Methods and products for treating or preventing melanoma

Abstract

The present invention relates to methods for treating or preventing melanoma in a subject, including a subject with Stage IV melanoma. In particular, the methods include administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and administering to the subject one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors. The melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof, the one or more immunotherapeutic agents, and / or the one or more cancer cell-specific inhibitors, can be administered to the subject in any order, and / or they can be administered concurrently. Also encompassed by the present invention is a combination product which includes a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, and includes one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors, wherein these components are provided in a form for separate administration to a subject, including a subject with melanoma, including Stage IV melanoma.

Description

METHODS AND PRODUCTS FOR TREATING OR PREVENTING MELANOMAPRIORITY CLAIM

[0001] This application claims priority from Australian provisional patent application number 2023902816 filed on 1 September 2023, the contents of which are to be taken as incorporated herein by this reference.FIELD OF THE INVENTION

[0002] The present invention relates to methods and products for treating or preventing melanoma (including Stage IV melanoma) in a subject, and to a combination product for treating or preventing melanoma.

[0003] The present invention further relates to methods for inhibiting formation and / or growth of tumours in a subject with melanoma, improving the rate of survival / life expectancy of a subject with melanoma, and inducing an anti-tumour immune response in a subject with melanoma.

[0004] The present invention also relates to a method of selecting a subject with melanoma suitable for treatment.BACKGROUND OF THE INVENTION

[0005] Melanoma is a malignant tumor of melanocytes, cells that are derived from the neural crest. Melanoma can spread by local extension (through lymphatics) and / or by hematogenous routes to distant sites. Any organ may be the site for metastases, but lungs and liver are common sites.

[0006] In human subjects, clinical staging of melanoma is based on whether the tumor has spread to regional lymph nodes or distant sites. Subjects with early stage melanoma (such as Stage I or Stage II) have cancer confined to the place of origin with no spread to nearby lymph nodes. In Stage III melanoma, the originating cancer has spread to one or more nearby lymph nodes, nearby skin and / or skin lymphatic channels around the tumour. Subjects with Stage IV, or metastatic melanoma, have cancer that has spread from its site of origin to distant lymph nodes and / or distant sites, such as the lungs, liver and brain. Additional prognostic factors for Stage IV melanoma include site of distant metastases and elevated blood enzyme LDH levels. Further details regarding clinical staging of lung cancercan be found at the American Cancer Society (www.cancer.org / cancer / types / melanoma- skin-cancer / detection-diagnosis-staging / melanoma-skin-cancer-stages.html).

[0007] The American Joint Committee on Cancer (AJCC) has designated staging by TNM classification to define melanoma. For example, Stage IV melanoma is defined by the following clinical stage grouping: any primary tumor (T), any metastasis to a regional lymph node (N), and a distant metastasis (M).

[0008] While 5-year survival rates for Stage I melanoma are promising (around 99½ for Stage I and Stage II), the survival rate for latter stages declines. For example, according to the American Cancer Society, Stage IV melanoma has a 5-year survival rate of only around 30½. Surgery for isolated metastases is an option for a subgroup of patients with Stage IV melanoma, and occasionally chemotherapy can be effective in specific instances, but these options are usually not available for widespread metastatic disease and response rates to any form of standard therapy are almost universally non-existent. Since about 2014, additional inhibitors and immunotherapies have been developed and registered and have improved the survival from Stage III and Stage IV advanced melanoma significantly. However, still about 70½ of Stage IV melonama patients will die from their disease. Therefore, a major unmet need in effective therapy for the treatment of stage IV melanoma sufferers remains.

[0009] Despite many studies relating to the development of therapeutic interventions for melanoma, there has been little progress in this field. Indeed, apart from surgical intervention and the use of chemotherapeutic agents, which have a relatively low percentage impact, there is no effective treatment for Stage IV melanoma. Even with the newer immunotherapeutic and small molecule inhibitory agents, longer-term survival is less than 40½ overall and the majority of patients with Stage IV melanoma still die. In light of the above, there is a real, present and pressing need for new treatments for melanoma, including Stage IV melanoma.

[0010] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any, or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.SUMMARY OF THE INVENTION

[0011] The present invention is predicated in part on the identification of a novel treatment method for melanoma. In this regard, in a first aspect the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) administering to the subject one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors.

[0012] In some embodiments, the subject is a human subject.

[0013] In some embodiments, the melanoma cell lysate includes fragmented melanoma cell membranes. In some embodiments, the melanoma cell lysate is a viral melanoma cell lysate. In some embodiments, the viral melanoma cell lysate is a vaccinia melanoma cell lysate. In some embodiments, the melanoma cell lysate is an allogeneic cell lysate. In some embodiments, the melanoma cell lysate includes a lysate from MM200 cells.

[0014] In some embodiments, the melanoma is Stage IV melanoma. In some embodiments, the Stage IV melanoma is advanced Stage IV melanoma.

[0015] In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject prior to administration of the one or more immunotherapeutic agents to the subject. In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject about every two weeks for one or more cycles prior to administration of the one or more immunotherapeutic agents to the subject. In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof is administered to the subject about every two weeks for at least about three months prior to administration of the one or more immunotherapeutic agents to the subject. In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject about every two weeks for at least about six months prior to administration of the one or more immunotherapeutic agents to the subject. In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject about every two weeks for about seven months prior to administration of the one or more immunotherapeutic agents to the subject.

[0016] In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject after administration of the one or more immunotherapeutic agents to the subject. In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject about every two weeks for one or more cycles after administration of the one or more immunotherapeutic agents to the subject. In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof is administered to the subject about every two weeks for at least about three months after administration of the one or more immunotherapeutic agents to the subject. In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject about every two weeks for at least about seven months after administration of the one or more immunotherapeutic agents to the subject. In some embodiments, the one or more immunotherapeutic agents are administered to the subject about every three weeks for at least about six weeks.

[0017] In some embodiments, the one or more cancer cell-specific inhibitors are administered to the subject after the one or more immunotherapeutic agents are administered to the subject, and prior to administration of the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof to the subject. In some embodiments, the one or more cancer cell-specific inhibitors are administered to the subject at least about two weeks after the one or more immunotherapeutic agents are administered to the subject. In some embodiments, the one or more cancer cell-specific inhibitors are administered to the subject about every four weeks for at least about one month.

[0018] In some embodiments, the one or more cancer cell-specific inhibitors are also administered to the subject during a period in which the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof is administered to the subject.

[0019] In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject after administration of the one or more cancer cell-specific inhibitors to the subject.

[0020] In some embodiments, the one or more immunotherapeutic agents comprise one or more immune checkpoint inhibitors.

[0021] In some embodiments, the one or more immune checkpoint inhibitors are selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, an OX40 inhibitor, an A2AR inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, an IDO inhibitor, a TIM3 inhibitor, a KIR inhibitor, and an IL-2R inhibitor.

[0022] In some embodiments, the PD-1 inhibitor is selected from one or more of Pembrolizumab, Cemiplimab, Pidilizumab, Nivolumab, and MEDI-0680; the PD-L1 inhibitor is selected from one or more of Atezolizumab, Avelumab, Durvalumab, and BMS-936559 / MDX- 1105; the CTLA-4 inhibitor is selected from one or more of Tremelimumab and Ipilimumab; the LAG-3 inhibitor is Relatlimab; the B7-H3 inhibitor is MGA271 ; the KIR inhibitor is Lirilumab; and the OX40 inhibitor is MEDI-6469.

[0023] In some embodiments, the immune check-point inhibitor is selected from one or more of Pembrolizumab, Nivolumab and Ipilimumab.

[0024] In some embodiments, the one or more cancer cell-specific inhibitors are selected from the group consisting of a receptor tyrosine kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a serine / threonine kinase inhibitor, an epigenetic inhibitor, a hedgehog pathway inhibitor, a proteasome inhibitor, and a poly (ADP-ribose) polymerase (PARP) inhibitor.

[0025] In some embodiments, the receptor tyrosine kinase inhibitor is selected from one or more of an anaplastic lymphoma kinase (ALK) inhibitor, a cellular-mesenchymal-epithelial transition factor (c-Met) inhibitor, a c-Kit (CD117) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a Fms-like tyrosine kinase 3 (FLT3) inhibitor, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a fibroblast growth factor receptor (FGFR) inhibitor, a platelet-derived growth factor receptor (PDGFR) inhibitor, and a tropomyosin receptor kinase (TRK) inhibitor.

[0026] In some embodiments, the receptor tyrosine kinase inhibitor is selected from one or more of crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, capmatinib, tepotinib, gefitinib, erlotinib, lapatinib, icotinib, afatinib, osimertinib, neratinib, dacomitinib, almonertinib, tucatinib, midostaurin, gilteritinib, quizartinib, pexidartinib, sorafenib, sunitinib, pazopanib, vandetanib, axitinib, cabozantinib, regorafenib, apatinib, Lenvatinib, tivozanib, fruquintinib, nintedanib,anlotinib, erdafitinib, pemigatinib, avapritinib, ripretinib, selpercatinib, pralsetinib, Larotrectinib and entrectinib.

[0027] In some embodiments, the non-receptor tyrosine kinase inhibitor is selected from one or more of a Bcr-Abl1 inhibitor, a Bruton’s agammglobulinemia tyrosine kinase (BTK) inhibitor, and a janus kinase (JAK) inhibitor.

[0028] In some embodiments, the non-receptor tyrosine kinase inhibitor is selected from one or more of imatinib, dasatinib, nilotinib, bosutinib, radotinib, ponatinib, ibrutinib, acalabrutinib, zanubrutinib, ruxolitinib, and fedratinib.

[0029] In some embodiments, the serine / threonine kinase inhibitor is selected from one or more of a BRAF inhibitor, a MEK inhibitor, an ERK inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, an AKT inhibitor, and a mammalian target of rapamycin (mTOR) inhibitor.

[0030] In some embodiments, the serine / threonine kinase inhibitor is selected from one or more of dabrafenib, trametinib, vemurafenib, encorafenib, cobimetinib, binimetinib, selumetinib, Palbociclib, ribociclib, abemaciclib, idelalisib, copanlisib, duvelisib, alpelisib, temsirolimus, everolimus, and sirolimus.

[0031] In some embodiments, the epigenetic inhibitor is selected from one or more of an enhancer of zeste homolog 2 (EZH2) inhibitor, a histone deacetylase (HDAC) inhibitor, and an isocitrate dehydrogenase 1 / 2 (IDH1 / 2) inhibitor.

[0032] In some embodiments, the epigenetic inhibitor is selected from one or more of tazemetostat, vorinostat, romidepsin, belinostat, tucidinostat, panobinostat, enasidenib, and ivosidenib.

[0033] In some embodiments, the hedgehog pathway inhibitor is selected from one or more of venetoclax, vismodegib, sonidegib, glasdegib, bortezomib, carfilzomib, ixazomib, olaparib, rucaparib, niraparib and talazoparib.

[0034] In some embodiments, the proteasome inhibitor is selected from one or more of bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, and delanzomib.

[0035] In some embodiments, the PARP inhibitor is selected from one or more of Nicotinamide, Olaparib, rucaparib, niraparib, talazoparib, pamiparib, veliparib, iniparib, amelparib, and fluzoparib.

[0036] In some embodiments, the one or more cancer cell-specific inhibitors are dabrafenib and trametinib.

[0037] In a second aspect, the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) subsequently administering to the subject one or more immunotherapeutic agents.

[0038] In a third aspect, the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more immunotherapeutic agents; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0039] In a fourth aspect, the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more immunotherapeutic agents; and(ii) subsequently administering to the subject one or more cancer cell-specific inhibitors; and(iii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0040] In a fifth aspect, the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more immunotherapeutic agents; and(ii) subsequently administering to the subject one or more cancer cell-specific inhibitors; and(iii) subsequently administering to the subject one or more cancer cell-specific inhibitors and a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0041] In a sixth aspect, the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more cancer cell-specific inhibitors; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0042] In a seventh aspect, the present invention provides a method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) subsequently administering to the subject Pembrolizumab.

[0043] In an eighth aspect, the present invention provides a method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject Nivolumab and Ipilimumab; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0044] In a ninth aspect, the present invention provides a method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject Nivolumab and Ipilimumab; and(ii) subsequently administering to the subject dabrafenib and trametinib; and(iii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0045] In a tenth aspect, the present invention provides a method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject Nivolumab and Ipilimumab; and(ii) subsequently administering to the subject dabrafenib and trametinib; and(iii) subsequently administering to the subject dabrafenib and trametinib, and a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0046] In some embodiments of the first to tenth aspects of the invention, the method includes the further step of resecting metastases from the subject. In some embodiments, the metastases are resected prior to administration of the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0047] In some embodiments of the first to tenth aspects of the invention, the method includes the step of selecting a subject suitable for treatment. In some embodiments, the subject is selected on the basis that the subject shows a T cell response to the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0048] In some embodiments of the first to tenth aspects of the invention, formation and / or growth of a secondary tumour in the subject is inhibited. In some embodiments, growth of a primary melanoma in the subject is inhibited. In some embodiments, rate of survival of the subject is improved. In some embodiments, life expectancy of the subject is improved. In some embodiments, an anti-tumour immune response in the subject is induced.

[0049] In some embodiments of the first to tenth aspects of the invention, one or more of an increase in the number of intratumoral dendritic cells is produced in the subject, dendritic cell activation is improved in the subject, and the number and / or activity of regulatory T cells is decreased in the subject.

[0050] In an eleventh aspect, the present invention provides a combination product including the following components:(i) a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof; and(ii) one or more immunotherapeutic agents and / or one or more cancer cell- specific inhibitors; wherein the components are provided in a form for separate administration to a subject.

[0051] In some embodiments of the eleventh aspect of the invention, the melanoma cell lysate includes fragmented melanoma cell membranes. In some embodiments, the melanoma cell lysate is a viral melanoma cell lysate. In some embodiments, the viral melanoma cell lysate is a vaccinia melanoma cell lysate. In some embodiments, the melanoma cell lysate is an allogeneic cell lysate. In some embodiments, the melanoma cell lysate includes a lysate from MM200 cells.

[0052] In some embodiments of the eleventh aspect of the invention, the one or more immunotherapeutic agents comprise one or more immune checkpoint inhibitors. In some embodiments, the one or more immune checkpoint inhibitors are selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, an OX40 inhibitor, an A2AR inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, an IDO inhibitor, a TIM3 inhibitor, a KIR inhibitor, and an IL-2R inhibitor.

[0053] In some embodiments of the eleventh aspect of the invention, the PD-1 inhibitor is selected from one or more of Pembrolizumab, Cemiplimab, Pidilizumab, Nivolumab, and MEDI-0680; the PD-L1 inhibitor is selected from one or more of Atezolizumab, Avelumab, Durvalumab, and BMS-936559 / MDX-1105; the CTLA-4 inhibitor is selected from one or more of Tremelimumab and Ipilimumab; the LAG-3 inhibitor is Relatlimab; the B7-H3 inhibitor is MGA271 ; the KIR inhibitor is Lirilumab; and the OX40 inhibitor is MEDI-6469.

[0054] In some embodiments of the eleventh aspect of the invention, the combination product includes a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, and Pembrolizumab.

[0055] In some embodiments of the eleventh aspect of the invention, the one or more cancer cell-specific inhibitors are selected from the group consisting of a receptor tyrosine kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a serine / threonine kinase inhibitor, an epigenetic inhibitor, a hedgehog pathway inhibitor, a proteasome inhibitor, and a poly (ADP- ribose) polymerase (PARP) inhibitor.

[0056] In some embodiments of the eleventh aspect of the invention, the receptor tyrosine kinase inhibitor is selected from one or more of an anaplastic lymphoma kinase (ALK) inhibitor, a cellular-mesenchymal-epithelial transition factor (c-Met) inhibitor, a c-Kit (CD117) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a Fms-like tyrosine kinase 3 (FLT3) inhibitor, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a fibroblast growth factor receptor (FGFR) inhibitor, a platelet-derived growth factor receptor (PDGFR) inhibitor, and a tropomyosin receptor kinase (TRK) inhibitor.

[0057] In some embodiments of the eleventh aspect of the invention, the receptor tyrosine kinase inhibitor is selected from one or more of crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, capmatinib, tepotinib, gefitinib, erlotinib, lapatinib, icotinib, afatinib, osimertinib, neratinib, dacomitinib, almonertinib, tucatinib, midostaurin, gilteritinib, quizartinib, pexidartinib, sorafenib, sunitinib, pazopanib, vandetanib, axitinib, cabozantinib, regorafenib, apatinib, Lenvatinib, tivozanib, fruquintinib, nintedanib, anlotinib, erdafitinib, pemigatinib, avapritinib, ripretinib, selpercatinib, pralsetinib, Larotrectinib and entrectinib.

[0058] In some embodiments of the eleventh aspect of the invention, the non-receptor tyrosine kinase inhibitor is selected from one or more of a Bcr-Abl1 inhibitor, a Bruton’s agammglobulinemia tyrosine kinase (BTK) inhibitor, and a janus kinase (JAK) inhibitor.

[0059] In some embodiments of the eleventh aspect of the invention, the non-receptor tyrosine kinase inhibitor is selected from one or more of imatinib, dasatinib, nilotinib, bosutinib, radotinib, ponatinib, ibrutinib, acalabrutinib, zanubrutinib, ruxolitinib, and fedratinib.

[0060] In some embodiments of the eleventh aspect of the invention, the serine / threonine kinase inhibitor is selected from one or more of a BRAF inhibitor, a MEK inhibitor, an ERK inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, an AKT inhibitor, and a mammalian target of rapamycin (mTOR) inhibitor.

[0061] In some embodiments of the eleventh aspect of the invention, the serine / threonine kinase inhibitor is selected from one or more of dabrafenib, trametinib, vemurafenib, encorafenib, cobimetinib, binimetinib, selumetinib, Palbociclib, ribociclib, abemaciclib, idelalisib, copanlisib, duvelisib, alpelisib, temsirolimus, everolimus, and sirolimus.

[0062] In some embodiments of the eleventh aspect of the invention, the epigenetic inhibitor is selected from one or more of an enhancer of zeste homolog 2 (EZH2) inhibitor, a histone deacetylase (HDAC) inhibitor, and an isocitrate dehydrogenase 1 / 2 (IDH1 / 2) inhibitor.

[0063] In some embodiments of the eleventh aspect of the invention, the epigenetic inhibitor is selected from one or more of tazemetostat, vorinostat, romidepsin, belinostat, tucidinostat, panobinostat, enasidenib, and ivosidenib.

[0064] In some embodiments of the eleventh aspect of the invention, the hedgehog pathway inhibitor is selected from one or more of venetoclax, vismodegib, sonidegib, glasdegib, bortezomib, carfilzomib, ixazomib, olaparib, rucaparib, niraparib and talazoparib.

[0065] In some embodiments of the eleventh aspect of the invention, the proteasome inhibitor is selected from one or more of bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, and delanzomib.

[0066] In some embodiments of the eleventh aspect of the invention, the PARP inhibitor is selected from one or more of Nicotinamide, Olaparib, rucaparib, niraparib, talazoparib, pamiparib, veliparib, iniparib, amelparib, and fluzoparib.

[0067] In some embodiments of the eleventh aspect of the invention, the combination product is used to inhibit growth of a primary melanoma in the subject. In some embodiments, the combination product is used to inhibit formation and / or growth of a secondary tumour in the subject.

[0068] In some embodiments of the eleventh aspect of the invention, the subject has melanoma and the combination product is used to treat melanoma in the subject. In some embodiments, the subject has melanoma, and the combination product is used to improve the rate of survival of the subject. In some embodiments, the subject has melanoma, and the combination product is used to improve the life expectancy of the subject. In some embodiments, the subject has melanoma, and the combination product is used to induce an anti-tumour immune response in the subject.

[0069] In some embodiments of the eleventh aspect of the invention, the combination product includes instructions for selecting a subject with melanoma suitable for treatment. In some embodiments, the combination product includes instructions for selecting a subject with melanoma suitable for treatment on the basis that the subject shows a T cell response to the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof. In some embodiments, the combination product includes instructions for selecting a subject with melanoma suitable for treatment on the basis that the subject has a decreased level and / or activity of regulatory T cells upon administration to the subject of the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof.

[0070] In some embodiments of the eleventh aspect of the invention, the subject has Stage IV melanoma. In some embodiments, the subject is a human subject.DETAILED DESCRIPTION OF THE INVENTION

[0071] The present invention arises from clinical studies into the use of a combination of a melanoma cell lysate with one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors and an examination of the effects of such combinations on melanoma cancer. Accordingly, amongst other applications, the present invention provides methods,and combination products for use in such methods, to treat patients with melanoma, including Stage IV metastatic melanoma.

[0072] Certain disclosed embodiments provide methods, combination products and compositions, that have one or more advantages. For example, some of the advantages of some embodiments disclosed herein include one or more of the following: new methods, combination products and compositions to treat melanoma; new methods, combination products and compositions to prevent melanoma; new methods, combination products and compositions to inhibit formation and / or growth of a secondary tumour; new methods, combination products and compositions to inhibit growth of a primary melanoma; new methods, combination products and compositions to increase the rate of survival of a subject with melanoma; new methods, combination products and compositions to increase life expectancy of a subject with melanoma; to provide one or more advantages, or to provide a commercial alternative. Other advantages of some embodiments of the present disclosure are provided herein.

[0073] Accordingly, in a first aspect the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) administering to the subject one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors.

[0074] In some embodiments, a Vaccinia Melanoma Cell Lysate (VMCL) vaccine has been used with an immune checkpoint inhibitor (a PD-1 inhibitor) to treat patients with Stage IV metastatic melanoma. In some embodiments, a Vaccinia Melanoma Cell Lysate (VMCL) vaccine has been used with cancer cell-specific inhibitors to treat patients with Stage IV metastatic melanoma. In these embodiments, the cell lysate was prepared from a melanoma cell line. These studies show that use of a VMCL vaccine in combination with an immunotherapeutic agent such as an immune checkpoint inhibitor, and / or in combination with one or more cancer cell-specific inhibitors, is effective in inducing complete regression of tumours in patients with advanced “incurable” Stage IV metastatic melanoma. These are patients for which no other effective treatment currently exists.

[0075] The term “subject” as used throughout the specification is to be understood to mean any human or animal subject. In this regard, the present invention includes within its scopeveterinary applications for the treatment of melanoma. For example, the animal subject may be a mammal, a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), a laboratory test animal (eg. a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.

[0076] It will also be appreciated that the term “subject” includes within its scope a subject originally diagnosed as suffering from a melanoma and subsequently treated for the melamona. Thus, the present invention extends to the treatment of those original subjects using a method as described herein. For example, the subject may be a human subject suffering from a melanoma treated for the melanoma (e.g. by chemotherapy, radiotherapy, surgery, and the like) before, after or during treatment using a method as described herein.

[0077] The terms “treat” and “treating”, and variants thereof, as used throughout the present specification are to be understood to mean therapeutic intervention with a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) and with one or more immune checkpoint inhibitors and / or or one or more cancer cell-specific inhibitors in a subject with melanoma. The terms "prevent", "preventing" and “prevention” as used herein are to be understood to include within their scope obtaining a desired pharmacologic and / or physiologic effect in terms of arresting or suppressing the appearance of one or more symptoms in the subject. To “prevent” may also encompass inhibiting recurrence of the cancer following treatment.

[0078] For example, the aforementioned terms include within their scope a therapeutic or preventative intervention which has one or more of the following outcomes in the subject: (i) inhibiting or preventing the growth of a primary melanoma in the subject, including reducing the growth of the primary tumour after resection; (ii) inhibiting or preventing the formation and / or growth of one or more secondary tumours in the subject, including tumours in the lymph nodes; (iii) inhibiting the recurrence of the cancer in the subject following treatment; (iv) increasing the life expectancy of the subject as compared to the untreated state; (v) improving the rate of survival of the subject; (vi) inducing an anti-tumour immune response in the subject; and (vii) improving the quality of life the subject as compared to the untreated state. Accordingly, certain disclosed embodiments of the present invention provide methods and compositions that have one or more advantages, and / or provide a commercial alternative to existing methods and compositions. Other advantages of some embodiments of the present disclosure are provided herein.

[0079] The term “inhibit” and the like as used throughout the specification is to be understood to mean a reduction in the progress of a process, including any one or more of the start, continuation or termination of a process.

[0080] The present invention is directed to methods of treating melanoma using a immunotherapeutic approach, and in particular, treating the melanoma by administering a vaccine derived from melanoma cells. In human subjects, assessment of the clinical staging of melanoma may generally be made by a suitably qualified practitioner using any tests that are generally known and accepted in the art. In some embodiments, the cancer staging can comprise that developed by the American Joint Committee on Cancer (AJCC) which has designated staging in humans by “TNM” classification. Generally, the TNM system provides results from various tests and scans in order to determine the size and location of the primary tumor (Tumor, T); whether the cancer has spread to the lymph nodes, and if it has, the location and number of the affected lymph nodes (Node, N); and whether the cancer has spread to other parts of the body, and if it has, the extent and location of the remote cancer (Metastasis, M). While each type of cancer may have its own specific system, the TNM staging system generally uses scaled scoring for each letter.

[0081] For Tumor, "T" is associated with a number (e.g., 0 to 4) to describe the general tumor size, location, and whether it intrudes into nearby tissues. Larger or more intrusive tumors are given a higher number and, depending on the cancer, a lowercase letter, such as "a," "b," or "m" (for multiple), may be added to provide more detail. Similarly, for Node, "N" is associated with a number (e.g., 0 to 3) to describe whether cancer has been found in the lymph nodes, and can also indicate the number of lymph nodes containing cancer. Larger numbers are assigned when more lymph nodes are involved with cancer. For Metastasis, "M" indicates whether or not the cancer has spread to other parts of the body and is labeled M0 for no spread, or M1 if it has spread.

[0082] The T, N, and M results are combined to determine the stage of cancer, typically one of four stages: stages I (one) to IV (four). Some cancers also have a stage 0 (zero). Stage 0 describes cancer in situ, remaining local to the original tissue without any spread to nearby tissues. Staging may include optional analysis of prognostic factors to provide chances of recovery and a recommended therapy. Prognostic factors may include grading the cancer based on appearance of the cancer cells; analysis of tumor marker expression; and analysis of tumor genetics. A cancer may be restaged using the same initial system in order to determine efficacy of a treatment or obtain more information about a recurrent cancer.

[0083] In some embodiments of the present invention, the melonama is Stage IV melanoma. Stage IV melanoma as defined herein includes the following clinical or pathological stage grouping: any primary tumor (T), any metastasis to a regional lymph node (N), and the presence of a distant metastasis (M). In some embodiments, the Stage IV melanoma in the various forms of the present invention is advanced Stage IV melanoma. Advanced Stage IV melanoma is generally non-resectable or where surgery is not able to be performed, whereas Stage IV melanoma is resectable.

[0084] The melanoma cell lysate in the various forms of the present invention is a lysate that is able to induce an anti-tumour response in the subject, so as to provide an immunotherapeutic effect in the subject. In this regard, the term “lysate” is to be understood to mean the cellular debris and / or fluid produced by lysis of a cell, or an extract, a component including a semi-purified or purified component, or antigen derived from the lysate. The immunotherapeutic approach of the present invention includes administration of one or more of a lysate, an extract of the lysate, a semi-purified or purified component derived from the lysate, and one or more immunotherapeutic antigens derived from the lysate.

[0085] Lysis of a melanoma cell may be achieved by a suitable method, for example, by viral lysis. Methods for producing an immunotherapeutic extract of the lysate, an immunotherapeutic component of the lysate (semi-pruified or purified), or one or more antigens present in the lysate with an immunotherapeutic effect are known in the art.

[0086] In some embodiments of the present invention, the melanoma cell lysate includes fragmented melanoma cell membranes.

[0087] It is appreciated that the lysate in the various forms of the present invention includes either or both of antigens that are, or were derived from, membrane-associated and non- membrane associated antigens. In this regard, the term “membrane-associated” will be understood to mean an antigen that normally is part of a membrane from a melanoma cell, an antigen that is normally bound to a membrane from a melanoma cell, or an antigen that co-purifies with a cell membrane so as to be associated with a cell membrane in the lysate of the present invention.

[0088] In some embodiments of the present invention, the lysate includes membrane- associated antigens.

[0089] In some embodiments, the melanoma cell lysate is an allogeneic cell lysate. For example, in the case of treating a human subject, the melanoma cell lysate may be an allogeneic human melanoma cell lysate.

[0090] In the case where the lysate includes fragmented cell membranes, the presence of fragmented cell membranes in the melanoma cell lysate may be determined by a suitable method known in the art. For example, the presence of fragmented cell membranes in the lysate may be confirmed by microscopic analysis of the lysate.

[0091] In some embodiments, the melanoma cell lysate is a viral melanoma cell lysate. In this regard, the virus used to produce the lysate may be a naturally occurring strain (or a derivative thereof), or may be a recombinant virus. In the case of a recombinant virus, the recombinant virus may also encode one or more gene products. For example, the recombinant virus may encode an immunostimulating molecule such as a cytokine, a hematopoietic growth factor or a melanoma immunogen.

[0092] Methods for producing cell lysates with viruses are known in the art. Generally, to prepare a viral melanoma cell lysate, melanoma cells are infected with a suitable virus resulting in lysis of a proportion of the cells. After a suitable period of time, the lysed cells are collected and processed to produce a lysate suitable for administration to a subject.

[0093] For example, the cells may be collected, homogenised and subject to centrifugation to produce a supernatant. The pellet resulting after centrifugation may be also further processed, such as by being subjected to one or more freeze-thaw cycles, and combined with the earlier supernatant. The resulting supernatant (either from the centrifugation or when combined with the freeze-thawed cells) may then be subjected to a further high-speed centrifugation to sediment the material. The pellet may then be resuspended in saline and the protein and viral content determined. The lysate is then in a form suitable for administration to a subject. The lysate may be further processed to produce an extract of the lysate, a component of the lysate, or purify one or more antigens present in the lysate, each with immunotherapeutic effect.

[0094] In some embodiments, the viral melanoma cell lysate is a vaccinia melanoma cell lysate. Methods for producing cell lysates with vaccinia virus are known in the art.

[0095] Accordingly, in a further aspect the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject a vaccinia melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) administering to the subject one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors.

[0096] In the case where a recombinant virus is used to prepare the cell lysate, the virus may also encode an antigen such as a melanoma immunogen (e.g. MAGE-1 , MAGE-3, BAGE, GAGE, PRAME and NY-ESO-1); melanocyte differentiation antigens (e.g. tyrosinase, Melan- A / MART-1 , gp100, TRP-1 and TRP-2); mutated or aberrantly expressed antigens (e.g. MUM- 1 , CDK4, beta-catenin, gp100 and N-acetylglucosaminyltransferase); and other antigens like B7-1 , TA-90, lysosome-associated membrane protein (LAMP), melanocyte-stimulating hormone receptor (MOIR), and p90 calnexin.

[0097] Methods for producing recombinant viruses encoding a desired gene product are known in the art. Methods for cloning nucleic acids into viral vectors are known in the art, for example as described in Green MR and Sambrook J, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012.

[0098] As discussed above, the melanoma cell lysate may be an allogeneic melanoma cell lysate. However, it will be appreciated that the melanoma cell lysate in the various forms of the present invention may also be a lysate produced (or derived) from melanoma cells or cell lines (primary or secondary) from the subject being treated (i.e. an autologous cell lysate), including melanoma cells derived from the primary tumour or a metastasis resected from the patient.

[0099] Methods for producing an autologous cell lysate are known in the art. For example, a suitable method for isolating a malignant cell suspension for use as an autologous antigen source is by excision of malignant tissue and mechanically disaggregated using a scalpel in RPMI 1640 medium in a Petri dish, a flask, or a vessel. The cell suspension may be rinsed with RPMI 1640 and transferred to a sterile tissue culture plate, RPMI 1640 containing 5½ FCS and antibiotics added and cells cultured in a 37°C, 5½ CO2humidified incubator.

[0100] Primary or secondary cell lines may also be produced from these cells by a method known in the art. Procedures for establishing melanoma cell lines are known in the art.

[0101] In the case of treatment of a human subject with the melanoma cell lysate of the present invention, the melanoma cell lysate may be for example an allogeneic cell line referred to as MM200, as described in Pope et al., 1979, Pathology, 11 : 191-195. This cell line has the HLA type A1 ,3; B7,35; DR2,4. Chromosomal analysis of this cell line showed a nodal number of 76, and a number of marker chromosomes were also revealed by karyotypic analysis (Muir and Gunz, 1979, Pathology, 11 : 597-606). Antigens known to be expressed in MM200 cells are Tyrosinase, gp100; MART-1 ; MAGE-A3; MAGE-A10; BAGE; GAGE; XAGE.

[0102] In some embodiments, the melanoma cell lysate of the present invention includes a lysate (and / or an immunotherapeutic extract, component or antigen thereof) from MM200 cells.

[0103] Other human melanoma cell lines are described in Satyamoorthy et al., 1997, Melanoma Res., 7 Suppl 2: S35-42.

[0104] Human melanoma cells are also available from from the American Tissue Culture Collection. Examples of a non-limiting list of such cells are shown in Table 1.TABLE 1 ATCC Melanoma Cell lines

[0105] In one form, the melanoma cells used to produce the lysate in the various forms of the present invention provide at least one HLA class I antigen.

[0106] In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) is administered to the subject in the absence of an adjuvant. As would be understood in the art, an adjuvant is any agent that acts to boost or augment an immune response with the aim of making the melanoma cell lysate more effective, or more active, in its intended action.

[0107] In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) is administered to the subject in the absence of a carrier, helper protein, or hapten.

[0108] In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), when administered to the subject, may include one or more adjuvants, carriers, helper proteins, haptens, and / or excipients, as described in more detail below. An example of an adjuvant is montanide.

[0109] The administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) to the subject in the various forms of the present invention may be performed by a suitable method known in the art and may be conducted over a suitable period of time.

[0110] In some embodiments, administration is performed using multiple injections administered over a time course which is selected to maximize an immune response in the subject. Accordingly, administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) to the subject may include multiple administrations to the subject during a treatment period.

[0111] In some embodiments of the methods encompassed herein, administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) to the subject includes a treatment period of administration about every one to four weeks for one or more cycles. In some embodiments, administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) to the subject includes a treatment period of administration about every two weeks for one or more cycles. In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) is administered to the subject about every one to four weeks for at least about one to six months. In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) is administered to the subject about every two weeks for at least about three to six months. In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) is administered to the subject about every two weeks for at least about four months. In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) is administered to the subject about every two weeks for at least about seven months. In some embodiments, administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) to the subject includes a treatment period of administration about every two weeks for one or more cycles, followed by a period of administration about once a month for one or more cycles. In some embodiments, administration of the melanoma cell lysate (and / or an immunotherapeutic extract, componentor antigen thereof) to the subject includes a treatment period of administration about every two weeks for one or more cycles, followed by a period of administration about once a month for one or more cycles, followed by a period of administration about once every three months for one or more cycles.

[0112] In some embodiments, the subject receives bi-weekly administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) (e.g. by injection) for about six weeks, then about every month for about six months, then about every three months for about six months. However, any suitable dosing regimen of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) with respect to the methods disclosed herein can be used as would be determined by a skilled physician.

[0113] It will be appreciated that administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) is not to be restricted to injection, and indeed the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) may be administered by any conventional route including parenteral and oral routes, as explained in more detail below. Examples of parenteral routes include subcutaneous, intradermal, transcutaneous, intra-tumoural, intravascular, intravascular, intravenous, intramuscular, intraorbital, intracapsular, intrathecal, intraspinal, intracranial, intraventricular, intracisternal, intranasal and intraperitoneal. In the case of injection, suitable sites of the injection in a human subject are on anterior thighs, anterior upper arms, or the anterior thorax.

[0114] It will also be appreciated that the administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) also includes within its scope ex vivo treatment of the subject’s cells, or a suitably matched donor’s cells, and re- introduction into the subject. For example, antigen presenting cells such as dendritic cells may be isolated from the subject and treated or contacted with the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof). Methods for isolating antigen presenting cells, such as dendritic cells, are known in the art.

[0115] The antigen presenting cells discussed above include antigen presenting cells isolated from a subject, or an antigen presenting cell formed in vitro from a precursor cell. The antigen presenting cells may be present in a mix of one or more types of cell, or alternatively may be substantially purified from other types of cells.

[0116] For example, dendritic cells may be isolated directly from a subject, as a mixture of cells enriched for dendritic cells prepared by leukapheresis or separation of peripheral blood from a subject. Alternatively, the isolated dendritic cells may be prepared by obtaining dendritic cell precursor cells and treating them in vitro to form immature dendritic cells, for example as described in Sallustro et al., 1994, J. Exp. Med., 179: 1109-18.

[0117] The introduction of treated antigen presenting cells into a subject may be by a suitable method known in the art, including the introduction intravenously into the subject. For example, treated dendritic cells may be introduced into a human subject as described in Lau et al., 2001 , J. Immunol., 24(1): 66-78. Typically, 1x106to 1x108dendritic cells will be introduced into a human subject. The time period over which introduction of the treated cells occurs will depend on a number of factors, including the extent of the immune response required, the age and body weight of the subject, and the administration of other active agents to the subject.

[0118] The dosage levels of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) administered to the subject in the various forms of the present invention are not particularly limited and depend on the mode of administration, the characteristics of the subject, and the nature of any carrier or adjuvant that may be included in a formulation.

[0119] It will be appreciated that the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) is administered to the subject in an effective amount. The term “effective amount” as used herein is the quantity of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) which, when administered to a subject, improves the prognosis and / or health state of the subject. The amount of melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) to be administered to a subject will depend on particular characteristics as intimated above, including (but not limited to) one or more of: the extent of the immune response required; the mode of administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof); and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors.

[0120] In the case of a vaccinia melanoma cell lysate, a suitable dose for administration is in the range of 0.1-1 .0 ml lysate. A suitable dose may therefore include, but is not limited to, 0.1-0.2 ml lysate, 0.1-0.3 ml lysate, 0.1-0.4 ml lysate, 0.1-0.5 ml lysate, 0.1-0.6 ml lysate, 0.1- 0.7 ml lysate, 0.1-0.8 ml lysate, 0.1-0.9 ml lysate, 0.1-1 .0 ml lysate, 0.2-0.3 ml lysate, 0.2-0.4 ml lysate, 0.2-0.5 ml lysate, 0.2-0.6 ml lysate, 0.2-0.7 ml lysate, 0.2-0.8 ml lysate, 0.2-0.9 ml lysate, 0.2-1 .0 ml lysate, 0.3-0.4 ml lysate, 0.3-0.5 ml lysate, 0.3-0.6 ml lysate, 0.3-0.7 ml lysate, 0.3-0.8 ml lysate, 0.3-0.9 ml lysate, 0.3-1 .0 ml lysate, 0.4-0.5 ml lysate, 0.4-0.6 ml lysate, 0.4-0.7 ml lysate, 0.4-0.8 ml lysate, 0.4-0.9 ml lysate, 0.4-1 .0 ml lysate, 0.5-0.6 ml lysate, 0.5-0.7 ml lysate, 0.5-0.8 ml lysate, 0.5-0.9 ml lysate, 0.5-1 .0 ml lysate, 0.6-0.7 ml lysate, 0.6-0.8 ml lysate, 0.6-0.9 ml lysate, 0.6-1 .0 ml lysate, 0.7-0.8 ml lysate, 0.7-0.9 ml lysate, 0.7-1 .0 ml lysate, 0.8-0.9 ml lysate, 0.8-1 .0 ml lysate, or 0.9-1.0 i nl lysate. In some embodiments, 0.3 ml of the lysate is administered to the subject per dose.

[0121] As would be appreciated by a person skilled in the art, the term “immunotherapeutic agent” as used herein refers to any therapeutic approaches aimed at mobilising or manipulating a subject’s immune system to treat or cure cancer. It includes targeting tumour cells by recognizing the immunogenic proteins or antigens expressed by said tumour cells, which can be accomplished by utilising either passively transferred immune molecules such as antibodies, or preparations designed to induce antibodies or T lymphocytes (T cells) recognising a localised region of an antigen or an epitope specific to the tumor cell. Examples of immunotherapeutic agents may therefore include, but are not limited to, immune checkpoint inhibitors, immunomodulators, activation immunotherapies, suppression immunotherapies, CAR T-cell therapies, and the like.

[0122] Immunomodulators may include interleukins (e.g. IL-2, IL-7, IL-12, IL-15, IL-17 and IL- 21), cytokines (e.g. interferons, and G-CSF), chemokines (e.g. CCL3, CCL26 and CXCL7), immunomodulatory imide agents (IMiDs), and others such as cytosine phosphate-guanosine, oligodeoxynucleotides, and glucans. Other immunomodulators are contemplated. Activation immunotherapies may include dendritic cell-based pump-priming or vaccination, and T-cell adoptive transfer, while suppression immunotherapies may include the use of immunosuppressive drugs and immune tolerance therapries.

[0123] Immune checkpoints are a normal part of the immune system. Generally, their role is to prevent an immune response from being too strong and destroying healthy cells in the body. As part of an effort to harness a cancer patient's own immune system to fight his / her cancer, immunotherapy drugs that belong to a category of immune checkpoint inhibitorshave been developed. Those drugs generally work by blocking immune checkpoint proteins, therefore preventing an inhibition of the immune response to cancer cells. For example, administration of immune checkpoint inhibitors may allow T cells to kill cancer ceils. Inhibition of immune checkpoints in cancer patients may also result in inducing the cancer patient’s B cells to generate antibodies against the cancer ceils.

[0124] The term “immune checkpoint protein” is known in the art. Within the known meaning of this term it will be clear to the person skilled in the art that on the level of “immune checkpoint proteins” the immune system provides inhibitory signals to its components in order to balance immune reactions. Known immune checkpoint proteins may comprise PD1 (also known as PD-1 and Programmed Death 1 receptor) and its ligands PD-L1 and PD-L2, CTLA-4 (Cytotoxic T-lymphocyte-Associated protein, CD152), LAG-3 (Lymphocyte Activation Gene-3), OX40, A2AR (Adenosine A2A Receptor), BTLA (B and T Lymphocyte Attenuator, CD272), B7-H3 (CD276), B7-H4 (VTCN1), IDO (Indoleamine 2,3-dioxygenase), TIM3 (T-cell Immunoglobulin domain and Mucin domain 3), KIR (Killer-cell Immunoglobulin-like Receptor), VISTA (V-domain Ig Suppressor of T cell Activation), and IL-2R (lnterleukin-2 Receptor). The pathways involving LAG3, BTLA, B7H3, B7H4, TIM3, and KIR are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012, Nature Rev. Cancer, 12: 252-264; and Mellman et al., 2011 , Nature, 480: 480-489).

[0125] Accordingly, as used herein, an “immune checkpoint inhibitor” is any agent inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. The designation “immune checkpoint” includes the experimental demonstration of stimulation of an antigen-receptor triggered T lymphocyte response by inhibition of the immune checkpoint protein in vitro or in vivo, e.g. mice deficient in expression of the immune checkpoint protein demonstrate enhanced antigen-specific T lymphocyte responses or signs of autoimmunity (such as disclosed in Waterhouse et al., 1995, Science, 270: 985-988; Nishimura et al., 1999, Immunity, 11 : 141-151). It may also include demonstration of inhibition of antigen-receptor triggered CD4+ or CD8+ T cell responses due to deliberate stimulation of the immune checkpoint protein in vitro or in vivo (e.g. Zhu et al., 2005, Nature Immunol. 6: 1245-1252).

[0126] Examples of immune checkpoint inhibitors include antibodies that specifically recognize immune checkpoint proteins. A number of PD1 , PD-L1 , PD-L2, CTLA-4, LAG-3, OX40, A2AR, BTLA, B7-H3, B7-H4, IDO, TIM3, KIR, and IL-2R inhibitors are known and inanalogy of these known immune checkpoint inhibitors, alternative immune checkpoint inhibitors may be developed in the future.

[0127] Examples of PD-1 inhibitors include humanized antibodies blocking human PD-1 , including but not limited to Pembrolizumab (Keytruda) (e.g. disclosed as hPD109A and its humanized derivatives h409A11 , h409A16 and h409A17 in WO2008 / 156712; Hamid et al., N. Engl. J. Med., 369: 134-144 2013), Cemiplimab (Libtayo), Pidilizumab (CureTech) (disclosed in Rosenblatt et al., 2011 , J. Imm another., 34: 409-18), and MEDI-0680 (AstraZeneca, previously known as AMP-514).

[0128] PD-1 inhibitors may also include fully human antibodies including but not limited to Nivolumab (Opdivo) (previously known as MDX-1106 or BMS-936558, Topalian et al., 2012, N. Eng. J. Med., 366: 2443-2454, disclosed in US Patent 8,008,449). Other PD-1 inhibitors may include presentations of soluble PD-1 ligand including without limitation PD-L2 Fc fusion protein also known as B7-DC-lg or AMP-244 (disclosed in Mkrtichyan M, et al. 2012, J. Immunol., 189: 2338-2347) and other PD-1 inhibitors presently under investigation and / or development for use in therapy.

[0129] Examples of PD-1 L inhibitors may include without limitation humanized or fully human antibodies such as Atezolizumab (MPDL3280A), Avelumab (MSB0010718C), Durvalumab (Imfinzi; MEDI-4736), BMS-936559 / MDX-1105 (Bristol-Myers Squibb), and other PD-L1 inhibitors presently under investigation.

[0130] Examples of CTLA-4 inhibitors include, but are not limited to, Tremelimumab (AstraZeneca; referenced in Ribas et al., 2013, J. Clin. Oncol. 31 : 616-22), and Ipilimumab (Yervoy; Bristol-Myers Squibb),

[0131] An Example of a LAG-3 inhibitor includes, but is not limited to, Relatlimab (Bristol- Myers Squibb; BMS-986016). An Example of a B7-H3 inhibitor includes, but is not limited to, MGA271 (Macrogenics). An Example of a KIR inhibitor includes, but is not limited to, Lirilumab (Bristol-Myers Squibb; IPH2102). An Example of an OX40 inhibitor includes, but is not limited to, MEDI-6469 (Medlmmune). An Example of an IL-2R inhibitor, for preferentially depleting Treg cells (e.g., FoxP-3+ CD4+ cells), comprises IL- 2-toxin fusion proteins, which include, but are not limited to, denileukin diftitox (Ontak; Eisai).

[0132] Accordingly, in some embodiments, the immune checkpoint inhibitor is selected from one or more of the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, an OX40 inhibitor, an A2AR inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, an IDO inhibitor, a TIM3 inhibitor, a KIR inhibitor, and an IL-2R inhibitor. Examples of such inhibitors are described above. Known inhibitors of these immune checkpoint proteins may be used as such or analogues may be used, in particular chimerized, humanized or human forms of antibodies, as described above.

[0133] As the person skilled in the art will know, alternative and / or equivalent names may be in use for certain antibodies mentioned above. Such alternative and / or equivalent names are interchangeable in the context of the present invention. For example, it is known that Pembrolizumab is also known under the alternative and equivalent names MK-3475 and Lambrolizumab.

[0134] In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject prior to administration of the one or more immunotherapeutic agents to the subject. For example, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject every two weeks for one or more cycles, prior to administration of the one or more immunotherapeutic agents to the subject. In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof is administered to the subject every two weeks for at least three months, every two weeks for at least six months, or every two weeks for about seven months, prior to administration of the one or more immunotherapeutic agents to the subject.

[0135] Accordingly, in a further aspect the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) subsequently administering to the subject one or more immunotherapeutic agents.

[0136] In some embodiments, the one or more immunotherapeutic agents are administered to the subject about one week to about six months or more after administration of the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof. For example, the one or more immunotherapeutic agents are administered to the subjectabout 1 , 2, or 3 weeks, or about 1 , 2, 3, 4, or 5 months after administration of the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0137] The dosage and frequency of dose of the immunotherapeutic agent(s) administered to the subject may vary depending on the half-life of the agent in the subject. It would be understood by a person skilled in the art that such guidelines would be adjusted for the molecular weight of the agent, the clearance from the blood, the mode of administration, and other pharmacokinetic parameters. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, subcutaneous, etc., or for systemic administration.

[0138] In some embodiments, the immunotherapeutic agent(s) are administered to the subject about once every one to about eight weeks or more for up to about one year or more. The administration may be continuous, or may be interrupted by one or more breaks depending on how the subject reacts to the treatment and any associated side-effects observed.

[0139] In some embodiments, the immunotherapeutic agent is an immune check-point inhibitor selected from one or more of Pembrolizumab, Nivolumab and Ipilimumab. Accordingly, in a further aspect, the present invention provides a method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) subsequently administering to the subject Pembrolizumab.

[0140] In some embodiments, the Pembrolizumab is administered to the subject about one month after administration of the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof. In some embodiments, the Pembrolizumab is administered about once every three weeks for up to about one year.

[0141] In some embodiments, the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof is administered to the subject after administering the one or more immunotherapeutic agents to the subject. Accordingly, in a further aspect the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more immunotherapeutic agents; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0142] In a further aspect, the present invention provides a method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject Nivolumab and Ipilimumab; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0143] As used herein, a cancer cell-specific inhibitor refers to a molecule that can specifically target cancer cells but spare normal (non-cancerous) cells. Cancer cell-specific inhibitors can therefore be distinguished from chemotherapeutic agents given that chemotherapeutic agents are unable to distinguish between cancer cells and normal cells, resulting in significant toxicity and side effects to the subject being treated. Cancer cell- specific inhibitors can be broadly classified into two categories, namely small molecules and macromolecules (e.g. monoclonal antibodies, polypeptides, antibody-drug conjugates, and nucleic acids). Accordingly, in some embodiments the cancer cell-specific inhibitor is a small molecule.

[0144] Examples of small molecule cancer cell-specific inhibitors include, but are not limited to, kinase inhibitors (such as receptor tyrosine kinase inhibitors, non-receptor tyrosine kinase inhibitors, and serine / threonine kinase inhibitors), epigenetic inhibitors, hedgehog pathway inhibitors, proteasome inhibitors, and poly (ADP-ribose) polymerase (PARP) inhibitors.

[0145] Protein kinases are a type of enzyme that catalyze the transfer of y-phosphate groups from ATP to protein residues containing hydroxyl groups. They have an important role in cell growth, proliferation, and differentiation. The human kinome comprises ~535 protein kinases which can be classified as tyrosine kinases (including both receptor and non-receptor tyrosine kinases), serine / threonine kinases, and tyrosine kinase-like enzymes, according to their substrate residues.

[0146] Examples of receptor tyrosine kinase inhibitors include anaplastic lymphoma kinase (ALK) inhibitors, cellular-mesenchymal-epithelial transition factor (c-Met) inhibitors, c-Kit (CD117) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, Fms-like tyrosine kinase 3 (FLT3) inhibitors, vascular endothelial growth factor receptor (VEGFR) inhibitors, fibroblast growth factor receptor (FGFR) inhibitors, platelet-derived growth factor receptor(PDGFR) inhibitors, and tropomyosin receptor kinase (TRK) inhibitors. In some embodiments, a receptor tyrosine kinase inhibitor may be selected from the group consisting of crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, capmatinib, tepotinib, gefitinib, erlotinib, lapatinib, icotinib, afatinib, osimertinib, neratinib, dacomitinib, almonertinib, tucatinib, midostaurin, gilteritinib, quizartinib, pexidartinib, sorafenib, sunitinib, pazopanib, vandetanib, axitinib, cabozantinib, regorafenib, apatinib, Lenvatinib, tivozanib, fruquintinib, nintedanib, anlotinib, erdafitinib, pemigatinib, avapritinib, ripretinib, selpercatinib, pralsetinib, Larotrectinib and entrectinib. Other receptor tyrosine kinase inhibitors are contemplated.

[0147] Examples of non-receptor tyrosine kinase inhibitors include Bcr-Abl1 inhibitors, Bruton’s agammglobulinemia tyrosine kinase (BTK) inhibitors, and janus kinase (JAK) inhibitors. In some embodiments, a non-receptor tyrosine kinase inhibitor may be selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, radotinib, ponatinib, ibrutinib, acalabrutinib, zanubrutinib, ruxolitinib, and fedratinib. Other non-receptor tyrosine kinase inhibitors are contemplated.

[0148] Examples of serine / threonine kinase inhibitors include BRAF inhibitors, MEK inhibitors, ERK inhibitors, cyclin-dependent kinase (CDK) inhibitors, phosphatidylinositol 3- kinase (PI3K) inhibitors, AKT inhibitors, and mammalian target of rapamycin (mTOR) inhibitors. In some embodiments, a serine / threonine kinase inhibitor may be selected from the group consisting of dabrafenib, trametinib, vemurafenib, encorafenib, cobimetinib, binimetinib, selumetinib, Palbociclib, ribociclib, abemaciclib, idelalisib, copanlisib, duvelisib, alpelisib, temsirolimus, everolimus, and sirolimus. Other serine / threonine kinase inhibitors are contemplated.

[0149] Examples of epigenetic inhibitors include enhancer of zeste homolog 2 (EZH2) inhibitors, histone deacetylase (HDAC) inhibitors, and isocitrate dehydrogenase 1 / 2 (IDH1 / 2) inhibitors. In some embodiments, an epigenetic inhibitor may be selected from the group consisting of tazemetostat, vorinostat, romidepsin, belinostat, tucidinostat, panobinostat, enasidenib, and ivosidenib. Other epigenetic inhibitors are contemplated.

[0150] Examples of hedgehog pathway inhibitors include venetoclax, vismodegib, sonidegib, glasdegib, bortezomib, carfilzomib, ixazomib, olaparib, rucaparib, niraparib and talazoparib. Other hedgehog pathway inhibitors are contemplated.

[0151] Examples of proteasome inhibitors include bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, and delanzomib. Other proteasome inhibitors are contemplated.

[0152] Examples of PARP inhibitors include Nicotinamide, Olaparib, rucaparib, niraparib, talazoparib, pamiparib, veliparib, iniparib, amelparib, and fluzoparib. Other PARP inhibitors are contemplated.

[0153] In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject after administration of the one or more cancer cell-specific inhibitors to the subject. Accordingly, in a further aspect the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more cancer cell-specific inhibitors; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0154] In some embodiments, the one or more cancer cell-specific inhibitors are administered to the subject about one week to about six months or more before administration of the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof. For example, the one or more cancer cell-specific inhibitors are administered to the subject about 1 , 2, or 3 weeks, or about 1 , 2, 3, 4, or 5 months before administration of the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0155] In some embodiments, the one or more cancer cell-specific inhibitors are dabrafenib and trametinib. Accordingly, in a further aspect the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject dabrafenib and trametinib; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0156] The dosage, length of dosing, and frequency of dosing of the cancer cell-specific inhibitor(s) may vary depending on the half-life of the inhibitor in the subject. It would be understood by a person skilled in the art that such guidelines would be adjusted for the molecular weight of the inhibitor, the clearance from the blood, the mode of administration,and other pharmacokinetic parameters. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, subcutaneous, etc., or for systemic administration.

[0157] In some embodiments, the one or more cancer cell-specific inhibitors are administered to the subject daily. Depending on the nature of the cancer cell-specific inhibitor, the inhibitor may be administered more than once per day. In some embodiments, the one or more cancer cell-specific inhibitors are administered to the subject once or more daily for a period of time, followed by no administration for a period of time (i.e. an “on-off’ dosing schedule). In some embodiments, the dosing schedule of the one or more cancer cell-specific inhibitors is 3 days on, 7 days off; 5 days on, 5 days off; or 6 days on, 4 days off.Other dosing schedules are contemplated as would be directed by a skilled physician.

[0158] In some embodiments, the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject after administration of the one or more immunotherapeutic agents and after administration of the one or more cancer cell-specific inhibitors to the subject.

[0159] Accordingly, in a further aspect the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more immunotherapeutic agents; and(ii) subsequently administering to the subject one or more cancer cell-specific inhibitors; and(iii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0160] In some embodiments, the immunotherapeutic agents are the immune checkpoint inhibitors Nivolumab and Ipilimumab, and the cancer cell-specific inhibitors are dabrafenib and trametinib. Accordingly, in a further aspect the present invention provides a method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject Nivolumab and Ipilimumab; and(ii) subsequently administering to the subject dabrafenib and trametinib; and(iii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0161] In some embodiments, the one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors may be administered to the subject over one or morecycles coordinated with the cyclic administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) as described above. For example, the subject may receive a dose of an immunotherapeutic agent and / or a cancer cell-specific inhibitor, then a dose of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), etc, repeated in a cyclic manner. In this instance, the immunotherapeutic agent, the cancer cell-specific inhibitor, and the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) are administered separately and at discreet time points from each other.

[0162] In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) may be administered to the subject at the same time as the subject is also receiving treatment with one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors. For example, the subject may receive a dose of the melanoma cell lysate (and / or an immunotherapeutic extract, component, or antigen thereof), at the same time as receiving immunotherapeutic agent and / or cancer cell-specific inhibitor treatment. The melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) may still be administered cyclicly as described above, but would be coordinated with the concurrent treatment with a dose of the immunotherapeutic agent(s) and / or the cancer cell-specific inhibitor(s). In this instance, the immunotherapeutic agent(s), the cancer cell-specific inhibitor(s), and the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), may be administered separately but are administered at the same time points as each other.

[0163] In some embodiments, the one or more cancer cell-specific inhibitors are also administered to the subject during a period in which the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof is administered to the subject. Accordingly, in a further aspect the present invention provides a method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more immunotherapeutic agents; and(ii) subsequently administering to the subject one or more cancer cell-specific inhibitors; and(iii) subsequently administering to the subject one or more cancer cell-specific inhibitors and a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0164] In some embodiments, the one or more immunotherapeutic agents are administeredto the subject about one week to about six months or more before administration of the one or more cancer cell-specific inhibitors. For example, the one or more immunotherapeutic agents are administered to the subject about 1 , 2, or 3 weeks, or about 1 , 2, 3, 4, or 5 months before administration of the one or more cancer cell-specific inhibitors.

[0165] In some embodiments, the immunotherapeutic agents are the immune checkpoint inhibitors Nivolumab and Ipilimumab, and the cancer cell-specific inhibitors are dabrafenib and trametinib. Accordingly, in a further aspect, the present invention provides a method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject Nivolumab and Ipilimumab; and(ii) subsequently administering to the subject dabrafenib and trametinib; and(iii) subsequently administering to the subject dabrafenib and trametinib, and a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0166] It will be appreciated that the one or more immunotherapeutic agents and / or cancer cell-specific inhibitors are administered to the subject in an effective amount. The term “effective amount” in this regard has been described above with respect to the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof). For example, particular characteristics dictating an effective amount of the agent or inhibitor to be administered include (but are not limited to) the mode of administration, the chemical nature, structure and size of the agent or inhibitor, relevant pharmacokinetic properties of the agent or inhibitor, and the characteristics of the subject as indicated above. A person skilled in the art will be able to determine appropriate dosages for each agent or inhibitor depending on these and other factors.

[0167] The manner in which the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) and the immunotherapeutic agent(s) and / or cancer cell- specific inhibitor(s) are administered to the subject is not limited to a particular mode, and indeed the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) may be administered in the same or a different mode to which the agent or inhibitor is administered. In this regard, one route of administration may be systemic administration and therefore the lysate, agent and / or inhibitor may be in the form of separate injectable solutions, or may be in another dosage form useful for systemic administration of agents. Another route of administration may include topical administration and therefore the lysate, agent and / or inhibitor, or a composition comprising the lysate, agent or inhibitor, may be in the form of a separate liquid, gel, suspension, paste, lotion, cream, solid, semi-solid,powder, and the like. Other forms of administration may include delivery by way of a scaffold, such as a biomaterial scaffold including a scaffold produced from collagen, hydroxyapatite, β- tricalcium phosphate or a combination thereof. Other routes of administration are contemplated.

[0168] The melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) and the immunotherapeutic agent(s) and / or cancer cell-specific inhibitor(s) may be administered alone or each may be delivered in the form of a suitable pharmaceutical composition, for example in a mixture with other therapeutic substances and / or other substances that enhance, stabilise or maintain the activity of each active component of the pharmaceutical composition. In some embodiments, an administration vehicle (e.g., injectable solution, liquid, gel, paste, powder, cream, pill, tablet, capsule, aerosol, etc) would contain the active component or a pharmaceutical composition comprising the active component and / or additional substance(s). In this regard, the pharmaceutical composition may also include the use of one or more pharmaceutically acceptable carriers or additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into consideration the particular physical and chemical characteristics of the active component to be administered.

[0169] In some embodiments, the carrier may be chosen based on various considerations including the route of administration, and the time course of delivery of the pharmaceutical composition. The term “pharmaceutically acceptable carrier” refers to a substantially inert solid, semi-solid or liquid filler, diluent, excipient, encapsulating material or formulation auxiliary of any type. An example of a pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known in the art. Some examples of materials which can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN 80; buffering agents such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; and phosphate buffer solutions, as well as other non- toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well ascolouring agents, releasing agents, coating agents, sweetening, flavouring and perfuming agents, preservatives and antioxidants can also be present.

[0170] The preparation of such pharmaceutical compositions is known in the art, for example as described in Remington’s Pharmaceutical Sciences, 18thed., 1990, Mack Publishing Co., Easton, Pa. and U.S. Pharmacopeia: National Formulary, 1984, Mack Publishing Company, Easton, Pa, which are incorporated herein by reference in their entirety.

[0171] As intimated above, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), the immunotherapeutic agent(s), the cancer cell-specific inhibitor(s), or a pharmaceutical composition comprising one of said agents, may be formulated for parenteral administration. The term “parenteral” as used herein includes, but is not limited to, subcutaneous, intradermal, transcutaneous, intravascular, intravenous, intramuscular, intraorbital, intracapsular, intrathecal, intraspinal, intracranial, intraventricular, intracisternal, intranasal and intraperitoneal injection or infusion techniques.

[0172] When administered parenterally, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), the immunotherapeutic agent(s), the cancer cell-specific inhibitor(s), or a pharmaceutical composition comprising one of said agents, will normally be in a unit dosage, sterile injectable, form (solution, suspension or emulsion) which is preferably isotonic with the blood of the recipient with a pharmaceutically acceptable carrier. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents, for example, as solutions in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, saline, Ringer’s solution, dextrose solution, isotonic sodium chloride solution, and Hanks’ solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil may be employed including synthetic mono- or di- glycerides, corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

[0173] The carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example anti-oxidants, buffers and preservatives.

[0174] In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), the immunotherapeutic agent(s), the cancer cell- specific inhibitor(s), or a pharmaceutical composition comprising one of said agents, may be formulated for topical administration, e.g. transdermal administration. Transdermal administrations are understood to include all administrations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administrations may be carried out using a liquid, gel, paste, lotion, cream, ointment, powder, foam, patch, suspension, solution, or other suitable formulation.

[0175] A cream is a formulation that contains water and oil and is stabilized with an emulsifier. Lipophilic creams are called water-in-oil emulsions, and hydrophilic creams oil-in- water emulsions. The cream base for water-in-oil emulsions are normally absorption bases such as aseline, ceresin or lanolin. The bases for oil-in-water emulsions are mono-, di-, and tri-glycerides of fatty acids or fatty alcohols with soaps, alkyl sulphates or alkyl polyglycol ethers as emulsifiers.

[0176] A lotion is an opaque, thin, non-greasy emulsion liquid dosage form for external application to the skin, which generally contains a water-based vehicle with greater than 50½ of volatiles and sufficiently low viscosity that it may be delivered by pouring. Lotions are usually hydrophilic and contain greater than 50½ of volatiles as measured by LOD (loss on drying). A lotion tends to evaporate rapidly with a cooling sensation when rubbed onto the skin.

[0177] A paste is an opaque or translucent, viscous, greasy emulsion or suspension semisolid dosage form for external application to the skin, which generally contains greater than 50½ of hydrocarbon-based or a polyethylene glycol-based vehicle and less than 20½ of volatiles. A paste contains a large proportion (20-50½) of dispersed solids in a fatty or aqueous vehicle.

[0178] An ointment is an opaque or translucent, viscous, greasy emulsion or suspension semisolid dosage form for external application to the skin, which generally contains greater than 50½ of hydrocarbon-based or a polyethylene glycol-based vehicle and less than 20½ ofvolatiles. An ointment is usually lipophilic and contains >50½ of hydrocarbons or polyethylene glycols as the vehicle and <20½ of volatiles as measured by LOD. An ointment tends not to evaporate or be absorbed when rubbed onto the skin.

[0179] A gel is usually a translucent, non-greasy emulsion or suspension semisolid dosage form for external application to the skin, which contains a gelling agent in quantities sufficient to impart a three-dimensional, cross-linked matrix. A gel is usually hydrophilic and contains sufficient quantities of a gelling agent such as starch, cellulose derivatives, carbomers, magnesium-aluminum silicates, xanthan gum, colloidal silica, aluminium or zinc soaps.

[0180] The melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), the immunotherapeutic agent(s), the cancer cell-specific inhibitor(s), or a pharmaceutical composition comprising one of said agents, when in a form for topical administration, may further include drying agents, anti-foaming agents, buffers, neutralizing agents, agents to adjust pH, colouring agents and decolouring agents, emollients, emulsifying agents, emulsion stabilizers and viscosity builders, humectants, odorants, preservatives, antioxidants, and chemical stabilizers, solvents, and thickening, stiffening, and suspending agents, and a balance of water or solvent.

[0181] Transdermal administration may also be accomplished through the use of a transdermal patch containing the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), or the immune checkpoint inhibitor, and a carrier that is inert to the active agents, is non-toxic to the skin, and allows delivery of the active agents for systemic absorption into the blood stream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active agents may also be suitable. A variety of occlusive devices may be used to release the active agents into the blood stream such as a semi-permeable membrane covering a reservoir containing the active agents with or without a carrier, or a matrix containing the active agents. Transdermal formulations are known in art and may be formulated by a skilled person.

[0182] The melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), the immunotherapeutic agent(s), the cancer cell-specific inhibitor(s), or a pharmaceutical composition comprising one of said agents, may also be formulated usingcontrolled release technology. For example, they may be administered as a sustained- release pharmaceutical. To further increase the sustained release effect, they may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty acid esters such as ethyl oleate; glycerol monooleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof (weight average molecular weight: ca. 80,000 to 2,000,000), carboxymethylcellulose sodium (weight average molecular weight: ca. 20,000 to 400,000), hydroxypropylcellulose (viscosity in 2½ aqueous solution: 3 to 4,000 cps), atherocollagen (weight average molecular weight: ca. 300,000), polyethylene glycol (weight average molecular weight: ca. 400 to 20,000), polyethylene oxide (weight average molecular weight: ca. 100,000 to 9,000,000), hydroxypropylmethylcellulose (viscosity in 1½ aqueous solution: 4 to 100,000 cSt), methylcellulose (viscosity in 2½ aqueous solution: 15 to 8,000 cSt), polyvinyl alcohol (viscosity: 2 to 100 cSt), polyvinylpyrrolidone (weight average molecular weight: 25,000 to 1 ,200,000).

[0183] Alternatively, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), the immunotherapeutic agent(s), the cancer cell-specific inhibitor(s), or a pharmaceutical composition comprising one of said agents, may be incorporated into a hydrophobic polymer matrix, scaffold or support (such as a biodegradable matrix or support), including for controlled release of the active agents over a period of days. Methods for delivering agents via scaffolds are known in the art. For example, a biomaterial scaffold including a scaffold produced from collagen, hydroxyapatite, β-tricalcium phosphate or a combination thereof may be used to deliver the active agent. Alternatively, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) the immunotherapeutic agent(s), the cancer cell-specific inhibitor(s), or a pharmaceutical composition comprising one of said agents, may be incorporated into a basement membrane matrix, such as that secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. Such a matrix is sold under the trade name Matrigel™ which resembles the laminin / collagen IV-rich basement membrane extracellular environment found in many tissues. Methods for incorporating agents into such substrates are known in the art.

[0184] The melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), the immunotherapeutic agent(s), the cancer cell-specific inhibitor(s), or a pharmaceutical composition comprising one of said agents, may also be moulded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of thecomposition over a prolonged period of time without the need for frequent re-dosing. Such controlled release films are well known in the art. Other examples of polymers commonly employed for this purpose that may be used include nondegradable ethylene-vinyl acetate copolymer or degradable lactic acid-glycolic acid copolymers which may be used externally or internally. Certain hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.

[0185] The carrier may also be a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time release characteristics and release kinetics. The melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), the immunotherapeutic agent(s), the cancer cell-specific inhibitor(s), or a pharmaceutical composition comprising one of said agents, may then be moulded into a solid implant suitable for providing efficacious concentrations of the active agent over a prolonged period of time without the need for frequent re-dosing. These can be incorporated into the biodegradable polymer or polymer mixture in any suitable manner known to one of skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be moulded into a solid implant.

[0186] The treatment of melanoma in the subject in the various forms of the present invention may also include one or more other interventions, including for example chemotherapy, radiation therapy, and / or surgery (such as surgical resection of primary or secondary tumours).

[0187] In the case where surgical interventions are used, the treatment methods encompassed by the present invention may include the further step of resecting metastases from the subject. In some embodiments, the metastases are resected prior to administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof). In some embodiments, the metastases are resected after administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) but before administering the immunotherapeutic agent(s) and / or cancer cell-specific inhibitor(s) to the subject. In some embodiments, the metastases are resected before or after administration of the immunotherapeutic agent(s) and / or cancer cell-specific inhibitors). Metastatic involvement of organs may include skin, subcutaneous tissues, muscle, lung, liver, brain, bowel, spleen and bone.

[0188] Examples of chemotherapeutic agents would be known in the art. For example, suitable chemotherapeutic agents could include DTIC (Dacarbazine), Temozolomide (Temodal®), and Fotemustine. Suitable dosages are Dacarbazine (DTIC) 850mg / m23- weekly intravenously; Fotemustine 100mg / m2weekly intravenously for 3 weeks, then 4 weekly thereafter; Temazolamide 200mg / m2orally day 1-5 initially, then continued if tolerated well. Other dosages and dosing regimens are contemplated and would ultimately be dictated by a skilled physician.

[0189] In some embodiments, the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) may be administered to the human subject two or more weeks prior to an administration of a chemotherapeutic agent. This is done so that an induction of an immune response in the patient may be achieved prior to the reduction in leukocytes due to treatment with the chemotherapeutic agent.

[0190] The method of treating the subject in the various forms of the present invention may also include the step of selecting a subject suitable for treatment. In one form, the subject is selected for treatment on the basis that the subject shows a T cell response to a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) prior to or after administration of the immunotherapeutic agent(s) and / or cancer cell-specific inhibitor(s).

[0191] Methods for assessing whether a subject shows a T cell response to a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, are known in the art. An antigen of the cell lysate will be understood to mean an antigen normally present in the melanoma cell lysate that is involved in the induction of an immune response to a melanoma in the subject.

[0192] Accordingly, in one aspect the present invention provides a method of selecting a subject with Stage IV melanoma suitable for treatment with the method of treatment as described herein, the method including the step of identifying a subject that shows a T cell response upon administration to the subject of a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

[0193] The subject may also be selected on the basis that the subject has a decreased level and / or activity of regulatory T cells upon administering to the subject a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) prior to or after administration of the immunotherapeutic agent(s) and / or cancer cell-specific inhibitor(s).

[0194] Accordingly, in one aspect the present invention provides a method of selecting a subject with melanoma suitable for treatment with the method of treatment as described herein, the method including the step of identifying a subject that has a decreased level and / or activity of regulatory T cells upon administration to the subject of a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof. Regulatory T cells are CD4+CD25+ T cells (reviewed in Terabe and Berzofsky, 2004, Curr. Opin. Immunol., 16(2): 157-162). Methods for assessing the level and / or activity of regulatory T cells are known in the art.

[0195] Additional agents may also be used in the various forms of the present invention in conjunction with the administration of the melanoma cell lysate. Such agents include for example interferons (e.g. a-interferon), and interleukins (e.g. IL-2).

[0196] As indicated above, the present invention may be used to inhibit, prevent and / or slow the formation and / or growth of secondary tumours in a subject. Therefore, the present invention may also be used to inhibit or prevent metastases in the subject. The extent of formation and growth of primary and secondary tumours in a subject may be monitored by a method known in the art. Monitoring methods include clinical examination and measurement, U / S scans, CT scans, chest X ray, bone X ray, MRI scans, PET scans, and bone scans.

[0197] The aforementioned methods of the present invention may also be used to inhibit or prevent the growth of the primary tumour in a subject, including reducing the growth of the primary tumour after resection or other treatment.

[0198] The aforementioned methods of the present invention may also be used to improve the rate of survival of a subject with a Stage IV melanoma. An improved rate of survival can be measured, for example, as an improved survival rate after a given amount of time (e.g. 2 to 10 years or longer), or an improved median survival time. Methods for determining the improvement in survival rate are known in the art.

[0199] The aforementioned methods of the present invention may also be used to improve the life expectancy of a subject. The aforementioned methods of the present invention may also be used to improve the outcome for a subject suffering from melanoma, and / or improve the quality of life of the subject. Methods for assessing outcome and quality of life parameters are known in the art. The aforementioned methods of the present invention mayalso be used to induce an anti-tumour immune response in a subject. Methods for assessing an anti-tumour response in a subject are known in the art.

[0200] Induction of anti-melanoma immunity may be determining by a delayed type hypersensitivity (DTH) response against melanoma antigens prior to and at periods after administration of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), one or more immunotherapeutic agents, and / or one or more cancer cell- specific inhibitors In addition, serum and peripheral blood lymphocytes may be obtained prior to, and at periods after, lysate or inhibitor administration to test the induction of anti- melanoma immunity by cytotoxicity assay, CTL precursor frequency (CTLp) assay and phenotypic analysis of lymphocytes.

[0201] In some embodiments of the aforementioned aspects of the present invention, the melanoma cell lysate includes fragmented melanoma cell membranes. In some embodiments, the melanoma cell lysate is a viral melanoma cell lysate. In some embodiments, the viral melanoma cell lysate is a vaccinia melanoma cell lysate.

[0202] In some embodiments of the aforementioned aspects of the present invention, the treatment method is capable of producing one or more of an increase in the number of intratumoral dendritic cells in the subject, an improvement in dendritic cell activation in the subject, and a modulation (i.e. increase or decrease) in the number and / or activity of regulatory T cells in the subject. Assays to determine these treatment outcomes would be known in the art.

[0203] The terms “increase” and “improve” and the like as used throughout the specification are to be understood to mean a greater level in the progress of a process, including any one or more of the start, continuation or termination of a process. For example, the terms “increase”, and “improve” can refer to an at least 1½, 5½, 10½, 20½, 30½, 40½, 50½, 60½, 70½, 80½, 90½, 100½, or greater, or by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6.0-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60- fold, 70-fold, 80-fold, 90-fold, 100-fold, 125-fold, 150-fold, 175-fold, 200-fold, 225-fold, 250- fold, 275-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 10,000-fold, 100,000-fold, or greater, level when compared to the level of intratumoral dendritic cells or dendritic cell activation in a subject which has not been treated using the method of the present invention, or when compared to the number and / or activity of regulatory T cells in a subject which has not been treated using the method of the present invention.

[0204] Similarly, the term “decrease” refers to an at least 1½, 5½, 10½, 20½, 30½, 40½, 50½, 60½, 70½, 80½, 90½, 100½, or greater, or by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6.0- fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 125-fold, 150-fold, 175-fold, 200-fold, 225- fold, 250-fold, 275-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 10,000-fold, 100,000-fold, or less, number and / or activity when compared to the number and / or activity of regulatory T cells in a subject which has not been treated using the method of the present invention.

[0205] In a further aspect, the present invention provides a combination product including the following components:(i) a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof; and(ii) one or more immunotherapeutic agents and / or one or more cancer cell- specific inhibitors, wherein the components are provided in a form for separate administration to a subject.

[0206] In the context of the present invention, the “combination product” may be in the form of a kit which includes each component.

[0207] In some embodiments, the combination product is used to treat or prevent melanoma, including Stage IV melanoma, in a subject. In some embodiments, the combination product may also be used to inhibit growth of a primary tumour in a subject, inhibit formation and / or growth of a secondary tumour in a subject, to improve the rate of survival of a subject with melanoma, to improve the life expectancy of a subject with melanoma, and / or to induce an anti-tumour immune response in a subject with melanoma.

[0208] Accordingly, in further aspects the present invention provides a combination product for use in any one or more of: treating or preventing melanoma, including Stage IV melanoma, in a subject; inhibiting growth of a primary tumour in a subject; inhibiting formation and / or growth of a secondary tumour in a subject; improving the rate of survival of a subject with melanoma; improving the life expectancy of a subject with melanoma; and inducing an anti-tumour immune response in a subject with melanoma, wherein the combination product includes the following components:(i) a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof; and(ii) one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors, and wherein the components are provided in a form for separate administration to the subject.

[0209] In further aspects, the present invention provides use of a combination product for any one or more of: treating or preventing melanoma, including Stage IV melanoma, in a subject; inhibiting growth of a primary tumour in a subject; inhibiting formation and / or growth of a secondary tumour in a subject; improving the rate of survival of a subject with melanoma; improving the life expectancy of a subject with melanoma; and inducing an anti- tumour immune response in a subject with melanoma, wherein the combination product includes the following components:(i) a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof; and(ii) one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors, and wherein the components are provided in a form for separate administration to the subject.

[0210] In some embodiments, the combination product includes a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, dabrafenib, trametinib, Nivolumab and Ipilimumab.

[0211] In some embodiments of the aforementioned combination product, the subject has Stage IV melanoma. In some embodiments of the aforementioned combination product, the is a human subject.

[0212] The combination product may also include instructions on when and how to administer each component of the combination product to the subject. The combination product may also include instructions for selecting a subject with melanoma suitable for treatment using the combination product. Such instructions may, for example, include instructions for selecting a subject with melanoma suitable for treatment on the basis that the subject shows a T cell response to a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), and / or for selecting a subject on the basis that the subject has a decreased level and / or activity of regulatory T cells upon administration to the subject of the melanoma cell lysate (and / or an immunotherapeutic extract, component orantigen thereof). In these instances, the combination product may be supplied in the form of a kit for performing the relevant function.

[0213] Methods for packaging the various components of the combination product are known in the art.

[0214] The nature of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), and the types of immunotherapeutic agents and / or cancer cell-specific inhibitors to be included in the combination product have been described in detail above. In some embodiments, the one or more immunotherapeutic agents are one or more immune checkpoint inhibitors, as described above.

[0215] In a further aspect the present invention provides use of a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) and one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors in the preparation of a medicament for treating melanoma in a subject.

[0216] In a further aspect the present invention provides use of a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) and one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors in the preparation of a medicament for inhibiting formation and / or growth of a secondary tumour in a subject with melanoma.

[0217] In a further aspect the present invention provides use of a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) and one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors in the preparation of a medicament for inhibiting growth of a primary melanoma in a subject with melanoma.

[0218] In a further aspect the present invention provides use of a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) and one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors in the preparation of a medicament for improving the rate of survival of a subject with melanoma.

[0219] In a further aspect the present invention provides use of a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) and one or moreimmunotherapeutic agents and / or one or more cancer cell-specific inhibitors in the preparation of a medicament for increasing life expectancy of a subject with melanoma.

[0220] In a further aspect the present invention provides use of a melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof) and one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors in the preparation of a medicament for inducing an anti-tumour immune response in a subject with melanoma.

[0221] The nature of the melanoma cell lysate (and / or an immunotherapeutic extract, component or antigen thereof), and the types of immunotherapeutic agents and / or cancer cell-specific inhibitors to be used in preparation of the aforementioned medicaments have been described in detail above.General Methods

[0222] Activation, phenotype and function of T cells may be tested through ELISPOT, cytokine assays, cytotoxicity assays and proliferation assays.

[0223] IFN-γ and IL4 ELISPOT, antigen specific3H-thymidine incorporation proliferation assay and flow cytometry may be used to analyse regulatory T cells.

[0224] For T cell assays, PBMC will be isolated from peripheral blood using standard Ficoll- Hypaque density centrifugation methods. PBMC may be frozen in aliquots suitable for the experiments and thawed when required. Blood from healthy or other volunteers may be used as controls to establish the ‘normal’ range of responses and background values for the various tests used.

[0225] IFN-γ and IL4 ELISPOT assays may be used to test for the frequency of tumour antigen reactive T cells (CTL and T helper cells). Commercially available antibody pairs (R&D systems for IFN-γ and BD Pharmingen for IL4) may be used in a standard ELISPOT protocol. PBMC may be cultured with various antigens in 96-well PVDF membrane microwell plates (Millipore), which have been coated before with the appropriate amount of antibody. Antigens that may be used include vaccinia melanoma cell lysate, MM200 lysate, autologous tumour cell lysate (were applicable), Tet-Tox as a recall antigen and PHA as a positive control. Peripheral blood mononuclear cells (PBMCs) without adding any antigen may be used as a background control.

[0226] Antigen specific proliferation of PBMCs may be tested using standard3H-thymidine incorporation assays. PBMCs are cultured in 96 well plates in the presence of antigen for 2 to 3 days.3H thymidine may be added for the last 16h of the culture.

[0227] The Miltenyi Cytokine Secretion Assay may be used to detect IFN-γ and IL4 producing T cells in the peripheral blood. This is a very sensitive assay giving information on the frequency of cytokine producing cells. Additionally, cells can be counterstained with cell surface markers to identify the subpopulation of cells. CD3 may be used as a general T cell marker and CD8 to distinguish between CD8 and CD4 T cells as cell surface markers. PBMCs cultured overnight (16h) in the presence of vaccinia melanoma cell lysate, MM200 cell lysate or autologous tumour cell lysate (where applicable) may be used in this assay.

[0228] Cytotoxicity of activated T cells may be tested with standard calcein-Acetoxymethyl (AM) (Molecular Probes) cytotoxicity assays (Lichtenfels et al., 1994, J. Immunol. Methods, 172(2): 227-239; Neri et al., 2001 , Clin. Diagn. Lab. Immunol., 8(6): 1131-1135). This a nonradioactive, very sensitive cytotoxicity assay with comparable results to the chromium release assay. Calcein-AM freely diffuses into most cells. Once inside the cell, this nonfluorescent substrate is converted by nonspecific intracellular esterases into a fluorescent product that are retained by cells with intact plasma membranes. In contrast, the dye will leak from cells with damaged cell membranes and can be detected in the supernatant.

[0229] PBMCs may be tested for cytotoxicity towards calcein-AM labelled cells either directly after being isolated from the peripheral blood (fresh) or after being cultured in vitro for one week with antigen. Target cells used in this assay would be autologous tumour cells (where applicable), MM200 cells or other HLA-matched melanoma cell lines. Autologous monocyte derived DC (MoDC) pulsed or unpulsed with tumour cell lysate as target cells may be used if no suitable HLA-matched melanoma cell line can be used. MoDC may be generated using standard protocols with IL4 and GMCSF as described in (Heinzel et al., 2001 , Cancer Immunol. Immunother., 49(12): 671-8). Target cells may be labelled with calcein-AM for 30 min at 37°C. 104target cells may be incubated with effector cells (50:1 to 1 :1 ratios) for 4 hours. Supernatants may then be transferred to new wells and measured using a Typhoon 9410 (Amersham Biosciences). (Excitation 488nm, emission 530nm). Percent lysis is calculated with the same standard formula used for chromium release assays.

[0230] Flow cytometry may be used to investigate the phenotype of activated T cells. Fresh PBMCs, or after 10-day culture in the presence of antigen, can be analysed for the expression of markers including CD3, CD4, CD8, CD25, CD16, CD56, CD69, CD45RO.

[0231] To test for the cytokine profile of activated T cells, PBMCs can be stimulated in vitro in the presence of antigen (MoDC pulsed with MM200 lysate or vaccinia melanoma cell lysate). After 10 days of culture, cells can be tested with intracellular cytokine staining and flow cytometric analysis for the production of cytokines. Cells may be harvested and stimulated for 4 hours with PHA / lonomycin in the presence of Brefeldin A. Cytokines suitable for testing include IL2, IL4, IL10, IL13, TNFa and IFN-g. Cells may also be stained with cell surface markers and analysed using three colour flow cytometry. Cell surface markers include CD3, CD4, CD8, CD69, CD25 and CD45RO.

[0232] The presence of anti-ganglioside antibodies in the sera from subjects may be tested. Antiganglioside antibodies may be detected by immunodot-blot. Strips of PVDF-P membranes (Millipore) are coated with purified commercial GM3, GM2, GD3, GM1 , GD1a, GD1 b, and GT1 b gangliosides (Sigma). The strips are incubated with 1 / 100, 1 / 200, and 1 / 500 dilutions of the patients’ sera. Bound antibodies can be detected with alkaline phosphatase conjugated antibodies to human IgG and IgM. BCIP / NBT will be used as substrate.

[0233] PBMCs activated in vitro with vaccinia melanoma cell lysate and tumour cell lysate may be tested to determine whether a vaccinia melanoma cell lysate or tumour specific response can be induced and to test whether the type of T cell activated by vaccinia melanoma cell lysate is comparable in vitro and in vivo. Activated T cells may be phenotyped as described above. PBMC can be stimulated in vitro with autologous MoDC pulsed with vaccinia melanoma cell lysate, MM200 cell lysate, irradiated MM200 cells, and autologous tumour cell lysate (where applicable). Tet-Tox can be used as a control. After 10 to 14 days of culture T cells may be restimulated once with antigen pulsed MoDC and cultured for another 10-14 days. IL2 may be added to the culture 2 days after restimulation. General activation can be tested with3H-thymidine proliferation assay. Frequency of vaccinia melanoma cell lysate or MM200 reactive T cells may be measured with ELISPOT and Miltenyi cytokine secretion assay.

[0234] The relative number of CD4+CD25+ Treg cells in the peripheral blood of patients may be determined. This is done by comparing the number of Tregs in patients before and aftervaccination; in addition, it is also possible to compare the number of CD4+CD25+ Treg cells between different patient groups, namely responders to the vaccine with non-responders, and Stage IV to stage III patients. The frequency of CD4+CD25 Treg cells can be determined using 3 colour flow cytometry. Cells may be stained with CD4 and CD25 antibodies plus other important markers to define the population as Treg cells. Suitable markers are CD45RA, CD45RO, CD62L, CD122. The population of interest is the CD4+, CD25high population. Most of these cells also express CD45RO, CD62L and CD122 and will not express CD45RA. The Treg (CD4+CD25+) fraction may be assessed in association with the induction of specific CD8+cells.

[0235] To test the functionality of the CD4+CD25high cell population to inhibit normal T cell responses, the CSFE cell division assay may be used (Lyons and Parish, 1994, Immunol. Methods 171 (1): 131-7). T cells from peripheral blood can be separated into CD8+and CD4+ fractions. The CD4+ cell may be further separated into the CD25neg and the CD25high population. The CD25neg population can be stained with CFSE, and activated non- specifically with anti-CD3 and anti-CD28 antibodies or specifically with vaccinia melanoma cell lysate or MM200 tumour cell lysate pulsed autologous MoDC and co-cultured for 4 days with or without the CD25high population. Cell division may be determined in the flow cytometer. CD4+CD25+cells can also be tested for their cytokine production, using intracellular cytokine staining as described above. Cytokines tested for include IL10 and TGF-β.

[0236] Unless otherwise indicated, the practice of many aspects of the present invention employs conventional techniques of molecular biology, recombinant DNA technology and immunology, which are within the skill of the art. Such techniques are described in more detail in the scientific literature, for example, Green MR and Sambrook J, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012, Ausubel, F. M. et al. Current Protocols in Molecular Biology, Wiley-lnterscience, New York, current volume; Albers, B. et al., Molecular Biology of the Cell, 2. sup. nd Ed., Garland Publishing, Inc., New York, N.Y. (1989); Lewin, B M, Genes IV, Oxford University Press, Oxford, (1990); Watson, J. D. et al., Recombinant DNA, Second Edition, Scientific American Books, New York, 1992; Darnell, J E et al., Molecular Cell Biology, Scientific American Books, Inc., New York, N.Y. (1986); Old, R. W. et al., Principles of Gene Manipulation. An Introduction to Genetic Engineering, 2.sup.nd Ed., University of California Press, Berkeley Calif. (1981); DNA Cloning Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., CurrentEdition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); Methods in Enzymology: Guide to Molecular Cloning Techniques, (Berger and Kimmel, eds., 1987); Hartlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan, J. E. et al., eds., Current Protocols in Immunology, Wiley-lnterscience, New York 1991.

[0237] It is to be noted that where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all numerical values or sub-ranges in between these limits as if each numerical value and sub- range is explicitly recited. The statement "about X½ to Y½" has the same meaning as "about X½ to about Y½," unless indicated otherwise.

[0238] The term “about” as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of + / - 10½ or less, + / - 5½ or less, + / - 1½ or less, or + / - 0.1½ or less of and from the numerical value or range recited or claimed.

[0239] As used herein, the singular forms “a,” “an,” and “the” may refer to plural articles unless specifically stated otherwise. Therefore, it is to be made clear that reference to an antimicrobial agent being present in the antimicrobial compositions described herein includes reference to use of a combination of antimicrobial agents in the composition.

[0240] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0241] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0242] The invention is further illustrated in the following examples. The examples are for the purpose of describing particular embodiments only and are not intended to be limiting with respect to the above description.EXAMPLE 1 Preparation of Melanoma Cell Lysate

[0243] Melanoma cell lysate preparation was carried out as in Hersey et al., 1987, Cancer Immunol. Immunother. 25: 257-265, using methods similar to those described by Wallack et al., 1998, J. Am. Coll. Surg. 187:69-77 and Wallack et al., 1986, Cancer 57:649-655.

[0244] A single allogeneic melanoma cell line, referred to as MM200 (Pope et al., 1979, Pathology 11 : 191 -195), was infected with vaccinia virus (prepared by Commonwealth Serum Laboratories, Melbourne, Australia) at 2.5 pock-forming units / cell. MM200 was originally isolated from a primary melanoma on a 43-year-old woman in 1972 but no other patient details are available. The HLA type was A1 ,3; B7,35; DR2,4. Chromosomal analysis showed a nodal number of 76, and a number of marker chromosomes were revealed by karyotypic analysis (Muir and Gunz, 1979, Pathology 11 :597-606).

[0245] The vaccinia virus was the strain referred to as strain O, which was derived in 1921 from two strains imported from the Lister Institute in 1912 and a strain from Japan imported in 1913. After 24 hours of incubation, the lysed cells were further homogenized with a sterile Dounce (type B pestle) homogenizer and centrifuged at 400 x g for 7 minutes. The supernatant (1) was kept and the pellet frozen and thawed in 1 to 3 mL of distilled sterile water. The latter was then made up to 20 mL, centrifuged at 400 x g for 7 minutes and the supernatant (2) added to supernatant (1). The pooled supernatant was centrifuged at 38,000 x g for 60 minutes and the sediment resuspended in saline to give an equivalent of 5 x 106MM200 cells / 0.5 mL saline. The lysate was tested for pathogenic viral, bacterial, or fungal contamination and kept at -80°C until use.EXAMPLE 2Administration Regimen

[0246] For the clinical study described below in Example 3 (i.e. VMCL treatment in the absence of immune checkpoint inhibitor or cancer cell-specific inhibitor treatment), the VMCL vaccine protocol (vaccination with 0.1 - 0.5 ml of the lysate) specified 2 weekly vaccinations for 5 cycles, monthly for 4 cycles and then 3 monthly for as long as a measurable observable clinical response was present. Chemotherapy was used for some patients who developed progressive disease despite vaccine therapy. Where chemotherapy was used with VMCL vaccine, the schedule was modified slightly to administer the vaccine in the 1-2 weeks prior to each dacarbazine dose (given in standard 3 weekly cycles), with the aim of induction of an immune response prior to leukocyte nadir at about 7-10 days. The use of chemotherapy wasdetermined by the patient’s condition and consent. The effect was assessed after 3 doses, with CT scans, before a further 3 doses were given.

[0247] Patients were given the vaccine by intradermal injection over the deltoids or on the anteromedial aspect of the thighs, rotating to a different site with each injection. The first injection was given at a site opposite to the site of LN dissection. As discussed above, injections were given every 2 weeks for the first 8 weeks, monthly for 4 months, and then 3 monthly for as long as a measurable observable clinical response was present.

[0248] For chemotherapy, the general approach was to administer VMCL vaccine therapy first, then if required, include chemotherapy using dacarbazine, fotemustine, temozolomide (Temodal®), or a combination sequentially, administered synchronously with the VMCL vaccine therapy. In some patients, the chemotherapeutic agents (detailed above) were given or commenced prior to the administration of the VMCL vaccine and this was allowable within the protocol. Standard doses of DTIC were 850mg / m23-weekly intravenously, Fotemustine 100mg / m2weekly intravenously for 3 weeks, then 4 weekly thereafter, and Temazolamide 200mg / m2orally day 1-5 initially, then continued if tolerated well.EXAMPLE 3VMCL Stage IV Melanoma Clinical Study

[0249] This pilot study was to test the effectiveness of VMCL administration in the absence of subsequent immune checkpoint inhibitor or cancer cell-specific inhibitor treatment. Data was obtained for 54 patients with advanced Stage IV Melanoma treated with the VMCL vaccine with or without chemotherapy. These patients would ordinarily be considered to have “incurable” metastatic disease, beyond the capacity for surgical resection of metastases. A range of metastatic sites were present in these patients including subcutaneous, lung, brain, liver, bone and bowel. Patients with brain metastases were excluded at entry. However, if cerebral metastases developed during the study these were then resected where possible and adjuvant radiotherapy was used post-surgery.TABLE 2Study statistics of VMCL clinical studyTABLE 3Results of VMCL clinical studyNOTE: * some “Slowing of Disease Progression”, “Partial Regression” and “Stabilisation of Disease” responses occurred in the same patient within different metastases, ie. overlapping populations, occurring either synchronously or chronologically.

[0250] As evidenced by this study, while administration of the VMCL alone had an early positive affect on slowing and stabilising disease progression, partial regression of tumours was observed in some subjects and 24 patients survived less than 12 months from initial treatment. This prompted a further study as described below in Examples 4 and 5 in which VMCL treatment was combined with administration of an immune checkpoint inhibitor (Example 4) or with cancer cell-specific inhibitors (Example 5).EXAMPLE 4VMCL + ICI - Stage IV Melanoma Case Study

[0251] An 80-year-old man presented with a primary Clark IV Breslow 1 ,4mm melanoma with 8 mitoses / mm2of his left neck skin in March 2015. The subject underwent Wider Local Excision + Sentinel Lymph Node Tracing and Dissection revealing no metastatic spread in one removed sentinel lymph node.

[0252] In 2017, the subject developed another primary Clark V Breslow 14.2mm thick melanoma with 5 mitoses / mm2of his right lower back. No mutations were detected in BRAF, NRAS or KIT. There was some suggestion that it could be a metastasis. RLL lung nodule biopsy showed metastatic malignant melanoma. PET scan showed active uptake in mediastinal lymph nodes and pulmonary metastatic melanoma deposits.

[0253] VMCL immunotherapy with fortnightly 0.3 ml dosing intradermally commenced in March 2017. The VMCL was prepared as described above in Example 1. Fortnightly doses were given for 3-months, initially, then PET on 26 June 2017 revealed complete resolution (Complete Response; CR) of pulmonary nodules and regression of mediastinal nodules, with loss of all activity on repeat PET. Treatment was continued fortnightly for a further 4-months, and the CR continued for 7-months, and then on a repeat PET scan at 7-months some activity occurred of the mediastinal lymph nodes. A CT scan 6 October 2017 revealed some increase in size of subcarinal lymph node.

[0254] In November 2017, the subject was treated with Pembrolizimab (anti-PD1 therapy) (3- weekly at 2mg / kg intravenously (160 mg three-weekly)) for metastatic melanoma of mediastinal lymph nodes and received a series of doses (14 in total) before developing eye issues. Given a break from treatment (7 weeks) The subject recovered and resumed with another four doses, before he developed pneumonitis and possibly some myocarditis. The subject then ceased all further anti-PD-1 therapy. The subject then developed a CR again, with complete resolution of all metastases on CT & PET scans for 4-years duration. The subject subsequently died of a separate cardiac event, off of anti-PD-1 therapy treatment and unrelated to any oncological treatment being administered at the time. Prior to death no melanoma was detectable on CT scans, indicating a fully established and maintained Complete Response. This demonstrates the effect of initial melanoma cell lysate immunotherapy followed by immunotherapeutic agent treatment (in this instance immune checkpoint inhibitor treatment). These results also show the effectiveness of the treatment protocol in preventing melanoma recurrence, preventing the growth of a further primarymelanoma, and preventing the formation and / or growth of one or more secondary tumours in the subject.EXAMPLE 5VMCL + Cancer cell-specific Inhibitors - Stage IV Melanoma Case Study

[0255] This case study relates to a 20-year-old man with metastatic melanoma extensively involving bone, liver and adrenal glands who failed initial standard immunotherapies (ipilimumab and nivolumab) with severe treatment related side-effects (visual changes from immune related inflammatory iritis / retinitis and severe bone pain) resulting in cessation of therapy. He was commenced on oral Braf / Mek inhibitor therapies (Dabrafenib & Trametinib) and had to cease both after the first dose due to severe vomiting, weight loss, fever, itchiness, rash, and nausea. This required a break of 4-weeks from all treatment. He commenced on VMCL treatment, and also a low and infrequent dose of oral Braf / Mek inhibitor therapies (Dabrafenib & Trametinib) therapies. Treatment details are provided below.

[0256] The subject initially noted right knee pain 15 June 2022, but an x-ray showed no changes. His pain worsened, he became tired and fatigued, and an MRI of his right knee showed malignant changes. His neck lymph nodes became enlarged, and 3 biopsies of his right knee showed metastatic melanoma. A PET scan showed a liver mass lesion, and biopsy showed metastatic melanoma (B-raf mutation positive). Further adrenal deposits and a right arm deposit was noted.

[0257] The subject was referred to a medical oncologist, and in September 2022 he was given three doses (on 22 / 09 / 2022, 13 / 10 / 2022, and 3 / 11 / 2022) of Nivolumab (65mg IV; 1 mg / kg) and Ipilimumab (195mg IV; 3mg / kg) checkpoint immunotherapy intravenous infusions. He developed severe pain and nausea with pituitary insufficiency and adrenal failure and eye inflammatory changes (after 1stdose) requiring immunosuppressive and replacement steroid therapy. A PET scan on 31 / 10 / 2022, just before receiving 3rddual doses of Nivolumab (1 mg / kg) and Ipilimumab (3mg / kg) on 3 / 11 / 2022, showed progressive disease notably in the liver, bones, adrenals and lymph nodes. His side-effects and condition worsened, and was unable to have further immunotherapy due to severe visual disturbances and deterioration (due to immunotherapy autoimmunity and effects of steroids on eyes). Oral Dabrafenib (75mg twice daily) and Trametinib (2mg daily) cancer cell-specific inhibitor treatment was commenced on 17 / 11 / 2022, wherein he developed severe itchiness, but his pain improved together with better opioid analgesia. The Dabrafenib and Trametinib cancercell-specific inhibitor treatment had to cease due to subsequent severe vomiting, weight loss, fever, itchiness, rash, and nausea, requiring two periods of hopsitlaisation for 7-10 days each, but was recommenced after a 5-week break at a less frequent and lower dose (3 days dosing, 7-days off with no dosing). Repeat PET (9 / 01 / 2023) showed some reduced bone and liver metastasis size and metabolic activity, but persistent widespread metastatic disease with some deposits enlarging.

[0258] VMCL immunotherapy with fortnightly dosing commenced on 20 / 1 / 2023. The VMCL was prepared as described above in Example 1 and was administered intradermally by injection. At a consultation on 3 / 2 / 2023, the subject showed significant improvement with reduced pain requirements (opiate dosage reduced by half), improved mobility, and he had been taken off dual Dabrafenib and Trametinib cancer cell-specific inhibitor treatment for 5 days due to side-effects. His cough was less frequent. He received a second dose of the VMCL immunotherapy on 3 / 2 / 2023. In a consult on 17 / 2 / 2023, the subject looked better, his pain requirements had decreased further, and his vision was stable with glasses. He received a third dose of the VMCL immunotherapy on 17 / 2 / 2023. In a consult on 3 / 3 / 2023, the subject was also receiving dual Dabrafenib and Trametinib cancer cell-specific inhibitor treatment with a 5-days on 5-days off schedule. He received a fourth dose of the VMCL immunotherapy on 3 / 3 / 2023. In a consult on 17 / 3 / 23, the subject continued to look well, had no cough, had no pain and was still not on any pain relief, and was on hydrocortisone steroid (prednisolone) replacement. The subject was also receiving dual Dabrafenib and Trametinib cancer cell-specific inhibitor treatment with a 6-days on 4-days off schedule. The subject also had good chest expansion. He received a fifth dose of the VMCL immunotherapy on 17 / 3 / 2023. In a consult on 31 / 3 / 2023, the subject again looked well, had no pain and was still not on any pain relief, and had no respiratory symptoms. He received a sixth dose of the VMCL immunotherapy on 31 / 3 / 2023. In a consult on 28 / 4 / 2023, the subject was off taking prednisolone, and was not taking any pain relief. He received a seventh dose of the VMCL immunotherapy on 28 / 4 / 2023. The subject received and eighth dose of the VMCL immunotherapy on 12 / 5 / 2023 and a ninth dose on 29 / 5 / 2023. He is back at work, doing running and rock climbing, looks very well, and is fully functional. Fortnightly VMCL vaccination is continuing, and he has now received 7-months of VMCL therapy and remains fully functional with no pain.

[0259] In summary, the subject received VMCL immunotherapy with fortnightly dosing commencing in January 2023. Fortnightly doses were given for 3-months, initially with marked clinical improvement. A PET done on 5 / 4 / 2023 revealed notable Partial resolution(Partial Response; PR) with regression of liver and bone metastases, and stabilisation of other metastases, and appearance of no new metastases. Fortnightly VMCL vaccination was continued. The CT, MRI and PET scans (on 25 / 7 / 2024) showed resolution of some metastases, with stabilisation of the remaining metastases and appearance of no new metastases. He was running 10-20km per week and almost fully functional. This demonstrates the effect of melanoma cell lysate immunotherapy together with cancer cell- specific inhibitor treatment. These results also show the effectiveness of the treatment protocol in preventing melanoma recurrence, preventing the growth of a further primary melanoma, and preventing the formation and / or growth of one or more secondary tumours in the subject.

[0260] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0261] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Claims

CLAIMS1 . A method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) administering to the subject one or more immunotherapeutic agents and / or one or more cancer cell-specific inhibitors.

2. The method according to claim 1 , wherein the subject is a human subject.

3. The method according to claim 1 or claim 2, wherein the melanoma cell lysate includes fragmented melanoma cell membranes.

4. The method according to any one of claims 1 to 3, wherein the melanoma cell lysate is a viral melanoma cell lysate.

5. The method according to claim 4, wherein the viral melanoma cell lysate is a vaccinia melanoma cell lysate.

6. The method according to any one of claims 1 to 3, wherein the melanoma cell lysate is an allogeneic cell lysate.

7. The method according to claim 6, wherein the melanoma cell lysate includes a lysate from MM200 cells.

8. The method according to any one of claims 1 to 7, wherein the melanoma is Stage IV melanoma.

9. The method according to claim 8, wherein the Stage IV melanoma is advanced Stage IV melanoma.

10. The method of any one of claims 1 to 9, wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject prior to administration of the one or more immunotherapeutic agents to the subject.

11. The method according to claim 10, wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject about every two weeks for one or more cycles prior to administration of the one or more immunotherapeutic agents to the subject.

12. The method according to claim 10 or claim 11 , wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof is administered to the subject about every two weeks for at least about three months prior to administration of the one or more immunotherapeutic agents to the subject.

13. The method according to any one of claims 10 to 12, wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject about every two weeks for at least about six months prior to administration of the one or more immunotherapeutic agents to the subject.

14. The method according to any one of claims 10 to 13, wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject about every two weeks for about seven months prior to administration of the one or more immunotherapeutic agents to the subject.

15. The method of any one of claims 1 to 9, wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject after administration of the one or more immunotherapeutic agents to the subject.

16. The method according to claim 15, wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject about every two weeks for one or more cycles after administration of the one or more immunotherapeutic agents to the subject.

17. The method according to claim 15 or claim 16, wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof is administered to the subject about every two weeks for at least about three months after administration of the one or more immunotherapeutic agents to the subject.

18. The method according to any one of claims 15 to 17, wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administeredto the subject about every two weeks for at least about seven months after administration of the one or more immunotherapeutic agents to the subject.

19. The method according to any one of claims 15 to 18, wherein the one or more immunotherapeutic agents are administered to the subject about every three weeks for at least about six weeks.

20. The method according to any one of claims 15 to 19, wherein the one or more cancer cell-specific inhibitors are administered to the subject after the one or more immunotherapeutic agents are administered to the subject, and prior to administration of the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof to the subject.

21. The method of claim 20, wherein the one or more cancer cell-specific inhibitors are administered to the subject at least about two weeks after the one or more immunotherapeutic agents are administered to the subject.

22. The method of claim 20 or claim 21 , wherein the one or more cancer cell-specific inhibitors are administered to the subject about every four weeks for at least about one month.

23. The method of any one of claims 20 to 22, wherein the one or more cancer cell- specific inhibitors are also administered to the subject during a period in which the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof is administered to the subject.

24. The method of any one of claims 1 to 9, wherein the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, is administered to the subject after administration of the one or more cancer cell-specific inhibitors to the subject.

25. The method according to any one of claims 1 to 24, wherein the one or more immunotherapeutic agents comprise one or more immune checkpoint inhibitors.

26. The method according to claim 25, wherein the one or more immune checkpoint inhibitors are selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD- L2 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, an OX40 inhibitor, an A2AR inhibitor, aBTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, an IDO inhibitor, a TIM3 inhibitor, a KIR inhibitor, and an IL-2R inhibitor.

27. The method according to claim 26, wherein: the PD-1 inhibitor is selected from one or more of Pembrolizumab, Cemiplimab, Pidilizumab, Nivolumab, and MEDI-0680; the PD-L1 inhibitor is selected from one or more of Atezolizumab, Avelumab, Durvalumab, and BMS-936559 / MDX-1105; the CTLA-4 inhibitor is selected from one or more of Tremelimumab and Ipilimumab; the LAG-3 inhibitor is Relatlimab; the B7-H3 inhibitor is MGA271 ; the KIR inhibitor is Lirilumab; and the OX40 inhibitor is MEDI-6469.

28. The method according to claim 26 or claim 27, wherein the immune check-point inhibitor is selected from one or more of Pembrolizumab, Nivolumab and Ipilimumab.

29. The method according to any one of claims 1 to 28, wherein the one or more cancer cell-specific inhibitors are selected from the group consisting of a receptor tyrosine kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a serine / threonine kinase inhibitor, an epigenetic inhibitor, a hedgehog pathway inhibitor, a proteasome inhibitor, and a poly (ADP- ribose) polymerase (PARP) inhibitor.

30. The method according to claim 29, wherein the receptor tyrosine kinase inhibitor is selected from one or more of an anaplastic lymphoma kinase (ALK) inhibitor, a cellular- mesenchymal-epithelial transition factor (c-Met) inhibitor, a c-Kit (CD117) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a Fms-like tyrosine kinase 3 (FLT3) inhibitor, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a fibroblast growth factor receptor (FGFR) inhibitor, a platelet-derived growth factor receptor (PDGFR) inhibitor, and a tropomyosin receptor kinase (TRK) inhibitor.

31. The method according to claim 29 or claim 30, wherein the receptor tyrosine kinase inhibitor is selected from one or more of crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, capmatinib, tepotinib, gefitinib, erlotinib, lapatinib, icotinib, afatinib, osimertinib, neratinib, dacomitinib, almonertinib, tucatinib, midostaurin, gilteritinib, quizartinib, pexidartinib, sorafenib, sunitinib, pazopanib, vandetanib, axitinib, cabozantinib, regorafenib, apatinib,lenvatinib, tivozanib, fruquintinib, nintedanib, anlotinib, erdafitinib, pemigatinib, avapritinib, ripretinib, selpercatinib, pralsetinib, Larotrectinib and entrectinib.

32. The method according to claim 29, wherein the non-receptor tyrosine kinase inhibitor is selected from one or more of a Bcr-Abl1 inhibitor, a Bruton’s agammglobulinemia tyrosine kinase (BTK) inhibitor, and a janus kinase (JAK) inhibitor.

33. The method according to claim 29 or claim 32, wherein the non-receptor tyrosine kinase inhibitor is selected from one or more of imatinib, dasatinib, nilotinib, bosutinib, radotinib, ponatinib, ibrutinib, acalabrutinib, zanubrutinib, ruxolitinib, and fedratinib.

34. The method according to claim 29, wherein the serine / threonine kinase inhibitor is selected from one or more of a BRAF inhibitor, a MEK inhibitor, an ERK inhibitor, a cyclin- dependent kinase (CDK) inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, an AKT inhibitor, and a mammalian target of rapamycin (mTOR) inhibitor.

35. The method according to claim 29 or claim 34, wherein the serine / threonine kinase inhibitor is selected from one or more of dabrafenib, trametinib, vemurafenib, encorafenib, cobimetinib, binimetinib, selumetinib, Palbociclib, ribociclib, abemaciclib, idelalisib, copanlisib, duvelisib, alpelisib, temsirolimus, everolimus, and sirolimus.

36. The method according to claim 29, wherein the epigenetic inhibitor is selected from one or more of an enhancer of zeste homolog 2 (EZH2) inhibitor, a histone deacetylase (HDAC) inhibitor, and an isocitrate dehydrogenase 1 / 2 (IDH1 / 2) inhibitor.

37. The method according to claim 29 or claim 36, wherein the epigenetic inhibitor is selected from one or more of tazemetostat, vorinostat, romidepsin, belinostat, tucidinostat, panobinostat, enasidenib, and ivosidenib.

38. The method according to claim 29, wherein the hedgehog pathway inhibitor is selected from one or more of venetoclax, vismodegib, sonidegib, glasdegib, bortezomib, carfilzomib, ixazomib, olaparib, rucaparib, niraparib and talazoparib.

39. The method according to claim 29, wherein the proteasome inhibitor is selected from one or more of bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, and delanzomib.

40. The method according to claim 29, wherein the PARP inhibitor is selected from one or more of Nicotinamide, Olaparib, rucaparib, niraparib, talazoparib, pamiparib, veliparib, iniparib, amelparib, and fluzoparib.

41. The method according to any one of claims 1 to 40, wherein the one or more cancer cell-specific inhibitors are dabrafenib and trametinib.

42. A method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) subsequently administering to the subject one or more immunotherapeutic agents.

43. A method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more immunotherapeutic agents; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

44. A method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more immunotherapeutic agents; and(ii) subsequently administering to the subject one or more cancer cell-specific inhibitors; and(iii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

45. A method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more immunotherapeutic agents; and(ii) subsequently administering to the subject one or more cancer cell-specific inhibitors; and(iii) subsequently administering to the subject one or more cancer cell-specific inhibitors and a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

46. A method of treating or preventing melanoma in a subject, the method including the steps of:(i) administering to the subject one or more cancer cell-specific inhibitors; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

47. A method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof; and(ii) subsequently administering to the subject Pembrolizumab.

48. A method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject Nivolumab and Ipilimumab; and(ii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

49. A method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject Nivolumab and Ipilimumab; and(ii) subsequently administering to the subject dabrafenib and trametinib; and(iii) subsequently administering to the subject a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

50. A method of treating or preventing Stage IV melanoma in a subject, the method including the steps of:(i) administering to the subject Nivolumab and Ipilimumab; and(ii) subsequently administering to the subject dabrafenib and trametinib; and(iii) subsequently administering to the subject dabrafenib and trametinib, and a melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

51. The method according to any one of claims 1 to 50, wherein the method includes the further step of resecting metastases from the subject.

52. The method according to claim 51 , wherein the metastases are resected prior to administration of the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

53. The method according to any one of claims 1 to 52, wherein the method includes the step of selecting a subject suitable for treatment.

54. The method according to claim 53, wherein the subject is selected on the basis that the subject shows a T cell response to the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

55. The method according to any one of claims 1 to 54, wherein formation and / or growth of a secondary tumour in the subject is inhibited.

56. The method according to any one of claims 1 to 55, wherein growth of a primary melanoma in the subject is inhibited.

57. The method according to any one of claims 1 to 56, wherein rate of survival of the subject is improved.

58. The method according to any one of claims 1 to 57, wherein life expectancy of the subject is improved.

59. The method according to any one of claims 1 to 58, wherein an anti-tumour immune response in the subject is induced.

60. The method according to any one of claims 1 to 59, wherein one or more of an increase in the number of intratumoral dendritic cells is produced in the subject, dendritic cell activation is improved in the subject, and the number and / or activity of regulatory T cells is decreased in the subject.

61. A combination product including the following components:(i) a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof; and(ii) one or more immunotherapeutic agents and / or one or more cancer cell- specific inhibitors;wherein the components are provided in a form for separate administration to a subject.

62. The combination product according to claim 61 , wherein the melanoma cell lysate includes fragmented melanoma cell membranes.

63. The combination product according to claim 61 or claim 62, wherein the melanoma cell lysate is a viral melanoma cell lysate.

64. The combination product according to claim 63, wherein the viral melanoma cell lysate is a vaccinia melanoma cell lysate.

65. The combination product according to claim 61 or claim 62, wherein the melanoma cell lysate is an allogeneic cell lysate.

66. The combination product according to claim 65, wherein the melanoma cell lysate includes a lysate from MM200 cells.

67. The combination product according to any one of claims 61 to 66, wherein the one or more immunotherapeutic agents comprise one or more immune checkpoint inhibitors.

68. The combination product according to claim 67, wherein the one or more immune checkpoint inhibitors are selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, an OX40 inhibitor, an A2AR inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, an IDO inhibitor, a TIM3 inhibitor, a KIR inhibitor, and an IL-2R inhibitor.

69. The combination product according to claim 68, wherein: the PD-1 inhibitor is selected from one or more of Pembrolizumab, Cemiplimab, Pidilizumab, Nivolumab, and MEDI-0680; the PD-L1 inhibitor is selected from one or more of Atezolizumab, Avelumab, Durvalumab, and BMS-936559 / MDX-1105; the CTLA-4 inhibitor is selected from one or more of Tremelimumab and Ipilimumab; the LAG-3 inhibitor is Relatlimab; the B7-H3 inhibitor is MGA271 ; the KIR inhibitor is Lirilumab; andthe OX40 inhibitor is MEDI-6469.

70. The combination product according to claim 69, wherein the combination product includes a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, and Pembrolizumab.

71. The combination product according to any one of claims 61 to 70, wherein the one or more cancer cell-specific inhibitors are selected from the group consisting of a receptor tyrosine kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a serine / threonine kinase inhibitor, an epigenetic inhibitor, a hedgehog pathway inhibitor, a proteasome inhibitor, and a poly (ADP-ribose) polymerase (PARP) inhibitor.

72. The combination product according to claim 71 , wherein the receptor tyrosine kinase inhibitor is selected from one or more of an anaplastic lymphoma kinase (ALK) inhibitor, a cellular-mesenchymal-epithelial transition factor (c-Met) inhibitor, a c-Kit (CD117) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a Fms-like tyrosine kinase 3 (FLT3) inhibitor, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a fibroblast growth factor receptor (FGFR) inhibitor, a platelet-derived growth factor receptor (PDGFR) inhibitor, and a tropomyosin receptor kinase (TRK) inhibitor.

73. The combination product according to claim 71 or claim 72, wherein the receptor tyrosine kinase inhibitor is selected from one or more of crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, capmatinib, tepotinib, gefitinib, erlotinib, lapatinib, icotinib, afatinib, osimertinib, neratinib, dacomitinib, almonertinib, tucatinib, midostaurin, gilteritinib, quizartinib, pexidartinib, sorafenib, sunitinib, pazopanib, vandetanib, axitinib, cabozantinib, regorafenib, apatinib, lenvatinib, tivozanib, fruquintinib, nintedanib, anlotinib, erdafitinib, pemigatinib, avapritinib, ripretinib, selpercatinib, pralsetinib, Larotrectinib and entrectinib.

74. The combination product according to claim 71 , wherein the non-receptor tyrosine kinase inhibitor is selected from one or more of a Bcr-Abl1 inhibitor, a Bruton’s agammglobulinemia tyrosine kinase (BTK) inhibitor, and a janus kinase (JAK) inhibitor.

75. The combination product according to claim 71 or claim 74, wherein the non-receptor tyrosine kinase inhibitor is selected from one or more of imatinib, dasatinib, nilotinib, bosutinib, radotinib, ponatinib, ibrutinib, acalabrutinib, zanubrutinib, ruxolitinib, and fedratinib.

76. The combination product according to claim 71 , wherein the serine / threonine kinase inhibitor is selected from one or more of a BRAF inhibitor, a MEK inhibitor, an ERK inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, an AKT inhibitor, and a mammalian target of rapamycin (mTOR) inhibitor.

77. The combination product according to claim 71 or claim 76, wherein the serine / threonine kinase inhibitor is selected from one or more of dabrafenib, trametinib, vemurafenib, encorafenib, cobimetinib, binimetinib, selumetinib, Palbociclib, ribociclib, abemaciclib, idelalisib, copanlisib, duvelisib, alpelisib, temsirolimus, everolimus, and sirolimus.

78. The combination product according to any one of claims 61 to 69 and 71 to 77, wherein the combination product includes a melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof, dabrafenib, trametinib, Nivolumab and Ipilimumab.

79. The combination product according to claim 71 , wherein the epigenetic inhibitor is selected from one or more of an enhancer of zeste homolog 2 (EZH2) inhibitor, a histone deacetylase (HDAC) inhibitor, and an isocitrate dehydrogenase ½ (IDH1 / 2) inhibitor.

80. The combination product according to claim 71 or claim 79, wherein the epigenetic inhibitor is selected from one or more of tazemetostat, vorinostat, romidepsin, belinostat, tucidinostat, panobinostat, enasidenib, and ivosidenib.

81. The combination product according to claim 71 , wherein the hedgehog pathway inhibitor is selected from one or more of venetoclax, vismodegib, sonidegib, glasdegib, bortezomib, carfilzomib, ixazomib, olaparib, rucaparib, niraparib and talazoparib.

82. The combination product according to claim 71 , wherein the proteasome inhibitor is selected from one or more of bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, and delanzomib.

83. The combination product according to claim 71 , wherein the PARP inhibitor is selected from one or more of Nicotinamide, Olaparib, rucaparib, niraparib, talazoparib, pamiparib, veliparib, iniparib, amelparib, and fluzoparib.

84. The combination product according to any one of claims 61 to 83, wherein the combination product is used to inhibit growth of a primary melanoma in the subject.

85. The combination product according to any one of claims 61 to 84, wherein the combination product is used to inhibit formation and / or growth of a secondary tumour in the subject.

86. The combination product according to any one of claims 61 to 85, wherein the subject has melanoma, and the combination product is used to treat melanoma in the subject.

87. The combination product according to any one of claims 61 to 86, wherein the subject has melanoma, and the combination product is used to improve the rate of survival of the subject.

88. The combination product according to any one of claims 61 to 87, wherein the subject has melanoma, and the combination product is used to improve the life expectancy of the subject.

89. The combination product according to any one of claims 61 to 88, wherein the subject has melanoma, and the combination product is used to induce an anti-tumour immune response in the subject.

90. The combination product according to any one of claims 61 to 89, wherein the combination product includes instructions for selecting a subject with melanoma suitable for treatment.

91. The combination product according to claim 90, wherein the combination product includes instructions for selecting a subject with melanoma suitable for treatment on the basis that the subject shows a T cell response to the melanoma cell lysate and / or an immunotherapeutic extract, component or antigen thereof.

92. The combination product according to claim 90 or claim 91 , wherein the combination product includes instructions for selecting a subject with melanoma suitable for treatment on the basis that the subject has a decreased level and / or activity of regulatory T cells upon administration to the subject of the melanoma cell lysate, and / or an immunotherapeutic extract, component or antigen thereof.

93. The combination product of any one of claims 61 to 92, wherein the subject has Stage IV melanoma.

94. The combination product of any one of claims 61 to 93, wherein the subject is a human subject.

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