RU2783535C2 - Treatment of cancer, using antibody to semaphorin 4d in combination with epigenetic modulating agent - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including a pharmaceutical composition for inhibition, delay, or reduction in growth of malignant cells in subject suffering from cancer (options), and a method for inhibition, delay, or reduction in growth of malignant cells in a subject suffering from cancer, including administration to the subject of the above-mentioned pharmaceutical composition. In one of implementations, the pharmaceutical composition contains effective amount of an antibody or its antigen-binding fragment, specifically binding to semaphorin 4D (SEMA4D), and epigenetic modulating agent selected from entinostat and azacytidine.

EFFECT: invention expands the arsenal of means for inhibition, delay, or reduction in growth of malignant cells in a subject suffering from cancer.

26 cl, 12 dwg, 4 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Application No. 62/473,731, filed March 20, 2017, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING STATEMENT

[0002] The contents of the electronically submitted sequence listing in an ASCII text file (name: 58008_172854_Seq-List_ST25.txt; size: 5936 bytes; creation date: February 28, 2018) filed with the application are incorporated herein by reference in their entirety .

PRIOR ART

[0003] Semaphorin 4D (SEMA4D), also known as CD100, is a transmembrane protein belonging to the semaphorin gene family. SEMA4D is expressed on the cell surface as a homodimer, but once activated, SEMA4D can be released from the cell surface via proteolytic cleavage to form sSEMA4D, a soluble form of the protein that is also biologically active. See Suzuki et al., Nature Rev. Immunol. 3:159-167 (2003); Kikutani et al., Nature Immunol. 9:17-23 (2008).

[0004] SEMA4D is highly expressed 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 widely expressed on resting T cells, but weakly expressed on resting B cells and antigen presenting cells (APCs) such as dendritic cells (DCs). However, its expression in these cells is activated after activation by various immunological stimuli. The release of soluble SEMA4D from immune cells is also increased by cell activation. SEMA4D has been shown to be involved in several cancers (Ch'ng et al., Cancer 110:164-72 (2007); Campos et al., Oncology Letters 5:1527-35 (2013); Kato et al., Cancer Sci. /02:2029-37 (2011)), and several reports suggested that one of the mechanisms of this influence is the role of SEMA4D in the development of 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 75:391-407 (2012 )). Tumor growth and metastasis involve a complex process of cross-talk between tumor cells, stroma and immune infiltrate, as well as endothelial cells and the vasculature. SEMA4D is overexpressed in a variety of tumor types and is also produced by inflammatory cells recruited into the tumor microenvironment. Recent studies suggest that SEMA4D plays a role in the migration, survival, differentiation, and organization of the various cell types that make up the tumor stroma (Evans et al., Cancer Immunol. Res. 3:689-701 (2015)).

[0005] To avoid recognition by the host's immune system, cancer cells can adapt by modifying expressed genes through epigenetic mechanisms, without altering the genomic DNA sequence. Epigenetic mechanisms are mainly focused on changes in genomic DNA methylation and post-translational histone modifications, leading, for example, to changes in chromatin structure and the availability of DNA for transcription. Through these epigenetic mechanisms, cancer cells can create altered inherited gene expression patterns that promote proliferation and evade recognition by the immune system. See, for example, Maio, et al., Clin. Cancer Res. 27:4040-4047 (2015).

[0006] Cells can control the coiling and unwinding of DNA around histones by histone acetyltransferases (HATs), which acetylate lysine residues in core histones, resulting in less compact and more transcriptionally active chromatin, and histone deacetylases (HDACs), which remove acetyl groups from lysine residues , leading to the formation of transcription-repressed condensed chromatin. Modulation of HAT/HDAC activity by tumor cells can lead to extensive epigenetic modulation of gene expression in tumor cells, allowing the cells to evade surveillance by the patient's immune system. See, for example, Maio, et al., Clin. Cancer Res. 27:4040-4047 (2015).

[0007] The possibility of using histone deacetylase inhibitors (HDACi (used in the present application both in the singular and in the plural)) as agents for the treatment of cancer has become known. HDACi can inhibit tumor cell proliferation by causing extensive transcriptional changes, activating and/or repressing various genes through modulation of histone acetylation/deacetylation 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, such as cell cycle, differentiation and apoptosis of cancer cells. HDACi are also able to enhance immunogenicity and antigen presentation by tumor cells, which justifies the combination of HDACi with immunotherapy (Gameiro SR et al., Oncotarget 7:7390-7402 (2016)). Several compounds are currently in the early stages of clinical research as potential agents for the treatment of solid and hematologic cancer, either alone or in combination with cytotoxic and differentiating agents, see e.g. Mottamal, M., et al., Molecules 20:3898-3941 (2015).

[0008] Genomic DNA methylation is a well-characterized epigenetic modification. DNA methylation leads to transcriptional passivity leading to 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. See same source. Hypermethylated DNA is often found in tumor and other cancer cells. Currently, various DNA methyltransferase inhibitors (DNMTi (used in both singular and plural in this application)) are being used or are being studied for the treatment of various types of cancer. These include deoxyribonucleoside analogs such as azacitidine and non-nucleoside analogs such as antisense oligonucleotides and small molecule enzyme inhibitors. See same source.

BRIEF DESCRIPTION OF THE INVENTION

[0009] The present invention relates to a method for inhibiting, delaying, or reducing the growth of malignant cells in a subject suffering from cancer, comprising administering to said subject a combination therapy comprising an effective amount of an isolated antibody or an antigen-binding fragment thereof that specifically binds to semaphorin 4D (SEMA4D), and an effective amount of an epigenetic modulating agent. According to the proposed method, the antibody to SEMA4D or its fragment can inhibit the interaction of SEMA4D with its receptor, for example, plexin B1, plexin B2 or CD72. For example, in the proposed method, an anti-SEMA4D antibody, or fragment thereof, can inhibit SEMA4D-mediated plexin B1 signaling.

[0010] In certain aspects, an anti-SEMA4D antibody or fragment thereof comprises a heavy chain variable region (VH) comprising VH CDRs 1-3 having the amino acid sequences of SEQ ID NOs: 2, 3 and 4, respectively, and a light chain variable region (VL ) comprising VL CDRs 1-3 having the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively. In certain aspects, the amino acid sequences of VH and VL include SEQ ID NO: 1 and SEQ ID NO: 5, or SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0011] In certain aspects, an epigenetic modulating agent may include a histone deacetylase (HDAC) inhibitor (HDACi), a DNA methyltransferase (DNMT) inhibitor (DNMTi), or any combination thereof.

[0012] In certain aspects, an epigenetic modulating agent may include a histone deacetylase inhibitor (HDAC) (HDACi). In certain aspects, the HDAC may be a Class I HDAC, Class IIA HDAC, Class PV HDAC, Class IV HDAC, or any combination thereof, or the HDAC may include a zinc-containing catalytic domain. In certain aspects, HDACi can bind to the zinc-containing catalytic domain of HDAC. In certain aspects, HDACi may include a chemical moiety selected from the group consisting of 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, HDACi may 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, HDACi is an entinostat (pyridin-3-ylmethyl-N-[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate).

[0013] In certain aspects, an epigenetic modulating agent may include a DNA methyltransferase (DNMT) inhibitor (DNMTi). In certain aspects, the DNMT may be DNMT1, DNMT-3a, DNMT-3b, or any combination thereof. In certain aspects, DNMTi can be a nucleoside analog, an antisense oligonucleotide, a small molecule enzyme inhibitor, or any combination thereof. For example, in certain aspects, DNMTi may be azacitidine, decitabine, zebularine, SGI-110, epigallocatechin gallate, MG98, RG108, procainamide, hydralazine, any combination thereof, or any salt thereof, crystal, amorphous structure, hydrate, derivative, metabolite, isomer or a prodrug. In certain aspects, DNMTi is azacitidine.

[0014] In a particular aspect of the proposed method, the isolated antibody or its antigen-binding fragment that specifically binds to semaphorin 4D (SEMA4D) contains a VH containing the amino acid sequence of SEQ ID NO: 1, and a VL containing the amino acid sequence of SEQ ID NO: 5, and an epigenetic the modulating agent includes an HDACi entinostat. In another particular aspect of the proposed method, the isolated antibody or its antigen-binding fragment that specifically binds to semaphorin 4D (SEMA4D) contains a VH containing the amino acid sequence of SEQ ID NO: 1 and a VL containing the amino acid sequence of SEQ ID NO: 5, and an epigenetic modulating agent includes DNMTi azacitidine.

[0015] According to the proposed method, the antibody or antigen-binding fragment and the epigenetic modulating agent can be administered separately, or they can be administered simultaneously.

[0016] In some aspects of the proposed method, the introduction of combination therapy can lead to an increase in therapeutic efficacy compared with the introduction of the antibody or its fragment or epigenetic modulating agent alone. For example, in some aspects, the increased therapeutic efficacy outweighs the additive effect, such as a synergistic effect.

[0017] In certain aspects of the proposed method, the cancer to be treated may be a solid tumor, a hematological malignancy, any of their metastases, or any combination of them. In certain aspects, a solid tumor may be, for example, a sarcoma, carcinoma, melanoma, any of their metastases, or any combination thereof. More specifically, the solid tumor may be, for example, squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, renal cell carcinoma, ductal breast carcinoma, soft tissue sarcoma, osteosarcoma, melanoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, peritoneal cancer. , 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, vulvar cancer, thyroid cancer, head and neck cancer, any metastases thereof, or any combination thereof.

[0018] In certain aspects of the proposed method, the cancer to be treated may be a hematological malignancy or its metastases. In certain aspects, the hematologic malignancy may be leukemia, lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, any of their metastases, or any combination thereof. .

[0019] In some aspects, the method proposed in this application may further include the introduction of additional anti-cancer therapy, for example, surgery, chemotherapy, radiation therapy, cancer vaccine, administration of an immunostimulatory agent, adoptive T-cell therapy or antibody therapy, administration of an inhibitor, blocking immune checkpoints, the introduction of a modulator of regulatory T cells (Treg) and their combination.

[0020] This invention also relates to a pharmaceutical composition comprising an effective amount of an isolated antibody or antigen-binding fragment specifically binding to semaphorin 4D (SEMA4D) and an effective amount of an epigenetic modulating agent. In certain aspects, the pharmaceutical composition may further include a carrier, excipient, or any combination thereof.

[0021] In some aspects, the antibody or its fragment in the composition of the proposed composition may include a heavy chain variable region (VH) containing CDR 1-3 VH containing SEQ ID NO: 2, 3 and 4, respectively, and a light chain variable region ( VL) containing CDR 1-3 VL containing SEQ ID NO: 6, 7 and 8, respectively. In some aspects, the antibody or its fragment in the proposed composition may include VH and VL, which include, respectively, SEQ ID NO: 1 and SEQ ID NO: 5 or SEQ ID NO: 9 and SEQ ID NO: 10.

[0022] In some aspects, the epigenetic modulating agent in the composition of the proposed pharmaceutical composition may include a histone deacetylase (HDAC) inhibitor (HDACi), a DNA methyltransferase (DNMT) inhibitor (DNMTi), or any combination thereof.

[0023] When the composition includes HDACi, it can be, for example, vorinostat, romidepsin, chidamide, panobinostat, belinostat, valproic acid or a salt thereof, mocetinostat, abexinostat, entinostat, pracinostat, resminostat, givinostat, quisinostat, kevethrin, CUDC-101, AR-42, tefinostat (CHR-2845), CHR-3996, 4SC-202, CG200745, ACY-1215, ACY-241, any combination thereof or any salt thereof, crystal, amorphous structure, hydrate, derivative, metabolite, isomer, or prodrug. In certain aspects, the HDACi may be an entinostat (pyridin-3-ylmethyl-N-[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate).

[0024] When the composition includes DNMTi, it can be, for example, azacitidine, 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. In certain aspects, DNMTi may be azacitidine.

[0025] In a particular aspect, the invention relates to a pharmaceutical composition comprising an effective amount of an isolated antibody or antigen-binding fragment specifically binding to semaphorin 4D (SEMA4D), where the antibody or fragment comprises a VH amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of VL, which is SEQ ID NO: 5; and an effective amount of HDACi entinostat. In yet another particular aspect, the invention relates to a pharmaceutical composition comprising an effective amount of an isolated antibody or antigen-binding fragment specifically binding to semaphorin 4D (SEMA4D), where the antibody or fragment comprises the amino acid sequence VH, which is SEQ ID NO: 1, and the amino acid a VL sequence which is SEQ ID NO: 5; and an effective amount of DNMTi of azacitidine.

BRIEF DESCRIPTION OF GRAPHICS / FIGURES

[0026] FIG. 1 shows the experimental design for the combination therapy study in Examples 1 and 2.

[0027] FIG. 2A shows the change in mean tumor volume over time for different treatment options in Example 1.

[0028] FIG. 2B shows the change in percent survival over time for various treatments in Example 1. Statistical significance was determined using the Cox-Mantel log-rank test. In Prism, the results are presented as non-significant (ns) at P>0.05, significant (indicated 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.

[0029] FIG. 2C is a bar graph showing percent complete tumor regression (tumor volume<50 mm ³ ) for various treatments in Example 1. Statistical significance was determined using Fisher's exact test, in Prism the results are reported as non-significant (ns) at P>0. 05, significant (marked with "*") 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.

[0030] FIG. 2D shows the same data as in FIG. 2C stratified by whether the tumor volume exceeded at least 100 mm ³ before regression.

[0031] FIG. 3A shows the change in mean tumor volume over time for various treatments in Example 2. Statistical significance was determined using two-way ANOVA. Results are reported in Prism as non-significant (ns) at P>0.05, significant (marked with "*") at 0.01<P≤0.05, very significant ("**") at 0.001<P≤0.01 .

[0032] FIG. 3B shows the time course of tumor volume for selected control animals in Example 2.

[0033] FIG. 3C shows the evolution of tumor volume over time for individual animals treated with anti-SEMA4D antibody in Example 2.

[0034] FIG. 3D shows the time course of tumor volume for individual animals treated with entinostat (ENT) and control antibody in Example 2.

[0035] FIG. 3E shows the evolution of tumor volume over time for individual animals treated with ENT and anti-SEMA4D antibody in Example 2.

[0036] FIG. 3F shows the change in percent survival over time for various treatments in Example 2. Statistical significance was determined using the Cox-Mantel log-rank test. Results are presented in Prism as significant (indicated by "*") at 0.01<P≤0.05 and very significant ("**") at 0.001<P≤0.01.

[0037] FIG. 4 shows the experimental design for the combination therapy study in Example 3.

[0038] FIG. 5A shows the change in mean tumor volume over time for various treatments in Example 3. Statistical significance was determined using two-way ANOVA. ** P≤0.01.

[0039] FIG. 5B shows the change in percent survival over time for various treatments in Example 3. Statistical significance was determined using the Cox-Mantel log-rank test. In Prism, results are reported as non-significant (ns) at P>0.05 or very significant ("**") at P≤0.01.

[0040] FIG. 5C is a bar graph showing percent complete tumor regression (tumor volume <50 mm ³ ) for various treatments in Example 3. Statistical significance was determined using Fisher's exact test, in Prism the results are presented as significant (indicated by "*") at P ≤0.05 or very significant (“**”) at P≤0.01.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0041] It should be noted that a term denoting an object in the singular refers to one or more specified objects; for example, a "binding molecule" is meant to be one or more binding molecules. Accordingly, the terms in the singular, the terms "one or more" and "at least one" can be used interchangeably in this application.

[0042] In addition, when used in this application, "and/or" should be considered as a specific description of each of the two specified features or components with or without the second. Thus, the term "and/or" used in this application in a phrase such as "A and/or B" is intended to include "A and B", "A or B", "A" (separately) and "B" (separately). Likewise, the term "and/or" as used herein in a phrase such as "A, B and/or C" is intended to include 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 (separately); B (separately); and C (separately).

[0043] Unless otherwise indicated, all technical and scientific terms used in this application have the meaning of the generally accepted meaning known to a person skilled in the art to which the present invention pertains. For example, in 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 Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, a general glossary of a large number of terms used in the description is provided for the skilled person.

[0044] Units, prefixes and symbols are given in their common form according to the Systeme International de Unites (SI system). Numeric ranges include numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino-terminal to carboxy-terminal orientation. The headings given in this application are not intended to limit the various aspects of the invention that may be embodied with reference to the description as a whole. Accordingly, the terms defined below are more fully defined by reference to the description as a whole.

[0045] Wherever embodiments are described using the term "comprising", similar embodiments described using the terms "consisting of" and/or "essentially consisting of" are also contemplated.

[0046] Amino acids are designated in this application by their well-known three-letter symbols or one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Nucleotides are also designated by their common single letter codes.

[0047] As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals in which a cell population is characterized by uncontrolled cell growth. Cancers can be classified, for example, as solid tumors or malignancies, or hematological cancers or malignancies. Both types can migrate to distant sites in the body as metastases. A solid tumor may be classified, for example, as a sarcoma, carcinoma, melanoma, or metastases thereof.

[0048] The terms "proliferative disorder" and "proliferative disease" refer to disorders associated with abnormal cell proliferation, such as cancer.

[0049] The terms "tumor" and "neoplasm" in the context of this application refer to any mass of tissue resulting from excessive growth or proliferation of cells, either benign (noncancerous) or malignant (cancerous), including precancerous lesions. In certain embodiments, the tumors described herein express the SEMA4D receptor, eg, plexin B1, plexin B2, and/or CD72, and/or may express SEMA4D.

[0050] The terms "metastasis", "metastases", "metastatic" and other grammatical equivalents in the context of this application refer to cancer cells that spread or move from their place of origin (for example, the primary tumor) to other areas of the body with the development of a similar cancerous lesion in a new place. A "metastatic" or "metastasizing" cell is a cell that loses adhesive contacts with neighboring cells and migrates through the bloodstream or lymph from the primary site of the disease, penetrating into neighboring body structures. The terms also refer to the process of metastasis, which includes, but is not limited to, separation of cancer cells from the primary tumor, entry of tumor cells into the bloodstream, their survival and migration to a distant site, attachment and exit to a new site from the bloodstream, and microcolonization at a remote site, and also the growth and development of a tumor in a distant place.

[0051] Examples of such solid tumors may include, for example, squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, renal cell carcinoma, ductal breast carcinoma, soft tissue sarcoma, osteosarcoma, melanoma, small cell lung cancer, non-small cell lung cancer (NSCLC), adenocarcinoma lung, peritoneal cancer, 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, vulvar cancer, thyroid cancer, head and neck cancer, any metastases thereof, or any combination thereof.

[0052] Examples of hematologic cancers or malignancies include, but are not limited to, leukemia, lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, any of their metastases or any combination of them.

[0053] In certain embodiments, cancers treatable with the methods provided herein include, but are not limited to, sarcomas, breast carcinomas, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, esophageal cancer, stomach cancer, kidney cancer, liver cancer, bladder cancer, colorectal cancer and pancreatic cancer. In certain embodiments, cancer or tumor cells treatable by the methods provided herein express plexin B1, plexin B2, or CD72 receptors for SEMA4D.

[0054] As used herein, the term "polypeptide" is intended to include "polypeptide" in the singular as well as several "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). connections). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific product length. 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 covered by the definition of "polypeptide" and the term "polypeptide" may 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 said polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation and derivatization with known protecting/blocking groups, proteolytic cleavage, or modification by unnatural amino acids. The polypeptide may be derived from a natural biological source or obtained using recombinant technology, but is not necessarily translated from a given nucleic acid sequence. It can be obtained by any method, including chemical synthesis.

[0055] A polypeptide described herein may have 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, 1000 or more, or 2000 or more amino acids. Polypeptides may have a specific three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are called folded, and polypeptides that do not have a defined three-dimensional structure, but are able to take on a large number of different conformations, are called unfolded. In the context of the present application, the term "glycoprotein" refers to a protein associated with at least one carbohydrate fragment attached to the protein through an oxygen-containing or nitrogen-containing amino acid side chain, for example, serine or asparagine.

[0056] By "isolated" polypeptide or fragment, variant or derivative thereof is meant a polypeptide that is not found in its natural environment. No specific level of cleansing is required. For example, an isolated polypeptide may simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of this application, as are native or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified using any suitable technique.

[0057] In the context of this application, the term "non-naturally occurring polypeptide" or any grammatical variants thereof is a conditional definition that expressly excludes only those forms of the polypeptide that are defined or can be defined or interpreted by a judge or an administrative or judicial authority as " found in nature."

[0058] Other polypeptides disclosed in this application are fragments, derivatives, analogs or variants of the above polypeptides and any combination thereof. The terms "fragment", "variant", "derivative" and "analogue", as disclosed in this application, include any polypeptides that retain at least some of the properties of the corresponding native antibody or polypeptide, for example, specific binding to an antigen. Polypeptide fragments include, for example, proteolytic fragments as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere in this application. Variants, for example, of a polypeptide include fragments as described above, as well as polypeptides with amino acid sequences altered due to amino acid substitutions, deletions, or insertions. In some aspects, the variants may not be naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives are polypeptides that have been modified to acquire additional features that the original polypeptide does not have. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogues". In the context of the present application, the term "derivative" of a polypeptide may also refer to the subject polypeptide in which one or more amino acids have been chemically derivatized by reaction of a functional side group. Also included as "derivatives" are peptides containing one or more derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace series; and ornithine can replace lysine.

[0059] A "conservative amino acid substitution" is one in which one amino acid is replaced by another amino acid with a similar side chain. Families of amino acids with similar side chains have been identified 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), non-polar 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 (eg tyrosine, phenylalanine, tryptophan, histidine). For example, the replacement of tyrosine with phenylalanine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present invention do not abolish the binding of the polypeptide or antibody containing the amino acid sequence to the antigen to which the binding molecule binds. Methods for identifying nucleotide and amino acid conservative substitutions that do not abolish antigen binding are well known in the art (see, for example, 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)).

[0060] The term "polynucleotide" is intended to encompass a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). The polynucleotide may contain a conventional phosphodiester bond or a non-covalent bond (eg, an amide bond such as that found in peptido nucleic acids (PNAs)). The terms "nucleic acid" or "nucleic acid sequence" refer to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0061] By "isolated" nucleic acid or polynucleotide is meant any form of nucleic acid or polynucleotide that is separated from its native environment. For example, a polynucleotide purified by gel permeation chromatography or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered "isolated". In addition, a polynucleotide segment, such as a PCR product, that has been designed to have restriction sites for cloning is considered "isolated". Additional 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 buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides where the transcript is not a naturally occurring transcript. Isolated polynucleotides or nucleic acids further include such synthetically produced molecules. In addition, the polynucleotide or nucleic acid may be or may include a regulatory element such as a promoter, a ribosome binding site, or a transcription terminator.

[0062] In the context of this application, the term "non-naturally occurring polynucleotide" or any of its grammatical variants is a conditional definition that expressly excludes only those forms of a nucleic acid or polynucleotide that are defined or can be defined or interpreted by a judge or an administrative or judicial body as "naturally occurring".

[0063] As used herein, a "coding region" is a portion of a nucleic acid consisting 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 part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, and the like, are not part of the coding region. areas. The two or more coding regions may be in the same polynucleotide construct, eg on the same vector, or in separate polynucleotide constructs, eg on separate (different) vectors. In addition, any vector may contain a single coding region, or may contain two or more coding regions, for example, one vector may individually encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, the vector, polynucleotide, or nucleic acid may include heterologous coding regions, either fused or not fused to another coding region. Heterologous coding regions include, but are not limited to, specialized elements or motifs such as a secretory signal peptide or a heterologous functional domain.

[0064] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide containing a nucleic acid encoding a polypeptide may typically include a promoter and/or other transcription or translation control elements operably linked to one or more coding regions. A functional link is present when the region encoding a gene product, eg a polypeptide, is linked to one or more regulatory regions such that expression of said gene product is under the influence or control of said regulatory region(s). Two DNA fragments (such as a polypeptide-coding region and its associated promoter) are "operably linked" if induction of promoter function results in transcription of an mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not preclude the ability of the regulatory sequences to expression control the expression of the gene product, or does not interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably linked to a nucleic acid encoding a polypeptide if the promoter were capable of transcribing that nucleic acid. The promoter may be a cell-specific promoter that directs basic transcription of DNA in predetermined cells. Transcription control elements other than the promoter, such as enhancers, operators, repressors, and transcription termination signals, may be operably linked to the polynucleotide to direct cell-specific transcription.

[0065] Specialists in the art are aware of various areas of transcriptional control. Such regions include, but are not limited to, transcription control regions functioning in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (an early early promoter along with intron A), simian virus 40 (an early promoter), and retroviruses (such like the Rous sarcoma virus). Other regions of transcriptional control 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 transcriptional control regions include tissue-specific promoters and enhancers, as well as promoters inducible by lymphokines (eg, promoters induced by interferons or interleukins).

[0066] Likewise, many translation control elements are known to those skilled in the art. Such elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (specifically, the internal ribosome entry site or IRES, also referred to as the CITE sequence).

[0067] In other embodiments, the implementation of the polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA or ribosomal RNA.

[0068] Regions encoding polynucleotides and nucleic acids can be associated with additional coding regions that encode secretory or signal peptides that control the secretion of the polypeptide encoded by the polynucleotide, as disclosed in this application. According to the signal sequence hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein after export of the growing protein chain through the rough endoplasmic reticulum has begun. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells may have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the full or "full length" polypeptide to form the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide is used, for example, an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of this sequence, which retains the ability to direct the secretion of an operably linked polypeptide. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild type leader may be replaced with a human tissue plasminogen activator (TPA) or mouse B-glucuronidase leader.

[0069] Disclosed herein are individual binding molecules or antigen-binding fragments, variants or derivatives thereof. The term "binding molecule", when not specifically referring to full-length antibodies, embraces full-length antibodies as well as antigen-binding subunits, fragments, variants, analogs or derivatives of such antibodies, for example, engineered antibody molecules or fragments that bind antigen in the same way, like antibody molecules, but using a different framework.

[0070] In the context of this application, the term "binding molecule" refers in the broadest sense to a molecule that specifically binds to a receptor, such as an epitope or antigenic determinant. As described later in this application, the binding molecule may contain one or more of the "antigen-binding domains" described in this application. A non-limiting example of a binding molecule is an antibody or fragment thereof that retains antigen-specific binding capability.

[0071] In the context of this application, the terms "binding domain" or "antigen-binding domain" refer to the region of the binding molecule that is necessary and sufficient for specific binding to an epitope. For example, "Fv", eg, the heavy chain variable region and the light chain variable region of an antibody, either as two separate polypeptide subunits or as a single chain, is considered a "binding domain". Other binding domains include, but are not limited to, the heavy chain variable region (VHH) of an antibody derived from a camelid species or the six immunoglobulin complementarity determining regions (CDRs) expressed in a fibronectin scaffold. A "binding molecule" as described herein may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more "antigen binding domains".

[0072] The terms "antibody" and "immunoglobulin" in this application can be used interchangeably. An antibody (or fragment, variant or derivative thereof as disclosed herein) comprises at least a heavy chain variable domain (for camelid species) or at least a heavy chain and a light chain variable domain. The basic structures of immunoglobulins in vertebrate systems are relatively well understood. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ^ed . 1988). Unless otherwise indicated, the term "antibody" encompasses everything from a small antigen-binding fragment of an antibody to a full-length antibody, e.g., an IgG antibody including two full heavy chains and two full light chains, an IgA antibody including four full heavy chains and four full light chains. chain, and optionally including a J chain and/or a secretory component, or an IgM antibody, including ten or twelve full heavy chains and ten or twelve full light chains, and optionally including a J chain.

[0073] As discussed in more detail below, the term "immunoglobulin" includes various broad classes of biochemically distinct polypeptides. Those skilled in the art will recognize that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) and there are some subclasses among them (e.g., γ ¹ -γ ⁴ or α1-α2 ). It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA IgG, or IgE, respectively. Subclasses (isotypes) of immunoglobulins, eg IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , IgA ₂ , etc., are well characterized and are known to provide functional specialization. Modified versions of each of these classes and isotypes are readily discernible by one of ordinary skill in the art given the present disclosure and are therefore within the scope of the present invention.

[0074] Light chains are subdivided into kappa or lambda (κ, λ). Each heavy chain class can be linked to a kappa or lambda light chain. Typically, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin molecules are generated by hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequences run from the N-terminus at the branched ends of the Y-shaped configuration to the C-terminus at the bottom of each chain. The basic structure of individual antibodies, eg, IgG antibodies, includes two heavy chain subunits and two light chain subunits covalently linked via disulfide bonds to form a "Y" shaped structure, also referred to herein as an "H2L2" structure.

[0075] Both light and heavy chains are divided into areas of structural and functional homology. The terms "constant" and "variable" are used in relation to function. In this regard, it should be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the light chain (CL) and heavy chain (CH1, CH2 or CH3) constant domains confer biological properties such as secretion, transplacental transfer, Fc receptor binding, complement fixation, and the like. It is accepted that the domain numbers of the constant region increase with distance from the antigen-binding site or amino-terminus of an antibody. The N-terminus is the variable region and the C-terminus is the constant region; the CH3 (or CH4 in the case of IgM) and CL domains actually contain the carboxy terminus of the heavy and light chains, respectively.

[0076] As noted above, the variable region (i.e., "binding domain") allows the binding molecule to selectively recognize and specifically bind epitopes on antigens. That is, a VL domain and a VH domain, or a subset of complementarity determining regions (CDRs) of a binding molecule, such as an antibody, combine to form a variable region defining 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. Some antibodies form larger structures. For example, IgA may form a molecule comprising two H2L2 units, a J chain and a secretory component, all of which are covalently linked by disulfide bonds, and IgM may form a pentameric or hexamer molecule comprising five or six H2L2 units and optionally a J chain, covalently linked by disulfide bonds.

[0077] The six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous sequences of amino acids arranged in a specific manner to form the antigen-binding domain when an antibody assumes its three-dimensional configuration in an aqueous environment. The rest of the amino acids in the binding domain, called "framework" regions, exhibit less intermolecular variability. The framework regions generally adopt a B-sheet conformation, and the CDRs form loops that connect, and in some cases are part of, the B-sheet structure. Thus, the framework regions contribute to the formation of a framework that ensures that the CDRs are positioned in the correct orientation through interstrand non-covalent interactions. The binding domain formed by the occupied CDRs determines surface complementarity to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its recognized epitope. The amino acids constituting the CDRs and framework regions, respectively, can be easily identified for any given heavy or light chain variable region by one of skill in the art, as they have been determined in a variety of 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., 79 (5:901-917 (1987), incorporated herein by reference in its entirety).

[0078] In the event that there are two or more definitions of a term that is used and/or accepted in the art, the definition of the term used in this application is intended to include all such meanings, unless expressly stated otherwise. A specific example is the use of the term "complementarity determining region" ("CDR") to describe non-contiguous antigen-binding sites present in the variable region of both heavy and light chain polypeptides. These specific areas 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 Chothia et al., J. Mol. Biol 79(5:901-917 (1987), which are incorporated herein by reference. In comparison to each other, definitions according to Kabat and Chothia include overlapping amino acids or subgroups of amino acids. However, the use of any of these definitions (or other definitions known to those skilled in the art) to refer to the CDR of an antibody or variant thereof is within the scope of the term as defined and used herein unless otherwise indicated. Corresponding amino acids comprising a CDR as defined in each of the above sources are shown below in Table 1 for comparison.The exact numbers of residues comprising a particular CDR will vary depending on the sequence and size of the CDR.It can be routinely determined by those skilled in the art which amino acid residues contain a particular CDR, given the amino acid sequence of the variable region antibodies.

[0079] Kabat et al. also defined a variable domain sequence numbering system that is applicable to any antibody. One skilled in the art can unambiguously apply this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. In the context of this application, the term "numbering according to Kabat" refers to the numbering system established by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest") (1983). However, unless explicitly stated to use the Kabat numbering system, sequential numbering is used for all amino acid sequences in this application.

[0080] Binding molecules, for example, 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, for example, Fab, Fab' and F(ab') ₂ , Fd, Fvs, single chain Fv (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), VL or VH domain containing fragments, Fab expression library derived fragments. ScFv molecules are known in the art and are described, for example, in US Pat. for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or a subclass of the immunoglobulin molecule.

[0081] By "specifically binds" is generally meant that the binding molecule, e.g., an antibody or fragment, variant or derivative thereof, binds to the epitope via its antigen-binding domain, and that the binding implies some degree of complementarity between the antigen-binding domain and the epitope. By this definition, a binding molecule "specifically binds" to an epitope when it binds to that epitope through its antigen-binding domain more easily than it binds to a random, unrelated epitope. The term "specificity" is used in this application to define the relative affinity with which a certain binding molecule binds to a certain epitope. For example, the "A" binding molecule is considered to have a higher specificity for a given epitope than the "B" binding molecule, or the "A" binding molecule can be said to bind to the "C" epitope with a higher specificity than its specificity for related epitope "D".

[0082] It can be said that the binding molecule disclosed in this application, for example, an antibody or fragment, variant or derivative thereof, binds the target antigen with a dissociation rate (k(off)) less than or equal to 5 X 10 ^-2 s ^-1 , 10 ^-2 s ^-1 , 5 X 10 ^-3 s ^-1 , 10 ^-3 s ^-1 , 5 X 10 ^-4 s ^-1 , 10 ^-4 s ^-1 , 5 X 10 ^-5 s ^-1 , or 10 ^-5 s ^-1 5 X 10 ^-6 s ^-1 , 10 ^-6 s ^-1 , 5 X 10 ^-7 s ^-1 , or 10 ^-7 s ^-1 .

[0083] It can be said that the binding molecule disclosed in this application, for example, an antibody or its antigen-binding fragment, variant or derivative, binds the target antigen with an association rate (k(on)) greater than or equal to 10 ³ M ^-1 s ^{- 1} , 5 X 10 ³ M ^-1 s ^-1 , 10 ⁴ M ^-1 s ^-1 , 5 X 10 ⁴ M ^-1 s ^-1 , 10 ⁵ M ^-1 s ^-1 , 5 X 10 ⁵ M ^-1 s ^-1 , 10 ⁶ M ^-1 s ^-1 , or 5 X 10 ⁶ M ^-1 s ^-1 , or 10 ⁷ M ^-1 s ^-1 .

[0084] A binding molecule, such as an antibody or fragment, variant, or derivative thereof, is said to competitively inhibit the binding of a reference antibody or antigen-binding fragment to a given epitope if it selectively binds to that epitope to the extent that it blocks, to some extent. 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, such as a competitive ELISA assay. The binding molecule can be said to competitively inhibit the 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%. %.

[0085] In the context of the present application, the term "affinity" refers to an indicator of the binding strength of an individual epitope to one or more binding domains, for example, an immunoglobulin molecule. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ^ed . 1988), pages 27-28. In the context of this application, the term "avidity" refers to the overall stability of the complex between the population of binding domains and the antigen. See, for example, Harlow, pages 29-34. Avidity is related both to the affinity of individual binding domains in a population for specific epitopes and to the valency of immunoglobulins and antigen. For example, an interaction between a divalent monoclonal antibody and an antigen with a highly repetitive epitope structure, such as a polymer, will have high avidity. An interaction between a divalent monoclonal antibody and a receptor present at high density on the cell surface will also have high avidity.

[0086] Binding molecules or antigen-binding fragments, variants or derivatives thereof, as disclosed in this application, can also be described or characterized in terms of their cross-reactivity. In the context of the present application, the term "cross-reactivity" refers to the ability of a binding molecule, for example, an antibody or fragment, variant or derivative thereof, specific to 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 usually contains a large number of complementary structural features contained in the inducing epitope, and in some cases may actually match better than the original.

[0087] A binding molecule, such as an antibody or fragment, variant or derivative thereof, can also be described or characterized in terms of its binding affinity for an antigen. For example, a binding molecule can bind to an antigen with a dissociation constant or K _d of at most 5 x 10 ^-2 M, 10 ^-2 M, 5 x 10 ^-3 M, 10 ^-3 M, 5 x 10 ^-4 M, 10 ^-4 M, 5 × 10 ^-5 M, 10 ^-5 M, 5 × 10 ^-6 M, 10 ^-6 M, 5 × 10 ^-7 M, 10 ^-7 M, 5 × 10 ^-8 M, 10 ^-8 M, 5 × 10 ^-9 M, 10 ^-9 M, 5 × 10 ^-10 M, 10 ^-10 M, 5 × 10 ^-11 M, 10 ^-11 M, 5 × 10 ^-12 M, 10 ^-12 M, 5 × 10 ^-13 M, 10 ^-13 M, 5 × 10 ^-14 M, 10 ^-14 M, 5 × 10 ^-15 M or 10 ^-15 M.

[0088] Antibody fragments, including single chain antibodies or other binding domains, may exist alone or in combination with one or more of the following: a hinge region, CH1, CH2, CH3, or CH4 domains, a J chain, or a secretory component. Also included are antigen binding fragments, which may 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, such as antibodies or antigen-binding fragments thereof, may be of any animal origin, including birds and mammals. The antibodies may be human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In yet another embodiment, the variable region may be derived from cartilaginous fish (eg, sharks). As used herein, "human" antibodies include antibodies containing 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 some of which may express endogenous immunoglobulins as described below. and, for example, in US patent No. 5939598 authors Kucherlapati et al.

[0089] In the context of the present application, the term "heavy chain subunit" includes amino acid sequences derived from the heavy chain of an immunoglobulin, a binding molecule, for example, an antibody containing a heavy chain subunit, includes at least one of: a VH domain, a CH1 domain, a hinge ( for example, upper, middle and/or lower hinge region) of a domain, CH2 domain, CH3 domain, CH4 domain, or a variant or fragment thereof. For example, a binding molecule, such as an antibody or fragment, variant or derivative thereof, may include, in addition to the VH domain, a CH1 domain; CH1 domain, hinge and CH2 domain; CH1 domain and CH3 domain; CH1 domain, hinge and CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In certain aspects, a binding molecule, for example, an antibody or fragment, variant or derivative thereof, may include, in addition to the VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain and a J chain. In addition, a binding molecule for use in the invention may lack certain portions of the constant region, such as all or part of the CH2 domain. One skilled in the art will recognize that these domains (eg, the heavy chain subunit) can be modified such that they differ in amino acid sequence from the original immunoglobulin molecule.

[0090] The heavy chain subunits of a binding molecule, such as an antibody or fragment thereof, may include domains derived from different immunoglobulin molecules. For example, a polypeptide heavy chain subunit may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain subunit may include a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain subunit may comprise a chimeric hinge derived partly from an IgG1 molecule and partly from an IgG4 molecule.

[0091] 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 the VL or CL domain (eg, Cκ or Cλ).

[0092] Binding molecules, such as antibodies or antigen-binding fragments, variants or derivatives thereof, can be described or characterized in the context of the epitope(s) or portion(s) of the 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 "antigenic determinant". The target antigen may contain one epitope, or at least two epitopes, and may include any number of epitopes, depending on the size, conformation, and type of the antigen.

[0093] As previously stated, the subunit structures and the three-dimensional configuration of the constant regions of the various classes of immunoglobulins are well known. In the context of the present application, 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) domain of an immunoglobulin heavy chain constant region. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the conventional immunoglobulin heavy chain molecule.

[0094] As used herein, the term “CH2 domain” includes the portion of a heavy chain molecule that extends, for example, from about amino acids 244 to amino acids 360 of an IgG antibody, according to conventional numbering systems (amino acids 244 to 360, Kabat numbering system). ; and amino acids 231-340, EU numbering system, see Kabat EA et al., cited above. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 amino acids. Some classes of immunoglobulins, such as IgM, further include region CH4.

[0095] In the context of this application, the term "hinge region" includes the part of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 amino acids and is flexible, allowing the two N-terminal antigen-binding regions to move independently.

[0096] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In some 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 positions 239 and 242 in the Kabat numbering system (position 226 or 229, EU numbering system).

[0097] In the context of this application, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is derived or derived from the first species, and the constant region (which may be intact, partial or modified) is derived from the second species. In some embodiments, the target binding region or site will be from a non-human source (eg, mouse or primate) and the constant region is human.

[0098] The terms "multispecific antibody" or "bispecific antibody" refer to an antibody containing binding domains for two or more different epitopes in a single antibody molecule. Other binding molecules, in addition to the canonical structure of an antibody, can be designed with two binding specificities. Binding of an epitope by bispecific or polyspecific 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 techniques. (Strohlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 73:543-9 (2010)). The bispecific antibody may also be a diabody.

[0099] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain in either the heavy chain or the light chain or both is altered by at least partial substitution of one or more amino acids in either the CDR or frame areas. In certain aspects, the full set of CDRs from an antibody of known specificity may be grafted onto the framework regions of a heterologous antibody. Although alternative CDRs may be derived from an antibody of the same class, or even subclass, as the antibody from which the framework regions are derived, CDRs may also be derived from an antibody of a different class, such as an antibody from another animal species. An engineered antibody in which one or more "donor" CDRs from an antibody of known non-human specificity is grafted onto a human heavy or light chain framework region is referred to herein as a "humanized antibody". In some aspects, not all CDRs are replaced by the full set of CDRs from the donor variable region, and yet the antigen-binding ability of the donor may still be transferred to the recipient's variable domains. Subject to the explanations set forth in, for example, US Pat.

[0100] In the context of this application, the term "engineered" includes the manipulation of nucleic acid or polypeptide molecules by synthetic means (for example, using recombinant technologies, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or any combination of these methods).

[0101] In the context of this application, the terms "related", "merged" or "merge", or other grammatical equivalents can be used interchangeably. These terms refer to the combination of two or more elements or components by any means, including chemical conjugation or recombinant techniques. "In-frame fusion" refers to the combination of two or more open reading frames (ORFs) of a polynucleotide to form a contiguous longer ORF such that the translational reading frame of the original ORFs is preserved. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to the polypeptides encoded by the original ORFs (these segments are not normally fused in this way in nature). Although the reading frame is thus made contiguous across all fused segments, the segments may be physically or spatially separated, for example, by an in-frame linker sequence. For example, polynucleotides encoding an immunoglobulin variable region CDR may be fused in-frame, but may be separated by a polynucleotide encoding at least one immunoglobulin framework or additional CDRs, provided that the "fused" CDRs are translated together as part continuous polypeptide.

[0102] In the context of polypeptides, a "linear sequence" or "sequence" is the amino-terminal to carboxy-terminal order of amino acids in a polypeptide, in which amino acids that are adjacent to each other in the sequence are adjacent in the primary structure of the polypeptide. The portion of a polypeptide that is "amino-terminal" or "N-terminal" to another portion of the polypeptide is the portion that is earlier in the sequential polypeptide chain. Similarly, the portion of a polypeptide that is "carboxy-terminal" or "C-terminal" to another portion of the polypeptide is the portion that is 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.

[0103] the Term "expression" in the context of this application refers to the process by which a gene produces a biochemical product, such as a polypeptide. This process includes any manifestation of the functional presence of a gene in a cell, including, but not limited to, gene knockdown, as well as both transient expression and stable expression. It includes, but is not limited to, transcription of a gene into messenger RNA (mRNA) and 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. The expression of a gene produces a "gene product". In the context of the present application, a gene product can be either a nucleic acid, such as messenger RNA, obtained by transcription of a gene, or a polypeptide translated from a transcript. The gene products described herein also include nucleic acids with post-transcriptional modifications such as polyadenylation, or polypeptides with post-translational modifications such as methylation, glycosylation, lipid addition, binding to other protein subunits or proteolytic cleavage, and the like.

[0104] In the context of this application, the term "epigenetic modification(s)" refers to a stably inherited phenotype(s) resulting from chromosomal changes without changes in the DNA sequence. Epigenetic modifications can in some cases 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 chromatin structure that can lead to overexpression and/or repression of genes that control cellular processes such as differentiation, proliferation and/or apoptosis. Such modifications may include, for example, DNA methylation and histone acetylation. See, for example, Gnyska, A., et al., Anticancer Res. 33:2989-2996 (2013).

[0105] As used herein, the term "epigenetic modulating agent" refers to an agent, e.g., a therapeutic agent, that can affect, e.g., block, reduce, reverse, or attenuate a disease-causing epigenetic modification, thereby providing treatment for a disease, e.g., cancer. Examples of epigenetic modulators include DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi). Various HDACi and DNMTi are approved or are being studied for the treatment of selected cancers. Examples of agents are disclosed elsewhere in this application.

[0106] As used herein, the terms "histone deacetylase inhibitor(s)" or "HDAC inhibitor(s)" or "HDACi" refer to a compound or compounds capable of inhibiting histone deacetylation in vivo, in vitro, or both. HDACs inhibit the activity of at least one histone deacetylase. As a result of inhibition of deacetylation of at least one histone, an increase in the amount of acetylated histone occurs, and the accumulation of acetylated histone can be used as a biological marker to assess HDAC activity.

[0107] As used herein, the terms "DNA methyltransferase inhibitor(s)" or "DNMT inhibitor(s)" or "DNMTi" refer to a compound or compounds capable of inhibiting DNA hypermethylation and/or DNA methyltransferase overexpression ("DNMT" ) in vivo, in vitro, or both. By inhibiting DNMT overexpression and/or DNMT activity, DNMTi can, for example, restore the expression of aberrantly silenced genes such as tumor suppressor genes.

[0108] In the context of the present application, the term "binding molecule to SEMA4D" refers to a molecule that specifically binds to SEMA4D, for example, an antibody or antigen-binding fragment, variant or derivative. The term “anti-SEMA4D antibody”, unless specifically referring to full-length antibodies such as naturally occurring antibodies, encompasses full-length antibodies as well as antigen-binding fragments, as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, such as those occurring naturally occurring antibody or immunoglobulin molecules, or engineered antibody molecules or fragments that bind an antigen in the same manner as antibody molecules.

[0109] Terms such as "treat" or "treat" or "alleviate" or "alleviate" refer to therapeutic measures that cure, slow, reduce symptoms and/or stop or slow the progression of an existing diagnosed pathological condition or disorder. Terms such as "prevent", "avoid", "avoid", "contain", and the like, refer to prophylactic or precautionary measures to prevent the development of an undiagnosed target pathological condition or disorder. Thus, "those in need of treatment" may include those who already have the disorder; those who are predisposed to developing the disorder; and those in whom the disorder needs to be prevented.

[0110] 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, a therapeutically effective amount of the drug may reduce the number of cancer cells; slow down or stop the division of cancer cells, reduce or slow down the growth of the tumor; inhibit, for example, suppress, slow down, prevent, stop, delay or reverse the infiltration of cancer cells into peripheral organs, including, for example, the spread of cancer to soft tissue and bone; inhibit, for example, suppress, slow down, prevent, reduce, stop, delay or reverse tumor metastasis; inhibit, for example, suppress, slow down, prevent, stop, delay or reverse tumor growth; alleviate to some extent one or more of the symptoms associated with cancer, reduce morbidity and mortality; improve the quality of life; or provide a combination of such effects. If a drug prevents the growth of existing cancer cells and/or kills them, it can be called cytostatic and/or cytotoxic.

[0111] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, in particular a mammal, for which diagnosis, prognosis, or therapy is desired. Mammals include humans, pets, farm and zoo animals, sports or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and so on.

[0112] In the context of this application, expressions such as "a subject that will benefit from therapy" and "an animal in need of treatment" include subjects, such as mammals, that would benefit from the administration of therapy, as described in this application, such as an antibody containing one or more antigen-binding domains, in combination with an epigenetic modulating agent, eg, HDACi, DNMTi, and/or a combination thereof.

[0113] The term "immunomodulatory therapy" or "immunotherapy" refers to treatment that affects a disease or disorder in a subject by inducing and/or enhancing an immune response in that subject. Immunomodulatory therapies include cancer vaccines, immunostimulatory agents, adoptive T-cell or antibody therapy, and immune checkpoint blocking (Lizee et al. Ann. Rev. Med. 64:71-90 (2013)).

[0114] The term "immunomodulatory agent" refers to active agents for immunotherapy. Immunomodulatory agents include a variety of recombinant, synthetic and natural preparations. Examples of immunomodulatory 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; immune checkpoint blocking antagonists such as anti-CTLA-4, anti-PD-1 or anti-PD-Ll (PD-1 ligand), anti-LAG3, anti-TIM3, anti-B7-H3, synthetic cytosine phosphate- guanosine (CpG) oligodeoxynucleotides, glucans; and regulatory T cell (Treg) modulators such as cyclophosphamide.

Description of the target polypeptide - SEMA4D

[0115] In the context of this application, the terms "semaphorin 4D", "SEMA4D" and "SEMA4D polypeptide" are used interchangeably, as well as "SEMA4D" and "Sema4D". In certain embodiments, SEMA4D is expressed on or secreted from the cell surface. In yet another embodiment, SEMA4D is membrane bound. In yet another embodiment, SEMA4D is soluble, such as sSEMA4D. In yet another embodiment, SEMA4D may include full-length SEMA4D or a fragment thereof, or a variant SEMA4D polypeptide, where the SEMA4D fragment or variant SEMA4D polypeptide retains some or all of the functional properties of full-length SEMA4D.

[0116] The full length human SEMA4D protein is a homodimeric transmembrane protein consisting of two 150 kDa polypeptide chains. SEMA4D belongs to the semaphorin family of cell surface receptors and is also referred to as CD100. Both human and murine SEMA4D/Sema4D can be proteolytically cleaved from its transmembrane form to form 120 kDa soluble forms, resulting in two isoforms of Sema4D (Kumanogoh et al., J. Cell Science 77(5:3464 ( 2003)).Semaphorins are composed of soluble and membrane-bound proteins that were originally identified as axonal guidance factors that play an important role in establishing precise connections between neurons and their respective target.SEMA4D, structurally considered a class IV semaphorin, consists of an amino-terminal signal sequence, followed by a characteristic "Sema" domain containing 17 conserved cysteine residues, an Ig-like domain, a lysine-rich region, a hydrophobic transmembrane region, and a cytoplasmic tail.

[0117] The SEMA4D polypeptide comprises a signal sequence of approximately 13 amino acids, followed by a semaphorin domain of approximately 512 amino acids, an immunoglobulin-like (Ig-like) domain of approximately 65 amino acids, a lysine-rich region of 104 amino acids, a hydrophobic transmembrane region of approximately 19 amino acids, and cytoplasmic tail of 110 amino acids. The tyrosine phosphorylation consensus site in the cytoplasmic tail supports the predicted association of SEMA4D with tyrosine kinase (Schlossman et al., Eds. (1995) Leucocyte Typing V (Oxford University Press, Oxford).

[0118] 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., Cell99:71-80 (1999)). Stimulation by SEMA4D of plexin B1 signaling has been shown to cause collapse of neuronal growth cones and cause collapse of process extension and apoptosis of oligodendrocytes (Giraudon et al., J. Immunol. 772:1246-1255 (2004); Giraudon et al., NeuroMolecular Med 7:207-216 (2005)). After binding to SEMA4D, plexin B1 signaling mediates R-Ras inactivation, resulting in decreased integrin-mediated attachment to the extracellular matrix, as well as RhoA activation, leading to cell collapse by rearrangement of the cytoskeleton (Kruger et al., Nature Rev. Mol. Cell Biol (5:789-800 (2005); Pasterkamp, TRENDS in Cell Biology 75:61-64 (2005)) Plexin B2 has a moderate affinity for SEMA4D and a recent report indicates that plexin B2 is expressed on keratinocytes and activates SEMA4D-positive γδ T cells, promoting epithelial regeneration (Witherden et al., Immunity 37:314-25 (2012)).

[0119] In lymphoid tissues, CD72 is used as a low affinity (300 nM) SEMA4D receptor (Kumanogoh et al, Immunity 73:621-631 (2000)). B cells and antigen presenting cells (APCs) express CD72, and anti-CD72 antibodies have many of the effects of sSEMA4D, such as enhancing CD40-induced B cell responses and shedding CD23 by B cells. CD72 is believed to act as a negative regulator of B cell responses by recruiting tyrosine phosphatase SHP-1, which can bind to many inhibitory receptors. The 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 antibody responses in vivo (Ishida et al., Inter. Immunol. 75:1027-1034 (2003); Kumanogoh and H. Kukutani , Trends in Immunol 22:670-676 (2001)). sSEMA4D enhances CD40-induced DC maturation, including activation of costimulatory molecules and increased secretion of IL-12. In addition, sSEMA4D can inhibit immune cell migration, which can be reversed by adding blocking mouse anti-SEMA4D antibodies (Elhabazi et al., J. Immunol. 166:4341-4347 (2001); Delaire et al., J. Immunol. 766 :4348-4354 (2001)).

[0120] Sema4D is highly expressed 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 widely expressed on resting T cells, but weakly expressed on resting B cells and antigen presenting cells (APCs) such as dendritic cells (DCs).

[0121] Cellular activation increases the surface expression of SEMA4D, as well as the formation of soluble SEMA4D (sSEMA4D). The SEMA4D expression profile suggests that it plays an important physiological and pathological role in the immune system. SEMA4D has been shown to promote B cell activation, aggregation, and survival; enhances CD40-induced proliferation and antibody production; enhances the response of antibodies to T-dependent antigens; increases T cell proliferation; enhances maturation of dendritic cells and the ability to stimulate T cells; and is directly involved in demyelination and axonal degeneration (Shi et al., Immunity 73:633-642 (2000); Kumanogoh et al., J. Immunol. 7(59:1175-1181 (2002); and Watanabe et al., J Immunol 767:4321-4328 (2001)).

Antibodies to SEMA4D

[0122] Antibodies that bind SEMA4D have been described in the prior art. See, for example, US Patent Application Publication 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), all of which are incorporated herein by reference.

[0123] The invention generally relates to a method for inhibiting, delaying, or reducing tumor growth or metastasis in a subject, e.g., a human cancer patient, comprising administering an antibody that specifically binds to SEMA4D, or an antigen-binding fragment, variant, or derivative thereof, and an effective amount of an epigenetic modulator an agent, for example, a histone deacetylase (HDAC) inhibitor (HDACi), a DNA methyltransferase (DNMT) inhibitor (DNMTi), or any combination thereof. HDACi and DNMTi are described in detail elsewhere in this application. In certain embodiments, an anti-SEMA4D antibody blocks the interaction of SEMA4D with one or more of its receptors, eg, plexin B1, plexin B2, and/or CD72. In certain embodiments, cancer cells express plexin B1 and/or plexin B2. Anti-SEMA4D antibodies having these properties can be used in the methods of this application. Antibodies that can be used include, but are not limited to, Mab (monoclonal antibodies) VX15/2503, 67, 76, 2282 and their antigen-binding fragments, variants or derivatives, which are fully described in US 2010/0285036 A1 and US 2008/0219971 A1. Additional antibodies that can be used in the methods proposed in this application 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 228 , MAb 2282, MAb 2283, MAb 2284 and MAb 2285, and 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 suitable are antibodies that bind to the same epitope as any of the above antibodies and/or antibodies that competitively inhibit the binding or activity of any of the above antibodies.

[0124] In certain embodiments, an anti-SEMA4D antibody or antigen-binding fragment, variant or derivative thereof suitable for the methods provided herein has an amino acid sequence of at least about 80%, about 85%, about 88% , approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, or approximately 95% identical to the amino acid sequence of an anti-SEMA4D reference antibody molecule, such as those described above. In a further embodiment, the sequence of the binding molecule is at least about 96%, about 97%, about 98%, about 99%, or 100% identical to that of the reference antibody.

[0125] In certain aspects, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof can inhibit the interaction of SEMA4D with its receptor, for example, plexin B1, plexin B2, or CD72. In certain aspects, an anti-SEMA4D antibody, or antigen-binding fragment, variant, or derivative thereof, can inhibit SEMA4D-mediated plexin B1 signaling.

[0126] In certain aspects, an anti-SEMA4D antibody or antigen-binding fragment, variant or derivative thereof competitively inhibits binding of a reference antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 5, with SEMA4D. In certain aspects, an anti-SEMA4D antibody or antigen-binding fragment, variant or derivative thereof binds to the same SEMA4D epitope as a reference antibody containing a VH containing the amino acid sequence of SEQ ID NO: 1 and a VL containing the amino acid sequence of SEQ ID NO: 5. In certain aspects, the VH of an anti-SEMA4D antibody, or antigen-binding fragment, variant, or derivative thereof, contains three complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3, and the VL contains three CDRs: LCDR1, LCDR2, and LCDR3, where the CDRs comprise the amino acid sequences of 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 CDRs. In certain aspects, the CDRs comprise the amino acid sequences of 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.

[0127] In certain aspects, the VH of an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof contains an amino acid sequence of at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%. identical to SEQ ID NO: 1 and the VL of an anti-SEMA4D antibody or antigen-binding fragment, variant or derivative thereof contains an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 5; or VH contains an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 9, and VL contains an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 10. In certain aspects, VH contains the amino acid sequence of SEQ ID NO: 1, and VL contains the amino acid sequence of SEQ ID NO: 5; or VH contains the amino acid sequence of SEQ ID NO: 9 and VL contains the amino acid sequence of SEQ ID NO: 10.

[0128] 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 containing such polynucleotides, and cells -hosts containing such vectors or polynucleotides, all of which are intended for the production of antibodies to SEMA4D or their antigen-binding fragments, variants or derivatives for use in the methods proposed in this application.

[0129] Suitable biologically active variants of antibodies to SEMA4D according to the invention can be used in the methods according to the present invention. Such variants retain the desired binding properties of the parental anti-SEMA4D antibody. Methods for producing antibody variants are generally available in the art.

[0130] Methods for mutagenesis and alteration of nucleotide sequences 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 52:488-492 (1985); Kunkel et al., Methods Enzymol. 154:367-382 (1987); Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); US Pat. No. 4,873,192; and references cited therein; these sources are incorporated into this application by reference. Guidance for the selection of suitable amino acid substitutions that do not affect the biological activity of the polypeptide of interest is provided in the model according to Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), pp. 345-352, incorporated herein by reference in its entirety. In the Dayhoff et al. an amino acid similarity matrix based on fixed point mutations (PAM) (PAM 250 matrix) is used to determine suitable conservative amino acid substitutions. In certain aspects, conservative substitutions are used, such as substituting one amino acid for another having similar properties. Examples of conservative amino acid substitutions, according to the PAM 250 matrix of the Dayhoff et al. model, include, but are not limited to, Gly↔Ala, Val↔Ile↔Leu, Asp↔Glu, Lys↔Arg, Asn↔Gln, and Phe↔Trp↔Tyr.

[0131] When constructing variants of an anti-SEMA4D binding molecule, such as an antibody or antigen-binding fragment thereof, polypeptides of interest, modifications are made such that the variants continue to have the desired properties, such as being able to specifically bind to SEMA4D, such as human, murine, or both human and murine SEMA4D, for example, expressed on the surface or secreted by the cell, and had the activity of blocking SEMA4D, as described in this application. In certain aspects, mutations introduced into the DNA encoding the variant polypeptide maintain the reading frame and do not create complementary regions that could form the secondary structure of the mRNA. See European Patent Application Publication No. 75444.

[0132] Methods for measuring the binding specificity of a binding molecule to SEMA4D, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, include, but are not limited to, standard competitive binding assays, assays to monitor immunoglobulin secretion by T cells or B cells, T proliferation assays cells, apoptosis assays, ELISA assays, and the like. See, for example, such assays disclosed in WO 93/14125; Shi et al., Immunity 73:633-642 (2000); Kumanogoh et al., J. Immunol. 7(59:1175-1181 (2002); Watanabe et al., J. Immunol. 7(57:4321-4328 (2001); Wang et al., Blood 97:3498-3504 (2001); and Giraudon et al. ., J. Immunol 772:1246-1255 (2004), all of which are incorporated herein by reference.

[0133] 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.

[0134] When discussing in this application whether any particular polypeptide, including constant regions, CDRs, VH domains or VL domains disclosed in this application, is at least about 65%, about 70%, about 75%, approximately 80%, approximately 85%, approximately 90% approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97 %, about 98%, about 99%, or even about 100% identical to another polypeptide, % identity can be determined using the method and computer programs/software known in the art, such as, but not limited to, the program BESTFIT (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm according to Smith and Waterman (1981) Adv to find the segment of best homology between two sequences. Appl. Math. 2:482-489. When using the BESTFIT program or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to the reference sequence according to the present invention, the parameters are of course set such that the percent identity is calculated over the entire length of the reference polypeptide sequence , and homology gaps were resolved up to 5% of the total number of amino acids in the reference sequence.

[0135] For purposes of the present invention, percent sequence identity can be determined using the Smith-Waterman homology search algorithm using an affine gap search with a gap opening penalty of 12 and a gap widening penalty of 2, with a BLOSUM matrix of 62. Algorithm the search for Smith-Waterman homology is set out in Smith and Waterman (1981) Adv. Appl. Math. 2:482-489. A variant may, for example, differ from a reference anti-SEMA4D antibody (e.g. MAb VX15/2503, 67, 76, or 2282) by as little as 1 to 15 amino acid residues, by as little as 1 to 10 amino acid residues, for example by 6-10 , for only 5, for only 4, 3, 2 or even 1 amino acid residue.

[0136] The constant region of an anti-SEMA4D antibody can be mutated to change effector function in several ways. See, for example, US Patent No. 6,737,056 B1 and US Patent Application Publication No. 2004/0132101 A1, which disclose Fc mutations that optimize antibody binding to Fc receptors.

[0137] In individual anti-SEMA4D antibodies or fragments, variants or derivatives thereof suitable for use in the methods provided herein, the Fc portion may be mutated to reduce effector function using methods known in the art. For example, deletion or inactivation (through point mutations or other means) of the constant region domain can reduce Fc receptor binding of the circulating modified antibody, thereby improving tumor localization. In other cases, constant region modifications consistent with the present invention reduce complement fixation and thus decrease serum half-life. Other constant region modifications can be used to modify disulfide bonds or oligosaccharide fragments that provide improved 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 be easily measured and quantified using well known immunological methods without undue experimentation.

[0138] Anti-SEMA4D antibodies for use in the methods provided herein include derivatives that are modified, for example, by covalently attaching any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, but not limited to, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, etc. Any of a variety of chemical modifications can be carried out by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, and so on. In addition, the derivative may contain one or more non-standard amino acids.

[0139] A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue containing a side chain with a similar charge. Families of amino acid residues containing side chains with similar charges have been defined in the prior 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 (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine). Alternatively, mutations can be introduced randomly across all or part of the coding sequence, e.g., by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind a polypeptide to SEMA4D, block the interaction SEMA4D with its receptor, or inhibit, delay or reduce metastasis in a subject, such as a cancer patient).

[0140] For example, you can introduce mutations only in the frame regions or only in the CDR regions of the antibody molecule. The introduced mutations can be silent or neutral missense mutations, that is, have little or no effect on the ability of the antibody to bind antigen. These types of mutations may be useful for optimizing codon usage or improving antibody production by hybridomas. Conversely, non-neutral missense mutations can alter the ability of an antibody to bind an antigen. One of skill in the art is able to design and test mutant molecules with desired properties, such as no change in antigen binding activity or change in binding activity (eg, improvements in antigen binding activity or change in antibody specificity). After mutagenesis, the encoded protein can be expressed in the usual way, and the functional and/or biological activity of the encoded protein can be determined (for example, the ability to immunospecifically bind at least one epitope of the SEMA4D polypeptide) using the methods described in this application, or by standard modification techniques. known in the art.

[0141] In certain embodiments, anti-SEMA4D antibodies for use in the methods provided herein comprise at least one optimized complementarity determining region (CDR). By "optimized CDR" is meant that the CDR has been modified and optimized to improve the binding affinity and/or anti-SEMA4D activity conferred by an anti-SEMA4D antibody containing the optimized CDR. "Anti-SEMA4D activity" or "SEMA4D blocking activity" may include an activity that modulates one or more of the following activities associated with SEMA4D: activation, aggregation, and survival of B cells; CD40-induced proliferation and antibody production; antibody response to T-dependent antigens; proliferation of T cells or other immune cells; maturation of dendritic cells; demyelination and axonal degeneration; apoptosis of pluripotent neutral progenitors and/or oligodendrocytes; induction of endothelial cell migration; inhibition of spontaneous migration of monocytes; inhibiting, delaying or reducing tumor cell growth or metastasis, binding to plexin B1 or another cell surface receptor, or any other activity associated with soluble SEMA4D or SEMA4D expressed on the surface of SEMA4D+ cells. In a particular embodiment, the anti-SEMA4D activity includes the ability to inhibit, delay or reduce tumor metastases, either in combination with inhibition, delay or reduction in the growth of primary tumor cells and tumor metastases, or independently of the growth of primary tumor cells and tumor metastases. Activity against SEMA4D may also be associated with a reduction in the incidence or severity of diseases associated with the expression of SEMA4D, including, but not limited to, certain cancers, including lymphomas, autoimmune diseases, inflammatory diseases, including inflammatory diseases of the central nervous system (CNS) and peripheral nervous system (PNS), transplant rejection and invasive angiogenesis. Examples of optimized antibodies based on the mouse anti-SEMA4D MAb BD16 antibody have been described in US Patent Application Publication No. 2008/0219971 A1, International Patent Application WO 93/14125, and Herold et al., Int. Immunol. 7:1-8 (1995), all of which are hereby incorporated by reference in their entirety. Modifications may include substitution of amino acid residues in the CDR such that the anti-SEMA4D antibody retains specificity for the SEMA4D antigen and has improved binding affinity and/or improved anti-SEMA4D activity.

Histone deacetylase inhibitors (HDACi)

[0142] In the context of this application, the terms "histone deacetylase inhibitor(s)," HDAC inhibitor(s), and "HDACi" are used interchangeably and may refer to a single HDACi or multiple HDACi.

[0143] Histone acetyltransferase (HAT) and histone deacetylase (HDAC) regulate the status of histone acetylation. Histone acetyltransferases are enzymes that acetylate lysine residues in core histones, resulting in less compact and more transcriptionally active chromatin, resulting in gene expression. HDACs, in contrast, are enzymes that catalyze the removal of acetyl groups from lysine residues in the amino-terminal tails of nucleosomal core histones. HDACs can be divided into three classes based on structural homology. Class I HDACs (HDACs 1, 2, 3 and 8) are associated with the yeast RPD3 gene. Class IIA (HDAC 4, 5, 7 and 9) is similar to the yeast Hda1 gene. Class III, also known as sirtuins, belongs to the Sir2 gene and includes SIRT1-7. Class IV, containing only HDAC 11, shares characteristics with both Class I and II. See, for example, Mottamal, M., et al., Molecules 20:3898-3941 (2015).

[0144] Some HDAC inhibitors act by binding to the zinc catalytic domain of HDAC. They can be classified based on the chemical moiety that binds to the zinc ion or as cyclic tetrapeptides that bind to the zinc ion via a thiol group. See, for example, Drummond, D.C., et al., Ann. Rev. Pharmacol. Toxicol. 45:495-528 (2005). These include, but are not limited to: hydroxamic acids (or hydroxamates) such as trichostatin A; cyclic tetrapeptides (such as trapoxin B) and depsipeptides; benzamides; electrophilic ketones; and aliphatic acid compounds (short chain fatty acids (SCFA) such as phenylbutyrate and valproic acid). See, for example, Porcu, M., and Chiarugi, A., Trends Pharmacolog. sci. 2(5:94-103 (2005). More specifically, HDAC inhibitors include, but are not limited to, hydroxamic acids vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589); and benzamides: entinostat (MS-275) , CI994 and mocetinostat (MGCD0103) Class III sirtuin HDACs are dependent on NAD+ (nicotinamide adenine dinucleotide) and are therefore inhibited by nicotinamide as well as NAD derivatives, dihydrocoumarin, naphthopyranone and 2-hydroxynaphthaldehydes See the same reference.

[0145] Non-limiting examples of HDAC inhibitors for use in inhibiting histone deacetylase, inducing terminal differentiation, arresting cell growth and/or apoptosis in malignant cells, and/or inducing differentiation, arresting cell growth and/or apoptosis of tumor cells in tumors are listed in Table 2 It is intended that HDAC inhibitors include any salts, crystal structures, amorphous structures, hydrates, derivatives, metabolites, stereoisomers, structural isomers and prodrugs of the HDAC inhibitors described herein.

[0146] In certain embodiments, HDAC inhibitors suitable for 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 currently in or in clinical trials, are listed below.

[0147] FDA approved HDAC inhibitors. Vorinostat was licensed by the US 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 the treatment of cutaneous T-cell lymphoma (CTCL). Hydamide was approved by China in 2015 for the treatment of peripheral T-cell lymphoma (PTCL). Panobinostat (trade name Faridak) 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.

[0148] In one non-limiting aspect, the HDAC inhibitor is an entinostat (ENT). Enthinostat 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 a class I HDAC inhibitor (Syndax Pharmaceuticals). ENT is a benzamide histone deacetylase inhibitor that inhibits HDAC1 and HDAC3 with IC50s of 0.51 µM and 1.7 µM. ENT is currently undergoing clinical trials for the treatment of various types of cancer. DNA methyltransferase inhibitors

[0149] In the context of this application, the terms "DNA methyltransferase inhibitor(s)," DNMT inhibitor(s), and "DNMTi" are used interchangeably and may refer to a single DNMTi or multiple DNMTi.

[0150] DNA methyltransferases catalyze the addition of methyl groups to the 5' carbon of cytosine residues. In cancer cells, silencing of tumor suppressor genes occurs via DNMT-mediated methylation. Gravina et al., Molecular Cancer 9:305-320 (2010). There are several isoforms of DNMT including DNMTI, DNMT-3a and DNMT-3b. See same source. Various DNMTi have been identified and two have been approved for cancer treatment in the United States. Classes of molecules include nucleoside analogs, antisense oligonucleotides, small molecule enzyme inhibitors, and agents that block DNMT interaction with DNA.

[0151] Non-limiting examples of DNMTi for use in inhibiting DNA methyltransferases, inducing terminal differentiation, arresting cell growth and/or apoptosis in malignant cells, and/or inducing differentiation, arresting cell growth and/or apoptosis of tumor cells in tumors are listed in Table 1. 3. DNMTi inhibitors are meant to include any salts, crystal structures, amorphous structures, hydrates, derivatives, metabolites, stereoisomers, structural isomers, and prodrugs of the DNMTi inhibitors described herein.

[0152] In certain embodiments, DNMT inhibitors suitable for the methods provided herein include azacitidine, 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 currently in or in clinical trials, are listed below.

[0153] DNMTi approved by the Food and Drug Administration (FDA) and currently in clinical trials. Azacitidine, sold under the name ^VIDAZA® , was approved by the FDA for the treatment of myelodysplastic syndrome (MDS) on May 19, 2004 and is in clinical trials for the treatment of advanced solid tumors. Azacitidine has been shown to enhance the activity of immune checkpoint (ICP) inhibitors in preclinical models of melanoma and colon cancer by activating the interferon (IFN) pathway and reversing viral antigen suppression, making the tumor more immunogenic and responsive to effective ICP therapy. (Li et al., Oncotarget 5:587 (2014) and Roulois et al., Cell 162:961 (2015)). In addition, azacitidine has been shown to increase the expression of testicular cancer antigens (CTAs) in tumors, leading to increased anti-CTA T cell activity in patients with MDS, acute myeloid leukemia (AML), and chronic myelomonocytic leukemia (CMML) (Gang , AO et al Blood Cancer J. doi: 10.1038/bcj.2014.14.(2014)). Juergens, R. A. et al., (Cancer Discov. 1:598-607 (2011)) used azacitidine and entinostat for refractory advanced NSCLC. Six patients from this study were included in an additional study (Wrangle, J., et al., Oncotarget 4:2067-2079 (2013)) with immune checkpoint inhibitors, where five patients showed responses. See also: blogs.biomedcentral.com/on-biology/2015/03/26/improving-lung-cancer-immunotherapy-using-epigenetic-approaches/ (accessed March 16, 2017).

[0154] Decitabine, sold under the name DACOGEN ^® , is approved for the treatment of MDS and is also approved in Europe for the treatment of AML and is undergoing clinical trials for the treatment of AML. SGI-110 is in phase II clinical trials for the treatment of AML. MG98 has completed phase I clinical trials for the treatment of advanced solid tumors. ECGC has completed phase II clinical trials for the treatment of prostate cancer.

[0155] In one non-limiting aspect, DNMTi is azacitidine. Azacitidine is a chemical analogue of the nucleoside cytosine. Azacitidine is able, among other things, to integrate into DNA, causing DNA hypomethylation through covalent binding to DNMT, preventing DNA synthesis, and leading to cytotoxicity and degradation of entrapped DNMT. Stresemann, C, and Lyko, F., Int. J. Cancer 123:8-13 (2008).

Methods of treatment using therapeutic binding molecules to SEMA4D and an epigenetic modulating agent, for example, HDACi and/or DNMTi

[0156] The present invention provides a method for inhibiting, delaying or reducing the growth of malignant cells in a subject suffering from cancer by administering to the subject a combination therapy containing an effective amount of an isolated binding molecule that specifically binds to SEMA4D, for example, an antibody to SEMA4D or an antigen-binding fragment thereof , variant or derivative, in combination with an effective amount of an epigenetic modulating agent, eg HDACi, DNMTi and/or combinations thereof. Examples of anti-SEMA4D antibodies and epigenetic modulators are described in detail elsewhere in this application. In certain aspects, administration of the combination therapy provided herein may partially or completely inhibit tumor or malignant cell growth, may delay tumor progression and malignant cell growth in a subject, may prevent the spread of metastases in a subject, may reduce the size of a subject's tumor, for example, to provide more successful surgical removal, may reduce the tumor vasculature in the subject, or may result in any combination of positive therapeutic responses in the subject. Examples of therapeutic responses that can be achieved are described in this application.

[0157] In some aspects, the introduction of combination therapy can lead to increased therapeutic efficacy compared with the introduction of antibodies to SEMA4D or its fragment or epigenetic modulating agent alone. In some aspects, the improved efficacy of treatment is synergistic and exceeds the additive efficacy of each individual agent. In certain aspects, the improved efficacy of treatment compared to either agent administered alone, as measured by, for example, increased tumor growth retardation (TGD), increased rate of tumor regression, such as 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 efficacy of treatment compared to the additive efficacy of both agents administered alone, as measured by, for example, increased tumor growth retardation (TGD), increased rate of tumor regression, such as 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 m ep 1000%.

[0158] In certain aspects, the anti-SEMA4D antibody, or fragment thereof, may be VX15/2503, mAb 67, or an antigen-binding fragment, variant, or derivative thereof. For example, an antibody or fragment may include a heavy chain variable region (VH) containing CDRs 1-3 VH containing SEQ ID NOs: 2, 3 and 4, respectively, and a light chain variable region (VL) containing CDRs 1-3 VL containing SEQ ID NO: 6, 7 and 8, respectively, or VH and VL containing, respectively, SEQ ID NO: 1 and SEQ ID NO: 5 or SEQ ID NO: 9 and SEQ ID NO: 10.

[0159] In certain aspects, the method provided herein comprises administering an anti-SEMA4D antibody, or antigen-binding fragment, variant, or derivative thereof, and an HDAC-inhibiting HDACi, which may be HDAC class I, class IIA, class IIB, class IV, and/or any combination thereof, for example, HDAC contains a zinc-containing catalytic domain. In certain aspects, the HDACi may include a moiety that binds to the zinc-containing catalytic domain of the HDAC. For example, in certain aspects, HDACi may include one or more or a combination of chemical moieties such as, but not limited to, 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 method of treatment proposed in this application include vorinostat, romidepsin, chidamide, panobinostat, belinostat, valproic acid or its salt, 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 may be an entinostat (pyridin-3-ylmethyl-N-[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate).

[0160] In certain aspects, the method provided herein comprises administering an anti-SEMA4D antibody, or antigen-binding fragment, variant, or derivative thereof, and DNMTi that inhibits DNMT1, DNMT-3a, DNMT-3b, and/or any combination thereof. For example, in certain aspects, DNMTi may include one or more or a combination of chemical moieties such as, but not limited to, azacitidine, decitabine, zebularine, SGI-110, epigallocatechin gallate, MG98, RG108, procainamide, hydralazine, and/or any combination thereof. . In certain aspects, DNMTi may be azacitidine.

[0161] According to the method proposed in this application, a binding molecule to SEMA4D, for example, an antibody or antigen-binding fragment, variant or derivative thereof, and an epigenetic modulating agent, for example, HDACi, DNMTi, or any combination thereof, can be administered separately or simultaneously. Any of the agents may be administered before the other, or the agents may be administered simultaneously, for example, in the same formulation. In addition, dosing methods, formulations of agents, and/or dosing regimens may be the same or different.

[0162] According to the method proposed in this application, a binding molecule to SEMA4D, for example, an antibody or its antigen-binding fragment, a variant or its derivative that specifically binds to SEMA4D, and an epigenetic modulating agent, for example, HDACi, DNMTi and / or any combination thereof , is administered to a subject suffering from cancer. The subject's cancer may be first diagnosed or, in certain aspects, the administration may follow more conventional cancer treatments. In certain aspects, the administration of the combination therapy of this application may be used as a prophylactic measure in a subject highly susceptible to developing a particular type of cancer, or to prevent the recurrence of a previously treated cancer. The subject's cancer may be, for example, a solid tumor, a hematological malignancy, any metastasis thereof, or any combination thereof.

[0163] In certain aspects, the subject's cancer is a solid tumor or its metastases. A solid tumor may be, for example, a sarcoma, carcinoma, melanoma, any of their metastases, or any combination thereof. Examples of solid tumors to be treated in accordance with the method proposed in this application include, but are not limited to, squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, renal cell carcinoma, ductal breast carcinoma, soft tissue sarcoma, osteosarcoma, melanoma, small cell carcinoma lung, non-small cell lung cancer, lung adenocarcinoma, peritoneal cancer, 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, vulvar cancer, thyroid cancer, head and neck cancer, any of these metastases, or any combination thereof.

[0164] In some aspects, the cancer is a hematological malignancy or its metastases. Examples of hematological malignancies to be treated in accordance with the method proposed in this application include, but are not limited to, leukemia, lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma , non-Hodgkin's lymphoma, multiple myeloma, any of their metastases, or any combination thereof.

[0165] In some aspects, the method of treating cancer, proposed in this application, may further include the introduction of additional anti-cancer therapy. Additional anti-cancer therapy can be carried out simultaneously with the introduction of the combination therapy proposed in this application, before the combination therapy proposed in this application, or after the combination therapy proposed in this application. In certain aspects, adjunctive therapy may include, but is not limited to, surgery, chemotherapy, radiation therapy, administration of a cancer vaccine, administration of an immunostimulating agent, adoptive T-cell therapy or antibody therapy, administration of an inhibitor that blocks immune checkpoints, administration of a regulatory T-regulatory modulator. cells (Treg) or a combination of these therapies.

[0166] Administration of an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or any combination thereof, in combination with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, may be effective in the treatment of a variety of cancers and non-malignancies. . By "antitumor activity" is meant a reduction in the rate of proliferation or accumulation of malignant cells and, therefore, a reduction in the growth rate of an existing tumor or a tumor that has arisen during therapy, and / or the destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells, and, therefore, a decrease in total tumor size during therapy. "Antineoplastic activity" may also include stimulation of tumor immune infiltration, a shift towards functional, tumor-specific, IFNγ-secreting CD8 ⁺ cytotoxic T cells, an increase in the ratio of effector T cells to regulatory T cells, increased T cell activity, infiltration, and activation of antigen-presenting T cells. cells that cross-present tumor antigens and locally activate tumor-specific T cells, reduced tumor-associated angiogenesis, inhibition of tumor progression, and increased survival. For example, therapy with an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or a combination thereof, and an anti-SEMA4D antibody may induce a physiological response, e.g., inhibition, delay, or reduction in tumor or malignant cell growth and metastases, which is useful in the treatment of cancer associated with cells expressing SEMA4D in humans.

[0167] In certain aspects, combination therapy with an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or a combination thereof, and an anti-SEMA4D binding molecule, e.g., an antibody or fragment thereof, can be used as a drug, in particular for use in the treatment or cancer prevention or for use in a precancerous condition or lesion. In certain aspects, combination therapy with an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or a combination thereof, and an anti-SEMA4D binding molecule, e.g., an antibody or fragment thereof, can be used to treat cancer overexpressing SEMA4D or cancer associated with cells expressing SEMA4D. .

[0168] In accordance with the methods of treatment proposed in this application, the introduction of an epigenetic modulating agent, for example, HDACi, DNMTi and / or combinations thereof, and a binding molecule to SEMA4D, for example, an antibody or its antigen-binding fragment, variant or derivative, as defined elsewhere in this application, may be used to stimulate a positive therapeutic response in a subject suffering from cancer or predisposed to developing cancer. A "positive therapeutic response" to cancer is intended to include an improvement in the disease due to "antitumor" activity, a reduction in the rate of proliferation or accumulation of malignant cells, and therefore a reduction in the rate of growth of an existing tumor or a tumor that has arisen during therapy, and/or destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells, and therefore a decrease in the overall size of the tumor during therapy. Such positive therapeutic responses are not limited to the route of administration. The methods provided herein can be used to inhibit, delay or reduce tumor growth, malignant cell growth and metastasis in a subject suffering from cancer. Thus, as a non-limiting example, improvement in disease can be characterized as reduced tumor growth or absence of tumors. As described in other parts of this application, the therapeutic response achieved with the introduction of the proposed combination therapy may exceed the corresponding therapeutic response achieved with the introduction of an epigenetic modulating agent or binding molecule to SEMA4D alone. In some aspects, the therapeutic response achieved is greater than the additive response expected when the two agents are administered. In other words, the therapeutic response achieved is synergistic. In certain aspects, a synergistic response may result in more effective treatment, faster treatment, or may allow treatment with lower doses of agents in combination therapy.

Pharmaceutical compositions and routes of administration

[0169] The present invention provides a composition, e.g., a pharmaceutical composition, comprising an effective amount of an anti-SEMA4D binding molecule, e.g., an antibody or an antigen-binding fragment, variant, or derivative thereof, and an effective amount of an epigenetic modulating agent, e.g., HDACi, DNMTi and/or their combinations. The composition may additionally contain one or more pharmaceutically acceptable carriers or excipients, and/or one or more additional therapeutic agents, examples of which are disclosed elsewhere in this application. Suitable binding molecules to SEMA4D and epigenetic modulators for use in such a pharmaceutical composition are proposed in this application.

[0170] In certain aspects, the pharmaceutical composition comprises an effective amount of an anti-SEMA4D antibody or antigen-binding fragment thereof, comprising VH and VL, where VH and VL contain the amino acid sequences of the CDRs contained in MAb VX15/2503 and MAb 67, namely, HCDR1 containing amino acid sequence of SEQ ID N0: 2, HCDR2 containing the amino acid sequence of SEQ ID NO: 3, HCDR3 containing the amino acid sequence of SEQ ID NO: 4, LCDR1 containing the amino acid sequence of SEQ ID NO: 6, LCDR2 containing the amino acid sequence of SEQ ID NO: 7, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 8. In certain aspects, the pharmaceutical composition comprises an effective amount of an antibody to SEMA4D or an antigen-binding fragment thereof, comprising the amino acid sequences VH and VL of MAb VX15/2503, namely VH, containing the amino acid sequence of SEQ ID NO: 1, and VL containing the amino acid sequence awn SEQ ID NO: 5.

[0171] In certain aspects, the pharmaceutical composition comprises an effective amount of an HDACi entinostat. In a separate aspect, the pharmaceutical composition comprises an effective amount of azacitidine DNMTi.

[0172] An exemplary pharmaceutical composition provided herein includes an effective amount of MAb VX15/2503 and an effective amount of an entinostat. Another illustrative pharmaceutical composition proposed in this application includes an effective amount of MAb VX15/2503 and an effective amount of azacitidine. In certain aspects, an "effective amount" of a particular agent included in the proposed pharmaceutical composition may differ from, for example, be less than the amount of the agent that would be effective as monotherapy. In certain aspects, the therapeutic efficacy of the pharmaceutical composition provided herein exceeds the additive effect of the agents included therein when administered alone.

[0173] The route of administration of an epigenetic modulating agent in combination with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, may be, for example, oral, parenteral, inhaled, or topical. The term "parenteral" in the context of the present application includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The modes of administration of the two agents may be the same or different. While all of these administration forms are expressly included within the scope of the methods provided herein, a non-limiting example of an administration form is an injection solution, in particular for intravenous or intra-arterial injection or drip infusion. A suitable pharmaceutical composition for injection may contain a buffer (eg, an acetate, phosphate or citrate buffer), a surfactant (eg, polysorbate), optionally a stabilizing agent (eg, human albumin), and the like. However, in other ways consistent with the scope of the present application, an epigenetic modulating agent and/or binding molecule to SEMA4D, for example, an antibody or antigen-binding fragment, variant or derivative thereof, can be delivered directly to the site of an undesired cell population, thereby increasing the exposure of the therapeutic agent. to the affected tissue.

[0174] Methods for preparing and administering a combination therapy as provided herein, comprising an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or a combination thereof, in combination with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof to a subject in need are well known or easily determined by those skilled in the art.

[0175] As discussed herein, an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or a combination thereof, in combination with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, can be administered in a pharmaceutically effective manner. amount for the treatment of cancer in vivo. In this regard, it should be understood that the disclosed epigenetic modulating agent and binding molecule may be formulated in such a way as to facilitate administration and promote stability of the active agent. In certain embodiments, the implementation of pharmaceutical compositions in accordance with the methods proposed in this application, include a pharmaceutically acceptable, non-toxic, sterile carrier such as saline, non-toxic buffers, preservatives, and the like. For the purposes of this application, a pharmaceutically effective amount of an epigenetic modulating agent and a binding molecule is an amount sufficient to affect the activity of at least one epigenetic modification in a cancer cell, for example, to reduce histone deacetylase activity or DNA methyltransferase activity, and to achieve effective binding to target and achieve benefits, such as antiproliferative effects, inhibiting, delaying or reducing the growth and metastasis of tumors and malignant cells, preventing further tumor growth, reducing tumor size, reducing tumor vasculature, and reducing the number of cancer cells, in a subject suffering from cancer, and /or in order to be able to observe the reduction of one or more symptoms associated with the disease.

[0176] Pharmaceutical compositions used in the methods of this application may include pharmaceutically acceptable carriers, such as, for example, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, block copolymers of polyethylene and polyoxypropylene, polyethylene glycol and wool grease.

[0177] Formulations for parenteral administration may 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, for example, 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, for example 0.05 M phosphate buffer or 0.8% saline. Other common carriers for parenteral administration include sodium phosphate solutions, dextrose Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Carriers for intravenous administration include fluid and nutrient replenishers, electrolyte replenishers such as those based on dextrose Ringer's solution, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, complexing agents, and inert gases, and the like.

[0178] Non-limiting examples of pharmaceutical compositions suitable for injection include sterile aqueous solutions (when water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition should be sterile and fluid to the extent that it can be easily injected with a syringe. The compositions are usually formulated so that they are stable under the conditions of manufacture and storage and can be protected from the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyhydric alcohol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, as well as 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).

[0179] Prevention of the action of microorganisms can be achieved by the use of various antibacterial and antimycotic agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents such as sugars, polyols such as mannitol, sorbitol, or sodium chloride may be included in the composition. Prolonged absorption of injectable compositions can be achieved by including an absorption delaying agent such as aluminum monostearate and gelatin in the composition.

[0180] Sterile injectable solutions can be prepared by incorporating active ingredients (e.g., an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or combinations thereof, in combination with an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof, alone or in combinations with other active agents) in the required amount in a suitable solvent with one or a combination of the ingredients listed in this application, as needed, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, two non-limiting examples of preparation methods are vacuum drying and lyophilization, which provide a powder of the active ingredient with any additional required ingredient from its solution, previously sterilized by filtration. Formulations for injection are processed, dispensed into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions in accordance with methods known in the art. In addition, preparations may be packaged and sold in kit form. Such finished products may preferably have labels or instructions for use indicating that the compositions are suitable for the treatment of a subject suffering from or predisposed to developing cancer.

[0181] Parenteral formulations can be a single bolus dose, an infusion, or a loading bolus dose followed by a maintenance dose. These compositions may be administered at certain fixed or variable intervals, such as once a day, or as needed.

[0182] Some useful pharmaceutical compositions may be administered orally in a suitable dosage form, including, for example, capsules, tablets, aqueous suspensions or solutions. Some pharmaceutical compositions may also be administered as an intranasal aerosol or inhaled formulation. Such compositions may be formulated as solutions in saline using benzyl alcohol or other suitable preservatives, absorption accelerators to increase bioavailability, and/or other conventional solubilizing or dispersing agents.

[0183] The amount of epigenetic modulating agent, e.g., HDACi, DNMTi, and/or combinations thereof, and an anti-SEMA4D binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, to combine with the carrier materials to prepare a single dose dosage form will vary depending on the subject to be treated and the particular route of administration. The composition may be administered as a single dose, multiple doses, or over a set period of time as an infusion. Dosing regimens can also be adjusted to obtain the optimal desired response (eg, therapeutic or prophylactic response).

[0184] Within the scope of the present invention, an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or a combination thereof, in combination with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, may be administered to a human or other animal in accordance with the above methods of treatment in an amount sufficient to achieve a therapeutic effect. The epigenetic modulating agent in combination with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative thereof, may be administered to such human or other animal in a conventional dosage form prepared by combining the antibody and the epigenetic modulating agent with a conventional pharmaceutically acceptable carrier or diluent. according to known methods. One of skill in the art will recognize that the form and nature 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 appreciate that a cocktail containing an epigenetic modulating agent and a binding molecule can be used.

[0185] By "effective amount" is meant the amount of an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or combinations thereof, and the amount of an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, that, when administered, elicits a positive therapeutic effect. a response to the treatment of a patient with a disease being treated, for example, an antiproliferative effect, prevention of further tumor growth, reduction in tumor size, reduction in tumor vasculature, reduction in the number of cancer cells, inhibition, delay or reduction in the growth of tumor and/or malignant cells, and/ or metastases in a subject suffering from cancer, and/or a reduction in one or more of the symptoms associated with the disease can be observed.

[0186] Therapeutically effective doses of the compositions described herein, for example, for antiproliferative action, preventing further tumor growth, reducing tumor size, reducing tumor vasculature, reducing the number of cancer cells, inhibiting, delaying or reducing the growth of tumor and/or malignant cells , and/or metastases in a subject suffering from cancer and/or reduction in one or more of the symptoms associated with the disease may vary depending on many different factors, including routes of administration, target site, physiological state of the patient, whether the patient is a human or other animals, whether other drugs are being administered, and whether the treatment is prophylactic or therapeutic. In certain embodiments, the patient is a human, but non-human animals, including transgenic animals, may also be treated. Dosages for treatment may be adjusted using routine methods known to those skilled in the art to optimize safety and efficacy.

[0187] The amount of epigenetic modulating agent, e.g., HDACi, DNMTi, and/or combinations thereof, in combination with an anti-SEMA4D binding molecule to be administered can be readily determined by one of skill in the art without undue experimentation, given the disclosure provided herein . Factors influencing the mode of administration and the appropriate amount of the epigenetic modulating agent and binding molecule include, but are not limited to, disease severity, medical history, and the age, height, body weight, health, and physical condition of the individual being treated. Likewise, the amount of epigenetic modulating agent and binding molecule to be administered will depend on the mode of administration and whether the subject will be subjected to single or multiple doses of the agent.

[0188] Also provided is the use of an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or combinations thereof, in combination with an anti-SEMA4D binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, in the manufacture of a medicament for the treatment of a subject suffering from cancer, and in some aspects further comprising prior treatment with at least one other therapy. By "pre-treated" or "pre-treated" is meant that the subject received one or more other therapies (e.g., received at least one other anti-cancer therapy) prior to receiving a drug containing an epigenetic modulating agent in combination with a binding molecule to SEMA4D, for example, an antibody or antigen-binding fragment, variant or derivative thereof. "Pre-treated" or "pre-treated" includes subjects who received at least one other therapy for 2 years, for 18 months, for 1 year, for 6 months, for 2 months, for 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 up to 1 day prior to treatment with a drug containing an epigenetic modulating agent such as HDACi such as entinostat and/or DNMTi such as azacitidine and an anti-SEMA4D binding molecule such as the VX15/2503 monoclonal antibody disclosed herein. application, or its antigennegative fragment, variant or derivative. It is not necessary for the subject to respond to pre-treatment with prior therapy or therapies. Thus, a subject receiving a drug containing an epigenetic modulating agent in combination with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, may or may not have responded to prior treatment with prior therapy, or to one or more previous therapies, if the previous treatment included several therapies.

[0189] The methods provided herein also involve the use of an epigenetic modulating agent, e.g., HDACi, DNMTi, and/or combinations thereof, in combination with an anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, in the manufacture of a medicament for treating a subject suffering from cancer, wherein the medicament is administered to a subject also receiving at least one other therapy. Available therapies disclosed in this application include, but are not limited to, surgery, radiation therapy, cancer vaccine, administration of an immunostimulatory agent, adoptive T-cell therapy or antibody therapy, administration of an inhibitor that blocks immune checkpoints, administration of a modulator of regulatory T- cells (Treg) or a combination thereof.

[0190] The present invention uses, unless otherwise indicated, conventional methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are known to those skilled in the art. Such methods are fully described 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. US patent No. 4683195; 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., NY); 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, NY, (1986); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0191] General principles for constructing antibodies are set forth in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed ^.; Oxford Univ. Press). General principles for constructing proteins are set forth in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). The 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, NY). In addition, standard immunology methods known in the art and not specifically described can be followed, as set forth 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 (WH Freeman and Co., NY).

[0192] Standard reference works that outline the 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 Analyzes (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).

[0193] All sources cited above, as well as all sources cited in this application, are fully incorporated by reference.

[0194] The following examples are for illustrative purposes only and not for limitation.

Examples

[0195] Example 1: Administration of Entinostat (ENT) and Anti-SEMA4D Antibody Increases Survival, Significantly Delays Tumor Growth, and Increases Complete Tumor Regression Rate

[0196] The effect of combined administration of ENT and MAb to SEMA4D on survival, tumor growth, and tumor regression was tested in a mouse tumor model. Colon26 cells (500,000 cells) were implanted subcutaneously in Balb/c mice. Mice received 1) control Ig (10 mg/kg, ip, weekly x 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 r/week × 2 weeks, started when the average tumor volume was ~300 mm ³ ) (n=21); 3) anti-SEMA4D/MAb 67 antibody (10 mg/kg, ip, weekly x 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 times a week × 2 weeks, started when the average volume tumor was ~300 mm ³ ) (n=26) (experimental scheme is shown in FIG. 1). The volume of tumors during treatment was the same among the control and experimental groups. A Kaplan-Meier survival analysis was performed to evaluate the survival function, a change in tumor volume was measured to determine the rate of tumor growth, and the rate of complete tumor regression was assessed by determining the percentage of mice without a tumor. The percentage of complete regression (% CR) was also stratified depending on whether the tumor volume exceeded at least 100 mm ³ before regression. Statistical significance was determined using the Cox-Mantel log-rank test for survival, two-way ANOVA for tumor volume, and Fisher's exact test for CR. Results are reported in Prism as non-significant (ns) at P>0.05, significant (marked with "*") at 0.01<P≤0.05, very significant ("**") at 0.001<P≤0.01 , extremely significant (“***”) at Р≤0.001, and most significant (“****”) at Р≤0.0001. Mice that did not develop tumors or that developed tumor ulceration before the tumors reached a volume of 700 mm ³ were excluded from the analysis.

[0197] A synergistic effect on tumor growth retardation was observed in the group treated with the combination of ENT and anti-SEMA4D/MAb 67 antibodies (maximum tumor growth retardation (TGD) 782%) compared with groups treated with either agent alone: 107% TGD with SEMA4D/MAb 67 and 214% TGD with ENT (FIG. 2A). Mice treated with ENT and anti-SEMA4D/MAb 67 also showed a significant improvement in survival compared to mice treated with ENT or anti-SEMA4D/MAb 67 (p<0.05) (FIG. 2B). In addition, combination treatment with ENT and anti-SEMA4D/MAb 67 significantly increased the rate of complete tumor regression (62%***), especially among tumors greater than 100 mm ³ before regression, compared with either agent alone (FIG. .2C and 2D). These results indicate that ENT and anti-SEMA4D MAb treatment improved survival, reduced tumor growth, and resulted in regression of larger established tumors.

[0198] Example 2: Administration of ENT and Anti-SEMA4D Antibody Increases Survival, Reduces Tumor Growth, and Increases Complete Regression Rate

[0199] Colon26 cells (500,000 cells) were subcutaneously implanted in Balb/c mice. Mice received 1) control Ig (10 mg/kg, weekly x 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 r/week × 2 weeks, started when the average tumor volume was ~250 mm ³ ) (n =9); 3) anti-SEMA4D/MAb 67 antibody (10 mg/kg, ip, weekly x 5 weeks starting on day 2) (n=8); or 4) anti-SEMA4D/MAb 67 antibody (10 mg/kg, weekly x 5 weeks, starting on day 2) and ENT (20 mg/kg, 3 times a week x 2 weeks, started when the mean tumor volume was ~250 mm ³ ) (n=13). The volume of tumors during treatment was the same among the control and experimental groups. Mean tumor volume, Kaplan-Meier survival curves, and complete tumor regression rates are shown for each group. Statistical significance was determined using two-way ANOVA, log-rank Cox-Mantel test, and Fisher's exact test, respectively. Results are reported in Prism as non-significant (ns) at P>0.05, significant (indicated by "*") at 0.01<P≤0.05, very significant ("**") at 0.001P≤0.01, and extremely significant (“***”) at Р≤0.001. Mice that did not develop tumors or that developed tumor ulceration before the tumors reached a volume of 700 mm ³ were excluded from the analysis.

[0200] Mice treated with ENT and anti-SEMA4D/MAb 67 antibody showed a maximum TGD of 705%, while mice treated with anti-SEMA4D/MAb67 antibody showed a tumor growth delay of 119% (FIG. 3A). Combined treatment of mice with ENT and anti-SEMA4D/MAb 67 antibody also increased the rate of complete tumor regression (62%**) compared to control Ig and ENT (25%*) groups (FIGS. 3B-3D). In addition, mice treated with ENT and anti-SEMA4D/MAb 67 showed 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 combined treatment with ENT and anti-SEMA4D MAb resulted in increased survival, decreased tumor growth, and tumor eradication.

[0201] Example 3: Administration of Azactidine (AZA) and Anti-SEMA4D Antibody Increases Survival, Significantly Delays Tumor Growth, and Increases Complete Tumor Regression Rate

[0202] The effect of combined administration of AZA and MAb to SEMA4D on survival, tumor growth, and tumor regression was tested in a mouse tumor model. Colon26 cells (500,000 cells) were implanted subcutaneously in Balb/c mice. Mice received: group 1: control Ig (10 mg/kg, ip, weekly × 4, starting on day 2 (n=12)); group 2: anti-SEMA4D/MAb 67 antibody (10 mg/kg, ip, weekly x 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 r/week X 4 doses, starting on days 22-24, when the average tumor volume was ~200 mm ³ (n=10)); and group 4: anti-SEMA4D/MAb 67 antibody (10 mg/kg, ip, weekly × 4 from day 2 + AZA (0.8 mg/ kg, 3 r/week X 4 doses starting from days 22-24, when the average tumor volume was ~200 mm ³ (n=9)). until tumor volumes reached approximately 1500 mm ³ .

[0203] The volumes of tumors during treatment were the same among the control and experimental groups. A Kaplan-Meier survival analysis was performed to evaluate the survival function, a change in tumor volume was measured to determine the rate of tumor growth, and the rate of complete tumor regression was assessed by determining the percentage of mice without a tumor. Statistical significance was determined using the Cox-Mantel log-rank test for survival, two-way ANOVA for tumor volume, and Fisher's exact test for complete tumor regression. In Prism, results are reported as non-significant at P>0.05, significant (marked "*") 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 the tumors reached a volume of 700 mm ³ were excluded from the analysis.

[0204] Mice treated with AZA and anti-SEMA4D/MAb67 antibody showed a maximum TGD of 705%, while mice treated with anti-SEMA4D/MAb67 antibody showed tumor growth inhibition of 119% (FIG. 5A). Mice treated with AZA and anti-SEMA4D/MAb 67 antibody also showed a significant improvement in survival compared to control mice (p<0.001) and a near-significant improvement in survival compared to the anti-SEDMA4D/MAb67 antibody agent alone (p=0.0697) (FIG. 5B). In addition, combination treatment with AZA and anti-SEMA4D/MAb 67 antibody significantly increased the rate of complete tumor regression to 78% (p<0.01) compared to control Ig (17%) (FIG. 5C). These results indicate that AZA and anti-SEMA4D MAb treatment improved survival, reduced tumor growth, and resulted in regression of larger established tumors.

[0205] The breadth and scope of the present invention is not limited by any of the illustrative embodiments described above, but should only be determined in accordance with the following claims and their equivalents.

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SEQUENCE LIST

<110> Vaccinex, Inc.

Evans, Elizabeth

Smith, Ernest

ZAUDERER, Maurice

<120> CANCER TREATMENT WITH ANTIBODY TO SEMAPHORINE 4D

IN COMBINATION WITH AN EPIGENETIC MODULATING AGENT

<130> 58008-172854

<150> 62/473.731

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<---

Claims (34)

1. Pharmaceutical composition for inhibiting, delaying or reducing the growth of malignant cells in a subject suffering from cancer, containing an effective amount of an isolated antibody or antigen-binding fragment specifically binding 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) and azacitidine, wherein said antibody or fragment thereof contains a variable heavy chain (VH) containing CDR 1-3 VH, containing SEQ ID NO: 2, 3 and 4, respectively, and a variable light chain (VL) containing CDR 1-3 VL, containing SEQ ID NO: 6, 7 and 8, respectively. 2. Pharmaceutical composition according to claim 1, additionally containing a carrier, excipient, or any combination thereof. 3. Pharmaceutical composition according to claim 1, where VH and VL contain, respectively, SEQ ID NO: 1 and SEQ ID NO: 5, or SEQ ID NO: 9 and SEQ ID NO: 10. 4. The pharmaceutical composition according to claim 1, wherein the epigenetic modulating agent is an entinostat (pyridin-3-ylmethyl-N-[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate). 5. The pharmaceutical composition of claim 1 wherein the epigenetic modulating agent comprises azacitidine. 6. Pharmaceutical composition for inhibiting, delaying or reducing the growth of malignant cells in a subject suffering from cancer, containing: (a) an effective amount of an isolated antibody or antigen-binding fragment thereof specifically binding to semaphorin 4D (SEMA4D), wherein the antibody or fragment thereof comprises the VH amino acid sequence of SEQ ID NO: 1 and the VL amino acid sequence of SEQ ID NO: 5, and (b) an effective amount of HDACi entinostat. 7. Pharmaceutical composition for inhibiting, delaying or reducing the growth of malignant cells in a subject suffering from cancer, containing: (a) an effective amount of an isolated antibody or antigen-binding fragment thereof specifically binding to semaphorin 4D (SEMA4D), wherein the antibody or fragment thereof comprises the VH amino acid sequence of SEQ ID NO: 1 and the VL amino acid sequence of SEQ ID NO: 5, and (b) an effective amount of DNMTi of azacitidine. 8. A method of inhibiting, delaying or reducing the growth of malignant cells in a subject suffering from cancer, comprising administering to said subject a pharmaceutical composition according to any one of paragraphs. 1-7. 9. The method of claim 8, wherein the antibody or fragment thereof inhibits the interaction of SEMA4D with its receptor. 10. The method of claim 9, wherein the receptor is plexin B1, plexin B2, or CD72. 11. The method according to any one of paragraphs. 9, 10, wherein the antibody or fragment thereof inhibits SEMA4D-mediated plexin B1 signaling. 12. The method according to any one of paragraphs. 8-11, where the antibody or fragment contains a variable heavy chain (VH) containing CDR 1-3 VH containing SEQ ID NO: 2, 3 and 4, respectively, and a variable light chain (VL) containing CDR 1-3 VL containing SEQ ID NO: 6, 7 and 8, respectively. 13. The method according to claim 12, where VH and VL contain, respectively, SEQ ID NO: 1 and SEQ ID NO: 5, or SEQ ID NO: 9 and SEQ ID NO: 10. 14. The method according to any one of paragraphs. 8-13, wherein the antibody or antigen-binding fragment thereof and the epigenetic modulating agent are administered separately or simultaneously. 15. The method according to any one of paragraphs. 8-14, where the administration of the combination therapy results in an increase in therapeutic efficacy compared to the administration of the antibody or fragment thereof or the epigenetic modulating agent alone. 16. The method of claim 15 wherein the increased therapeutic efficacy outweighs the additive effect. 17. The method according to any one of paragraphs. 8-16, wherein the cancer includes a solid tumor, a hematological malignancy, any metastasis thereof, or any combination thereof. 18. The method of claim 17 wherein the cancer is a solid tumor or its metastases. 19. The method of claim 18, wherein the solid tumor is a sarcoma, carcinoma, melanoma, any of their metastases, or any combination thereof. 20. The method of claim 18 wherein the solid tumor is squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, renal cell carcinoma, ductal breast carcinoma, soft tissue sarcoma, osteosarcoma, melanoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, peritoneal cancer, 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 colon, colorectal, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, any metastasis thereof, or any combination thereof. 21. The method of claim 17 wherein the cancer is a hematological malignancy or metastases thereof. 22. The method of claim 21 wherein the hematologic malignancy is leukemia, lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, any of them. metastases, or any combination thereof. 23. The method according to any one of paragraphs. 8-22, further comprising administering additional anti-cancer therapy. 24. The method according to claim 23, where the additional therapy includes surgery, chemotherapy, radiation therapy, cancer vaccine, administration of an immunostimulatory agent, adoptive T-cell therapy or antibody therapy, administration of an inhibitor that blocks immune checkpoints, administration of a regulatory T-cell modulator (Treg) and their combination. 25. The method according to any one of paragraphs. 8-24 where: (a) an isolated antibody or antigen-binding fragment thereof specifically binding to semaphorin 4D (SEMA4D) contains a VH containing the amino acid sequence of SEQ ID NO: 1 and a VL containing the amino acid sequence of SEQ ID NO: 5, and (b) the epigenetic modulating agent comprises an HDACi entinostat. 26. The method according to any one of paragraphs. 8-24 where: (a) an isolated antibody or antigen-binding fragment thereof specifically binding to semaphorin 4D (SEMA4D) contains the VH amino acid sequence of SEQ ID NO: 1, and the VL amino acid sequence of SEQ ID NO: 5, and (b) the epigenetic modulating agent includes DNMTi azacitidine.

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