USE OF SEMAPHORIN-4D INHIBITOR MOLECULES IN COMBINATION WITH AN IMMUNOMODULATIVE THERAPY TO INHIBIT

专利摘要:
abstract "use of semaphorin-4d inhibitory molecules in combination with an immunomodulatory therapy to inhibit tumor growth and metastases" provided herein are methods to inhibit, delay, or reduce tumor growth and cancer cell metastases expressing plexin b1 in a patient, comprising administering to the patient an effective amount of an isolated ligand molecule that specifically binds semaphorin-4d (sema4d) in combination with an effective amount of at least one other immunomodulatory therapy.
公开号:BR112015032690B1
申请号:R112015032690-0
申请日:2014-06-20
公开日:2020-03-10
发明作者:Elizabeth E. Evans；Ernest S. Smith；Maurice Zauderer
申请人:Vaccinex, Inc.；
IPC主号:

专利说明:

“USE OF SEMAPHORIN-4D INHIBITOR MOLECULES IN COMBINATION WITH AN IMMUNOMODULATIVE THERAPY TO INHIBIT TUMOR GROWTH AND METASTASIS” REFERENCE TO THE ELECTRONICALLY SUBMITTED SEQUENCE LISTING
[001] The content of the sequence listing electronically submitted in the text file (Name: 58008_133124_SEQ_LST.txt; Size: 37,171 bytes; and Creation Date: June 20, 2014) deposited with the application is incorporated by reference in its entirety.
BACKGROUND
[002] Semaphorin 4D (SEMA4D), also known as CD100, is a transmembrane protein (for example, SEQ ID NO: 1 (human); SEQ ID NO: 2 (muzzle)) that belongs to the semaphorin gene family. SEMA4D is expressed on the cell surface as a homodimer, but with cell activation SEMA4D can be released from a cell surface through proteolytic cleavage to generate sSE-MA4D, 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).
[003] 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 in resting T cells, but only weakly expressed in resting B cells and antigen presenting cells (APCs), such as dendritic cells (CDs). Its expression, however, is regulated positively in these cells following activation by various immune 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 and et al., Oncology Letters, 5: 1527-35 (2013); Kato et al., Cancer Sci. 102: 2029-37 (2011)) and several reports suggest that a 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 involves a complex process of cross-talk between tumor cells, stroma and immune infiltrate, as well as endothelial cells and vasculature. SEMA4D is overexpressed in a wide range of tumor types and is also produced by inflammatory cells recruited to the tumor microenvironment, the question of what role SEMA4D can play in the migration, survival, differentiation and organization of different cell types that constitute the tumor stroma remains for be addressed.
Brief Summary [004] This application addresses the need for safe and effective cancer treatments that serve either as a single agent that inhibits, reduces, suppresses, prevents, slows down or slows down the progression of, decreases, or directly attacks tumor cells or that can act in combination with other immunomodulatory therapies to increase their therapeutic benefit. In particular, SEMA4D has been shown to play a role in the infiltration, maturation and organization of immune cells and macrophages that either promote or inhibit tumor growth, which can contribute to the development of effective methods to reduce tumor growth and metastasis in a cancer patient.
[005] Certain aspects of the application are directed to a method for inhibiting, delaying or reducing tumor growth or metastasis or both, tumor growth and metastasis in a cancer patient comprising administering to the patient an effective amount of an isolated binding molecule which specifically binds semaphorin-4D (SEMA4D) and an effective amount of at least one immunomodulatory therapy.
[006] In some embodiments, the ligand molecule inhibits interaction of SEMA4D with its receptor (for example, Plexin-B1). In some embodiments, the ligand molecule inhibits SEMA4D-mediated Plexin-B1 signal transduction. In some embodiments, inhibition, delay, or reduction of metastases occurs regardless of inhibition, delay, or reduction of primary tumor growth. In some modalities, cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, cancer 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, vulvar cancer, thyroid cancer, head and neck cancer, and a combination of them. In some modalities, the patient has elevated levels of either B cells, T cells, or both, B cells and T cells, when compared to other cancer patients.
[007] In some embodiments, the isolated binding molecule specifically binds to the same SEMA4D epitope as a reference monoclonal antibody selected from the group consisting of VX15 / 2503 and 67. In some embodiments, the isolated binding molecule comprises an antibody or fragment antigen linker. In some embodiments, the antibody or antigen-binding fragment thereof comprises the six complementarity-determining regions (CDRs) of monoclonal antibody VX15 / 2503 or 67.
[008] In some embodiments, immunomodulatory therapy is selected from the group consisting of a cancer vaccine, an immunostimulatory agent, adoptive T-cell or antibody therapy, immune checkpoint block and a combination thereof. In some embodiments, the immune modulating agent is selected from the group consisting of interleukins, cytokines, chemokines, immune checkpoint block antagonists and a combination of them. In some embodiments, immunomodulatory therapy may be cancer therapy. In some modalities, cancer therapy is selected from the group consisting of surgery or surgical procedures, radiation therapy, chemotherapy or a combination thereof. In some embodiments, the isolated binding molecule and the immunomodulating agent or immunomodulatory therapy are administered separately or concurrently.
[009] In some embodiments, methods for inhibiting, delaying or reducing tumor growth in a cancer patient are provided that comprise administering to the patient an effective amount of an isolated binding molecule that specifically binds to semaphorin-4D (SEMA4D) and a effective amount of at least one other immunomodulatory therapy. In some embodiments, the ligand molecule inhibits interaction of SEMA4D with its receptor. In some embodiments, the receptor is Plexin-B1. In some embodiments, the linker molecule inhibits SEMA4D-mediated Plexin-B1 signal transduction. In some embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma , peritoneum cancer, hepatocellular cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblas-toma, 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, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, and a combination thereof. In some modalities, vulvar cancer, thyroid cancer, head and neck cancer, and a combination of them. In some embodiments, the isolated binding molecule specifically binds to the same SEMA4D epitope as a VX15 / 2503 or 67 reference monoclonal antibody. In some embodiments, the competitively isolated binding molecule inhibits a VX15 / 2503 or 67 reference monoclonal antibody from specifically binding to SEMA4D. In some embodiments, the isolated binding molecule comprises an antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising VHCDRs 1-3 comprising SEQ ID Nos: 6, 7, and 8, respectively, and a variable light chain (VL) comprising VLCDRs 1-3 comprising SEQ ID Nos 14, 15, and 16, respectively. In some embodiments, VH and VL comprise SEQ ID NO: 9 and SEQ ID NO: 17 or SEQ ID NO: 10 and SEQ ID NO: 18, respectively. In some embodiments, immunomodulatory therapy is selected from the group consisting of administration of a cancer vaccine, administration of an immunomodulatory agent, adoptive T cell therapy or antibody, administration of an immune checkpoint block inhibitor, administration of a regulatory T cell modulator (Treg), and a combination thereof. In some embodiments, immunomodulatory therapy comprises an inhibitor of the immune checkpoint block. In some embodiments, where the immune checkpoint block inhibitor is an anti-CTLA4 antibody, an anti-PD-1 antibody, or a combination thereof. In some embodiments, immunomodulatory therapy involves administering a cancer vaccine. In some embodiments, the Treg modulator is cyclophosphamide. In some embodiments, the isolated binding molecule and immunomodulatory therapy are administered separately or concurrently. In some embodiments, administration of the combination of the isolated binding molecule and immunomodulatory therapy results in increased therapeutic efficacy relative to administration of the isolated binding molecule or immunomodulatory therapy alone. In some modalities, the patient who has a high level of B cells, T cells or both, B cells and T cells when compared to other cancer patients. In some embodiments, the level of B cells and / or T cells per microliter of blood in the patient is about 1.5; 2; 2.5; 3; 3.5; 4; 4.5; or 5 times the average number of B cells and / or T cells in the circulation in other cancer patients. In some embodiments, the level of B cells and / or T cells per microliter of blood in patients ranges from about 147 to about 588 and from about 1173 to about 3910, respectively, for example, when compared to other patients with cancer. In some modalities, the patient has B cell and / or T cell levels that fall within or above the range of B cells and / or T cells of healthy patients without cancer. In some embodiments, the levels of B cell and / or T cell per microliter of blood in the patient range from about 225 to about 275 or more and from about 1350 to 1650 or more, respectively, for example, when compared to patients healthy without cancer.
[010] In some embodiments, methods for treating a patient having cancer with immunotherapy are provided that comprise: (a) determining the number of B cells and / or T cells in a cancer patient; and (b) administering to the patient an effective amount of an isolated molecular ligand that specifically binds semaphorin-4D (SEMA4D) and an effective amount of at least one other immunomodulatory therapy if the number of B cells and / or T cells in the patient exceeds a predetermined threshold level. In some embodiments, the pre-determined limit levels of B cells and / or T cells per microliter of blood in the patient is about 1.5; 2; 2.5; 3; 3.5; 4; 4.5 or 5 times the average number of B cells and / or T cells in the circulation in other cancer patients. In some embodiments, the predetermined threshold levels of B cells and / or T cells per microliter of blood in the patient range from about 147 to about 588 and from about 1173 to about 3910, respectively, for example, when compared to other cancer patients. In some embodiments, the pre-determined threshold levels of B cells and / or T cells per microliter of blood in the patient fall within or above the range of B cells and / or T cells of healthy patients without cancer. In some embodiments, the predetermined threshold levels of B cells and / or T cells per microliter of blood in the patient range from about 225 to about 275 or more and from about 1350 to about 1650, respectively, for example, when compared to healthy cancer-free patients.
[011] In some embodiments, methods of treating a patient having cancer with immunotherapy are provided which comprise: administration of a combination of an effective amount of an isolated binding molecule that specifically binds semaforian-4D (SEMA4D) and an effective amount of hair at least one other immunomodulatory therapy to a cancer patient, wherein administration of the combination results in increased therapeutic efficacy relative to administration of the isolated binding molecule or other immunomodulatory therapy alone. In some embodiments, immunomodulatory therapy is selected from the group consisting of administration of a cancer vaccine, administration of an immunostimulatory agent, adoptive T cell therapy or antibody, administration of an immune checkpoint blocking inhibitor, administration of a regulatory T cell modulator (Treg), and a combination thereof. In some embodiments, immunomodulatory therapy comprises an immune checkpoint block inhibitor. In some embodiments, the immune checkpoint blocking inhibitor is an anti-CTLA4 antibody, an anti-PD-1 antibody, or a combination thereof. In some embodiments, immunomodulatory therapy involves administering a cancer vaccine. In some embodiments, the Treg modulator is cyclophosphamide. In some embodiments, the isolated binding molecule and immunomodulatory therapy are administered separately or concurrently. In some modalities, the patient has elevated levels of either B cells, T cells, or both, B cells and T cells when compared to other cancer patients. In some embodiments, the levels of B cells and / or T cells per microliter of blood in the patient is about 1.5; 2; 2.5; 3; 3.5; 4; 4.5; or 5 times the average number of B cells and / or T cells in the circulation in other cancer patients. In some embodiments, the levels of B cells and / or T cells per microliter of blood in the patient range from about 147 to about 588 and from about 1173 to about 3910, respectively, for example, when compared to other patients with cancer. In some modalities, the patient has levels of B cells and / or T cells that fall within or above the range of B cells and / or T cells of healthy patients without cancer. In some embodiments, the levels of B cells and / or T cells per microliter of blood in the patient range from about 225 to about 275 or more and from about 1350 to about 1650, or more, respectively, for example , when compared to healthy patients without cancer. In some embodiments, cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma , peritoneum cancer, hepatocellular cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepa-toma, breast cancer, colon cancer , colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, and a combination thereof. In some embodiments of any of the aforementioned methods, the isolated binding molecule specifically binds to the same SEMA4D epitope as a reference monoclonal antibody selected from the group consisting of VX15 / 2503 or 67. In some embodiments of any of the above methods, the competitively isolated binding molecule inhibits a reference monoclonal antibody selected from the group consisting of VX15 / 2503 or 67 to specifically bind SEMA4D. In some embodiments, the isolated binding molecule comprises an antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises the six complementary determining regions (CDRs) of the VX15 / 2503 monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment thereof is VX15 / 2503 or 67 monoclonal antibody.
[012] Also provided are methods for inhibiting, delaying, or reducing growth of tumor cells expressing Her2 and Plexin B1, Plexin B2, or a combination thereof, comprising bringing tumor cells into contact with an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D), in which the growth of tumor cells is inhibited, delayed, or reduced. In some embodiments, putting in contact comprises administering the SEMA4D binding molecule to a cancer patient, in which the patient's cancer cells express Her2 and Plexin B1, Plexin B2, or a combination thereof. In some modalities, the cancer is breast cancer, ovarian cancer, lung cancer or prostate cancer.
[013] Also provided are methods for treating a patient having cancer comprising: (a) testing the patient's cancer cells for expression of Her2 and Plexin B1, Plexin B2, or a combination thereof; and (b) administering to the patient an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) if the patient's cancer cells express Her2 and Plexin B1, Plexin B2, or a combination thereof. The method of claim 48 or claim 49, further comprising administering an effective amount of an anti-HER2 / neu binding molecule. The method of claim 48 or claim 49, wherein the isolated binding molecule specifically binds to the same SEMA4D epitope as a reference monoclonal antibody VX15 / 2503 or 67. The method of claim 48 or claim 49, wherein the isolated binding molecule competitively inhibits a reference monoclonal antibody VX15 / 2503 or 67 from specifically binding SEMA4D. The method of claim 48 or claim 49, wherein the isolated binding molecule comprises an antibody or antigen binding fragment thereof. The method of claim 53, wherein the antibody or antigen binding fragment thereof comprises a variable heavy chain (VH) comprising VHCDRs 1-3 comprising SEQ ID Nos 6, 7 and 8, respectively, and a variable light chain (VL) comprising VLCDRs 1-3 comprising SEQ ID Nos: 14, 15 and 16, respectively. The method of claim 54, wherein VH and VL comprise SEQ ID NO: 9 and SEQ ID NO: 17 or SEQ IDNO: 10 and SEQ IDNO: 18, respectively.
BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES
[014] Figures 1A-1B: Tumor volume measurement in mice implanted with syngeneic Colon26 tumor cells. FIG. 1st shows measurement of Colon26 tumor volume in Balb / c and SCID mice treated twice a week with either 1 mg (50 mg / kg) of anti-SEMA4D antibody (Ab) 67 or isotype 2B8 control immunoglobulin (2B8 Ig control). FIG. 1B shows survival time, as defined in Example 1 below, of Balb / c and SCID mice treated with either anti-SEMA4D 67 or Ig Control 2B8.
[015] FIGURE 2: Shows measurement of Colon26 tumor volume in Balb / c mice implanted with tumor cells and treated first with anti-CD8 depleting antibody (Clone 2.43, bioXCell) or Rat Control Ig (150 mg / kg) and then treated as in FIG. 1A with or Control Ig 2B8 or Ab anti-SEMA4D 67.
[016] FIGURES 3A-3B: Measurement of immune cell density in the Co-lon26 tumor of grafted mice. FIG. 3A shows density of CD8 + T cells as determined by% tumor area stained with anti-CD8 antibody after treatment with Ig Control or anti-SEMA4D 67. FIG. 3B shows density of CD20 + B cells as determined by% of tumor area stained with anti-CD20 antibody after treatment with Ig Control or Ab anti-SEMA4D 67.
[017] FIGURES 4A-4D: Measurement of the distribution of macrophage and CD8 + T cell at the edges of the tumor in grafted Colon26 mice. FIG. 4A shows images of representative Colon26 tumors from mice grafted 27 days ago and treated with either Ig Control or anti-SEMA4D 67 as described in FIG. 1. FIG. 4B shows a measurement of type M1 macrophage density at the edges of the tumor, defined as a wide region of 300 pixels (250 microns) from the edge of the tumor, as determined by% pixel per area stained with anti-F4 / 80 antibody. FIG. 4C shows measurement of type M2 macrophage density at the tumor edge as determined by% pixel per area with anti-CD206 antibody. FIG. 4D shows a measurement of the density of CD8 + T cells at the edge of the tumor, as determined by% pixel in the area stained with anti-CD8 cytotoxic T cell antibody.
[018] FIGURES 5A-5D: Tumor volume measurement in mice implanted with syngeneic Colon26 tumor cells. FIG. 5A shows measurement of Colon26 tumor volume in Balb / c mice treated with either mouse IgG1 / 2B8 control or anti-SEMA4D 67-2 (50 mg / kg, IP, weekly), with or without anti-CTLA / MAb UC10-4F10 -11 (100 gg on day 8 and 50 gg on day 11 and 14 post tumor inoculation), and with anti-PD1 / RPMI-14 (100 gg on day 3, twice weekly) in combination with anti-CTLA4 / Mab UC10-4F10-11. FIG. 5B shows survival time of Balb / c mice treated with either control mouse IgG1 / 2B8 or anti-SEMA4D / MAb 67-2, with or without anti-CTLA4 / MAb UC10-4F10-11, and with anti-PD1 / RPM1 -14 (100 gg on day 3, twice weekly) in combination with anti-CTLA4 / MAb UC10-4F10-11. FIG. 5C shows the frequency of tumor regression in Balb / c mice treated with either mouse IgG1 / 2B8 control or anti-SEMA4D / MAb 67-2, with or without anti-CTLA4 / MAb UC10-4F10-11, and with anti-PD1 / RPMI1-14 (100 pg on day 3, twice a week) in combination with anti-CTLA4 / Mab UC10-4F10-11 (p values, * 0.05 and ** 0.001). FIG. 5D shows measurements of proinflammatory cytokines IFNy in tumor infiltrating lymphocytes of mice treated with the combination of anti-SEMA4D / Mab and anti-CTLA4 / Mab UC10-4F10-11 compared with or control mouse IgG1-2B8 or monotherapy (or anti-SEMA4D / Mab 67-2 or anti-CTLA4 / Mab UC10-4F10-11). FIG. 5E shows frequencies of secretory peptide-specific IFNy responders among tumor infiltrating lymphocytes recovered from the spleen of mice treated with the combination of anti-SEMA4D / Mab 67-2 and anti-CTLA4 / Mab UC10-4F10-11 compared a or mouse IgG1 / 2B8 control or monotherapy (or anti-SEMA4D / Mab 67-2 or anti-CTLA4 / Mab UC10-4F10-11).
[019] FIGURES 6A-6E: Measurement of an anti-SEMA4D antibody to affect tumor infiltration of tumor specific CD8 + cytotoxic T cells. FIG. 6A shows measurement of IFNy secreting cells in mice treated with MAb 67 in both, in the presence and absence of peptide. FIG. 6B shows representative ELISPOT images. FIG. 6C shows measurement of antitumor cytokines, such as IFNy and TNFa, in tumor infiltrating lymphocytes (TIL). FIG. 6D shows measurements of anti-inflammatory cytokines IFNy and TNFa in TIL in mice treated with anti-SEMA4D / MAb 67 antibody. FIG. 6E shows frequency of responders secreting peptide-specific IFNy in tumor infiltrating lymphocytes of mice treated with anti-SEMA4D / MAb 67 antibody.
[020] FIGURES 7A-7D: Tumor volume measurement in mice implanted with syngeneic Colon26 tumor cells. FIG. 7A shows a measurement of Colon26 tumor volume in Balb / c mice treated with either IgG1 / 2B8 from control mice or anti-SEMA4D / MAb 67-2 (50 mg / kg, IP, weekly), along with either Ig from control mice or anti-PD1 / MAbRPMI1-14 of rat (100 pg, twice a week, for 2 weeks starting 3 days after tumor inoculation). FIG. 7B shows survival time of Balb / c mice treated with either mouse IgG1 / 2B8 control or anti-SEMA4D / MAb 67-2 along with either mouse control Ig or mouse anti-PD1 / MAbRPMI1-14. FIGS. 7C and 7D show the frequency of tumor regression in Balb / c mice treated with either control mouse IgG1 / 2B8 or anti-SEMA4D / MAb 67-2 together with either control mouse Ig or anti-PD1 / MAbRPMI1-14.
[021] FIGURES 8A-8E: Measurement of tumor volume in mice implanted with syngeneic Colon26 tumor cells. FIG. 8A shows average measurement of Colon26 tumor volume in Balb / c mice treated with either mouse IgG1 / 2B8 control or anti-SEMA4D / MAb 67-2 (50 mg / kg, IP, weekly), with or without cyclophosphamide (CY ) (50 mg / kg, IP). FIG. 8B shows the average measurement of Colon26 tumor volume in Balb / c mice treated with either mouse IgG1 / 2B8 control or anti-SEMA4D / MAb 67-2 (50 mg / kg, IP, weekly), with or without cyclophosphamide (CY ) (50 mg / kg, IP). FIG. 8C shows survival time of Balb / c mice treated with either control mouse IgG1 / 2B8 or anti-SEMA4D / MAb 67-2, with or without cyclophosphamide. FIGS. 8D and 8E show the frequency of tumor regressions in Balb / c mice treated with either mouse IgG1 / 2B8 control or anti-SEMA4D / MAb 67-2, with or without cyclophosphatides (CY).
[022] FIGURES 9A-9C: Measurement of tumor volume in mice implanted with Tubo.A5 tumor cells. FIG. 9A shows measurement of tumor volume in Balb / c mice treated with either IgG1 / 2B8 from control mice or anti-SEMA4D / MAb 67-2 (50 mg / kg, IP, weekly), with or without anti-Neu / MAb7 .16.4 (aNeu) (200 gg IP weekly X2 starting when Tumor Volume (TV) is approximately 200 mm3, on days 21 and 28). FIG. 9B shows survival time of Balb / c mice treated with either control mouse IgG1 / 2B8 or anti-SEMA4D / MAb 67-2, with or without anti-Neu / MAb7.16.4 (aNeu). FIG. 9C shows the frequency of tumor regression in Balb / c mice treated with either control mouse IgG1 / 2B8 or anti-SEMA4D / MAb 67-2, with or without anti-Neu / MAb7.16.4 (aNeu).
[023] FIGURES 10A-10E: Tumor volume measurement in Balb / c mice implanted with Tubo.A5 tumor cells. FIG. 10A shows a measurement of tumor volume in Balb / c mice treated with either IgG1 / 2B8 from control mice or anti-SEMA4D / MAb 67-2 (50 mg / kg, IP, weekly). FIG. 10B shows survival time for Balb / c mice treated with either control mouse IgG1 / 2B8 or anti-SEMA4D / MAb 67-2. FIGS. 10C-10E show the frequency of tumor regressions in the Tubo.A5 tumor model. Specifically, FIG. 10C shows control mice grafted with the Tubo.A5 tumor. FIG. 10D shows mice that have Tubo.A5 tumor grafts rejected following treatment with anti-SEMA4D / MAb 67-2 and that were challenged again with Tu-bo.A5 tumor on day 90 following the original graft. FIG. 10E shows virgin mice challenged with the same tumor graft as in FIG. 10D to demonstrate tumor viability in vivo.
[024] FIGURES 11A-11B: Measurement of T cell infiltration and MDSC in the Tubo.A5 tumor models. FIG. 11A shows measurement of CD3 + T cells in tumors of Balb / c mice treated with either mouse IgG1 / 2B8 control or anti-SEMA4D / MAb 67-2 (50 mg / kg, IP, weekly). FIG. 11B shows a measurement of CD11b + Gr1 + MDSC in tumors of Balb / c mice treated with either mouse IgG1 / 2B8 control or anti-SEMA4D / MAb 67-2 (50 mg / kg, IP, weekly).
[025] FIGURES 12A-12D: Measurement of tumor volume in mice implanted with either Colon 26 or Tubo.A5 tumor cells. FIG. 12A shows measurement of tumor volume Tubo.A5 in Balb / c mice treated with either mouse IgG1 / 2B8.IE7 (50 mg / kg, IP, weekly x 6) or levels ranging from anti-SEMA4D / MAb 67-2 (1, 10 or 50 mg / kgm IP, weekly x 6). FIG. 12B shows survival time of Balb / c mice treated with either IgG1 / 2B8.IE7 from control mice (50 mg / kg, IP, weekly x6) or varying levels of anti-SEMA4D / MAb 67-2 (1, 10 or 50 mg / kg, PI, weekly x6). FIG. 12C shows measurement of tumor volume 26 in Balb / c mice treated with IgG1 / 2B8 / IE7 from control mice (50 mg / kg, IP, weekly x5), anti-SEMA4D / MAb 67-2 (50 mg / kg, IP , weekly x 5), anti-CTLA4 / MAb UC10-4F10-11 (5mg / kg, IP, weekly x 5), or a combination of anti-CTLA4 / MAb UC10-4F10-11 (5mg / kg, IP, weekly x 5) and variant levels of anti-SEMA4D / MAb 67-2 (0.3, 3, 10, or 50 mg / kg, IP, weekly x 5). FIG. 12D shows survival time of Balb / c mice treated with IgG1 / 2B8.IE7 from control mice (50 mg / kg, IP, weekly x 5), anti-SEMA4D / MAb 67-2 (50 mg / kg IP, weekly x5 ), anti-CTLA4 / MAb UC10-4F10-11 (5 mg / kg, IP, weekly x 5), or a combination of anti-CTLA4 / MAb UC10-4F10-11 (5 mg / kg, IP, weekly x 5 ) and variant levels of anti-SEMA4D / MAb 67-2 (0.3, 3, 10, or 50 mg / kg, IP, weekly x 5).
[026] FIGURE 13: Summary of experiments conducted in the figures above showing tumor regressions and growth after a new tumor challenge in the Colon26 and Tubo.A5 tumor models.
DETAILED DESCRIPTION I. Definitions [027] It is to be noted that the term "one" or "one" entity refers to one or more of this entity; for example, "a polynucleotide", is understood to represent one or more polynucleotides. Likewise, the terms "one" (or "one"), "one or more", and "at least one" can be used interchangeably.
[028] In addition, "and / or" where used herein is to be taken as specific disclosure of each of the two characteristics or components specified with or without the other. So, the term "and / or" as used in a phrase such as "A and / or B" here is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a sentence such as "A, B, and / or C" is intended to comprise each of the following modalities: 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).
[029] Unless otherwise defined, all technical and scientific terms used here have the same meaning as commonly understood by one skilled in the art to which this revelation 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, provides an expert on the technique with a general dictionary of many of the terms used in this revelation.
[030] Units, prefixes, and symbols are denoted in their accepted form in the International System of Units (SI). Numerical variations are inclusive of the numbers defining the variation. Unless otherwise indicated, amino acid sequences are written from left to right in the amino orientation for carboxy. The sections provided here are not limitations on the various aspects or modalities of the disclosure, which can be taken by reference to the specification as an integer. Consequently, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
[031] Wherever modalities are described with the language "comprising", otherwise analogous modalities described in terms of "consisting of" and / or "consisting essentially of" are also provided.
[032] Amino acids are referred to here by their commonly known three-letter symbols or by a one-letter symbol recommended by the Biochemical Nomenclature Commission IUPAC-IUB. Nucleotides, likewise, are referred to by their commonly accepted one-letter codes.
[033] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a cell population is 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, lung adenocarcinoma, lung squamous carcinoma, peritoneum cancer, hepa-cellular cancer, gastrointestinal cancer, gastric cancer, 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, cancer liver, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma and various types of head and neck cancers.
[034] In certain embodiments, metastatic cancers that are amenable to treatment using the methods provided herein include, but are not limited to metastatic sarcomas, breast carcinomas, ovarian cancer, head and neck cancer, and pancreatic cancer. In certain modalities metastatic cancers or tumor cells that are amenable to treatment using methods provided herein express Plexin-B1 and / or Plexin-B2 receptors for SEMA4D.
[035] “Angiogenesis” refers to a complex multi-step morphogenic event during which endothelial cells, stimulated by major determinants of vascular re-modeling, dynamically modify their cell-to-cell and cell-to-matrix contacts and move directly to be recognized in a mature vascular tree (Bussolino et al., Trends Biochem Sci. 22: 251-256 (1997); Risau, Nature 386: 671-674 (1997); Jain, Nat. Med. 9: 685- 693 (2003)). The formation of new blood vessels is a key step during embryonic development, but it also occurs in adults in physiological and pathological conditions, such as retinopathy, rheumatoid arthritis, ischemia, and particularly tumor growth and metastasis (Carmeliet, Nat. Med. 9 : 653-660 (2003)).
[036] As used herein, the term “clinical laboratory” refers to the facility for examining or processing materials derived from a living patient, for example, a human being. Non-limiting examples of processing include biological, biochemical, serological, chemical, immunohematological, hematological, biophysical, cytological, pathological, genetic or other examination of materials derived from the human body for the purpose of providing information, for example, for diagnosis, prevention, or treatment of any disease or impairment, or assessment of the health of living patients, for example, humans. These tests may also include procedures for collecting or otherwise obtaining a sample, preparing, determining, measuring, or otherwise describing the presence or absence of various substances in the body of a living patient, for example, a human being, or a sample obtained from the body of a living patient, for example, a human being.
[037] The terms "proliferative disorder" and "proliferative disease" refer to disorders associated with abnormal cell proliferation such as cancer.
[038] "Tumor" and "neoplasm" as used herein refer to any mass of tissue that results from cell growth or excessive proliferation, either benign (non-cancerous) or malignant (cancerous) including precancerous lesions. In certain embodiments, tumors described herein express Plexin-B1 and / or Plexin-B2, and can express SEAM4D and activated Met.
[039] As used herein, the term “health care benefit provider” includes individual parties, organizations, or groups providing, presenting, offering, paying in full or in part, or being otherwise associated with giving access to the patient of one or more health care benefits, benefit plans, health insurance, and / or health care expense counting programs.
[040] The term "immunomodulatory therapy" or "immunotherapy" refers to treatment that impacts a disease or disorder on a patient by inducing and / or increasing an immune response in that patient. Immunomodulatory therapies include cancer vaccines, immunostimulatory agents, adoptive T cell or antibody therapy, and immune checkpoint blocking (Lizée et al. 2013. Harnessing the Power of the Immune System to Target Cancer. Annu. Rev. Med. Vol 64 No. 71-90).
[041] The term "immunomodulating agent" refers to active immuno-therapy agents. Immunomodulating agents include a diverse network of re-combining, synthetic and natural preparations. Examples of immunomodulatory agents include, but are not limited to, interleukins such as IL-2, IL-7, IL-12; cytokines such as granulocyte colony stimulating factor (G-CSF), interferons; various chemokines such as CXCL13, CCL26, CXCL7; immune checkpoint block antagonists such as anti-CTLA4, anti-PDI or anti-PD-L1 (PD-1 ligand), anti-LAG3, anti-B7-H3, synthetic cytosine phosphate-guanosine (CpG) oligodeoxynucleotides , glucans; and regulatory T cell modulators (T regs) such as cyclophosphamide.
[042] The terms "metastasis", "metastasis", "metastatic", and other grammatical equivalents as used herein refer to cancer cells that spread or transfer from the site of origin (eg, a primary tumor) to other regions of the body with the development of a similar cancerous lesion in a new location. A "metastatic" or "metastatic" cell is one that loses adhesive contact with neighboring cells and migrates through the bloodstream or lymph from a primary disease site to invade neighboring body structures. The terms also refer to processes of metastasis, which include, but are not limited to, the detachment of cancer cells from a primary tumor, intravasation of tumor cells into the circulation, their survival and migration to a distant site, attachment and extravasation at a new site from the circulation, and microcolonization at the distant site, and tumor growth and development at a distant site.
[043] The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other effective drug to "treat" a disease or disorder in a patient or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; slow or stop the division of cancer cells, reduce or delay an increase in tumor size; inhibiting, for example, suppressing, delaying, preventing, stopping, delaying, or reversing cancer cell infiltration into peripheral organs including, for example, the spread of cancer in mold and bone tissue; inhibit, for example, suppress, delay, prevent, retract, stop, delay, or reverse tumor metastasis; inhibit, for example, suppress, delay, prevent, stop, delay, or reverse tumor growth; relieve to some extent one or more symptoms associated with cancer, reduce morbidity and mortality; improve the amount of life; or a combination of such effects. To an extent the drug that prevents growth and / or death of existing cancer cells, can be referred to as cytostatic and / or cytotoxic.
[044] Terms such as "treat" or "treatment" or "to treat" or "relieve" or "to relieve" refer to both, 1) therapeutic measures that heal, slow down, lessen symptoms of, reverse, and / or halt the progression of a condition or pathological disorder diagnosed and 2) prophylactic or preventive measures that prevent and / or slow the development of a condition or pathological disorder al l. So, those in need of treatment include those already with the disorder; those prone to having the disorder; and those in which the disorder is to be prevented. A patient is successfully "treated" according to the methods of the present invention 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 the size of the tumor; or delay or inversion of tumor growth, inhibition, for example, suppression, prevention, delay, shrinkage, delay, or reversal of metastasis, for example, of the infiltration of the cancer cell in the peripheral organs including, for example, the spread of the cancer in soft and bone tissue; inhibition of, for example, suppression of, delay of, prevention of, shrinkage of, reversal of, delay of, or absence of tumor metastasis, inhibition of, for example, suppression of, delay of, prevention of, shrinkage of, reversal of , delayed, 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. Desired beneficial or clinical results include, but are not limited to, symptom relief, decrease in disease extent, stabilized (that is, not worsen) disease, delay or decrease in disease progression, improvement or palliation of disease state , and remission (if partial or total), if detectable or undetectable. "Treatment" can also mean prolonged 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 likely to have the condition or disorder or those in which the condition or disorder is to be prevented.
[045] By "subject" or "individual" or "animal" or "patient" or "mammal", any patient, particularly a mammalian patient, is meant for which diagnosis, prognosis, or therapy is desired. Mammalian patients include humans, domestic animals, farm animals, and zoo, sports or domestic animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, and the like.
[046] As used herein, phrases such as "a patient who may benefit from administering an anti-SEMA4D antibody as a single agent or in combination with at least one other immunomodulatory therapy" "an animal in need of treatment" includes patients, such as mammalian patients, who may benefit from the administration of an anti-SEMA4D antibody as a single agent or in combination with at least one other immunomodulatory therapy.
[047] A "binding molecule" or "antigen binding molecule" of the present disclosure refers in its greater sense to a molecule that specifically binds to an antigenic determinant. In one embodiment, the linker molecule binds to SEMA4D, for example, a transmembrane SEMA4D polypeptide of about 150 kDa or a soluble SEMA4D polypeptide of about 120 kDa (commonly referred to as sSEMA4D). In another embodiment, a linker molecule of the disclosure is an antibody or antigen-binding fragment thereof. In another embodiment, a linker molecule of the disclosure comprises at least a heavy or light chain Complementarity Determining Region (CDR) of an antibody molecule. In another embodiment, a linker molecule of the disclosure comprises at least two CDRs of one or more antibody molecules. In another embodiment, a linker molecule of the disclosure comprises at least three CDRs of one or more antibody molecules. In another embodiment, the linker molecule of the disclosure comprises at least four CDRs from one or more antibody molecules. In another embodiment, a linker molecule of the disclosure comprises at least five CDRs from one or more antibody molecules. In another embodiment, a linker molecule of the disclosure comprises at least six CDRs of one or more antibody molecules. In another embodiment, the linker molecule may be a Plexin-BI receptor antagonist for SEMA4D. By antagonist is meant a ligand molecule that interferes with the signaling function of the receptor. The antagonist can competitively block the binding of a natural ligand, but fail to initiate the normal physiological response. Binding molecules can be antibodies or antigen-binding fragments thereof as described above or they can be other biological or small molecule drugs that act as competitive inhibitors or interfere with signaling by natural ligands. The present disclosure is directed to a method of inhibiting tumor growth and metastasis in a patient, for example, a cancer patient, comprising administering to the patient an anti-SEMA4D binding molecule, for example, an antibody, or binding fragment antigen, variant, or derivatives thereof, as a single agent or in combination with at least one other immunomodulatory therapy. Unless specifically referring to full-length antibodies such as naturally occurring anti-corpora, the term "anti-SEMA4D antibody" comprises full-length antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, for example, antibody naturally occurring or immunoglobulin molecule or constructed antibody molecules or fragments that bind antigen in a similar way to antibody molecules. Also included in the SEMA4D binding molecules are other biological or small molecules that bind and inhibit the activity of SEMA4D or its Plexin-B1 receptor.
[048] 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 transgenic animals to one or more human immunoglobulins, as described above and , for example, in 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) ( is) has (have) the immunoglobulin variable domain (s) amino acid sequence.
[049] "Human" or "completely human" antibodies also include "human" or "fully human" antibodies, as described above, which comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (for example, example, the VH regions and / or VL regions) described herein, whose antibodies or fragments of the same immunospecifically bind to a SEMA4D polypeptide or fragment or variant thereof. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding a human anti-SEMA4D antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis that results in amino acid substitutions. In certain respects, variants (including derivatives) encoding 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 than 2 amino acid substitutions, relative to the reference Vh region, VHCDR1, VHCDR2, VHCDR3, VL region, VLCDR1, VLCDR2, or VLCDR3 .
[050] In certain embodiments, amino acid substitutions are conserved amino acid substitutions, discussed further below. Alternatively, mutations can be introduced at random along with all or part of the coding sequence, such as by mutagenesis saturation, and the resulting mutants can be selected for biological activity that retain activity (for example, the ability to bind to the SEMA4D polypeptide, for example, SEMA4D human, mouse, or both, human and mouse). 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 ligand" and include antibodies that have improved affinity for the antigen .
[051] The terms "antibody" or "immunoglobulin" are used interchangeably here. 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, for example, Harlow and collaborators. (1988) Antibodies: A Laboratory Manual (2nd ed .; Cold Spring Harbor Laboratory Press).
[052] As used herein, the term "immunoglobulin" comprises several broad classes of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that heavy chains are classified as gam-ma, um, alpha, delta and epsilon (γ, μ, α, δ, ε) with some subclasses between them (for example, γ1-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgGor IgE, respectively. The immunoglobulin subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotopes are easily discernible to those skilled in the art in view of this present disclosure and, consequently, are within the scope of this present disclosure. All classes of immunoglobulins are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of approximately 23000 Daltons molecular weight, and two identical heavy chain polypeptides of 53000-70000 molecular weight. The four chains are typically joined by disulfide bridges in a "Y" configuration in which the light chains support the heavy chains starting at the "Y" entrance and continuing through the variable region.
[053] Light chains are classified as either kappa or lambda ((κ, λ). Each heavy chain class can be linked with either a kappa or lambda light chain. In general, light or heavy chains are covalently linked to each other , and the "terminal" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when immunoglobulins are generated, or by hybridomas, B cells or genetically engineered host cells. from an N-terminal in the forked termination of the Y configuration to the C terminal at the bottom of each chain.
[054] Both 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 light (VL or VK) and heavy (VH) portions determine antigen recognition and specifically. On the other hand, the light chain (CL) and heavy chain (CH1, CH2 or CH3) constant domains confer important biological properties such as secretion, transplacental mobility, binding to the Fc receptor, complement binding, and the like. By convention, the numbering of constant region domains increases when it becomes more distal from the antigen or amino-terminal binding site of the antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and Cl domains actually comprise heavy and light chain carboxy terminals, respectively.
[055] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitope or antigens. That is, the VL domain and the VH domain, or subgroup of 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 examples, for example, certain immunoglobulin molecules derived from camelid or immuno-globulin species constructed based on camelids, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, for example, Hamers-Casterman et al., Nature 363: 446-448 (1993).
[056] 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 rest of the amino acids in the antigen-binding domains, referred to as "structural" regions, show less intermolecular variability. The structural regions widely adopt a β-sheet conformation and the CDRs form loops that connect, and in some cases form part of the β-sheet structure. Then, structural regions act to form a structure that provides for positioning the CDRs in the correct orientation by non-knitting interchange interactions. The antigen-binding domain by the positioned CDRs defines a surface complementarity to the epitope in the immunoreactive antigen. This complementary surface promotes the non-covalent attachment of the antibody to its cognate epitope. Amino acids comprising CDRs and structural regions, respectively, can be easily identified for any given heavy or light chain variable domain by one skilled in the art, as long as they have been precisely defined (see below).
[057] In the case where there are two or more definitions of a term that is used and / or accepted within the technique, the definition of the term as used herein is intended to include all meanings unless explicitly stated otherwise. 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 and colleagues. (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 definitions include overlap or subgroups of amino acid residues when compared between them. However, application of or definition to refer to a CDR of an antibody or variants of it is intended to be within the scope of the term as defined and used herein. The appropriate residues that comprise the CDRs as defined by each of the references cited above are revealed below in Table 1 as a comparison. The exact residue numbers that comprise 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.
Table 1. CDR1 definitions 1Numeration of all CDR definitions in Table 1 is in accordance with the numbering conventions revealed by Kabat and collaborators (see below).
[058] Kabat and colleagues also defined a numbering system for variable domain strings that is applicable to any antibody. One skilled in the art can unequivocally assign this “Kabat numbering” system to any variable domain sequence, without checking any experimental data beyond its own sequence. As used herein, "Kabat numbering" refers to the numbering system disclosed 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, are in accordance with the Kabat numbering system.
[059] 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, binding fragments of epitope, for example, Fab, Fab 'and F (ab') 2, Fd, Fvs, Fvs (scFv) single chain, Fvs (sdFv) linked to disulfide, fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to anti-SEMA4D antibodies disclosed herein). ScFv molecules are known in the art and are described, for example, in U.S. Pat. North American No. 5,892,019. The immunoglobulin or antibody molecules of the disclosure can be of any type (for example, IgG, IgE, IgD, IgA, and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 etc.), or subclass immunoglobulin molecule.
[060] As used herein, the term "heavy chain moiety" includes amino acid sequences derived from an immunoglobulin heavy chain. In certain embodiments, a polypeptide comprising a heavy chain moiety comprises at least one of: a VH domain, a CH1 domain, a hinge domain (e.g., upper, middle, and / or lower loop region), a CH2 domain , a CH3 domain, or a variant or fragment thereof. For example, a polypeptide linker for use in the disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 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. In addition, a linker polypeptide for use in the disclosure may be missing at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As disclosed above, it will be understood by one skilled in the art that these domains (e.g., the heavy chain moieties) can be modified such that they vary in the amino acid sequence from the naturally occurring immunoglobulin molecule.
[061] In certain anti-SEMA4D antibodies, or antigen-binding fragments, variants or derivatives thereof disclosed herein, the heavy chain portions of a polypeptide chain of a multimer are identical to those in a second polypeptide chain of the multimer. Alternatively, monomers containing the heavy chain portion 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 therefore binds to two different types of antigen. Variations in the bispecific antibody format are contemplated within the scope of the present disclosure. Bispecific antibodies can be generated using techniques that are well known, for example, see, Ghayur et al., Expert Review of Clinicai Pharmacology 3.4 (July 2010): p491; 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.
[062] The heavy chain portions of a binding molecule for use in the methods disclosed here can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion may comprise a hinge region drained, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion may comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.
[063] As used herein, the term "light chain moiety" includes amino acid sequences derived from an immunoglobulin light chain, for example, a kappa or lambda light chain. In certain aspects, the light chain portion comprises at least one VL or CL domain.
[064] Anti-SEMA4D antibodies, or variant antigen-binding fragments, or derivatives thereof disclosed herein can be described or specified in terms of the epitope (s) or portion (s) of an antigen, for example, an antigen target polypeptide disclosed herein (e.g., SEMA4D) that they specifically recognize or 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. In addition, it should be noted that an "epitope" in a target polypeptide can be or can include non-polypeptide elements, for example, an epitope can include a carbohydrate side chain.
[065] 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, amino acids comprising an epitope need not be contiguous, and in some cases, may not even be in the same peptide chain. A polypeptide peptide or epitope recognized by anti-SEMA4D antibodies of the present disclosure may contain a sequence of at least 4, at least 5, at least 7, at least 8, at least 9, at least 10, at least at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous SEMA4D amino acids.
[066] By "specifically binding" it is generally meant that an antibody binds to an epitope through its antigen-binding domain, and that binding brings about some complementarity between the antigen-binding domain and epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to this epitope, via its antigen-binding domain more easily than it can bind to an unrelated random epitope. The term "specificity" is used here to describe the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" can be considered to have a greater specificity or affinity for a given epitope than antibody "B", or antibody "A" can be said to bind to epitope "C" with a greater specificity or affinity than it has for the related “D” epitope.
[067] By "binding preferentially" it is meant that the antibody specifically binds to an epitope more easily than it can bind to a related, similar, homologous or analogous epitope. Therefore, an antibody that "preferably binds" to a given epitope can more likely bind that epitope than to a related epitope, although such an antibody may cross-react with the related epitope.
[068] By way of non-limiting example, an antibody can be considered to bind a first epitope preferably if it binds to said epitope with a dissociation constant (KD) that is less than the KD of the antibody to the second epitope. In another non-limiting example, an antibody can be considered to bind a first antigen preferably if it binds to the first epitope with an affinity that is at least an order of magnitude less than the KD of the antibody to the second epitope. In another non-limiting example, an antibody can be considered to bind a first epitope preferably if it binds to the first epitope with an affinity that is at least two orders of magnitude less than the KD of the antibody to the second epitope.
[069] In another non-limiting example, an antibody can be considered to bind a first epitope preferably if it binds to the first epitope with an off rate (k (off)) which is less than the k (off) of the antibody to the second epitope. In another non-limiting example, an antibody can be considered to bind a first epitope preferably if it binds to the first epitope with an affinity that is at least an order of magnitude less than the k (off) of the antibody to the second epitope. In another non-limiting example, an antibody can be considered to bind a first epitope preferably if it binds to the first epitope with an affinity that is at least two orders of magnitude less than the k (off) of the antibody to the second epitope.
[070] An antibody is said to competitively inhibit the binding of a reference antibody to a given epitope if it preferably binds 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, ELISA competition assays. An antibody can be said to competitively bind the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
[071] As used herein, the term "affinity" refers to a measure of the strength of binding of an individual epitope with the CDR of an immunoglobulin molecule. See, for example, Harlow and collaborators. (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 an immunoglobulin population and an antigen, that is, the strength of the functional combination of an immunoglobulin mixture with the antigen. See, for example, Harlow on pages 29-34. Avidity is related to both, the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valences of immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeated epitope structure, such as a polymer, can be highly avid.
[072] Anti-SEMA4D antibodies or antigen-binding fragments, variants or derivatives of the disclosure memos 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 an antigen, to react with a second antigen; a measure of the relationship between two different antigenic substances. Therefore, an antibody is cross-reactive if it binds to an epitope other than the one that induced its formation. The cross-reactivity epitope generally contains many of the same complementary structural features as the induced epitope, and in some cases, may actually fit better than the original.
[073] For example, certain antibodies have the same degree of cross-reactivity, in that they bind to related but not identical epitopes, for example, 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%, 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 to 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 considered "highly specific" for a certain epitope, if it does not bind to any other analog, orthologist, or homologue to that epitope.
[074] Anti-SEMA4D binding molecules, for example, antibodies or antigen-binding moieties, variants or derivatives thereof of the disclosure can also be described or specified in terms of that binding affinity to a disclosure polypeptide, for example, SEMA4D, for example example, SEMA4D human, mouse, or both, human and mouse. In certain respects, binding affinities include those with a dissociation constant or Kd less than 5 x 10-2 M, 10-2 M, 5 x 10-3 M, 10-3 M, 5 x 10-4 M, 10 -4 M, 5 x 10-5 M, 10-5 M, 5 x 10-6 M, 10-6 M, 5 x 10-7 M, 10-7 M, 5 x 10-8 M, 10-8 M, 5 x 10-9 M, 10-9 M, 5 x 10-10 M, 10-10 M, 5 x 10-11 M, 10-11 M, 5 x 10-12 M, 10-12 M, 5 x 10-13 M, 10-13 M, 5 x 10-14 M, 10-14 M, 5 x 10-15 M, or 10-15 M. In some cases, the anti-SEMA4D binding molecule, for example, a antibody or antigen-binding fragment thereof, from the disclosure binds human SEMA4D with a Kd of about 5 x 10-9 to about 6 x 10-9. In another embodiment, the anti-SEMA4D binding molecule, for example, an antibody or antigen binding fragment thereof, discloses murine SEMA4D binding with a Kd of about 1 x 10-9 to about 2 x 10-9.
[075] As used herein, the term "chimeric antibody" will be retained to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial or modified) is obtained from a second species. In some embodiments, the target binding region or site will be from a non-human source (for example, mouse or primate) and the constant region is human.
[076] As used herein, the term "constructed antibody" refers to an antibody in which the variable domain in either the heavy or light chains or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, if necessary, replacement of the partial structure region and change of sequence. Although CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the structural regions are derived, it is anticipated that CDRs will be derived from an antibody of a different class or an antibody from a different species. An antibody constructed in which one or more "donors" CDRs from a non-human antibody of known specificity is grafted into a human heavy and light chain structural region is referred to here as a "humanized antibody". In some respects, it is not necessary to replace all CDRs with full CDRs from a donor variable domain to transfer the antigen binding capacity from one variable domain to another. Predictably, only residues that are necessary to maintain the activity of the binding site against the target antigen can be transferred.
[077] It is further recognized that the structural regions within the variable domain in a heavy or light chain, or both, of a humanized antibody can comprise only residues of human origin, in which case these structural regions of the humanized antibody are referred to as “regions completely human structural ”(for example, MAb VX15 / 2503, disclosed in U.S. Patent Application Publication No. UA 2010/0285036 A1 as MAb 2503, hereby incorporated by reference in its entirety). Alternatively, one or more residues from the structural region (s) of the donor variable domain (s) can be constructed within the corresponding position of the human structural region (s) ) of a variable domain in a heavy and / or light chain, or both, of a humanized antibody if necessary to maintain proper binding or to increase binding to the SEMA4D antigen. A human structural region that has been constructed in this way must then comprise a mixture of human and donor structural waste, and is referred to here as a "partially human structural region".
[078] For example, humanizing an anti-SEMA4D antibody can essentially be done following the method of Winter and collaborators (Jones and collaborators., Nature 321: 522-525 (1986); Riechmann and collaborators., Nature 332: 323327 ( 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, Pat. North American Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205; incorporated herein by reference. The resulting humanized anti-SEMA4D antibody can 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 examples, residues within the structural regions of one or more variable domains of the humanized anti-SEMA4D antibody are replaced by corresponding non-human residues (eg, rodents) (see, for example, U.S. Pat. No. 5,585,089 ; 5,693,761; 5,693,762; and 6,180,370), in which case the humanized anti-SEMA4D antibody may comprise partially human structural regions within the variable domain of the heavy and / or light chain. Similar methods can be used to humanize an anti-VEGF antibody.
[079] In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further define the performance of the antibody (for example, to obtain the desired affinity). In general, the humanized antibody will comprise substantially all of at least, and typically two, variable domains, where all or substantially all of the CDRs correspond to those of non-human immunoglobulin and all or substantially all of the framework regions are those of a sequence of human immunoglobulin. The humanized antibody optionally will also 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); Riech-mann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992); incorporated herein by reference. Consequently, such "humanized" antibodies may include antibodies in which less substantially than an intact human variable domain has been replaced 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 structural residues are replaced by residues from analogous sites on rodent antibodies. See, for example, Pat. North American Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. See also Pat. North American 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 revealed.
II. Description Target Polypeptide - SEMA4D
[080] As used herein, the terms "semaphorin-4D", "SEMA4D", and "SEMA4D polypeptide" are used interchangeably, as are "SEMA4D" and "Se-ma4D". In certain embodiments, SEMA4D is expressed on the surface of or secreted by a cell. In another embodiment, SEMA4D is membrane bound. In another embodiment, SEAM4D is soluble, for example, sSEMA4D. In another embodiment, SEAM4D may include a full-length SEMA4D or fragment thereof, or a SEMA4D variant polypeptide, wherein the SEMA4D fragment or SEMA4D variant polypeptide retains some or all of the full-length SEMA4D functional properties.
[081] The full-length human SEMA4D protein is a homodimeric trans-membrane protein consisting of two 150 kDa polypeptide chains. SEMA4D belongs to the semaphorin family of cell surface receptors and is also referred to as CD100. Both human and mouse SEMA4D / Sema4D are proteolytically cleaved from their transmembrane form to generate soluble 120 kDa forms, giving rise to two Sema4D isoforms (Kumano-goh et al., J. Cell Science 116 (7) : 3464 (2003)). Semaphorins consist of soluble and membrane-bound proteins that were originally defined as axonal guiding factors that play an important role in establishing precise connections between neurons and their appropriate target. Structurally considered a class IV semaphorin, SEMA4D consists of a terminal signal sequence followed by a characteristic “Sema” domain, which contains 17 conserved cysteine residues, an Ig-type domain, a lysine-rich stretch, a hydrophobic transmit-white region, and a cytoplasmic tail.
[082] The SEMA4D polypeptide includes a signal sequence of about 13 amino acids followed by a semaphorin domain of about 512 amino acids, an immunoglobulin-like domain (type Ig) of about 65 amino acids, a lysine-rich stretch of 104 amino acids, a hydrophobic transmembrane region of about 19 amino acids, and a cytoplasmic tail of 110 amino acids. A consensus site for tyrosine phosphorylation in the cytoplasmic tail supports the predicted association of SEMA4D with a tyrosine kinase (Schlossman et al., Eds. (1995) Leucocyte Typing V (Oxford University Press, Oxford).
[083] SEMA4D is known to have at least three functional receptors, Plexi-na-B1, Plexin-B2 and CD72. Plexin-B1 is expressed in non-lymphoid tissues and has been shown to be a high affinity receptor (1 nM) for SEMA4D (Tamagnone et al., Cell 99: 71-80 (1999)). SEMA4D stimulation of Plexin B1 signaling has been shown to induce collapse of neuron cone growth, and to induce oligodendrocyte exsession collapse and process (Giraudon et al., J. Immunol. 172: 1246-1255 (2004); Giraudon et al., Neu-roMolecular Med. 7: 207-216 (2005)). After binding to SEMA4D, Plexin B1 signaling mediates the inactivation of R-Ras, leading to a decrease in intgrin-mediated binding to the extracellular matrix, as well as activation of RhoA, leading to cell collapse by cytoskeleton reorganization. See, Kruger et al., Nature Rev. Mol. Cell Biol. 6: 789-800 (2005); Pasterkamp, TRENDS in Cell Biology 15: 61-64 (2005)). Plexin-B2 has an intermediate affinity for SEMA4D and a recent report indicates that PLXNB2 is expressed in keratinocytes and activates SEMA4D-positive γδ cells to contribute to epithelial repair (Witherden et al., Immunity. 2012 Aug 24; 37 (2): 314-25).
[084] In lymphoid tissues, CD72 is used as a low affinity SEMA4D (300 nM) receptor (Kumanogoh et al., Immunity 13: 621-631 (2000)). B cells and Antigen-Presenting Cells (APC) express CD72, and anti-CD72 antibodies have many of the same effects as sSEMA4D, such as increased CD40-induced B cell response and CD23 B cell suppression. CD72 is thought to act as a negative regulator of B cell responses by recruiting tyrosine phosphatase SHP-1, which can associate with many inhibitory receptors. Interaction of SEMA4D with CD72 results in the dissociation of SHP-1, and the loss of this negative activation signal. SEMA4D has been shown to promote T cell stimulation and B cell aggregation and in vitro survival. The addition of cells expressing SEMA4D or sSEMA4D increases CD40-induced B cell proliferation and immunoglobulin production in vitro, and accelerates antibody responses in vivo (Ishida et al., Inter. Immunol. 15: 1027-1034 (2003); Kuma -nogoh and H. Kukutani, Trends in Immunol. 22: 670-676 (2001)). sSEMA4D increases CD40-induced maturation of CDs, including positive regulation of co-stimulatory molecules and increased IL-12 secretion. In addition, sSEMA4D can inhibit immune cell migration, which can be reversed by adding anti-SEMA4D mouse antibody blocking (Elhabazi et al., J. Immunol. 166: 4341-4347 (2001); Delaire et al., J. Immunol 166: 4348-4354 (2001)).
[085] Sema4D is expressed at high levels in lymphoid organs, including the spleen, thymus, and lymph nodes, and non-lymphoid organs, such as the brain, heart and kidney. In lymphoid organs, Sema4D is abundantly expressed in T cells in respiration, but only weakly expressed in B cells and antigen presenting cells (APCs), such as dendritic cells (CDs).
[086] Cell activation increases the surface expression of SEMA4D as well as the generation of soluble SEMA4D (sSEMA4D). The expression pattern of SEMA4D suggests that it plays an important physiological as well as pathological role in the immune system. SEMA4D has been shown to promote B cell activation, aggregation and survival; increased CD40-induced proliferation and antibody production; increased anti-chlorine response to T cell-dependent antigens; increased T cell proliferation; increased dendritic cell maturation and ability to stimulate T cells; and is directly implicated in demyelination and axonal degeneration (Shi et al., Immunity 13: 633-642 (2000); Kumanogoh et al., J Immunol 169: 1175-1181 (2002); and Watanabe et al., J Immunol 167: 4321-4328 (2001)).
[087] SEMA4D knockout mice (SEMA4D - / -) have additional evidence provided that SEMA4D plays an important role in both humoral and cellular immune responses. There are no known abnormalities of non-lymphoid tissues in SEMA4D - / - mice. Dendritic cells (CDs) from SEMA4D - / - mice have poor allostimulatory ability and show defects in the expression of co-stimulatory molecules, which can be rescued by the addition of sSEMA4D. SEMA4D-deficient mice (SEMA4D - / -) fail to develop experimental autoimmune encephalomyelitis induced by oligodendrocyte myelin glycoprotein peptide, because specific T cells for myelin-oligodendrocyte glycoprotein are poorly generated in the absence of SEMA4D collaborators (Kumanogo and Kumanogo. 169: 1175-1181 (2002)). A significant amount of soluble SEMA4D is also detected in the soto of MRL / lpr mice prone to autoimmunity (model of systemic autoimmune diseases such as SLE), but not in normal mice. In addition, sSEMA4D levels correlate with levels of autoantibodies and increase with age (Wang et al., Blood 97: 3498-3504 (2001)). Soluble SEMA4D has also been shown to accumulate in the cerebrospinal fluid and serum of patients with demyelinating disease, and sSE-MA4D induces apoptosis of human pluripotent neural precursors (Dev cells), and both inhibit extension process and induce apoptosis of rat oligodendrocytes in vitro (Giraudon et al., J Immunol 172 (2): 1246-1255 (2004)). This apoptosis was blocked by an anti-SEMA4D monoclonal antibody (MAb).
III. Anti-SEMA4D antibodies
[088] Antibodies that bind SEMA4D have been described in the art. See, for example, North American Publication Nos. 2008/0219971 A1, US 2010/0285036 A1, and US 2006/0233793 A1, International Patent Applications WO 93/14125, WO 2008/100995, and WO 2010/129917, and Herold et al., Int. Immunol. 7 (1): 1-8 (1995), each of which is incorporated herein in its entirety by reference.
[089] The disclosure generally relates to a method of inhibiting, delaying, or reducing tumor growth or metastasis in a patient, for example, a human cancer patient, comprising administration of an antibody that specifically binds SEMA4D, or a fragment antigen linker, variant, or derivative thereof. In certain embodiments, the antibody blocks the interaction of SEMA4D with one or more of these receptors, for example, Plexin-B1 and / or Plexin-B2. In certain embodiments, cancer cells express Plexin-B1 and / or Plexin-B2. Anti-SEMA4D antibodies having these properties can be used in the methods provided herein. Antibodies that can be used include, but are not limited to, MAbs VX15 / 2503, 67, 76, 2282 and antigen-binding fragments, variants, or derivatives thereof that are fully described in US 2010/0285036 A1 and US 2008/0219971 A1 . Additional antibodies that can be used in the methods provided herein include the BD16 antibody described in US 2006/0233793 A1 as well as antigen-binding fragments, variants, or derivatives thereof; or any of MAb 301, MAb 1893, MAb 657, MAb 1807, MAb 1656, MAb 1808, Mab 59, MAb 2191, MAb 2274, MAb 2275, MAb 2276, MAb 2277, MAb 2278, MAb 2279, MAb 2280, MAb 2281 , MAb 2282, MAb 2283, MAb 2284, and MAb 2285, as well as any fragments, variants or derivatives thereof as described in US 2008/0219971 A1. In certain embodiments, an anti-SEMA4D antibody for use in the methods provided herein binds human, murine, or both SEMA3D, human and murine. Also useful are antibodies that bind to the same epitope as the antibodies mentioned above and / or antibodies that competitively inhibit binding or activity of any antibodies mentioned above.
[090] In certain embodiments, an anti-SEMA4d antibody or antigen-binding fragment, variant or derivative of the same useful in the methods provided here has an amino acid sequence that is 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 for the amino acid sequence for a molecule of anti-SEMA4D antibody, for example, those described here. In an additional embodiment, the linker molecule divides at least about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to a reference antibody.
[091] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative of the same 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 VH domain CDRs 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 CDR1, CDR2 or CDR3 of SEQ ID NO: 9, 10, 25 or 48.
[092] In another embodiment, an anti-SEMA4D antibody or variant antigen-binding fragment, or derivative thereof useful in the methods provided herein comprise, consist essentially of, or consist of, an immunoglobulin heavy chain variable domain (VH domain), where at least one of the VH domain CDRs 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 SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.
[093] In another embodiment, an anti-SEMA4Dor antigen-binding fragment, variant, or derivative of the same 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 VH domain CDRs has an identical amino acid sequence, except for 1,2, 3, 4, or 5 conserved amino acid substitutions, for SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.
[094] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative of the same 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 SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 25 or SEQ ID NO: 48, wherein an anti-SEMA4D antibody comprising the specifically encoded VH domain or preferably binds SEMA4D.
[095] 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 in 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 CDR1, CDR2, or CDR3 of SEQ ID NO: 17, 18, 29 or 47.
[096] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative of the same 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 in 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 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.
[097] In another embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or useful derivative thereof 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 VR domain CDRs has an identical amino acid sequence, except for 1, 2, 3, 4 or 5 conserved amino acid substitutions for SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.
[098] In an additional embodiment, an anti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof useful in the methods herein comprises, essentially consists of, or consists of a VL domain that has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91% m about 92% m about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 29, or SEQ ID NO: 47, where an anti-SEMA4D antibody comprises the domain VL specifically encoded or preferably binds to SEMA4D.
[099] Also included for use in the methods provided herein are polypeptides encoding anti-SEMA4D antibodies, or antigen-binding fragments, or derivatives thereof as described herein, polynucleotides encoding such polypeptides, vectors comprising such polynucleotides, and host cells comprising such vectors or polynucleotides, all to produce anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof for use in the methods described herein.
[0100] Suitable biologically active variants of the anti-SEMA4D antibodies of the disclosure can be used in the methods for the present disclosure. Such variants will retain the desired binding properties of the parental anti-SEMA4D antibody. Methods for making variant antibodies are generally available in the art.
[0101] Methods for mutagenesis and nucleotide sequence changes 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 and collaborators. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.I.); Pat. North American No. 4,873,192; and the references cited therein; incorporated herein by reference. A guide to appropriate amino acid substitutions that does not affect the biological activity of the polypeptide of interest can be found in the model by Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), pp. 345-352, incorporated herein by reference in its entirety. The model of Dayhoff and collaborators. Uses the Accepted Point of Mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine suitable conserved amino acid substitutions. In certain respects, conservative substitutions, such as exchanging one amino acid with another having similar properties are used. Examples of conserved 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 ^ Ile ^ Leu, Asp ^ Glu, Lys ^ Arg, Asn ^ Gln, and Phe ^ Trp ^ Tyr.
[0102] In variant constructions of the anti-SEMA4D binding molecule, for example, an antibody or antigen binding fragment thereof, polypeptides of interest, modifications are made such that variants continue to have the desired properties, for example, being able to specifically bind to a SEMA4D, for example, human SEMA4D, mouse, or both, human and mouse, for example, expressed on the surface of or secreted by a cell and having SEMA4D blocking activity, as described herein. In some respects, mutations in the DNA encoding the variant polypeptide maintain the reading structure and do not create complementary regions that can produce secondary mRNA structure. See EP Patent Application Publication No. 75,444.
[0103] Methods for measuring anti-SEMA4D binding molecule, for example, an antibody or antibody binding fragment, variant, or derivative thereof, binding specificity includes, but is not limited to, standard competitive binding assays, assays to monitor immunoglobulin secretion by T cells or B cells, T cell proliferation assays, apoptosis assay, ELISA assay, 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 incorporated herein by reference.
[0104] Methods for measuring the anti-angiogenic ability of an an-ti-SEMA4D antibody or antigen-binding fragment, variant, or derivative thereof, are well known in the art.
[0105] When discussed here whether any particular polypeptide, including the constant regions, CDRs, VH domains, or VL domains disclosed herein, is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% identical to another polypeptide, the% identity can be determined using methods and computer programs / software known in the art such as, but not limited to, BESTFIT (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 homology segment 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 adjusted, of course, such that the percentage of identity is calculated over the entire length of the reference polypeptide sequence and gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
[0106] For the purpose of the present disclosure, percent sequence identity can be determined using the Smith-Waterman homology search algorithm using a related gap search with an open gap 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 may, for example, differ from a reference anti-SEMA4D antibody (for example, 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.
[0107] The anti-SEMA4D antibody constant region can be mutated to alter effector function in a number of ways. For example, see Pat. No. 6,737,056B1 and U.S. Patent Application Publication No. 2004 / 013210A1, which discloses Fc mutations that optimize antibody binding to Fc receptors.
[0108] 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 (via mutation points or other means) of a constant region domain can reduce binding to the Fc receptor of the modified antibody circulating in this way by increasing the tumor location. In other cases, modifications of the constant region consistent with the moderate complement binding of the present disclosure and then reduction in serum half-life. Still other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide fractions that allow for increased localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor location, biodistribution and serum half-life, can easily be measured and quantified using well-known immunological techniques without undue experimentation.
[0109] Anti-SEMA4D antibodies for use in the methods provided herein include derivatives that are modified, for example, by covalent binding of any type of molecule to the antibody such that covalent binding does not prevent the antibody from specific binding to its cognate epitope. For example, but not by way of limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivation by protective / blocking groups, proteolytic cleavage, binding to a cell ligand or other protein etc. Any of the numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, etc. In addition, the derivative may contain one or more non-classical amino acids.
[0110] A "conserved 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 (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutaminia, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and chains aromatic sides (eg tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along with all or part of the coding sequence, such as by mutagenesis saturation, and the resulting mutants can be selected for biological activity to identify mutants that retain activity (for example, the ability to bind a anti-SEMA4D polypeptide, to block SEMA4D interaction with its receptor, or to inhibit, delay, or reduce metastasis in a patient, for example, a cancer patient).
[0111] For example, it is possible to introduce mutations only in the structural regions or only in the CDR regions of an antibody molecule. Introduced mutations can be silent or neutral sense-shifting mutations, that is, having no, or having little effect on an antibody's ability to bind antigen. These types of mutations can be useful to optimize the codon for use, or improve hybridoma antibody production. Alternatively, mutations with non-neutral sense changes can alter an antibody's ability to bind to the antigen. One skilled in the art may be able to design and test mutant molecules with desired properties such as no change in antigen binding activity or change in binding activity (for example, 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 (eg, ability to immunospecifically bind at least an epitope of a SEMA4D polypeptide) can be determined using techniques described here or by routinely modifying known techniques in art.
[0112] In certain embodiments, the anti-SEMA4D antibodies for use in the methods provided here comprise at least an optimized complementarity determining region (CDR). By "optimized CDR" it is intended that the CDR has been modified and optimized to improve binding affinity and / or anti-SEMA4D activity that is prevented for an anti-SEMA4D antibody comprising the optimized CDR. “Anti-SEMA4D activity” or “SEMA4D blocking activity” may include activity that 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; cell proliferation of T cell or other immune cell; 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 of cell surface B1 plexin or other receptor, or any other activity associated 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, or in combination with inhibition, delay, or reduction of primary tumor cell growth and tumor metastases, or regardless of cell growth primary tumor and tumor metastasis. Anti-SEMA4D activity can also be attributed to a decrease in the 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 inflammatory diseases of the central nervous system (CNS) ) and peri-faith system (SNP), and invasive angiogenesis. Examples of optimized antibodies based on MAb BD16 anti-SEMA4D have been described in Pub. North American No. 2008/0219971 A1, International Patent Application WO 93/14125 and Herold et al., Int. Immunol. 7 (1): 1-8 (1995), each of which is incorporated herein by reference in its entirety. The modifications may 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.
IV. Binding Characteristics of Anti-SEMA4D Antibodies
[0113] In certain embodiments, the binding molecule is an antibody that specifically binds to SEMA4D, or a binding fragment of the antigen, variant, or derivative thereof. In certain embodiments, the linker molecule binds to an SEMA4D epitope. The nucleotide and amino acid sequences for one SEMA4D variant are revealed in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and for another SEMA4D variant are revealed in SEQ ID NO: 15 and SEQ ID NO: 16. In some embodiments, the anti-SEMA4D antibody designated as VX15 / 2503 is provided. Antibodies that have the binding characteristics of the VX15 / 2503 antibody are also disclosed here. Such antibodies include, but are not limited to, antibodies that compete in VX15 / 2503 competitiveness binding assays, as well as antibodies that bind to an epitope (as defined below) capable of binding VX15 / 2503. Methods for assessing whether antibodies have the same or similar binding characteristics include traditional quantitative methods such as, for example, determining and comparing antibody affinity or avidity for the antigenic epitope (e.g., SEMA4D peptide). Other exemplary methods for comparing antibody binding characteristics include competitive western blotting, enzyme immunoassays, ELISA, and flow cytometry. Methods for assessing and comparing antibody-antigen binding characteristics are well known in the art. VX15 / 2503 variants and fragments that retain the ability to specifically link to SEMA4D are also provided. VX15 / 2503 and 67 antibodies share the same 6 CDRs and bind to the same SEMA4D epitope.
[0114] In some embodiments, anti-SEMA4D antibodies, or antigen-linking fragments, variants or derivatives thereof disclosed herein may be described or specified in terms of the epitope (s) or portion (s) of an ant -gen, for example, a target polypeptide disclosed herein (for example, SEMA4D) that they specifically recognize or 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".
[0115] In some embodiments, an "epitope" is intended to be part of an antigenic molecule that is used to produce an antibody and / or that an antibody will specifically bind. An "SEMA4D" epitope comprises the part of the SEMA4D protein to which an anti-SEMA4D antibody binds. Epitopes can comprise linear amino acid residues (that is, residues within the epitope that are arranged sequentially one after another in a linear form), non-linear amino acid residues (referred to here as "non-linear epitopes" or "conformational epitopes", these epitopes are not arranged sequentially), or both linear or non-linear amino acid residues. Non-linear epitopes or conformational epitopes can also include amino acid residues that contribute to the overall conformation of the antibody's recognition structure. Typically, epitopes short amino acid sequences, for example, about five amino acids in length. Systematic techniques for identifying epitopes are known in the art and are described, for example, in the examples disclosed below.
[0116] 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. In addition, it should be noted that an "epitope" in a target polypeptide can be or can include non-polypeptide elements, for example, an epitope can include a carbohydrate side chain.
[0117] 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, or at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, amino acids comprising an epitope need not be contiguous, and in some cases, they may not even be in the same peptide chain. A polypeptide peptide or epitope recognized by anti-SEMA4D antibodies of the present disclosure may contain a sequence of at least 4, at least 4, 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 SEMA4D amino acids.
[0118] In some embodiments, the epitope has at least 80%, 85%, 90%, 95% or 100% identity to a target polypeptide amino acid sequence (for example, the sequence disclosed in SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46).
[0119] In some embodiments, the epitope is identical to a target polypeptide amino acid sequence (for example, the sequence disclosed in SEQ ID NO: 42, SEQ ID NO: 44, or SEQ ID NO: 46) except 4, 3 , 2, 1 or 0 amino acid substitutions. In another embodiment, the epitope is identical to a target polypeptide amino acid sequence (for example, the sequence disclosed in SEQ ID NO: 42, SEQ ID NO: 44, or SEQ ID NO: 46) except for conserved amino acid substitutions (for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 conserved amino acid substitutions).
[0120] In some embodiments, the epitope comprises a sequence disclosed in SEQ ID NO: 42, SEQ ID NO: 44, or SEQ ID NO: 46. In another embodiment, the epitope is the sequence disclosed in SEQ ID NO: 42, SEQ ID NO: 44, or SEQ ID NO: 46. In some embodiments, the epitope is a linear epitope. In some embodiments, the epitope is a conformational epitope.
[0121] In some embodiments, the epitope comprises, consists essentially of, or consists of LKVPVFYALFTPQLNNV (SEQ ID NO: 42, corresponding to residues 304 to 320 of the full-length SEMA4D sequence disclosed in SEQ ID NO: 1), KWTSFLKARLIASRP (SEQ ID NO: 44, corresponding to residues 270 to 284 of the full-length SEMA4D amino acid sequence disclosed in SEQ ID NO: 1, where position 281 can be a cysteine or an alanine), or EFVFRVLIPRIARV (SEQ ID NO: 46; corresponding to residues 243 to 256 of the full-length SEMA4D amino acid sequence disclosed in SEQ ID NO: 1). In some embodiments, the epitope comprises one or more of the amino acid sequences disclosed in SEQ ID NO: 42, 44 and 46. In some embodiments, the epitope is a discontinuous epitope comprised in amino acid residues spanning domain 243 to 320 of SEQ ID NO :1. V. Treatment Methods Using Therapeutic Anti-SEMA4D Antibodies As A Single Agent Or In Combination With At Least One Immunomodulatory Therapy [0122] Disclosure methods are directed to the use of anti-SEMA4d or anti-Plexin-B1 binding molecules, for example for example, antibodies, including antigen-binding fragments, variants, derivatives thereof, or as single agents or in combination with at least one other immunomodulatory therapy, to inhibit, delay, or reduce tumor growth or metastasis in a patient in need of such inhibition, delay, or reduction, for example, a cancer patient. In certain modalities the cancer cells express a SEMA4D receptor, in certain modalities the receptor is Plexin-B1. Although the following discussion concerns the administration of an anti-SEMA4D antibody, the methods described herein are equally applicable to the antigen-binding fragments, variants, and derivatives of those antibodies that retain the desired properties of the antibodies of the disclosure, for example, capable to specifically bind SEMA4D, for example, human, mouse, or human and mouse SEMA4D, having neutralizing activity of SEMA4D, and / or blocking the interaction of SEMA4D with its receptors. The methods described herein are also applicable to other biological products or small molecule drugs that retain the desired properties of the disclosure antibodies, for example, capable of specifically binding SEMA4D, for example, human, mouse, or human and mouse SEMA4D, having neutralizing activity of SEMA4D, and / or blocking the interaction of SEMA4D with its receptors.
[0123] In one embodiment, anti-SEMA4D molecules, for example, antibodies, including antigen-binding fragments, variants, and derivatives thereof, can be used as a single agent to inhibit, delay, or reduce tumor growth in a patient in need for such inhibition, delay, or reduction, for example, a cancer patient. In certain embodiments, cancer cells express a SEMA4D receptor, such as, for example, Plexin-B1 or Plexin-B2. In other embodiments, cancer cells express other receptors that can work in conjunction with a SEMA4D receptor. An example of such a receptor is HER2 (ErbB2). Examples of cancers in which expression of Plexin-B1 or Plexin-B2 in combination with Her2 have been observed to include lung cancer, breast cancer, prostate cancer, and ovarian cancer. As, in certain embodiments, anti-SEMA4D molecules, for example, antibodies, including antigen-binding fragments, variants, and derivatives thereof, can be used as a single agent to inhibit, delay, or reduce tumor growth in a patient having lung cancer, breast cancer, prostate cancer, or ovarian cancer.
[0124] In one embodiment, immunomodulatory therapy may include cancer vaccines, immunostimulatory agents, adoptive T cell therapy or antibody, and immune checkpoint blocking inhibitors (Lizée et al. 2013. Harnessing the Power of the Immune System to Target Cancer, Annu. Rev. Med. Vol. 64 No. 71-90).
[0125] Cancer vaccines. Cancer vaccines activate the body's immune system and natural resistance to an abnormal cell, such as cancer, resulting in eradication or disease control. 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 tumoral antigens, heterologous tumor cells transfected with an immunostimulating agent, such as GM-CSF, recombinant virus, or proteins or peptides that are usually administered with a potent immune adjuvant such as CpG .
[0126] Immunostimulatory Agents. Immunostimulatory agents work to improve or increase the immune response to tumors, which is suppressed in many cancer patients through various mechanisms. Immunomodulatory therapies may include lymphocytes, macrophages, dendritic cells, natural killer cells (NK cell), or subgroups of these cells such as cytotoxic T lymphocytes (CTL) or natural killer T cells (NKT). Because of the interaction of immune cascades, an effect of a group of immune cells will always apply by spreading other cells, for example, increased antigen presenting cell activity promotes T and B lymphocyte response. Examples of immunostimulatory agents include, but are not are limited to, HER2, cytokines such as G-CSF, GM-CSF and IL-2, cell membrane fractions from bacteria, glycolipids that associate with CD1d to activate natural killer T cells (NKT), CpG oligonucleotides.
[0127] Macrophages, myelophagocytic cells of the immune system, are a fundamental part of innate defense mechanisms, which can promote specific immunity by inducing T cell recruitment and activation. Despite this, their presence within the tumor environment has been associated with tumor progression increased and shown to promote cancer cell growth and dispersion, angiogenesis and immunosuppression. Key participants in adjusting their phenotype are the microenvironment signals to which the macrophages are exposed, which selectively tune their functions within a functional spectrum comprising the extremes M1 (tumor-inhibiting macrophages) and M2 (tumor-promoting macrophages). Sica et al., Seminars in Cancer Biol. 18: 349-355 (2008). Increased macrophage numbers during cancer generally correlate with poor prognosis (Qualls and Murray, Curr. Topics in Develop. Biol. 94: 309-328 (2011)). Of the multiple types of unique stromal cells common to solid tumors, tumor-associated macrophages (TAMs) are meant to stimulate tumor progression. Target molecular pathways regulating TAM polarization hold great promise for anticancer therapy. Ruffell et al., Trends in Immunol. 33: 119-126 (2012).
[0128] Adoptive Cell Transfer. Adoptive cell transfer may employ cytotoxic and T cell based 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 to the cancer patient. One study showed that adoptive transfer of autologous tumor infiltrating lymphocytes expanded in vitro 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 the patient's resected 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 alloreactive feeder cells. These T cells are then transferred back to the patient along with exogenous IL-2 administration to further challenge its anti-cancer activity. In other studies, autologous T cells have been transduced with a chimeric antigenic receptor that cause it to react to a target tumor antigen (Liddy et al., Nature Med. 18: 980-7, (2012); Grupp et al., New England J. Med. 368: 1509-18, (2013)).
[0129] Another adoptive cell transfer therapy employs autologous denitic cells exposed to natural or modified tumor antigens ex vivo that are re-infused into the patient. Provenge is such that an FDA-approved therapy in which autologous cells are incubated with a prostatic acid phosphatase fusion protein 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)).
[0130] Immune Checkpoint Blocking: Immune checkpoint blocking therapies increase T cell immunity by removing a negative feedback control that limits continuous immune responses. These types of therapies targeting inhibitory pathways in the immune system that are crucial for modulating the duration and range of physiological immune responses in peripheral tissues (anti-CTLA4) or in tumor tissue expressing PD-L1 (anti-PD1 or anti-PD- L1) in order to minimize collateral tissue damage. Tumors can evolve to explore 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 the 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 otherwise in the immunosuppressive tumor microenvironment. Examples of immune checkpoint block therapies are those targeting antigen associated with cytotoxic T lymphocyte 4 (CTLA-4), PD-1, its ligand PD-L1, LAG3 and B7-H3.
[0131] Cyclophosphamide. Cyclophosphamide, a commonly used chemotherapeutic agent, can enhance immune responses. Cyclophosphamides differently suppress the function of regulatory T cells (T regs) relative to effector T cells. T regs are important in regulating anti-cancer immune responses. Tumor infiltrating regions have previously been associated with poor prognosis. While agents that specifically target T regs are currently unavailable, cyclophosphamide has emerged as a clinically viable agent that can preferably suppress T regs relative to other T cells and thus allow more effective induction of anti-tumor immune responses.
[0132] Other Immunomodulatory Therapies: In another embodiment, therapy with a SEMA4D or Plexin-B1 binding molecule, for example, an antibody or antigen binding fragment, variant, or derivative thereof, may be combined with or low dose of chemotherapy or radiation therapy. Although standard chemotherapy is always immunosuppressive, low doses of chemotherapeutic agents such as cyclophosphamide, doxorubicin, and paclitaxel have been shown to increase responses to cancer vaccine therapy (Machiels et al., Cancer Res. 61: 3689-3697 (2001) ). In some cases, chemotherapy can differently inactivate regulatory T cells (Treg) and suppressor cells of myeloid origin (MDSC) that negatively regulate immune responses in the tumor environment. Radiation therapy has generally been employed to explore the direct tumoricidal effect of ionizing radiation. In fact, high doses of radiation can, like chemotherapy, be self-suppressing. Numerous observations, however, suggest that under appropriate conditions of fractionation and dose sequencing, radiation therapy can increase tumor-specific immune responses and the effects of immunomodulatory agents. One of the several mechanisms that contribute to this effect is cross-presentation by dendritic cells and other antigen presenting cells of tumor antigens released by radiation-induced tumor cell death (Higgins et al., Cancer Biol. Ther. 8: 1440-1449 ( 2009)). In effect, radiation therapy can induce in situ vaccination against a tumor (Ma et al., Seminar Immunol. 22: 113-124 (2010)) and this can be amplified by combining it with therapy with a SEAM4D or Plexin-binding molecule B1, for example, an antibody or antigen-binding fragment, variant, or derivative thereof.
[0133] In one embodiment, immunomodulatory therapy can be an immunomodulatory agent, including, but not limited to, interleukins such as IL-2, IL-7, IL-12; cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), interferon; various chemokines such as CXCL13, CCL26, CXCL7; immune checkpoint block antagonists such as anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-LAG3 and anti-B7-H3; synthetic cyanosine guanosine-phosphate (CpG), oli-godeoxynucleotides, glucans, regulatory T cell modulators (T regs) such as cyclophosphamide, or other immunomodulatory agents. In one embodiment, the immunomodulatory agent is a 4-1BB agonist antibody (CD137). As recently reported, such an agonist antibody for 4-1BB may give rise to a new class of KLRG1 + T cells that are highly cytotoxic to tumors (Curran et al., J. Exp. Med. 210: 743-755 (2013)). In all cases, additional immunomodulatory therapy is administered prior to, during, or subsequent to the anti-SEMA4D or anti-Plexin-B1 binding molecule, for example, co-antibody therapy or antigen binding fragment, variant, or derivative thereof. Where the combined therapies comprise administration of an anti-SEMA4D binding molecule, for example, an antibody or antigen binding fragment, variant, or derivative thereof, in combination with administration of another immunomodulatory agent, the methods of the disclosure comprise co-administration, using formulations separate or a single pharmaceutical formulation, with simultaneous or consecutive administration in both orders.
[0134] In one embodiment, immunomodulatory therapy may be a cancer therapy agent, including, but not limited to, surgery or surgical procedures (for example, splenectomy, hepatectomy, lymphadenoctomy, leukophoresis, bone marrow transplantation, and the like) ; radiation therapy; chemotherapy, optionally in combination with autologous bone marrow transplantation, or other cancer therapy; where additional cancer therapy is administered prior to, during, or subsequent to the anti-SEMA4D binding molecule for example, antibody therapy or antigen binding fragment, variant, or derivative thereof. Where the combined therapies comprise administration of an anti-SEMA4D binding molecule, for example, an antibody or antigen binding fragment, variant, or derivative thereof, in combination with administration of another therapeutic agent, the methods of the disclosure comprise co-administration, using formulations separate or a single pharmaceutical formulation, with simultaneous or consecutive administration in both orders.
[0135] In another embodiment, the disclosure is directed to the use of anti-SEMA4D or anti-Plexin-B1 binding molecules, for example, antibodies, including antigen binding fragments, variants, and derivatives thereof, either as single agents or in combination with at least one other immunomodulatory therapy, to treat cancer patients with elevated levels of either B cells, T cells or both, B cells and circulating T cells when compared to other patients with solid tumors, as found in the brain, ovary , breast, colon, and other tissues but excluding hematological cancers. As used herein, the term "elevated" refers to cancer patients who are at least 1.5 times, for example, about 1.5 to about 5 times, for example, about 1.5; 2; 2.5; 3; 3.5; 4; 4.5; or 5 times the average number of B cells and / or T cells in the circulation than other cancer patients. In a non-limiting example, in a group of 34 patients with solid tumors, the average number of B cells was 98 per microliter of blood and the average number of T cells was 782 per microliter of blood. Consequently, the average number of B cells and T cells per microliter of blood observed in this subset of patients with high levels of cancer with B cells and T cells ranging from about 147 to about 588 and from about 1173 to about 3910, respectively, when compared to other cancer patients.
[0136] In another embodiment, the disclosure is directed to the use of anti-SEMA4D or anti-Plexin-B1 binding molecules, for example, antibodies, including antigen-binding fragments, variants, and derivatives thereof, either as single agents or in combination with at least one other immunomodulatory therapy, to treat cancer patients with levels of either B cells, t cells, or both B cells and T cells in the circulation that fall within or above the range of normal individuals. As used herein, the term "normal" refers to the levels of B and / or T cells that are found in healthy patients without cancer. As used herein, the term "inside" refers to a ten (10) percent difference in B and / or T cell levels. In a non-limiting example, the variation from normal levels includes, for example, a cell count B of about 250 cells per microliter or more and / or a T cell count of about 1500 cells per microliter or more. Thus, the average number of B cells or T cells per microliter of blood in cancer patients with levels of B cell and T cell can vary from about 225 to about 275 or more and from about 1350 to about 1650 and more, respectively, when compared to healthy patients without cancer. Of course, one skilled in the art may appreciate that B and T cell levels may vary depending on a variety of factors, for example, type of cancer, stage of cancer etc., and thus, levels that are below one provided above may also constitutes elevated levels by a certain type or stage of cancer.
[0137] In some embodiments, absolute T and B cell counts are measured using an immunophenotypic assay based on validated flow cytometry (MD Mutitest 6-color TBNK Reagent), which is a six-color direct immunofluorescence assay that also uses BD Trucount tubes and a BD FACScanto flow cytometer. This assay is used routinely to determine the percentages and absolute counts of T, B and NK cells, as well as CD4 and CD8 subpopulations of T cells in peripheral blood. Peripheral blood cells are first selected (gate) in the CD45 + lymphocytes. T cells are defined as CD3 + cells within this gate and B cells are defined as CD19 + CD3- cells within this gate. Percentages are simply taken directly from the flow cytometer after the appropriate gate is adjusted, and absolute counts are calculated using the following formula (taken directly from the BD manual procedure): [(#events in the cell population / # events in the region spherical absolute count)] * [(beads / test) / test volume] = absolute count of the cell population, where “a” is the value found in the foil and aluminum marking of the BD Tru-count tube.
[0138] It should also be appreciated that the methods described herein are also applicable to the replacement of anti-Plexin-B1 binding molecules for anti-SEMA4D binding molecules. In some embodiments, an anti-Plexin-B1 binding molecule can be used to inhibit the interaction of SEMA4D with Plexin-B1 by blocking binding of SEMA4D to Plexin-B1 and / or by preventing activation of Plexin-B1 by SEMA4D. It should also be appreciated that the methods described herein are also applicable to the use of small molecule drugs or other biological products to inhibit the activity of SEMA4D or Plexin-B1. In some embodiments, a small molecule drug or biological product other than an anti-SEMA4D binding molecule can be used to inhibit the interaction of SEMA4D with Plexin-B1 by binding blockage of SEMA4D to Plexin-B1 and / or by preventing activation of Plexin-B1 by SEMA4D.
[0139] In one embodiment, treatment includes the application or administration of an anti-SEMA4D binding molecule, for example, an antibody or antigen-binding fragment thereof as described herein as a single agent or in combination with at least one other immunomodulatory therapy to a patient, or application or administration of the anti-SEMA4D binding molecule as a single agent or in combination with at least one other immunomodulatory therapy to an isolated tissue or cell line from a patient, where the patient has, or has the risk of metastasis from cancer cell development. In another embodiment, treatment is also intended to include the application or administration of a pharmaceutical composition comprising the anti-SEMA4D binding molecules, for example, an antibody or antigen binding fragment thereof to a patient, in combination with at least one other immunotherapy therapy. or applying or administering a pharmaceutical composition comprising the anti-SEMA4D binding molecule and at least one immunomodulatory therapy to an isolated tissue or cell line from a patient, where the patient has, or is at risk of developing cancer cell metastasis.
[0140] Anti-SEMA4D binding molecules, for example, antibodies or binding fragments thereof as described herein, as single agents or in combination with at least one other immunomodulatory therapy are useful for the treatment of various malignant and non-malignant tumors. By "anti-tumor activity" is intended a reduction in the rate of production or accumulation of SEMA4D associated directly with the tumor or indirectly with stromal cells in the tumor environment, and then a decline in the growth rate of an existing tumor or an tumor that increases during therapy, and / or destruction of existing neoplastic cells (tumors) or newly formed neoplastic cells, and then 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 or in combination with at least one other immunomodulatory therapy causes a physiological response, for example, a reduction in metastases, which is beneficial with respect to the treatment of disease states associated with cells expressing SEMA4D in a human.
[0141] In one embodiment, the disclosure relates to the use of anti-SEMA4D binding molecules, for example, antibodies or antigen binding fragments, variants, or derivatives thereof, as a single agent or in combination with at least an immunomodulatory therapy as a medicine, in the treatment or prophylaxis of cancer or for use in a precancerous condition or injury to inhibit, reduce, prevent, delay, or minimize the growth or metastasis of tumor cells.
[0142] According to the methods of the present disclosure, at least one anti-SEMA4D binding molecule, for example, an antibody or antigen binding fragment, variant, or derivative thereof, as a single agent or in combination with at least one another immunomodulatory 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, an improvement in the disease is associated with the anti-tumor activity of these binding molecules, for example, antibodies or fragments thereof, and / or an improvement in the symptoms associated with the disease. In particular, the methods provided herein are directed to inhibit, prevent, reduce, alleviate, delay, or slow the growth of a tumor and / or the development of metastases from primary tumors in a patient. This is to prevent the consequences of distal tumors can be observed. So, for example, an improvement in the disease can be characterized as a complete response. By "complete response" is meant an absence of clinically detectable metastases with normalization of any previous abnormal radiographic studies, for example, at the site of the primary tumor or the presence of tumor metastasis in the bone marrow. Alternatively, an improvement in the disease can be categorized as a partial response. By "partial response" is intended at least about 50% decrease in all measurable metastases (that is, the number of tumor cells present in the patient at a remote site from the primary tumor). Alternatively, an improvement in the disease can be categorized as relapse-free survival or "survival-free progression". “Relapse-free survival” means the time for tumor recurrence anywhere. "Progression-free survival" is the time before additional tumor growth at a site being monitored is detected.
[0143] Inhibition, delay, or metastasis reduction can be evaluated using selection techniques such as imaging, for example, fluorescent antibody imaging, bone scanning image, and tumor biopsy sample including bone marrow aspiration (BMA), or immunohistochemistry. In addition to these positive therapeutic responses, the patient undergoing therapy with the anti-SEMA4D binding molecule, for example, an antibody or antigen binding fragment, variant, or derivative thereof, may experience the beneficial effect of an associated symptom improvement. with the disease.
[0144] Clinical response can be assessed using selection techniques such as magnetic resonance imaging (MRI) scanning, X-radiographic imaging, computed tomography (CT) scanning, flow cytometry or fluorescence activated cell separation analysis ( FACS), histology, gross pathology, and blood chemistry, including but not limited to detectable changes by ELISA, RIA, chromatography, and the like.
[0145] To apply the methods and systems of the disclosure in certain modalities, samples from a patient can be obtained before or after administration of a therapy comprising either: (1) an effective amount of an isolated binding molecule that specifically binds to semaphorin-4D (SEMA4D) and an effective amount of at least one other immunomodulatory therapy; or (2) an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) to a patient having a tumor that is Her2 + and or Plexin B1 + or Plexin B2 +. In some cases, successive samples may be obtained from the patient after therapy has started or therapy has ceased. Samples may, for example, be requested by a health care provider (for example, a doctor) or health care benefits provider, obtained and / or processed by the same provider or a different health care provider (for example , a nurse, a hospital) or a clinical laboratory, and after processing, results can be forwarded to yet another health care provider, health care benefit provider or the patient. Similarly, the measurement / determination of one or more scores, comparisons between scores, assessment of scores and treatment decisions can be made by one or more health care providers, health care benefit providers, and / or clinical laboratories.
[0146] As used herein, the term "health care provider" refers to individuals or institutions that directly interact and administer for living patients, for example, human patients. Non-limiting examples of health care providers include doctors, nurses, technicians, therapists, pharmacists, counselors, alternative medicine practitioners, medical facilities, doctor's offices, hospitals, emergency rooms, clinics, urgent care centers, clinics / health facilities. alternative medicine, and any other entity providing general and / or specialized treatment, assessment, maintenance, therapy, medication, and / or advice regarding all, or any portion of, a patient's health status, including, but not limited to, general practitioner , specialized doctor, surgery and / or any other type of treatment, evaluation, maintenance, therapy, medication and / or device.
[0147] In some respects, the health care provider may administer or instruct another health care provider to administer a therapy comprising either: (1) an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D ) and an effective amount of at least one other immunomodulatory therapy; or (2) an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D), where the patient has, or is suspected to have, tumor cells that are Her2 + and either Plexin B1 + or Plexin B2 +. A health care provider can implement or instruct another health care provider or patient to do the following: take a sample, process a sample, submit a sample, receive a sample, transfer a sample, analyze or measure a sample, quantify a sample, provide the results obtained after analyzing / measuring / quantifying a sample, receiving the results obtained after analyzing / measuring / quantifying a sample, comparing / punctuating the results obtained after analyzing / measuring / quantifying one or more samples, providing comparing / scoring one or more samples, obtaining a comparison / scoring for one or more samples, administering a therapy (eg, (1) an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D ) and an effective amount of at least one other immunomodulatory therapy, or (2) an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) to a patient, where the patient has, or is suspected of having, tumor cells that are Her2 + and or Plexin B1 + or Plexin B2 +, starting the administration of a therapy, interrupting the administration of a therapy, continuing the administration of a therapy, temporarily interrupting the administration of a therapy, increasing the amount of a therapeutic agent administered, decreasing the amount of a therapeutic agent administered, continuing to administer a quantity of a therapeutic agent, increasing the frequency of administration of a therapeutic person, decrease the frequency of administration of a therapeutic agent, maintain the same dose frequency in a therapeutic agent, replace a therapy or therapeutic agent with at least another therapy or therapeutic agent, combine a therapy or therapeutic agent with at least another therapy or therapeutic agent additional. In some respects, a health care benefits provider may authorize or deny, for example, collecting a sample, processing a sample, submitting a sample, receiving a sample, transferring a sample, analyzing or measuring a sample. sample, quantification of a sample, provision of results obtained after analysis / measurement / quantification of a sample, transfer of results obtained after analysis / measurement / quantification of a sample, comparison / score of results obtained after analysis / measurement / quantification of one or more samples, transfer of comparison / scoring of one or more samples, administration of a therapy or therapeutic agent, start administration of a therapy or therapeutic agent, interruption of administration of a therapy or therapeutic agent, continuation of administration of a therapy or therapeutic agent, temporary interruption of administration of a therapy or the therapeutic people, increase in the amount of therapeutic agent administered, decrease in the amount of therapeutic agent administered, continued administration of a quantity of a therapeutic person, increase in the frequency of administration of a therapeutic agent, decrease in the frequency of administration of a therapeutic agent, maintaining the same dose frequency in a therapeutic person, substitution or therapeutic agent for at least another therapy or therapeutic agent, or combining a therapy or therapeutic agent with at least one additional therapy or therapeutic agent.
[0148] In addition, a health care benefit provided may, for example, authorize or deny the prescription of a therapy, authorize or deny coverage for therapy, authorize or deny reimbursement for the cost of therapy, determine or deny eligibility for therapy etc.
[0149] In some respects, a clinical laboratory may, for example, collect or obtain a sample, process a sample, submit a sample, receive a sample, transfer a sample, analyze or measure a sample, quantify a sample, provide the results obtained after analysis / measurement / quantification of a sample, receive the results obtained after analysis / measurement / quantification of a sample, compare / score the results obtained after analysis / measurement / quantification of one or more samples, provide the comparison / score of one or more samples, obtain the comparison / score of one or more samples, or other related activities. SAW. Diagnostic and Treatment Methods [0150] In certain embodiments, this disclosure provides methods of treating a patient, for example, a cancer patient, where the patient has elevated levels of either B cells, T cells or both, B cells and T cells , comprising administering a combination of an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) and an effective amount of at least one other immunomodulatory therapy whether levels of B cell, T cell or both B cells and patient's T cells are above a predetermined threshold level of B cells, T cells or both B cells and T cells, or are elevated relative to the level of B cells, T cells or both, B cells and T cells, in one or more control samples that may include, but not be limited to, samples from other cancer patients or healthy patients without cancer. Levels of B cell, T cell, or B cell or T cell can be measured by a healthcare provider or by a clinical laboratory, where a sample, for example, a blood sample, is obtained from the patient or by health care provider or clinical laboratory. In one aspect, the patient's level of B cells, T cells, or both, B cells and T cells, can be measured in an immunophenotypic assay based on cytometry.
[0151] In certain embodiments, this disclosure also provides a method of treating a patient, for example, a cancer patient, comprising administering to the patient an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) if expression of Her2 and or Plexin B1 or Plexin B2 in a sample taken from the patient's tumor cells is above predetermined threshold levels, or is high relative to the expression of Her2 and or Plexin B1 or Plexin B2 in one or more samples control. Her2, Plexin B1, and / or Plexin B2 expression in the patient's tumor cells can be measured by a healthcare provider or by a laboratory at the protein level and / or the mRNA level. In some ways, expression of Her2, Plexin B1. and / or Plexin B2 can be measured in situ, for example, using imaging techniques. In certain aspects, expression of Her2, Plexin B1, and / or Plexin B2 can be measured in a tumor cell sample from the patient via a biopsy. In one aspect, expression of Her2, Plexin B1, and / or Plexin B2 in tumor cells can be measured in an immunoassay employing antibodies or antigen-binding fragments thereof that recognize Her2, Plexin B1, and / or Plexin B2 proteins, or antigen-binding fragments, variants or derivatives thereof. In another aspect, expression of Her2, Plexin B1, and / or Plexin B2 can be measured using a quantitative gene expression assay, for example, an RT-PCR assay.
[0152] This disclosure also provides methods, assays, and kits to facilitate a determination by a health care provider, a health care benefit provider, or a clinical laboratory as to whether a patient, for example, a patient with cancer, will benefit from treatment with either: (1) an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) and an effective amount of at least one other immunomodulatory therapy; or (2) an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMQ4D), where the patient has, or is suspected to have, tumor cells that are Her2 + and either Plexin B1 + or Plexin B2 +. The methods, assays, and kits provided here will also facilitate a determination by a health care provider, a health care benefit provider, or a clinical laboratory if a patient, for example, a cancer patient, will benefit from treating (1) an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) and an effective amount of at least one other immunomodulatory therapy; (2) an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) (for example, where the patient's tumor cells express, or can be determined to express, Her2 and or Plexin B1 or Plexin B2).
[0153] The present disclosure provides a method of treating a patient, for example, a cancer patient, comprising administering an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) and an effective amount of at least another immunomodulatory therapy; whether the level of B cells, T cells, or T cells and B cells in a sample taken from the patient is above a predetermined threshold level, or is above the level of B cells, T cells, or T cells and B cells in one or more control samples. In some respects, the sample is obtained from the patient and is submitted for measurement of the level of B cells, T cells, or T cells and B cells in the sample, for example, to a clinical laboratory.
[0154] Also provided is a method of treating a patient, for example, a cancer patient, comprising (a) submitting a sample taken from the patient to measure the level of B cells, T cells, or T cells and B cells in the sample ; and, (b) administering an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) and an effective amount of at least one other immunomodulatory therapy to the patient if the level of B cells, T cells, or T cells and the patient's B cells is above a predetermined threshold level, or is above the level of B cells, T cells, or T cells and B cells in one or more control samples.
[0155] The disclosure also provides a method of treating a patient, for example, a cancer patient, comprising (a) measuring the level of B cells, T cells, or T cells and B cells in a sample obtained from a patient, for example, a cancer patient, where the level of B cells, T cells, or T cells and B cells of the patient in the sample is measured, for example, in an immunophenotypic assay based on cytometry; (b) determining whether the level of B cells, T cells, or T cells and B cells in the sample is above a predetermined threshold level, or is above the level of B cells, T cells or T cells and B cells in one or more control samples; and (c) warning, instructing, or authorizing a health care provider to administer an effective amount of an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D) and an effective amount of at least one other immunomodulatory therapy to the patient if the patient's B cell, T cell, or T cell and B cell level is above a predetermined threshold level, or it is above the B cell, T cell, or T cell and B cell level in one or more samples control.
[0156] In some ways, the patient's level of B cells, T cells, or T cells and B cells can be measured in an immunophenotypic assay based on cytometry. In certain aspects, the test can be done on a sample obtained from the patient, by the health care professional treating the patient, for example, using a test as described here, formulated as a “point of care” diagnostic kit. In some respects, a sample can be obtained from the patient and can be submitted, for example, to a clinical laboratory, to measure the level of B cells, T cells, or T cells and B cells in the sample according to the instructions health care professional, including, but not limited to, use of an immunophenotypic cytometry-based assay as described herein. In certain respects, the clinical laboratory conducting the trial may advise the health care provider or a health care benefit provider if the patient can benefit from treatment with an effective amount of an isolated ligand molecule that specifically binds semaphorin -4D (SEMA4D0 and an effective amount of at least one other immunomodulatory therapy, if the patient's level of B cells, T cells, or T cells and B cells is above a predetermined threshold level, or is above the level of B cells, T cells, or T cells and B cells in one or more control samples.
[0157] In certain respects, results of an immunoassay as provided herein may be submitted to a health care benefit provider to determine whether patient insurance will cover treatment with an isolated binding molecule that specifically binds semaphorin-4D (SEMA4D ) and at least one other immunomodulatory therapy. VII. Pharmaceutical Compositions and Methods of Administration [0158] Methods of preparing and administering anti-SEMA4D binding molecules, for example, antibodies, or antigen binding fragments, variants, or derivatives thereof as a single agent or in combination with at least one other therapy immunomodulatory to a patient in need of it are well known to or are easily determined by those skilled in the art. The route of administration of the anti-SEMA4D binding molecule, for example, or antigen binding fragment, variant, or derived from it as a single agent or in combination with at least one other immunomodulatory therapy, can be, for example, oral, parenteral , by inhalation or topical at the same time or different for each therapeutic agent. The term parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all of these forms of administration are clearly contemplated to be within the scope of the disclosure, an example of a form for administration may be a solution for injection, in particular for intravenous or intraarterial injection or drops. A pharmaceutical composition suitable for injection may comprise a buffer (for example, acetate, phosphate or citrate buffer), a surfactant (for example, polysorbate), optionally a stabilizing agent (for example, human albumin) etc. However, in other methods compatible with the teachings here, anti-SEMA4D binding molecules, for example, antibodies, or antigen-binding fragments, variants, or derivatives thereof as a single agent or in combination with at least one other immunomodulatory therapy may be used. distributed directly to the site of the adverse cell population thus increasing the exposure of the diseased tissue to the therapeutic agent.
[0159] As discussed herein, anti-SEMA4D binding molecules, for example, antibodies, or antigen binding fragments, variants, or derivatives thereof as a single agent or in combination with at least one other immunomodulatory 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 binding molecules can be formulated in order to facilitate administration and promote stability of the active agent. In certain embodiments, pharmaceutical compositions according to the present disclosure comprise a sterile, non-toxic, pharmaceutically acceptable carrier such as non-toxic physiological saline buffers, preservatives and the like. For the purposes of the present application, a pharmaceutically effective amount of an anti-SEMA4D binding molecule, for example, or an antigen-binding fragment, variant, or derivative thereof, as a single agent or in combination with at least one other immunomodulatory therapy should be maintained to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, that is, to inhibit, delay, or reduce metastasis in a cancer patient.
[0160] The pharmaceutical compositions used in this disclosure comprise pharmaceutically acceptable carriers, including, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acids, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, hydrogen potassium phosphate, sodium chloride, zinc salts, colloidal silica , magnesium trisilicate, polyvinyl pyrrolidone, substances based on cellulose, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, blocking polymers, polyethylene-polyoxypropylene, polyethylene glycol, and lanolin.
[0161] 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 ethyl oleate. Aqueous vehicles include, for example, water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered medium. Pharmaceutically acceptable vehicles may 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, Ringer's lactate, or fixed oils. Intravenous vehicles include replenishing nutrients, replenishing electrolytes, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
[0162] More particularly, pharmaceutical compositions suitable for injectable use include aqueous solutions (where water-soluble) or dispersions and post-sterols for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition may be sterile and must be fluid to the extent that easy syringability exists. It must be stable in conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, glycol propylene, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining a certain particle size in the case of dispersion and by using surfactants. Formulations suitable for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).
[0163] Prevention of the action of microorganisms can be achieved by the 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 injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
[0164] In any case, sterile injectable solutions can be prepared by incorporating an active compound (for example, an anti-SEMA4D antibody, or antigen-binding fragment, variant, or derivative thereof, by itself or in combination with at least another immunomodulatory therapy) in a certain amount in an appropriate solvent with one or a combination of ingredients listed here, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation may include vacuum drying or lyophilization, which can produce a powder of an active ingredient plus any desired additional ingredient from a previously sterile filtered solution of the same. Injection preparations are processed, placed in containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. In addition, the preparations can be packaged and kept in the form of a kit. Such articles of manufacture may have labels or packaging inserted indicating that the associated compositions are useful for treating a patient suffering from, or predisposed to, a disease or disorder.
[0165] Parenteral formulations can be a single bolus dose, an infusion or a loaded bolus dose followed by a maintenance dose. These compositions can be administered at specific or variable fixed intervals, for example, once a day, or on an "as needed" basis.
[0166] Certain pharmaceutical compositions can be administered orally in an acceptable dosage form including, for example, capsules, tablets, suspensions or aqueous solutions. Certain pharmaceutical compositions can also be administered by nasal spray or inhalation. Such compositions can be prepared as saline solutions, using benzyl alcohol or other suitable preservatives, absorption enhancers to increase bioavailability, and / or other conventional solubilizing or dispersing agents.
[0167] The amount of an anti-SEMA4D binding molecule, for example, antibody, or fragment, variant, or derivative thereof, as a single agent or in combination with at least one other immunomodulatory therapy to be combined with carrier materials for producing a single dosage form will vary depending on the treated host 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. Dose regimens can also be adjusted to provide the optimal desired response (for example, a therapeutic or prophylactic response).
[0168] In keeping with the purpose of the present disclosure, anti-SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof as a single agent or in combination with at least one other immunomodulatory therapy can be administered to a human or another animal according to the aforementioned methods of treatment in an amount sufficient to produce a therapeutic effect. Anti-SEMA4D antibodies, or antigen-binding fragments, variants or derivatives thereof as a single agent or in combination with at least one other immunomodulating therapy can be administered to such a human or other animal in a conventional dosage form prepared by combining the antibody provided herein with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one skilled in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will still appreciate that a cocktail comprising one or more species of anti-SEMA4D binding molecules, for example, antibodies, or antigen binding fragments, variants, or derivatives thereof as proved herein can be used.
[0169] By "therapeutically effective dose or amount" or "effective amount" is meant an amount of anti-SEMA4D binding molecule, for example, antibody or antigen binding fragment, variant, or derivative thereof, as a single agent or in combination with at least one other immunomodulatory therapy which when administered brings a positive therapeutic response with respect to treatment of a patient with a disease to be treated, for example, an inhibition, delay, or reduction of metastasis in the patient.
[0170] Therapeutically effective doses of the compositions of the present disclosure, for inhibition, delay, or reduction of metastasis, vary depending on many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or a animal, other medications administered, and whether treatment is prophylactic or therapeutic. In certain embodiments the patient is a human, but non-human mammals including transfenational mammals can also be treated. Treatment doses can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.
[0171] The amount of anti-SEMA4D binding molecule, for example, antibody or binding fragment, variant, or derivative thereof, administered as a single agent or in combination with at least one other immunomodulatory therapy is readily determined by an expert. in the technique without undue experimentation given the disclosure of the present disclosure. Factors influencing the mode of administration and respective amount of anti-SEMA4D binding molecule, for example, antibody, antigen binding fragment, variant or derivative thereof to be administered as a single agent or in combination with at least one other immunomodulatory therapy includes, but the severity of the disease, the history of the disease, the potential for metastasis, and the age, height, weight, health, and physical condition of the individual undergoing therapy are not limited. Similarly, the amount of anti-SEMA4D binding molecule, for example, antibody, or fragment, variant, or derivative thereof, as a single agent or in combination with at least one other immunomodulatory therapy to be administered will be dependent on the mode of administration and whether the patient will undergo a single dose or multiple doses of this agent.
[0172] The disclosure also provides for the use of an anti-SEMA4D binding molecule, for example, antibody, or antibody binding fragment, variant, or derivative thereof, as a single agent or in combination with at least one other immunomodulatory therapy. in the manufacture of a drug to treat a patient with cancer, where the drug is used on a patient who has been pretreated with at least one therapy. By "pretreatment" or "pretreatment" the patient is intended to have received one or more other therapies (for example, been treated with at least one other cancer therapy) before receiving the drug comprising the anti-SEMA4D binding molecule , for example, antibody or antigen-binding fragment, variant, or derived therefrom as a single agent or in combination with at least one other immunomodulatory therapy. “Pre-treated” or “pre-treatment” includes patients who have been treated with at least one other therapy within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, within 6 weeks, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 month, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or less within 1 day prior to the start of treatment with the drug comprising the anti-SEMA4D binding molecule, for example, the monoclonal antibody VX15 / 2503 disclosed herein, or antigen binding fragment, variant, or derived from it as a single agent or in combination with at least one other immunomodulatory therapy. It is not necessary for the patient to have responded to pre-treatment with previous therapy or therapy. Then, the patient receiving the drug comprising the anti-SEMA4D binding molecule, for example, an antibody or antibody binding fragment, variant, or derivative thereof as a single agent or in combination with at least one other immunomodulatory therapy may have responded , or may have failed to respond (for example, the cancer was retractable), for pretreatment with the previous therapy, or to one or more of the previous therapies where pretreatment comprised multiple therapies. Examples of other cancer therapies for which a patient may have received pretreatment prior to receiving the drug comprising the anti-SEAM4D binding molecule, for example, antibody or antigen binding fragment, variant, or derived from it as a single agent or in combination with at least one other immunomodulatory therapy includes, but is not limited to, surgery; radiation therapy; chemotherapy, optionally in combination with autologous bone marrow transplantation, where suitable chemotherapeutic agents include, but are not limited to, those listed here above; another anti-cancer monoclonal antibody therapy; small molecule-based cancer therapy, including, but not limited to, small molecules listed here above; vaccine / immunotherapy-based cancer therapies; steroid therapy; another cancer therapy; or any combination thereof.
[0173] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiolody, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully explained 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 and collaborators. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., Eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.I., (1986); and in Ausubel and collaborators. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).
[0174] General principles of antibody construction are revealed in Bor-rebaeck, ed. (1995) Antibody Engineering (2nd ed .; Oxford Univ. Press). General principles of protein construction are revealed in Rickwood et al., Eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General principles of antibody construction and anti-body-hapten binding are disclosed 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.I.). In addition, 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 and collaborators., 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., NI).
[0175] Standard reference works containing 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, Hybrid-oma: A New Dimension in Biological Analyzes (Plenum Press, NY); Campbell (1984) "Monoclonal Antibody Technology" in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al., (Elsevere, Amsterdam); Goldsby et al., Eds. (2000) Kuby Immunnology (4th ed .; H. Freemand &Co.); Roitt and collaborators. (2001) Immunology (6th ed .; London: Mosby); Abbas and collaborators. (2005) Cellular and Molecular Immunology (5th ed .; Elsevier Health Sciences Division); Kontermann and Dubel (2001) Antibody Engineering (Springer Verlan); Sam-brook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall2003); Harlow and Lane (1988) Anti-bodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).
[0176] All references cited above, as well as all references cited here, are incorporated here by reference in their entirety.
[0177] The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES
Example 1: Test of the ability of an anti-SEMA4D antibody to delay tumor growth in immunologically competent mice [0178] Experimental Design: The basic experimental design is as follows. Colon26 tumor cells were implanted subcutaneously in the flank of immunocompetent syngeneic Balb / c mice (5χ10Λ5 cells) or immunologically deficient SCID mice (1χ10Λ5 cells) in 0.2 mL of saline. Treatment with Ig 2B8 Control or anti-SEMA4D ab 67 was started on day 2 after tumor implantation. Mice (n = 20) were treated twice a week with 1.0 mg (approximately 50 mg / kg) each of the monoclonal antibody via intraperitoneal (IP) injection. Tumors were measured with calibrators 3x / week starting 3 days after implantation. Mice were weighed 2x / week starting on day 3. Animals were sacrificed when the tumor volume reached 1000 mm3.
[0179] Anti-SEMA4D treatment delayed tumor growth in mice with competent immune system. Tumor growth was measured by calipers and measures were used to calculate the tumor volume using the formula (l2 x c) / 2, where l = width, smaller measure and l = length, in mm, of the tumor. Average tumor volume (FIG1A) and Kaplan Meier survival curves (FIG 1B), defined as time to the end point where the tumor volume = 1000 mm3, are shown in FIGS. 1A and 1B. Statistical analysis was conducted using Two-tailed Variance Analysis (ANOVA) and Log Rank analysis, respectively, which showed a statistically significant treatment effect with anti-SEMA4D antibody in Balb / c mice.
[0180] Twenty-nine percent tumor growth delay (29%) was achieved in Balb / c mice, however, no treatment related to tumor growth was observed in SCID mice. Delayed tumor growth (TGD) is defined as the increase in the mean time to end point (TTE) in a treatment group compared to the control group:% TGD - [(TC) / C] x 100, where T = mean TTE for a treatment group, C = mean TTE for the treatment group. Balb / c animals treated with anti-SEMA4D 67 antibody showed a statistically significant reduction in primary tumor volume in sacrifice time over control animals (p <0.0001). This finding shows that anti-SEMA4D anti-chlorine was effective in delaying tumor growth in mice with a competent immune system, but not in immune-deficient mice.
Example 2: Testing the ability of an anti-SEMA4D antibody to delay tumor growth in the presence of CD8 + T effector cells [0181] Experimental Design: Colon26 tumor cells were implanted subcutaneously in the flank of Balb / c mice (5x10A5 in 0.2 mL saline).
Anti-CD8 depleting antibody (Clone 2.43, BioXCell) or control rat Ig (Clone LTF-2, BioXCell) (150 mg / kg) were administered via intraperitoneal (IP) injection on days -1, 0, 1, 11 and weekly thereafter. Treatment with Ig Control 2B8 or Ab 67 anti-SEMA4D was started on day 2. Mice (n = 20) were treated twice weekly with 1.0 mg (approximately 50 mg / kg) of monoclonal antibody by intraperitoneal injection. Tumors were measured with calibrators 3x / week starting 3 days after implantation. Animals were sacrificed when the mean tumor volume of the control group reached 1000 mm3, day 30 for groups treated with rat Ig, and day 26 for groups treated with anti-CD8.
[0182] Anti-SEMA4D treatment delayed tumor growth in the presence of CD8 + T lymphocytes. Tumor volume was measured by calibrators using the formula (l2 x c) / 2, where l = width, smaller measurement and c = length, in mm, of the tumor. Statistical differences in tumor volume were determined using a two-way One-Way Analysis of Variance (ANOVA) comparing groups treated with antibodies with the control group Ig 2B8. Average tumor volume is shown in FIG. 2.
[0183] Inhibition of tumor growth was also determined. Tumor growth inhibition (TGI) was measured using the following formula:% TGI = 1 - [(Tf-Ti) / mean (Cf-Ci)]; % TGI reported is the average% TGI for each tumor treated. Statistical differences in tumor volume were determined using a two-way One-Way Analysis of Variance (ANOVA) followed by Dunnett's multiple comparison test comparing treated groups with the 2B8 control group. Thirty percent (30%) of tumor growth inhibition was achieved following treatment with anti-SEMA4D antibody, however no treatment-related effect was observed when CD8 + T cells were depleted. These results show that the inhibition of tumor growth with anti-SEMA4D was dependent on the presence of effector CD8 + T cells.
Example 3: Testing the ability of an anti-SEMA3D antibody to increase the density of Tumor Infiltrating Lymphocytes (TIL) [0184] Experimental Design: Colon26 tumor cells were implanted subcutaneously in the flank of Balb / c mice (5χ10Λ5 cells in 0, 2 mL of saline). Treatment with Ig Control 2B8 or Ab 67 anti-SEMA4D was started on day 2 (50 mg / kg IP, twice a week, n = 10), Tumors were measured with 3x / week calipers starting 3 days after implantation. Animals were sacrificed on day 27, when the average tumor volume of the control group reached 1000 mm3. Tumors, including stroma and skin, were collected and fixed in formalin for 24 hours, then transferred to 70% ethanol. Samples were then processed by incorporation into paraffin, and 5 micron sections were cut from the resulting blocks.
[0185] Adjacent sections were stained for Sema4D, CD8 and CD20 using the following methods: [0186] a. To detect Sema4D, slides were heated to 60 ° C for 1 hour, then deparaffinized and rehydrated through xylonic and graduated ethanol baths. Epitope recovery was performed by boiling for 20 min with Target Recovery Solution (Dako, Carpinteria, CA) followed by 30 min of cooling. Slides were washed twice with PBS containing 0.0% Tween-20 (TPBS), then endogenous peroxidases were inactivated with a 10 min block with Dual Blocking Enzyme (Dako, Carpinteria, CA). Slides were washed with TPBS twice, then non-specific binding was blocked by a 20 min incubation with 2.5% normal goat serum in TPBS. Following a single wash with TPBS, slides were incubated for 60 min with rabbit anti-Sema4d at 2 pg / mL in TPBS, followed by 2 washes with TPBS. Slides were then incubated for 20 min with goat anti-rabbit polymer labeled with Envision HRP (Dako, Carpinteria, CA), followed by 2 washes with TPBS and a 5 min incubation with DAB + (Dako, Carpinteria, CA). Sections were counterstained with Harris hematoxylin, discolored, bluish under running water, dehydrated, and mounted non-aqueous with Permount.
[0187] b. CD8 was detected using the method above, but using a commercial rabbit polygonal antibody (Abbiotec) at 2 pg / ml.
[0188] c. CD20 was detected using the above method, but using normal donkey serum for blocking, and using a primary goat anti-CD20 antibody (Santa Cruz) at 1 pg / mL followed by a 20 min incubation with anti-goat antibody labeled with HRP (Golden Bridge).
[0189] d. Slides were photographed at a 20x magnification using a Retiga WICAM-12 bit camera attached to an Olympus Ix50 microscope.
[0190] Anti-SEM4D treatment increased the frequency of tumor-filtering immune cells (TIL). Immune cell density was measured by scanning sections of the entire tumor, quantifying areas of Tumor Infiltrating Lymphocytes CD8 + or CD20 + (TIL), and then normalizing the total area of the tumor. Sections from 9 (Ig Control) or 10 (Ab 67 anti-SEMA4D) mice per group were used for analysis. Statistical significance was calculated for CD8 and CD20 using two-tailed unpaired T test for 95% CI.
[0191] Treatment of Colon26 tumors with anti-SEMA4D antibody 67 resulted in an increase in both CD8 + T cell density and CD20 + Y cell density, as compared to the control group. The increase in density of CD20 + T cells was statistically significant at 95% with a value of 0.0388. The increase in the density of CD8 + T cells showed a trend, but not statistical significance. These findings show that anti-SEMA4D treatment of Colon26 tumors resulted in an increased frequency of immune cells infiltrating the tumor. The results are shown graphically in FIGS. 3A and 3B.
[0192] Example 4. Testing the ability of an anti-SEMA4D antibody to affect migration and distribution of subgroups of M1 and M2 macrophages and CD8 + T cells at the frontal ends of the tumor.
[0193] Anti-SEMA4d treatment altered macrophage distribution and CD8 + T cells in the frontal extremity of the tumor. Macrophage distribution was measured by scanning sections of the entire tumor, quantifying the area of M1 (staining with Alexa67 conjugated with mouse anti-F4 / 80 (Biolegend BM8 clone) at 2 pg / mL) and M2 (staining with biotin conjugated with anti-mouse CD206 (Biolegend, clone C068C2) at 2 pg / ml), and then normalized to the total tumor area to determine density of M1 and M2 within the tumor. Sections of 9 (Ig Control) or 10 (Ab 67 anti-SEMA4D) mice treated by group were used for analysis. To determine cell density in frontal tumor growth, a 300 pixel wide (250 micron) region was defined from the tumor tip. Statistical significance for M1 and M2 was calculated using One-Way ANOVA with Dunn's post-test for 95% CI. Change in density of normalized M1 macrophage to the frontal end was significant.
[0194] CD8 + T cell numbers were measured in the entire tumor sections stained with CD8 antibody (Abbiotec Cat # 250596 at 1: 250) and DAB detection system. The number of CD8 + events in the entire tumor sections was enumerated after a positive signal threshold using the Imagepro Program. CD8 + density for each animal was calculated by dividing the number of CD8 + events by pixel area of entire tumor. Individual CD8 density averages were taken to arrive at the CD8 + T cell distribution in animals treated with 2E8 and mAb67 (n = 10). Statistical significance was calculated using One-Way ANOVA with Kruskal-Wallace and Dunn post-test for 95% CI.
[0195] SEMA4D distribution was measured by scanning sections of the entire tumor stained for SEMA4D with an antibody to a distinct epitope from that recognized by Ab 67 and analysis for Sema4D distribution. Sections of 9 (Control Ig) or 10 (Ab 67 anti-SEMA4D) of mice treated per group were used for analysis.
[0196] Colon26 tumor cells expressed low levels of SEMA4D when cultured in vitro, but SEMA4D upregulated in vivo at the front end of the tumor. This leads to the establishment of a gradient of expression of SEMA4D with high concentration in the periphery of the tumor. Treatment with anti-SEMA4D antibody neutralized SEMA4D and broke the expression gradient. This resulted in a noticeable change in macrophage migration and distribution, as shown in FIG. 4A. In particular, tumors treated with anti-SEMA4D Ab 67 had higher levels of pro-inflammatory M1 + macrophages at the front end of the tumor as shown in FIG. 4B. The increase in the M1 + macrophage was statistically significant. Tumors treated with anti-SEMA4D Ab 67 also showed a decrease in the frequency of the M2 pro-tumor macrophage at the front end of the tumor as shown in FIG. 4C. These findings showed that treatment with anti-SEMA4D Ab 67 altered distribution and macrophages in a way that increased the density of tumor-inhibiting macrophage, i.e., M1, at the front end of the tumor while decreasing the presence of the tumor-promoting macrophage, i.e. , M2, in the same region. In addition, these findings showed an overall increase in CD8 + T cell density within tumors isolated from MAb 67-treated mice, as shown in FIG. 4D. These discoveries suggest that neutralization of SEMA4D with MAb 67-2 facilitates the entrapment of anti-tumor M1 macrophage in the zone of high proliferation of tumor cells and CD8 + T cells through the zone and extension at the front end (insertion).
Example 5: Test of the ability of an anti-SEMA4D antibody to delay tumor growth in mice when used in combination with anti-CTLA4 antibodies [0197] Experimental Design: Colon26 5x105 tumor cells were implanted subcutaneously in the flank of female Balb / c mice. Treatment with IgG1 / 2B8 from control mice or anti-SEMA4D / MAb67-2 was started 1 day post-inoculation (50 mg / kg, IP, weekly x5), with or without anti-CTLA4 / MAb UC10-4F10-11 (100 pg on days 8 and 50 pg on days 11 and 14 post tumor inoculation). Treatment with anti-PD1 / RPM1-14 was started 1 day after inoculation (100 pg on day 3, twice a week) in combination with anti-CTLA4 / MAb UC10-4F10-11. There were 20 mice per group. Tumors were measured with calibrators 2x / week starting 5 days post-implant. Animals were sacrificed when the tumor volume reached 1000 mm3.
[0198] Combination of anti-SEMA4D and anti-CTLA4 antibodies delayed tumor growth in mice. Tumor growth was measured by calibrators and measures were used to calculate tumor volume using the formula (l2 x c) / 2, where l = width, smallest measure and c = length, in mm, of the tumor. Average tumor volume and Kaplan Meier survival curves, defined as end point time where tumor volume = 1000 mm3, are shown in FIGS. 5A and 5B, respectively. Statistical analysis was conducted using Two-tailed Analysis of Variance (ANOVA) and Log Rank analysis, respectively, which showed a statistically significant treatment effect with anti-SEMA4D antibody (9% Tumor Growth Delay, TGD **) and anti-CTLA4 antibody (2% TGD, ns), and a highly significant increase in tumor growth delay with the combination of anti-SEMA4D and anti-CTLA4 antibodies (maximum TGD, 114% ****). The responses were durable for at least 60 days.
[0199] The frequency of tumor regression in the Colon26 tumor model was also determined. Regression is the absence of a palpable tumor, defined by a tumor measurement <50 mm3 for at least two consecutive measurements. As shown in FIG. 5C, combination of anti-SEMA4D and anti-CTLA4 antibodies increases the number of regressions in the Colon26 tumor model. Regressions to combination therapy (anti-SEMA4D + antiCTLA4 antibodies) are statistically significant compared to Ig Control (p <0.0001) and compared to anti-CTLA4 or anti-SEMA4D monotherapies (p = 0.0022), as determined Fisher's exact test. Importantly, these findings show that the combination of anti-SEMA4D and anti-CTLA4 antibodies was synergistic: that is, the combination was significantly more effective, resulting in an increased frequency of durable tumor regressions, so treatment with anti-SEMA4D antibody alone or with antibody anti-CTLA4 alone. In addition, these results demonstrate that the combination of anti-SEMA4D and anti-CTLA4 antibodies is at least as effective as, or better than, the combination of anti-PD1 and anti-CTLA4.
[0200] It was further determined that treatment with anti-SEMA4D antibody increases tumor-reactive CTL activity, and also increases anti-CTLA4-mediated CTL activity. A follow-up of the study was conducted to examine the effect of anti-CTLA monotherapy, compared to combination anti-CTLA4 and anti-SEMA4D therapy on the frequency of tumor-specific infiltrating leukocytes (TIL) and secretion of pro-inflammatory cytokines. In this follow-up study, immune cells were isolated from tumors and spleens of mice with Colon26 tumor treated in vivo with IgG1 / MAb2b8 Control, anti-CTLA4 / MabUC10-4F10, or the combination of anti-CTLA4 / MabUC10-4F10 and anti -SEMA4D / MAb67. Tissues were collected on day 15, day 1 post-final dose of anti-CTLA4 antibody and just before tumor regression. Total CD45 + TIL was assessed for secreted cytokine levels, and frequency of IFNg-secreting CD8 + T cells in the presence of MHC-I restricted specific Colon26 tumor-specific g70 peptide was determined by ELISPOT. The frequency of specific MHCI responders was calculated by subtracting the control mean from wells containing peptide.
[0201] As shown in FIG. 5D, increased levels of proinflammatory cytokine IFNg was observed in tumors of mice treated with anti-CTLA4 antibody monotherapy (p = 0.0135), which was further and significantly increased following treatment with anti-CTLA4 combination therapy and anti-SEMA4D (p = 0.0002 compared to control or monotherapy). In FIG. 5E, increased frequency of peptide-specific IFNg secreting responders was observed in the spleens of mice treated with anti-CTLA4 antibody. This discovery was expected because anti-CTLA4 is reported to induce T cell activation in the periphery. Combination therapy of anti-CTLA4 and anti-SEAM4D has not been found for yet increased activity in the spleen. In contrast, a substantial increase in the frequency of responders secreting peptide-specific IFNg was observed in TIL following treatment with anti-CTLA4 monotherapy, which was further and significantly increased following treatment with anti-CTLA4 and anti-SEMA4D combination therapy. This finding suggests that the addition of anti-SEMA4D treatment may significantly improve tumor-specific CD8 + T cell activity in a specific localized tumor form.
Example 6: Testing the ability of an anti-SEMA4D antibody to affect tumor infiltration of tumor-specific cytotoxic CD8 + T cells [0202] Treatment with MAb 67-2 increases the frequency of tumor-specific TIL and secretion of pro-inflammatory cytokines. Following four weeks of anti-SEMA4D treatment in vivo, tumors were dissociated and enriched for CD45 + cells by magnetic separation. CD45 + TIL, grouped of 5 mice, were incubated in the presence and absence of immunodominant tumor peptide, AH-1 at various cell densities. IFNy secreting cells were measured by ELISPOT; specific response to peptide was determined by removing the average of wells without peptide. Each sample was tested in replicates of 6 and is plated above. Statistical significance was determined using the Mann-Whitney non-parametric T test.
[0203] An increase in IFNy secreting cells was observed in mice treated with MAb 67 both, in the presence and absence of the peptide, as shown in FIG. 6A. CD45 + TIL, especially MHC-I restricted peptide-specific CD8 + cytotoxic T cells, represents active effector cells following treatment with MAb 67-2. FIG. 6B shows representative ELISPOT images. CD45 + TIL were then cultured ex vivo for 48 hours and evaluated by cytokine secretion using CBA analysis. As shown in FIG. 6C, MAb 67-2 promotes secretion of anti-tumor cytokines, such as IFNy and TNFa, in TIL. Statistical significance was determined using the Mann-Whitney non-parametric T test.
[0204] A follow-up study was conducted to examine the effect of MAb 67-2 treatment on the frequency of tumor-specific infiltrating leukocytes (TIL) for tumor and secretion and pro-inflammatory cytokines. In this follow-up study, immune cells were isolated from mouse tumors having Colon26 tumor treated in vivo with IgG1 / MAb2B8 control or anti-SEMA4D / MAb67. Total CD45 + TIL was assessed for levels of secreted cytokine, and frequency of CD8 + T cells secreting IFNg in the presence of MHC-I-restricted Colon 26 tumor-specific gp70 peptide was determined by ELISPOT. The frequency of specific MHCI responders was calculated by subtracting the control mean from wells containing peptide.
[0205] As shown in FIG. 6D, increased levels of pro-inflammatory cytokines IFNg and TNFa were observed in TIL of mice treated with anti-SEMA4D antibody. In addition, as shown in FIG. 6E, increased frequency of responders secreting peptide-specific IFNg was observed in TIL of mice treated with anti-SEMA4D antibody. This finding suggests that the addition of anti-SEMA4D treatment can significantly improve tumor-specific CD8 + T cell activity in a specific form for localized tumor.
[0206] Example 7: Testing the ability of an anti-SEMA4D antibody to delay tumor growth in mice when used in combination with anti-PD1 antibodies.
[0207] Experimental Design: 5x105 Colon26 tumor cells were implanted subcutaneously in the flank of female Balb / c mice. Treatment with IgG1 / 2B8 from control mice or anti-SEMA4D / MAb 67-2 was started 1 day post-inoculation (50 mg / kg, IP, weekly). Each group of mice was also treated with either control rat Ig or rat anti-PD1 / MAbRMP1-14 (100 pg, twice a week, X 2 weeks starting at 3 days post tumor inoculation). They had 20 mice per group. Tumors were measured with calibrators 3x / week starting 5 days after implantation. Animals were sacrificed when the tumor volume reached 1000 mm3.
[0208] Combination of anti-SEMA4D and anti-PD1 antibodies delayed tumor growth in mice. Tumor growth was measured by calibrators and measures were used to calculate tumor volume using the formula (l2 x c) / 2, where l = width, smallest measure, and c = length, in mm, of the tumor. Average tumor volume and Kaplan Meier survival curves, defined with time to end point where tumor volume = 1000 mm3, are shown in FIGS. 7A and 7B, respectively. Statistical analysis was conducted using Bicau-dal Analysis of Variance (ANOVA) and Log Rank analysis, respectively, which showed a statistically significant treatment effect with anti-SEMA4D antibody combined with anti-PD1 antibodies in Balb / c mice. These findings show that the combination of anti-SEMA4D and anti-PD1 antibodies was more effective than treatment with anti-SEMA4D antibody or with anti-PD1 alone.
[0209] The frequency of regressions in the Colon26 tumor model was also measured and is shown in FIGS. 7C and 7D. Regression is the absence of a palpable tumor, defined by a tumor measurement <50 mm3 for at least two consecutive measurements. Combination of anti-SEMA4D and anti-PD1 antibodies increases the number of regressions in the Colon26 tumor model. Regressions to combination therapy (aSEMA4D + α PD1 antibodies) are statistically significant compared to aSEMA4D + α PD1 to Ig control (p = 0.0083) or a single anti-PD-1 agent (p = 0.02), as determined by Fisher's exact test.
Example 8: Test of the ability of an anti-SEMA4D antibody to delay tumor growth in mice when used in combination with cyclophosphamide [0210] Experimental Design. Colon26 5x105 tumor cells were implanted subcutaneously in the flank of Balb / c mice. Treatment with IgG1 / 2B8 from control mice or anti-SEMA4D / MAb 67-2 was started 1 day post-inoculation (50 mg / kg, IP, weekly). Treatment with cyclophosphamide (50 mg / kg, IP) was administered on days 12 and 20. They had 20 mice per group. Tumors were measured with calibrators 3x / week starting 5 days after implantation. Animals were sacrificed when the tumor volume reached 1000 mm3.
[0211] Combination of anti-SEMA4D antibodies and cyclophosphamide delayed tumor growth in mice. Tumor growth was measured by calipers and measures were used to calculate tumor volume using the formula (l2 x c) / 2, where l = width, smaller measurement, and c = length, in mm, of the tumor. Average tumor volume, median tumor volume, and Kaplan Meier survival curves, defined with time to end point where tumor volume = 1000 mm3, are shown in FIGS. 8â, 8B and 8C, respectively. Statistical analysis was conducted using Two-tailed Analysis of Variance (ANOVA) and Log Rank analysis, respectively, which showed a statistically significant treatment effect with anti-SEMA4D antibody combined with cyclophosphamide in Balb / c mice.
[0212] Specifically, the findings show a 232% Tumor Growth Delay (TGD) when anti-SEMA4D antibodies were used in combination with cyclophosphamide. This finding was statistically significant compared to Ig Control (p <0.0001), as determined by Mantel Cox Log Rank analysis. There was also a 3% TGD when treatment with anti-SEMA4D antibody was used alone (statistically significant compared to the control Ig (p = 0.0282)) and a 96% TGD when treatment with cyclophosphamide was used alone (statistically significant compared to Control Ig (p <0.0001), as determined by Mantel Cox Log Rank analysis. Responses were durable for at least 81 days. These findings show that the combination of anti-SEMA4D antibodies and cyclophosphamide was more effective in delaying tumor growth than treatment with anti-SEMA4D antibody or cyclophosphamide alone.
[0213] The frequency of regression in the Colo26 tumor model was also measured and shown in FIGS. 8D and 8E. Regression is the absence of a palpable tumor, defined by a tumor measurement <50 mm3 for at least two consecutive measurements. Combination of anti-SEMA4D antibodies and cyclophosphamide increases the number of regressions in the Colon26 tumor model. Regressions to combination therapy (aSEMA4D antibodies + cyclophosphamide) are statistically significant compared to control Ig (p <0.003), as determined by Fisher's exact test. These data demonstrate increased efficacy and response to treatment with cyclophosphamide when combined with anti-SEMA4D anti-chlorine.
Example 9: Testing the ability of an anti-SEMA4D anti-chlorine to delay tumor growth in mice when used in combination with anti-HER2 / neu antibodies [0214] Experimental Design. Tubo.A5 3x104 tumor cells were implanted subcutaneously in the mammary fat of female Balb / c mice. Treatment with IgG1 / 2B8 from control mice or anti-SEMA4D / MAb 67-2 was started 7 days post-inoculation (50 mg / kg, IP, weekly x6). Treatment with anti-Neu / MAb7.16.4 (200 pg Ip weeklyX2, started when tumor volume was approximately 200 mm3, on days 21 and 28) There were 15 mice per group. Tumors were measured with calibrators 2x / week starting 11 days after implantation. Animals were sacrificed when the tumor volume reached 800 mm3.
[0215] Combination of anti-SEMA4D and anti-HER2 / Neu antibodies delayed tumor growth in mice. Tumor growth was measured by calipers and measures were used to calculate tumor volume using the formula (l2 x c) / 2, where l = width, smaller measurement, and c = length, in mm, of the tumor. Average tumor volume, and Kaplan Meier survival curves, defined with time to end point where tumor volume = 800 mm3, are shown in FIGS. 9â and 9B, respectively. Statistical analysis was conducted using Two-tailed Analysis of Variance (ANOVA) and Log Rank analysis, respectively, which showed a statistically significant treatment effect with anti-SEMA4D antibody combined with anti-Her2 / Neu antibody in Balb / c mice. The findings show Tumor Growth Delay of 48% when the anti-SEMA4D antibody is used in combination with anti-Neu antibody and which is statistically significant compared to the use of an irrelevant control antibody (p = 0.017) or anti-Neu monotherapy ( p = 0.006), as determined by Mantel Cox Log Rank analysis.
[0216] The frequency of tumor regressions in the Tubo tumor model was also measured and is shown in FIG. 9C. Regression is the absence of a palpable tumor, defined as a measurable tumor <50 mm3 by at least two consecutive measurements. Combination of anti-SEMA4D and anti-Neu antibodies increases the number of regressions in mice having a tube. Regressions to combination therapy (aSEMA4D + aNeu antibodies) are statistically significant for Ig Control (p = 0.016), as determined by Fisher's Exact test.
[0217] Example 10: Testing the ability of an anti-SEMA4D antibody to delay the growth of a breast carcinoma model in vivo [0218] Experimental Design. Tubo.A5 3x104 tumor cells were implanted subcutaneously in the mammary fat of female Balb / c mice. Treatment with IgG1 / 2B8 from control mice or anti-SEMA4D / MAb 67-2 was started 6 days post-inoculation (50 mg / kg, IP, weekly x6). They had 20 mice per group, however, some mice were excluded from the analysis due to premature death before reaching the end point resulting from the ulceration or general health problem. Tumors were measured with calibrators 2x / week starting 13 days after implantation. Animals were sacrificed when the tumor volume reached 800 mm3.
[0219] Treatment with anti-SEMA4D antibodies delayed tumor growth in mice. Tumor growth was measured by calibrators and measures were used to calculate tumor volume using the formula (l2 x c) / 2, where l = width, smallest measure, and c = length, in mm, of the tumor. Average tumor volume, and Kaplan Meier survival curves, defined with time to end point where tumor volume = 800 mm3, are shown in FIGS. 10A and 10 B, respectively. Statistical analysis was conducted using Two-tailed Analysis of Variance (ANOVA) and Log Rank analysis, respectively, which showed a statistically significant treatment effect with anti-SEMA4D antibody. The findings show Tumor Growth Delay (133%) with anti-SEMA4D antibody treatment; this is statistically significant compared to using an irrelevant control antibody (p <0.0001), as determined by Mantel Cox Log Rank analysis.
[0220] The frequency of tumor regressions in the Tubo.A5 tumor model was also measured and is shown in FIG. 10C-10E. Regression is the absence of a palpable tumor, defined as a measurable tumor <50 mm3 by at least two consecutive measurements. In the 90 days post-implantation, 85% (12/14) of MAb67-treated mice were tumor-free regressors and one of the 14 never developed a measurable tumor, compared to 0/14 regressions in the mice treated with Ig control. On day 90, mice that had complete rejection of their primary tumors (13/14 of mice treated with MAb67) were challenged with viable Tu bo.A5 (30,000) on the contralateral side; Naive mice were included as controls for grafting. As shown in FIG. 10D, all 13 mice that were treated with anti-SEMA4D rejected challenge with subsequent tumor, suggesting an immune memory response, in contrast to naive mice that did not reject the tumor challenge as shown in FIG. 10E. The regression frequency is statistically significant compared to the control Ig (p <0.0001), as determined by Fisher's exact test.
Example 11: Effect of anti-SEMA4D antibody on T cell and MDSC infiltration in the Tubo.A5 tumor models.
[0221] Experimental Design. Tubo.A5 tumor cells were implanted in singenene Balb / c mice. Treatment with control Ig or anti-SEMA4D MAb 67 was started on day 6 (50 mg / kg, IP, weekly). Tumors were collected on day 39, just before the tumor regressed. FACS was performed on lymphocytic cell fractions grouped from tumors of 14-21 mice / group. Average of replicate assays is shown; Significance was determined using the two-tailed T test.
[0222] As shown in FIGS. 11A and 11B, anti-SEMA4D antibody therapy increases CD3 + T cell infiltration and decreases CD11b + Gr1 + MDSC in mouse tumors treated with anti-SEMA4D. These data suggest an increase in anti-tumorigenic T cell response and a decrease in immunosuppressive cells, such as MDSC. These data are consistent with the modulation of the immune balance observed in the Colon model26.
Example 12: Dose titration of MAb67 in Tumor Models Tubo.A5 and Colon 26 [0223] Experimental Design for Tumor Model Tubo.A5. 3x104 Tubo.A5 tumor cells were implanted subcutaneously in the breast fat of female Balb / c mice. Treatment with IgG1 / 2B8.1 E7 from control mice (50 mg / kg, IP, weekly, x6) or anti-SEMA4D / MAb 67-2 (1, 10 or 50 mg / kg, IP, weekly x6) was started 6 days post-inoculation. They had 20 mice per group, however, some mice were excluded from the analysis due to premature death before reaching the end point resulting from ulceration or general health problems. Tumors were measured with calibrators 2x / week starting 13 days after implantation. Animals were sacrificed when the tumor volume reached 800 mm3.
[0224] Experimental Design for Colon 26 Tumor Model. 5x105 Colon26 tumor cells were implanted subcutaneously in the flank of female Balb / c mice. Treatment with IgG1 / 2B8.1E7 from control mice (50 mg / kg, IP, weekly, x6) or anti-SEMA4D / MAb 67-2 (1, 10 or 50 mg / kg, IP, weekly x5) was started 1 day post-inoculation, with or without anti-CTLA4-MAb UC10-4F10-11 (100 pg ~ 5 mg / kg on day 8, and 50 pg ~ 2.5 mg / kg on days 11 and 14 post tumor inoculation ). They had 15 mice per group. Tumors were measured with calibrators 2x / week starting 5 days after implantation. Animals were sacrificed when the tumor volume reached> 1000 mm3.
[0225] Minimum Effective Dose is Approximately 3 mg / kg. Tubo.A5 tumor treatment with 50 or 10 mg / kg MAb67 resulted in delayed tumor growth that was statistically significant compared to control IgG (p <0.0001 and p = 0.0015, respectively), but not significantly different from of another. Regression frequencies of 38% (9/24) and 54% (6/13) in the Tubo.A5 tumor treated with 50 or 10 mg / kg Mab67 were also significantly significant (p = 0.0069 and p = 0.0014). In contrast, 1 mg / kg MAb67 was ineffective and did not significantly delay tumor growth (p = 0.01441). In this model, the minimum effective dose was determined to be between 1 to 10 mg / kg. Tumor growth was measured by calibrators and measures were used to calculate the tumor volume using the formula (l2 x c) / 2, where l = width, smallest measure, and c = length, in mm, of the tu mor. Average tumor volume and Kaplan Meier survival curves, defined as end point time where tumor volume = 800 mm3, are shown in FIGS. 12A and 12B, respectively. Statistical analysis was conducted using two-tailed analysis of variance (ANOVA) and log rank analysis, respectively.
[0226] Further refinement of the effective MAb67 dose was investigated in the Colon26 model and was determined to be> 3 mg / kg. Treatment of Colon26 tumors with anti-CTLA4 + anti-SEMA4D resulted in maximum tumor growth delay (119%) compared to anti-CTLA4 monotherapy when anti-SEMA4D / MAb 67 doses were> 3 mg / kg; at 10 mg / kg MAb67, p = 0.0101 and at 3 mg / kg, p = 0.571, as compared to anti-CTLA4 monotherapy and determined using Mantel Cos Log Rank analysis. All doses between 3-50 mg / kg were not significantly different than one other. In cotraste, when anti-CTLA4 was administered in combination with 0.3 mg / kg MAb67, the difference was statistically different than treatment with 10 mg / kg MAb 67 (p = 0.0325) but was statistically significant compared to treatment with monotherapy anti-CTLA4 (p = 0.4945). Tumor growth was measured by calibrators and measures were used to calculate tumor volume using the formula (l2 x c) / 2, where l = width, smallest measure, and c = length, in mm, of the tumor. Average tumor volume and Kaplan Meier survival curves, defined as end point time where tumor volume = 1000 mm3, are shown in FIGS. 12C and 12D, respectively. Statistical analysis was conducted using two-tailed analysis of variance (ANOVA) and log rank analysis, respectively.
Example 13: Effect of anti-SEMA4D antibody on tumor growth delay in Colon26 and Tubo.A5 Tumor Models. [0227] Experimental Design. FIG. 13 is a summary of experiments conducted in the Examples above showing tumor regressions and growth after a new tumor challenge in the Colon26 and Tubo.A5 tumor models. The experimental design of the respective experiments is summarized in the Examples above.
[0228] Anti-SEMA4D antibody therapy results in complete and durable tumor regression. As shown in FIG. 13, treatment with anti-SEMA4D antibody therapy results in a statistically significant increase in tumor regression when compared to treatment with IgG1 in control mice in both Colon26 and Tubo.A5 models, 7% (P <0.001 ***) and 85 % (P <0.0001 ****), respectively. In addition, treatment with anti-SEMA4D antibody therapy is not significantly different than treatment with anti-PD1 alone (7% with anti-SEMA4D alone, vs. 8% with anti-PD1 alone, ns), but it is significantly increased used in combination with anti-PD1 therapy (28% for combination therapy vs. 7% for anti-SEMA4D or 8% for anti-PD1 monotherapy, P <0.0001 ****). In addition, treatment with anti-SEMA4D antibody therapy in combination with anti-CTLA4 therapy results in a statistically significant increase in tumor regression when compared to treatment with anti-CTLA4 alone (74% for combination therapy vs. 20% for anti monotherapy) -CTLA4, P <0.0001 ****). In addition, treatment with anti-SEMA4D antibody therapy in combination with anti-CTLA4 therapy results in a statistically significant increase in tumor regression when compared to treatment with anti-SEMA4D in combination with anti-PD1 (74% for anti-SEMA4D combination therapy / anti-CTLA4 vs. 60% for anti-SEMA4D / anti-PD1 combination therapy, P <0.001 ****). The greater apparent synergy between anti-SEMA4D in combination with anti-CTLA4 as compared to ati-SEMA4D in combination with anti-PD1 indicates that not all immune checkpoint blocking inhibitors are equivalent in this concept and that differences in the mechanism can be associated with differential therapeutic benefit. Finally, treatment with anti-SEMA4D in combination with cyclophosphamide results in a statistically significant increase in tumor regression when compared to treatment with cyclophosphamide alone (40% for combination therapy vs. 10% for cyclophosphamide monotherapy, P <0.01 ** ).
[0229] Many modifications and other modalities of the modalities disclosed here will come to mind to a person versed in the technique to which this revelation belongs, having the benefit of the teachings presented in the aforementioned descriptions and the associated drawings. Therefore, it is to be understood that the disclosure is not limited to the specific modalities disclosed and that modifications and other modalities are intended to be included within the scope of the appended claims and list of modalities disclosed herein. Although specific terms are used here, they are used in a generic and descriptive sense only and not for purposes of limitation.

权利要求:
Claims (9)
[1]
1. Use of an isolated antibody or antigen-binding fragment that specifically binds to semaphorin-4D (SEMA4D) in combination with at least one other immunomodulatory therapy CHARACTERIZED by the fact that it is for the manufacture of a drug to inhibit delay, or reduce solid tumor growth in an individual with cancer, where the anti-SEMA4D antibody or fragment thereof: (a) comprises a variable heavy chain (VH) comprising VHCDRs 1 to 3 defined by SEQ ID NOs: 6, 7 and 8, respectively, and a variable light chain (VL) comprising VLCDRs 1 to 3 defined by SEQ ID NOs: 14, 15 and 16, respectively; or (b) comprises a VH and VL defined by, respectively, SEQ ID NO: 9 and SEQ ID NO: 17, or SEQ ID NO: 10 and SEQ ID NO: 18; and wherein at least one other immunomodulatory therapy comprises an immunological checkpoint blocking inhibitor comprising a protein 4 antibody associated with anti-cytotoxic T lymphocytes (CTLA4), an anti-programmed cell death antibody (PD- 1), an anti-programmed 1-binding death antibody (PD-L1), an anti-lymphocyte gene 3 activation antibody (LAG3), an anti-B7-H3 antibody, an antigen binding fragment thereof, or a combination thereof; or the regulatory T cell modulator (Treg) comprises cyclophosphamide; and wherein the combination with at least one other immunomodulatory therapy results in improved therapeutic efficacy relative to the antibody or fragment thereof or the immonomodulatory therapy alone.
[2]
2. Use, according to claim 1, CHARACTERIZED by the fact that the anti-SEMA4D antibody or fragment thereof and at least one immunomodulatory therapy is: (a) formulated to be administered separately; or (b) formulated to be administered concurrently.
[3]
3. Use, according to claim 1 or 2, CHARACTERIZED by the fact that the individual has high levels of B cells, T cells or both B cells and T cells in the circulation when compared to other patients with solid tumors.
[4]
4. Use according to claim 3, CHARACTERIZED by the fact that: (a) the level of B cells and / or T cells per microliter of blood in an individual is 1.5 to 5 times the average number of B cells and / or T cells in the circulation in other patients with solid tumors; and / or (b) the individual has levels of B cells and / or T cells that lie within or above the range of B cells and / or T cells of healthy individuals.
[5]
5. Use according to any of claims 1 to 4, CHARACTERIZED by the fact that the anti-SEMA4D antibody or fragment thereof inhibits SEMA4D interaction with its receptor, in which the receptor is Plexin-B1, Plexin-B2 or CD72.
[6]
6. Use according to any one of claims 1 to 5, CHARACTERIZED by the fact that the anti-SEMA4D antibody or fragment thereof inhibits SEMA4D-mediated Plexin-B1 signal transduction.
[7]
7. Use according to any one of claims 1 to 6, CHARACTERIZED by the fact that the cancer is carcinoma, lymphoma, blastoma, sar-coma, squamous cell cancer, small cell lung cancer, non-small cell lung cancer , lung adenocarcinoma, lung squamous carcinoma, peritoneum cancer, hepatocellular cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, cancer breast, colon cancer, colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, or a combination thereof.
[8]
8. Use, according to claim 7, CHARACTERIZED by the fact that the cancer expresses Her2 and Plexin B1 or Plexin B2.
[9]
9. Use according to any one of claims 1 to 8, CHARACTERIZED by the fact that cancer is a solid tumor.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

NZ201918A|1981-09-18|1987-04-30|Genentech Inc|N-terminal methionyl analogues of bovine growth hormone|

---

US4683195B1|1986-01-30|1990-11-27|Cetus Corp|

---

US5225539A|1986-03-27|1993-07-06|Medical Research Council|Recombinant altered antibodies and methods of making altered antibodies|

---

US4873192A|1987-02-17|1989-10-10|The United States Of America As Represented By The Department Of Health And Human Services|Process for site specific mutagenesis without phenotypic selection|

---

US5892019A|1987-07-15|1999-04-06|The United States Of America, As Represented By The Department Of Health And Human Services|Production of a single-gene-encoded immunoglobulin|

---

US5070192A|1988-03-23|1991-12-03|The Johns Hopkins University|Cloned human topoisomerase i: cdna expression, and use for autoantibody detection|

---

US5530101A|1988-12-28|1996-06-25|Protein Design Labs, Inc.|Humanized immunoglobulins|

---

US5859205A|1989-12-21|1999-01-12|Celltech Limited|Humanised antibodies|

---

AU633698B2|1990-01-12|1993-02-04|Amgen Fremont Inc.|Generation of xenogeneic antibodies|

---

FR2686087B1|1992-01-13|1994-04-22|Institut Nal Sante Recherc Medic|

---

US5639856A|1993-09-13|1997-06-17|The Regents Of The University Of California|Semaphorin gene family|

---

US5595756A|1993-12-22|1997-01-21|Inex Pharmaceuticals Corporation|Liposomal compositions for enhanced retention of bioactive agents|

---

US6576754B2|1995-11-09|2003-06-10|Dana-Farber Cancer Institute|CD100 antigen and uses therefor|

---

EP1325932B9|1997-04-07|2006-07-19|Genentech, Inc.|Anti-vegf antibodies|

---

US6884879B1|1997-04-07|2005-04-26|Genentech, Inc.|Anti-VEGF antibodies|

---

AU6897998A|1997-04-11|1998-11-11|Dendreon Corporation|Composition and method for inducing an immune response against tumour-rela ted antigens|

---

EP0892047A3|1997-07-09|2000-03-08|Hoechst Marion Roussel Deutschland GmbH|Human and murine semaphorin L|

---

US20020032315A1|1997-08-06|2002-03-14|Manuel Baca|Anti-vegf antibodies|

---

CA2350207C|1998-11-10|2011-02-08|University Of Rochester|T cells specific for target antigens and methods and vaccines based thereon|

---

US6737056B1|1999-01-15|2004-05-18|Genentech, Inc.|Polypeptide variants with altered effector function|

---

US6849425B1|1999-10-14|2005-02-01|Ixsys, Inc.|Methods of optimizing antibody variable region binding affinity|

---

JP3787473B2|1999-11-30|2006-06-21|独立行政法人科学技術振興機構|Semaphorin receptor|

---

US6635742B1|2000-01-25|2003-10-21|Nuvelo, Inc.|Antibodies specific for semaphorin-like polypeptides|

---

WO2002102854A2|2001-06-20|2002-12-27|Morphosys Ag|Antibodies that block receptor protein tyrosine kinase activation, methods of screening for and uses thereof|

---

EP1365018A1|2002-05-23|2003-11-26|Institut National De La Sante Et De La Recherche Medicale |CD100 semaphorin in myelination|

---

CA2487932A1|2002-05-30|2003-12-11|Macrogenics, Inc.|Cd16a binding proteins and use for the treatment of immune disorders|

---

US9809654B2|2002-09-27|2017-11-07|Vaccinex, Inc.|Targeted CD1d molecules|

---

US20040132101A1|2002-09-27|2004-07-08|Xencor|Optimized Fc variants and methods for their generation|

---

EP1442749A1|2003-01-31|2004-08-04|Institut National De La Sante Et De La Recherche Medicale |Use of anti-CD100 antibodies for the treatment and the diagnosis of inflammatory disorder affecting the central nervous system|

---

CA2526085A1|2003-05-30|2005-01-06|Genentech, Inc.|Treatment with anti-vegf antibodies|

---

EP1708751B1|2003-12-04|2011-09-28|Vaccinex, Inc.|Methods of killing tumor cells by targeting internal antigens exposed on apoptotic tumor cells|

---

US20050233960A1|2003-12-11|2005-10-20|Genentech, Inc.|Methods and compositions for inhibiting c-met dimerization and activation|

---

DK1773885T3|2004-08-05|2010-08-16|Genentech Inc|Humanized anti-c-met antagonists|

---

US20060147449A1|2004-11-15|2006-07-06|Brass Lawrence F|Method of using CD100 to mediate platelet activation and inflammatory responses|

---

US7919246B2|2005-04-07|2011-04-05|Novartis Vaccines And Diagnostics, Inc.|SEMA4D in cancer diagnosis, detection and treatment|

---

US9296816B2|2005-04-15|2016-03-29|Macrogenics, Inc.|Covalent diabodies and uses thereof|

---

JP2007308465A|2006-05-15|2007-11-29|Boehringer Ingelheim Internatl Gmbh|Method for treatment of inflammatory disease, autoimmune disease or bone resorption abnormality|

---

US9382327B2|2006-10-10|2016-07-05|Vaccinex, Inc.|Anti-CD20 antibodies and methods of use|

---

WO2008100995A1|2007-02-14|2008-08-21|Vaccinex, Inc.|Humanized anti-cd100 antibodies|

---

WO2008103392A2|2007-02-21|2008-08-28|Vaccinex, Inc.|Modulation of nkt cell activity with antigen-loaded cdid molecules|

---

KR101642846B1|2007-12-26|2016-07-26|백시넥스 인코포레이티드|Anti-C35 antibody combination therapies and methods|

---

WO2009089461A1|2008-01-10|2009-07-16|Genentech, Inc.|Plexind1 agonists and their use|

---

NO2427212T3|2009-05-08|2018-01-20|

---

US9539320B2|2009-05-15|2017-01-10|Irx Therapeutics, Inc.|Vaccine immunotherapy|

---

EP2579895A4|2010-06-14|2013-12-18|Vaccinex Inc|Anti-vegf antibodies and uses thereof|

---

AU2011295902B2|2010-09-02|2014-12-04|Vaccinex, Inc.|Anti-CXCL13 antibodies and methods of using the same|

---

CA2835599C|2011-05-13|2019-03-26|National University Corporation Tokyo Medical And Dental University|Osteogenesis promoter|

---

CA2851244A1|2011-10-06|2013-04-11|The Board Of Regents, The University Of Texas System|Anti-human sema4a antibodies useful to treat disease|

---

EP2766093B1|2011-10-11|2018-02-21|Vaccinex, Inc.|Use of semaphorin-4d binding molecules for modulation of blood brain barrier permeability|

---

CA2762446C|2011-12-06|2021-02-23|Vaccinex, Inc.|Use of the combination of semaphorin-4d inhibitory molecules and vegf inhibitory molecules to inhibit angiogenesis|

---

US8790652B2|2011-12-06|2014-07-29|Vaccinex, Inc.|Use of the combination of semaphorin-4D inhibitory molecules and VEGF inhibitory molecules to inhibit angiogenesis|

---

WO2013130959A1|2012-03-02|2013-09-06|Vaccinex, Inc.|Methods for the treatment of b cell-mediated inflammatory diseases|

---

US9090709B2|2012-03-28|2015-07-28|Vaccinex, Inc.|Anti-SEMA4D antibodies and epitopes|

---

US9708601B2|2012-04-26|2017-07-18|Vaccinex, Inc.|Fusion proteins to facilitate selection of cells infected with specific immunoglobulin gene recombinant vaccinia virus|

---

US10494440B2|2012-05-11|2019-12-03|Vaccinex, Inc.|Use of semaphorin-4D binding molecules to promote neurogenesis following stroke|

---

CA2899344A1|2013-01-31|2014-08-07|Vaccinex, Inc.|Methods for increasing immunoglobulin a levels|

---

US9371352B2|2013-02-08|2016-06-21|Vaccinex, Inc.|Modified glycolipids and methods of making and using the same|

---

NZ631015A|2013-06-25|2019-08-30|Vaccinex Inc|Use of semaphorin-4d inhibitory molecules in combination with an immune modulating therapy to inhibit tumor growth and metastases|

---

NZ630881A|2013-10-10|2016-03-31|Vaccinex Inc|Use of semaphorin-4d binding molecules for treatment of atherosclerosis|

---

NZ630892A|2013-10-21|2016-03-31|Vaccinex Inc|Use of semaphorin-4d binding molecules for treating neurodegenerative disorders|

---

RU2759846C2|2016-04-22|2021-11-18|Вэксинекс, Инк.|Display of an integral membrane protein on extracellular enveloped poxvirus virions|

---

SG11201900595YA|2016-08-02|2019-02-27|Vaccinex Inc|Improved methods for producing polynucleotide libraries in vaccinia virus/eukaryotic cells|

---

US20190383836A1|2017-02-22|2019-12-19|Vaccinex, Inc.|Early detection of glial cell activation in neurodegenerative or neuroinflammatory diseases|NO2427212T3|2009-05-08|2018-01-20|

---

US9090709B2|2012-03-28|2015-07-28|Vaccinex, Inc.|Anti-SEMA4D antibodies and epitopes|

---

US9708601B2|2012-04-26|2017-07-18|Vaccinex, Inc.|Fusion proteins to facilitate selection of cells infected with specific immunoglobulin gene recombinant vaccinia virus|

---

US10494440B2|2012-05-11|2019-12-03|Vaccinex, Inc.|Use of semaphorin-4D binding molecules to promote neurogenesis following stroke|

---

NZ631015A|2013-06-25|2019-08-30|Vaccinex Inc|Use of semaphorin-4d inhibitory molecules in combination with an immune modulating therapy to inhibit tumor growth and metastases|

---

NZ630881A|2013-10-10|2016-03-31|Vaccinex Inc|Use of semaphorin-4d binding molecules for treatment of atherosclerosis|

---

NZ630892A|2013-10-21|2016-03-31|Vaccinex Inc|Use of semaphorin-4d binding molecules for treating neurodegenerative disorders|

---

EP3136858A4|2014-05-02|2018-01-03|Nitor Therapeutics|Guanosine as an immunepotentiator mediated through toll receptors|

---

WO2017132541A1|2016-01-27|2017-08-03|University Of Maryland, Baltimore|A method for monitoring cancer and for regulation of semaphorin 4d to improve cancer immunotherapy regimens|

---

EP3213768A1|2016-03-01|2017-09-06|LODOCO CLINICAL Kft|Combination of low dose immune checkpoint blockade with high dose il-2 for treating metastatic cancer|

---

RU2759846C2|2016-04-22|2021-11-18|Вэксинекс, Инк.|Display of an integral membrane protein on extracellular enveloped poxvirus virions|

---

JP6656661B2|2016-06-16|2020-03-04|国立大学法人 東京大学|Plexin binding regulator|

---

SG11201900595YA|2016-08-02|2019-02-27|Vaccinex Inc|Improved methods for producing polynucleotide libraries in vaccinia virus/eukaryotic cells|

---

USD889258S1|2016-08-04|2020-07-07|Carolin McKie|Beverage container identification marker kit|

---

US20190194333A1|2016-09-16|2019-06-27|The Brigham And Women's Hospital, Inc.|Klrg1 depletion therapy|

---

AU2017342364A1|2016-10-12|2019-05-23|Board Of Regents, The University Of Texas System|Methods and compositions for TUSC2 immunotherapy|

---

EP3600419A4|2017-03-20|2020-12-02|Vaccinex, Inc.|Treatment of cancer with a semaphorin-4d antibody in combination with an epigenetic modulating agent|

---

EP3693380A1|2019-02-11|2020-08-12|Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V.|Dual inhibition of plexin-b1 and plexin-b2|

---

EP3946624A1|2019-03-28|2022-02-09|Vaccinex, Inc.|Semaphorin-4d antagonists for use in cancer therapy|

---

BR112021025795A2|2019-06-21|2022-02-01|H Lee Moffitt Cancer Center And Res Institute Inc A Florida Non Profit Corporation|Combination therapy with semaphorin-4d blockadeand dc1 therapy|

---

WO2021021991A1|2019-08-01|2021-02-04|Vaccinex,Inc.|Combined inhibition of semaphorin-4d and tgfb and compositions therefor|

---

CN111158663B|2019-12-26|2021-07-02|深圳逻辑汇科技有限公司|Method and system for handling references to variables in program code|

---

WO2021262564A1|2020-06-25|2021-12-30|Vaccinex, Inc.|Use of semaphorin-4d binding molecules for the treatment of rett syndrome|

法律状态:
2017-08-29| B15G| Petition not considered as such [chapter 15.7 patent gazette]|

---

2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

---

2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2018-03-20| B65X| Notification of requirement for priority examination of patent application|

---

2018-05-02| B65Y| Grant of priority examination of the patent application (request complies with dec. 132/06 of 20061117)|

---

2019-01-02| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

---

2019-03-06| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2019-09-03| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

---

2019-12-31| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2020-03-10| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 20/06/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361839170P| true| 2013-06-25|2013-06-25|

---

US61/839.170|2013-06-25|

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US201361874241P| true| 2013-09-05|2013-09-05|

---

US61/874.241|2013-09-05|

---

US201361884771P| true| 2013-09-30|2013-09-30|

---

US61/884.771|2013-09-30|

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US201361907845P| true| 2013-11-22|2013-11-22|

---

US61/907.845|2013-11-22|

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PCT/US2014/043466|WO2014209802A1|2013-06-25|2014-06-20|Use of semaphorin-4d inhibitory molecules in combination with an immune modulating therapy to inhibit tumor growth and metastases|

---