. at 2 mg/kg every three weeks beginning on week 2.
In some embodiments, IMO-2125 is administered intratumorally to a metastatic
melanoma patient exhibiting no measurable expression of HLA-A, HLA-B, and HLA-
C, e.g., in a tumor biopsy. In some embodiments, IMO-2125 is administered
intratumorally to a metastatic melanoma patient exhibiting no measurable
expression of
B2M, the f32-microglobulin gene, e.g., in a tumor biopsy. In another aspect.
IMO-2125
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Date Recue/Date Received 2022-02-10
is administered to metastatic cancer patients exhibiting elevated levels of
serum PD-L2.
In another aspect, IMO-2125 is administered to metastatic cancer patients with
tumors
enriched for dendritic cells as determined by pre-treatment biopsy analysis.
In some embodiments, IMO-2125 is administered intratumorally to a metastatic
melanoma patient exhibiting high baseline expression of CTLA4, e.g., in a
tumor biopsy.
For example, IMO-2125 is administered at a dose of from 4 to 32 mg per dose in
weeks
1, 2, 3, 5, 8, and 11, with ipilimumab i.v. at 3 mg/kg. Ipilimumab can be
administered
every three weeks, beginning in week 2. Alternatively, pembrolizumab can be
administered i.v. at 2 mg/kg every three weeks beginning on week 2.
The present methods in various embodiments allow for a robust anti-tumor
immune response (which in some embodiments is a complete response), and which
does
not come at the expense of significant side effects, e.g. relative to side
effects observed
when one or more immunotherapies are used in the absence of the TLR9 agonist.
Such
side effects include commonly observed immune-related adverse events that
affect
various tissues and organs including the skin, the gastrointestinal tract, the
kidneys,
peripheral and central nervous system, liver, lymph nodes, eyes, pancreas, and
the
endocrine system; such as hypophysitis, colitis, hepatitis, pneumonitis, rash,
and
rheumatic disease (among others).
In an embodiment of the invention, a method for treating a tumor in a patient
having low tumor expression of MHC Class I genes comprising intratumoral
administration of a TLR 9 agonist is disclosed. In some embodiments, a method
for
treating a tumor in a patient comprising: (a) determining MHC Class I gene
expression
in a tumor sample and (b)
administering a TLR9 agonist if said gene
expression is present in less than 50% of the tumor cells is disclosed.
In some embodiments according to the present invention, a method for treating
a
tumor in a patient having increased serum PD-L2 levels comprising intratumoral
administration of a TLR 9 agonist is disclosed. In some embodiments according
to the
present invention, a method for treating a tumor in a patient having increased
serum PD-
L2 levels comprising: (a) determining serum PD-L2 levels in said patient and
(b)
administering a TLR 9 agonist if said PD-L2 levels are increased in said
patient as
compared with a control level. In some embodiments, the PD-L2 level is between
about
750 pg/mL and 5000 pg/mL. In some embodiments, the PD-L2 level is between
about
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Date Recue/Date Received 2022-02-10
1100 pg/mL and about 3000 pg/mL. In some embodiments, the PD-L2 level is
between
about 1100 pg/mL and 2100 pg/mL. In some embodiments, the patient is selected
based
on a baseline tumor biopsy enriched in dendritic cells.
In an embodiment of the invention, a method for treating a tumor in a patient
having high baseline tumor expression of CTLA4 comprising intratumoral
administration of a TLR9 agonist and a CTLA4 checkpoint inhibitor (e.g., an
antibody)
is disclosed. In some embodiments, a method for treating a tumor in a patient
comprising: (a) determining CTLA4 expression in a tumor sample, (b)
administering a
TLR9 agonist if said CTLA4 expression is greater than or equal to 20% greater
than
mean tumor baseline expression of CTLA4 in the patient population, and 3)
administering an inhibitor of CTLA4 if said CTLA4 expression is greater than
or equal
to 20% greater than mean tumor baseline expression of CTLA4 in the patient
population.
For example, upon determining that a cancer patient's baseline tumor
expression of
CTLA4 is greater than or equal to 20% greater than mean tumor baseline
expression of
CTLA4 in the patient population, IMO-2125 may be administered at a dose of
from 4 to
32 mg per dose in weeks 1, 2, 3, 5, 8, and 11, with ipilimumab i.v. at 3
mg/kg. Ipilimumab
can be administered every three weeks, beginning in week 2. Alternatively,
pembrolizumab can be administered i.v. at 2 mg/kg every three weeks beginning
on week
2.
In any of the methods disclosed herein, the TLR9 agonist has the structure: 5'-
TCG1AACG1TTCG1-X- G1CTTG1CAAGICT-5' (5' SEQ ID NO:4-X-SEQ ID NO:4 5'),
wherein Gi is 2'-deoxy-7-deazaguanosine and X is a glycerol linker. In some
embodiments, the TLR9 agonist is tilsotolimod (IMO-2125). In another
embodiment,
any of the methods disclosed herein further comprise administering at least
one immune
checkpoint inhibitor. In another embodiment, any of the methods disclosed
herein
further comprise first sensitizing the tumor microenvironment with
intratumoral
administration of the TLR9 agonist.
In some embodiments according to the present invention, the immune checkpoint
inhibitor is co-administered with the TLR9 agonist. In some embodiments,
immune
checkpoint inhibitor is administered after the TLR9 agonist. In some
embodiments, the
immune checkpoint inhibitor is administered at least one day after the TLR9
agonist. In
some embodiments, the immune checkpoint inhibitor is administered at least one
week
after the TLR9 agonist.
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Date Recue/Date Received 2022-02-10
In some embodiments according to the present invention, the immune checkpoint
inhibitor is selected from a checkpoint inhibitor that targets PD-1, PD-L1,
cytotoxic T-
lymphocyte-associated protein 4 (CTLA-4), LAG3, B7-H3, B7-H4, KIR, 0X40, IgG,
IDO-1, IDO-2, CEACAM1, TNFRSF4, BTLA, OX4OL, and TIM3. In some
embodiments, the checkpoint inhibitor targets CTLA-4 and is a monoclonal
antibody
against CTLA-4. In some embodiments, the checkpoint inhibitor is selected from
the
group consisting of ipilimumab, tremelimumab, or biosimilars thereof. In some
embodiments, the checkpoint inhibitor targets PD-1 and is selected from the
group
consisting of nivolumab, pembrolizumab, and biosimilars thereof.
In some embodiments according to the present invention, the checkpoint
inhibitor
is administered beginning on week 2 after a first administration of TLR9
agonist. In
some embodiments, the checkpoint inhibitor is administered beginning on week 3
after
a first administration of TLR9 agonist. In some embodiments, the checkpoint
inhibitor
is administered every three weeks. In some embodiments, the checkpoint
inhibitor is
administered at least 2 to 6 times.
In some embodiments according to the present invention, the TLR9 agonist is
administered at a dose of from about 1 mg to about 20 mg. In some embodiments,
the
dose is about 8 mg.
In some embodiments, the TLR 9 agonist is IMO-2125 and the immune
checkpoint inhibitor therapy is an anti-CTLA4 inhibitor.
In some embodiments according to the present invention, the tumor is a
metastatic tumor. In some embodiments, the tumor is selected from melanoma,
lung
tumor, kidney tumor, prostate tumor, cervical tumor, colorectal tumor, colon
tumor,
pancreatic tumor, ovarian tumor, urothelial tumor, gastric/GEJ tumor, head and
neck
tumor, glioblastoma, Merkel cell tumor, head and neck squamous cell carcinoma
(HNSCC), non-small cell lung carcinoma (NSCLC), small cell lung tumor (SCLC),
or
bladder tumor. In some embodiments, the tumor is metastatic melanoma. In some
embodiments, the tumor is a colorectal tumor or a colon tumor. In some
embodiments,
the tumor is a head and neck tumor or a head and neck squamous cell carcinoma
(HNSCC).
In some embodiments according to the present invention, the low expression of
tumor MHC Class I gene expression is less than 25% of the expression in
healthy tissue.
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Date Recue/Date Received 2022-02-10
In some embodiments, the low expression of tumor MHC Class I gene expression
is less
than 50% of the expression in healthy tissue.
In some embodiment according to the present invention, a method for treating
metastatic melanoma in a patient having 50% or lower tumor expression of MHC
Class
I genes, the method comprising: (a) sensitizing the tumor microenvironment
with
intratumoral administration of tilsotolimod (IMO-2125) at a dose of about 8 mg
and (b)
systemically administering ipilimumab at least one week after the
administration of
tilsotolimod is disclosed.
In some embodiments according to the present invention, the high baseline
expression of tumor CTLA4 is greater than or equal to 20% greater than the
mean
baseline CTLA4 expression of the patient population (e.g., baseline tumor
CTLA4
expression is 30% higher than mean baseline tumor expression in a patient
population
having metastatic melanoma).
In some embodiment according to the present invention, a method for treating
metastatic melanoma in a patient having high baseline tumor expression of
CTLA4, the
method comprising: (a) sensitizing the tumor microenvironment with
intratumoral
administration of tilsotolimod (IMO-2125) at a dose of about 8 mg and (b)
systemically
administering ipilimumab at least one week after the administration of
tilsotolimod is
disclosed.
Other aspects and embodiments will be apparent from the following detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows IRF7 gene expression levels before an intratumoral dose of
tilsotolimod and 24 hours after an intratumoral dose.
FIG. 2 shows a volcano plot of genes upregulated following an intratumoral
dose
of tilsotolimod. Among the upregulated genes are IRF7, MX1, IFIT1, IFIT2,
TAP1, and
TAP2.
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Date Recue/Date Received 2022-02-10
FIG. 3 shows the Dendritic Cell (DC) score (10g2) of the baseline tumor
lesions
for patients showing either a complete response (CR) or a partial response
(PR), those
with progressive disease (PD), and those with stable disease (SD) after
treatment.
FIG. 4 shows the results of flow cytometry analysis of tumor biopsy tissues
both
before an intratumoral dose of tilsotolimod and 24 hours after an intratumoral
dose. The
percentage of cells expressing HLA-DR are reported.
FIGS. 5A-5C show HLA-A, HLA-B, and HLA-C gene expression, respectively,
in tumors from patients showing either a complete response (CR) or a partial
response
(PR), those with progressive disease (PD), and those with stable disease (SD)
after
treatment.
FIG. 6 shows a heatmap of the cytotoxicity gene expression profile in baseline
tumor samples. The heatmap is shaded based on clinical response.
FIG. 7 comprises FIGS. 7A-7E. FIG. 7A shows imaging guided intratumoral
injection of IMO-2125. FIG. 7B shows two pre- and post-therapy injected
(yellow
arrow) and distant (red arrow) lesions. FIG. 7C depicts the RECIST v1.1
classification
of the decrease in target lesion diameters for the study participants in
change from
baseline (percentage, %). FIG. 7D plots the best response as of the cut-off
date for study
subjects with at least one post-baseline disease evaluation. FIG. 7E is a
waterfall plot
showing the maximum percentage reduction from baseline sum of the individual
longest
lesion diameters (mm) by injection status, where, at each point, the bars on
the left
represent injected measurable lesions and the bars on the right represent non-
injected
measurable lesions.
FIG. 8 comprises FIGS. 8A-8E, and demonstrates IMO-2125 induces a local
type 1 IFN response gene signature, macrophage influx and DC1 maturation. FIG.
8A
shows a schematic of the tissue and blood samples collected from subjects
during the
course of the study. Light arrows depict tumor biopsy collection and dark
arrows show
collection of peripheral blood mononuclear cells (PBMCs). FIG. 8B is a volcano
plot of
RNA extracted from the local injected lesion at 24h post IMO-2125 as compared
to the
same lesion at baseline (predose). The adjusted p-value is shown. FIG. 8C
shows the
macrophage score as determined using the nSolver advanced analysis tool and is
shown
on a 10g2 scale. FIG. 8D shows the predose (baseline) and 24-hour postsdose
percentage
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Date Recue/Date Received 2022-02-10
HDR-DR expressed on live, lineage neg, CD lc+ mDC1 cells. A minimum of 100
events
was required for subgating. Finally, FIG. 8E shows the number of cells/mm2
expressing
IDO as assessed by a chromogenic immunohistochemistry (IHC) assay.
FIG. 9 comprises FIGS. 9A-9E and illustrate that local DC presence at baseline
and combination therapy overcomes known mechanisms of resistance to single
agent
anti-CTLA4. Fig. 9A plots the DC (dendritic cell) score determined using the
nSolver
advanced analysis tool and shown on a 10g2 scale. FIG. 9B plots the
concentration of
soluble PD-L2 in patient plasma measured before treatment. FIG. 9C plots the
cell type
score of each major cell type at baseline in local lesions as determined by
the nSolver
advanced analysis tool and is plotted based on patient clinical response.
Complete
Response (CR) + Partial Response (PR); Progressed Disease (PD); and Stable
Disease
(SD). FIG. 9D is a heatmap produced by hierarchical clustering using the T-
cell
functionality gene set at baseline in both local and distant lesions. FIG. 9E
is a second
hierarchal clustering heatmap based on the cytotoxicity gene set at baseline
in both local
and distant lesions.
FIG. 10 comprises FIGS. 10A-10D and illustrate data from banked PBMCs
collected prior to-and-during treatment. The PBMCs were thawed and stained for
memory/differentiation status and sorted using flow cytometry. The horizontal
line in
each of FIGS. 10B-10D indicates the median frequency across all the patients
at a given
time point. Each patient is indicated by their study ID. FIG. 10A is a
representative dot
plot showing memory subset identification by co-expression patterns of CCR7
and
CD45RA with the live, CD45+CD3+CD8+ subset. FIGS. 10B through 10D show the
frequency of the TEm subset over time in responding patients (PR+CR) (FIG.
10B), SD
patients (FIG. 10C), and PD patients (FIG. 10D).
FIG. 11 comprises FIGS. 11A-11D and illustrates that tumor infiltrating
lymphocyte (TIL) activation and proliferation correlates with response to
combination
therapy. Unsupervised hierarchical clustering based on Nanostring gene
expression
profiling: FIG. 11A shows a T-cell functional gene signature; and FIG. 11B
shows a
cytotoxicity gene signature. FIGS. 11C and 11D depict proliferation as
measured using
Ki67 staining and sorting by flow cytometry of CD8+ TILs at baseline, 24h
after
intratumoral injection and at C3W8 in either tumor lesions ( p=0.0071) or in
PBMCs (
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Date Recue/Date Received 2022-02-10
p>0.05) at baseline and C3W8, for responders (FIG. 11C) and non-responders
(FIG.
11D).
FIG. 12 comprises FIGS. 12A-12C. FIG. 12A and 12B show the frequency of
the top 50 clones in the distant tumor lesions at C3W8 compared to their
initial frequency
at baseline for responding (FIG. 12A) and non-responding (FIG. 12B) patients.
FIG. 12C
illustrates individual T-cell clones (top 50) identified at C3W8 in the
distant lesions of
individual responding patients assessed for presence in the local/injected
lesion (baseline
and C3W8) and at baseline in the distant lesion. Each image represents
individual
patients with each circle representing an individual T cell clone. Clones
shared between
lesions at all time points are shown in blue. The size of the circle indicates
relative
frequency at C3W8 and the numbers indicate the frequency relative to initial
baseline
presence.
FIG. 13 Compares PD-Li staining prior to therapy (at baseline) to that 24h
post
injection in the injected lesion. Chromogenic IHC staining PD-Li of injected
tumor
lesions prior to therapy and 24h post IMO-2125 injection. PD-Li is indicated
as a
percentage of the tumor cells present as indicated by H&E.
FIG. 14 comprises FIGS. 14A and 14B and shows Immunohistochemistry (IHC)
staining of CD3+ and CD8+ of injected and distant tumor lesions prior to
therapy. The
closed circles indicate stable disease (SD) and progressive disease (PD)
patients. Open
circles indicate partial response (PR) and compete response (CR) patients.
Each point
represents the mean of the total areas assessed in cells/mm2. FIG. 14A shows
CD3
staining and FIG. 14B shows CD8 staining. Only samples with tumor presence as
indicated by H&E are shown.
FIG. 15 comprises FIGS. 15A-15C and illustrates normalized, linear reads of
the
baseline tumor lesions (both local/injected and distant): FIG. 15A shows HLA-A
expression; FIG. 15B shows HLA-B expression; and FIG. 15C HLA-C expression
stratified based upon subsequent confirmed clinical response.
FIG. 16 comprises FIGS. 16A and 16B. FIG. 16A is a heatmap of the global
pathways assessed. The induction of a macrophage function score at cycle 3
week 8 as
compared to baseline tumor tissue is shown in FIG. 16B. Each spot represents a
single
patient sample and is in a 10g2 scale.
Date Recue/Date Received 2022-02-10
FIG. 17A shows the CTLA4 expression score (10g2) at baseline and at Week 8 of
treatment for tumor lesions (both treated and distant) for patients showing
either a
complete response (CR) or a partial response (PR), those with progressive
disease (PD),
and those with stable disease (SD) after treatment.
FIG. 17B shows the change in CTLA4 expression in tumor lesions (both treated
and distant) at Week 8 of treatment compared to the initial CTLA4 expression
in the
tumor lesions at baseline for patients showing either a complete response (CR)
or a partial
response (PR), those with progressive disease (PD), and those with stable
disease (SD)
after treatment.
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Date Recue/Date Received 2022-02-10
DETAILED DESCRIPTION
Definitions
The term "3", when used directionally, generally refers to a region or
position in
a polynucleotide or oligonucleotide 3' (toward the 3' position of the
oligonucleotide) from
another region or position in the same polynucleotide or oligonucleotide.
The term "5", when used directionally, generally refers to a region or
position in
a polynucleotide or oligonucleotide 5' (toward the 5' position of the
oligonucleotide) from
another region or position in the same polynucleotide or oligonucleotide.
The term "about" generally means plus or minus 10% of an associated numerical
value or numerical value range.
The term "agonist" generally refers to a substance that binds to a receptor of
a
cell and induces a response. Such response may be an increase in the activity
mediated
by the receptor. An agonist often mimics the action of a naturally occurring
substance
such as a
ligand.
The term "antagonist" or "inhibitor" generally refers to a substance that can
bind
to a receptor, but does not produce a biological response upon binding. The
antagonist
or inhibitor can block, inhibit, or attenuate the response mediated by an
agonist and may
compete with agonist for binding to a receptor. Such antagonist or inhibitory
activity may
be reversible or
irreversible.
The term "antigen" generally refers to a substance that is recognized and
selectively bound by an antibody or by a T cell antigen receptor. Antigens may
include
but are not limited to peptides, proteins, nucleosides, nucleotides and
combinations
thereof. Antigens may be natural or synthetic and generally induce an immune
response
that is specific for that
antigen.
The term "cancer" generally refers to, without limitation, any malignant
growth
or tumor caused by abnormal or uncontrolled cell proliferation and/or
division. Cancers
may occur in humans and/or animals and may arise in any and all tissues.
Treating a
patient having cancer with the invention may include administration of a
compound,
pharmaceutical formulation or vaccine according to the invention such that the
abnormal
or uncontrolled cell proliferation and/or division is affected.
The term an "effective amount" generally refers to an amount sufficient to
affect
a desired biological effect, such as a beneficial result. Thus, an "effective
amount" will
12
Date Recue/Date Received 2022-02-10
depend upon the context in which it is being administered. A effective amount
may be
administered in one or more prophylactic or therapeutic administrations.
The term "in combination with" generally means administering a first agent and
another agent useful for treating the disease or condition.
The term "individual", "patient", or "subject" are used interchangeably and
generally refers to a mammal, such as a human. Mammals generally include, but
are not
limited to, humans, non-human primates, rats, mice, cats, dogs, horses,
cattle, cows, pigs,
sheep and rabbits.
The term "linker" generally refers to any moiety that can be attached to an
oligonucleotide by way of covalent or non-covalent bonding through a sugar, a
base, or
the backbone. The linker can be used to attach two or more nucleosides or can
be attached
to the 5' and/or 3' terminal nucleotide in the oligonucleotide. In certain
embodiments of
the invention, such linker may be a non-nucleotidic linker.
The term "non-nucleotidic linker" generally refers to a chemical moiety other
than a nucleotidic linkage that can be attached to an oligonucleotide by way
of covalent
or non-covalent bonding. Preferably such non-nucleotidic linker is from about
2
angstroms to about 200 angstroms in length, and may be either in a cis or
trans
orientation.
The term "nucleotidic linkage" generally refers to a chemical linkage to join
two
nucleosides through their sugars (e.g. 3'-3', 2'-3',2'-5', 3'-5') consisting
of a phosphorous
atom and a charged, or neutral group (e.g., phosphodiester, phosphorothioate
or
phosphorodithioate)between adjacent nucleosides.
The term "treatment" generally refers to an approach intended toobtain a
beneficial or desired result, which may include alleviation of symptoms, or
delaying or
ameliorating a disease progression.
As used herein, the term "TLR9 agonist" generally refers to
animmunostimulatory oligonucleotide compound comprising a CpG
dinucleotidemotif
and is able to enhance or induce an immune stimulation mediated by TLR9. In
some
embodiments the CpG dinucleotide is selected from the groupconsisting of CpG,
C*pG,
CpG*, and C*pG*, wherein C is 2'-deoxycytidine,C* is an analog thereof, G is
2'-
deoxyguanosine, and G* is an analogthereof, and p is an internucleoside
linkage selected
from the groupconsisting of phospho di ester,
phosphorothioate, .. and
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Date Recue/Date Received 2022-02-10
phosphorodithioate.In preferred embodiments C* is selected from the group
consisting
of2'-deoxythymidine, arabinocytidine, 2'-
deoxythymidine,2'-deoxy-2'-
substitutedarabinocytidine, 2'-0-
substitutedarabinocytidine,2'-deoxy-5-
hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine,2'-deoxy-4-thiouridine. In
preferred
embodiments, G* is 2'deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine,
arabinoguanosine,2'-deoxy-2'substituted-arabinoguanosine,2'-0-substituted-
arabinoguanosine, 2'-deoxyinosine. In certain preferredembodiments, the
immunostimulatory dinucleotide is selected from thegroup consisting of C*pG,
CpG*,
and C*pG*.
As used herein, an immunomer refers to a compound comprising a tleast two
oligonucleotides linked together through their 3' ends, such that the
immunomer has more
than one accessible 5' end, wherein at least one of the oligonucleotides is an
immunostimulatory oligonucleotide. The linkage at the 3' ends of the component
oligonucleotides is independent of the other oligonucleotide linkages and may
be directly
via 5', 3' or2' hydroxyl groups, or indirectly, via a non-nucleotide linker or
a nucleoside,
utilizing either the 2' or 3' hydroxyl positions of the nucleoside. Linkages
may also utilize
a functionalized sugar or nucleobase of a 3' terminal nucleotide. The term
"accessible 5'
end "means that the 5' end of the oligonucleotide is sufficiently available
such that the
factors that recognize and bind to immunomers and stimulate the immune system
have
access to it. Optionally, the 5' OH can be linked to a phosphate,
phosphorothioate, or
phosphorodithioate moiety, an aromatic or aliphatic linker, cholesterol, or
another entity
which does not interfere with accessibility.
As used herein, an immunostimulatory oligonucleotide is an
oligodeoxyribonucleotide that comprises a CpG dinucleotide motif and is
capable of
enhancing or inducing a TLR9-mediated immune response. In some embodiments the
CpG dinucleotide is selected from the group consisting of CpG, C*pG, CpG*, and
C*pG*, wherein C is 2'-deoxycytidine, C* is an analog thereof, G is 2'-
deoxyguanosine,
and G* is an analog thereof, and p is an intemucleoside linkage selected from
the group
consisting of phosphodiester, phosphorothioate, and phosphorodithioate. In
preferred
embodiments C* is selected from the group consisting of 2'-deoxythymidine,
arabinocytidine, 2'-deoxythymidine,2'-deoxy-2'-substitutedarabinocytidine, 2'-
0-
substitutedarabinocytidine,2' -deoxy-5-hydroxycyti dine, 2'-deoxy-N4-alkyl-
cytidine,2'-
deoxy-4-thiouridine. In preferred embodiments, G* is 2'deoxy-7-deazaguanosine,
2'-
14
Date Recue/Date Received 2022-02-10
deoxy-6-thioguanosine, arabinoguanosine,2'-deoxy-2'substituted-
arabinoguanosine,2'-
0-substituted-arabinoguanosine, 2'-deoxyinosine. In certain preferred
embodiments, the
immunostimulatory dinucleotide is selected from the group consisting of C*pG,
CpG*,
and C*pG*.
In some embodiments, the immunomer comprises two or more
immunostimulatory oligonucleotides which may be the same or different.
Preferably,
each such immunostimulatory oligonucleotide has at least one accessible 5'
end.
In some embodiments, the oligonucleotides of the immunomer each
independently have from about 3 to about 35 nucleoside residues, preferably
from about
4 to about 30 nucleoside residues, more preferably from about 4 to about 20
nucleoside
residues. In some embodiments, the oligonucleotides have from about 5 to about
18, or
from about 5 to about 14, nucleoside residues. As used herein, the term
"about" implies
that the exact number is not critical. Thus, the number of nucleoside residues
in the
oligonucleotides is not critical, and oligonucleotides having one or two fewer
nucleoside
residues, or from one to several additional nucleoside residues are
contemplated as
equivalents of each of the embodiments described above. In some embodiments,
one or
more of the oligonucleotides have 11 nucleotides.
In certain embodiments of the invention, the immunomers comprise two
oligonucleotides covalently linked by a nucleotide linkage, or anon-nucleotide
linker, at
their 3'-ends or by functionalized sugar or by functionalized nucleobase via a
non-
nucleotide linker or a nucleotide linkage. As a non-limiting example, the
linker may be
attached to the 3'-hydroxyl. In such embodiments, the linker comprises a
functional
group, which is attached to the 3'-hydroxyl by means of a phosphate-based
linkage like,
for example, phosphodiester, phosphorothioate,
phosphorodithioate,
methylphosphonate, or by non-phosphate-based linkages. Possible sites of
conjugation
for the ribonucleotide are indicated in Formula I, below, wherein B represents
a
heterocyclic base and wherein the arrow pointing to P indicates any attachment
to
phosphorous.
15
Date Recue/Date Received 2022-02-10
1 /
VW - 0 0
0
Et
.-----4e
Formula I
0 01{
....-----v I .......,õ.s,
0=P¨s-
1 Ow
In some embodiments, the non-nucleotide linker is a small molecule,
macromolecule or biomolecule, including, without limitation, polypeptides,
antibodies,
lipids, antigens, allergens, and oligosaccharides. In some other embodiments,
the non-
nucleotidic linker is a small molecule. For purposes of the invention, a small
molecule is
an organic moiety having a molecular weight of less than 1,000 Da. In some
embodiments, the small molecule has a molecular weight of less than 750 Da.
In some embodiments, the small molecule is an aliphatic or aromatic
hydrocarbon, either of which optionally can include, either in the linear
chain connecting
the oligoribonucleotides or appended to it, one or more functional groups
including, but
not limited to, hydroxy, amino, thiol, thioether, ether, amide, thioamide,
ester, urea, or
thiourea. The small molecule can be cyclic or acyclic. Examples of small
molecule
linkers include, but are not limited to, amino acids, carbohydrates,
cyclodextrins,
adamantane, cholesterol, haptens and antibiotics. However, for purposes of
describing
the non-nucleotidic linker, the term "small molecule" is not intended to
include a
nucleoside.
In some embodiments, the non-nucleotidic linker is an alkyl linker or amino
linker. The alkyl linker may be branched or unbranched, cyclic or acyclic,
substituted or
unsubstituted, saturated or unsaturated, chiral, achiral or racemic mixture.
The alkyl
linkers can have from about 2 to about 18 carbon atoms. In some embodiments
such alkyl
linkers have from about 3 to about 9 carbon atoms. Some alkyl linkers include
one or
more functional groups including, but not limited to, hydroxy, amino, thiol,
thioether,
ether, amide, thioamide, ester, urea, and thioether. Such alkyl linkers can
include, but are
not limited to, 1,2 propanediol, 1,2,3 propanetriol, 1,3 propanediol,
triethylene glycol
hexaethylene glycol, polyethylene glycol linkers (e.g. [--0--CH2-CH2-In (n=1-
16
Date Recue/Date Received 2022-02-10
9)),methyl linkers, ethyl linkers, propyl linkers, butyl linkers, or hexyl
linkers. In some
embodiments, such alkyl linkers may include peptides or amino acids.
In various aspects, the present invention provides a method for treating a
tumor,
e.g. a metastatic tumor (including, without limitation, metastatic melanoma)
comprising
intratumorally administering an oligonucleotide TLR9 agonist (e.g., IM0-2125)
to a
cancer patient, in combination with immunotherapy with an immune checkpoint
inhibitor
therapy, such as a therapy targeting CTLA-4, PD-1/PD-Li/PD-L2, LAG3, TIM3,
and/or
IDO, wherein the tumor has low MHC Class I expression.
In various aspects, the present invention provides a method for treating a
tumor,
e.g. a metastatic tumor (including, without limitation, metastatic melanoma)
comprising
intratumorally administering an oligonucleotide TLR9 agonist (e.g., IM0-2125)
to a
cancer patient, in combination with immunotherapy with an immune checkpoint
inhibitor
therapy, such as a therapy targeting CTLA-4, PD-1/PD-Li/PD-L2, LAG3, TIM3,
and/or
IDO, wherein the tumor has high baseline CTLA-4 expression.
In some embodiments, the immune checkpoint inhibitor is an inhibitor of
Programmed Death-Ligand 1 (PD-L1, also known as B7-H1,CD274), Programmed
Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3,TIM3, 2B4, A2aR, B7H1,
B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40,CD70, CD80, CD86, CD137,
CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2,HVEM, ID01, ID02, ICOS
(inducible T cell costimulator), KIR, LAIR1,LIGHT, MARCO (macrophage receptor
with collageneous structure), PS(phosphatidylserine), OX-40, SLAM, TIGHT,
VISTA,
VTCN1, or any combinations thereof. In some embodiments, the immune checkpoint
inhibitor is an inhibitor of ID01, CTLA4, PD-1, LAG3, PD-L1, TIM3, or
combinations
thereof. In some embodiments, the immune checkpoint inhibitor is an inhibitor
of PD-
Li. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-
1. In
some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4.
In some
embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3. In some
embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3. In some
embodiments, the immune checkpoint inhibitor is an inhibitor of ID01. In some
embodiments, the one or more checkpoint inhibitors are administered by any
suitable
route. In some embodiments, the route of administration of the one or more
checkpoint
inhibitors is parenteral, mucosal delivery, oral, sublingual, transdermal,
topical,
inhalation, intranasal, aerosol, intratumoral, intraocular, intratracheal,
intrarectal,
17
Date Recue/Date Received 2022-02-10
intragastric, vaginal, by gene gun, dermal patch or in eye drop or mouthwash
form. In
some embodiments, the one or more TLR9 agonists and the one or more checkpoint
inhibitors are each administered in a pharmaceutically effective amount.
Exemplary immune checkpoint inhibitors include anti-PD-1, anti-PD-L1, anti-
PD-L2, and anti-CTLA-4 agents. PD-1/PD-Li/PD-L2 antibodies inhibit the
interaction
between PD-1 and its ligands (PD-Li and PD-L2) on tumor cells to promote
immune-
mediated tumor destruction. CTLA-4 antibodies block the inhibitory signals to
T-cells
transmitted by CTLA-4. While PD-1 antibodies and CTLA-4 antibodies have
emerged
as important therapeutic options for a variety of cancers, many patients fail
to respond.
.. For example, some melanoma patients show no response to anti-PD-1
treatment, or even
progress, after 12 weeks of treatment. Further, immune checkpoint blockade is
associated
with various immune-related adverse events, which can affect various tissues
and organs
including the skin, the gastrointestinal tract, the kidneys, peripheral and
central nervous
system, liver, lymph nodes, eyes, pancreas, and the endocrine system. These
immune-
related adverse events (irAEs) can be severe, or even fatal, and may require
discontinuation of therapy. Examples of common irAEs are hypophysitis,
colitis,
hepatitis, pneumonitis, rash, and rheumatic disease.
Expression of the various immune checkpoint molecules on cells of the immune
system induces a complex series of events that determines whether an immune
response
will be effective to combat the tumor, or otherwise result in immune
tolerance. For
example, increased expression of PD-1 on dendritic cells (DCs) promotes
apoptosis of
activated DCs, a critical antigen presenting cell for anti-tumor immune
responses. Park
SJ, Negative role of inducible PD-1 on survival of activated dendritic cells,
J. Leukocyte
Biology 95(4):621-629 (2014). Further, expression of IDO, PD-L1, and CTLA-4 in
the
peripheral blood of melanoma patients and can be associated with advanced
disease and
negative outcomes, and are interconnected, suggesting that multiple immune
checkpoints
might require targeting to improve therapy in some cases. Chevolet I, et al.,
Characterization of the in vivo immune networks of IDO, tryptophan metabolism,
PD-
L1, and CTLA-4 in circulating immune cells in melanoma, Oncoimmunology 4(3)
e982382-7 (2015).
In some embodiments, the metastatic tumor has a high proportion of dendritic
cells (DC) at baseline. In some embodiments, the metastatic tumor is enriched
for
18
Date Recue/Date Received 2022-02-10
dendritic cells before treatment with tilsotolimod (IMO-2125). Enrichment for
dendritic
cells in baseline metastatic tumors may be determined, for example, by
analyzing a
biopsy specimen with immunohistochemistry (IHC) or by disaggregating fresh
biopsy
specimens and using flow cytometry sorting cells bearing DC markers, for
example,
CD209, CCL13, HSD11B1, and CD1 1 c+. Figure 9A shows the level of dendritic
cells
in baseline tumor biopsy specimens. Metastatic tumors that responded to
intratumoral
IMO-2125 treatment in combination with systemic anti-CTLA4 treatment were
enriched
at baseline for dendritic cells in the tumor biopsy. In another aspect,
metastatic melanoma
patients who have progressive disease following treatment with one or more
checkpoint
inhibitors are selected for intratumoral IMO-2125 treatment based on dendritic
cell
enrichment in one or more progressive disease tumors.
In another aspect, the patient with metastatic cancer has elevated levels of
PD-L2
protein in serum. In some embodiments, the elevated level of PD-L2 protein
between
about 750 pg/mL and about 5000 pg/mL. In some embodiments, the elevated level
of
PD-L2 protein above about 1000 pg/mL. In some embodiments, the elevated level
is
above about 1100 pg/mL, above about 1200 pg/mL, above about 1300 pg/mL, above
about 1400 pg/mL, above about 1500 pg/mL, above about 1600 pg/mL, above about
1700 pg/mL, above about 1800 pg/mL, above about 1900 pg/mL, above about 2000
pg/mL, above about 2100 pg/mL, above about 2200 pg/mL, above about 2300 pg/mL,
above about 2400 pg/mL, above about 2500 pg/mL, above about 2600 pg/mL, above
about 2700 pg/mL, above about 2800 pg/mL, above about 2900 pg/mL, and above
about
3000 pg/mL.
PD-L2 protein may be detected by methods known to the art; for example ELISA,
surface plasmon resonance (SPR) binding assays, quantitative fluorescent
competition
assays, and mass spectrometry methods.
In another aspect, serum PD-L2 protein levels may be estimated by
quantitatively
detecting and measuring serum PD-L2 mRNA, for example, using Quantitative RT-
PCR
(qRT-PCR).
The identification of metastatic tumor patients that will benefit from
treatment
with checkpoint inhibitors and benefit from immunooncology therapies generally
has
been particularly difficult. In particular identifying patients for whom
durable responses
19
Date Recue/Date Received 2022-02-10
are possible has been particularly difficult. E.g., Snyder et al., Genetic
Basis for Clinical
Response to CTLA-4 Blockade in Melanoma, NF,JM371:2189-2199 (2014).
Figure 14 shows that, surprisingly, the presence of T-cells in baseline tumors
and
the level of activation of T-cells in baseline tumor specimens does not
correlate with
response to immunooncology therapy. It is broadly believed that baseline TIL
infiltration
is a prognostic marker, with more infiltration correlating with better
clinical outcomes.
Gooden et al., The prognostic influence of tumour-infiltrating lymphocytes in
cancer: a
systematic review with meta-analysis, Br. J Cancer 105:93-103 (2011).
Surprisingly,
clinical response, both in injected tumors (local) and non-injected tumors
(remote),
correlates with a high proportion of dendritic cells (DC) in baseline tumors.
Further,
surprisingly, clinical response, both in injected tumors (local) and non-
injected tumors
(remote), correlates with elevated serum levels of PD-L2. Figure 9B shows the
increased
levels of serum PD-L2 in patients responding to intratumoral IMO-2125 in
combination
with systemic anti-CTLA4 treatment. Furthermore, tumors with a higher
neutrophil
score and greatly reduced mast cell score at baseline did not respond to
combinatorial
therapy, as shown in Figure 9C.
In one aspect, the TLR9 agonist is the oligonucleotide known as IMO-2125,
which is described more fully herein, upon intratumoral injection induces
global
increases in expression of checkpoint genes, including IDO1 (5.3 fold), PDL1
(2.6 fold),
PD1 (2.5 fold), IDO2 (5.9 fold), CEACAM1 (2.1 fold), 0X40 (1.4 fold), TIM3
(2.9 fold),
LAG3 (1.9 fold), CTLA4 (1.8 fold), and OX4OL (1.5 fold). See FIG. 6B. By
altering
immune signaling in the tumor microenvironment, such changes in gene
expression
provide opportunities to improve responsiveness with checkpoint inhibitor
therapy, and
to achieve lasting anti-tumor immunity. Further, by targeting a single immune
checkpoint
molecule selected from the stronger inhibitory signals of PD-1 or CTLA-4, in
connection
with the robust activation of antigen presenting cells (e.g., DCs) and priming
of T cells
with IMO-2125, the invention provides the opportunity to balance anti-tumor
responses
with inhibitory signals, thereby also minimizing irAEs of checkpoint inhibitor
therapy.
In another aspect, intratumoral administration of IMO-2125 in conjunction with
systemic checkpoint inhibitor administration results in proliferation of T-
cells in both
treated tumors and untreated tumors. In another aspect, IT administration of
IMO-2125
in conjunction with systemic ipilimumab administration results in T-cell
proliferation in
Date Recue/Date Received 2022-02-10
the IMO-2125 injected tumor and in remote tumors that have not been treated
with IMO-
2125. See Example 5.
In various embodiments, the patient has a cancer that was previously
unresponsive to, or had become resistant to, a checkpoint inhibitor therapy.
In some
embodiments, the cancer is refractory or relapsed. For example, the cancer may
be
refractory or insufficiently responsive to an immunotherapy, such as anti-CTLA-
4, anti-
PD-1, or anti-PD-Li and/or PD-L2 agent, including for example, one or more of
ipilimumab, tremelimumab, pembrolizumab and nivolumab. In various embodiments,
the cancer patient has progressed after or during treatment with an anti-CTLA-
4, anti-
PD-1, or anti-PD-Li and/or PD-L2 agent, including for example, one or more of
ipilimumab, tremelimumab, pembrolizumab and nivolumab (or agents related
thereto) or
shown no response to such treatment for at least about 12 weeks.
Other immune checkpoint inhibitors can be administered alone (e.g, in place
of)
or in combination with anti-CTLA4 or anti-PD-1/anti-PD-L1, such as an
inhibitor of IDO
(e.g., IDO-1 or IDO-2), LAG3, TIM3, among others. These and other immune
checkpoint inhibitors are described in US 2016-0101128, which is hereby
incorporated
by reference in its entirety. For example, the patient may further receive a
regimen of an
IDO-1 inhibitor such as Epacadostat.
In various embodiments, the cancer is a primary cancer or a metastatic cancer.
A
primary cancer refers to cancer cells at an originating site that become
clinically
detectable, and may be a primary tumor. "Metastasis" refers to the spread of
cancer from
a primary site to other places in the body. Cancer cells can break away from a
primary
tumor, penetrate into lymphatic and blood vessels, circulate through the
bloodstream,
and grow in a distant focus (metastasize) in normal tissues elsewhere in the
body.
Metastasis can be local or distant. In some embodiments, the cancer is a
relapsed or
refractory cancer, for example, a sarcoma or a carcinoma.
The cancer may have an origin from any tissue. The cancer may originate from
skin, colon, breast, or prostate, and thus may be made up of cells that were
originally
skin, colon, breast, or prostate, respectively. The cancer may also be a
hematological
malignancy, which may be lymphoma. In various embodiments, the primary or
metastatic cancer is lung cancer, kidney cancer, prostate cancer, cervical
cancer,
colorectal cancer, colon cancer, pancreatic cancer, ovarian cancer, urothelial
cancer,
21
Date Recue/Date Received 2022-02-10
gastric/GEJ cancer, head and neck cancer, glioblastoma, Merkel cell cancer,
head and
neck squamous cell carcinoma (HNSCC), non-small cell lung carcinoma (NSCLC),
small cell lung cancer (SCLC), bladder cancer, prostate cancer (e.g. hormone-
refractory)
and hematologic malignancies.
In some embodiments, the cancer is progressive, locally advanced, or
metastatic
carcinoma. In some embodiments, the cancer is metastatic melanoma, and may be
recurrent. In some embodiments, the metastatic melanoma is stage III or IV,
and may be
stage IVA, IVB, or IVC. The metastasis may be regional or distant.
In various embodiments, the metastatic tumor is a low MHC Class I expressing
tumor. In various embodiments, the low MHC Class I expressing tumor expresses
less
than 50% of normal MHC Class I mRNA expression. In some embodiments, the low
MHC Class I expressing tumor expresses less than 35% of normal MHC Class I
mRNA
expression. In some embodiments, the low MHC Class I expressing tumor
expresses
less than 30% of normal MHC Class I mRNA expression. In some embodiments, the
low MHC Class I expressing tumor expresses less than 25% of normal MHC Class I
mRNA expression. In some embodiments, the low MHC Class I expressing tumor
expresses no detectable levels of at least one MHC Class I mRNA.
In various embodiments, the metastatic tumor is a low MHC Class I expressing
tumor. In various embodiments, the low MHC Class I expressing tumor expresses
less
than 50% of normal MHC Class I protein expression. In some embodiments, the
low
MHC Class I expressing tumor expresses less than 35% of normal MHC Class I
protein
expression. In some embodiments, the low MHC Class I expressing tumor
expresses
less than 30% of normal MHC Class I protein expression. In some embodiments,
the
low MHC Class I expressing tumor expresses less than 25% of normal MHC Class I
protein expression. In some embodiments, the low MHC Class I expressing tumor
expresses no detectable levels of at least one MHC Class I protein.
In various embodiments, the metastatic tumor is a high baseline CTLA4
expressing tumor. In various embodiments, the high baseline CTLA4 expressing
tumor
expresses greater than or equal to 20% of mean baseline tumor CTLA4 expression
in the
patient population. In some embodiments, the high baseline CTLA4 expressing
tumor
expresses greater than or equal to 25% of mean baseline tumor CTLA4 expression
in the
patient population.. In some embodiments, the high baseline CTLA4 expressing
tumor
22
Date Recue/Date Received 2022-02-10
expresses greater than or equal to 30% of mean baseline tumor CTLA4 expression
in the
patient population. In some embodiments, the high baseline CTLA4 expressing
tumor
expresses greater than or equal to 35% of mean baseline tumor CTLA4 expression
in the
patient population.
In various embodiments, the metastatic tumor is a high baseline CTLA4
expressing tumor. In various embodiments, the high baseline CTLA4 expressing
tumor
expresses greater than or equal to 20% of mean baseline tumor CTLA4 expression
in the
patient population. In some embodiments, the high baseline CTLA4 expressing
tumor
expresses greater than or equal to 25% of mean baseline tumor CTLA4 expression
in the
patient population. In some embodiments the high baseline CTLA4 expressing
tumor
expresses greater than or equal to 30% of mean baseline tumor CTLA4 expression
in the
patient population. In some embodiments, the high baseline CTLA4 expressing
tumor
expresses greater than or equal to 35% of mean baseline tumor CTLA4 expression
in the
patient population.
In various embodiments, the metastatic tumor has no measurable expression of
B2M, the 132-microglobulin gene. In various embodiments, the B2M mRNA is
detected,
but there is no 132-microglobulin protein detected.
Gene expression of MHC Class I, CTLA4, and B2M may be measured by any
suitable technique in the art, such as, and without limitation, reverse
transcriptase
polymerase chain reaction (rtPCR) or quantitative PCR (qPCR), to detect mRNA
presence or absence, or to quantitate mRNA expression level. Expression of MHC
Class
I proteins HLA-A. HLA-B, and HLA-C. CTLA4 protein, and 132-microglobulin
protein
may be measured by any suitable technique in the art, such as, and without
limitation,
immunohistochemistry staining of pretreatment tumor biopsy samples. Rodig et
al., Sci.
Transl. Med., "MHC proteins confer differential sensitivity to CTLA-4 and PD-1
blockade in untreated metastatic melanoma," 10, eaar3342 (2018), discloses
exemplary
immunohistochemistry methods quantitating protein expression of each of the
MHC
Class I genes HLA-A, HLA-B, and HLA-C.
In some embodiments, patients are identified for treatment with methods of the
invention by assessing the percentage of baseline CTLA4 expression in a tumor
biopsy
specimen for CTLA4 protein expression. In some embodiments, a patient with 20%
or
23
Date Recue/Date Received 2022-02-10
greater baseline expression of CTLA4 in the biopsied tumor cells in comparison
to the
mean baseline expression of CTLA4 in the patient population's tumor samples is
treated.
In some embodiments, patients are identified for treatment with methods of the
invention by assessing baseline CTLA4 expression in a tumor biopsy specimen
for
CTLA4 protein expression. In some embodiments, a patient with 20% or greater
baseline
expression of CTLA4 in the biopsied tumor cells in comparison to the mean
baseline
expression of CTLA4 in the patient population's tumor samples is treated.
In some embodiments, patients are identified for treatment with methods of the
invention by assessing the expression level of the B2M gene. In some
embodiments,
patients with metastatic tumors expressing no detectable levels of B2M mRNA
are
selected for treatment.
IMO-2125 and related immunostimulatory oligonucleotides target TLR9, and act
as TLR9 agonists to alter immune signaling in the tumor microenvironment, and
induce
anti-tumor T cell responses.
In accordance with various embodiments, the TLR9 agonist comprises at least
two oligonucleotides linked together through their 3' ends, so as to have
multiple
accessible 5' ends. The linkage at the 3' ends of the component
oligonucleotides is
independent of the other oligonucleotide linkages and may be directly via 3'
or 2'
hydroxyl groups, or indirectly, via a non-nucleotide linker or a nucleoside,
utilizing either
the 2' or 3 ' hydroxyl positions of the nucleoside. Linkages may also employ a
functionalized sugar or nucleobase of a 3' terminal nucleotide. Exemplary TLR9
agonists
are described in US Patent Nos. 8,420,615, 7,566,702, 7,498,425, 7,498,426,
7,405,285,
7,427,405, including Tables 1 and 2A-2D of each, the entire contents of which
are hereby
incorporated by reference in their entireties. Exemplary TLR9 agonists are
also
described in US Patent Nos. 7,745,606 and 8,158,768, the entire contents of
which are
hereby incorporated by reference in their entireties.
In various embodiments, the TLR agonist is selected from:
5'-TCTGACG1TTCT-X-TCTTGICAGTCT-5' (SEQ ID NO:1)
5'-TCTGTCG1TTCT-X-TCTTGICTGTCT-5' (SEQ ID NO :2)
5'-TCGiTCGiTTCTG-X-GTCTTGiCTGiCT-5' (SEQ ID NO: 3)
5'-TCGiAACGiTTCGi-X-GICTTGICAAGICT-5' (SEQ ID NO :4)
24
Date Recue/Date Received 2022-02-10
5'-CTGTCoG2TTCTC-X-CTCTTG2oCTGTC-5' (SEQ ID NO :5)
5'-CTGTCG2TTCTCo-X-oCTCTTG2CTGTC-5' (SEQ ID NO :6)
5'-TCG1AACG1TTCG1-X-TCTTG2CTGTCT-5' (SEQ ID NO :7)
5'-TCG1AACG1TTCG1-Y-GACAG1CTGTCT-5' (SEQ ID NO:8)
5'-CAGTCG2TTCAG-X-GACTTG2CTGAC-5' (SEQ ID NO:9)
5'-CAGTCG1TTCAG-X-GACTTG1CTGAC-5' (SEQ ID NO:10)
5'-TCGiAACGiTTCoG-Z-GoCTTGiCAAGiCT-5' (SEQ ID NO:11)
5'-TCG1AACG1TTCG1-Y2-TCTTG1CTGTCTTG1CT-5' (SEQ ID NO:12)
5'-TCG1AACG1TTCG1-Y2-TCTTG1CTGUCT-5' (SEQ ID NO:13)
5'-TCGiAACGiToTCoG-m-GoCToTGiCAAGiCT-5' (SEQ ID NO:14)
5'-TCG1AACG1TTCoG-Y3-GACTTG2CTGAC-5' (SEQ ID NO:15)
5'-TCG1AACG1TTCG1-Y4-TGTTG1CTGTCTTG1CT-5' (SEQ ID NO:16)
5'-TCG2TCG2TTU1Y-M-YU1TTG2CTG2CT-5' (SEQ ID NO:17)
5'-CAGTCG2TTCAG-Y3-TCTTG1CTGTCT-5' (SEQ ID NO:18)
5'-TCGiTACGiTACGi-X-GiCATGiCATGiCT-5' (SEQ ID NO:19)
5'-TCG1AACG1TTCG-Z-GCTTG1CAAG1CT-5' (SEQ ID NO :20)
5'-TCG1AACG1TTCoG-Y3-CTTG2CTGACTTG1CT-5' (SEQ ID NO :21)
5'-TCGiAACGioTTCGi-X2-GiCTToGiCAAGiCT-5' (SEQ ID NO :22)
5'-TCG1AACG1TTCG1-Y4-CATTG1CTGTCTTG1CT-5' (SEQ ID NO: 23)
5'-TCGiAACGiTTCGi-m-GiCTTGiCAAGiCT-5' (SEQ ID NO:24)
5'-TCoGioAACoGiTTCoGio-X2-oGioCTTGioCAAoGioCT-5' (SEQ ID
NO:25)
5'-ToCGioAACoGiTTCoGio-X2-oGioCTTGioCAAoGiCoT-5' (SEQ ID
NO:26)
5'-TCoGioAACoGiTTCoGio-m-oGioCTTGioCAAoGioCT-5' (SEQ ID
NO:27)
Date Recue/Date Received 2022-02-10
5'-TCoG2oAACoG2TTCoG2o-X2-oG2oCTTG2oCAAoG2oCT-5' (SEQ ID
NO:28)
5'-TCoGioAACoGiTTCoGo-Z-oGoCTTGioCAAoGioCT-5' (SEQ ID NO:29)
and
5'-ToCGioAACoGiTTCoGo-Z-oGoCTTGioCAAoGiCoT-5' (SEQ ID NO:30),
where Gi is 2'-deoxy-7-deazaguanosine; G2 is 2'-deoxy-arabinoguanosine; G, C,
or U are
T-0-methylribonucleotides; Ui is 2'-deoxy-U; o is a phosphodiester linkage; X
is a
glycerol linker; X2 is a isobutanetriol linker, Y is C3-linker; m is cis,trans-
1,3,5-
cyclohexanetriol linker; Y2 is 1,3-propanediol linker; Y3 is 1,4-butanediol
linker; Y4 is
1,5-pentandiol linker; Z is 1,3,5-pentanetriol linker; and M is cis,cis-1,3,5-
cyclohexanetriol linker.
In various embodiments, the TLR9 agonist is selected from 5'-
TCGiAACGiTTCGi-X-GiCTTGiCAAGiCT-5' (SEQ ID NO: 4), 5'-
CTGTCoG2TTCTC-X-CTCTTG2oCTGTC-5' (SEQ ID NO :5), 5'-CTGTCG2TTCTCo-
X-oCTCTTG2CTGTC-5' (SEQ ID NO:6), 5'-TCG1AACG1TTCG1-Y-
TCTTG2CTGTCT-5' (SEQ ID NO:7), and 5'-TCG1AACG1TTCG1-Y-
GACAG1CTGTCT-5' (SEQ ID NO:8), wherein X is a glycerol linker, Y is a C3-
linker,
Gi is 2'-deoxy-7-deazaguanosine. G2 is arabinoguanosine, and o is a
phosphodiester
linkage.
In various embodiments, the TLR9 agonist is 5'-TCGiAACGiTTCGi-X-
GiCTTGiCAAGiCT-5' (SEQ ID NO:4), wherein X is a glycerol linker and Gi is 2'-
deoxy-7-deazaguanosine, otherwise known as IMO-2125.
Alternative TLR9 agonists are immune stimulatory oligonucleotides disclosed in
US 8,871,732, which is hereby incorporated by reference in its entirety. Such
agonists
comprise a palindromic sequence of at least 8 nucleotides and at least one CG
dinucleotide.
In accordance with embodiments of the invention, the immunostimulatory
oligonucleotide (e.g., IMO-2125) is administered intratumorally. In some
embodiments,
the intratumoral administration is in a primary or secondary tumor (e.g.,
metastatic
melanoma lesion). Intratumoral administration alters immune signaling in the
tumor
microenvironment, priming the immune system for an effective anti-tumor
response,
26
Date Recue/Date Received 2022-02-10
while inducing changes that are compatible with more effective checkpoint
inhibitor
therapy.
Illustrative dosage forms suitable for intratumoral administration include
solutions, suspensions, dispersions, emulsions, and the like. The TLR9 agonist
may be
provided in the form of sterile solid compositions (e.g. lyophilized
composition), which
can be dissolved or suspended in sterile injectable medium immediately before
use. They
may contain, for example, suspending or dispersing agents known in the art.
In various embodiments, the TLR9 agonist is IMO-2125 and is administered
intratumorally at from about 1 mg to about 20 mg, from about 4 mg to about 64
mg per
dose, or in some embodiments from about 8 mg to about 64 mg per dose, or from
about
12 mg to about 64 mg per dose, or from about 16 mg to about 64 mg per dose, or
from
about 20 mg to about 64 mg per dose. In some embodiments, IMO-2125 is
administered
at from about 20 mg to about 48 mg per dose, or about 20 mg to about 40 mg per
dose.
For example, in various embodiments, IMO-2125 is administered at about 4 mg,
or about
8 mg, or about 12 mg, or about 16 mg, or about 20 mg, or about 24 mg, or about
28 mg,
or about 32 mg, or about 36 mg, or about 40 mg, or about 44 mg, or about 48
mg, or
about 52 mg, or about 56 mg, or about 60 mg, or about 64 mg per dose, e.g.
intratumorally.
In various embodiments, about 1, about 2, or about 3 to about 12 doses of the
TLR9 agonist (e.g. IMO-2125) are administered (e.g. about 1 dose, or about 2
doses, or
about 3 doses, or about 4 doses, or about 5 doses, or about 6 doses, or about
7 doses, or
about 8 doses, or about 9 doses, or about 10 doses, or about 11 doses, or
about 12 doses).
In various embodiments, about 4 to about 8 doses are administered over 10 to
12 weeks.
In some embodiments, about 6 doses are administered over 10 to 12 weeks. In
some
embodiments, therapy is initiated with 3 to 5 weekly doses of IMO-2125,
optionally
followed by 3 to 8 maintenance doses, which are administered about every three
weeks.
In some embodiments, an IMO-2125 dose is administered in weeks 1, 2, 3, 5, 8,
and 11.
The IMO-2125 doses may be administered in the same or different lesions.
During the regimen of IMO-2125 (or other TLR9 agonist), one or more
checkpoint inhibitor therapies are administered to take advantage of the
changes in
immune signaling. The one or more checkpoint inhibitors can be administered
parenterally, including intravenously, intratumorally, or subcutaneously,
among other
27
Date Recue/Date Received 2022-02-10
methods. In some embodiments, the patient receives an anti-CTLA-4 agent. For
example,
the anti-CTLA-4 agent may be an antibody that targets CTLA-4, for instance an
antagonistic antibody. In various embodiments, the anti-CTLA-4 is ipilimumab
(e.g.
YERVOY, BMS-734016, MDX-010, MDX-101). In various embodiments, the anti-
CTLA-4 is tremelimumab (e.g. CP-675,206, MEDIMMUNE). In other embodiments,
the immunotherapy agent is an anti-PD-1 agent. For example, the anti-PD-1
agent may
be an antibody that targets the PD-1, for instance, inhibiting the interaction
between PD-
1 and PD-Li (and/or PD-L2). In various embodiments, the anti-PD-1 agent is
nivolumab
(ON0-4538/BMS-936558, MDX1106 or OPDIVO). In various embodiments, the anti-
PD-1 agent is pembrolizumab (KEYTRUDA or MK-3475). In various embodiments, the
anti-PD-1 agent is pidilizumab (CT-011 or MEDIVATION).
In some embodiments, the present immunotherapy agent is an anti-PD-Li and/or
PD-L2 agent. For example, in various embodiments, the anti-PD-Li and/or PD-L2
agent
is an antibody that targets PD-Li and/or PD-L2, for instance, inhibiting the
interaction
between PD-1 and PD-Li and/or PD-L2. In various embodiments, the anti-PD-Li
and/or
PD-L2 agent is atezolizumab (TECENTRIQ, ROCHE) BMS 936559 (BRISTOL
MYERS SQUIBB), or MPDL3280A (ROCHE).
In various embodiments, the anti-CTLA-4, anti-PD-1, or anti-PD-Li and/or PD-
L2 agent (e.g. YERVOY, OPDIVO, or KEYTRUDA, or comparable agents thereto) is
administered at a dose of about 1 mg/kg, or about 2 mg/kg, or about 3 mg/kg,
or about 4
mg/kg, or about 5 mg/kg, e.g. intravenously. For example, in some embodiments,
the
dose of an anti-CTLA-4 agent, e.g. YERVOY, is about 3 mg/kg. For example, in
some
embodiments, the dose of an anti-PD-1 agent, e.g. OPDIVO, is about 3 mg/kg.
For
example, in some embodiments, the dose of an anti- PD-1 agent, e.g. KEYTRUDA,
is
about 2 mg/kg. In various embodiments, the initial dose of the anti-CTLA-4,
anti-PD-1,
or anti-PD-Li and/or PD-L2 agent (e.g. YERVOY, OPDIVO, or KEYTRUDA, or
comparable agents thereto) is administered at least one week after the initial
TLR9
agonist dose, for example in about weeks 2, 3 or 4.
In some embodiments, the immunotherapy agent is anti-CTLA-4 (e.g.
YERVOY), anti-PD-1 (e.g. OPDIVO or KEYTRUDA), or anti-PD-Li and/or anti-PD-
L2 agent, which is administered from about 2 to about 6 times (e.g. about 2
times, or
about 3 times, or about 4 times, or about 5 times, or about 6 times). In some
embodiments,
28
Date Recue/Date Received 2022-02-10
the immunotherapy agent, e.g. anti-CTLA-4 (e.g. YERVOY), anti-PD-1 (e.g.
OPDIVO
or KEYTRUDA), or anti-PD-Li and/or PD-L2 agent is administered about 4 times.
In some embodiments, the immunotherapy agent is an anti-CTLA-4 agent such
as YERVOY and is dosed at 3 mg/kg i.v. over about 90 minutes about every 3
weeks. In
some embodiments, the immunotherapy agent is an anti-PD-1 agent such as OPDIVO
and is dosed at about 3 mg/kg i.v. over about 60 minutes about every 2 weeks.
In some
embodiments, the immunotherapy agent is an anti-PD-1 agent such as KEYTRUDA
and
is dosed at about 2 mg/kg i.v. over about 30 minutes about every 3 weeks.
In some embodiments, maintenance doses of the TLR9 agonist (e.g. IMO-2125),
along with dosing of anti-CTLA-4, anti-PD-1, or anti-PD-Li and/or PD-L2 agent
(e.g.
YERVOY, OPDIVO, or KEYTRUDA, or comparable agents thereto) are administered
about every 3 weeks.
In various embodiments, the present immunostimulatory oligonucleotides allow
for a dose reduction of the immunotherapy to about 10%, or about 20%, or about
30%,
or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about
90%,
or about 100% of a monotherapy dose. For example, in some embodiments, an
immunotherapy dose is about 0.1 mg/kg, or about 0.3 mg/kg, or about 0.5 mg/kg,
or
about 0.7 mg/kg, or about 1 mg/kg, or about 1.5 mg/kg, or about 2 mg/kg, or
about 2.5
mg/kg, or about 3 mg/kg.
In some embodiments, IMO-2125 is administered intratumorally to a metastatic
melanoma patient previously found to be unresponsive or only partially
responsive to
PD-1 blockade therapy. IMO-2125 is administered at a dose of from 4 to 32 mg
per dose
(e.g., about 16 mg, about 20 mg, about 24 mg, about 28 mg, or about 32 mg) in
weeks 1,
2, 3, 5, 8, and 11, with ipilimumab i.v. at 3 mg/kg. Ipilimumab can be
administered every
three weeks, beginning in week 2 (e.g., weeks 2, 5, 8, and 11). Alternatively,
pembrolizumab can be administered i.v. at 2 mg/kg every three weeks beginning
on week
2 (e.g., weeks 2, 5, 8, and 11).
In some embodiments, the patient further receives a regimen of Epacadostat (an
IDO-1 inhibitor), which may be administered at from 25 mg to 300 mg orally,
about
twice daily. The regimen may be administered for about 5 day cycles. The first
dose of
Epacadostat may be administered starting at about one week following the
initial IMO-
2125 (or other TLR9 agonist) intratumoral injection.
29
Date Recue/Date Received 2022-02-10
In various embodiments, without wishing to be bound by theory, the invention
provides for a more balanced immune response in a cancer patient, including
cancer
patients with advanced, metastatic disease. The combination therapy described
herein
can eliminate or reduce deficiencies that are observed in the respective
monotherapies.
For example, various patients are refractory to immunotherapies, or such
monotherapies
are hampered by extensive side effect profiles. Further as the field is moving
to
combinations of immunotherapies (e.g. YERVOY and OPDIVO), such side effects
are
likely to be more problematic.
In various embodiments, the combination therapy allows for activation and/or
maturation of dendritic cells, e.g. plasmacytoid dendritic cells, and
modulates the tumor
microenvironment (TME) in both treated and distant tumors. For example, in
various
embodiments, the combination therapy provides for improvements in the amount
or
quality of TILs and/or CD8+ T cells to promote anti-tumor activities. For
example,
primed T cells are observed to invade both the proximal and distal tumors.
Such primed
T cells are suited for tumor invasion, particularly at distal sites (e.g.
secondary tumors),
and, without wishing to be bound by theory, encounter a tumor environment that
has
reduced tolerance mechanisms in place. In various embodiments, the combination
therapy provides for stimulation of interferons (e.g. IFN-a) and various Thl
type
cytokines (e.g. IFN-y, IL-2, IL-12, and TNF-f3). See Example 4.
The invention provides, in various embodiments, methods for treating cancers,
including metastatic cancers, in which the overall host immune milieu is
reengineered
away from tumor tolerance. For example, a local TME is created that both
disrupts
pathways of immune tolerance and suppression and allow for tumor regression.
The
present methods provide in some embodiments, a TME capable of propagating a
robust
immune response.
In various embodiments, a cancer patient's DCs are immature and unable to take
up, process, or present antigens. These DCs may also be inhibited from
migrating to
regional lymph nodes or may induce tolerance, especially when presenting self-
antigens.
The cancer patient's tumor site may also be infiltrated with regulatory T
cells that are
able to mediate suppression of antigen-primed T cells. The helper CD4 T cell
response
may also be skewed toward a Th2 phenotype, which inhibits the initiation of
Thl T cells
and effective cellular immunity. The tumor cells may express aberrant MHC
class I
molecules or f32-microglobulin, resulting in inadequate antigen presentation
and, thus,
Date Recue/Date Received 2022-02-10
inefficient recognition of tumors by effector T cells. Finally, tumor cells
and the
surrounding stroma may release a number of suppressive cytokines, such as IL-
6, IL-10,
and TGF-f3. This creates an environment that is not conducive to local
immunity, which
allows tumor cells to escape. In various embodiments, the present methods
allow for an
environment that is conducive to local immunity against tumors, e.g., without
limitation,
maturation of DCs and/or reduction of regulatory T cells and Th2 CD4 T cells.
In some embodiments, the combination therapy according to the invention alters
the balance of immune cells in favor of immune attack of a tumor. For
instance, in some
embodiments, the present methods shift the ratio of immune cells at a site of
clinical
importance, e.g. at the site of agent administration or a distal site, in
favor of cells that
can kill and/or suppress a tumor (e.g. T cells, cytotoxic T lymphocytes, T
helper cells,
natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor
macrophages (e.g. M1
macrophages), B cells, dendritic cells, or subsets thereof) and in opposition
to cells that
protect tumors (e.g. myeloid-derived suppressor cells (MDSCs), regulatory T
cells
(Tregs); tumor associated neutrophils (TANs), M2 macrophages, tumor associated
macrophages (TAMs), or subsets thereof). In some embodiments, the present
methods
increase a ratio of effector T cells to regulatory T cells. In various
embodiments, this
altered balance of immune cells is affected locally/proximally and/or
systemically/distally. In various embodiments, this altered balance of immune
cells is
affected in the TME.
Further, in various embodiments, the present methods allow for a robust anti-
tumor immune response that does not come at the expense of significant side
effects (e.g.,
irAEs), e.g. relative to side effects observed when one or more
immunotherapies are used
in the absence of the TLR9 agonist.
For example, the combination therapy reduces one or more side effects of an
immunotherapy, e.g an anti-CTLA-4, anti-PD-1, or anti-PD-Li and/or PD-L2
agent,
including for example, one or more of YERVOY, OPDIVO, and KEYTRUDA or agents
related thereto. Such side effects include: fatigue, cough, nausea, loss of
appetite, skin
rash, itching pruritus, rash, and colitis. In some embodiments, the side
effects are
intestinal problems (e.g. colitis) that can cause perforations in the
intestines. Signs and
symptoms of the colitis may include: diarrhea or more bowel movements than
usual;
blood in the stools or dark, tarry, sticky stools; and abdominal pain or
tenderness. In some
embodiments, the side effects are liver problems (e.g. hepatitis) that can
lead to liver
31
Date Recue/Date Received 2022-02-10
failure. Signs and symptoms of hepatitis may include: yellowing of skin or the
whites of
the eyes; dark urine; nausea or vomiting; pain on the right side of the
stomach; and
bleeding or bruising more easily than normal. In some embodiments, the side
effects are
skin problems that can lead to severe skin reactions. Signs and symptoms of
severe skin
reactions may include: skin rash with or without itching; sores in the mouth;
and the skin
blisters and/or peels. In some embodiments, the side effects are nerve
problems that can
lead to paralysis. Symptoms of nerve problems may include: unusual weakness of
legs,
arms, or face; and numbness or tingling in hands or feet. In some embodiments,
the side
effects are hormone gland problems (e.g. pituitary, adrenal, and thyroid
glands). Signs
and symptoms include: persistent or unusual headaches; unusual sluggishness;
feeling
cold all the time; weight gain; changes in mood or behavior such as decreased
sex drive,
irritability, or forgetfulness; and dizziness or fainting. In some
embodiments, the side
effects are ocular problems. Symptoms may include: blurry vision, double
vision, or
other vision problems; and eye pain or redness.
In some embodiments, patients experience fewer incidences of colitis, crohn's
disease, or other GI involved irAE in accordance with the present invention.
In some embodiments, the patient achieves longer progression-free interval or
longer survival (e.g., as compared to monotherapy), or in some embodiments,
achieves
remission or complete response. A complete response refers to the
disappearance of all
signs of cancer in response to treatment.
This invention is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1 Identification of Patients Likely to Respond to Tilsotolimod in
Combination
with Ipilimumab
Fresh metastatic melanoma tumor tissue was disaggregated to generate a single
cell suspension for staining. PBMCs were thawed, washed and resuspended for
staining.
Surface staining was performed in FACS Wash Buffer (1X DPBS with 1% Bovine
Serum
Albumin) for 30 min on ice using fluorochrome-conjugated monoclonal antibodies
from
BD Biosciences, Biolegend, or eBioscience, as described previously. Cells were
fixed in
1% paraformaldehyde solution for 20 minutes at room temperature following
surface
staining. For panels containing transcription factors, cells were fixed and
permeabilized
32
Date Recue/Date Received 2022-02-10
using the eBioscience FoxP3 kit according to the manufacturer's instructions.
Samples
were acquired using the BD FACSCanto II or BD Fortessa X20 and analyzed using
FlowJo Software v 7.6.5 (Tree Star). Dead cells were stained using AQUA
live/dead dye
(Invitrogen) and excluded from the analysis.
RNA was extracted from core needle biopsies preserved in RNA later using the
Qiagen AllPrep Universal Kit (Cat # 80224) according to the manufacturers'
instructions.
Purity and concentration were assessed using Nanodrop. RNA was assayed using
the
Nanostring Pan Cancer Immune Panel and analyzed using the nSolver Advanced
Analysis Software.
Gene expression profiling of tumor tissue collected from the injected tumor
lesion
at baseline and the same lesion 24 hours post injection demonstrated that
intratumoral
tilsotolimod triggered activation of a Type-I interferon response profile.
Figure 1 shows
the induction of a key gene in this pathway, IRF-7. The volcano plot shown in
Figure 2
demonstrates that several Type-I IFN pathway genes that are elevated and
indicates the
level of significance (p<0.01). In addition, the presence of a dendritic cell
expression
profile (DC score) was found to be higher prior to therapy in patients that
subsequently
respond (Figure 3, p<0.017) and local DCs were found to have gained the
maturation
marker HLA-DR (MHC class II) in some patients after i.t. tilsotolimod (Figure
4, p=0.07)
indicating that this drug was able induce maturation of local DCs which in
turn have
better antigen presentation capacity and could induce a more favorable
environment for
subsequent T cell activation.
Unexpectedly, this combination treatment was able to overcome a known
mechanism of resistance to single agent ipilimumab which has been linked to
the need
for high levels of the antigen presentation molecule MHC class I which is
comprised of
three major genes (HLA-A, HLA-B, HLA-C). Gene expression profiling of baseline
tumor
biopsies revealed that the combination of tilsotolimod and ipilimumab is able
to
overcome this mechanism of resistance as some responding patients had a lower
expression level of HLA-A, HLA-B, HLA-C. Figure 5 shows the gene expression
profile
of each gene individually and Figure 6 shows the cumulative expression of all
the genes
in this cytotoxicity signature. The observed clinical data are surprising and
unexpected
because the expectation of one skilled in the art is that normal or higher
levels of MHC
Class I expression is required for response to immune checkpoint inhibitors.
33
Date Recue/Date Received 2022-02-10
Example 2 Clinical Stratification of Patients Based on MHC Class I Expression
A pretreatment biopsy sample is collected from a patient with metastatic
melanoma. The sample is sectioned and the sections fixed for chromogenic
immunohistochemistry (IHC). A dual IHC for MHC class I (HLA-A, HLA-B, and HLA-
C, clone EMR8-5, 1:6000; Abeam) is used to identify cells in the section
expressing a
MHC Class I protein. MHC class I. MHC class II, and f32M staining is scored
for the
percentage of malignant cells in 10% increments (0 to 100%) with positive
membrane
staining within the entire tissue section, as determined by the consensus of
two
pathologists. The result of the visual analysis is a percentage of malignant
cells
expressing a MHC Class I protein.
Patients whose tumors comprise less than 50% malignant cells expressing a MHC
Class I protein are selected preferentially for tilsotolimod co-administered
with
ipilimumab.
Example 3 Clinical Stratification of Patients Based on Dendritic Cell
Enrichment in
Baseline Tumor Specimens
A pretreatment biopsy sample is collected from a patient with metastatic
melanoma.
The sample is disaggregated and the myeloid cells separated from the bulk
specimen.
Fresh tumor tissue is disaggregated using a medimachine followed by filtering
to
generate a single cell suspension. Flow cytometry is used to identify live
cells that
possess one or more dendritic cell surface markers, for example, CD lc, CD1
lc, CD141,
and CD141. The number of live dendritic cells per 100,000 cells is determined.
This
value is compared to the number of live dendritic cells per 100,000 cells in
the patient's
peripheral blood mononuclear cells (circulating DC level). The tumor biopsy is
considered enriched for dendritic cells if the tumor DCs are 8% or more above
the
circulating DC level. Patients with tumor biopsy specimens enriched for DCs
are
selected for treatment with IMO-2125 in combination with an immune checkpoint
inhibitor.
Example 4 Intratumoral Administration of IMO-2125 Stimulates Type 1 Interferon
Response
34
Date Recue/Date Received 2022-02-10
As depicted in Figure 8A, tumor tissue and peripheral blood were collected
from
patients participating in the NCT02644967 clinical trial. To determine the
impact of
intratumoral administration of IMO-2125 (tilsotolimod) on the injected tumor,
tumor
tissue was collected at baseline, before IMO-2125 administration, and 24-hours
after
administration. The gene expression profile was determined for each tumor
sample using
NanoString gene profiling.
RNA was extracted from core needle biopsies preserved in RNAlater using the
Qiagen AllPrep Universal Kit (catalog # 80224) according to the manufacturer's
instructions. Purity and concentration of the resulting RNA preparation was
assessed
using Nanodrop. RNA was assayed using the Nanostring Pan Cancer Immune Panel
and
the resulting data analyzed using the nSolver Advanced Analysis Software.
Figure 8B shows the comparison of the baseline gene expression compared to the
24-hour post-injection gene expression profile. Intratumoral injection of IMO-
2125
induces a type 1 interferon response, illustrated by the significant
upregulation of IRF7 ,
IL 12A, IL 1RN , CCL8, and CCL8 (adjusted p < 0.01) genes. The upregulated
gene profile
includes both type I and type II interferon (IFNy) response, e.g. IDO and PD-
Li
(CD27 4), but did not result in the upregulation of -classical" IFNy genes
such as the
MHC Class I genes or IRF 1.
Markers of DC activation, for example, CD80 and IL 12, and chemoattractants
CCL7 and CCL8 were found to be upregulated at the 24-hour sampling time after
IMO-
2125 administration. See Table 1 below. These data correlate with the increase
in the
macrophage gene expression score (CD163, CD68, CD84, MS4A4A) (p = 0.0003, n =
12 paired samples) as depicted in Figure 8C. Maturation of the CD 1c+ subset
is further
demonstrated by the upregulation of MHC class II (HLA-DR) in a subset of
patients as
detected by flow cytometry on fresh tumor tissue (p = 0.07, n = 12; shown in
Figure 8D).
Also, IDO expression was induced by IMO-2125 administration as detected by IHC
and
RNA expression (p = 0.0012; n = 13, shown in Figure 8E).
Table 1 shows the top 70 enriched mRNAs of the 600 measured, sorted by p-
value.
Log2 std Linear
BY p-
Gene Name fold error fold p-value
probe.ID
value
change (10g2) change
Date Recue/Date Received 2022-02-10
ISG15-mRNA 4.85 0.476 28.8
8.81E-10 3.68E-06 NM_005101.3:305
IFIT1-mRNA 4.9 0.508 29.9
2.32E-09 4.85E-06 NM_001548.3:1440
MX1-mRNA 3.79 0.489 13.8
9.76E-08 0.000136 NM_002462.2:1485
DDX58-mRNA 3.32 0.441 10
1.54E-07 0.000161 NM_014314.3:2130
OAS3-mRNA 3.45 0.472 11
2.48E-07 0.000196 NM_006187.2:4980
IFITM1-mRNA 2.93 0.403 7.62
2.81E-07 0.000196 NM _003641.3:482
IRF7-mRNA 2.87 0.405 7.29
4.16E-07 0.000234 NM _001572.3:1763
11_1 RN-mRNA 4.71 0.668 26.2
4.49E-07 0.000234 NM_000577.3:480
CCL7-mRNA 4.53 0.714 23.1
2.21E-06 0.00103 NM_006273.2:120
S100Al2-mRNA 3.65 0.597 12.6
3.71E-06 0.00125 NM_005621.1:260
ISG20-mRNA 3.33 0.545 10
3.81E-06 0.00125 NM_002201.4:358
IFIH1-mRNA 2.67 0.437 6.37
3.81E-06 0.00125 NM_022168.2:185
IFIT2-mRNA 3.47 0.569 11.1
3.89E-06 0.00125 NM_001547.4:1995
CCL8-mRNA 3.27 0.544 9.68 4.68E-
06 0.0014 NM _005623.2:689
IF135-mRNA 2.44 0.427 5.44 9.17E-
06 0.00255 NM _005533.3:415
LAM P3-mRNA 3.37 0.603 10.3 1.27E-
05 0.00331 NM_014398.3:1400
LI LRA5-mRNA 3.02 0.55 8.12 1.60E-05
0.00393 NM_181879.2:545
SELL-mRNA 2.29 0.434 4.9
2.68E-05 0.00602 NR_029467.1:1585
FPR2-mRNA 2.82 0.535 7.06 2.74E-
05 0.00602 NM _001462.3:1200
CXCL11-mRNA 3.62 0.756 12.3
8.74E-05 0.0183 NM_005409.4:282
TNFSF10-m RNA 2.79 0.592 6.9
0.000108 0.0215 NM_003810.2:115
TNFSF18-mRNA 3.1 0.655 8.56
0.000114 0.0216 NM _005092.2:175
ILI R2-mRNA 2.42 0.515 5.34
0.000125 0.0227 NM _173343.1:113
CXCL10-mRNA 2.76 0.613 6.79
0.000174 0.0304 NM_001565.1:40
SOCS1-mRNA 2.01 0.459 4.03
0.000242 0.0404 NM_003745.1:1025
STAT2-mRNA 1.45 0.342 2.73
0.000333 0.0535 NM_005419.2:1965
IF116-mRNA 1.15 0.297 2.21
0.000841 0.13 NM_005531.1:2255
36
Date Recue/Date Received 2022-02-10
CCL19-mRNA 2.47 0.647 5.55
0.000928 0.138 NM_006274.2:401
S100A8-mRNA 2.36 0.636 5.12
0.00124 0.179 NM_002964.3:115
BTLA-m RNA 2.03 0.554 4.09
0.00134 0.185 NM_181780.2:305
BST2-m RNA 1.7 0.464 3.25 0.00137 0.185
NM_004335.2:560
TAP1-m RNA 1.44 0.397 2.71
0.00154 0.201 NM_000593.5:2075
CD38-mRNA 1.59 0.446 3.01
0.00173 0.213 NM_001775.2:460
CXCR2-m RNA 2.22 0.618 4.65
0.00174 0.213 NM_001557.2:2055
IFITM2-mRNA 1.45 0.412 2.73
0.00191 0.228 NM_006435.2:390
TAP2-m RNA 1.41 0.403 2.67
0.00197 0.229 NM_000544.3:909
CCL2-mRNA 1.77 0.515 3.42
0.00232 0.262 NM_002982.3:123
CCR7-mRNA 1.88 0.56 3.68 0.00303 0.333
NM_001838.2:1610
IF127-mRNA 1.86 0.581 3.62
0.00414 0.438 NM_005532.3:390
SIGLEC1-mRNA 1.45 0.453 2.73
0.00419 0.438 NM_023068.3:5165
LAG3-m RNA 1.64 0.521 3.12
0.00468 0.477 NM_002286.5:1735
CCR1-mRNA 1.49 0.484 2.82
0.00542 0.539 NM_001295.2:535
PPBP-mRNA 2.13 0.692 4.37
0.00559 0.543 NM_002704.2:330
CCL13-mRNA 1.54 0.509 2.9
0.00632 0.6 NM_005408.2:320
PTGS2-m RNA 1.87 0.623 3.66 0.0067
0.615 NM_000963.1:495
CD80-mRNA 1.25 0.418 2.38
0.00677 0.615 NM_005191.3:1288
ID01-mRNA 2.12 0.714 4.35
0.00712 0.633 NM_002164.3:50
CD274-mRNA 1.53 0.517 2.88
0.00741 0.636 NM_014143.3:1245
STAT1-m RNA 1.16 0.392 2.23
0.00747 0.636 NM_007315.2:205
CD1D-mRNA 1.44 0.489 2.72
0.00767 0.641 NM_001766.3:1428
LI LRB2-mRNA 1.43 0.491 2.69
0.00815 0.668 NM_005874.1:595
NOD2-mRNA 1.48 0.51 2.78 0.00841 0.676
NM_022162.1:4080
STAT4-m RNA 0.949 0.33 1.93 0.00869 0.685
NM_003151.2:789
37
Date Recue/Date Received 2022-02-10
TNFSF13B-
mRNA 1.33 0.479 2.52 0.0108
0.834 NM_006573.4:1430
CD48-mRNA 1.02 0.367 2.03 0.0111
0.837 NM_001778.2:270
CD47-mRNA 0.881 0.318 1.84 0.0112
0.837 NM_001777.3:897
CCND3-mRNA 0.978 0.367 1.97 0.0142
1 NM_001760.2:1215
IL15RA-mRNA 1.29 0.486 2.45 0.0144
1 NM_002189.2:505
IL12A-mRNA 1.37 0.515 2.58 0.0149
1 NM_000882.2:775
TLR3-mRNA 1.45 0.558 2.72 0.0167
1 NM_003265.2:230
TFRC-m RNA -1.02 0.4 0.493 0.0181 1
NM_003234.1:1220
CXCL13-mRNA 1.48 0.582 2.78 0.0187
1 NM_006419.2:210
MRC1-mRNA -1.37 0.541 0.387 0.019
1 NM_002438.2:525
CCL11-mRNA 1.79 0.705 3.45 0.0192
1 NM_002986.2:378
LILRB1-mRNA 1.07 0.431 2.11
0.0206 1 NM_001081637.1:2332
IRF2-mRNA 0.957 0.384 1.94 0.0206
1 NM_002199.3:1624
CSF2RB-mRNA 1.21 0.496 2.32 0.0229
1 NM_000395.2:3300
GZMB-mRNA 1.16 0.474 2.23 0.0232
1 NM_004131.3:540
PSM B8-m RNA 0.884 0.364 1.85 0.0239
1 NM_004159.4:1215
Example 5 Combination IT IMO-2125 and Systemic CPI Therapy Induces Local and
Remote T-Cell Proliferation
As depicted in Figure 9D and 9E, baseline tumor tissue in responding and non-
responding patients in the NCT02644967 clinical trial show similar T-cell
functional
gene signatures and cytotoxic gene signatures. However, intratumoral
administration of
IMO-2125 shows a significant up regulation of T-cell functional genes (IFNy,
Tbx21,
perform, granzymes) as well as antigen presenting cell activation (CD86,
IL12), and
genes associated with response to IFNy (PD-L1, HLA-A, HLA-B, HLA-C) in
responding
patients at C3W8. Such up regulation was not observed in non-responding
patients (n =
13, as shown in Figure 11A and 11B). Treatment also induced other types of
cellular
functions, including macrophage function, again by C3W8, and more enriched in
responding patients (depicted in Figure 16A and 16B).
38
Date Recue/Date Received 2022-02-10
Furthermore, combination therapy drives expansion of the T-cell clones that
are
shared between intratumoral injected (local) and non-injected (distant)
tumors. Figure
12A shows that such parallel expansion was not observed in those patients that
did not
respond (that is, patients with stable disease (SD) or progressive disease
(PD)). Figure
12B shows that the comparison between baseline and C3W8 of the local lesion.
Example 6 Identification and Clinical Stratification of Patients Likely to
Respond to
Tilsotolimod in Combination with Ipilimumab based on Baseline CTLA4 Expression
Fresh metastatic melanoma tumor tissue was disaggregated to generate a single
cell suspension for staining. PBMCs were thawed, washed and resuspended for
staining.
Surface staining was performed in FACS Wash Buffer (1X DPBS with 1% Bovine
Serum
Albumin) for 30 min on ice using fluorochrome-conjugated monoclonal antibodies
from
BD Biosciences, Biolegend, or eBioscience, as described previously. Cells were
fixed in
1% paraformaldehyde solution for 20 minutes at room temperature following
surface
staining. For panels containing transcription factors, cells were fixed and
permeabilized
using the eBioscience FoxP3 kit according to the manufacturer's instructions.
Samples
were acquired using the BD FACSCanto II or BD Fortessa X20 and analyzed using
FlowJo Software v 7.6.5 (Tree Star). Dead cells were stained using AQUA
live/dead dye
(Invitrogen) and excluded from the analysis.
RNA was extracted from core needle biopsies and preserved in RNA later using
the Qiagen AllPrep Universal Kit (Cat # 80224) according to the manufacturers'
instructions. Purity and concentration were assessed using Nanodrop. RNA was
assayed
using the Nanostring Pan Cancer Immune Panel and analyzed using the nSolver
Advanced Analysis Software.
Gene expression profiling of tumor tissue collected from injected and non-
injected tumor lesions at baseline and 8 weeks after institution of treatment
demonstrated
that intratumoral tilsotolimod was carried out. Figure 17A shows the baseline
expression
profile of CTLA4 in both treated and distant tumors was found to be higher
prior to
therapy in patients that subsequently respond (Figure 17A, p<0.0001).
Figure 17B shows the change in CTLA4 gene expression profile in treated and
distant tumors from baseline to Week 8 of treatment. The observed clinical
data are
surprising and unexpected because, until now, baseline levels of CTLA4
expression in
39
Date Recue/Date Received 2022-02-10
tumors have not been correlated with likelihood of treatment response in
metastatic
melanoma or other cancers.
Patients whose tumors at baseline express greater than or equal to 20% higher
levels of CTLA4 in comparison to the baseline tumor expression of CTLA4 of the
patient
population are selected preferentially for tilsotolimod co-administered with
ipilimumab.
EQUIVALENTS
While the invention has been described in connection with specific embodiments
thereof, it will be understood that it is capable of further modifications and
this
application is intended to cover any variations, uses, or adaptations of the
invention
following, in general, the principles of the invention and including such
departures from
the present disclosure as come within known or customary practice within the
art to
which the invention pertains and as may be applied to the essential features
hereinbefore
set forth and as follows in the scope of the appended claims.
Those skilled in the art will recognize, or be able to ascertain, using no
more than
routine experimentation, numerous equivalents to the specific embodiments
described
specifically herein. Such equivalents are intended to be encompassed in the
scope of the
following claims.
INCORPORATION BY REFERENCE
All patents and publications referenced herein are hereby incorporated by
reference in their entireties. PCT/U517/51742, filed on September 17, 2017, is
incorporated by reference in its entirety. U.S. application 15/703,631, filed
on September
15, 2017, is incorporated by reference in its entirety.
Date Recue/Date Received 2022-02-10
