Use of sEphB4-HSA fusion protein as a first-line therapy in cancer treatment

Abstract

Disclosed herein are methods of using sEphB4-HSA as an effective first-line therapy for cancers where current therapies are ineffective, result in recurrence, or are not even considered for use due to the type of cancer and associated tumor.

Description

[Technical field]

[0001] The disclosure provides, in part, compositions and methods comprising soluble ephrin-HSA fusion proteins and their uses, including methods for treating cancer.

[0002] Related Applications This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 162,691, filed March 18, 2021, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Copy of the Sequence Listing in Computer Readable Format (Filename: "VAS-002PC_ST25.txt", Recorded Date: March 17, 2022, File Size: 14,127 bytes). [Background technology]

[0004] Today, despite the development of numerous advanced diagnostic and therapeutic methods, cancer remains the leading cause of death worldwide. In humans, cancer forms by many mechanisms, including but not limited to, increased cell metabolism and proliferation rate, increased blood supply to the tumor through stimulation of angiogenesis, and dysregulation of signaling pathways and tumor suppressors, after a primary genetic event. Definitive treatment protocols in clinical oncology still rely on a combination of surgical resection, ionizing radiation, and cytotoxic chemotherapy. A major obstacle to successful cancer treatment and prevention remains the fact that many cancers do not respond to current chemotherapeutic and immunotherapeutic interventions, and many experience recurrence or death even with aggressive treatment. In addition, tumors can become resistant to anticancer drugs by many mechanisms, including but not limited to, efflux of drugs from cells, the occurrence of mutations that prevent the binding of drugs to their targets, and the occurrence of further mutations in genes and their protein products that are unrelated to the drug target. To address these shortcomings, there is a trend in drug discovery to develop targeted therapies that can modulate signaling axes that are dysregulated in cancer. There are now a large number of FDA-approved antibodies and small molecules that provide therapeutic control of a myriad of clinically important targets.

[0005] Eph (erythropoietin-producing hepatocellular carcinoma) receptors and ligands are part of the largest family of receptor tyrosine kinases (RTKs). The family is subdivided into class A and class B based on sequence homology and binding affinity for two different types of membrane-anchored ephrin ligands. Each Eph receptor and ligand can bind multiple ligands and receptors, with certain receptors hypothesized 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 induced in tumor vasculature and regulate immune cell trafficking. Inhibition of ephrinB2-EphB4 interaction shows direct inhibitory effects on tumor cell proliferation in vitro and ex vivo. Summary of the Invention

[0006] In an aspect, the disclosure relates to a method for treating cancer comprising administering to a patient in need thereof an effective amount of a polypeptide agent that inhibits EphB4 or Ephrin B2-mediated function, wherein the polypeptide agent is used as a first line therapy in the treatment.

[0007] In an aspect, the disclosure relates to the use of a polypeptide agent that inhibits EphB4 or EphrinB2-mediated function in the preparation of a medicament for use as a first-line therapy in treating cancer. In an embodiment, the cancer is selected from, but is not limited to, head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), Kras-mutated non-small cell lung adenocarcinoma, and Kaposi's sarcoma (KS).

[0008] In embodiments, the subject has previously responded to anti-cancer treatment but experienced a relapse upon cessation of treatment (hereinafter "recurrent cancer"). In embodiments, the subject has a resistant or refractory cancer. In embodiments, the cancer is refractory to platinum-based chemotherapy. In embodiments, the cancer is refractory to immunotherapy. In embodiments, the cancer is refractory to treatment with chemotherapeutic agents. In embodiments, the cancer is refractory to treatment with an ablative antibody against a specific tumor antigen. In embodiments, the cancer is refractory to treatment with an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint). In embodiments, the cancer is refractory to targeted therapy with an immunoconjugate, antibody drug conjugate (ADC), or fusion molecule comprising an ablative antibody against a specific tumor antigen and a cytotoxic agent. In embodiments, the cancer is refractory to targeted therapy with a small molecule kinase inhibitor. In embodiments, the cancer is refractory to treatment with surgery. In embodiments, the cancer is refractory to treatment with stem cell transplantation. In an embodiment, the cancer is refractory to treatment with radiation. In an embodiment, the cancer is refractory to combination therapy with two or more of immunotherapy, treatment with platinum-based chemotherapy, treatment with tumor antigen-specific depletion antibody, treatment with immunoconjugate, ADC, or fusion molecule containing tumor antigen-specific depletion antibody and cytotoxic agent, targeted therapy with small molecule kinase inhibitor, treatment with surgery, treatment with stem cell transplantation, and treatment with radiation. In an embodiment, the subject has a form of cancer that is determined not to be considered for use of various anti-cancer therapies.

[0009] In an embodiment, the use of the present invention relates to a method for treating squamous cell carcinoma of the head and neck (HNSCC) in a subject, comprising administering to the subject a therapeutically effective amount of sEphB4-HSA polypeptide as a first line therapy. In an embodiment, the HNSCC is refractory to treatment with platinum-based chemotherapy and / or radiation therapy. In an embodiment, the HNSCC is refractory to treatment with a checkpoint inhibitor. In an embodiment, the subject has recurrent HNSCC.

[0010] In an embodiment, the use of the present invention relates to a method for treating hepatocellular carcinoma (HCC) in a subject, comprising administering to the subject a therapeutically effective amount of sEphB4-HSA polypeptide as a first-line therapy. In an embodiment, the HCC is refractory to treatment with platinum-based chemotherapy and / or radiation therapy. In an embodiment, the HCC is refractory to treatment with checkpoint inhibitors. In an embodiment, the subject has recurrent HCC.

[0011] In an embodiment, the use of the present invention relates to a method for treating Kras-mutated non-small cell lung adenocarcinoma in a subject, comprising administering to the subject a therapeutically effective amount of sEphB4-HSA polypeptide as a first line therapy. In an embodiment, the checkpoint inhibitor is a PD-1 inhibitor. In an embodiment, the adenocarcinoma is refractory to treatment with platinum-based chemotherapy and / or radiation therapy. In an embodiment, the adenocarcinoma is refractory to treatment with a checkpoint inhibitor. In an embodiment, the subject has recurrent adenocarcinoma.

[0012] In an embodiment, the use of the present invention relates to a method for treating Kaposi's sarcoma (KS) in a subject, comprising administering to the subject a therapeutically effective amount of sEphB4-HSA polypeptide as a first line therapy. In an embodiment, the KS is refractory to treatment with platinum-based chemotherapy and / or radiation therapy. In an embodiment, the KS is refractory to treatment with a checkpoint inhibitor. In an embodiment, the subject has relapsed KS.

[0013] In embodiments, a soluble extracellular fragment of EphB4 fused to albumin (sEphB4-HSA) blocks the interaction of ephrin-B2 and EphB4, blocks bidirectional signaling, and consequently promotes immune cell trafficking and induces anti-tumor immune responses in various cancers. Thus, the present disclosure provides, in various embodiments, an ephrinB2-EphB4 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 binds to the transmembrane protein ephrin-B2, which is the ligand of the EphB4 tyrosine kinase receptor. This binding prevents endogenous EphB tyrosine kinase receptors from interacting with ephrinB2. Data show that sEphB4-HSA reduces tumor vascularization, thereby starving tumors of blood and inhibiting the ability of ephrinB2 to suppress T cell recruitment to tumors, resulting in increased T cell recruitment.

[0014] In an embodiment, the sEphB4 polypeptide agent for use in the treatment of cancer further comprises the use of an anti-EGFR antibody or antibody fragment thereof or a taxane. In an embodiment, a composition comprising a soluble EphB4-HSA fusion protein (sEphB4-HSA) and an anti-EGFR antibody or fragment thereof for use in the treatment of cancer. In an embodiment, the anti-EGFR antibody is cetuximab.

[0015] In an embodiment, the method of the invention provides for combination therapy with a taxane, optionally paclitaxel (taxol) or docetaxel (taxotere).In an embodiment, the method of the invention provides for combination therapy with an anti-EGFR antibody, optionally cetuximab.

[0016] The data presented herein indicate that sEphB4-HSA may be an effective first-line therapy for many cancers where current therapies are ineffective, result in recurrence, or are not even considered for use due to the type of cancer and associated tumor. [Brief description of the drawings]

[0017] [Figure 1] Figure 1 shows scans of a patient with tonsillar SCC who received 10 mg / kg of sEphB4-HSA weekly, showing a partial response at 8 weeks of treatment and no sign of tumor at 16 weeks. [Diagram 2] Figure 2 shows a scan of a patient with laryngeal SCC receiving 10 mg / kg weekly sEphB4-HSA, showing a partial response at week 8 of treatment. [Diagram 3] Figure 3 shows scans of a patient with tonsillar SCC (HPV-) who received 10 mg / kg of sEphB4-HSA weekly. The scan shows a partial response at week 8 of treatment and no signs of tumor at week 16. The patient was taken off the study during the response and has been in stable disease. [Figure 4] Figure 4 shows a scan of a patient with hepatocellular carcinoma (HCC) who received weekly doses of 10 mg / kg sEphB4-HSA. The scan shows a partial response at 16 weeks of treatment with no evidence of tumor at week 16. The patient has remained disease-free and treatment-free for 8+ months. [Diagram 5] Figure 5 shows a scan of a patient with hepatocellular carcinoma (HCC) treated weekly with 10 mg / kg sEphB4-HSA. The scan shows a partial response at 16 weeks of treatment. The patient was taken off the study during the response and has stable disease. At 18+ months since study enrollment, the patient remains on treatment and in stable disease. [Figure 6] Figure 6 shows a scan of a patient with lung and brain metastases from a Kras-mutated multifocal adenocarcinoma who progressed from prior treatment with whole brain radiation and received 10 mg / kg weekly sEphB4-HSA followed by three cycles of carboplatin, paclitaxel, and Avastin. The patient is stable at 11+ months on sEphB4-HSA therapy. [Figure 7] 7 shows a photograph of a Kaposi's sarcoma (KS) patient who received weekly administration of 10 mg / kg sEphB4-HSA, and the patient experienced complete disappearance of the tumor and complete disappearance of the swelling in the lower extremities. [Figure 8] FIG. 8 shows a graphical representation of overall survival in newly diagnosed, advanced bladder cancer patients treated with regimens containing sEphB4-albumin fusion proteins. [Figure 9] FIG. 9 shows a graphic representation of neoadjuvant therapy for muscle-invasive bladder cancer, a chemotherapy-free sEphB4-albumin-containing regimen. [Figure 10] Figure 10 shows the response of an in vivo spontaneous breast tumor mouse model (MMTV-neu / Her2) to sEphB4-HSA treatment. Mice were treated with 7.5 mg / kg IP injection three times a week for five weeks. Tumor tissues were analyzed for total protein expression and phosphorylation of Her2 / ERBB2. Lungs were analyzed for metastasis. [Figure 11] FIG. 11 shows the response of an exemplary ERBB2 exon 20 duplication to sEphB4 therapy. [Figure 12] Figure 12 is a Western blot analysis showing how EphB4 binds and stabilizes EGFR. EphB4 binds to EGFR; knockdown of EphB4 reduces EGFR; EphB4 increases EGFR. [Figure 13] Figure 13 shows an in vivo efficacy study of sEphB4-HSA and EGFR antibody. Control mice received sEphB4 + anti-EGFR antibody (cetuximab) on day 42 and showed synergistic effects. [Figure 14] Figure 14 shows in vivo tumor regression of cholangiocarcinoma in a human patient (JG 64F). sEpHB4-HSA was administered at 15 mg / kg every 2 weeks for 15 months. The patient survived 24+ months from the start of sEphB4-HSA treatment. [Figure 15]Figures 15A-15F show that EphB4 expression confers a growth advantage to Kras mutant cells, with heat maps showing the effect of mRNA-mediated knockdown of tyrosine kinases on cell lines (Figure 15A), the effect of Kras ablation on six cell lines (Figure 15B), and the effect of EphB4 on Kas-mediated cell lines (Figure 15C). EphB4 protein was enhanced by Kras in a dose-dependent manner (Figure 15D), both EphB4 and its ligand ephrinB2 were increased in tumors (Figure 15E), and overexpression of EphB4 and ephrinB2 was also observed in tumors (Figure 15F). [Figure 16] Figures 16A-16D show that gene ablation of EphB4 increases survival in Kras mutant mice, which creates a premature stop codon in the ephB4 gene after cre-mediated recombination (Figure 16A) and EphB4 reconstitution (Figure 16B). Figure 16C shows that K14KB4 (n=9) mice had significantly less tumor growth and longer survival. Lung adenocarcinoma carcinogenesis was dramatically reduced in AdKPB4 (Figure 16D). [Figure 17] Figures 17A-17D show the effect of EphB4 knockdown attenuating AKT and ERK signaling in Kras-driven tumors. Signaling indicators, except for p-ERK1 / 2, were detected in oral papillomas (Figure 17A) but not in lung adenocarcinoma EphB4 knockout mouse tissues (Figure 17B). Figure 17C shows that overexpressed EphB4 mRNA and protein were found in tumors by in situ staining and immunofluorescence staining, respectively. Figure 17D shows protein expression in Ad-Cre mice. [Figure 18]Figures 18A-18G show that pharmacological inhibition of EphB4 effectively inhibits Kras-driven tumor formation in vivo. Figure 18A shows Western blots of EphB4 p-Tyr signal in sEphB4-treated tumors. Figure 18B shows survival rates of both sEphB4-treated groups compared to control K14K mice. Figure 18C shows the effect of prophylactic administration of sEphB4 to K14KP on tumor formation and survival. Figure 18D shows the effect of combined administration of taxol and sEphB4. Figures 18E and 18F show the effect of sEphB4 administration on tumor apoptosis and cell proliferation by TUNEL and Ki67 staining. Figure 18G shows the abundance of P-AKT and P-S6 after EphB4 administration. [Figure 19] Figures 19A-19F show the effect of EphB4 on β-TrCP1-mediated Kras ubiquitination and degradation. Figure 19A shows that knockdown of EphB4 by siRNA reduced the half-life of endogenous Kras protein. Figure 19B shows Kras levels in tumors of K14K mice after sEphB4 administration. Figure 19C shows the effect of siRNA knockdown of EphB4 and EphB4 overexpression on Kras ubiquitination. Figure 19D shows the effect of siRNA knockdown of β-TrCP1 and β-TrCP1 overexpression on Kras ubiquitination. Figure 19E shows IP / Western blot analysis of the effect of overexpressed EphB4 on β-TrCP-mediated Kras polyubiquitination. Figure 19F shows co-IP studies of protein-protein interactions between Kras and β-TrCP1, between EphB4 and β-TrCP1, and between EphB4 and Kras. [Figure 20]Figures 20A-20F show that the presence of a C-terminal EphB4 fragment modulates β-TrCP1 ligase activity and promotes monoubiquitination of Kras at Cys118. Figure 20A shows bacterially purified His-tagged Kras proteins (wild type, WT or G12V mutant) subjected to in vitro ubiquitination in the presence or absence of the in vitro transcribed and translated C-terminal fragments of EphB4 indicated in the figure, using b-TrCP1-GFP immunoprecipitated from HEK293 cell lysates. After 2 h of incubation, the reaction was stopped by adding sample loading dye and subjected to immunoblotting with the antibodies indicated in the figure. Figure 20B shows MS / MS spectra of peptides revealing ubiquitination of Kras at Cys118. Peptides isolated by in-gel digestion were separated on a reversed-phase column and collision-induced dissociation spectra were obtained using an Orbitrap XL mass spectrometer. FIG. 20C shows validation of the importance of C118 monoubiquitination, where a Cys118Ser mutant of Kras (designated GC mutant) in a G12D mutant background was subjected to in vitro ubiquitination together with WT and G12D (GD) mutants as described in panel (a) and processed / analyzed by immunoblotting with the antibodies indicated in the figure. FIG. 20D shows steady-state levels of different KRAS (wild type, WT; G12D, GD; C118S, CS, and G12D+C118S, GC) mutants in either the presence or absence of EphB4 overexpression. Relative band intensities (arbitrary units) were quantified using Image J, with wild type Kras levels taken as "1". Relative band intensities were calculated as above for -TrCP1 as above. Figure 20E shows the protein half-life of WT, GD, CS and GC Kras mutants calculated in the presence and absence of EphB4 by adding cycloheximide (CHX, 50 μg / ml). Samples were collected at the indicated time points, and band intensities were calculated and plotted over time. Figure 20F shows a hypothetical model showing the importance of EphB4 in promoting monoubiquitination of C118, which is required for the hyperactivation of mutant Kras. Targeting EphB4 or genetic modification of the C118 site to serine (S) can attenuate the oncogenic activity of mutant Kras. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In an embodiment, a method is provided for treating cancer comprising administering to a patient in need thereof an effective amount of a polypeptide agent that inhibits EphB4 or Ephrin B2, e.g., sEphB4, wherein the treatment is a first line therapy.

[0019] In one embodiment, a method of treating cancer is provided comprising administering an effective amount of a polypeptide agent that inhibits EphB4 or Ephrin B2, e.g., sEphB4, to a patient in need thereof, where the patient has not been treated with another anti-cancer agent.

[0020] In an embodiment, there is provided a polypeptide agent that inhibits EphB4 or Ephrin B2-mediated function for use in the preparation of a medicament for use as a first line therapy in treating cancer.In an embodiment, there is provided a polypeptide agent that inhibits EphB4 or Ephrin B2-mediated function for use as a first line therapy in treating cancer.

[0021] EphB4-ephrin B2 inhibitors The methods of the disclosure include treating, reducing, or preventing primary tumor growth or primary cancer formation, or cancer metastasis, as a first line therapy, by administering a polypeptide agent that inhibits EphB4 or Ephrin B2-mediated function.

[0022] The type 1 receptor tyrosine kinase EphB4 and its membrane-localized ligand ephrinB2 induce bidirectional signaling (forward signaling in receptor-expressing cells and reverse signaling in ligand-expressing cells). EphB4 belongs to the largest family of receptor tyrosine kinases, and upon interaction with ephrinB2 ligand, it has been reported to regulate neuronal migration, bone remodeling, angiogenesis, cancer progression, and metastasis (Pasquale EB, Cell, 133: 38-52, 2008). Expression of EphB4 and ephrinB2 is downregulated in most adult normal tissues as early as postnatal development, but 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 defective PTEN, induce EphB4 expression. EphB4 expression correlates with disease stage, grade, and survival, as knockdown of EphB4 leads to apoptotic cell death. Overexpression of its ligand, ephrinB2, has been reported to correlate with poor prognosis in several cancer types. ICT increases ephrinB2 in tumor vasculature (and tumors), and high levels of ephrinB2 impede immune cell recruitment, thus resulting in resistance to therapy.

[0023] Inhibition of EphB4-EphrinB2 interaction shows direct inhibitory effects on tumor cell proliferation in vitro and extracellularly. Polypeptide agents that inhibit EphB4 or EphrinB2-mediated functions have been previously described by the inventors of the present invention (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 composed of the soluble EphB4 extracellular domain fused to albumin at the C-terminus when expressed as a single seamless protein of 123.3 kDa. sEphB4-HSA specifically binds to EphrinB2. Preliminary studies of sEphB4-HSA in tumor models have shown increased migration of T and NK cells into tumors. This is accompanied by induction of ICAM-1 on tumor vasculature. ICAM-1 is an integrin that promotes adhesion of T and NK cells to the endothelium and subsequent migration of cells into tumors. sEphB4-HSA also downregulates PI3K signaling by blocking EphB-ephrinB2 interactions on tumor cells and tumor vasculature. By downregulating the PI3K pathway, sEphB4-HSA blocks signaling, promotes trafficking of immune cells into tumors, and inhibits survival signals in tumor cells.

[0024] Targeting EphB4-ephrinB2 is a therapeutic strategy that has survived clinical trials and has been shown to be safe with minimal to no toxicity in multiple clinical trials (A. El-Khoueiry BG, et al., Eur J Cancer, 69, 2016), likely due to its low expression levels in normal tissues. Although there is no direct evidence implicating EphB4-ephrinB2 interactions in cancer-associated immune responses, several papers have reported that members of the Eph / ephrin gene family regulate immune cell processes in inflammatory models such as atherosclerosis 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 have also been reported to regulate monocyte adhesion to the vascular wall, transepithelial migration, T cell chemotaxis, activation, proliferation and apoptosis, and hematopoietic cell mobilization from bone marrow sinusoidal cells.

[0025] In an embodiment of the disclosure, the polypeptide agent that inhibits EphB4 or EphrinB2-mediated function is a monomeric ligand-binding portion of an EphB4 protein or an EphrinB2 protein, or an antibody that binds to and affects EphB4 or EphrinB2. In an embodiment, the polypeptide agent is a soluble EphB4 (sEphB4) polypeptide that specifically binds to an EphrinB2 polypeptide and comprises the amino acid sequence of the extracellular domain of an EphB4 protein. In an embodiment, the sEphB4 polypeptide comprises the globular domain of an EphB4 protein.

[0026] In an embodiment, the sEphB4 polypeptide comprises a sequence selected from a sequence that is at least 90% identical to residues 1-522, a sequence that is at least 90% identical to residues 1-412, and a sequence that is at least 90% identical to residues 1-312 of the amino acid sequence of SEQ ID NO:1. In an embodiment, the sEphB4 polypeptide can comprise a sequence that includes a globular (G) domain (amino acids 29-197 of SEQ ID NO:1), and optionally further 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), a second fibronectin type 3 domain (amino acids 434-526 of SEQ ID NO:1). In an embodiment, the sEphB4 polypeptide will comprise amino acids 1-537 of SEQ ID NO:1. In an embodiment, the sEphB4 polypeptide will comprise amino acids 1-427 of SEQ ID NO:1. In an embodiment, the sEphB4 polypeptide will comprise amino acids 1-326 of SEQ ID NO:1. In an embodiment, the sEphB4 polypeptide will comprise amino acids 1 to 197, 29 to 197, 1 to 312, 29 to 132, 1 to 321, 29 to 321, 1 to 326, 29 to 326, 1 to 412, 29 to 412, 1 to 427, 29 to 427, 1 to 429, 29 to 429, 1 to 526, 29 to 526, 1 to 537, and 29 to 537 of SEQ ID NO: 1. In an embodiment, the sEphB4 polypeptide will comprise amino acids 16 to 197, 16 to 312, 16 to 321, 16 to 326, 16 to 412, 16 to 427, 16 to 429, 16 to 526 of SEQ ID NO: 1. In embodiments, an sEphB4 polypeptide can comprise an amino acid sequence at least 90%, optionally 95% or 99% identical to any of the foregoing amino acid sequences while retaining EphrinB2 binding activity. In embodiments, variations in the amino acid sequence from the sequence shown in SEQ ID NO:1 are conservative changes or deletions of not more than 1, 2, 3, 4 or 5 amino acids, particularly in the surface loop regions.

[0027] In embodiments, the soluble polypeptides can be prepared in multimeric form, for example, by expressing them as Fc fusion proteins or fusing them to another multimerization domain.

[0028] In an embodiment, the sEphB4 polypeptide will further comprise an additional component that provides an increased serum half-life while retaining EphrinB2 binding activity. In an embodiment, the sEphB4 polypeptide is monomeric and covalently linked to one or more polyoxyacrylene groups (e.g., polyethylene, polypropylene). In an embodiment, the sEphB4 polypeptide is covalently linked to a single polyethylene glycol (PEG) group (hereinafter "sEphB4-PEG"). In an embodiment, the sEphB4 polypeptide is covalently linked to two, three, or more PEG groups.

[0029] In embodiments, the one or more PEGs can have molecular weights 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 or branched PEG. In embodiments, the soluble monomeric EphB4 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 about 30 kDa, for example, via the s-amino group of an sEphB4 lysine or the N-terminal amino group. In embodiments, sEphB4 is randomly PEGylated at one of the amino groups of the s-amino group of an sEphB4 lysine and the N-terminal amino group.

[0030] In embodiments, the sEphB4 polypeptide is stably associated with a second stabilizing polypeptide that improves half-life without substantially reducing EphrinB2 binding. In embodiments, the stabilizing polypeptide will be immunocompatible with a human patient (or an animal patient if veterinary use is contemplated) and will have little or no significant biological activity. In embodiments, the sEphB4 polypeptide is covalently or non-covalently associated with an albumin selected from human serum albumin (HSA) (hereinafter "sEphB4-HSA") and bovine serum albumin (BSA) (hereinafter "sEphB4-BSA").

[0031] In embodiments, covalent binding can be achieved by expression of the sEphB4 polypeptide as a co-translational fusion with human serum albumin. The albumin sequence can be fused at the N-terminus, C-terminus, or at a non-disruptive internal position within the sEphB4 polypeptide. Exposed loops of sEphB4 seem to be suitable positions for the insertion of albumin sequences. Albumin can also be post-translationally linked to the sEphB4 polypeptide, for example, by chemical cross-linking. In embodiments, the sEphB4 polypeptide can also be stably linked to two or more albumin polypeptides.

[0032] In embodiments, the sEphB4-HSA fusion inhibits the interaction between EphrinB2 and EphB4, clustering of EphrinB2 or EphB4, phosphorylation of EphrinB2 or EphB4, or a combination thereof. In embodiments, the sEphB4-HSA fusion has enhanced in vivo stability compared to the unmodified wild-type polypeptide.

[0033] In an embodiment, sEphB4-HSA comprises residues 16-197 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In an embodiment, sEphB4-HSA comprises residues 16-312 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In an embodiment, sEphB4-HSA comprises residues 16-321 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In an embodiment, sEphB4-HSA comprises residues 16-326 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In an embodiment, sEphB4-HSA comprises residues 16-412 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In an embodiment, sEphB4-HSA comprises residues 16-427 of SEQ ID NO:1 directly fused to residues 25-609 of SEQ ID NO:2. In an embodiment, sEphB4-HSA comprises residues 16-429 of SEQ ID NO: 1 directly fused to residues 25-609 of SEQ ID NO: 2. In an embodiment, sEphB4-HSA comprises residues 16-526 of SEQ ID NO: 1 directly fused to residues 25-609 of SEQ ID NO: 2. In an embodiment, sEphB4-HSA comprises residues 16-537 of SEQ ID NO: 1 directly fused to residues 25-609 of SEQ ID NO: 2.

[0034] Head and neck squamous cell carcinoma (HNSCC) In embodiments, the present disclosure relates to methods and uses for treating HNSCC, e.g., as first-line therapy; and / or as treatment of subjects who previously responded to treatment with an anti-cancer therapy but experienced recurrent cancer upon cessation of treatment; and / or as treatment of subjects with resistant or refractory cancer.

[0035] Head and neck squamous cell carcinoma (HNSCC) accounts for nearly 90% of cancers involving the upper aerodigestive tract (UADT). In the United States, cancers of the oral cavity, pharynx, and larynx are expected to account for nearly 3% of incident cancers and 2% of cancer deaths in 2005. Approximately 500,000 new cases are diagnosed each year worldwide. Men are affected more than twice as often as women. More than half of these cancers arise in the oral cavity. The remainder are equally divided between the larynx and pharynx. Numerous clinical trials are testing the benefits of immunotherapy in human cancers, including head and neck squamous cell carcinoma (HNSCC). The objective response rate is 6-20% (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 exhibit innate or acquired resistance to immunotherapy. Attempts to simply combine more immune checkpoint inhibitors have also proven disappointing due to increased toxicity and lack of added benefit for patients (Clinical Trial Number NCT02205333). In orthotopic mouse models of HNSCC, it has recently been shown that tumor regrowth occurs even after combination treatment with anti-PDL1 antibodies and radiation therapy (RT) (Oweida A, et al., Clin Cancer Res, 2018; Messenheimer DJ, et al., Clin Cancer Res, 23:6165-77, 2017).

[0036] Although radiotherapy remains the standard of care in the definitive management of patients with locally advanced HNSCC and can serve as an adjuvant therapy for immunotherapy, there are several undesirable effects that occur in response to RT, which consequently impair the efficacy of immunotherapy drugs. RT cannot overcome the accumulation of immunosuppressive populations such as Tregs in the late (repair) phase. Therefore, it is important to find other therapies that act synergistically with RT and counteract its adverse effects in order to overcome harmful side effects, treatment resistance, and tumor regrowth.

[0037] The 5-year survival rate for HNSCC is poor and has not improved for several decades. In addition, patients with the disease experience severe morbidity, including disfigurement, speech disorders, swallowing disorders, and respiratory problems. Late diagnosis and propensity for recurrence are challenges that impede efforts to improve outcomes for 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. On August 5, 2016, the U.S. Food and Drug Administration (FDA) approved pembrolizumab (Keytruda®) for the treatment of some patients with advanced forms of head and neck cancer. The approval is intended for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) that continues to progress despite standard treatment with chemotherapy. According to the FDA approval summary, 28 patients (16%) experienced tumor responses after treatment with pembrolizumab. In 23 of these patients (82%), tumor responses lasted for more than 6 months, and several lasted for more than 2 years. HNSCC patients whose tumors are human papillomavirus (HPV) positive typically have better outcomes after chemotherapy treatment than patients whose tumors are HPV negative. Responses were seen not only in patients with HPV-negative tumors, but also in patients with HPV-positive tumors (24% and 16%, respectively), according to the FDA approval summary.

[0038] Recurrent, locally advanced, or metastatic head and neck squamous cell carcinoma (HNSCC) is a life-threatening disease. Head and neck squamous cell carcinoma is a heterogeneous tumor with prognosis that varies depending on the site of origin. There are two categories: oral cavity / pharyngeal and laryngeal. HPV-negative tumors are associated with high risk, while HPV is associated with a low risk group in the oropharynx. In 2016, more than 48,000 new cases of oral cavity and pharyngeal cancer and more than 13,000 cases of laryngeal cancer were diagnosed in the United States, and approximately 13,000 deaths were attributed to these cancers. At the time of initial diagnosis, approximately 18% of patients with oral cavity / pharyngeal cancer and approximately 19% of patients with laryngeal cancer have distant metastases. Furthermore, at presentation, approximately 47% of patients with oral / pharyngeal cancer and 22% with laryngeal cancer have regional lymph node disease (without distant metastases); in such patients with locally advanced disease, 20%-30% will develop a local recurrence and an additional 10%-15% can be expected to develop distant metastases. In most clinical series, the median survival of patients with recurrent or metastatic HNSCC is 6-10 months.

[0039] Standard treatment for locally advanced HNSCC includes platinum-containing chemotherapy combined with radiation (e.g., as induction therapy, as concurrent therapy with radiation, or as part of adjuvant therapy with radiation after surgical resection). First-line chemotherapy for metastatic HNSCC consists of multiagent platinum-containing chemotherapy regimens, such as cisplatin or carboplatin + 5-fluorouracil + cetuximab. Recently, PD1 antibodies have been approved for patients with recurrent or refractory HNSCC after failure of platinum agents and cetuximab. Response rates to PD1 antibodies alone are 16%, with DORs ranging from 2.4+ to 27.7+ months, indicating durable responses. However, there remains a need for new therapies for patients who have failed or experienced unacceptable toxicity to chemotherapy, cetuximab, and PD1 antibodies.

[0040] Hepatocellular carcinoma (HCC) In embodiments, the present disclosure relates to methods and uses for treating HCC, e.g., as first-line therapy; and / or as treatment of subjects who previously responded to treatment with an anti-cancer therapy but experienced recurrent cancer upon cessation of treatment; and / or as treatment of subjects with resistant or refractory cancer.

[0041] Liver cancer accounts for over 850,000 new cancer cases annually, with approximately 90% of these being hepatocellular carcinoma (HCC). Chronic infection with hepatitis C virus (HCV) or hepatitis B virus (HBV) is the leading cause of HCC. HCC is the most frequent cancer in certain parts 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, for example, Parkin DM, Lancet Oncology, 2:533-43, 2001). Because HCC is often diagnosed late in the clinical course, only 10-15% of patients are candidates for curative surgery. Multiple modalities are available for local therapy, including surgery, chemical ablation, radioablation, and chemoembolization, with local disease control in a significant patient population. Systemic chemotherapy or supportive care are the mainstay treatment options for the majority of HCC patients. HCC generally responds poorly to treatment, and most chemotherapeutic agents have limited effectiveness and have failed to improve patient survival (see, e.g., Gish RG et al., J. of Clinical Oncology 25:3069-75, 2007; Ramanathan RK et al., J. of Clinical Oncology 24:4010, 2006).

[0042] Sorafenib is a small molecule multikinase inhibitor and was the first systemic therapy approved for advanced hepatocellular carcinoma. In some patients who tolerated sorafenib but progressed on alternative treatments, another multikinase inhibitor, regorafenib, was approved and provided a survival benefit compared with placebo controls (10.6 vs. 7.8 months). More recently, the combination of atezolizumab and bevacizumab was shown to be superior to sorafenib as first-line treatment. After a median follow-up of 8.6 months, median overall survival was not reached in the combination arm compared with 13.2 months in the sorafenib arm. Overall response rate was 27% in the combination arm compared with 12% in the sorafenib arm.

[0043] Recent studies evaluating the Programmed Death 1 (PD-1) antibody nivolumab (OPDIVO®) have 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). Overall survival (OS) at 6 months is 72%. Nivolumab demonstrated a manageable AE profile and demonstrated durable responses across all dose levels and HCC cohorts, with favorable 6-month OS rates. PD-1 antibodies have also been granted accelerated approval in second-line treatment. Additional therapies are needed for patients who fail currently approved therapies.

[0044] Non-small cell lung cancer (NSCLC) In embodiments, the present disclosure relates to methods and uses for treating NSCLC, e.g., as first line therapy; and / or as treatment of subjects who previously responded to treatment with an anti-cancer therapy but experienced recurrent cancer upon cessation of treatment; and / or as treatment of subjects with resistant or refractory cancer.

[0045] 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 approximately 85% of all lung cancers. As a class, NSCLC is relatively less sensitive to chemotherapy compared to small cell carcinoma. It is primarily treated by surgical resection with curative intent, when possible, but chemotherapy is increasingly being used both before (neoadjuvant chemotherapy) and after (adjuvant chemotherapy) surgery. 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 as a first-line treatment for NSCLC when the cancer overexpresses PDL1 and the cancer does not have EGFR or ALK mutations; if chemotherapy has already been administered, pembrolizumab can be used as a second-line treatment, but if the cancer has EGFR or ALK mutations, drugs targeting those mutations should be used first. PDL1 should be assessed using a validated and approved companion diagnostic. The Keynote-001 study (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. The objective response rate for all patients 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 value from the training group. In patients with a proportion score of at least 50% in the validation group, the response rate was 45.2%. In 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).KRAS-mutated lung adenocarcinomas, which account for 30% of non-small cell lung cancers, exhibit a high degree of heterogeneity whose clinical implications are becoming evident. Tumor heterogeneity can be influenced by genetic and / or epigenetic alterations that occur concomitantly with KRAS, while heterogeneous tumor subsets may also be the product of cells of different origin. Transgenic mouse models based on the spatial and temporal activation of oncogenic Kras have helped to address these issues. Indeed, most tissues, except the lung, express Kras. G12V The observation that these cells are resistant to oncogenic signals highlights the exquisite cell-type dependency of transformation driven by oncogenic Kras.

[0046] Kaposi's Sarcoma (KS) In embodiments, the present disclosure relates to methods and uses for treating KS, e.g., as a first line therapy; and / or as treatment of subjects who previously responded to treatment with an anti-cancer therapy but experienced recurrent cancer upon cessation of treatment; and / or as treatment of subjects with resistant or refractory cancer.

[0047] Kaposi's sarcoma (KS) is a multifocal angioproliferative 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 many epidemiological and pathophysiological factors. KS is classified into four clinical types: classical Mediterranean KS, African endemic KS, immunosuppressant-associated KS, and HIV-associated KS. HIV-associated KS, a rare disease prior to the era of HIV and AIDS, is the most frequent malignancy in HIV-infected patients. KS can affect many organs. KS most frequently manifests as a skin disorder. In many advanced cases, KS affects organs such as the lungs, liver, and gastrointestinal tract. At present, KS is incurable. Available treatments are palliative. Systemic chemotherapy is generally used for patients with more advanced disease or evidence of rapid disease progression. The main 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 advanced cutaneous KS includes cytotoxic chemotherapy, such as liposomal anthracyclines and paclitaxel. Liposomal doxorubicin has superior efficacy and good 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, response rates to liposomal doxorubicin may be higher. However, complete responses are rare, and there is no cure. At present, targeted therapies for KS are not well developed.

[0048] In embodiments, the cancer is selected from, but is not limited to, head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), Kras-mutated non-small cell lung adenocarcinoma, and Kaposi's sarcoma (KS).

[0049] In an embodiment, the patient has previously responded to treatment with an anti-cancer therapy, but experienced a recurrence upon cessation of treatment (hereinafter "recurrent proliferative disease").

[0050] In embodiments, the patient has a resistant or refractory cancer. In embodiments, the cancer is refractory to immunotherapy. In embodiments, the cancer is refractory to treatment with a chemotherapeutic agent. In embodiments, the cancer is refractory to treatment with an ablative antibody against a specific tumor antigen. In embodiments, the cancer is refractory to treatment with an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint). In embodiments, the cancer is refractory to targeted therapy with an immunoconjugate, antibody drug conjugate (ADC), or fusion molecule comprising an ablative antibody against a specific tumor antigen and a cytotoxic agent. In embodiments, the cancer is refractory to targeted therapy with a small molecule kinase inhibitor. In embodiments, the cancer is refractory to combination therapy involving two or more of, for example, immunotherapy, treatment with a chemotherapeutic agent, treatment with an ablative 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 an ablative antibody against a specific tumor antigen and a cytotoxic agent, targeted therapy with a small molecule kinase inhibitor, treatment with surgery, treatment with stem cell transplant, and treatment with radiation.

[0051] Bladder cancer In embodiments, the present disclosure relates to methods and uses for treating bladder cancer, e.g., as a first line therapy; and / or as treatment of subjects who previously responded to treatment with an anti-cancer therapy but experienced recurrent cancer upon cessation of treatment; and / or as treatment of subjects with resistant or refractory cancer.

[0052] In an embodiment, bladder cancer is newly diagnosed, locally advanced (beyond the bladder or urinary system, ureter, renal pelvis) bladder cancer and urothelial cancer. In an embodiment, the bladder cancer patient has not received systemic therapy or is within 12 months of neoadjuvant systemic chemotherapy.

[0053] In an embodiment, the bladder cancer patient is ineligible to receive a standard regimen that includes cisplatin.

[0054] In embodiments, the bladder cancer tumor has a TP53, ARID-1, BAP-1, RAS, PBRM1, PI3K, and / or PIK3CA mutation. In embodiments, the bladder cancer tumor has a HER2 and / or EGFR2 mutation.

[0055] In an embodiment, the bladder cancer is muscle-invasive bladder cancer.

[0056] Cholangiocarcinoma (CCA) In embodiments, the present disclosure relates to methods and uses for treating CCA, e.g., as a first line therapy; and / or as treatment of subjects who previously responded to treatment with an anti-cancer therapy but experienced recurrent cancer upon cessation of treatment; and / or as treatment of subjects with resistant or refractory cancer.

[0057] CCAs constitute a diverse group of malignancies arising in the biliary tree. CCAs are divided into three subtypes, depending on their anatomic site of origin: intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA). HCC-CCA mixed tumors are considered independent entities and are a rare type of hepatic malignancy that share features of both iCCA and HCC, with an aggressive disease course and poor prognosis. iCCA arises above the secondary bile ducts, while the anatomical point of distinction between pCCA and dCCA is the insertion of the cystic duct. pCCA and dCCA are sometimes collectively referred to as "extrahepatic" (eCCA). In the United States, pCCA is the single largest group, accounting for approximately 50-60% of all CCAs, followed by dCCA (20-30%) and iCCA (10-20%). CCA is the second most common primary liver malignancy after hepatocellular carcinoma (HCC), accounting for approximately 15% of all primary liver tumors and 3% of gastrointestinal cancers. CCA is usually asymptomatic in the early stages and is therefore often diagnosed when the disease is already at an advanced stage, greatly limiting treatment options and resulting in a poor prognosis. Although CCA is a rare cancer, its incidence (0.3-6 per 100,000 inhabitants per year) and mortality (1-6 per 100,000 inhabitants per year worldwide, not taking into account certain regions such as Korea, China, and Thailand, where the incidence is >6 per 100,000 inhabitants) have been increasing worldwide in recent decades, making it a global health problem. Despite advances in awareness, knowledge, diagnosis, and treatment of CCA, patient prognosis has not improved substantially in the past decade, with 5-year survival rates (7-20%) and tumor recurrence rates after resection still disappointing. Large-bore iCCA, like pCCA and dCCA, has a high frequency of mutations in the KRAS and / or TP53 genes. As discussed in Examples 1-2, tumors with mutations in TP53, ARID-1, BAP-1, RAS, PBRM1, PI3K, PIK3CA did not prevent response to sEphB4-HSA therapy. Furthermore, co-administration of sEphB4-HSA + anti-EGFR (cetuximab) may have synergistic anti-tumor effects, particularly in anti-EGFR resistant cancers, possibly due to factors such as HER2 overexpression.

[0058] In an embodiment, the CCA patient is resistant to cisplatin and / or gemcitabine.

[0059] Compositions for treating cholangiocarcinoma, among other cancer types, include co-administration of sEphB4-HSA fusion protein and anti-EGFR antibody or antibody fragment thereof (e.g., VHH, nanobody, scFv, etc.). In embodiments, the anti-EGFR antibody can be the monoclonal antibody (mAb) cetuximab. The antibody can be a humanized antibody, a human antibody, a chimeric antibody, among other antibody formats.

[0060] HER2 / EGFR2 Mutated Cancer In embodiments, the disclosure relates to methods and uses for treating HER2 / EGFR2 mutant cancers, for example, as first line therapy; and / or as treatment of subjects who previously responded to treatment with an anti-cancer therapy but experienced recurrent cancer upon cessation of treatment; and / or as treatment of subjects with resistant or refractory cancer.

[0061] In embodiments, the HER2 / EGFR2 mutant cancer is lung cancer, head and neck cancer, or bladder cancer.

[0062] In an embodiment, the HER2 / EGFR2 mutant cancer patient has failed chemotherapy and / or kinase inhibitor therapy and / or Her2 antibody therapy (including, for example, ADC).

[0063] In an embodiment, the HER2 / EGFR2 mutant cancer patient has an exon 20 p^772_A775 duplication Her2 mutation. In an embodiment, the HER2 / EGFR2 mutant cancer patient has a simultaneous mutation in RB1 exon 20 pL700X and TP53 exon 4 p.S116fs.

[0064] In an embodiment, the HER2 / EGFR2 mutant cancer patient has an ERBB2 exon 17 V659E mutation. In an embodiment, the HER2 / EGFR2 mutant cancer patient simultaneously has a PIK3CA E 545K, a TP53 exon 5 R158fs, and an ATM G2891D NF1 E2143 mutation.

[0065] In an embodiment, the HER2 / EGFR2 mutant cancer patient has an ERBB2 mutation. In an embodiment, the HER2 / EGFR2 mutant cancer patient has ATM, RICTOR, CCNE1, CDKN18, IRS2, PMS2, TERT, and TP53 mutations simultaneously.

[0066] In embodiments, EGFR-mutated cancers, such as head and neck cancer, lung cancer, colon cancer, and bladder cancer, exhibit high EGFR levels.

[0067] KRAS-mutated cancer In embodiments, the disclosure relates to methods and uses for treating kras mutant cancers, for example, as first line therapy; and / or as treatment of subjects who previously responded to treatment with an anti-cancer therapy but experienced recurrent cancer upon cessation of treatment; and / or as treatment of subjects with resistant or refractory cancer.

[0068] In embodiments, the kras mutant cancer is selected from lung, colorectal and pancreatic cancer. In embodiments, the kras mutant cancer is selected from pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC).

[0069] In an embodiment, the kras mutation is selected from G12C, G12D, and G12R.

[0070] In an embodiment, the method of the invention causes a reduction or inhibition of Kras nucleic acid or protein levels, hi an embodiment, the method of the invention causes an increase in proteolysis of Kras protein.

[0071] In an embodiment, the methods of the invention result in a reduction or inhibition of tumor formation caused by Kras, for example compared to an untreated state.

[0072] Pharmaceutical Compositions In embodiments, the polypeptide therapeutics of the present disclosure are often administered as pharmaceutical compositions containing an active therapeutic agent, i.e., and various other pharma- ceutically acceptable ingredients (see Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended method of administration and therapeutic application. The composition may also contain pharma- ceutically acceptable non-toxic carriers or diluents, defined as vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans, 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, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, and the like.

[0073] In embodiments, the pharmaceutical compositions for the treatment of primary or metastatic cancer may be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular means.

[0074] For parenteral administration, the disclosed pharmaceutical compositions can be administered as injectable doses of a solution or suspension of the substance in a physiologically acceptable diluent, with the pharmaceutical carrier being capable of being a sterile liquid, such as water, an oil, saline, glycerol, ethanol, and the like. Additionally, 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 those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols, such as propylene glycol and polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. The antibody and / or polypeptide can be administered in the form of a depot injectable or implantable preparation, formulated in such a manner as to permit sustained release of the active ingredient. Typically, the pharmaceutical compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid vehicles prior to injection can also be prepared. The preparations can also be emulsified or encapsulated in liposomes or microparticles, such as polylactides, polyglycolides, copolymers, to enhance adjuvant effect, as discussed above. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The polypeptide agents of the present disclosure can be administered in the form of a depot injection or implant preparation, which can be formulated in such a way as to permit sustained or pulsatile release of the active ingredient.

[0075] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

[0076] In embodiments, the methods of the disclosure include administering to a patient in need of treatment a therapeutically effective amount or effective amount of a sEphB4-HSA polypeptide of the disclosure. In embodiments, the effective amount of a polypeptide of the disclosure described herein, for example for the treatment of primary or metastatic cancer, will vary depending 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, other medications administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, although non-human mammals, including transgenic mammals, can also be treated. Therapeutic dosages should be titrated to optimize safety and efficacy.

[0077] In embodiments, the dosage can range from about 0.0001 to 100 mg / kg of host body weight, more usually 0.01 to 10 mg / kg. For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight, or in the range of 1 to 10 mg / kg. In embodiments, the dosage of the polypeptide administered to the patient is selected from about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, and about 10.0 mg / kg. In embodiments, the regime involves weekly administration. In embodiments, the regime involves biweekly or monthly or 3 to 6 monthly administrations. The therapeutic entities of the present disclosure are typically administered multiple times. The interval between single doses can be weekly, biweekly, monthly, or yearly. Intervals can also be irregular, if indicated, by measuring the blood levels of the therapeutic entity in the patient. Alternatively, the therapeutic entities of the present disclosure can be administered as sustained release formulations, in which case less frequent administration is required. Dosage and frequency will vary depending on the half-life of the polypeptide in the patient.

[0078] The toxicity of the polypeptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example by measuring the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating a non-toxic dosage range for use in humans. The dosage of the polypeptides described herein is preferably within a range of circulating concentrations that include an effective dose with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be selected by the subject physician in view of the patient's condition (see, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1).

[0079] In an embodiment, the method of the present invention includes one or more additional anti-cancer therapies selected from immunotherapy, chemotherapy, treatment with ablative antibodies against target specific tumor antigens, treatment with agonistic, antagonistic or blocking antibodies against target costimulatory or co-inhibitory molecules (immune checkpoints), targeted therapy with immunoconjugates, ADCs or fusion molecules comprising ablative antibodies against specific tumor antigens and cytotoxic agents, targeted therapy with small molecule kinase inhibitors, surgery, radiation therapy and stem cell transplantation. Combinations may have synergistic effects. Combinations may increase the therapeutic index of anti-cancer therapies.

[0080] In embodiments, immunotherapy includes treatment with agonistic, antagonistic, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints) such as PD-1, PD-L1, PD-L2, CTLA-4, OX-40, CD137, GITR, LAG3, TIM-3, VISTA, etc.; treatment with bispecific T cell engaging antibodies (BiTE®) such as blinatumomab; treatment with biological responses such as IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, IFN-γ, etc. The immunotherapy is selected from treatment with administration of a modulator; treatment with a therapeutic vaccine such as sipuleucel-T; treatment with a dendritic cell vaccine or a tumor antigen peptide vaccine; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor infiltrating lymphoid tumors (TIL); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with an immunostimulatory agent such as the Toll-like receptor (TLR) agonist CpG or imiquimod. In an embodiment, the immunotherapy is selected from treatment with an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; and treatment with a bispecific T cell-inducing antibody (BiTE®). In an embodiment, the immunotherapy is treatment with an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule. In an embodiment, the immunotherapy is a treatment with chimeric antigen receptor (CAR)-T cells. In an embodiment, the immunotherapy is a treatment with CAR-NK cells. In an embodiment, the immunotherapy is also a treatment with bispecific T cell-engaging antibodies (BiTE®).

[0081] Depending on the nature of the combination therapy, administration of the polypeptide therapeutic of the present disclosure can be continued while the other therapy is being administered and / or after. The polypeptide therapeutic can 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 can be delivered in a single dose or divided into multiple doses, and can be delivered over a period of time, including, for example, daily, every other day, semi-weekly, or weekly. The effective amount will vary depending on the route of administration, the particular agent, the dose of anti-cancer agent, etc., and can be determined empirically by one of skill in the art.

[0082] The following examples are presented to further illustrate the present disclosure. EXAMPLES

[0083] Example 1: Phase I / II study of sEphB4-HSA monotherapy in head and neck SCC The study enrolled 18 patients with HNSCC, including 7 in the dose escalation cohort and 11 in the expansion cohort. Eight patients were HPV negative and 10 patients were HPV positive. One patient with adenocystic carcinoma of the parotid gland was excluded from the analysis. One patient discontinued treatment within the first 3 weeks of treatment.

[0084] Sixteen patients were evaluated for response. Patients received weekly intravenous sEphB4-HSA at 10 mg / kg. Fifteen patients had previously received definitive radiation therapy and chemotherapy. Ten patients had undergone surgery either at the time of diagnosis or at recurrence. All patients had received chemotherapy for recurrent HNSCC. Prior systemic therapy ranged from two to six different regimens. Additionally, 12 patients had previously received cetuximab and one patient had received a PD1 antibody. The results are shown in Table 1 below and in Figures 1, 2, and 3. TIFF2024511995000002.tif69170

[0085] Among these patients, overall responses included 2 PRs, 2 tumor regressions, 1 mixed response, and 5 stable disease for >4 months. Six patients progressed. One of the responding patients underwent biopsy with no evidence of viable tumor (Figure 1). One patient decided to stop treatment after 10 months and has remained disease-free for 16 months since. Figure 2 shows a scan of a patient with laryngeal SCC treated weekly with 10 mg / kg sEphB4-HSA. The scan shows a partial response at week 8 of treatment.

[0086] Examples of patients showing tumor response or disease control are shown in Figures 1, 2, and 3 and Tables 2-4. TIFF2024511995000003.tif30170TIFF2024511995000004.tif38170TIFF2024511995000005.tif54170

[0087] This study demonstrated that sEphB4-HSA has activity in recurrent refractory HNSCC as a single agent, suggesting that sEphB4-HSA could be used as a first-line therapy for the treatment of HNSCC.

[0088] Example 2: sEphB4-HSA monotherapy in hepatocellular carcinoma A cohort of patients with histologically confirmed advanced HCC (≥18 years of age) was studied. Patients previously treated with sorafenib and / or PD1 antibody were eligible. Primary endpoints were safety and tolerability and objective response rate (Response Evaluation Criteria in Solid Tumors version 1.1), duration of response, duration of stable disease, and time to progression. Patients received 10 mg / kg sEphB4-HSA intravenously weekly. Fifteen eligible patients were enrolled in the study. Most patients were Asian males, and most patients had an ECOG performance status of 1. All patients had received prior systemic therapy: PD1 antibody in 10 patients, Nexavar in 9, surgery in 6, and radiation in 5. Most patients had received two or more regimens previously. To date, objective response rate has been observed in 1 of 15 patients (7%), with stable disease for >4 months in 8 patients (>20+ months in 2 patients). In conclusion, sEphB4-HSA can be safely administered long-term. Grade 3 toxicities included fatigue in 1 patient, nausea in 1 patient, neutropenia in 1 patient, and hypertension in 6 patients. Five patients experienced hypertension, which required dose reduction. The sustained objective responses and long-term stable disease after failure of Nexavar and PD-1 support its development as a single agent and in combination with PD-1 antibodies. TIFF2024511995000006.tif74170TIFF2024511995000007.tif63170TIFF2024511995000008.tif88170

[0089] In total, eight patients achieved stable disease for ≥4 months, and two patients achieved stable disease for >20 months. Grade 3 / 4 treatment-related adverse events were observed in seven patients (47%), including six patients with hypertension. Two patients required dose reduction. No patients experienced complications due to hypertension.

[0090] One patient (FN) was a 79-year-old woman with HCV-associated HCC who was previously treated with TACE for 15 months with a partial response. She was then treated with PD1 antibody and progressed after 5 months. She was subsequently treated with sEphB4-HSA for 11 months and had two small residual nodules treated with stereotactic radiotherapy. The patient currently remains treatment-free and disease-free for 8+ months (or 22+ months from initial study enrollment) (Figure 4). TIFF2024511995000009.tif41170

[0091] Another patient (BW) was a male with HCV-associated HCC who had been previously treated with TACE and had been stable for 7 months. He then received PD1 antibody and had tumor regression. He subsequently experienced disease progression, including lung metastases. PD1 antibody was continued for 18 months, but slowly progressed after the first 8 months. He has been on sEphB4-HSA for 20+ months and has been stable. The third patient (TN) underwent liver transplantation and developed recurrent HCC. He had previously received Nexavar, TACE, Gemzar, oxaliplatin, and yttrium-90. He had been stable for 8 months (Figure 5). TIFF2024511995000010.tif41170

[0092] This phase 2 study indicates that after failure of Nexavar and PD1 antibody, treatment with sEphB4-HSA resulted in tumor regression and durable disease control, with improved responses, duration of response, and survival, and that sEphB4-HSA can be used as first-line therapy for the treatment of HCC.

[0093] Example 3: sEphB4-HSA monotherapy in Kras-mutated non-small cell lung adenocarcinoma A cohort of patients with Kras-mutated lung adenocarcinoma was evaluated in the single-agent sEphB4-HSA trial. Patients were included if they had a diagnosis of KRAS-mutated lung cancer, had failed prior therapy, and had evidence of progressive disease. Nine patients were enrolled. Two of the nine patients were withdrawn from the study within 4 weeks of treatment and were not assessed for response. Patient profiles are also included in the clinical profile data. Each of the five cases is summarized below.

[0094] Patient AC: Kras-mutated multifocal lung adenocarcinoma and brain metastases. The patient underwent brain irradiation and then received three cycles of carboplatin, paclitaxel, and Avastin. The patient was intolerant and progressed at 7 months. He was treated with sEphB4-HSA and remained stable for 11 months (Figure 6). TIFF2024511995000011.tif31170

[0095] Patient TC: Kras-mutated right upper lung adenocarcinoma, treated with Alimta and Carboplatin, progressed after 10 months, then nanosphere docetaxel for 6 months, stable, then Taxotere for 4 months, progressed, then sEphB4-HSA, right shoulder pain disappeared, stable for 8 months. TIFF2024511995000012.tif32170

[0096] Patient PS: Treated with Kras-mutated lung cancer with 6 cycles of Alimta, carboplatin, and Avastin, followed by maintenance Alimta for a total of 21 months. At relapse, treated with Taxotere for 8 months, progressed on Avastin, intolerant to novelvin and Avastin, progressed on Avastin alone, and progressed on etoposide + cisplatin + gemcitabine. Then received gemcitabine and Avastin for 6 months. Underwent sEphB4-HSA testing at progression. Stable for 4 months. TIFF2024511995000013.tif40170

[0097] Patient JC: A 76-year-old female with moderately differentiated adenocarcinoma of the right upper lung, Kras mutant, underwent surgery and 4 cycles of adjuvant cisplatin and Alimta. The patient's disease progressed. As the tumor was PD-L1 positive (70%), she was placed on a trial of pembrolizumab. The tumor progressed after 3 months. She was placed on a trial of sEphB4-HSA with stable disease for 4 months. TIFF2024511995000014.tif34170

[0098] Patient HW: This patient with Kras-mutated lung cancer received Avastin, Alimta, and Carboplatin. After 6 months, the patient progressed and entered the sEphB4-HSA trial. The patient had stable disease for 32 weeks. TIFF2024511995000015.tif33170

[0099] Of the 7 of 9 patients evaluable for response, 5 patients had stable disease for 11, 8, 8, 4, and 4 months on EphB4-HSA monotherapy, respectively.

[0100] Example 4: sEphB4-HSA monotherapy in Kaposi's sarcoma Three patients with KS were included in the study; two were HIV-infected and one was HIV-negative. All three patients had received multiple prior therapies. One of the two HIV KS patients who had received six prior therapies had disease progression involving the entire leg and long-standing extensive associated edema that did not completely resolve after three prior therapies.

[0101] Another patient had previously been treated with cytotoxic chemotherapy and multiple investigational drugs. sEphB4-HSA was administered. The tumor completely disappeared, as did leg edema (Figure 7). Remission has continued for more than 2 years. Treatment frequency was reduced to 10 mg / kg once every 2 weeks, and remission has been maintained for more than 6 months. Based on preclinical studies, tumor target expression, and clinical response, a phase II trial is currently underway through the NCI-CTEP-AMC (AIDS Malignancy Consortium). TIFF2024511995000016.tif73170

[0102] Example 5: First-line or frontline therapy for sEphB4-HSA bladder cancer Advanced disease: Locally advanced (beyond the bladder or urinary system, ureters, or renal pelvis) bladder and urothelial cancers newly diagnosed prior to the use of systemic therapy or within 12 months of neoadjuvant systemic chemotherapy (Figure 8). Eight patients were ineligible to receive standard cisplatin-containing regimens and had very poor survival as a result. Ten patients had been treated with a regimen containing 10 mg / kg sEphB4-albumin weekly without receiving cytotoxic chemotherapy such as cisplatin, carboplatin, gemcitabine, or methotrexate. Six patients completed the first 6 weeks of treatment and had one or more tumor evaluations by radiological methods (computed tomography). Each of the six patients had a response as defined by RECIST response criteria (version 1.0). Additionally, each of the six patients achieved a complete remission. Furthermore, none of the patients recurred after 4 to 16 months of follow-up. Two patients died after discontinuing treatment for unrelated reasons within the first 3 weeks. Eight patients are alive.

[0103] Tumors with mutations in TP53, ARID-1, BAP-1, RAS, PBRM1, PI3K, and PIK3CA did not prevent response to treatment.

[0104] Standard chemotherapy includes cisplatin, typically combined with gemcitabine. The best treatment regimens produce an overall response in about 40% of cases, with progression-free survival of 6-7 months and a median overall survival of about 16 months. Patients who could not receive cisplatin (typically 40-60%) had a much worse prognosis.

[0105] Example 6: Muscle-invasive bladder cancer Figure 8 graphs the overall survival rate for newly diagnosed, advanced bladder cancer patients treated with regimens containing sEphB4-albumin fusion proteins.

[0106] Patients with newly diagnosed bladder cancer were treated with standard-of-care cisplatin plus gemcitabine chemotherapy, which resulted in nearly 30% pathological complete response at the time of definitive surgery (radical cystectomy), predicting long-term disease-free survival. The median time to recurrence was approximately 14–17 months. Seventeen patients with newly diagnosed muscle-invasive bladder cancer were treated with sEphB4-albumin fusion protein (Figure 9). The 10 patients who expressed the drug's target, ephrin B2, had a pathological complete response rate of 70%. None of these seven patients recurred during the maximum follow-up period of 36 months. Furthermore, two patients who refused cystectomy remained disease-free after more than 2 years of follow-up, indicating that organ preservation can be achieved in patients with biomarker-positive bladder cancer. These results were highly unexpected. Furthermore, of the seven biomarker ephrinB2-negative patients, none relapsed after up to 36 months of follow-up, but only two achieved pathological complete remission. These data suggested that ephrinB2 could be induced during treatment, potentially resulting in biological benefits and even memory responses.

[0107] Example 7: Non-muscle invasive or superficial bladder cancer Patients with biomarker-positive tumors, such as muscle-invasive bladder cancer, are more likely to have tumors that respond to immunotherapy and obtain durable responses than those with muscle-invasive and metastatic bladder cancer. In particular, BCG shows high activity in non-muscle-invasive bladder cancer. HER2 / EGFR2 mutant cancers of various organs: lung, head and neck, bladder, Her2 mutant tumors are incurable. Standard therapies include chemotherapy, kinase inhibitors, and Her2 antibody drug conjugates. These treatments have produced partial responses in a subgroup of patients.

[0108] Her2 is localized to the cell membrane along with EphB4. Her2 induces EphB4. Her2-specific antibodies reduced EphB4 levels. Studies have shown that EphB4 regulates downstream signaling and phosphorylation of Her2. First, we examined Her2 transgenic mice, which have high expression of EphB4 as expected. Administration of soluble EphB4 to transgenic mice prevented tumor formation and metastasis, including to the lungs (Figure 10). Mice were administered a dose of 7.5 mg / kg by IP injection three times a week for 5 weeks. Tumor tissues were analyzed for Her2 / ERBB2 total protein and protein phosphorylation. Histological analysis showed a decrease in Her2 phosphorylation by fluorescent confocal microscopy staining analysis. Thus, we treated Her2-overexpressing tumors where chemotherapy had failed. Rapid and often complete remissions were observed and were sustained for a long period of time. Treatment of mice with sEphB4-HSA showed a statistically significant reduction in tumor volume (p=0.005) and tumor burden per mouse (p<0.005). A statistically significant reduction in the mean number of tumors per mouse was observed after treatment with sEphB4-HSA (p<0.01). Furthermore, the amount of lung metastases observed was greatly reduced.

[0109] In humans, Her2 mutant tumors pose an even greater challenge. We treated a patient with Her2 mutant cancer who had failed standard of care chemotherapy and kinase inhibitors. This state of the disease represents an unmet need. We treated five patients with Her2 mutations including exon 20 p^772_A775 duplication, RB1 exon 20 pL700X, TP53 exon 4 p.S116fs; ERBB2 exon 17 V659E co-mutations, co-mutations PIK3CA E 545K, TP53 exon 5 R158fs, ATM G2891D NF1 E2143, in another case, ERBB2 mutations co-mutated ATM, RICTOR, CCNE1, CDKN18, IRS2, PMS2, TERT, TP53. The patient went into complete remission and remained disease-free and treatment-free 2 years later. FIG. 11 shows the response of ERBB2 exon 20 duplication to sEphB4 therapy.

[0110] Another patient had an ERBB2 mutation and co-mutation of ALK and ROS1 rearrangements.The tumor was confined to the lung, head and neck, bone, brain, and lymph nodes.

[0111] Rapid responses were observed in individual patients following treatment with sEphB4-albumin fusion protein at 10 mg / kg once weekly, with complete responses observed in three of five patients. Four of five patients responded, with three achieving complete remissions that ranged from 6 months to more than 2 years in duration.

[0112] Example 8: EGFR mutations and high expression are major clinical challenges Gain in EGFR expression is seen in many cancers, including head and neck, lung, colon, and bladder cancers. Response rates to single-agent antibody therapy are relatively low and of short duration. EGFR mutations pose an even greater challenge, and kinase inhibitors typically fail. Additional therapies are needed in combination with EGFR-targeted therapy, especially early in the treatment process.

[0113] We have shown that EGFR and EphB4 enhance each other's expression. As shown in Figure 12, immunoprecipitation (IP) with anti-EphB4 pulled down EGFR, and similarly, IP with anti-EGFR pulled down EphB4, indicating that the two colocalize via direct binding. Knockdown of EphB4 reduced EGFR protein levels as shown by immunoblotting of cell lysates. Each targeted agent was shown to be effective in EphB4-overexpressing NSCLC cells (H358 non-small cell lung cancer (NSCLC) cell line harboring a KRAS mutation), and when used in combination, their activity was enhanced even in H661 (Her2-overexpressing) NSCLC cells. Thus, sEphB4 and EGFR-targeted therapies showed strong synergistic activity, providing a rationale for their combination.

[0114] In vivo efficacy studies using sEphB4 and an anti-EGFR antibody (cetuximab) demonstrated synergistic efficacy, as shown in Figure 13. In tumors resistant to anti-EGFR treatment, sEphB4 was effective, and sEphB4 + cetuximab was more effective than either treatment alone.

[0115] Example 9: Outcome of sEphB4-HSA treatment of cholangiocarcinoma Cholangiocarcinoma responds poorly to treatment. Standard chemotherapy is cisplatin and gemcitabine. New therapies, especially targeted therapies, are needed. Recently, treatment of FGFR-mutated cholangiocarcinoma with kinase inhibitors has shown tumor regression, but response rates are low and complete remission is even less likely. Novel therapies are needed. Cholangiocarcinoma has been treated with sEphB4, and some patients have shown durable responses. This was a solution to an unmet need.

[0116] For example, a 64-year-old woman with progressing lung metastases who had previously been treated with gemcitabine, cisplatin, mitomycin C, surgery, radiation therapy, and high-frequency ultrasound was treated with sEphB4-albumin fusion protein at 15 mg / kg every 2 weeks, with substantial tumor regression lasting for over a year, as shown in Figure 14, which illustrates an example of a response in cholangiocarcinoma. TIFF2024511995000017.tif61170

[0117] Example 10: EphB4 expression confers a growth advantage to Kras mutant cells We investigated the role of tyrosine kinases in regulating survival of Kras mutant cancer cells using a human tyrosine kinase siRNA library (Thermo Scientific). Three Kras mutant cancer cell lines (H358, H727, and Mia Paca-2) and two Kras wild-type cell lines (293T and LTC) were transfected with a SMARTpool siRNA library (a mixture of four siRNAs per gene) targeting 85 tyrosine kinases. The results of the MTT assay are shown as a heat map showing the effect of siRNA-mediated tyrosine kinase knockdown on cell line survival (Figure 15A). Inhibition of the receptor tyrosine kinase EphB4 resulted in the greatest reduction in survival in the three Kras mutant cells, with the best p-value (P=0.018) compared to the controls (293T and LTC), making EphB4 a target of interest (Figure 15A).

[0118] The present inventors confirmed that EphB4 is a key regulator of cell survival in Kras mutation-dependent cancer cell lines. The following cell lines with oncogenic Kras mutations shown in Table 17 were analyzed for Kras mutation dependency: non-small cell lung cancer cell lines (NSCLC) (H358, H727 and H2009), pancreatic cancer cell lines (Mia Paca-2), and colon cancer cell lines (HCT116 and SW620). All six cell lines were sensitive to the removal of Kras mutations (Figure 15B). Furthermore, EphB4 was knocked down in cells by two shRNAs that target different regions of EphB4. The results showed that EphB4 is required for the survival of Kras mutant cell lines, regardless of the presence or absence of TP53 gene abnormality (Figure 15C and Table 17). TIFF2024511995000018.tif71170

[0119] EphB4 protein expression is induced in various human cancers and is associated with tumor stage progression. KrasG12D-myc was overexpressed in HCT116 cells, and it was shown that the level of endogenous EphB4 protein was enhanced by Kras in a dose-dependent manner (Figure 15D). We also examined EphB4 expression in tumors of two different mouse cancer models (oral papilloma and NSCLC) driven by Kras. K14-CreERtam;LSL-KrasG12D mice express tamoxifen-inducible Cre recombinase (CreERtam) driven by the cytokeratin 14 (K14) promoter. These mice also carry a Kras mutation (LSL-KrasG12D) and develop oral papillomas one month after tamoxifen induction (Cre-mediated removal of the loxP-flanked STOP cassette (LSL) upstream of the mutated KrasG12D). Immunostaining demonstrated that both EphB4 and its ligand ephrin B2 were increased in the tumors (Fig. S4E). Elevated EphB4 staining was observed in the basal and intermediate layers of the papillomas, whereas the ephrin B2 ligand was expressed in a complementary manner to the EphB4 receptor in more differentiated tumor regions.

[0120] In a mouse model of NSCLC, adenovirus was used to deliver Cre recombinase (adeno-Cre) to lung cells of LSL-KrasG12D;P53F / F mice. The mice develop lung adenocarcinoma after intratracheal adenovirus infection. Overexpression of EphB4 and ephrinB2 was also observed in the tumors (Figure S15F). These results suggested that EphB4 signaling is induced by oncogenic KrasG12D.

[0121] Example 11: Genetic ablation of EphB4 increases survival of Kras mutant mice To investigate the role of EphB4 in tumor development, we generated EphB4 conditional knockout mice targeted to exons 2 and 3 of the ephB4 gene. This mutant creates a premature stop codon in ephB4 after cre-mediated recombination (Figure 16A). To determine tissue-specific knockout of EphB4F / F, we crossed the mutant with K14-CreERtam mice and administered tamoxifen to the mice. DNA samples from the lip, tongue, lung, and heart were collected and genotyped 1 month after tamoxifen treatment. As expected, EphB4 rearrangement (EphB4 RA in Figure 16B) was detected only in the lip and tongue of K14-CreERtam;EphB4F / F mice. Removal of the STOP cassette upstream of the Kras gene in K14-CreERtam;LSL-KrasG12D mice was also confirmed in lip tumors.

[0122] Previously reported conventional EphB4 knockout mice show embryonic lethality at E10 due to cardiac defects. Therefore, we crossed the conditional EphBb4 mutant with ubiquitously expressed CMV-Cre deletion mice, which resulted in complete gene knockout. Growth retardation of CMV-Cre;EphB4F / F embryos was observed at E10.5 and E11.5 stages. We also crossed EphB4F / F with tamoxifen-induced CMV-Cretam mice to investigate the significance of EphB4 in adults. Pathological analysis of major organs including lung, heart, kidney, liver, and small intestine of CMV-Cretam;EphB4F / F mice was performed 1 month after tamoxifen induction. The induced mice were healthy and viable, and no significant phenotype was observed in the mutant organs compared to the controls. These results suggested that EphB4 is important for embryonic development, but does not have any significant function in normal adults.

[0123] In addition to the oncogenic properties of overexpressed EphB4 in many human cancers, knockdown of EphB4 reduced cell viability in Kras-dependent cell lines. These results prompted us to investigate whether EphB4 influences tumorigenesis in Kras-driven cancer models. To this end, K14-CreERtam;LSL-K-rasG12D;EphB4F / F (K14KB4) mice were generated and compared with K14-CreERtam;LSL-K-rasG12D (K14K) mice. Four weeks after tamoxifen treatment, oral squamous cell papillomas were detected in 100% (n = 10) of K14K mice. The results showed that K14KB4 (n = 9) mice had significantly less tumor growth and longer survival compared with K14K mice (Figure 16C) (P < 0.005). An even greater survival difference was observed in the NSCLC mouse model. LSL-KrasG12D;p53F / F or LSL-K-rasG12D;p53F / F;EphB4F / F mice were challenged with Ad-Cre (AdKP and AdKPB4, respectively). Lung adenocarcinoma development was dramatically reduced with AdKPB4 (Figure 16D). More than half (56%) of AdKPB4 (n=18) survived until day 150 after Ad-Cre infection, whereas all AdKP (n=11) mice died before day 98 (P<0.0001).

[0124] Example 12: Knockdown of EphB4 attenuated AKT and ERK signaling in Kras-driven tumors It is well known that oncogenic Kras activates the PI3K / AKT and MAPK / ERK signaling pathways, both of which serve as important therapeutic targets in cancer treatment. We therefore examined the expression levels of p-AKT and p-S6 for activated PI3K / AKT pathway, and p-ERK1 / 2 for activated MAPK / ERK pathway in mouse oral papillomas and lung adenocarcinomas. All signaling indicators, except for p-ERK1 / 2, which was undetectable in oral papillomas, were significantly increased in the tumor area but not in tissues from EphB4 knockout mice (Fig. S17A for oral papillomas and Fig. S17B for lung adenocarcinomas).

[0125] We observed that EphB4F / F delayed tumor formation in both oral papilloma and lung adenocarcinoma mouse models, whereas K14KB4 or AdKPB4 mice eventually developed various levels of tumors. We wondered whether this could be the result of incomplete knockout of EphB4 in tumors. First, we examined EphB4 expression in lung tissues of AdKPB4 mice. Both in situ and immunofluorescence staining showed overexpression of EphB4 mRNA and protein, respectively (Figure 17C). We also microdissected lung cryosections of AdKPB4 mice and found that rearranged (RA)-Kras, RA-P53, and RA-EphB4 could be detected by PCR in both tumor and nontumor regions, indicating Ad-Cre activity throughout the lung (Figure 17D, upper panel). However, when all loxP-transfected alleles were deleted, the loxP-transfected EphB4 band was completely lost, providing evidence of incomplete knockout, with the presence of the loxP-transfected EphB4 gene. Furthermore, RT-PCR clearly demonstrated overexpression of EphB4 mRNA in tumors compared with nontumor areas, suggesting that EphB4 expression could not be successfully reduced in certain regions of the lung, and as a result, AdKPB4 mice were unable to prevent tumor formation induced by mutant Kras and p53 (Figure 17D, lower panel).

[0126] We analyzed the RNA expression of EphB4 and ephrinB2 in oral papillomas using in situ hybridization. The results showed that the RNA expression of EphB4 and ephrinB2 was rather weak but still detectable in the tumor area of ​​K14KB4 mice. Immunostaining revealed partial expression of EphB4 protein in K14KB4 tumors.

[0127] Example 13: Pharmacological inhibition of EphB4 effectively blocks Kras-driven tumorigenesis in vivo To further demonstrate the efficiency of sEphB4, we examined the tyrosine autophosphorylation status of EphB4 protein activated under sEphB4 administration. Tamoxifen-induced K14K mice were intraperitoneally administered sEphB4 (50 mg / kg mouse body weight). Oral papillomas were harvested 3 days after sEphB4 administration, and tumor lysates were immunoprecipitated with anti-EphB4 antibody. Western blotting showed that the p-Tyr signal of EphB4 was significantly decreased in sEphB4-treated tumors, but not in controls (Figure 18A). The results showed that sEphB4 could block both autophosphorylation and activation of EphB4 receptor in vivo.

[0128] We investigated the therapeutic potency of sEphB4 using oral papilloma and NSCLC mouse models. K14K mice were administered sEphB4 (20 mg / kg, every other day) either simultaneously with tamoxifen induction (prophylactic group) or 2 weeks later (reversal group). The survival rates of both sEphB4-treated groups were significantly increased compared to control K14K mice (Figure 18B). Addition of an additional P53 knockout mutation in K14K mice (K14KP) promoted tumor development. Prophylactic administration of sEphB4 to K14KP also delayed tumor formation and extended survival (Figure 18C).

[0129] The chemotherapy drug paclitaxel (Taxol) is widely used to treat NSCLC. It has been shown to have synergistic interactions with other cancer drugs. sEphB4 in combination with Taxol was administered to NSCLC model mice AdKP. Single-agent administration of Taxol and sEphB4 showed similar significant survival advantages compared to controls, but no significant differences were observed between Taxol- and sEphB4-treated mice. Combined administration of Taxol and sEphB4 improved survival even more significantly compared to each monotherapy (Figure 18D).

[0130] Kras mutations have been suggested to be associated with decreased apoptosis and increased proliferation in tumors. To understand the effect of sEphB4 in Kras-driven cancer, the apoptosis and proliferation status of sEphB4-treated K14K mice bearing spontaneous tumors were examined using TUNEL assay and Ki67 staining, respectively. We found that administration of sEphB4 every other day for 20 days significantly increased apoptosis and decreased tumor proliferation (Figure 18E and Figure 18F). Kras downstream signaling molecules p-AKT and P-S6 were also significantly decreased by sEphB4 treatment (Figure 18G). Furthermore, short-term (44 h) tumor tissue culture confirmed the remarkable effect of sEphB4 on the induction of apoptosis in tumor cells.

[0131] Example 14: EphB4 prevents β-TrCP1-mediated ubiquitination and degradation of Kras It was shown that knockdown of EphB4 signaling, either by genetic modification of mice or administration of the antagonist sEphB4, effectively abolished tumor formation caused by Kras. Meanwhile, knockdown of EphB4 with siRNA in HCT116 cell line reduced the level of CMV promoter-driven overexpressed Ras protein, suggesting that EphB4 may affect the stability of Ras protein. We examined whether EphB4 regulates the half-life of endogenous Kras protein. We selected human oral squamous cell carcinoma cell line SCC71 harboring wild-type Kras and mouse NSCLC cell line 4B-GFP harboring oncogenic KrasG12D to examine whether both wild-type and mutant Kras could be regulated by EphB4. We found that knockdown of EphB4 with siRNA reduced the half-life of endogenous Kras protein from 30.7 h to 8.9 h in SCC71 and from 41.1 h to 7.1 h in 4B-GFP cells (Figure 19A). Furthermore, the decrease in Kras half-life could be reversed by the proteasome inhibitor MG132, suggesting that the ubiquitin-proteasome machinery plays a major role in regulating Kras proteolysis.We further examined Kras levels in tumors from K14K mice and found that Kras expression was decreased in the basal and intermediate zones of papillomas, which correspond to EphB4-overexpressing cells, after sEphB4 treatment (Figure 19B).

[0132] Prompted by the results of the Kras half-life study, and without wishing to be bound by theory, we hypothesize that EphB4 affects Kras protein stability through regulating Kras ubiquitination. Ub-Flag and Kras-myc were expressed in 293T cells, while EphB4 levels were altered by knockdown or overexpression. Immunoprecipitation was performed with anti-Myc antibody (Kras-myc), followed by immunoblotting with anti-Flag antibody (Ub-Flag). The results showed that knockdown of EphB4 by siRNA greatly increased Kras polyubiquitination, consistent with decreased stability, whereas overexpression of EphB4 decreased Kras ubiquitination (Figure 19C).

[0133] Kras polyubiquitination has been shown to be regulated through the F-box family E3 ligase β-transducing repeat-containing protein 1 (β-TrCP1). We confirmed that overexpression of β-TrCP1 promoted Kras polyubiquitination, whereas knockdown of β-TrCP1 reduced Kras ubiquitination (Figure 19D). To gain insight into how EphB4 participates in this process, EphB4 was overexpressed in the presence of Flag-β-TrCP1, HA-Ub and Kras-myc in 293T cells. IP / Western blot analysis showed that overexpressed EphB4 abolished β-TrCP-mediated polyubiquitination (Figure 19E). Co-immunoprecipitation (Co-IP) studies further demonstrated that the protein-protein interaction between Kras and β-TrCP1 could be specifically disrupted by overexpressed EphB4, but not by EphB4-eGFP (an intracellular domain truncated form of EphB4), ephrinB2, or Her2 (Figure 19F, upper panel). Co-IP between EphB4 and β-TrCP1 or between EphB4 and Kras showed that EphB4 competes with β-TrCP1-Kras interaction by binding to β-TrCP1 instead of Kras protein (Figure 19F, middle panel).

[0134] Example 15: The presence of a C-terminal EphB4 fragment modulates β-TrCP1 ligase activity promoting Kras monoubiquitination at Cys118 To elucidate the mechanism by which EphB4 inhibits β-TrCP1-mediated Kras polyubiquitination, we performed in vitro ubiquitination assays. As shown in Figure 20A, both wild-type (WT) and G12V mutant Kras showed a slower migrating band of Kras, indicating possible monoubiquitination, but only in the presence of higher concentrations of EphB4. Mass spectrometry analysis then demonstrated specific binding of the ubiquitin moiety at cysteine ​​118 (118-CggDLPSR-123) in both WT and G12V mutant Kras (Figure 20B). Based on the results, we generated a Cys118 to serine (S) mutant of Kras in the G12D mutant background, named the GC mutant, and further performed in vitro ubiquitination and mass spectrometry analysis as described above. Only in the presence of EphB4, C118 monoubiquitination was confirmed in both WT and GD mutants of Kras, whereas the GC mutant did not show any mobility shift indicative of monoubiquitination (Figure 20C), nor did we identify any ubiquitin-modified peptides after mass spectrometry analysis. Interestingly, while analyzing the protein expression data, we consistently observed β-TrCP1 fragments only in the presence of EphB4. We next measured the steady-state levels of various Kras mutants (WT, GD, CS, GC) and found increased levels of the KrasGC mutant (Figure 20D). To confirm the role of EphB4 on Kras protein stability, we performed siRNA-mediated knockdown of EphB4 and measured the half-life of various Kras proteins. We found that loss of EphB4 negatively affected Kras protein stability in WT, GD, and CS mutants. The data showed increased GC mutant protein stability even in the absence of EphB4 (Figure 20E), suggesting the importance of C118 in EphB4-mediated Kras regulation. The GC mutant, despite being more stable, was found to be less active compared to the GD Kras mutant, as indicated by lower pERK1 / 2 levels. This study also observed a decrease in β-TrCP1 levels upon EphB4 overexpression, suggesting a negative correlation between the EphB4-β-TrCP1 axis.Taken together, a model was proposed in which overexpression of EphB4 could result in enhanced β-TrCP1-mediated monoubiquitination of Kras, which is required for the hyperactivation of mutant Kras proteins (Figure S2F). As a result, either loss of EphB4 or site-specific mutation of C118 in Kras could attenuate mutant Kras activity, suggesting cooperativity between mutant Kras and EphB4.

[0135] Example 16: Clinical efficacy of sEphB4-HSA targeting Ras-mutated human tumors Preclinical data have demonstrated that EphB4-EphrinB2 pathway targeting may be effective in human tumors, and with a conserved mechanism of action and key residues conserved across all forms of Ras (KRas, HRas, NRas), all mutations within each Ras isoform are subject to interference with EphB4-EphrinB2 targeting.

[0136] We have treated several patients with Ras mutations. 1. A 57-year-old female with lung adenocarcinoma harboring a KRas12D mutation and co-mutations ATM G2891D and PIK3CA E545K. The patient had previously undergone radiation therapy with failure. The tumor showed ephrin B2 expression. The patient received sEphB4-albumin fusion protein therapy and achieved a complete remission. The patient has been tumor free for >2 years. The patient has been treatment-free for >1 year. 2. Patient CB, a 62-year-old female with adenocarcinoma of the lung with a KRasG12C mutation and co-mutation in DKN2A. The patient had previously received chemotherapy including carboplatin, Alimta, and Avastin. The patient had a short-term response lasting 5 months. The patient's tumor showed ephrin B2 expression and was treated with sEphB4-albumin-containing therapy without the addition of chemotherapy. The patient responded to treatment for 6 months. The patient decided to stop treatment and eventually progressed. 3. A 42-year-old patient with JK NRAS G13R mutation and concurrent GNAS and TP53 mutations had bladder cancer. The patient was treated with cisplatin and etoposide but showed primary resistance. Tumor analysis showed ephrin B2 expression. The patient then received sEphB4-albumin fusion protein therapy. The patient showed a tumor response lasting 4 months but eventually progressed. 4. A 79-year-old male with RG NRAS mutation, CYLC L227fs, and FBXWY R49Q co-mutations. The patient had head and neck cancer and was treated with radiation therapy and EGFR antibody, achieving a response that lasted for 9 months. The patient experienced tumor recurrence in the lungs and lymph nodes. Tumor biopsy showed ephrin B2 expression. The patient received sEphB4-albumin fusion protein therapy. The patient achieved a complete remission and maintained remission without treatment for more than 2.5 months.

[0137] array The amino acid sequences provided in the accompanying sequence listing are shown using the standard three letter code for amino acids.

[0138] 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)

[0139] 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)

[0140] Incorporation by Reference 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 Application No. 2020 / 018160, International Application No. 2020 / 023215, and all references disclosed herein are hereby incorporated by reference in their entirety for all purposes.

[0141] Others All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the articles and methods of the present invention have been described in terms of embodiments, it will be apparent to one of ordinary skill in the art that modifications can be made to the articles and methods without departing from the spirit and scope of the invention. All such modifications and equivalents, whether now existing or later developed, that are apparent to those skilled in the art are deemed to be within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification are indicative of the level of skill in the art to which the invention pertains. All patents, patent applications, and publications are incorporated herein by reference in their entirety for all purposes and to the same extent as if each of the subject publications was specifically and subjectively indicated to be incorporated by reference in its entirety for all purposes. The invention illustratively described herein can be practiced in the absence of any element(s) not specifically disclosed herein. The terms and expressions employed are used as terms of description, not of limitation, and in the use of such terms and expressions there is no intention to exclude the features shown and described or equivalents of portions thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Thus, although the present invention has been specifically disclosed by embodiments and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be employed by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

Claims

A pharmaceutical composition for use in the treatment of cancer, the pharmaceutical composition comprising a polypeptide agent that inhibits a function mediated by EphB4 or ephrin B2, and the pharmaceutical composition being used as a first-choice therapy in the treatment. Claim 2 The pharmaceutical composition according to claim 1, wherein the cancer is selected from head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), Kras-mutant non-small cell lung adenocarcinoma (NSCLC), Kaposi's sarcoma (KS), bladder, and cholangiocarcinoma (CCA). Claim 3 The pharmaceutical composition according to claim 1 or 2, wherein the cancer is refractory to an anticancer therapy selected from immunotherapy, treatment with chemotherapeutic agents, treatment with depleting antibodies against specific tumor antigens, treatment with agonist antibodies, antagonist antibodies, or blocking antibodies against costimulatory molecules or coinhibitory molecules, optionally immunotherapy checkpoint inhibitors, depleting antibodies against specific tumor antigens and immunocomplexes containing cytotoxic agents, antibody-drug conjugates (ADCs), targeted therapy using fusion molecules, targeted therapy using small molecule kinase inhibitors, treatment using surgery, treatment using stem cell transplantation, and treatment using radiation. Claim 4 The pharmaceutical composition according to claim 3, wherein the cancer is refractory to treatment with an immunotherapy checkpoint inhibitor. Claim 5 The pharmaceutical composition according to claim 3, wherein the cancer is refractory to treatment with radiation therapy. Claim 6 The pharmaceutical composition according to claim 3, wherein the cancer is refractory to treatment with platinum-based chemotherapy. Claim 7 The pharmaceutical composition according to any one of claims 1 to 6, wherein the cancer includes a tumor that expresses ephrin B2. Claim 8 The pharmaceutical composition according to any one of claims 1 to 7, wherein the polypeptide agent is a ligand-binding portion of the EphB4 protein and includes a modification that increases the half-life in serum. Claim 9 The pharmaceutical composition according to any one of claims 1 to 8, wherein the polypeptide agent is covalently or non-covalently bound to an albumin selected from human serum albumin (HSA) ("sEphB4-HSA") and bovine serum albumin (BSA) ("sEphB4-BSA"), and contains the sequences of amino acids 1 to 197, 16 to 197, 29 to 197, 1 to 312, 16 to 312, 29 to 312, 1 to 321, 16 to 321, 29 to 321, 1 to 326, 16 to 326, 29 to 326, 1 to 412, 16 to 412, 29 to 412, 1 to 427, 16 to 427, 29 to 427, 1 to 429, 16 to 429, 29 to 429, 1 to 526, 16 to 526, 29 to 526, 1 to 537, 16 to 537, and 29 to 537 of SEQ ID NO: 1 ("sEphB4 polypeptide").

10. The pharmaceutical composition according to claim 9, wherein the sEphB4-HSA contains residues 16 to 326 of SEQ ID NO: 1 directly fused to residues 25 to 609 of SEQ ID NO:

2.

11. The pharmaceutical composition according to claim 10, wherein the sEphB4-HSA contains residues 16 to 537 of SEQ ID NO: 1 directly fused to residues 25 to 609 of SEQ ID NO:

2.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the treatment further comprises administering an anti-EGFR antibody or an antibody fragment thereof, optionally cetuximab.

13. The pharmaceutical composition according to claim 12, wherein the treatment further comprises administering a taxane, optionally paclitaxel (Taxol) or docetaxel (Taxotere).

14. A pharmaceutical composition for treating cancer, the pharmaceutical composition comprising (i) the ligand-binding portion of the EphB4 protein containing the sequence of amino acids 16 to 537 of SEQ ID NO: 1 and (ii) human serum albumin (HSA) containing the sequence of amino acids 25 to 609 of SEQ ID NO: 2 and comprising a polypeptide agent, the pharmaceutical composition being used as a first-choice therapy in the treatment and / or wherein the cancer is a recurrent, resistant, or refractory cancer A pharmaceutical composition characterized by the above.

15. The pharmaceutical composition according to claim 14, wherein the cancer is selected from head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), Kras mutant non-small cell lung adenocarcinoma (NSCLC), Kaposi's sarcoma (KS), bladder, and cholangiocarcinoma (CCA).

16. The pharmaceutical composition according to claim 14 or 15, wherein the cancer is resistant or unresponsive to immune checkpoint inhibitors, radiotherapy, and / or chemotherapy.

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