Use of Anti-semaphorin-4d inhibitory molecules in combination with FLT3 ligand to inhibit tumor growth and metastasis

Abstract

A combination therapy comprising a semaphorin-4D inhibitory molecule and Flt3 ligand or a FLT3 agonist with or without a checkpoint inhibitor for treating cancer is disclosed.

Description

USE OF ANTI- SEMAPHORING INHIBITORY MOLECULES IN COMBINATION WITH FLT3 LIGAND TO INHIBIT TUMOR GROWTH AND METASTASISFIELD OF THE DISCLOSURE

[0001] The disclosure relates to a combination therapy comprising a semaphorin-4D inhibitory molecule and Flt3 ligand or a FLT3 agonist with or without a checkpoint inhibitor for treating cancer.BACKGROUND

[0002] Semaphorin 4D (SEMA4D), also known as CD 100, is a transmembrane protein that belongs to the semaphorin gene family. SEMA4D is expressed on the cell surface as a homodimer, but upon cell activation SEMA4D can be released from the cell surface via proteolytic cleavage to generate sSEMA4D, a soluble form of the protein, which is also biologically active. See Suzuki et al., Nature Rev. Immunol. 3: 159-167 (2003); Kikutani et al., Nature Immunol. 9: 17-23 (2008).

[0003] SEMA4D is expressed at high levels in lymphoid organs, including the spleen, thymus, and lymph nodes, and in non-lymphoid organs, such as the brain, heart, and kidney. In lymphoid organs, SEMA4D is abundantly expressed on resting T cells but only weakly expressed on resting B cells and antigen-presenting cells (APCs), such as dendritic cells (DCs). Its expression, however, is upregulated in these cells following activation by various immunological stimuli. The release of soluble SEMA4D from immune cells is also increased by cell activation. SEMA4D has been implicated in the development of certain cancers (Ch’ng et al., Cancer 110: 164-72 (2007); Campos et al., Oncology Letters, 5: 1527-35 (2013); Kato et al., Cancer Sci. 102:2029-37 (2011)) and several reports suggest that one mechanism of this influence is the role of SEMA4D in promoting tumor angiogenesis (Conrotto et al., Blood 105:4321-4329 (2005). Basile et al., J Biol. Chem. 282: 34888-34895 (2007); Sierra et.al. J. Exp. Med. 205: 1673 (2008); Zhou et al., Angiogenesis 15:391-407 (2012)). Tumor growth and metastasis involve a complex process of cross talk amongst the tumor cells, stroma and immune infiltrate, as well as the endothelial cells and vasculature. SEMA4D is over-expressed in a wide array of tumor types and is also produced by inflammatory cells recruited to the tumor microenvironment. Strong expression of SEMA4D at the invasive margins of actively growing tumors negatively influences the infiltration and distribution of leukocytes in the tumor microenvironment (TME). (Evans et al., Antibody Blockadeof Semaphorin 4D Promotes Immune Infiltration into Tumor and Enhances Response to Other Immunomodulatory Therapies, Cancer Immunol . Res. 2015 Jun;3(6):689-701).

[0004] The cytokine Fms-like tyrosine kinase 3 ligand (FL or Flt3L) is an important regulator of hematopoiesis. Its receptor, FLT3, is expressed on myeloid, lymphoid and dendritic cell progenitors and is considered to be an important growth and differentiation factor for several hematopoietic lineages. Flt3L binds to an FLT3 receptor and activates a signaling pathway, promoting the growth and development of hematopoietic stem cells, progenitor cells, and the immune system. Fit- 3 ligand is critical to the expansion of dendritic cells (DCs), which are central to inducing T cell responses.

[0005] To mount an effective anti-tumor immune response capable of controlling or eliminating a cancer, sufficient numbers of lymphocytes (T cells and B cells) and antigen-presenting cells must be recruited to malignant tissue and allowed to sustain their effector functions within the tumor microenvironment (TME). A higher infiltration of T and B cells in tumor tissue, often referred to as “hot tumors,” is prognostic for patient survival and predictive of response to immunotherapy in almost all cancer types. The organization of immune cells into tertiary lymphoid structures (TLS) in solid tumors is an example of a hot tumor in which T and B lymphocytes aggregate with antigen presenting cells, e.g., DCs. The presence of high density mature DC within mature TLS, characterized by a strong memory Thl and cytotoxic orientation, confers a lower risk of tumor progression and death. (Cancer Res (2014) 74 (3): 705-715). TLS have been observed in almost every solid tumor type and correlate with improved survival in cancer patients (Fridman WH, et al., B cells and tertiary lymphoid structures as determinants of tumor immune contexture and clinical outcome. Nat Rev Clin Oncol (2022) 7:441-57).

[0006] Thus, there is a need in the art for methods of disrupting the gradient of expression of SEMA4D at the tumor margins and enhancing recruitment of activated monocytes and lymphocytes into the tumor and inducing the formation of TLS. The present disclosure addresses this need by providing a combination therapy including an anti-SEMA4D antibody and Flt3L or a FLT3 agonist, and optionally other immunotherapeutic agents, such as immune checkpoint inhibitors (ICI).SUMMARY OF THE DISCLOSURE

[0007] The present disclosure relates to the use of a combination immunotherapy containing an anti-SEMA4D antibody and Flt3L or a FLT3 agonist to treat cancer.

[0008] In one aspect, the embodiments of the disclosure provide for a method of treating a subject having or suspected of having cancer comprising administering to the subject a combination therapy comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to semaphorin-4D (SEMA4D) and an effective amount of Fms-like tyrosine kinase 3 ligand (Flt3L) or a Fms-like tyrosine kinase 3 (FLT3) agonist, and optionally at least one other immune modulating therapy, preferably at least one immune checkpoint inhibitor.

[0009] In some embodiments of any of the methods and uses described herein, the anti- SEMA4D antibody or antigen-binding fragment thereof inhibits SEMA4D interaction with its receptor. In some of these embodiments, the receptor is Plexin-Bl, Plexin-B2, CD72, or any combination thereof. In some of these embodiments, the anti-SEMA4D antibody or fragment thereof inhibits SEMA4D-mediated signal transduction.

[0010] In some embodiments of any of the methods and uses described herein, the anti- SEMA4D antibody or antigen-binding fragment thereof is selected from the group consisting of

[0011] (i) an antibody or antigen binding fragment thereof comprising a variable heavy chain (VH) region having VH CDRs 1-3 comprising amino acid sequences SEQ ID NOS: 1, 2, and 3, respectively, and a variable light chain (VL) region comprising VL CDRs 1-3 comprising SEQ ID NOS: 4, 5, and 6, respectively;

[0012] (ii) the antibody of (i), or antigen binding fragment thereof, wherein the VH and VL chains comprise, respectively, SEQ ID NO: 7 and SEQ ID NO: 8 (human), or SEQ ID NO: 9 and SEQ ID NO: 10 (mouse);

[0013] (iii) an antibody or antigen binding fragment thereof comprising a VH region having VH CDRs 1-3 comprising amino acid sequences SEQ ID NOS: 11, 12, and 13, respectively; and a VL region comprising VL CDRs 1-3 comprising amino acid sequences SEQ ID NOS: 14, 15, and 16, respectively; and

[0014] (iv) the antibody of (ii), or antigen binding fragment thereof, wherein the VH and VL chains comprise, respectively, SEQ ID NO: 17 and SEQ ID NO: 18.

[0015] For ease of understanding, (i) provides the six CDRs for mouse / human MAbVXl 5 / 2503; (ii) provides the antigen binding domains for mouse / human MAbVXl 5 / 2503; (iii) provides the six CDRs for MAb D2517; and (iv) provides the antigen binding domains for MAb D2517.

[0016] In some embodiments of any of the methods and uses described herein, the anti- SEMA4D antibody is pepinemab or an antigen-binding fragment thereof.

[0017] In some embodiments of any of the methods and uses described herein, the combination therapy comprises Flt3L. In certain embodiments, the Flt3L is a human recombinant protein. In some embodiments, the Flt3L is a Flt3L fusion protein (Flt3L-Fc). In certain embodiments the Flt3L-Fc is encoded by a vector, such as an adenovirus or poxvirus vector. In other embodiments, the combination therapy comprises a FLT3 agonist, such as a Flt3L fusion protein, which can be encoded by a vector.

[0018] In some embodiments of any of the methods and uses described herein, the isolated anti- SEMA4D antibody, or antigen-binding fragment thereof, the Flt3L or FLT3 agonist and the optional one or more other immune modulating therapy, preferably an immune checkpoint inhibitor, are administered separately or concurrently.

[0019] In some embodiments, the other immune modulating therapy is selected from the group consisting of administration of a cancer vaccine, administration of an immunostimulatory agent, adoptive T cell or antibody therapy, administration of an immune checkpoint inhibitor, administration of a regulatory T cell (Treg) modulator, and a combination thereof.

[0020] In some embodiments, the immune modulating therapy comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of an antibody or antigen-binding fragment thereof that specifically binds to CTLA4, PD-1, PD-L1, LAG3, TIM3, B7-H3, TIGIT or any combination thereof. In some embodiments, the antibody or antigen-binding fragment of the immune checkpoint inhibitor comprises the anti- PD-1 antibody pembrolizumab (Keytruda®), nivolumab (Opdivo®), cemiplimab (Libtayo®), retifanlimab (Zynyz®), dostarlimab (Jemperli®), or anti-PD-Ll antibody avelumab, atezolizumab, durvalumab.

[0021] In some embodiments, the immune modulating therapy comprises administration of a cancer vaccine.

[0022] In some embodiments, the immune modulating therapy comprises administration of a Treg modulator. In some embodiments, the Treg modulator is cyclophosphamide

[0023] In some embodiments of any of the methods and uses described herein, the cancer is carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, head and neck cancer, melanoma, or any combination thereof. In certain embodiments of any of the methods and uses described herein, the cancer is locally advanced, recurrent or metastatic cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Fig. 1 is a graph showing the probability of survival of Colon26-tumor bearing mice treated with control Ig, MAb67 (anti-SEMA4D MAb), Flt3L plus control Ig, or Flt3L plus MAb67.

[0025] Fig.2A-D are graphs showing tumor volume of individual Colon26-tumor bearing mouse after treatment with control Ig (2A), MAb67 (anti-SEMA4D MAb), Flt3L plus control Ig (C), and Flt3L plus MAb67 (2D).

[0026] Fig. 3 is a series of bar graphs showing infiltration of dendritic cells (DC)(3A), B cells (3B) or T cells (3C) into tissue of tumors resected from Colon26-tumor bearing mice treated with control Ig, anti-SEMA4D MAb67, Flt3L plus control Ig, or combination of Flt3L plus anti- SEMA4D MAb67

[0027] Figure 4 is a graph showing probability of survival of TUBO.B 1 mice treated with control Ig, MAb67 (anti-SEMA4D MAb), Flt3LPlus control Ig, or combination of Flt3L plus MAb67.

[0028] Figure 5A-D are graphs showing tumor volumes of individual TUBO.B 1 -tumor bearing mice after treatment with control Ig (5 A), MAb67 (anti-SEMA4D MAb) (5B), Flt3L plus control Ig (5C), and Flt3L plus MAb67 (anti-SEMA4D MAb) (5D). Combination therapy significantly increased the number of complete tumor responses (CR).

[0029] Figure 6 is a graph showing probability of survival of BCA34 fibrosarcoma implanted mice treated with control Ig, MAb67 (anti-SEMA4D MAb), Flt3L plus control Ig, or a combination of Flt3L plus MAb67 (anti-SEMA4D MAb).

[0030] Figure 7A-D are graphs showing tumor volume of BCA34 fibrosarcoma implanted mice after treatment with control Ig (2A), MAb67 (2B), Flt3L plus control Ig, (C), and Flt3L plus MAb67 (2D).

[0031] Figure 8 is a graph showing tumor volume of B16.F1 melanoma-implanted mice after treatment with control Ig (2A), pepinemab (2B), Flt3L plus control Ig (C), and Flt3L plus MAb67 (2D).

[0032] Figure 9 is a graph showing probability of survival of B16.F1 melanoma-implanted mice treated with control Ig, MAb67 (pepinemab), Flt3L, or a combination of MAb67 and Flt3LDETAILED DESCRIPTION OF THE DISCLOSUREDefinitions

[0033] In order that the present disclosure may be more readily understood, certain terms are defined below. Additional definitions may be found within the detailed description of the disclosure.

[0034] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0035] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0037] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0038] Wherever embodiments are described with the language "comprising," otherwise analogous embodiments described in terms of "consisting of' and / or "consisting essentially of' are also provided.

[0039] Amino acids are referred to herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

[0040] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, gastric, pancreatic 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, sarcoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, melanoma, and various types of head and neck cancers.

[0041] In certain embodiments, metastatic cancers that are amenable to treatment via the methods provided herein include, but are not limited to metastatic sarcomas, breast carcinomas, ovarian cancer, head and neck cancer, lung cancers, pancreatic cancer, and melanoma. In certain embodiments metastatic cancers or tumor cells that are amenable to treatment via the methods provided herein express Plexin-Bl and / or Plexin-B2 receptors for SEMA4D.

[0042] The terms “proliferative disorder” and “proliferative disease” refer to disorders associated with abnormal cell proliferation such as cancer.

[0043] “ Tumor” and “neoplasm” as used herein refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions. In certain embodiments, tumors described herein express Plexin- B1 and / or Plexin-B2 and can express SEMA4D.

[0044] The term “immune modulating therapy” or “immunotherapy” refers to treatment that impacts a disease or disorder in a subject by inducing and / or enhancing an immune response in that subject. Immune modulating therapies include cancer vaccines, immunostimulatory agents, adoptive T cell or antibody therapy, and immune checkpoint inhibitors (Lizee et al. 2013. Harnessing the Power of the Immune System to Target Cancer. Annu. Rev. Med. Vol. 64 No. 71- 90).

[0045] The term “immune modulating agent” refers to the active agents of immunotherapy. Immune modulating agents include a diverse array of recombinant, synthetic and natural preparations. Examples of immune modulating agents include, but are not limited to, interleukins such as IL-2, IL-7, IL- 12; cytokines such as granulocyte colony-stimulating factor (G-CSF), interferons; various chemokines such as CXCL13, CCL26, CXCL7; antagonists of immune checkpoint blockades such as anti-CTLA-4, anti-PDl or anti-PD-Ll (ligand of PD-1), anti-LAG3, anti-B7-H3, synthetic cytosine phosphate-guanosine (CpG) oligodeoxynucleotides, glucans; and modulators of regulatory T cells (Tregs) such as cyclophosphamide.

[0046] The terms “metastasis,” "metastases," "metastatic," and other grammatical equivalents as used herein refer to cancer cells which spread or transfer from the site of origin (e.g., a primary tumor) to other regions of the body with the development of a similar cancerous lesion at the new location. A “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures. The terms also refer to the process of metastasis, which includes, but is not limited to detachment of cancer cells from a primary tumor, intravasation of the tumor cells to circulation, their survival and migration to a distant site, attachment and extravasation into a new site from the circulation, and microcolonization at the distant site, and tumor growth and development at the distant site.

[0047] The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of thedrug can reduce the number of cancer cells; retard or stop cancer cell division, reduce or retard an increase in tumor size; inhibit, e.g., suppress, retard, prevent, stop, delay, or reverse cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit, e.g., suppress, retard, prevent, shrink, stop, delay, or reverse tumor metastasis; inhibit, e.g., suppress, retard, prevent, stop, delay, or reverse tumor growth; relieve to some extent one or more of the symptoms associated with the cancer, reduce morbidity and mortality; improve quality of life; or a combination of such effects. To the extent the drug prevents growth and / or kills existing cancer cells, it can be referred to as cytostatic and / or cytotoxic.

[0048] Terms such as "treating" or "treatment" or “to treat” or "alleviating" or “to alleviate” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, reverse, and / or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. A subject is successfully "treated" according to the methods of the present disclosure if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in tumor size; or retardation or reversal of tumor growth, inhibition, e.g., suppression, prevention, retardation, shrinkage, delay, or reversal of metastases, e.g., of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of, e.g., suppression of, retardation of, prevention of, shrinkage of, reversal of, delay of, or an absence of tumor metastases; inhibition of, e.g., suppression of, retardation of, prevention of, shrinkage of, reversal of, delay of, or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; or some combination of effects. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0049] By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, and so on.

[0050] As used herein, phrases such as "a subject that would benefit from administration of an anti-SEMA4D antibody in combination with Flt3L or optionally administration of an anti- SEMA4D antibody in combination with Flt3L and at least one immune checkpoint inhibitor" and "an animal in need of treatment" include subjects, such as mammalian subjects, that would benefit from administration of an anti-SEMA4D antibody or antigen-binding fragment thereof in combination with Flt3L or optionally administration of an anti-SEMA4D antibody in combination with Flt3L and at least one immune checkpoint inhibitor.

[0051] As used herein, "human" or "fully human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins, as described infra and, for example, in U.S. Pat. No. 5,939,598 by Kucherlapati et al. "Human" or "fully human" antibodies also include antibodies comprising at least the variable domain of a heavy chain, or at least the variable domains of a heavy chain and a light chain, where the variable domain(s) have the amino acid sequence of human immunoglobulin variable domain(s).

[0052] "Human" or "fully human" antibodies also include "human" or "fully human" antibodies, as described above, that comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (e.g., the VH regions and / or VL regions) described herein, which antibodies or fragments thereof immunospecifically bind to a SEMA4D polypeptide or fragment or variant thereof. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a human anti-SEMA4D antibody, including, but not limited to, site directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. In certain aspects, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than2 amino acid substitutions relative to the reference VH region, VHCDR1, VHCDR2, VHCDR3, VL region, VLCDR1, VLCDR2, or VLCDR3.

[0053] In certain embodiments, the amino acid substitutions are conservative amino acid substitution, discussed further below. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind a SEMA4D polypeptide, e.g., human, murine, or both human and murine SEMA4D). Such variants (or derivatives thereof) of "human" or "fully human" antibodies can also be referred to as human or fully human antibodies that are "optimized" or "optimized for antigen binding" and include antibodies that have improved affinity to antigen.

[0054] The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press).

[0055] As used herein, the term “immunoglobulin” comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, with some subclasses among them (e.g., yl-y4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgD, or IgE, respectively. The immunoglobulin subclasses (isotypes) e g., IgGl, IgG2, IgG3, IgG4, IgAl, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable region.

[0056] Light chains are classified as either kappa or lambda. Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0057] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL or VK) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

[0058] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs) within these variable domains, of an antibody combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains. In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule can consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0059] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acidsin the antigen binding domains, referred to as "framework" regions, show less inter-molecular variability. The framework regions largely adopt a [l-shcct conformation and the CDRs form loops that connect, and in some cases form part of, the P-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non- covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable domain by one of ordinary skill in the art, since they have been precisely defined (see below).

[0060] In the case where there are two or more definitions of a term that is used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al. (1983) U.S. Dept, of Health and Human Services, "Sequences of Proteins of Immunological Interest" and by Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0061] Table 1. CDR Definitions1Kabat ChothiaVH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-10295-102VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96 Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et al. (see below).

[0062] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al. (1983) U.S. Dept, of Health and Human Services, "Sequence of Proteins of Immunological Interest." Unless otherwise specified, references to the numbering of specific amino acid residue positions in an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof of the present disclosure are according to the Kabat numbering system.

[0063] Antibodies or antigen-binding fragments, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, bispecific, human, humanized, primatized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e g., anti-Id antibodies to anti-SEMA4D antibodies disclosed herein). ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2, etc.), or subclass of immunoglobulin molecule.

[0064] As used herein, the term "heavy chain portion" includes amino acid sequences derived from an immunoglobulin heavy chain. In certain embodiments, a polypeptide comprising a heavy chain portion comprises at least one of: a VH domain, a CHI domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the disclosure can comprise a polypeptide chain comprising a CHI domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHI domain and a CH3 domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, anda CH3 domain, or a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain. Further, a binding polypeptide for use in the disclosure can lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain portions) can be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.

[0065] In certain anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein, the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer. Alternatively, heavy chain portion-containing monomers of the disclosure are not identical. For example, each monomer can comprise a different target binding site, forming, for example, a bispecific antibody. A bispecific antibody is an artificial protein that is composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigen. Variations on the bispecific antibody format are contemplated within the scope of the present disclosure. Bispecific antibodies can be generated using techniques that are well known in the art for example, see, for example, Ghayur et al., Expert Review of Clinical Pharmacology 3.4 (July 2010): p. 491; Lu et al., J. Biological Chemistry Vol. 280, No. 20, p. 19665-19672 (2005); Marvin et al., Acta Pharmacologic Sinica 26(6):649-658 (2005); and Milstein C et al., Nature 1983; 305: 537-40; 30 Brennan M et al., Science 1985; 229: 81-3; Thakur et al., Curr Opin Mol Ther. 2010 Jun;12(3):340-9; and U.S. Patent Publication No. 2007 / 0004909.

[0066] The heavy chain portions of a binding molecule for use in the methods disclosed herein can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide can comprise a CHI domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.

[0067] As used herein, the term “light chain portion” includes amino acid sequences derived from an immunoglobulin light chain, e.g., a kappa or lambda light chain. In certain aspects, the light chain portion comprises at least one of a VL or CL domain.

[0068] Anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein can be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target polypeptide disclosed herein (e.g., SEMA4D) that they recognize or specifically bind. The portion of a target polypeptide that specifically interacts with the antigen binding domain of an antibody is an "epitope," or an "antigenic determinant." A target polypeptide can comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen. Furthermore, it should be noted that an "epitope" on a target polypeptide can be or can include non-polypeptide elements, e g., an epitope can include a carbohydrate side chain.

[0069] The minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids. Peptide or polypeptide epitopes can contain at least seven, at least nine and, in some cases, between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain. A peptide or polypeptide epitope recognized by anti-SEMA4D antibodies of the present disclosure can contain a sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids of SEMA4D.

[0070] By "specifically binds," it is generally meant that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" can be deemed to have a higher specificity or affinity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with a higher specificity or affinity than it has for related epitope "D."

[0071] By "preferentially binds," it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody that "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody can cross-react with the related epitope.

[0072] By way of non-limiting example, an antibody can be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (KD) that is less than the antibody's KD for the second epitope. In another non-limiting example, an antibody can be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's KD for the second epitope. In another nonlimiting example, an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's KD for the second epitope.

[0073] In another non-limiting example, an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's k(off) for the second epitope.

[0074] An antibody is said to competitively inhibit binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. An antibody can be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0075] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interactionbetween a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity.

[0076] Anti-SEMA4D antibodies or antigen-binding fragments, variants, or derivatives thereof of the disclosure can also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, an antibody is cross reactive if it binds to an epitope other than the one that induced its formation. The cross-reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original.

[0077] For example, certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody can be said to have little or no cross-reactivity if it does not bind epitopes with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody can be deemed "highly specific" for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope.

[0078] Anti-SEMA4D binding molecules, e.g., antibodies or antigen-binding fragments, variants or derivatives thereof of the disclosure can also be described or specified in terms of their binding affinity to a polypeptide of the disclosure, e.g., SEMA4D, e.g., human, murine, or both human and murine SEMA4D. In certain aspects, binding affinities include those with a dissociation constant or Kd less than 5 x 10’2M, 10'2M, 5 x 10'3M, 10’3M, 5 x 10'4M, 10’4M, 5 x 10‘5M, 10’5M, 5 x IO'6M, 10‘6M, 5 x IO’7M, 10‘7M, 5 x IO’8M, IO’8M, 5 x 10‘9M, IO’9M, 5 x IO’10M, IO’10M, 5 x 10’11M, 10’11M, 5 x IO’12M, 10’12M, 5 x 10’13M, 10’13M, 5 x IO’14M, 10'14M, 5 x IO’15M, or 10'15M. In certain embodiments, the anti-SEMA4D binding molecule, e.g., an antibody or antigen binding fragment thereof, of the disclosure binds human SEMA4D with a Kd of about 5 x 10'9to about 6 x 10'9. In another embodiment, the anti-SEMA4D binding molecule, e.g., an antibody or antigen binding fragment thereof, of the disclosure binds murine SEMA4D with a Kd of about 1 x 10’9to about 2 x 10'9.

[0079] As used herein, the term "chimeric antibody" will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial or modified) is obtained from a second species. In some embodiments the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0080] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain in either the heavy or light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, if necessary, by partial framework region replacement and sequence changing. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class or from an antibody from a different species. An engineered antibody in which one or more "donor" CDRs from a non- human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody." In certain aspects it is not necessary to replace all of the CDRs with the complete CDRs from the donor variable domain to transfer the antigen binding capacity of one variable domain to another. Rather, only those residues that are necessary to maintain the activity of the binding site against the targeted antigen can be transferred.

[0081] It is further recognized that the framework regions within the variable domain in a heavy or light chain, or both, of a humanized antibody can comprise solely residues of human origin, in which case these framework regions of the humanized antibody are referred to as "fully human framework regions" (for example, MAb VX15 / 2503, disclosed in U.S. Patent Appl. Publication No. U.S. 2010 / 0285036 Al as MAb 2503, incorporated herein by reference in its entirety). Alternatively, one or more residues of the framework region(s) of the donor variable domain can be engineered within the corresponding position of the human framework region(s) of a variable domain in a heavy or light chain, or both, of a humanized antibody if necessary to maintain proper binding or to enhance binding to the SEMA4D antigen. A human framework region that has been engineered in this manner would thus comprise a mixture of human and donor framework residues and is referred to herein as a "partially human framework region."

[0082] For example, humanization of an anti-SEMA4D antibody can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)),by substituting rodent or mutant rodent CDRs or CDR sequences for the corresponding sequences of a human anti-SEMA4D antibody. See also U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205; herein incorporated by reference. The resulting humanized anti-SEMA4D antibody would comprise at least one rodent or mutant rodent CDR within the fully human framework regions of the variable domain of the heavy and / or light chain of the humanized antibody. In some instances, residues within the framework regions of one or more variable domains of the humanized anti-SEMA4D antibody are replaced by corresponding non-human (for example, rodent) residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762; and 6,180,370), in which case the resulting humanized anti-SEMA4D antibody would comprise partially human framework regions within the variable domain of the heavy and / or light chain. Similar methods can be used for humanization of an anti-VEGF antibody.

[0083] Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain desired affinity). In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones et al., Nature 331 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); herein incorporated by reference. Accordingly, such "humanized" antibodies can include antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. See also U.S. Pat. No. 6,180,370, and International Publication No. WO 01 / 27160, where humanized antibodies and techniques for producing humanized antibodies having improved affinity for a predetermined antigen are disclosed.Anti-SEMA4D Antibodies

[0084] Antibodies that bind SEMA4D have been described in the art. See, for example, U.S. Patent No. 8,496,938; U.S. Patent No. 11,427,634, U.S. Publ. Nos. 2008 / 0219971, U.S. 2010 / 0285036, and U.S. 2006 / 0233793, U.S. Publ. No. 2021 / 0032329, International Patent Applications WO 93 / 14125, WO 2008 / 100995, and WO 2010 / 129917, and Herold et al., Int. Immunol. 7(1): 1-8 (1995), each of which is herein incorporated in its entirety by reference.

[0085] In certain embodiments, the antibody blocks the interaction of SEMA4D with one or more of its receptors, e.g., Plexin-Bl, Plexin-B2, and CD72. In certain embodiments the cancer cells and tumor infiltrating immune cells express Plexin-Bl, Plexin-B2 and / or CD72. Anti- SEMA4D antibodies having these properties can be used in the methods provided herein. Antibodies that can be used include but are not limited to MAbs VX15 / 2503 (pepinemab), 67, 76, 2282, VX18, D2517, or D2585, and antigen-binding fragments, variants, or derivatives thereof which are fully described in U.S. 2010 / 0285036, U.S. 2008 / 0219971 and U.S. Patent No. 11,427,634. The amino acid sequences of the VH and VL regions, as well as the associated CDRs for pepinemab (with VH / VL SEQ ID NOS: 7 and 8) and VX18 are provided in Table 2A and Table 2B, respectively.Table 2ATable 2B

[0086] Additional antibodies which can be used in the methods provided herein include the BD16 antibody described in U.S. 2006 / 0233793 Al as well as antigen-binding fragments, variants, or derivatives thereof; or any of MAb 301, MAb 1893, MAb 657, MAb 1807, MAb 1656, MAb 1808, Mab 59, MAb 2191 , MAb 2274, MAb 2275, MAb 2276, MAb 2277, MAb 2278, MAb 2279, MAb 2280, MAb 2281, MAb 2282, MAb 2283, MAb 2284, and MAb 2285, as well as any fragments, variants or derivatives thereof as described in U.S. 2008 / 0219971 Al. In certain embodiments an anti-SEMA4D antibody for use in the methods provided herein binds human, murine, or both human and murine SEMA4D. Also useful are antibodies which bind to the same epitope as any of the aforementioned antibodies and / or antibodies which competitively inhibit binding or activity of any of the aforementioned antibodies.

[0087] In certain embodiments, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof useful in the methods provided herein has an amino acid sequence that has at least about 80%, about 85%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% sequence identity to the amino acid sequence for a reference anti- SEMA4D antibody molecule, for example, those described above. In a further embodiment, the binding molecule shares at least about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to a reference antibody.

[0088] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin heavy chain variable domain (VH domain), where at least one ofthe CDRs of the VH domain has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or identical to any CDR1, CDR2 or CDR3 set forth in Tables 2A and 2B.

[0089] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin heavy chain variable domain (VH domain), where at least one of the CDRs of the VH domain has an amino acid sequence identical, except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, to any CDR1, CDR2 or CDR3 set forth in Tables 2A and 2B.

[0090] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof useful in the methods provided herein comprises, consists essentially of, or consists of a VH domain that has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to those VH domains set forth in Tables 2A and 2B, wherein an anti-SEMA4D antibody comprising the encoded VH domain specifically or preferentially binds to SEMA4D.

[0091] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin light chain variable domain (VL domain), where at least one of the CDRs of the VL domain has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or identical to any CDR1, CDR2 or CDR3 set forth in Tables 2A and 2B.

[0092] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof useful in the methods provided herein comprises, consists essentially of, or consists of an immunoglobulin light chain variable domain (VL domain), where at least one of the CDRs of the VL domain has an amino acid sequence identical, except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, to any CDR1, CDR2 or CDR3 set forth in Tables 2A and 2B.

[0093] In a further embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof useful in the methods provided herein comprises, consists essentially of, or consists of a VL domain that has an amino acid sequence that is at least about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to those VL domains set forth in Tables 2A and 2B, wherein an anti-SEMA4D antibody comprising the encoded VL domain specifically or preferentially binds to SEMA4D.

[0094] Suitable biologically active variants of the anti-SEMA4D antibodies of the disclosure can be used in the methods of the present disclosure. Such variants will retain the desired binding properties of the parent anti-SEMA4D antibody. Methods for making antibody variants are generally available in the art.

[0095] Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Methods Enzymol. 154:367-382 (1987); Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the polypeptide of interest can be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), pp. 345-352, herein incorporated by reference in its entirety. The model of Dayhoff et al. uses the Point Accepted Mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine suitable conservative amino acid substitutions. In certain aspects, conservative substitutions, such as exchanging one amino acid with another having similar properties are used. Examples of conservative amino acid substitutions as taught by the PAM 250 matrix of the Dayhoff et al. model include, but are not limited to, Gly^Ala, Val -Mle«->Leu, Asp^Glu, Lys«->Arg, Asn^GIn, and Phe^Trp^Tyr.

[0096] In constructing variants of the anti-SEMA4D binding molecule, e.g., an antibody or antigen-binding fragment thereof, polypeptides of interest, modifications are made such that variants continue to possess the desired properties, e.g., being capable of specifically binding to a SEMA4D, e.g., human, murine, or both human and murine SEMA4D, e.g., expressed on the surface of or secreted by a cell and having SEMA4D blocking activity, as described herein. In certain aspects, mutations made in the DNA encoding the variant polypeptide maintain the reading frame and do not create complementary regions that could produce secondary mRNA structure. See EP Patent Application Publication No. 75,444.

[0097] Methods for measuring anti-SEMA4D binding molecule, e.g., an antibody or antigenbinding fragment, variant, or derivative thereof, binding specificity include, but are not limited to, standard competitive binding assays, assays for monitoring immunoglobulin secretion by T cells or B cells, T cell proliferation assays, apoptosis assays, ELISA assays, and the like. See, for example, such assays disclosed in WO 93 / 14125; Shi et al., Immunity 13:633-642 (2000); Kumanogoh et al., J Immunol 169:1175-1181 (2002); Watanabe et al., J Immunol 167:4321-4328 (2001); Wang et al., Blood 97:3498-3504 (2001); and Giraudon et al., J Immunol 172(2): 1246- 1255 (2004), all of which are herein incorporated by reference.

[0098] When discussed herein whether any particular polypeptide, including the constant regions, CDRs, VH domains, or VL domains disclosed herein, is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% identical to another polypeptide, the % identity can be determined using methods and computer program s / software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0099] For purposes of the present disclosure, percent sequence identity can be determined using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math. 2:482-489. A variant can, for example, differ from a reference anti-SEMA4D antibody (e g., MAb VX15 / 2503, 67, 76, or 2282) by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

[0100] The constant region of an anti-SEMA4D antibody can be mutated to alter effector function in a number of ways. For example, see U.S. Pat. No. 6,737,056Bl and U.S. PatentApplication Publication No. 2004 / 0132101 Al , which disclose Fc mutations that optimize antibody binding to Fc receptors.

[0101] In certain anti-SEMA4D antibodies or fragments, variants or derivatives thereof useful in the methods provided herein, the Fc portion can be mutated to decrease effector function using techniques known in the art. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization. In other cases, constant region modifications consistent with the instant disclosure moderate complement binding and thus reduce the serum half-life. Yet other modifications of the constant region can be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor localization, biodistribution and serum half-life, can easily be measured and quantified using well known immunological techniques without undue experimentation.

[0102] Anti-SEMA4D antibodies for use in the methods provided herein include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc. Additionally, the derivative can contain one or more non-classical amino acids.

[0103] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e g., tyrosine, phenylalanine, tryptophan,T1histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind an anti-SEMA4D polypeptide, to block SEMA4D interaction with its receptor, or to inhibit, delay, or reduce metastases in a subject, e.g., a cancer patient).

[0104] For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. Introduced mutations can be silent or neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind antigen. These types of mutations can be useful to optimize codon usage or improve a hybridoma's antibody production. Alternatively, non-neutral missense mutations can alter an antibody's ability to bind antigen. One of skill in the art would be able to design and test mutant molecules with desired properties such as no alteration in antigen binding activity or alteration in binding activity (e g., improvements in antigen binding activity or change in antibody specificity). Following mutagenesis, the encoded protein can routinely be expressed and the functional and / or biological activity of the encoded protein, (e.g., ability to immunospecifically bind at least one epitope of a SEMA4D polypeptide) can be determined using techniques described herein or by routinely modifying techniques known in the art.

[0105] In certain embodiments, the anti-SEMA4D antibodies for use in the methods provided herein comprise at least one optimized complementarity-determining region (CDR). By "optimized CDR" is intended that the CDR has been modified and optimized to improve binding affinity and / or anti-SEMA4D activity that is imparted to an anti-SEMA4D antibody comprising the optimized CDR. "Anti-SEMA4D activity" or "SEMA4D blocking activity" can include activity which modulates one or more of the following activities associated with SEMA4D: B cell activation, aggregation and survival; CD40-induced proliferation and antibody production; antibody response to T cell dependent antigens; T cell or other immune cell proliferation; dendritic cell maturation; demyelination and axonal degeneration; apoptosis of pluripotent neural precursors and / or oligodendrocytes; induction of endothelial cell migration; inhibition of spontaneous monocyte migration; inhibition, delay, or reduction of tumor cell growth or metastasis, binding to cell surface plexin Bl or other receptor, or any other activity association with soluble SEMA4D or SEMA4D that is expressed on the surface of SEMA4D+ cells. In a particular embodiment, anti- SEMA4D activity includes the ability to inhibit, delay, or reduce tumor metastases, either incombination with inhibition, delay, or reduction of primary tumor cell growth and tumor metastases, or independently of primary tumor cell growth and tumor metastases. Anti-SEMA4D activity can also be attributed to a decrease in incidence or severity of diseases associated with SEMA4D expression, including, but not limited to, certain types of cancers including lymphomas, autoimmune diseases, inflammatory diseases including central nervous system (CNS) and peripheral nervous system (PNS) inflammatory diseases, transplant rejections, and invasive angiogenesis. Examples of optimized antibodies based on murine anti-SEMA4D MAb BD16 were described in U.S. Publ. No. 2008 / 0219971 Al, International Patent Application WO 93 / 14125 and Herold et al., Int. Immunol. 7(1): 1-8 (1995), each of which are herein incorporated by reference in their entirety. The modifications can involve replacement of amino acid residues within the CDR such that an anti-SEMA4D antibody retains specificity for the SEMA4D antigen and has improved binding affinity and / or improved anti-SEMA4D activity.Flt3 Ligand (Flt3L)

[0106] In humans, Flt3L (Fms-related tyrosine kinase 3 ligand) is encoded by the FLT3LG gene. Recombinant Flt3L is a therapeutic agent which is chemically identical to or similar to the endogenous cytokine Flt3L. It is a hematopoietic four helical bundle cytokine and is structurally homologous to SCF (stem cell factor) and CSF-1 (colony stimulating factor 1).

[0107] Flt3 ligand binds to and activates the Flt3 tyrosine kinase receptor (FLT3) on terminally differentiated DCs and, synergistically with other growth factors, stimulates the proliferation and mobilization of certain bone marrow precursor cells, including CD34+ cells, and dendritic cells. Flt3L controls the development of dendritic cells (DCs), which provide the key link between innate and adaptive immunity by recognizing pathogens and priming pathogen-specific immune responses. DCs are the most efficient antigen-presenting cells for T cells and have been investigated as cellular immunotherapeutic agents in cancer. Targeted disruption of the Flt3L gene in mice is associated with significant impairment of the immune system, as well as a reduction in myeloid progenitor cells, B-cell progenitors, dendritic cells (DCs), and natural killer (NK) cells. (McKenna, HJ et al . , Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood (2002) 100 (5): 1532-1542).

[0108] Flt3L, in particular an effective amount of recombinant Flt3L, such as CDX-301, a soluble recombinant human protein form of Flt3L (Celldex Therapeutics, Inc.) or a Flt3L fusionprotein (Flt3L-Fc) (He D, Duval A, Exbrayat S, et al., Human stem cell derived dendritic cells provide a physiologically relevant system to evaluate the pharmacology of a FLT3L-Fc fusion protein for cancer immunotherapy. Journal for ImmunoTherapy of Cancer 2023;! 1). In some embodiments of the disclosure, an effective amount of a FLT3 agonist is used in combination with an anti-SEMA4D antibody or antigen-binding fragment thereof for treatment of cancer, such as the FLT3 agonist Fc fusion protein, GS-3583. In some embodiments of the methods and uses disclosed herein, an effective amount of Flt3L, a FLT3 agonist , or Flt3L / Flt3L-fc-encoding construct is used in combination with an anti-SEMA4D antibody or antigen-binding fragment thereof and another immune modulating therapy, such as an immune checkpoint inhibitor (ICI), e.g., an anti-PDl, anti-PDLl, anti-CTLA4, anti-TIGIT, an anti-B7-H3 or anti-LAG3 inhibitory molecule or other immunomodulatory agent or therapy.In some embodiments of the disclosure, the combination of an anti-SEMA4D antibody or antigenbinding fragment thereof and Flt3L, Flt3 fusion protein , Flt3L / Flt3L-fc-encoding construct, or a FLT3 agonist further comprises an adjuvant, such as CD40L (CD154) / CD40 adjuvant, polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC), or imiquimod for example.Treatments Using Therapeutic Anti-SEMA4D Antibodies in Combination With Flt3L and, Optionally, at Least One Immune Modulating Therapy

[0109] Certain methods of the disclosure are directed to the use of anti-SEMA4D antibodies, including antigen-binding fragments, variants, and derivatives thereof, in combination with Flt3L or a FLT3 agonist and optionally, at least one other immune modulating therapy, to inhibit, delay, or reduce tumor growth or metastases in a subject in need of such inhibition, delay, or reduction, e.g., a cancer patient. More particularly, such methods of the disclosure for treating, inhibiting, delaying, or reducing malignant cell growth in a subject with cancer comprise administering to the subject an effective amount of an isolated antibody or antigen-binding fragment thereof that specifically binds to SEMA4D and an effective amount Flt3L or a FLT3 agonist and optionally, at least one immune checkpoint inhibitor or at least one other immune modulating therapy, thereby treating the subject.

[0110] In any of the embodiments herein, the isolated antibody or antigen-binding fragment thereof that specifically binds to SEMA4D variants, and derivatives of these antibodies are asdescribed in Tables 2 A and B above. In some embodiments herein, at least one immune checkpoint inhibitor (ICI), such as an anti-PDl antibody, an anti-PD-Ll antibody, an anti-LAG3 antibody, an anti-TIGIT antibody, an anti-B7-H3 antibody, an anti CTLA4 antibody, or an antigen-binding fragment of any of these ICI antibodies is used in combination with an anti-SEMA4D antibody and Flt3L or a FLT3 agonist.

[0111] Though the following discussion refers to administration of an anti-SEMA4D antibody, the methods described herein are equally applicable to the antigen-binding fragments, variants, and derivatives of these antibodies that retain the desired properties of the antibodies of the disclosure, e.g., capable of specifically binding SEMA4D, e.g., human, mouse, or human and mouse SEMA4D, having SEMA4D neutralizing activity, and / or blocking the interaction of SEMA4D with its receptors. In embodiments of any of the methods and uses described herein, the anti-SEMA4D, its fragments, variants and derivatives are described in Tables 2A and B.

[0112] In one embodiment, the immune modulating therapy can include cancer vaccines, immunostimulatory agents, adoptive T cell or antibody therapy, and immune checkpoint inhibitors (Lizee et al. 2013. Harnessing the Power of the Immune System to Target Cancer. Annu. Rev. Med. Vol. 64 No. 71-90).

[0113] Cancer Vaccines. Cancer vaccines activate the body’s immune system and natural resistance to an abnormal cell, such as cancer, resulting in eradication or control of the disease. Cancer vaccines generally consist of a tumor antigen in an immunogenic formulation that activates tumor antigen-specific helper cells and / or CTLs and B cells. Vaccines can be in a variety of formulations, including, but not limited to, dendritic cells, especially autologous dendritic cells pulsed with tumor cells or tumor antigens, heterologous tumor cells transfected with an immune stimulating agent such as GM-CSF, recombinant virus, or proteins or peptides that are usually administered together with a potent immune adjuvant such as CpG.

[0114] Immunostimulatory Agents. Immunostimulatory agents act to enhance or increase the immune response to tumors, which is suppressed in many cancer patients through various mechanisms. Immune modulating therapies can target lymphocytes, macrophages, dendritic cells, natural killer cells (NK Cell), or subsets of these cells such as cytotoxic T lymphocytes (CTL) or Natural Killer T (NKT) cells. Because of interacting immune cascades, an effect on one set of immune cells will often be amplified by spreading to other cells, e.g. enhanced antigen presenting cell activity promotes response of T and B lymphocytes. Examples of immunostimulatory agentsinclude, but are not limited to, HER2, cytokines such as G-CSF, GM-CSF and IL-2, cell membrane fractions from bacteria, glycolipids that associate with CD Id to activate Natural Killer T (NKT) cells, CpG oligonucleotides.

[0115] Macrophages, myelophagocytic cells of the immune system, are a fundamental part of the innate defense mechanisms, which can promote specific immunity by inducing T cell recruitment and activation. Despite this, their presence within the tumor microenvironment has been associated with enhanced tumor progression and shown to promote cancer cell growth and spread, angiogenesis and immunosuppression. Key players in the setting of their phenotype are the microenvironmental signals to which macrophages are exposed, which selectively tune their functions within a functional spectrum encompassing the Ml (tumor inhibiting macrophage) and M2 (tumor promoting macrophage) extremes. Sica et al., Seminars in Cancer Biol. 18:349-355 (2008). Increased macrophage numbers during cancer generally correlates with poor prognosis (Qualls and Murray, Curr. Topics in Develop. Biol. 94:309-328 (2011)). Of the multiple unique stromal cell types common to solid tumors, tumor-associated macrophages (TAMs) are significant for fostering tumor progression. Targeting molecular pathways regulating TAM polarization holds great promise for anticancer therapy. Ruffell et al., Trends in Immunol. 33: 119-126 (2012).

[0116] Adoptive Cell Transfer. Adoptive cell transfer can employ T cell-based cytotoxic responses to attack cancer cells. Autologous T cells that have a natural or genetically engineered reactivity to a patient's cancer are generated and expanded in vitro and then transferred back into the cancer patient. One study demonstrated that adoptive transfer of in vitro expanded autologous tumor-infiltrating lymphocytes was an effective treatment for patients with metastatic melanoma. (Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME (April 2008). "Adoptive cell transfer: a clinical path to effective cancer immunotherapy". Nat. Rev. Cancer 8 (4): 299-308). This can be achieved by taking T cells that are found within resected patient tumor. These T cells are referred to as tumor-infiltrating lymphocytes (TIL) and are presumed to have trafficked to the tumor because of their specificity for tumor antigens. Such T cells can be induced to multiply in vitro using high concentrations of IL-2, anti-CD3 and allo-reactive feeder cells. These T cells are then transferred back into the patient along with exogenous administration of IL-2 to further boost their anti-cancer activity. In other studies, autologous T cells have been transduced with a chimeric antigen receptor that renders them reactive to a targeted tumor antigen (Liddy et al., Nature Med. 18:980-7, (2012); Grupp et al., New England J. Med. 368: 1509-18, (2013)).

[0117] Other adoptive cell transfer therapies employ autologous dendritic cells exposed to natural or modified tumor antigens ex vivo that are re-infused into the patient. Provenge® is such an FDA approved therapy in which autologous cells are incubated with a fusion protein of prostatic acid phosphatase and GM-CSF to treat patients with prostate tumors. GM-CSF is thought to promote the differentiation and activity of antigen presenting dendritic cells (Small et al., J. Clin. Oncol. 18: 3894-903(2000); US Patent 7,414,108)).

[0118] Immune Checkpoint Inhibitors. Immune checkpoint inhibitors enhance T-cell immunity by removing a negative feedback control that limits ongoing immune responses. These types of therapies target inhibitory pathways in the immune system that are crucial for modulating the duration and amplitude of physiological immune responses in peripheral tissues (anti-CTLA4) or in tumor tissue expressing PD-L1 (anti-PD-1 or anti-PD-Ll) to minimize collateral tissue damage. Tumors can evolve to exploit certain immune-checkpoint pathways as a major mechanism of immune resistance against T cells that are specific for tumor antigens. Since many immune checkpoints are initiated by ligand-receptor interactions, these checkpoints can be blocked by antibodies to either receptor or ligand or can be modulated by soluble recombinant forms of the ligands or receptors. Neutralization of immune checkpoints allows tumor-specific T cells to continue to function in the otherwise immunosuppressive tumor microenvironment. Examples of immune checkpoint blockade therapies are those which target cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), PD-1, its ligand PD-L1, LAG3, TIGIT, and B7-H3.

[0119] Cyclophosphamide. Cyclophosphamide, a commonly used chemotherapeutic agent, can enhance immune responses. Cyclophosphamide differentially suppresses the function of regulatory T cells (Tregs) relative to effector T cells. Tregs are important in regulating anticancer immune responses. Tumor-infiltrating Tregs have previously been associated with poor prognosis. While agents that target Tregs specifically are currently unavailable, cyclophosphamide has emerged as a clinically feasible agent that can preferentially suppress Tregs relative to other T cells and, therefore, allows more effective induction of antitumor immune responses.

[0120] The methods of the disclosure encompass co-administration, using separate formulations or a single pharmaceutical formulation, with simultaneous or consecutive administration in either order of the anti-SEMA4D antibody or antigen-binding fragment thereof and Flt3L or FLT3 agonist, and optionally another immune modulating therapy.

[0121] The anti-SEMA4D antibodies or binding fragments thereof as described herein, in combination with Flt3L or FLT3 agonist and optionally, at least one other immune modulating therapy are useful for the treatment of various malignant and non-malignant tumors. By "antitumor activity" is intended a reduction in the rate of SEMA4D production or accumulation associated directly with the tumor or indirectly with stromal cells of the tumor environment, and hence a decline in growth rate of an existing tumor or of a tumor that arises during therapy, and / or destruction of existing neoplastic (tumor) cells or newly formed neoplastic cells, and hence a decrease in the overall size of a tumor and / or the number of metastatic sites during therapy. For example, therapy with at least one anti-SEMA4D antibody in combination with Flt3L or a FLT3 agonist and optionally, at least one other immune modulating therapy causes a physiological response, for example, an increase in immune activity that results a reduction of tumor growth or metastases, that is beneficial with respect to treatment of disease states associated with SEMA4D- expressing cells in a human.

[0122] In one embodiment, the disclosure relates to the use of anti-SEMA4D antibodies or antigen-binding fragments, variants, or derivatives thereof, in combination with Flt3L or a FLT3 agonist and optionally, at least one other immune modulating therapy as a medicament, in the treatment or prophylaxis of cancer or for use in a precancerous condition or lesion to inhibit, reduce, prevent, delay, or minimalize the growth or metastases of tumor cells.

[0123] In accordance with the methods of the present disclosure, at least one anti-SEMA4D antibody or antigen binding fragment, variant, or derivative thereof, in combination with Flt3L or a FLT3 agonist and optionally, at least one other immune modulating therapy can be used to promote a positive therapeutic response with respect to a malignant human cell. By "positive therapeutic response" with respect to cancer treatment is intended an improvement in the disease in association with the anti-tumor activity of these binding molecules, e.g., antibodies or fragments thereof, and / or an improvement in the symptoms associated with the disease. In particular, the methods provided herein are directed to inhibiting, preventing, reducing, alleviating, delaying, or lessening growth of a tumor and / or the development of metastases of primary tumors in a patient. That is the prevention of distal tumor outgrowths, can be observed. Thus, for example, an improvement in the disease can be characterized as a complete response. By "complete response" is intended an absence of clinically detectable metastases with normalization of any previously abnormal radiographic studies, e g. at the site of the primary tumor or the presence of tumormetastases in bone marrow. Alternatively, an improvement in the disease can be categorized as being a partial response. By "partial response" is intended at least about a 50% decrease in all measurable metastases (i.e., the number of tumor cells present in the subject at a remote site from the primary tumor). Alternatively, an improvement in the disease can be categorized as being relapse free survival or “progression free survival”. By “relapse free survival” is intended the time to recurrence of a tumor at any site. “Progression free survival” is the time before further growth of tumor at a site being monitored can be detected.

[0124] Inhibition, delay, or reduction of tumor growth or metastases can be assessed using screening techniques such as imaging, for example, fluorescent antibody imaging, bone scan imaging, and tumor biopsy sampling including bone marrow aspiration (BMA), or immunohistochemistry. In addition to these positive therapeutic responses, the subject undergoing therapy can experience the beneficial effect of an improvement in the symptoms associated with the disease.

[0125] Clinical response can be assessed using screening techniques such as magnetic resonance imaging (MRI) scan, x-radiographic imaging, computed tomographic (CT) scan, flow cytometry or fluorescence-activated cell sorter (FACS) analysis, histology, gross pathology, and blood chemistry, including but not limited to changes detectable by ELISA, RIA, chromatography, and the like.Pharmaceutical Compositions and Administration Methods

[0126] Methods of preparing and administering anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof in combination with Flt3L or a FLT3 agonist and optionally, at least one other immune modulating therapy to a subject in need thereof are well known to or are readily determined by those skilled in the art. The route of administration of the anti-SEMA4D antibody, or antigen-binding fragment, variant, or derivative thereof in combination with Flt3L or an FLT3 agonist and optionally, at least one other immune modulating therapy, can be, for example, oral, parenteral, by inhalation or topical at the same or different times for each therapeutic agent. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all these forms of administration are clearly contemplated as being within the scope of the disclosure, an example of a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. A suitable pharmaceutical composition for injection can comprisea buffer (e g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. However, in other methods compatible with the teachings herein, anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof in combination with at least one other immune modulating therapy can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0127] As discussed herein, anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof in combination with Flt3L or a FLT3 agonist and optionally, at least one other immune modulating therapy can be administered in a pharmaceutically effective amount for the in vivo treatment of diseases such as neoplastic disorders, including solid tumors. In this regard, it will be appreciated that the disclosed molecules can be formulated to facilitate administration and promote stability of the active agent. In certain embodiments, pharmaceutical compositions in accordance with the present disclosure comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the instant application, a pharmaceutically effective amount of an anti- SEMA4D antibody, or antigen-binding fragment, variant, or derivative thereof, in combination with Flt3L or a FLT3 agonist and optionally, at least one other immune modulating therapy shall be held to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, i.e., to inhibit, delay, or reduce metastases in a cancer patient.

[0128] The pharmaceutical compositions used in this disclosure comprise pharmaceutically acceptable carriers, including, e.g., ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0129] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyloleate. Aqueous carriers include, e.g., water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Pharmaceutically acceptable carriers can include, but are not limited to, 0.01-0.1 M, or 0.05 M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0130] More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition can be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a certain particle size in the case of dispersion and by the use of surfactants. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

[0131] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In certain embodiments, isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride can be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0132] In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., an anti-SEMA4D antibody, or antigen-binding fragment, variant, or derivative thereof, in combination with at least one other immune modulating therapy) in a certain amount in an appropriate solvent with one or a combination of ingredients enumerated herein, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle, which contains a basic dispersion medium and the other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying or freeze-drying, which can yield a powder of an active ingredient plus any additional desired ingredient from a previously sterile- fdtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations can be packaged and sold in the form of a kit. Such articles of manufacture can have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from or predisposed to a disease or disorder.

[0133] Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed with a maintenance dose. These compositions can be administered at specific fixed or variable intervals, e.g., once a day, or on an "as needed" basis.

[0134] Certain pharmaceutical compositions can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other conventional solubilizing or dispersing agents.

[0135] The amount of an anti-SEMA4D antibody, or fragment, variant, or derivative thereof, and the amount of Flt3L or FLT3 agonist and any optional other immune modulating therapies to be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0136] By "therapeutically effective dose or amount" or "effective amount" is intended an amount of anti-SEMA4D antibody or antigen binding fragment, variant, or derivative thereof, in combination with an amount of Flt3L or FLT3 agonist and any optional other immune modulating therapy that when administered brings about a positive therapeutic response with respect to treatment of a patient with a disease to be treated, e.g., an inhibition, delay, or reduction of metastases in the patient.

[0137] Therapeutically effective doses for the compositions of the present disclosure, for the inhibition, delay, or reduction of metastases, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. In certain embodiments the patient is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0138] The amount of anti-SEMA4D antibody or binding fragment, variant, or derivative thereof, administered in combination with Flt3L or a FLT3 agonist and any optional other immune modulating therapy is readily determined by one of ordinary skill in the art without undue experimentation given the disclosure of the present disclosure. Factors influencing the mode of administration and the respective amounts of the components include, but are not limited to, the severity of the disease, the history of the disease, the potential for metastases, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Similarly, the amount of the components to be administered will be dependent upon the mode of administration and whether the subject will undergo a single dose or multiple doses of this agent.

[0139] In some embodiments of any of the methods and uses described herein, the dose of the anti-SEMA4D antibody is about 20 mg / kg, or an equivalent thereof for antigen binding fragments, variants or derivatives thereof. Such equivalents can readily be calculated by those of skill in the art.

[0140] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRE Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, MethodsIn Enzymology (Academic Press, Inc., N. Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0141] General principles of antibody engineering are set forth in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). General principles of protein engineering are set forth in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General principles of antibodies and antibody-hapten binding are set forth in: Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y.). Additionally, standard methods in immunology known in the art and not specifically described are generally followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY).

[0142] Standard reference works setting forth general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell (1984) "Monoclonal Antibody Technology" in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) Kuby Immunnology (4th ed.; H. Freemand & Co.); 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 Verlan); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

[0143] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.EXAMPLES

[0144] The examples presented herein represent certain embodiments of the present disclosure. However, it is to be understood that these examples are for illustration purposes only and do not intend, nor should any be construed, to be wholly definitive as to conditions and scope of this disclosure. The examples were carried out using standard techniques, which are known and routine to those of skill in the art, except where otherwise described in detail.Example 1. Anti-SEMA4D Monoclonal Antibody and Flt3L Treatment in a Colon26 Mouse Model

[0145] Experimental design. The purpose of this study was to determine the effects of an anti- SEMA4D antibody, Mab67, a murine antibody surrogate of pepinemab, in combination with Flt3L on the growth of Colon26 tumors grafted onto 8 weeks old Balb / c J mice. The endpoints of the study were determination of tumor volume, survival, and complete response (CR) rate.

[0146] Tumor-bearing mice were generated by implanting 500,000 Colon26 cells subcutaneously into one flank of eight weeks old Balb / c J mice. Tumors were allowed to generate for two days prior to treatment with either Control Ig (MAb2B8) or SEMA4D blocking antibody (MAb67). Tumor bearing mice were separated into four treatment groups as described below.

[0147] At two days and nine days following implant, the mice were interperitoneally (ip) administered control Ig or Mab67 at 10 mg / kg. At days 7, 8, 9, 10, 11, 12, 13, and 14, Flt3L was administered ip at 0.125 mg / kg to the different groups of mice according to the table above. Caliper measurements of the tumors were made biweekly for up to 60 days starting at day 0. The endpoints were set at 1000 mm3, tumor growth delay / survival.

[0148] The results are shown in Figures 1, 2A-D and 3. The combination of MAb67 and Flt3L significantly delayed tumor growth and improved survival. 134% tumor growth delay was observed (p=0.0013) compared to control and (p=0.029) compared to MAb67 single agent.The combination therapy also significantly increased the infiltration of DC, B cells and T cells into the treated tumors (Figure 3). Mice were treated as above and tumors were collected on day 16from 3 mice / group. Tumor tissues were formalin fixed, paraffin embedded and stained using immunohistochemistry. Cell densities within the tumor area were quantified for the presence of DC, B cells and T cells using Visiopharm® software. Quantification of immune cells in tumors was determined by immunohistochemical staining of tumor tissue for a cellular differentiation marker of DCs (CDl lc, Fig 3A), ofB cells (B220, Fig. 3B) and of T cells (CD3, Fig. 3C).

[0149] * One-way ANOVA Tukey’s multiple comparison test.

[0150] Example 2. Effects of Anti-SEMA4D Monoclonal Antibody and Flt3-L combination therapy on breast cancer tumors.

[0151] Experimental design. The purpose of this study is to determine the effects of an anti-SEMA4D antibody MAb67, in combination with Flt3L on the growth of breast cancer tumors grafted onto 8 weeks old Balb / c J mice, i.e., breast cancer tumor model TUBO.B1. The endpoints of the study are determination of at least the following: size of the tumor, tumor growth delay and survival.

[0152] Tumor-bearing mice are generated by orthotopic implantation of 25,000 TUBO.B1 cells in mammary fat pads, followed by treatment with control Ig (Mab 2B8), MAb67, Flt3-L plus control Ig, or a combination of Mab 67 and Flt3-L administered interperitoneally.Amounts and timing of administration of each of the control Ig, MAb67, and Flt3L, alone and in combination, is shown in Table 3 below. Caliper measurements of the tumors were made biweekly for up to 60 days starting at day 0. The endpoints were set at 1000 mm3, tumor growth delay / survival.4. 15. I 1131.. + aSEMA4D . Flt3L: 125ug / kg;

[0153] The results are shown in Figures 4 and 5 A-D. Combination therapy delayed tumor growth and improved survival 27%. Tumor growth delay (p=0.0001) compared to control and (p=0.02) compared to Flt3L single agent. Of the 15 mice treated with combination therapy, 6 showed a complete response (CR) (p=0.015).

[0154] Example 3. Effects of Anti-SEMA4D Monoclonal Antibody and Flt3-L combination therapy on fibrosarcoma.

[0155] Experimental design. The purpose of this study is to determine the effects of an anti- SEMA4D antibody MAb67, i.e., pepinemab, in combination with Flt3L on the growth of fibrosarcomas grafted onto 8 weeks old Balb / c mice. The endpoints of the study are determination of at least the following: size of the tumor, tumor growth delay and survival.

[0156] Tumor-bearing mice were generated by implanting 30,000 BCA34 cells intramuscularly into eight weeks old Balb / c mice, an aggressive tumor model. Tumors were allowed to generate for two days prior to treatment with either Control Ig (MAb2B8) or pepinemab (MAb67). Tumor bearing mice were separated into four treatment groups as shown in Table 4 below. Caliper measurements of the tumors were made biweekly for up to 40 days starting at day 0. The endpoints were set at 1000 mm3, tumor growth delay / survival.Table 4

[0157] The results are shown in Figures 6 and 7A-D. The combination therapy delayed tumor growth and improved survival in this aggressive tumor model. 1 of the 15 mice tested showed a complete response (CR) to the combination therapy in this aggressive tumor model.

[0158] Example 4. Effects of Pepinemab and Flt3-L combination therapy on melanoma.

[0159] Experimental design. The purpose of this study is to determine the effects of an anti- SEMA4D antibody MAb67, i.e., pepinemab, in combination with Flt3L on the growth of melanoma B16F.1 cells grafted onto 8 weeks old C57B1 / 6 mice. The endpoints of the study are determination of at least the following: size of the tumor, tumor growth delay and survival.

[0160] Tumor-bearing mice were generated by implanting 30,000 B16 cells subcutaneously into eight weeks old mice C57B1 / 6 mice. Tumor bearing mice were separated into four treatment groups as shown in Table 5 below. Caliper measurements of the tumors were made biweekly for up to 40 days starting at day 0. The endpoints were set at 800 mm3, tumor growth del ay / survival.Table 5

[0161] The results are shown in Figures 8 (Mean Tumor Volume) and 9 (Survival). The combination therapy delayed tumor growth and improved survival. A 20% tumor growth delay was observed for mice treated with the combination therapy (p=0.1073) , compared to 9% and 12%, respectively, for single agents MAb67 (aSEMA) and Flt3L.Calculations and Statistics.

[0162] Tumor volume calculation:Tumors were calipered in two dimensions and tumor size was calculated using the formula: Tumor Volume (mm3) = (w2 x l) / 2 where w = width and 1 = length, in mm, of the tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3of tumor volume.Mean tumor volumes were calculated and graphed for each group at each timepoint.Mean calculations are displayed only when >50% animals / group remain on study.Final tumor volume for animals sacrificed due to excess tumor growth is “carried over” if an animal is sacrificed when >50% animals / group remain on study.Animals sacrificed due to accidental death or unexplained death are not included in mean and median calculations, unless tumors reach endpoint volume before death.CR = # Complete Responders (tumor volume < 50 mm3for >2 consecutive measurements) Differences in regression rates were determined using fisher’s exact test, compared to Control.

[0163] Time to Endpoint (TTE) for each mouse was calculated with the following equation: TTE = [log 10 (endpoint volume) - b] / m where TTE is expressed in days, endpoint volume is in mm3, b is the intercept, and m is the slope of the line obtained by linear regression of a log-transformed tumor growth data set. Note: linear regression analysis was also performed using raw tumor volume data.The data set is comprised of the first observation that exceeded the study endpoint volume and the four consecutive observations that immediately preceded the attainment of the endpoint volume. The calculated TTE is usually less than the day on which an animal is euthanized for tumor size.Animals whose tumors do not reach the endpoint are to be assigned a TTE value equal to the last day of the study. Animals that died from nontreatment-related (NTR) causes were excluded from TTE calculations.Stats: Significance of TTE values were determined by Applying D'Agostino & Pearson omnibus normality test to TTE values of control and treatment groups. If both groups pass normality test, a parametric t test was applied.Validity of parametric t test was confirmed if data are normally distributed and variances are significantly different. If variance and / or distribution are ns, apply a non-parametric test, such as Mann Whitney t-test. Generally, a parametric t test is preferred and expected to be more sensitive in determining significant differences, (a p < 0.05 for both tests simply supports the statistical significance of the result)

[0164] Tumor growth delay (TGD), is defined as the increase in the median TTE in a treatment group compared to the control group: TGD = T - C, expressed in days, or as a percentage of the median TTE of the control group: % TGD - [(T-C) / C] x 100Where T = median TTE for a treatment group, C = median TTE for the control group.

[0165] Survival was analyzed by the Kaplan-Meier method, employing the logrank test to assess the significance of the difference between the overall survival experiences (survival curves) of two groups, based on their TTE values.Stats: The two-tailed statistical analyses were conducted at P = 0.05.Since the logrank test is a test of significance and does not provide an estimate of the magnitude of the difference between groups, all levels of significance are reported as either significant or non-significant within the text of this report.

[0166] Prism reports results as non-significant (ns) at P > 0.05, significant (symbolized by “*”) at 0.01 < P < 0.05, very significant (“**”) at 0.01 < P <0.01, and extremely significant (“***”) at P < 0.001; **** p<0.0001

[0167] Many modifications and other embodiments of the embodiments set forth herein will come to mind to one skilled in the art to which this disclosure pertains, having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosure is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims and list of embodiments disclosed herein. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

We claim:

1. Use of an isolated antibody or antigen-binding fragment thereof which specifically binds to semaphorin-4D (SEMA4D) in combination with an effective amount of a Fms-like tyrosine kinase 3 ligand (Flt3L) or a Fms-like tyrosine kinase 3 (FLT3) agonist for treatment of cancer in a subject in need thereof.

2. A combination therapy for use in treating cancer in a subject in need thereof, wherein the combination therapy comprises an isolated antibody or antigen-binding fragment thereof that specifically binds to semaphorin-4D (SEMA4D) and an effective amount of a Fms-like tyrosine kinase 3 ligand (Flt3L) or a Fms-like tyrosine kinase 3 (FLT3) agonist.

3. The use claim 1 or the combination therapy of claim 2, wherein said anti-SEMA4D antibody or antigen-binding fragment thereof inhibits SEMA4D interaction with its receptor.

4. The use or the combination therapy of claim 3 wherein the receptor is Plexin-Bl, Plexin- B2, CD72, or any combination thereof.

5. The use of claim 1 or 3 or the combination therapy of claim 2, wherein the antibody or fragment thereof inhibits SEMA4D-mediated signal transduction.

6. The use of any one of claims 1, 3, 4, and 5 or the combination therapy of any one of claims 2-5, wherein the Flt3L is recombinant Flt3L.

7. The use or the combination therapy of claim 6, wherein the recombinant Flt3L is a Flt3L fusion protein (Flt3L-Fc).

8. The use or the combination therapy of claim 6 or 7, wherein the recombinant Flt3L or Flt3L-Fc is encoded by a vector.

9. The use of any one of claims 1 and 3-8 or the combination therapy of any one of claims 2- 8, wherein said isolated antibody or antigen-binding fragment thereof is selected from the group consisting of(i) an antibody or antigen binding fragment thereof comprising a variable heavy chain (VH) region having VH CDRs 1-3 comprising amino acid sequences SEQ ID NOS: 1, 2, and 3, respectively, and a variable light chain (VL) region comprising VL CDRs 1-3 comprising SEQ ID NOS: 4, 5, and 6, respectively;(ii) the antibody of (i), or antigen binding fragment thereof, wherein the VH and VL chains comprise, respectively, SEQ ID NO: 7 and SEQ ID NO: 8 (human), or SEQ ID NO: 9 and SEQ ID NO: 10 (mouse);(iii) an antibody or antigen binding fragment thereof comprising a VH region having VH CDRs 1-3 comprising amino acid sequences SEQ ID NOS: 11, 12, and 13, respectively; and a VL region comprising VL CDRs 1-3 comprising amino acid sequences SEQ ID NOS: 14, 15, and 16, respectively; and(iv) the antibody of (ii), or antigen binding fragment thereof, wherein the VH and VL chains comprise, respectively, SEQ ID NO: 17 and SEQ ID NO: 18.

10. The combination therapy of any one of claims 2-9, further comprising an immune modulating therapy selected from the group consisting of a cancer vaccine, an immunostimulatory agent, adoptive T cell or antibody therapy, immune checkpoint inhibitor, a regulatory T cell (Treg) modulator, and a combination thereof.

11. The combination therapy of claim 10, wherein the immune modulating therapy is at least one immune checkpoint inhibitor.

12. The combination therapy of claim 11, wherein the at least one immune checkpoint inhibitor is an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti- LAG3 antibody, an anti-TIGIT antibody, an anti-B7-H3 antibody, an anti-TIM3 antibody, or a combination thereof.

13. The combination therapy of claim 10, wherein the Treg modulator is cyclophosphamide.

14. The isolated use of any one of claims 1 and 3 -9 or the combination therapy of any one of claims 2-13, wherein the cancer is carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma,kidney cancer, prostate cancer, vulval cancer, thyroid cancer, head and neck cancer, melanoma, or any combination thereof.

15. The use of or the combination therapy of claim 14, wherein the cancer is locally advanced or recurrent or metastatic cancer.

16. A method of treating a subject having or suspected of having cancer comprising administering to the subject a combination therapy comprising an isolated antibody or antigenbinding fragment thereof that specifically binds to semaphorin-4D (SEMA4D) and an effective amount of Fms-like tyrosine kinase 3 ligand (Flt3L) or a Fms-like tyrosine kinase 3 (FLT3) agonist, and optionally at least one other immune modulating therapy.

17. The method of claim 16, wherein said anti-SEMA4D antibody or antigen-binding fragment thereof inhibits SEMA4D interaction with its receptor.

18. The method of claim 17, wherein the receptor is Plexin-Bl, Plexin-B2, CD72, or any combination thereof.

19. The method of claim 18, wherein the antibody or fragment thereof inhibits SEMA4D- mediated signal transduction.

20. The method of claim 16, wherein the Flt3L is recombinant Flt3L.

21. The method of claim 16, wherein the Flt3L is a Flt3L fusion protein (e.g. Flt3L-Fc).

22. The method of claim 21, wherein the Flt3L or Flt3L-Fc is encoded by a vector.

23. The method of any one of claims 16-22, wherein said anti-SEMA4D antibody or antigenbinding fragment thereof is selected from the group consisting of(i) an antibody or antigen binding fragment thereof comprising a variable heavy chain (VH) region having VH CDRs 1-3 comprising amino acid sequences SEQ ID NOS: 1, 2, and 3,respectively, and a variable light chain (VL) region comprising VL CDRs 1-3 comprising SEQ ID NOS: 4, 5, and 6, respectively;(ii) the antibody of (i), or antigen binding fragment thereof, wherein the VH and VL chains comprise, respectively, SEQ ID NO: 7 and SEQ ID NO: 8 (human), or SEQ ID NO: 9 and SEQ ID NO: 10 (mouse);(iii) an antibody or antigen binding fragment thereof comprising a VH region having VH CDRs 1-3 comprising amino acid sequences SEQ ID NOS: 11, 12, and 13, respectively; and a VL region comprising VL CDRs 1-3 comprising amino acid sequences SEQ ID NOS: 14, 15, and 16, respectively; and(iv) the antibody of (ii), or antigen binding fragment thereof, wherein the VH and VL chains comprise, respectively, SEQ ID NO: 17 and SEQ ID NO: 18.

24. The method of any one of claims 1-23, wherein the at least one other immune modulating therapy is an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-LAG3 antibody, an anti-TIGIT antibody, an anti-B7-H3 antibody or a combination thereof.

25. The method of any one of claims 1-24, wherein the isolated antibody or antigen-binding fragment thereof, the Flt3L or FLT3 agonist, and the at least one other immune modulating therapy, are administered separately or concurrently.

26. The method of any one of claims 1-25, wherein the at least one other immune modulating therapy is selected from the group consisting of administration of a cancer vaccine, administration of an immunostimulatory agent, adoptive T cell or antibody therapy, administration of an immune checkpoint inhibitor, administration of a regulatory T cell (Treg) modulator, and a combination thereof.

27. The method of claim 26, wherein the at least one other immune modulating therapy comprises administration of a cancer vaccine.

28. The method of claim 26, wherein the Treg modulator is cyclophosphamide.

29. The method of claim 16, wherein the cancer is carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, head and neck cancer, melanoma, or any combination thereof.

30. The method of any one of claims 16-29, wherein the cancer is locally advanced, recurrent or metastatic cancer.

31. The method of any one of claims 16-30, wherein the combination therapy comprises Flt3L.

32. The method of any one of claims 31, wherein the Flt3L is a human recombinant Flt3L.

33. The method of claim 16, wherein the combination therapy comprises a FLT3 agonist.

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