Method for diagnosing cancer using ephrin B2 expression

Abstract

The present invention describes the use of ephrinB2 expression as a biomarker to evaluate treatment efficacy and assist physicians in deciding on treatment course in individuals suffering from platinum-resistant metastatic cancer.In one embodiment, the present invention provides a method for diagnosing and selecting subjects suffering from cancer for treatment with EphB4-ephrinB2 inhibitors, or EphB4-ephrinB2 inhibitors in combination with immunostimulants, as first-line treatment for cancer treatment.In another embodiment, the present invention provides a method for diagnosing and selecting subjects suffering from cancer for treatment with EphB4-ephrinB2 inhibitors, or EphB4-ephrinB2 inhibitors in combination with immunostimulants, for treatment of some cancers where standard treatments are found to be ineffective, cause recurrence, or are not even considered for use due to the type of cancer and associated tumor. In another aspect, the invention provides methods for diagnosing and selecting a subject suffering from cancer for treatment with an EphB4-Ephrin B2 inhibitor, or an EphB4-Ephrin B2 inhibitor in combination with an immunostimulant, for treatment of some cancers where the subject is currently undergoing a course of treatment with an immunostimulant.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 241,448, filed September 7, 2021, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Array List This application contains a sequence listing in the form of a "paper copy" (PDF file) and a file containing the reference sequences (SEQ ID NOs: 1-5) submitted herein in computer readable form (ST25 format text file). The sequence listing is presented using the standard three letter code for amino acids as defined in 37 CFR 1.822. [Background technology]

[0003] Today, cancer remains the leading cause of death worldwide, despite the many advanced diagnostic and therapeutic methods that have been developed. In humans, cancer takes hold after initial genetic events by several mechanisms, including but not limited to increased cell metabolism and proliferation rates, increased blood supply to the tumor through stimulation of angiogenesis, and dysregulation of signaling pathways and tumor suppressors. Curative treatment protocols in clinical oncology continue to rely on a combination of surgical resection, ionizing radiation, and cytotoxic chemotherapy. A major barrier to successful cancer treatment and prevention lies in the fact that many cancers are unresponsive to current chemotherapeutic and immunotherapeutic interventions, with many individuals experiencing recurrence or death even after active treatment. Furthermore, tumors can become resistant to anticancer drugs by several mechanisms, including but not limited to efflux of drugs from cells, the occurrence of mutations that prevent the drug from binding to its target, and the occurrence of further mutations in genes and their protein products that are not associated with the drug target. To address these shortcomings, there has been a trend in drug discovery to develop targeted therapies that can modulate the signaling axes that are dysregulated in cancer. A key feature of targeted therapeutic approaches are reliable diagnostic and prognostic biomarkers. There are now many FDA-approved antibodies and small molecules that enable the therapeutic manipulation of a myriad of clinically relevant targets.

[0004] Immunotherapy using agonistic, antagonistic, or blocking antibodies against costimulatory or coinhibitory molecules (immune checkpoints) has been an area of ​​extensive research and clinical evaluation. Immune checkpoint proteins include CTLA-4, PD-1, PD-L1, LAG-3, and TIM-3, as well as several others (Sharpe et al., Nat Immunol, 8:239-45, 2007). Under normal physiological conditions, immune checkpoints are essential for maintaining self-tolerance (i.e., preventing autoimmunity) and protecting tissues from damage when the immune system responds to pathogenic infections. It is now clear that tumors also co-opt certain immune checkpoint pathways as a major mechanism of immune tolerance, especially to T cells that are specific for tumor antigens (Pardoll DM., Nat Rev Cancer, 12:252-64, 2012). Thus, for example, CTLA-4 (ipilimumab), PD-1 (nivolumab; pembrolizumab; pidilizumab) and PD-L1 (BMS-936559; MPLD3280A; MEDI4736; MSB0010718C) (see, e.g., Philips and Atkins, International Immunology, 27(1); 39-46, Oct 2014), as well as OX-40, CD137, GITR, LAG3, TIM-3, and VISTA (see, e.g., Sharon et al., Chin J Cancer., 33(9):434-444, Sep 2014; Hodi et al., N Engl J Med, 2010; Topalian et al., N Engl J Therapies utilizing antibodies against immune checkpoint molecules, including those against inflammatory cytokines (see, for example, inflammatory bowel disease (IG), immune checkpoint receptors (ICR), and immune activators (IL-1, IL-2, and IL-3), are being evaluated as novel immunotherapeutic alternatives for treating patients with proliferative diseases such as cancer, particularly those with refractory and / or recurrent cancers. Despite significant advances in diagnosis and treatment, cancer remains a major common cause of morbidity and mortality.

[0005] Currently, checkpoint inhibitor therapy has become the first-, second-, or third-line treatment for many cancers, and PD1 / PDL1 antibodies have changed the treatment paradigm for several cancers. Despite these great advances, there is still a great need to improve upon the current state of the art. For example, checkpoint inhibitor therapy remains limited by concerns about some possible side effects and the fact that many tumors lack the target antigen and therefore evade treatment. Overall, about 20% of patients with various cancers respond to PD-1 / PD-L1 or CTLA-4 antibodies, and overall survival is still at most less than one year, with modest objective response rates, highlighting the unmet need for about 70-80% of these patients.

[0006] Eph (Erythropoietin Producing Hepatoma) receptors and ligands are part of the largest family of receptor tyrosine kinases (RTKs). This family is further classified into Class A and Class B based on sequence homology and binding affinity to two distinct types of membrane-anchored ephrin ligands. Each Eph receptor and ligand can bind multiple ligands and receptors, with certain receptors postulated as putative tumor suppressors and others as tumor promoters (Vaught et al, Breast Cancer Res, 10(6):217-224, 2008). EphrinB2 and its high-affinity cognate receptor, EphB4, are transmembrane proteins that are directed to tumor vasculature and regulate immune cell trafficking. Inhibition of EphB4-EphrinB2 interaction has a direct inhibitory effect on tumor cell proliferation in vitro and ex-vivo. Polypeptide agents that inhibit EphB4-mediated or EphrinB2-mediated functions have been previously described by the present inventors (see, e.g., U.S. Patent Nos. 7,381,410; 7,862,816; 7,977,463; 8,063,183; 8,273,858; 8,975,377; 8,981,062; and 9,533,026; each of which is incorporated herein by reference in its entirety for all purposes).

[0007] The inventors have identified that a soluble extracellular fragment of EphB4 fused to albumin (sEphB4-HSA) blocks the interaction of ephrin-B2 with EphB4 and blocks bidirectional signaling, thus promoting immune cell trafficking and inducing anti-tumor immune responses in various cancers. As such, the inventors have developed an EphB4-ephrinB2 inhibitor, "sEphB4-HSA" (a soluble extracellular fragment of the EphB4 tyrosine kinase receptor fused to human serum albumin) for the treatment of various cancers. sEphB4-HSA consists of the extracellular domain of the human EphB4 receptor (sEphB4) fused in-frame with human serum albumin (HSA). This fusion with HSA enhances the pharmacokinetics of sEphB4.sEphB4-HSA binding to the ligand of the EphB4 tyrosine kinase receptor: the transmembrane protein ephrin-B2. This binding blocks endogenous EphB tyrosine kinase receptors from interacting with ephrinB2. Evidence to date indicates that sEphB4-HSA reduces angiogenesis in tumors, thus inhibiting the ability of ephrinB2 to starve tumors of blood vessels and suppress T cell recruitment to tumors, thus increasing T cell recruitment. EphB4 is a survival factor in several tumor types, such as squamous cell carcinoma, urothelial carcinoma, colon cancer, lung cancer, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, and others. EphrinB2 is a survival factor in several tumors, such as Kaposi's sarcoma. Blockade of bidirectional signaling blocks activation of EphB4, and thus anterograde signaling, leading to cell quiescence or cell death.

[0008] The present invention is directed, in part, to the use of Ephrin B2 expression as a biomarker to assess treatment efficacy and assist physicians in making decisions regarding the course of treatment in individuals with metastatic cancer.

[0009] References The patent documents, U.S. Patent No. 7,381,410; U.S. Patent No. 7,862,816; U.S. Patent No. 7,977,463; U.S. Patent No. 8,063,183; U.S. Patent No. 8,273,858; U.S. Patent No. 8,975,377; U.S. Patent No. 8,981,062; U.S. Patent No. 9,533,026; International Patent Application (PCT / US) No. 2020 / 018160; International Patent Application (PCT / US) No. 2020 / 023215, and all references disclosed herein are hereby incorporated by reference in their entirety for all purposes. Summary of the Invention

[0010] The present invention is based in part on surprising discoveries including: 1) that EphrinB2 expression appears to correlate with response to EphrinB2 targeted therapy, either as a single agent and / or in combination therapy with a polypeptide agent that inhibits EphB4 or EphrinB2 mediated function ("EphB4-EphrinB2 inhibitor") (e.g., sEphB4-HSA) in combination with an immune stimulant (including but not limited to, an antagonist antibody against PD-1 / PD-L1, CTLA-4, LAG3, TIM3, TIGIT, OX40 ligand) as first line treatment in locally advanced or metastatic urothelial / bladder cancer; and 2) that higher EphrinB2 expression appears to correlate with lower response to monotherapy with an immune stimulant (e.g., a PD-1 / PD-L1 antagonist antibody) in metastatic urothelial cancer following systemic chemotherapy trials.

[0011] Thus, the present invention describes the use of EphrinB2 expression as a biomarker to evaluate treatment efficacy and assist physicians in deciding on treatment course in individuals suffering from metastatic cancer.In various aspects, the present invention provides a method for diagnosing and selecting subjects suffering from cancer for treatment with EphB4-EphrinB2 inhibitors, or EphB4-EphrinB2 inhibitors in combination with immune stimulants, as first-line treatment for cancer treatment.In various aspects, the present invention provides a method for diagnosing and selecting subjects suffering from cancer for treatment with EphB4-EphrinB2 inhibitors, or EphB4-EphrinB2 inhibitors in combination with immune stimulants, for treatment of some cancers where standard treatments are found to be ineffective, to result in recurrence, or to not even be considered for use due to the type of cancer and associated tumor. In various aspects, the present invention provides methods for diagnosing and selecting subjects suffering from cancer for treatment with an EphB4-EphrinB2 inhibitor, or an EphB4-EphrinB2 inhibitor in combination with an immunostimulant, for the treatment of some cancers where the subject is currently undergoing treatment with an immunostimulant. In some embodiments, the individual suffers from platinum-resistant metastatic cancer. In some embodiments, the individual has not previously received platinum chemotherapy or is not healthy enough to receive platinum chemotherapy.

[0012] In some embodiments, a method for diagnosing and selecting a subject suffering from cancer for treatment with an EphB4-EphrinB2 inhibitor in combination with an immunostimulant comprises: i) detecting an EphrinB2 expression level in a biological sample from a subject diagnosed with cancer; and ii) selecting the subject for treatment with an EphB4-EphrinB2 inhibitor in combination with an immunostimulant as a first line treatment if EphrinB2 expression is 1% or greater.

[0013] In some embodiments, a method for diagnosing and selecting a subject suffering from cancer for treatment with an EphB4-Ephrin B2 inhibitor comprises: i) detecting Ephrin B2 expression levels in a biological sample from a subject diagnosed with cancer; and ii) selecting the subject for treatment with an EphB4-Ephrin B2 inhibitor as a first line treatment if Ephrin B2 expression is 1% or greater.

[0014] In some embodiments, a method for diagnosing a subject suffering from cancer comprises: i) detecting Ephrin B2 expression levels in a biological sample from a subject diagnosed with cancer and currently undergoing treatment with an immunostimulant; and ii) modifying treatment if Ephrin B2 expression is 1% or greater.

[0015] In some embodiments, the polypeptide agent that inhibits EphB4 or EphrinB2-mediated function is a monomeric ligand-binding portion of EphB4 protein or EphrinB2 protein, or an antibody that binds and acts with EphB4 or EphrinB2. In some embodiments, the polypeptide agent is a soluble EphB4 (sEphB4) polypeptide that specifically binds to EphrinB2 polypeptide and comprises the amino acid sequence of the extracellular domain of EphB4 protein. In some embodiments, the sEphB4 polypeptide comprises the globular domain of EphB4 protein. In some embodiments, the agent that inhibits EphB4 or EphrinB2-mediated function is a nucleic acid therapeutic agent. In some embodiments, the nucleic acid therapeutic agent that inhibits EphB4 or EphrinB2-mediated function is an oligonucleotide DNA or siRNA that targets EphrinB2 or EphB4.

[0016] In some embodiments, the sEphB4 polypeptide comprises a sequence selected from the group consisting of a sequence that is at least 90% identical to residues 1-522, at least 90% identical to residues 1-412, and at least 90% identical to residues 1-311 of the amino acid sequence of SEQ ID NO:1. In some embodiments, the sEphB4 polypeptide may comprise a sequence that includes a globular (G) domain (amino acids 29-197 of SEQ ID NO:1), and optionally additional domains, such as a cysteine-rich domain (amino acids 239-321 of SEQ ID NO:1), a first fibronectin type 3 domain (amino acids 324-429 of SEQ ID NO:1), and a second fibronectin type 3 domain (amino acids 434-526 of SEQ ID NO:1). In some embodiments, the sEphB4 polypeptide will comprise amino acids 1-537 of SEQ ID NO:1. In some embodiments, the sEphB4 polypeptide will comprise amino acids 1-427 of SEQ ID NO:1. In some embodiments, the sEphB4 polypeptide will comprise amino acids 1-326 of SEQ ID NO:1. In some embodiments, an sEphB4 polypeptide will include amino acids 1-197, 29-197, 1-312, 29-132, 1-321, 29-321, 1-326, 29-326, 1-412, 29-412, 1-427, 29-427, 1-429, 29-429, 1-526, 29-526, 1-537, and 29-537 of SEQ ID NO: 1. In some embodiments, an sEphB4 polypeptide will include amino acids 16-197, 16-312, 16-321, 16-326, 16-412, 16-427, 16-429, 16-526, and 16-537 of SEQ ID NO: 1.

[0017] In some embodiments, sEphB4 polypeptides may be prepared in multimeric form, for example, by expressing them as an Fc fusion protein or a fusion with another multimerization domain.

[0018] In some embodiments, the sEphB4 polypeptide further comprises an additional moiety that confers increased serum half-life while still retaining EphrinB2 binding activity. In some embodiments, the sEphB4 polypeptide is monomeric and covalently linked to one or more polyoxyalkylene groups (e.g., polyethylene, polypropylene). In some embodiments, the sEphB4 polypeptide is covalently linked to a polyethylene glycol (PEG) group (hereinafter "sEphB4-PEG").

[0019] In some embodiments, the sEphB4 polypeptide is stably associated with a second stabilizing polypeptide that confers improved half-life without substantially compromising EphrinB2 binding. In some embodiments, the stabilizing polypeptide is immunocompatible with a human patient (or an animal patient, if veterinary use is intended) and will have little or no significant biological activity. In some embodiments, the sEphB4 polypeptide is covalently or non-covalently associated with an albumin selected from the group consisting of human serum albumin (HSA) (hereinafter "sEphB4-HSA") and bovine serum albumin (BSA) (hereinafter "sEphB4-BSA"). In some embodiments, the sEphB4-HSA comprises residues 16-197 of SEQ ID NO:1 directly fused with residues 25-609 of SEQ ID NO:2. In some embodiments, the sEphB4-HSA comprises residues 16-312 of SEQ ID NO:1 directly fused with residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-321 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-326 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-412 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-427 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-429 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-526 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-537 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, sEphB4-HSA comprises the amino acid sequence set forth in SEQ ID NO:4.In some embodiments, the sEphB4-HSA comprises the amino acid sequence set forth in SEQ ID NO:5.

[0020] In some embodiments, sEphB4 polypeptides may be prepared in multimeric form by expressing them as fusion proteins with molecules that block signaling with Eph receptors or block Ephrin B2 interactions, including, but not limited to, antisense oligonucleotides, siRNA, and gene editing such as CRISPR / CAS.

[0021] In some embodiments, the immune stimulant is: SIRP (expressed by macrophages, monocytes, dendritic cells), CD47 (highly expressed by tumor cells and other cell types), TIGIT (an immune receptor present on some T cells and natural killer cells), VISTA (expressed by monocytes, dendritic cells, B cells, T cells), CD152 (expressed by activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed by tumor-infiltrating lymphocytes, activated T CD274 (expressed by T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, pancreatic islet cells), and CD223 (expressed by activated T cells, regulatory T cells, anergic T cells, NK cells, NKT cells, and plasmacytoid dendritic cells). In some embodiments, the immunostimulatory agent is selected from the group consisting of anti-PD-1 Ab, anti-PD-L1 Ab, anti-CTLA Ab, anti-TIGIT Ab, anti-LAG3 antibody, anti-TIM3 antibody, and combinations thereof.

[0022] In some embodiments, EphrinB2 expression is determined by protein expression using a method selected from the group consisting of immunohistochemistry (IHC), immunofluorescence, flow cytometry, and Western blot. In some embodiments, mRNA expression levels are determined using a method selected from the group consisting of quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), RNA sequencing, microarray analysis, in situ hybridization, and serial analysis of gene expression (SAGE).

[0023] In some embodiments, the biological sample is selected from the group consisting of a tissue sample, a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid (CSF) sample, a peritoneal fluid sample, and a cell culture sample.

[0024] In some embodiments, the cancer is selected from the group consisting of: B-cell lymphoma; lung cancer (small cell lung cancer and non-small cell lung cancer); bronchial cancer; colorectal cancer; prostate cancer; breast cancer; pancreatic cancer; gastric cancer; ovarian cancer; bladder cancer; brain or central nervous system cancer; peripheral nervous system cancer; esophageal cancer; cervical cancer; melanoma; uterine or endometrial cancer; oral or pharyngeal cancer; liver cancer; renal cancer; biliary tract cancer; small intestine or appendix cancer; salivary gland cancer; thyroid cancer; adrenal cancer; osteosarcoma; chondrosarcoma; liposarcoma; testicular cancer; and malignant fibrous histiocytoma; skin cancer; head and neck cancer; lymphoma; sarcoma; multiple myeloma; and leukemia. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is a RAS (e.g., KRAS, HRAS, NRAS) mutated cancer. In some embodiments, the cancer is a PTEN deleted cancer.

[0025] In some embodiments, the subject has previously responded to treatment with an anti-cancer therapy, but has relapsed upon cessation of treatment (hereinafter "relapsed cancer"). In some embodiments, the subject has a resistant or refractory cancer. In some embodiments, the cancer is refractory to platinum-based chemotherapy. In some embodiments, the cancer is refractory to immunotherapy treatment. In some embodiments, the cancer is refractory to treatment with a chemotherapeutic agent. In some embodiments, the cancer is refractory to treatment with a depleting antibody against a specific tumor antigen. In some embodiments, the cancer is refractory to treatment with an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint). In some embodiments, the cancer is refractory to targeted therapy with an antibody-drug conjugate (ADC), or a fusion molecule comprising a depleting antibody against a specific tumor antigen and a cytotoxic agent. In some embodiments, the cancer is refractory to targeted therapy with a small molecule kinase inhibitor. In some embodiments, the cancer is refractory to treatment with surgery. In some embodiments, the cancer is refractory to treatment with stem cell transplantation. In some embodiments, the cancer is refractory to treatment with radiation. In some embodiments, the cancer is refractory to combination therapy including, for example, two or more of immunotherapy treatment, platinum-based chemotherapy treatment, tumor antigen-specific depleting antibody treatment, immunoconjugate, ADC, or fusion molecule including tumor antigen-specific depleting antibody and cytotoxic agent treatment, targeted therapy with small molecule kinase inhibitor, surgery treatment, stem cell transplantation treatment, and radiation treatment.

[0026] In some embodiments, the method of treating cancer in a subject further comprises a second therapy selected from the group consisting of: small molecule kinase inhibitor targeted therapy, surgery, cytoreductive therapy, cytotoxic chemotherapy, and immunotherapy. In some embodiments, the combination therapy may have a synergistic effect. In some embodiments, the second therapy is a cytoreductive therapy, and the combination may increase the therapeutic index of the cytoreductive therapy. In some embodiments, the cytoreductive therapy may act on a DNA repair pathway. In some embodiments, the cytoreductive therapy is radiation therapy. In some embodiments, the combination may have a synergistic effect.

[0027] In some embodiments, the second treatment is selected from the group consisting of daunorubicin, adriamycin (doxorubicin), epirubicin, idarubicin, annamycin, MEN10755, etoposide, teniposide, vinblastine, vincristine, vinorelbine (NAVELBINE); vindesine, vindoline, vincamine, mechlorethamine, cyclophosphamide, melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, cytarabine (CYTOSAR-U), cytosine, cyclospor ... and the chemotherapeutic agent is selected from the group consisting of tetracycline arabinoside, fluorouracil (5-FU), floxuridine (FUdR), thioguanine (6-thioguanine), mercaptopurine (6-MP), pentostatin, fluorouracil (5-FU), methotrexate, 10-propargyl-5.8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolate (DDATHF), leucovorin, cisplatin (cis-DDP), carboplatin, oxaliplatin, hydroxyurea, gemcitabine, and N-methylhydrazine.

[0028] In some embodiments, the second treatment will include administering a poly(ADP-ribose) polymerase inhibitor (PARP inhibitor). In some embodiments, the PARP inhibitor is selected from the group consisting of ABT-767, AZD2461, BGB-290, BGP15, CEP9722, E7016, E7449, fluzoparib, INO1001, JPI289, MP124, niraparib, olaparib, ONO2231, rucaparib, SC101914, talazoparib, veliparib, WW46, or salts or derivatives thereof. Olaparib, rucapari, niraparib, talazoparib and veliparib. In some embodiments, the combination may have a synergistic effect.

[0029] In some embodiments, the method of treatment will include administering sEphB4-HSA in combination with PEGylated liposomal doxorubicin (PLD). In some embodiments, the method of treatment will include administering sEphB4-HSA in combination with paclitaxel. In some embodiments, the combination may have a synergistic effect.

[0030] In some embodiments, the second treatment includes, but is not limited to, treatment with a depleting antibody against a specific tumor antigen; treatment with an antibody-drug conjugate; treatment with an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint), such as CTLA-4, PD-1, OX-40, CD137, GITR, LAG3, TIM-3, and VISTA; treatment with a bispecific T cell engaging antibody (BiTE®); treatment including administration of IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ biological response modifiers; treatment with a therapeutic vaccine, such as sipuleucel-T; treatment with a dendritic cell vaccine or a tumor antigen peptide vaccine; treatment with oncolytic virus therapy (T-VEC); treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor infiltrating lymphocytes (TIL); treatment with matched transplanted anti-tumor T cells (ex and treatment with immunostimulants such as the Toll-like receptor (TLR) agonist CpG and imiquimod, wherein the combination therapy enhances effector cell killing of tumor cells, i.e., there is a synergistic effect between sEphB4-HSA and the immunotherapy when co-administered.

[0031] In some embodiments, the additional therapy comprises administering an antibody that specifically binds to an immune checkpoint protein antigen from the list including, but not limited to, CD276, CD272, CD152, CD223, CD279, CD274, TIM-3 and B7-H4; or any immune checkpoint protein antigen antibody taught in the art. In some embodiments, the PD-1 inhibitor is selected from the group consisting of, but not limited to, nivolumab (Bristol-Myers Squibb) (Drugbank 09035; Drugbank 06132), pembrolizumab (Merck) (Drugbank 09037) and pidilizumab (Medivation) (Drugbank 15383). In some embodiments, the CTLA-4 inhibitor is selected from the group consisting of, but not limited to, ipilimumab (Bristol-Myers Squibb) (Drugbank 06186) and tremelimumab (MedImmune) (Drugbank 11771). [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 provides photographs of ephrinB2 immunohistochemistry (IHC) and ephrinB2 in situ hybridization (ISH / RNAScope) of an ephrinB2 positive non-responder. [Diagram 2] FIG. 2 provides photographs of ephrin B2 immunohistochemistry (IHC) and ephrin B2 in situ hybridization (ISH / RNAScope) of five responders. [Diagram 3] FIG. 3 provides pictures of cell pellets of various isogenic CHO cell lines, controls: wild type or ectopically expressed human ephrin B2, and ephrin B2 immunohistochemistry (IHC) and ephrin B2 in situ hybridization (ISH / RNAScope) of two urothelial cancer tissues. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Unless otherwise defined herein, scientific and technical terms used in the context of the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms include plurals and plural terms include singulars. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and these techniques, are commonly used and well known in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout this specification. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0034] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of two or more amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids, as well as natural and non-natural amino acid polymers. The terms "antibody" and "antibodies" are used interchangeably herein to refer to a polypeptide that can interact and / or bind to another molecule, often referred to as an antigen. Antibodies can also include, for example, "antigen-binding polypeptides" or "target molecule-binding polypeptides". Antigens of the present invention can also include, for example, any of the polypeptides described in the present invention.

[0035] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same base chemical structure as natural amino acids, i.e., an α-carbon bonded with a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same base chemical structure as natural amino acids. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of amino acids, but function similarly to natural amino acids. All one-letter abbreviations used in the present invention to represent amino acids are used according to the commonly accepted amino acid symbols commonly used in the art, such as A for alanine, C for cysteine, and so on. Amino acids are represented by a single letter before or after the relevant position to reflect the change from the original amino acid (before) to the changed amino acid (after), e.g., A19T means that the amino acid alanine at position 19 has been changed to threonine.

[0036] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0037] The terms "cancer," "neoplasm," and "tumor" are used interchangeably herein to refer to cells that exhibit autonomous unregulated growth and thus exhibit an abnormal growth phenotype characterized by a profound loss of control over cell proliferation. In general, cells of interest for detection, analysis, classification, or treatment herein include precancerous (e.g., benign), malignant, premetastatic, and nonmetastatic cells.

[0038] The term "primary tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues located at the anatomical site where autonomous unregulated cell proliferation begins, e.g., the organ of origin of the cancerous tumor. Primary tumors do not include metastases.

[0039] The "pathology" of cancer includes all phenomena that impair the well-being of the patient, including, but not limited to, abnormal or uncontrolled cell proliferation, primary tumor growth and formation, metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretory products, suppression or exacerbation of inflammatory or immune responses, neoplasia, precancerous conditions, malignant tumors, invasion of surrounding or distant tissues or organs such as lymph nodes, etc.

[0040] As used herein, the terms "cancer recurrence" and "tumor recurrence" and grammatical variations thereof refer to the further proliferation of neoplastic or cancerous cells after cancer diagnosis. In particular, recurrence can occur when further cancerous cell proliferation occurs within cancerous tissue. Similarly, "tumor spread" occurs when cells of a tumor spread to local or distant tissues and organs; thus, tumor spread encompasses tumor metastasis. "Tumor invasion" occurs when tumor growth spreads locally, impairing the function of diseased tissues by compressing, destroying, or preventing normal organ function.

[0041] As used herein, the term "metastasis" refers to the growth of a cancerous tumor in an organ or body part that is not directly connected to the organ of the original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of an undetectable amount of cancerous cells in an organ or body part that is not directly connected to the organ of the original cancerous tumor (e.g., the organ that contains the primary tumor). Metastasis can also be defined as a process of several steps, including the detachment of cancer cells from the original tumor site (e.g., the primary tumor site) and the migration and / or invasion of cancer cells to other parts of the body.

[0042] Depending on the nature of the cancer, obtain a suitable patient sample. As used herein, the phrase "cancerous tissue sample" refers to any cell obtained from a cancerous tumor. For solid tumors that have not metastasized (e.g., primary tumors), tissue samples are usually obtained from tumors that have been surgically removed and prepared for testing by conventional techniques.

[0043] "Early stage cancer" or "early stage tumor" means a cancer that has not invasively or metastasized, or is classified as stage 0, 1, or 2 cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumor, gastrinoma, and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukocyte or lymphoid malignancies. More particularly, the present invention relates to a method for treating cancer of the eye, including bladder cancer (e.g., urothelial bladder cancer (e.g., transitional cell or urothelial carcinoma, non-muscle invasive bladder cancer, muscle invasive bladder cancer, and metastatic bladder cancer) and non-urothelial bladder cancer), squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung cancer, including adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic These include cancer of the anus, cancer of the penis, Merkel cell carcinoma, mycoses fungoids, testicular cancer, esophageal cancer, tumors of the bile duct, as well as head and neck cancer and hematological malignancies.

[0044] Tumors of interest for treatment using the methods of the present invention include solid tumors, such as carcinomas, gliomas, melanomas, sarcomas, and the like. Ovarian and breast cancers are of particular interest. Carcinomas include, for example, various adenocarcinomas in the prostate, lung, and the like; adrenal cortical carcinoma; hepatocellular carcinoma; hepatocellular carcinoma, ovarian carcinoma, intraepithelial carcinoma, ductal carcinoma, breast carcinoma, basal cell carcinoma; squamous cell carcinoma; transitional cell carcinoma; colon carcinoma; nasopharyngeal carcinoma; multilocular cystic renal cell carcinoma; oat cell carcinoma, large cell lung carcinoma; small cell lung carcinoma; and others. Carcinomas may be found in the prostate, pancreas, colon, brain (e.g., glioblastoma), lung, breast, skin, and the like. Neoplastic tissue formations derived from fibroblasts, myofibroblasts, histiocytes, vascular / endothelial cells, and nerve sheath cells are included in the designation of soft tissue tumors. Tumors of connective tissue include sarcoma, histiocytoma, fibroma, skeletal chondrosarcoma, extraskeletal myxoid chondrosarcoma, clear cell sarcoma, fibrosarcoma, etc. Hematological cancers include leukemias and lymphomas, such as cutaneous T-cell lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), etc.

[0045] "Resistant or refractory cancer" refers to tumor cells or cancer that are unresponsive to previous anti-cancer therapies, including, for example, chemotherapy, surgery, radiation therapy, hepatocyte transplantation, and immunotherapy. Tumor cells may be resistant or refractory at the beginning of treatment, or may become resistant or refractory during treatment. Refractory tumor cells include tumors that are unresponsive at the beginning of treatment, or that respond poorly to treatment after a short initial period of response. Refractory tumor cells also include tumors that respond to treatment with anti-cancer therapy, but do not respond well to subsequent rounds of treatment. For purposes of the present invention, refractory tumor cells also include tumors that appear to be inhibited by treatment with anti-cancer therapy, but recur up to 5 years, and sometimes up to 10 years or more after treatment is stopped. Anti-cancer therapy can use chemotherapy alone, radiation alone, targeted therapy alone, surgery alone, or a combination thereof. For ease of explanation and not limitation, it will be understood that refractory tumor cells can be replaced with resistant tumor cells. In some embodiments, the cancer is resistant to standard treatment. In some embodiments, the cancer is a chemotherapy-resistant cancer, hi some embodiments, the cancer is a platinum-resistant cancer.

[0046] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated, and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0047] As used herein, the term "sample" refers to a composition obtained or derived from a subject and / or individual of interest that contains cells and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase "disease sample" and variations thereof refer to any sample obtained from a subject of interest that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0048] "Tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue from fresh, frozen and / or preserved organs, tissue samples, biopsies, and / or aspirates; blood or blood components such as plasma; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time point in the subject's pregnancy or development. The tissue sample may be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue / organ. For example, a "tumor sample" is a tissue sample obtained from a tumor or other cancerous tissue. The tissue sample may contain a mixed population of cell types (e.g., tumor cells and non-tumor cells, cancerous cells and non-cancerous cells). The tissue sample may contain compounds that are not naturally mixed with tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

[0049] The term "detection" includes any means of detection, including direct and indirect detection.

[0050] As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, e.g., a predictive, diagnostic, and / or prognostic indicator. A biomarker can serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by specific molecular, pathological, histological, and / or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotide (e.g., DNA and / or RNA), polynucleotide copy number variation (e.g., DNA copy number), polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrate, and / or glycolipid-based molecular markers.

[0051] As used herein, when assessing ephrin B2 expression as a biomarker, ephrin B2 expression is positive in 1% or more of the cells indicating membrane signaling.

[0052] As used herein, "treatment" is an approach to obtain an effective or desired clinical result. For the purposes of the present invention, effective or desired clinical results include, but are not limited to, alleviation of one or more symptoms; reduction in the extent of disease; prevention or delay of disease spread (e.g., metastasis, e.g., to lungs or lymph nodes); prevention or delay of disease recurrence; stabilization; delay or slowing of disease progression; remission of disease; remission (whether partial or total); and improvement of quality of life. Reduction of pathological consequences of proliferative diseases is also encompassed by "treatment". The method of the present invention contemplates any one or more of these aspects of treatment.

[0053] Treatment may refer to any indicator of successful treatment or remission or prevention, including any objective or subjective parameter, such as alleviation; remission; reduction of symptoms or making the condition more tolerable to the patient; slowing the rate of degeneration or decline; or making the degeneration end point less severe. Treatment or remission of symptoms can be based on objective or subjective parameters, including the results of examination by a physician. Thus, the term "treatment" includes administering a compound or agent of the present invention to prevent or delay, ameliorate, or stop or inhibit the onset of symptoms or pathology. The term "therapeutic effect" refers to the reduction, elimination, or prevention of disease, symptoms of disease, or side effects of disease in a subject.

[0054] The phrase "synergistic effect" describes the effect achieved when active ingredients used together are greater than the sum of the effects resulting from the active ingredients used separately.

[0055] "Sustained response" refers to a sustained effect on tumor growth reduction after treatment is stopped. For example, the size of the tumor may remain the same or become smaller compared to the size at the beginning of the administration phase. In some embodiments, the sustained response has a treatment period of at least the same duration as the treatment period, or at least 1.5 times, 2.0 times, 2.5 times, or 3.0 times the treatment period.

[0056] As used herein, "reducing or inhibiting cancer recurrence" refers to reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression. As disclosed herein, cancer recurrence and / or cancer progression includes, but is not limited to, cancer metastasis.

[0057] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions.

[0058] As used herein, "partial response" or "PR" refers to at least a 30% reduction in the sum of the longest diameters (SLD) of target lesions relative to baseline SLD.

[0059] As used herein, "stable disease" or "SD" refers to target lesions that have not sufficiently shrunk to the quality of PR or increased to the quality of PD, based on the minimum SLD since the start of treatment.

[0060] As used herein, "progression" or "PD" refers to at least a 20% increase in the SLD of a target lesion, based on the smallest SLD recorded since the start of treatment or the presence of one or more new lesions.

[0061] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not worsen. Progression-free survival may include the amount of time a patient experiences a complete or partial response, as well as the amount of time a patient experiences stable disease.

[0062] As used herein, "overall response rate" or "objective response rate" (ORR) refers to the sum of the complete response (OR) rate and the partial response (PR) rate.

[0063] As used herein, "overall survival" (OS) refers to the percentage of individuals in a population who are likely to be alive after a particular period of time.

[0064] As used herein, polypeptide agents that inhibit EphB4 or EphrinB2-mediated functions ("EphrinB2 / EphB4 inhibitors") are used to refer to inhibitors, activating molecules or regulatory molecules, respectively, identified using in vitro and in vivo assays for receptor or ligand binding or signaling, e.g., ligands, receptors, agonists, antagonists, and homologs and mimetics thereof.

[0065] EphrinB2 / EphB4 inhibitors having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host to inhibit EphB4 or EphrinB2 mediated functions. Therapeutic agents may be administered in a variety of ways, including parenterally, e.g., intravenously, subcutaneously, intraperitoneally, by viral infection, intravascularly, and others. Intravenous delivery is of particular interest. Depending on the method of introduction, the compounds may be formulated in a variety of ways. The concentration of the therapeutically active compound in the formulation may vary from about 0.1 to 100% by weight.

[0066] Pharmaceutical compositions can be formulated in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, ointments, lotions and the like. Pharmaceutical grade organic or inorganic carriers and / or diluents suitable for oral and topical use can be used to make compositions containing therapeutically active compounds. Diluents known in the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting agents and emulsifying agents, salts for modifying osmotic pressure or salts of buffers for ensuring sufficient pH value, and skin penetration enhancers can be used as auxiliary agents.

[0067] "Pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and useful for formulating a pharmaceutical composition, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0068] The terms "pharmaceutical acceptable," "physiologically acceptable," and grammatical variations thereof, when used interchangeably to refer to compositions, carriers, diluents, and reagents, indicate that these materials can be administered to or to a human without producing undesirable physiological effects to an extent that would interfere with administration of the composition.

[0069] "Dosage unit" refers to a physically discrete unit suited as a unitary dosage for a particular individual to be treated. Each unit can contain a predetermined quantity of active compound calculated to obtain the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms can be determined by (a) the inherent characteristics of the active compound and the particular therapeutic effect to be obtained, and (b) the constraints inherent in the technology for compounding such active compounds.

[0070] The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a mammal being evaluated for and / or treated for treatment. In an embodiment, the mammal is a human. Thus, the terms "subject", "individual" and "patient" include individuals with cancer who have undergone or are candidates for resection (surgery) to remove cancerous tissue, including, but not limited to, ovarian or prostate adenocarcinoma, breast cancer, glioblastoma, etc. The subject may be a human, but also includes mammals, particularly mammals useful as experimental models for human disease, such as mice, rats, etc.

[0071] The term "diagnosis" is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of a viral infection.

[0072] A "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject for the treatment of breast or ovarian cancer, is sufficient to affect such cancer treatment. A "therapeutically effective amount" may vary depending, for example, on the sEphB4-HSA polypeptide or immunostimulant selected, the stage of the cancer, the age, weight and / or health of the patient, and the judgment of the prescribing physician. The appropriate amount in any given case may be readily ascertained by one of ordinary skill in the art or can be determined by routine experimentation.

[0073] The phrase "determining therapeutic efficacy" and variations thereof can include any method for determining that a treatment provides a benefit to a subject. The term "therapeutic efficacy" and variations thereof is generally indicated by the alleviation of one or more symptoms or signs associated with a disease, and can be easily determined by one of skill in the art. "Therapeutic efficacy" can also refer to the prevention or amelioration of toxic signs and symptoms normally associated with standard or non-standard treatment of a disease. Determining therapeutic efficacy is usually symptom and disease specific, and can include any method known or available in the art for determining that a treatment provides a beneficial effect to a patient. For example, evidence of therapeutic efficacy includes, but is not limited to, the amelioration of a disease or symptoms. Additionally, therapeutic efficacy can also include a general improvement in the overall health of a subject, such as an enhanced quality of life for a patient, an increase in the predicted survival rate of a subject, an inhibition or reduction in the recurrence rate of symptoms (prolonged remission time), etc. (see, e.g., Physicians' Desk Reference (2010)).

[0074] In the case of cancer or tumors, an effective amount of a drug may have the effect of reducing the number of cancer cells; reducing the size of the tumor; inhibiting (i.e., slowing or desirably stopping to some extent) cancer cell invasion into surrounding organs; inhibiting (i.e., slowing or desirably stopping to some extent) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As will be understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be administered in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in terms of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if it is likely or will achieve a desired result in combination with one or more other agents.

[0075] As used herein, "in conjunction with" refers to the administration of one therapy in addition to another therapy. As such, "in conjunction with" refers to the administration of one therapy before, during, or after the administration of the other therapy to an individual.

[0076] As used herein, "in combination with," "combination therapy," and "combination product" refer, in certain embodiments, to simultaneous administration to a first medical patient and the compounds used herein. In some embodiments, the combination product is not administered simultaneously. When administered in combination, each component can be administered sequentially in any order at the same time or at different times. Thus, each component can be administered separately but close enough in time to achieve the desired therapeutic effect.

[0077] "Co-administration" of a pharmaceutical composition of the present invention and a known cancer therapeutic agent refers to administration of the drug and AXL variant at a time such that both the known drug and the composition of the present invention have a therapeutic effect. Such co-administration may include administration of the drug simultaneously (i.e., at the same time), before, or after administration of the drug in relation to administration of the compound of the present invention. One of ordinary skill in the art would have no difficulty in determining the appropriate timing, sequence, and dosage of administration of a particular drug and composition of the present invention.

[0078] As used herein, the term "correlating" or "correlating with" and similar terms refer to a statistical association between two instances of events, where the events include numbers, data sets, and the like. For example, where the events include numbers, a positive correlation (also referred to herein as "direct correlation") means that when one increases, the other also increases. A negative correlation (also referred to herein as "inverse correlation") means that when one increases, the other decreases.

[0079] Exemplary embodiments The use of EphrinB2 expression as a biomarker to evaluate treatment efficacy and assist physicians in deciding on treatment course in individuals suffering from metastatic cancer is described.In various aspects, the present invention provides a method for diagnosing and selecting subjects suffering from cancer for treatment with EphB4-EphrinB2 inhibitors, or EphB4-EphrinB2 inhibitors in combination with immune stimulants, as first-line treatment for cancer treatment.In various aspects, the present invention provides a method for diagnosing and selecting subjects suffering from cancer for treatment with EphB4-EphrinB2 inhibitors, or EphB4-EphrinB2 inhibitors in combination with immune stimulants, for treatment of some cancers where standard treatments are found to be ineffective, cause recurrence, or are not even considered for use due to the type of cancer and associated tumor. In various aspects, the present invention provides methods for diagnosing and selecting subjects suffering from cancer for treatment with an EphB4-EphrinB2 inhibitor, or an EphB4-EphrinB2 inhibitor in combination with an immunostimulant, for the treatment of some cancers where the subject is currently undergoing treatment with an immunostimulant. In some embodiments, the individual suffers from platinum-resistant metastatic cancer. In some embodiments, the individual has not previously received platinum chemotherapy or is not healthy enough to receive platinum chemotherapy.

[0080] In some embodiments, a method for diagnosing and selecting a subject suffering from cancer for treatment with an EphB4-EphrinB2 inhibitor in combination with an immunostimulant comprises: i) detecting an EphrinB2 expression level in a biological sample from a subject diagnosed with cancer; and ii) selecting the subject for treatment with an EphB4-EphrinB2 inhibitor in combination with an immunostimulant as a first line treatment if EphrinB2 expression is 1% or greater.

[0081] In some embodiments, a method for diagnosing and selecting a subject suffering from cancer for treatment with an EphB4-Ephrin B2 inhibitor comprises: i) detecting Ephrin B2 expression levels in a biological sample from a subject diagnosed with cancer; and ii) selecting the subject for treatment with an EphB4-Ephrin B2 inhibitor as a first line treatment if Ephrin B2 expression is 1% or greater.

[0082] In some embodiments, a method for diagnosing a subject suffering from cancer comprises: i) detecting Ephrin B2 expression levels in a biological sample from a subject diagnosed with cancer and currently undergoing treatment with an immunostimulant; and ii) modifying treatment if Ephrin B2 expression is 1% or greater.

[0083] In some embodiments, EphrinB2 expression is determined by protein expression using a method selected from the group consisting of immunohistochemistry (IHC), immunofluorescence, flow cytometry, and Western blot. In some embodiments, mRNA expression levels are determined using a method selected from the group consisting of quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), RNA sequencing, microarray analysis, in situ hybridization, and serial analysis of gene expression (SAGE).

[0084] In some embodiments, the biological sample is selected from the group consisting of a tissue sample, a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid (CSF) sample, a peritoneal fluid sample, and a cell culture sample.

[0085] EphB4-ephrin B2 inhibitors One type of receptor tyrosine kinase EphB4 and the membrane-localized ligand ephrinB2 involve bidirectional signaling (anterograde in receptor-expressing cells and retrograde in ligand-expressing cells). EphB4 belongs to the largest family of receptor tyrosine kinases and was reported to regulate neuronal migration, bone remodeling, angiogenesis, cancer progression, and metastasis through interaction with ephrinB2 ligand (Pasquale EB, Cell, 133:38-52, 2008). EphB4 and ephrinB2 expression are downregulated as early as early postnatal development in the vast majority of adult normal tissues, whereas EphB4 is overexpressed in multiple epithelial cancers, including lung, bladder, head and neck, and pancreatic cancers (Ferguson BD, et el., Growth Factors, 32:202-6, 2014). Oncogenes, including mutant Kras and PTEN deletion, induce EphB4 expression. EphB4 expression correlates with stage, grade and survival, as knockdown of EphB4 leads to cell death by apoptosis. Overexpression of the ligand ephrinB2 and its correlation with poor outcome have been reported in several cancer types. ICT increases ephrinB2 in tumor vasculature (and tumors), and high ephrinB2 prevents immune cell recruitment and therefore resistance to therapy.

[0086] Inhibition of EphB4-EphrinB2 interaction has a direct inhibitory effect on tumor cell growth in vitro and ex-vivo. In various embodiments, the EphB4-EphrinB2 pathway antagonist or agonist is selected from: (i) a soluble polypeptide comprising the extracellular domain of EphrinB2; (ii) a soluble polypeptide comprising the extracellular domain of EphB4; (iii) an antibody or fragment thereof that binds to EphB4; (iv) an antibody or fragment thereof that binds to EphrinB2; (v) a nucleic acid compound that hybridizes with EphB4 transcripts under physiological conditions and reduces EphB4 expression in cells; or (vi) a nucleic acid compound that hybridizes with EphrinB2 transcripts under physiological conditions and reduces EphrinB2 expression in cells.

[0087] In various embodiments, the EphB4-EphrinB2 pathway antagonist inhibits the interaction between EphrinB2 and EphB4. In various embodiments, the EphrinB2 / EphB4 pathway antagonist inhibits the clustering of EphrinB2 or EphB4. In various embodiments, the EphB4-EphrinB2 pathway antagonist inhibits the phosphorylation of EphrinB2 or EphB4. In various embodiments, the EphB4-EphrinB2 pathway agonist stimulates the kinase activity of EphrinB2 or EphB4. In various embodiments, the agent that inhibits EphB4 or EphrinB2-mediated function is a nucleic acid therapeutic. In some embodiments, the nucleic acid therapeutic that inhibits EphB4 or EphrinB2-mediated function is an oligonucleotide DNA or siRNA that targets EphrinB2 or EphB4.

[0088] Polypeptide agents that inhibit EphB4-mediated or ephrinB2-mediated functions have been previously described by the present inventors (see, e.g., U.S. Patent No. 7,381,410; U.S. Patent No. 7,862,816; U.S. Patent No. 7,977,463; U.S. Patent No. 8,063,183; U.S. Patent No. 8,273,858; U.S. Patent No. 8,975,377; U.S. Patent No. 8,981,062; U.S. Patent No. 9,533,026; each of which is incorporated herein by reference in its entirety for all purposes). sEphB4-HSA is a fully human fusion protein consisting of a soluble EphB4 extracellular domain fused at the C-terminus to albumin by expression as a single seamless protein of 123.3 kDa. sEphB4-HSA specifically binds to ephrinB2. Preliminary testing of sEphB4-HSA in tumor models has shown increased T-cell and NK cell migration to tumors. This is done by inducing ICAM-1 in tumor blood vessels. ICAM-1 is an integrin that promotes T cell and NK cell attack on endothelium and then migration of cells to tumors. sEphB4-HSA also shows downregulation of PI3K signaling by blocking EphB-ephrinB2 interaction in tumor cells and tumor blood vessels. sEphB4-HSA blocks the signaling, promotes immune cell trafficking to tumors, and inhibits survival signals in tumor cells by downregulating the PI3K pathway.

[0089] Targeting EphB4-ephrinB2 is a therapeutic strategy that has survived clinical trials and has been found to be safe in multiple clinical trials with minimal to no toxicity, due to low levels of expression in normal tissues (A. El-Khoueiry BG, et al., Eur J Cancer, 69, 2016). Although there is no direct evidence implicating EphB4-ephrinB2 interactions in cancer-associated immune responses, multiple reports have demonstrated that Eph / ephrin gene family members regulate immune cell processes in inflammatory models such as arteriosclerosis and wound healing (Braun J,et al.,Arterioscler Thromb Vasc Biol,31:297-305,2011;Poitz DM,et al.,Mol Immunol,68:648-56,2015;Yu G,et al.,J Immunol,171:106-14,2003;Funk SD,et al.,Arterioscler Thromb Vasc Biol,32:686-95,2012). Eph / ephrin interactions were also reported to regulate monocyte adhesion to the vascular wall transendothelial migration, T cell chemotaxis, activation, proliferation and apoptosis, and hematopoietic cell mobilization from bone marrow sinusoids.

[0090] The inventors have queried TCGA database for the expression of ephrin B2 ("EFNB2"). The expression of EFNB2 was significantly higher in tumor tissues compared to normal tissues based on the data analyzed using the Oncomin microarray database. High EFNB2 expression was significantly correlated with poor survival in bladder urothelial cancer (median OS 23.19 vs. 44.28 months). In addition, the expression of EFNB2 was correlated with decreased disease-free survival in the same population. Data validation at the protein level remains.

[0091] In some embodiments of the present invention, the polypeptide drug that inhibits EphB4 or EphrinB2-mediated function is a monomeric ligand-binding portion of EphB4 protein or EphrinB2 protein, or an antibody that binds and acts with EphB4 or EphrinB2. In some embodiments, the polypeptide drug is a soluble EphB4 (sEphB4) polypeptide that specifically binds to EphrinB2 polypeptide and comprises the amino acid sequence of the extracellular domain of EphB4 protein. In some embodiments, the sEphB4 polypeptide comprises the globular domain of EphB4 protein. In some embodiments, the drug that inhibits EphB4 or EphrinB2-mediated function is an oligonucleotide DNA or siRNA that targets EphrinB2 or EphB4.

[0092] In some embodiments, the sEphB4 polypeptide comprises a sequence selected from the group consisting of a sequence that is at least 90% identical to residues 1-522, at least 90% identical to residues 1-412, and at least 90% identical to residues 1-312 of the amino acid sequence of SEQ ID NO:1. In some embodiments, the sEphB4 polypeptide may comprise a sequence that includes a globular (G) domain (amino acids 29-197 of SEQ ID NO:1), and optionally additional domains, such as a cysteine-rich domain (amino acids 239-321 of SEQ ID NO:1), a first fibronectin type 3 domain (amino acids 324-429 of SEQ ID NO:1), and a second fibronectin type 3 domain (amino acids 434-526 of SEQ ID NO:1). In some embodiments, the sEphB4 polypeptide will comprise amino acids 1-537 of SEQ ID NO:1. In some embodiments, the sEphB4 polypeptide will comprise amino acids 1-427 of SEQ ID NO:1. In some embodiments, the sEphB4 polypeptide will comprise amino acids 1-326 of SEQ ID NO:1. In some embodiments, an sEphB4 polypeptide will include amino acids 1-197, 29-197, 1-312, 29-132, 1-321, 29-321, 1-326, 29-326, 1-412, 29-412, 1-427, 29-427, 1-429, 29-429, 1-526, 29-526, 1-537, and 29-537 of SEQ ID NO: 1. In some embodiments, an sEphB4 polypeptide will include amino acids 16-197, 16-312, 16-321, 16-326, 16-412, 16-427, 16-429, 16-526 of SEQ ID NO: 1. In some embodiments, an sEphB4 polypeptide may comprise an amino acid sequence that is at least 90%, and optionally 95% or 99% identical to any of the foregoing amino acid sequences while retaining EphrinB2 binding activity, hi some embodiments, any variation of the amino acid sequence from the sequence shown in SEQ ID NO:1 is a conservative replacement or deletion of no more than 1, 2, 3, 4 or 5 amino acids, particularly in the surface loop regions.

[0093] In some embodiments, soluble polypeptides may be prepared in multimeric form, for example, by expressing them as an Fc fusion protein or a fusion with another multimerization domain.

[0094] In some embodiments, the sEphB4 polypeptide further comprises an additional moiety that confers increased serum half-life while still retaining EphrinB2 binding activity. In some embodiments, the sEphB4 polypeptide is monomeric and covalently linked to one or more polyoxyalkylene groups (e.g., polyethylene, polypropylene). In some embodiments, the sEphB4 polypeptide is covalently linked to a single polyethylene glycol (PEG) group (hereinafter "sEphB4-PEG"). In some embodiments, the sEphB4 polypeptide is covalently linked to two, three, or more PEG groups.

[0095] In some embodiments, the one or more PEGs may have a molecular weight ranging from about 1 kDa to about 100 kDa, about 10 to about 60 kDa, and about 10 to about 40 kDa. The PEG group may be a linear PEG or a branched PEG. In some embodiments, the soluble monomeric sEphB4 complex comprises an sEphB4 polypeptide covalently linked to one PEG group of about 10 to about 40 kDa (mono-PEGylated EphB4) or about 15 to 30 kDa, preferably via the s-amino group of an sEphB4 lysine or the N-terminal amino group. In some embodiments, the sEphB4 is randomly PEGylated at one amino group selected from the group consisting of the s-amino group of an sEphB4 lysine and the N-terminal amino group.

[0096] In some embodiments, the sEphB4 polypeptide is stably associated with a second stabilizing polypeptide that confers improved half-life without substantially compromising EphrinB2 binding. In some embodiments, the stabilizing polypeptide is immunocompatible with a human patient (or an animal patient, if veterinary use is intended) and will have little or no significant biological activity. In some embodiments, the sEphB4 polypeptide is covalently or non-covalently associated with an albumin selected from the group consisting of human serum albumin (HSA) (hereinafter "sEphB4-HSA") and bovine serum albumin (BSA) (hereinafter "sEphB4-BSA").

[0097] In some embodiments, covalent binding may be achieved by expressing sEphB4 polypeptide as a co-translational fusion with human serum albumin. Albumin sequence may be fused at N-terminus, C-terminus or non-disruptive internal position in sEphB4 polypeptide. Exposed loop of sEphB4 may be suitable position for inserting albumin sequence. Albumin may be post-translationally linked to sEphB4 polypeptide, for example, by chemical cross-linking. In some embodiments, sEphB4 polypeptide may also stably associate with two or more albumin polypeptides.

[0098] In some embodiments, the sEphB4-HSA fusion inhibits the interaction of EphrinB2 with EphB4, the clustering of EphrinB2 or EphB4, the phosphorylation of EphrinB2 or EphB4, or a combination thereof. In some embodiments, the sEphB4-HSA fusion has enhanced in vivo stability relative to the unmodified wild-type polypeptide.

[0099] In some embodiments, sEphB4-HSA comprises residues 16-197 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-312 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-321 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-326 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-412 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-427 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-429 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-526 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises residues 16-537 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In some embodiments, sEphB4-HSA comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, sEphB4-HSA comprises the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, sEphB4-HSA comprises the amino acid sequence set forth in SEQ ID NO:5.

[0100] In some embodiments, sEphB4 polypeptides may be prepared in multimeric form by expressing them as fusion proteins with molecules that block signaling with Eph receptors or block Ephrin B2 interactions, including, but not limited to, antisense oligonucleotides, siRNA, and gene editing such as CRISPR / CAS.

[0101] In various embodiments, the method of the present invention may utilize a nucleic acid therapeutic agent that inhibits or reduces the gene expression of ephrin ligand and / or Eph in cancer. As used herein, the term "nucleic acid therapeutic agent" or "nucleic acid drug" or "nucleic acid compound" refers to any nucleic acid-based compound that contains nucleotides and has a desired effect on a target gene. Examples of nucleic acid therapeutic agents that are intended for use include, but are not limited to, antisense nucleic acids, dsRNA, siRNA, and enzymatic nucleic acid compounds.

[0102] In various embodiments, the present disclosure relates to antisense nucleic acid. "Antisense nucleic acid" refers to a non-enzymatic nucleic acid compound that binds to a target nucleic acid by RNA-RNA, RNA-DNA or RNA-PNA (protein nucleic acid) interactions and modifies the activity of the target nucleic acid (for review, see Stein and Cheng, 1993 Science 261,1004 and Woolf et al. US Patent No. 5,849,902). Usually, an antisense molecule is complementary to a target sequence along a single continuous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can form a loop and bind to a substrate nucleic acid that forms a loop. Thus, an antisense molecule can be complementary to two (or more) non-contiguous substrate sequences, or two (or more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence, or both. For reviews of current antisense strategies, see, e.g., Schmajuk et al., 1999, J. Biol. Chem., 274:21783-21789; Delihas et al., 1997, Nature, 15:751-753; Stein et al., 1997, Antisense NA Drug Dev., 7:151; Crooke, 2000, Methods Enzymol., 313:3-45; and Crooke, 1998, Biotech. Genet. Eng. Rev., 15:121-157.

[0103] In various embodiments, the antisense nucleic acid of the present disclosure can be delivered as, for example, an expression plasmid that produces RNA that is complementary to at least a unique portion of the cellular mRNA that codes for EphrinB2 or EphB4 polypeptide when transcribed in a cell. Alternatively, the construct is an oligonucleotide that is made ex vivo and that hybridizes with the mRNA and / or genomic sequence that codes for EphrinB2 or EphB4 polypeptide when introduced into a cell, thereby causing inhibition of expression. Such oligonucleotide probes are optionally modified oligonucleotides that are resistant to endogenous nucleases, such as exonucleases and / or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid compounds for use as antisense oligonucleotides are phosphoramidate, phosphorothioate and methylphosphonate analogs of DNA (see also U.S. Patent Nos. 5,176,996; 5,264,564; and 5,256,775). In addition, general approaches to the construction of oligomers useful in nucleic acid therapy are reviewed by, for example, van der Krol et al., (1988) Biotechniques 6:958-976; and Stein et al., (1988) Cancer Res 48:2659-2668.

[0104] In various embodiments, the present disclosure relates to double-stranded RNA (dsRNA) and RNAi constructs.As used herein, the term "dsRNA" refers to double-stranded RNA molecules that can induce RNA interference (RNAi), including siRNA (see, for example, Bass, 2001, Nature, 411:428-429; Elbashir et al., 2001, Nature, 411:494-498; and Kreutzer et al., WO 00 / 44895; Zernicka-Goetz et al., WO 01 / 36646; and Li et al., WO 00 / 44914).In addition, RNAi is a term that was first applied to the phenomenon observed in plants and worms, where double-stranded RNA (dsRNA) specifically and post-transcriptionally blocks gene expression.RNAi provides a useful method of inhibiting gene expression in vitro or in vivo.

[0105] As used herein, the term "short interfering RNA", "siRNA" or "short interfering nucleic acid" refers to any nucleic acid compound that can mediate RNAi or gene silencing when properly processed into cells. For example, siRNA can be a double-stranded polynucleotide molecule that comprises self-complementary sense and antisense regions, and the antisense region comprises complementarity with a target nucleic acid compound (e.g., Ephrin B2 or EphB4). siRNA can be a single-stranded hairpin polynucleotide with self-complementary sense and antisense regions, and the antisense region comprises complementarity with a target nucleic acid compound. siRNA can be a circular single-stranded polynucleotide with a stem that comprises two or more loop structures and self-complementary sense and antisense regions, and the antisense region comprises complementarity with a target nucleic acid compound, and the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA that can mediate RNAi. The siRNA can also comprise a single-stranded polynucleotide having complementarity to a target nucleic acid compound, which can further comprise a terminal phosphate group, such as a 5'-phosphate (see, e.g., Martinez et al., 2002, Cell., 110, 563-574), or a 5',3'-diphosphate.

[0106] The applicants have produced several monoclonal antibodies against EphB4 as well as hybridoma cell lines producing EphB4 monoclonal antibodies. These antibodies have been further characterized in a number of ways, such as their ability to inhibit the interaction of EphB4 with its ligand (e.g., EphrinB2), their ability to inhibit the dimerization or multimerization of EphB4 receptor, their ability to induce tyrosine phosphorylation of EphB4, their cross-reactivity with other Eph family members, their ability to inhibit angiogenesis, and their ability to inhibit tumor growth. Furthermore, epitope mapping studies reveal that these EphB4 antibodies can specifically bind to one or more regions of EphB4 (e.g., globular domain, cysteine-rich domain, or fibronectin type III domain). For example, EphB4 antibodies can bind to both fibronectin type III domains. Such antibodies are described, for example, in US Patent Application Publication No. 2005 / 0249736, US Patent Application Publication No. 2009 / 0196880, US Patent No. 8,273,858, US Patent No. 8,981,062 and US Patent No. 8,975,377.

[0107] In various embodiments, the method of the present invention comprises administering a therapeutically effective amount or effective dose of the sEphB4-HSA polypeptide of the present invention to a patient in need of treatment. In various embodiments, the effective dose of the polypeptide of the present invention, for example for primary or metastatic cancers as described herein, will depend on many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, whether other drugs are administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Therapeutic dosages need to be titrated to optimize safety and efficacy.

[0108] In various embodiments, the dosage may range from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5.0 mg / kg of host body weight. For example, the dosage may be 1 mg / kg or 10 mg / kg body weight or within the range of 1 to 10 mg / kg body weight. In various embodiments, the dosage of the polypeptide administered to the patient is selected from the group consisting of about 0.5 mg / kg, about 1.0 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 3.0 mg / kg, about 3.5 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 7.0 mg / kg, about 8.0 mg / kg, about 9.0 mg / kg, and about 10.0 mg / kg. In various embodiments, the treatment regime entails administration once every two weeks or once a month or once every 3 to 6 months. The therapeutic entity of the present invention is usually administered multiple times. The interval between single administrations can be weekly, biweekly, monthly or yearly. The interval can be irregular, as indicated by measuring the blood level of the therapeutic entity in the patient. Alternatively, the therapeutic entity of the present invention can be administered as a sustained release formulation, in which case it does not need to be administered as frequently. The dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0109] Immunostimulants Some immune checkpoint protein antigens are, for example, SIRP (expressed by macrophages, monocytes, dendritic cells), CD47 (highly expressed by tumor cells and other cell types), VISTA (expressed by monocytes, dendritic cells, B cells, T cells), TIGIT (an immune receptor present on some T cells and natural killer cells), CD152 (expressed by activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed by tumor-infiltrating lymphocytes, activated CD274 (expressed by T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, pancreatic islet cells), and CD223 (expressed by activated T cells, regulatory T cells, anergic T cells, NK cells, NKT cells, and plasmacytoid dendritic cells) have been reported to be expressed on various immune cells (see, e.g., Pardoll, D., Nature Reviews Cancer, 12:252-264, 2012), including activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, anergic T cells, monocytes, and dendritic cells). LAG3, TIM3, TIGIT, OX40 ligand, interferon, and IL-2.

[0110] Antibodies that bind to antigens that have been determined to be immune checkpoint proteins are known to those of skill in the art. For example, various anti-CD276 antibodies have been described in the art (e.g., US Patent Application Publication No. 2012 / 0294796 (Johnson et al) and references cited therein); various anti-CD272 antibodies have been described in the art (e.g., US Patent Application Publication No. 2014 / 0017255 (Mataraza et al) and references cited therein); various anti-CD152 / CTLA-4 antibodies have been described in the art (e.g., US Patent Application Publication No. 2013 / 0136749 (Korman et al) and references cited therein); various anti-LAG-3 / CD223 antibodies have been described in the art (e.g., US Patent Application Publication No. 2011 / 0150892 (Thudium et al) and references cited therein); various anti-CD279 (PD-1) antibodies have been described in the art (e.g., US Patent Application Publication No. 7,488,802 (Collins et al) and references cited therein). al) and the references cited therein); various anti-PD-L1 antibodies have been described in the art (e.g., US Patent Application Publication No. 2013 / 0122014 (Korman et al) and the references cited therein); various anti-TIM-3 antibodies have been described in the art (e.g., US Patent Application Publication No. 2014 / 0044728 (Takayanagi et al) and the references cited therein); and various anti-B7-H4 antibodies have been described in the art (e.g., US Patent Application Publication No. 2011 / 0085970 (Terrett et al) and the references cited therein). Each of these references is incorporated herein by reference in its entirety with respect to the specific antibodies and sequences taught therein.

[0111] Immune checkpoint inhibitor targeted agents are effective in patients with tumors that express an inflammatory signature and contain resident immune cells, commonly referred to as "hot tumors." Tumors with few or no immune cells (cold tumors) are less likely or unlikely to respond. Immune checkpoint PD-1 and CTLA inhibitors are effective in some cancers that express an interferon-gamma signature, are enriched in tumor-infiltrating immune cells, and express PD-L1. Tumor vasculature regulates the egress of immune cells into tumors, and thus tumor vasculature modulation may provide an avenue to alter the tumor environment.

[0112] PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1 expressed on the cell surface of activated T cells under healthy conditions is to downregulate unwanted or excessive immune responses, including autoimmune responses. Ligands for PD-1 (PD-L1 and PD-L2) can be constitutively expressed or induced in various cell types, including non-hematopoietic tissues, as well as in various tumors. Binding of any of the PD-1 ligands to PD-1 inhibits T cell activation triggered by the T cell receptor. PD-1 has been suggested to regulate tumor-specific T cell expansion in subjects with melanoma (MEL). This suggests that the PD-1 / PD-L1 pathway plays an important role in tumor immune evasion and should be considered as an attractive target for therapeutic intervention.

[0113] Pembrolizumab (KEYTRUDA®) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4 / κ isotype designed to directly block the interaction of PD-1 with its ligands, PD-L1 and PD-L2. The U.S. Food and Drug Administration (FDA) approved KEYTRUDA® on August 5, 2016 for the treatment of some patients with advanced forms of head and neck cancer. The approval is for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) that has continued to progress despite standard treatment with chemotherapy agents. KEYTRUDA® was recently approved in the U.S. for the treatment of patients with unresectable or metastatic melanoma and disease progression after ipilimumab and, if BRAF V600 mutation-positive, after a BRAF inhibitor.

[0114] Nivolumab (OPDIVO®) is a human IgG4 anti-PD-1 monoclonal antibody that acts as a checkpoint inhibitor, blocking signals that would prevent activated T cells from attacking the cancer, thus allowing the immune system to get rid of the cancer. OPDIVO® is used as a first-line treatment for inoperable or metastatic melanoma, in combination with ipilimumab as a second-line treatment after treatment with ipilimumab when the cancer does not have a mutation in BRAF, and in combination with a BRAF inhibitor as a second-line treatment for squamous non-small cell lung cancer and for renal cell carcinoma when the cancer has a mutation in BRAF.

[0115] In various embodiments, the PD-1 inhibitor used in the combination treatment method is selected from the group consisting of: but not limited to, nivolumab (Bristol-Myers Squibb) (Drugbank 09035; Drugbank 06132), pembrolizumab (Merck) (Drugbank 09037) and pidilizumab (Medivation) (Drugbank 15383).

[0116] In various embodiments, the CTLA-4 inhibitor is selected from the group consisting of, but not limited to, ipilimumab (Bristol-Myers Squibb) (Drugbank 06186) and tremelimumab (MedImmune) (Drugbank 11771).

[0117] cancer Urothelial cancer, with an incidence of 80,470 cases per year, is responsible for 17,670 deaths per year and remains a significant health challenge in the United States (Siegel RL, et al., Cancer J Clin. 2019;69(1):7-34, 2019). If untreated, patients have a median survival of approximately 4.5 months. If treated with cytotoxic chemotherapy, survival increases to approximately 7.5 months, with an ORR of approximately 15% and a PFS of 3 to 3.5 months. However, cytotoxic chemotherapy results in substantial toxicity. Combination cytotoxic chemotherapy moderately improves response rates without improving survival, but with worsening toxicity. As a result, before the advent of immunotherapy, single-agent therapy was more effective than combination therapy for previously treated metastatic urothelial patients. The most commonly used single agents in the United States included gemcitabine, paclitaxel, and docetaxel.

[0118] In the metastatic setting, the standard of care in the first-line setting remains cisplatin-based chemotherapy, unchanged since 2000. Several single agents, such as vinflunine (ORR 18%, OS 6.6 months), gemcitabine (ORR 11%, OS 8.7 months), pemetrexed (ORR 28%, OS 9.6 months), paclitaxel (ORR 10%, OS 7.2 months), as well as combination regimens, such as paclitaxel with methotrexate (ORR 32%, OS 5 months) or gemcitabine (ORR 47%, OS 7.5 months) or docetaxel with ifosfamide (ORR 25%, OS 4 months), have been tested after failure of first-line therapy. Based on safety and efficacy, the most commonly used agents are paclitaxel, docetaxel, and carboplatin. The response rate is about 10-15%, and the overall survival is 6-9 months. Combination therapy has resulted in higher response rates, greater toxicity, but no improvement in survival (Raggi D, et al., Ann Oncol.27(1):49-61,2016). Only with the approval of anti-PD1 / PDL1 antibodies, a more durable second-line option with survival benefit became available for previously treated metastatic urothelial carcinoma patients. Five different drugs, including pembrolizumab, nivolumab, atezolizumab, avelumab, and durvalumab, are used in clinical practice, with a median survival of 10.3 months or less and a response rate of 21.1% expected. Pembrolizumab has been approved for this patient population and has been effective in only a small number of patients with a median overall survival (OS) of 10.3 months (95% CI, 8 to 11.8), median overall progression-free survival of 2.1 months (95% CI, 2.0 to 2.2), an overall response rate (ORR) of 21.1% (95% CI, 16.4 to 26.5), and a complete response rate of 7% in this patient population.

[0119] Hepatocellular carcinoma (HCC) is the most common cancer in certain regions of the world and the fifth most common cancer worldwide. Globally, it is the second leading cause of cancer death in men and the sixth leading cause of cancer death in women (see, e.g., Parkin DM, Lancet Oncology, 2:533-43, 2001). HCC is often diagnosed late in the clinical course, and only 10-15% of patients are candidates for curative surgery. For the majority of HCC patients, systemic chemotherapy or supportive care are the mainstay treatment options. Overall, HCC is highly refractory to treatment, with most chemotherapeutic agents showing limited efficacy and failing to improve patient survival (see, e.g., Gish RGet al., J.of Clinical Oncology 25:3069-75, 2007; Ramanathan RKet al., J.of Clinical Oncology 24:4010, 2006). Recent studies evaluating the programmed death 1 (PD-1) antibody nivolumab (OPDIVO®) demonstrated response rates of approximately 10-20%. Response durations ranged from 14-17+ months for CR, <1-8+ months for PR, and 1.5-17 months for stable disease (SD). The 6-month overall survival (OS) rate is 72%. Nivolumab demonstrated a manageable AE profile, yielded durable responses across all dose levels and HCC cohorts, and had a favorable 6-month OS rate.

[0120] Head and neck squamous cell carcinoma (HNSCC) accounts for nearly 90% of cancers involving the upper aerodigestive tract (UADT). In 2005, cancers of the oral cavity, pharynx, and larynx are estimated to account for nearly 3% of incident cancers and 2% of cancer deaths in the United States. Approximately 500,000 new cases are diagnosed worldwide each year. Men are affected more than twice as often as women. More than half of these cancers involve the oral cavity. The remainder are divided evenly between the larynx and pharynx. Numerous clinical trials are testing the effects of immunotherapy in human cancers, including head and neck squamous cell carcinoma (HNSCC). The objective response rate is between 6 and 20% (Szturz P,et al.,BMC Med,15:110,2017;Ferris RL,et al.,Oral Oncol,81:45-51,2018;Postow MA,et al.,J Clin Oncol,33:1974-82,2015;Chow LQM,et al.,J Clin Oncol,34:3838-45,2016;Siu LL,et al.,JAMA Oncol 2018), and the majority of patients show either innate or adaptive resistance to immunotherapy. Attempts to simply combine more immune checkpoint inhibitors have also proven disappointing due to increased toxicity and lack of further benefit to patients (https: / / clinicaltrials.gov / ct2 / show / NCT02205333). In an orthotopic mouse model of HNSCC, we recently demonstrated that tumor growth occurred even after combined treatment with anti-PDL1 antibodies and radiation therapy (RT) (7, 8; Oweida A, et al., Clin Cancer Res, 2018; Messenheimer DJ, et al., Clin Cancer Res, 23:6165-77, 2017).

[0121] Although radiation therapy remains the standard of care in the reliable management of patients with locally advanced HNSCC and can act as an adjuvant for immunotherapy, there are several undesirable effects that occur in response to RT that in turn impair the efficacy of immunotherapeutic drugs. RT cannot overcome the accumulation of immune suppressive populations such as Tregs in the late (convalescent) phase (7). Therefore, the discovery of other treatments that synergize with RT and attenuate its adverse effects is crucial to overcome adverse side effects, treatment regimes, and tumor regrowth.

[0122] The 5-year survival rate for HNSCC is poor and has not improved in decades. In addition, patients with the disease experience several morbidities, including disfiguring, speaking, swallowing, and breathing problems. The late stage of diagnosis and tendency to recur are challenges that frustrate efforts to improve outcomes in these patients. Pembrolizumab is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4 / κ isotype designed to directly block the interaction of PD-1 with its ligands, PD-L1 and PD-L2. The U.S. Food and Drug Administration (FDA) approved pembrolizumab (KEYTRUDA®) on August 5, 2016, for the treatment of some patients with advanced forms of head and neck cancer. The approval is for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) that has continued to progress despite standard treatment with chemotherapy drugs. According to the FDA approval summary, 28 patients (16%) experienced a tumor response following treatment with pembrolizumab. In 23 of 28 patients (82%), tumor responses lasted for more than 6 months, and some lasted for more than 2 years. HNSCC patients whose tumors are positive for human papillomavirus (HPV) usually have better outcomes after treatment with chemotherapy than patients whose tumors are HPV-negative. Responses were seen in patients with HPV-positive tumors as well as in patients with HPV-negative tumors (24% and 16%, respectively), according to the FDA approval summary.

[0123] Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Squamous cell carcinoma, adenocarcinoma, and large cell carcinoma are all subtypes of NSCLC. NSCLC accounts for 85% of all lung cancers. As a classification, NSCLC is relatively insensitive to chemotherapy compared to small cell lung cancer. They are primarily treated by surgical resection with curative intent, although chemotherapy is increasingly being used both before surgery (neoadjuvant chemotherapy) and after surgery (adjuvant chemotherapy) when possible. On October 2, 2015, the FDA approved pembrolizumab for the treatment of metastatic non-small cell lung cancer (NSCLC) in patients whose tumors express PD-L1 and who have failed treatment with other chemotherapy agents. In October 2016, pembrolizumab became the first immunotherapy used in the treatment of NSCLC if the cancer overexpresses PDL1 and the cancer does not have a mutation in EGFR or ALK; chemotherapy has already been administered and then pembrolizumab can be used as a second-line treatment, but if the cancer has an EGFR or ALK mutation, an agent targeting these mutations should be used first. Assessment of PDL1 must be performed with a validated and approved companion diagnostic. The KEYNOTE-001 trial (NTC01295827) evaluated the efficacy and safety of programmed cell death 1 (PD-1) inhibition with pembrolizumab in patients with advanced non-small cell lung cancer. Among all patients, the objective response rate was 19.4%, with a median duration of response of 12.5 months. The median progression-free survival was 3.7 months, and the median overall survival was 12.0 months. PD-L1 expression on at least 50% of tumor cells was selected as the cutoff from the training group. Among patients with a proportion score of at least 50% in the validation group, the response rate was 45.2%. Among all patients with a proportion score of at least 50%, the median progression-free survival was 6.3 months; the median overall survival was not reached. PD-L1 expression on at least 50% of tumor cells correlated with improved efficacy of pembrolizumab (Garon et al., N Engl J Med, 372:2018-2028, 2015).

[0124] Prostate cancer is the most common non-skin malignant tumor in men and the second leading cause of cancer death in men in the Western world. Prostate cancer results from the uncontrolled proliferation of abnormal cells in the prostate. Once prostate cancer tumors develop, androgens such as testosterone promote prostate cancer tumor growth. In its early stages, localized prostate cancer is often treated with localized treatments, including, for example, surgical removal of the prostate and radiation therapy. However, if localized treatments are unsuccessful in curing prostate cancer, as is the case in less than one-third of men, the disease progresses to incurable metastatic disease (i.e., disease in which the cancer has spread from one part of the body to another). As used herein, the term "prostate cancer" is used in a broad sense to refer to all stages and all forms of cancer that originate from the tissues of the prostate. The term "prostate cancer" includes any type of malignant (i.e., non-benign) tumor localized in prostate tissue, such as, for example, prostatic carcinoma, prostatic sarcoma, undifferentiated prostate cancer, prostatic squamous cell carcinoma, prostatic ductal transitional carcinoma, and prostatic intraepithelial neoplasia.

[0125] Kaposi's sarcoma (KS) is a multifocal vascular proliferative disease of the vascular endothelium most commonly associated with infection with Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV-8). KS is associated with several epidemiological and pathophysiological factors. KS is classified into four distinct clinical types: classical Mediterranean KS, African endemic KS, immunosuppressant-associated KS, and HIV-associated KS. A rare disease before the era of HIV and AIDS, HIV-associated KS is the most frequent malignancy in HIV-infected patients. KS can affect many organs. KS most frequently manifests as a skin disease. In many advanced cases, KS involves organs such as the lungs, liver, or gastrointestinal tract. At this point, KS is incurable. Available treatments are palliative. Systemic chemotherapy is generally used in patients with more advanced disease or evidence of rapid disease progression. The primary goals of treatment are symptom relief, prevention of disease progression, and reduction of tumor burden to relieve lymphedema, organ damage, and psychological stress. Standard treatment for visceral or cutaneous KS includes cytotoxic chemotherapy, such as liposomal anthracyclines and paclitaxel. Liposomal doxorubicin has superior efficacy and favorable tolerability and toxicity compared with the combination of nonliposomal doxorubicin, vincristine, and bleomycin, with an overall response rate of 59% in HIV patients. In classical KS, the response rate to liposomal doxorubicin may be higher. However, complete response rates are rare, and there is no cure. At this time, there is no fully developed targeted therapy for KS.

[0126] In 2014, 46,420 new cases of pancreatic cancer are expected to be diagnosed in the United States, with an estimated 39,590 deaths from the disease. Surgical resection is the only potentially curative treatment, but only 15-20% of patients have respectable disease at diagnosis, and treatment for unresectable locally advanced and metastatic pancreatic cancer remains primarily palliative. Gemcitabine monotherapy has been used as the reference regimen for the treatment of advanced pancreatic cancer following randomized trials and has shown clinical efficacy as well as a survival benefit of approximately one month when compared with single-agent fluorouracil. Combination therapy with gemcitabine-based regimens for locally advanced and metastatic pancreatic cancer has been shown in meta-analyses to provide a modest benefit in overall survival (OS) despite more frequent toxicities, with some evidence suggesting improved efficacy with combination regimens in patients with good performance status. One such combination regimen is gemcitabine plus albumin-bound paclitaxel (nab-paclitaxel). In the phase 3, open-label MPACT trial, 861 patients were randomized 1:1 to receive gemcitabine (1000 mg per square meter of body surface area or 1000 mg / m 2 ) alone or gemcitabine (1000mg / m 2 ) + nab-paclitaxel (125 mg / m 2 ) intravenous infusion. The combination group had an increased median overall survival of 8.5 months compared with 6.7 months in the single-agent group, but higher grade neutropenia, fatigue, and neuropathy were seen in the former. In the combination group, 41% of patients had their nab-paclitaxel dose reduced, and 47% had their gemcitabine dose reduced. With an increase in OS of 1.8 months, this trial led to the U.S. Food and Drug Administration (FDA) approval of nab-paclitaxel for the treatment of late-stage pancreatic cancer in 2013. An updated OS analysis of the MPACT trial published in 2015 confirmed a longer median OS of 8.7 months in the nab-paclitaxel and gemcitabine combination group compared with 6.6 months in the gemcitabine monotherapy group.

[0127] In various embodiments, the cancer is selected from the group consisting of, but not limited to, non-small cell lung cancer (NSCLC), colon cancer, metastatic urothelial carcinoma, breast cancer, hepatocellular carcinoma (HCC), mesothelioma, pancreatic cancer, prostate cancer, bladder cancer, head and neck squamous cell carcinoma (HNSCC), Kaposi's sarcoma, and leukemia.

[0128] In various embodiments, the patient previously responded to treatment with an anti-cancer therapy, but relapsed upon cessation of treatment (hereinafter "recurrent proliferative disease").

[0129] In various embodiments, the patient has a resistant or refractory cancer. In various embodiments, the cancer is refractory to treatment with an immunotherapy. In various embodiments, the cancer is refractory to treatment with a chemotherapeutic agent. In various embodiments, the cancer is refractory to treatment with a depleting antibody against a specific tumor antigen. In various embodiments, the cancer is refractory to treatment with an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint). In various embodiments, the cancer is refractory to targeted therapy with an antibody-drug conjugate (ADC) or a fusion molecule comprising a depleting antibody against a specific tumor antigen and a cytotoxic agent. In various embodiments, the cancer is refractory to targeted therapy with a small molecule kinase inhibitor. In various embodiments, the cancer is refractory to combination therapy including, for example, two or more of: immunotherapy treatment, treatment with a chemotherapeutic agent, treatment with a depleting antibody against a specific tumor antigen, treatment with an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint), treatment with an immunoconjugate, ADC, or fusion molecule comprising a depleting antibody against a specific tumor antigen and a cytotoxic agent, targeted therapy with a small molecule inhibitor, treatment with surgery, treatment with a therapeutic vaccine, treatment with hepatocyte transplantation, and treatment with radiation.

[0130] Combination therapy In some embodiments, the method of treating or delaying the progression of cancer in a subject further comprises a second therapy selected from the group consisting of: small molecule kinase inhibitor targeted therapy, surgery, cytoreductive therapy, cytotoxic chemotherapy, and immunotherapy. In some embodiments, the combination therapy will have a synergistic effect. In some embodiments, the second therapy is a cytoreductive therapy, and the combination may increase the therapeutic index of the cytoreductive therapy. In some embodiments, the cytoreductive therapy may act on a DNA repair pathway. In some embodiments, the cytoreductive therapy is radiation therapy. In some embodiments, the combination may have a synergistic effect.

[0131] In some embodiments, the combination therapy includes anti-proliferative or cytoreductive therapy. Anti-proliferative or cytoreductive therapy is used to therapeutically eliminate tumor cells and other undesirable cells in the host, and includes the use of treatments such as delivery of ionizing radiation and administration of chemotherapy drugs. For example, ionizing radiation (IR) is used to treat about 60% of cancer patients by depositing energy that injures or destroys cells in the treated area, and for the purposes of the present invention, it may be delivered at conventional doses and regimens, or at reduced doses. Radiation damage to cells is non-specific and has complex effects on DNA. The effectiveness of the treatment depends on the cytotoxicity to cancer cells, which are larger than normal cells. Radiotherapy may be used to treat all types of cancer. Some types of radiotherapy include photons, such as X-rays or gamma rays. Another technique for radiation delivery to cancer cells is internal therapy, which places radioactive implants directly into the tumor or body cavity so that the radiation dose is concentrated in a small area. A suitable dose of ionizing radiation may range from at least about 2 Gy to no more than about 10 Gy, and is usually about 5 Gy. A suitable dose of UV radiation is at least about 5 J / m 2 ~about 50J / m 2 can range from 10 J / m to 100 J / m. 2 Samples may be collected at least about 4 hours and up to about 72 hours after exposure to UV light, typically about 4 hours.

[0132] Chemotherapeutic drugs are well known in the art and are used in conventional doses and regimens or reduced doses or regimens, including topoisomerase inhibitors such as anthracyclines, including compounds daunorubicin, adriamycin (doxorubicin), epirubicin, idarubicin, MEN10755, and the like. Other topoisomerase inhibitors include podophyllotoxin analogs etoposide and teniposide, as well as anthracenedione, mitoxantrone, and amsacrine. Other antiproliferative agents interfere with microtubule polymerization, such as the family of vinca alkaloids. Examples of vinca alkaloids include vinblastine, vincristine; vinorelbine (NAVELBINE); vindesine; vindoline; vincamine; and others. DNA damaging agents include nucleotide analogs, alkylating agents, and others. Alkylating agents include nitrogen mustards, such as mechlorethamine, cyclophosphamide, melphalan (L-sarcolysin), and the like; and nitrogen sources, such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, and the like. Nucleotide analogs include pyrimidines, such as cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FUdR), etc.; purines, such as thioguanine (6-thioguanine), mercaptopurine (6-MP), pentostatin, fluorouracil (5-FU), etc.; and folic acid analogs, such as methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, etc. Other chemotherapeutic agents of interest include metal complexes, such as cisplatin (cis-DDP), carboplatin, oxaliplatin, etc.; ureas, such as hydroxyurea; gemcitabine, and hydrazines, such as N-methylhydrazine. In some embodiments, the dosage of such chemotherapy agents is approximately 10 mg / m 2 , 20 mg / m 2 , 30 mg / m2 , 40 mg / m 2 , 50 mg / m 2 , 60 mg / m 2 , 75 mg / m 2 , 80 mg / m 2 , 90 mg / m 2 , 100 mg / m 2 , 120 mg / m 2 , 150 mg / m 2 , 175 mg / m 2 , 200 mg / m 2 , 210 mg / m 2 , 220 mg / m 2 , 230 mg / m 2 , 240 mg / m 2 , 250 mg / m 2 , 260 mg / m 2 , and 300 mg / m 2 These include, but are not limited to, any of the following:

[0133] In some embodiments, the combination therapy will include immunotherapy. As used herein, the term "immunotherapy" refers to, but is not limited to, treatment with depleting antibodies against specific tumor antigens (see, e.g., reviews by Blattman and Greenberg, Science, 305:200, 2004; Adams and Weiner, Nat Biotech, 23:1147, 2005; Vogal et al. J Clin Oncology, 20:719, 2002; Colombat et al., Blood, 97:101, 2001); treatment with antibody-drug conjugates (see, e.g., Ducrry, Laurent (Ed.) Antibody Drug Conjugates In: Methods in Molecular Biology. Book 1045. New York (NY), Humana Press, 2013; Nature Reviews Drug Discovery 12, 259-260, April 2001); 2013); CTLA-4 (ipilimumab), PD-1 (nivolumab; pembrolizumab; pidilizumab and PD-L1 (BMS-936559; MPLD3280A; MEDI4736; MSB0010718C) (see, e.g., Philips and Atkins, International Immunology, 27(1); 39-46, Oct 2014), OX-40, CD137, GITR, LAG3, TIM-3, and VISTA (see, e.g., Sharon et al., Chin J Cancer., 33(9):434-444, Sep 2014; Hodi et al., N Engl J Med, 2010; Topalian et al.,N Engl J Med,366:2443-54,2012); treatment with bispecific T cell engaging antibodies (BiTE®) such as blinatumomab (see, e.g., U.S. Pat. No. 9,260,522; U.S. Patent Application Publication No. 2014 / 0302037); treatment involving administration of IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β and IFN-γ biological response modifiers (see, e.g., Sutlu T et al.,,Journ of Internal Medicine,266(2):154-181,2009; Joshi S PNAS USA,106(29):12097-12102,2009; Li Y et al.,Journal of Translational Medicine,266(2):154-181,2009; Medicine, 7:11, 2009); treatment with therapeutic vaccines such as sipuleucel-T (see, e.g., Kantoff PW New England Journal of Medicine, 363(5):411-422, 2010; Schlom J., Journal of the National Cancer Institutes, 104(8):599-613, 2012); treatment with dendritic cell vaccines, treatment with oncolytic virus therapy (e.g., T-VEC); treatment with tumor antigen peptide vaccines; treatment with chimeric antigen receptor (CAR)-T cells (see, e.g., Rosenberg SA Nature Reviews Cancer, 8(4):299-308, 2008; Porter DL et al, New England Journal of Medicine, 365(8):725-733, 2011; Grupp SA et al., New England Journal of Medicine, 368(16):1509-151, 2013; U.S. Patent No. 9,102,761; U.S. Patent No. 9,101,584); treatment with CAR-NK cells (see, e.g., Glienke et al., Front Pharmacol, 6(21):1-7, Feb 2015); treatment with tumor infiltrating lymphocytes (TIL) (see, e.g., Wu et al, Cancer J., 18(2):160-175, 2012); treatment with matched transplanted anti-tumor T cells (ex vivo expanded and / or TCR transgenic) (see, e.g., Wrzesinski et al., J Immunother, 33(1):1-7, 2010); treatment with TALL-104 cells; and treatment with immune stimulants such as the Toll-like receptor (TLR) agonists CpG and imiquimod (see, e.g., Krieg, Oncogene, 27:161-167, 2008; Lu, Front Immunol, 5(83):1-4, March 2014).

[0134] Immunotherapy focused on the use of depleting antibodies against specific tumor antigens has been investigated with much success (see, e.g., reviews by Blattman and Greenberg, Science, 305:200, 2004; Adams and Weiner, Nat Biotech, 23:1147, 2005). Some examples of such tumor antigen-specific depleting antibodies are HERCEPTIN® (anti-Her2 / neu mAb) (Baselga et al., J Clin Oncology, Vol 14:737, 1996; Baselga et al., Cancer Research, 58:2825, 1998; Shak, Semin. Oncology, 26 (Suppl12):71, 1999; Vogal et al. J Clin Oncology, 20:719, 2002); and RITUXAN® (anti-CD20 mAb) (Colombat et al., Blood, 97:101, 2001). Unfortunately, although apparently successful in tumor treatment as monotherapy, only about 30% of individuals have been successful in total, with partial responses. Moreover, many individuals ultimately fail or relapse after treatment with these antibody-containing regimens.

[0135] Treatment with agonistic, antagonistic, or blocking antibodies against costimulatory or coinhibitory molecules (immune checkpoints) has been an area of ​​extensive research and clinical evaluation. Under normal physiological conditions, immune checkpoints are essential for maintaining self-tolerance (i.e., preventing autoimmunity) and protecting tissues from damage when the immune system responds to pathogenic infections. It is now clear that tumors also co-opt specific immune checkpoint pathways as a major mechanism of immune tolerance, especially to T cells that are specific for tumor antigens (Pardoll DM., Nat Rev Cancer, 12:252-64, 2012). Thus, for example, CTLA-4 (ipilimumab), PD-1 (nivolumab; pembrolizumab; pidilizumab) and PD-L1 (BMS-936559; MPLD3280A; MEDI4736; MSB0010718C) (see, e.g., Philips and Atkins, International Immunology, 27(1);39-46, Oct 2014), as well as OX-40, CD137, GITR, LAG3, TIM-3, and VISTA (see, e.g., Sharon et al., Chin J Cancer., 33(9):434-444, Sep 2014; Hodi et al., N Engl J Med, 2010; Topalian et al., N Engl J Therapies utilizing antibodies against immune checkpoint molecules, including those responsible for immune checkpoints (see, e.g., IL-1, IL-2, and IL-3), are being evaluated as novel immunotherapeutic alternatives for treating patients with proliferative diseases such as cancer, particularly those with refractory and / or recurrent cancers.

[0136] In various embodiments, about 0.1 mg / kg to about 10 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 1 mg / kg to about 15 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 3 mg / kg to about 12 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 1 mg / kg to about 10 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 3 mg / kg to about 10 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 1 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 2 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 3 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 5 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 10 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 10 mg / kg to about 400 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 50 mg / kg to about 400 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 10 mg / kg to about 300 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 50 mg / kg to about 300 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 10 mg / kg to about 250 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 50 mg / kg to about 250 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 50 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 100 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 150 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 200 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 250 mg / kg of the PD-1 inhibitor is administered. In various embodiments, at least about 300 mg / kg of the PD-1 inhibitor is administered. In various embodiments, the PD-1 inhibitor is administered at least once per cycle. In various embodiments, the PD-1 inhibitor is administered at least twice per cycle. In various embodiments, the cycle is 21 days. In various embodiments, the cycle is 28 days.In various embodiments, the PD-1 inhibitor is administered at least once per week. In various embodiments, the PD-1 inhibitor is administered at least once every two weeks. In various embodiments, the PD-1 inhibitor is administered at least once every three weeks. In various embodiments, the PD-1 inhibitor is administered at least once every four weeks.

[0137] Treatment with chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy in which the patient's own T cells are isolated in the laboratory, redirected with a synthetic receptor to recognize a specific antigen or protein, and then reinfused back into the patient. CARs are synthetic molecules that minimally contain (1) an antigen-binding region derived from a conventional antibody, (2) a transmembrane domain to anchor the CAR to the T cell, and (3) one or more intracellular T-cell signaling domains. CARs rely on human leukocyte antigens (HLA) to redirect T-cell specificity to the antigen, overcoming issues with T-cell tolerance (Kalos M and June CH, Immunity, 39(1):49-60, 2013). Over the last 5 years, at least 15 clinical trials of CAR-T-cell therapy have been published. A new wave of excitement around CAR T cell therapy began in August 2011, when researchers at the University of Pennsylvania (Penn) published a report of three patients with refractory chronic lymphocytic leukemia (CLL) who were in long-lasting remission after a single injection of CD19-directed CAR T cells (Porter DL, et al., N Engl J Med., 365(8):725-733, 2011).

[0138] In contrast to donor T cells, natural killer (NK) cells have been found to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). Therefore, alloreactive NK cells are also a focus of considerable interest as suitable and potent effector cells for cell therapy of cancer. Several human NK cell lines have been established, e.g., NK-92, HANK-1, KHYG-1, NK-YS, NKG, YT, YTS, NKL and NK3.3 (Kornbluth, J., et al., J. Immunol. 134, 728-735, 1985; Cheng, M. et al., Front. Med. 6:56, 2012), and various CAR-expressing NK cells (CAR-NK) have been generated. Immunotherapy using CAR-expressing NK cells (CAR-NK) is an active area of ​​research and clinical evaluation (see, e.g., Glienke et al., Front Pharmacol, 6(21):1-7, Feb 2015).

[0139] Bispecific T cell inducer molecules (BiTEs) constitute a class of bispecific single chain antibodies for polyclonal activation and redirection of cytotoxic T cells against pathogenic target cells. BiTEs are bispecific for surface target antigens on cancer cells and for CD3 on T cells. BiTEs can connect any type of cytotoxic T cell to cancer cells, regardless of T cell receptor specificity, costimulation, or peptide antigen presentation, a unique property that has not yet been reported for any other type of bispecific antibody construct, namely, exceptional potency and efficacy against target cells at low T cell numbers without the need for T cell costimulation (Baeuerle et al., Cancer Res, 69(12):4941-4, 2009). BiTE antibodies have been constructed to date against over 10 different target antigens, including CD19, EpCAM, Her2 / neu, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or HMW-MAA). (Id.) Treatment with BiTE® antibodies such as blinatumomab (Nagorsen, D. et al., Leukemia & Lymphoma 50(6):886-891, 2009) and solitomab Amann et al., Journal of Immunotherapy 32(5):452-464, 2009) is being clinically evaluated.

[0140] In some embodiments, the second treatment will include administering a PARP inhibitor. Poly (ADP-ribose) polymerase (PARP) is a family of enzymes involved in various activities in response to DNA damage. PARP-1 is a key DNA repair enzyme that mediates single-strand break (SSB) repair by base excision repair (BER) pathway. PARP inhibitors have been demonstrated to selectively kill tumor cells that fix BRCA1 and BRCA2 mutations. In addition, preclinical and preliminary clinical data suggest that PARP inhibitors are selectively cytotoxic to tumors with homologous recombination repair deficiency caused by the dysfunction of genes other than BRCA1 or BRCA2. In some embodiments, the PARP inhibitor is selected from the group consisting of ABT-767, AZD2461, BGB-290, BGP15, CEP9722, E7016, E7449, fluzoparib, INO1001, JPI289, MP124, niraparib, olaparib, ONO2231, rucaparib, SC101914, talazoparib, veliparib, WW46, or salts or derivatives thereof. In some embodiments, the anti-PARP therapy is administered at a dose equivalent to about 100 mg, about 200 mg, or about 300 mg of niraparib or a salt or derivative thereof. In some embodiments, the anti-PARP therapy is administered at a dose equivalent to about 100 mg of niraparib or a salt or derivative thereof. In some embodiments, the anti-PARP therapy is administered at a dose equivalent to about 200 mg of niraparib or a salt or derivative thereof. In certain embodiments, the anti-PARP therapy is administered at a dose equivalent to about 300 mg of niraparib, or a salt or derivative thereof.

[0141] In some embodiments, the second treatment will include administering a therapeutic cancer vaccine. Therapeutic cancer vaccines are designed to be used in people who already have cancer and work against cancer cells that contain substances called tumor-associated antigens that are not present in normal cells or, if present, are present at low levels. Therapeutic vaccines can help the immune system learn to recognize and respond to these antigens, destroying the cancer cells that contain these antigens. Currently, there are two FDA-approved vaccines to prevent cancer; human papillomavirus (HPV) to prevent cervical, vaginal and vulvar cancer; and a hepatitis B vaccine to prevent liver cancer. The first FDA-approved oncolytic virus therapy is talimogene laherparepvec (T-VEC, or Imlygic®) against herpes simplex virus type 1. In various embodiments, the vaccine therapy is selected from, but is not limited to, treatment with a therapeutic vaccine, such as sipuleucel-T; treatment with a dendritic cell vaccine; treatment with an oncolytic virus therapy; and treatment with a tumor antigen peptide vaccine.

[0142] Depending on the nature of the combination therapy, administration of the polypeptide therapeutic of the present invention may continue while and / or after the administration of the other therapy. The polypeptide therapeutic may be administered before, simultaneously with, or after the additional anti-cancer therapy, usually within at least about one week, at least about five days, at least about three days, at least about one day. The polypeptide therapeutic may be delivered in a single dose or divided into multiple doses, and may be delivered over a period including, for example, once a day, once every other day, twice a week, once a week, etc. Effective doses vary depending on the route of administration, the specific agent, the dose of the anti-cancer agent, and the like, and may be determined empirically by one of skill in the art.

[0143] In some embodiments, the treatment regime requires administration once every two weeks or once a month or once every 3-6 months. The therapeutic entities of the present invention are usually administered multiple times. The interval between single administrations can be weekly, monthly or yearly. The intervals can be irregular as indicated by measuring blood levels of the therapeutic entity in the patient. Alternatively, the therapeutic entities of the present invention can be administered as sustained release formulations, in which case less frequent administration is required. The dosage and frequency will vary depending on the half-life of the polypeptide in the patient.

[0144] Furthermore, in some embodiments, the therapeutic entities of the present invention are often administered as pharmaceutical compositions containing an active therapeutic agent, i.e., and various other pharma- ceutical acceptable ingredients. (See Remington's Pharmaceutical Science, 15.sup.th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended method of administration and the therapeutic application. The composition may also contain pharma-ceutical-acceptable non-toxic carriers or diluents, defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration, depending on the desired formulation. The diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

[0145] In yet some other embodiments, the pharmaceutical compositions of the invention can also include large slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex-functionalized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulants (i.e., adjuvants).

[0146] In preventative applications, lower dosages are administered less frequently over a longer period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, higher dosages may be required at shorter intervals until the progression of the disease is slowed or halted, preferably until the patient shows partial or complete remission of symptoms of the disease. The patient can then be administered a preventative regimen.

[0147] In still other embodiments, for prophylactic applications, the pharmaceutical composition or medicament is administered to a patient susceptible to or otherwise at risk of a disease or condition in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, including the biochemical, histological and / or behavioral manifestations of the disease, its complications, and intermediate pathological phenotypes exhibited during disease development.

[0148] In still some other embodiments, for therapeutic applications, the therapeutic entities of the present invention are administered to patients suspected of or already suffering from such diseases in an amount sufficient to cure or at least partially arrest the symptoms (biochemical, histological and / or behavioral) of the disease, including its complications in disease development and intermediate pathological phenotypes. An amount sufficient to perform therapeutic or prophylactic treatment is defined as a therapeutically effective or prophylactically effective dose. In both prophylactic and therapeutic regimes, the agent is usually administered in several doses until a sufficient response is obtained. The response is usually monitored, and repeated doses are given if the cancer recurs.

[0149] In accordance with the present invention, compositions for the treatment of primary or metastatic cancers can be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular means. Although other routes may be similarly effective, the most typical routes of administration are intravenous or intratumoral.

[0150] For parenteral administration, the compositions of the present invention can be administered as an injectable preparation of a solution or suspension of the substance in a physiologically acceptable diluent together with a pharmaceutical carrier, which can be a sterile liquid, such as water, oil, saline, glycerol, or ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances, and the like, can be present in the composition. Other components of the pharmaceutical composition are of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. The antibody and / or polypeptide can be administered in the form of a depot injection or implant preparation, which can be formulated to allow sustained release of the active ingredient. In some embodiments, the composition comprises 1 mg / mL of the polypeptide formulated in an aqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mM L-arginine, 0.01% polysorbate 20, adjusted to pH 7.4 with HCl or NaOH.

[0151] Usually, the composition is formulated as either an injectable liquid solution or suspension; solid forms suitable for solution or suspension in liquid medium before injection can also be formulated.As mentioned above, it can also be emulsified or encapsulated in liposomes or microparticles such as polylactic acid, polyglycolic acid, or copolymers for enhancing adjuvant effect.Langer, Science 249:1527,1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119,1997.The agent of the present invention can be administered in the form of depot injection or implant preparation, which can be formulated to allow sustained release of active ingredient.

[0152] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

[0153] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations contain excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders and contain 10% to 95%, preferably 25% to 70%, of the active ingredient.

[0154] Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or its detoxified derivatives or subunits or other similar bacterial toxins. Glenn et al., Nature 391:851, 1998. Co-administration can be achieved by using the components as a mixture or as combined molecules obtained by chemical cross-linking or expression as a fusion protein. Alternatively, transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et al., Eur. J. Immunol. 25:3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368:201-15, 1998.

[0155] Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Preferably, a therapeutically effective dose of the polypeptide compositions described herein will provide a therapeutic effect without causing substantial toxicity.

[0156] Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., LD 50 (the dose lethal to 50% of the population) or LD 100The dose ratio between toxic and therapeutic effects is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a non-toxic dose range for use in humans. The dosage of the proteins described herein is preferably within a range of blood concentrations that includes the effective dose with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage are selected by the individual physician in view of the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1).

[0157] A kit comprising the composition of the present invention and instructions for use is also within the scope of the present invention. The kit further comprises at least one additional reagent, such as a tumor reducing agent. The composition may be provided in a unit dose formulation. The kit usually comprises a label indicating the intended use of the kit contents. The term label includes any written or recorded material provided on or with the kit or otherwise attached to the kit.

[0158] Example 1 Pembrolizumab is the current therapy for patients with locally advanced or metastatic urothelial carcinoma who have failed platinum-treated metastatic urothelial carcinoma. This was based on a phase 3 trial in which patients were randomized to pembrolizumab (270) or chemotherapy (paclitaxel, docetaxel, or vinflunine (272)). Pembrolizumab vs chemotherapy had a median OS of 10.3 months (95% CI, 8-11.8) vs 7.4 months, 2.1 months (95% CI, 2.0-2.2) vs 3.1 months, and an ORR of 21.1% (95% CI, 16.4-26.5) vs 11.4%. Competitive remissions with pembrolizumab were 7%. Pembrolizumab has been granted FDA approval.

[0159] Immune checkpoint inhibitor targeted agents such as pembrolizumab are effective in patients with tumors that express an inflammatory signature and contain resident immune cells, commonly referred to as "hot tumors." Tumors with few or no immune cells (cold tumors) are unlikely to respond. Thus, novel agents that recruit immune cells are likely to improve patient response to the current state of immunotherapy. Immune cell recruitment is limited in part by the tumor-vascular barrier. EphrinB2 and its high-affinity cognate receptor, -EphB4-, are transmembrane proteins that are directed to tumor vasculature and regulate immune cell trafficking. A soluble extracellular fragment of EphB4 fused to albumin (sEphB4-HSA) blocks the interaction of endogenously expressed ephrinB2 with EphB4, blocking bidirectional signaling. sEphB4-HSA promotes immune cell trafficking.

[0160] In this example, eight patients with naive locally advanced or metastatic urothelial carcinoma of the bladder were treated with sEphB4-HSA in combination with an anti-PD-1 antibody. The treatment regimen consisted of sEphB4-HSA 10 mg / kg weekly intravenous infusion + pembrolizumab (KEYTRUDA®) 200 mg intravenous infusion every 3 weeks. Tumor response was measured every 6 weeks. Baseline or archival tissues were collected for biomarkers (specifically PD-L1 IHC 22C3 PharmDx, a companion marker for pembrolizumab / KEYTRUDA®).

[0161] Patient eligibility required that patients had no prior immunotherapy, were ineligible for platinum-based chemotherapy, and declined or refused to receive chemotherapy previously. Age ranged from 53 to 90 years. Six of the eight patients were male. Two had a prior radical cystectomy (cystectomy) for bladder / urothelial cancer. Two patients had disease in the upper urinary tract. Eight patients had Vermunt 1 risk factors and one patient had Vermunt 2 risk factors. One case had a hemoglobin below 10. Seven patients were not suitable for platinum-based chemotherapy. One patient refused to receive chemotherapy. Pathology: variants (micropapillary in one, squamous and sarcomatoid variants in one each). All patients had lymph node metastases and seven had primary bladder cancer.

[0162] Results: The combination of soluble EphB4-albumin fusion and PD-1 antibody is well tolerated. When correlated with biomarkers, 3 patients were PD-L1 positive (expression ≥1%) and all 8 patients were ephrin B2 positive (expression ≥1%). 6 patients were assessed for response by CT scan. CT scan is pending in 1 case and 1 patient discontinued treatment early. Remarkably, 5 of 6 patients with CT scan at the time of treatment had remission, with all remissions being complete. The overall response rate for the purposes of treatment is 71.4%. Among the PD-L1 positive cases, 2 of 3 patients responded to treatment. Duration of response was remarkable, with no patient progressing after 10 months or less of follow-up from response. Thus, the combination of soluble EphB4-albumin fusion and PD-1 antibody had a high response rate of 71.4%, and ephrin B2 expression as a biomarker appears to be highly predictive of response with combination therapy.

[0163] Example 2 We previously reported on a Phase II clinical trial of sEphB4-HSA in combination with pembrolizumab. Patient eligibility criteria were locally advanced or metastatic urothelial carcinoma, and patients had previously failed (relapsed or ineffective or intolerant) a cisplatin-containing regimen for locally advanced or metastatic disease, or had relapsed within 12 months of a cisplatin-containing neoadjuvant therapy. Exclusion criteria were patients who had received prior checkpoint inhibitor targeted therapy.

[0164] In this study, the treatment regimen consisted of sEphB4-HSA 10 mg / kg weekly intravenous infusion plus pembrolizumab (KEYTRUDA®) 200 mg intravenous infusion every 3 weeks. Tumor response was measured every 6 weeks. Baseline or archival tissue was collected for biomarkers, specifically PD-L1 IHC 22C3 PharmDx, a companion marker for pembrolizumab. Independent assessment of response was assessed by blinded radiological review. PD-L1 staining was performed in a reference laboratory. All patients were eligible for toxicity assessment. The primary endpoint of the study was OS, with secondary endpoints being ORR and PFS. Analysis of high-risk subsets included squamous cell mutations, upper urinary tract disease, liver metastases, hemoglobin <10 mg / dl, levels, and performance status >0. 69 patients were enrolled in the study.

[0165] Sixty-nine patients were acquired. The median age was 67 years. 80% of patients were male. Four cases were excluded due to lack of tissue for biomarker analysis (two cases had no tissue available and two cases did not contain tumor). Sixty-five patients with available tissue, biomarkers (ephrin B2 and PD-L1) were analyzed. Fifty patients were positive for either ephrin B2 or PD-L1. Fifteen patients were negative for both ephrin B2 and PD-L1. Forty-five patient tissues were ephrin B2 positive (32 patient tissues were only ephrin B2 positive), 18 patients were PD-L1 positive (5 patient tissues were only PD-L1 positive), and 13 patients expressed both ephrin B2 and PD-L1. Fifty-two patients (35 ephrin B2 positive) had prior chemotherapy treatment.

[0166] Ephrin B2-positive patients were determined to have an overall response rate (ORR) of 51%, compared with an overall of 37% for all patients. Importantly, both response rates were higher than the expected response rate of 21% (based on historical data) if patients were treated with pembrolizumab alone. Four patients with metastatic urothelial carcinoma were treated with sEphB4-HSA during the Phase I portion of the study, with no patients experiencing an objective response. Thus, the combinatorial activity of sEphB4-HSA in combination with PD-1 antibodies appears to arise through complementary functions, whereby sEphB4-HSA promotes migration of T cells to the tumor, while PD-1 antibodies activate newly recruited resident immune cells to achieve a sustained response.

[0167] In this trial, sEphB4-HSA and pembrolizumab demonstrated true synergy, with an overall survival of 21 months, double the 10 months observed with monotherapy with pembrolizumab, and 69% of secondary responders remained in remission 2 years later.

[0168] Example 3 Patients with recurrent and refractory head and neck squamous cell carcinoma after chemotherapy have low response rates using cetuximab, systemic chemotherapy or checkpoint inhibitor targeted therapy. Patients with HPV-negative HNSCC have poorer outcomes compared to HPV-positive patients, including responses to both checkpoints. The 2-year overall survival rate for patients with HPV-associated head and neck cancer is 95%, while the 2-year overall survival rate for non-HPV-associated HNSCC is 62%. Thus, there is a large unmet need for novel therapies.

[0169] This clinical trial was a phase IIa, single-arm, non-randomized, open-label trial of sEphB4-HSA in combination with pembrolizumab (MK-7435) in patients with squamous cell carcinoma (SCC) of the head and neck. Patients aged ≥18 years with SCC of the head and neck met other eligibility criteria for study enrollment. Patients had locally advanced or metastatic disease that had progressed after two or more prior lines of therapy, including radiochemotherapy. Patients must have been treated with systemic chemotherapy for first recurrence, had adequate organ function, had measurable disease based on RECIST 1.1 (Immune-Related Response Criteria in Solid Tumors) of 0 or 1, ECOG (Eastern Cooperative Oncology Group) performance status, and had baseline tumor tissue available for analysis. Endpoints were toxicity, overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). HPV status and ephrin B2 biomarker expression were assessed for outcome.

[0170] Planned enrollment was 25 patients. The treatment regimen was sEphB4-HSA 10 mg / kg once weekly on days 1, 8, and 15, with pembrolizumab 200 mg given by intravenous infusion on day 1 of each 3-week cycle, once every 3 weeks. Treatment was given for a maximum of 24 months. Treatment was discontinued upon radiographic disease progression, unacceptable adverse events, intercurrent illness, or noncompliance with treatment, and patient or tumor imaging was performed every 6 weeks (every 2 cycles) with computed tomography (CT) scans of the chest, abdomen, and pelvis plus soft tissue CT scans of the neck. Investigator decision to withdraw from the study. Tumor response was assessed according to RECIST. Patients with confirmed complete response (CR) who were treated with pembrolizumab for ≥24 weeks and had at least 2 treatments with pembrolizumab beyond the date of initial CR had the option to discontinue treatment. Such patients were eligible for up to 1 year of additional treatment if they experienced radiographic progression and had not received any cancer therapy since their last dose of pembrolizumab, at the investigator's discretion.

[0171] Immunohistochemical staining (IHC) was performed on patient tumor samples obtained at baseline and week 8 (cycle 2) on treatment. Biomarkers analyzed included ephrinB2, PD-L1, immune cell markers CD3, and CD8 for both collections. Patient tissue samples at baseline were sent to Caris Life Sciences for comprehensive tumor sequencing and PD-L1 using monoclonal rabbit anti-PD-L1 clone 28-8. Scoring of PD-L1 on both baseline and on-treatment tissue samples included tumor and immune cell membrane PD-L1 staining. Patients were determined to be PD-L1 positive if their tissue samples at baseline showed a ≥ 1% combined positive score (CPS). Scoring procedures and staining protocols are described in the instructions for the commercially available assay for squamous cell carcinoma of the head and neck (SCCHN). IHC for ephrinB2 and immune markers was performed in a CLIA-certified core laboratory and analyzed by an independent pathologist (IS). EphrinB2 assay used rabbit monoclonal anti-ephrinB2 antibody. Scoring and analysis of ephrinB2 positivity in baseline and on-treatment biopsies was based on tumor cell membrane staining for ephrinB2. Patients were determined to be ephrinB2 positive if their tissue sample at baseline showed ≥1% TPS. Scoring of both ephrinB2 and PD-L1 was based on a scale of 0 to 100%. p16 staining was performed in a CLIA-certified clinical laboratory as a routine service. IHC of immune markers was performed to assess immune cell infiltration into the tumor. This included staining for CD3, and CD8.

[0172] The demographics of the study population are shown in Table 1 below. TIFF2024534935000001.tif236170TIFF2024534935000002.tif140170

[0173] Efficacy data are shown in Table 2. TIFF2024534935000003.tif140170

[0174] The combination of sEphB4-HSA + pembrolizumab resulted in an overall response in seven patients, six of which were in the HPV-negative patient population. The majority of HPV-negative patients have high ephrin B2 expression. Despite the small study population, it is clear that targeting ephrin B2 in combination with a PD-1 antibody (pembrolizumab) offers a higher likelihood of response. Overall survival (OS) among HPV-negative patients is substantially higher with the combination therapy than that expected with pembrolizumab alone (12.4 months vs. 8 months, respectively).

[0175] Approximately 60% of patients were HPV negative by p16 biomarker analysis. When comparing efficacy endpoints within HPV-negative patients, the combination also compared favorably with pembrolizumab alone; the ORR among HPV-negative patients receiving sEphB4-HSA + pembrolizumab was 40% and the ORR among HPV-negative patients receiving pembrolizumab alone was 14%. This substantial difference in response rates suggests that non-HPV-associated HN SCC patients, who usually have a worse prognosis than HPV-associated HN SCC patients, may benefit significantly from the combination of sEphB4-HSA + pembrolizumab. Given the effects of single-agent sEphB4-HSA on immune cell migration and activation in the tumor microenvironment, plus the demonstrated efficacy of pembrolizumab in enhancing immune-mediated tumor killing, it is plausible that the combination of the two treatments would result in greater immune cell infiltration into the tumor and more effective responses in patients.

[0176] Seventeen of 25 patients (68%) were ephrin B2 positive, whereas 12 of 15 HPV negative cases were ephrin B2 positive. All responding patients were ephrin B2 positive. Twelve of 25 patients (48%) were PD-L1 positive with a CPS of 1% or more. Of 13 patients with PD-L1 less than 1%, 3 patients responded (23%). Further analysis performed for ORR based on ephrin B2 and PD-L1 positivity showed that: 4 of 9 patients (44%) responded in the ephrin B2 positive and PD-L1 positive group, whereas responses were observed in 4 of 8 patients (50%) in the ephrin B2 positive and PD-L1 negative group. These data suggest that ephrin B2 expression provides a predictive value for better response. The lack of response in both the ephrinB2-negative and PD-L1-positive (N=3) or PD-L1-negative (N=5) groups is consistent with these findings.

[0177] PD-L1 ≥1% was less common in this study population (11 of 25), which is consistent with the 3105 patient meta-analysis, showing 42% positivity, but substantially lower than reported in Keynote 012 (85%). The ORR in ≥1% PD-L1 was similar to that in <1%, 3 of 11 and 3 of 13, respectively. PD-L1 <1% shows a very low response rate (4 of 46, 8 in Keynote 048), with the responses in the 13 PD-L1 negative patients (3 of 13 or 23%) being surprising. The results from the current study reflect an opportunity to address minority proposed exceptions as well as unmet needs.

[0178] Findings from this study provide support for the safety and efficacy of the combination of sEphB4-HSA + pembrolizumab. There were no grade 4 adverse events or treatment-related deaths, and both toxicities experienced by patients were manageable and did not require discontinuation of treatment except for one case of cardiomyopathy, a known toxicity of pembrolizumab. The combination therapy significantly improved the ORR vs. pembrolizumab alone, especially in the HPV-negative subgroup. Finally, biomarker analysis showed a clear difference in the objective response rate in patients with tumors expressing ephrinB2 vs. ephrinB2-negative patients; in contrast, differences in response rates between PD-L1-positive vs. PD-L1-negative patients were not observed as well. Further studies are warranted to evaluate the tumor suppressive process behind EphB4-ephrinB2 inhibition in the context of PD-1 / PD-L1 inhibition, especially in patients who do not express PD-L1.

[0179] Example 4 Patients with metastatic urothelial carcinoma have a poor prognosis after failure of standard first-line chemotherapy. Immune checkpoint programmed death 1-programmed death ligand 1 antibodies have low response rates and therefore a large unmet need exists.

[0180] Patients with metastatic urothelial carcinoma who relapsed or progressed after platinum-based chemotherapy received soluble EphB4-human serum albumin (sEphB4-HSA) in combination with pembrolizumab. Patients aged 18 years or older with histologically confirmed carcinoma of the urinary tract (renal pelvis, ureter, bladder, or urethra) predominantly urothelial with or without mutations, including squamous cell carcinoma, poorly differentiated carcinoma, met eligibility for enrollment. Eligible patients had disease progression after platinum-based chemotherapy for advanced disease or relapsed within 12 months of receiving platinum-based adjuvant or neoadjuvant therapy for locally muscle-invasive disease. Patients may have received one or more systemic chemotherapy regimens for advanced disease prior to study enrollment. Primary endpoints were tolerability and overall survival (OS). Secondary endpoints were progression-free survival (PFS), objective response rate (ORR), duration of response, and toxicity. Expression of the sEphB4-HSA target ephrinB2 correlated with outcome.

[0181] Patients were assigned to one of two cohorts: Cohort A patients received only one prior platinum-based chemotherapy and Cohort B patients received one additional systemic chemotherapy regimen, with planned enrollment of 36 and 24 patients, respectively. Treatment regimens consisted of pembrolizumab 200 mg intravenously on day 1 and sEphB4-HSA 10 mg / m2 intravenously once daily on days 1, 8, and 15 every 3 weeks. Treatment continued until RECIST-defined disease progression, unacceptable toxicity, patient withdrawal of consent, investigator decision to discontinue treatment, or completion of 2 years of treatment.

[0182] The median age was 67 years, and the male:female ratio was 59:11. Sixty-four patients enrolled had only one prior treatment, and six patients had two or more prior treatments. The sites of disease at baseline were lymph nodes 45 (64%), lung 24 (34%), liver 18 (26%), and bone 9 (13%). Thirty-nine patients (56%) had ECOG performance status 0, and 31 (44%) patients had ECOG performance status 1. The Bellmunt risk group distribution was as follows: 27% no risk factor, 37% one risk factor, and 36% two or more risk factors. Fourteen patients (20%) had upper urinary tract disease as their primary site of disease. Forty-six patients were ephrin B2 positive.

[0183] Efficacy data are shown in Tables 3 and 4. TIFF2024534935000004.tif115170 TIFF2024534935000005.tif239170

[0184] Median follow-up was 22.9 months (range, 1.3 to 54.7). The regimen had acceptable toxicity. In the intention-to-treat analysis (N5 70), median OS was 14.6 months (95% CI, 9.2 to 21.5). Twenty-six (37%) patients had an objective response (95% CI, 26 to 48). Median PFS was 4.1 months (95% CI, 1.5 to 5.7). Among 46 (66%) patients expressing ephrinB2, median OS was 21.5 months (95% CI, 12.4 to not reached) and ORR was 52% (95% CI, 37 to 67), including a complete response rate of 24% (11 of 46; 95% CI, 12 to 36). Median PFS was 5.7 months (95% CI, 2.7 to 27.9). Responses were maintained at 6, 12, and 24 months in 88%, 74%, and 69% of patients, respectively.

[0185] The combination exceeded expectations across all endpoints in the intention-to-treat analysis. In the ephrin B2-positive subgroup (n 5 46, 66%), all endpoints showed improvement across the intention-to-treat population. The combination of sEphB4-HSA and pembrolizumab appears synergistic with improved OS and ORR compared to historical data for programmed death 1 / programmed death ligand 1 single agents.

[0186] Example 5 To determine whether ephrinB2 expression is a prognostic marker for urothelial carcinoma and a predictor of response to checkpoint inhibitor monotherapy, a retrospective study of patients diagnosed with metastatic urothelial carcinoma treated with PD1 / PD-L1 antibody monotherapy analyzed ephrinB2 expression and response among these patients. Briefly, tissue samples were collected and stained for ephrinB2, with >1% expression considered biomarker positive. Biomarker expression was correlated with reported outcomes of PD1 / PD-L1 monotherapy. Eligible patient selection required the following: 1) at least one pathology specimen obtained prior to PD1 / PD-L1 antibody therapy must be available, with at least three unstained slides required for tissue analysis; 2) radiographically measurable disease must be present at the start of PD1 / PD-L1 monotherapy; 3) treatment outcome must be documentable as radiographic progression (PD), including death, stable disease, partial response, and complete response; and 4) if available, at the time of enrollment, ECOG status 0, or 1, or 2. It was understood that by measuring expression of ephrinB2 in tissues from these patients, it would be possible to determine whether ephrinB2 expression was simply a surrogate for response to checkpoint blockade.

[0187] To conduct the study, the investigators obtained IRB approval for the retrospective evaluation of tissue from patients with locally advanced or mUC who had disease progression after systemic chemotherapy with cisplatin or carboplatin and were subsequently treated with anti-PD1 / PD-L1 therapy. Patients included in the study had to have tissue available for biomarker analysis and radiographic assessment of response to immunotherapy (protocol and IRB approval are included in the appendix). Demographic, patient-specific, and disease-specific data were collected. Tumor tissue blocks were freshly sectioned, stained for ephrin B2 and PD-L1, and blinded reviewed by a pathologist experienced in the assessment of ephrin B2 and PD-L1 status (Dr. Imran Siddiqi, Norris Cancer Hospital, Keck School of Medicine).

[0188] Of the 41 patients identified for the study, 28 patients met the study inclusion requirements. Demographics of the study population are shown in Table 5 below. TIFF2024534935000006.tif163170TIFF2024534935000007.tif151170

[0189] Of the 28 patients included in this study, 5 patients were responders; 2 patients with complete response and 3 patients with partial response. Overall response 5 out of 28 (17.9%). An additional 3 patients had best response as stable disease based on radiographic assessment. Most patients in this series were treated with pembrolizumab. These data are in line with literature values ​​and expectations based on many prospective clinical trials with PD-1 or PD-L1 antagonist antibodies in this patient population. A meta-analysis of FDA-approved PD-1 / PD-L1 antagonist antibodies tested for relapsed / refractory urothelial carcinoma after systemic chemotherapy has a response rate of 18%. (Tafuri et al. Clin GU Cancer. 2020, 18, 351-360).

[0190] EphrinB2 was expressed in 18 of 28 cases and PD-L1 was expressed >1% in 8 patients. Among the 5 responders, ephrinB2 was expressed moderately to high levels in 2 patients (2 / 18=11%). One of these patients had PD-L1 positivity >1% and had multiple sites of disease (liver, lung, lymph nodes). The second ephrinB2 positive patient with a partial response had PD-L1 levels <1% and had lung as a site of metastatic disease. Three of the 5 responders did not express ephrinB2 (3 / 10=30% ORR). Two of these patients had complete remissions. Two of the three patients expressed PD-L1. All three patients had disease localized to lymph nodes. TIFF2024534935000008.tif56170

[0191] EphrinB2 positivity had an 11% response rate, whereas ephrinB2 negativity had a 30% response rate, thus a three-fold difference, suggesting that ephrinB2 expression predicts a poorer response to PD-L1 monotherapy.

[0192] Figures 1-3 are photographs of ephrinB2 immunohistochemistry (IHC) and ephrinB2 in situ hybridization (ISH / RNAScope) of all 5 responders. Non-responders with high ephrinB2 and low ephrinB2 are also shown. Immunohistochemistry was performed with a recombinant monoclonal antibody made in rabbits (Abcam 201512). The antibody showed specificity for ephrinB2 analyzed in syngeneic CHO cell lines, wild type or ectopically expressed human ephrinB2 full length (CHO-WT, CHO-ephrinB2). It should be noted that ephrinB2 is a membrane protein and therefore requires membrane localization for positive IHC staining. Absence of any staining or staining restricted to signals in the nucleus or cytoplasm was considered negative (or nonspecific). EphrinB2 IHC positivity is defined by membrane localization. Membrane localization in the presence of nuclear or cytoplasmic staining is considered a positive result. We also verified expression using in situ hybridization (RNAScope), a technology developed by Advanced Cell Diagnostics (ACD). The assay was validated in syngeneic cell lines (CHO-WT as a negative control, CHO-EphrinB2 as a positive control, CHO-EphrinB1 as a negative control) showing specificity even within its closely related proteins.

[0193] A secondary retrospective study of patients diagnosed with metastatic urothelial carcinoma treated with PD1 / PD-L1 antibody monotherapy (56) analyzed ephrin B2 expression and response among these patients. Demographics of the study population are shown in Table 7 below. TIFF2024534935000009.tif204170TIFF2024534935000010.tif51170

[0194] Ephrin B2 positivity had a 14% response rate, whereas ephrin B2 negativity had a 35% response rate. And importantly, patients with very high ephrin B2 (7 cases) did not respond. Thus, this study validates the first study and collectively demonstrates that there were at least 12 patients with high ephrin B2 expression who again did not respond, and that ephrin B2 expression predicts a poorer response to PD-L1 monotherapy.

[0195] All publications and patents herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and as if incorporated by reference to disclose and describe the methods and / or materials in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0196] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any described method can be carried out in the order of events described or in any other order that is logically possible. It will also be understood that the techniques used herein are for the purpose of describing particular embodiments.

[0197] Although the foregoing invention has been described in some detail by way of figures and examples for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the invention that certain changes and modifications thereto may be made without departing from the spirit or merely are not intended to limit the scope of the invention, which is limited only by the appended claims.

[0198] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the scope of the claims appended hereto.

[0199] Array List The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter abbreviations for amino acids as defined in 37 CFR 1.822.

[0200] SEQ ID NO: 1 is the amino acid sequence of human ephrin type B receptor precursor (NP_004435.3). Amino acid residues 1 to 15 encode a signal sequence. (SEQ ID NO:1)

[0201] SEQ ID NO: 2 is the amino acid sequence of human serum albumin preproprotein (NP_000468.1). Amino acid residues 25 to 609 encode the mature peptide. (SEQ ID NO:2)

[0202] SEQ ID NO:3 is the amino acid sequence of the human Ephrin type-B receptor-human serum albumin protein. LEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCDVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFYYESDADTATALT PAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYC REDGQWAEQPVTGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCTTPPSADAHKSEVAHRFKDLGEENFKALVLI AFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGD LLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCC AAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRV TKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0203] SEQ ID NO:4 is the amino acid sequence of the human Ephrin type-B receptor-human serum albumin protein.

[0204] SEQ ID NO:5 is the amino acid sequence of the human Ephrin type-B receptor-human serum albumin protein.

Claims

1. A pharmaceutical composition for the treatment of a subject suffering from cancer with an EphB4-ephrinB2 inhibitor in combination with an immunostimulant as a first-line treatment, comprising: The pharmaceutical composition comprises an EphB4-EphrinB2 inhibitor, and the method for diagnosing and selecting a subject suffering from cancer comprises: i) detecting an EphrinB2 expression level in a biological sample from a subject diagnosed with cancer; and ii) selecting the subject for treatment with an EphB4-EphrinB2 inhibitor in combination with an immunostimulant if EphrinB2 expression is 1% or greater.

2. A pharmaceutical composition for the treatment of a subject suffering from cancer with an EphB4-ephrinB2 inhibitor as a first-line treatment, comprising: The pharmaceutical composition comprises an EphB4-EphrinB2 inhibitor, and the method for diagnosing and selecting a subject suffering from cancer comprises: i) detecting the expression level of EphrinB2 in a biological sample from a subject diagnosed with cancer; and ii) selecting the subject for treatment with the EphB4-EphrinB2 inhibitor if EphrinB2 expression is 1% or greater.

3. A pharmaceutical composition according to claim 1 or 2, characterized in that the cancer is one for which standard anticancer therapy is ineffective.

4. A pharmaceutical composition according to claim 1 or 2, characterized in that the subject has relapsed from previous treatment with a standard anti-cancer therapy.

5. A pharmaceutical composition according to claim 1 or 2, wherein the EphB4-ephrinB2 inhibitor is a monomeric ligand-binding portion of the EphB4 protein and contains modifications that increase its serum half-life.

6. The pharmaceutical composition of claim 1, wherein the EphB4-ephrinB2 inhibitor comprises amino acids 1-197, 16-197, 29-197, 1-312, 16-312, 29-312, 1-313, 1-314, 1-315, 1-316, 1-317, 1-318, 1-319, 1-320, 1-321, 1-322, 1-323, 1-324, 1-325, 1-326, 1-327, 1-328, 1-329, 1-330, 1-331, 1-332, 1-333, 1-334, 1-335, 1-336, 1-337, 1-338, 1-339, 1-439, 1-440, 1-441, 1-442, 1-443, 1-444, 1-445, 1-446, 1-447, 1-448, 1-449, 1-450, 1-451, 1-452, 1-453, 1-454, 1-455, 1-456, 1-457, 1-458, 1-459, 1-460, 1-461, 1-462, 1-463, 1-464, 1-465, 1-470, 1-471, 1-472, 1-473, 1-474, 1-475, 1-476, 1-477, 1-4 16-526, 29-526, 1-537, 16-537 and 29-537 (an "sEphB4 polypeptide").

7. The pharmaceutical composition of claim 6, wherein the sEphB4-HSA comprises residues 16-326 of SEQ ID NO: 1 directly fused to residues 25-609 of SEQ ID NO:

2.

8. The pharmaceutical composition of claim 6, wherein the sEphB4-HSA comprises residues 16-526 of SEQ ID NO: 1 directly fused to residues 25-609 of SEQ ID NO:

2.

9. The pharmaceutical composition of claim 6, wherein the sEphB4-HSA comprises residues 16-537 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:

2.

10. The pharmaceutical composition according to claim 6, wherein the sEphB4-HSA comprises an amino acid sequence selected from the group of sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

5.

11. The pharmaceutical composition according to claim 1, wherein the immunostimulant is selected from the group consisting of anti-PD-1 Ab, anti-PD-L1 Ab, anti-CTLA Ab, anti-TIGIT Ab, anti-LAG3 antibody, and anti-TIM3 antibody.

12. A pharmaceutical composition according to claim 1 or 2, characterized in that ephrin B2 expression is determined by protein expression using a method selected from the group consisting of immunohistochemistry (IHC), immunofluorescence, flow cytometry, and Western blot.

13. The pharmaceutical composition according to claim 1 or 2, wherein the biological sample is selected from the group consisting of a tissue sample, a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid (CSF) sample, an ascites sample, and a cell culture medium sample.

14. A pharmaceutical composition according to claim 1 or 2, wherein the cancer is selected from the group consisting of B-cell lymphoma; lung cancer (small cell lung cancer and non-small cell lung cancer); bronchial cancer; colorectal cancer; prostate cancer; breast cancer; pancreatic cancer; gastric cancer; ovarian cancer; bladder cancer; brain or central nervous system cancer; peripheral nervous system cancer; esophageal cancer; cervical cancer; melanoma; uterine or endometrial cancer; oral or pharyngeal cancer; liver cancer; renal cancer; biliary tract cancer; small intestine or appendix cancer; salivary gland cancer; thyroid cancer; adrenal cancer; osteosarcoma; chondrosarcoma; liposarcoma; testicular cancer; and malignant fibrous histiocytoma; skin cancer; head and neck cancer; lymphoma; sarcoma; multiple myeloma; and leukemia.

15. The pharmaceutical composition according to claim 14, wherein the cancer is bladder cancer.

16. The pharmaceutical composition of claim 3, wherein the refractory cancer is selected from the group consisting of: cancers that are resistant to platinum-based chemotherapy, cancers that are resistant to immunotherapy, cancers that are resistant to treatment with chemotherapy drugs, cancers that are resistant to treatment with depleting antibodies against specific tumor antigens, cancers that are resistant to treatment with agonist antibodies, antagonist antibodies, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), cancers that are resistant to targeted therapy with immune conjugates, antibody-drug conjugates (ADCs), or fusion molecules comprising a depleting antibody against a specific tumor antigen and a cytotoxic agent, cancers that are resistant to targeted therapy with small molecule kinase inhibitors, cancers that are resistant to treatment with surgery, cancers that are resistant to treatment with stem cell transplantation, cancers that are resistant to treatment with therapeutic vaccines, and cancers that are resistant to treatment with radiation.

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