Treatment Of Cancer With A Semaphorin-4D Antibody In Combination With An

Epigenetic Modulating Agent

DOCUMENT ID DATE PUBLISHED

US 11572408 B2 2023-02-07

INVENTOR INFORMATION
NAME CITY STATE ZIP CODE COUNTRY Evans;
Elizabeth
Bloomfield NY N/A US Smith; Ernest West Henrietta
NY N/A US Zauderer; Pittsford NY
Maurice N/A US

ASSIGNEE INFORMATION
NAME CITY STATE ZIP CODE COUNTRY TYPE CODE
Vaccinex, Inc. Rochester NY N/A US 02

APPLICATION NO DATE FILED

16/496286 2018-03-14

DOMESTIC PRIORITY (CONTINUITY DATA)

us-provisional-application US 62473731 20170320

US CLASS CURRENT:

1/1

CPC CURRENT
TYPE CPC DATE
CPCI A 61 K 39/3955 2013-01-01
CPCI A 61 P 35/00 2018-01-01
CPCI A 61 K 31/4406 2013-01-01
CPCI C 07 K 16/2803 2013-01-01
CPCI A 61 K 31/706 2013-01-01
CPCA C 07 K 2317/76 2013-01-01
CPCA A 61 K 2039/505 2013-01-01

KWIC Hits

Abstract

This disclosure provides a method for inhibiting, delaying, or reducing malignant cell growth in a
subject with cancer, comprising administering to the subject a combination therapy comprising an
effective amount of an isolated antibody or antigen-binding fragment thereof that specifically binds to
semaphorin-4D (SEMA4D) and an effective amount of an epigenetic modulating agent, e.g., a histone
deacetylase (HDAC) inhibitor (HDACi) a DNA methyltransferase (DNMT) inhibitor (DNMTi), or any
combination thereof. The disclosure further provides a pharmaceutical composition comprising the
combination therapy.

Background/Summary

CROSS-REFERENCE TO RELATED APPLICATIONS

(1) This application is a US National Stage Entry of PCT Application No. PCT/US2018/022414, filed
Mar. 14, 2018, which claims the benefit of U.S. Provisional Patent Application Ser. No. 62/473,731,
filed Mar. 20, 2017, which are each hereby incorporated by reference in their entireties.

SEQUENCE LISTING STATEMENT

(1) The content of the electronically submitted sequence listing in ASCII text file (Name:
58008_172854_Seq-List_ST25.txt; Size: 5936 bytes; Date of Creation: Feb. 28, 2018) filed with the
application is incorporated herein by reference in its entirety.

BACKGROUND

(2) Semaphorin 4D (SEMA4D), also known as CD100, is a transmembrane protein that belongs to the
semaphorin gene family. SEMA4D is expressed on the cell surface as a homodimer, but upon cell
activation SEMA4D can be released from the cell surface via proteolytic cleavage to generate
sSEMA4D, a soluble form of the protein, which is also biologically active. See Suzuki et al., Nature
Rev. Immunol. 3:159-167 (2003); Kikutani et al., Nature Immunol. 9:17-23 (2008).

(3) SEMA4D is expressed at high levels in lymphoid organs, including the spleen, thymus, and lymph
nodes, and in non-lymphoid organs, such as the brain and kidney. In lymphoid organs, SEMA4D is
abundantly expressed on resting T cells but only weakly expressed on resting B cells and antigen-
presenting cells (APCs), such as dendritic cells (DCs). Its expression, however, is upregulated in these
cells following activation by various immunological stimuli. The release of soluble SEMA4D from
immune cells is also increased by cell activation. SEMA4D has been implicated in the development of
certain cancers (Ch'ng et al., Cancer 110:164-72 (2007); Campos et al., Oncology Letters 5:1527-35
(2013); Kato et al., Cancer Sci. 102:2029-37 (2011)) and several reports suggest that one mechanism
of this influence is the role of SEMA4D in promoting tumor angiogenesis (Conrotto et al., Blood
105:4321-4329 (2005). Basile et al., J Biol. Chem. 282: 34888-34895 (2007); Sierra et. al. J. Exp.
Med. 205:1673 (2008); Zhou et al., Angiogenesis 15:391-407 (2012)). Tumor growth and metastasis
involve a complex process of cross talk amongst the tumor cells, stroma and immune infiltrate, as well
as the endothelial cells and vasculature. SEMA4D is over-expressed in a wide array of tumor types
and is also produced by inflammatory cells recruited to the tumor microenvironment. Recent work
suggests that SEMA4D plays a role in migration, survival, differentiation and organization of the
different cell types that constitute the tumor stroma (Evans et al., Cancer Immunol. Res. 3:689-701
(2015)).

(4) Cancer cells can adapt to avoid host immune recognition through modification of gene expression
via epigenetic mechanisms, without altering the sequence of the genomic DNA. Epigenetic
mechanisms largely center on alterations of genomic DNA methylation and posttranslational
modifications of histones, resulting in, e.g., alterations in chromatin structure and availability of DNA for
transcription. Through these epigenetic mechanisms cancer cells can establish altered, heritable
gene-expression profiles that promote proliferation and immune evasion. See, e.g., Maio, et al., Clin.
Cancer Res. 21:4040-4047 (2015).

(5) Cells can control the coiling and uncoiling of DNA around histones via histone acetyl transferases
(HAT), which acetylate the lysine residues in core histones leading to a less compact and more
transcriptionally active chromatin and histone deacetylases (HDAC), which remove the acetyl groups
from the lysine residues leading to the formation of a condensed and transcriptionally silenced
chromatin. Modulation of HAT/HDAC activity by tumor cells can results in extensive epigenetic
modulation of gene expression in tumor cells, allowing the cells to evade surveillance by the patient's
immune system. See, e.g., Maio, et al., Clin. Cancer Res. 21:4040-4047 (2015).
(6) Histone deacetylase inhibitors (HDACi (used herein both as the singular and the plural)) have
emerged as cancer therapeutic agents. HDACi can inhibit the proliferation of tumor cells by inducing
extensive transcriptional changes, activating and/or repressing various genes through modulating the
acetylation/deacetylation of histones and/or non-histone proteins such as transcription factors. Chueh,
A. C., et al, Antioxidants & Redox Signaling 23:66-84 (2015). HDACi can interfere with cell proliferation
and survival, e.g., with cell cycle, differentiation, and apoptosis of cancer cells. HDACi can also
enhance immunogenicity and antigen presentation by tumor cells and provides rationale for combining
HDACi with immunotherapy (Gameiro S R et al., Oncotarget 7:7390-7402 (2016)). Several
compounds are currently in early phase clinical development as potential treatments for solid and
hematological cancers both as monotherapy and in combination with cytotoxics and differentiation
agents. See, e.g., Mottamal, M., et al., Molecules 20:3898-3941 (2015).

(7) Methylation of genomic DNA is an extensively characterized epigenetic modification. Methylation of

DNA leads to transcriptional quiescence, resulting in gene silencing. Gravina G. L., et al., Mol. Cancer
9:305-320 (2010). Methylation is facilitated by DNA methyltransferases (DNMTs), which catalyze the
addition of a methyl group to the 5′ carbon of cytosine residues. Id. Hypermethylated DNA is common
in tumor and other cancer cells. A variety of DNA methyltransferase inhibitors (DNMTi (used herein
both as the singular and the plural)) are currently in use or are being tested for treatment of various
cancers. These include deoxyribonucleoside analogs such as azacytidine, and non-nucleoside
analogs such as antisense oligonucleotides and small molecule enzyme inhibitors. Id.

SUMMARY

(8) This disclosure provides a method for inhibiting, delaying, or reducing malignant cell growth in a
subject with cancer, where the method includes administering to the subject a combination therapy
including an effective amount of an isolated antibody or antigen-binding fragment thereof that
specifically binds to semaphorin-4D (SEMA4D) and an effective amount of an epigenetic modulating
agent. According to the provided method, the anti-SEMA4D antibody or fragment thereof can inhibit
SEMA4D interaction with its receptor, e.g., Plexin-B1, Plexin-B2, or CD72. For example, according to
the provided method the anti-SEMA4D antibody or fragment thereof can inhibit SEMA4D-mediated
Plexin-B1 signal transduction.

(9) In certain aspects, the anti-SEMA4D antibody or fragment thereof includes a variable heavy chain
(VH) that includes VH CDRs 1-3 having the amino acid sequences SEQ ID NOS: 2, 3, and 4,
respectively, and a variable light chain (VL) that includes VL CDRs 1-3 having the amino acid
sequences SEQ ID NOS: 6, 7, and 8, respectively. In certain aspects the amino acid sequences of the
VH and VL include, respectively, SEQ ID NO: 1 and SEQ ID NO: 5, or SEQ ID NO: 9 and SEQ ID NO:
10.

(10) In certain aspects, the epigenetic modulating agent can include a histone deacetylase (HDAC)
inhibitor (HDACi), a DNA methyltransferase (DNMT) inhibitor (DNMTi), or any combination thereof.

(11) In certain aspects, the epigenetic modulating agent can include a histone deacetylase (HDAC)
inhibitor (HDACi). In certain aspects the HDAC can be a Class I HDAC, a Class IIA HDAC, a Class IIB
HDAC, a Class IV HDAC, or any combination thereof, or the HDAC can include a zinc-containing
catalytic domain. In certain aspects, the HDACi can bind to the zinc-containing catalytic domain of the
HDAC. In certain aspects, the HDACi can include a chemical moiety selected from the group
consisting of a hydroxamic acid or a salt thereof, a cyclic tetrapeptide, a depsipeptide, a benzamide,
an electrophilic ketone, an aliphatic acid or a salt thereof, or any combination thereof. For example, in
certain aspects, the HDACi can be Vorinostat, Romidepsin, Chidamide, Panobinostat, Belinostat,
Valproic acid or a salt thereof, Mocetinostat, Abexinostat, Entinostat, Pracinostat, Resminostat,
Givinostat, Quisinostat, Kevetrin, CUDC-101, AR-42, Tefinostat (CHR-2845), CHR-3996, 4SC-202,
CG200745, ACY-1215, ACY-241, any combination thereof, or any salt, crystal, amorphous structure,
hydrate, derivative, metabolite, isomer, or prodrug thereof. In certain aspects, the HDACi is Entinostat
(Pyridin-3-ylmethyl N-[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate).
(12) In certain aspects, the epigenetic modulating agent can include a DNA methyltransferase (DNMT)
inhibitor (DNMTi). In certain aspects the DNMT can be DNMT1, DNMT-3a, DNMT-3b, or any
combination thereof. In certain aspects, the DNMTi can be a nucleoside analog, an antisense
oligonucleotide, a small molecule enzyme inhibitor, or any combination thereof. For example, in certain
aspects the DNMTi can be azacytidine, decitabine, zebularine, SGI-110, epigallocatechin gallate,
MG98, RG108, procainamide, hydralazine, any combination thereof, or any salt, crystal, amorphous
structure, hydrate, derivative, metabolite, isomer, or prodrug thereof. In certain aspects the DNMTi is
azacytidine.

(13) In a particular aspect of the provided method, the isolated antibody or antigen-binding fragment
thereof that specifically binds to semaphorin-4D (SEMA4D) comprises a VH comprising the amino acid
sequence SEQ ID NO: 1 and a VL comprising the amino acid sequence SEQ ID NO: 5, and the
epigenetic modulating agent comprises the HDACi Entinostat. In another particular aspect of the
provided method, the isolated antibody or antigen-binding fragment thereof that specifically binds to
semaphorin-4D (SEMA4D) comprises a VH comprising the amino acid sequence SEQ ID NO: 1 and a
VL comprising the amino acid sequence SEQ ID NO: 5, and the epigenetic modulating agent
comprises the DNMTi azacytidine.

(14) According to the provided method, the antibody or antigen-binding fragment thereof and the
epigenetic modulating agent can be administered separately, or they can be administered
simultaneously.

(15) In certain aspects of the provided method, administration of the combination therapy can result in
enhanced therapeutic efficacy relative to administration of the antibody or fragment thereof or the
epigenetic modulating agent alone. For example, in certain aspects the enhanced therapeutic efficacy
is greater than an additive effect, e.g., a synergistic effect.

(16) In certain aspects of the provided method, the cancer to be treated can be a solid tumor, a
hematological malignancy, any metastasis thereof, or any combination thereof. In certain aspects, the
solid tumor can be, e.g., a sarcoma, a carcinoma, a melanoma, any metastases thereof, or any
combination thereof. More specifically, the solid tumor can be, e.g., squamous cell carcinoma,
adenocarcinoma, basal cell carcinoma, renal cell carcinoma, ductal carcinoma of the breast, soft
tissue sarcoma, osteosarcoma, melanoma, small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastrointestinal
cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer,
ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer,
colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma,
kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, head and neck cancer, any
metastases thereof, or any combination thereof.

(17) In certain aspects of the provided method, the cancer to be treated can be a hematologic
malignancy or metastasis thereof. In certain aspects, the hematologic malignancy can be leukemia,
lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia,
chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma,
multiple myeloma, any metastases thereof, or any combination thereof.

(18) In certain aspects, the method provided herein can further include administration of an additional
cancer therapy, e.g., surgery, chemotherapy, radiation therapy, a cancer vaccine, administration of an
immunostimulatory agent, adoptive T cell or antibody therapy, administration of an immune checkpoint
blockade inhibitor, administration of a regulatory T cell (Treg) modulator, and a combination thereof.

(19) This disclosure further provides a pharmaceutical composition that includes an effective amount
of an isolated antibody or antigen-binding fragment thereof that specifically binds to semaphorin-4D
(SEMA4D) and an effective amount of an epigenetic modulating agent. In certain aspects, the
pharmaceutical composition can further include a carrier, an excipient, or any combination thereof.
(20) In certain aspects, the antibody or fragment thereof of the provided composition can include a
variable heavy chain (VH) comprising VH CDRs 1-3 comprising SEQ ID NOS: 2, 3, and 4,
respectively, and a variable light chain (VL) comprising VL CDRs 1-3 comprising SEQ ID NOS: 6, 7,
and 8, respectively. In certain aspects, the antibody or fragment thereof of the provided composition
can include a VH and VL that include, respectively, SEQ ID NO: 1 and SEQ ID NO: 5, or SEQ ID NO:
9 and SEQ ID NO: 10.

(21) In certain aspects, the epigenetic modulating agent included in the provided pharmaceutical
composition can include a histone deacetylase (HDAC) inhibitor (HDACi), a DNA methyltransferase
(DNMT) inhibitor (DNMTi), or any combination thereof.

(22) Where the composition includes a HDACi, the HDACi can be, e.g., Vorinostat, Romidepsin,
Chidamide, Panobinostat, Belinostat, Valproic acid or a salt thereof, Mocetinostat, Abexinostat,
Entinostat, Pracinostat, Resminostat, Givinostat, Quisinostat, Kevetrin, CUDC-101, AR-42, Tefinostat
(CHR-2845), CHR-3996, 4SC-202, CG200745, ACY-1215, ACY-241, any combination thereof, or any
salt, crystal, amorphous structure, hydrate, derivative, metabolite, isomer, or prodrug thereof. In certain
aspects the HDACi can be Entinostat (Pyridin-3-ylmethyl N-[[4-[(2-
aminophenyl)carbamoyl]phenyl]methyl]carbamate).

(23) Where the composition includes a DNMTi, the DNMTi can be, e.g., azacytidine, decitabine,
zebularine, SGI-110, epigallocatechin gallate, MG98, RG108, procainamide, hydralazine, any
combination thereof, or any salt, crystal, amorphous structure, hydrate, derivative, metabolite, isomer,
or prodrug thereof. In certain aspects the DNMTi can be azacytidine.

(24) In a particular aspect, the disclosure provides a pharmaceutical composition that includes an
effective amount of an isolated antibody or antigen-binding fragment thereof that specifically binds to
semaphorin-4D (SEMA4D), where the antibody or fragment thereof includes the VH amino acid
sequence SEQ ID NO: 1 and the VL amino acid sequence SEQ ID NO: 5; and an effective amount of
the HDACi Entinostat. in another particular aspect, the disclosure provides a pharmaceutical
composition that includes an effective amount of an isolated antibody or antigen-binding fragment
thereof that specifically binds to semaphorin-4D (SEMA4D), where the antibody or fragment thereof
includes the VH amino acid sequence SEQ ID NO: 1 and the VL amino acid sequence SEQ ID NO: 5;
and an effective amount of the DNMTi azacytidine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

(1) FIG. 1 shows the experimental design for combination therapy experiments in Examples 1 and 2.

(2) FIG. 2A shows the mean tumor volume over time for the various treatments in Example 1.

(3) FIG. 2B shows percent survival over time for the various treatments in Example 1. Statistical
significance was determined using Mantel Cox Log Rank test. Prism reports results as non-significant
(ns) at P>0.05, significant (symbolized by “*”) at 0.01<P≤0.05, very significant (“**”) at 0.001<P≤0.01,
and extremely significant (“***”) at P≤0.001 or (“****”) at P≤0.0001.

(4) FIG. 2C is a bar graph showing the percent of complete tumor regression (tumor volume <50
mm.sup.3) for the various treatments in Example 1. Statistical significance was determined using
Fisher's exact test, Prism reports results as non-significant (ns) at P>0.05, significant (symbolized by
“*”) at 0.01<P≤0.05, very significant (“**”) at 0.001<P≤0.01, and extremely significant (“***”) at P≤0.001
or (“****”) at P≤0.0001.

(5) FIG. 2D shows the same data as FIG. 2C, stratified as to whether or not the tumor exceeded at
least 100 mm.sup.3 before regressing.

(6) FIG. 3A shows mean tumor volume over time for the various treatments in Example 2. Statistical
significance was determined using 2-way ANOVA. Prism reports results as non-significant (ns) at
P>0.05, significant (symbolized by “*”) at 0.01<P≤0.05, very significant (“**”) at 0.001<P≤0.01

(7) FIG. 3B shows tumor volume for individual control animals over time in Example 2.

(8) FIG. 3C shows tumor volume for individual animals treated with anti-SEMA4D antibody over time in
Example 2.

(9) FIG. 3D shows tumor volume for individual animals treated with Entinostat (ENT) and control
antibody over time in Example 2.

(10) FIG. 3E shows tumor volume for individual animals treated with ENT and anti-SEMA4D antibody
over time in Example 2.

(11) FIG. 3F shows percent survival over time for the various treatments in Example 2. Statistical
significance was determined using the Mantel Cox Log Rank test. Prism reports results as significant
(symbolized by “*”) at 0.01<P≤0.05 and very significant (“**”) at 0.001<P≤0.01.

(12) FIG. 4 shows the experimental design for combination therapy experiment in Example 3.

(13) FIG. 5A shows the mean tumor volume over time for the various treatments in Example 3.
Statistical significance was determined using 2-way ANOVA. **P≤0.01

(14) FIG. 5B shows percent survival over time for the various treatments in Example 3. Statistical
significance was determined using Mantel Cox Log Rank test. Prism reports results as non-significant
at P>0.05, or very significant (“**”) at P≤0.01.

(15) FIG. 5C is a bar graph showing the percent of complete tumor regression (tumor volume <50
mm.sup.3) for the various treatments in Example 3. Statistical significance was determined using
Fisher's exact test, Prism reports results as significant (symbolized by “*”) at P≤0.05, or very significant
(“**”) at P≤0.01.

DETAILED DESCRIPTION

Definitions

(16) It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a
binding molecule,” is understood to represent one or more binding molecules. As such, the terms “a”
(or “an”), “one or more,” and “at least one” can be used interchangeably herein.

(17) Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two
specified features or components with or without the other. Thus, the term and/or” as used in a phrase
such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass
each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B
and C; A (alone); B (alone); and C (alone).

(18) Unless defined otherwise, technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which this disclosure is related. For
example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002,
CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the
Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press,
provide one of skill with a general dictionary of many of the terms used in this disclosure.

(19) Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted
form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated,
amino acid sequences are written left to right in amino to carboxy orientation. The headings provided
herein are not limitations of the various aspects or aspects of the disclosure, which can be had by
reference to the specification as a whole. Accordingly, the terms defined immediately below are more
fully defined by reference to the specification in its entirety.

(20) Wherever embodiments are described with the language “comprising,” otherwise analogous
embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.

(21) Amino acids are referred to herein by their commonly known three letter symbols or by the one-
letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, are referred to by their commonly accepted single-letter codes.

(22) As used herein, the terms “cancer” and “cancerous” refer to or describe the physiological
condition in mammals in which a population of cells are characterized by unregulated cell growth.
Cancers can be categorized, e.g., as solid tumors or malignancies, or hematological cancers or
malignancies. Both types can migrate to remote sites as metastases. A solid tumor can be
categorized, e.g., as a sarcoma, a carcinoma, a melanoma, or a metastasis thereof.

(23) The terms “proliferative disorder” and “proliferative disease” refer to disorders associated with
abnormal cell proliferation such as cancer.

(24) “Tumor” and “neoplasm” as used herein refer to any mass of tissue that result from excessive cell
growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-
cancerous lesions. In certain embodiments, tumors described herein express a SEMA4D receptor,
e.g., Plexin-B1, Plexin-B2, and/or CD72, and/or can express SEMA4D.

(25) The terms “metastasis,” “metastases,” “metastatic,” and other grammatical equivalents as used
herein refer to cancer cells which spread or transfer from the site of origin (e.g., a primary tumor) to
other regions of the body with the development of a similar cancerous lesion at the new location. A
“metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and
migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body
structures. The terms also refer to the process of metastasis, which includes, but is not limited to
detachment of cancer cells from a primary tumor, intravasation of the tumor cells to circulation, their
survival and migration to a distant site, attachment and extravasation into a new site from the
circulation, and microcolonization at the distant site, and tumor growth and development at the distant
site.

(26) Examples of such solid tumors can include, e.g., squamous cell carcinoma, adenocarcinoma,
basal cell carcinoma, renal cell carcinoma, ductal carcinoma of the breast, soft tissue sarcoma,
osteosarcoma, melanoma, small-cell lung cancer, non-small cell lung cancer (NSCLC),
adenocarcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastrointestinal
cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer,
ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer,
colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma,
kidney cancer, prostate cancer, vulval cancer, thyroid cancer, head and neck cancer, any metastases
thereof, or any combination thereof.

(27) Examples of hematologic cancers or malignancies include without limitation leukemia, lymphoma,
myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic
lymphocytic leukemia, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple
myeloma, any metastases thereof, or any combination thereof.

(28) In certain embodiments, cancers that are amenable to treatment via the methods provided herein
include, but are not limited to sarcomas, breast carcinomas, ovarian cancer, cervical cancer, head and
neck cancer, NSCLC, esophageal cancer, gastric cancer, kidney cancer, liver cancer, bladder cancer,
colorectal cancer, and pancreatic cancer. In certain embodiments cancers or tumor cells that are
amenable to treatment via the methods provided herein express Plexin-B1, Plexin-B2, or CD72
receptors for SEMA4D.
(29) As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well
as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked
by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains
of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides,
dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a
chain or chains of two or more amino acids are included within the definition of “polypeptide,” and the
term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term
“polypeptide” is also intended to refer to the products of post-expression modifications of the
polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and
derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-
naturally occurring amino acids. A polypeptide can be derived from a biological source or produced by
recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It
can be generated in any manner, including by chemical synthesis.

(30) A polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20
or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more,
or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although
they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure
are referred to as folded, and polypeptides which do not possess a defined three-dimensional
structure, but rather can adopt a large number of different conformations, and are referred to as
unfolded. As used herein, the term glycoprotein refers to aprotein coupled to at least one carbohydrate
moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of
an amino acid, e.g., a serine or an asparagine.

(31) By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide

that is not in its natural milieu. No particular level of purification is required. For example, an isolated
polypeptide can be removed from its native or natural environment. Recombinantly produced
polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are
native or recombinant polypeptides which have been separated, fractionated, or partially or
substantially purified by any suitable technique.

(32) As used herein, the term “a non-naturally occurring polypeptide” or any grammatical variants
thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the
polypeptide that are, or might be, determined or interpreted by a judge or an administrative or judicial
body, to be “naturally-occurring.”

(33) Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the
foregoing polypeptides, and any combination thereof. The terms “fragment,” “variant,” “derivative” and
“analog” as disclosed herein include any polypeptides which retain at least some of the properties of
the corresponding native antibody or polypeptide, for example, specifically binding to an antigen.
Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments,
in addition to specific antibody fragments discussed elsewhere herein. Variants of, e.g., a polypeptide
include fragments as described above, and also polypeptides with altered amino acid sequences due
to amino acid substitutions, deletions, or insertions. In certain aspects, variants can be non-naturally
occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques.
Variant polypeptides can comprise conservative or non-conservative amino acid substitutions,
deletions or additions. Derivatives are polypeptides that have been altered so as to exhibit additional
features not found on the original polypeptide. Examples include fusion proteins. Variant polypeptides
can also be referred to herein as “polypeptide analogs.” As used herein a “derivative” of a polypeptide
can also refer to a subject polypeptide having one or more amino acids chemically derivatized by
reaction of a functional side group. Also included as “derivatives” are those peptides that contain one
or more derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be
substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be
substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for
lysine.
(34) A “conservative amino acid substitution” is one in which one amino acid is replaced with another
amino acid having a similar side chain. Families of amino acids having similar side chains have been
defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For
example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain
embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the
present disclosure do not abrogate the binding of the polypeptide or antibody containing the amino
acid sequence, to the antigen to which the binding molecule binds. Methods of identifying nucleotide
and amino acid conservative substitutions which do not eliminate antigen-binding are well-known in
the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al., Protein Eng.
12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

(35) The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural
nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA
(mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can comprise a conventional
phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide
nucleic acids (PNA)). The terms “nucleic acid” or “nucleic acid sequence” refer to any one or more
nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

(36) By an “isolated” nucleic acid or polynucleotide is intended any form of the nucleic acid or
polynucleotide that is separated from its native environment. For example, gel-purified polynucleotide,
or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered to
be “isolated.” Also, a polynucleotide segment, e.g., a PCR product, which has been engineered to
have restriction sites for cloning is considered to be “isolated.” Further examples of an isolated
polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified
(partially or substantially) polynucleotides in a non-native solution such as a buffer or saline. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not
one that would be found in nature. Isolated polynucleotides or nucleic acids further include such
molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a
regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

(37) As used herein, the term “a non-naturally occurring polynucleotide” or any grammatical variants
thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the nucleic
acid or polynucleotide that are, or might be, determined or interpreted by a judge, or an administrative
or judicial body, to be “naturally-occurring.”

(38) As used herein, a “coding region” is a portion of nucleic acid which consists of codons translated
into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it
can be considered to be part of a coding region, but any flanking sequences, for example promoters,
ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding
region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a
single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors.
Furthermore, any vector can contain a single coding region, or can comprise two or more coding
regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region
and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid
can include heterologous coding regions, either fused or unfused to another coding region.
Heterologous coding regions include without limitation, those encoding specialized elements or motifs,
such as a secretory signal peptide or a heterologous functional domain.

(39) In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a
polynucleotide comprising a nucleic acid which encodes a polypeptide normally can include a
promoter and/or other transcription or translation control elements operably associated with one or
more coding regions. An operable association is when a coding region for a gene product, e.g., a
polypeptide, is associated with one or more regulatory sequences in such a way as to place
expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA
fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably
associated” if induction of promoter function results in the transcription of mRNA encoding the desired
gene product and if the nature of the linkage between the two DNA fragments does not interfere with
the ability of the expression regulatory sequences to direct the expression of the gene product or
interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be
operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of
effecting transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs
substantial transcription of the DNA in predetermined cells. Other transcription control elements,
besides a promoter, for example enhancers, operators, repressors, and transcription termination
signals, can be operably associated with the polynucleotide to direct cell-specific transcription.

(40) A variety of transcription control regions are known to those skilled in the art. These include,
without limitation, transcription control regions which function in vertebrate cells, such as, but not
limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in
conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous
sarcoma virus). Other transcription control regions include those derived from vertebrate genes such
as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences
capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control
regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters
(e.g., promoters inducible by interferons or interleukins).

(41) Similarly, a variety of translation control elements are known to those of ordinary skill in the art.
These include, but are not limited to ribosome binding sites, translation initiation and termination
codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or
IRES, also referred to as a CITE sequence).

(42) In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA
(mRNA), transfer RNA, or ribosomal RNA.

(43) Polynucleotide and nucleic acid coding regions can be associated with additional coding regions
which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a
polynucleotide as disclosed herein. According to the signal hypothesis, proteins secreted by
mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature
protein once export of the growing protein chain across the rough endoplasmic reticulum has been
initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells can
have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete
or “full length” polypeptide to produce a secreted or “mature” form of the polypeptide. In certain
embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal
peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion
of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal
peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence
can be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-
glucuronidase.

(44) Disclosed herein are certain binding molecules, or antigen-binding fragments, variants, or
derivatives thereof. Unless specifically referring to full-sized antibodies, the term “binding molecule”
encompasses full-sized antibodies as well as antigen-binding subunits, fragments, variants, analogs,
or derivatives of such antibodies, e.g., engineered antibody molecules or fragments that bind antigen
in a manner similar to antibody molecules, but which use a different scaffold.

(45) As used herein, the term “binding molecule” refers in its broadest sense to a molecule that
specifically binds to a receptor, e.g., an epitope or an antigenic determinant. As described further
herein, a binding molecule can comprise one of more “antigen binding domains” described herein. A
non-limiting example of a binding molecule is an antibody or fragment thereof that retains antigen-
specific binding.
(46) As used herein, the terms “binding domain” or “antigen binding domain” refer to a region of a
binding molecule that is necessary and sufficient to specifically bind to an epitope. For example, an
“Fv,” e.g., a variable heavy chain and variable light chain of an antibody, either as two separate
polypeptide subunits or as a single chain, is considered to be a “binding domain.” Other binding
domains include, without limitation, the variable heavy chain (VHH) of an antibody derived from a
camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a
fibronectin scaffold. A “binding molecule” as described herein can include one, two, three, four, five,
six, seven, eight, nine, ten, eleven, twelve or more “antigen binding domains.”

(47) The terms “antibody” and “immunoglobulin” can be used interchangeably herein. An antibody (or
a fragment, variant, or derivative thereof as disclosed herein) includes at least the variable domain of a
heavy chain (for camelid species) or at least the variable domains of a heavy chain and a light chain.
Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g.,
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
Unless otherwise stated, the term “antibody” encompasses anything ranging from a small antigen-
binding fragment of an antibody to a full sized antibody, e.g., an IgG antibody that includes two
complete heavy chains and two complete light chains, an IgA antibody that includes four complete
heavy chains and four complete light chains and optionally includes a J chain and/or a secretory
component, or an IgM antibody that includes ten or twelve complete heavy chains and ten or twelve
complete light chains and optionally includes a J chain.

(48) As will be discussed in more detail below, the term “immunoglobulin” comprises various broad
classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate
that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with some
subclasses among them (e.g., γ1-γ4 or α1-α2). It is the nature of this chain that determines the “class”
of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes)
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, IgA.sub.2, etc. are well characterized and
are known to confer functional specialization. Modified versions of each of these classes and isotypes
are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within
the scope of this disclosure.

(49) Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be bound
with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded
to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent
disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by
hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid
sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the
bottom of each chain. The basic structure of certain antibodies, e.g., IgG antibodies, includes two
heavy chain subunits and two light chain subunits covalently connected via disulfide bonds to form a
“Y” structure, also referred to herein as an “H2L2” structure.

(50) Both the light and heavy chains are divided into regions of structural and functional homology.
The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the
variable domains of both the variable light (VL) and variable heavy (VH) chain portions determine
antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the
heavy chain (CH1, CH2 or CH3) confer biological properties such as secretion, transplacental mobility,
Fc receptor binding, complement binding, and the like. By convention the numbering of the constant
region domains increases as they become more distal from the antigen binding site or amino-terminus
of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant
region; the CH3 (or CH4 in the case of IgM) and CL domains actually comprise the carboxy-terminus
of the heavy and light chain, respectively.

(51) As indicated above, a variable region (i.e., the “binding domain”) allows the binding molecule to
selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH
domain, or subset of the complementarity determining regions (CDRs), of a binding molecule, e.g., an
antibody combine to form the variable region that defines a three dimensional antigen binding site.
More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains.
Certain antibodies form larger structures. For example, IgA can form a molecule that includes two
H2L2 units, a J chain, and a secretory component, all covalently connected via disulfide bonds, and
IgM can form a pentameric or hexameric molecule that includes five or six H2L2 units and optionally a
J chain covalently connected via disulfide bonds.

(52) The six “complementarity determining regions” or “CDRs” present in an antibody antigen-binding
domain are short, non-contiguous sequences of amino acids that are specifically positioned to form
the binding domain as the antibody assumes its three dimensional configuration in an aqueous
environment. The remainder of the amino acids in the binding domain, referred to as “framework”
regions, show less inter-molecular variability. The framework regions largely adopt a n-sheet
conformation and the CDRs form loops which connect, and in some cases form part of, the n-sheet
structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in
correct orientation by inter-chain, non-covalent interactions. The binding domain formed by the
positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This
complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The
amino acids that make up the CDRs and the framework regions, respectively, can be readily identified
for any given heavy or light chain variable region by one of ordinary skill in the art, since they have
been defined in various different ways (see, “Sequences of Proteins of Immunological Interest,” Kabat,
E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol.,
196:901-917 (1987), which are incorporated herein by reference in their entireties).

(53) In the case where there are two or more definitions of a term which is used and/or accepted within
the art, the definition of the term as used herein is intended to include all such meanings unless
explicitly stated to the contrary. A specific example is the use of the term “complementarity determining
region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable
region of both heavy and light chain polypeptides. These particular regions have been described, for
example, by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of
Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are
incorporated herein by reference. The Kabat and Chothia definitions include overlapping or subsets of
amino acids when compared against each other. Nevertheless, application of either definition (or other
definitions known to those of ordinary skill in the art) to refer to a CDR of an antibody or variant thereof
is intended to be within the scope of the term as defined and used herein, unless otherwise indicated.
The appropriate amino acids which encompass the CDRs as defined by each of the above cited
references are set forth below in Table 1 as a comparison. The exact amino acid numbers which
encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled
in the art can routinely determine which amino acids comprise a particular CDR given the variable
region amino acid sequence of the antibody.

(54) TABLE-US-00001 TABLE 1 CDR Definitions.sup.1 Kabat Chothia VH CDR1 31-35 26-32 VH
CDR2 50-65 52-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL
CDR3 89-97 91-96 .sup.1Numbering of all CDR definitions in Table 1 is according to the numbering
conventions set forth by Kabat et al. (see below).

(55) Kabat et al. also defined a numbering system for variable domain sequences that is applicable to
any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat
numbering” to any variable domain sequence, without reliance on any experimental data beyond the
sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat
et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest”
(1983). Unless use of the Kabat numbering system is explicitly noted, however, consecutive
numbering is used for all amino acid sequences in this disclosure.

(56) Binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof
include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies,
single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′).sub.2, Fd, Fvs, single-
chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a
VL or VH domain, fragments produced by a Fab expression library. ScFv molecules are known in the
art and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules
encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g.,
IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

(57) By “specifically binds,” it is generally meant that a binding molecule, e.g., an antibody or fragment,
variant, or derivative thereof binds to an epitope via its antigen binding domain, and that the binding
entails some complementarity between the antigen binding domain and the epitope. According to this
definition, a binding molecule is said to “specifically bind” to an epitope when it binds to that epitope,
via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The
term “specificity” is used herein to qualify the relative affinity by which a certain binding molecule binds
to a certain epitope. For example, binding molecule “A” can be deemed to have a higher specificity for
a given epitope than binding molecule “B,” or binding molecule “A” can be said to bind to epitope “C”
with a higher specificity than it has for related epitope “D.”

(58) A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof disclosed herein
can be said to bind a target antigen with an off rate (k(off)) of less than or equal to 5×10.sup.−2
sec.sup.−1, 10.sup.−3 sec.sup.−1, 5×10.sup.−3 sec.sup.−1, 10.sup.−3 sec.sup.−1, 5×10.sup.−4
sec.sup.−1, 10.sup.−4 sec.sup.−1, 5×10.sup.−5 sec.sup.−1, or 10.sup.−5 sec.sup.−1 5×10.sup.−6
sec.sup.−1, 10.sup.−6 sec.sup.−1, 5×10.sup.−7 sec.sup.−1, or 10.sup.−7 sec.sup.−1.

(59) A binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative disclosed
herein can be said to bind a target antigen with an on rate (k(on)) of greater than or equal to 10.sup.3
M.sup.−1 sec.sup.−1, 5×10.sup.3 M.sup.−1 sec.sup.−1, 10.sup.4 M.sup.−1 sec.sup.−1, 5×10.sup.4
M.sup.−1 sec.sup.−1, 10.sup.5 M.sup.−1 sec.sup.−1, 5×10.sup.5 M.sup.−1 sec.sup.−1, 10.sup.6
M.sup.−1 sec.sup.−1, or 5×10.sup.6 M.sup.−1 sec.sup.−1, or 10.sup.7 M.sup.−1 sec.sup.−1.

(60) A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof is said to
competitively inhibit binding of a reference antibody or antigen binding fragment to a given epitope if it
preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference
antibody or antigen binding fragment to the epitope. Competitive inhibition can be determined by any
method known in the art, for example, competition ELISA assays. A binding molecule can be said to
competitively inhibit binding of the reference antibody or antigen binding fragment to a given epitope
by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

(61) As used herein, the term “affinity” refers to a measure of the strength of the binding of an
individual epitope with one or more binding domains, e.g., of an immunoglobulin molecule. See, e.g.,
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)
at pages 27-28. As used herein, the term “avidity” refers to the overall stability of the complex between
a population of binding domains and an antigen. See, e.g., Harlow at pages 29-34. Avidity is related to
both the affinity of individual binding domains in the population with specific epitopes, and also the
valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent
monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer,
would be one of high avidity. An interaction between a bivalent monoclonal antibody with a receptor
present at a high density on a cell surface would also be of high avidity.

(62) Binding molecules or antigen-binding fragments, variants or derivatives thereof as disclosed

herein can also be described or specified in terms of their cross-reactivity. As used herein, the term
“cross-reactivity” refers to the ability of a binding molecule, e.g., an antibody or fragment, variant, or
derivative thereof, specific for one antigen, to react with a second antigen; a measure of relatedness
between two different antigenic substances. Thus, a binding molecule is cross reactive if it binds to an
epitope other than the one that induced its formation. The cross reactive epitope generally contains
many of the same complementary structural features as the inducing epitope, and in some cases, can
actually fit better than the original.

(63) A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof can also be
described or specified in terms of their binding affinity to an antigen. For example, a binding molecule
can bind to an antigen with a dissociation constant or K.sub.D no greater than 5×10.sup.−2 M, 10.sup.
−2 M, 5×10.sup.−3 M, 10.sup.−3 M, 5×10.sup.−4M, 10.sup.−4M, 5×10.sup.−5 M, 10.sup.−5 M,
5×10.sup.−6 M, 10.sup.−6 M, 5×10.sup.−7 M, 10.sup.−7 M, 5×10.sup.−8M, 10.sup.−8 M, 5×10.sup.
−9M, 10.sup.−9M, 5×10.sup.−10 M, 10.sup.−10 M, 5×10.sup.−11M, 10.sup.−11 M, 5×10.sup.−12M,
10.sup.−12M, 5×10.sup.−13M, 10.sup.−13M, 5×10.sup.−14M, 10.sup.−14M, 5×10.sup.−15M, or
10.sup.−15M.

(64) Antibody fragments including single-chain antibodies or other binding domains can exist alone or
in combination with one or more of the following: hinge region, CH1, CH2, CH3, or CH4 domains, J
chain, or secretory component. Also included are antigen-binding fragments that can include any
combination of variable region(s) with one or more of a hinge region, CH1, CH2, CH3, or CH4
domains, a J chain, or a secretory component. Binding molecules, e.g., antibodies, or antigen-binding
fragments thereof can be from any animal origin including birds and mammals. The antibodies can be
human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another
embodiment, the variable region can be condricthoid in origin (e.g., from sharks). As used herein,
“human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin
and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for
one or more human immunoglobulins and can in some instances express endogenous
immunoglobulins and some not, as described infra and, for example in, U.S. Pat. No. 5,939,598 by
Kucherlapati et al.

(65) As used herein, the term “heavy chain subunit” includes amino acid sequences derived from an
immunoglobulin heavy chain, a binding molecule, e.g., an antibody comprising a heavy chain subunit
includes at least one of: a VH domain, a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge
region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant or fragment thereof. For
example, a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof can include,
in addition to a VH domain, a CH1 domain; CH1 domain, a hinge, and a CH2 domain; a CH1 domain
and a CH3 domain; a CH1 domain, a hinge, and a CH3 domain; or a CH1 domain, a hinge domain, a
CH2 domain, and a CH3 domain. In certain aspects a binding molecule, e.g., an antibody or fragment,
variant, or derivative thereof can include, in addition to a VH domain, a CH3 domain and a CH4
domain; or a CH3 domain, a CH4 domain, and a J chain. Further, a binding molecule for use in the
disclosure can lack certain constant region portions, e.g., all or part of a CH2 domain. It will be
understood by one of ordinary skill in the art that these domains (e.g., the heavy chain subunit) can be
modified such that they vary in amino acid sequence from the original immunoglobulin molecule.

(66) The heavy chain subunits of a binding molecule, e.g., an antibody or fragment thereof, can
include domains derived from different immunoglobulin molecules. For example, a heavy chain subunit
of a polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived
from an IgG3 molecule. In another example, a heavy chain subunit can include a hinge region derived,
in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain
subunit can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an
IgG4 molecule.

(67) As used herein, the term “light chain subunit” includes amino acid sequences derived from an
immunoglobulin light chain. The light chain subunit includes at least one of a VL or CL (e.g., Cκ or Cλ)
domain.

(68) Binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof
can be described or specified in terms of the epitope(s) or portion(s) of an antigen that they recognize
or specifically bind. The portion of a target antigen that specifically interacts with the antigen binding
domain of an antibody is an “epitope,” or an “antigenic determinant.” A target antigen can comprise a
single epitope or at least two epitopes, and can include any number of epitopes, depending on the
size, conformation, and type of antigen.

(69) As previously indicated, the subunit structures and three dimensional configuration of the constant
regions of the various immunoglobulin classes are well known. As used herein, the term “VH domain”
includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1
domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy
chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of a
typical immunoglobulin heavy chain molecule.

(70) As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that
extends, e.g., from about amino acid 244 to amino acid 360 of an IgG antibody using conventional
numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340),
EU numbering system; see Kabat E A et al. op. cit. The CH3 domain extends from the CH2 domain to
the C-terminal of the IgG molecule and comprises approximately 108 amino acids. Certain
immunoglobulin classes, e.g., IgM, further include a CH4 region.

(71) As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins
the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acids and is
flexible, thus allowing the two N-terminal antigen binding regions to move independently.

(72) As used herein the term “disulfide bond” includes the covalent bond formed between two sulfur
atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a
second thiol group. In certain IgG molecules, the CH1 and CL regions are linked by a disulfide bond
and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242
using the Kabat numbering system (position 226 or 229, EU numbering system).

(73) As used herein, the term “chimeric antibody” refers to an antibody in which the immunoreactive
region or site is obtained or derived from a first species and the constant region (which can be intact,
partial or modified) is obtained from a second species. In some embodiments the target binding region
or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.

(74) The terms “multispecific antibody, or “bispecific antibody” refer to an antibody that has binding
domains for two or more different epitopes within a single antibody molecule. Other binding molecules
in addition to the canonical antibody structure can be constructed with two binding specificities.
Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential. Triomas
and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific
antibodies can also be constructed by recombinant means. (Strohlein and Heiss, Future Oncol.
6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). A bispecific antibody can also be a
diabody.

(75) As used herein, the term “engineered antibody” refers to an antibody in which the variable domain
in either the heavy and light chain or both is altered by at least partial replacement of one or more
amino acids in either the CDR or framework regions. In certain aspects entire CDRs from an antibody
of known specificity can be grafted into the framework regions of a heterologous antibody. Although
alternate CDRs can be derived from an antibody of the same class or even subclass as the antibody
from which the framework regions are derived, CDRs can also be derived from an antibody of different
class, e.g., from an antibody from a different species. An engineered antibody in which one or more
“donor” CDRs from a non-human antibody of known specificity are grafted into a human heavy or light
chain framework region is referred to herein as a “humanized antibody.” In certain aspects not all of
the CDRs are replaced with the complete CDRs from the donor variable region and yet the antigen
binding capacity of the donor can still be transferred to the recipient variable domains. Given the
explanations set forth in, e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will
be well within the competence of those skilled in the art, either by carrying out routine experimentation
or by trial and error testing to obtain a functional engineered or humanized antibody.

(76) As used herein the term “engineered” includes manipulation of nucleic acid or polypeptide
molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by
enzymatic or chemical coupling of peptides or some combination of these techniques).

(77) As used herein, the terms “linked,” “fused” or “fusion” or other grammatical equivalents can be
used interchangeably. These terms refer to the joining together of two more elements or components,
by whatever means including chemical conjugation or recombinant means. An “in-frame fusion” refers
to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer
ORF, in a manner that maintains the translational reading frame of the original ORFs. Thus, a
recombinant fusion protein is a single protein containing two or more segments that correspond to
polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.)
Although the reading frame is thus made continuous throughout the fused segments, the segments
can be physically or spatially separated by, for example, in-frame linker sequence. For example,
polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but
be separated by a polynucleotide encoding at least one immunoglobulin framework region or
additional CDR regions, as long as the “fused” CDRs are co-translated as part of a continuous
polypeptide.

(78) In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a
polypeptide in an amino to carboxyl terminal direction in which amino acids that neighbor each other in
the sequence are contiguous in the primary structure of the polypeptide. A portion of a polypeptide that
is “amino-terminal” or “N-terminal” to another portion of a polypeptide is that portion that comes earlier
in the sequential polypeptide chain. Similarly a portion of a polypeptide that is “carboxy-terminal” or “C-
terminal” to another portion of a polypeptide is that portion that comes later in the sequential
polypeptide chain. For example in a typical antibody, the variable domain is “N-terminal” to the
constant region, and the constant region is “C-terminal” to the variable domain.

(79) The term “expression” as used herein refers to a process by which a gene produces a
biochemical, for example, a polypeptide. The process includes any manifestation of the functional
presence of the gene within the cell including, without limitation, gene knockdown as well as both
transient expression and stable expression. It includes without limitation transcription of the gene into
messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired
product is a biochemical, expression includes the creation of that biochemical and any precursors.
Expression of a gene produces a “gene product.” As used herein, a gene product can be either a
nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is
translated from a transcript. Gene products described herein further include nucleic acids with post
transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational
modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein
subunits, proteolytic cleavage, and the like.

(80) As used herein, the term “epigenetic modification(s)” refers to stably-heritable phenotype(s)
resulting from chromosomal alterations without alterations to the DNA sequence. Epigenetic
modifications can, in some instances lead to a disease or disorder such as cancer. Berger, S L, et al.,
Genes Dev. 23:781-783 (2009). Epigenetic modifications typically involve changes in the chromatin
structure that can result in overexpression and/or repression of genes that control cellular processes
such as differentiation, proliferation, and/or apoptosis. Such modifications can involve, e.g., DNA
methylation and histone acetylation. See, e.g., Gnyska, A., et al., Anticancer Res. 33:2989-2996
(2013).

(81) As used herein, the term “epigenetic modulating agent” refers to an agent, e.g., a therapeutic
agent, which can affect, e.g., block, reduce, reverse, or alleviate, a disease-causing epigenetic
modification, thereby treating the disease, e.g., cancer. Exemplary epigenetic modulating agents
include DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi). A
variety of HDACi and DNMTi are approved for or are being tested for treatment of certain cancers.
Exemplary agents are disclosed elsewhere herein.

(82) As used herein, the terms “histone deacetylase inhibitor(s)” or “HDAC inhibitor(s)” or “HDACi”
refer to a compound or compounds that are capable of inhibiting the deacetylation of histones in vivo,
in vitro, or both. HDAC inhibit the activity of at least one histone deacetylase. As a result of inhibiting
the deacetylation of at least one histone, an increase in acetylated histone occurs and accumulation of
acetylated histone can provide a biological marker for assessing the activity of HDAC.

(83) As used herein, the terms “DNA methyltransferase inhibitors(s)” or “DNMT inhibitor(s)” or “DNMTi”
refer to a compound or compounds that are capable of inhibiting DNA hypermethylation and/or DNA
methyltransferase (“DNMT”) overexpression in vivo, in vitro, or both. As a result of inhibiting DNMT
overexpression and/or DNMT activity, DNMTi can, e.g., restore expression of aberrantly silenced
genes such as tumor suppressor genes.

(84) As used herein, the term “anti-SEMA4D binding molecule” refers to a molecule that specifically
binds to SEMA4D, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof. Unless
specifically referring to full-sized antibodies such as naturally occurring antibodies, the term “anti-
SEMA4D antibody” encompasses full-sized antibodies as well as antigen-binding fragments as well as
antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally
occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that
bind antigen in a manner similar to antibody molecules.

(85) Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to
therapeutic measures that cure, slow down, lessen symptoms of, and/or halt or slow the progression
of an existing diagnosed pathologic condition or disorder. Terms such as “prevent,” “prevention,”
“avoid,” “deterrence” and the like refer to prophylactic or preventative measures that prevent the
development of an undiagnosed targeted pathologic condition or disorder. Thus, “those in need of
treatment” can include those already with the disorder; those prone to have the disorder; and those in
whom the disorder is to be prevented.

(86) The term “therapeutically effective amount” refers to an amount of an antibody, polypeptide,
polynucleotide, small organic molecule, or other drug effective to “treat” a disease or disorder in a
subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce
the number of cancer cells; retard or stop cancer cell division, reduce or retard an increase in tumor
size; inhibit, e.g., suppress, retard, prevent, stop, delay, or reverse cancer cell infiltration into
peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit, e.g.,
suppress, retard, prevent, shrink, stop, delay, or reverse tumor metastasis; inhibit, e.g., suppress,
retard, prevent, stop, delay, or reverse tumor growth; relieve to some extent one or more of the
symptoms associated with the cancer, reduce morbidity and mortality; improve quality of life; or a
combination of such effects. To the extent the drug prevents growth and/or kills existing cancer cells, it
can be referred to as cytostatic and/or cytotoxic.

(87) By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly
a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects
include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats,
guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.

(88) As used herein, phrases such as “a subject that would benefit from therapy” and “an animal in
need of treatment” includes subjects, such as mammalian subjects, that would benefit from
administration of a therapy as described herein, such as an antibody, comprising one or more antigen
binding domains, in combination with an epigenetic modulating agent, e.g., an HDACi, a DNMTi,
and/or a combination thereof.

(89) The term “immune modulating therapy” or “immunotherapy” refers to treatment that impacts a
disease or disorder in a subject by inducing and/or enhancing an immune response in that subject.
Immune modulating therapies include cancer vaccines, immunostimulatory agents, adoptive T cell or
antibody therapy, and immune checkpoint blockade (Lizée et al. Ann. Rev. Med. 64:71-90 (2013)).

(90) The term “immune modulating agent” refers to the active agents of immunotherapy. Immune
modulating agents include a diverse array of recombinant, synthetic and natural, preparation.
Examples of immune modulating agents include, but are not limited to, interleukins such as IL-2, IL-7,
IL-12; cytokines such as granulocyte colony-stimulating factor (G-CSF), interferons; various
chemokines such as CXCL13, CCL26, CXCL7; antagonists of immune checkpoint blockades such as
anti-CTLA-4, anti-PD-1 or anti-PD-L1 (ligand of PD-1), anti-LAG3, anti-TIM3, anti-B7-H3, synthetic
cytosine phosphate-guanosine (CpG) oligodeoxynucleotides, glucans; and modulators of regulatory T
cells (Tregs) such as cyclophosphamide.

(91) Target Polypeptide Description—SEMA4D

(92) As used herein, the terms “semaphorin-4D”, “SEMA4D”, and “SEMA4D polypeptide” are used
interchangeably, as are “SEMA4D” and “Sema4D.” In certain embodiments, SEMA4D is expressed on
the surface of or secreted by a cell. In another embodiment, SEMA4D is membrane bound. In another
embodiment, SEMA4D is soluble, e.g., sSEMA4D. In another embodiment, SEMA4D can include a
full-sized SEMA4D or a fragment thereof, or a SEMA4D variant polypeptide, where the fragment of
SEMA4D or SEMA4D variant polypeptide retains some or all functional properties of the full-sized
SEMA4D.

(93) The full-sized human SEMA4D protein is a homodimeric transmembrane protein consisting of two
polypeptide chains of 150 kDa. SEMA4D belongs to the semaphorin family of cell surface receptors
and is also referred to as CD100. Both human and mouse SEMA4D/Sema4D can be proteolytically
cleaved from their transmembrane form to generate 120-kDa soluble forms, giving rise to two Sema4D
isoforms (Kumanogoh et al., J. Cell Science 116:3464 (2003)). Semaphorins consist of soluble and
membrane-bound proteins that were originally defined as axonal-guidance factors which play an
important role in establishing precise connections between neurons and their appropriate target.
Structurally considered a class IV semaphorin, SEMA4D consists of an amino-terminal signal
sequence followed by a characteristic ‘Sema’ domain, which contains 17 conserved cysteine residues,
an Ig-like domain, a lysine-rich stretch, a hydrophobic transmembrane region, and a cytoplasmic tail.

(94) The SEMA4D polypeptide includes a signal sequence of about 13 amino acids followed by a
semaphorin domain of about 512 amino acids, an immunoglobulin-like (Ig-like) domain of about 65
amino acids, a lysine-rich stretch of 104 amino acids, a hydrophobic transmembrane region of about
19 amino acids, and a cytoplasmic tail of 110 amino acids. A consensus site for tyrosine
phosphorylation in the cytoplasmic tail supports the predicted association of SEMA4D with a tyrosine
kinase (Schlossman et al., Eds. (1995) Leucocyte Typing V (Oxford University Press, Oxford).

(95) SEMA4D is known to have at least three functional receptors, Plexin-B1, Plexin-B2 and CD72.
Plexin-B1, is expressed in non-lymphoid tissues and has been shown to be a high affinity (1 nM)
receptor for SEMA4D (Tamagnone et al., Cell 99:71-80 (1999)). SEMA4D stimulation of Plexin-B1
signaling has been shown to induce growth cone collapse of neurons, and to induce process
extension collapse and apoptosis of oligodendrocytes (Giraudon et al., J. Immunol. 172:1246-1255
(2004); Giraudon et al., NeuroMolecular Med. 7:207-216 (2005)). After binding to SEMA4D, Plexin-B1
signaling mediates the inactivation of R-Ras, leading to a decrease in the integrin mediated
attachment to the extracellular matrix, as well as to activation of RhoA, leading to cell collapse by
reorganization of the cytoskeleton (Kruger et al., Nature Rev. Mol. Cell Biol. 6:789-800 (2005);
Pasterkamp, TRENDS in Cell Biology 15:61-64 (2005)). Plexin-B2 has an intermediate affinity for
SEMA4D and a recent report indicates that Plexin-B2 is expressed on keratinocytes and activates
SEMA4D-positive γδ T cells to contribute to epithelial repair (Witherden et al., Immunity 37:314-25
(2012)).

(96) In lymphoid tissues, CD72 is utilized as a low affinity (300 nM) SEMA4D receptor (Kumanogoh et
al., Immunity 13:621-631 (2000)). B cells and Antigen Presenting Cells (APC) express CD72, and anti-
CD72 antibodies have many of the same effects as sSEMA4D, such as enhancement of CD40-
induced B cell responses and B cell shedding of CD23. CD72 is thought to act as a negative regulator
of B cell responses by recruiting the tyrosine phosphatase SHP-1, which can associate with many
inhibitory receptors. Interaction of SEMA4D with CD72 results in the dissociation of SHP-1, and the
loss of this negative activation signal. SEMA4D has been shown to promote T cell stimulation and B
cell aggregation and survival in vitro. The addition of SEMA4D-expressing cells or sSEMA4D
enhances CD40-induced B cell proliferation and immunoglobulin production in vitro, and accelerates in
vivo antibody responses (Ishida et al., Inter. Immunol. 15:1027-1034 (2003); Kumanogoh and H.
Kukutani, Trends in Immunol. 22:670-676 (2001)). sSEMA4D enhances the CD40 induced maturation
of DCs, including up-regulation of costimulatory molecules and increased secretion of IL-12. In
addition, sSEMA4D can inhibit immune cell migration, which can be reversed by addition of blocking
anti-SEMA4D mouse antibodies (Elhabazi et al., J. Immunol. 166:4341-4347 (2001); Delaire et al., J.
Immunol. 166:4348-4354 (2001)).
(97) Sema4D is expressed at high levels in lymphoid organs, including the spleen, thymus, and lymph
nodes, and in non-lymphoid organs, such as the brain, heart, and kidney. In lymphoid organs,
Sema4D is abundantly expressed on resting T cells but only weakly expressed on resting B cells and
antigen-presenting cells (APCs), such as dendritic cells (DCs).

(98) Cellular activation increases the surface expression of SEMA4D as well as the generation of
soluble SEMA4D (sSEMA4D). The expression pattern of SEMA4D suggests that it plays an important
physiological as well as pathological role in the immune system. SEMA4D has been shown to promote
B cell activation, aggregation and survival; enhance CD40-induced proliferation and antibody
production; enhance antibody response to T cell dependent antigens; increase T cell proliferation;
enhance dendritic cell maturation and ability to stimulate T cells; and is directly implicated in
demyelination and axonal degeneration (Shi et al., Immunity 13:633-642 (2000); Kumanogoh et al., J.
Immunol. 169:1175-1181 (2002); and Watanabe et al., J. Immunol. 167:4321-4328 (2001)).

(99) Anti-SEMA4D Antibodies

(100) Antibodies that bind SEMA4D have been described in the art. See, for example, US Publ. Nos.
2008/0219971 A1, US 2010/0285036 A1, and US 2006/0233793 A1, International Patent Applications
WO 93/14125, WO 2008/100995, and WO 2010/129917, and Herold et al., Int. Immunol. 7:1-8 (1995),
each of which is herein incorporated in its entirety by reference.

(101) The disclosure generally relates to a method of inhibiting, delaying, or reducing tumor growth or
metastases in a subject, e.g., a human cancer patient, comprising administration of an antibody which
specifically binds to SEMA4D, or an antigen-binding fragment, variant, or derivative thereof and an
effective amount of an epigenetic modulating agent, e.g., a histone deacetylase (HDAC) inhibitor
(HDACi), a DNA methyltransferase (DNMT) inhibitor (DNMTi), or any combination thereof. HDACi and
DNMTi are described in detail elsewhere herein. In certain embodiments, the anti-SEMA4D antibody
blocks the interaction of SEMA4D with one or more of its receptors, e.g., Plexin-B1, Plexin-B2, and/or
CD72. In certain embodiments the cancer cells express Plexin-B1 and/or Plexin-B2. Anti-SEMA4D
antibodies having these properties can be used in the methods provided herein. Antibodies that can be
used include, but are not limited to MAbs VX15/2503, 67, 76, 2282 and antigen-binding fragments,
variants, or derivatives thereof which are fully described in US 2010/0285036 A1 and US
2008/0219971 A1. Additional antibodies which can be used in the methods provided herein include the
BD16 antibody described in US 2006/0233793 A1 as well as antigen-binding fragments, variants, or
derivatives thereof or any of MAb 301, MAb 1893, MAb 657, MAb 1807, MAb 1656, MAb 1808, Mab
59, MAb 2191, MAb 2274, MAb 2275, MAb 2276, MAb 2277, MAb 2278, MAb 2279, MAb 2280, MAb
2281, MAb 2282, MAb 2283, MAb 2284, and MAb 2285, as well as any fragments, variants or
derivatives thereof as described in US 2008/0219971 A1. In certain embodiments an anti-SEMA4D
antibody for use in the methods provided herein binds human, murine, or both human and murine
SEMA4D. Also useful are antibodies which bind to the same epitope as any of the aforementioned
antibodies and/or antibodies which competitively inhibit binding or activity of any of the aforementioned
antibodies.

(102) In certain embodiments, an anti-SEMA4D antibody or antigen-binding fragment, variant, or

derivative thereof useful in the methods provided herein has an amino acid sequence that has at least
about 80%, about 85%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about
94%, or about 95% sequence identity to the amino acid sequence for a reference anti-SEMA4D
antibody molecule, for example, those described above. In a further embodiment, the binding molecule
shares at least about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to a
reference antibody.

(103) In certain aspects, the anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative
thereof can inhibit SEMA4D interaction with its receptor, e.g., Plexin-B1, Plexin-B2, or CD72. In certain
aspects the anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof can
inhibit SEMA4D-mediated Plexin-B1 signal transduction.
(104) In certain aspects, the anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative
thereof competitively inhibits a reference antibody comprising a variable heavy chain region (VH)
comprising the amino acid sequence SEQ ID NO: 1 and a variable light chain region (VL) comprising
the amino acid sequence SEQ ID NO: 5 from binding to SEMA4D. In certain aspects, the anti-
SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof binds to the same
SEMA4D epitope as a reference antibody comprising a VH comprising the amino acid sequence SEQ
ID NO: 1 and a VL comprising the amino acid sequence SEQ ID NO: 5. In certain aspects, the VH of
the anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof comprises three
complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises
three CDRs LCDR1, LCDR2, and LCDR3, the CDRs comprising the amino acid sequences SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively
except for at least one, two, three, four, five, or six single conservative amino acid substitutions in one
or more of the CDRs. In certain aspects the CDRs comprise the amino acid sequences SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

(105) In certain aspects the VH of the anti-SEMA4D antibody or antigen-binding fragment, variant, or
derivative thereof comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or
100% identical to SEQ ID NO: 1 and the VL of the anti-SEMA4D antibody or antigen-binding fragment,
variant, or derivative thereof comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%,
95%, or 100% identical to SEQ ID NO: 5; or the VH comprises an amino acid sequence at least 70%,
75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 9 and the VL comprises an amino acid
sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 10. In certain
aspects, the VH comprises the amino acid sequence SEQ ID NO: 1 and the VL comprises the amino
acid sequence SEQ ID NO: 5; or the VH comprises the amino acid sequence SEQ ID NO: 9 and the
VL comprises the amino acid sequence SEQ ID NO: 10.

(106) Also included for use in the methods provided herein are polypeptides encoding anti-SEMA4D
antibodies, or antigen-binding fragments, variants, or derivatives thereof as described herein,
polynucleotides encoding such polypeptides, vectors comprising such polynucleotides, and host cells
comprising such vectors or polynucleotides, all for producing anti-SEMA4D antibodies, or antigen-
binding fragments, variants, or derivatives thereof for use in the methods described herein.

(107) Suitable biologically active variants of the anti-SEMA4D antibodies of the disclosure can be used
in the methods of the present disclosure. Such variants will retain the desired binding properties of the
parent anti-SEMA4D antibody. Methods for making antibody variants are generally available in the art.

(108) Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for
example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing
Company, New York); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Methods
Enzymol. 154:367-382 (1987); Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by
reference. Guidance as to appropriate amino acid substitutions that do not affect biological activity of
the polypeptide of interest can be found in the model of Dayhoff et al. (1978) in Atlas of Protein
Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), pp. 345-352, herein
incorporated by reference in its entirety. The model of Dayhoff et al. uses the Point Accepted Mutation
(PAM) amino acid similarity matrix (PAM 250 matrix) to determine suitable conservative amino acid
substitutions. In certain aspects, conservative substitutions, such as exchanging one amino acid with
another having similar properties are used. Examples of conservative amino acid substitutions as
taught by the PAM 250 matrix of the Dayhoff et al. model include, but are not limited to, Gly.Math.Ala,
Val .Math.Ile.Math.Leu, Asp.Math.Glu, Lys.Math.Arg, Asn.Math.Gln, and Phe.Math.Trp.Math.Tyr.

(109) In constructing variants of the anti-SEMA4D binding molecule, e.g., an antibody or antigen-
binding fragment thereof, polypeptides of interest, modifications are made such that variants continue
to possess the desired properties, e.g., being capable of specifically binding to a SEMA4D, e.g.,
human, murine, or both human and murine SEMA4D, e.g., expressed on the surface of or secreted by
a cell and having SEMA4D blocking activity, as described herein. In certain aspects, mutations made
in the DNA encoding the variant polypeptide maintain the reading frame and do not create
complementary regions that could produce secondary mRNA structure. See EP Patent Application
Publication No. 75,444.

(110) Methods for measuring anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding
fragment, variant, or derivative thereof, binding specificity include, but are not limited to, standard
competitive binding assays, assays for monitoring immunoglobulin secretion by T cells or B cells, T
cell proliferation assays, apoptosis assays, ELISA assays, and the like. See, for example, such assays
disclosed in WO 93/14125; Shi et al., Immunity 13:633-642 (2000); Kumanogoh et al., J. Immunol.
169:1175-1181 (2002); Watanabe et al., J. Immunol. 167:4321-4328 (2001); Wang et al., Blood
97:3498-3504 (2001); and Giraudon et al., J. Immunol. 172:1246-1255 (2004), all of which are herein
incorporated by reference.

(111) Methods for measuring the anti-angiogenic ability of an anti-SEMA4D antibody or antigen-
binding fragment, variant, or derivative thereof are well known in the art.

(112) When discussed herein whether any particular polypeptide, including the constant regions,
CDRs, VH domains, or VL domains disclosed herein, is at least about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
96%, about 97%, about 98%, about 99%, or even about 100% identical to another polypeptide, the %
identity can be determined using methods and computer programs/software known in the art such as,
but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711).
BESTFIT uses the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-
489, to find the best segment of homology between two sequences. When using BESTFIT or any
other sequence alignment program to determine whether a particular sequence is, for example, 95%
identical to a reference sequence according to the present disclosure, the parameters are set, of
course, such that the percentage of identity is calculated over the full length of the reference
polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the
reference sequence are allowed.

(113) For purposes of the present disclosure, percent sequence identity can be determined using the
Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search
algorithm is taught in Smith and Waterman (1981)Adv. Appl. Math. 2:482-489. A variant can, for
example, differ from a reference anti-SEMA4D antibody (e.g., MAb VX15/2503, 67, 76, or 2282) by as
few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5,
as few as 4, 3, 2, or even 1 amino acid residue.

(114) The constant region of an anti-SEMA4D antibody can be mutated to alter effector function in a
number of ways. For example, see U.S. Pat. No. 6,737,056 B1 and U.S. Patent Application Publication
No. 2004/0132101 A1, which disclose Fc mutations that optimize antibody binding to Fc receptors.

(115) In certain anti-SEMA4D antibodies or fragments, variants or derivatives thereof useful in the
methods provided herein, the Fc portion can be mutated to decrease effector function using
techniques known in the art. For example, the deletion or inactivation (through point mutations or other
means) of a constant region domain can reduce Fc receptor binding of the circulating modified
antibody thereby increasing tumor localization. In other cases, constant region modifications
consistent with the instant disclosure moderate complement binding and thus reduce the serum half-
life. Yet other modifications of the constant region can be used to modify disulfide linkages or
oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or
antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of
the modifications, such as tumor localization, biodistribution and serum half-life, can easily be
measured and quantified using well known immunological techniques without undue experimentation.

(116) Anti-SEMA4D antibodies for use in the methods provided herein include derivatives that are
modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent
attachment does not prevent the antibody from specifically binding to its cognate epitope. For
example, but not by way of limitation, the antibody derivatives include antibodies that have been
modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by
known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein,
etc. Any of numerous chemical modifications can be carried out by known techniques, including, but
not limited to specific chemical cleavage, acetylation, formylation, etc. Additionally, the derivative can
contain one or more non-classical amino acids.

(117) A “conservative amino acid substitution” is one in which the amino acid residue is replaced with
an amino acid residue having a side chain with a similar charge. Families of amino acid residues
having side chains with similar charges have been defined in the art. These families include amino
acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine)
and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations
can be introduced randomly along all or part of the coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that
retain activity (e.g., the ability to bind an anti-SEMA4D polypeptide, to block SEMA4D interaction with
its receptor, or to inhibit, delay, or reduce metastases in a subject, e.g., a cancer patient).

(118) For example, it is possible to introduce mutations only in framework regions or only in CDR
regions of an antibody molecule. Introduced mutations can be silent or neutral missense mutations,
i.e., have no, or little, effect on an antibody's ability to bind antigen. These types of mutations can be
useful to optimize codon usage, or improve a hybridoma's antibody production. Alternatively, non-
neutral missense mutations can alter an antibody's ability to bind antigen. One of skill in the art would
be able to design and test mutant molecules with desired properties such as no alteration in antigen
binding activity or alteration in binding activity (e.g., improvements in antigen binding activity or change
in antibody specificity). Following mutagenesis, the encoded protein can routinely be expressed and
the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecifically bind
at least one epitope of a SEMA4D polypeptide) can be determined using techniques described herein
or by routinely modifying techniques known in the art.

(119) In certain embodiments, the anti-SEMA4D antibodies for use in the methods provided herein
comprise at least one optimized complementarity-determining region (CDR). By “optimized CDR” is
intended that the CDR has been modified and optimized to improve binding affinity and/or anti-
SEMA4D activity that is imparted to an anti-SEMA4D antibody comprising the optimized CDR. “Anti-
SEMA4D activity” or “SEMA4D blocking activity” can include activity which modulates one or more of
the following activities associated with SEMA4D: B cell activation, aggregation and survival; CD40-
induced proliferation and antibody production; antibody response to T cell dependent antigens; T cell
or other immune cell proliferation; dendritic cell maturation; demyelination and axonal degeneration;
apoptosis of pluripotent neural precursors and/or oligodendrocytes; induction of endothelial cell
migration; inhibition of spontaneous monocyte migration; inhibition, delay, or reduction of tumor cell
growth or metastasis, binding to cell surface plexin-B1 or other receptor, or any other activity
association with soluble SEMA4D or SEMA4D that is expressed on the surface of SEMA4D+ cells. In
a particular embodiment, anti-SEMA4D activity includes the ability to inhibit, delay, or reduce tumor
metastases, either in combination with inhibition, delay, or reduction of primary tumor cell growth and
tumor metastases, or independently of primary tumor cell growth and tumor metastases. Anti-SEMA4D
activity can also be attributed to a decrease in incidence or severity of diseases associated with
SEMA4D expression, including, but not limited to, certain types of cancers including lymphomas,
autoimmune diseases, inflammatory diseases including central nervous system (CNS) and peripheral
nervous system (PNS) inflammatory diseases, transplant rejections, and invasive angiogenesis.
Examples of optimized antibodies based on murine anti-SEMA4D MAb BD16 were described in US
Publ. No. 2008/0219971 A1, International Patent Application WO 93/14125 and Herold et al., Int.
Immunol. 7:1-8 (1995), each of which are herein incorporated by reference in their entirety. The
modifications can involve replacement of amino acid residues within the CDR such that an anti-
SEMA4D antibody retains specificity for the SEMA4D antigen and has improved binding affinity and/or
improved anti-SEMA4D activity.
(120) Histone Deacetylase Inhibitors (HDACi)

(121) As used herein, the terms “histone deacetylase inhibitor(s),” “HDAC inhibitor(s),” and “HDACi”
are used interchangeably, and can refer to singular HDACi or plural HDACi.

(122) Histone acetyl transferases (HATs) and histone deacetylases (HDACs) regulate the acetylation
status of histones. Histone acetyl transferases are enzymes that acetylate the lysine residues in core
histones resulting in less compact and more transcriptionally active chromatin, which leads to gene
expression. HDACs, conversely, are enzymes that catalyze the removal of acetyl groups from lysine
residues in the amino terminal tails of the nucleosomal core histones. HDACs can be divided into three
classes based on structural homology. Class I HDACs (HDACs 1, 2, 3, and 8) are related to the yeast
RPD3 gene. Class IIA (HDACs 4, 5, 7, and 9) bear similarity to the yeast Hdal gene. Class III, also
known as sirtuins, are related to the Sir2 gene and include SIRT1-7. Class IV, which only contains
HDAC11, shares features of both Class I and II. See, e.g., Mottamal, M., et al., Molecules 20:3898-
3941 (2015).

(123) Certain HDAC inhibitors act by binding to the zinc-containing catalytic domain of the HDACs.
These can be classified based on the chemical moiety that binds to the zinc ion, or as cyclic
tetrapeptides, which bind to the zinc ion with a thiol group. See, e.g., Drummond, D. C., et al., Ann.
Rev. Pharmacol. Toxicol. 45:495-528 (2005). These include, without limitation: hydroxamic acids (or
hydroxamates), such as trichostatin A; cyclic tetrapeptides (such as trapoxin B) and the depsipeptides;
benzamides; electrophilic ketones; and aliphatic acid compounds (short-chain fatty acids (SCFAs)
such as phenylbutyrate and valproic acid. See, e.g., Porcu, M., and Chiarugi, A., Trends Pharmacolog.
Sci. 26:94-103 (2005). More specifically, HDAC inhibitors include, without limitation, hydroxamic acids
Vorinostat (SAHA), Belinostat (PXD101), LAQ824, and Panobinostat (LBH589); and the benzamides:
entinostat (MS-275), CI994, and Mocetinostat (MGCD0103). The sirtuin Class III HDACs are
dependent on NAD+ and are, therefore, inhibited by nicotinamide, as well as derivatives of NAD,
dihydrocoumarin, naphthopyranone, and 2-hydroxynaphthaldehydes. Id.

(124) Non-limiting examples of HDAC inhibitors for use in inhibiting histone deacetylase, inducing
terminal differentiation, cell growth arrest and/or apoptosis in malignant cells, and/or inducing
differentiation, cell growth arrest, and/or apoptosis of tumor cells in a tumor are listed in Table 2. It is
understood that the HDAC inhibitors include any salts, crystal structures, amorphous structures,
hydrates, derivatives, metabolites, stereoisomers, structural isomers and prodrugs of the HDAC
inhibitors described herein.

(125) TABLE-US-00002 TABLE 2 Non-limiting list of HDAC inhibitors Name Structure Reference
Entinostat Bracker, et al. Int J Oncol. 35:909-20 (2009). Vorinostat Lee, J.-H:,et al.. Proc. Natl. Acad.
Sci. USA 110: 15704-9 (2013). Romidepsin Nakajima H, et al., Exp. Cell Res. 241A26-33 (1998).
Chidamide Qiao, Z et al., Biochem Biophys Res Commun. 434:95-101 (2013). Panobinostat Prince,
HM; and M Bishton Hematology Meeting Reports. Parkville, Australia: Peter MacCallum Cancer
Centre and Univ, of Melbourne. 3 (1): 33-38 (2009). Belinostat Plumb JA, et al. Mol Cancer Ther
2:721-728 (2003). Valproic Acid Phiel et al. J. Biol. Chem. 276:36734- 36741 (2001); Gottlicher et al.
EMBO J 20:6969- 6978 (2001). Mocetinostat Foumel et al. Mol. Cancer Ther. 7:759-768 (2008).
Abexinostat Buggy, JJ, et al., Mol. Cancer Ther. 5:1309- 1317 (2006). Pracinostat 0 Novotny-
Diermayr; V., et al. Mol Cancer Ther. doi:10.1158/ 1535-7163.MCT- 09-0689 (2010). Resminostat
Mandi-Weber S, et al., Br. J. Haematol. 149:518-528 (2010). Givinostat Leoni F, and Fossati G. Mol.
Med. 11:1 (2005). Quisinostat Arts. J et al., Clin. Cancer Res. 15:6841-51 (2009). Kevetrin
clinicaltrials.gov /ct2/show/ NCT01664000?term= kevetrin&rank=1. CUDC-101 (Curis) Lai, et al.,
Cancer Res. 70:3647-3656 (2010). 4SC-202 Zhijun, H., et al., Tumor Biol. 37:10257 (2016). ACY-241
Niesvizky et al. Blood 126:3040 (2015). AR-42 Lin TY et al., Blood 115:4217-4225 (2010); Sargeant
AM et al., Cancer Res. 68:3999- 4009 (2008). Tefinostat Zabkiewicz, J., et al., Blood 122:1297 (2013).
CHR-3996 0 Moffat, D., et al. J. Med. Chem. 53:8663-78 (2010). Ricolinostat (ACY-1215) Yee AJ et
al., Blood 126:3055 (2015); Santo L., et al., Blood. 119:2579-89 (2012). CG200745 Chun SM, et al.,
PLoS One. 10:e0119379. doi:10.1371/joumal. pone.0119379. eCollection 2015.
(126) In certain embodiments, HDAC inhibitors useful in the methods provided herein include
Entinostat, Vorinostat, Romidepsin, Chidamide, Panobinostat, Belinostat, Valproic acid, Mocetinostat,
Abexinostat, Pracinostat, Resminostat, Givinostat, Quisinostat, Kevetrin, CUDC-101, 4SC-202, ACY-
241, AR-42, Tefinostat, CHR-3996, Ricolinostat (ACY-1215), and/or CD200745. Some FDA approved
HDAC inhibitors for cancer treatment, as well as HDAC inhibitors that are currently in pending or
completed clinical trials, are listed below.

(127) Food and Drug Administration (FDA) Approved HDAC Inhibitors. Vorinostat was licensed by the
U.S. FDA in October 2006 for the treatment of cutaneous T cell lymphoma (CTCL). Romidepsin (trade
name Istodax) was licensed by the US FDA in November 2009 for cutaneous T-cell lymphoma
(CTCL). Chidamide was approved by China in 2015 for peripheral T-cell lymphoma (PTCL).
Panobinostat (trade name Farydak) was licensed by the US FDA in February 2015 for the treatment of
multiple myeloma. Belinostat (PXD101) was licensed by the FDA for the treatment of peripheral T-cell
lymphoma in 2014.

(128) In one non-limiting aspect, the HDAC inhibitor is Entinostat (ENT). Entinostat has the IUPAC
chemical name pyridin-3-ylmethyl N-[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl] carbamate. ENT,
also known as SNDX-275 and MS-275, is an inhibitor of class I HDACs (Syndax Pharmaceuticals).
ENT is a benzamide histone deacetylase inhibitor that inhibits HDAC1 and HDAC3 with IC50 of 0.51
μM and 1.7 μM. ENT is currently undergoing clinical trials for treatment of various cancers.

(129) DNA Methyltransferase Inhibitors

(130) As used herein, the terms “DNA methyltransferase inhibitor(s),” “DNMT inhibitor(s),” and
“DNMTi” are used interchangeably, and can refer to singular DNMTi or plural DNMTi.

(131) DNA methyltransferases catalyze the addition of methyl groups to the 5′ carbon of cytosine
residues. In cancer cells, silencing of tumor suppressor genes occurs though DNMT-mediated
methylation. Gravina et al., Molecular Cancer 9:305-320 (2010). Several isoforms of DNMTs exist,
including DNMT1, DNMT-3a and DNMT-3b. Id. A variety of DNMTi have been identified, and two are
approved for cancer therapy in the United States. The classes of molecules include nucleoside
analogs, antisense oligonucleotides, small molecule enzyme inhibitors, and agents that block the
interaction of DNMTs with DNA.

(132) Non-limiting examples of DNMTi for use in inhibiting DNA methyltransferases, inducing terminal
differentiation, cell growth arrest and/or apoptosis in malignant cells, and/or inducing differentiation,
cell growth arrest, and/or apoptosis of tumor cells in a tumor are listed in Table 3. It is understood that
the DNMTi include any salts, crystal structures, amorphous structures, hydrates, derivatives,
metabolites, stereoisomers, structural isomers and prodrugs of the DNMT inhibitors described herein.

(133) TABLE-US-00003 TABLE 3 Non-limiting list of DNMT inhibitors Name and Synonyms Structure
Reference Azacytidine (trade name VIDAZA ®); 5- azacytidine; Azacytidine; 320-67-2; Ladakamycin;
4-Amino-1-beta-D- ribofuranosyl-s-triazin- 2(1H)-one; U-18496 Cihák A., Oncology 30:405-422 (1974)
Decitabine (trade name: DACOGEN ®); 5-aza-2′- deoxycytidine; 5-aza- dCyd; Deoxyazacytidine;
dezocitidine; 4-amino-1- [(2R,4S,5R)-4-hydroxy-5- (hydroxymethyl)oxolan-2- yl]-1,3,5-triazin-2-one
Kantarjian H., et al., Cancer 106:1794-1803 (2006) Zebularine; 1-(β-D- Ribofuranosyl)-2(1H)-
pyrimidinone; Pyrimidin- 2-one β-D-ribofuranoside; l-[(2R,3R,4S,5R)-3,4- dihydroxy-5-
(hydroxymethyl)oxolan-2- yllpyrimidin-2-one Zhou, L., et al., J. Mol. Biol. 321:591- 599 (2002) SGI-
110; 2′-Deoxy-5′-O- [(2′-deoxy-5-azacytidin-3′- O- yl)(hydroxy)phosphoryl] guanosine sodium salt;
guadecitabine sodium Lavelle, D., et al., J. Transl. Med. 8:92 (2010) Epigallocatechin gallate; (-)-
epigallocatechin-3- gallate; EGCG Fang, MZ., et al., Cancer Res. 63:7563-7570 (2003); Li, Y. and
Tollefsbol, TO., Curr. Med. Chem 17:2141-2151 (2010) MG98 2′O-CH3—substituted phosphorothioate
Klisovic, TB., et al., oligodeoxynucleotide antisense molecule Clin. Cancer Res. designed to hybridize
to the 3′ untranslated 14:2444-2449 region of the human DNMT1 mRNA. (2008) RG108; N-Phthalyl-L-
tryptophan; 2-(1,3-dioxo- 1,3-dihydro-2H-isoindol- 2-yl)-3-(1H-indol-3- yl)propanoic acid Brueckner, B.,
et al., Cancer Res. 65:6305-6311 (2005) Procainamide; 4-amino- N-2-(diethylamino)ethyl- benzamide
Lee, BH., et al.,J Biol. Chem. 280:40749-40756 (2007) Hydralazine; phthalazin- 1-ylhydrazine; 0
Zambrano, P., et al., BMC Cancer 5:44 (2005)

(134) In certain embodiments, DNMT inhibitors useful in the methods provided herein include
azacytidine, decitabine, zebularine, SGI-110, epigallocatechin gallate, MG98, RG108, procainamide,
and/or hydralazine. Some FDA approved DNMT inhibitors for cancer treatment, as well as DNMT
inhibitors that are currently in pending or completed clinical trials, are listed below.

(135) Food and Drug Administration (FDA) Approved and in trial DNMTi. Azacytidine, marketed as
VIDAZA®, was approved by the FDA for treatment of myelodysplastic syndrome (MDS) on May 19,
2004, and is in clinical trials for advanced solid tumors. Azacytidine has been shown to enhance
activity of immune checkpoint inhibitors (ICP) in preclinical melanoma and colon cancer models by
upregulating the interferon (IFN) pathway and by de-repressing viral antigens, thereby making the
tumor more immunogenic and susceptible to effective ICP therapy. (Li et al., Oncotarget 5:587 (2014)
and Roulois et al., Cell 162:961 (2015)). Azacytidine was further shown to upregulate expression of
cancer testes antigens (CTA) in tumors leading to increased T cell activity against CTA in patients with
MDS, acute myeloid leukemia (AML), and chronic myelomonocytic leukemia (CMML) (Gang, A O. et
al. Blood Cancer J. doi:10.1038/bcj.2014.14. (2014)). Juergens, R A. et al., (Cancer Discov. 1:598-607
(2011)) used azacytidine and entinostat in refractory advanced NSCLC. Six patients from this trial
were enrolled in an additional trial (Wrangle, J., et al., Oncotarget 4:2067-2079 (2013)) with immune
checkpoint inhibitors, with five patients developing responses. See also: blogs.biomedcentral.com/on-
biology/2015/03/26/improving-lung-cancer-immunotherapy-using-epigenetic-approaches/(visited Mar.
16, 2017).

(136) Decitabine, marketed as DACOGEN®, is approved for the treatment of MDS, and is also
approved in Europe for the treatment of AML and is in clinical trials for AML. SGI-110 is in phase II
clinical trials for the treatment of AML. MG98 completed phase I clinical trials for advanced solid
tumors. ECGC completed phase II clinical trials for prostate cancer.

(137) In one non-limiting aspect the DNMTi is Azacytidine. Azacytidine is a chemical analog of the
nucleoside cytosine. Azacytidine can, among other activities, incorporate into DNA causing
hypomethylation of DNA through covalent binding with DNMT, preventing DNA synthesis and leading
to cytotoxicity and degradation of the trapped DNMT. Stresemann, C., and Lyko, F., Int. J Cancer
123:8-13 (2008).

(138) Treatment Methods Using Therapeutic Anti-SEMA4D Binding Molecules and Epigenetic
Modulating Agent, e.g., an HDACi and/or a DNMTi

(139) This disclosure provides a method for inhibiting, delaying, or reducing malignant cell growth in a
subject with cancer by administering to the subject a combination therapy comprising an effective
amount of an isolated binding molecule that specifically binds to SEMA4D, e.g., an anti-SEMA4D
antibody or antigen-binding fragment, variant, or derivative thereof, in combination with an effective
amount of an epigenetic modulating agent, e.g., an HDACi, a DNMTi, and/or a combination thereof.
Exemplary anti-SEMA4D antibodies and exemplary epigenetic modulating agents are described in
detail elsewhere herein. In certain aspects, administration of the combination therapy provided herein
can inhibit tumor or malignant cell growth partially or completely, can delay the progression of tumor
and malignant cell growth in the subject, can prevent metastatic spread in the subject, can reduce the
subject's tumor size, e.g., to allow more successful surgical removal, can decrease tumor vasculature
in the subject, or can result in any combination of positive therapeutic responses in the subject.
Exemplary therapeutic responses that can be achieved are described herein.

(140) In certain aspect, administration of the combination therapy can result in enhanced therapeutic
efficacy relative to administration of the anti-SEMA4D antibody or fragment thereof or the epigenetic
modulating agent alone. In certain aspects the improved treatment efficacy is synergistic, and is
greater than the additive efficacy of each individual agent. In certain aspects the improved treatment
efficacy over either agent administered alone, measured, e.g., in increased tumor growth delay (TGD),
increased frequency of tumor regression, e.g., complete tumor regression, or increased survival is at
least 5%, at least 10%, at least 20%, at least 5%, at least 10%, at least 20%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least
150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at
least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%,
at least 850%, at least 900%, at least 950%, or at least 1000%. In certain aspects the improved
treatment efficacy over the additive efficacy of both agents administered individually, measured, e.g.,
in increased tumor growth delay (TGD), increased frequency of tumor regression, e.g., complete tumor
regression, or increased survival is at least 5%, at least 10%, at least 20%, at least 5%, at least 10%,
at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%,
at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least
700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, or at least 1000%.

(141) In certain aspects, the anti-SEMA4D antibody or fragment can be VX15/2503, mAb 67, or an
antigen-binding fragment, variant or derivative thereof. For example the antibody or fragment thereof
can include a variable heavy chain (VH) comprising VH CDRs 1-3 comprising SEQ ID NOS: 2, 3, and
4, respectively, and a variable light chain (VL) comprising VL CDRs 1-3 comprising SEQ ID NOS: 6, 7,
and 8, respectively, or the VH and VL comprise, respectively, SEQ ID NO: 1 and SEQ ID NO: 5, or
SEQ ID NO: 9 and SEQ ID NO: 10.

(142) In certain aspects the method provided herein comprises administration of an anti-SEMA4D
antibody or antigen-binding fragment, variant, or derivative thereof and an HDACi that inhibits an
HDAC that can be a Class I, Class IIA, Class IIB, Class IV HDAC, and/or any combination thereof,
e.g., an HDAC comprises a zinc-containing catalytic domain. In certain aspects, the HDACi can
include a moiety that binds to the zinc-containing catalytic domain of the HDAC. For example, in
certain aspects, the HDACi can include one or more, or a combination of chemical moieties such as,
but not limited to, a hydroxamic acid or a salt thereof, a cyclic tetrapeptide, a depsipeptide, a
benzamide, an electrophilic ketone, and/or an aliphatic acid or a salt thereof. Non-limiting examples of
HDACi that can be used in the treatment method provided herein include Vorinostat, Romidepsin,
Chidamide, Panobinostat, Belinostat, Valproic acid or a salt thereof, Mocetinostat, Abexinostat,
Entinostat, Pracinostat, Resminostat, Givinostat, Quisinostat, Kevetrin, CUDC-101, AR-42, Tefinostat
(CHR-2845), CHR-3996, 4SC-202, CG200745, ACY-1215, ACY-241, and/or any combination thereof.
In certain aspects, the HDACi can be Entinostat (Pyridin-3-ylmethyl N-[[4-[(2-
aminophenyl)carbamoyl]phenyl]methyl]carbamate).

(143) In certain aspects the method provided herein comprises administration of an anti-SEMA4D
antibody or antigen-binding fragment, variant, or derivative thereof and a DNMTi that inhibits DNMT1,
DNMT-3a, DNMT-3b, and/or any combination thereof. For example, in certain aspects, the DNMTi can
include one or more, or a combination of chemical moieties such as, but not limited to, azacytidine,
decitabine, zebularine, SGI-110, epigallocatechin gallate, MG98, RG108, procainamide, hydralazine,
and/or any combination thereof. In certain aspects, the DNMTi can be azacytidine.

(144) According to the method provided herein, the anti-SEMA4D binding molecule, e.g., antibody or
antigen-binding fragment, variant, or derivative thereof, and the epigenetic modulating agent, e.g., the
HDACi, the DNMTi, or any combination thereof can be administered separately or simultaneously.
Either agent can be administered before the other or the agents can be administered simultaneously,
e.g., in a single formulation. Moreover, the routes of dosing, formulation of the agents, and/or
schedules of dosing can be the same or different.

(145) According to the method provided herein, the anti-SEMA4D binding molecule, e.g., the antibody
or antigen-binding fragment, variant, or derivative thereof that specifically binds to SEMA4D, and the
epigenetic modulating agent, e.g., the HDACi, the DNMTi, and/or any combination thereof are
administered to a subject with cancer. The subject's cancer can be newly diagnosed, or in certain
aspects, the administration can follow more traditional cancer treatments. In certain aspects,
administration of the combination therapy provided herein can be used as a preventative measure in a
subject with high susceptibility to develop a certain cancer, or to prevent recurrence of a cancer that
has been previously treated. The subject's cancer can be, e.g., a solid tumor, a hematological
malignancy, any metastasis thereof, or any combination thereof.

(146) In certain aspects, the subject's cancer is a solid tumor or metastasis thereof. The solid tumor
can be, e.g., a sarcoma, a carcinoma, a melanoma, any metastases thereof, or any combination
thereof. Examples of solid tumors that can be treated according to the method provided herein include,
without limitation, squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, renal cell
carcinoma, ductal carcinoma of the breast, soft tissue sarcoma, osteosarcoma, melanoma, small-cell
lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, cancer of the peritoneum,
hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine
cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer,
hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma,
esophageal cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval
cancer, thyroid cancer, head and neck cancer, any metastases thereof, or any combination thereof.

(147) In certain aspects, the cancer is a hematologic malignancy or metastasis thereof. Examples of
hematologic malignancies that can be treated according to the method provided herein include,
without limitation, leukemia, lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia,
acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin lymphoma,
non-Hodgkin lymphoma, multiple myeloma, any metastases thereof, or any combination thereof.

(148) In certain aspects, the cancer treatment method provided herein can further include
administration of an additional cancer therapy. The additional cancer therapy can take place
simultaneously with the administration of combination therapy provided herein, before the combination
therapy provided herein, or after the combination therapy provided herein. In certain aspects the
additional therapy can include, without limitation, surgery, chemotherapy, radiation therapy,
administration of a cancer vaccine, administration of an immunostimulatory agent, adoptive T cell or
antibody therapy, administration of an immune checkpoint blockade inhibitor, administration of a
regulatory T cell (Treg) modulator, or a combination of such therapies.

(149) Administration of the epigenetic modulating agent, e.g., the HDACi, the DNMTi, and/or any
combination thereof, in combination with the anti-SEMA4D binding molecule, e.g., an antibody or
antigen-binding fragment, variant, or derivative thereof can be useful for the treatment of various
malignant and non-malignant tumors. By “anti-tumor activity” is intended a reduction in the rate of
malignant cell proliferation or accumulation, and hence a decline in growth rate of an existing tumor or
in a tumor that arises during therapy, and/or destruction of existing neoplastic (tumor) cells or newly
formed neoplastic cells, and hence a decrease in the overall size of a tumor during therapy. “Anti-
tumor activity” can also comprise promotion of immune infiltration into the tumor, a shift toward
functional, tumor-specific, IFNγ-secreting CD8.sup.+ cytotoxic T cells, an increase in the ratio of T
effector cells to T regulatory cells, increased T-cell activity, infiltration and activation of antigen
presenting cells that cross-present tumor antigens and locally activate tumor-specific T cells, reduced
tumor-associated angiogenesis, inhibition of tumor progression, and enhanced survival. For example,
therapy with an epigenetic modulating agent, e.g., an HDACi, a DNMTi, and/or a combination thereof
and an anti-SEMA4D antibody can elicit a physiological response, for example, inhibition, delay or
reduction of tumor or malignant cell growth and metastases, which is beneficial with respect to
treatment of cancer associated with SEMA4D-expressing cells in a human.

(150) In certain aspects, combination therapy with an epigenetic modulating agent, e.g., an HDACi, a
DNMTi, and/or a combination thereof, and an anti-SEMA4D binding molecule, e.g., an antibody or
fragment thereof, can be used as a medicament, in particular for use in the treatment or prophylaxis of
cancer or for use in a precancerous condition or lesion. In certain aspects, combination therapy with
an epigenetic modulating agent, e.g., an HDACi, a DNMTi, and/or a combination thereof, and an anti-
SEMA4D binding molecule, e.g., an antibody or fragment thereof, can be used for the treatment of a
SEMA4D over-expressing cancer, or cancer associated with SEMA4D-expressing cells.

(151) In accordance with the treatment methods provided herein, administration of an epigenetic
modulating agent, e.g., an HDACi, a DNMTi, and/or a combination thereof, and an anti-SEMA4D
binding molecule, e.g., an antibody or antigen binding fragment, variant, or derivative thereof, as
defined elsewhere herein can be used to promote a positive therapeutic response in the subject with
cancer or predisposed to contract cancer. A “positive therapeutic response” with respect to cancer is
intended to include an improvement in the disease in association with the “anti-tumor” activity is
intended a reduction in the rate of malignant cell proliferation or accumulation, and hence a decline in
growth rate of an existing tumor or in a tumor that arises during therapy, and/or destruction of existing
neoplastic (tumor) cells or newly formed neoplastic cells, and hence a decrease in the overall size of a
tumor during therapy. Such positive therapeutic responses are not limited to the route of
administration. The methods provided herein can be drawn to inhibiting, delaying, or reducing tumor
growth, malignant cell growth, and metastases in a subject with cancer. Thus, as a non-limiting
example, an improvement in the disease can be characterized as a reduction in tumor growth or
absence of tumors. As described elsewhere herein, the therapeutic response achieved upon
administration of the provided combination therapy can be greater than the corresponding therapeutic
response achieved upon administering an epigenetic modulating agent or anti-SEMA4D binding
molecule individually. In certain aspects the therapeutic response achieved is greater than the additive
response expected upon administration of the two agents. In other words, the therapeutic response
achieved is synergistic. In certain aspects, the synergistic response can result in a more effective
treatment, a faster treatment, or can allow treatment with reduced dosages of the agents in the
combination therapy.

(152) Pharmaceutical Compositions and Administration Methods

(153) This disclosure provides a composition, e.g., a pharmaceutical composition, which includes an
effective amount of an anti-SEMA4D binding molecule, e.g., an antibody, or antigen-binding fragment,
variant, or derivative thereof, and an effective amount of an epigenetic modulating agent, e.g., an
HDACi, a DNMTi, and/or a combination thereof. The composition can further comprise one or more
pharmaceutically acceptable carriers or excipients, and/or one or more additional therapeutic agents,
examples of which are disclosed elsewhere herein. Suitable anti-SEMA4D binding molecules and
epigenetic modulating agents for use in such a pharmaceutical composition are provided herein.

(154) In certain aspects the pharmaceutical composition includes an effective amount of an anti-
SEMA4D antibody or antigen-binding fragment thereof that includes a VH and a VL, where the VH and
VL comprises the CDR amino acid sequences contained in MAbs VX15/2503 and in MAb 67, namely,
an HCDR1 comprising the amino acid sequence SEQ ID NO: 2, an HCDR2 comprising the amino acid
sequence SEQ ID NO: 3, an HCDR3 comprising the amino acid sequence SEQ ID NO: 4, an LCDR1
comprising the amino acid sequence SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence
SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence SEQ ID NO: 8. In certain aspects
the pharmaceutical composition includes an effective amount of an anti-SEMA4D antibody or antigen-
binding fragment thereof that includes the VH and VL amino acid sequences of MAb VX15/2503,
namely, a VH comprising the amino acid sequence SEQ ID NO: 1 and a VL comprising the amino acid
sequence SEQ ID NO: 5.

(155) In certain aspects the pharmaceutical composition includes an effective amount of the HDACi
entinostat. In certain aspect the pharmaceutical composition includes an effective amount of the
DNMTi azacytidine.

(156) An exemplary pharmaceutical composition provided herein includes an effective amount of MAb
VX15/2503 and an effective amount of entinostat. Another exemplary pharmaceutical composition
provided herein includes an effective amount of MAb VX15/2503 and an effective amount of
azacytidine. In certain aspects the “effective amount” of a particular agent included in the provided
pharmaceutical composition can be different, e.g., lower, than the amount of the agent that would be
effective as an individual therapy. In certain aspects, the therapeutic efficacy of the pharmaceutical
composition provided herein is greater than the additive effect of the included agents were they to be
administered individually.

(157) The route of administration of the epigenetic modulating agent in combination with the anti-
SEMA4D binding molecule, e.g, antibody, or antigen-binding fragment, variant, or derivative thereof,
can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein
includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal
administration. The modes of administration of the two agents can be the same or different. While all
these forms of administration are clearly contemplated as being within the scope of the methods
provided herein, a non-limiting example of a form for administration would be a solution for injection, in
particular for intravenous or intraarterial injection or drip. A suitable pharmaceutical composition for
injection can comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g.
polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. However, in other methods
compatible with the teachings herein, the epigenetic modulating agent and/or the anti-SEMA4D
binding molecule, e.g., the antibody, or antigen-binding fragment, variant, or derivative thereof can be
delivered directly to the site of the adverse cellular population thereby increasing the exposure of the
diseased tissue to the therapeutic agent.

(158) Methods of preparing and administering the combination therapy provided herein that includes
the epigenetic modulating agent, e.g., an HDACi, a DNMTi, and/or a combination thereof in
combination with the anti-SEMA4D binding molecule, e.g., the antibody, or antigen-binding fragment,
variant, or derivative thereof, to a subject in need thereof are well known to or are readily determined
by those skilled in the art.

(159) As discussed herein, the epigenetic modulating agent, e.g., an HDACi, a DNMTi, and/or a
combination thereof in combination with the anti-SEMA4D binding molecule, e.g., an antibody, or
antigen-binding fragment, variant, or derivative thereof can be administered in a pharmaceutically
effective amount for the in vivo treatment of cancer. In this regard, it will be appreciated that the
disclosed epigenetic modulating agent and binding molecule can be formulated so as to facilitate
administration and promote stability of the active agent. In certain embodiments, pharmaceutical
compositions in accordance with the methods provided herein comprise a pharmaceutically
acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and
the like. For the purposes of the instant application, a pharmaceutically effective amount of the
epigenetic modulating agent and binding molecule is an amount sufficient to affect the activity of at
least one epigenetic modification in a cancer cell, e.g., to reduce histone deacetylase activity or DNA
methyltransferase activity, and to achieve effective binding to a target and to achieve a benefit, e.g., an
anti-proliferative effect, an inhibition, delay or reduction in tumor and malignant cell growth and
metastases, the prevention of further tumor outgrowths, a reduction in tumor size, a decrease in tumor
vasculature, and a reduction in the number of cancer cells, in a subject with cancer, and/or a decrease
in one or more symptoms associated with the disease can be observed.

(160) The pharmaceutical compositions used in the methods provided herein can include
pharmaceutically acceptable carriers such as, e.g., ion exchangers, alumina, aluminum stearate,
lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates,
glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids,
water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool
fat.

(161) Preparations for parenteral administration can include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Non-limiting examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
Aqueous carriers include, e.g., water, alcoholic/aqueous solutions, emulsions or suspensions,
including saline and buffered media. Pharmaceutically acceptable carriers include, but are not limited
to, 0.01-0.1 M, e.g., 0.05 M phosphate buffer or 0.8% saline. Other common parenteral vehicles
include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte
replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other
additives can also be present such as, for example, antimicrobials, antioxidants, chelating agents, and
inert gases and the like.
(162) Non-limiting examples of pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile
and should be fluid to the extent that easy syringability exists. Compositions are typically formulated to
be stable under the conditions of manufacture and storage and can be preserved against the
contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of surfactants. Suitable formulations for use in
the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences
(Mack Publishing Co.) 16th ed. (1980).

(163) Prevention of the action of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the
like. Isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride
can be included in the composition. Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent which delays absorption, for example,
aluminum monostearate and gelatin.

(164) Sterile injectable solutions can be prepared by incorporating the active ingredients (e.g., an
epigenetic modulating agent, e.g., an HDACi, a DNMTi, and/or a combination thereof in combination
with an anti-SEMA4D antibody, or antigen-binding fragment, variant, or derivative thereof, by itself or
in combination with other active agents) in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a
basic dispersion medium and the required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable solutions, two non-limiting examples of
methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active
ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The
preparations for injections are processed, filled into containers such as ampoules, bags, bottles,
syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further,
the preparations can be packaged and sold in the form of a kit. Such articles of manufacture can have
labels or package inserts indicating that the associated compositions are useful for treating a subject
suffering from, or predisposed to cancer.

(165) Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed
with a maintenance dose. These compositions can be administered at specific fixed or variable
intervals, e.g., once a day, or on an “as needed” basis.

(166) Certain pharmaceutical compositions used can be orally administered in an acceptable dosage
form including, e.g., capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical
compositions also can be administered by nasal aerosol or inhalation. Such compositions can be
prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption
promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.

(167) The amount of an epigenetic modulating agent, e.g., an HDACi, a DNMTi, and/or a combination
thereof, and an anti-SEMA4D binding molecule, e.g., antibody, or fragment, variant, or derivative
thereof, to be combined with the carrier materials to produce a single dosage form will vary depending
upon the subject to be treated and the particular mode of administration. The composition can be
administered as a single dose, multiple doses or over an established period of time in an infusion.
Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic
or prophylactic response).

(168) In keeping with the scope of the present disclosure, the epigenetic modulating agent, e.g., the
HDACi, the DNMTi, and/or a combination thereof in combination with the anti-SEMA4D binding
molecule, e.g., the antibody, or antigen-binding fragment, variant, or derivative thereof can be
administered to a human or other animal subject in accordance with the aforementioned methods of
treatment in an amount sufficient to produce a therapeutic effect. The epigenetic modulating agent in
combination with the anti-SEMA4D binding molecule, e.g., the antibody, or antigen-binding fragment,
variant, or derivative thereof can be administered to such human or other animal in a conventional
dosage form prepared by combining the antibody and epigenetic modulating agent with a conventional
pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by
one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is
dictated by the amount of active ingredient with which it is to be combined, the route of administration
and other well-known variables. Those skilled in the art will further appreciate that a cocktail
comprising epigenetic modulating agent and binding molecule can be used.

(169) By “effective amount” is intended an amount of the epigenetic modulating agent, e.g., the
HDACi, the DNMTi, and/or a combination thereof, and an amount of the anti-SEMA4D binding
molecule, e.g., the antibody, or antigen-binding fragment, variant, or derivative thereof, that when
administered brings about a positive therapeutic response with respect to treatment of a patient with a
disease to be treated, e.g., an anti-proliferative effect, the prevention of further tumor outgrowths, a
reduction in tumor size, a decrease in tumor vasculature, a reduction in the number of cancer cells, the
inhibition, delay, or reduction of tumor and/or malignant cell growth and/or metastases in a subject with
cancer, and/or a decrease in one or more symptoms associated with the disease can be observed.

(170) Therapeutically effective doses of the compositions described herein, e.g., for an anti-
proliferative effect, the prevention of further tumor outgrowths, a reduction in tumor size, a decrease in
tumor vasculature, a reduction in the number of cancer cells, the inhibition, delay, or reduction of tumor
and/or malignant cell growth and/or metastases in a subject with cancer, and/or a decrease in one or
more symptoms associated with the disease, can vary depending upon many different factors,
including means of administration, target site, physiological state of the patient, whether the patient is
human or other animal, other medications administered, and whether treatment is prophylactic or
therapeutic. In certain embodiments the patient is a human, but non-human animals, including
transgenic animals, can also be treated. Treatment dosages can be titrated using routine methods
known to those of skill in the art to optimize safety and efficacy.

(171) The amount the epigenetic modulating agent, e.g., the HDACi, the DNMTi, and/or a combination
thereof in combination with the anti-SEMA4D binding molecule to be administered is readily
determined by one of ordinary skill in the art without undue experimentation given the disclosure
provided herein. Factors influencing the mode of administration and the respective amount epigenetic
modulating agent and binding molecule include, but are not limited to, the severity of the disease, the
history of the disease, and the age, height, weight, health, and physical condition of the individual
undergoing therapy. Similarly, the amount of epigenetic modulating agent and binding molecule to be
administered will be dependent upon the mode of administration and whether the subject will undergo
a single dose or multiple doses of this agent.

(172) The use of an epigenetic modulating agent, e.g., an HDACi, a DNMTi, and/or a combination
thereof, in combination with an anti-SEMA4D binding molecule, e.g., antibody or fragment, variant, or
derivative thereof, in the manufacture of a medicament for treating a subject with cancer, and in some
aspects further including pretreatment with at least one other therapy, is also provided. By “pretreated”
or “pretreatment” is intended the subject has received one or more other therapies (e.g., been treated
with at least one other cancer therapy) prior to receiving the medicament comprising the epigenetic
modulating agent in combination with the anti-SEMA4D binding molecule, e.g., antibody or antigen-
binding fragment, variant, or derivative thereof “Pretreated” or “pretreatment” includes subjects that
have been treated with at least one other therapy within 2 years, within 18 months, within 1 year,
within 6 months, within 2 months, within 6 weeks, within 1 month, within 4 weeks, within 3 weeks,
within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days,
or even within 1 day prior to initiation of treatment with the medicament comprising the epigenetic
modulating agent, e.g., HDACi, for example, Entinostat and/or DNMTi, for example, azacytidine, and
an anti-SEMA4D binding molecule, for example, the monoclonal antibody VX15/2503 disclosed
herein, or antigen-binding fragment, variant, or derivative thereof. It is not necessary that the subject
was a responder to pretreatment with the prior therapy or therapies. Thus, the subject that receives
the medicament comprising the epigenetic modulating agent in combination with an anti-SEMA4D
binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof could
have responded, or could have failed to respond, to pretreatment with the prior therapy, or to one or
more of the prior therapies where pretreatment comprised multiple therapies.

(173) The methods provided herein also provide for the use of an epigenetic modulating agent, e.g.,
an HDACi, a DNMTi, and/or a combination thereof, in combination with an anti-SEMA4D binding
molecule, e.g., antibody, or antigen-binding fragment, variant, or derivative thereof, in the manufacture
of a medicament for treating a subject with cancer, where the medicament is used in a subject that is
also being treated with at least one other therapy. Available therapies disclosed herein include, without
limitation, surgery, radiation therapy, a cancer vaccine, administration of an immunostimulatory agent,
adoptive T cell or antibody therapy, administration of an immune checkpoint blockade inhibitor,
administration of a regulatory T cell (Treg) modulator, or combination thereof.

(174) This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which
are within the skill of the art. Such techniques are explained fully in the literature. See, for example,
Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor
Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs
Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)
Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984)
Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney
(1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press)
(1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian
Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155;
Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic
Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-
IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
(1986); and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons,
Baltimore, Md.).

(175) General principles of antibody engineering are set forth in Borrebaeck, ed. (1995) Antibody
Engineering (2nd ed.; Oxford Univ. Press). General principles of protein engineering are set forth in
Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ.
Press, Oxford, Eng.). General principles of antibodies and antibody-hapten binding are set forth in:
Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and
Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y.).
Additionally, standard methods in immunology known in the art and not specifically described can be
followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al., eds.
(1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and
Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY).

(176) Standard reference works setting forth general principles of immunology include Current
Protocols in Immunology, John Wiley & Sons, New York; Klein J., Immunology: The Science of Self-
Nonself Discrimination (John Wiley & Sons, NY (1982)); Kennett et al., eds. (1980) Monoclonal
Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell
(1984) “Monoclonal Antibody Technology” in Laboratory Techniques in Biochemistry and Molecular
Biology, ed. Burden et al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) Kuby Immunology (4th
ed.; W.H. Freeman and Co., NY); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et
al. (2005) Cellular and Molecular Immunology (5th ed.; Elsevier Health Sciences Division);
Kontermann and Dubel (2001) Antibody Engineering (Springer Verlag); Sambrook and Russell (2001)
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII
(Prentice Hall, 2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor
Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).
(177) All of the references cited above, as well as all references cited herein, are incorporated herein
by reference in their entireties.

(178) The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1: Administration of Entinostat (ENT) and Anti-SEMA4D Antibody Increases Survival,

Significantly Delays Tumor Growth, and Increases Percent Complete Tumor Regression

(179) The effect of combined ENT and anti-SEMA4D MAb administration on survival, tumor growth,
and tumor regression was tested in a tumor mouse model. Colon26 cells (500,000 cells) were
implanted subcutaneously into Balb/c mice. Mice were treated with 1) control Ig (10 mg/kg, ip,
weekly×5, starting on day 2) (n=20); 2) control Ig (10 mg/kg, ip, weekly×5, starting on day 2)+ENT (20
mg/kg, 3×/weekly×2 weeks, initiated when average tumor volume was ˜300 mm.sup.3) (n=21); 3) anti-
SEMA4D/MAb 67 (10 mg/kg, IP, weekly×5 weeks, starting on day 2) (n=24); or 4) anti-SEMA4D/MAb
67 (10 mg/kg, IP, weekly×5 weeks, starting on day 2) and ENT (20 mg/kg, 3×/weekly×2 weeks,
initiated when average tumor volume was ˜300 mm.sup.3)(n=26) (Experimental Design depicted in
FIG. 1). Tumor volume at time of treatment was similar among control and experimental groups.
Kaplan-Meier survival analysis was performed to assess survival function, change in tumor volume
was measured to determine tumor growth rates, and frequency of complete tumor regression was
evaluated by determining the percentage of tumor-free mice. Percent complete regression (% CR)
was also stratified by tumors that exceeded at least 100 mm.sup.3 before regressing. Statistical
significance was determined using Mantel Cox Log Rank test for survival, 2-way ANOVA for tumor
volume, and Fisher's exact test for CR. Prism reports results as non-significant (ns) at P>0.05,
significant (symbolized by “*”) at 0.01<P≤0.05, very significant (“**”) at 0.001<P≤0.01, extremely
significant (“***”) at P≤0.001, and highest significance (“****”) at P≤0.0001. Mice that did not develop
tumors or that developed tumor ulceration before reaching a tumor volume of 700 mm.sup.3 were
excluded from analysis.

(180) A synergistic effect on tumor growth delay was seen for the combined ENT and anti-
SEMA4D/MAb 67 administered group (782% maximal tumor growth delay (TGD)) compared to groups
administered either agent alone: 107% TGD with SEMA4D/MAb 67 and 214% TGD with ENT (FIG.
2A). ENT and anti-SEMA4D/MAb 67 treated mice also exhibited a significant improvement in survival
compared to mice treated with ENT or anti-SEMA4D/MAb 67 (p<0.05) (FIG. 2B). Furthermore,
combined ENT and anti-SEMA4D/MAb 67 treatment significantly increased the frequency of complete
tumor regression (62%***), especially among tumors that exceeded 100 mm.sup.3 before regressing,
compared to either single agent (FIGS. 2C and 2D). These results show that treatment with ENT and
anti-SEMA4D MAb improved survival, reduced tumor growth, and resulted in regression of larger
established tumors.

Example 2: Administration of ENT and Anti-SEMA4D Antibody Enhances Survival, Reduces Tumor
Growth, and Increases Frequency of Complete Regression

(181) Colon26 cells (500,000 cells) were implanted subcutaneously into Balb/c mice. Mice were
treated with 1) Control Ig (10 mg/kg, weekly×5 weeks, starting on day 2 (n=12)); 2) Control Ig (10
mg/kg, weekly×5 weeks, starting on day 2) and ENT (20 mg/kg, 3×/week×2 weeks, initiated when
average tumor volume ˜250 mm.sup.3) (n=9); 3) anti-SEMA4D/MAb 67 (10 mg/kg, IP, weekly×5
weeks, starting on day 2) (n=8); or 4) anti-SEMA4D/MAb 67 (10 mg/kg, weekly×5 weeks, starting on
day 2) and ENT (20 mg/kg, 3×/week×2 weeks, initiated when average tumor volume ˜250 mm.sup.3)
(n=13). Tumor volume at the time of treatment was similar among control and experimental groups.
Mean tumor volume, Kaplan Meier Survival curves, and frequency of complete tumor regression are
shown for each group. Statistical significance was determined using 2-way ANOVA, Mantel Cox Log
Rank test, and Fisher's exact test, respectively. Prism reports results as non-significant (ns) at P>0.05,
significant (symbolized by “*”) at 0.01<P≤0.05, very significant (“**”) at 0.001<P≤0.01, and extremely
significant (“***”) at P≤0.001. Mice that did not develop tumors or that developed tumor ulceration
before reaching a tumor volume of 700 mm.sup.3 were excluded from analysis.
(182) The mice that received ENT and anti-SEMA4D/MAb 67 exhibited maximal 705% TGD, while
mice treated with anti-SEMA4D/MAb67 showed a 119% delay in tumor growth (FIG. 3A). Combined
treatment of mice with ENT and anti-SEMA4D/MAb 67 also increased the frequency of complete
tumor regression (62%**), compared to Control Ig and ENT treatment groups (25%*) (FIGS. 3B-3D).
Moreover, mice that received ENT and anti-SEMA4D/MAb 67 exhibited a significant improvement in
survival compared to mice treated with Control Ig (P≤0.001) or Control Ig and ENT (p<0.01) (FIG. 3E).
These results demonstrate that a combined treatment with ENT and anti-SEMA4D MAb resulted in
enhanced survival, decreased tumor growth, and eradication or tumors.

Example 3: Administration of Azacytidine (AZA) and Anti-SEMA4D Antibody Increases Survival,

Significantly Delays Tumor Growth, and Increases Percent Complete Tumor Regression

(183) The effect of combined AZA and anti-SEMA4D MAb administration on survival, tumor growth,
and tumor regression was tested in a tumor mouse model. Colon26 cells (500,000 cells) were
implanted subcutaneously into Balb/c mice. Mice were treated with: Group 1: control Ig (10 mg/kg, ip,
weekly×4, starting on day 2 (n=12)); Group 2: anti-SEMA4D/MAb 67 (10 mg/kg, ip, weekly×4, starting
on day 2 (N=8)); Group 3: control Ig (10 mg/kg, ip, weekly×4, starting on day 2+AZA (0.8 mg/kg,
3×/week×4 doses, starting on day 22-24, when average tumor volume is ˜200 mm.sup.3 (n=10)); and
Group 4: anti-SEMA4D/MAb 67 (10 mg/kg, ip, weekly×4, starting on day 2+AZA (0.8 mg/kg,
3×/week×4 doses, starting on day 22-24, when average tumor volume is ˜200 mm.sup.3 (n=9)). The
Experimental Design is depicted in FIG. 4). The mice were monitored for 60 days or until the tumor
volumes reached about 1,500 mm.sup.3.

(184) Tumor volumes at time of treatment were similar among control and experimental groups.
Kaplan-Meier survival analysis was performed to assess survival function, change in tumor volume
was measured to determine tumor growth rates, and frequency of complete tumor regression was
evaluated by determining the percentage of tumor-free mice. Statistical significance was determined
using Mantel Cox Log Rank test for survival, 2-way ANOVA for tumor volume, and Fisher's exact test
for complete tumor regression. Prism reports results as non-significant P>0.05, significant (symbolized
by “*”) at 0.01<P≤0.05, or very significant (“**”) at 0.001<P≤0.01. Mice that did not develop tumors or
that developed tumor ulceration before reaching a tumor volume of 700 mm.sup.3 were excluded from
analysis.

(185) The mice that received AZA and anti-SEMA4D/MAb 67 exhibited maximal 705% TGD, while
mice treated with anti-SEMA4D/MAb67 showed a 119% delay in tumor growth (FIG. 5A). AZA and
anti-SEMA4D/MAb 67 treated mice also exhibited a significant improvement in survival compared to
control mice (p<0.001) and nearly significant improvement in survival compared to single agent anti-
SEDMA4D/MAb67 (p=0.0697) (FIG. 5B). Furthermore, combined AZA and anti-SEMA4D/MAb 67
treatment significantly increased the frequency of complete tumor regression to 78% (p<0.01),
compared to Control Ig (17%) (FIG. 5C). These results show that treatment with AZA and anti-
SEMA4D MAb improved survival, reduced tumor growth, and resulted in regression of larger
established tumors.

(186) The breadth and scope of the present disclosure should not be limited by any of the above-
described exemplary embodiments, but should be defined only in accordance with the following claims
and their equivalents.

(187) TABLE-US-00004 TABLE 4 Sequences SEQ ID NO Description Sequence 1 VX15/2503

QVQLVQSGAEVKKPGSSVKVSCKASGYSFSDYYMHW VH
VRQAPGQGLEWMGQINPTTGGASYNQKFKGKATITV
DKSTSTAYMELSSLRSEDTAVYYCARYYYGRHFDVW GQGTTVTVSS 2 VX15/2503 GYSFSDYYMH
HCDR1 3 VX15/2503 QINPTTGGASYNQKFKG HCDR2 4 VX15/2503 YYYGRHFDV HCDR3 5
VX15/2503 DIVMTQSPDSLAVSLGERATINCKASQSVDYDGDSYM VL
NWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTD
FTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK 6 VX15/2503 KASQSVDYDGDSYMN LCDR1 7
VX15/2503 AASNLES LCDR2 8 VX15/2503 QQSNEDPYT LCDR3 9 Mab 67 VH
QVQLQQSGPELVKPGASVKISCKASGYSFSDYYMHW
VKQSPENSLEWIGQINPTTGGASYNQKFKGKATLTVD
KSSSTAYMQLKSLTSEESAVYYCTRYYYGRHFDVWG QGTTVTVSS 10 Mab 67 VL
DIVMTQSPASLAVSLGQRATISCKASQSVDYDGDSYM
NWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDF
TLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK

Claims

1. A method for inhibiting, delaying, or reducing malignant cell growth in a subject with colon cancer,
comprising administering to the subject a combination therapy comprising an effective amount of an
isolated antibody or antigen-binding fragment thereof that specifically binds to semaphorin-4D
(SEMA4D) and an effective amount of an epigenetic modulating agent selected from Entinostat
(Pyridin-3-ylmethyl N-[[4-[(2-aminophenyl) carbamoyl] phenyl] methyl]carbamate), azacytidine and a
combination thereof, wherein the antibody or fragment thereof comprises a variable heavy chain (VH)
comprising VH CDRs 1-3 comprising SEQ ID NOS: 2, 3, and 4, respectively, and a variable light chain
(VL) comprising VL CDRs 1-3 comprising SEQ ID NOS: 6, 7, and 8, respectively.

2. The method of claim 1, wherein the antibody or fragment thereof inhibits SEMA4D interaction with
its receptor.

3. The method of claim 1, wherein the antibody or fragment thereof inhibits SEMA4D-mediated Plexin-
B1 signal transduction.

4. The method of claim 1, wherein the VH and VL comprise, respectively, SEQ ID NO: 1 and SEQ ID
NO: 5, or SEQ ID NO: 9 and SEQ ID NO: 10.

5. The method of claim 1, wherein the epigenetic modulating agent is Entinostat (Pyridin-3-ylmethyl N-
[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate).

6. The method of claim 1, wherein the epigenetic modulating agent comprises azacytidine.

7. The method of claim 1, wherein: the isolated antibody or antigen-binding fragment thereof that
specifically binds to semaphorin-4D (SEMA4D) comprises a VH comprising the amino acid sequence
SEQ ID NO: 1 and a VL comprising the amino acid sequence SEQ ID NO: 5, and the epigenetic
modulating agent comprises the HDACi Entinostat or the DNMTi azacytidine.

8. A pharmaceutical composition for the treatment of colon cancer comprising an effective amount of
an isolated antibody or antigen-binding fragment thereof that specifically binds to semaphorin-4D
(SEMA4D) and an effective amount of an epigenetic modulating agent selected from Entinostat
(Pyridin-3-ylmethyl N-[[4-[(2-aminophenyl) carbamoyl] phenyl] methyl]carbamate), azacytidine and a
combination thereof, wherein the antibody or fragment thereof comprises a variable heavy chain (VH)
comprising VH CDRs 1-3 comprising SEQ ID NOS: 2, 3, and 4, respectively, and a variable light chain
(VL) comprising VL CDRs 1-3 comprising SEQ ID NOS: 6, 7, and 8, respectively.

9. The pharmaceutical composition of claim 8, wherein the VH and VL comprise, respectively, SEQ ID
NO: 1 and SEQ ID NO: 5, or SEQ ID NO: 9 and SEQ ID NO: 10.

10. The pharmaceutical composition of claim 8, wherein the epigenetic modulating agent is Entinostat
(Pyridin-3-ylmethyl N-[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate).

11. The pharmaceutical composition of claim 8, wherein the epigenetic modulating agent is
azacytidine.

12. A pharmaceutical composition for the treatment of colon cancer comprising: an effective amount of
an isolated antibody or antigen-binding fragment thereof that specifically binds to semaphorin-4D
(SEMA4D), wherein the antibody or fragment thereof comprises the VH amino acid sequence SEQ ID
NO: 1 and the VL amino acid sequence SEQ ID NO: 5, and an effective amount of the HDACi
Entinostat or an effective amount of the DNMTi azacytidine.
Sequence Listing

CWU

SEQUENCE LISTING NEW RULES

101118PRTArtificial SequenceVX15/2503 VH 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Asp Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gln Ile Asn Pro Thr Thr Gly Gly
Ala Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Tyr Tyr
Gly Arg His Phe Asp Val Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser 115210PRTArtificial
SequenceVX15/2503 HCDR1 2Gly Tyr Ser Phe Ser Asp Tyr Tyr Met His1 5 10317PRTArtificial
SequenceVX15/2503 HCDR2 3Gln Ile Asn Pro Thr Thr Gly Gly Ala Ser Tyr Asn Gln Lys Phe Lys1 5 10
15Gly49PRTArtificial SequenceVX15/2503 HCDR3 4Tyr Tyr Tyr Gly Arg His Phe Asp Val1
55111PRTArtificial SequenceVX15/2503 VL 5Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30Gly Asp Ser
Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu
Glu Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65
70 75 80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110615PRTArtificial SequenceVX15/2503 LCDR1
6Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn1 5 10 1577PRTArtificial
SequenceVX15/2503 LCDR2 7Ala Ala Ser Asn Leu Glu Ser1 589PRTArtificial SequenceVX15/2503
LCDR3 8Gln Gln Ser Asn Glu Asp Pro Tyr Thr1 59118PRTArtificial SequenceMab 67 VH 9Gln Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Ser Phe Ser Asp Tyr 20 25 30Tyr Met His Trp Val Lys Gln Ser Pro Glu Asn Ser Leu Glu Trp Ile 35
40 45Gly Gln Ile Asn Pro Thr Thr Gly Gly Ala Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr
Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Lys Ser Leu Thr Ser Glu Glu Ser
Ala Val Tyr Tyr Cys 85 90 95Thr Arg Tyr Tyr Tyr Gly Arg His Phe Asp Val Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser 11510111PRTArtificial SequenceMab 67 VL 10Asp Ile Val Met Thr Gln Ser
Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp
Tyr Asp 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu
Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser
Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110