Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Fe-ENHANCED ANTI-WT1/HLA ANTIBODY
Cross-Reference to Related Applications
[0001] The benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent
Application Serial No. 61/901,210 filed November 7, 2013, is hereby claimed,
and the
disclosure of the priority document is incorporated herein by reference in its
entirety.
[0002] This application contains subject matter that is related to the subject
matter of
U.S. Provisional Application No. 61/470,635, filed April 1, 2011, U.S.
Provisional
Application No. 61/491,392 filed May 31, 2011 and U.S. Application serial no.
14/008,447, which is a national stage entry of PCT International Application
No.
PCT/US2012/031892 filed April 1, 2012. These applications are hereby
incorporated by
reference in their entirety into the present disclosure.
Statement of Rights Under Federally-Sponsored Research
[0003] This invention was made with government support under grants
P01CA23766, RO1CA55349 and T32 CA062948 awarded by the U.S. National
Institutes
of Health. The government has certain rights in the invention.
Sequence Listing
[0004] This application contains a Sequence Listing, created on November
7,
2014; the file, in ASCII format, is designated 48316_SeqListing.txt and is
46,083 bytes
in size . The file is hereby incorporated by reference in its entirety into
the application
Technical Field
[0005] The present invention relates generally to antibodies against
cytosolic
proteins. More particularly, the invention relates to antibodies against Wilms
tumor
oncogene protein (WT1), specifically antibodies that recognize a WT1 peptide
in
conjunction with a major histocompatibility antigen.
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Background of the Invention
[0006] Therapeutic monoclonal antibodies (mAbs) are highly specific and
effective drugs, with pharmacokinetics suitable for infrequent dosing.
However, all
current marketed therapeutic anticancer mAbs target extracellular or cell-
surface
molecules, whereas many of the most important tumor-associated and oncogenic
proteins are nuclear or cytoplasmic (Sensi M and Anichini A. Clinical cancer
research:
an official journal of the American Association for Cancer Research 2006
;12(17):5023-
32; Kessler JH and Melief CJ. Leukemia 2007;21(9):1859-74).
[0007] Intracellular proteins are processed by the proteasome and
presented on
the cell surface as small peptides in the pocket of major histocompatibility
complex
(MHC) class I molecules (in humans, also called human leukocyte antigen, HLA)
allowing recognition by T-cell receptors (TCRs) (Morris E et al. Blood Rev
2006;20(2):61-9; Konig R. Curr Opin Immunol 2002;14(1):75-83). Therefore, mAbs
that
mimic the specificity of TCRs (that is, recognizing a peptide presented in the
context of
a specific HLA-type) can bind cell-surface complexes with specificity for an
intracellular
protein. A "TCR-mimic" (TCRm) antibody was first reported by Andersen et. al.
(Andersen PS et al. Proceedings of the National Academy of Sciences of the
United
States of America 1996;93(5):1820-4), and several have since been developed by
various groups (Epel M et al. European journal of immunology 2008;38(6):1706-
20;
Wittman VP et al. J Immunol 2006;177(6):4187-95; Klechevsky et al. Cancer
research
2008;68(15):6360-7; Bhattacharya R et al. Journal of cellular physiology
2010;225(3):664-72; Verma B, et al. J Immunol 2010;184(4):2156-65; Sergeeva et
al.
Blood 2011 ;117(16):4262-72).
[0008] The first fully human TCRm mAb, called ESK1, that specifically
targets
RMFPNAPYL (RMF), a peptide derived from Wilms' tumor gene 1 (WT1), presented
in
the context of HLA-A0201 was recently reported (Dao T et al. Science
translational
medicine 2013;5(176):176ra33). WT1 is an important, immunologically validated
oncogenic target that has been the focus of many vaccine trials (Dao T et al.
Best
practice & research Clinical haematology 2008;21(3):391-404). WT1 is a zinc
finger
transcription factor with limited expression in normal adult tissues, but is
over expressed
in the majority of leukemias and a wide range of solid tumors (Sugiyama H.
Japanese
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journal of clinical oncology 2010;40(5):377-87). WT1 was ranked as the top
cancer
antigenic target for immunotherapy by a National Institutes of Health-convened
panel
(Cheever MA et al. Clinical cancer research : an official journal of the
American
Association for Cancer Research 2009;15(17):5323-37); further, WT1 expression
is a
biomarker and a prognostic indicator in leukemia (Inoue K et al. Blood
1994;84(9):3071-
9, Ogawa H et al. Blood 2003;101(5):1698-704). ESK1 mAb specifically bound to
leukemias and solid tumor cell lines that are both WT1+ and HLA-A0201+ and
showed
efficacy in mouse models in vivo against several WT1+ HLA-A0201+ leukemias
(Dao T
et al. Science translational medicine 2013;5(176):176ra33). Therefore, ESK1 is
a useful
therapeutic platform for further clinical development, and improvements to the
native
antibody could help potentiate its effect and improve clinical efficacy.
Summary of the Invention
[0009] In one aspect, the invention relates to an antibody comprising:
(A) a heavy
chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3
respectively, comprising amino acid sequences SEQ ID NOS: 2, 3, and 4; 18, 19
and
20; 34, 35, and 36; 50, 51, and 52; 66, 67, and 68 or 82, 83, and 84; and a
light chain
(LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively,
comprising amino acid sequences SEQ ID NOS: 8, 9 and 10; 24, 25 and 26; 40, 41
and
42; 56, 57 and 58; 72, 73 and 74 or 88, 89 and 90; or (B) a VH and VL
comprising the
amino acid sequence of SEQ ID NO: 14 and SEQ ID NO: 16; 30 and 32; 46 and 48;
62
and 64; 78 and 80 or 94 and 96, respectively, wherein said antibody has no
detectable
fucose or galactose. As a result of the modification in glycosylation, the
altered
antibody exhibits between 50-100% (80%) higher affinity for activating human
FcyRIlla
(158V variant) than normally glycosylated antibody, has 3-4-fold (3.5-fold)
higher affinity
for a FcyRIlla 158F variant than normally glycosylated antibody, and has
between 30
and 70% (50%) reduced affinity for inhibitory FcyRIlb than normally
glycosylated
antibody.
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[00010] In one embodiment, the antibody comprises a light chain consisting
essentially of the amino acid sequence of SEQ ID NO: 100 and a heavy chain
consisting essentially of the amino acid sequence of SEQ ID NO: 101.
[00011] In a related aspect, the invention relates to isolated nucleic
acids, vectors
and cells comprising a nucleic acid that encodes an antibody as described
herein.
[00012] In yet another aspect, the invention relates to the use of an
antibody as
disclosed herein for the treatment of a WT1 positive disease and therefore, to
a
pharmaceutical composition comprising an antibody of the invention and a
pharmaceutically acceptable carrier.
[00013] In a related aspect, the invention relates to a method for
treatment of a
subject having a WT1-positive disease, comprising administering to the subject
a
therapeutically effective amount of an antibody disclosed herein. WT1-positive
disease
amenable to treatment with the antibody of the invention includes chronic
leukemia or
acute leukemia or WT1+ cancer, including, for example, chronic myelocytic
leukemia,
multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute
myeloid/myelogenous leukemia (AML), myelodysplastic syndrome (MDS),
mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer,
prostate cancer
and glioblastoma.
[00014] The foregoing summary is not intended to define every aspect of
the
invention, and other features and advantages of the present disclosure will
become
apparent from the following detailed description, including the drawings. The
present
disclosure is intended to be related as a unified document, and it should be
understood
that all combinations of features described herein are contemplated, even if
the
combination of features are not found together in the same sentence,
paragraph, or
section of this disclosure. In addition, the disclosure includes, as an
additional aspect,
all embodiments of the invention narrower in scope in any way than the
variations
specifically mentioned above. With respect to aspects of the disclosure
described or
claimed with "a" or "an," it should be understood that these terms mean "one
or more"
unless context unambiguously requires a more restricted meaning. With respect
to
elements described as one or more within a set, it should be understood that
all
combinations within the set are contemplated. If aspects of the disclosure are
described
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as "comprising" a feature, embodiments also are contemplated "consisting of"
or
"consisting essentially of" the feature. Additional features and variations of
the
disclosure will be apparent to those skilled in the art from the entirety of
this application,
and all such features are intended as aspects of the disclosure.
Brief Description of the Drawings
[00015] Figure 1 shows that ESKM has a modified Fc glycosylation pattern,
altering binding to FcyRs but not to the RMF/A2 target. (1A) Comparison of the
oligosaccharide profile of ESK1 and ESKM. Peak assignment is based on the
retention
time and the monosaccharide composition analysis. G# indicates the number of
terminal galactoses (0, 1, or 2), F indicates presence of core fucose,
Hex5GIcNAc2
denotes (GIcNAc)2 core with terminal Hexose 5 glycan structure (terminating in
mannose and/or glucose). (1B) Summary of ESK1 and ESKM binding to mouse and
human FcyRs. Anti-mouse FcR binding was assessed by ELISA, while anti-human
FcR
binding was determined by FCM titration on FcyR-expressing CHO cells.
Representative binding curves of ESK1 and ESKM against human FcRn (1C), mouse
FcyRIV (10), and mouse FcyRIlb (1E). 1251-labeled ESK1 (1F) and ESKM (1G) mAbs
were titrated against JMN cells. All curves were fit with a non-linear single-
site total
binding saturation curve, and Ka was calculated using Prism software. ESKM
having
100% reduced fucose content relative to ESK1 wildtype IgG1 showed improved
reverse
signaling through FcyRIlla compared to ESK1 wildtype IgG1 and ESK1 containing
D265A/P329A mutations in the Fc domain (ESK1-DAPA) (1H).
[00016] Figure 2 shows that ESKM is more efficacious and potent in ADCC
assays with human PBMC effectors at the indicated mAb concentrations and
effector/target (E:T) ratios. Cytotoxicity was measured by 4-hour 51Cr release
assay.
(2A) T2 cells were pulsed with RMF peptide and incubated with 3 pg/mL mAb. HLA-
A0201+ human leukemia cell lines: (2B) BA25 ALL, (2C) AML14 and (20) SET2 AML
(2E) HLA-A0201 negative HL60 promyelocytic leukemia. HLA-A0201+ human
mesothelioma cell lines: (2F) JMN, (2G) Meso37 and (2H) Meso56. Data presented
are
averages of triplicate measurements from representative experiments, all with
isolated
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PBMCs from the same donor. All cell lines, with exception of Meso37 and
Meso56,
were repeated 3 or more times with multiple donors. Both ESKM having 100%
reduced
fucose content relative to ESK1 wildtype IgG1 and ESKM having 70% reduced
fucose
content relative to ESK1 wildtype IgG1 resulted in greater ADCC killing of
0V56 ovarian
cancer cells compared to ESK1 wildtype IgG1 and ESK1 containing D265A/P329A
mutations in the Fc domain (ESK1-DAPA) (21).
[00017] Figure 3 shows that ESKM more effectively treats JMN mesothelioma
in
SCID mice. Tumor burden was determined by luciferase imaging of mice in the
supine
position (n=5 per group). Where noted, signal was normalized to the day 4
signal for
each mouse. Arrows indicate treatment with mAb. (3A) ESKM significantly
reduced
mean tumor growth as assessed by total luminescence (*p<0.05, multiple T-tests
on
and after day 18). (3B) ESKM reduced individual tumor burden during the
treatment
course in 3 of 5 mice. This effect was reproduced in a second experiment in
the same
model. (3C) ESKM also significantly improved survival (p=0.016 vs isotype,
p=0.095 vs
ESK1), with events representing death or terminal morbidity as assessed on
protocol by
veterinarians. (3D) ESKM is effective against SET2 AML (*p<0.05, multiple T-
tests).
(3E-3F) ESKM is effective against a disseminated fresh, patient-derived pre-B
ALL
(*p<0.05, "p<0.01, multiple T-tests). (3G) Bone marrow cells were harvested
from mice
in F, and transplanted as subcutaneous tumors into NSG mice. Bone marrow cells
from
the isotype-treated mice were injected into the right shoulder flank (viewed
from above),
while an equal number of bone marrow cells from the ESKM-treated mice were
injected
into the left shoulder. (3H) Quantitation of total bone-marrow signal from
mice in 3F,
before harvesting. (31) Quantitation of subcutaneous tumors in 30, 28 days
post
transplantation.
[00018] Figure 4 shows that ESKM and native ESK1 display similar
pharmacokinetics and biodistribution. 1251-labeled mAb was injected IV and
activity was
measured in blood or harvested organs (n=3 per group). Pharmacokinetics (4A)
and
biodistribution (4B) of ESKM or native ESK1 (3 pg each) in C57BL6/J mice. (4C)
Pharmacokinetics of ESKM (2 pg) in C57BL6/J or HLA-A0201+ transgenic mice.
(40)
Biodistribution of ESKM (100 pg) in C57BL6/J or HLA-A0201+ transgenic mice,
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harvested after 1 day. ESKM (4E) or hIgG1 isotype control (4F) (2 pg each) in
C57BL6/J or HLA-A0201+ transgenic mice, harvested after 1 day.
[00019] Figure 5 shows that ESKM treatment does not affect leukocyte or
hematopoietic stem cell (HSC) counts in HLA-A0201+ transgenic mice. Animals
(n=5
per group) were treated with 100 pg ESKM or hIgG1 isotype control on days 0
and 4;
blood and bone marrow were harvested on day 5. (5A) Total white blood cell
(WBC)
and WBC subset cell counts. Absolute number (5B) and frequency (5C) of lineage-
SCA1+ KIT+ (LSK) cells. Absolute number (5D) and frequency (5E) of long-term
HSCs
(Slamf1+ CD34- LSK cells).
[00020] Figure 6 shows that ESKM has no significant effect against
intraperitoneal
JMN mesothelioma in NOG mice. Mice were engrafted intraperitoneally with 3x106
luciferase+ JMN cells, then treated with 50 pg ESKM or hIgG1 isotype control
antibody
twice weekly starting on day 4 via intraperitoneal injections.
[00021] Figure 7 shows that all human antibodies tested accumulated more
in
spleens of HLA-A2+ transgenic mice, but ESK1 did not bind specifically to
isolated HLA-
A2+ spleen, bone marrow or thymus cells. (7A) Accumulation of 1251-labeled
antibodies
in spleens of C57BL6/J or HLA-A2+ transgenic mice relative to antibody level
in the
blood. Mice were injected retroorbitally with 2 pg indicated antibody, then
sacrificed after
24 hours for blood and spleen collection. (7B) Specific binding of 1251-
labeled ESK1 to
bone marrow, spleen, or thymus cells isolated from C57BL6/J or HLA-A2+
transgenic
mice. Tissues were collected from 2 (C57) or 3 (HLA-A2+ transgenic) mice, then
bound
by 1 pg/mL 1251-labeled ESK1 either alone or after blocking with 50-fold
excess
unlabeled ESK1. Specific binding was determined, and #ESK1 bound per cell was
calculated.
Detailed Description of the Invention
[00022] All publications, patents and other references cited herein are
incorporated by reference in their entirety into the present disclosure.
[00023] Unless otherwise defined herein, scientific and technical terms
used in
connection with the present invention shall have the meanings that are
commonly
understood by those of ordinary skill in the art. Further, unless otherwise
required by
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context, singular terms shall include pluralities, and plural terms shall
include the
singular. Generally, nomenclatures used in connection with, and techniques of,
cell and
tissue culture, molecular biology, immunology, microbiology, genetics and
protein and
nucleic acid chemistry and hybridization described herein are those well-known
and
commonly used in the art. In practicing the present invention, many
conventional
techniques in molecular biology, microbiology, cell biology, biochemistry, and
immunology are used, which are within the skill of the art. These techniques
are
described in greater detail in, for example, Molecular Cloning: a Laboratory
Manual 3rd
edition, J.F. Sambrook and D.W. Russell, ed. Cold Spring Harbor Laboratory
Press
2001; Recombinant Antibodies for lmmunotherapy, Melvyn Little, ed. Cambridge
University Press 2009; "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984);
"Animal Cell
Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press,
Inc.);
"Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987,
and periodic
updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A
Practical
Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A
Laboratory
Manual" (Barbas et al., 2001). The contents of these references and other
references
containing standard protocols, widely known to and relied upon by those of
skill in the
art, including manufacturers' instructions are hereby incorporated by
reference as part
of the present disclosure. The following abbreviations are used throughout the
application:
[00024] Ab: Antibody
[00025] ADCC: Antibody-dependent cellular cytotoxicity
[00026] ALL: Acute lymphocytic leukemia
[00027] AML: Acute myeloid leukemia
[00028] CDC: Complement dependent cytotoxicity
[00029] CMC: Complement mediated cytotoxicity
[00030] CDR: Complementarity determining region (see also HVR below)
[00031] CL: Constant domain of the light chain
[00032] CH1: 1st constant domain of the heavy chain
[00033] CH1, 2, 3: 1st, 2nd and 3rd constant domains of the heavy chain
[00034] CH2, 3: 2nd and 3rd constant domains of the heavy chain
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[00035] CHO: Chinese hamster ovary
[00036] CTL: Cytotoxic T cell
[00037] EC50: Half maximal effective concentration
[00038] E:T Ratio: Effector:Target ratio
[00039] Fab: Antibody binding fragment
[00040] FACS: Flow assisted cytometric cell sorting
[00041] FBS: Fetal bovine serum
[00042] FR: Framework region
[00043] HC: Heavy chain
[00044] HLA: Human leukocyte antigen
[00045] HVR-H: Hypervariable region-heavy chain (see also CDR)
[00046] HVR-L: Hypervariable region-light chain (see also CDR)
[00047] Ig: lmmunoglobulin
[00048] IRES: Internal ribosome entry site
[00049] KD: Dissociation constant
[00050] koff: Dissociation rate
[00051] kon: Association rate
[00052] MHC: Major histocompatibility complex
[00053] MM: Multiple myeloma
[00054] VH: Variable heavy chain includes heavy chain hypervariable region
and
heavy chain variable framework region
[00055] VL: Variable light chain includes light chain hypervariable region
and light
chain variable framework region
[00056] WT1: Wilms tumor protein 1
[00057] In the description that follows, terms used herein are intended to
be
interpreted consistently with the meaning of those terms as they are known to
those of
skill in the art. The definitions provided herein below are meant to clarify,
but not limit,
the terms defined.
[00058] As used herein, "administering" and "administration" refer to the
application of an active ingredient to the body of a subject.
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[00059] "Antibody" and "antibodies" as those terms are known in the art
refer to
antigen binding proteins of the immune system. The term "antibody" as referred
to
herein includes whole, full length antibodies having an antigen-binding
region, and any
fragment thereof in which the "antigen-binding portion" or "antigen-binding
region" is
retained, or single chains, for example, single chain variable fragment
(scFv), thereof. A
naturally occurring "antibody" is a glycoprotein comprising at least two heavy
(H) chains
and two light (L) chains inter-connected by disulfide bonds. Each heavy chain
is
comprised of a heavy chain variable region (abbreviated herein as VH) and a
heavy
chain constant (CH) region. The heavy chain constant region is comprised of
three
domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain
variable
region (abbreviated herein as VL) and a light chain constant CL region. The
light chain
constant region is comprised of one domain, CL. The VH and VL regions can be
further
subdivided into regions of hypervariability, termed complementarity
determining regions
(CDR), interspersed with regions that are more conserved, termed framework
regions
(FR). Each VH and VL is composed of three CDRs and four FRs arranged from
amino-
terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3,
CDR3, FR4. The variable regions of the heavy and light chains contain a
binding
domain that interacts with an antigen. The constant regions of the antibodies
may
mediate the binding of the immunoglobulin to host tissues or factors,
including various
cells of the immune system (e.g., effector cells) and the first component
(C1q) of the
classical complement system.
[00060] The term "antigen-binding portion" or "antigen-binding region" of
an
antibody, as used herein, refers to that region or portion of the antibody
that binds to the
antigen and which confers antigen specificity to the antibody; fragments of
antigen-
binding proteins, for example, antibodies includes one or more fragments of an
antibody
that retain the ability to specifically bind to an antigen (e.g., an
peptide/HLA complex). It
has been shown that the antigen-binding function of an antibody can be
performed by
fragments of a full-length antibody. Examples of antigen-binding fragments
encompassed within the term "antibody fragments" of an antibody include a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains;
a
F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a
disulfide
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bridge at the hinge region; a Fd fragment consisting of the VH and CH1
domains; a Fv
fragment consisting of the VL and VH domains of a single arm of an antibody; a
dAb
fragment (Ward et al., Nature 1989;341:544-546), which consists of a VH
domain; and
an isolated complementarity determining region (CDR).
[00061] Furthermore, although the two domains of the Fv fragment, VL and
VH,
are coded for by separate genes, they can be joined, using recombinant
methods, by a
synthetic linker that enables them to be made as a single protein chain in
which the VL
and VH regions pair to form monovalent molecules. These are known as single
chain Fv
(scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al.,
1988 Proc.
Natl. Acad. Sci. 85:5879-5883. These antibody fragments are obtained using
conventional techniques known to those of skill in the art, and the fragments
are
screened for utility in the same manner as are intact antibodies.
[00062] As used herein, the term "effective amount" means that amount of a
compound or therapeutic agent that will elicit the biological or medical
response of a
tissue, system, animal, or human that is being sought, for instance, by a
researcher or
clinician.
[00063] The term "therapeutically effective amount" means any amount
which, as
compared to a corresponding subject who has not received such amount, results
in
improved treatment, healing, prevention, or amelioration of a disease,
disorder, or side
effect, or a decrease in the rate of advancement of a disease or disorder. The
term also
includes within its scope amounts effective to enhance normal physiological
function.
[00064] The present disclosure provides compositions and methods of
treatment
relating to recombinant antibodies. TCRm antibodies are potentially limited by
the
extremely low number of epitopes presented on the target cell, which may be as
few as
several hundred sites (Dao T et al. Science translational medicine
2013;5(176):176ra33). Therefore, mechanisms to enhance potency may be
essential to
their success in humans as therapeutic agents against cancer.
[00065] The mechanisms of action of mAbs can be enhanced through Fc region
protein engineering (Desjarlais JR et al. Drug discovery today 2007;12(21-
22):898-910),
or by modification of Fc-region glycosylation (Jefferis R. Biotechnology
progress
2005;21(1):11-6; Hodoniczky J et al. Biotechnology progress 2005;21(6):1644-
52).
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Removal of fucose from the carbohydrate chain increases mAb binding affinity
for the
activating FcyRIlla receptor and enhances ADCC (de Romeuf C et al. British
journal of
haematology 2008;140(6):635-43; Masuda K, et al. Molecular immunology
2007;44(12):3122-31; Shields RL et al. The Journal of biological chemistry
2002;277(30):26733-40; Shinkawa T et al. The Journal of biological chemistry
2003;278(5):3466-73). The addition of bisecting N-acetyl-D-glucosamine
(GIcNAc) can
also significantly enhance ADCC (Shinkawa T et al. The Journal of biological
chemistry
2003;278(5):3466-73; Davies J et al. Biotechnology and bioengineering
2001;74(4):288-
94; Umana P et al. Nature biotechnology 1999;17(2):176-80). However, removal
or
replacement of the terminal galactose residues present on endogenous IgG
reduces
complement dependent cytotoxicity (CDC) activity (Hodoniczky J et al.
Biotechnology
progress 2005;21(6):1644-52; Boyd PN et al. Molecular immunology 1995;32(17-
18):1311-8).
[00066] An Fc-modified antibody can be generated by expressing a construct
encoding an anti-WT1/HLA/A2 antibody, for example, as disclosed in WO
2012/135854,
in MACE 1.5 CHO cells in accordance with methodology disclosed in U.S.
8,025,879
(Eureka Therapeutics, Inc), resulting in a consistent pattern of
defucosylation and
exposed terminal hexose (mannose and/or glucose), allowing higher affinity for
activating human FcyRIlla and murine FcyRIV while decreasing affinity for
inhibitory
FcyRIlb. A modified antibody of the present disclosure, designated herein as
"ESKM",
has reduced fucose content and/or galactose content, e.g., relative to a wild-
type
antibody. The fucose content and/or galactose content can be reduced by 30% to
100% using any method known in the art.
[00067] ESKM mediated ADCC at lower doses than native ESK1 and was more
potent in human tumor models in vivo. Further, ESKM had similar
pharmacokinetics and
biodistribution to the native antibody. ESKM showed no observable off-target
tissue sink
in wild-type mice, and at therapeutic doses there was no difference in half-
life or
biodistribution in HLA-A2.1+ transgenic mice compared to the parent strain.
Importantly,
therapeutic doses of ESKM in these mice caused no depletion of total WBCs or
hematopoietic stem cells (HSCs), or pathologic tissue damage. The retained
specificity,
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enhanced potency, favorable pharmacokinetics and distribution, and lack of
toxicity in
these models support ESKM as to treat a wide variety of cancers and leukemias.
[00068] In one embodiment, the antibody of the invention is an anti-
WT1/HLA-A2
antibody having an antigen binding region that specifically binds to a WT1
peptide with
the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-
A0201.
[00069] In some embodiments, the antibody of the invention comprises one
of the
combinations of amino acid sequences for CDRs, and heavy and light chain
variable
regions from Tables 1-6.
Table 1
Antigen WT1 (Ext002 #3)
Peptide RMFPNAPYL (SEQ ID NO: 1)
CDRs: 1 2 3
VH GGTFSSYAIS GIIPI FGTANYAQKFQG RIPPYYGMDV
(SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ
ID NO: 4)
DNA ggaggcaccttcagcag gggatcatccctatctttggtac cggattcccccgtactacggtat
ctatgctatcagc agcaaactacgcacagaagtt ggacgtc (SEQ ID NO: 7)
(SEQ ID NO: 5) ccagggc (SEQ ID NO:
6)
VL SGSSSNIGSNYVY RSNQRPS
AAWDDSLNGVV
(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ
ID NO: 10)
tctggaagcagctccaac aggagtaatcagcggccctca gcagcatgggatgacagcctg
DNA atcggaagtaattatgtat (SEQ ID NO: 12)
aatggtgtggta
ac (SEQ ID NO: 11) (SEQ ID NO: 13)
Full QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE
VH WMGGIIPI FGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYY
CARRIPPYYGMDVWGQGTTVTVSS (SEQ ID NO: 14)
DNA
caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgc
aaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagg
gcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccaggg
cagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgag
atctgaggacacggccgtgtattactgtgcgagacggattcccccgtactacggtatggacgtctgg
ggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 15)
Full QTVVTQP PSASGTPGQRVTISCSGSSSN IGSNYVYWYQQLPGTAP KL
VL LIYRSNQRPSGVPDRFSGSKSGTSASLAISGPRSVDEADYYCAAWDD
SLNGVVFGGGTKLTVLG (SEQ ID NO: 16)
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Antigen WT1 (Ext002 #3)
Peptide RMFPNAPYL (SEQ ID NO: 1)
DNA
cagactgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgtt
ctggaagcagctccaacatcggaagtaattatgtatactggtaccaacagctcccaggaacggcc
cccaaactcctcatctataggagtaatcagcggccctcaggggtccctgaccgattctctggctcca
agtctggcacctcagcctccctggccatcagtgggccccggtccgtggatgaggctgattattactgt
gcagcatgggatgacagcctgaatggtgtggtattcggcggagggaccaagctgaccgtcctagg
t (SEQ ID NO: 17)
Table 2
Antigen WT1 (Ext002 #5)
Peptide RMFPNAPYL (SEQ ID NO: 1)
CDRs 1 2 3
VH GDSVSSNSAAWN RTYYGSKWYNDYAVS GRLGDAFDI
(SEQ ID NO: 18) VKS (SEQ ID NO: 19) (SEQ ID NO: 20)
DNA ggggacagtgtctctagc aggacatactacgggtccaag ggtcgcttaggggatgcttttga
aacagtgctgcttggaac tggtataatgattatgcagtatct tatc (SEQ ID NO: 23)
(SEQ ID NO: 21) gtgaaaagt (SEQ ID NO:
22)
VL RASQSISSYLN AASSLQS QQSYSTPLT
(SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ ID NO: 26)
DNA cgggcaagtcagagcatt gctgcatccagtttgcaaagt caacagagttacagtacccct
agcagctatttaaat (SEQ ID NO: 28)
ctcact (SEQ ID NO: 29)
(SEQ ID NO: 27)
Full QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGL
VH EWLG RTYYGSKWYNDYAVSVKSRITINP DTSKNQFSLQLNSVTPEDTA
VYYCARGRLGDAFDIWGQGTMVTVSS (SEQ ID NO: 30)
DNA
caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgt
gccatctccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcg
agaggccttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctg
tgaaaagtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactct
gtgactcccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctgg
ggccaagggacaatggtcaccgtctcttca (SEQ ID NO: 31)
Full DIQMTQSPSSLSASVG DRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
VL AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLT
FGGGTKVDIKR (SEQ ID NO: 32)
DNA
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttg
ccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagccccta
agctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatct
gggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaaca
gagttacagtacccctctcactttcggcggagggaccaaagtggatatcaaacgt (SEQ ID
NO: 33)
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Table 3
Antigen WT1 (Ext002 #13)
Peptide RMFPNAPYL (SEQ ID NO: 1)
CDRs: 1 2 3
VH GYSFTNFWIS RVDPGYSYSTYSPSF VQYSGYYDWFDP
(SEQ ID NO: 34) QG (SEQ ID NO: 35) (SEQ ID NO: 36)
DNA ggatacagcttcaccaact agggttgatcctggctactctta
gtacaatatagtggctactatg
tctggatcagc tagcacctacagcccgtccttc actggttcgacccc
(SEQ ID NO: 37) caaggc (SEQ ID NO: 39)
(SEQ ID NO: 38)
VL SGSSSNIGSNTVN SNNQRPS AAWDDSLNGWV
(SEQ ID NO: 40) (SEQ ID NO: 41) (SEQ ID NO: 42)
DNA tctggaagcagctccaac agtaataatcagcggccctca gcagcatgggatgacagcct
atcggaagtaatactgtaa (SEQ ID NO: 44) gaatggttgggtg
ac (SEQ ID NO: 43) (SEQ ID NO: 45)
Full VH QMQLVQSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLE
WMGRVDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTA
MYYCARVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 46)
DNA cagatgcagctggtgcagtccggagcagaggtgaaagagcccggggagtctctgaggatctcct
gtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccgggaaa
ggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttccaag
gccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctgaag
gcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcgacc
cctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 47)
Full QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK
VL LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD
DSLNGWVFGGGTKLTVLG (SEQ ID NO: 48)
DNA
caggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt
tctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacgg
cccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctc
caagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattac
tgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct
aggt (SEQ ID NO: 49)
Table 4
Antigen WT1 (Ext002 #15)
Peptide RMFPNAPYL (SEQ ID NO: 1)
CDRs 1 2 3
VH GYNFSNKWIG IlYPGYSDITYSPSFQG HTALAGFDY
(SEQ ID NO: 50) (SEQ ID NO: 51) (SEQ ID NO: 52)
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Antigen WT1 (Ext002 #15)
Peptide RMFPNAPYL (SEQ ID NO: 1)
DNA ggctacaactttagcaaca atcatctatcccggttactcgga
cacacagctttggccggctttg
agtggatcggc catcacctacagcccgtccttc actac (SEQ ID NO: 55)
(SEQ ID NO: 53) caaggc (SEQ ID NO:
54)
VL RASQNINKWLA KASSLES
QQYNSYAT
(SEQ ID NO: 56) (SEQ ID NO: 57) (SEQ ID NO: 58)
DNA Cgggccagtcagaatatc aaggcgtctagtttagaaagt caacaatataatagttatgcga
aataagtggctggcc (SEQ ID NO: 60) cg (SEQ ID NO: 61)
(SEQ ID NO: 59)
Full QVQLVQSGAEVKKPGESLKISCKGSGYNFSNKWIGWVRQLPGRGLE
VH WIAllYPGYSDITYSPSFQGRVTISADTSINTAYLHWHSLKASDTAMYYC
VRHTALAGFDYWGLGTLVTVSS (SEQ ID NO: 62)
DNA caggtgcagctggtgcagtctggagcagaggtgaaaaagcccggagagtctctgaagatctcctg
taagggttctggctacaactttagcaacaagtggatcggctgggtgcgccaattgcccgggagagg
cctggagtggatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggc
cgcgtcaccatctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggc
ctcggacaccgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgg
gcaccctggtcaccgtctcctca
(SEQ ID NO: 63)
Full DIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLI
VL YKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYAT
FGQGTKVEIKR (SEQ ID NO: 64)
DNA
gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcacaatcacttg
ccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccct
cagctcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatc
tgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaac
aatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgt (SEQ ID NO:
65)
Table 5
Antigen WT1 (Ext002 #18)
Peptide RMFPNAPYL (SEQ ID NO: 1)
CDRs: 1 2 3
VH GFTFDDYGMS GINWNGGSTGYADS ERGYGYHDPHDY
(SEQ ID NO: 66) VRG (SEQ ID NO: 67) (SEQ ID NO: 68)
DNA gggttcacctttgatgattat ggtattaattggaatggtggt
gagcgtggctacgggtacca
ggcatgagc agcacaggttatgcagactc tgatccccatgactac
(SEQ ID NO: 69) tgtgaggggc (SEQ ID (SEQ
ID NO: 71)
NO: 70)
VL GRNNIGSKSVH DDSDRPS QVWDSSSDHVV
(SEQ ID NO: 72) (SEQ ID NO: 73) (SEQ ID NO: 74)
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Antigen WT1 (Ext002 #18)
Peptide RMFPNAPYL (SEQ ID NO: 1)
DNA gggagaaacaacattgg gatgatagcgaccggccctc caggtgtgggatagtagtagt
aagtaaaagtgtgcac a gatcatgtggta
(SEQ ID NO: 75) (SEQ ID NO: 76) (SEQ ID NO: 77)
Full EVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKG
VH LEWVSGINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAE
DTALYYCARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 78)
DNA
gaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtccctgagactctcct
gtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaagctccagggaag
gggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgcagactctgtgagg
ggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctg
agagccgaggacacggccttgtattactgtgcgagagagcgtggctacgggtaccatgatccc
catgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO: 79)
Full QSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL
VL VVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW
DSSSDHVVFGGGTKLTVLG (SEQ ID NO: 80)
DNA cagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattac
ctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccag
gcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctgg
ctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggcc
gactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagct
gaccgtcctaggt (SEQ ID NO: 81)
Table 6
Antigen WT1 (Ext002 #23)
Peptide RMFPNAPYL (SEQ ID NO. 1)
CDRs 1 2 3
VH GFSVSGTYMG LLYSGGGTYHPASLQ GGAGGGHFDS
(SEQ ID NO. 82) G
(SEQ ID NO. 84)
(SEQ ID NO. 83)
DNA gggttctccgtcagtggcac cttctttatagtggtggcggcac gaggggcaggaggtggcc
ctacatgggc(SEQ ID
ataccacccagcgtccctgca actttgactcc (SEQ ID
NO. 85) gggc NO. 87)
(SEQ ID NO. 86)
VL TGSSSNIGAGYDVH GNSNRPS
AAWDDSLNGYV
(SEQ ID NO. 88) (SEQ ID NO. 89)
(SEQ ID NO. 90)
DNA actgggagcagctccaac ggtaacagcaatcggccctca gcagcatgggatgacagcct
atcggggcaggttatgatgt (SEQ ID NO. 92) gaatggttatgtc
acac (SEQ ID NO. 93)
(SEQ ID NO. 91)
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Antigen WT1 (Ext002 #23)
Peptide RMFPNAPYL (SEQ ID NO. 1)
Full EVQLVETGGGLLQPGGSLRLSCAASGFSVSGTYMGWVRQAPGKGLE
VH WVALLYSGGGTYHPASLQGRFIVSRDSSKNMVYLQMNSLKAEDTAVY
YCAKGGAGGGHFDSWGQGTLVTVSS (SEQ ID NO. 94)
DNA gaggtgcagctggtggagaccggaggaggcttgctccagccgggggggtccctcagactctcctg
tgcagcctctgggttctccgtcagtggcacctacatgggctgggtccgccaggctccagggaaggg
actggagtgggtcgcacttctttatagtggtggcggcacataccacccagcgtccctgcagggccg
attcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaagccgag
gacacggccgtctattactgtgcgaaaggaggggcaggaggtggccactttgactcctggggcca
aggcaccctggtgaccgtctcctca
(SEQ ID NO. 95)
Full QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK
VL LLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD
DSLNGYVFGTGTKLTVLG (SEQ ID NO. 96)
DNA
cagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgc
actgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaac
agcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggc
tccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattatta
ctgtgcagcatgggatgacagcctgaatggttatgtcttcggaactgggaccaagctgaccgtccta
ggt (SEQ ID NO. 97)
[00070] In constructing a recombinant immunoglobulin containing the
desired
antigen-binding region, the sequences shown above can be used in combination
with
appropriate amino acid sequences for constant regions of various
immunoglobulin
isotypes using methods for the production of a wide array of antibodies that
are known
to those of skill in the art. The light chain constant region can be, for
example, a kappa-
or lambda-type light chain constant region, e.g., a human kappa- or lambda-
type light
chain constant region. The heavy chain constant region can be, for example, an
alpha-,
delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a
human
alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In
one
aspect, the light or heavy chain constant region is a fragment, derivative,
variant, or
mutein of a naturally occurring constant region. In one embodiment, however,
light and
heavy chain constant regions may have the amino acid sequences (as shown in
Table
7) of SEQ ID NO. 98 and SEQ ID NO. 99, respectively.
Table 7
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LC constant QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSP
region VKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGS
TVEKTVAPTECS (SEQ ID NO: 98)
HC constant TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
region VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 99)
[00071] In one embodiment, the antibody of the invention comprises a light
chain
and heavy chain with amino acid sequences as follows:
Table 8
Complete light MOW5CiiiitiFLVATATOQAVVTQP P SASGT P GQ RVT I SCSGSSSNIGSN
chain TVNWYQQVPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGL
QSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKANPTVTLFPPS
SEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQS
NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
(SEQ ID NO: 100)
Complete IVIGWSOULFILVATATGQMQLVQSGAEVKEPG ES L R I SCKG SGYSFTN
heavy chain FWISWVRQMPGKGLEWMGRVDPGYSYSTYSPSFQGHVTISADKSTS
TAYLQWNSLKASDTAMYYCARVQYSGYYDWFDPWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
(SEQ ID NO: 101)
[00072] A leader sequence, MGWSCIILFLVATATG (SEQ ID NO: 102), as shown
in gray may be included. CDRs are bolded in Table 8 and correspond to the CDRs
listed in Table 3.
[00073] In each of Tables 1-6, a nucleic acid that encodes for the
variable and
hypervariable (CDR) regions of the heavy or light chain is also shown. Vectors
and
other nucleic acid constructs which comprise a nucleic acid that encodes the
antibody
and which can be used for expression of the antibodies from MACE 1.5 CHO cells
are
also encompassed by the invention. The antibodies of the present disclosure
also
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include substantially homologous polypeptides having antigen-binding portions
that are
at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at
least 97%, at least 98%, or at least 99% identical to the peptides described
in Tables 1-
6 or 8. In one aspect, an antibody of the present disclosure comprises a heavy
chain
variable region comprising CDR1, CDR2, and CDR3 from a VH sequence in any of
Tables 1-6 or 8 that is at least 90% identical to that VH sequence and/or
comprises a
light chain variable region comprising CDR1, CDR2, and CDR3 from a VL sequence
in
Tables 1-6 or 8 that is at least 90% identical to that VL sequence. For
example, in one
aspect, an antibody according to the present disclosure comprises a heavy
chain
variable region comprising CDR1, CDR2, and CDR3 from SEQ ID NO: 101 that is at
least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical
to SEQ ID
NO: 101 and/or comprises a light chain variable region comprising CDR1, CDR2,
and
CDR3 from SEQ ID NO: 100 that is at least 90%, at least 95%, at least 97%, at
least
98%, or at least 99% identical to SEQ ID NO: 100.
[00074] In one aspect, the present disclosure provides an antibody
comprising: (A)
a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3
respectively, comprising amino acid sequences SEQ ID NOS: 2, 3, and 4; 18, 19
and
20; 34, 35, and 36; 50, 51, and 52; 66, 67, and 68 or 82, 83, and 84; and a
light chain
(LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively,
comprising amino acid sequences SEQ ID NOS: 8, 9 and 10; 24, 25 and 26; 40, 41
and
42; 56, 57 and 58; 72, 73 and 74 or 88, 89 and 90; or (B) a VH and VL
comprising the
amino acid sequence of SEQ ID NO: 14 and SEQ ID NO: 16; 30 and 32; 46 and 48;
62
and 64; 78 and 80 or 94 and 96, respectively, wherein said antibody has no
detectable
fucose or galactose.
[00075] In one aspect, the antibody comprises a heavy chain (HC) variable
region
comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid
sequences SEQ ID NOS: 2, 3, and 4; and a light chain (LC) variable region
comprising
LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences
SEQ ID NOS: 8,9 and 10.
[00076] In another aspect, the antibody comprises a heavy chain (HC)
variable
region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino
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acid sequences SEQ ID NOS: 18, 19 and 20; and a light chain (LC) variable
region
comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid
sequences SEQ ID NOS: 24, 25 and 26.
[00077] In another aspect, the antibody comprises a heavy chain (HC)
variable
region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino
acid sequences SEQ ID NOS: 34, 35, and 36; and a light chain (LC) variable
region
comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid
sequences SEQ ID NOS: 40, 41 and 42.
[00078] In another aspect, the antibody comprises a heavy chain (HC)
variable
region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino
acid sequences SEQ ID NOS: 50, 51, and 52; and a light chain (LC) variable
region
comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid
sequences SEQ ID NOS: 56, 57 and 58.
[00079] In another aspect, the antibody comprises a heavy chain (HC)
variable
region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino
acid sequences SEQ ID NOS: 66, 67, and 68; and a light chain (LC) variable
region
comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid
sequences SEQ ID NOS: 72, 73 and 74.
[00080] In another aspect, the antibody comprises a heavy chain (HC)
variable
region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino
acid sequences SEQ ID NOS: 82, 83, and 84; and a light chain (LC) variable
region
comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid
sequences SEQ ID NOS: 88, 89 and 90.
[00081] In one embodiment, the antibody comprises a light chain consisting
essentially of the amino acid sequence of SEQ ID NO: 100 and a heavy chain
consisting essentially of the amino acid sequence of SEQ ID NO: 101.
[00082] In one aspect, an antibody of the present disclosure specifically
binds to
WT-1 peptide RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA/A2. Optionally,
the HLA-A2 is HLA-A0201. In another aspect, the antibody exhibits between 50-
100%
(80%) higher affinity for activating human FcyRIlla (158V variant) than
normally
glycosylated antibody, has 3- to 4-fold (3.5-fold) higher affinity for a
FcyRIlla 158F
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variant than normally glycosylated antibody, and has between 30 and 70% (50%)
reduced affinity for inhibitory FcyRI lb than normally glycosylated antibody.
[00083] In other aspects, the present disclosure provides an isolated
nucleic acid
that encodes an antibody described herein, a vector comprising said nucleic
acid, and a
cell comprising said nucleic acid or said vector.
In another aspect, the present
disclosure provides a kit comprising an antibody described herein.
[00084] In another aspect, the invention relates to a derivative or analog
of an
antibody of the present disclosure. A derivative can comprise any molecule or
substance that imparts a desired property, such as increased half-life in a
particular use.
Examples of molecules that can be used to form a derivative include, but are
not limited
to, albumin (e.g., human serum albumin) and polyethylene glycol (PEG).
Derivatives
such as albumin-linked and PEGylated derivatives of antibodies can be prepared
using
techniques well known in the art. An analog may be a non-peptide analog of an
antibody described herein. Non-peptide analogs are commonly used in the
pharmaceutical industry as drugs with properties analogous to those of the
template
peptide. These types of non-peptide compound are termed "peptide mimetics" or
"peptidomimetics," (Fauchere, J. Adv. Drug Res 1986;15:29; Veber and
Freidinger TINS
1985;p. 392; Evans et al. J. Med. Chem 1987;30:1229). Peptide mimetics that
are
structurally similar to the antibodies of the present disclosure may be used
to produce
an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics
are
structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a
desired
biochemical property or pharmacological activity), such as a human antibody,
but have
one or more peptide linkages optionally replaced by a linkage selected from
the group
consisting of: ¨CH2NH¨, ¨CH2S¨, ¨CH2¨CH2¨, ¨CH=CH-(cis and trans), ¨
COCH2¨, ¨CH(OH)CH2¨, and ¨CH2S0¨, by methods well known in the art.
[00085] Methods for the recovery and purification of antibodies are well
known in
the art. Antibodies according to the present disclosure may be prepared by any
of a
number of conventional techniques. For example, they may be produced in
recombinant
expression systems, using any technique known in the art. See, for example,
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses,
Kennet
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et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory
Manual,
Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
N.Y., (1988). Certain of the techniques involve isolating a nucleic acid
encoding a
polypeptide chain (or portion thereof) of an antibody of interest, and
manipulating the
nucleic acid through recombinant DNA technology. The nucleic acid may be fused
to
another nucleic acid of interest, or altered (e.g., by mutagenesis or other
conventional
techniques) to add, delete, or substitute one or more amino acid residues.
[00086] Any expression system known in the art can be used to make the
recombinant antibodies of the present disclosure. In general, host cells are
transformed
with a recombinant expression vector that comprises DNA encoding a desired
polypeptide. Among the host cells that may be employed are prokaryotes, yeast
or
higher eukaryotic cells. Prokaryotes include gram negative or gram positive
organisms,
for example E. coli or bacilli. Higher eukaryotic cells include insect cells
and established
cell lines of mammalian origin. Examples of suitable mammalian host cell lines
include
the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell
1981;23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163),
Chinese
hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, and the
CVI/EBNA cell line derived from the African green monkey kidney cell line CVI
(ATCC
CCL 70) as described by McMahan et al., EMBO J 1991; 10: 2821. Appropriate
cloning
and expression vectors for use with bacterial, fungal, yeast, and mammalian
cellular
hosts are described in the art, e.g., by Pouwels et al., Cloning Vectors: A
Laboratory
Manual, Elsevier, N.Y., 1985.
[00087] The transformed cells can be cultured under conditions that
promote
expression of the polypeptide, and the polypeptide recovered by conventional
protein
purification procedures. One such purification procedure includes the use of
affinity
chromatography, e.g., over a matrix having all or a portion of the antigen
bound thereto.
Polypeptides contemplated for use herein include substantially homogeneous
recombinant antibodies substantially free of contaminating endogenous
materials.
[00088] The resulting antibody has an amino acid sequence as described
above
and no detectable fucose or galactose as part of the carbohydrate of the
antibody. For
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example, the fucose content and/or galactose content of the antibody can be
reduced
by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at
least 90%, or 100% compared to the wildtype antibody.
[00089] In another aspect, the present disclosure provides a
pharmaceutical
composition comprising an antibody described herein and a physiologically
acceptable
diluent, excipient, or carrier. In one aspect, a pharmaceutical composition of
the present
disclosure comprises a antibody described herein with one or more substances
selected
from the group consisting of a buffer, an antioxidant such as ascorbic acid, a
low
molecular weight polypeptide (such as those having fewer than 10 amino acids),
a
protein, an amino acid, a carbohydrate, a chelating agent such as EDTA,
glutathione, a
stabilizer, and an excipient. Neutral buffered saline or saline mixed with
serum albumin
are examples of appropriate diluents. In accordance with appropriate industry
standards,
preservatives such as benzyl alcohol may also be added. A liquid
pharmaceutical
composition may include, for example, one or more of the following: a sterile
diluent
such as water for injection, saline solution, preferably physiological saline,
Ringer's
solution, isotonic sodium chloride, fixed oils that may serve as the solvent
or suspending
medium, polyethylene glycols, glycerin, propylene glycol or other solvents;
antibacterial
agents; antioxidants; chelating agents; buffers and agents for the adjustment
of tonicity
such as sodium chloride or dextrose. A parenteral preparation can be enclosed
in
ampoules, disposable syringes or multiple dose vials made of glass or plastic.
The use
of physiological saline is preferred, and an injectable pharmaceutical
composition is
preferably sterile. In one aspect, the composition may be formulated as a
lyophilizate
using appropriate excipient solutions (e.g., sucrose) as diluents. Suitable
components
are nontoxic to recipients at the dosages and concentrations employed. Further
examples of components that may be employed in pharmaceutical formulations are
presented in Remington's Pharmaceutical Sciences, 16th Ed. (1980) and 20th Ed.
(2000), Mack Publishing Company, Easton, Pa.
[00090] As is understood in the art, pharmaceutical compositions
comprising the
antibodies of the present disclosure are administered to a subject in a manner
appropriate to the indication. A pharmaceutical composition of the present
disclosure
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comprising an antibody described herein may be formulated for delivery by any
route
that provides an effective dose of the antibody. Pharmaceutical compositions
may be
administered by any suitable technique, including but not limited to,
parenterally,
topically, or by inhalation. If injected, the pharmaceutical composition can
be
administered, for example, via intra-articular, intravenous, intramuscular,
intralesional,
intraperitoneal or subcutaneous routes, by bolus injection, or continuous
infusion.
Localized administration, e.g., at a tumor site, is contemplated, as are
transdermal
delivery and sustained release from implants. Delivery by inhalation includes,
for
example, nasal or oral inhalation, use of a nebulizer, inhalation of the
antagonist in
aerosol form, and the like. Other alternatives include eyedrops; oral
preparations
including tablets, capsules, syrups, lozenges or chewing gum; and topical
preparations
such as lotions, gels, sprays, patches, and ointments.
[00091] In one aspect, the present disclosure provides use of an antibody
described herein, e.g., in the preparation of a medicament, for the treatment
of a WT1
positive disease. In another aspect, the present disclosure provides a method
for
treatment of a subject having a WT1-positive disease, comprising administering
to the
subject a therapeutically effective amount of an antibody or antigen binding
fragment
described herein. In one aspect, the WT1-positive disease is a chronic
leukemia or
acute leukemia or a WT1+ cancer, for example, a WT1-positive disease selected
from
the group consisting of chronic myelocytic leukemia, multiple myeloma (MM),
acute
lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML),
myelodysplastic syndrome (MDS), mesothelioma, ovarian cancer, gastrointestinal
cancers, breast cancer, prostate cancer and glioblastoma
[00092] The methods of treatment and uses of the present disclosure
encompass
alleviation or prevention of at least one symptom or other aspect of a
disorder, or
reduction of disease severity, and the like. In one aspect, a therapeutically
effective
amount of an antibody or pharmaceutical composition of the invention is an
amount
effective to inhibit growth of WT1-positive cells, reduce tumor size/burden,
prevent
tumor cell metastasis/infiltration, and/or result in cell death, e.g., via
apoptosis or
necrosis. An antibody or pharmaceutical composition described herein need not
effect
a complete cure, or eradicate every symptom or manifestation of a disease, to
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constitute a viable therapeutic agent. As is recognized in the art,
therapeutic agents
may reduce the severity of a given disease state, but need not abolish every
manifestation of the disease to be regarded as useful. Simply reducing the
impact of a
disease (for example, by reducing the number or severity of its symptoms, or
by
increasing the effectiveness of another treatment, or by producing another
beneficial
effect), or reducing the likelihood that the disease will occur or worsen in a
subject, is
sufficient.
[00093] Dosages and the frequency of administration for use in the methods
of the
present disclosure may vary according to such factors as the route of
administration, the
particular antibodies employed, the nature and severity of the disease to be
treated,
whether the condition is acute or chronic, and the size and general condition
of the
subject. Appropriate dosages can be determined by procedures known in the
pertinent
art, e.g., in clinical trials that may involve dose escalation studies.
[00094] An antibody of the present disclosure may be administered, for
example,
once or more than once, e.g., at regular intervals over a period of time. In
general, the
antibody or pharmaceutical composition is administered to a subject until the
subject
manifests a medically relevant degree of improvement over baseline for the
chosen
indicator or indicators.
[00095] In general, the amount of an antibody described herein present in
a dose,
or produced in situ by an encoding polynucleotide present in a dose, ranges
from about
pg per kg to about 20 mg per kg of host. The use of the minimum dosage that is
sufficient to provide effective therapy is usually preferred. Patients may
generally be
monitored for therapeutic or prophylactic effectiveness using assays suitable
for the
condition being treated or prevented; assays will be familiar to those having
ordinary
skill in the art and some are described herein.
[00096] The methods disclosed herein may include oral administration of an
antibody described herein or delivery by injection of a liquid pharmaceutical
composition.
When administered in a liquid form, suitable dose sizes will vary with the
size of the
subject, but will typically range from about 1 ml to about 500 ml (comprising
from about
0.01 pg to about 1000 pg per kg) for a 10kg to 60 kg subject. Optimal doses
may
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generally be determined using experimental models and/or clinical trials. The
optimal
dose may depend upon the body mass, body area, weight, or blood volume of the
subject. As described herein, the appropriate dose may also depend upon the
patient's
condition, that is, stage of the disease, general health status, age, gender,
weight, and
other factors familiar to a person skilled in the medical art.
[00097] In particular embodiments of the methods and uses described
herein, the
subject is a human or non-human animal. A subject in need of the treatments
described
herein may exhibit symptoms or sequelae of a disease, disorder, or condition
described
herein or may be at risk of developing the disease, disorder, or condition.
Non-human
animals that may be treated include mammals, for example, non-human primates
(e.g.,
monkey, chimpanzee, gorilla), rodents (e.g., rats, mice, gerbils, hamsters,
ferrets,
rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine,
feline, bovine, and
other domestic farm and zoo animals.
[00098] The present disclosure will be more readily understood by
reference to the
following Examples, which are provided by way of illustration and are not
intended to be
limiting.
EXAMPLES
Oligosaccharide analysis and FcR binding assays
[00099] N-Glycan from ESK1 or ESKM antibodies was cleaved from antibody by
PNGase F, and measured by HPAEC-PAD using PA200 column. Binding of
ESK1/ESKM antibodies to mouse FcyR4 and mouse FcyRI lb were measured by ELISA.
Briefly, 2 pg/mL recombinant mouse FcyR4 or FcyRIlb were coated onto ELISA
plate.
Various concentrations of ESK1 or ESKM antibodies were added to the wells for
1 hour
at room temperature, then detected by secondary antibody (HRP conjugated anti-
human IgG Fab'2 fragment). Binding of ESK1/ESKM to human FcyRs was measured by
Flow Cytometry (Guava easyCyte HT, Millipore) against CHO cells expressing
appropriate human FcyR. Binding of ESK1/ESKM to human FcyRI, FcyRIla, FcyRIlla-
158V, FcyRIlla-158F and human FcRn were measured directly using ESK1 or ESKM
antibody, followed by the 2nd antibody (FITC conjugated Fab'2 fragment anti-
human IgG
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Fab'2). For human FcyRIlb, dimers were formed first by mixing ESK1 or ESKM to
a PE-
conjugated Fab'2 fragment anti-human Fab'2 at 2:1 ratio at RT for 2 hour.
Binding of
dimeric complex of ESK1 or ESKM to human FcyRIlb were measured directly by
Flow
Cytometry using the immunocomplex.
[000100] In another assay, ESK1 was expressed in Chinese Hamster Ovary
cells
using the GlymaxXO technology (ProBioGen, Berlin, Germany) to reduce the
fucose
content of the antibody. ESKM antibody was purified from two separate pools of
cells,
and fucose reduction confirmed by mass spectrometry to be 70% reduced or 100%
reduced. Both the ESKM having 70% reduction in fucosylation and the ESKM
having
100% reduction in fucosylation (completely a-fucosylated) batches were
compared to
ESK1 as a wildtype IgG1 and to ESK1 containing D265A/P329A mutations in the Fc
domain (ESK1 DAPA) that eliminated binding to human FcgRIlla in ADCC killing
assays. T2 cells were pulsed with 25 mg/ml RMFPNAPYL (SEQ ID NO:1) peptide
overnight. The next day, 15,000 pulsed cells were added to serially diluted
ESK-1
antibodies. Then 90,000 Jurkat cells transduced to express CD16A and an NFAT-
luciferase reporter were added. Plates were gently mixed and spun at 200 x g
for 4
minutes and then incubated at 37 C for 4 hours. At the end of the incubation,
the plates
were brought to room temp for -15 minutes, 60 I of BrightliteTM (Perkin
Elmer) was
then added to each well. Plates were shaken for 3 minutes and analyzed on the
EnVision Multilabel Reader (PerkinElmer).
Cell lines and reagents
[000101] Cell lines were from laboratory stocks, and were maintained in
RPMI with
10% FBS. Peptides for T2 pulsing assays were purchased and synthesized by
Genemed Synthesis, Inc. (San Antonio, TX). Peptides were > 90% pure. GFP+
luciferase-expressing SET2 and JMN cells were generated as described
previously
(Dao T et al. Science translational medicine 2013;5(176):176ra33). All cells
were HLA
typed.
Animals
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[000102]
C57BL/6 and C57BL/-Tg (HLA-A2.1) 1 Enge/J (6- 8 week-old male), and
NOD.Cg-Prkdcscid//2dmiwP/SzJ mice (6-8 week-old male), known as NOD scid gamma
(NSG), were purchased from Jackson Laboratory (Bar Harbor, ME). NOD.Cg-
Prkdcsc'd 112rgtmlsug/JicTac (NOG), and C.B-Igh-lb/IcrTac-Prkdcscid
(SC ID) were
purchased from Taconic (Hudson, NY). All studies were conducted in accordance
with
IACUC approved protocols.
Antibody-dependent cellular cytotoxicity (ADCC)
[000103] Peripheral blood mononuclear cells (PBMCs) from healthy donors were
obtained by Ficoll density centrifugation. Target cells used for ADCC were T2
cells
pulsed with or without WT1 or RHAMM-3 peptides, and cancer cell lines without
peptide
pulsing. ESK1, ESKM or isotype control human IgG1 at various concentrations
were
incubated with target cells and fresh PBMCs at different effector: target
(E:T) ratio.
Cytotoxicity was measured by standard 4 hour 51Cr-release assay.
[000104] In another assay, 5 x 106 0V56 human ovarian cancer cells were
collected,
washed and resuspended in CalceinAM. After a 50-minute incubation, cells were
washed twice with PBS and added to the assay plate containing serially diluted
antibodies. Purified human NK cells from leukopak (HemaCare, Van Nuys, CA)
were
added in an effector to target ratio of 20:1. Cells were incubated at 37 C
for 3.5 hours
and Calcein release was measured on EnVision Multilabel Reader (PerkinElmer,
Waltham, MA). Specific lysis was calculated as (sample ¨ spontaneous
release)/(max
release ¨ spontaneous release)*100%.
Therapy of ESK1 and ESKM in human mesothelioma, AML and ALL xenograft
mouse models
[000105]
Luciferase-expressing JMN cells (3 x 105) were injected into the
intraperitoneal cavity of CB17 SCID mice. On day 4, tumor engraftment was
confirmed
by luciferase imaging, signal was quantified with Living Image software
(Xenogen),
and mice were sorted into groups with similar average signal from the supine
position.
Mice were injected intraperitoneally with 50 pg ESK1, ESKM or human isotype
IgG1
antibody twice weekly beginning on day 4.
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[000106] For AML leukemia studies, luciferase-expressing BV173 (Ph+ ALL) or
SET2 (AML) cells (3 x 106) were injected intravenously via tail vein into NSG
mice.
Animals were sorted, and, where indicated, treated with intraperitoneal
injections of
100 g ESKM twice weekly beginning on day 6.
[000107] For ALL leukemia studies, fresh pre-B cell ALL cells were obtained
under
IRB approved protocols from the CNS relapse of a female pediatric patient
after
treatment with a chemotherapy induction regimen and bone-marrow transplant.
Leukemia cells were transduced with a lentiviral vector containing a plasmid
encoding
luciferase/GFP. Luciferase+/GFP+ leukemia was then expanded in NSG mice,
luciferase signal was confirmed by bioluminescent imaging, and tumor cells
were
harvested and sorted for CD45. Leukemia cells (5.5 x 106/animal) were then
injected
intravenously into NSG mice, and engraftment was confirmed by bioluminescent
imaging on day 2 post-injection. Animals were sorted into two groups (n = 5
each) so
that average signal in each group was equal. ESKM or isotype control antibody
(100
g/animal) was administered via retro-orbital injection on days 2, 5, 9, 12, 14
and 23,
and leukemia growth was followed by bioluminescent imaging. On day 41, animals
were
sacrificed and bone marrow cells were harvested and pooled: after dissection
and
homogenization, cells were centrifuged, subjected to Ficoll density
centrifugation, and
counted after red blood cell lysis (acetic acid). An equal number of cells
from each
treatment group was resuspended in matrigel (200 L/injection) and engrafted
SC into
the opposite shoulders of NSG mice (n = 4). No further treatment was given,
and tumor
growth was followed by bioluminescent imaging.
Pharmacokinetic and biodistribution studies
[000108] Antibody was labeled with 1261 (PerkinElmer) using the chloramine-
T
method. 100 lig antibody was reacted with 1mCi 1261 and 20 g chloramine-T,
quenched
with 200 lig Na metabisulfite, then separated from free 1261 using a 10DG
column
equilibrated with 2% bovine serum albumin in PBS. Specific activities of
products were
in the range of 4-8 mCi/mg. Radiolabeled mAb was injected into mice retro-
orbitally, and
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blood and/or organs were collected at various time points, weighed and
measured on a
gamma counter.
Toxicity studies
[000109] For isolated cell binding studies, C57BL6/J or HLA-A2.1+
transgenic mice
were sacrificed, and cells were harvested from spleen, thymus and bone-marrow.
After
red blood cell lysis, cells (106 per tube, in duplicate) were incubated with
1261-labeled
ESK1 (1 pg/ml) for 45 minutes on ice, then washed extensively with 1% bovine
serum
albumin in PBS on ice. To determine specific binding, a set of cells was
assayed after
pre-incubation in the presence of 50-fold excess unlabeled ESK1 for 20 minutes
on ice.
Bound radioactivity was measured by a gamma counter, specific binding was
determined, and the number of bound antibodies per cell was calculated from
specific
activity.
[000110] For toxicity studies, 100 pg of ESKM or isotype control mAb was
injected
into human HLA-A0201 transgenic mice (Jackson Labs) on days 0 and 4, to mimic
the
maximum dose and therapeutic schedule used in the therapy experiments. Mice
were
sacrificed on day 5 for collection and analysis of whole blood and bone marrow
leukocytes. Whole blood was analyzed with a Hemavet system (Drew Scientific).
Bone
marrow cells were harvested from both femurs and tibias of mice and subjected
to red
blood cell lysis, then analyzed by flow cytometry (see Antibodies and flow
cytometry
analysis).
[000111] Alternatively, mice treated as above were sacrificed on day 6 for
histolo-
pathologic examination of major organs and possible WT1 positive target organs
(spleen, bone and bone marrow, liver, thymus, kidney) as well as heart, lung,
and ileum.
Mice were sacrificed and whole organs were collected, fixed (4%
paraformaldehyde),
decalcified in EDTA where necessary (femurs only), embedded in paraffin,
sectioned
and stained with H&E.
Antibodies and flow cytometry analysis
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[000112] For cell surface staining, cells were incubated with appropriate mAbs
for 30-
60 minutes on ice, washed, and incubated with secondary antibody reagents when
necessary. Flow cytometry data were collected on a FACS Calibur or LSRFortessa
(Becton Dickinson) and analyzed with FlowJo software. AFC-labeled ESK1 and
hIgG1
isotype antibodies were generated with Lightning-Link kit (Innova
Biosciences).
[000113] For HSC toxicity studies, mouse bone marrow cells were stained with
the
following antibodies: (Lineage; CD3, CD4, CD8, Gr1, B220, CD19, TER119, all
conjugated with PE-Cy5), Sca-Pacific Blue, CD34-FITC, SLAM-AFC, CD48-PE and c-
KIT-AlexaFluor 780. The stained cells were analyzed for flow cytometry on the
BD
LSRII instrument.
[000114] For mouse immunophenotyping, cells were isolated from the
intraperitoneal
cavity by washing with complete media, or from spleen by dissection and red
blood cell
lysis (RBC Lysis Solution, Qiagen). Samples were then analyzed by flow
cytometry after
multi-color staining with well-characterized lineage-specific markers: CD335
(NKp46)-
PE and F4/80 (BM8)-AlexaFluor700 (BioLegend), CD49b/VLA-2a (DX5)-FITC (Life
Technologies), CD3e-PE-Cy7 and Cr-1/Ly-6G/Ly-6C (RB6-8C5)-PerCP-Cy5.5 (BD
Pharmingen).
ESKM antibody has enhanced binding affinity for Fcyllilla and reduced affinity
for
Fcyllilb
[000115] ESKM mAb was produced in MACE 1.5 CHO cells, with the
homogeneous oligosaccharide structure (Fig 1A) and no detectable fucose or
galactose. ESKM had 80% higher affinity for activating human FcyRIlla (158V
variant),
3.5-fold higher affinity for the FcyRIlla 158F variant, and 50% reduced
affinity for
inhibitory FcyRI lb (Table 9)
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Table 9
n.Ratio of Affioity
fid filloh3) Constargh
jESKM#S..K1)
. ...............
.....................................
.........................................................................
.Motsto
fty911b 32.0 7. 0.454 62,3 7. 7,27 03:1
figyitiV 334= 0:193 2.21 ciss Lst
"moo
ktiki 11,S81 &6113 66.4:Z. 0,i2S.
rtyRkt JOS y 133 583 14 RIO L6:1
Ry91111 1$36 7, :253 2644 '7. 43$.
92.6 15,0 50.4 7, 135 1.21,
(ISSV)
la
19.0 '7: 2:39. 5.S3 0341 3,4:S
Fan 624 7. 102 7:4 7 , vs NL[000116]
Importantly, ESKM affinity for FcRn was unchanged (Fig. 1B and 1C).
Similarly, ESKM had 51% higher affinity for activating mouse FcyRIV, and half
the
affinity for inactivating mouse FcyRIlb (Fig. 1B, 1D and 1E). Changes in Fc
glycosylation pattern should not be expected to affect antigen binding, and
indeed,
avidity of ESKM against WT1+ HLA-A0201+ JMN cells was nearly identical to the
native
ESK1 (0.2-0.4 nM) (Fig. 1F and 10).
[000117] ESKM showed enhanced reverse signaling through FcyRIIIA (CD16A)
compared to wildtype ESK1, indicating improved binding interaction. An
approximate 5-
fold decrease in EC50 was observed with ESKM relative to wildtype ESK1 (EC50
values: 0.17 for ESKM; 0.88 for ESK1 wildtype; >10000 for ESK1 DAPA) (Fig.
1H).
ADCC mediated by ESKM in vitro.
[000118] The relationship of cell surface antigen density with ESKM ADCC
efficacy
was investigated using T2, a TAP-deficient cell line that expresses HLA-A0201,
but
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does not present peptides through the ER pathway, and thus can be loaded with
exogenous peptides for presentation in a dose-dependent manner. To determine
whether ESKM could better mediate ADCC against cells with low antigen density,
the
dose of ESK1 and ESKM mAbs was fixed and tested against T2 cells loaded with
titrated RMF peptide. Both antibodies were effective against T2 cells pulsed
with high
peptide concentrations (achieving 40-50% specific lysis), but ESKM was able to
mediate greater ADCC against cells with fewer RMF/A2 complexes (Fig. 2A).
[000119] The in vitro ADCC activity of ESK1 and ESKM against cell lines
presenting
a range of levels of cell surface RMF/A2 was determined (Dao T et al. Science
translational medicine 2013;5(176):176ra33). ESKM showed both increased
potency
and efficacy against six leukemia and mesothelioma cell lines in an HLA-A2-
restricted
manner. ESKM effectively mediated ADCC against BA-25, an acute lymphoblastic
leukemia (ALL) cell line expressing approximately 1000-2000 RMF/A2 targets per
cell;
both antibodies were similarly effective at ADCC at concentrations above 1
pg/mL, but
ESKM was more potent at concentrations down to 100 ng/ml of mAb (Fig 2B).
Against
AML-14 and SET2 acute myeloid leukemia (AML) cell lines, which both bind -5000
mAb
per cell, ESKM mediated higher specific cell lysis than ESK1 at the highest
antibody
concentrations, and showed cytolytic efficacy down to doses as low as 100
ng/ml (Fig.
1C-1D). Further, the maximal specific lysis achieved against the AML cell
lines was
generally twice that shown against BA25 (30-45% vs 18%), supporting the
hypothesis
that increased RMF/A2 levels lead to improved mAb efficacy, regardless of the
Fc
construct. As was shown previously for ESK1 (Dao T et al. Science
translational
medicine 2013;5(176):176ra33), ESKM did not kill leukemia cells not expressing
HLA-
A2 (Fig 1E). Further, ESKM mediated higher specific lysis at nearly all doses
tested
against 3 HLA-A0201+ mesothelioma cell lines: JMN (Fig. 1F), Meso-37 (Fig. 10)
and
Meso-56 (Fig 1H). These data show that ESKM is both more potent-as illustrated
by its
ability to kill cells with lower mAb concentrations and fewer cell surface
targets¨and
more effective than ESK1, as demonstrated by higher specific lysis attained at
equal
concentrations.
[000120] The effect of different levels of a-fucosylation was evaluated.
Both 70%
fucose-reduced and 100% fucose-reduced ESKM resulted in greater ADCC activity
than
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the wildtype ESK1 IgG1. An approximate 6-fold decrease in EC50 was observed
with
ESKM relative to wildtype ESK1 (EC50 values: 2.9 for 100% a-fucosylated ESKM;
3.9
for 70% a-fucosylated ESKM; 18.9 for ESK1 wildtype; >10000 for ESK1 DAPA)
(Fig. 21).
Potency of ESKM against human mesothelioma and leukemia models in mice.
[000121] It was previously reported that ESKM is effective at a low dose
against
bcr/abl+ BV173 ALL in a NSG mouse model. Data from several in vitro and in
vivo
experiments provided strong evidence that ADCC was the dominant mechanism of
therapeutic action of the ESK1 mAb, even in mice lacking functional NK-cells,
which
might be expected to provide substantial effector function (Dao T et al.
Science
translational medicine 2013;5(176):176ra33). To investigate whether ESKM
offered a
consistent and significant improvement over native ESK1 in vivo in mice with
more
complete effector cell repertoire, SCID mice, which have intact NK cell
functionality,
were used, and both antibodies in treatment of human JMN mesothelioma were
investigated. Human Fc can engage murine FcyRIV (Pietzsch J et al. Proceedings
of
the National Academy of Sciences of the United States of America 2012
;109(39):15859-
64), therefore murine NK cells should serve as potent effectors in vivo; as
ESKM has
enhanced binding to murine FcyRIV, it was expected to be more efficacious than
the
native mAb in this model. Mice were engrafted with luciferase+ JMN
mesothelioma cells
in the intraperitoneal cavity (simulating this serosal cavity cancer). To
determine the
relative abundance of effector cell populations in the intraperitoneal cavity,
extracted
cells with common murine immunophenotyping markers (Lai L et al. J Immunol
1998;160(8):3861-8) were analyzed. SCID mice contain intraperitoneal
macrophages,
neutrophils and NK-cells. Intraperitoneal cells were isolated from mice (n = 3
each
strain) and analyzed by multi-color flow cytometry. Cell type was determined
by the
indicated markers, and quantified as percentage of total leukocytes isolated.
(Table 10)
Table 10
Cells (% of parent population +/- SD)
liNiii.#11=11111111111111110iiiiiiiilEillialbIREIIIIIIIIMME11111111111111aft$01
011111111111111111061111111111111111
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Populatton ___________________ 6tyoemmggmmggmmmerrmmgmnwrmmmgm9m
Granulocyte GR-1 F4/80- ......46.6 +/- 28.3.........7.45
Macrophage GR lowF4/8o+ 105+/687 190+/i 29
963+/i 06
Monocyte GR-1- F4/80- 39.2 +/- 21.1 70.2 +/-2.73
1.25 +/-0.562
¨CD/ 1 b- NK cell NKp46+ 1.68 +1- 0.250
36.2 +1- 12.0 0.278 +/-0413
CD3E-
[000122] This flow cytometry analysis also confirmed the presence of murine
monocytes, macrophages and NK cells, but lack of B- and T-cells in the spleen
and
peripheral blood, as expected (Bosma MJ and Carroll AM. Annual review of
immunology 1991;9:323-50). Biweekly 50 pg treatment with ESKM was more
effective
than ESK1 against intraperitoneal JMN (Fig. 3A). Further, ESKM was able to
reduce
tumor burden during the treatment course, whereas ESK1 merely slowed growth
(Fig.
3B). ESKM treated mice survived significantly longer than isotype-treated, and
had
improved survival over ESK1-treated groups (Fig. 3C). In a third experiment at
the same
dose and schedule, neither antibody construct showed efficacy against
intraperitoneal
JMN in NOG mice (Fig. 6), which lack NK-cells and intraperitoneal neutrophils,
indicating that these cell populations likely play an important role in
efficacy in these
models. These studies provide further evidence that ESKM is a more potent mAb
construct.
[000123] ESKM was also investigated in the luciferase+ SET2 mouse model of
AML. The SET2 cell line grew much faster than BV173 in the NSG mouse model and
disseminated throughout the mouse bone marrow. ESKM was able to significantly
reduce tumor growth (Fig. 3D). To further address the clinical utility of
ESKM, a fresh
human pre-B-cell ALL derived from a CNS-relapse was engrafted into NSG mice.
ESKM significantly reduced initial leukemia burden and slowed leukemia
outgrowth (Fig.
3E and 3F). Leukemia relapsed after treatment was stopped (Fig. 3F), allowing
for
leukemia cells to be collected from the bone marrow and transplanted to new
animals to
assess outgrowth from remaining progenitors (Fig. 30). Total bone marrow
signal in
ESKM-treated mice was lower at time of transplant (Fig. 3H), but equal numbers
of
ESKM-treated and isotype-treated bone marrow cells were engrafted into
recipient
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animals. Subcutaneous leukemia tumors from isotype-treated leukemia cells grew
20
times faster than from ESKM-treated cells (Fig. 31).
Pharmacokinetics and biodistribution of ESK1 and ESKM
[000124] Altering Fc glycosylation could potentially change pharmacokinetic
properties of the mAb, thereby affecting its therapeutic utility. To determine
mAb
pharmacokinetics, trace 1251-labeled antibodies were injected intravenously
into C57
BL6/J mice and blood levels of mAb were measured over 7 days. Both ESK1 and
ESKM exhibited biphasic clearance common of monoclonal antibodies, with
initial tissue
distribution and an alpha half-life of 1.1 ¨ 2.4hr, followed by a slower beta
half-life of
several days (Fig. 4A). ESKM had a shorter beta half-life than ESK1 (4.9 days
vs 6.5
days). The biodistribution patterns of the antibodies were determined using
the same
radiolabeled constructs. Both antibodies displayed similar patterns of organ
distribution
and clearance (Fig. 4B).
[000125] While increased FcyRIV binding or manose receptor binding could
create
a sink for ESKM in FcyRIV-expressing tissues, no increase in ESKM distribution
to the
liver, spleen, thymus or bone marrow was found that could account for the
shortened
serum half-life.
[000126] As the ESKM antibody targets a human-specific epitope, the
C57BL6/J
mouse model cannot recapitulate possible on-target binding to normal tissues
that could
alter antibody pharmacokinetics and biodistribution. Therefore, a transgenic
mouse
model based on the C57BL6/J background that expresses human HLA-A201 driven by
a lymphoid promoter was used. The 9-mer RMF sequence is identical in human and
mouse, and therefore this transgenic model could recapitulate antigen
presentation of
the RMF/HLA-A0201 epitope in various healthy organs. There was no difference
between wild-type and HLA-A0201 transgenic mice in blood pharmacokinetics of
ESKM, indicating that there was no significant antibody sink (Fig. 4C).
Further, at a
therapeutic dose of antibody (100 pg), there was no difference in
biodistribution in
transgenic compared to wild-type mice (Fig. 4D).
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[000127] At low doses of trace-labeled ESKM (2 pg) there was a small yet
detectable increase in uptake of antibody in the spleen of transgenic mice
compared to
wild-type (Fig. 4E). The additional binding in HLA-A0201 transgenic spleens
accounted
for only 16 ng of antibody. This small uptake could be due to RMF presentation
in HLA-
A0201+ cells, or to an unknown cross-reacting epitope. Splenic uptake was not
due to
Fc glycosylation pattern alone, as the native ESK1 mAb also showed increased
uptake
in transgenic spleens at 24 hours (Fig. 7A). Additionally, increased spleen
uptake in
transgenic mice appeared to be partly related to strain differences in
clearance, as the
isotype control antibody also showed 57% increased splenic uptake at 24 hours
in
transgenic compared to wild-type mice (Fig. 4F). Further, no binding of ESK1
to cells
isolated from A0201 transgenic mouse spleen cells was observed by either flow
cytometry (Dao T et al. Science translational medicine 2013;5(176):176ra33) or
specific
binding assay with 125 I-labeled ESK1 (Fig. 7B), suggesting that if a cross-
reacting
epitope was present, it was not expressed in detectable amounts on a specific
cell type.
Toxicity of ESKM in HLA-A0201 transgenic mouse model
[000128] WT1 is reported to be expressed in hematopoetic stem cells (HSC)
(Ariyaratana S and Loeb DM. Expert reviews in molecular medicine 2007;9(14):1-
17),
so the C57 BL6/J transgenic mouse model with human HLA-A0201 driven by a
lymphoid promoter provides an opportunity to assess possible toxicity against
progenitor cells in the hematopoetic compartment that, given the high potency
of ESKM,
might occur even at low epitope density. To assess toxicity, white blood cell
and bone-
marrow cell counts were measured one day after the final of two therapeutic
doses of
ESKM or isotype control mAb on the same schedule as previously described
therapy
experiments (Dao T et al. Science translational medicine 2013;5(176):176ra33).
There
were no differences in total white blood cell count, or lymphocytes,
neutrophils,
monocytes, eosinophil or basophil cell counts (Fig. 5A). Within the bone-
marrow
compartment, equivalent absolute number and frequency of hematopoetic stem
cell
(HSC) progenitors (LSK: Lineage, c-kit, Sca1+) (Fig. 5B-5C) and HSCs (CD150hi,
CD48-, LSK) (Fig. 5D-5E) were found in the ESKM treated and isotype control-
treated
mice.
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[000129] Finally, gross and microscopic pathology of lymphoid and major
organs of
A0201 transgenic mice treated with ESKM or isotype mAb on the same schedule
were
assessed. No striking differences between ESKM- and isotype-treated groups
were
observed by a trained hematopathologist (Table 11). Almost all of the target
organs
were present with good histology.
Table 11
spleen thymus liver kidney lung GI heart bone
marrow
hIgG1-2 Nml N/A* Nml Nml Nml Nml Nml Nml
hIgG1-4 Nml Nml Nml Nml Nml Nml Nml Nml
6.01111111119
ESKM-1 Nml Nml Nml Nml Nml Nml Nml Nml
ESKM-3 Nml Nml** Nml Nml Nm Nml Nml Nml
ESKM-5 Nml Nml Nml Nml Nml Nml Nml Nml
Nml - Normal
*Thymus specimen is missing from specimen IgG-2A
[000130] The bone marrow sections of both groups showed trilineage
hematopoiesis with adequate maturation of the myeloid and erythroid lineages.
The
megakaryocytes were adequate in number with normal morphology. Thymus sections
showed a well-defined cortex and medulla with few Hassall's corpuscles, which
is
normal for rodent histology. The kidney sections showed no pathologic findings
such as
glomerulosclerosis, congestion, or inflammation. Liver sections showed normal
lobular
architecture without congestion or inflammation. All spleen sections showed a
normal
distribution of red and white pulp. Occasional scattered megakaryocytes were
seen in
the red pulp consistent with extramedullary hematopoiesis (Cesta MF.
Toxicologic
pathology 2006;34(5):455-65). Heart sections of the control groups and
treatment
groups had normal myocytes without inflammation or fibrosis. Small intestine
sections
showed normal villi and crypts, and incidentally sampled pancreas sections for
some of
the control and treatment groups showed no pathologic findings. Finally, the
lung
sections of both treatment groups were unremarkable.
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[000131] The generation of TCRm antibodies allows use of the mAb to target
cell-
surface fragments of intracellular proteins, provided that they are processed
and
presented on MHC class I molecules. ESK1 was the first TCRm antibody reported
against a peptide derived from WT1, an important oncogene expressed in a wide
variety
of cancers, but not normal adult tissues. WT1 appears to be expressed in
leukemic
stem cells (Ariyaratana S and Loeb DM. Expert reviews in molecular medicine
2007;9(14):1-17), raising the possibility that the mAb could ultimately
eliminate
clonogenic leukemia cells in patients. Other therapeutic TCRm mouse
antibodies,
human ScFy and Fab fragments have been previously described (Epel M et al.
European journal of immunology 2008;38(6)1 706-20; Wittman VP et al. J Immunol
2006;177(6):4187-95; Klechevsky et al. Cancer research 2008;68(15):6360-7;
Verma B,
et al. J Immunol 2010;184(4):2156-65; Sergeeva et al. Blood 2011;117(16):4262-
72).
However, ESK1 is the first and only fully human therapeutic TCRm mAb reported.
[000132] The features of the RMF/A2 epitope, especially the low levels of
expression on the cell surface, require selection of a highly potent and
effective ESK1
construct. The ESK1 construct was improved by altering Fc glycosylation as a
means to
enhance ADCC, the major mechanism of ESK1 action in vitro and in vivo (Dao T
et al.
Science translational medicine 2013;5(176)1 76ra33). Several ADCC-enhanced
mAbs
to highly expressed cell-surface antigens, produced, either by glyco-
engineering or point
mutations, are in clinical trials in the U.S. with promising results (Kubota T
et al. Cancer
science 2009;100(9):1566-72; lshida T et al. Journal of clinical oncology :
official journal
of the American Society of Clinical Oncology 2012;30(8):837-4; Subramaniam JM
et al.
Drugs 2012;72(9):1293-8). The ESKM mAb had a homogeneous glycosylation pattern
lacking N-linked fucose and with terminal hexose (mannose and/or glucose)
structure.
This engineering strategy modulates mAb binding to Fcy receptors in two ways:
a
higher affinity for activating human FcyRIlla (and murine FcyRIV) increases
ADCC
activity; while diminished affinity for both human and murine FcyRIlb should
reduce
inhibitory receptor activation. As expected, ESKM was both more potent and
effective in
vitro even at very low epitope density.
[000133] ESKM was also more effective than ESK1 in vivo, and was able to
treat
peritoneal mesothelioma in SCID mice, modeling the clinical situation. Three
of 5
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animals displayed absolute reduction in tumor burden over the two-week
treatment
course, whereas none of the ESK1-treated mice achieved more than a slowing of
initial
tumor growth. After termination of therapy, ESKM-treated mice survived longer
than
ESK1 treatment groups, with 1 of 5 animals surviving without disease. Further,
ESKM
significantly slowed leukemia growth of disseminated SET2, an AML cell line
with much
more aggressive in vivo leukemia growth kinetics than BV173, and a fresh
patient-
derived pre-B-cell ALL in xenograft models. In the fresh ALL model, tumor
relapsed
after mAb therapy was stopped, but leukemia cells extracted from the bone
marrow of
ESKM-treated mice and transplanted as subcutaneous tumors showed minimal
outgrowth. This suggests that ESKM may target a progenitor population of
leukemia
cells, which is consistent with the hypothesis that WT1 expression in HSCs
could allow
ablation of this population. However, cells collected from the bone marrow
were not
phenotyped and sorted, so the exact cell population targeted was not
determined.
These data provide further evidence that ESKM is a potent agent in diverse
mouse
models of human cancer.
[000134] ESKM therapy was not effective against peritoneal mesothelioma in
NSG
or NOG mice, which lack NK-cells, though naked ESK1 did previously show potent
activity against a disseminated leukemia model in these mice. This discrepancy
could
be due both to the tumor model¨leukemia cells could have different sensitivity
to
effector-mediated cytotoxicity¨and to the availability of effector cells in
the NSG/NOG
model. Access to ESK-bound target cells is likely more optimal in the
circulation and
hematopoetic compartments, where the leukemia grew, than in the peritoneal
cavity;
further, assays indicated that the intraperitoneal cavity of NOG mice
contained
predominately macrophages, while neutrophils were present in the blood and
spleen.
The marked improvement in efficacy with ESKM in SCID mice indicated that NK
cells
and/or monocytes (both with FcyRIV) are important to therapy in this model.
[000135] Altering Fc glycosylation could potentially change pharmacokinetic
properties of the mAb through a number of mechanisms, including: altered FcRn
binding and antibody recycling, modified binding to circulating effector
cells, and
differential engagement with clearance mechanisms, such as mannose receptors.
Similar afucosylated, Fc-modified antibodies with improved ADCC have been
41
CA 02934033 2016-06-15
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investigated in pharmacokinetic studies in vivo (Gasdaska JR et al. Molecular
immunology 201 2 ;50(3):134-41, Junttila TT et al. Cancer research 201 0
;70(11):4481-9).
ESKM had nearly identical biodistribution to ESK1, but a shortened blood half-
life. No
change in biodistribution pattern that could account for this altered half-
life was seen.
IgG half-life is regulated by the neonatal Fc receptor, FcRn (Raghavan M and
Bjorkman
PJ. Annual review of cell and developmental biology 1996;12:181-220; Roopenian
DC
and Akilesh S. Nature reviews Immunology 2007;7(9):715-25); however, ESKM had
identical affinity for FcRn as ESK1. The altered pharmacokinetics is possibly
due to
interaction with mannose receptor on macrophages, a known mechanism of
glycoprotein clearance (Allavena P et al. Critical reviews in immunology
2004;24(3)1 79-
92; Lee SJ et al. Science 2002;295(5561):1898-901; Stahl PD. Curr Opin Immunol
1992 ;4(1):49-52)..
[000136] Since ESK mAbs target a human HLA-specific epitope, the human HLA-
A0201+ transgenic mouse strain was utilized for toxicology studies. WT1 is
reportedly
expressed in HSCs, yet a therapeutic dose of ESKM that cleared leukemia in the
models had no effect on LSK cells or early HSCs. Further, after this same
treatment
schedule, organ histology was normal. Importantly, ESKM did not affect the
architecture
or cell coverage in the bone marrow, thymus or spleen, where WT1+ HSCs could
be
expected, and where HLA-A0201 expression is highest because the transgene is
driven
by a lymphoid promoter. There was also no observed pathology in the kidney,
where
WT1 expression might be expected in mature podocytes.
[000137] ESKM has moderately decreased half-life yet increased potency and
broader applicability. The potential enhanced efficacy against tumors
expressing fewer
RMF/A2 sites could expand the number of patients and cancer types eligible for
this
therapy as well as increase efficacy. In addition, the MACE 1.5 CHO
engineering
technology generates mAbs that effectively engage FcyRIlla (CD16), regardless
of
amino acid 158 polymorphism. Carriers of CD16-158F are less responsive than
CD16-
158V/V individuals to human IgG1 therapeutics such as rituximab and
trastuzumab
(Cartron G et al. Blood 2002;99(3):754-8, Musolino A et al. Journal of
clinical oncology:
official journal of the American Society of Clinical Oncology 2008
;26(11):1789-96). WT1
is expressed in multiple cancers (Sugiyama H. Japanese journal of clinical
oncology
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2010;40(5):377-87), making RMF/A2 a potential therapeutic target for many
indications.
In preclinical models, efficacy against bcr/abl+ ALL and B-ALL (Dao T et al.
Science
translational medicine 2013;5(176):176ra33), and here, AML and mesothelioma
xenografts, was shown. In summary, ESKM is a potent therapeutic mAb against a
widely expressed oncogenic target with a restricted normal cell expression
profile, and
has shown efficacy against multiple human tumor models in mice.
[000138] Although the foregoing invention has been described in some detail
by
way of illustration and example for purposes of clarity of understanding, it
will be readily
apparent to those of ordinary skill in the art in light of the teachings of
this disclosure
that certain changes and modifications may be made thereto without departing
from the
spirit or scope of the appended claims.
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