WO2016168361A1 - Polyvalent vaccines and combination therapy for the treatment of melanoma - Google Patents

Abstract

The present invention features a polyvalent vaccine for inducing an immune response and methods for treating patients with melanoma or preventing the recurrence of melanoma in patients in need. The vaccine can also be administered in combination with one or more active pharmaceutical ingredients, such as one or more checkpoint inhibitors. Also provided are methods for using and making the compositions.

Description

POLYVALENT VACCINES AND COMBINATION THERAPY FOR THE

TREATMENT OF MELANOMA

FIELD OF THE INVENTION

[0001] The present invention relates to compositions and methods for treating patients with melanoma or preventing the recurrence of melanoma in patients in need. More particularly, the invention includes compositions and methods related to the administration of a vaccine comprising partially purified melanoma antigens shed from multiple human melanoma cell lines, optionally in combination with one or more active pharmaceutical ingredients, such as one or more checkpoint inhibitors or immunopotentiators.

BACKGROUND

[0002] Malignant melanoma is the most dangerous form of skin cancer. The incidence of melanoma is increasing each year. The American Cancer Society estimates that there will be more than 76,000 new cases and more than 9,000 deaths from this cancer in the United States in 2014. Surgical resection of early stage localized (Stages 1 and 2) disease is the only curative treatment. Once melanoma metastasizes to regional nodes, only a little over one-half (62%) of the patients survive for 5 years. After the melanoma metastasizes to distant organs and/or tissues, the 5 year survival rate in patients declines drastically to 16%. The morbidity and cost of treatment are significant. A continued need exists for additional treatments for patients with melanoma at risk for recurrence.

[0003] Surgical resection is the first line of treatment for localized (Stages 1 and 2) cutaneous melanoma. Patients that have a primary tumor larger than 4 mm and/or have a tumor that involves the regional lymph nodes or a tumor with both regional extension and involves the regional lymph nodes have an increases risk for recurrence of melanoma. Only two FDA-approved drugs (ipilimumab (Yervoy™), a monoclonal antibody, and vemurafenib (Zelboraf®), a B-Raf enzyme inhibitor which only works in patients who have a V600E BRAF mutation, have shown extended survival rates in advanced melanoma. Thus, many patients with advanced metastatic melanoma have a high risk of mortality. SUMMARY

[0004] The present invention features compositions and methods useful in treating melanoma. In some aspects, the invention features a method of treating melanoma by administering a therapeutically effective amount of a polyvalent melanoma cancer vaccine to a patient in need thereof. The polyvalent vaccine is not patient-specific in that it does not require the patient's own tumor to make the vaccine. Thus, the polyvalent vaccine can be used to treat all patients with melanoma or at risk for recurrence of melanoma regardless of the patient's genetic make-up or melanoma type.

[0005] In one aspect, the method comprises administering a therapeutically effective amount of a soluble polyvalent melanoma cancer vaccine comprising multiple partially purified soluble melanoma antigens released or shed from their external surface from melanoma cells. Three or more human melanoma cell lines can be used to generate tumor- associated antigens (comprising e.g., MAGE-1, MAGE-2, MAGE-3, S100B, HLA-1 (β-2 microglobulin), Melan-A/MART-1, gplOO, tyrosinase, melanocortin receptor (MC1R), and dopachrome tautomerase (TRP-2)) that are useful in preparing polyvalent melanoma vaccines. Exemplary human melanoma cells lines, include but are not limited to: HM2 (also referred to as SFHM2; previously designated as M20), HM4 (also referred to as SFHM4; previously designated as M14) and HM8 (also referred to as SFHM8; previously designated as SK Mel28 or Mel 28). The tumor-associated antigens are collected and partially purified to produce the melanoma polyvalent vaccine. By using antigens from multiple tumors or cell lines, a "polyvalent" vaccine can be produced.

[0006] In some aspects this invention features a polyvalent melanoma cancer vaccine comprising shed antigens from three or more human melanoma cell lines. In some aspects the melanoma vaccine comrpises shed antigens from HM2, HM4 and HM8 human melanoma cell lines. The pooled antigens present in a trivalent vaccine prepared from shed antigens from the HM2, HM4 and HM8 melanoma cell lines (the "Tri" vaccine) were surprisingly found to be sufficiently representative of melanoma antigens to ensure that the Tri vacccine contains one or more antigens present on the tumor of a patient in need of treatment, resulting in a therapeutically effective anti-tumor response. In some aspects, the therapeutic effects of the polyvalent vaccine include extending the period of recurrence free survival in post- resection melanoma patients. In some aspects, the methods of treating post resection melanoma patients by administering a therapeutically effective amount of a Tri vaccine comprising equal portions (w/w/) of multiple partially purified soluble shed melanoma antigens from the HM2, HM4 and HM8 cell lines extends recurrence-free survival by at least 20 months compared to patients receiving no treatment, treatment with a null vaccine, or standard of care. In some embodiments, the Tri vaccine extends median RFS by at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or by at least about 50 or more months, or by any time range between any two of the extension times set forth above.

[0007] Another aspect of the invention features a method of treating melanoma, comprising the administration of a polyvalent melanoma vaccine in combination with an agent that boosts the immune response to the vaccine. In some aspects, the agent targets one or more immune system checkpoints. Immune system checkpoints are inhibitory signaling pathways by which checkpoint proteins can turn off immune system effectors cells, for example, T cells. Because checkpoint proteins can dampen the immune response to the vaccine, inhibiting one or more checkpoint proteins will increase the immune response to the vaccine, thereby increasing its therapeutic effect. Some embodiments of the invention can include checkpoint inhibiting agents (also called immune-checkpoint inhibitors or checkpoint inhibitors) that target checkpoint proteins. The checkpoint inhibiting agents include, but are not limited to, indoleamine (2, 3)-dioxygenase (IDO); programmed cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death-1 (PD-1); programmed death-ligand 1 (PD-L1); PD- L2; lymphocyte activation gene 3 (LAG3); and B7 homolog 3 (B7-H3).

[0008] In another aspect, an immunopotentiator that stimulates the immune response may be used in combination with the vaccine. A variety of adjuvants and immunopotentiators are known by those having ordinary skill in the art. Such immunopotentiators may be immunosuppressors or immunostimulants. In some embodiments, the immune response may be decreased by the immunopotentiator. In some embodiments, the immune response may be increased by the immunopotentiator or adjuvant.

[0009] In another aspect, the invention features a pharmaceutical composition comprising an adjuvant or an additional substance to further enhance the immune system's response to the tumor-associated antigens or other vaccine ingredients. Useful adjuvants include aluminum hydroxide, aluminum phosphate and potassium aluminum sulfate (also called 'alum'). [0010] Thus, in some aspects, the present invention features methods of administering a polyvalent melanoma vaccine to a subject in combination with one or more active pharmaceutical agents, including one or more checkpoint inhibitors, adjuvants or

immunopotentiators. In some aspects treatment with a polyvalent melanoma vaccine in combination with one or more checkpoint inhibitors, adjuvants or immunopotentiators extends RFS by more than 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, or by more than at least about 100 months.

[0011] In another aspect, the checkpoint inhibitors, adjuvants and/or

immunopotentiators administered in combination with the vaccine may be coadministered with the vaccine, or administered separately from the vaccine according to the same or a different dosage regimen.

[0012] In some aspects, the polyvalent melanoma vaccine stimulates an anti- melanoma antibody and peptide-specific CD8+ T-cell immune responses. For instance, one aspect of the invention features a method of modulating (e.g., increasing or decreasing) the effector T cell response level and subsets in vitro or in vivo comprising contacting a cell in vitro, or administering to a patient a therapeutically effective amount of a polyvalent melanoma cancer vaccine comprising multiple partially purified soluble shed melanoma antigens from at least three human melanoma cell lines, and a therapeutically effective amount of a checkpoint inhibitor; and, modulating effector T cell response levels and subsets thereof, as compared to a baseline control. Some aspects of the invention further features a method of increasing the number (or value) of the effector T cell response level and subsets thereof, as compared to a reference level.

[0013] In another aspect, the invention features methods of treating a patient who is suffering from melanoma or was previously diagnosed with melanoma. The method can include the step of identifying a patient in need of treatment (e.g., a patient in which the cancer was resected or a patient that is considered at risk for melanoma or the recurrence of melanoma; or a patient who has a primary or secondary tumor), and includes the step of administering to the patient a therapeutically effective amount of a pharmaceutical composition that includes a polyvalent melanoma cancer vaccine in combination with a checkpoint inhibitor as described herein. The patient's melanoma can be associated with other types of cancer (e.g., breast cancer; colon cancer; several types of 'lymph' cancers often referred to as non-Hodgkins lymphoma; lung cancer; and/or prostate cancer). In some instances identifying a patient at risk for melanoma may include determining the presence of genetic markers associated with an increased risk of melanoma or the presence of melanoma.

[0014] The present invention also features methods of producing the soluble polyvalent melanoma cancer vaccine comprising multiple partially purified soluble melanoma antigens released or shed from their external surface from melanoma cells as described herein; methods of producing pharmaceutical compositions that include them; and the use of the polyvalent vaccine and compositions in treating melanoma and/or reducing or preventing the recurrence of melanoma.

[0015] In another aspect, the invention features pharmaceutical compositions comprising multiple partially purified soluble shed melanoma antigens comprising at least three human melanoma cell lines, a checkpoint inhibitor, and one or more pharmaceutically acceptable diluents and/or excipients, and an adjuvant. These compositions can be formulated for intradermal, intramuscular, parenteral, intranasal, or other modes of administration.

[0016] Another aspect of the invention features pharmaceutical compositions comprising partially purified soluble tumor-associated antigens shed from human melanoma cells: HM2, HM4 and HM8. In some aspects, the polyvalent Tri vaccine contains a mixture of at least 300 proteins as determined by 2D-SDS-PAGE. In some aspects, the polyvalent vaccine comprises shed melanoma antigens having a pi ranging between 3.5 to 9 with the majority of the proteins having a pi between 5 and 7. The isoelectric point (pi) value is a standard measure for distinguishing between proteins.

[0017] Alternatively, the pharmaceutical compositions can be produced using tumor- associated antigens shed from HM2, HM4 and HM8 combined with tumor-associated antigens shed from additional human or animal cancer cell lines.

[0018] In some aspects, the vaccine and/or shed antigens before or after pooling are stored at between 2 to 8 °C. In some aspects, the vaccine and/or shed antigens before or after pooling may be stored in a controlled rate freezer at -70 to -80 °C for 12 hours or less.

Storage at between 2 to 8 °C or for no more than 12 hours at a time in a controlled rate freezer at -80 °C maintains the conformational and/or biochemical integrity of the shed antigens, and promotes a clinically relevant immune response. In some aspects, the polyvalent compositions and polyvalent vaccines of the invention disclosed herein may have one or more of the following advantages: freeze-thaw stability, degree of sterility, little or no precipitation at dosing concentrations and high solubility (producing few if any aggregates in solution). In addition, the antigens shed from human melanoma cell lines can lead to protective immunity across a patient population, despite the heterogeneity of the antigens and HLA polymorphisms present in a patient population with melanoma. Further, the polyvalent compositions administered along with one or more checkpoint inhibitor can stimulate or activate responses to multiple targets present on the patient's melanoma cells.

[0019] The pharmaceutical composition of the present invention can include a step of identifying a patient or subject in need, as disclosed herein, and administering the pharmaceutical composition to reduce the likelihood or to prevent the recurrence of melanoma. In one embodiment, the invention features a method of treating a patient with resected melanoma, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition as described herewith.

[0020] In some aspects, the course of administration can be controlled. In some aspects, the course of administration can be administering a therapeutically effective amount of a polyvalent melanoma cancer vaccine in doses at treatment time periods of once every 3 weeks times 4 injections, then every month times 3, every 3 months times 2, and then every 6 months up to 2 years.

[0021] In some aspects, the polyvalent vaccine administration can modulate effector T cell response levels and subsets in vitro or in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows the baseline disease characteristics and demographic of the patients with resected stage II-IV cutaneous melanoma receiving one of two formulations of a polyvalent, shed-antigen vaccine as adjuvant therapy in a randomized Phase 2 study.

[0023] Figure 2 shows the Kaplan-Meier estimate of recurrence-free survival by treatment. The top data line represents the trivalent-vaccine treatment group, while the middle and bottom data lines represent the quadravalent- and -null vaccine treatment groups, respectively. [0024] Figure 3 reports the recurrence-free survival results in patients receiving the trivalent vaccine, including those patients that did and did not have a delay ed-type hypersensitivity response, and compares these groups to the patients receiving the null vaccine.

[0025] Figure 4 is the TRP-2 Western Blot of the Tri Vaccine.

DETAILED DESCRIPTION

[0026] An alternative approach to the administration of monoclonal antibody therapy for the treatment of melanoma is the use of a polyvalent melanoma cancer vaccine.

Accordingly, in some embodiments, featured herein is a polyvalent melanoma cancer vaccine comprising multiple melanoma antigens shed from three or more human melanoma cells lines, where the cell lines comprise, for example, HM2, HM4, and HM8. The polyvalent vaccine is not patient-specific in that it does not require the patient's own tumor to make the vaccine, and can be used to treat all patients with melanoma or at risk for melanoma regardless of their genetic make-up or tumor type. In some embodiments the vaccine is a trivalent vaccine comprising multiple melanoma antigens shed from HM2, HM4, and HM8 cell lines. In some embodiments, the vaccine stimulates an anti-melanoma antibody and peptide-specific CD8+ T-cell immune response without causing substantial toxicity.

[0027] The vaccines and compositions of this invention are useful for treating melanoma and/or for preventing, inhibiting or treating recurrence in post-resection melanoma patients. In some embodiments the invention features methods of extending recurrence-free survival time of a post-resection melanoma patient, comprising administering a

therapeutically effective amount of a polyvalent melanoma cancer vaccine comprising equal portions (w/w/) of multiple partially purified soluble shed melanoma antigens from three human melanoma cell lines. In some embodiments, the trivalent melanoma cancer vaccine comprises shed antigens from HM2, HM4 and HM8 human melanoma cell lines ('Tri" vaccine).

[0028] The cell lines are used to produce tumor-associated antigens for use in a melanoma cancer vaccine. The inclusion of antigens from three unique melanoma cell lines (HM2, HM4, and HM8) in the Tri vaccine was surprisingly found to increase recurrence free survival compared to a null vaccine or a Quad vaccine. In some embodiments, the trivalent vaccine extends median recurrence free survival ("RFS" or "average RFS") by at least 20 months compared to treatment with a null vaccine, no treatment, or treatment with standard of care. In some embodiments, the trivalent vaccine extends median RFS by at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or by at least about 50 or more months, or by any time range between any two of the extension times set forth above. In some embodiments, the Tri vaccine extends median RFS by at least about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more months compared to treatment with the Quad vaccine.

[0029] Thus, it was surprisingly found that the shed antigens from HM2, HM4 and HM8 alone provided a sufficient pool of antigens to ensure that the vaccine contains one or more antigens present on the tumor of a patient in need of treatment, resulting in a therapeutically effective anti -tumor response which does not depend on the patient's genetic make-up, type of melanoma, or somatic mutations in the melanoma.

[0030] Also featured herein are methods of treating melanoma by administering to a patient in need thereof a polyvalent melanoma cancer vaccine in combination with one or more active pharmaceutical agents, including one or more checkpoint inhibitors, adjuvants or immunopotentiators.

[0031] Accordingly, in some aspects, the invention features a method of treating the recurrence of melanoma in a patient in need by administering a therapeutically effective amount of a polyvalent melanoma cancer vaccine. In one aspect, the method comprises administering a therapeutically effective amount of a soluble polyvalent melanoma cancer vaccine comprising multiple partially purified soluble melanoma antigens released or shed from their external surface from melanoma cells. Three human melanoma cell lines can be used to generate tumor-associated antigens that are useful in preparing polyvalent melanoma vaccines. The antigens may comprise, for example, MAGE-1, MAGE-2, MAGE-3, S100B, HLA-1 (β-2 microglobulin), Melan-A/MART-1, gplOO, tyrosinase, melanocortin receptor (MC1R), and dopachrome tautomerase (TRP-2). Exemplary human melanoma cells lines, include but are not limited to: HM2 (also referred to as SFHM2; previously designated as M20), HM4 (also referred to as SFHM4; previously designated as M14) and HM8 (also referred to as SFHM8; previously designated as SK Mel28 or Mel 28). The tumor-associated antigens are collected and partially purified to produce the melanoma polyvalent vaccine. By using antigens from multiple tumors or cell lines, a "polyvalent" vaccine can be produced. [0032] Another aspect of the invention features a method comprising the

administration of a polyvalent melanoma vaccine along with an agent that boosts the immune response to the vaccine. In some aspects, the agent targets one or more immune system checkpoints. Immune system checkpoints are inhibitory signaling pathways by which checkpoint proteins can turn off immune system effectors cells, for example, T cells.

Because checkpoint proteins can dampen the immune response to the vaccine, inhibiting one or more checkpoint proteins will increase the immune response to the vaccine, thereby increasing its therapeutic effect. Some embodiments of the invention can include checkpoint inhibiting agents (also called immune-checkpoint inhibitors or checkpoint inhibitors) that target checkpoint proteins. The checkpoint inhibiting agents include, but are not limited to, indoleamine (2, 3)-dioxygenase (IDO); programmed cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death- 1 (PD-1); programmed death-ligand 1 (PD-L1); PD-L2; lymphocyte activation gene 3 (LAG3); and B7 homolog 3 (B7-H3).

[0033] In another aspect, an immunopotentiator that stimulates the immune response may be administered in combination with the vaccine. A variety of immunopotentiators are known by those having ordinary skill in the art. Such immunopotentiators may be immunosuppressors or immunostimulants. In some embodiments, the immune response may be decreased by the immunopotentiator. In some embodiments, the immune response may be increased by the immunopotentiator. In some embodiments, the immune response may initially be increased by an immunostimulant, then later decreased by an immunosuppressor.

[0034] In another aspect, the adjuvants and/or immunopotentiators may be coadministered with the vaccine, or administered separately from the vaccine according to the same or a different dosage regimen.

[0035] The term "polyvalent vaccine" refers to a suspension formed from melanoma antigens released or shed from melanoma cells. The melanoma cells can be human, humanized, from animals or a combination thereof. In the present invention, the antigens are released from human melanoma cells (e.g., HM2, HM4, and HM8). In some embodiments, the polyvalent melanoma cancer vaccine can be produced using tumor-associated antigens shed from HM2, HM4 and HM8 combined with tumor-associated antigens shed from additional human or animal cancer cell lines. The polyvalent vaccine can be formulated for administration by any appropriate means, including intradermal, intramuscular, parenteral, oral, buccal, submucosal, intranasal, or intraabdominal administration. [0036] The term "stimulates," or "generates," means eliciting an immune response to an antigen in the vaccine and/or enhancing an immune response already present in the patient. In some instances the vaccine may stimulate an increase in the immune response in the patient to the antigens present in the vaccine and melanoma cells, for example, an increase in antibody levels to an antigen, thereby producing clinically effective amounts of antibodies.

[0037] As used herein, the term "treating" refers to both therapeutic treatment and prophylactic, or preventative treatment, or administering an agent having therapeutic potential or effects, or inhibiting the progression of a colony of cancer cells or a disease. The term includes preventative (e.g., prophylactic and/or preventing or extending the time before onset or recurrence) and palliative treatment. "Treating" also refers to the resectioning of melanoma, at a local site or in a general area. The term also includes its definition as known in the art.

[0038] The term "a pharmaceutically effective amount," or "clinically effective amount" as used herein, means an amount of active compound, or pharmaceutical agent, that elicits the biological, or medicinal, response in a tissue, system, animal, or human that is being sought, which includes alleviation or palliation of the symptoms of the disease being treated and/or an amount sufficient to have utility and provide desired therapeutic endpoint. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; slow recurrence or onset; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can be measured, e.g., by assessing the time to disease progression and/or determining the response rate. Potency is determined by measuring antibody responses following administration using a microtiter based assay, for example, an ELISA assay.

[0039] In some embodiments the polyvalent vaccine has no apparent, or very limited, signs of toxicity over a broad dose range. For example, the polyvalent vaccine may exhibit no apparent, or very limited signs of toxicity at doses of 40 μg or 100 μg human equivalent concentrations, or about 0.57 to 1.43 μg /kg for a 70 kg human) evaluated in the Brown Norway rats. In some aspects, the dose may range from 20 μg to 150 μg, and may be, for example, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 μg, or any range between any two of these doses.

[0040] In some embodiments, the vaccine and/or shed antigens before or after pooling are stored at between 2 to 8 °C. In some aspects, the vaccine and/or shed antigens before or after pooling may be stored at -80 °C for 12 hours or less. Storage at between 2 to 8 °C or for no more than 12 hours at a time at -80 °C maintains the conformational and/or biochemical integrity of the shed antigens, and promotes and/or stimulates a clinically relevant immune response.

[0041] Certain polyvalent vaccines comprising shed antigens from melanoma cells have been reported. Much of the early research has focused on a quadravalent vaccine formulation including three human cells lines, SFHM2, SFHM4 and SFHM8, and one hamster cell line (HM54). Reynolds et al, J. Immunology, 1998, 161 :6970-76 also reported the characterization of patients' CD8+ T cell responses to antigens including MAGE-3, Melan-A/MART-1, gplOO, Tyrosinase, MC1R, and TRP-2 in melanoma patients vaccinated with a melanoma vaccine (SFM14, SFM20 and SFSKMel28) frozen at -70 to -80 °C until used. Reynolds et al. measured the CD8+ T Cell responses in 22 post-resection patients after the fourth vaccination, and reported that 54% of patients who exhibited a CD8+ T cell response remained progression free after one year, whereas only 22% of patients who did not exhibit a CD8+ T cell response remained progression free after one year. Most of the patients received the vaccine encapsulated in liposomes along with interleukin 2 ("IL-2"), a signaling molecule in the immune system, further complicating the clinical outcome results. Other studies that assessed patient survival after immunization with a polyvalent vaccine failed to distinguish between the trivalent and quadravalent vaccine groups (Miller et al., Cancer, 1995; 75:495-502; Bystryn et al, Cancer, 1992; 69: 1157-1164), furthering complicating the interpretation of their findings. Although these studies demonstrated biologic activity in human patients with resected stage 2, stage 3 or stage 4 melanoma, they have not shown significant recurrence free survival ("RSF") after immunization with the trivalent vaccine, either alone or in combination with a checkpoint inhibitor and/or immunopotentiator. Furthermore, variations in the vaccine preparation and storage process may also affect the clinical outcome of the vaccine. [0042] It has been found that treatment of resected melanoma patients with a trivalent vaccine (SFHM2, SFHM4 and SFHM8) stored at 2 to 8 °C (or at -70 to -80 °C in a controlled rate freezer, optionally for less than 12 hours) results in longer recurrence free survival. Median RFS was 36.5 months following treatment with the Tri vaccine, compared to a median RFS of only 13.2 months in patients treated with the Quad vaccine, and only 11.0 months in patients administered a null vaccine in a clinical trial.

[0043] The trivalent vaccine containing shed antigens from only three human melanoma cells (SFHM2, SFHM4 and SFHM8) also reduced potential contamination of the preparation with kenogenic proteins shed from the hamster cell line HM54.

[0044] Also featured herein in some embodiments are methods for producing an antigenically heterogeneous human melanoma antigen vaccine and the method of administering the melanoma vaccine comprising multiple melanoma antigens shed from three human melanoma cell lines (e.g., HM2, HM4 and HM8) to treat patients with melanoma and prevent or reduce the recurrence of melanoma in patients at risk for melanoma or in need thereof. The descriptions provided herein serve to illustrate, but do not limit, the invention.

[0045] In some embodiments, the shed antigens in a polyvalent vaccine may have a pi ranging from 3.5 to 9 with the majority of the proteins having a pi between 5 and 7. In some embodiments, the pH of the vaccine and/or shed antigens may be from 5-8. In some embodiments, the pH of the vaccine and/or shed antigens may be from about 5.4 to 7.2, or from about 5.8 to about 7.0.

[0046] The MAGE-3, Melan- A/MART- 1 , gplOO, Tyrosinase, MC1R, HLA-1, S100B, and TRP-2 proteins profiled in a Tri vaccine were immunogenic and may be responsible for generating or stimulating the clinically effective immune responses induced by administration of the trivalent vaccine prepared from shed antigens from melanoma cultured cells. Immune system cells that may be assayed include, but are not limited to: B lymphocytes (B cells), T lymphocytes (T cells), natural killer (NK) cells, lymphokine- activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhan's cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor-infiltrating (TIL) cells, drug modified immune system cells, antigen presenting cells (APC) and derivatives, precursors or progenitors of the above cell types. [0047] Immune system effector cells refers to cells, and subsets thereof (e.g., Treg, Thl, Th2) that can bind an antigen or lead to a response after contact with APC, thereby stimulating the immune system in such a way that the immune response is selective for the antigen. Examples of immune system effector cells include, but are not limited to: T cells and their subsets (e.g., Treg, Thl, Th2, and Thl7); B cells, APC (e.g., dendritic cells, monocytes, macrophages); myeloid suppressor cells, NK cells, cytotoxic T lymphocytes.

[0048] Effector T cells (also called regulatory T cells, or Treg cells, or suppressor T cells) are immune system cells that can inhibit a T cell response and are important in leading to cell-mediated immunity. Effector T cells can contain cell surface markers including CD25 (the IL-2 receptor), CD45RB, CTLA4 and FOXP3. A T cell response is an immune-type response that mediated through T cells. When T cells are activated, they will divide and generate more T cells (e.g. Thl or Th2 cells, also called CD4+ cells or T helper cells;

memory T cells; or cytotoxic T cells; also referred to as CTLs or CD8+ T cells). Once activated, T helper cells divide and generate cytokines that can regulate or otherwise assist other immune system cells to elicit an immune system response. Memory T cells also generate additional effector T cells upon activation after previous exposure to a known antigen. Cytotoxic T cells kill tumor cells when presented with a target that they recognize.

[0049] The immune system can adapt to a changing environment and thereby lead to pathogen-specific immunity. Innate immunity refers to the natural defense an individual is bom with to protect itself from harmful pathogens. Adaptive immunity can occur through direct exposure to a pathogen including vaccines. Adaptive immunity can be further subdivided into humoral immunity and cell-mediated immunity. Humoral immunity is a type of immune response mediated by antibodies in the serum. Cell-mediated immunity confers protective that is mediated through cells (i.e., through the activation of T cells, phagocytes and cytokines). Adaptive immunization is established during the life of the subject, patient, individual or animal, is specific for activation of a particular antigen and leads to an enhanced immune response upon additional exposure to an antigen. T cells can detect small concentrations of a given pathogen displayed on APCs.

[0050] The immune response can be measured as described herein and according to a variety of methods known by those skilled in the art. Clinical methods that assess the stage or degree of a particular condition or disease can be used to determine whether a particular response is induced. For example, in a melanoma patient, clinical methods can include a physical exam to identify lumps, lymph node mapping, biopsy (e.g., sentinel lymph node, tumor), imaging (e.g., CT (CAT) scan, PET scan, MRI with gadolinium), and blood chemistry studies to measure the levels of lactate dehydrogenase or other blood tests as determined by attending physician.

[0051] The methods to determine if a specific type of response is induced can include comparing a subject's or patient's sample with a standard reference sample or level for a specific marker, cell-type or assay or a pre-existing or baseline sample from the subject or patient receiving treatment as described herein. A standard reference sample or reference sample often indicates the level from a large population of subjects or patients. Further, the reference sample can include subjects of similar age, ethnicity, gender, disease state, height and/or weight as the patient or subject receiving treatment with compositions or vaccines as described herein. For example, the marker level (e.g., CD8+ T cell response; also referred to as response level) can be compared to values or levels from subjects or patients 1) who have not received any treatment for melanoma and/or a null treatment such as a null vaccine, including patients diagnosed for melanoma, healthy subjects, and those who are at risk for melanoma; 2) who have had resected melanoma; 3) who have received some type of cancer treatment, for example, standard of care for treating melanoma; 4) who are any stage of melanoma cancer; 5) who have received the treatment described herewith; and/or the subject's or patient's own pre-existing or baseline control sample that can include any measurements taken before starting treatment with the trivalent vaccine, or polyvalent vaccines in combination with one or more checkpoint inhibitors, adjuvants and/or immunopotentiators, and then again at any time during treatment and at the conclusion of such treatment or to any other type of treatment. Any population size between 2 and 250 or more subjects or individuals can be used to determine the average reference levels.

[0052] While the disposition of the antigens in the polyvalent vaccine from the site of injection has not been determined, the following non-limiting theory is considered the most likely mechanism for clearing the polyvalent vaccine antigens from the body. First, the bulk of the antigens in the polyvalent vaccine that reach systemic circulation from the site of injection will most likely be conveyed to regional nodes where these antigens will be ingested by antigen-presenting cells, macrophages, and other phagocytic cells. Prior to this clearing mechanism, some antigens in the polyvalent vaccine, or their peptides, are expected so to interact with class I HLA antigen receptors on the external surface of antigen-presenting cells and thus are expected to stimulate CD8+ T cell responses (one of the mechanisms of pharmacological activity possible for the Tri vaccine). Second, some of the polyvalent vaccine administered with adjuvant is expected to precipitate at the site of injection and this material is projected to be involved in the generation of anti- Tri vaccine antibodies that will bind with the antigens expressed on the melanoma cells, resulting in cell death (a second proposed mechanism of pharmacological activity for the polyvalent vaccine). This precipitated material will have limited solubility and thus the material will most likely be cleared from the site of injection by macrophages and other phagocytic cells. It is believed that the polyvalent vaccine could also enhance an immune response already present in the patient with resected melanoma, and that the introduction of the polyvalent vaccine increases the immune response in the patient to the antigens present in the vaccine and melanoma cells.

[0053] Checkpoint inhibiting agents are agents that target checkpoint proteins or a derivative thereof, and may be referred to as "checkpoint inhibitors." Checkpoint inhibitors can be, for example, proteins, polypeptides, amino acid residues, and monoclonal or polyclonal antibodies. The polyvalent vaccine can include or be administered along with one or more checkpoint inhibitor. The checkpoint inhibitors can bind, for example, to ligands or proteins that are found on any of the family of T cell regulators, such CD28/CTLA-4.

Targets of checkpoint inhibitors include, but are not limited to, receptors or co-receptors (e.g., CTLA-4; CD8) expressed on immune system effector or regulator cells (e.g., T cells);

proteins expressed on the surface of antigen-presenting cells (e.g., expressed on the surface of activated T cells, including PD-1, PD-2, PD-L1 PD-L2, 4-1BB, and OX40); metabolic enzymes or metabolic enzymes that are expressed by both tumor and tumor-infiltrating cells (e.g., indoleamine (IDO), including isoforms, such as IDOl and ID02); proteins that belong to the immunoglobulin superfamily (e.g., lymphocyte-activation gene 3, also known as LAG3); proteins that belong to the B7 superfamily (e.g., B7-H3 or homologs thereof;). B7 proteins can be found on both activated antigen presenting cells and T cells. In some embodiments, two or more checkpoint inhibitors can be combined or paired together. For example, a B7 family checkpoint inhibitor, found on an antigen presenting cell, can be paired with a CD28 or CTLA-4 inhibitor, expressed on surface of a T cell, to produce a co- inhibitory signal to decrease the activity between these two types of cells. A co-receptor refers to the presence of two different receptors located on the same cell that after binding to an external ligand can regulate internal cellular processes. Co-receptors can be stimulatory or inhibitory. Co-receptors are sometimes called accessory receptors or co-signally receptors. The term "co-inhibitory," in large part, is context dependent, but generally, is the result of more than one molecule binding to its respective receptor on the surface of a cell thereby slowing down or preventing an intracellular process from occurring.

[0054] Pharmaceutical formulations, doses, and administration: Pharmaceutical compositions for use in accordance with the present invention may be formulated using one or more physiologically acceptable carriers or excipients. Any suitable concentration of the polyvalent vaccine or polyvalent composition may be used, and any active pharmaceutical ingredient will be administered in an amount effective to achieve its intended purpose, a "therapeutically effective amount." A therapeutically effective amount of polyvalent vaccine or composition and/or a second active ingredient or agent may be determined in view of the disclosure herein by those of ordinary skill in the art. In some embodiments, the dose may range from 20 μg to 150 μg, and may be, for example, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 μg, or any range between any two of these doses.

[0055] The specific, therapeutically effective dose level for any particular patient will depend upon a variety of factors including the activity of any other specific compounds or agents administered; the specific composition administered; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, the duration of the treatment; drugs used in combination or coincidentally with the polyvalent vaccine and checkpoint inhibitor; and other factors arrived at by those in the medical arts.

[0056] The therapeutically effective dose of the polyvalent vaccine or compositions can be administered using any medically acceptable mode of administration. The polyvalent vaccine or composition can be administered with a second agent in a single dosage form or otherwise administered in combination (e.g., by sequential administration through the same or a different route of administration and according to the same or a different dosage regimen). Suitable formulations will depend on the method of administration. The pharmaceutical composition is preferably administered by intradermal administration, but other routes of administration include for example oral, buccal, sublingual, rectal, parenteral, intramuscular, subcutaneous, intraperitoneal, transdermal, intrathecal, nasal, intracheal. The polyvalent vaccine can also be administered to the lymph nodes such as axillary, inguinal or cervial lymph nodes. The active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation. [0057] The polyvalent vaccine or composition can be administered at a single site on the patient or a plurality of sites on the patient. In some embodiments, the vaccine may be administered, for example, intradermally or intramuscularly into all four extremities. In some aspects, the course of administration can be controlled. In some aspects, the course of administration can be administering a therapeutically effective amount of a polyvalent melanoma cancer vaccine in doses at treatment time periods of once every 3 weeks times 4 injections, then every month times 3, every 3 months times 2, and then every 6 months up to 2 years.

[0058] Therapeutic agents, as non-limiting examples such as agents that target immune system checkpoints, can be incorporated into a variety of formulations for therapeutic administration by combination with appropriate pharmaceutically acceptable carriers or diluents. Such therapeutic agents may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. A variety of suspending fluids or carriers known to one of ordinary skill in the art may be employed to suspend the polyvalent vaccine composition. Such fluids include without limitation: sterile water, saline, buffer, or complex fluids derived from growth medium or other biological fluids. Preservatives, stabilizers and antibiotics known to one of ordinary skill in the art may be employed in the polyvalent vaccine composition.

[0059] Methods of making a pharmaceutical composition include admixing at least one active compound or agent, as defined above, together with one or more other pharmaceutically acceptable ingredients, such as carriers, diluents, excipients, and the like. When formulated as discrete units, such as tablets or capsule or suspension, each unit contains a predetermined amount of the active compound or agent. The liquid compositions of one aspect of the invention may be stored in a cooled state for several months prior to use. In some embodiments liquid compositions comprising the vaccine or shed antigens, either before or after pooling, are stored at 2-8 °C for several months prior to use. In some embodiments, liquid compositions comprising the vaccine or shed antigens, either before or after pooling, are stored at -70 to -80 °C in a controlled rate freezer. In some embodiments, storage at -70 to -80 °C in a controlled rate freezer is for no longer than 12 hours. Storage at -70 to -80 °C in a non-controlled rate freezer may degrade or denature the protein antigens during the cooling and/or thawing process which would reduce the clinical efficacy of the vaccine.

[0060] Pharmaceutical compositions described herein may be administered directly. The compositions may also be formulated to include at least one pharmaceutically - acceptable, nontoxic carriers of diluents, adjuvants, immunopotentiators, checkpoint protein inhibitors, or non-toxic, nontherapeutic, fillers, buffers, preservatives, lubricants, solubilizers, surfactants, wetting agents, masking agents, coloring agents, flavoring agents, and sweetening agents. The formulations may also include other active agents, for example, other therapeutic or prophylactic agents, nonimmunogenic stabilizers, excipients and the like. Several non-limiting examples of nonimmunogenic stabilizers are thiomersal (ethyl(2- mercaptobenzoato-(2-)-0,S)mercurate (1-) sodium), Phenol, Phemerol (benzethonium chloride), and 2-phenoxyethanol. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.

[0061] A variety of adjuvants are known in the medicinal arts. Such adjuvants include, but are not limited to: polymers, copolymers such as polyoxyethylene- polyoxypropylene co-polymers, including block co-polymers; polymer PI 005; monotide ISA72; Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300 adjuvant; alum; Detox, QS 21, muramyl dipeptide; trehalose; bacterial extracts, including

mycobacterial extracts; detoxified endotoxins; membrane lipids, lipopolysaccharides; water- in-oil mixtures, water-in-oil-in-water mixtures, sodium phosphate, poly-ICLC or

combinations thereof.

[0062] A variety of immunopotentiators are known by those having ordinary skill in the art. Such immunopotentiators may be immunosuppressors or immunostimulants. Such immunostimulants include, but are not limited to: CpG motif olignucleotide sequences, T-cell costimulatory molecules such as B7-1, ICAM-1, and LFA-3; cyclophosphamide, Flt31, M1P- la chemokine, IL-2 cytokine, IL-12 cytokine, IL-15 cytokine, TGF cytokine, granulocyte- macrophase colony stimulating factor (GM-CSF) cytokine, antitumor Mab-IL-2, and

Myobacterium bovis Bacille Calmette-Guerin (BCG) vector, and combinations thereof.

[0063] The adjuvants and/or immunopotentiators may be coadministered with the vaccine, or administered separately from the vaccine according to the same or a different administration regimen. The coadministration of the vaccine with the immunopotentiators and/or adjuvants may occur simultaneously or sequentially. In some embodiments, an immunostimulant can be administered in conjunction with the vaccine administration, and an immunosuppressor may be administered following the elicited immune response, for example, to treat delayed hypersensitivity.

[0064] The formulations can be in unit-dose or multi-dose containers. Non-limiting examples of such containers include sealed ampules and vials. The polyvalent vaccine can be stored at 2° to 8° C (refrigerated) until use.

[0065] The polyvalent vaccine of the present invention may be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes. The polyvalent vaccine can be administered from about 1 to 15 dosages per immunization regimen. Each dose may comprise one or a plurality of administrations. As a non-limiting example, one dose of the vaccine may comprise from 1 to 4 injections of the vaccine at the same or different injection sites on the patient. The polyvalent vaccine dose per administration (e.g., injection) can range from about less than 1 ng to 1 gram based on total antigens shed from three human melanoma cell lines (e.g., HM2, HM4 and HM8). In some embodiments, the dose may range from 10 ng to 1 mg, from 100 ng to 1 mg, from 1 μg to 1 mg, from 20 μg to 150 μg, or may be, for example, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 μg, or any range between any two of these doses. The volume of administration may vary depending on the administration route. A non-limiting range for the volume of the dose may be 0.01 ml to 100 ml. The effective dose of the polyvalent (i.e., the effective concentration and frequency of administration) can depend on the specific type and concentration of the checkpoint inhibitor, the severity of the condition or cancer, the age of the patient and the overall general state of the patient's health. The timing between doses can also be controlled. In one aspect of the invention, the vaccine will be administered every 2 weeks for five intervals, then monthly for four intervals, and then every 3 months through Month 24, for a maximum of 15 injection days per patient. In some aspects of the invention, the routes of administration may comprise inhalation of a lyophilized vaccine.

[0066] Combination therapies: The methods of the present invention can be carried out in combination with other treatments (e.g., chemotherapy, radiation) and the polyvalent vaccine comprising multiple shed antigens from three human melanoma cell lines described herein can be administered together with other active pharmaceutical agents, such as one or more checkpoint inhibitors. For ease of administration, the polyvalent vaccine and a second pharmaceutical agent can be combined in the same dosage form. Checkpoint inhibitors useful in the present invention include but are not limited to are Ipilimumab (Yervoy);

Tremelimumab (formerly ticilimumab); Pidilizumab (CT-01 1); Nivolumab (BMS-936558); Lambrolizumab (MK-3475); Pembrolizumab; MPDL3280A; BMS-936559; BMS-663513; AMP-224; IMP321 (ImmuFact); MGA271 ; Indoximod; and INCB024360. Other checkpoint inhibitors are those which are antibodies to 4- IBB and OX40. Combinations of the above may also be used in the course of the administration of the polyvalent vaccine.

[0067] In some embodiments, the combination of a polyvalent vaccine with a checkpoint inhibitor further extends the RFS. While the use of checkpoint inhibitors alone can result in an increase in RFS over placebo, and, as disclosed herein, the Tri vaccine surprisingly extends RFS over Quad vaccine or a Null vaccine, the use of both a checkpoint inhibitor and Tri vaccine can result in an RFS longer than that observed in either treatment with the checkpoint inhibitor alone or the Tri vaccine alone due to synergistic effects of the two therapies acting by different mechanisms and thus working in concert to treat the melanoma. As described above, checkpoint inhibitors are primarily antibodies to T cells presenting checkpoint modulators. As described above, polyvalent vaccines are comprised of multiple antigens present in melanoma cells which will induce multiple antibodies in the patient against the antigens. By presenting both a checkpoint inhibitor and a polyvalent vaccine, a variety of antibodies will be present to treat the resected melanoma. It should be noted that because both of these mechanisms operate via the immune response, the further addition of immunopotentiators can further increase the immune response effect, thereby further increasing the RFS in patients treated with the combination of checkpoint inhibitor, polyvalent vaccine, and immunopotentiator over patients treated with placebo. Practitioners in the art will appreciate that immunopotentiators can induce a clinically effective response. In some embodiments, the immune response may be decreased, for example, to reduce autoimmune responses from the presentation of the Tri vaccine. A suitable therapeutically effective dose of a checkpoint inhibitor or immunopotentiator may be from about 0.001 to about 1 mg/kg body weight such as about 0.01 to about 1 mg/kg, 0.01 to about 0.5 mg/kg, or 0.1 to about 1 mg/kg body weight. A suitable dose may however be from about 0.001 to about 0.1 mg/kg body weight such as about 0.01 to about 0.050 mg/kg body weight. [0068] In some embodiments, the RFS may be extended in patients treated with the Tri vaccine with additional characteristics compared to patients treated with the Tri vaccine which did not possess the additional characteristics. In some embodiments, the RFS difference may be 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more months for those patients treated with the Tri vaccine with additional characteristics compared to those patients treated with the Tri vaccine which did not possess the additional characteristics. In some embodiments, the additional characteristics of the Tri vaccine are one or more of the following selected characteristics: the addition of one or a plurality of immunopotentiator types, the addition of one or a plurality of checkpoint inhibitor types, and the storage condition of 2-8 C° before dosings.

[0069] Conditions amenable to evaluation and treatment: As noted above, the present invention involves an immunotherapeutic approach for the prevention and/or treatment of cancer. The administration to a patient or subject in need of a polyvalent vaccine along with a checkpoint inhibitor in accordance with this invention for the prevention and/or treatment of cancer can take place before or after a surgical procedure to remove the cancer, before or after a chemotherapeutic procedure for the treatment of cancer, and before or after radiation therapy for the treatment of cancer and/or any combination thereof. The cancer immunotherapy in accordance with this invention would be a treatment for the prevention and/or for the treatment of cancer, particularly since the risk and side effects involved are substantially minimal compared with the other treatments (e.g., surgery, chemotherapy and radiation therapy). A unique aspect of the present invention is that the polyvalent vaccine in combination with a checkpoint inhibitor described herewith can prevent or reduce the recurrence of cancer in a subject without cancer but which is at risk of developing cancer and improve recurrence free survival. The pharmaceutical compositions of some aspects of the invention can be administered to the subject (patient) in an amount sufficient to delay, reduce, or prevent the onset of melanoma or the onset of any other cancer or clinical disease.

[0070] Conditions amenable to evaluation and, if merited, treatment, with a polyvalent vaccine along with a checkpoint inhibitor include, without limitation, and which may increase the risk of subject or patient of being diagnosed with melanoma, excessive or too much exposure to the sun's ultraviolet rays, including subjects that have or have had blistering sunburns at any time of life; getting intense sun exposure, even from time to time; fair skin that does not tan and tends to sunburn or freckle, along with having blue or green eyes or red or blonde hair; numerous moles and/or more than one atypical mole; a large mole since birth; a personal or family history of melanoma; Xeroderma pigmentosum; and surgical excision of melanoma, including without limitation, melanoma of the sole of the foot or palm of the hand or nail bed. Additional conditions for evaluation are patients with melanoma who have a weakened immune system, such as subjects who have had an organ transplant or who are infected with or a carrier of HIV. In some therapeutic applications, the compositions or vaccines along with a checkpoint inhibitor are administered to a subject or patient currently suffering from melanoma or other cancer in an amount sufficient to at least partially improve a sign or symptom or to inhibit the progression or arrest the symptoms of the condition, disease, its complications and consequences. According to the invention, the compositions or vaccines described herein can be administered to a subject or patient in need during any stage (e.g., 0, 1, 2, 3, 4 or 5) or any sub-stage (e.g., 1A, IB, 2A, 2B, 2C, 3A, 3B, 3C, 3D, etc.) of melanoma.

[0071] The administration of a polyvalent vaccine prepared in accordance with this invention, in combination with a checkpoint inhibitor, is generally applicable to the prevention or treatment or reducing the recurrence of cancer. Cancers suitably treated in accordance with the practices of this invention include cancers of the lung, breast, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone liver, esophagus, brain, testicle, uterus and the various leukemias and lymphomas.

[0072] The present methods can include a step of identifying a patient in need of treatment as described herein.

[0073] The polyvalent compositions and checkpoint inhibitors provided herein can be administered in conjunction with other therapeutic modalities to a subject or patient in need of therapy. The present polyvalent composition, and optional checkpoint inhibitors, adjuvants and immunopotentiators can be given prior to, simultaneously with or after treatment with other agents or regimes. For example, the polyvalent vaccine and checkpoint inhibitor, adjuvant and/or immunopotentiator can be give along with or following standard chemotherapy.

[0074] The polyvalent vaccine administration can extend recurrence-free survival time of a post-resection melanoma patient by at least 34 months by administering a therapeutically effective amount of a polyvalent melanoma cancer vaccine comprising equal portions (w/w/) of multiple partially purified soluble shed melanoma antigens from SFHM2, SFHM4 and SFHM8 human melanoma cell lines.

[0075] The polyvalent vaccine administration can modulate effector T cell response levels and subsets in vitro or in vivo, by a method comprising: contacting a cell in vitro, or administering to a patient a therapeutically effective amount of a polyvalent melanoma cancer vaccine comprising multiple partially purified soluble shed melanoma antigens from at least human melanoma cell lines, and a therapeutically effective amount of a checkpoint inhibitor adjuvant and/or immunopotentiator; and, modulating effector T cell response levels and subsets thereof, as compared to a baseline control.

[0076] Kits. The kits of some aspects of the invention can include any combination of the compositions and/or vaccines disclosed above and suitable instructions (e.g., written, audiovisual). In one embodiment, the kit includes a pharmaceutical composition or vaccine that is packaged along with instructions for use and any instrument useful in administering the compositions. For example, the kits of some aspects of the invention can include one or more of: diluents, gloves, vials or other containers, pipettes, needles, syringes, tubing, sterile cloths or drapes, positive and/or negative controls, and the like. In some embodiments the kit may also comprise a checkpoint inhibitor, adjuvant and/or immunopotentiator packaged in the same or a separate vessel from the vaccine. In embodiments where the vaccine is packaged in the same vessel as a checkpoint inhibitor, adjuvant and/or immunopotentiator, there may be a removable barrier between the vaccine and the checkpoint inhibitor, adjuvant and/or immunopotentiator

EXAMPLES

Example 1. Vaccine preparation and analysis.

[0077] Vaccine preparation: Vaccines made from whole tumor cells or their lysate and vaccines made from tumor DNA, RNA, or genetically modified tumor cells contain numerous antigens. However, the bulk of the material in these vaccines consists of nuclear and cytoplasmic material (whole cell material) that is irrelevant, dilutes the concentration of relevant antigens, and/or may contain material that is detrimental or interferes with the action of the vaccine, thereby increasing its toxicity. One embodiment of the claimed invention comprises a vaccine which was partially purified, being enriched in soluble shed antigens expressed on the external surface of melanoma cells, to exclude the bulk of unrelated cellular material that is present in the cytoplasm or nucleus of cells.

[0078] The Tri human melanoma antigen vaccine was prepared from material shed by three lines of human melanoma cells: HM2, HM4 and HM8 (all were obtained from ATCC, Manassas, Virginia, and grown at Goodwin Biotechnology, Inc.). The Quad vaccine contained pooled antigens from the same 3 human cell lines plus a hamster melanoma cell line (HM54). The melanoma cells were selected because they rapidly release or "shed" numerous antigens (e.g., gplOO, MART-1, MAGE-1, MAGE-2, MAGE3, MAGE- A3, S100B, MC1R, HLA-1 and TRP-2). Shed antigens were used because they were partially purified. The shed antigens were separated from the bulk of cytoplasmic components, which were slowly released ( <5%) during the short collection period; surface components, however, were rapidly released. In addition, the shed material was enriched in surface antigens which were more likely to be relevant for immunotherapy and could be repeatedly harvested from the same cells, thus reducing culture requirements. Cells were obtained from metastatic nodules of patients presumed to have stage IV melanoma. No information was available about the identity of the donors. The cells were adapted to grow and were maintained in a serum-free medium for at least eight weeks prior to use. The composition and concentration of the cell growth medium are as follows: RPMI-1640 (with glutamine and HEPES), L-glutamine (200 mM), human recombinant insulin (4 mg/mL) and human transferrin (0.5 mg/mL).

[0079] Melanoma cells were incubated at a concentration of 2 x 10⁶/ml serum-free cell growth medium as described above. After 3 hours at 37° C, the medium was collected and the cells were removed by centrifugation at 2,000 g for 10 minutes, and larger particles were removed by recentrifugation at 12,000 g for 15 minutes. Each of the cell lines was processed as a separate batch to create shed antigen pools followed by further downstream processes that included, but were not limited to a series of steps, comprising concentration and diafiltration, NP-40 treatment, and ultracentrifugation. Equal volumes of the shed material was collected, concentrated and diafiltrated, pooled, treated with a non-ionic surfactant, 0.5% P-40 to break up aggregates and then ultracentrifuged at 100,000 x g for 90 min to deplete alloantigens. Equal volumes of medium from the three cell lines were pooled, concentrated 10-fold by vacuum ultrafiltration, bound to alum as an adjuvant and made up to a final concentration and stored in sterile glass vials. [0080] The shed antigen pools were created and further processed as follows.

Supernatant pools from each cell line were thawed and each pool was concentrated to about 10X. Equal quantities of each of the three cell line shed released harvest, as measured by ELISA, and were then pooled to create a mixed antigen pool. The pool was further sampled. The combined pool was then concentrated using flow filtration with a 10 kDa molecular weight cut-off.

[0081] The concentrated protein mixture solution was diafiltered against 12 volumes of 0.9% saline solution using continuous TFF at around 4°C to remove the salts and amino acids from the medium and other low molecular weight materials from the shed antigens collection. The material was then concentrated to approximately 10 L and collected. The system was flushed with two hold-up volumes of 0.9% saline, collected, and added to the retentate. The concentrated material was diluted to approximately 1 mg/mL with 0.9% saline. The material was then process using routine bioassays (e.g., ELISA, SDS-PAGE).

[0082] Next, NP-40 was added to the concentrated material to yield a final NP-40 concentration of 0.5%. The detergent treatment step served to inactivate enveloped viruses and to solubilize protein aggregates.

[0083] The solubilized material was ultracentrifuged at 100,000 g. The supernatant was collected and total protein in the supernatant was then determined. The concentration was adjusted by dilution to approximately 1 mg/mL aluminum in aluminum hydroxide suspensions in 4 mM phosphate-buffered saline and 0.9% NaCl. The product was packaged in sterile, pyrogen free vials and stored at 2° to 8°C (refrigerated) until use. The vaccine was used as is and required no reconstitution after it was warmed to room temperature.

[0084] Protein profile of vaccine: The polyvalent compositions of one embodiment of the present invention were a mixture of soluble proteins (e.g., the protein mixture was solubilized by 0.5% NP-40 in 0.9% NaCl). The mixtures contained at least 300 proteins as determined by 2D-SDS-PAGE with only a few proteins over 2%. The molecular weight of the proteins ranged from 6 to 200 kDaltons with the majority of proteins between 10 and 100 kDaltons. The pi of the proteins ranged from 3.5 to 9 with the majority of the proteins between 5 and 7. The identity and structures of most of the proteins (except S100B, HLA-I, and TRP-2) were not known. The pH was between 5 and 8.

Example 2. S100B and TP-2 antigens. [0085] The Tri vaccine was prepared as described in Example 1.

[0086] The S100B antigen concentration was measured by ELISA assay using methods known to those skilled in the art. Dilutions of S-100B standards, which vary across kits but generally range from 4000 to 50 pg/mL, were used for the standard curve. A sample from the Tri vaccine and one of each cell line lysate lot (HM2, HM4 & HM8) were analyzed at one dilution each, both unspiked and spiked with 3 different dilutions of S-100B standard. It was found that the vaccine solution contained 0.3 ug/mg S-100B.

[0087] The TP-2 antigen concentration was measured by the intensity of the Western blot corresponding to the TP-2 protein. Tyrosinase-related protein 2 (TRP-2) is a highly immunogenic tumor-associated antigen associated with glioma cells and is also highly expressed in melanoma cells. It is a membrane-bound DOPAchrome (3,4-dihydroxy- phenylalanine) tautomerase catalyzing a distal step in the melanin synthesis pathway. It most likely exists as a high molecular weight multimer (200-700 kDa) in its native form. The molecular weight of the monomer is around 60-75 kDa. A 150 ug sample from both the Tri vaccine was separately analyzed with the TRP-2 standard, molecular weight standards, and other control cell lysates. The analytes were loaded into a 12-well pre-cast 4-12% Bis-Tris gel. SDS-PAGE electrophoresis was carried out at 200 volts for approximately 38 minutes. The gel was removed from the apparatus and protein was transferred to PVDF membrane via electroblotting. After transfer, the blot membrane was blocked with 1% BSA and incubated with mouse anti Trp-2 monoclonal antibody. The membrane was then incubated with 3 mL of goat anti-mouse IgG HRP diluted to 1 : 10,000 for 10 minutes. Finally, the image was developed using X-ray film and scanned with a densitometer. The results are shown in Figure 4. In Figure 4, the leftmost lane with signal is the MW control lane. The next two lanes to the right with signal are the reference standard of TRP-2. The next two lanes to the right are TRP-2 signals from different batches of the vaccine process. The next two lanes to the right with signal are additional reference standards of TRP-2. The results indicate that the TRP-2 is present.

[0088] The potency of the tri-vaccine was measured in mice. Two different dosings were used for each sample preparation method: 40 ug (low dose), and 100 ug (high dose). Female BALB/c mice (5-6 weeks old) were be immunized with the mixture on days 1, 7, 14, and 21 by subcutaneous injection into the abdominal region (switching sides weekly to minimize local reactions). Mice weights were evaluated pre- and post-immunization for a general health assessment. Blood was collected from the retro-orbital sinus plexus prior to immunization and at days 20, 28, and 35 after initial immunization. Blood was frozen until testing for antibody response. Mice were terminated at day 28 or 35.

[0089] Antibody responses were measured using an ELISA assay as follows: Mice sera test samples were prepared in dilutions from 1 : 100 to 1 :3200. Microtiter plates were coated with diluted test sample mixed with PBS, sealed, and incubated. The next day positive control serum was tested at serial 2-fold dilutions from 1 : 100 to 1 :3200. Negative controls were prepared at 1 : 100 dilution. Plates were then washed with PBS, BSA is added, and the plates were incubated. Plates were then washed again and controls, standards, precision samples, and assay blanks were added to 3 wells each. Plates were sealed and incubated. Plates were then washed again and diluted goat anti-mouse IgG-HRP conjugate was added to all wells. Plates were again incubated, then washed. TMB substrate was then added to all wells and plates were incubated. TMB Stop Solution was then added to all wells and absorbance was read with a microtiter plate reader at 450 nm. Standard curves were plotted using a 4-parameter logistic curve-fitting algorithm of the average absorbance at 450 nm against the corresponding concentrations of calibration standards in ng/mL.

[0090] The % potency of the test samples was calculated to be % ± SD compared to a positive control. The potency of the vaccine composition was 67.39%±2.0% for the low dose and 58.9%±3.2% for the high dose.

Example 3 : A Randomized Phase 2 Study of a Polyvalent. Shed- Antigen Vaccine as Adjuvant Therapy for Resected Stage II -IV Cutaneous Melanoma.

[0091] This study measured the efficacy of the trivalent vaccine and further evaluated recurrence-free survival (RFS) in patients with resected, high-risk, cutaneous melanoma.

[0092] Patients. Adult patients ("subjects") (> 18 years of age) with surgically resected American Joint Committee on Cancer (AJCC) Stage II, III, or IV cutaneous melanoma or Stage I disease with lesions > 4mm thick who were surgically disease free and within 16 weeks of definitive surgery were eligible. Eligible patients were required to have a life expectancy > 4 months and an intact cellular immune response as evidenced by reactivity to standard recall antigens such as tuberculosis purified protein derivative (PPD), trichophyton, mumps, or Candida, or the ability to be sensitized to dinitrochlorobenzene (DNCB). Eligible patients were also required to have adequate baseline renal, hepatic, and bone marrow function (white blood cell count > 3,000 cells/mL, platelet count 10 >

100,000/μΕ, serum creatinine < 1.5 mg/dL, and bilirubin < 2.0 mg/dL). Patients with concurrent infection or severe cardiac, pulmonary, renal, or hepatic co-morbidities were excluded.

[0093] Study Design. In this randomized, double-blind, phase 2 study, patients were randomly assigned to either the polyvalent vaccine group or the null vaccine group.

Treatment was initiated within 8 weeks after 16 resection of regional lymph nodes. Patients received a fixed dose of 40 μg vaccine administered intradermally into 4 injection sites on the extremities (10 μg per site) once every 3 weeks times 4 injections. Thereafter, booster injections were given every month times 3, every 3 months times 2, and then every 6 months until disease recurrence or for a maximum of 2 years.

[0094] This study assessed the induction of humoral, cellular, delayed type hypersensitivity (DTH) responses to the vaccine at predefined time points as well conduct an exploratory post-hoc assessment of recurrence-free survival (RFS). RFS was defined as time from randomization to first documented recurrence of melanoma or death from any cause. A humoral response was defined as development of serum antibodies (immunoglobulin-M

[IgM] or immunoglobulin-G [IgG]) to melanoma antigens contained in the vaccine preparation (1 week after the first, second, and fourth vaccination and every 3-6 months thereafter). A cellular response was defined as development of cytotoxic T lymphocyte (CTL)-mediated cytotoxicity against the patient's own tumor tissue. A DTH response was defined as a skin test reaction to vaccine 1 week after the 3 fourth vaccination that was at least 15 mm (diameter of induration) greater than the DTH 4 response within 1 week of the first vaccination.

[0095] Study Assessments. Clinical and laboratory assessments were conducted every 3 weeks for the first 3 months, monthly for 3 months, and thereafter at 3- to 6-month intervals until disease recurrence or study withdrawal. The humoral immune response was assessed by ELISA assay, immunoprecipitation, and complement fixation, and the CTL response was assessed by an ELISPOT assay in a subset of 8 evaluable patients. Disease recurrence was assessed by chest X-ray and CT scan of head, chest, and abdomen every 3-6 months. After study initiation, several changes from protocol-specified assessments were instituted. The humoral IgM and IgG response was assessed at only 2 time points (at baseline and after the fourth vaccination) and only qualitative assessments were conducted.

[0096] Statistical Methods. As originally designed, no direct comparison between the treatment arms was planned. The Quad arm was intended only as an active internal control to determine whether the Tri vaccine was active. The criterion for declaring the Tri vaccine active was an expected DTH response rate of approximately 35% but not < 20%. If at least 1 DTH response occurred within the first 19 patients, this criterion would be met based on the upper limit of the 95% confidence interval (CI). Antibody responses are reported as means (95% CI). In the final analysis, recurrence-free survival ("RFS"), a secondary endpoint, was estimated by the Kaplan-Meier method (unadjusted log-rank) and compared between the Tri, Quad, and Null vaccine groups. Descriptive statistics were used to describe the IgM, IgG, CTL and DTH response to vaccine.

[0097] Patients. A total of 116 patients were enrolled at a single center (New York University) between January 1991 and December 1995, and all were randomized. Among 87 patients who were randomized to active vaccine, 42 received the Tri vaccine and 45 received the Quad vaccine. The other 29 patients received an inactive vaccine preparation (Null vaccine). Overall, 105 patients (91%) completed the 2-year study; 6 patients withdrew before the fourth vaccination, 4 withdrew before the seventh vaccination, and 1 was lost to follow- up. Demographic and baseline disease characteristics are shown in Figure 1. Median age was approximately 54 years, and the majority of the patients had AJCC stage II or III disease and fewer than 2 positive lymph nodes; 22 patients with fully resected stage IV disease were also enrolled. The randomized treatment groups were well balanced with respect to baseline characteristics. In comparison to patients randomized to active vaccine, the group receiving Null vaccine had a lower proportion of patients with resected stage IV disease and may have had thinner lesions, although 41% of patients in this group did not have data available on primary tumor thickness. Otherwise this group appeared to be comparable to the groups receiving active vaccine.

[0098] Immune Response. After the fourth vaccination, an IgM response was observed in 24% of patients receiving the Tri vaccine and 15% of patients receiving the Quad vaccine. An IgG response was observed in 27% of patients receiving the Tri vaccine and 31% of patients receiving the Quad vaccine. ADTH response was observed in 9 of 38 evaluable patients (24%) receiving the Tri vaccine, 11 of 38 evaluable patients (29%) receiving the Quad vaccine, and 4 of 27 evaluable patients (15%) receiving the 23 Null vaccine. In addition, analysis of the CTL response in a subset of 8 patients who received 24 active vaccine (4 in each group) indicated that 2 evaluable patients (50%) receiving the Tri vaccine and 2 evaluable patients receiving the Quad vaccine had a CTL response to their own tumor tissue.

[0099] Recurrence-Free Survival. Kaplan-Meier analysis of RFS (Figure 2) confirmed the clinical benefit of the Tri vaccine, which was more effective than the Quad vaccine and significantly more effective than the Null vaccine. Median RFS was 36.5 months with the Tri vaccine, 13.2 months with the Quad vaccine, and 11.0 months with the null vaccine. Compared with the Null vaccine, the Tri vaccine reduced the relative risk of recurrence by nearly 60% (HR = 0.41; 95% CI: 0.23, 0.72; 9 P = 0.002). Compared with the Quad vaccine, the Tri vaccine reduced the risk of recurrence by 35% (HR = 0.65; 95% CI: 0.39, 1.17; P = 0.17). Although not statistically significant, the Quad vaccine improved RFS by nearly 40% compared with the Null vaccine (HR = 0.63; 95% CI: 12 0.37, 1.09; P = 0.095). Subgroup analysis of RFS showed consistent benefit of Tri versus Null vaccine regardless of gender, age, tumor stage, primary tumor thickness, or number of positive nodes (Figure 3).

[0100] An exploratory landmark analysis of RFS for all patients who remained on study at Day 100 was conducted to compare RFS in patients receiving the Tri vaccine who did or did not have a DTH response, and each of those groups was compared to the Null vaccine group (Figure 3). This analysis included 38 patients who received the Tri vaccine, of whom 9 (24%) had a DTH response, and 22 patients who received Null vaccine, of whom 2 (9%) had a DTH response. This analysis suggests a correlation between DTH response to the Tri vaccine and improved RFS. Median RFS was not reached in DTH responders compared with 40.3 months in DTH non-responders, and both groups had prolonged RFS compared with the Null vaccine group.

[0101] Safety. Adverse events were not collected; however, no serious adverse events were reported.

[0102] Summary and Conclusion. This study yielded the surprising result that the Tri vaccine was equally immunogenic compared with the Quad vaccine. Qualitative analysis of the humoral, cellular, and DTH response indicated that both vaccine preparations have similar immunogenicity. More importantly, both vaccine preparations improved RFS compared with the Null vaccine, and the Tri vaccine surprisingly demonstrated a statistically and clinically significant improvement in RFS compared with the Null vaccine. This is surprising as the Tri vaccine would be expected to yield fewer diversity of antigens than the Quad vaccine, yet the RFS was longer for the Tri vaccine than the Quad vaccine.

[0103] A comparison of the outcomes of the current study with previously reported adjuvant studies suggest that the Tri vaccine was highly effective at delaying or preventing disease recurrence, yielding a median RFS of 36.5 months (3 years) in this high-risk patient population. In the randomized phase 2 study comparing the Quad vaccine with placebo (Bystryn et al, Cancer Res, 2001 ; 7(7): 1882-7, median RFS in the Quad vaccine arm was 1.6 years (slightly better than the 13 months observed in the current study). Similar outcomes were reported in the adjuvant trials of high-dose IFN-a (E1684 and E1690), which demonstrated a median RFS in the range of 18-20 months (1.5-1.7 years) in the high-dose arm. Although such comparisons must be interpreted with caution, particularly in light of the small sample size in the current study, it suggests that the Tri vaccine was at least as effective as high-dose IFN-a2b. It should also be emphasized that none of the prior studies yielded a RFS as high as 34.5 months, as in the present disclosure.

[0104] As previously discussed, the observed clinical benefit of the Tri vaccine was consistent with the reported biologic activity of this polyvalent, shed antigen vaccine.

Previously reported studies have demonstrated that biomarkers associated with an immune response to the Tri vaccine correlated with improved RFS. For example, vaccine-induced CD8+ T-cell responses to MAGE- A3 and vaccine-induced clearance of circulating melanoma cells were shown to correlate with improved RFS (Reynolds et al, Clin Cancer Res, 2003; 9(2):657-62; Reynolds et al, Clin Cancer Res, 2003; 9(4): 1497-502) Similarly, the current study has shown that a DTH response to the Tri vaccine appears to be associated with improved RFS.

[0105] Surprisingly, the trivalent vaccine formulation was as biologically active as the quadravalent vaccine formulation, and it significantly improved RFS compared with a Null vaccine.

Example 4. Treatment with a Polyvalent. Shed- Antigen Vaccine in Combination with a Checkpoint Inhibitor or Immunopotentiator as Adjuvant Therapy for Resected Stage II-IV Cutaneous Melanoma

[0106] The Tri vaccine is prepared as described above.

[0107] Subgroups of patients are administered the vaccine. The vaccine may further comprises an adjuvant of polyoxyethylene-polyoxypropylene co-polymers; polymer P1005; monotide ISA72; Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300 adjuvant; alum; Detox, QS 21, muramyl dipeptide; trehalose; membrane lipids,

lipopolysaccharides; water-in-oil mixtures, water-in-oil-in-water mixtures, sodium phosphate, poly-ICLC or combinations thereof. The Tri vaccine may further comprise an

immunopotentiator of CpG motif olignucleotide sequences, T-cell costimulatory molecules such as B7-1, ICAM-1, and LFA-3; cyclophosphamide, Fms-like tyrosine kinase receptor-3 ligand ("Flt31"), MlP-Ια chemokine, IL-2 cytokine, IL-12 cytokine, IL-15 cytokine, TGFfi cytokine, granulocyte-macrophase colony stimulating factor (GM-CSF) cytokine, antitumor Mab-IL-2, and Myobacterium bovis Bacille Calmette-Guerin (BCG) vector, or combinations thereof. The Tri vaccine further comprises a checkpoint inhibitor of Ipilimumab (Yervoy); Tremelimumab (formerly ticilimumab); Pidilizumab (CT-011); Nivolumab (BMS-936558); Lambrolizumab (MK-3475); Pembrolizumab; MPDL3280A; BMS-936559; BMS-663513; AMP-224; IMP321 (ImmuFact); MGA271 ; Indoximod; and INCB024360; antibodies to 4- 1BB; antibodies to OX40; or combinations thereof. The amount of the checkpoint inhibitor per dose is 1 mg/kg, 2 mg/kg, 5 mg/kg, 7 mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg. The amount of the immunopotentiator per dose is 10 ug, 20 ug, 30 ug, 40 ug, 50 ug, 60 ug, 70 ug, 80 ug, 90 ug, or 100 ug. For the immunopotentiators administered in units of IU, the amount of the immunopotentiator per dose is 200,000 IU/kg; 300,000 IU/kg; 400,000 IU/kg; 500,000 IU/kg; 600,000 IU/kg; 700,000 IU/kg; 800,000 IU/kg; 900,000 IU/kg; or 1,000,000 IU/kg.

[0108] More than n=20 patients are selected for this treatment study. All patients are previously screened to be one at risk of recurrence following melanoma resection or in need of treatment for melanoma.

[0109] Subgroups of patients are administered with doses of 0 μg (as a control), 10 μg, 20 μg, 30 μg, 40 μg, 50-100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg of the Tri vaccine. Each dosing comprises multiple deliveries of the Tri vaccine per dose, and subgroups of the patients are dosed in the amounts of: 2 injections per dose, 3 injections per dose, 4 injections per dose, or 5 injections per dose. Subgroups of the patients are given administrations at varying time intervals, at times of: weekly, biweekly, triweekly, monthly, bimonthly, trimonthly, every six months, and combinations thereof.

[0110] The recurrence free survival time (as determined by a Kaplan-Meier analysis) yields an average RFS of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100 months, or longer for patients who are administered the Tri vaccine at doses of 40 ug or greater compared to patients who are administered 0 μg of the Tri vaccine. The surprising discovery that the RFS is longer than about 15 months (the average RFS in prior studies of administration of the Tri vaccine with no checkpoint inhibitor or immunipotentiator) is due to the combination of the dosing schedule, addition of checkpoint inhibitors, storage condition of the prepared composition prior to use, processing steps, addition of immunopotentiators, or an increase dosing amounts over 40 ug (the dose given in the prior studies).

[0111] All of the references cited herein are incorporated by reference.

[0112] The inventions illustratively described and claimed herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein, or described herein,, as essential. Thus, for example, the terms

"comprising," "including," "containing," "for example", etc., shall be read expansively and without limitation. The term "including" means "including but not limited to." The phrase "for example" is not limited to, or by, the items that follow the phrase. All references to things "known in the art" include all those things and equivalents and substitutes, whether now known, or later discovered.

[0113] In claiming their inventions, the inventors reserve the right to substitute any transitional phrase with any other transitional phrase, and the inventions shall be understood to include such substituted transitions and form part of the original written description of the inventions. Thus, for example, the term "comprising" may be replaced with either of the transitional phrases "consisting essentially of or "consisting of."

[0114] The methods and processes illustratively described herein may be suitably practiced in differing orders of steps. They are not necessarily restricted to the orders of steps indicated herein, or in the claims.

[0115] Under no circumstances may the patent be interpreted to be limited to the specific examples, or embodiments, or methods, specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any Statement made by any Examiner, or any other official or employee of the Patent and Trademark Office, unless such Statement was specifically, and without qualification or reservation, expressly adopted by Applicants in a responsive writing specifically relating to the application that led to this patent prior to its issuance.

[0116] The terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions, or any portions thereof, to exclude any equivalents now know or later developed, whether or not such equivalents are set forth or shown or described herein or whether or not such equivalents are viewed as predictable, but it is recognized that various modifications are within the scope of the invention claimed, whether or not those claims issued with or without alteration or amendment for any reason. Thus, it shall be understood that, although the present invention has been specifically disclosed by some embodiments and optional features, modifications and variations of the inventions embodied therein or herein disclosed can be resorted to by those skilled in the art, and such modifications and variations are considered to be within the scope of the inventions disclosed and claimed herein.

[0117] Specific methods and compositions described herein are representative of some embodiments and are exemplary of, and not intended as limitations on, the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. Where examples are given, the description shall be construed to include, but not to be limited to, only those examples. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein, without departing from the scope and spirit of the invention, and from the description of the inventions, including those illustratively set forth herein, it is manifest that various modifications and equivalents can be used to implement the concepts of the present invention, without departing from its scope. A person of ordinary skill in the art will recognize that changes can be made in form and detail without departing from the spirit and the scope of the invention. The described embodiments are to be considered in all respects as illustrative and not restrictive. Thus, for example, additional embodiments are within the scope of the invention and within the following claims.

[0118] While this invention has been disclosed with reference to specific

embodiments, it is apparent that other embodiments and variations of this invention can be devised by those skilled in the art, without departing from the true spirit and scope of the invention. The appended claims include all such embodiments and equivalent variations.

Claims

WHAT IS CLAIMED IS:

1. A method of treating melanoma in a patient in need thereof comprising administering a therapeutically effective amount of a polyvalent melanoma cancer vaccine comprising multiple partially purified soluble shed melanoma antigens from at least three human melanoma cell lines, and a therapeutically effective amount of a checkpoint inhibitor.

2. The method of claim 1 , wherein the human melanoma cell lines are SFHM2, SFHM4 and SFHM8.

3. The method of claim 1, wherein the patient is a patient with resected melanoma.

4. The method of claim 1, wherein the vaccine is administered intradermally.

5. The method of claim 1, wherein the administering a therapeutically effective amount of a polyvalent melanoma cancer vaccine occurs in doses at treatment time periods of once every 3 weeks times 4 injections, then every month times 3, every 3 months times 2, and then every 6 months up to 2 years.

6. The method of claim 5, wherein the doses at treatment time period doses is comprised of 4 intradermal injections with an equal amount per injection per time period.

7. The method of claim 5, further comprising the treating results in an average recurrence-free survival time of over 34 months.

8. The method of claim 1, wherein the administering a therapeutically effective amount of a polyvalent melanoma cancer vaccine occurs at a dose of 40 ug or 100 ug per patient.

9. The method of claim 1, wherein the multiple partially purified soluble shed melanoma antigens from at least three human melanoma cell lines further comprise one or more of the following selected antigens: gplOO, MART-1, MAGE-1, MAGE-2, MAGE3, MAGE- A3, S100B, MC1R, HLA-1 and TRP-2.

10. The method of claim 1, further comprising the coadministration of the therapeutically effective amount of a polyvalent melanoma cancer vaccine with an immunopotentiator.

11. The method of claim 1 , wherein the checkpoint inhibitor targets one or more of the following proteins: indoleamine (2, 3)-dioxygenagse (IDO); programmed death-1 (PD-1); programmed death-ligand 1 (PD-L1); PD-L2; OX40; 4- IBB; lymphocyte activation gene 3 (LAG3); and B7 homolog 3 (B7-H3).

12. The method of claim 11, wherein the checkpoint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, pidilizumab (CT-011), nivolumab (BMS- 936558), lambrolizumab (MK-3475), Pembrolizumab, BMS-936559, BMS-663513, MPDL3280A, Amplimmune (AMP-224), immutep (IMP321), MGA271, indoximod, or INCB024360).

13. A method of modulating effector T cell response levels and subsets in vitro or in vivo, comprising:

contacting a cell in vitro, or administering to a patient a therapeutically effective amount of a polyvalent melanoma cancer vaccine comprising multiple partially purified soluble shed melanoma antigens from at least human melanoma cell lines, and a

therapeutically effective amount of a checkpoint inhibitor; and, modulating effector T cell response levels and subsets thereof, as compared to a baseline control.

14. The method of claim 13, wherein the baseline control is the recurrence-free survival time of patients treated with the Quad-vaccine, and the at least human melanoma cell lines consists of three cell lines.

15. The method of claim 13, wherein an effector T cell is one or more cell surface markers comprising: CTLA4 or CD8+.

16. The method of claim 13, where the human melanoma cell lines comprise SFHM2, SFHM4 and SFHM8.

17. A method of extending recurrence-free survival time of a post-resection melanoma patient by at least 34 months comprising administering a therapeutically effective amount of a polyvalent melanoma cancer vaccine comprising equal portions (w/w/) of multiple partially purified soluble shed melanoma antigens from SFHM2, SFHM4 and SFHM8 human melanoma cell lines.

18. A pharmaceutical composition comprising multiple partially purified soluble shed melanoma antigens comprising at least human melanoma cell lines, a checkpoint inhibitor, and one or more pharmaceutically acceptable diluents and/or excipients, and an adjuvant.

19. The pharmaceutical composition of claim 18, further comprising human melanoma cell lines SFHM2, SFHM4 and SFHM8.

20. The pharmaceutical composition of claim 18, wherein the checkpoint inhibitor targets one or more of the following proteins: indoleamine (2, 3)-dioxygenagse (IDO); programmed death- 1 (PD-1); programmed death-ligand 1 (PD-L1); PD-L2; lymphocyte activation gene 3 (LAG3); OX40; 4-1BB; and B7 homolog 3 (B7-H3).

21. The pharmaceutical composition of claim 18, wherein the composition is administered to prevent recurrence of melanoma in a patient in need thereof.

22. The pharmaceutical composition of claim 18, wherein the adjuvant is alum.

23. The pharmaceutical composition of claim 18, wherein the shed melanoma antigens have a isoelectric point value between 3.5 to 9.

24. The pharmaceutical composition of claim 18, wherein the shed melanoma antigens comprise one or more of the following selected antigens: gplOO, MART-1, MAGE- 1, MAGE-2, MAGE3, MAGE- A3, S100B, MC1R, HLA-1 and TRP-2.

25. The pharmaceutical composition of claim 18, wherein the checkpoint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, pidilizumab (CT-011), nivolumab (BMS-936558), lambrolizumab (MK-3475), Pembrolizumab, BMS-936559, BMS-663513, MPDL3280A, Amplimmune (AMP-224), immutep (IMP321), MGA271, indoximod, INCB024360, and combinations thereof.

26. The pharmaceutical composition of claim 18, wherein the adjuvant is IL-2.

27. The pharmaceutical composition of claim 18, wherein the adjuvant is an immunopotentiator selected from one of the following: CpG motif olignucleotide sequences, T-cell costimulatory molecules such as B7-1, ICAM-1, and LFA-3; cyclophosphamide, Flt31, MlP-Ια chemokine, IL-2 cytokine, IL-12 cytokine, IL-15 cytokine, TGF cytokine, granulocyte-macrophase colony stimulating factor (GM-CSF) cytokine, antitumor Mab-IL-2, Myobacterium bovis Bacille Calmette-Guerin (BCG) vector, and combinations thereof.

28. A method of treating a patient with resected melanoma, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of any of claims 18-28.