In SITU tumor-infiltrating lymphocyte therapeutics for cancer

Abstract

This invention relates to compositions, uses, and methods for use in treating or ameliorating the symptoms of cancer in a subject. A composition can include an inducer of TLR10 and a vascular disrupting agent. Compositions, uses, and methods may further include an immune checkpoint inhibitor. Patients may be selected with one or more biomarkers.

Description

IN SITU TUMOR-INFILTRATING LYMPHOCYTE THERAPEUTICS FOR CANCERSEQUENCE LISTING

[0001] This application includes a sequence listing submitted electronically as an ST.26 file created on May 25, 2025, named 018988-016W01_SL.xml, which is 2,755 bytes in size.TECHNICAL FIELD

[0002] This invention relates to compositions, uses and methods for treating cancer with an in situ therapeutic. The in situ therapeutic can include an agent for inducing tumor TLR10 and tumor-infiltrating lymphocytes in combination with a vascular disrupting agent for producing tumor necrosis antigens, and optionally an immunotherapy agent such as a checkpoint inhibitor. One or more biomarkers can be used to select subjects.BACKGROUND

[0003] Cancer is a complex pathology involving multiple variant cellular pathways. Because of this complexity, it has been difficult to find effective therapeutic strategies that can have antitumor effects in various cancers.

[0004] Conventional efforts include cell immunotherapy approaches in which tumorinfiltrating lymphocytes are extracted from a cancer patient’s tumor and expanded in vitro to prepare a medication for treating the patient.

[0005] Drawbacks of conventional cell-based therapies include limited ability for identifying and obtaining the necessary cells from the patient, as well as lack of methods for expanding the cells in vitro to retain their potency.

[0006] Further drawbacks of such conventional cell-infusing therapies include the need for preparing the patient with additional immune-suppressing drugs to allow the infused cells to take hold.

[0007] Additional drawbacks of cell infusion immunotherapy include high toxicity at required levels of therapeutic administration which can generate extreme side effects.

[0008] What is needed are in situ methods, agents and uses for cancer diseases to increase efficacy, and reduce toxicity and unwanted side effects.

[0009] For example, what is needed are compositions of agents having multiple anti -tumor and cancer immunotherapeutic effects, which can reduce side effects and adverse healtheffects. There is a need for improved guidance of such compositions using appropriate biomarkers to select synergistic effects of the compositions.BRIEF SUMMARY

[0010] This invention provides a form of tumor-infiltrating lymphocyte (TIL) therapy for cancer. The therapy includes in situ vaccination compositions and methods which can harness endogenous immune effectors. As such, the compositions and methods of this invention may be likened to “death-therapeutics” which kill tumors and cancer cells by combining a vascular disrupting agent (VDA) with an inducer of TLR10. This invention can provoke controlled tumor necrosis by releasing a rich repertoire of unfiltered, chemically modified intracellular proteins called neoantigens that are rapidly taken up by antigen-presenting cells for enhancing the ability of a subject’s immune system to kill tumors and cancer cells.

[0011] In some embodiments, the compositions and methods of this disclosure may be combined with immune checkpoint inhibitor therapeutics.

[0012] Without wishing to be bound by theory, compositions and methods of this invention can provide necrotic milieu enriched in DAMPs such as HMGB 1 and ATP, inducing dendritic cell maturation and cross-presentation. Concurrently, membrane-bursting modalities such as necroptosis and pyroptosis further amplify T-cell priming by broadening the neo-epitope landscape. These effects provide deep CD8+T-cell infiltration into previously inaccessible tumor cores.

[0013] This invention relates to compositions, uses and methods for treating cancer with an in situ therapeutic. The in situ therapeutic can include an agent for inducing tumor necrosis and tumor-infiltrating lymphocytes in combination with a vascular disrupting agent, and optionally an immunotherapy agent such as a checkpoint inhibitor. The in situ therapeutic will upregulate one or more genes associated with tumorinfiltrating lymphocytes and / or tumor necrosis. The agent for inducing tumor necrosis and tumor-infiltrating lymphocytes may be a toll-like receptor inducing-ligand, a tolllike receptor upregulating cytokine, or a toll-like receptor agonist. One or more biomarkers can be used to select subjects who benefit from the compositions, uses and methods, wherein the biomarkers can include a level of one or more tumor necrosis- associated genes. The in situ therapeutic can be used in combination with chemotherapy and other standard of care therapies for cancer.

[0014] In further aspects, compositions and methods of this disclosure can provide upregulation of TLR10, which enhances necrotic antigen immunogenicity and may provide a surprising increase in median overall survival in certain cancer patients.

[0015] In some embodiments, TLR10 can further be used as a prognostic biomarker for clinical response.

[0016] Embodiments of this invention contemplate modalities for pharmacologically-induced necrosis as a scalable “off-the-shelf’ cancer therapy by in situ vaccination, bridging the gap between oncolytic and cellular therapies to achieve durable antitumor immunity.

[0017] This invention relates to in situ therapeutics which contain agents to stimulate tumor cell death in patients. Tumor cell necrosis can release tumor-associated antigens and enhance immune response to malignancies by activating tumor infiltrating lymphocytes.

[0018] Compositions, uses and methods of this invention can further include agents to attack and disrupt vascularization of tumors. The vascular disrupting agents can shut down tumor blood flow and further induce tumor necrosis.

[0019] Compositions, uses and methods of this invention can advantageously reduce tumors by stimulating patient immune response in situ without the need for ex vivo methods for isolating and re-infusing a patient’s tumor cells.

[0020] Embodiments of this invention can advantageously stimulate a robust immune response to malignancy in a patient because a broad, polyclonal range of tumor neoantigens can be stimulated in situ, which are not limited by ex vivo manipulations.

[0021] This invention relates to compositions, uses, and methods for treating or ameliorating the symptoms of cancer with an in situ therapeutic containing agents for inducing tumor cell necrosis. The in situ therapeutic can include an agent for inducing TLR10 and tumor-infiltrating lymphocytes in combination with a vascular disrupting agent and optionally an immunotherapy agent such as a checkpoint inhibitor.

[0022] An in situ therapeutic of this invention can upregulate one or more genes associated with tumor-infiltrating lymphocytes and / or tumor necrosis.

[0023] In some embodiments, the agent for inducing tumor necrosis and tumorinfiltrating lymphocytes may be a toll-like receptor inducing-ligand, a toll-like receptor upregulating cytokine, or a toll-like receptor agonist.

[0024] In further embodiments, one or more biomarkers can be used to select subjects who benefit from the compositions, uses and methods, wherein the biomarkers can include a level of one or more tumor necrosis-associated genes.

[0025] In some embodiments, tumor bacterial flora cell counts can be used as biomarkers to select subjects who benefit from the compositions, uses and methods.

[0026] An in situ therapeutic of this invention can be used in combination with chemotherapy and other standard-of-care therapies for cancer.

[0027] In some embodiments, methods and therapeutic strategies of this invention can increase efficacy, as well as reduce toxic side effects and adverse health effects found with conventional cancer treatment.

[0028] In certain embodiments, methods and therapeutic strategies of this invention can improve guidance of in situ therapy using appropriate biomarkers to select synergistic effects of the compositions.

[0029] Embodiments of this invention include the following:

[0030] A composition for use in treating or ameliorating the symptoms of cancer in a subject, the composition comprising: an inducer of TLR10; a vascular disrupting agent; and a carrier.

[0031] A composition for use in the preparation of a medicament for treating or ameliorating the symptoms of cancer in a subject, the composition comprising: an inducer of TLR10; and a vascular disrupting agent.

[0032] A method for treating or ameliorating the symptoms of cancer in a subject in need, the method comprising: preparing a composition comprising an inducer of TLR10; and a vascular disrupting agent; and administering a therapeutically sufficient amount of the composition to the subject.

[0033] The composition or method above, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, melanoma, sarcoma, ocular cancer, retinoblastoma, ocular melanoma, or intraocular lymphoma.

[0034] The composition or method above, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, melanoma, sarcoma, ocular cancer, retinoblastoma, ocular melanoma, or intraocularlymphoma, and wherein the subject’s cancer presents tumors having bacterial flora levels above median.

[0035] The composition or method above, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, or colon cancer, and wherein the subject’s cancer presents tumors having bacterial flora levels above median.

[0036] The composition or method above, wherein the inducer of TLR10 is a TLR10, TLR2, or TLR1 agonist or ligand.

[0037] The composition or method above, wherein the inducer of TLR10 is N- palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3 -lysine (Pam3CSK4), N-palmitoyl-S-[2-hydroxy-3-(palmitoyloxy)propyl]-(R)-cysteinyl-(lysyl)3 -lysine (PamCysPamSK4), or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer thereof.

[0038] The composition or method above, wherein the vascular disrupting agent is fosbretabulin having at least 70, 80, 90, or 99% cis-isomer, or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer thereof.

[0039] The composition or method above, wherein the vascular disrupting agent is fosbretabulin and the inducer of TLR10 is Pam3CSK4, or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer of each.

[0040] The composition or method above, wherein the vascular disrupting agent is fosbretabulin and the inducer of TLR10 is PamCysPamSK4, or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer of each.

[0041] The composition or method above, wherein the cancer is pancreatic cancer and the subjects have biomarker PNPLA6 elevated.

[0042] The composition or method above, wherein the cancer is lung cancer and the subjects have biomarker MROH2B elevated.

[0043] The composition or method above, wherein the cancer is bladder cancer and the subjects have biomarker BLNK elevated.

[0044] The composition or method above, wherein the cancer is uterine cancer and the subjects have biomarker CD7 or LRRC45 elevated.

[0045] The composition or method above, wherein the cancer is uveal melanoma and the subjects have biomarker CD7 elevated.

[0046] The composition or method above, wherein the cancer is sarcoma, the subjects have biomarker AK7 elevated, and the sarcoma size is at least 1 cm3.

[0047] The composition or method above, wherein the subject presents a sign of neutropenia and the dosage of the vascular disrupting agent is 10-45 mg / m2and exhibits an AUC of 12-19 uM*hr.

[0048] The composition or method above, wherein the inducer of TLR10 and the vascular disrupting agent are singly-formulated and administered concurrently, simultaneously, sequentially, or separately in time.

[0049] The composition or method above, in combination with a singly-formulated immune checkpoint inhibitor, wherein the immune checkpoint inhibitor and the composition comprising the inducer of TLR10 and the vascular disrupting agent are administered concurrently, simultaneously, sequentially, or separately in time.

[0050] The composition or method above, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA-4, or PD-L1 .

[0051] The composition or method above, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab, or durvalumab.

[0052] The composition or method above, in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor and the composition comprising the inducer of TLR10 and the vascular disrupting agent are co-formulated.

[0053] The composition or method above, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA-4, or PD-L1 .

[0054] The composition or method above, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab, or durvalumab.

[0055] The composition or method above, in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor, the inducer of TLR10, and the vascular disrupting agent are singly-formulated and administered concurrently, simultaneously, sequentially, or separately in time.

[0056] The composition or method above, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA-4, or PD-L1 .

[0057] The composition or method above, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab, or durvalumab.

[0058] The composition or method above, wherein the composition or method is combined with a standard of care treatment for the cancer or one or more additional therapeutics for treatment of cancer selected from TMZ, radiation, and bevacizumab.

[0059] The composition or method above, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is co-formulated with an immune checkpoint inhibitor for subcutaneous injection or intravenous injection or infusion.

[0060] The composition or method above, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated for subcutaneous injection.

[0061] The composition or method above, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated for intravenous injection or infusion.

[0062] The composition or method above, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated for ocular use as a drop, an intraocular injection, or a subconjunctival injection.

[0063] The composition or method above, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated with an amino acid selected from ornithine, phenylalanine, histidine and arginine and an anionic excipient selected from benzenesulfonic acid, pyridoxine, and thiamine phosphoric acid ester.

[0064] The composition or method above, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated with an immune checkpoint inhibitor, an amino acid selected from ornithine, phenylalanine, histidine and arginine, and an anionic excipient selected from benzenesulfonic acid, pyridoxine, and thiamine phosphoric acid ester.

[0065] The composition or method above, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated with a buffer selected from histidine, succinate, citrate, acetate, phosphate, glutamate, adipic acid, aspartic acid, lactic acid, tromethamine, and 2-(N-morpholino)-ethanesulfonic acid and a surfactant selected from polysorbate 20, polysorbate 80, and poloxamer 188.

[0066] The composition or method above, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated in a prefilled autoinjector or syringe.

[0067] The composition or method above, wherein the composition or medicament is administered guided by imaging.

[0068] The composition or method above, wherein the inducer of TLR10 induces tumor-infiltrating lymphocytes.

[0069] The composition or method above, wherein the composition or method upregulates expression of one or more genes selected from TLR10, CD3, CD4, CD8, CD7, BLNK, AK7, PNPLA6, MROH2B, LRRC45, SECTM1, CACNA1I, CENPX, HEXD, KRAS, NXPH1, RAC3, SLC12A3, and ZNF518A.

[0070] The composition or method above, wherein the composition, agent, medicament or administration increases a survival rate of subjects at month 6, 12, 18, 24, 30, or 36.BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1 shows results of a study of clinical outcomes for melanoma cancer patients and the beneficial impact on overall survival of patients for therapeutic use of a TLR10 agonist agent in combination with an immune checkpoint inhibitor.

[0072] The TLR10 agonist agent enhances immune response to tumor necrosis antigens. Patients in this study included those with multiple tumor types. The Kaplan- Meier chart of FIG. 1 shows that median overall survival of melanoma cancer patients was significantly improved (logrank P=9.6e-5) for higher expression of TLR10. Upper quartile overall survival of cancer patients increased more than threefold from 6 months to 20 months. This result is surprising because TLR10 is not recognized as being important for survival. This study established a basis for therapeutic use of a TLR10 agonist agent for inducing necrosis and treating cancer in combination with an immune checkpoint inhibitor.

[0073] FIG. 2 shows results of a study of clinical outcomes for cancer patients and the beneficial impact on overall survival of patients for therapeutic use of a necrosis inducing agent, a TLR10 agonist, in combination with an immune checkpoint inhibitor.

[0074] The Kaplan-Meier chart of FIG. 2 shows that median overall survival of melanoma cancer patients treated with pembrolizumab was significantly improved(logrank P=0.00078) for higher expression of TLR10 gene in the presence of high PNPLA6, which was evidence of necrosis. This study established a basis for therapeutic use of a vascular disrupting agent for inducing necrosis and treating cancer in combination with the immune checkpoint inhibitor pembrolizumab.

[0075] FIG. 3 shows results of a study of clinical outcomes for melanoma cancer patients and the beneficial impact on overall survival of patients for therapeutic use of a necrosis inducing agent, a TLR10 agonist, in combination with an immune checkpoint inhibitor.

[0076] The Kaplan-Meier chart of FIG. 3 shows that median overall survival of cancer patients treated with ipilimumab was significantly improved (logrank P=0.00075) for higher expression of TLR10 gene in the presence of high PNPLA6, which was evidence of necrosis. This study established a basis for therapeutic use of a TLR10 agonist agent for inducing necrosis and treating cancer in combination with the immune checkpoint inhibitor ipilimumab.

[0077] FIG. 4 shows results of a study of clinical outcomes for sarcoma patients and the impact on overall survival of necrosis gene AK7.

[0078] The Kaplan-Meier chart of FIG. 4 shows that median overall survival of sarcoma patients was significantly improved (logrank P=0.00097) for higher expression of necrosis gene AK7, which was evidence of tumor necrosis. The association of AK7 to cancer may not be limited to sarcoma.

[0079] FIG. 5 shows results of a study of clinical outcomes for bladder cancer patients and the impact on overall survival of necrosis gene BLNK.

[0080] The Kaplan-Meier chart of FIG. 5 shows that median overall survival of bladder cancer patients was significantly improved (logrank P=0.0035) for higher expression of necrosis gene BLNK, which was evidence of tumor necrosis. The association of BLNK to cancer may not be limited to bladder cancer.

[0081] FIG. 6 shows results of a study of clinical outcomes for uterine cancer patients and the impact on overall survival of necrosis gene CD7.

[0082] The Kaplan-Meier chart of FIG. 6 shows that median overall survival of uterine cancer patients was significantly improved (logrank P=0.00075) for higher expression of necrosis gene CD7, which was evidence of tumor necrosis. The association of CD7 to cancer may not be limited to uterine cancer.

[0083] FIG. 7 shows results of a study of clinical outcomes for ovarian cancer patients and the impact on overall survival of necrosis gene LRRC45.

[0084] The Kaplan-Meier chart of FIG. 7 shows that median overall survival of ovarian cancer patients was significantly improved (logrank P=0.0071) for higher expression of necrosis gene LRRC45, which was evidence of tumor necrosis. The association of LRRC45 to cancer may not be limited to ovarian cancer.

[0085] FIG. 8 shows results of a study of clinical outcomes for lung cancer patients and the impact on overall survival of necrosis gene MR0H2B.

[0086] The Kaplan-Meier chart of FIG. 8 shows that median overall survival of lung cancer patients was significantly improved (logrank P=0.0012) for higher expression of necrosis gene MR0H2B, which was evidence of tumor necrosis. The association of MR0H2B to cancer may not be limited to lung cancer.

[0087] FIG. 9 shows results of a study of clinical outcomes for pancreatic cancer patients and the impact on overall survival of necrosis gene PNPLA6.

[0088] The Kaplan-Meier chart of FIG. 9 shows that median overall survival of pancreatic cancer patients was significantly improved (logrank P=0.00025) for higher expression of necrosis gene PNPLA6, which was evidence of tumor necrosis. The association of PNPLA6 to cancer may not be limited to pancreatic cancer.

[0089] FIG. 10 shows results of a breast cancer model study (EMT-6) and the beneficial impact on reducing tumor volume for use of a vascular disrupting agent, combretastatin CA4P, alone and in combination with an immune checkpoint inhibitor (anti-CTLA-4).

[0090] The mean tumor volume chart of FIG. 10 shows that the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor (anti-CTLA-4) significantly reduced tumor volume as compared to negative control and either CA4P or anti-CTLA-4 alone. In vivo survival studies confirmed the potency of the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor. This study established a basis for therapeutic use of a vascular disrupting agent for inducing necrosis and treating cancer in combination with an immune checkpoint inhibitor.

[0091] FIG. 11 shows results of a colon cancer model study (murine CT-26) and the beneficial impact on reducing tumor volume for use of a vascular disrupting agent,combretastatin CA4P, alone and in combination with an immune checkpoint inhibitor (anti-CTLA-4).

[0092] The mean tumor volume chart of FIG. 11 shows that the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor (anti-CTLA-4) significantly reduced tumor volume as compared to negative control and either CA4P or anti-CTLA-4 alone. In vivo survival studies confirmed the potency of the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor. This study established a basis for therapeutic use of a vascular disrupting agent for inducing necrosis and treating cancer in combination with an immune checkpoint inhibitor.

[0093] FIG. 12 shows results of an in vivo cancer study and the impact on inducing necrosis and eliciting immune response for use of a vascular disrupting agent, combretastatin CA4P, in combination with an immune checkpoint inhibitor (anti-CTLA- 4).

[0094] The chart of FIG. 12 shows that the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor (anti-CTLA-4) induced a large increases in tumor-associated T-cells and CD8+ T-cells as compared to negative control and either CA4P or anti-CTLA-4 alone. This study established a basis for therapeutic use of a vascular disrupting agent for inducing necrosis and treating cancer in combination with an immune checkpoint inhibitor.

[0095] FIG. 13 shows results of an in vivo cancer study and the impact on inducing necrosis and eliciting immune response for use of a vascular disrupting agent, combretastatin CA4P, in combination with an immune checkpoint inhibitor (anti-CTLA- 4).

[0096] The chart of FIG. 13 shows that the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor (anti-CTLA-4) induced a large increases in tumor necrosis area as compared to negative control and either CA4P or anti-CTLA-4 alone. This study established a basis for therapeutic use of a vascular disrupting agent for inducing necrosis and treating cancer in combination with an immune checkpoint inhibitor.

[0097] FIG. 14 shows results of a preclinical study of uveal melanoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P. Administration by injection and topical routes are compared.

[0098] FIG. 15 shows results of a preclinical study of uveal melanoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P. Administration by injection and topical routes are compared.

[0099] FIG. 16 shows results of a preclinical study of retinoblastoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P.

[0100] Transgenic retinoblastoma mice were administered via subconjunctival route of exposure CA4P via 6 injections, 2x / week for 3 weeks, of 0.02, 0.2, or 2.0 mg / pL at 12 weeks of age. Eyes enucleated post-treatment at 16-weeks of age. Eyes were histopathologically examined for presence of tumor. The results show that subconjunctival CA4P inhibited tumor vasculature and was associated with tumor inhibition in murine retinoblastoma. FIG. 16 shows a large tumor is present.

[0101] FIG. 17 shows results of a preclinical study of retinoblastoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P.

[0102] As compared to the previous figure, FIG. 17 shows a moderate tumor is present.

[0103] FIG. 18 shows results of a preclinical study of retinoblastoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P.

[0104] As compared to the previous figure, FIG. 18 shows minimal tumor is present.

[0105] FIG. 19 shows results of a preclinical study of retinoblastoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P.

[0106] As compared to the previous figure, FIG. 19 shows no tumor is present.

[0107] FIG. 20 shows results of a study of clinical outcomes for 68 uveal melanoma patients and the impact on overall survival of necrosis gene SECTM1.

[0108] FIG. 21 shows results of a study of clinical outcomes for 68 uveal melanoma patients and the impact on overall survival of necrosis gene CD7.

[0109] FIG. 22 shows results of a study of clinical outcomes for 68 uveal melanoma patients and the impact on overall survival of necrosis gene PNPLA6.

[0110] FIG. 23 shows results of a study of clinical outcomes for 68 uveal melanoma patients and the impact on overall survival of necrosis gene CACNA1L.

[0111] FIG. 24 shows results of a study of AUC for ocular administration of CA4P in eye drops, minitablets, and by injection.

[0112] FIG. 25 shows results of a study of AUC for ocular administration of CA4P in eye drops, minitablets, and by injection.

[0113] FIG. 26 shows results of a clinical study of pharmacokinetics of CA4P therapy in ovarian cancer.

[0114] FIG. 27 shows results of a clinical study of pharmacokinetics of CA4P therapy in ovarian cancer.DETAILED DESCRIPTION OF THE DISCLOSURE

[0115] This invention relates to methods, compositions and uses thereof for treating or ameliorating the symptoms of cancer in a human or animal subj ect with compositions and methods designed to promote anti-tumor effects.

[0116] This invention further relates to in situ therapeutics which contain agents to stimulate tumor cell death in patients. Tumor cell necrosis can release tumor-associated antigens and enhance immune response to malignancies by activating tumor infiltrating lymphocytes.

[0117] Compositions, uses and methods of this invention can further include agents to attack and disrupt vascularization of tumors. The vascular disrupting agents can shut down tumor blood flow and further induce tumor necrosis.

[0118] In certain embodiments, the compositions, uses and methods of this invention can employ a vascular disrupting agent in combination with a TLR10 agonist agent to stimulate immunity against tumor cells. The combination of agents can advantageously induce immune response to attack tumors in a patient by activating a patient’s own tumor-infiltrating lymphocytes. This is a vastly different approach than conventional therapeutics which must rely on ex vivo preparations. An “in situ” “death therapeutic” composition of this invention can produce superior results over conventional therapies.

[0119] Compositions, uses and methods of this invention can advantageously reduce tumors by stimulating patient immune response in situ without the need for ex vivo methods for isolating and re-infusing a patient’s tumor cells.

[0120] Embodiments of this invention can advantageously stimulate a robust immune response to malignancy in a patient because a broad, polyclonal range of tumor neoantigens can be stimulated in situ, which are not limited by ex vivo manipulations.

[0121] In some embodiments, innate TIL cells are driven to tumors, ultimately triggering an antitumor immune effect. Without wishing to be bound by theory, the induction of necrotic cell death and activation of “death genes” can expose tumor-reactive antigens. Such antigens initiatean immune response and activate antigen-presenting cells (APCs) to achieve large-scale antigen presentation, thereby activating T cells and inducing strong and sustained cytotoxic T lymphocyte responses. This antitumor immune effect can be provided by use of a vascular disrupting agent (VDA) in combination with an inducer of TLR10, optionally with an immune checkpoint inhibitor (ICI). This therapeutic composition induces and utilizes the immunogenicity of necrotic antigens.

[0122] Examples of an inducer of TLR10 include N-palmitoyl-S-[2,3-bis(palmitoyloxy)- propyl]-(R)-cysteinyl-(lysyl)3-lysine (Pam3CSK4), N-palmitoyl-S-[2-hydroxy-3- (palmitoyloxy)propyl]-(R)-cysteinyl-(lysyl)3-lysine (PamCysPamSK4), or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer thereof.

[0123] This invention relates to compositions, uses, and methods for treating or ameliorating the symptoms of cancer with an in situ therapeutic containing agents for inducing tumor cell necrosis. The in situ therapeutic can include an agent for inducing tumor necrosis and tumor-infiltrating lymphocytes in combination with an inducer of TLR10 and optionally an immunotherapy agent such as a checkpoint inhibitor.

[0124] An in situ therapeutic of this invention can upregulate one or more genes associated with tumor-infiltrating lymphocytes and / or tumor necrosis.

[0125] In some embodiments, the agent for inducing tumor necrosis and tumorinfiltrating lymphocytes may be a toll-like receptor inducing-ligand, a toll-like receptor upregulating cytokine, or a toll-like receptor agonist.

[0126] In further embodiments, one or more biomarkers can be used to select subjects who benefit from the compositions, uses and methods, wherein the biomarkers can include a level of one or more tumor necrosis-associated genes.

[0127] In some embodiments, the cancer can be pancreatic cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, melanoma, sarcoma, ocular cancer, retinoblastoma, ocular melanoma, or intraocular lymphoma, wherein the subject’s cancer presents tumors having bacterial flora levels above median. The median can be determined by bacterial cell counts of tumor biopsies from a group of patients having the same cancer. In these embodiments, bacterial cell counts can be used as biomarkers to select subjects who benefit from the compositions, uses and methods.

[0128] An in situ therapeutic of this invention can be used in combination with chemotherapy and other standard-of-care therapies for cancer.

[0129] In some embodiments, methods and therapeutic strategies of this invention can increase efficacy, as well as reduce toxic side effects and adverse health effects found with conventional cancer treatment.

[0130] In certain embodiments, methods and therapeutic strategies of this invention can improve guidance of in situ therapy using appropriate biomarkers to select synergistic effects of the compositions.Agents for inducing tumor necrosis

[0131] This invention includes agents for inducing immune response to neoantigens released during tumor necrosis.

[0132] Examples of agents for inducing immune response to tumor necrosis by inducing tumor-infiltrating lymphocytes include toll-like receptor inducing-ligands, toll-like receptor upregulating cytokines, and toll-like receptor agonists.

[0133] Examples of toll-like receptor inducing-ligands include certain bacterial lipoproteins and viral components.

[0134] Examples of agents for inducing immune response to tumor necrosis include tolllike receptor agonists selected from TLR10 agonists, TLR9 agonists, TLR2 agonists, TLR4 agonists, TLR7 agonists, and TLR8 agonists.

[0135] Without wishing to be bound by theory, the ability of toll-like receptor agonists for inducing immune response to tumor necrosis is surprising because toll-like receptor genes may be downregulated in tumor cell necrosis. For example, in some embodiments of this invention an agonist of TLR10 can induce immune response to tumor necrosis.Therapeutics of this invention can utilize an agonist of TLR10 to stimulate immune response to tumor necrosis and further expression of TLR10. The immune response to tumor necrosis therefore further destroys the tumor. Enhanced expression of TLR10 can enhance activation of antigen-presenting cells to achieve large-scale neoantigen presentation, activate T cells, induce strong and sustained cytotoxic T lymphocyte responses, and create an anti-tumor immune effect.

[0136] In certain embodiments, therapeutics of this invention utilizing an agonist of TLR10 may surprisingly improve cancer patient survival for multiple cancer types, indications, andstages. Use of a TLR10 agonist can have significant impact on cancer patient survival by inducing tumor necrosis.

[0137] Examples of agents for inducing tumor necrosis include toll-like receptor agonists selected from Pam3CSK4 (triacyl lipopeptide, Pam3CSK4, palmitoyl-3-cysteine-serine-lysine-4), lipopolysaccharide (LPS), and CpG oligodeoxynucleotides.

[0138] The structure of PAM3CSK4 is as follows:

[0139] S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoyl-L-cysteinyl-L-(seryl)-(L-lysyl)3-L- lysine. The structure is {(propyl)Cys-Ser-Lys-Lys-Lys-Lys] represented as CSKKKK (SEQ ID NO:1). For example, PAM3CSK4, hydrochloride, is CAS 112208-00-1. For example, PAM3CSK4, TFA salt, is CAS 112208-01-2.

[0140] The structure of PAMCYSPAMSK4 is as follows:

[0141] S-[2-OH-3-(palmitoyloxy)propyl]-N-palmitoyl-L-cysteinyl-L-(seryl)-(L-lysyl)3-L- lysine. The structure is {(propyl)Cys-Ser-Lys-Lys-Lys-Lys] represented as CSKKKK (SEQ ID NO:2).

[0142] Examples of agents for inducing tumor necrosis include toll-like receptor agonist selected from imidazoquinoline derivatives, imiquimod, and resiquimod (R848).

[0143] In some embodiments, agents for inducing tumor cell necrosis can increase expression of tumor necrosis-associated genes.

[0144] In further embodiments, agents for inducing tumor cell necrosis can advantageously increase cancer patient survival in combination with an inducer of TLR10. Tumor necrosis-associated genes

[0145] This invention can utilize tumor necrosis-associated genes.

[0146] Clinical analysis of genetic alterations can be performed across multiple cancer indications and types for comparing the association of necrosis genes to tumor necrosis. Monitoring expression of necrosis genes identified by such analysis can be used to guide a successful therapy.

[0147] Expression of tumor necrosis-associated genes can be correlated with cancer type, indication, or stage as a prognostic factor.

[0148] In some embodiments, elevated expression of a necrosis gene can be used as a biomarker to select patients who will benefit from a therapeutic of this invention.

[0149] In certain embodiments, biomarkers may be used to select the subj ects who benefit from the composition, use or method. Examples of biomarkers include a level of one or more tumor necrosis-associated genes selected from TLR10, CD7, BLNK, AK7, PNPLA6, MR0H2B, LRRC45, SECTM1, CACNA1I, CENPX, HEXD, KRAS, NXPH1, RAC3, and SLC 12A3.Vascular disrupting agents

[0150] This invention includes vascular disrupting agents. Vascular disrupting agents can destroy vascularization of a tumor and cause reduction of tumor mass or volume.

[0151] Examples of vascular disrupting agents include combretastatin CA4P.

[0152] In some embodiments, this invention utilizes vascular disrupting agents for increasing cancer patient survival.

[0153] In additional embodiments, vascular disrupting agents can reduce tumor mass or volume.

[0154] In certain embodiments, vascular disrupting agents can attack tumor vascularization and reduce tumors, which can further increase tumor cell necrosis. Use oradministration of vascular disrupting agents can also correlate with elevated expression of a necrosis gene.

[0155] In further embodiments, vascular disrupting agents can increase cancer patient survival.

[0156] In certain embodiments, vascular disrupting agents can increases tumor T-cells, CD8+ cytotoxic T-Cells, CD4+ T-helper-Cells, CD3+ T lymphocytes, as well as tumor necrosis. In situ therapeutics

[0157] This invention further includes compositions and methods for in situ immunization. Use of an in situ vaccine of this disclosure in a cancer patient may induce immunogenic cell death, expose tumor-reactive antigens to initiate an immune response, activate antigen-presenting cells to achieve large-scale antigen presentation, activate T cells, induce strong and sustained cytotoxic T lymphocyte responses, and create an anti-tumor immune effect.

[0158] An in situ vaccine of this disclosure may further use biomarkers to identify a patient population most likely to respond and benefit from the therapy.

[0159] In certain embodiments, the compositions, uses and methods of this invention can employ a tumor vascular disrupting agent in combination with an agent as inducer of TLR10. The combination of agents can advantageously induce immune response to attack tumors in a patient by activating a patient’s own tumor-infiltrating lymphocytes. Methods of this invention do not utilize ex vivo cell preparations. An “in situ” “death therapeutic” composition of this invention can produce superior results over conventional therapies.

[0160] Without wishing to be bound by theory, in situ death therapeutics of this invention increase tumor necroptotic cell death to drive a patient’s tumor-infiltrating lymphocytes to invade tumors. Such in situ vaccination does not require ex vivo cell extractions, expansions, or preparations. An in situ vaccination formulation of this invention can induce a patient’s immune system to increase immunogenic cell death, expose tumor-reactive antigens to initiate immune response, activate antigen-presenting cells to achieve large-scale neoantigen presentation, fully activate patient T cells, induce strong and sustained cytotoxic T-lymphocyte response, induce the anti-tumor immune effect.

[0161] In some embodiments, inducing tumor necroptotic cell death can be monitored and confirmed by elevation of cell death genes, namely the tumor necrosis-associated genes.Methods and compositions in ocular oncology

[0162] Therapeutics of this invention can be used beneficially in indications including retinoblastoma and uveal melanoma.

[0163] In some embodiments, retinoblastoma can be treated by a subconjunctival route of exposure.

[0164] A vascular disrupting agent or combination therapeutic of this invention can be used in a regimen of subconjunctival injection. This therapy can inhibit tumor vasculature and inhibit retinoblastoma.

[0165] In further embodiments, uveal melanoma can be treated with combined routes of exposure.

[0166] A vascular disrupting agent or combination therapeutic of this invention can be used in a delivery regimen. This therapy can inhibit growth of tumors and inhibit uveal melanoma. Anti-cancer methods and compositions

[0167] This invention includes compositions, uses, and methods based on combination therapeutics which may utilize one or more agents for inducing TLR10, and one or more vascular disrupting agents.

[0168] In some embodiments, a combination of agents for inducing TLR10 and vascular disrupting agents can further be combined with an immune checkpoint inhibitor.

[0169] In certain embodiments, the immune checkpoint inhibitor and the composition comprising the inducer and the vascular disrupting agent can be administered concurrently, simultaneously, sequentially, or separately in time.

[0170] In some embodiments, an immune checkpoint inhibitor can be used to induce tumor necrosis, as shown by elevated expression of certain tumor necrosis-associated genes. In further embodiments, a combination therapeutic with an immune checkpoint inhibitor can increase cancer patient overall survival. In certain embodiments, the immune checkpoint inhibitor can be a PD1 inhibitor.

[0171] This invention includes methods for treating or ameliorating the symptoms of cancer in a human or animal subject in need, by administering a therapeutically sufficient amount of a pharmaceutical composition comprising an inducer of TLR10 and a vascular disrupting agent.

[0172] As referred to herein, checkpoint inhibitors as known in the art are immune checkpoint inhibitor agents. Checkpoint inhibitors are immunotherapy drugs whichblock checkpoint proteins from binding with their partner proteins. This prevents an “off’ signal from being sent, which allows T cells to kill cancer cells. More particularly, checkpoint proteins, such as PD-1 on T cells, keep immune responses in check. Binding of PD-L1 to PD-1 keeps T cells from killing tumor cells. Thus, blocking the binding of PD-L1 to PD-1 with an immune checkpoint inhibitor may allow the T cells to kill tumor cells. The immune system is essentially turned back on so that T cells can attack cancer cells.

[0173] In some embodiments, a checkpoint inhibitor of this disclosure may be an inhibitor of CTLA-4, PD-1, or PD-L1.

[0174] In certain embodiments, a checkpoint inhibitor of this disclosure may be an inhibitor of PD-1.

[0175] In certain embodiments, a checkpoint inhibitor of this disclosure may be pembrolizumab.

[0176] In certain embodiments, a checkpoint inhibitor of this disclosure may be pembrolizumab, ipilimumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, or durvalumab.

[0177] Without wishing to be bound by theory, the PD-1 receptor-ligand interaction can be a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1, expressed on the cell surface of activated T cells under healthy conditions, is to down-modulate unwanted or excessive immune responses, including autoimmune reactions. Following T-cell stimulation, PD-1 recruits the tyrosine phosphatases, SHP-1 and SHP-2, to the immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, leading to the dephosphorylation of effector molecules such as CD3 zeta (CD3Q, protein kinase C-theta (PKC0), and zeta-chain-associated protein kinase (ZAP70), which are involved in the CD3 T-cell signaling cascade.Formulations and administration

[0178] Fosbretabulin (CAS 222030-63-9) [(Z)-combretastatin-A4 monophosphate tri s(hydoxymethyl)aminom ethane salt] (herein, “Fosbretabulin tris salt”) is a white to pale yellow solid and is a salt form of the pro-drug called CA4P. CA4P is a water soluble pro-drug of cis-combretastatin-A4 (cis-CA4), which binds to tubulin at the colchicine binding site. A chemical name for Combretastatin-A4 phosphate (CA4P) is cis-combretastatin-A4 phosphate. A chemical name for the salt form of the pro-drug is Combretastatin-A4 monophosphate tris salt.The chemical formula of the tris salt form of the pro-drug is Gs^oCkPfCLH^NCh ] and the molecular weight is 517.46.(Z)-combretastatin-A4 monophosphate tris(hydoxymethyl)aminomethane salt

[0179] Fosbretabulin tris salt for Injection is supplied as a sterile lyophilized drug product. It is a white to pale yellow, whole or fragmented cake for reconstitution. Fosbretabulin tris salt for Injection is supplied as 90 mg / vial (as the free acid) and packaged in 10 mL type I amber FD glass vial, stoppered with 20 mm Fluotec FD stopper and sealed with 20 mm aluminum Flip Top over-seal.

[0180] Fosbretabulin tris salt for Injection is manufactured and packaged for clinical studies in compliance with the applicable current Good Manufacturing Practice (cGMP) Regulations for Finished Pharmaceuticals and should be stored in a secure area according to institutional and Good Clinical Practice (GCP) guidelines.

[0181] Fosbretabulin tris salt for Injection is light sensitive and should be stored protected from light in a refrigerator maintained at 2-8°C. Light promotes the conversion of active CA4P to the less active trans-isomer. The study drug solution can be protected from light either by using light sensitive infusion bags or by covering the infusion bags with aluminum foil or other appropriate light resistant covers.

[0182] Aseptic technique must be employed when preparing the drug for administration. Each vial of Fosbretabulin tris salt should be reconstituted with 10 mL Water for Injection (USP / EP), to yield a concentration of 9 mg / mL of drug product as the free acid. The appropriate volume will then be withdrawn from the vial and dispensed into a 50, 100, or 150 mL standard saline infusion bag. Vioflex or Viaflo bags from Baxter were found suitable to use with fosbretabulin.

[0183] The PES and PVDF filters from Millipore have been found suitable to use with fosbretabulin tris salt. The target infusion time is 10 minutes. The maximum infusion time should not exceed 20 minutes, with a minimum flow rate of 5 mL / min.

[0184] Drug product in amber vials is stable as a lyophilized solid for more than 18 months at controlled room temperature (25°C). A reconstituted solution with water is stable for up to 30 minutes under room light and ambient temperature. Once diluted and placed into the infusion bag, the study drug solution can be stored up to 4 hours at ambient temperatures.

[0185] This disclosure further provides ophthalmic formulations for ocular administration comprising a pharmaceutically effective amount of a combretastatin, from 60% to 95% w / w pregelatinized starch, from 1% to 10% w / w hydrophilic matrix forming polymer, and from 0.2% to 5% lubricant.

[0186] This disclosure further provides ocular bioadhesive tablets comprising a pharmaceutically effective amount of a combretastatin, from 60% to 95% w / w pre-gelatinized starch, from 1% to 10% w / w hydrophilic matrix forming polymer, and from 0.2% to 5% lubricant.

[0187] In certain embodiments, a composition may be a pharmaceutical composition containing a therapeutically effective amount of one or more active compounds. Some examples of excipients are given in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975, and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980. Methods for determining a therapeutically effective amount of a compound are known in the art.

[0188] In certain embodiments, a composition may be a pharmaceutical composition containing a therapeutically effective amount of a Fosbretabulin salt form with at least 70, 80, 90, or 99% cis-isomer.

[0189] In some embodiments, a composition may be a pharmaceutical composition containing a therapeutically effective amount of a Fosbretabulin salt form with at least 70, 80, 90, or 99% cis-isomer and PAM3CSK4. This formulation may include a pharmaceutically acceptable carrier.

[0190] In some embodiments, a composition may be a pharmaceutical composition containing a therapeutically effective amount of a Fosbretabulin salt form with at least 70, 80, 90, or 99% cis-isomer and PAMCYSPAMSK4. This formulation may include a pharmaceutically acceptable carrier.

[0191] In additional embodiments, a composition may be a pharmaceutical composition containing a therapeutically effective amount of a Fosbretabulin salt form and a pharmaceutically acceptable carrier.

[0192] Embodiments of this disclosure include compositions which may be pharmaceutical compositions and contain a therapeutically effective amount of an agent and a pharmaceutically acceptable carrier.

[0193] In some embodiments, a composition may be a pharmaceutical composition containing a therapeutically effective amount of a Fosbretabulin salt form with at least 70, 80, 90, or 99% cis-isomer, PAM3CSK4, and an immune checkpoint inhibitor such as pembrolizumab, ipilimumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, or durvalumab. This formulation may include a pharmaceutically acceptable carrier, and may include an amino acid selected from ornithine, phenylalanine, histidine and arginine, and an anionic excipient selected from benzenesulfonic acid, pyridoxine, and thiamine phosphoric acid ester.

[0194] In some embodiments, a composition may be a pharmaceutical composition containing a therapeutically effective amount of a Fosbretabulin salt form with at least 70, 80, 90, or 99% cis-isomer, PAMCYSPAMSK4, and an immune checkpoint inhibitor such as pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab or durvalumab. This formulation may include a pharmaceutically acceptable carrier, and may include an amino acid selected from ornithine, phenylalanine, histidine and arginine, and an anionic excipient selected from benzenesulfonic acid, pyridoxine, and thiamine phosphoric acid ester.

[0195] In certain embodiments, the active agents can be singly-formulated and administered. For example, CA4P can be singly formulated with a carrier and excipients. In another example, Pam3CSK4 can be singly formulated with a carrier and excipients. Each can be separately administered concurrently, simultaneously, sequentially, or separately in time.

[0196] Embodiments of this invention further contemplate co-formulating active agents with carrier and excipients. For example, CA4P and Pam3CSK4 can be combined in co-formulation with carrier and excipients for use. In another example, CA4P, Pam3CSK4, and pembrolizumab can be combined with carrier and excipients in co-formulation for use.

[0197] Further embodiments include ophthalmic formulations which may include additional agents such as analgesics, anesthetics, or anti-inflammatory agents such as hydrocortisone, dexamethasone, fluocinolone, prednisone, prednisolone, methylprednisolone, fluorometholone, betamethasone and triamcinolone.

[0198] Ophthalmic formulations may further include antibiotics such as tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, gramicidin, cephalexin, oxytetracycline, chloramphenicol, rifampicin, ciprofloxacin, levofloxacin, Gatifloxacin, moxifloxacin, aminosides, gentamycin, erythromycin, penicillin, quinolone, ceftazidime, vancomycin, imipeneme, sulfonamides, sulfadiazine, sulfacetamide, sulfamethizole, sulfisoxazole, nitrofurazone and sodium propionate, antifungals such as amphotericin B, fluconazole, ketoconazole and miconazole, anti-allergics such as sodium cromoglycate, antazoline, methapyriline, chlorpheniramine, cetirizine, pyrilamine and prophenpyridamine, antiprotozoal agents, antiviral agents, antifungal agents, anti-infective agents, antimetabolites, and antiangiogenic agents.

[0199] In some embodiments, a total amount of a combretastatin administered can be about 20 pg to about 4000 pg, or from about 10 pg to about 2000 pg, or from about 10 pg to 1750 pg, or about 1500 pg to 1000 pg, or from about 10 pg to 1000 pg.

[0200] Ophthalmic formulations of this disclosure may include ocular minitablets.

[0201] Ophthalmic formulations of this disclosure may include an ophthalmic formulation for ocular administration comprising (a) a pharmaceutically effective amount of a combretastatin; (b) from 60% to 95% w / w pre-gelatinized starch; (c) from 1% to 10% w / w hydrophilic matrix forming polymer; and (d) from 0.2% to 5% lubricant. The ophthalmic formulation may contain about 0.1% to 10% combretastatin A4 phosphate or a pharmaceutically acceptable salt thereof.Combinations and dosages

[0202] Examples of pharmaceutically acceptable excipients and components are given in, for example, Remington, The Science and Practice of Pharmacy, 21st ed., 2005; Rowe et al., Handbook of Pharmaceutical Excipients, 6th ed., 2012; Ash, Handbook of Pharmaceutical Additives, 3rd ed., 2007; Gibson, Pharmaceutical Preformulation and Formulation, 2nd ed., 2009.

[0203] The actives in a composition of this invention can be administered at an initial dosage of from about 0.0001 mg / kg to about 1,000 mg / kg daily.

[0204] In some embodiments, a daily dose range may comprise about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg.

[0205] Embodiments of this invention contemplate a dose administered to a patient sufficient to effect a beneficial therapeutic response.

[0206] Determination of dosage may normally be within the skill of a health care practitioner.

[0207] Embodiments of this invention also contemplate various modalities of administration including parenteral, intravenous, infusion, intradermal, subcutaneous, intramuscular, colonical, rectal, and intraperitoneal.

[0208] A therapeutic composition of this invention can be administered in combination with a checkpoint inhibitor immunotherapeutic agent for use in treating or ameliorating the symptoms of cancer in a human subject or animal.

[0209] A therapeutic composition of this invention can be administered in combination with other drugs, actives, or agents, as well as standard of care treatments for the same disease, each of which can be administered concurrently, simultaneously, sequentially, or separately in time.

[0210] A therapeutic composition of this invention can include pharmaceutically acceptable salt forms, esters, polymorphs or stereoisomers of ingredients, as well as a solvent carrier.

[0211] Examples of a solvent or suspension carrier include sterile water for injection, saline, isotonic saline, and combinations thereof.

[0212] For example, clinical outcomes for pancreatic cancer are unexpectedly improved with a composition containing an inducer of TLR10 and a vascular disrupting agent. The composition contains a pharmaceutically-acceptable carrier buffer. The composition contains a histidine buffer and polysorbate 80. The inducer of TLR10 is Pam3CSK4 and the vascular disrupting agent is CA4P. The patients present a sign of neutropenia and the dosage of the vascular disrupting agent is 45 mg / m2. The patients have biomarker PNPLA6 elevated. Overall survival of cancer patients is surprisingly increased more than three-fold.

[0213] For example, clinical outcomes for breast cancer are unexpectedly improved with a composition containing an inducer of TLR10, a vascular disrupting agent, and animmune checkpoint inhibitor. The composition contains a pharmaceutically-acceptable carrier buffer. The composition contains a histidine buffer and polysorbate 80. The inducer of TLR10 is Pam3CSK4, the vascular disrupting agent is CA4P, and the immune checkpoint inhibitor is pembrolizumab. The composition is formulated with ornithine and benzenesulfonic acid. The patients present a sign of neutropenia and the dosage of the vascular disrupting agent is 10-45 mg / m2. Overall survival of cancer patients is surprisingly increased more than three-fold.

[0214] In some embodiments, a composition may be stable for at least one hour, or at least one day, or 14 days, or at least 21 days, or at least 28 days in a solvent carrier at 25°C.

[0215] In certain embodiments, a formulation for pharmaceutical use can have a concentration of from 0.05 to 50 pM, or 0.1 to 25 pM, or 0.1 to 10 pM, or 0.1 to 7.5 pM, or 0. 1 to 5 pM.

[0216] In certain embodiments, a formulation for pharmaceutical use can have an effective dosage amount of from 0.01 to 1000 mg / m2 / day, or from 1 to 500 mg / m2 / day, or from 1 to 250 mg / m2 / day, or from 1 to 100 mg / m2 / day, or from 1 to 50 mg / m2 / day. Mean human body surface area can be about 1.6 to 1.9 m2.

[0217] In additional embodiments, a formulation for pharmaceutical use can have an effective dosage amount of from 0.05 to 40 mg / kg / day, or from 0.1 to 30 mg / kg / day, or from 0.2 to 20 mg / m2 / day, or from 0.3 to 10 mg / m2 / day, or from 0.5 to 5 mg / m2 / day. Mean human body weight can be about 60 kg.Additional anti-cancer therapeutics

[0218] Additional therapeutics which can be used in combination therapies of this invention include immune checkpoint inhibitors.

[0219] Additional therapeutics which can be used in combination therapies of this invention include immunotherapeutics interferon-gamma and an interleukin immunotherapeutic agent based on IL-2.

[0220] Additional therapeutics which can be used in combination therapies of this invention include a targeted cancer drug, a cancer growth blocker, an EGFR inhibitor, erlotinib, gefitinib, afatinib, osimertinib, dacomitininb, and combinations thereof.

[0221] Additional therapeutics which can be used in combination therapies of this invention include targeted cancer drugs selected from bevacizumab, everolimus, belzutifan, dabrafenib, trametinib, and combinations thereof.

[0222] Additional therapeutics which can be used in combination therapies of this invention include cancer growth blockers selected from an angiogenesis inhibitor, a histone deacetylase inhibitor, a hedgehog blocker, an mTOR inhibitor, a p53 inhibitor, a PARP inhibitor, a proteasome inhibitor, a tyrosine kinase inhibitor, and combinations thereof.

[0223] Additional therapeutics which can be used in combination therapies of this invention include TMZ, radiation, and bevacizumab.Additional embodiments

[0224] Compounds of this disclosure may be made by methods known in the art.

[0225] As used herein, the term "alkyl" can refer to a saturated, branched or unbranched, substituted or unsubstituted aliphatic group containing from 1 to 22 carbon atoms. This definition applies to the alkyl portion of other groups. As used herein, the term “C(l-5)alkyl,” for example, includes C(l)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, and C(5)alkyl. Likewise, the term “C(3-22)alkyl,” for example, includes C(l)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, C(5)alkyl, C(6)alkyl, C(7)alkyl, C(8)alkyl, C(9)alkyl, C(10)alkyl, C(l l)alkyl, C(12)alkyl, C(13)alkyl, C(14)alkyl, C(15)alkyl, C(16)alkyl, C(17)alkyl, C(18)alkyl, C(19)alkyl, C(20)alkyl, C(21)alkyl, and C(22)alkyl.

[0226] As used herein, the term "alkenyl" can refer to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to 22 carbon atoms and at least one carbon-carbon double bond.

[0227] As used herein, the term "alkynyl" can refer to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to 22 carbon atoms and at least one carbon-carbon triple bond.

[0228] As used herein, the term "substituted" can refer to an atom having one or more substitutions or substituents which can be the same or different and may include a hydrogen substituent. Thus, the terms alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino, alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, and aralkyl as used herein refer to groups which include substituted variations. Substituted variations include linear, branched, and cyclic variations, and groups having a substituent or substituents replacing one or more hydrogens attached to any carbon atom of the group. Substituents that may be attached to acarbon atom of the group include alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino, alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, aralkyl, acyl, hydroxyl, cyano, halo, haloalkyl, amino, aminoacyl, alkylaminoacyl, acyloxy, aryloxy, aryloxyalkyl, mercapto, nitro, carbamyl, carbamoyl, and heterocycle. For example, the term ethyl includes without limitation -CH2CH3, -CHFCH3, -CF2CH3, -CHFCH2F, -CHFCHF2, -CHFCF3, -CF2CH2F, — CF2CHF2, -CF2CF3, and other variations as described above. In general, substituents may be further substituted with any atom or group of atoms.

[0229] In general, a compound may contain one or more chiral centers. Compounds containing one or more chiral centers may include those described as an "isomer," a "stereoisomer," a "diastereomer," an "enantiomer," an "optical isomer," or as a "racemic mixture." Conventions for stereochemical nomenclature, for example the stereoisomer naming rules of Cahn, Ingold and Prelog, as well as methods for the determination of stereochemistry and the separation of stereoisomers are known in the art. See, for example, Michael B. Smith and Jerry March, March’s Advanced Organic Chemistry, 5th edition, 2001. The compounds and structures of this disclosure are meant to encompass all possible isomers, stereoisomers, diastereomers, enantiomers, and / or optical isomers that would be understood to exist for the specified compound or structure, including any mixture, racemic or otherwise, thereof.

[0230] Some compounds, peptides and / or protein compositions of this invention may have one or more chiral centers and / or geometric isomeric centers (E- and Z-isomers), and it is to be understood that the invention encompasses all such optical isomers, diastereoisomers, geometric isomers, and mixtures thereof.

[0231] This invention encompasses any and all tautomeric, solvated or unsolvated, hydrated or unhydrated forms, as well as any atom isotope forms of the compounds or compositions disclosed herein.

[0232] Numbered embodiments of this invention include the following:

[0233] (1) A composition for use in treating or ameliorating the symptoms of cancer in a subj ect, the composition comprising: an inducer of TLR10; a vascular disrupting agent; and a carrier.

[0234] (2) A composition for use in the preparation of a medicament for treating or ameliorating the symptoms of cancer in a subject, the composition comprising: an inducer of TLR10; and a vascular disrupting agent.

[0235] (3) A method for treating or ameliorating the symptoms of cancer in a subject in need, the method comprising: preparing a composition comprising an inducer of TLR10; and a vascular disrupting agent; and administering a therapeutically sufficient amount of the composition to the subject.

[0236] (4) The composition or method of any of embodiments 1-3, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, melanoma, sarcoma, ocular cancer, retinoblastoma, ocular melanoma, or intraocular lymphoma.

[0237] (5) The composition or method of any of embodiments 1-4, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, melanoma, sarcoma, ocular cancer, retinoblastoma, ocular melanoma, or intraocular lymphoma, and wherein the subject’s cancer presents tumors having bacterial flora levels above median.

[0238] (6) The composition or method of any of embodiments 1-5, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, or colon cancer, and wherein the subject’s cancer presents tumors having bacterial flora levels above median.

[0239] (7) The composition or method of any of embodiments 1-6, wherein the inducer of TLR10 is a TLR10, TLR2, or TLR1 agonist or ligand.

[0240] (8) The composition or method of any of embodiments 1-7, wherein the inducer of TLR10 is N-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl- (lysyl)3 -lysine (Pam3CSK4), N-palmitoyl-S-[2-hydroxy-3-(palmitoyloxy)propyl]-(R)- cysteinyl-(lysyl)3 -lysine (PamCysPamSK4), or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer thereof.

[0241] (9) The composition or method of any of embodiments 1-8, wherein the vascular disrupting agent is fosbretabulin having at least 70, 80, 90, or 99% cis-isomer, or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer thereof.

[0242] (10) The composition or method of any of embodiments 1-9, wherein the vascular disrupting agent is fosbretabulin and the inducer of TLR10 is Pam3CSK4, or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer of each.

[0243] (11) The composition or method of any of embodiments 1-10, wherein the vascular disrupting agent is fosbretabulin and the inducer of TLR10 is PamCysPamSK4, or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer of each.

[0244] (12) The composition or method of any of embodiments 1-11, wherein the cancer is pancreatic cancer and the subjects have biomarker PNPLA6 elevated.

[0245] (13) The composition or method of any of embodiments 1-12, wherein the cancer is lung cancer and the subjects have biomarker MR0H2B elevated.

[0246] (14) The composition or method of any of embodiments 1-13, wherein the cancer is bladder cancer and the subjects have biomarker BLNK elevated.

[0247] (15) The composition or method of any of embodiments 1-14, wherein the cancer is uterine cancer and the subjects have biomarker CD7 or LRRC45 elevated.

[0248] (16) The composition or method of any of embodiments 1-15, wherein the cancer is uveal melanoma and the subjects have biomarker CD7 elevated.

[0249] (17) The composition or method of any of embodiments 1-16, wherein the cancer is sarcoma, the subjects have biomarker AK7 elevated, and the sarcoma size is at least 1 cm3.

[0250] (18) The composition or method of any of embodiments 1-17, wherein the subject presents a sign of neutropenia and the dosage of the vascular disrupting agent is 10-45 mg / m2and exhibits an AUC of 12-19 uM*hr.

[0251] (19) The composition or method of any of embodiments 1-18, wherein the inducer of TLR10 and the vascular disrupting agent are singly-formulated and administered concurrently, simultaneously, sequentially, or separately in time.

[0252] (20) The composition or method of any of embodiments 1-19, in combination with a singly-formulated immune checkpoint inhibitor, wherein the immune checkpoint inhibitor and the composition comprising the inducer of TLR10 and the vascular disrupting agent are administered concurrently, simultaneously, sequentially, or separately in time.

[0253] (21) The composition or method of embodiment 20, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA-4, or PD-L1.

[0254] (22) The composition or method of embodiment 20, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab, or durvalumab.

[0255] (23) The composition or method of any of embodiments 1-19, in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor and the composition comprising the inducer of TLR10 and the vascular disrupting agent are coformulated.

[0256] (24) The composition or method of embodiment 23, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA-4, or PD-L1.

[0257] (25) The composition or method of embodiment 23, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab, or durvalumab.

[0258] (26) The composition or method of any of embodiments 1-19, in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor, the inducer of TLR10, and the vascular disrupting agent are singly-formulated and administered concurrently, simultaneously, sequentially, or separately in time.

[0259] (27) The composition or method of embodiment 26, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA-4, or PD-L1.

[0260] (28) The composition or method of embodiment 26, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab, or durvalumab.

[0261] (29) The composition or method of any of embodiments 1-28, wherein the composition or method is combined with a standard of care treatment for the cancer or one or more additional therapeutics for treatment of cancer selected from TMZ, radiation, and bevacizumab.

[0262] (30) The composition or method of any of embodiments 1-29, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is coformulated with an immune checkpoint inhibitor for subcutaneous injection or intravenous injection or infusion.

[0263] (31) The composition or method of any of embodiments 1-30, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated for subcutaneous injection.

[0264] (32) The composition or method of any of embodiments 1-31, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated for intravenous injection or infusion.

[0265] (33) The composition or method of any of embodiments 1-32, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated for ocular use as a drop, an intraocular injection, or a subconjunctival injection.

[0266] (34) The composition or method of any of embodiments 1-33, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated with an amino acid selected from ornithine, phenylalanine, histidine and arginine and an anionic excipient selected from benzenesulfonic acid, pyridoxine, and thiamine phosphoric acid ester.

[0267] (35) The composition or method of any of embodiments 1-34, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated with an immune checkpoint inhibitor, an amino acid selected from ornithine, phenylalanine, histidine and arginine, and an anionic excipient selected from benzenesulfonic acid, pyridoxine, and thiamine phosphoric acid ester.

[0268] (36) The composition or method of any of embodiments 1-35, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated with a buffer selected from histidine, succinate, citrate, acetate, phosphate, glutamate, adipic acid, aspartic acid, lactic acid, tromethamine, and 2-(N-morpholino)- ethanesulfonic acid and a surfactant selected from polysorbate 20, polysorbate 80, and poloxamer 188.

[0269] (37) The composition or method of any of embodiments 1-36, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated in a prefilled autoinjector or syringe.

[0270] (38) The composition or method of any of embodiments 1-37, wherein the composition or medicament is administered guided by imaging.

[0271] (39) The composition or method of any of embodiments 1-38, wherein the inducer of TLR10 induces tumor-infiltrating lymphocytes.

[0272] (40) The composition or method of any of embodiments 1-39, wherein the composition or method upregulates expression of one or more genes selected fromTLR10, CD3, CD4, CD8, CD7, BLNK, AK7, PNPLA6, MROH2B, LRRC45, SECTM1, CACNA1I, CENPX, HEXD, KRAS, NXPH1, RAC3, SLC12A3, and ZNF518A.

[0273] (41) The composition or method of any of embodiments 1-40, wherein the composition, agent, medicament or administration increases a survival rate of subjects at month 6, 12, 18, 24, 30, or 36.

[0274] All publications including patents, patent application publications, and nonpatent publications referred to in this description, as well as the sequence listing are each expressly incorporated herein by reference in their entirety for all purposes.

[0275] Although the foregoing disclosure has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the artisan that certain changes and modifications are comprehended by the disclosure and may be practiced without undue experimentation within the scope of the appended claims, which are presented by way of illustration not limitation. This invention includes all such additional embodiments, equivalents, and modifications. This invention includes any combinations or mixtures of the features, materials, elements, or limitations of the various illustrative components, examples, and claimed embodiments.EXAMPLES

[0276] As used herein, for a valid stratification without any bias, the median of the population data is defined as the cutoff. For each gene of interest, the median expression level across all the samples is calculated. The median is the middle value in a list of numbers sorted in ascending or descending order and is used because it is less affected by outliers than the mean. Samples may then be stratified into two groups based on whether the expression level of a particular gene is above or below the median. This creates a "high expression" group and a "low expression" group.

[0277] Example 1 This example demonstrated that an inducer of tumor necrosis can be used in combination with a immune checkpoint inhibitor to improve cancer patient survival.

[0278] FIG. 1 shows results of a study of clinical outcomes for cancer patients and the beneficial impact on overall survival of patients for therapeutic use of a TLR10 agonist agent in combination with an immune checkpoint inhibitor.

[0279] The TLR10 agonist agent induces tumor necrosis and upregulation of necrosis genes. Patients in this study included those with multiple tumor types. The Kaplan-Meier chart of FIG. 1 shows that median overall survival of cancer patients was significantly improved (logrank P=9.6e-5) for higher expression of TLR10. Upper quartile overall survival of cancer patients surprisingly increased more than threefold from 6 months to 20 months. This result is surprising because TLR10 is not known to synergize with immune response to tumor necrosis. This study established a basis for therapeutic use of a TLR10 agonist agent for inducing immune response to tumor necrosis and treating cancer in combination with an immune checkpoint inhibitor.

[0280] Example 2. This example demonstrated that an inducer of TLR10 can be used in combination with a immune checkpoint inhibitor to improve cancer patient survival.

[0281] FIG. 2 shows results of a study of clinical outcomes for cancer patients and the beneficial impact on overall survival of patients for therapeutic use of a TLR10 agonist in combination with immune checkpoint inhibitor pembrolizumab.

[0282] The Kaplan-Meier chart of FIG. 2 shows that median overall survival of cancer patients was significantly improved (logrank P=0.00078) for higher expression of necrosis gene PNPLA6, which was evidence of necrosis. This study established a basis for therapeutic use of a TLR10 agonist agent for inducing immune response in combination with an immune checkpoint inhibitor.

[0283] Example 3. This example demonstrated that an inducer of tumor necrosis can be used in combination with a immune checkpoint inhibitor to improve cancer patient survival.

[0284] FIG. 3 shows results of a study of clinical outcomes for cancer patients and the beneficial impact on overall survival of patients for therapeutic use of a TLR10 agonist, in combination with immune checkpoint inhibitor ipilimumab.

[0285] The Kaplan-Meier chart of FIG. 3 shows that median overall survival of cancer patients was significantly improved (logrank P=0.00075) for higher expression of necrosis gene PNPLA6, which was evidence of necrosis. This study established a basis for therapeutic use of a TLR10 agonist agent for inducing immune response and treating cancer in combination with an immune checkpoint inhibitor.

[0286] Example 4. This example demonstrated that expression of tumor necrosis- associated genes can be used as biomarkers to guide in situ cancer therapeutics to improve cancer patient survival.

[0287] FIG. 4 shows results of a study of clinical outcomes for sarcoma patients and the impact on overall survival of necrosis gene AK7.

[0288] The Kaplan-Meier chart of FIG. 4 shows that median overall survival of sarcoma patients was significantly improved (logrank P=0.00097) for higher expression of necrosis gene AK7, which was evidence of tumor necrosis. The association of AK7 to cancer may not be limited to sarcoma.

[0289] Example 5. This example demonstrated that expression of tumor necrosis- associated genes can be used as biomarkers to guide in situ cancer therapeutics to improve cancer patient survival.

[0290] FIG. 5 shows results of a study of clinical outcomes for bladder cancer patients and the impact on overall survival of necrosis gene BLNK.

[0291] The Kaplan-Meier chart of FIG. 5 shows that median overall survival of bladder cancer patients was significantly improved (logrank P=0.0035) for higher expression of necrosis gene BLNK, which was evidence of tumor necrosis. The association of BLNK to cancer may not be limited to bladder cancer.

[0292] Example 6 This example demonstrated that expression of tumor necrosis- associated genes can be used as biomarkers to guide in situ cancer therapeutics to improve cancer patient survival.

[0293] FIG. 6 shows results of a study of clinical outcomes for uterine cancer patients and the impact on overall survival of necrosis gene CD7.

[0294] The Kaplan-Meier chart of FIG. 6 shows that median overall survival of uterine cancer patients was significantly improved (logrank P=0.00075) for higher expression of necrosis gene CD7, which was evidence of tumor necrosis. The association of CD7 to cancer may not be limited to uterine cancer.

[0295] Example 7 This example demonstrated that expression of tumor necrosis- associated genes can be used as biomarkers to guide in situ cancer therapeutics to improve cancer patient survival.

[0296] FIG. 7 shows results of a study of clinical outcomes for ovarian cancer patients and the impact on overall survival of necrosis gene LRRC45.

[0297] The Kaplan-Meier chart of FIG. 7 shows that median overall survival of ovarian cancer patients was significantly improved (logrank P=0.0071) for higherexpression of necrosis gene LRRC45, which was evidence of tumor necrosis. The association of LRRC45 to cancer may not be limited to ovarian cancer.

[0298] Example 8. This example demonstrated that expression of tumor necrosis- associated genes can be used as biomarkers to guide in situ cancer therapeutics to improve cancer patient survival.

[0299] FIG. 8 shows results of a study of clinical outcomes for lung cancer patients and the impact on overall survival of necrosis gene MR0H2B.

[0300] The Kaplan-Meier chart of FIG. 8 shows that median overall survival of lung cancer patients was significantly improved (logrank P=0.0012) for higher expression of necrosis gene MR0H2B, which was evidence of tumor necrosis. The association of MR0H2B to cancer may not be limited to lung cancer.

[0301] Example 9. This example demonstrated that expression of tumor necrosis- associated genes can be used as biomarkers to guide in situ cancer therapeutics to improve cancer patient survival.

[0302] FIG. 9 shows results of a study of clinical outcomes for pancreatic cancer patients and the impact on overall survival of necrosis gene PNPLA6.

[0303] The Kaplan-Meier chart of FIG. 9 shows that median overall survival of pancreatic cancer patients was significantly improved (logrank P=0.00025) for higher expression of necrosis gene PNPLA6, which was evidence of tumor necrosis. The association of PNPLA6 to cancer may not be limited to pancreatic cancer.

[0304] Example 10. This example identified tumor necrosis-associated genes.

[0305] A study of gene expression in cancer patients was performed across multiple tumor types, indications and stages (cBioPortal). Expression levels of cancer-associated genes was used to confirm the identity of tumor necrosis-associated genes. Cancer- associated genes with elevated expression in tumors were confirmed as tumor necrosis- associated genes.

[0306] Expression of certain tumor necrosis-associated genes was correlated with improved cancer patient survival.

[0307] Expression of tumor necrosis-associated genes across multiple tumor types, indications and stages was classified as being high or low. Highly expressed tumor necrosis-associated genes can be used as biomarkers to guide cancer therapeutics of this invention.

[0308] In general, a patient having a positively-correlated tumor necrosis-associated gene had elevated expression of the gene. P-values can be used to evaluate the relative significance of high gene expression for a patient to the cancer patient group as a whole.

[0309] The results of the study are shown in Table 1.Table 1 : Patient expression of tumor necrosis-associated genes

[0310] Example 11 An in vivo study established a basis for therapeutic use of a vascular disrupting agent for treating cancer in combination with an immune checkpoint inhibitor.

[0311] FIG. 10 shows results of a breast cancer model study (EMT-6) and the beneficial impact on reducing tumor volume for use of a vascular disrupting agent, combretastatin CA4P, alone and in combination with an immune checkpoint inhibitor (anti-CTLA-4).

[0312] The mean tumor volume chart of FIG. 10 shows that the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor (anti-CTLA-4) significantly reduced tumor volume as compared to negative control and either CA4P or anti-CTLA-4 alone. In vivo survival studies confirmed the potency of the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor.

[0313] Example 12 An in vivo study established a basis for therapeutic use of a vascular disrupting agent for treating cancer in combination with an immune checkpoint inhibitor.

[0314] FIG. 11 shows results of a colon cancer model study (murine CT-26) and the beneficial impact on reducing tumor volume for use of a vascular disrupting agent, combretastatin CA4P, alone and in combination with an immune checkpoint inhibitor (anti-CTLA-4).

[0315] The mean tumor volume chart of FIG. 11 shows that the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor (anti-CTLA-4) significantly reduced tumor volume as compared to negative control and either CA4P or anti-CTLA-4 alone. In vivo survival studies confirmed the potency of the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor.

[0316] Clinical data demonstrate that CA4P induces coagulative and hemorrhagic necrosis within 24 hours, quantitatively shown by decreases in DCE-MRI perfusion metrics (Ktrans, IAUGC parameters), and produces necrotic tumor fractions exceeding 50 percent. In the clinic,CA4P in combination with an immune checkpoint inhibitor can yield surprising levels of tumor regression, as well as improved overall survival.

[0317] Example 13 An in vivo study established a basis for therapeutic use of a vascular disrupting agent for treating cancer in combination with an immune checkpoint inhibitor.

[0318] FIG. 12 shows results of an in vivo cancer study and the impact on inducing necrosis and eliciting immune response for use of a vascular disrupting agent, combretastatin CA4P, in combination with an immune checkpoint inhibitor (anti-CTLA- 4).

[0319] The chart of FIG. 12 shows that the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor (anti-CTLA-4) induced a large increases in tumor-associated T-cells and CD8+ T-cells as compared to negative control and either CA4P or anti-CTLA-4 alone.

[0320] Example 14 An in vivo study established a basis for therapeutic use of a vascular disrupting agent for treating cancer in combination with an immune checkpoint inhibitor.

[0321] FIG. 13 shows results of an in vivo cancer study and the impact on inducing necrosis and eliciting immune response for use of a vascular disrupting agent, combretastatin CA4P, in combination with an immune checkpoint inhibitor (anti-CTLA- 4).

[0322] The chart of FIG. 13 shows that the combination of vascular disrupting agent CA4P and immune checkpoint inhibitor (anti-CTLA-4) induced large increases in tumor necrosis area as compared to negative control and either CA4P or anti-CTLA-4 alone.

[0323] Example 15. A clinical study established a basis for therapeutic use of a vascular disrupting agent for treating uveal cancer.

[0324] FIG. 14 shows results of a clinical study of uveal melanoma patients and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P. Administration by injection and topical routes are compared.

[0325] Example 16. A clinical study established a basis for therapeutic use of a vascular disrupting agent for treating uveal cancer.

[0326] FIG. 15 shows results of a clinical study of uveal melanoma patients and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P. Administration by injection and topical routes are compared.

[0327] Example 17 A previously-published study with FIG. 16 shows results of a preclinical study of retinoblastoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P.

[0328] Transgenic retinoblastoma mice were administered via subconjunctival route of exposure CA4P via 6 injections, 2x / week for 3 weeks, of 0.02, 0.2, or 2.0 mg / pL at 12 weeks of age. Eyes enucleated post-treatment at 16-weeks of age. Eyes were histopathologically examined for presence of tumor. The results show that subconjunctival CA4P inhibited tumor vasculature and was associated with tumor inhibition in murine retinoblastoma. FIG. 16 shows a large tumor is present.

[0329] Example 18 A previously-published study with FIG. 17 shows results of a preclinical study of retinoblastoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P.

[0330] As compared to the previous figure, FIG. 17 shows a moderate tumor is present.

[0331] Example 19 A previously-published study with FIG. 18 shows results of a preclinical study of retinoblastoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P.

[0332] As compared to the previous figure, FIG. 18 shows minimal tumor is present.

[0333] Example 20. A previously-published study with FIG. 19 shows results of a preclinical study of retinoblastoma and the impact on reducing tumor volume with a vascular disrupting agent, combretastatin CA4P.

[0334] As compared to the previous figure, FIG. 19 shows no tumor is present.

[0335] Example 21. This example study investigated therapeutic use of CA4P for treating uveal melanoma.

[0336] FIG. 20 shows results of a study of clinical outcomes for 68 uveal melanoma patients and the impact on overall survival of necrosis gene SECTM1.

[0337] The Kaplan-Meier chart of FIG. 20 shows that overall survival of melanoma patients was significantly worse (logrank P=2.783e-5) for higher expression of necrosisgene SECTM1, which was evidence of tumor necrosis. This study showed that the use of CA4P alone did not improve overall survival for uveal melanoma.

[0338] Example 22. This example study investigated uveal melanoma cancer.

[0339] FIG. 21 shows results of a study of clinical outcomes for 68 uveal melanoma patients and the impact on overall survival of necrosis gene CD7.

[0340] The Kaplan-Meier chart of FIG. 21 shows that overall survival of uveal melanoma patients was significantly worse (logrank P=5.060e-5) for higher expression of necrosis gene CD7, which was evidence of tumor necrosis. This study confirmed that expression of necrosis gene CD7 is a biomarker for melanoma cancer.

[0341] Example 23 This example study investigated uveal melanoma cancer.

[0342] FIG. 22 shows results of a study of clinical outcomes for 68 uveal melanoma patients and the impact on overall survival of necrosis gene PNPLA6.

[0343] The Kaplan-Meier chart of FIG. 22 shows that overall survival of uveal melanoma patients appears worse for higher expression of necrosis gene PNPLA6, which was evidence of tumor necrosis. This study confirmed that expression of necrosis gene PNPLA6 is a biomarker for melanoma cancer.

[0344] Example 24. This study investigated uveal melanoma cancer.

[0345] FIG. 23 shows results of a study of clinical outcomes for 68 uveal melanoma patients and the impact on overall survival of necrosis gene CACNA1L.

[0346] The Kaplan-Meier chart of FIG. 23 shows that overall survival of uveal melanoma patients was significantly worse (logrank P=8.685e-3) for higher expression of necrosis gene CACNA1L, which was evidence of tumor necrosis. This study confirmed that expression of necrosis gene CACNA1L is a biomarker for melanoma cancer.

[0347] Example 25 This example shows results of a clinical study in FIG. 24 of AUC for ocular administration of CA4P in eye drops, minitablets, and by injection. This study demonstrated a formulation for ocular use in treating cancer.

[0348] FIG. 25 shows results of a clinical study of AUC for ocular administration of CA4P in eye drops, minitablets, and by injection.

[0349] Example 26: This example study of clinical outcomes demonstrated effectiveness of CA4P in treating cancer. The PK / PD analyses of four previous clinical trials were analyzed to determine dose-limiting toxicity (DLT) for CA4P. FIG. 26shows pharmacokinetics of CA4P and CA4 in three clinical monotherapy studies (CA4P-101, CA4P-102, Phl_066). FIG. 27 shows pharmacokinetics of CA4P and CA4 in a combination study (CA4P-103).

[0350] In addition, the dose-limiting toxicity (DLT) data from 15 previous clinical studies including five monotherapy and ten combination therapy were re-analyzed. In five monotherapy studies, DLTs were observed when CA4P was administrated at and above 45 mg / m2. The percentage of DLT at each dose level was much higher when CA4P was administered at a dose above 70 mg / m2. When separated by the dosing schedules, DLTs were observed at 60 mg / m2and above when administered every 21 days (q21 d) or daily x 5 for 1 week every 21 days (qdx5 / 21d). However, when CA4P was administered weekly, DLTs were observed at 45 mg / m2and above.

[0351] In sum, this example shows why previous attempts failed. Overcoming these previous failures

[0352] In the ten combination therapy studies, DLTs were observed when CA4P was administrated at and above 45 mg / m2. The percentage of DLT was much higher when CA4P was administered at a dose of 72 mg / m2. When separated by the dosing schedules, DLTs were observed at 60 mg / m2and 72 mg / m2when administered every 21 days (q21d). However, when CA4P was administered weekly, DLTs were observed at 45 mg / m2and above.

Claims

WHAT IS CLAIMED IS:

1. A composition for use in treating or ameliorating the symptoms of cancer in a subject, the composition comprising: an inducer of TLR10; a vascular disrupting agent; and a carrier.

2. A composition for use in the preparation of a medicament for treating or ameliorating the symptoms of cancer in a subject, the composition comprising: an inducer of TLR10; and a vascular disrupting agent.

3. A method for treating or ameliorating the symptoms of cancer in a subject in need, the method comprising: preparing a composition comprising an inducer of TLR10; and a vascular disrupting agent; and administering a therapeutically sufficient amount of the composition to the subject.

4. The composition or method of any of claims 1-3, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, melanoma, sarcoma, ocular cancer, retinoblastoma, ocular melanoma, or intraocular lymphoma.

5. The composition or method of any of claims 1-3, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, melanoma, sarcoma, ocular cancer, retinoblastoma, ocular melanoma, or intraocular lymphoma, and wherein the subject’s cancer presents tumors having bacterial flora levels above median.

6. The composition or method of any of claims 1-3, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, or colon cancer, and wherein the subject’s cancer presents tumors having bacterial flora levels above median.

7. The composition or method of any of claims 1-3, wherein the inducer of TLR10 is a TLR10, TLR2, or TLR1 agonist or ligand.

8. The composition or method of any of claims 1-3, wherein the inducer of TLR10 is N- palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine (Pam3CSK4), N-palmitoyl-S-[2-hydroxy-3-(palmitoyloxy)propyl]-(R)-cysteinyl-(lysyl)3-lysine(PamCysPamSK4), or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer thereof.

9. The composition or method of any of claims 1-3, wherein the vascular disrupting agent is fosbretabulin having at least 70, 80, 90, or 99% cis-isomer, or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer thereof.

10. The composition or method of any of claims 1-3, wherein the vascular disrupting agent is fosbretabulin and the inducer of TLR10 is Pam3CSK4, or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer of each.

11. The composition or method of any of claims 1-3, wherein the vascular disrupting agent is fosbretabulin and the inducer of TLR10 is PamCysPamSK4, or a pharmaceutically acceptable salt form, ester, polymorph, racemic mixture, or stereoisomer of each.

12. The composition or method of any of claims 1-3, wherein the cancer is pancreatic cancer and the subjects have biomarker PNPLA6 elevated.

13. The composition or method of any of claims 1-3, wherein the cancer is lung cancer and the subjects have biomarker MR0H2B elevated.

14. The composition or method of any of claims 1-3, wherein the cancer is bladder cancer and the subjects have biomarker BLNK elevated.

15. The composition or method of any of claims 1-3, wherein the cancer is uterine cancer and the subjects have biomarker CD7 or LRRC45 elevated.

16. The composition or method of any of claims 1-3, wherein the cancer is uveal melanoma and the subjects have biomarker CD7 elevated.

17. The composition or method of any of claims 1-3, wherein the cancer is sarcoma, the subjects have biomarker AK7 elevated, and the sarcoma size is at least 1 cm3.

18. The composition or method of any of claims 1-3, wherein the subject presents a sign of neutropenia and the dosage of the vascular disrupting agent is 10-45 mg / m2and exhibits an AUC of 12-19 uM*hr.

19. The composition or method of any of claims 1-3, wherein the inducer of TLR10 and the vascular disrupting agent are singly-formulated and administered concurrently, simultaneously, sequentially, or separately in time.

20. The composition or method of any of claims 1-3, in combination with a singly-formulated immune checkpoint inhibitor, wherein the immune checkpoint inhibitor and the composition comprising the inducer of TLR10 and the vascular disrupting agent are administered concurrently, simultaneously, sequentially, or separately in time.

21. The composition or method of claim 20, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA-4, or PD-L1.

22. The composition or method of claim 20, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab, or durvalumab.

23. The composition or method of any of claims 1-3, in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor and the composition comprising the inducer of TLR10 and the vascular disrupting agent are co-formulated.

24. The composition or method of claim 23, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA-4, or PD-L1.

25. The composition or method of claim 23, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab, or durvalumab.

26. The composition or method of any of claims 1-3, in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor, the inducer of TLR10, and the vascular disrupting agent are singly-formulated and administered concurrently, simultaneously, sequentially, or separately in time.

27. The composition or method of claim 26, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, CTLA-4, or PD-L1.

28. The composition or method of claim 26, wherein the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, spartalizumab, atezolizumab, avelumab, ipilimumab, or durvalumab.

29. The composition or method of any of claims 1-3, wherein the composition or method is combined with a standard of care treatment for the cancer or one or more additional therapeutics for treatment of cancer selected from TMZ, radiation, and bevacizumab.

30. The composition or method of any of claims 1-3, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is co-formulated with an immune checkpoint inhibitor for subcutaneous injection or intravenous injection or infusion.

31. The composition or method of any of claims 1-3, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated for subcutaneous injection.

32. The composition or method of any of claims 1-3, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated for intravenous injection or infusion.

33. The composition or method of any of claims 1-3, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated for ocular use as a drop, an intraocular injection, or a subconjunctival injection.

34. The composition or method of any of claims 1-3, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated with an amino acid selected from ornithine, phenylalanine, histidine and arginine and an anionic excipient selected from benzenesulfonic acid, pyridoxine, and thiamine phosphoric acid ester.

35. The composition or method of any of claims 1-3, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated with an immune checkpoint inhibitor, an amino acid selected from ornithine, phenylalanine, histidine and arginine, and an anionic excipient selected from benzenesulfonic acid, pyridoxine, and thiamine phosphoric acid ester.

36. The composition or method of any of claims 1-3, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated with a buffer selected from histidine, succinate, citrate, acetate, phosphate, glutamate, adipic acid, aspartic acid, lactic acid, tromethamine, and 2-(N-morpholino)-ethanesulfonic acid and a surfactant selected from polysorbate 20, polysorbate 80, and poloxamer 188.

37. The composition or method of any of claims 1-3, wherein the composition comprising the inducer of TLR10 and the vascular disrupting agent is formulated in a prefilled autoinjector or syringe.

38. The composition or method of any of claims 1-3, wherein the composition or medicament is administered guided by imaging.

39. The composition or method of any of claims 1-3, wherein the inducer of TLR10 induces tumor-infiltrating lymphocytes.

40. The composition or method of any of claims 1-3, wherein the composition or method upregulates expression of one or more genes selected from TLR10, CD3, CD4, CD8, CD7, BLNK, AK7, PNPLA6, MROH2B, LRRC45, SECTM1, CACNA1I, CENPX, HEXD, KRAS, NXPH1, RAC3, SLC12A3, and ZNF518A.

41. The composition or method of any of claims 1-3, wherein the composition, agent, medicament or administration increases a survival rate of subjects at month 6, 12, 18, 24, 30, or36.

Images