Survivin and MAGE-A9 dual-targeted immunotherapy

Abstract

This application relates generally to methods for treating tumors, and in particular to methods for using both survivin and MAGE-A9 peptides in dual-targeted immunotherapy to generate targeted T cell responses against these two tumor antigens.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 250,130, filed September 29, 2021, the disclosure of which is incorporated by reference in its entirety herein.

[0002] The present application relates generally to pharmaceutical compositions and methods for treating tumors, and in particular to compositions and methods for delivering survivin and MAGE-A9 therapeutic antigens for dual targeted immunotherapy. [Background technology]

[0003] Bladder cancer is the fifth most common cancer in Canada. Men are three times more susceptible to the disease than women (Brenner et al., 2020). The majority of patients (70-75%) have non-muscle invasive bladder cancer (NMIBC) (stages Ta, T1, Tis), which are generally treated by transurethral resection (TUR); after TUR, tumors recur with a frequency of over 60% in 10-20% of cases and progress to muscle invasive bladder cancer (MIBC) (stages T2-T4) (Kamat et al., 2016). Recurrence of NMIBC after TUR is prevented by intravesical therapy using chemotherapy or immunotherapy. Since the late 1970s, nonspecific immunotherapy using intravesical instillation of Bacillus Calmette-Guérin (BCG) (live attenuated mycobacteria tuberculosis) has been the best treatment available to prevent recurrence in patients with high-risk NMIBC (Gandhi et al., 2013). However, there are often adverse side effects associated with BCG that affect the patient's quality of life. Specifically, numerous grade 3 toxicities as well as urinary and genital domains of health-related quality of life are of greatest concern to bladder cancer patients (Botteman et al., 2003). Although this therapy fails in 30-40% of patients and is associated with significant side effects, the success of this nonspecific immunotherapy indicates that bladder cancer is amenable to immunotherapy (Gandhi et al., 2013). Furthermore, BCG is difficult to manufacture reliably due to the variability in clinical efficacy documented between BCG-Connaught and BCG-Tice products (Rentsch et al., 2014). The manufacturer of BCG-Connaught discontinued its production, resulting in increased demand for the less effective BCG-Tice and recurrent shortages of the product over the past few years. For patients with high-grade NMIBC in whom intravesical BCG has failed, radical cystectomy is currently the primary alternative; however, this is a major surgery with a high mortality rate.Thus, there is a critical need for the development of more effective immunotherapeutic approaches for NMIBC and for the identification of biomarkers that can aid in the selection of patients who will most benefit from these therapies and predict response to these treatments.

[0004] Recent advances in cancer immunotherapy have allowed researchers to gain further insight into the complexities of the immune environment, leading to a better understanding of the mechanisms by which tumor cells evade the immune system. However, despite the promising results shown, there are still some patients who do not benefit from current treatment options. One of the reasons why these approaches have limited success in some patients is that the levels of immune cells, especially T lymphocytes, that target cancer are insufficient. Therefore, there is a need in the art for new and effective therapies or combinations of therapies that can increase the number of tumor-directed T cells, better control tumors, and ultimately improve clinical outcomes. Summary of the Invention

[0005] The present disclosure provides, inter alia, a lipid composition suitable for delivery of at least two T cell activating therapeutics to a subject and a method of treating a tumor in a subject by administering to the subject a composition for delivering at least two T cell activating therapeutics.

[0006] In one aspect, the present invention relates to a pharmaceutical composition for delivery of at least two T cell activation therapeutics to a subject, comprising: i) at least two T cell activation therapeutics; ii) one or more lipid-based structures; and iii) a carrier, wherein the at least two T cell activation therapeutics comprise at least one survivin antigen and at least one melanoma associated antigen 9 (MAGE-A9) antigen.

[0007] In certain embodiments of the pharmaceutical compositions disclosed herein, the survivin antigen is a survivin peptide antigen comprising the amino acid sequence FEELTLGEF (SEQ ID NO:1); FTELTLGEF (SEQ ID NO:2); LTLGEFLKL (SEQ ID NO:3); LMLGEFLKL (SEQ ID NO:4); RISTFKNWPF (SEQ ID NO:5); RISTFKNWPK (SEQ ID NO:6); STFKNWPFL (SEQ ID NO:7); or LPPAWQPFL (SEQ ID NO:8), or any combination thereof, or a nucleic acid molecule encoding said survivin peptide antigen.

[0008] In certain embodiments of the pharmaceutical compositions disclosed herein, the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising the amino acid sequence of SEQ ID NOs: 9-12, 26-44, 46-52, 54-62, 64-75, or 79-93, or any combination thereof, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen. In certain embodiments of the pharmaceutical compositions disclosed herein, the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising at least one amino acid sequence in Table 17, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen.

[0009] In certain embodiments of the pharmaceutical compositions disclosed herein, the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising the amino acid sequence KVAELVHFL (SEQ ID NO:9); GLMGAQEPT (SEQ ID NO:10); ALSVMGVYV (SEQ ID NO:11); FLWGSKAHA (SEQ ID NO:12); FMFQEALKL (SEQ ID NO:26); EVDPAGHSY (SEQ ID NO:27); NYKRYFPVI (SEQ ID NO:28); VYYTLWSQF (SEQ ID NO:29); SYILVTALG (SEQ ID NO:30); MPKAALLII (SEQ ID NO:31); SVMGVYVGK (SEQ ID NO:32); ALLIIVLGV (SEQ ID NO:33); FLLHKYRVK (SEQ ID NO:34); or IVLGVILTK (SEQ ID NO:35), or any combination thereof, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen.

[0010] In certain embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition comprises at least one MAGE-A9 antigen that binds to HLA-A1, HLA-A2, HLA-A3, HLA-A24, and / or HLA-B7. In certain embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition comprises at least one MAGE-A9 antigen that binds to each of HLA-A1, HLA-A2, HLA-A3, HLA-A24, and HLA-B7 individually.

[0011] In certain embodiments of the pharmaceutical compositions disclosed herein, the survivin antigen is a survivin peptide antigen comprising the amino acid sequence FEELTLGEF (SEQ ID NO:1); FTELTLGEF (SEQ ID NO:2); LTLGEFLKL (SEQ ID NO:3); LMLGEFLKL (SEQ ID NO:4); RISTFKNWPF (SEQ ID NO:5); RISTFKNWPK (SEQ ID NO:6); STFKNWPFL (SEQ ID NO:7); and / or LPPAWQPFL (SEQ ID NO:8), or a nucleic acid molecule encoding said survivin peptide antigen, and the MAGE-A9 antigen is a survivin peptide antigen comprising the amino acid sequence KVAELVHFL (SEQ ID NO:9); GLMGAQEPT (SEQ ID NO:10); or a nucleic acid molecule encoding said MAGE-A9 peptide antigen, which is a MAGE-A9 peptide antigen comprising: SEQ ID NO:10; ALSVMGVYV (SEQ ID NO:11); FLWGSKAHA (SEQ ID NO:12); FMFQEALKL (SEQ ID NO:26); EVDPAGHSY (SEQ ID NO:27); NYKRYFPVI (SEQ ID NO:28); VYYTLWSQF (SEQ ID NO:29); SYILVTALG (SEQ ID NO:30); MPKAALLII (SEQ ID NO:31); SVMGVYVGK (SEQ ID NO:32); ALLIIVLGV (SEQ ID NO:33); FLLHKYRVK (SEQ ID NO:34); and / or IVLGVILTK (SEQ ID NO:35).

[0012] In certain embodiments of the pharmaceutical compositions disclosed herein, the survivin antigen is a survivin peptide antigen comprising the amino acid sequence LMLGEFLKL (SEQ ID NO: 4) and / or STFKNWPFL (SEQ ID NO: 7), or a nucleic acid molecule encoding said survivin peptide antigen, and the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising the amino acid sequence KVAELVHFL (SEQ ID NO: 9); GLMGAQEPT (SEQ ID NO: 10); ALSVMGVYV (SEQ ID NO: 11); or FLWGSKAHA (SEQ ID NO: 12), or any combination thereof, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen.

[0013] In certain embodiments of the pharmaceutical compositions disclosed herein, the survivin antigen is a survivin peptide antigen comprising the amino acid sequence LMLGEFLKL (SEQ ID NO: 4) and / or STFKNWPFL (SEQ ID NO: 7), or a nucleic acid molecule encoding said survivin peptide antigen, and the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising the amino acid sequence KVAELVHFL (SEQ ID NO: 9); GLMGAQEPT (SEQ ID NO: 10); or FLWGSKAHA (SEQ ID NO: 12), or any combination thereof, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen.

[0014] In certain embodiments of the pharmaceutical compositions disclosed herein, the survivin antigen is two survivin antigens comprising amino acid sequences LMLGEFLKL (SEQ ID NO: 4) and STFKNWPFL (SEQ ID NO: 7), or a nucleic acid molecule encoding said survivin peptide antigens, and the MAGE-A9 antigen is four MAGE-A9 antigens comprising amino acid sequences KVAELVHFL (SEQ ID NO: 9); GLMGAQEPT (SEQ ID NO: 10); ALSVMGVYV (SEQ ID NO: 11); and FLWGSKAHA (SEQ ID NO: 12), or a nucleic acid molecule encoding said MAGE-A9 peptide antigens.

[0015] In certain embodiments of the pharmaceutical compositions disclosed herein, the survivin antigen is two survivin antigens comprising amino acid sequences LMLGEFLKL (SEQ ID NO: 4) and STFKNWPFL (SEQ ID NO: 7), or a nucleic acid molecule encoding said survivin peptide antigens, and the MAGE-A9 antigen is three MAGE-A9 antigens comprising amino acid sequences KVAELVHFL (SEQ ID NO: 9); GLMGAQEPT (SEQ ID NO: 10); and FLWGSKAHA (SEQ ID NO: 12), or a nucleic acid molecule encoding said peptide antigens.

[0016] In certain embodiments of the pharmaceutical compositions disclosed herein, each of the survivin and MAGE-A9 peptide antigens is independently at a concentration of about 0.1 μg / μl to about 5.0 μg / μl. In certain embodiments, each of the survivin and MAGE-A9 peptide antigens is at a concentration of at least about 1.0 μg / ml. In certain embodiments, each of the survivin and MAGE-A9 peptide antigens is at a concentration of about 1.0 μg / ml.

[0017] In certain embodiments of the pharmaceutical compositions disclosed herein, the composition further comprises a T-helper epitope. In certain embodiments, the T-helper epitope is a peptide comprising the amino acid sequence AQYIKANSKFIGITEL (SEQ ID NO: 13).

[0018] In certain embodiments of the pharmaceutical composition disclosed herein, the composition further comprises an adjuvant.In certain embodiments, the adjuvant is polyIC polynucleotide.In certain embodiments, the polyIC polynucleotide is DNA (e.g., SEQ ID NO: 22) or RNA-based.

[0019] In certain embodiments of the pharmaceutical composition disclosed herein, carrier comprises hydrophobic carrier.In certain embodiments, hydrophobic carrier is vegetable oil, nut oil or mineral oil.In certain embodiments, hydrophobic carrier is mineral oil or mannide oleate in mineral oil solution.In certain embodiments, hydrophobic carrier is Montanide ISA51.

[0020] In certain embodiments of the pharmaceutical composition disclosed herein, one or more lipid-based structures comprise monolayer lipid assemblies.In certain embodiments, one or more lipid-based structures having monolayer lipid assemblies comprise lipid aggregates in which the hydrophobic part of the lipid is oriented outward toward the hydrophobic carrier and the hydrophilic part of the lipid aggregates as a core.In certain embodiments, one or more lipid-based structures having monolayer lipid assemblies comprise reverse micelles.In certain embodiments, the size of the lipid-based structures is about 2 nm to about 20 nm in diameter.In certain embodiments, the size of the lipid-based structures is about 5 nm to about 10 nm in diameter.

[0021] In certain embodiments of the pharmaceutical compositions disclosed herein, one or more of the T cell activation therapeutics are inside the lipid-based structure. In certain embodiments, one or more of the T cell activation therapeutics are outside the lipid-based structure.

[0022] In one aspect, the present invention relates to a method of treating a tumor in a subject, the method comprising administering to the subject a composition for delivering at least two T cell activating therapeutics, the composition comprising: i) at least two T cell activating therapeutics; ii) one or more lipid-based structures; and iii) a carrier, wherein the at least two T cell activating therapeutics comprise at least one survivin antigen and at least one melanoma associated antigen 9 (MAGE-A9) antigen.

[0023] In certain embodiments of the methods disclosed herein, the methods further comprise administering an effective amount of at least one active agent. In certain embodiments, the effective amount of the active agent is an amount sufficient to provide an immune modulating effect.

[0024] In certain embodiments of the methods disclosed herein, the active agent is administered prior to the T cell activation therapeutic agent. In certain embodiments, the method includes administering a first dose of the active agent at least two days prior to administration of the T cell activation therapeutic agent. In certain embodiments, the method includes administering a first dose of the active agent about one week prior to administration of the T cell activation therapeutic agent. In certain embodiments, the method includes administering a first dose of the active agent, followed by administration of one or more maintenance doses of the active agent. In certain embodiments, the active agent is administered twice daily for a period of about one week. In certain embodiments, the active agent is administered in a low-dose metronomic regimen. In certain embodiments, the metronomic regimen includes administering the active agent every other week for a period of about one week. In certain embodiments, the active agent is administered twice daily. In certain embodiments, the metronomic regimen comprises administering an active agent over a two-week cycle, where the active agent is administered during the first week of the cycle and the active agent is not administered during the second week of the cycle, and the metronomic regimen comprises at least two cycles.

[0025] In certain embodiments of the methods disclosed herein, the T cell activation therapy is administered to the subject about once every three weeks. In certain embodiments, administering the active agent begins about one week prior to administering the first dose of the T cell activation therapy, and the T cell activation therapy is administered about once every three weeks.

[0026] In certain embodiments of the method disclosed herein, the active agent is an agent that interferes with DNA replication.In certain embodiments, the active agent is an alkylating agent.In certain embodiments, the alkylating agent is a nitrogen mustard alkylating agent, and optionally cyclophosphamide.In certain embodiments, the active agent is a) at least one of gemcitabine, 5-FU, cisplatin, oxaliplatin, temozolomide, paclitaxel, capecitabine, methotrexate, epirubicin, idarubicin, mitoxantrone, bleomycin, decitabine, or docetaxel;b) at least one of thalidomide, bortezomib, IL-2, IL-12, IL-15, IFN-gamma, IFN-alpha, or TNF-alpha, metformin, or lenalidomide;and / or c) at least one inhibitor of VEGF, VEGFR, or CD40.

[0027] In certain embodiments of the methods disclosed herein, the active agent improves the efficacy of T cell activation therapy by directly enhancing the immune response to the antigen, for example, by increasing the activity or number of antigen-specific CD8+ T cells. In certain embodiments, increasing the activity or number of antigen-specific CD8+ T cells includes enrichment of antigen-specific CD8+ T cells with a relative reduction of total CD8+ T cells. In certain embodiments, the active agent improves the efficacy of T cell activation therapy by reducing the number or activity of suppressive immune cells, such as CD4+FoxP3+ regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and / or CD19+CD1d+CD5+ B cells (Bregs).

[0028] In certain embodiments of the methods disclosed herein, the method further comprises administering at least one additional therapeutic agent.In certain embodiments, the at least one additional therapeutic agent is a) one or more checkpoint agents;b) one or more of rapalogs, histone deacetylase (HDAC) inhibitors, parp inhibitors, or indoleamine 2,3-dioxygenase enzyme inhibitors;and / or c) doxorubicin, trastuzumab, bevacizumab, sunitinib, sorafenib, or combinations thereof. In certain embodiments, the checkpoint agent is an inhibitor of an immune checkpoint protein, said immune checkpoint protein being programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1, CD279), CTLA-4 (CD154), LAG3 (CD223), TIM3 (HAVCR2, CD366), 41BB (CD137), ICOS (inducible T cell costimulatory molecule), killer inhibitory receptor (KIR), CD27, OX-40, GITR, or phosphatidylserine (PS). In certain embodiments of the methods disclosed herein, the inhibitor of PD-1 is an antibody, optionally pembrolizumab.

[0029] In certain embodiments of the methods disclosed herein, the method comprises administering a first dose of the additional therapeutic agent, followed by one or more maintenance doses of the additional therapeutic agent. In certain embodiments, the additional therapeutic agent is administered about every 1 to 4 weeks. In certain embodiments, the additional therapeutic agent is administered every 3 weeks.

[0030] In certain embodiments of the methods disclosed herein, the survivin antigen is a survivin peptide antigen comprising the amino acid sequence FEELTLGEF (SEQ ID NO:1); FTELTLGEF (SEQ ID NO:2); LTLGEFLKL (SEQ ID NO:3); LMLGEFLKL (SEQ ID NO:4); RISTFKNWPF (SEQ ID NO:5); RISTFKNWPK (SEQ ID NO:6); STFKNWPFL (SEQ ID NO:7); or LPPAWQPFL (SEQ ID NO:8), or any combination thereof, or a nucleic acid molecule encoding said survivin peptide antigen.

[0031] In certain embodiments of the methods disclosed herein, the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising the amino acid sequence of SEQ ID NOs: 9-12, 26-44, 46-52, 54-62, 64-75, or 79-93, or any combination thereof, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen. In certain embodiments of the methods disclosed herein, the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising at least one amino acid sequence in Table 17, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen.

[0032] In certain embodiments of the methods disclosed herein, the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising the amino acid sequence KVAELVHFL (SEQ ID NO:9); GLMGAQEPT (SEQ ID NO:10); ALSVMGVYV (SEQ ID NO:11); FLWGSKAHA (SEQ ID NO:12), FMFQEALKL (SEQ ID NO:26); EVDPAGHSY (SEQ ID NO:27); NYKRYFPVI (SEQ ID NO:28); VYYTLWSQF (SEQ ID NO:29); SYILVTALG (SEQ ID NO:30); MPKAALLII (SEQ ID NO:31); SVMGVYVGK (SEQ ID NO:32); ALLIIVLGV (SEQ ID NO:33); FLLHKYRVK (SEQ ID NO:34); or IVLGVILTK (SEQ ID NO:35), or any combination thereof, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen.

[0033] In certain embodiments of the methods disclosed herein, at least one MAGE-A9 antigen binds to HLA-A1, HLA-A2, HLA-A3, HLA-A24, and / or HLA-B7. In certain embodiments of the methods disclosed herein, at least one MAGE-A9 antigen binds individually to each of HLA-A1, HLA-A2, HLA-A3, HLA-A24, and HLA-B7.

[0034] In certain embodiments of the methods disclosed herein, the survivin antigen is a survivin peptide antigen comprising the amino acid sequence FEELTLGEF (SEQ ID NO:1); FTELTLGEF (SEQ ID NO:2); LTLGEFLKL (SEQ ID NO:3); LMLGEFLKL (SEQ ID NO:4); RISTFKNWPF (SEQ ID NO:5); RISTFKNWPK (SEQ ID NO:6); STFKNWPFL (SEQ ID NO:7); and / or LPPAWQPFL (SEQ ID NO:8), or a nucleic acid molecule encoding said survivin peptide antigen, and the MAGE-A9 antigen is a survivin peptide antigen comprising the amino acid sequence KVAELVHFL (SEQ ID NO:9); GLMGAQEPT (SEQ ID NO:10); or a nucleic acid molecule encoding said MAGE-A9 peptide antigen, which includes LSVMGVYV (sequence number 11); FLWGSKAHA (sequence number 12); FMFQEALKL (sequence number 26); EVDPAGHSY (sequence number 27); NYKRYFPVI (sequence number 28); VYYTLWSQF (sequence number 29); SYILVTALG (sequence number 30); MPKAALLII (sequence number 31); SVMGVYVGK (sequence number 32); ALLIIVLGV (sequence number 33); FLLHKYRVK (sequence number 34); and / or IVLGVILTK (sequence number 35) or any combination thereof.

[0035] In certain embodiments of the methods disclosed herein, the survivin antigen is a survivin peptide antigen comprising the amino acid sequence LMLGEFLKL (SEQ ID NO: 4) and / or STFKNWPFL (SEQ ID NO: 7), or a nucleic acid molecule encoding said survivin peptide antigen, and the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising the amino acid sequence KVAELVHFL (SEQ ID NO: 9); GLMGAQEPT (SEQ ID NO: 10); ALSVMGVYV (SEQ ID NO: 11); or FLWGSKAHA (SEQ ID NO: 12), or any combination thereof, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen.

[0036] In certain embodiments of the methods disclosed herein, the survivin antigen is a survivin peptide antigen comprising the amino acid sequence LMLGEFLKL (SEQ ID NO: 4) and / or STFKNWPFL (SEQ ID NO: 7), or a nucleic acid molecule encoding said survivin peptide antigen, and the MAGE-A9 antigen is a MAGE-A9 peptide antigen comprising the amino acid sequence KVAELVHFL (SEQ ID NO: 9); GLMGAQEPT (SEQ ID NO: 10); or FLWGSKAHA (SEQ ID NO: 12), or any combination thereof, or a nucleic acid molecule encoding said MAGE-A9 peptide antigen.

[0037] In certain embodiments of the methods disclosed herein, the survivin antigen is two survivin antigens comprising amino acid sequences LMLGEFLKL (SEQ ID NO: 4) and STFKNWPFL (SEQ ID NO: 7), or a nucleic acid molecule encoding said survivin peptide antigens, and the MAGE-A9 antigen is four MAGE-A9 antigens comprising amino acid sequences KVAELVHFL (SEQ ID NO: 9); GLMGAQEPT (SEQ ID NO: 10); ALSVMGVYV (SEQ ID NO: 11); and FLWGSKAHA (SEQ ID NO: 12), or a nucleic acid molecule encoding said MAGE-A9 peptide antigens.

[0038] In certain embodiments of the methods disclosed herein, the survivin antigen is two survivin antigens comprising amino acid sequences LMLGEFLKL (SEQ ID NO: 4) and STFKNWPFL (SEQ ID NO: 7), or a nucleic acid molecule encoding said survivin peptide antigens, and the MAGE-A9 antigen is three MAGE-A9 antigens comprising amino acid sequences KVAELVHFL (SEQ ID NO: 9); GLMGAQEPT (SEQ ID NO: 10); and FLWGSKAHA (SEQ ID NO: 12), or a nucleic acid molecule encoding said peptide antigens.

[0039] In certain embodiments of the methods disclosed herein, each of the survivin and MAGE-A9 peptide antigens is independently at a concentration of about 0.1 μg / μl to about 5.0 μg / μl. In certain embodiments, each of the survivin and MAGE-A9 peptide antigens is at a concentration of at least about 1.0 μg / ml. In certain embodiments, each of the survivin and MAGE-A9 peptide antigens is at a concentration of about 1.0 μg / ml.

[0040] In certain embodiments of the methods disclosed herein, the tumor is a solid tumor.In certain embodiments, the tumor is a hematological malignancy.In certain embodiments, the tumor is breast cancer, ovarian tumor, fallopian tube tumor, peritoneal tumor, bladder tumor, diffuse large B-cell lymphoma, glioma, non-small cell lung tumor, or hepatocellular carcinoma.In certain embodiments, the tumor is bladder cancer.In certain embodiments, the tumor is breast cancer.In certain embodiments, the tumor is ovarian cancer.

[0041] In certain embodiments of the methods disclosed herein, the composition further comprises a T helper epitope. In certain embodiments, the T helper epitope is a peptide comprising the amino acid sequence AQYIKANSKFIGITEL (SEQ ID NO: 13).

[0042] In certain embodiments of the methods disclosed herein, the composition further comprises an adjuvant. In certain embodiments, the adjuvant is a PolyIC polynucleotide. In certain embodiments, the PolyIC polynucleotide is DNA or RNA based.

[0043] In certain embodiments of the method disclosed herein, the carrier comprises a hydrophobic carrier.In certain embodiments, the hydrophobic carrier is vegetable oil, nut oil, or mineral oil.In certain embodiments of the method disclosed herein, the hydrophobic carrier is mineral oil or mannide oleate in mineral oil solution.In certain embodiments of the method disclosed herein, the hydrophobic carrier is Montanide ISA51.

[0044] In certain embodiments of the method disclosed herein, the one or more lipid-based structures comprise monolayer lipid assemblies.In certain embodiments, the one or more lipid-based structures with monolayer lipid assemblies comprise lipid aggregates, in which the hydrophobic part of the lipids is oriented outward toward the hydrophobic carrier, and the hydrophilic part of the lipids is aggregated as a core.In certain embodiments, the one or more lipid-based structures with monolayer lipid assemblies comprise reverse micelles.In certain embodiments, the size of the lipid-based structures is about 2 nm to about 20 nm in diameter.In certain embodiments, the size of the lipid-based structures is about 5 nm to about 10 nm in diameter.

[0045] In certain embodiments of the methods disclosed herein, one or more of the T cell activation therapeutics are inside the lipid-based structure. In certain embodiments of the methods disclosed herein, one or more of the T cell activation therapeutics are outside the lipid-based structure. [Brief description of the drawings]

[0046] [Figure 1] FIG. 1 shows a schematic of the Flex-T HLA binding assay. [Diagram 2]FIG. 2 shows the results of an in vitro assay assessing the binding of each of the peptides to HLA-A2, and shows that all peptides demonstrated binding to HLA-A2. [Figure 3-1] Figures 3A-3C show that T cell activation therapeutics targeting survivin and dual T cell activation therapeutics targeting both survivin and MAGE-A9 elicited comparable immune responses against the common survivin. Figure 3A: Treatment schedule for A2 / DR1 transgenic mice that express human HLA-A2 and HLA-DR1 molecules and lack expression of mouse MHC class I and II molecules. Figure 3B: IFN-γ responses to in vitro peptide stimulation of splenocytes from A2 / DR1 mice immunized with T cell activation therapeutics targeting survivin or dual T cell activation therapeutics targeting both survivin and MAGE-A9, as determined by ELISPOT assay. [Figure 3-2] Figures 3A-3C show that the T cell activation therapeutics targeting survivin and the dual T cell activation therapeutics targeting both survivin and MAGE-A9 elicited comparable immune responses to the common survivin. Figure 3C: IFN-γ responses to in vitro peptide stimulation of lymph node cells from A2 / DR1 mice immunized with the T cell activation therapeutics targeting survivin or the dual T cell activation therapeutics targeting both survivin and MAGE-A9, as determined by ELISPOT assay. [Figure 4]Figure 4 provides a non-limiting schematic of the administration mode of the present invention. The study was performed as two equal arms. In each arm, a group of six A2 / DR1 mice was treated three times with the dual T cell activation therapeutic targeting both survivin and MAGE-A9, or with empty lipid vesicle particles or PBS as a control. One group of mice treated with the dual T cell activation therapeutic targeting both survivin and MAGE-A9 was also subjected to intermittent low-dose cyclophosphamide (CPA) for one week on and one week off. Mice from the first arm (primary / acute phase) were sacrificed 8 days after the last treatment, and mice from the second arm (recovery / chronic phase) were sacrificed 3 weeks after the last treatment to evaluate immunogenicity and toxicity. During the study, mice were monitored for safety signals including weekly detailed clinical examinations (DCE), body weight, and injection site reactions. [Figure 5-1] Figures 5A and 5B show that dual T cell activation therapeutics targeting both survivin and MAGE-A9 induce robust peptide-specific T cell responses. Figure 5A: IFN-γ responses to in vitro peptide stimulation of splenocytes from each treatment group as determined by ELISPOT assay in the primary (acute) phase. [Figure 5-2] Figures 5A and 5B show that dual T cell activation therapeutics targeting both survivin and MAGE-A9 induce robust peptide-specific T cell responses. Figure 5B: IFN-γ responses to in vitro peptide stimulation of splenocytes from each treatment group as determined by ELISPOT assay in the recovery (chronic) phase. [Figure 6] Figure 6 shows that the preliminary safety profile of the dual T cell activation therapeutic targeting both survivin and MAGE-A9 with and without intermittent low-dose CPA showed no signs of toxicity. [Figure 7] FIG. 7 shows weekly changes in A2 / DR1 and body weight in male and female mice treated with a dual T cell activation therapeutic targeting both survivin and MAGE-A9 (combined primary and recovery phases) with or without intermittent low-dose CPA, as well as the control group. [Figure 8] FIG. 8 provides a schematic of the clinical trial master protocol design. [Figure 9] FIG. 9 provides a non-limiting schematic diagram of an overview of a clinical trial study. [Figure 10] FIG. 10 provides a non-limiting schematic diagram of the study procedures for DPX-SurMAGE, with or without low-dose CPA studies. [Figure 11] Figures 11A and 11B show the results of IFN-γ ELISPOT of the first experiment for MAGE-A9 immunogenic HLA-A2 peptides. Induction of IFN-γ responses against HLA-A2 peptides by MAGE-A9 detected by IFN-γ ELISPOT assay. Groups of 10 mice were immunized with MAGE-A9 full-length recombinant protein mixed with poly(dI:dC). Mice were sacrificed 34 days after the first immunization and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 11A) or after direct stimulation with individual candidate or control peptides (Figure 11B). DCE: empty dendritic cells; NS: no stimulation; INF, gp100 and EBV: irrelevant HLA-A2 control peptides. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 12]Figures 12A and 12B show the results of IFN-γ ELISPOT of the second experiment for MAGE-A9 immunogenic HLA-A2 peptides. Induction of IFN-γ responses against HLA-A2 peptides by MAGE-A9 detected by IFN-γ ELISPOT assay. Groups of 10 mice were immunized with MAGE-A9 full-length recombinant protein mixed with poly(dI:dC). Mice were sacrificed 34 days after the first immunization and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 12A) or after direct stimulation with individual candidate or control peptides (Figure 12B). DCE: empty dendritic cells; NS: no stimulation; INF, gp100 and EBV: irrelevant HLA-A2 control peptides. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 13] Figures 13A and 13B show the results of IFN-γ ELISPOT of the third experiment for MAGE-A9 immunogenic HLA-A2 peptides. Induction of IFN-γ responses against HLA-A2 peptides by MAGE-A9 detected by IFN-γ ELISPOT assay. Groups of 10 mice were immunized with MAGE-A9 full-length recombinant protein mixed with poly(dI:dC). Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 13A) or after direct stimulation with individual candidate or control peptides (Figure 13B). DCE: empty dendritic cells; NS: no stimulation; INF, gp100 and EBV: irrelevant HLA-A2 control peptides. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 14]Figures 14A and 14B show the results of IFN-γ ELISPOT of the first experiment for MAGE-A9 immunogenic HLA-A1 peptides. IFN-γ responses induced by MAGE-A9 against HLA-A1 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 5 mice were immunized with poly(dI:dC) alone (as control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 14A) or after direct stimulation with individual candidate or control peptides (Figure 14B). DCE: empty dendritic cells; NS: no stimulation; Ctrl HLA-A1: irrelevant HLA-A1 peptide. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 15] Figures 15A and 15B show the results of IFN-γ ELISPOT of the second experiment on MAGE-A9 immunogenic HLA-A1 peptides. IFN-γ responses induced by MAGE-A9 against HLA-A1 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 5 mice were immunized with poly(dI:dC) alone (as control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 15A) or after direct stimulation with individual candidate or control peptides (Figure 15B). DCE: empty dendritic cells; NS: no stimulation; Ctrl HLA-A1: irrelevant HLA-A1 peptide. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 16]Figures 16A and 16B show the results of IFN-γ ELISPOT of the first experiment for MAGE-A9 immunogenic HLA-A24 peptides. IFN-γ responses induced by MAGE-A9 against HLA-A24 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 5 mice were immunized with poly(dI:dC) alone (as control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 16A) or after direct stimulation with individual candidate or control peptides (Figure 16B). DCE: empty dendritic cells; NS: no stimulation; A24-Ctrl: irrelevant control HLA-A24 peptide. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 17] Figures 17A and 17B show the results of IFN-γ ELISPOT of the second experiment on MAGE-A9 immunogenic HLA-A24 peptides. IFN-γ responses induced by MAGE-A9 against HLA-A24 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 5 mice were immunized with poly(dI:dC) alone (as control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 17A) or after direct stimulation with individual candidate or control peptides (Figure 17B). DCE: empty dendritic cells; NS: no stimulation; A24-Ctrl: irrelevant control HLA-A24 peptide. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 18]Figures 18A and 18B show the results of IFN-γ ELISPOT of the third experiment for MAGE-A9 immunogenic HLA-A24 peptides. IFN-γ responses induced by MAGE-A9 against HLA-A24 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 5 mice were immunized with poly(dI:dC) alone (as control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 18A) or after direct stimulation with individual candidate or control peptides (Figure 18B). DCE: empty dendritic cells; NS: no stimulation; A24-Ctrl: irrelevant control HLA-A24 peptide. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 19] Figures 19A and 19B show the results of IFN-γ ELISPOT of the first experiment for MAGE-A9 immunogenic HLA-B7 peptides. IFN-γ responses induced by MAGE-A9 against HLA-B7 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 5 mice were immunized with poly(dI:dC) alone (as control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 19A) or after direct stimulation with individual candidate or control peptides (Figure 19B). DCE: empty dendritic cells; NS: no stimulation; B7-Ctrl: irrelevant control HLA-B7 peptide. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 20]Figures 20A and 20B show the results of IFN-γ ELISPOT of the second experiment on MAGE-A9 immunogenic HLA-B7 peptides. IFN-γ responses induced by MAGE-A9 against HLA-B7 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 5 mice were immunized with poly(dI:dC) alone (as control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 20A) or after direct stimulation with individual candidate or control peptides (Figure 20B). DCE: empty dendritic cells; NS: no stimulation; B7-Ctrl: irrelevant control HLA-B7 peptide. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 21] Figures 21A and 21B show the results of IFN-γ ELISPOT of the third experiment for MAGE-A9 immunogenic HLA-B7 peptides. IFN-γ responses induced by MAGE-A9 against HLA-B7 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 5 mice were immunized with poly(dI:dC) alone (as control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 21A) or after direct stimulation with individual candidate or control peptides (Figure 21B). DCE: empty dendritic cells; NS: no stimulation; B7-Ctrl: irrelevant control HLA-B7 peptide. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 22]Figures 22A and 22B show the results of IFN-γ ELISPOT of the fourth experiment for MAGE-A9 immunogenic HLA-B7 peptides. IFN-γ responses induced by MAGE-A9 against HLA-B7 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 5 mice were immunized with poly(dI:dC) alone (as control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 34 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 22A) or after direct stimulation with individual candidate or control peptides (Figure 22B). DCE: empty dendritic cells, NS: no stimulation; B7-Ctrl: irrelevant control HLA-B7 peptide. Student T test *p<0.05;**p<0.005;***p<0.0005;****p<0.00005. [Figure 23] Figures 23A and 23B show the results of IFN-γ ELISPOT of the experiment on MAGE-A9 immunogenic HLA-A3 / A11 peptides. IFN-γ responses induced by MAGE-A9 against HLA-A3 candidate peptides detected by IFN-γ ELISPOT assay. Two groups of 3 and 10 HLA-A11 transgenic mice were immunized with DPX-Empty alone (as a control: left bar) and MAGE-A9 full-length recombinant protein mixed with poly(dI:dC) (right bar), respectively. Mice were sacrificed 36 days after the first immunization, and splenocytes were tested in IFN-γ ELISPOT after stimulation with dendritic cells loaded with individual candidate or control peptides (Figure 23A) or after direct stimulation with individual candidate or control peptides (Figure 23B). DCE: empty dendritic cells; NS: no stimulation; A3-Ctrl: irrelevant control HLA-A3 peptide. [Figure 24]Figure 24 shows the reactivity of HLA-A2 candidate peptides in a CTL assay. Splenocytes from A2 / DR1 mice immunized twice with full-length recombinant MAGE-A9 protein were restimulated for 3 days with irradiated splenocytes loaded with HLA-A2 candidate peptides or with an irrelevant HLA-A2 HA-58 peptide as a control. CTL activity upon stimulation was determined by standard 51Cr release using irradiated RMA-S HHD cells loaded with various peptides as target cells. Three different effector:target cell ratios were tested (75:1, 50:1, and 25:1). For each condition, the % of cytotoxicity (=(CPM-STR) / (MTR-STR) x 100) is presented. CPM: counts per minute; STR: spontaneous target release; MTR: maximum target release. Experiments were repeated at least three times. Results presented are from one representative experiment. Bars represent the mean plus or minus standard deviation (SD) of triplicate determinations. Adjusted p values ​​were determined using Tukey's multiple comparison test: *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Diagram 25] Figure 25 shows cytokine responses upon stimulation with reactive HLA-A2 MAGE-A9 peptides. Groups of three A2 / DR1 mice were either not immunized (naive mice), or immunized twice with a pool of four peptides reactive in the CTL assay (M9-A2-24;, M9-A2-111; M9-A2-223; M9-A2-270), or with adjuvant alone as a control. Mice were sacrificed 28 days later and spleens were harvested for isolation of splenocytes. Splenocytes were restimulated with individual peptides or pools of peptides for 72 hours. Supernatants were collected and assayed by ELISA for secretion of mouse IFN-γ. Bars represent the mean plus or minus standard deviation (SD) of triplicate determinations. Adjusted p-values ​​were determined using Tukey's multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Before the present invention is described, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0048] In one aspect, the present invention relates to a pharmaceutical composition for delivering at least two T cell activation therapeutics to a subject, comprising i) at least two T cell activation therapeutics and ii) a carrier, wherein the at least two T cell activation therapeutics are at least one survivin antigen and at least one Melanoma Antigen Gene (MAGE) antigen (e.g., MAGE-A9, MAGE-A1, MAGE-A2, MAGE-A2B, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, MAGE-A14, MAGE-A15, MAGE-A16, MAGE-A17, MAGE-A18, MAGE-A19, MAGE-A20, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE-A27, MAGE-A28, MAGE-A29, MAGE-A30, MAGE-A31, MAGE-A32, MAGE-A33, MAGE-A34, MAGE-A35, MAGE-A36, MAGE-A37, MAGE-A38, MAGE-A3 ... , MAGE-A11, MAGE-A12, MAGE-A13P, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B18, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D3, MAGE-D4, MAGE-D4B, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-G1, MAGE-H1, MAGE-L2, and NDN) antigens. In certain embodiments, at least one MAGE is MAGE-A9. MAGE-A9 is used as an example of MAGE antigen in this disclosure, but this is not meant to be limiting.

[0049] In another aspect, the present invention relates to a method of treating a tumor in a subject, the method comprising administering to a subject a composition for delivering at least two T cell activation therapeutics, the composition comprising: i) at least two T cell activation therapeutics; ii) a carrier, wherein the at least two T cell activation therapeutics are directed to at least one survivin antigen and at least one Melanoma Antigen Gene (MAGE) antigen (e.g., MAGE-A9, MAGE-A1, MAGE-A2, MAGE-A2B, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-B10, MAGE-B20, MAGE-B30, MAGE-B40, MAGE-B50, MAGE-B60, MAGE-B70, MAGE-B80, MAGE-B90, MAGE-B101, MAGE-B102, MAGE-B103, MAGE-B104, MAGE-B105, MAGE-B106, MAGE-B107, MAGE-B108, MAGE-B109, MAGE-B210, MAGE-B220, MAGE-B230, MAGE-B240, MAGE-B250, MAGE-B260, MAGE-B270, MAGE-B280, MAGE-B310, MAGE-B320, MAGE-B330, MAGE-B340, MAGE-B350, MAGE-B360, MAGE-B370, MAGE-B410, MAGE-B420, MAGE-B430, MAGE-B440, MAGE-B450, MAGE-B460, MAGE-B470, MAGE-B480, MAGE-B490, MAGE-B510, MAGE-B52 MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13P, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B18, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D3, MAGE-D4, MAGE-D4B, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-G1, MAGE-H1, MAGE-L2, and NDN) antigens. In certain embodiments, at least one MAGE is MAGE-A9. MAGE-A9 is used as an example of MAGE antigen in this disclosure, but this is not meant to be limiting.

[0050] definition It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0051] The phrase "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements connected thereby, i.e., in some cases conjunctively, and in other cases disjunctively. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements connected thereby. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to such elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used with open-ended language such as "comprising", may refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements), etc.

[0052] The term "about" as used throughout this specification means reasonably close. For example, "about" can mean within an acceptable standard deviation and / or acceptable error range for a particular value determined by a person skilled in the art, which is expected to depend on how the particular value is measured. Furthermore, when an integer is expressed, about can refer to a decimal value on either side of the integer. When used in the context of a range, the term "about" includes all exemplary values ​​between one particular value at one end of the range and the other particular value at the other end of the range, as well as values ​​reasonably close beyond each end.

[0053] As used herein, the transitional terms "comprising," "including," "carrying," "having," "containing," "involving," and the like, whether recited in the specification or the appended claims, shall be understood to be inclusive or open-ended (i.e., meaning including, but not limited to), and they do not exclude unrecited elements, materials, or method steps. Only the transitional phrases "consisting of" and "consisting essentially of" are exclusive or part-exclusive transitional phrases with respect to the claims and the exemplary embodiment paragraphs herein, respectively.

[0054] "Treating" or "treatment of" or "preventing" or "prevention of," as used herein, refers to an approach to obtain a beneficial or desired result. Beneficial or desired results may include, but are not limited to, alleviating or ameliorating one or more symptoms or conditions, reducing the extent of the disease, stabilizing the disease state, preventing the development of the disease, preventing the spread of the disease, delaying or slowing (e.g., suppressing) the progression of the disease, delaying or slowing the onset of the disease, conferring protective immunity against disease-causing agents, and alleviating or palliating the disease state. "Treating" or "preventing" may also mean prolonging the survival of a patient beyond that expected in the absence of treatment, or may mean temporarily inhibiting the progression of a disease or preventing the appearance of a disease, for example, by preventing an infection in a subject. "Treating" or "preventing" may also refer to a reduction in the size of a tumor mass, a reduction in tumor burden, a reduction in target tumor burden, a reduction in tumor aggressiveness, and the like.

[0055] "Treat" can be distinguished from "prevent" in that "treat" is typically performed on a subject that already has a disease or disorder, whereas "prevent" is typically performed on a subject that does not have a disease or disorder or is known to have been exposed to a cancer-causing agent. As is expected to be understood, there may be overlap in treatment and prevention. For example, it is possible to "treat" a disease in a subject while simultaneously "preventing" the symptoms or progression of the disease. Furthermore, "treat" and "prevent" may overlap in that treating a subject to induce an immune response (e.g., vaccination) may have a subsequent effect of preventing infection by a pathogen or preventing the underlying disease or symptoms caused by infection with a pathogen. These preventative aspects are encompassed herein by expressions such as "tumor treatment" or "cancer treatment".

[0056] As used herein, the terms "cancer," "cancer cell," "tumor," and "tumor cell" (used interchangeably) refer to cells exhibiting abnormal growth characterized by a marked loss of control over cell proliferation, or immortalized cells. The term "cancer" or "tumor" includes metastatic cancers or tumors, as well as non-metastatic cancers or tumors. Cancer can be diagnosed using criteria generally accepted in the art, including the presence of malignant tumors.

[0057] As used herein, a "therapeutically effective amount" refers to an amount of a T cell activating therapeutic, active agent, and / or any additional therapeutic agent effective to provide a therapeutic, prophylactic, or diagnostic benefit to a subject, and / or an amount sufficient to modulate an immune and / or humoral response in a subject. As used herein, "modulating" an immune and / or humoral response is distinct from and different from activating an immune and / or humoral response. "Modulating" refers to an active agent and / or additional therapeutic agent described herein enhancing an immune and / or humoral response that is activated by another mechanism or compound (e.g., by an antigen or immunogen). In one embodiment, the immune and / or humoral response was activated prior to administration of an effective active agent, T cell activating therapeutic, and / or any additional therapeutic agent described herein. In another embodiment, the immune and / or humoral response may be activated in response to administration of an effective active agent, T cell activating therapeutic, and / or any additional therapeutic agent described herein. In another embodiment, the immune and / or humoral response may be activated following administration of an effective active agent described herein, a T cell activation therapeutic, and / or any additional therapeutic agent.

[0058] In some embodiments, the therapeutically effective amount of the composition is the amount that can induce clinical response in the subject in the treatment of a particular disease or disorder.The determination of the therapeutically effective amount of the composition is well within the capabilities of those skilled in the art, especially in light of the disclosure provided herein.The therapeutically effective amount can be changed according to various factors such as the condition, weight, sex and age of the subject.

[0059] In the methods of the invention, the agent can "improve the efficacy of a T cell activation therapy (e.g., a survivin and / or MAGE-A9 therapy)" by directly or indirectly enhancing the immune response to the survivin and / or MAGE-A9 antigens in the T cell activation therapy. This can be accomplished, for example, by reducing the number and / or activity of inhibitory immune cells. The tumor microenvironment has been reported to upregulate many factors that promote the development of, for example, inhibitory immune cells, such as CD4+FoxP3+regulatory T cells (Treg) (Curiel et al., Nat Med 10(9): 942-949, 2004), myeloid-derived suppressor cells (MDSC) (Nagaraj and Gabrilovich, Cancer Res 68(8): 2561-3, 2008), and CD19+CD5+CD1dhiIL-10+B cells (Breg) (Balkwill et al., Trends Immunol, 3 Dec. 2012, 10.1016 / j.it.2012.10.007 (Epub ahead of print)). Therefore, the ability to reduce the number or activity of these inhibitory immune cells represents an embodiment for improving the therapeutic efficacy of T cell activation.

[0060] "Improving the efficacy of T cell activation therapeutics" (e.g., survivin therapeutics and / or MAGE-A9) can also be achieved, for example, by increasing the number and / or activity of antigen-specific CD8+ T cells. In this regard, it has been reported that the tumor microenvironment contributes to the direct suppression of activated CD8+ T cells, for example, by releasing immunosuppressive cytokines, such as TNF-α and TGF-β (Yang et al., Trends Immunol 31(6): 220-227, 2010). Therefore, the ability to increase the activity of antigen-specific CD8+ T cells represents a mechanism that may improve the therapeutic efficacy of T cell activation. The increase in antigen-specific CD8+ T cells may be the result of an increase in the number of such cells, an increase in the activity of such cells, and / or the generation of an enriched population of antigen-specific CD8+ T cells relative to total CD8+ T cells, for example, such generation by a relative decrease in total CD8+ T cells.

[0061] More generally, "improving the efficacy of a T cell activating therapeutic" refers to the ability of the methods of the invention to enhance the immunogenicity of a survivin and / or MAGE-A9 therapeutic by enhancing the cell-mediated and / or humoral immune response elicited by the survivin therapeutic; to increase the number of immune cells and / or antibodies at the injection site or tumor site; or to improve the therapeutic effect provided by a survivin and / or MAGE-A9 therapeutic of the invention, for example, by enhancing preventative and / or therapeutic treatment of cancer and / or by alleviating symptoms of, delaying the progression of, or inhibiting disease. Improving the efficacy of a survivin and / or MAGE-A9 therapeutic may also be associated with improved quality of life or reduced morbidity compared to monotherapy treatment.

[0062] "Improving the efficacy of a T cell activation therapy" can also mean that lower doses of the active ingredients of the combination of the invention are required to bring about the desired result. This includes both embodiments in which the dosage itself is lower, and embodiments in which the survivin and / or MAGE-A9 therapy, active agent and / or additional therapeutic agent (e.g., one that interferes with DNA replication and / or an immunomodulator) is applied less frequently.

[0063] The terms "subject," "patient," "individual," and "animal" are used interchangeably herein and refer to mammals, such as, but not limited to, humans and domestic animals (e.g., primates, cats, dogs, cows, horses, sheep, pigs, rabbits, mice, rats, etc.), and experimental animal models. In a preferred embodiment, the subject is a human.

[0064] Composition for T cell activation therapy In one aspect, the present invention relates to a pharmaceutical composition for delivering at least two T cell activation therapeutics to a subject comprising i) at least two T cell activation therapeutics and ii) a carrier, wherein the at least two T cell activation therapeutics comprise at least one survivin antigen and at least one melanoma associated antigen 9 (MAGE-A9) antigen.

[0065] The term "antigen" includes any substance, drug, molecule, element, compound, or combination thereof that is intended to be administered to a subject. An antigen is incorporated into the composition of the present invention as a hydrophobic phase antigen when it is contained in the hydrophobic phase of the composition, or as a hydrophilic phase antigen when it is contained in the hydrophilic phase of the composition. The antigen may be a natural product, a synthetic compound, or a combination of two or more substances. The antigen may be a peptide antigen, a DNA polynucleotide encoding the antigen, or an RNA polynucleotide encoding the antigen, or a functional equivalent or functional fragment of any one of them. In some embodiments, the antigen is a DNA polynucleotide or an RNA polynucleotide encoding the antigen. In some embodiments, the antigen is a peptide antigen. In some embodiments, the peptide antigen is glycosylated.

[0066] The T cell activation therapeutic composition of the present invention may have any form suitable for delivery of survivin and MAGE-A9 antigens to a subject. The T cell activation therapeutic composition according to the present invention can be formulated according to known methods, for example, by mixing one or more survivin antigens and one or more MAGE-A9 antigens with one or more pharma- ceutically acceptable excipients or carriers, preferably acceptable for administration to humans. Examples of such excipients, carriers and formulation methods can be found, for example, in Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, PA). To formulate a pharma-ceutically acceptable T cell activation therapeutic composition suitable for effective administration, such compositions will typically contain a therapeutically effective amount of a survivin antigen, for example, a survivin polypeptide, survivin peptide, or survivin peptide variant described herein, or a nucleic acid molecule or vector encoding such a survivin antigen, and a MAGE-A9 antigen, for example, a MAGE-A9 polypeptide, MAGE-A9 peptide, or MAGE-A9 peptide variant described herein, or a nucleic acid molecule or vector encoding such a MAGE-A9 antigen.

[0067] Once one or more suitable survivin antigens and one or more suitable MAGE-A9 antigens have been selected for inclusion in a T cell activation treatment composition according to the present invention, the agents may be delivered by a variety of suitable means known in the art. T cell activation therapeutic compositions for use in the methods described herein include, for example, but are not limited to, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991; Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAP) (see, e.g., Tarn, JP, Proc. Natl. Acad. Sci. USA 85:5409-5413, 1988;Tarn, JP, J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; ballistic delivery systems, typically crystallized peptides, peptides for use in viral delivery vectors (Perkus, ME et al., In: Concepts in vaccine development, Kaufmann, SHE, ed., p. 379, 1996;Chakrabarti, S. et al., Nature 320:535, 1986;Hu, SL et al., Nature 320:537, 1986;Kieny, M.-P. et al., AIDS Bio / Technology 4:790,1986;Top, FH et al., J. Infect. Dis. 124:148,1971;Chanda, PK et al., Virology 175:535,1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25,1996;Eldridge, JH et al., Sem. Hematol. 30:16,1993;Falo, LD, Jr. et al., Nature Med. 7:649,1995), adjuvants (Warren, HS, Vogel, FR, and Chedid, LA Annu. Rev. Immunol. 4:369,1986;Gupta, RK et al., Vaccine 1 1 :293, 1993), liposomes (Reddy, R. et al, J. Immunol. 148:1585, 1992;Rock, K. L, Immunol. Today 17:131, 1996), or naked or particle-adsorbed cDNA (Ulmer, JB et al., Science 259:1745, 1993;Robinson, H. L, Hunt, LA, and Webster, RG, Vaccine 1 1 :957, 1993;Shiver, JW et al., In: Concepts in vaccine development, Kaufmann, SHE, ed., p. 423, 1996;Cease, KB, and Berzofsky, JA, Annu. Rev. Immunol. 12:923, 1994 and Eldridge, JH et al., Sem. Hematol. 30:16, 1993). Each reference disclosed in this paragraph is incorporated herein by reference for all intended purposes.

[0068] The T cell activation therapeutic composition of the present invention also encompasses nucleic acid-mediated modes.For example, DNA or RNA encoding one or more of the survivin antigens described herein and DNA or RNA encoding one or more of the MAGE-A9 antigens may be administered to a subject.Such approaches are described, for example, in Wolff et al., Science 247:1465 (1990), as well as U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO98 / 04720.Examples of DNA-based delivery techniques include "naked DNA", facilitated (bupivicaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, for example, U.S. Patent No. 5,922,687). Each reference disclosed in this paragraph is incorporated herein by reference for all intended purposes.

[0069] In further embodiments of the T cell activation therapeutic composition, the survivin and MAGE-A9 antigens (e.g., survivin and MAGE-A9 peptides) can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector for expressing nucleotide sequences encoding, for example, survivin and MAGE-A9 peptides described herein. When introduced into an acutely or chronically infected host, or into a non-infected host, the recombinant vaccinia virus expresses the antigenic peptides, thereby eliciting a host immune response. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, such as adeno- and adeno-associated viral vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those of skill in the art and are encompassed within the T cell activation therapeutic compositions described herein. Each of the references disclosed in this paragraph is incorporated herein by reference for all intended purposes.

[0070] The T cell activation therapeutic agent according to the present invention also encompasses compositions containing one or more survivin antigens and one or more MAGE-A9 antigens, where the antigens may be present individually or as constructs containing multiple copies of the same or different survivin and MAGE-A9 antigens. For example, the survivin antigen may be present as a single nucleic acid molecule (e.g., vector) encoding several of the same or different survivin antigens, and the MAGE-A9 antigen may be present as a single nucleic acid molecule (e.g., vector) encoding several of the same or different MAGE-A9 antigens. Or in other embodiments, homopolymers containing multiple copies of the same survivin antigen, or heteropolymers of various different survivin antigens, and homopolymers containing multiple copies of the same MAGE-A9 antigen, or heteropolymers of various different MAGE-19 antigens may be used. Such polymers may have the advantage of providing an increased immune response because they contain multiple copies of the survivin and MAGE-A9 antigens, such that the resulting effect may be an enhanced ability to induce an immune response with one or more antigenic determinants of survivin and MAGE-A9. The composition may include naturally occurring regions of one or more survivin antigens and / or one or more MAGE-A9 antigens, or may include prepared antigens, for example antigens prepared recombinantly or by chemical synthesis.

[0071] The T cell activation therapeutic agent of the present invention may comprise an antigen-presenting cell (APC), such as a dendritic cell (DC), as a vehicle for presenting one or more survivin antigens (e.g., survivin peptides) and one or more MAGE-A9 antigens (e.g., MAGE-A9 peptides). Such a T cell activation therapeutic composition may be produced in vitro after mobilization and collection of dendritic cells, thereby causing loading of dendritic cells in vitro. For example, dendritic cells are transfected with DNA or RNA encoding one or more survivin antigens and DNA or RNA encoding one or more MAGE-A9 antigens, or pulsed with survivin and MAGE-A9 peptide antigens. The dendritic cells can then be administered to a subject to elicit an immune response in vivo.

[0072] The T cell activation therapeutic agent according to the present invention can be administered by any suitable means, such as injection (e.g., intramuscular, intradermal, subcutaneous, intravenous or intraperitoneal), aerosol, oral, nasal, topical, intravaginal, transdermal, transmucosal, or any other suitable route. The T cell activation therapeutic agent may be formulated for systemic or local distribution within the subject's body. Formulations for systemic administration include those designed for administration by injection as well as those designed for transdermal, transmucosal or oral administration.

[0073] For injection, the T cell activating therapeutic agent may be formulated in a carrier that includes a continuous phase of a hydrophobic substance as described herein, for example, in a water-in-oil emulsion or an oil-based carrier. In some embodiments, liposomes or lipid vesicle particles can be used with the carrier. The T cell activating therapeutic agent may also be formulated in an aqueous solution, for example, Hank's solution, Ringer's solution, or physiological buffered saline.

[0074] As expected from the above, the T cell activation therapeutic composition of the present invention is meant to encompass any composition or antigen delivery means (e.g., viral vectors, virus-like particles, lipid vesicle particles, etc.) useful in the treatment of cancer, including compositions capable of stimulating an immune response in a subject, e.g., a specific cytotoxic T cell response when administered. In some embodiments, the lipid vesicle particles used are bilayer vesicle structures, such as liposomes. Lipid vesicle particles are completely closed lipid bilayer membranes that contain an enclosed aqueous volume. Lipid vesicle particles can be unilamellar vesicles (having a single bilayer membrane) or multilamellar vesicles (characterized by multiple membrane bilayers, whereby each bilayer may or may not be separated from the next by an aqueous layer). A general discussion of liposomes can be found in Gregoriadis 1990; and Frezard 1999. Lipid vesicle particles can adsorb to virtually any type of cell and then release the incorporated agent (e.g., survivin and MAGE-A9 antigens). Alternatively, lipid vesicle particles can fuse with target cells, thereby releasing the contents of lipid vesicle particles into the target cells. Alternatively, lipid vesicle particles can be taken up by phagocytic cells. Lipid vesicle particles have been used in the preparation of compositions that include hydrophobic carriers, as vesicles for encapsulating antigens, and as emulsifiers for stabilizing formulations (see, for example, WO2002 / 038175, WO2007 / 041832, WO2009 / 039628, WO2009 / 146523, and WO2013 / 049941, each of which is incorporated herein by reference for all intended purposes). Hydrophilic antigens are typically trapped in the hydrophilic interior, while hydrophobic antigens can be intercalated in the lipid bilayer or dispersed in the oil phase. In another embodiment, pre-formed lipid vesicle particles can be used in the vaccine compositions disclosed herein.In embodiments in which the composition is water-free, one or more of the components of the composition (e.g., survivin and MAGE-A9 antigens, adjuvants, and / or T-helper epitopes) may be encapsulated or mixed or suspended in lipid vesicle particles in a hydrophilic phase, lyophilized, and then reconstituted in a hydrophobic carrier. In such embodiments, the lipid vesicle particles may be reorganized to form alternative structures in the hydrophobic carrier.

[0075] To obtain the T cell activation therapeutic composition of the present invention, it may be suitable to combine the survivin and MAGE-A9 antigens, which may be relatively small survivin or MAGE-A9 peptides, with various materials, such as adjuvants, excipients, surfactants, immunostimulatory components, and / or carriers. In certain embodiments, the peptides may be about 8 to about 24 amino acids in length. In certain embodiments, the peptides may be about 8 to about 11 amino acids in length. In certain embodiments, the peptides may be about 15 to about 24 amino acids in length. In certain embodiments, the peptides may be 5 to 120 amino acids in length, 5 to 100 amino acids in length, 5 to 75 amino acids in length, 5 to 50 amino acids in length, 5 to 40 amino acids in length, 5 to 30 amino acids in length, 5 to 20 amino acids in length, or 5 to 10 amino acids in length. In certain embodiments, the peptide antigen can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. To enhance specific immune response, adjuvants can be included in the T cell activation therapeutic composition. Depending on the desired route of administration or the desired distribution in the subject, for example, systemic or local distribution, various carriers can be used.

[0076] In certain embodiments, the T cell activation therapeutic composition for use in the methods of the invention is a composition comprising at least one survivin antigen and at least one MAGE-A9 antigen, lipid vesicle particles, and a carrier comprising a continuous phase of a hydrophobic substance (e.g., mineral oil, incomplete Freund's adjuvant (IFA), Montanide® ISA51, VG) for reorganization in the hydrophobic carrier to form alternative structures (reverse micelles). In further embodiments, the composition may additionally comprise an adjuvant. In further embodiments, the composition may additionally comprise a T helper epitope or antigen.

[0077] That is, in one embodiment, the T cell activation therapeutic composition comprises one or more survivin antigens and one or more MAGE-A9 antigens; a T helper epitope; an adjuvant; a lipid vesicle particle; and a carrier comprising a continuous phase of a hydrophobic material. The T helper epitope can be, for example, a peptide comprising the amino acid sequence AQYIKANSKFIGITEL (SEQ ID NO: 13). The adjuvant can be, for example, but not limited to, polyI:C or polydIdC polynucleotide (e.g., SEQ ID NO: 22).

[0078] In a further embodiment, the T cell activation therapeutic composition for use in the methods of the present invention is a composition comprising at least one survivin antigen and at least one MAGE-A9 antigen in conjunction with a lipid vesicle particle-based and / or amphiphilic compound-based vaccine adjuvant platform, such as, but not limited to, VacciMax®, DepoVax™, and DPX™ platform technologies (see, e.g., U.S. Pat. Nos. 6,793,923 and 7,824,686; U.S. Patent Publication No. 20160067335; WO2002 / 038175; WO2007 / 041832; WO2009 / 039628; WO2009 / 043165; WO2009 / 146523, WO2013049941, WO2014 (See WO2016 / 153636, WO2016 / 176761, WO2016 / 109880, WO2017 / 190242, WO2017 / 083963, WO2018 / 058230, WO2019 / 010560, WO2019 / 090411, or WO2021 / 072535). The DepoVax™ / DPX™ platform is a T cell activating therapeutic delivery formulation that provides controlled and prolonged exposure of antigens plus adjuvants to the immune system. This platform can provide a strong, specific, sustained immune response and can result in single dose efficacy.

[0079] In certain embodiments, the T cell activation therapy of the present invention comprises at least one survivin antigen, each of which is at a concentration of about 0.01 mg / ml to about 10 mg / ml, about 0.025 mg / ml to about 9 mg / ml, about 0.05 mg / ml to about 8 mg / ml, about 0.75 mg / ml to about 7 mg / ml, about 0.1 mg / ml to about 6 mg / ml, about 0.25 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 4 mg / ml, about 0.75 mg / ml to about 3 mg / ml, or about 1 mg / ml to about 2 mg / ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one survivin antigen, each of which is at a concentration of about 0.1 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 3 mg / ml, or about 0.5 mg / ml to about 2 mg / ml. In certain embodiments, a T cell activation therapy of the invention comprises at least one survivin antigen, and each survivin antigen is at a concentration of about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml. In certain embodiments, the T cell activation therapy of the invention comprises at least one survivin antigen, each survivin antigen being at a concentration of about 1 mg / ml.

[0080] In certain embodiments, the T cell activation therapy of the present invention comprises at least one MAGE-A9 antigen, each MAGE-A9 antigen being at a concentration of about 0.01 mg / ml to about 10 mg / ml, about 0.025 mg / ml to about 9 mg / ml, about 0.05 mg / ml to about 8 mg / ml, about 0.75 mg / ml to about 7 mg / ml, about 0.1 mg / ml to about 6 mg / ml, about 0.25 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 4 mg / ml, about 0.75 mg / ml to about 3 mg / ml, or about 1 mg / ml to about 2 mg / ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one MAGE-A9 antigen, each MAGE-A9 antigen being at a concentration of about 0.1 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 3 mg / ml, or about 0.5 mg / ml to about 2 mg / ml. In certain embodiments, the T cell activation therapy of the invention comprises at least one MAGE-A9 antigen, and each MAGE-A9 antigen is at a concentration of about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml , about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml. In certain embodiments, the T cell activation therapy of the invention comprises at least one MAGE-A9 antigen, and each MAGE-A9 antigen is at a concentration of about 1 mg / ml.

[0081] In certain embodiments, the T cell activation therapy of the present invention comprises at least one survivin antigen, and the T cell activation therapy is administered in a dose of about 0.01 to about 3 ml, about 0.05 ml to about 2 ml, about 0.075 ml to about 1.75 ml, about 0.1 ml to about 1.5 ml, about 0.125 ml to about 1.25 ml, about 0.15 ml to about 1 ml, about 0.175 ml to about 0.75 ml, about 0.2 ml to about 0.5 ml, or about 0.25 ml to about 0.5 ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one survivin antigen, and the T cell activation therapy is administered in a dose of about 0.01 ml to about 1 ml, about 0.5 ml to about 0.75, or about 0.25 ml to about 0.5 ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one survivin antigen, and the T cell activation therapy is administered in a volume of about 0.05 ml, about 0.06 ml, about 0.07 ml, about 0.08 ml, about 0.09 ml, about 0.1 ml, about 0.125 ml, about 0.15 ml, about 0.175 ml, about 0.2 ml, about 0.225 ml, about 0.25 ml, about 0.275 ml, about 0.3 ml, about 0.325 ml, about 0.35 ml, about 0.375 ml, about 0.4 ml, about 0.425 ml, about 0.45 ml 1, about 0.475 ml, about 0.5 ml, about 0.525 ml, about 0.55 ml, about 0.575 ml, about 0.6 ml, about 0.625 ml, about 0.65 ml, about 0.675 ml, about 0.7 ml, about 0.725 ml, about 0.75 ml, about 0.775 ml, about 0.8 ml, about 0.825 ml, about 0.85 ml, about 0.875 ml, about 0.9 ml, about 0.925 ml, about 0.95 ml, about 0.975 ml, about 1 ml, about 1.25 ml, about 1.5 ml, about 1.75 ml, or about 2 ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one survivin antigen, and the T cell activation therapy is administered in a dose of about 0.25 ml or about 0.5 ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one survivin antigen, and the T cell activation therapy is administered in a dose of about 0.1 ml. In certain embodiments, the dose is a priming dose. In certain embodiments, the dose is a booster dose.

[0082] In certain embodiments, the T cell activation therapy of the present invention comprises at least one MAGE-A9 antigen and the T cell activation therapy is administered in a dose of about 0.01 to about 3 ml, about 0.05 ml to about 2 ml, about 0.075 ml to about 1.75 ml, about 0.1 ml to about 1.5 ml, about 0.125 ml to about 1.25 ml, about 0.15 ml to about 1 ml, about 0.175 ml to about 0.75 ml, about 0.2 ml to about 0.5 ml, or about 0.25 ml to about 0.5 ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one MAGE-A9 antigen and the T cell activation therapy is administered in a dose of about 0.01 ml to about 1 ml, about 0.5 ml to about 0.75, or about 0.25 ml to about 0.5 ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one MAGE-A9 antigen, and the T cell activation therapy is at about 0.05 ml, about 0.06 ml, about 0.07 ml, about 0.08 ml, about 0.09 ml, about 0.1 ml, about 0.125 ml, about 0.15 ml, about 0.175 ml, about 0.2 ml, about 0.225 ml, about 0.25 ml, about 0.275 ml, about 0.3 ml, about 0.325 ml, about 0.35 ml, about 0.375 ml, about 0.4 ml, about 0.425 ml, about 0.45 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0. ... ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0.1 ml, about 0. ml, about 0.475 ml, about 0.5 ml, about 0.525 ml, about 0.55 ml, about 0.575 ml, about 0.6 ml, about 0.625 ml, about 0.65 ml, about 0.675 ml, about 0.7 ml, about 0.725 ml, about 0.75 ml, about 0.775 ml, about 0.8 ml, about 0.825 ml, about 0.85 ml, about 0.875 ml, about 0.9 ml, about 0.925 ml, about 0.95 ml, about 0.975 ml, about 1 ml, about 1.25 ml, about 1.5 ml, about 1.75 ml, or about 2 ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one MAGE-A9 antigen, and the T cell activation therapy is administered in a dose of about 0.25 ml or about 0.5 ml. In certain embodiments, the T cell activation therapy of the present invention comprises at least one MAGE-A9 antigen, and the T cell activation therapy is administered in a dose of about 0.1 ml. In certain embodiments, the dose is a priming dose. In certain embodiments, the dose is a booster dose.

[0083] (i) Survivin antigen The T cell activation therapeutic composition of the present invention comprises at least one survivin antigen. The expression "at least one" is used synonymously with the expression "one or more". These expressions refer to the number of different survivin antigens in the T cell activation therapeutic, and not the amount of any particular survivin antigen, unless expressly stated otherwise herein. In accordance with the ordinary meaning of "at least one" or "one or more", the T cell activation therapeutic composition of the present invention contains at least one survivin antigen.

[0084] Survivin, also called baculovirus inhibitor of apoptosis repeat-containing 5 (BIRC5), is a protein involved in the negative regulation of apoptosis. It is classified as a member of the family of inhibitors of apoptosis proteins (IAPs). Survivin is a 16.5 kDa cytoplasmic protein that contains a single BIR motif and a highly charged carboxy-terminal coiled region instead of a RING finger. The gene encoding survivin is nearly identical to the sequence of effector cell protease receptor-1 (EPR-1), but is oriented in the opposite direction. The coding sequence of survivin (human) is 429 nucleotides long (SEQ ID NO: 14), including a stop codon. The encoded protein, survivin (human), is 142 amino acids long (SEQ ID NO: 15).

[0085] [Table 1]

[0086] It is hypothesized that survivin protein functions to inhibit caspase activation, thereby resulting in the negative regulation of apoptosis or programmed cell death.Consistent with this function, survivin has been identified as one of the top genes that is consistently upregulated in many types of cancer, but not in normal tissues (see, for example, Altieri et al., Lab Invest, 79: 1327-1333, 1999; and U.S. Patent No. 6,245,523).Therefore, this fact makes survivin an ideal target for cancer therapy, since it targets cancer cells but not normal cells.In fact, survivin is reported to be highly expressed in many tumor types, including the majority of human cancers, and to be useful for prognostic prediction.

[0087] The T cell activation therapeutic of the present invention comprises one or more survivin antigens. The term "survivin antigen" as used herein includes any peptide, polypeptide, or variant thereof (e.g., survivin peptide variant) derived from survivin protein or fragment thereof. The term "survivin antigen" also includes polynucleotides encoding survivin peptides, survivin peptide variants, or functional equivalents of survivin peptides described herein.

[0088] The polynucleotides may be DNA (e.g., genomic DNA or cDNA) or RNA (e.g., mRNA) or a combination thereof. They may be naturally occurring or synthetic (e.g., chemically synthesized). It is contemplated that the polynucleotides may contain modifications of one or more nitrogenous bases, pentose sugars, or phosphate groups in the nucleotide chain. Such modifications are well known in the art and may be for purposes of, for example, improving the stability of the polynucleotide.

[0089] In one embodiment, the survivin antigen may comprise a full-length survivin polypeptide or a nucleic acid encoding a full-length survivin polypeptide. Alternatively, the survivin antigen may be a survivin peptide, including fragments of any length of the survivin protein. Exemplary embodiments include survivin peptides that include at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues. In specific embodiments, the survivin peptide consists of a heptapeptide, octapeptide, nanopeptide, decapeptide, or undecapeptide, each of 7, 8, 9, 10, or 11 consecutive amino acid residues of the survivin protein (e.g., SEQ ID NO: 15). Particular embodiments of the survivin antigen include survivin peptides of about 9 or 10 amino acids.

[0090] The survivin antigen of the present invention also includes the variant and functional equivalent of survivin peptide.Variants or functional equivalents of survivin peptide include peptides that show differences compared to the specific sequence of survivin protein, such as the amino acid sequence that contains one or more amino acid substitutions, deletions or additions, or any combination thereof.This difference can be measured as the reduction in identity between survivin protein sequence and survivin peptide variants or functional equivalents of survivin peptide.

[0091] The identity between amino acid sequences can be calculated using algorithms well known in the art.Survivin peptide variants or functional equivalents shall be considered to be included within the meaning of the "survivin antigen" of the present invention if they are at least 50% identical, for example at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, preferably over their entire length, to the peptide sequence of survivin protein, including 96%, 97%, 98% or 99% identical to the peptide sequence of survivin protein.In a specific embodiment, the survivin peptide variant has a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the consecutive amino acid sequence of SEQ ID NO: 15.

[0092] The survivin protein from which the survivin antigen may be derived is a survivin protein from any animal species in which this protein is expressed. A particular embodiment is a survivin protein from human (SEQ ID NO: 15). Based on the sequence of the selected survivin protein, the survivin antigen may be obtained by any suitable chemical or enzymatic treatment of the survivin protein or the encoding nucleic acid. Alternatively, the survivin antigen may be synthesized by any conventional peptide or nucleic acid synthesis procedure well known to those skilled in the art.

[0093] The survivin antigen (peptide or nucleic acid) of the present invention may have a sequence that is the native sequence of survivin. Alternatively, the survivin antigen may be a peptide or nucleic acid sequence that has been modified by one or more substitutions, deletions or additions, such as, for example, the survivin peptide variants or functional equivalents described herein. Exemplary procedures and modifications of survivin peptides that increase the immunogenicity of the peptide include those described in WO2004 / 067023 (incorporated herein in its entirety for all intended purposes), including amino acid substitutions introduced at anchor positions that increase the binding of the peptide to HLA class I molecules.

[0094] In one embodiment, the survivin antigen is any peptide derived from the survivin protein, or any survivin peptide variant thereof, capable of binding to MHC class I HLA molecules. Along these lines, the survivin antigen may be any survivin peptide, or any survivin peptide variant thereof, capable of inducing or enhancing an immune response in a subject.

[0095] In one embodiment, the survivin antigen is a peptide antigen comprising an amino acid sequence derived from the survivin protein (sequence number 15) capable of eliciting a cytotoxic T lymphocyte (CTL) response in a subject, or a nucleic acid molecule encoding said peptide.

[0096] In one embodiment, the T cell activation therapy comprises one or more synthetic survivin peptides based on the amino acid sequence of a survivin protein, such as the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof.

[0097] Survivin peptides, survivin peptide variants and functional equivalents of survivin, and their uses for diagnostic and therapeutic purposes, particularly in cancer, are described, for example, in WO2004 / 067023 and WO2006 / 081826, each of which is incorporated herein in its entirety for all intended purposes.It has been found that the novel peptides disclosed in these publications can elicit cytotoxic T lymphocyte (CTL) responses in cancer patients.In particular, in WO2004 / 067023, it has been found that MHC class I restricted peptides can be derived from survivin protein, which can bind to MHC class I HLA molecules, thereby eliciting both ex vivo and in situ CTL immune responses in patients suffering from various cancer diseases.

[0098] In one embodiment, the T cell activation therapeutic of the invention may comprise any one or more of the survivin peptides, survivin peptide variants or functional equivalents of survivin peptides disclosed in WO2004 / 067023 and WO2006 / 081826.

[0099] In another embodiment, the T cell activation therapeutic of the present invention may comprise one or more survivin peptides, survivin peptide variants or functional equivalents of survivin peptides having the ability to bind to any of the MHC class I molecules selected from HLA-A, HLA-B or HLA-C molecules.

[0100] Exemplary MHC class I HLA-A molecules to which survivin peptides, survivin peptide variants, or functional equivalents of survivin peptides may bind include, but are not limited to, HLA-A1, HLA-A2, HLA-A3, HLA-A9, HLA-A10, HLA-A11, HLA-A19, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68, and HLA-A69.

[0101] Exemplary MHC class I HLA-B molecules to which survivin peptides, survivin peptide variants, or functional equivalents of survivin peptides may bind include, but are not limited to, HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA-B15, HLA-B16, HLA-B17, HLA-B18, HLA-B21, HLA-B22, HLA-B27, HLA-B35, HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-B41, HLA-B42, HLA-B44, HLA-B45, HLA-B46, and HLA-B47.

[0102] Exemplary MHC class I HLA-C molecules to which survivin peptides, survivin peptide variants, or functional equivalents of survivin peptides may bind include, but are not limited to, HLA-C1, HLA-C2, HLA-C3, HLA-C4, HLA-C5, HLA-C6, HLA-C7, and HLA-C16.

[0103] In some embodiments, the T cell activation therapy may comprise one or more of survivin peptide antigens selected from i) FEELTLGEF (SEQ ID NO: 1) [HLA-A1]; ii) FTELTLGEF (SEQ ID NO: 2) [HLA-A1]; iii) LTLGEFLKL (SEQ ID NO: 3) [HLA-A2]; iv) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2]; v) RISTFKNWPF (SEQ ID NO: 5) [HLA-A3]; vi) RISTFKNWPK (SEQ ID NO: 6) [HLA-A3]; vii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24]; or viii) LPPAWQPFL (SEQ ID NO: 8) [HLA-B7], or a nucleic acid molecule encoding a survivin peptide antigen.

[0104] In some embodiments, the T cell activation therapy may comprise one or more survivin peptide antigens selected from i) FTELTLGEF (SEQ ID NO:2) [HLA-A1]; ii) LMLGEFLKL (SEQ ID NO:4) [HLA-A2]; iii) RISTFKNWPK (SEQ ID NO:6) [HLA-A3]; iv) STFKNWPFL (SEQ ID NO:7) [HLA-A24]; or v) LPPAWQPFL (SEQ ID NO:8) [HLA-B7], or a nucleic acid molecule encoding a survivin peptide antigen.

[0105] In some embodiments, the T cell activation therapy may comprise the following five survivin peptide antigens: i) FTELTLGEF (SEQ ID NO:2) [HLA-A1]; ii) LMLGEFLKL (SEQ ID NO:4) [HLA-A2]; iii) RISTFKNWPK (SEQ ID NO:6) [HLA-A3]; iv) STFKNWPFL (SEQ ID NO:7) [HLA-A24]; and v) LPPAWQPFL (SEQ ID NO:8) [HLA-B7], or a nucleic acid molecule encoding a survivin peptide antigen.

[0106] In some embodiments, the T cell activation therapy may comprise one or more survivin peptide antigens selected from i) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2] or ii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24], or a nucleic acid molecule encoding a survivin peptide antigen.

[0107] In some embodiments, the T cell activation therapy may comprise the following two survivin peptide antigens: i) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2] and ii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24] or a nucleic acid molecule encoding the survivin peptide antigens.

[0108] In a further embodiment, the T cell activation therapy comprises two survivin peptides: i) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2] and ii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24].

[0109] The survivin peptides listed above represent exemplary MHC class I restricted peptides encompassed by the present invention, but are not limited thereto. The specific MHC class I HLA molecules to which each of the survivin peptides is believed to bind are shown in brackets to the right. The T cell activation therapeutics of the present invention may comprise one or more of these survivin peptides in any suitable combination.

[0110] (ii) MAGE-A9 antigen The T cell activation therapeutic composition of the present invention comprises at least one MAGE-A9 antigen. The expression "at least one" is used synonymously with the expression "one or more". These expressions refer to the number of different MAGE-A9 antigens in the T cell activation therapeutic, and not the amount of any particular MAGE-A9 antigen, unless expressly stated otherwise herein. In accordance with the ordinary meaning of "at least one" or "one or more", the T cell activation therapeutic composition of the present invention contains at a minimum one MAGE-A9 antigen.

[0111] MAGE-A9 is a member of the melanoma-associated antigen (MAGE) group of proteins expressed in a wide variety of malignant tumors. The coding sequence of MAGE-A9 (homo sapiens) is 1814 nucleotides long (SEQ ID NO: 16). The encoded protein MAGE-A9 (homo sapiens) is 315 amino acids long (SEQ ID NO: 17).

[0112] [Table 2]

[0113] MAGE-A9 is a well-characterized cancer / testis antigen (CTA) that has been reported to be highly expressed in various human cancers, including lung, bladder, liver, ovarian, colon, breast, kidney, and liver cancers (Wei et al., 2018). For example, in a comparative study, MAGE-A9 was expressed more strongly and more frequently than NY-ESO-1 or MAGE-A3 in bladder tumors (Fradet et al., 2006; Picard et al., 2007). This fact therefore makes MAGE-A9 an ideal target for cancer therapy, as cancer cells but not normal cells are targeted. Moreover, it has been reported that MAGE-A9 expression has prognostic value.

[0114] MAGE-A9 is a member of the Melanoma Antigen Gene (MAGE) protein family. Human MAGE family members can be divided into two categories: i) type I MAGEs, which are thought to include the MAGE-A, -B, and -C subfamily members that cluster on the X chromosome, and ii) type II MAGEs (MAGE-D, -E, -F, -G, -H, -L subfamilies and necdin). Both type I and type II MAGEs contain a MAGE homology domain (MHD) of approximately 170 amino acids that is on average 46% conserved among all human MAGEs.

[0115] In certain embodiments, the T cell activation therapy of the present invention comprises at least one MAGE antigen. The following MAGE genes encode MAGE proteins having peptide sequences that can be incorporated as antigens into the T cell activation therapy of the present invention, including, but not limited to: MAGE-A1, MAGE-A2, MAGE-A2B, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13P, MAGE-A14, MAGE-A15, MAGE-A16, MAGE-A17, MAGE-A18, MAGE-A19, MAGE-A20, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE-A27, MAGE-A28, MAGE-A29, MAGE-A30, MAGE-A31, MAGE-A32, MAGE-A33, MAGE-A34, MAGE-A35, MAGE-A36, MAGE-A37, MAGE-A38, MAGE-A39, MAGE-A3A, MAGE-A3B, MAGE-A3C, MAGE-A3D, MAGE-A3D, MAGE-A3E, MAGE-A3F, MAGE-A3G, MAGE-A3H ... E-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B18, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D3, MAGE-D4, MAGE-D4B, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-G1, MAGE-H1, MAGE-L2, and NDN. In certain embodiments, the MAGE protein is MAGE-A9.

[0116] In certain embodiments, the T cell activation therapeutic of the present invention comprises one or more MAGE-A9 antigens. The term "MAGE-A9 antigen" as used herein includes any peptide, polypeptide, or variant thereof (e.g., MAGE-A9 peptide variant) derived from MAGE-A9 protein or fragment thereof. The term "MAGE-A9 antigen" also includes polynucleotides encoding MAGE-A9 peptides, MAGE-A9 peptide variants, or functional equivalents of MAGE-A9 peptides described herein.

[0117] The polynucleotides may be DNA (e.g., genomic DNA or cDNA) or RNA (e.g., mRNA) or a combination thereof. They may be naturally occurring or synthetic (e.g., chemically synthesized). It is contemplated that the polynucleotides may contain modifications of one or more nitrogenous bases, pentose sugars, or phosphate groups in the nucleotide chain. Such modifications are well known in the art and may be for purposes of, for example, improving the stability of the polynucleotide.

[0118] In one embodiment, the MAGE-A9 antigen may comprise a full-length MAGE-A9 polypeptide or a nucleic acid encoding a full-length MAGE-A9 polypeptide. Alternatively, the MAGE-A9 antigen may be a MAGE-A9 peptide, including fragments of any length of MAGE-A9 protein. Exemplary embodiments include MAGE-A9 peptides that include at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues. In a specific embodiment, the survivin peptide is a heptapeptide, octapeptide, nanopeptide, decapeptide, or undecapeptide, each of which consists of 7, 8, 9, 10, or 11 consecutive amino acid residues of the MAGE-A9 protein (e.g., SEQ ID NO: 17). Particular embodiments of the MAGE-A9 antigen include MAGE-A9 peptides of about 9 or 10 amino acids.

[0119] The MAGE-A9 antigen of the present invention also includes variants and functional equivalents of MAGE-A9 peptides.Variants or functional equivalents of MAGE-A9 peptides include peptides that exhibit differences compared to the specific sequence of MAGE-A9 protein, such as amino acid sequences that include one or more amino acid substitutions, deletions or additions, or any combination thereof.These differences can be measured as the reduction in identity between the MAGE-A9 protein sequence and the MAGE-A9 peptide variants or functional equivalents of MAGE-A9 peptides.

[0120] The identity between amino acid sequences can be calculated using algorithms well known in the art. MAGE-A9 peptide variants or functional equivalents shall be considered to be included within the meaning of "MAGE-A9 antigen" of the present invention if they are at least 50% identical, such as at least 60% identical, such as at least 70% identical, such as at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, preferably over their entire length, to the peptide sequence of MAGE-A9 protein, including 96%, 97%, 98% or 99% identical to the peptide sequence of MAGE-A9 protein. In a particular embodiment, the MAGE-A9 peptide variant has a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the consecutive amino acid sequence of SEQ ID NO: 17.

[0121] The MAGE-A9 protein from which the MAGE-A9 antigen may be derived is a MAGE-A9 protein from any animal species in which this protein is expressed. A particular embodiment is a MAGE-A9 protein from human (SEQ ID NO: 17). Based on the sequence of the selected MAGE-A9 protein, the MAGE-A9 antigen may be obtained by any suitable chemical or enzymatic treatment of the MAGE-A9 protein or the encoding nucleic acid. Alternatively, the MAGE-A9 antigen may be synthesized by any conventional peptide or nucleic acid synthesis procedure well known to those skilled in the art.

[0122] The MAGE-A9 antigens (peptides or nucleic acids) of the invention may have a sequence which is the native sequence of MAGE-A9. Alternatively, the MAGE-A9 antigens may be peptide or nucleic acid sequences which have been modified by one or more substitutions, deletions or additions, such as, for example, the MAGE-A9 peptide variants or functional equivalents described herein.

[0123] In one embodiment, the MAGE-A9 antigen is any peptide derived from the MAGE-A9 protein, or any MAGE-A9 peptide variant thereof, capable of binding to an MHC class I HLA molecule. Along these lines, the MAGE-A9 antigen may be any MAGE-A9 peptide, or any MAGE-A9 peptide variant thereof, capable of inducing or enhancing an immune response in a subject.

[0124] In one embodiment, the MAGE-A9 antigen is a peptide antigen comprising an amino acid sequence derived from the MAGE-A9 protein (sequence number 17) capable of eliciting a cytotoxic T lymphocyte (CTL) response in a subject, or a nucleic acid molecule encoding said peptide.

[0125] In one embodiment, the T cell activation therapy comprises one or more synthetic MAGE-A9 peptides based on the amino acid sequence of the MAGE-A9 protein, such as the amino acid sequence set forth in SEQ ID NO: 17, or a variant thereof.

[0126] In another embodiment, the T cell activation therapeutic of the present invention may comprise one or more MAGE-A9 peptides, MAGE-A9 peptide variants or functional equivalents of MAGE-A9 peptides having the ability to bind to any of the MHC class I molecules selected from HLA-A, HLA-B or HLA-C molecules.

[0127] Exemplary MHC class I HLA-A molecules to which the MAGE-A9 peptides, MAGE-A9 peptide variants, or functional equivalents of the MAGE-A9 peptides may bind include, but are not limited to, HLA-A1, HLA-A2, HLA-A3, HLA-A9, HLA-A10, HLA-A11, HLA-A19, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68, and HLA-A69.

[0128] Exemplary MHC class I HLA-B molecules to which the MAGE-A9 peptides, MAGE-A9 peptide variants, or functional equivalents of the MAGE-A9 peptides may bind include, but are not limited to, HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA-B15, HLA-B16, HLA-B17, HLA-B18, HLA-B21, HLA-B22, HLA-B27, HLA-B35, HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-B41, HLA-B42, HLA-B44, HLA-B45, HLA-B46 and HLA-B47.

[0129] Exemplary MHC class I HLA-C molecules to which MAGE-A9 peptides, MAGE-A9 peptide variants, or functional equivalents of MAGE-A9 peptides may bind include, but are not limited to, HLA-C1, HLA-C2, HLA-C3, HLA-C4, HLA-C5, HLA-C6, HLA-C7 and HLA-C16.

[0130] In certain embodiments, the T cell activation therapy may comprise one or more MAGE-A9 peptide antigens that bind to any one of HLA-A1, HLA-A2, HLA-A3, HLA-A24, and / or HLA-B7. In certain embodiments, the T cell activation therapy may comprise one or more MAGE-A9 peptide antigens that bind to HLA-A2. In certain embodiments, the T cell activation therapy may comprise at least five MAGE-A9 peptide antigens that bind to each of HLA-A1, HLA-A2, HLA-A3, HLA-A24, and HLA-B7.

[0131] In certain embodiments, the T cell activation therapy may comprise at least one MAGE-A9 peptide antigen that binds to one or more of HLA-A1, HLA-A2, HLA-A3, HLA-A24, or HLA-B7, respectively. In certain embodiments, the T cell activation therapy may comprise at least one MAGE-A9 peptide antigen that collectively binds to only one of HLA-A1, HLA-A2, HLA-A3, HLA-A24, or HLA-B. In certain embodiments, the T cell activation therapy may comprise at least two MAGE-A9 peptide antigens that collectively bind to two of HLA-A1, HLA-A2, HLA-A3, HLA-A24, or HLA-B7. In certain embodiments, the T cell activation therapy may comprise at least three MAGE-A9 peptide antigens that collectively bind to three of HLA-A1, HLA-A2, HLA-A3, HLA-A24, or HLA-B7. In certain embodiments, the T cell activation therapy may comprise at least four MAGE-A9 peptide antigens that collectively bind to four of HLA-A1, HLA-A2, HLA-A3, HLA-A24, or HLA-B7. In certain embodiments, the T cell activation therapy may comprise at least five MAGE-A9 peptide antigens that collectively bind to each of HLA-A1, HLA-A2, HLA-A3, HLA-A24, and HLA-B7.

[0132] In certain embodiments, the T cell activation therapy may comprise one or more of the MAGE-A9 peptide antigens selected from SEQ ID NOs: 9-12, 26-44, 46-52, 54-62, 64-75, or 79-93, or any combination thereof, or a nucleic acid molecule encoding said MAGE-A9 peptide antigens. In certain embodiments, the T cell activation therapy may comprise one or more of the MAGE-A9 peptide antigens selected from the MAGE-A9 antigens cited in Table 17.

[0133] In one embodiment, the T cell activation therapy is selected from the group consisting of i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO: 11) [MAGE-A9 223]; iv) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270]; v) FMFQEALKL (SEQ ID NO: 26) [MAGE-A9 102]; vi) EVDPAGHSY (SEQ ID NO: 27) [MAGE-A9 167]; vii) NYKRYFPVI (SEQ ID NO: 28) [MAGE-A9 141]; viii) VYYTLWSQF (SEQ ID NO: 29) [MAGE-A9 71]; ix) SYILVTALG (SEQ ID NO: 30) [MAGE-A9 xii) ALLIIVLGV (SEQ ID NO: 33) [MAGE-A9 199]; xiii) FLLHKYRVK (SEQ ID NO: 34) [MAGE-A9 118]; or xiv) IVLGVILTK (SEQ ID NO: 35) [MAGE-A9 203], or a nucleic acid molecule encoding a survivin peptide antigen.

[0134] In one embodiment, the T cell activation therapy is selected from the group consisting of i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO: 11) [MAGE-A9 223]; iv) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270] v) FMFQEALKL (SEQ ID NO: 26) [MAGE-A9 102]; vi) EVDPAGHSY (SEQ ID NO: 27) [MAGE-A9 167]; vii) NYKRYFPVI (SEQ ID NO: 28) [MAGE-A9 141]; viii) MPKAALLII (SEQ ID NO: 31) [MAGE-A9 195]; or ix) SVMGVYVGK (SEQ ID NO: 32) [MAGE-A9 225], or a nucleic acid molecule encoding a survivin peptide antigen.

[0135] In one embodiment, the T cell activation therapy is selected from the group consisting of i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) FMFQEALKL (SEQ ID NO: 26) [MAGE-A9 102]; iii) EVDPAGHSY (SEQ ID NO: 27) [MAGE-A9 167]; iv) NYKRYFPVI (SEQ ID NO: 28) [MAGE-A9 141]; v) VYYTLWSQF (SEQ ID NO: 29) [MAGE-A9 71]; vi) SYILVTALG (SEQ ID NO: 30) [MAGE-A9 174]; vii) MPKAALLII (SEQ ID NO: 31) [MAGE-A9 195]; viii) SVMGVYVGK (SEQ ID NO: 32) [MAGE-A9 225]; ix) ALLIIVLGV (SEQ ID NO: 33) [MAGE-A9 x) FLLHKYRVK (SEQ ID NO: 34) [MAGE-A9 118]; or xi) IVLGVILTK (SEQ ID NO: 35) [MAGE-A9 203], or a nucleic acid molecule encoding a survivin peptide antigen.

[0136] In one embodiment, the T cell activation therapy is selected from the group consisting of i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO: 11) [MAGE-A9 223]; iv) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270] v) FMFQEALKL (SEQ ID NO: 26) [MAGE-A9 102]; vi) EVDPAGHSY (SEQ ID NO: 27) [MAGE-A9 167]; vii) NYKRYFPVI (SEQ ID NO: 28) [MAGE-A9 141]; viii) MPKAALLII (SEQ ID NO: 31) [MAGE-A9 195]; or ix) SVMGVYVGK (SEQ ID NO: 32) [MAGE-A9 225], or a nucleic acid molecule encoding a survivin peptide antigen.

[0137] In one embodiment, the T cell activation therapy may comprise a nucleic acid molecule encoding one or more of the MAGE-A9 peptide antigens selected from i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) FMFQEALKL (SEQ ID NO: 26) [MAGE-A9 102]; iii) EVDPAGHSY (SEQ ID NO: 27) [MAGE-A9 167]; iv) NYKRYFPVI (SEQ ID NO: 28) [MAGE-A9 141]; v) MPKAALLII (SEQ ID NO: 31) [MAGE-A9 195]; or vi) SVMGVYVGK (SEQ ID NO: 32) [MAGE-A9 225], or a survivin peptide antigen.

[0138] In one embodiment, the T cell activation therapy may comprise one or more of the MAGE-A9 peptide antigens selected from i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) FMFQEALKL (SEQ ID NO: 26) [MAGE-A9 102]; iii) EVDPAGHSY (SEQ ID NO: 27) [MAGE-A9 167]; iv) NYKRYFPVI (SEQ ID NO: 28) [MAGE-A9 141]; v) SYILVTALG (SEQ ID NO: 30) [MAGE-A9 174]; vi) MPKAALLII (SEQ ID NO: 31) [MAGE-A9 195]; or vii) SVMGVYVGK (SEQ ID NO: 32) [MAGE-A9 225], or a nucleic acid molecule encoding a survivin peptide antigen.

[0139] In further embodiments, the T cell activation therapy comprises the following four MAGE-A9 peptide antigens: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO: 11) [MAGE-A9 223]; and iv) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270], or a nucleic acid molecule encoding the survivin peptide antigen.

[0140] In a further embodiment, the T cell activation therapy comprises a nucleic acid molecule encoding the following three MAGE-A9 peptide antigens: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; and iii) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270], or survivin peptide antigens.

[0141] In a further embodiment, the T cell activation therapy comprises the following four MAGE-A9 peptide antigens: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO: 11) [MAGE-A9 223]; and iv) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270].

[0142] In a further embodiment, the T cell activation therapy comprises the three MAGE-A9 peptide antigens i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; and iii) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270].

[0143] The MAGE-A9 peptides listed above represent exemplary, but not limited to, MHC class I restricted peptides encompassed by the present invention. The specific MHC class I HLA molecules to which each of the MAGE-A9 peptides is believed to bind are shown to the right in brackets. The T cell activation therapeutics of the present invention may include one or more of these MAGE-A9 peptides in any suitable combination.

[0144] (iii) Non-limiting examples of dual T cell activation therapeutics targeting both survivin and the MAGE-A9 antigen In certain embodiments, the T cell activation therapeutic comprises a nucleic acid molecule encoding any one or more survivin peptides or survivin peptide antigens comprising the amino acids of SEQ ID NOs: 1-8, and a nucleic acid molecule encoding one or more MAGE-A9 peptides or survivin peptide antigens comprising the amino acids of SEQ ID NOs: 9-12, 26-44, 46-52, 54-62, 64-75, or 79-93. In certain embodiments, the T cell activation therapeutic comprises any one or more survivin peptides in Table 17 and one or more MAGE-A9 peptides.

[0145] In further embodiments, the T cell activation therapy comprises any one or more of the eight survivin peptides listed below: i) FEELTLGEF (SEQ ID NO: 1) [HLA-A1]; ii) FTELTLGEF (SEQ ID NO: 2) [HLA-A1]; iii) LTLGEFLKL (SEQ ID NO: 3) [HLA-A2]; iv) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2]; v) RISTFKNWPF (SEQ ID NO: 5) [HLA-A3]; vi) RISTFKNWPK (SEQ ID NO: 6) [HLA-A3]; vii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24]; or viii) LPPAWQPFL (SEQ ID NO: 8) [HLA-B7] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen, and any one of the nine MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO: 11) [MAGE-A9 223]; iv) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270] v) FMFQEALKL (SEQ ID NO: 26); vi) EVDPAGHSY (SEQ ID NO: 27); vii) NYKRYFPVI (SEQ ID NO: 28); viii) VYYTLWSQF (SEQ ID NO: 29); or ix) SYILVTALG (SEQ ID NO: 30) in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen.

[0146] In further embodiments, the T cell activation therapy comprises any one or more of the five survivin peptides listed below: i) FTELTLGEF (SEQ ID NO:2) [HLA-A1]; ii) LMLGEFLKL (SEQ ID NO:4) [HLA-A2]; iii) RISTFKNWPK (SEQ ID NO:6) [HLA-A3]; iv) STFKNWPFL (SEQ ID NO:7) [HLA-A24]; or v) LPPAWQPFL (SEQ ID NO:8) [HLA-B7], in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen, and one of the fourteen MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO:9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO:10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO:11) [MAGE-A9 223]; iv) FLWGSKAHA (SEQ ID NO:12) [MAGE-A9 v) FMFQEALKL (SEQ ID NO: 26) [MAGE-A9 102]; vi) EVDPAGHSY (SEQ ID NO: 27) [MAGE-A9 167]; vii) NYKRYFPVI (SEQ ID NO: 28) [MAGE-A9 141]; viii) VYYTLWSQF (SEQ ID NO: 29) [MAGE-A9 71]; or ix) SYILVTALG (SEQ ID NO: 30) [MAGE-A9 174]; x) MPKAALLII (SEQ ID NO: 31) [MAGE-A9 195]; xi) SVMGVYVGK (SEQ ID NO: 32) [MAGE-A9 225]; xii) ALLIIVLGV (SEQ ID NO: 33) [MAGE-A9 199]; xiii) FLLHKYRVK (SEQ ID NO: 34) [MAGE-A9 118]; or xiv) IVLGVILTK (SEQ ID NO: 35) [MAGE-A9 203] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen.

[0147] In further embodiments, the T cell activation therapy comprises any one or more of the five survivin peptides listed below: i) FTELTLGEF (SEQ ID NO:2) [HLA-A1]; ii) LMLGEFLKL (SEQ ID NO:4) [HLA-A2]; iii) RISTFKNWPK (SEQ ID NO:6) [HLA-A3]; iv) STFKNWPFL (SEQ ID NO:7) [HLA-A24]; or v) LPPAWQPFL (SEQ ID NO:8) [HLA-B7] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen, and any one of the eleven MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO:9) [MAGE-A9 111]; ii) FMFQEALKL (SEQ ID NO:26) [MAGE-A9 102]; iii) EVDPAGHSY (SEQ ID NO:27) [MAGE-A9 167]; iv) NYKRYFPVI (SEQ ID NO: 28) [MAGE-A9 141]; v) VYYTLWSQF (SEQ ID NO: 29) [MAGE-A9 71]; vi) SYILVTALG (SEQ ID NO: 30) [MAGE-A9 174]; vii) MPKAALLII (SEQ ID NO: 31) [MAGE-A9 195]; viii) SVMGVYVGK (SEQ ID NO: 32) [MAGE-A9 225]; ix) ALLIIVLGV (SEQ ID NO: 33) [MAGE-A9 199]; x) FLLHKYRVK (SEQ ID NO: 34) [MAGE-A9 118]; or xi) IVLGVILTK (SEQ ID NO: 35) [MAGE-A9 203] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen.

[0148] In further embodiments, the T cell activation therapy comprises any one or more of the five survivin peptides listed below: i) FTELTLGEF (SEQ ID NO:2) [HLA-A1]; ii) LMLGEFLKL (SEQ ID NO:4) [HLA-A2]; iii) RISTFKNWPK (SEQ ID NO:6) [HLA-A3]; iv) STFKNWPFL (SEQ ID NO:7) [HLA-A24]; or v) LPPAWQPFL (SEQ ID NO:8) [HLA-B7] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen, and the nine MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO:9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO:10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO:11) [MAGE-A9 223]; iv) FLWGSKAHA (SEQ ID NO:12) [MAGE-A9 270] v) FMFQEALKL (SEQ ID NO:26) [MAGE-A9 102]; vi) EVDPAGHSY (SEQ ID NO:27) [MAGE-A9 167]; vii) NYKRYFPVI (SEQ ID NO:28) [MAGE-A9 141]; viii) MPKAALLII (SEQ ID NO:31) [MAGE-A9 195]; or ix) SVMGVYVGK (SEQ ID NO:32) [MAGE-A9 225] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen.

[0149] In further embodiments, the T cell activation therapy comprises any one or more of the five survivin peptides listed below: i) FTELTLGEF (SEQ ID NO:2) [HLA-A1]; ii) LMLGEFLKL (SEQ ID NO:4) [HLA-A2]; iii) RISTFKNWPK (SEQ ID NO:6) [HLA-A3]; iv) STFKNWPFL (SEQ ID NO:7) [HLA-A24]; or v) LPPAWQPFL (SEQ ID NO:8) [HLA-B7] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen, and the six MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO:9) [MAGE-A9 111]; ii) FMFQEALKL (SEQ ID NO:26) [MAGE-A9 102]; iii) EVDPAGHSY (SEQ ID NO:27) [MAGE-A9 167]; iv) NYKRYFPVI (SEQ ID NO:28) [MAGE-A9 141]; v) MPKAALLII (SEQ ID NO:31) [MAGE-A9 195]; or vi) SVMGVYVGK (SEQ ID NO:32) [MAGE-A9 225] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen.

[0150] In further embodiments, the T cell activation therapy comprises any one or more of the five survivin peptides listed below: i) FTELTLGEF (SEQ ID NO:2) [HLA-A1]; ii) LMLGEFLKL (SEQ ID NO:4) [HLA-A2]; iii) RISTFKNWPK (SEQ ID NO:6) [HLA-A3]; iv) STFKNWPFL (SEQ ID NO:7) [HLA-A24]; or v) LPPAWQPFL (SEQ ID NO:8) [HLA-B7] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen, and the seven MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO:9) [MAGE-A9 111]; ii) FMFQEALKL (SEQ ID NO:26) [MAGE-A9 102]; iii) EVDPAGHSY (SEQ ID NO:27) [MAGE-A9 167]; iv) NYKRYFPVI (SEQ ID NO:28) [MAGE-A9 141]; v) SYILVTALG (SEQ ID NO: 30) [MAGE-A9 174]; vi) MPKAALLII (SEQ ID NO: 31) [MAGE-A9 195]; or vii) SVMGVYVGK (SEQ ID NO: 32) [MAGE-A9 225] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen.

[0151] In a further embodiment, the T cell activation therapy comprises any one or more of the two survivin peptides listed below: i) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2] or ii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen, and any one of the four MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO: 11) [MAGE-A9 223]; or iv) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen.

[0152] In a further embodiment, the T cell activation therapy comprises any one or more of the two survivin peptides listed below: i) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2] or ii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen, and any one of the three MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; or iii) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270] in any suitable combination, or a nucleic acid molecule encoding a survivin peptide antigen.

[0153] In certain embodiments, the T cell activation therapy comprises two survivin peptides, listed below: i) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2] and ii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24], or a nucleic acid molecule encoding the survivin peptide antigens, and four MAGE-A9 peptides, listed below: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO: 11) [MAGE-A9 223]; and iv) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270], or a nucleic acid molecule encoding the survivin peptide antigens.

[0154] In certain embodiments, the T cell activation therapy comprises two survivin peptides, listed below: i) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2] and ii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24], or nucleic acid molecules encoding survivin peptide antigens, and three MAGE-A9 peptides, listed below: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; and iii) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270], or nucleic acid molecules encoding survivin peptide antigens.

[0155] In certain embodiments, the T cell activation therapy comprises two survivin peptides listed below: i) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2] and ii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24], and four MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; iii) ALSVMGVYV (SEQ ID NO: 11) [MAGE-A9 223]; and iv) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270].

[0156] In certain embodiments, the T cell activation therapy comprises two survivin peptides listed below: i) LMLGEFLKL (SEQ ID NO: 4) [HLA-A2] and ii) STFKNWPFL (SEQ ID NO: 7) [HLA-A24], and three MAGE-A9 peptides listed below: i) KVAELVHFL (SEQ ID NO: 9) [MAGE-A9 111]; ii) GLMGAQEPT (SEQ ID NO: 10) [MAGE-A9 24]; and iii) FLWGSKAHA (SEQ ID NO: 12) [MAGE-A9 270].

[0157] In addition to at least one survivin antigen and at least one MAGE-A9 antigen, further embodiments of the T cell activation therapeutics of the present invention may include one or more additional antigens useful in the treatment of cancer or in inducing or enhancing an immune response against cancer.

[0158] In a further embodiment, the dual T cell activation therapeutic composition targeting both survivin and MAGE-A9 may further comprise a carrier comprising a T helper epitope; an adjuvant; a lipid vesicle particle; and a continuous phase of a hydrophobic material. The T helper epitope may be, for example, a peptide comprising the amino acid sequence AQYIKANSKFIGITEL (SEQ ID NO: 13). The adjuvant may be, for example, an RNA or DNA-based polynucleotide adjuvant (e.g., poly I:C, poly dIdC, SEQ ID NO: 22, etc.). The lipid vesicle particle may be, for example, composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; a synthetic phospholipid) and cholesterol. The hydrophobic carrier may be, for example, Montanide® ISA51VG.

[0159] In certain embodiments, the T cell activation therapy comprises at least one survivin antigen, each survivin antigen being at a concentration of about 0.01 mg / ml to about 10 mg / ml, about 0.025 mg / ml to about 9 mg / ml, about 0.05 mg / ml to about 8 mg / ml, about 0.075 mg / ml to about 7 mg / ml, about 0.1 mg / ml to about 6 mg / ml, about 0.25 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 4 mg / ml, about 0.75 mg / ml to about 3 mg / ml, about 1 mg / ml to about 2 mg / ml. In certain embodiments, the T cell activation therapy comprises at least one survivin antigen, each survivin antigen being at a concentration of about 0.1 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 3 mg / ml, or about 0.5 mg / ml to about 2 mg / ml. In certain embodiments, a T cell activation therapy of the invention comprises at least one survivin antigen, and each survivin antigen is at a concentration of about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml. In certain embodiments, the T cell activation therapy comprises at least one survivin antigen, each survivin antigen being at a concentration of about 1 mg / ml.

[0160] In certain embodiments, the T cell activation therapy comprises at least one MAGE antigen, each MAGE antigen being at a concentration of about 0.01 mg / ml to about 10 mg / ml, about 0.025 mg / ml to about 9 mg / ml, about 0.05 mg / ml to about 8 mg / ml, about 0.075 mg / ml to about 7 mg / ml, about 0.1 mg / ml to about 6 mg / ml, about 0.25 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 4 mg / ml, about 0.75 mg / ml to about 3 mg / ml, about 1 mg / ml to about 2 mg / ml. In certain embodiments, the T cell activation therapy comprises at least one MAGE antigen, each MAGE antigen being at a concentration of about 0.1 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 3 mg / ml, or about 0.5 mg / ml to about 2 mg / ml. In certain embodiments, the T cell activation therapy comprises at least one MAGE antigen, and each MAGE antigen is at a concentration of about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml. In certain embodiments, the T cell activation therapeutic comprises at least one MAGE antigen, each MAGE antigen being at a concentration of about 1 mg / ml.

[0161] In certain embodiments, the composition comprises at least one T helper epitope, and the T helper epitope is at a concentration of about 0.01 mg / ml to about 5 mg / ml, about 0.01 mg / ml to about 10 mg / ml, about 0.025 mg / ml to about 9 mg / ml, about 0.05 mg / ml to about 8 mg / ml, about 0.075 mg / ml to about 7 mg / ml, about 0.1 mg / ml to about 6 mg / ml, about 0.25 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 4 mg / ml, about 0.75 mg / ml to about 3 mg / ml, or about 1 mg / ml to about 2 mg / ml. In certain embodiments, the composition comprises at least one T helper epitope, and the T helper epitope is at a concentration of about 0.1 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 3 mg / ml, or about 0.5 mg / ml to about 2 mg / ml. In certain embodiments, the composition comprises at least one T helper epitope, and the T helper epitope is at a concentration of about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml.

[0162] In certain embodiments, the composition comprises at least one adjuvant, and the adjuvant is in a concentration of about 0.01 mg / ml to about 4 mg / ml, about 0.01 mg / ml to about 10 mg / ml, about 0.025 mg / ml to about 9 mg / ml, about 0.05 mg / ml to about 8 mg / ml, about 0.075 mg / ml to about 7 mg / ml, about 0.1 mg / ml to about 6 mg / ml, about 0.25 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 4 mg / ml, about 0.75 mg / ml to about 3 mg / ml, about 1 mg / ml to about 2 mg / ml. In certain embodiments, the composition comprises at least one adjuvant, and the adjuvant is in a concentration of about 0.1 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 3 mg / ml, or about 0.5 mg / ml to about 2 mg / ml. In certain embodiments, the composition comprises at least one adjuvant, and the adjuvant is at a concentration of about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml.

[0163] In certain embodiments, the composition comprises at least one lipid, and the lipid is at a concentration of about 30mg / ml to about 240mg / ml, about 0.01mg / ml to about 10mg / ml, about 0.025mg / ml to about 9mg / ml, about 0.05mg / ml to about 8mg / ml, about 0.075mg / ml to about 7mg / ml, about 0.1mg / ml to about 6mg / ml, about 0.25mg / ml to about 5mg / ml, about 0.5mg / ml to about 4mg / ml, about 0.75mg / ml to about 3mg / ml, about 1mg / ml to about 2mg / ml. In certain embodiments, the composition comprises at least one lipid, and the lipid is at a concentration of about 0.1mg / ml to about 5mg / ml, about 0.5mg / ml to about 3mg / ml, or about 0.5mg / ml to about 2mg / ml. In certain embodiments, the composition comprises at least one lipid, and the lipid is at about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml 1, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 150 mg / ml, about 200 mg / ml, about 240 mg / ml, about 250 mg / ml, or about 300 mg / ml. In certain embodiments, the lipid is a synthetic DOPC phospholipid.

[0164] In certain embodiments, the composition comprises cholesterol, and the cholesterol is in a concentration of about 3 mg / ml to about 24 mg / ml, about 0.01 mg / ml to about 10 mg / ml, about 0.025 mg / ml to about 9 mg / ml, about 0.05 mg / ml to about 8 mg / ml, about 0.075 mg / ml to about 7 mg / ml, about 0.1 mg / ml to about 6 mg / ml, about 0.25 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 4 mg / ml, about 0.75 mg / ml to about 3 mg / ml, or about 1 mg / ml to about 2 mg / ml. In certain embodiments, the composition comprises cholesterol, and the cholesterol is in a concentration of about 0.1 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 3 mg / ml, or about 0.5 mg / ml to about 2 mg / ml. In certain embodiments, the composition comprises cholesterol, and the cholesterol is about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml l, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 150 mg / ml, about 200 mg / ml, about 240 mg / ml, about 250 mg / ml, or about 300 mg / ml.

[0165] In certain embodiments, the composition includes an oil and the therapeutic agent is administered in a volume of about 0.01 ml, about 0.02 ml, about 0.03 ml, about 0.04 ml, about 0.05 ml, about 0.06 ml, about 0.07 ml, about 0.08 ml, about 0.09 ml, about 0.1 ml, about 0.2 ml, about 0.3 ml, about 0.4 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about Contains about 0.9 ml, about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 20 ml, about 30 ml, about 40 ml, about 50 ml, about 60 ml, about 70 ml, about 80 ml, or about 90 ml, about 100 ml, about 150 ml, about 200 ml, about 240 ml, about 250 ml, or about 300 ml of oil.

[0166] In certain embodiments, the composition comprises sodium acetate, and the sodium acetate is at a concentration of about 0.025M to about 10M, about 0.025M to about 9M, about 0.05M to about 8M, about 0.075M to about 7M, about 0.1M to about 6M, about 0.25M to about 5M, about 0.5M to about 4M, about 0.75M to about 3M, about 1M to about 2M. In certain embodiments, the composition comprises sodium acetate, wherein the sodium acetate is at a concentration of about 0.01M, about 0.02M, about 0.03M, about 0.04M, about 0.05M, about 0.06M, about 0.07M, about 0.08M, about 0.09M, about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about 2M, about 3M, about 4M, about 5M, about 6M, about 7M, about 8M, about 9M, or about 10M.

[0167] Exemplary amounts of each component (per ml of T cell activation therapeutic composition) include, but are not limited to, about 0.01 mg / ml to about 10 mg / ml of each survivin and MAGE-A9 antigen; about 0.01 mg / ml to about 5 mg / ml of a T helper epitope (e.g., SEQ ID NO: 13); about 0.01 mg to about 4 mg / ml of an adjuvant (e.g., poly I:C polynucleotide (e.g., SEQ ID NO: 22)); about 30 mg to about 240 mg / ml of synthetic DOPC phospholipid; about 3 mg / ml to about 24 mg / ml of cholesterol; and about 0.5 to about 0.9 ml of a hydrophobic carrier (e.g., mineral oil, mannide oleate in mineral oil solution, Montanide® ISA51 VG). In certain embodiments, the composition further comprises about 0.025 M to about 0.1 M sodium acetate.

[0168] Exemplary amounts of each component (per ml of T cell activation therapeutic composition) include, but are not limited to, about 0.05 mg / ml to about 5 mg / ml of each survivin and MAGE-A9 antigen; about 0.05 mg / ml to about 2 mg / ml of a T helper epitope (e.g., SEQ ID NO: 13); about 0.04 mg to about 2 mg / ml of an adjuvant (e.g., poly I:C polynucleotide (e.g., SEQ ID NO: 22)); about 45 mg to about 210 mg / ml of synthetic DOPC phospholipid; about 4.5 mg / ml to about 21 mg / ml of cholesterol; and about 0.5 to about 0.9 ml of a hydrophobic carrier (e.g., mineral oil, mannide oleate in mineral oil solution, Montanide® ISA51 VG). In certain embodiments, the composition further comprises about 0.025 M to about 0.1 M sodium acetate.

[0169] Exemplary amounts of each component (per ml of T cell activation therapeutic composition) include, but are not limited to, about 0.1 mg / ml to about 2 mg / ml of each survivin and MAGE-A9 antigen; about 0.1 mg / ml to about 1 mg / ml of a T helper epitope (e.g., SEQ ID NO: 13); about 0.05 mg to about 1 mg / ml of an adjuvant (e.g., poly I:C polynucleotide (e.g., SEQ ID NO: 22)); about 60 mg to about 180 mg / ml of synthetic DOPC phospholipid; about 6 mg / ml to about 18 mg / ml of cholesterol; and about 0.5 to about 0.9 ml of a hydrophobic carrier (e.g., mineral oil, mannide oleate in mineral oil solution, Montanide® ISA51 VG). In certain embodiments, the composition further comprises about 0.025 M to about 0.1 M sodium acetate.

[0170] Exemplary amounts of each component (per ml of T cell activation therapeutic composition) include, but are not limited to, about 1.0 mg of each survivin and MAGE-A9 antigen; about 0.5 mg of a T helper epitope (e.g., SEQ ID NO: 13); about 0.4 mg of an adjuvant (e.g., poly I:C polynucleotide (e.g., SEQ ID NO: 22)); about 120.0 mg of synthetic DOPC phospholipid; about 12.0 mg of cholesterol; and about 0.7 ml of a hydrophobic carrier (e.g., mineral oil, mannide oleate in mineral oil solution, Montanide® ISA51 VG). In certain embodiments, the hydrophobic carrier is about 0.9 ml.

[0171] Further exemplary amounts of each component (per ml of T cell activation therapeutic composition) include, but are not limited to, about 1.0 mg of each survivin and MAGE-A9 antigen; about 0.5 mg of a T helper epitope (e.g., SEQ ID NO: 13); about 0.4 mg of an adjuvant (e.g., poly I:C polynucleotide (e.g., SEQ ID NO: 22)); about 120.0 mg of synthetic DOPC phospholipid; about 12.0 mg of cholesterol; and about 0.1 M of sodium acetate, and about 0.7 ml of a hydrophobic carrier (e.g., mineral oil, mannide oleate in mineral oil solution, Montanide® ISA51 VG). In certain embodiments, the hydrophobic carrier is about 0.9 ml.

[0172] The composition may optionally further comprise additional ingredients, such as, for example, an emulsifier. A more detailed disclosure of exemplary embodiments of the composition and its components is set forth below.

[0173] (iv) Additional antigens Other antigens that may be useful in the compositions of the invention include, but are not limited to, antigens capable of inducing or enhancing an immune response in a subject that is expected to be beneficial in the treatment of tumors or cancer, such as a cell-mediated or humoral-mediated immune response.

[0174] Cell-mediated immunity is an immune response that does not involve antibodies, but does involve the activation of macrophages and natural killer cells, the production of antigen-specific cytotoxic T lymphocytes in response to antigens, and the release of various cytokines. Cytotoxic T lymphocytes are a subgroup of T lymphocytes (a type of white blood cell) that can induce the death of infected somatic or tumor cells; they kill cells that are infected with viruses (or other pathogens) or that are otherwise damaged or dysfunctional.

[0175] Most cytotoxic T cells express a T cell receptor that can recognize a specific peptide antigen bound to a class I MHC molecule. These CTLs also express CD8 (CD8+ T cells), which are attracted to parts of the class I MHC molecule. This affinity keeps the CTL and target cell tightly bound during antigen-specific activation.

[0176] Cellular immunity protects the body, for example, by activating antigen-specific cytotoxic T lymphocytes that can lyse somatic cells that present epitopes of foreign antigens on their surface, such as virus-infected cells, cells that harbor intracellular bacteria, and cancer cells that present tumor antigens; by activating macrophages and natural killer cells so that they can destroy intracellular pathogens; and by stimulating cells to secrete various cytokines that affect the function of other cells involved in adaptive and innate immune responses.

[0177] Therefore, in a further embodiment, the T cell activation therapeutic composition of the present invention may comprise additional antigens in addition to one or more survivin antigens. For example, the additional antigen may be, but is not limited to, a peptide, a suitable natural, non-natural, recombinant or denatured protein or polypeptide, or a fragment or epitope thereof, capable of inducing or enhancing a CTL immune response in a subject.

[0178] The additional antigen may also be a polynucleotide that codes for a polypeptide that functions as an antigen. Nucleic acid-based vaccination strategies are known, in which a T cell activation therapeutic composition containing a polynucleotide is administered to a subject. The antigenic polypeptide encoded by the polynucleotide is expressed in the subject, so that the antigenic polypeptide is ultimately present in the subject as if the T cell activation therapeutic composition itself contained the polypeptide. For the purposes of the present invention, the additional antigen includes such polynucleotides that code for a polypeptide that functions as an antigen, when the context indicates.

[0179] The term "polypeptide" encompasses any chain of amino acids, regardless of length (e.g., at least 6, 8, 10, 12, 14, 16, 18, or 20 amino acids) or post-translational modification (e.g., glycosylation or phosphorylation), including, for example, natural proteins, synthetic or recombinant polypeptides and peptides, epitopes, hybrid molecules, variants, homologs, analogs, peptoids, peptidomimetics, and the like. Variants or derivatives therefore include deletions, such as truncations and fragments; insertions and additions, such as conservative substitutions, site-directed mutants, and allelic variants; and modifications, such as peptoids having one or more non-aminoacyl groups (e.g., sugars, lipids, and the like) covalently linked to the peptide and post-translational modifications. The term "conserved amino acid substitution" or "conservative substitution," as used herein, refers to the substitution of one amino acid with another amino acid at a given position in a peptide, where the substitution can be made without substantial loss of the associated function. In making such changes, substitutions of like amino acid residues can be made on the basis of the relative similarity of the side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, etc., and such substitutions can be assayed for their effect on the function of the peptide by routine testing. Specific non-limiting examples of conservative substitutions include the following:

[0180] [Table 3]

[0181] Polypeptides or peptides with substantial identity to preferred antigen sequences may be used. Two sequences are considered to have substantial identity if, when optimally aligned (gaps allowed), they have at least approximately 50% sequence identity, or if the sequences have a defined functional motif. In alternative embodiments, optimally aligned sequences can be considered to be substantially identical (i.e., have substantial identity) if they have at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity over a specified region. The term "identity" refers to the sequence similarity between two polypeptide molecules. Identity can be determined by comparing each position in the aligned sequences. The degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences, for example, over a specified region. Optimal alignment of sequences for identity comparison can be carried out using various algorithms, which are known in the art and include, for example, the ClustalW program available at http: / / clustalw.qenome.ad.jp, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, and computerized implementations of these algorithms (e.g. GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wl, USA).Sequence identity can also be determined using the BLAST algorithm (using published default settings) described in Altschul et al., 1990, J. Mol. Biol. 215:403-10. For example, the "BLAST2 sequence" tool available through the National Center for Biotechnology Information (via the Internet at http: / / www.ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cqi) can be used with the "blastp" program selected with the following default settings: expectation threshold 10; character size 3; matrix BLOSUM 62; gap cost presence 11, extension 1. In another embodiment, one skilled in the art can easily and appropriately align any given sequence and infer sequence identity and / or homology by simple visual inspection.

[0182] The polypeptides and peptides used as additional antigens in the T cell activation therapeutic composition of the present invention may be isolated from natural sources, synthetic, or recombinantly produced polypeptides. The peptides and proteins may be recombinantly expressed in vitro or in vivo. The peptides and polypeptides used to practice the present invention may be produced and isolated using any method known in the art. The polypeptides and peptides used to practice the present invention may also be wholly or partially synthesized using chemical methods well known in the art. See, for example, Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K, Therapeutic Peptides and Proteins, Formulation.

[0183] Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa. For example, peptide synthesis can be carried out using a variety of solid-phase techniques (see, e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13), and automated synthesis may be accomplished using, for example, an ABI 431A peptide synthesizer (Perkin Elmer) following instructions provided by the manufacturer.

[0184] In some embodiments, the additional antigen may be a purified antigen, e.g., about 25% to 50% pure, about 50% to about 75% pure, about 75% to about 85% pure, about 85% to about 90% pure, about 90% to about 95% pure, about 95% to about 98% pure, about 98% to about 99% pure, or greater than 99% pure.

[0185] As mentioned above, additional antigens include polynucleotides that code for polypeptides that function as antigens. The term "polynucleotide" as used herein includes chains of nucleotides of any length (e.g., 9, 12, 18, 24, 30, 60, 150, 300, 600, 1500 or more nucleotides) or number of strands (e.g., single-stranded or double-stranded). Polynucleotides may be DNA (e.g., genomic DNA or cDNA) or RNA (e.g., mRNA) or combinations thereof. They may be naturally occurring or synthetic (e.g., chemically synthesized). It is contemplated that polynucleotides may contain modifications of one or more nitrogenous bases, pentose sugars, or phosphate groups in the nucleotide chain. Such modifications are well known in the art and may be for the purpose of improving the stability of the polynucleotide, for example.

[0186] Polynucleotides can be delivered in a variety of forms. In some embodiments, naked polynucleotides can be used, either in linear form or inserted into a plasmid, such as an expression plasmid. In other embodiments, live vectors, such as viral or bacterial vectors, can be used.

[0187] There may be one or more regulatory sequences that aid in the transcription of DNA into RNA and / or the translation of RNA into polypeptides. In some cases, such as in the case of a polynucleotide that is a messenger RNA (mRNA) molecule, regulatory sequences (e.g., promoters) for the transcription process are not necessary, and protein expression may be affected in the absence of a promoter. Those skilled in the art can include suitable regulatory sequences as the environment requires.

[0188] In some embodiments, the polynucleotide is present in an expression cassette that is operably linked to a regulatory sequence that will allow expression of the polynucleotide in a subject to which the composition of the invention is administered. The choice of expression cassette will depend on the subject to which the composition is administered, as well as the characteristics desired for the expressed polypeptide.

[0189] Typically, the expression cassette comprises a promoter that is functional in the subject and may be constitutive or inducible; a ribosome binding site; an initiation codon (ATG) if necessary; a polynucleotide that encodes a polypeptide of interest; a stop codon; and optionally a 3' end region (translation and / or transcription terminator). Additional sequences may be included, such as a region that encodes a signal peptide. The polynucleotide that encodes a polypeptide of interest may be homologous or heterologous to any of the other regulatory sequences in the expression cassette. The sequence that is to be expressed together with the polypeptide of interest, such as a region that encodes a signal peptide, is typically placed adjacent to the polynucleotide that encodes the protein that is to be expressed and placed in a suitable reading frame. The open reading frame constituted by the polynucleotide that encodes the protein that is to be expressed alone or together with any other sequences that are to be expressed (e.g., signal peptide) is placed under the control of the promoter so that transcription and translation occur in the subject to which the composition is administered.

[0190] The amount of additional antigen used in a single treatment with the T cell activation therapeutic composition described herein can vary depending on the type of antigen and the size of the subject.Those skilled in the art will be able to determine the effective amount of additional antigen to be used in a particular application without undue experimentation.

[0191] In some embodiments, the additional antigen may be at least one CTL epitope capable of inducing a CTL response. For example, the additional antigen may be a CTL epitope derived from a protein identified as being upregulated in cancer cells.

[0192] In one embodiment, the CTL epitope may be an epitope of a tumor-associated protein, such as an epitope of a melanoma-associated protein, etc. In some embodiments, the melanoma-associated protein is tyrosine-related protein-2 (TRP-2) or p53, which can be obtained by a variety of methods, including recombinant technology or chemical synthesis.

[0193] The following genes code for tumor-associated proteins having peptide sequences that can be incorporated as additional antigens into the T cell activation therapeutic compositions of the present invention, including, but not limited to, p53, HPVE6 and E7, ART-4, CAMEL, CEA, Cyp-B, HER2 / neu, hTERT, hTRT, iCE, MUC1, MUC2, PRAME, P15, RUI, RU2, SART-1, SART-3, WT1, PSA, tyrosinase, TRP-1, TRP-2, gp100, MART-1 / Melan A, MAGE-A1.MAGE-A2, MAGE-A3, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, NA88-A, NY-ESO-1, NY-ESO-1a(CAG-3), AFP, β-catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, Ras, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, survivin, TRP-2 / INT2, and 707-AP.

[0194] In one embodiment, the T cell activation therapeutic composition may comprise a mixture of CTL epitopes associated with cancer as antigens for inducing CTL responses.For example, the antigen may comprise at least one or more of the survivin antigens described herein, such as, but not limited to, the survivin peptide antigens having the following amino acid sequences: FEELTLGEF (SEQ ID NO: 1); FTELTLGEF (SEQ ID NO: 2); LTLGEFLKL (SEQ ID NO: 3); LMLGEFLKL (SEQ ID NO: 4); RISTFKNWPF (SEQ ID NO: 5); RISTFKNWPK (SEQ ID NO: 6); STFKNWPFL (SEQ ID NO: 7); and LPPAWQPFL (SEQ ID NO: 8); at least one or more of the MAGE-A9 antigens described herein, such as, but not limited to, the MAGE-A9 peptide antigens having the following amino acid sequences: KVAELVHFL (SEQ ID NO: 9); GLMGAQEPT (SEQ ID NO: 10); ALSVMGVYV (SEQ ID NO: 11); FLWGSKAHA (SEQ ID NO: 12), together with at least one additional antigen of tumor-associated protein.

[0195] (v) T helper epitope In some embodiments, the T cell activation therapeutic composition of the present invention comprises at least one T helper epitope or T helper antigen.

[0196] T helper epitopes are sequences of amino acids (natural or non-natural) that have T helper activity. T helper epitopes are recognized by T helper lymphocytes, which play an important role in establishing and maximizing the performance of the immune system, and are involved in activating and directing other immune cells, such as cytotoxic T lymphocytes.

[0197] T helper epitopes may consist of continuous or discontinuous epitopes. Thus, not all but all amino acids of T helper are necessarily part of the epitope. Thus, T helper epitopes, including analogs and segments of T helper epitopes, can enhance or stimulate immune responses. Immunodominant T helper epitopes have broad reactivity with widely diversified MHC types in animal and human populations (Celis et al., (1988) J. Immunol. 140:1808-1815; Demotz et al., (1989) J. Immunol. 142:394- 402; Chong et al., (1992) Infect. Immun. 60:4640-4647). The T helper domain of the subject peptide has about 10 to about 50 amino acids, preferably about 10 to about 30 amino acids. When multiple T helper epitopes are present, each T helper epitope acts independently.

[0198] In some embodiments, T helper epitopes constitute part of the antigens described herein.In particular, antigens may contain epitopes that function as T helper epitopes when they have sufficient size.In other embodiments, T helper epitopes are molecules that are separate from antigens.

[0199] In another embodiment, the T helper epitope analogs may include substitutions, deletions and insertions of 1 to about 10 amino acid residues in the T helper epitope. A T helper segment is a contiguous portion of a T helper epitope sufficient to enhance or stimulate an immune response. An example of a T helper segment is a series of overlapping peptides derived from a single longer peptide.

[0200] In certain embodiments, the compositions of the invention may include as a T helper epitope or antigen a modified tetanus toxin peptide, A16L(830-844; AQYIKANSKFIGITEL (SEQ ID NO: 13), in which an alanine residue has been added to its amino terminus to enhance stability (Slingluff et al, Clin Cancer Res., 7: 3012-3024, 2001).

[0201] Other sources of T helper epitopes that can be used in the compositions of the invention include, for example, Hepatitis B surface antigen helper T cell epitopes, pertussis toxin helper T cell epitopes, measles virus F protein helper T cell epitopes, Chlamydia trachomitis major outer membrane protein helper! cell epitopes, diphtheria toxin helper T cell epitopes, Plasmodium falciparum circumsporozoite helper T cell epitopes, Schistosoma mansoni triosephosphate isomerase helper T cell epitopes, Escherichia coli TraT helper T cell epitopes, and immune enhancing analogs and segments of any of these T helper epitopes.

[0202] In some embodiments, the T helper epitope may be a universal T helper epitope. Universal T helper epitope, as used herein, refers to a peptide or other immunogenic molecule, or fragment thereof, that binds to multiple MHC class II molecules in a manner that activates T cell function in a class II (CD4+ T cell) restricted manner. An example of a universal T helper epitope is PADRE (pan-DR epitope), which comprises the peptide sequence AKXVAAWTLKAAA (SEQ ID NO: 18), where X may be cyclohexylalanyl. PADRE specifically has a CD4+ T helper epitope, i.e., stimulates the induction of PADRE-specific CD4+ T helper response.

[0203] In addition to the modified tetanus toxoid peptide A16L described above, tetanus toxoid has other T helper epitopes that act in a similar manner to PADRE. Tetanus and diphtheria toxins have universal epitopes on human CD4+ cells (Diethelm-Okita, BM et al., J. Infect. Diseases, 181:1001-1009, 2000). In another embodiment, the T helper epitope may be a tetanus toxoid peptide, such as F21E, which contains the peptide sequence FNNFTVSFWLRVPKVSASHLE (amino acids 947-967; SEQ ID NO: 19).

[0204] In certain embodiments, the T helper epitope is fused to at least one of the one or more survivin antigens in the T cell activation therapeutic composition of the present invention, or is fused to an additional antigen that may be included in the T cell activation therapeutic composition (e.g., a fusion peptide).

[0205] (vi) Adjuvant In some embodiments, the T cell activation therapeutic composition of the present invention comprises one or more pharma- ceutically acceptable adjuvants.Many adjuvants have been described and are known to those skilled in the art.See, for example, Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985) and the United States Pharmacopeia: National Formulary, published in 1999 (USP24NF19).

[0206] Exemplary adjuvants include, but are not limited to, alum, other aluminum compounds, Bacillus of Calmette and Guerin (BCG), TiterMax™, Ribi™, Freund's complete adjuvant (FCA), CpG-containing oligodeoxynucleotides (CpG ODN), lipopeptides and polynucleotides (e.g., poly I:C, poly dIdC, etc.). An exemplary CpG ODN is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 20). One of skill in the art can readily select other suitable CpG ODNs based on the target species and efficacy. Exemplary lipopeptides include, but are not limited to, Pam3Cys-SKKK (SEQ ID NO: 21) (EMC Microcollections, Germany), or variants, homologs and analogs thereof. The Pam2 family of lipopeptides has been shown to be an effective alternative to the Pam3 family of lipopeptides.

[0207] "Poly I:C" or "Poly I:C polynucleotide," as used herein, is a polynucleotide molecule (RNA or DNA or a combination of DNA and RNA) containing inosinic acid residues (I) and cytidylic acid residues (C) that is capable of inducing or enhancing the production of at least one inflammatory cytokine, such as an interferon, in a mammalian subject.

[0208] Poly I:C polynucleotides may have a length of about 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 500, 1000 or more residues. No upper limit is believed to be essential. Preferred poly I:C polynucleotides may have a minimum length of about 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 nucleotides, and a maximum length of about 1000, 500, 300, 200, 100, 90, 80, 70, 60, 50, 45, or 40 nucleotides. In certain embodiments, poly I:C polynucleotides are about 20 or more residues in length (typically 22, 24, 26, 28 or 30 residues in length). When made semisynthetically (e.g., using enzymes), the chains may be 500, 1000 or more residues in length.

[0209] In some embodiments, the poly I:C polynucleotide is double-stranded. In such embodiments, it may be composed of one strand entirely of cytosine-containing nucleotides and one strand entirely of inosine-containing nucleotides, although other structures are possible. For example, each strand may contain both cytosine-containing and inosine-containing nucleotides. Non-limiting examples include those in which each strand contains at least six consecutive inosinic or cytidylic acid residues, or six consecutive residues selected from inosinic and cytidylic acid in any order (e.g., IICIIC, ICICIC, or IIICCC). In some cases, either or both strands may additionally contain one or more non-cytosine or non-inosine nucleotides.

[0210] In other embodiments, the poly I:C polynucleotide may be a single-stranded molecule containing inosinic acid residues (I) and cytidylic acid residues (C). By way of example, and not by way of limitation, the single-stranded poly I:C may be a sequence of dIdC repeats. In certain embodiments, the sequence of the single-stranded poly I:C may be a 26-mer sequence with an (IC) of 13, i.e., ICICICICICICICICICICICICICICIC (SEQ ID NO: 22). As would be understood by one of skill in the art, these single-stranded molecules of dIdC repeats are expected to naturally form homodimers due to their nature (e.g., complementarity), and therefore are expected to be conceptually similar to poly I / poly C dimers.

[0211] In certain embodiments, each strand of the polyI:C polynucleotide may be a homopolymer of inosinic or cytidylic acid residues, or each strand may be a heteropolymer containing both inosinic and cytidylic acid residues. In either case, the polymer may be interrupted by one or more non-inosinic or non-cytidylic acid residues (e.g., uridine), provided that there is at least one continuous region of six I, six C, or six I / C residues as described above. Typically, each strand of the polyI:C polynucleotide is expected to contain no more than one non-I / C residue per six I / C residues, and more preferably no more than one non-I / C residue per 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 I / C residues, respectively.

[0212] Inosinic or cytidylic acid (or other) residues in poly I:C polynucleotides may be derivatized or modified as known in the art, provided that the ability of the poly I:C polynucleotide to promote the production of inflammatory cytokines, such as interferons, is maintained. Non-limiting examples of derivatives or modifications include, for example, azide modification, fluoro modification, or the use of thioester (or similar) linkages instead of natural phosphodiester bonds to enhance stability in vivo. Poly I:C polynucleotides may also be modified to enhance their resistance to degradation in vivo, for example, by complexing the molecule with positively charged polylysine and carboxymethylcellulose, or positively charged synthetic peptides.

[0213] In certain embodiments, the T cell activation therapeutic composition comprises a poly I:C polynucleotide as an adjuvant, such as, but not limited to, a 26-mer deoxyinosine / cytosine synthetic polynucleotide. In certain embodiments, the T cell activation therapeutic composition comprises a dIdC DNA polynucleotide as an adjuvant.

[0214] Poly I:C polynucleotides will typically be included in the compositions of the invention in an amount of about 0.001 mg to 1 mg per unit dose of the composition, hi certain embodiments, the amount of Poly I:C polynucleotides will be about 0.04 mg / mL of the T cell activation therapeutic composition.

[0215] Other suitable adjuvants for T cell activation therapeutic compositions are those that activate or increase the activity of TLR2. As used herein, adjuvants that "activate" or "increase the activity" of TLR include any adjuvant, including lipid-based adjuvants that act as TLR agonists in some embodiments. Furthermore, activating or increasing the activity of TLR2 encompasses its activation in any monomeric, homodimeric or heterodimeric form, and in particular includes the activation of TLR2 as a heterodimer with TLR1 or TLR6 (i.e., TLR1 / 2 or TLR2 / 6).

[0216] An exemplary embodiment of an adjuvant that activates or increases the activity of TLR2 is a lipid-based adjuvant that includes at least one lipid moiety or component.

[0217] The phrase "lipid moiety" or "lipid component" as used herein refers to any fatty acid (e.g., fatty acyl) or derivatives thereof, such as triglycerides, diglycerides, and monoglycerides. Exemplary fatty acids include, but are not limited to, palmitoyl, myristoyl, stearoyl, and decanoyl groups, or any C2-C30 saturated or unsaturated fatty acyl group, preferably any C14-C22 saturated or unsaturated fatty acyl group, more preferably a C16 saturated or unsaturated fatty acyl group. Thus, as referred to herein, the phrase "lipid-based adjuvant" encompasses any adjuvant that includes a fatty acyl group or derivatives thereof.

[0218] A lipid-based adjuvant contains, at a minimum, at least one lipid moiety, or synthetic / semi-synthetic lipid moiety analog, which may be coupled onto an amino acid, oligopeptide, or other molecule (e.g., carbohydrate, glycan, polysaccharide, biotin, rhodamine, etc.) Thus, without being limited thereto, a lipid-based adjuvant may be, for example, a lipoamino acid, lipopeptide, lipoglycan, lipopolysaccharide, or lipoteichoic acid.

[0219] Additionally, lipid moieties or structures containing lipid moieties can be covalently or non-covalently coupled to antigens to generate antigenic compounds with embedded auxiliary properties. For example, but not limited to, lipid-based moieties can include a cation (e.g., nickel) to provide a positive charge for non-covalent coupling.

[0220] In some embodiments, the lipid moiety or lipid component may be naturally occurring, such as a component of the cell wall (e.g., lipoproteins) from gram-positive or gram-negative bacteria, Rhodopseudomonas viridis, or mycoplasma, while in other embodiments, the lipid moiety or lipid component may be synthetic or semi-synthetic.

[0221] A lipid-based adjuvant may include palmitic acid (PAM) as at least one of the lipid moieties or components of the adjuvant. Such lipid-based adjuvants are referred to herein as "palmitic acid adjuvants." Palmitic acid is a low molecular weight lipid found in the immunologically reactive Braun lipoproteins of Escherichia coli. Other common chemical names for palmitic acid include, for example, the IUPAC nomenclature hexadecanoic acid and 1-pentadecanecarboxylic acid. The molecular formula for palmitic acid is CH3(CH2) 14It is CO2H.It is considered that the lipid chain of palmitic acid can be modified, as would be understood by those skilled in the art.The exemplary compounds that can be used herein as palmitic acid adjuvants and their synthesis methods are described in, for example, US Patent Publications US2008 / 0233143;US2010 / 0129385;and US2011 / 0200632, each of which is incorporated herein in its entirety for all intended purposes.

[0222] As generally described above for lipid moieties, palmitic acid adjuvants contain at least one palmitic acid moiety, which may be coupled onto an amino acid, oligopeptide or other molecule. Palmitic acid moieties or palmitic acid-containing structures may also be covalently or non-covalently coupled to antigens to generate antigenic compounds with embedded adjuvant properties. Palmitic acid moieties or palmitic acid-containing chemical structures may be conjugated to cysteine ​​peptides (Cys), allowing for a variety of structural configurations of adjuvants, including linear and branched structures. Typically, the cysteine ​​residue is extended at the C-terminus by polar residues such as serine (Ser) and / or lysine (Lys), resulting in adjuvant compounds with improved solubility. Palmitic acid-containing adjuvant compounds may be mixed with antigens, associated with antigens via non-covalent interactions, or alternatively covalently linked to antigens, either directly or using linkers / spacers, to generate enhanced immune responses. Most commonly, two palmitic acid moieties are attached to a glyceryl backbone and a cysteine ​​residue to give dipalmitoyl-S-glyceryl-cysteine ​​(PAM2Cys) or tripalmitoyl-S-glyceryl-cysteine ​​(PAM3Cys), which can also be used in multiple configurations as described above.

[0223] Thus, in one embodiment, the adjuvant of the composition may comprise a palmitic acid moiety or component that can be modified or engineered to improve its stability in vitro or in vivo, to enhance its binding to a receptor (such as, for example, a Toll-like receptor, as described below), or to enhance its biological activity.

[0224] In certain embodiments, the palmitic acid adjuvant may comprise PAM2Cys or PAM3Cys. In other specific embodiments, the palmitic acid adjuvant may be Pam-2-Cys-Ser-(Lys)4 (SEQ ID NO:23) or Pam-3-Cys-Ser-(Lys)4 (SEQ ID NO:24). Such palmitic acid adjuvants are available, for example as research reagents, from EMC Microcollections GmbH (Germany) and InvivoGen (San Diego, California, USA). Various analogs of Pam-2-Cys-Ser-(Lys)4 (SEQ ID NO:23) and Pam-3-Cys-Ser-(Lys)4 (SEQ ID NO:34), including labeled analogs, are also available from EMC Microcollections.

[0225] The composition of the present invention may include an adjuvant as described above in combination with at least one other suitable adjuvant. Exemplary embodiments of the at least one other adjuvant include, but are not limited to, organic and inorganic compounds, polymers, proteins, peptides, sugars (including, but not limited to, virosomes, virus-like particles, viruses and bacteria or components thereof) from synthetic, non-biological or biological sources.

[0226] Further examples of compatible adjuvants include, but are not limited to, chemokines, Toll-like receptor agonists, colony stimulating factors, cytokines, 1018ISS, aluminum salts, Amplivax, AS04, AS15, ABM2, Adjumer, Algammulin, AS01B, AS02 (SBASA), AS02A, BCG, calcitriol, chitosan, cholera toxin, CP-870,893, CpG, polyIC, CyaA, dimethyldioctadecylammonium bromide (DDA), dibutyl phthalate (DBP), dSLIM, gamma inulin, GLA-SE, GM-CSF, GMDP, glycerol, IC30, IC31, imiquimod, ImuFact IMP321, IS Examples include Patch, ISCOM, ISCOMATRIX, Juvlmmune, LipoVac, LPS, lipid core protein, MF59, monophosphoryl lipid A, Montanide® IMS 1312, Montanide® based adjuvants, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, other palmitoyl-based molecules, PLG microparticles, resiquimod, squalene, SLR172, YF-17DBCG, QS21, QuilA, P1005, poloxamer, saponin, synthetic polynucleotides, zymosan, and pertussis toxin.

[0227] Thus, the composition may include one or more pharma- ceutically acceptable adjuvants. In some embodiments, at least one of the one or more survivin antigens or additional antigens may be coupled to at least one of the adjuvants.

[0228] The amount of adjuvant used depends on the amount of antigen and the type of adjuvant. One skilled in the art can easily determine by empirical testing the amount of adjuvant required for a particular application.

[0229] (vii) Lipids For use in the compositions described herein, any lipid may be used so long as it is a membrane-forming lipid.

[0230] While any lipid as defined above may be used, particularly suitable lipids include those having at least one fatty acid chain containing at least 4 carbons, typically about 4 to 28 carbons. The fatty acid chain may contain any number of saturated and / or unsaturated bonds. The lipid may be a natural lipid or a synthetic lipid. Non-limiting examples of lipids include phospholipids, sphingolipids, sphingomyelins, celloside, gangliosides, ether lipids, sterols, cardiolipin, cationic lipids, and lipids modified with poly(ethylene glycol) and other polymers. Synthetic lipids may contain, but are not limited to, the following fatty acid moieties: lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fatty acids. In some embodiments, the lipid or lipid of the lipid vesicle particle is an amphoteric lipid, meaning that they have both hydrophilic and hydrophobic (lipophilic) properties.

[0231] Lipids suitable for use in the compositions of the present disclosure include, but are not limited to, phospholipids, cationic lipids, cholesterol, and / or cholesterol derivatives, or combinations thereof. It should be understood that the terms "phospholipid", "cationic lipid", or "cholesterol derivative" are not necessarily meant to be mutually exclusive.

[0232] Broadly defined, a "phospholipid" is a member of a group of lipid compounds that upon hydrolysis yield phosphoric acid, alcohol, fatty acid, and nitrogenous base. Phospholipids preferably used in the preparation of the compositions of the present disclosure are those having at least one head group selected from the group consisting of phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine, and phosphoinositol. More preferred are lipids that are about 94-100% phosphatidylcholine. Such lipids are commercially available as lecithin Phospholipon® 90G (Phospholipid GmBH, Germany) or lecithin S100 (Lipoid GmBH, Germany). In some embodiments, the phospholipid used in the preparation of the composition of the present disclosure is dioleoylphosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-succinate (DGS), or a combination thereof.In one embodiment, the phospholipid used in the preparation of the composition of the present disclosure is dioleoylphosphatidylcholine (DOPC).In some embodiments, a mixture of DOPC and non-esterified cholesterol may be used.In other embodiments, a mixture of Lipoid S 100 lecithin and non-esterified cholesterol may be used.

[0233] In one embodiment, the lipid vesicle particle comprises a synthetic lipid. In one embodiment, the lipid vesicle particle comprises a synthetic DOPC. In another embodiment, the lipid vesicle particle comprises a synthetic DOPC and cholesterol.

[0234] Another common phospholipid is sphingomyelin. Sphingomyelin contains sphingosine, an amino alcohol with a long unsaturated hydrocarbon chain. A fatty acyl side chain is linked to the amino group of sphingosine by an amide bond to form a ceramide. The hydroxyl group of sphingosine is esterified to phosphocholine. Like phosphoglycerides, sphingomyelin is amphiphilic.

[0235] Lecithin can also be used and is a natural mixture of phospholipids, typically derived from chicken eggs, sheep's wool, soybeans and other vegetable sources.

[0236] All of these and other phospholipids can be used in the practice of the present invention. Phospholipids can also be purchased, for example, from Avanti lipids (Alabastar, AL, USA), Lipoid LLC (Newark, NJ, USA) and Lipoid GmbH (Germany), among a variety of other sources.

[0237] Cholesterol and / or cholesterol derivatives can be used in the compositions of the present disclosure. When non-esterified cholesterol is used in the composition, cholesterol is usually used in an amount equivalent to about 10% of the amount of phospholipid. When a compound other than cholesterol is used to stabilize the composition, a person skilled in the art can easily determine the amount required for the composition. Cholesterol derivatives suitable for use in the present disclosure include positively charged cholesterol such as cholesterol β-D-glucoside, cholesterol 3-sulfate sodium salt, DC-cholesterol, and other cholesterol-like molecules such as campesterol, ergosterol, betulin, lupeol, β-sitosterol, α,β-amyrin, and bile acids.

[0238] In some embodiments, the lipid vesicle particles comprise DOPC and cholesterol in a DOPC:cholesterol ratio of about 10:1 (w / w).In some embodiments, the lipid vesicle particles comprise DOPC and cholesterol in a DOPC:cholesterol ratio of about 8:1 (w / w), about 9:1 (w / w), about 11:1, or about 12:1 (w / w).

[0239] In one embodiment, the compositions disclosed herein contain about 66 mg / ml of DOPC and cholesterol. In other embodiments, the compositions disclosed herein contain about 55 mg / ml, 56 mg / ml, 57 mg / ml, 58 mg / ml, 59 mg / ml, 60 mg / ml, 61 mg / ml, 62 mg / ml, 63 mg / ml, 64 mg / ml, 65 mg / ml, 67 mg / ml, 68 mg / ml, 69 mg / ml, 70 mg / ml, 71 mg / ml, 72 mg / ml, 73 mg / ml, 74 mg / ml, or 75 mg / ml of DOPC and cholesterol.

[0240] In one embodiment, the composition disclosed herein comprises about 60 mg / ml DOPC and about 6 mg / ml cholesterol.

[0241] In some embodiments, positively charged lipid (i.e. cationic lipid) is used in the composition of the present disclosure.Exemplary cationic lipid suitable for use in the composition of the present disclosure includes, but is not limited to, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolium chloride (DOTIM), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dioctadecylaminoglycylspermine-4 tri ... Fluoroacetic acid (DOGS), dioleyldimethylammonium chloride (DODAC), dimethyldioctadecylammonium bromide (DDAB), 1,2-distearoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-dipalmitoyl-sn-glycero-3-succinate (DGS), N-palmitoylhomocysteine ​​ammonia nium salt (PHC), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), dimethyldioctadecylammonium bromide salt (DDAB), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine chloride salt (EPC), N4-cholesteryl-spermine HCl salt (GL67), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORI), N-(3-aminopropyl)-N,N-dimethyl-2,3 -bis(dodecyloxy)-1-propane ammonium bromide (GAP-DLRIE), 2,3-dioleyloxy-N-[2[sperminecarboxaminino]ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and SAINT 2.Further examples of cationic lipids include those described, for example, in Audouy and Hoekstra, Mol Membr Biol, Apr-Jun 2001;18(2):129-43; Shim et al., Asian Journal of Pharmaceutical Sciences 8(2):72-80, April 2013; and Faneca et al (2013) Cationic Liposome-Based Systems for Nucleic Acid Delivery: From the Formulation Development to Therapeutic Applications. In: Coelho J. (eds) Drug Delivery Systems: Advanced Technologies Potentially Applicable in Personalised Treatment. Advances in Predictive, Preventive and Personalised Medicine, vol 4. Springer, Dordrecht, which are incorporated herein by reference in their entireties.

[0242] Lipid vesicle particles may have closed vesicle structure.They are typically spherical in shape, but other shapes and conformations can also be formed and are not excluded.Exemplary embodiments of lipid vesicle particles include, but are not limited to, unilamellar vesicle structure (e.g., micelles) and bilayer vesicle structure (e.g., unilamellar or multilamellar vesicles), or various combinations thereof.

[0243] "Monolayer" means that the lipids do not form a bilayer but remain in a layer with the hydrophobic portion oriented on one side and the hydrophilic portion oriented on the opposite side. "Bilayer" means that the lipids form two sheets, typically with the hydrophobic portion of each layer oriented inward toward the center of the bilayer and the hydrophilic portion oriented outward. However, the reverse arrangement is also possible. The term "multilayer" is meant to encompass any combination of monolayer and bilayer structures. The form adopted may depend on the specific lipids used.

[0244] In one embodiment, the lipid vesicle particle is a bilayer vesicle structure, such as a liposome. A liposome is a completely closed lipid bilayer membrane. A liposome may be a unilamellar vesicle (having a single bilayer membrane), a multilamellar vesicle (characterized by multiple membrane bilayers, whereby each bilayer may or may not be separated from the next by an aqueous layer) or a multivesicular vesicle (having one or more vesicles within a vesicle). A general discussion of liposomes can be found in Gregoriadis 1990 and Frezard 1999, which are incorporated herein by reference in their entirety.

[0245] That is, in one embodiment, the lipid vesicle particle is a liposome. In one embodiment, the liposome is unilamellar, multilamellar, multivesicular, or a mixture thereof.

[0246] (viii) Carrier In some embodiments, the T cell activation therapeutic composition of the present invention comprises a pharma- ceutically acceptable carrier, excipient or diluent. Pharmaceutically acceptable carrier, as used herein, refers to any material suitable for delivering the T cell activation therapeutic composition of the present invention and useful in the method of the present invention.

[0247] Carriers that can be used with the T cell activating therapeutics of the invention are well known in the art and include, but are not limited to, water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oil-in-water emulsions, oils, water-in-oil emulsions, esters, poly(ethylene-vinyl acetate), copolymers of lactic and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), cellulose, albumin, starch, casein, dextran, polyesters, ethanol, mathacrylate, polyurethanes, polyethylene, vinyl polymers, glycols, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly-L-lysine, poly-L-glutamic acid, influenza, Hepatitis B virus core protein, mixtures thereof, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, 2000, Gennaro, AR ed., Eaton, Pa.: Mack Publishing Co.

[0248] In certain embodiments, the carrier of the T cell activation therapeutic composition is a carrier that comprises a continuous phase of hydrophobic material, preferably a liquid hydrophobic material. The continuous phase may be essentially pure hydrophobic material or a mixture of hydrophobic materials. In addition, the carrier may be an emulsion of water in a hydrophobic material or an emulsion of water in a mixture of hydrophobic materials, provided that the hydrophobic material constitutes the continuous phase. Furthermore, in another embodiment, the carrier may function as an adjuvant.

[0249] The hydrophobic substances useful in the compositions described herein are medicamentally and / or immunologically acceptable. Preferably, the carrier is liquid, but certain hydrophobic substances that are not liquid at ambient temperature may be liquefied, for example by warming, and are also useful in this invention. In one embodiment, the hydrophobic carrier may be a phosphate buffered saline / incomplete Freund's adjuvant (PBS / FIA) emulsion.

[0250] Oil emulsions or water-in-oil emulsions are particularly suitable carriers for use in the T cell activation therapeutic composition of the present invention. The oil is expected to be medicamentously and / or immunologically acceptable. Suitable oils include, for example, mineral oils (especially light or low viscosity mineral oils, such as Drakeol® 6VR), vegetable oils (e.g. soybean oil), nut oils (e.g. peanut oil), or mixtures thereof. Thus, in certain embodiments, the carrier is a hydrophobic material such as vegetable oil, nut oil or mineral oil. Animal fats and artificial hydrophobic polymeric materials can also be used, especially those that are liquid at ambient temperature or can be relatively easily liquefied.

[0251] To enhance the immunogenicity of cancer T cell activation therapeutics, an adjunct T cell activation therapeutic composition platform was designed to facilitate a strong and robust immune response to peptide antigens. DepoVax™ or DPX™ is a water-free lipid-based drug that contains TLR-adjuvants and universal T helper peptides and can be formulated with any epitope or mixture of epitopes to induce cytotoxic T lymphocyte-mediated immune responses (Karkada et al., J Immunother 33(3):2050-261, 2010) and / or humoral immune responses. DPX™, which prolongs the exposure of antigens to the immune system, is removed from the injection site by phagocytic antigen-presenting cells.

[0252] It has been shown that a single injection of peptide in DPX™ results in an immune response equivalent to or superior to multiple injections of peptide in other conventional formulations such as Montanide ISA51VG emulsion, which is similar to VacciMax, a first generation emulsion-based T cell activation therapeutic composition platform (Daftarian et al., J Transl Med 5:26, 2007; Mansour et al., J Transl Med 5:20, 2007). A DPX™-based peptide-T cell activation therapeutic composition called DPX-0907 has completed Phase I clinical trials in breast, ovarian and prostate cancer patients and has demonstrated safety and immunogenicity in these ongoing patients (Berinstein et al., J Transl Med 10(1): 156, 2012).

[0253] Therefore, in certain embodiments, the carrier of the T cell activation therapeutic composition of the present invention may be a liposome-based auxiliary system.Unlike water-in-oil emulsion-based T cell activation therapeutics that rely on oil to entrap water droplets containing antigen and adjuvant, DepoVax™ / DPX™-based formulations rely on lipids and lipid mixtures to facilitate the incorporation of antigen and adjuvant directly into oil without the need for emulsification.The advantages of this approach include (1) enhancing the solubility of hydrophilic antigens / adjuvants in oil-based diluents that would otherwise normally have maximum solubility in hydrophilic-based diluents, and (2) eliminating cumbersome emulsification procedures before administration of the T cell activation therapeutic composition.

[0254] In a preferred embodiment, the carrier is mineral oil or a mannide oleate in mineral oil solution, such as that commercially available, for example Montanide® ISA51 (SEPPIC, France).

[0255] In certain embodiments, the composition may be substantially free of water (e.g., "water-free"). It is believed that the hydrophobic carriers of these "water-free" compositions may still contain small amounts of water, as long as water is present in the non-continuous phase of the carrier. For example, individual components of the composition may be bound with water that cannot be completely removed by processes such as lyophilization or evaporation, and certain hydrophobic carriers may contain small amounts of water dissolved therein. In general, the "water-free" compositions of the present invention contain less than about 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05% or less than 0.01% water, for example, based on the weight / weight of the total weight of the carrier components of the composition.

[0256] Active Agents and Additional Therapeutic Agents The method disclosed herein comprises administering a T cell activation therapeutic composition comprising at least one survivin antigen and at least one MAGE-A9 antigen to a subject with cancer. In certain embodiments, the invention further comprises administering at least one active agent. In certain embodiments, the invention further comprises administering an additional therapeutic agent. In certain embodiments, the active agent and the additional therapeutic agent are administered in the same regimen. In certain embodiments, the active agent and the additional therapeutic agent are administered in different regimens.

[0257] The active agents and / or additional therapeutic agents disclosed herein may be administered to a subject in a therapeutically effective amount. In certain embodiments, an effective amount of an active agent and / or additional therapeutic agent is an amount sufficient to provide an immune modulating effect.

[0258] "Active agent" or "additional therapeutic agent" as used herein refers to a pharmaceutical or therapeutic agent. The active agent and / or additional therapeutic agent may each individually be a small molecule drug, an antibody, an antibody mimetic, or a functional equivalent or functional fragment of any one of these.

[0259] In the methods disclosed herein, the amount of any particular active agent and / or additional therapeutic agent may depend on the type of agent, the disease or disorder being treated, and / or the particular characteristics of the subject (e.g., age, weight, sex, immune status, etc.) One of skill in the art can readily determine by empirical testing the amount of active agent and / or additional therapeutic agent required for a particular application.

[0260] In certain embodiments, the active agent and / or additional therapeutic agent is a small molecule drug. The term "small molecule drug" refers to an organic or inorganic compound that can be used to treat, cure, prevent, or diagnose a disease, disorder, or condition.

[0261] The term "small molecule" as used herein refers to a low molecular weight compound, which may be synthetically produced or obtained from natural sources, and has a molecular weight of less than 2000 Daltons (Da), less than 1500 Da, less than 1000 Da, less than 900 Da, less than 800 Da, less than 700 Da, less than 600 Da, or less than 500 Da. In one embodiment, the small molecule drug has a molecular weight of about 900 Da or less than 900 Da. More specifically, in one embodiment, the small molecule drug has a molecular weight of less than 600 Da, and even more specifically, less than 500 Da.

[0262] In one embodiment, the small molecule drug has a molecular weight between about 100 Da to about 2000 Da; about 100 Da to about 1500 Da; about 100 Da to about 1000 Da; about 100 Da to about 900 Da; about 100 Da to about 800 Da; about 100 Da to about 700 Da; about 100 Da to about 600 Da; or about 100 Da to about 500 Da. In one embodiment, the small molecule drug has a molecular weight of about 100 Da, about 150 Da, about 200 Da, about 250 Da, about 300 Da, about 350 Da, about 400 Da, about 450 Da, about 500 Da, about 550 Da, about 600 Da, about 650 Da, about 700 Da, about 750 Da, about 800 Da, about 850 Da, about 900 Da, about 950 Da, or about 1000 Da. In one embodiment, the small molecule drug may have a size of approximately 1 nm.

[0263] In one embodiment, a small molecule drug is a chemically manufactured active substance or compound (i.e., it is not produced by a biological process). Generally, these compounds are synthesized in a classical manner by chemical reactions between different organic and / or inorganic compounds. The term "small molecule drug" as used herein does not include larger structures, such as polynucleotides, proteins, and polysaccharides, that are produced by biological processes.

[0264] A small molecule drug can exert its activity in the form in which it is administered, or it can be a prodrug. In this regard, the term "small molecule drug" as used herein encompasses both the active form and the prodrug.

[0265] The term "prodrug" refers to a compound or substance that is converted into a therapeutically active drug under physiological conditions. In one embodiment, a prodrug is a compound or substance that is metabolized (e.g., by enzymatic activity in the subject's body) into a medicament that is medicamentally active after administration. A common method for making a prodrug is to include a selected component that is hydrolyzed under physiological conditions to produce a medicamentally active form.

[0266] In one embodiment, the active agent and / or additional therapeutic agent is an antibody, a functional equivalent of an antibody, or a functional fragment of an antibody.

[0267] "Antibody" broadly refers to a polypeptide or protein consisting of or comprising an antibody domain, understood as the constant and / or variable domain of an immunoglobulin heavy and / or light chain, with or without a linker sequence. In one embodiment, a polypeptide is understood as an antibody domain if it comprises a beta barrel sequence consisting of at least two beta strands of an antibody domain structure connected by a loop sequence. The antibody domain may be of native structure or may be modified by mutagenesis or derivatization, for example to modify binding specificity or any other property.

[0268] The term "antibody" refers to an intact antibody. In one embodiment, "antibody" may include complete (i.e., full-length) immunoglobulin molecules, such as polyclonal, monoclonal, chimeric, humanized and / or human versions with full-length heavy and / or light chains. The term "antibody" encompasses all manner of isotypes and subclasses, including, but not limited to, the major classes IgA, IgD, IgE, IgG and IgM, and subclasses IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In one embodiment, the antibody is an IgG. Antibodies may be naturally occurring or prepared by any means available to the skilled artisan, such as by using animals or hybridomas and / or by immunoglobulin gene fragment recombinatorial processes. Antibodies are reviewed, for example, in Greenfield, 2014.

[0269] In one embodiment, the antibody is in isolated form, meaning that the antibody is substantially free of other antibodies against different target antigens and / or contains a different structural arrangement of antibody domains. In one embodiment, the antibody may be an antibody isolated from a mammalian serum sample. In one embodiment, the antibody is in purified form, e.g., provided in the form of a preparation that contains only the isolated and purified antibody as an active agent. This preparation can be used in the preparation of the composition of the invention. In one embodiment, the antibody is an affinity purified antibody.

[0270] The antibody may be of any origin, including natural, recombinant and / or synthetic sources. In one embodiment, the antibody may be of animal origin. In one embodiment, the antibody may be of mammalian origin, including but not limited to human, mouse, rabbit and goat. In one embodiment, the antibody may be a recombinant antibody.

[0271] In one embodiment, the antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody or a fully human antibody. The meanings applied to these terms and the types of antibodies encompassed therein will be well understood by those of skill in the art.

[0272] Briefly, but without limitation, the term "chimeric antibody" as used herein refers to a recombinant protein that contains the variable domains (including the complementarity determining regions (CDRs)) of an antibody from one species, e.g., rodent, but the constant domains of the antibody are from a different species, e.g., human. For veterinary applications, the constant domains of the chimeric antibody may be from an animal, e.g., cat or dog.

[0273] Without limitation, a "humanized antibody," as used herein, refers to a recombinant protein in which the CDRs from an antibody of one species, e.g., a rodent, have been transferred from the variable heavy and light chains of the rodent antibody into human heavy and light chain variable domains containing human framework region (FR) sequences. The constant domains of a humanized antibody are likewise derived from a human antibody.

[0274] Without limitation, "human antibody" as used herein refers to an antibody obtained from a transgenic animal (e.g., a mouse) that has been genetically engineered to produce specific human antibodies in response to antigenic challenge. In this technology, elements of human heavy and light chain loci are introduced into a mouse strain derived from an embryonic stem cell line that contains targeted disruption of endogenous heavy and light chain loci. The transgenic animal is capable of synthesizing human antibodies specific to human antigens, and the animal can be used to produce hybridomas that secrete human antibodies. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green, 1994; Lonberg, 1994; and Taylor, 1994. Fully human antibodies may also be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. (See, e.g., McCafferty, 1990, for the production of human antibodies and fragments thereof in vitro from immunoglobulin variable domain gene repertoires from unimmunized donors). In this technique, genes for antibody variable domains are cloned in frame into either a major or minor coat protein gene of a filamentous bacteriophage and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of genes encoding antibodies exhibiting such properties. In this way, the phage mimics some of the properties of B cells. Phage display may also be performed, for a review see, e.g., Johnson and Chiswell, 1993. Human antibodies can also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0275] The term "functional fragment" as used herein with respect to an antibody refers to the antigen-binding site of the antibody. In this context, "functional" means that the fragment retains its ability to bind to the target antigen. In one embodiment, the binding affinity may be similar to or greater than that of the parent antibody. In one embodiment, the binding affinity may be less than that of the parent antibody, but the functional fragment nevertheless retains specificity and / or selectivity for the target antigen.

[0276] In one embodiment, in addition to a functional fragment maintaining its ability to bind to the target antigen of the parent antibody, a functional fragment also maintains, where appropriate, the effector functions of the antibody (e.g., activation of the classical complement pathway; antibody-dependent cellular cytotoxicity (ADCC); other downstream signaling processes).

[0277] Functional fragments of antibodies include, but are not limited to, portions of antibodies such as F(ab')2, F(ab)2, Fab', Fab, Fab2, Fab3, single domain antibodies (e.g., Dab or VHH), including half molecules of IgG4 (van der Neut Kolfschoten, 2007). Regardless of structure, functional fragments of antibodies bind to the same antigen recognized by the intact antibody. The term "functional fragment" also includes, with respect to antibodies, isolated fragments consisting of the variable regions, such as "Fv" fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which the light and heavy chain variable regions are connected by a peptide linker ("scFv protein"). The term "functional fragment" as used herein does not include fragments such as Fc fragments that do not contain an antigen binding site.

[0278] Antibody fragments, such as those described herein, may be incorporated into single domain antibodies (e.g., nanobodies), single chain antibodies, maxibodies, evibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, vNARs, bis-scFvs and other similar structures (see, e.g., Hollinger and Hudson, 2005). Antibody polypeptides, including fibronectin polypeptide monobodies, are also disclosed in U.S. Patent No. 6,703,199. Other antibody polypeptides are disclosed in U.S. Patent Publication No. 20050238646. Each reference cited herein is incorporated by reference in its entirety for all purposes.

[0279] Another form of a functional fragment is a peptide comprising one or more CDRs or one or more portions of a CDR of an antibody, provided that the resulting peptide retains the ability to bind to the target antigen.

[0280] Functional fragments may be synthetic or engineered proteins. For example, functional fragments include isolated fragments consisting of the light chain variable region, "Fv" fragments consisting of the heavy and light chain variable regions, and recombinant single chain polypeptide molecules in which the light and heavy chain regions are connected by a peptide linker (scFv protein).

[0281] The terms "antibody" and "functional fragment" of an antibody, as used herein, encompass any derivatives thereof. "Derivative" refers to any modification to an antibody or functional fragment, including both naturally occurring (e.g., in vivo) or artificially introduced (e.g., by experimental design). Non-limiting examples of such modifications include, for example, sequence modifications (e.g., amino acid substitutions, insertions or deletions), post-translational modifications (e.g., phosphorylation, N-linked glycosylation, O-linked glycosylation, acetylation, hydroxylation, methylation, ubiquitination, amidation, etc.), or any other covalent attachment or otherwise incorporation of a heterologous molecule (e.g., a polypeptide, a localization signal, a label, a targeting molecule, etc.). In one embodiment, the antibody or functional fragment thereof may be modified to generate a bispecific antibody or fragment (i.e., having binding specificity for more than one antigen) or a bivalent antibody or fragment (i.e., having more than one effector function).

[0282] "Functional equivalent," as used herein in the context of antibodies, refers to a polypeptide or other compound or molecule that has similar binding characteristics as an antibody for a particular target, but is not necessarily a recognizable "fragment" of an antibody. In one embodiment, a functional equivalent is one that binds to a particular target at least 10 -7 From 10 -12 The equilibrium dissociation constant (K D In one embodiment, a functional equivalent is a polypeptide having a specific target activity of 10 -8 or lower K D In one embodiment, the functional equivalent has a specific target binding domain of 10 -10 or lower K D In one embodiment, the functional equivalent has a specific target binding domain of 10 -11 or lower K D In one embodiment, the functional equivalent has a specific target binding domain of 10 -12 or lower KD The equilibrium constant (K D ) is the ratio of the dissociation rate (K-off) and the association rate (K-on) of a compound for its target.

[0283] In one embodiment, the antibody, functional fragment or functional equivalent thereof binds to a target on an immune cell, binds to a protein or polypeptide produced by an immune cell, or binds to a protein or polypeptide that interacts with or exerts a function (e.g., a ligand) on an immune cell.

[0284] In one embodiment, the antibody, functional fragment or functional equivalent thereof has an immunomodulatory activity or function. By "immunomodulatory activity or function" is meant that the antibody, functional fragment or functional equivalent thereof can enhance (upregulate), suppress (downregulate), direct, redirect or reprogram the immune response.

[0285] In one embodiment, the antibody, functional fragment or functional equivalent thereof binds to a stimulatory and / or inhibitory checkpoint molecule, such as, but not limited to, those described herein. In one embodiment, the antibody, functional fragment or functional equivalent thereof is an agonist or antagonist of the stimulatory and / or inhibitory checkpoint molecule. In one embodiment, the antibody, functional fragment or functional equivalent thereof is an antagonist of the inhibitory checkpoint molecule. In one embodiment, the antibody, functional fragment or functional equivalent thereof is an agonist or superagonist of the stimulatory checkpoint molecule.

[0286] In one embodiment, the active agent is an antibody mimetic, a functional equivalent of an antibody mimetic, or a functional fragment of an antibody mimetic.

[0287] The term "antibody mimic" as used herein refers to a compound that can specifically and / or selectively bind to an antigen or other target like an antibody, but is not structurally related to an antibody. Antibody mimics are typically artificial peptides or proteins, but are not limited to such embodiments. Typically, antibody mimics are smaller than antibodies, having a molar mass of about 3-20 kDa (whereas antibodies are generally about 150 kDa). Non-limiting examples of antibody mimics include peptide aptamers, affimers, affilins, affibodies, affitins, alphabodies, anticalins, avimers, DARPins™, fynomers, Kunitz domain peptides, nanoCLAMPs™, affinity reagents, and scaffold proteins. Nucleic acids and small molecules can also be antibody mimics.

[0288] The term "peptide aptamer", as used herein, refers to peptides or proteins that are designed to interfere with the interactions of other proteins inside the cell. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of peptide aptamers to levels comparable to that of antibodies (nanomolar range). The variable peptide loop typically contains 10 to 20 amino acids, and the scaffold can be any protein with good solubility properties. Currently, the bacterial protein thioredoxin-A is a commonly used scaffold protein, in which the variable peptide loop is inserted into the redox active site, which in the wild-type protein is a -Cys-Gly-Pro-Cys-loop (SEQ ID NO: 25), and the two cysteine ​​side chains can form a disulfide bridge. The selection of peptide aptamers can be made using a variety of systems, but currently the most widely used is the yeast two-hybrid system.

[0289] The term "affimer" as used herein refers to an evolved form of peptide aptamers. Affimers are small, highly stable proteins engineered to present peptide loops that provide a high affinity binding surface for a specific target protein or antigen. Affimers may have the same specificity advantages of antibodies, but are smaller, can be chemically synthesized or chemically modified, and are free of cell culture contaminants. Affimers are low molecular weight, typically 12 to 14 kDa proteins from the cystatin cysteine ​​protease inhibitor family. The affimer scaffold is a stable protein based on the cystatin protein fold. It presents two peptide loops and an N-terminal sequence that may be randomized to bind different target proteins with high affinity and specificity.

[0290] The term "affilin" as used herein refers to an antibody mimic developed by using either gamma B crystallin or ubiquitin as a scaffold and modifying the amino acids on the surface of these proteins by random mutagenesis. Selection of affilins with desired target specificity is performed, for example, by phage display or ribosome display technology. Depending on the scaffold, affilins have a molecular weight of approximately 10 kDa (ubiquitin) or 20 kDa (gamma B crystallin). The term affilin as used herein also refers to dimerized or multimerized forms of affilin (Weidle, 2013).

[0291] The term "affibody" as used herein refers to a family of antibody mimics derived from the Z domain of Staphylococcus aureus protein A. Structurally, affibody molecules are based on a three-helix bundle domain, which may be incorporated into a fusion protein. The affibody itself has a molecular weight of about 6 kDa and is stable at high temperature and under acidic or alkaline conditions. Target specificity is obtained by randomization of 13 amino acids located in two alpha helices involved in the binding activity of the parent protein domain (Feldwisch and Tolmachev, 2012, which is incorporated herein by reference in its entirety for all intended purposes). In one embodiment, it is an Affibody™ supplied by Affibody AB, Stockholm, Sweden.

[0292] "Affitins" (also known as nanofitins) are antibody mimetic proteins derived from the DNA-binding protein Sac7d of Sulfolobus acidocaldarius. Affitins typically have a molecular weight of about 7 kDa and are designed to specifically bind target molecules by randomizing amino acids on the binding surface (Mouratou, 2012). In one embodiment, the affitin is as described in WO2012 / 085861, which is incorporated by reference in its entirety for all intended purposes.

[0293] The term "alphabody" as used herein refers to a small 10 kDa protein engineered to bind various antigens. Alphabodies were developed as scaffolds with a series of amino acid residues that can be modified to bind protein targets while maintaining correct folding and thermal stability. Alphabody scaffolds are computationally designed based on a coiled-coil structure, but have no known counterpart in nature. Initially, the scaffold was made of three peptides non-covalently associated to form a parallel coiled-coil trimer (US Patent Publication No. 20100305304), but was later redesigned as a single peptide chain containing three alpha helices connected by a linker region (Desmet, 2014).

[0294] The term "anticalin" as used herein refers to engineered proteins derived from lipocalins (Beste, 1999; Gebauer and Skerra, 2009). Anticalins have an eight-stranded β-barrel that forms a highly conserved core unit among lipocalins and naturally forms a binding site for ligands by four structurally variable loops at the open end. Although anticalins are not homologous to the IgG superfamily, they exhibit features that have been considered typical of antibody binding sites: (i) high structural plasticity as a result of sequence variation, and (ii) enhanced conformational flexibility leading to induction of adaptation to targets with different shapes.

[0295] The term "avimer" (avimolecular multimer), as used herein, refers to a class of antibody mimics consisting of two or more peptide sequences of 30 to 35 amino acids each, derived from the A-domains of various membrane receptors and connected by a linker peptide. Binding of the target molecule occurs via the A-domains, and domains with the desired binding specificity can be selected, for example, by phage display technology. The binding specificities of the different A-domains contained in an avimer may be identical, but are not necessarily so (Weidle, 2013).

[0296] The term "DARPin™" as used herein refers to a designed ankyrin repeat domain (166 residues) that provides a rigid boundary that typically results from three repeated β-turns. DARPins usually have three repeats that correspond to an artificial consensus sequence, with six positions per repeat randomized. As a result, DARPins lack structural flexibility (Gebauer and Skerra, 2009).

[0297] The term "Fynomer™" as used herein refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain. Fyn SH3-derived polypeptides are well known in the art and are described, for example, in Grabulovski, 2007; WO2008 / 022759; Bertschinger, 2007; Gebauer and Skerra, 2009; and Schlatter, 2012).

[0298] A "Kunitz domain peptide" is derived from the Kunitz domain of a Kunitz-type protease inhibitor, such as the Kunitz domain of bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI). Kunitz domains have a molecular weight of approximately 6 kDA, and domains with the required target specificity can be selected by display techniques, such as phage display (Weidle, 2013).

[0299] The term "monobody" (also referred to as "adnectin"), as used herein, refers to a molecule based on the tenth extracellular domain of human fibronectin III (10Fn3), which adopts a 94-residue Ig-like β-sandwich fold with two to three exposed loops but lacking a central disulfide bridge (Gebauer and Skerra, 2009). Monobodies with desired target specificity may be engineered by introducing modifications into specific loops of the protein. In one embodiment, the monobody is ADNECTIN™ (Bristol-Myers Squibb, New York, New York).

[0300] The term "nanoCLAMP" (CLostridal Antibody Mimetic Proteins), as used herein, refers to affinity reagents that are 15 kDa proteins that bind tightly, selectively, gently and reversibly to target molecules. The nanoCLAMP scaffold is based on the IgG-like thermostable carbohydrate-binding module family 32 (CBM32) from Clostridium perfringens hyaluronidase (mu toxin). The shape of nanoCLAMPs approximates a cylinder with a length of approximately 4 nm and a diameter of 2.5 nm, roughly the same size as nanobodies. NanoCLAMPs for specific targets are generated by varying the amino acid sequence and sometimes the length of three solvent-exposed adjacent loops that connect the beta strands that make up the beta-sandwich fold to confer binding affinity and target specificity (Suderman, 2017).

[0301] The term "affinity reagent" as used herein refers to any compound or substance that binds to a larger target molecule to identify, track, capture, or affect its activity. Many different types of affinity reagents are available to those skilled in the art, although antibodies and peptide aptamers are common examples. In one embodiment, the affinity reagent provides a potential scaffold that can be engineered to specifically bind to a target (e.g., Top7 is a scaffold engineered to specifically bind CD4; Boschek, 2009).

[0302] The term "scaffolding proteins" as used herein refers to polypeptides or proteins that interact with and / or bind to multiple members of a signaling pathway. They are regulators of many major signaling pathways. In such pathways, they regulate signal transduction and localize pathway components. Herein, they are encompassed by the term "antibody mimics" due to their ability to specifically and / or selectively bind target proteins similar to antibodies. Scaffolding proteins may also have enzymatic activity in addition to their binding function and specificity. Exemplary scaffolding proteins include, but are not limited to, kinase suppressor of Ras1 (KNS), MEK kinase 1 (MEKK1), B-cell lymphoma / leukemia 10 (BCL-10), A kinase anchoring protein (AKAP), neuroblast differentiation associated protein AHNAK, HOMER1, pellino protein, NLRP family, disc large homolog 1 (DLG1) and spinophillin (PPP1R9B).

[0303] Other embodiments of antibody mimetics include, but are not limited to, the Z domain of protein A, gamma B crystallin, ubiquitin, cystatin, Sac7D from Sulfolobus acidocaldarius, lipocalin, a domain of a membrane receptor, ankyrin repeat motif, the SH3 domain of Fyn, the Kunitz domain of a protease inhibitor, the 10th type III domain of fibronectin, a three or four helix bundle protein, an armadillo repeat domain, a leucine-rich repeat domain, a PDZ domain, a SUMO or SUMO-like domain, an immunoglobulin-like domain, a phosphotyrosine binding domain, a pleckstrin homology domain, or a src homology 2 domain.

[0304] The term "functional fragment" as used herein, in reference to an antibody mimic, refers to any portion or fragment of an antibody mimic that maintains its ability to bind to a target molecule. A functional fragment of an antibody mimic may be, for example, any portion of the antibody mimics described herein. In one embodiment, the binding affinity may be equal to or greater than the binding affinity of the parent antibody mimic. In one embodiment, the binding affinity may be lower than the parent antibody mimic, yet the functional fragment maintains specificity and / or selectivity to the target antigen.

[0305] In one embodiment, in addition to a functional fragment of an antibody mimetic that maintains its ability to bind to a target molecule of the parent antibody mimetic, the functional fragment also maintains, where appropriate, an effector function (e.g., downstream signaling) of the antibody mimetic.

[0306] "Functional equivalent," as used herein, in the context of an antibody mimetic, refers to a polypeptide or other compound or molecule that has similar binding characteristics to the antibody mimetic, but is not necessarily a recognizable "fragment" of the antibody mimetic. In one embodiment, a functional equivalent is one that binds to a particular target at least 10 -7 From 10 -12The equilibrium dissociation constant (K D In one embodiment, a functional equivalent is a polypeptide having a specific target activity of 10 -8 or lower K D In one embodiment, the functional equivalent has a specific target binding domain of 10 -10 or lower K D In one embodiment, the functional equivalent has a specific target binding domain of 10 -11 or lower K D In one embodiment, the functional equivalent has a specific target binding domain of 10 -12 or lower K D The equilibrium constant (K D ) is the ratio of the dissociation rate (K-off) and the association rate (K-on) of a compound for its target.

[0307] In one embodiment, the antibody mimetic, functional fragment thereof or functional equivalent thereof, binds to a target on an immune cell, binds to a protein or polypeptide produced by an immune cell, or binds to a protein or polypeptide that interacts with or exerts a function (e.g., a ligand) on an immune cell.

[0308] Non-limiting examples In one embodiment, without limitation, the small molecule drug is a cytotoxic agent, an anti-cancer agent, an anti-tumor agent, a chemotherapeutic agent, an anti-neoplastic agent, an immunomodulatory agent (e.g., an immune enhancer), an immune response checkpoint inhibitor, an anti-angiogenic agent, an anti-osteoclastogenic, an enzyme modulator, a biological response modifier, a prodrug, a cytokine, a chemokine, a vitamin, a steroid, a ligand, a targeting agent, a radiopharmaceutical, or a radioisotope.

[0309] Small molecule drugs as used herein may be their pharmaceutically acceptable salts.The term "pharmaceutically acceptable salts" as used herein refers to any salt form of the active agent and / or immunomodulator described herein that is safe and effective for administration to the intended subject and has the desired biological, pharmaceutical and / or therapeutic activity.Pharmaceutically acceptable salts include salts of acidic or basic groups. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. A review of pharma- ceutically acceptable salts can be found, for example, in Berge, 1977, which is incorporated herein by reference in its entirety for all intended purposes.

[0310] In one embodiment, the small molecule drug is an agent that interferes with DNA replication. The expression "interferes with DNA replication" as used herein is intended to encompass any action that prevents, inhibits, or slows the biological process of copying (i.e., replicating) the DNA of a cell. Those skilled in the art will understand that there are various mechanisms that prevent, inhibit, or slow DNA replication, such as DNA cross-linking, DNA methylation, base substitution, and the like. The disclosure of the present invention encompasses the use of any agent that interferes with DNA replication. Exemplary, non-limiting embodiments of such agents that can be used are described, for example, in WO2014 / 153636 and WO2017 / 190242, each of which is incorporated herein in its entirety for all purposes. In one embodiment, the agent that interferes with DNA replication is an alkylating agent, such as a nitrogen mustard alkylating agent (e.g., cyclophosphamide, bendamustine, chlorambucil, ifosfamide, mechlorethamine, melphalan), a nitrosourea alkylating agent (e.g., carmustine, lomustine, streptozocin), a sulfonate alkylating agent (e.g., busulfan), a triazine alkylating agent (e.g., dacarbazine, temozolomide), or an ethylenimine alkylating agent (e.g., altretamine, thiotepa). In a particular embodiment, the agent that interferes with DNA replication is cyclophosphamide.

[0311] In one embodiment, the small molecule drug is cyclophosphamide or a pharma- ceutically acceptable salt thereof. Cyclophosphamide (N,N-bis(2-chloroethyl)-1,3,2-oxazaphosphinan-2-amine 2-oxide). The chemical structure of cyclophosphamide is:

[0312] [ka] It is.

[0313] Cyclophosphamide is also known and referred to under the trademarks Endoxan®, Cytoxan®, Neosar®, Procytox®, and Revimmune®. Cyclophosphamide (CPA) is a prodrug that is converted to its active metabolites, 4-hydroxy-cyclophosphamide and aldophosphamide, by oxidation by P450 enzymes. Intracellular 4-hydroxy-cyclophosphamide spontaneously degrades to the ultimate active metabolite, phosphoramide mustard.

[0314] The active metabolites of CPA are lipid soluble and enter cells via passive diffusion. Intracellular 4-OH-CPA spontaneously degrades to the final active metabolite, phosphoramide mustard, which catalyzes intrastrand and interstrand DNA crosslinks as well as DNA-protein crosslinks that inhibit DNA replication and cause cell death (de Jonge, Huitema et al. 2005). Phosphoramide mustard is eliminated by enzymatic conversion to carboxyphoshphamide by cytoplasmic aldehyde dehydrogenase (ALDH) (Emmenegger, Shaked et al., 2007; 2011). Cells with low levels of ALDH tend to accumulate CPA metabolites and are more sensitive to its effects, and indeed tumor upregulation of ALDH is one mechanism of CPA resistance (Zhang, Tian et al. 2005). Besides ALDH, low intracellular ATP levels also correlated with the selectivity of CPA for certain cell types (Zhao, Cao et al. 2010). At high doses, typically in the range of 1-5 g / im2, CPA action is most cytotoxic against rapidly dividing cells, regardless of cell type, and CPA is myelosuppressive since most hematopoietic cells divide rapidly (Bruce, Meeker et al. 1966; Smith and Sladek 1985).

[0315] Other nitrogen mustard alkylating agents in the same class as cyclophosphamide include, but are not limited to, palifosfamide, bendamustine, and ifosfamide.

[0316] In one embodiment, the small molecule drug is selected from the group consisting of, but not limited to, gemcitabine, 5-fluorouracil, cisplatin, oxaliplatin, temozolomide, paclitaxel, thalidomide, capecitabine, methotrexate, epirubicin, idarubicin, mitoxantrone, bleomycin, bortezomib, decitabine, docetaxel, ifosfamide, afosfamide, melphalan, bendamustine, uramustine, palifosfamide, chlorambucil, It may be cil, busulfan, 4-hydroxycyclophosphamide, bis-chloroethylnitrosourea (BCNU), mitomycin C, yondelis, procarbazine, dacarbazine, carboplatin, acyclovir, cytosine arabinoside, ganciclovir, camptothecin, topotecan, irinotecan, doxorubicin, daunorubicin, etoposide, teniposide, or pixantrone, or a pharma- ceutically acceptable salt of any one of them.

[0317] In one embodiment, the small molecule drug may be cyclophosphamide, gemcitabine, 5-fluorouracil, cisplatin, oxaliplatin, temozolomide, paclitaxel, thalidomide, capecitabine, methotrexate, epirubicin, idarubicin, mitoxantrone, bleomycin, bortezomib, decitabine, or docetaxel.

[0318] In one embodiment, the active agent or additional therapeutic agent may be an immune response checkpoint inhibitor. "Immune response checkpoint inhibitor" as used herein refers to any compound or molecule that fully or partially modulates (e.g., inhibits or activates) the activity or function of one or more checkpoint molecules (e.g., proteins). Checkpoint molecules are involved in the costimulatory or inhibitory interactions of T cell responses. Checkpoint molecules regulate and maintain the self-tolerance and duration and breadth of physiological immune responses. In general, there are two types of checkpoint molecules: stimulatory checkpoint molecules and inhibitory checkpoint molecules.

[0319] Stimulatory checkpoint molecules play a role in strengthening immune response. Numerous stimulatory checkpoint molecules are known, including but not limited to CD27, CD28, CD40, CD122, CD137, CD137 / 4-1BB, ICOS, IL-10, OX40 TGF-beta, TOR receptor, and glucocorticoid-induced TNFR-related protein GITR. In one embodiment, checkpoint molecule is an agonist or superagonist of one or more stimulatory checkpoint molecules. Those skilled in the art will be familiar with the checkpoint molecules that can be used to modulate stimulatory checkpoint molecules.

[0320] Inhibitory checkpoint molecules play a role in reducing or blocking immune responses (e.g., negative feedback loops). Numerous inhibitory checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2. Other inhibitory checkpoint molecules include, but are not limited to, adenosine A2A receptor (A2AR); B7-H3 (CD276); B7-H4 (VTCN1); BTLA (CD272); killer cell immunoglobulin-like receptor (KIR); lymphocyte activation gene-3 (LAG3); V-domain Ig suppressor of T cell activation (VISTA); and T cell immunoglobulin domain and mucin domain 3 (TIM-3); as well as their ligands and / or receptors. In one embodiment, the checkpoint molecule is an antagonist (i.e., an inhibitor) of one or more inhibitory checkpoint molecules. Those of skill in the art will be familiar with checkpoint molecules that can be used to modulate inhibitory checkpoint molecules.

[0321] In one embodiment, the checkpoint molecule is an immune response checkpoint inhibitor, which may be any of the following: programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1, CD279), CTLA-4 (CD154), PD-L2 (B7-DC, CD273), LAG3 (CD223), TIM3 (HAVCR2, CD366), 41BB (CD137), 2B4, A2aR, B7H1, B7H3, B7H4, B and T lymphocyte attenuator (BTLA), CD2, CD27, CD28, CD30, CD3 3, an inhibitor of CD40, CD70, CD80, CD86, CD160, CD226, CD276, DR3, GAL9, GITR, HVEM, ICOS (inducible T cell costimulatory molecule), killer inhibitory receptor (KIR), LAG-3, LAIR1, LIGHT, MARCO (macrophage receptor with collagen-like structure), phosphatidylserine (PS), OX-40, Siglec-5, Siglec-7, Siglec-9, Siglec-11, SLAM, TIGIT, TIM3, TNF-α, VISTA, VTCN1, or any combination thereof.

[0322] In one embodiment, the checkpoint molecule is an immune response checkpoint agent that is an inhibitor of PD-L1, PD-1, CTLA-4, LAG3, TIM3, 41BB, ICOS, KIR, CD27, OX-40, GITR, or PS, or any combination thereof.

[0323] In one embodiment, the checkpoint molecule may be one or more inhibitors of indoleamine 2,3-dioxygenase enzymes (e.g., IDO1 and / or IDO2). In certain embodiments, the indoleamine 2,3-dioxygenase inhibitor is epacadostat.

[0324] In one embodiment, the checkpoint molecule may be epacadostat, rapamycin, doxorubicin, valproic acid, mitoxantrone, vorinostat, irinotecan, cisplatin, methotrexate, tacrolimus, or a pharma- ceutically acceptable salt of any one of them.

[0325] In one embodiment, the checkpoint molecule is epacadostat:

[0326] [ka] or a pharma- ceutically acceptable salt thereof.

[0327] Those skilled in the art will be familiar with other active agents and additional therapeutic agents that can be used in the practice of the present invention. An example includes, but is not limited to, DrugBank™ (Wishart, 2017). Version 5.0.11 of DrugBank™, released on December 20, 2017, contains 10,990 drug entries, including over 2,500 approved active agents and additional therapeutic agents, which are incorporated herein by reference in their entirety for all purposes. Another example includes, but is not limited to, the A-Z list of cancer drugs provided by the National Cancer Institute (www.cancer.gov / about-cancer / treatment / drugs), which are incorporated herein by reference in their entirety for all purposes.

[0328] In one embodiment, the active agent and / or additional therapeutic agent is a cancer drug approved by the Food and Drug Administration (FDA) for bladder cancer. In one embodiment, the cancer drug includes, but is not limited to, atezolizumab, avelumab, Balversa® (erdafitinib), Bavencio® (avelumab), cisplatin, doxorubicin hydrochloride, enfortumab vedotin-ejfv, erdafitinib, Jelmyto® (mitomycin), Keytruda® (pembrolizumab), mitomycin, nivolumab, Opdivo® (nivolumab), cisplatin, doxorubicin hydrochloride, enfortumab vedotin-ejfv, erdafitinib, Jelmyto® (mitomycin), Keytruda® (mitomycin), nivolumab, Opdivo® (mitomycin), cisplatin, doxorubicin hydrochloride ... volumab), Padcev® (enfortumab vedotin-ejfv), pembrolizumab, sacituzumab, govitecan-hziy, Tecentriq® (atezolizumab), Tepadina® (thiotepa), thiotepa, Trodelvy® (sacituzumab govitecan-hziy), valrubicin, Valstar® (valrubicin), or a pharma- ceutical acceptable salt of any one of them.

[0329] In one embodiment, the active agent and / or additional therapeutic agent may be an antibody drug conjugate (ADC). An ADC is an antibody chemically linked to a drug, such as, but not limited to, a therapeutic compound or a cytotoxic agent. In one embodiment, the ADC may be, but is not limited to, sacituzumab govitecan, enfortumab vedotin, ASG-15ME, oportuzumab monatox (VB4-845), or a pharma- ceutically acceptable salt of any one thereof.

[0330] In one embodiment, the antibody may be an anti-PD-1 antibody, a functional fragment thereof or a functional equivalent thereof, or any combination thereof. PD-1 (CD279) is a cell surface receptor that functions as an immune checkpoint to downregulate immune responses and promote self-tolerance. In one embodiment, the PD-1 antibody may be, but is not limited to, nivolumab (Opdivo™; Bristol-Myers Squibb), pembrolizumab (Keytruda™; Merck), pidilizumab (Cure Tech), AMP-224 (MedImmune & GSK), or RMP1-4 or J43 (BioXCell) or their human or humanized counterparts. In certain embodiments, the PD-1 antibody may be pembrolizumab.

[0331] In one embodiment, the antibody may be an anti-PD-L1 antibody, a functional fragment or functional equivalent thereof, or any combination thereof. PD-L1 is a ligand for the PD-1 receptor, and binding to the receptor can transmit inhibitory signals that reduce proliferation of CD8+ T cells and can also induce apoptosis. In one embodiment, the PD-L1 antibody may be, but is not limited to, BMS-936559 (Bristol Myers Squibb), atezolizumab (MPDL3280A; Roche), avelumab (Merck & Pfizer), or durvalumab (MEDI4736; MedImmune / AstraZeneca).

[0332] In other embodiments, without limitation, the antibody, functional fragment or functional equivalent thereof may be an anti-PD-1 or anti-PD-L1 antibody, such as those disclosed in WO2015 / 103602, which is incorporated by reference in its entirety for all intended purposes.

[0333] In one embodiment, the antibody may be an anti-CTLA-4 antibody, its functional fragment or its functional equivalent, or any combination thereof. CTLA-4 (CD152) is a protein receptor that functions as an immune checkpoint to downregulate immune responses. In one embodiment, the anti-CTLA-4 antibody inhibits CTLA-4 activity or function, thereby enhancing immune responses. In one embodiment, the anti-CTLA-4 antibody may be, but is not limited to, ipilimumab (Bristol-Myers Squibb), tremelimumab (Pfizer; AstraZeneca) or BN-13 (BioXCell). In another embodiment, the anti-CTLA-4 antibody may be UC10-4F10-11, 9D9 or 9H10 (BioXCell), or their human or humanized counterparts.

[0334] In one embodiment, the antibody mimic, its functional fragment or functional equivalent has an immunomodulatory activity or function. In one embodiment, the antibody mimic, its functional fragment or functional equivalent binds to a stimulatory checkpoint molecule and / or an inhibitory checkpoint molecule, such as, but not limited to, those described herein. In one embodiment, the antibody mimic, its functional fragment or functional equivalent is an agonist or antagonist of a stimulatory checkpoint molecule and / or an inhibitory checkpoint molecule. In one embodiment, the antibody mimic, its functional fragment or functional equivalent is an antagonist of an inhibitory checkpoint molecule (e.g., CTLA-4, PD-1 or PD-L1). In one embodiment, the antibody mimic, its functional fragment or functional equivalent is an agonist or superagonist of a stimulatory checkpoint molecule.

[0335] The amount of any particular active agent described herein may depend on the type of agent (e.g., small molecule drug, antibody, functional fragment, etc.) One of ordinary skill in the art can readily determine by empirical testing the amount of active agent required for a particular application.

[0336] Immunomodulators In certain embodiments, the active agent and / or additional therapeutic agent is an immunomodulator. An "immunomodulator" as used herein is a compound or molecule that modulates the activity and / or effectiveness of immune response. "Modulate" as used herein means to enhance (upregulate), direct, re-direct, or reprogram immune response. The term "modulate" is not intended to mean activation or induction. This means that an immunomodulator modulates (enhances or directs) the immune response that is activated, initiated, or induced by a particular substance (e.g., an antigen), but the immunomodulator is not itself a substance that directs an immune response against it, nor is it an immunomodulator derived from that substance.

[0337] In one embodiment, an immunomodulatory agent modulates myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) or lymphoid cells (T cells, B cells, and natural killer (NK) cells). In a specific embodiment, an immunomodulatory agent modulates only lymphoid cells. In one embodiment, an immunomodulatory agent is a therapeutic agent that, when administered, stimulates immune cells to proliferate or become activated.

[0338] In one embodiment, the immunomodulatory agent enhances the immune response. The immune response may be one that has been previously activated or initiated, but with insufficient efficacy to provide an adequate or desired therapeutic benefit. Alternatively, the immunomodulatory agent may be provided in advance to prime the immune system, thereby enhancing the subsequent activated immune response.

[0339] In one embodiment, immunomodulatory agents that enhance the immune response can be selected from cytokines (e.g., certain interleukins and interferons), stem cell growth factors, lymphotoxins, costimulatory molecules, hematopoietic factors, colony stimulating factors, erythropoietin, thrombopoietin, and the like, and synthetic analogs of these molecules.

[0340] In one embodiment, immunomodulators that enhance the immune response may be selected from the following non-limiting examples: lymphotoxins, such as tumor necrosis factor (TNF); hematopoietic factors, such as interleukins (IL); colony-stimulating factors, such as granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF); interferons, such as interferon alpha, beta, or lambda; and stem cell growth factors, such as those termed "SI factors."

[0341] Among cytokines, growth hormones, including but not limited to human growth hormone, human N-methionyl growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, including but not limited to follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factors; prostaglandins, fibroblast growth factors; prolactin; placental lactogen, OB protein; tumor necrosis factors alpha and beta; Mullerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor (VEGF); integrins; thrombopoietin (TPO); nerve growth factors, including but not limited to NGF-beta; platelet growth factor; transforming growth factors. (TGF), such as, but not limited to, TGF alpha and TGFP; insulin-like growth factor I and II; erythropoietin (EPO); bone morphogenetic factor; interferons, such as, but not limited to, interferon alpha, beta, and gamma; colony stimulating factors (CSF), such as, but not limited to, macrophage-CSF (M-CSF); interleukins (IL), such as, but not limited to, IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, and tumor necrosis factor.

[0342] In one embodiment, the immunomodulatory agent may be an agent that modulates a checkpoint molecule. Checkpoint molecules are discussed in more detail above.

[0343] In one embodiment, an immunomodulatory agent is any compound, molecule, or substance that is an immune checkpoint inhibitor, examples of which include, but are not limited to, programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1, CD279), CTLA-4 (CD154), PD-L2 (B7-DC, CD273), LAG3 (CD223), TIM3 (HAVCR2, CD366), 41BB (CD137), 2B4, A2aR, B7H1, B7H3, B7H4, B and T lymphocyte attenuator (BTLA), CD2, CD27, CD28, CD30, CD40, CD50, CD60, CD80, CD90, CD100, CD110, CD120, CD135, CD140, CD150, CD160, CD170, CD180, CD190, CD290, CD281, CD291, CD292, CD293, CD294, CD295, CD296, CD297, CD298, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD308, CD309 ... and inhibitors of immune checkpoint proteins selected from D33, CD40, CD70, CD80, CD86, CD160, CD226, CD276, DR3, GAL9, GITR, HVEM, ICOS (inducible T cell costimulatory molecule), killer inhibitory receptor (KIR), LAG-3, LAIR1, LIGHT, MARCO (macrophage receptor with collagen-like structure), phosphatidylserine (PS), OX-40, Siglec-5, Siglec-7, Siglec-9, Siglec-11, SLAM, TIGIT, TIM3, TNF-α, VISTA, VTCN1, or any combination thereof.

[0344] In one embodiment, the immunomodulatory agent is any compound, molecule, or substance that inhibits or blocks CTLA-4. CTLA-4 signaling inhibits T cell activation, particularly during strong T cell responses. Blocking CTLA-4 using CTLA-4 inhibitors, such as anti-CTLA-4 monoclonal antibodies, is highly attractive because suppression of inhibitory signals leads to the generation of anti-tumor T cell responses. Both clinical and preclinical data show that blocking CTLA-4 leads to the direct activation of CD4+ and CD8+ effector cells, and anti-CTLA-4 monoclonal antibody therapy shows promise in many cancers.

[0345] In one embodiment, the immunomodulator is any compound, molecule, or substance that inhibits or blocks PD-1. Like CTLA-4 signaling, PD-1 / PD-L1 modulates T cell responses. The normal function of PD-1, expressed on the cell surface of activated T cells in healthy conditions, down-modulates unwanted or excessive immune responses, including autoimmune responses. The PD-1 pathway represents a major immune control switch that can be engineered into tumor cells to overcome immune surveillance of active T cells, which is usually hijacked by tumors to suppress immune control. Tregs expressing PD-1 have been shown to have immune inhibitory responses, and therefore PD-1 / PD-L1 expression is believed to play a role in self-tolerance. In the context of cancer, tumor cells overexpress PD-1 and PD-L1 to escape recognition by the immune system. Anti-cancer therapies that block PD-L1 / PD-1 increase effector T cell activity and decrease suppressive Treg activity, thereby enabling tumor recognition and destruction by an individual's immune system.

[0346] A variety of checkpoint inhibitors can be used. For example, the checkpoint inhibitor can be an antibody that binds to and antagonizes an inhibitory checkpoint protein. Exemplary antibodies include anti-PD-1 antibodies (pembrolizumab, nivolumab, pidilizumab, AMP-224, RMP1-4 or J43), anti-PD-L1 antibodies (atezolizumab, avelumab, BMS-936559 or durvalumab), anti-CTLA-4 antibodies (ipilimumab, tremelimumab, BN-13, UC10-4F10-11, 9D9 or 9H10), and the like. In some embodiments, the checkpoint inhibitor can be a small molecule or RNAi that targets an inhibitory checkpoint protein. In some embodiments, the checkpoint inhibitor can be a peptidomimetic or a polypeptide.

[0347] In one embodiment, the immunomodulatory agent may be an immune co-stimulatory molecule agonist.Immune co-stimulatory molecules are signaling proteins that play a role in regulating immune response.Some immune co-stimulatory molecules are receptors located on the surface of cells that respond to extracellular signaling.When activated, immune co-stimulatory molecules produce pro-inflammatory responses that may include the suppression of regulatory T cells and the activation of cytotoxic or killer T cells.Therefore, immune co-stimulatory molecule agonists can be used to activate the immune system in individuals to kill cancer cells.

[0348] Exemplary immune co-stimulatory molecules include CD27, CD28, CD40, CD122, CD137, CD137 / 4-1BB, ICOS, IL-10, OX40 TGF-beta, TOR receptor, and glucocorticoid-induced TNFR-related protein GITR. For example, OX40 stimulation suppresses the function of Treg cells while enhancing the survival and activity of effector T cells, thereby increasing anti-tumor immunity.

[0349] In one embodiment, an immunomodulatory agent is any compound, molecule or substance that is an agonist of a costimulatory immune molecule, including but not limited to, a costimulatory immune molecule selected from CD27, CD28, CD40, CD122, CD137, CD137 / 4-1BB, ICOS, IL-10, OX40, TGF-beta, TOR receptor, and glucocorticoid-inducible TNFR-related protein GITR.

[0350] A variety of immune costimulatory molecule agonists can be used. For example, the immune costimulatory molecule agonist can be an antibody that binds to and activates the immune costimulatory molecule. In a further embodiment, the immune costimulatory molecule agonist can be a small molecule that targets and activates the immune costimulatory molecule.

[0351] In one embodiment, the immunomodulatory agent may be any compound, molecule or substance that is an immunosuppressive cytotoxic agent, hi one embodiment, the immunosuppressive cytotoxic agent is a glucocorticoid, a cytostatic agent (e.g., an alkylating agent, an antimetabolite), an antibody, a drug acting on an immunophilin, an interferon, an opioid, or a TNF binding protein. Immunosuppressive cytotoxic drugs include, but are not limited to, nitrogen mustards (e.g., cyclophosphamide), nitrosoureas, platinum compounds, folic acid analogs (e.g., methotrexate), purine analogs (e.g., azathioprine and mercaptopurine), pyrimidine analogs (e.g., fluorouracil), protein synthesis inhibitors, cytotoxic antibiotics (e.g., dactinomycin, anthracyclines, mitomycin C, bleomycin, and mithramycin), cyclosporine, tacrolimus, sirolimus / rapamycin, everolimus, prednisone, dexamethasone, hydrocortisone, mechlorethamine, chlorambucil, mycopholic acid, fingolimod, myriocin, infliximab, etanercept, or adalimumab.

[0352] In one embodiment, the immunomodulatory agent may be an anti-inflammatory agent. In one embodiment, the anti-inflammatory agent may be a nonsteroidal anti-inflammatory agent. In one embodiment, the nonsteroidal anti-inflammatory agent may be a Cox-1 and / or Cox-2 inhibitor. In one embodiment, the anti-inflammatory agent may include, but is not limited to, aspirin, salsalate, diflunisal, ibuprofen, fenoprofen, flubiprofen, fenamate, ketoprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, or celecoxib. In one embodiment, the anti-inflammatory agent may be a steroidal anti-inflammatory agent. In one embodiment, the steroidal anti-inflammatory agent may be a corticosteroid.

[0353] In one embodiment, the immunomodulatory agent is any one or more of the active agents described herein (e.g., a small molecule drug, an antibody, an antibody mimetic, or a functional equivalent or fragment thereof), such that the active agent has an immunomodulatory function.

[0354] In one embodiment, the immunomodulatory agent is an additional therapeutic agent (e.g., a small molecule drug, an antibody, an antibody mimic, or a functional equivalent or fragment thereof) described herein, whereby the active agent has an immunomodulatory function. In certain embodiments, the additional therapeutic agent is any one or more of epacadostat, rapamycin, doxorubicin, valproic acid, mitoxantrone, vorinostat, cyclophosphamide, irinotecan, cisplatin, methotrexate, tacrolimus, anti-CTLA-4 antibody, or anti-PD-1 antibody (e.g., pembrolizumab).

[0355] Those skilled in the art will be familiar with other immunomodulatory agents encompassed by the above. In particular, the term "immunomodulatory agent" as used herein does not include compounds or compositions that function to enhance the immunogenicity of antigens by prolonging the exposure of the antigen to immune cells (i.e., via a delivery platform such as Freund's™ complete or incomplete adjuvant, Montanide™ ISA, or other oil-based carriers).

[0356] The amount of any particular immunomodulatory agent described herein may depend on the type of agent (e.g., small molecule drug, antibody, etc.) One of skill in the art can readily determine by empirical testing the amount of immunomodulatory agent required for a particular application.

[0357] Methods of Preparing Exemplary T Cell Activation Treatment Compositions The T cell activation therapeutic composition can be prepared by methods known in the art in view of the present disclosure. Exemplary embodiments for preparing the compositions disclosed herein are described below, but are not limited thereto.

[0358] In certain embodiments, the T cell activation therapeutic composition of the present invention comprises at least one survivin antigen and at least one MAGE-A9 antigen, lipid vesicle particles, and a carrier comprising a continuous phase of a hydrophobic material.

[0359] The method for making lipid vesicle particles, such as liposomes, is well known in the art.See, for example, Gregoriadis (1990) and Frezard (1999), both of which have been cited above.Any suitable method for making lipid vesicle particles may be used in the practice of the present invention, or lipid vesicle particles may be obtained from commercial sources.Lipid vesicle particles are typically prepared by hydrating lipid vesicle particle components that are expected to form lipid bilayers (e.g., phospholipids and cholesterol) with an aqueous solution, which may be pure water or a solution in which one or more components are dissolved in water, such as phosphate-buffered saline (PBS), phosphate-free saline, or any other physiologically compatible aqueous solution.

[0360] In one embodiment, lipid vesicle particle components or mixtures of lipid vesicle particle components, such as phospholipids (e.g., Phospholipon® 90G) or DOPC and cholesterol, can be solubilized in an organic solvent, such as a mixture of chloroform and methanol, followed by filtration (e.g., PTFE 0.2 μm filter) and drying, such as by rotary evaporation, to remove the solvent. Hydration of the resulting lipid mixture can be carried out, for example, by injecting the lipid mixture into an aqueous solution or by sonicating the lipid mixture and the aqueous solution. During the formation of lipid vesicle particles, the lipid vesicle particle components form a single bilayer (monolayer) or multiple bilayers (multilayer) that surround the volume of the aqueous solution that hydrates the lipid vesicle particle components.

[0361] In some embodiments, the lipid vesicle particles are then dehydrated, for example, by freeze-drying or lyophilization.

[0362] In some embodiments, the lipid vesicle particles are combined with a suitable carrier, such as a carrier that includes a continuous hydrophobic phase. This can be done in a variety of ways.

[0363] If the carrier is composed solely of a hydrophobic material or a mixture of hydrophobic materials (e.g., using a 100% mineral oil carrier), the lipid vesicle particles can be mixed solely with the hydrophobic material, or, if multiple hydrophobic materials are present, can be mixed with any one or combination of them.

[0364] Instead, when the carrier containing a continuous phase of hydrophobic material contains a discontinuous aqueous phase, the carrier is expected to typically take the form of an emulsion of the aqueous phase in the hydrophobic phase, for example, a water-in-oil emulsion. Such compositions may contain an emulsifier to stabilize the emulsion and promote even distribution of lipid vesicle particles. In this regard, even when a non-water-containing carrier is used, an emulsifier may be useful for the purpose of promoting even distribution of lipid vesicle particles in the carrier. Typical emulsifiers include mannide oleate (Arlacel™ A), lecithin (e.g., S100 lecithin), phospholipids, Tween™ 80, and Spans™ 20, 80, 83 and 85. Typically, the volume ratio (v / v) of hydrophobic material to emulsifier ranges from about 5:1 to about 15:1, with a ratio of about 10:1 being preferred.

[0365] In some embodiments, the lipid vesicle particles may be added to a completed emulsion, or the lipid vesicle particles may be present in either the aqueous or hydrophobic phase prior to emulsification.

[0366] The survivin antigen, MAGE-A9 antigen, or additional antigens described herein may be introduced at various different stages of the formulation process. More than one type of antigen may be incorporated into the composition. As used in this section, the term "antigen" is used generally and may refer to the survivin or MAGE-A9 antigens described herein, one or more survivin antigens or one or more MAGE-A9 antigens, the additional antigens described herein, or one or more additional antigens, or any combination thereof. This term is used generally to describe how any antigen can be formulated into the T cell activation therapeutic composition of the present invention. The term "antigen" encompasses both the singular "antigen" and the plural "antigens". It is not necessary that all antigens are introduced into the T cell activation therapeutic composition in the same way.

[0367] In some embodiments, the antigen is present in the aqueous solution used to hydrate the components (e.g., phospholipids and cholesterol) used to form the lipid bilayer of the lipid vesicle particle. In this case, the antigen will be encapsulated in the vesicle particle or liposome present in its aqueous interior. If the resulting lipid vesicle particle is not washed or dried, and therefore there is residual aqueous solution that is ultimately mixed with the carrier containing a continuous phase of hydrophobic material, it is possible that additional antigen may be present outside the lipid vesicle particle in the final product. In related techniques, the antigen may be mixed with the components used to form the lipid bilayer of the lipid vesicle particle before hydration with the aqueous solution. The antigen may also be added to preformed lipid vesicle particles, in which case the antigen may be actively loaded or bound to the surface of the lipid vesicle particle, or the antigen may remain outside the lipid vesicle particle. In such embodiments, prior to addition of the antigen, the preformed lipid vesicle particles may be empty lipid vesicle particles (e.g., containing no encapsulated antigen or lipid-based adjuvant) or the preformed lipid vesicle particles may contain a lipid-based adjuvant incorporated into or associated with the lipid vesicle particles. These steps may preferably be performed prior to mixing with a carrier comprising a continuous phase of a hydrophobic material.

[0368] In an alternative approach, the antigen may instead be mixed with the carrier, which comprises a continuous phase of hydrophobic material, before, during, or after combining the carrier with the lipid vesicle particles. If the carrier is an emulsion, the antigen may be mixed with either the aqueous phase or the hydrophobic phase, or both, before emulsification. Alternatively, the antigen may be mixed with the carrier after emulsification.

[0369] The technique of combining an antigen with a carrier can be used in conjunction with encapsulation of the antigen in lipid vesicle particles, as described above, such that the antigen is present both within the lipid vesicle particle and in a carrier comprising a continuous phase of hydrophobic material.

[0370] The above-mentioned procedure for introducing antigen into composition also applies to the T helper epitope and / or adjuvant of the composition described herein in the embodiment in which they are included.That is, T helper epitope and / or adjuvant can be introduced into one or more of the following, for example: (1) the aqueous solution used to hydrate the components used to form the lipid bilayer of lipid vesicle particles; (2) the aqueous solution after the formation of the lipid bilayer of lipid vesicle particles; (3) the components used to form the lipid bilayer of lipid vesicle particles; or (4) the carrier containing a continuous phase of hydrophobic material before, during, or after the carrier is combined with lipid vesicle particles.When the carrier is an emulsion, T helper epitope and / or adjuvant can be mixed with either the aqueous phase or the hydrophobic phase or both before, during, or after emulsification.

[0371] Techniques for combining T helper epitopes and / or adjuvants with a carrier can be used along with encapsulation of these components in or addition of these components to lipid vesicle particles such that the T helper epitopes and / or adjuvants are present inside and / or outside the lipid vesicle particle and in a carrier comprising a continuous phase of hydrophobic material.

[0372] The T helper epitope and / or adjuvant may be incorporated into the composition together with the antigen in the same processing step, or separately from the antigen in a different processing step.For example, the antigen, the T helper epitope and the adjuvant may all be present in the aqueous solution used to hydrate the lipid vesicle particle components forming the lipid bilayer, so that all three components become encapsulated in the lipid vesicle particle.Alternatively, the antigen and the T helper epitope may be encapsulated in the lipid vesicle particle, and the adjuvant may be mixed with a carrier comprising a continuous phase of a hydrophobic material.In a further embodiment, the T helper epitope and / or adjuvant may be incorporated into the composition after the antigen encapsulation step by passing the lipid vesicle particle-antigen preparation through a manual mini-extruder, and then mixing the resulting lipid vesicle particle-antigen preparation with a lipid-based adjuvant, for example, in a phosphate buffer solution. T helper epitopes and / or adjuvants may also be incorporated into the composition after lipid vesicle particles are formed, either alone or together with antigen, so that T helper epitopes and adjuvants can be associated with lipid vesicle particles or remain outside of them.T helper epitopes and / or adjuvants may also be incorporated into or associated with lipid vesicle particles before the addition of antigen, and antigen may remain outside of preformed lipid vesicle particles or be loaded / associated with lipid vesicle particles by further processing.In such an embodiment, the preparation obtained may be lyophilized and then reconstituted with a carrier that comprises a continuous phase of hydrophobic material.It is expected that many such combinations are possible.

[0373] If the composition contains one or more further adjuvants, such additional adjuvants may be incorporated into the composition in a manner similar to that described above for the adjuvants, or by combining some of the methods that may be suitable for the additional adjuvants.

[0374] Stabilizers that maintain biological activity or improve chemical stability, such as sugars, antioxidants, or preservatives, may be added to such compositions to extend the shelf life of the antigen, adjuvant, lipid vesicle particle, or continuous hydrophobic carrier.

[0375] In some embodiments, an antigen / adjuvant mixture can be used in cases where the antigen and adjuvant are incorporated into the composition at the same time. An "antigen / adjuvant mixture" refers to an embodiment in which the antigen and adjuvant are in the same diluent at least prior to incorporation into the composition. The antigen and adjuvant in the antigen / adjuvant mixture can be, but are not required to be, chemically linked, for example by covalent bonding.

[0376] In one embodiment for preparing the composition, the lipid preparation is prepared by dissolving the lipid or lipid mixture in a suitable solvent by gentle shaking. The T cell activation therapeutic agent can then be added to the lipid preparation either directly (e.g., by adding a dried active agent and / or immunomodulatory agent) or by first preparing a stock of the T cell activation therapeutic agent dissolved in a suitable solvent. In certain embodiments, the T cell activation therapeutic agent is added to or combined with the lipid preparation by gentle shaking. The T cell activation therapeutic agent preparation is then dried to form a dry cake, and the dry cake is resuspended in a hydrophobic carrier. The drying step can be carried out by various means known in the art, such as freeze-drying, lyophilization, rotary evaporation, evaporation under pressure, etc. Drying at low heat that does not damage the integrity of the components can also be used.

[0377] A "suitable solvent" is one that is capable of dissolving each component (eg, lipid, drug, or both) and can be determined by one of skill in the art.

[0378] For lipids, in one embodiment, suitable solvent is polar protic solvent, such as alcohol (e.g., tertbutanol, n-butanol, isopropanol, n-propanol, ethanol or methanol), water, acetate buffer, formic acid or chloroform.In one embodiment, suitable solvent is 40% tert-butanol.Those skilled in the art can determine other suitable solvents according to the lipid used.

[0379] In a particular embodiment for preparing the composition, a lipid mixture containing DOPC and cholesterol in a ratio of 10:1 (w:w) (Lipoid GmBH, Germany) can be dissolved in 40% tertiary butanol by shaking at 300 RPM at room temperature until dissolved. An active agent / immunomodulatory agent stock can be prepared in DMSO and diluted in 40% tertiary butanol before mixing with the dissolved lipid mixture. A T cell activation therapeutic agent stock can then be added to the dissolved lipid mixture with shaking at 300 RPM for about 5 minutes. The preparation can then be freeze-dried. The freeze-dried cake is then reconstituted with Montanide® ISA51VG (SEPPIC, France) to obtain a clear solution. Typically, the freeze-dried cake is stored (e.g., at 20° C.) until the time of administration, when the freeze-dried cake is reconstituted with a hydrophobic carrier.

[0380] In another embodiment for preparing the composition, the T cell activation therapeutic agent is dissolved in sodium phosphate or sodium acetate buffer together with S100 lipid and cholesterol (Lipoid, Germany).These components are then lyophilized to form a dry cake.Right before injection, the dry cake is resuspended in ISA51VG oil (SEPPIC, France) to prepare a water-free oil composition.

[0381] In another embodiment for preparing the composition, the active agent and / or immunomodulatory agent is dissolved in sodium phosphate or sodium acetate buffer with DOPC and cholesterol (Lipoid, Germany).These components are then lyophilized to form a dry cake.Right before injection, the dry cake is resuspended in ISA51VG oil (SEPPIC, France) to prepare a water-free oil composition.

[0382] In another embodiment for preparing the composition, the dry cake is mixed with lipid vesicle particles (e.g., particle size ≦110 nm, PDI ≦0.1) in sodium phosphate or sodium acetate buffer (100 mM, pH range 5.5 to 10.0). The lipid can be DOPC, DOPC / cholesterol. These components are then freeze-dried to form a dry cake. Just before injection, the dry cake is resuspended in ISA51VG oil (SEPPIC, France) to prepare a water-free oily composition.

[0383] In some embodiments, it may be appropriate to include an emulsifier in the hydrophobic carrier to help stabilize the dry cake components when they are resuspended in the hydrophobic carrier.The emulsifier is provided in an amount sufficient to resuspend the dry mixture of active agent and / or immunomodulator and lipid in the hydrophobic carrier and to maintain the active agent and / or immunomodulator and lipid dissolved in the hydrophobic carrier.For example, the emulsifier may be present at about 5% to about 15% weight per weight of the hydrophobic carrier or about 5% to about 15% weight per volume of the hydrophobic carrier.

[0384] Stabilizers that maintain biological activity or improve chemical stability to extend the shelf life of any of the components, such as sugars, antioxidants, or preservatives, may be added to the composition.

[0385] In one embodiment, the method for preparing the compositions described herein may include those disclosed in WO2009 / 043165, as appropriate in the context of the present disclosure. In such an example, the active agent and / or immunomodulatory agent described herein is expected to be incorporated into the composition in a manner similar to that described for the antigen in WO2009 / 043165.

[0386] In one embodiment, methods for preparing the compositions described herein can include those disclosed in publications WO2019090411 and WO2019010560, including the use of sized lipid vesicle particles. In such instances, the active agents and / or immunomodulatory agents described herein are expected to be incorporated into the compositions in a manner similar to that described for therapeutic agents in publications WO2019090411 and WO2019010560, both of which are incorporated by reference in their entirety for all intended purposes.

[0387] An exemplary method for preparing a T cell activation therapeutic targeting both survivin and MAGE-A9 is shown below. However, it will be understood that alternative embodiments are also encompassed herein, including, for example, the above-mentioned embodiments in which the antigen, adjuvant, and T helper epitope can be introduced at any stage in the formulation of the T cell activation therapeutic, in any order, and ultimately found inside, outside, or both inside and outside the lipid vesicle particle.

[0388] In certain embodiments, to prepare a T cell activation therapeutic targeting both survivin and MAGE-A9, two survivin antigens (SEQ ID NO: 4 and 7), three or four MAGE-A9 antigens (SEQ ID NO: 9, 10, and 12, or SEQ ID NO: 9, 10, 11, and 12); adjuvant (e.g., polyI:C or polydIdC polynucleotide), and lipid or lipid mixture (DOPC and cholesterol) are complexed in aqueous buffer (sodium acetate, 100 mM, pH 9.5) by a process of mixing and hydrating lipid components in the presence of survivin and MAGE-A9 antigens and adjuvant, extrusion to achieve particle size, which is sterile filtered, then filled into vials and lyophilized to dry cake. The dry cake is then resuspended in hydrophobic carrier Montanide ISA51VG prior to injection. This exemplary preparation method can be used with any combination of survivin and MAGE-A9 antigens, any suitable adjuvant and any suitable T-helper epitope.

[0389] In certain embodiments, to prepare a T cell activation therapeutic targeting both survivin and MAGE-A9, two survivin antigens (SEQ ID NO: 4 and 7), three or four MAGE-A9 antigens (SEQ ID NO: 9, 10, 11, and 12, or SEQ ID NO: 9, 10, and 12), and an adjuvant (e.g., polyI:C or polydIdC polynucleotide) are added to pre-prepared lipid vesicle particles (DOPC / Chol 132mg / mL in sodium acetate 50mM, pH 7.5 or pH 9.0, particle size <100nm, pdi <0.1), sterile filtered, and freeze-dried. The dried cake is then resuspended in hydrophobic carrier Montanide ISA51VG prior to injection. This exemplary preparation method can be used with any combination of survivin and MAGE-A9 antigens, any suitable adjuvant, and any suitable T helper epitope.

[0390] Stock solutions of peptides may be added and mixed with the prepared lipid vesicle particles in any order and in any combination. In some embodiments, MAGE-A9 peptides (e.g., MAGE-A9 111, MAGE-A9 24, MAGE-A9 270, and / or MAGE-A9 223) may be added before adding survivin-derived peptides (e.g., SurA24 and / or SurA2M). In some embodiments, survivin-derived peptides (e.g., SurA24 and / or SurA2M) may be added before adding MAGE-A9 peptides (e.g., MAGE-A9 111, MAGE-A9 24, MAGE-A9 270, and / or MAGE-A9 223). In some embodiments, MAGE-A9 peptides (e.g., MAGE-A9 111, MAGE-A9 24, MAGE-A9 270, and / or MAGE-A9 223) and survivin-derived peptides (e.g., SurA24 and / or SurA2M) may be added simultaneously.

[0391] The T helper epitope can be introduced at any stage in the formulation. In some embodiments, the T helper epitope is added simultaneously with the MAGE-A9 peptide (e.g., MAGE-A9 111, MAGE-A9 24, MAGE-A9 270, and / or MAGE-A9 223). In some embodiments, the T helper epitope is added simultaneously with the survivin-derived peptide (e.g., SurA24 and / or SurA2M). In some embodiments, the T helper epitope is added simultaneously with the MAGE-A9 peptide (e.g., MAGE-A9 111, MAGE-A9 24, MAGE-A9 270, and / or MAGE-A9 223) and the survivin-derived peptide (e.g., SurA24 and / or SurA2M). In some embodiments, the T helper epitope is added prior to the addition of either the MAGE-A9 or survivin-derived peptide. In some embodiments, the T helper epitope is added subsequent to the addition of either the MAGE-A9 or survivin derived peptide.

[0392] In one embodiment, the formulation can be prepared by separately preparing stock solutions of each of the DNA-based poly I:C polynucleotide adjuvants (dIdC) (e.g., SEQ ID NO: 22); MAGE-A9 24, MAGE-A9 111, and MAGE-A9 270 in an aqueous solution (e.g., sterile water); MAGE-A9 223, SurA2M, and SurA24 in a basic solution (e.g., 0.1 M sodium hydroxide); and T helper (e.g., A16L (SEQ ID NO: 13)) in an acidic solution (e.g., 0.125% acetic acid). In certain embodiments, the prepared peptide stock solutions of MAGE-A9 111, MAGE-A9 24, MAGE-A9 270, and / or MAGE-A9 223, and the T helper peptide A16L can be added to a buffer solution (e.g., sodium acetate buffer, 0.5 M, pH 9.75). In certain embodiments, pre-prepared lipid vesicle particles (e.g., 132 mg / mL DOPC / Chol, particle size <100 nm, PDI <0.1 in sodium acetate (50 mM, pH 7.5)) may be added to the diluted peptide stock solution above and gently mixed well (e.g., by hand or vortex for 30 seconds, or using a magnetic stir plate depending on the batch volume). In certain embodiments, peptide-loaded lipid vesicle particles may be added to the remaining peptide stock solution of SurA24 and SurA2M and mixed well as disclosed above. In certain embodiments, the pH of the formulation may be adjusted (e.g., to pH 7.0). In certain embodiments, a DNA-based poly I:C polynucleotide adjuvant (dIdC) may be added. In certain embodiments, the final formulation volume is made up to 1.0 mL by adding sterile water, mixed well, for example by vortexing for 30 seconds, and sterile filtered using a single or continuous 0.22 μm sterile filter membrane (e.g., PVDF, PES, PTFE). In certain embodiments, the vial may then be partially plugged and lyophilized.In certain embodiments, the lyophilized cake can be reconstituted with a carrier comprising a water-free continuous hydrophobic phase, for example 0.45 mL of Montanide® ISA 51 oil diluent, to give final concentrations of 132 mg / mL DOPC / Chol, 1 mg / mL each of survivin and MAGE-A9 peptides, 0.4 mg / mL dIdC adjuvant, 0.5 mg / mL T helper peptide A16L, and 0.1 M sodium acetate.

[0393] In certain embodiments, the order of preparation may be: addition of MAGE-A9 peptide stock (e.g., 10 mg / ml stock), addition of T helper stock (e.g., 10 mg / ml stock), addition of liposomes (e.g., DOPC:Chol 10:1, 66 mg / vial <110 nm), addition of survivin peptide (e.g., 10 mg / ml stock or 5 mg / ml stock), adjustment of pH, addition of adjuvant (e.g., poly dIdC (10 mg / ml stock)), and lyophilization. In certain embodiments, the pH is adjusted to about 6 to about 10, about 7.5 to about 9.5, or about 8 to about 9. In certain embodiments, adjustment of the pH to an alkaline pH range of about 8.0 to about 10 or about 9.0 is necessary to avoid precipitation.

[0394] Mode of Administration The method disclosed herein comprises administering a T cell activation therapeutic composition comprising at least one survivin and at least one MAGE-A9 antigen to a subject with cancer. In certain embodiments, the invention further comprises administering an additional therapeutic agent. In certain embodiments, the invention further comprises administering an active agent. In certain embodiments, the active agent and the additional therapeutic agent are administered in the same regimen. In certain embodiments, the active agent and the additional therapeutic agent are administered in different regimens.

[0395] The terms "combination," "co-administration," or "combined administration," and the like, as used herein, are meant to encompass administration of an active agent and a T cell activation therapy to a single patient, and are intended to include instances in which the agent and the T cell activation therapy are not necessarily administered by the same route of administration or at the same time. For example, the active agent and the T cell activation therapy may be administered separately, sequentially, or using alternating administration.

[0396] In certain embodiments, the active agent is administered prior to, concurrently with, and / or following administration of a T cell activating therapy.

[0397] Active agents are typically administered in amounts sufficient to provide an immune modulating effect.

[0398] In certain embodiments, the active agent is present in an amount of about 5 mg to about 5 g, about 10 mg to about 4.5 g, about 15 mg to about 4 g, about 20 mg to about 3.5 g, about 25 mg to about 3 g, about 30 mg to about 2.5 g, about 35 mg to about 2 g, about 40 mg to about 1.5 g, about 45 mg to about 1 g, about 50 mg to about 900 mg, about 55 mg to about 850 mg, about 60 mg to about 800 mg, about 65 mg to about 750 mg, about 70 mg to about 700 mg, about 75 mg to about 650 mg, about 80 mg to about 600 mg, about 80 mg to about 900 mg, about 90 mg to about 1000 mg, about 100 mg to about 1500 mg, about 150 mg to about 2000 mg, about 100 mg to about 2000 mg, about 100 mg to about 3000 mg, about 100 mg to about 3000 mg, about 100 mg to about 4000 mg, about 100 mg to about 5000 mg, about 100 mg to about 5000 mg, about 100 mg to about 6000 mg, about 100 mg to about 7000 mg, about 100 mg to about 8000 mg, about 100 mg to about 9000 mg, about 100 mg to about 1000 mg, about 100 mg to about 1500 mg, about 100 mg to about 2000 mg, about 100 mg to about 2000 mg, about 100 mg to about 3000 mg, about 100 mg to about 3000 mg, about 10 about 85 mg to about 550 mg, about 90 mg to about 500 mg, about 95 mg to about 450 mg, about 100 mg to about 400 mg, about 110 mg to about 350 mg, about 120 mg to about 300 mg, about 130 mg to about 290 mg, about 140 mg to about 280 mg, about 150 mg to about 270 mg, about 160 mg to about 260 mg, about 170 mg to about 250 mg, about 180 mg to about 240 mg, about 190 mg to about 230 mg, or about 200 mg to about 220 mg.In certain embodiments, the active agent is at least about 5 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 125 mg, at least about 150 mg, at least about 175 mg, at least about 200 mg, at least about 225 mg, at least about 250 mg, at least about 275 mg, at least about 300 mg, at least about 325 mg, at least about 350 mg, at least about 375 mg, at least about 40 ... at least about 25 mg, at least about 450 mg, at least about 475 mg, at least about 500 mg, at least about 525 mg, at least about 550 mg, at least about 575 mg, at least about 600 mg, at least about 625 mg, at least about 650 mg, at least about 675 mg, at least about 700 mg, at least about 725 mg, at least about 750 mg, at least about 775 mg, at least about 800 mg, at least about 825 mg, at least about 850 mg, at least about 875 mg, at least about 900 mg, at least about 925 mg, at least about 950 mg, at least about 975 mg, at least about 1 g, at least about 2 g, at least about 3 g, at least about 4 g, or at least about 5 g.

[0399] In certain embodiments, an "amount sufficient to provide an immune modulating effect" may be a "low dose." Thus, in certain embodiments, the methods of the invention include the use of low doses of an active agent in combination with a T cell activating therapeutic.

[0400] For certain embodiments of the present invention, a "low dose" is about 300 mg / m 2 Less than about 100-300 mg / m 2In some cases, the term "low dose" refers to a dose of active agent. In terms of daily administration, a "low dose" of active agent is about 25-300 mg / day or about 50-150 mg / day. In certain embodiments, the daily dosage is about 100 mg of active agent. In certain embodiments, the daily dosage is about 50 mg of active agent per dose.

[0401] With respect to certain embodiments of the invention in which the active agent is the alkylating agent cyclophosphamide, the expression "low dose" typically refers to a dose of about 300 mg / m 2 Less than about 100-300 mg / m 2 In terms of daily administration, a "low dose" of cyclophosphamide is about 25-300 mg / day or about 50-150 mg / day. In certain embodiments, the daily dosage is about 100 mg of cyclophosphamide. In certain embodiments, the daily dosage is about 50 mg of cyclophosphamide per dose. In some embodiments, cyclophosphamide enhances survivin-based T cell responses.

[0402] "Low dose" amounts of other active agents, as encompassed herein, would be expected to be known to those of skill in the art or can be determined by routine techniques.

[0403] In certain embodiments, the methods of the invention include administration of at least two doses of an active agent prior to the first administration of a T cell activating therapy. In connection with these embodiments, an active agent may be additionally administered to the subject at any other time before, during, or after the course of treatment with the T cell activating therapy, so long as at least two doses are administered prior to the first administration of the T cell activating therapy.

[0404] The expression "at least two doses" as used herein is intended to encompass any number of doses greater than a single dose. In one embodiment, the at least two doses include 2-50 doses, more particularly 2-28 doses, more particularly 2-14 doses. In one embodiment, the at least two doses are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 doses. The at least two doses may be separated by any suitable time. In a particular embodiment, the at least two doses include 2 doses per day for a period of one week, for a total of 14 doses.

[0405] In certain embodiments, the method of the present invention includes administering at least two doses of an active agent, and then subsequently administering a T cell activation therapy of the present invention. "Subsequently administering" refers to administering the active agent before the first administration of the T cell activation therapy (e.g., at least one or at least two doses of the agent are given to the subject before the T cell activation therapy). However, as described herein, administration of the active agent to the subject can continue after administration with the T cell activation therapy begins. In an alternative embodiment, administration of the active agent is stopped before the first administration of the T cell activation therapy.

[0406] In certain embodiments, the method of the present invention is such that the first dose of active agent is administered before any treatment of the subject with T cell activation therapeutic agent.In one embodiment, the minimum time separating the administration of the first active agent and the first administration of the T cell activation therapeutic agent can be any time that is sufficient to provide immune modulation effect.Those skilled in the art will understand and take into account the time that is sufficient to provide immune modulation based on the active agent and subject.

[0407] In some embodiments, the first dose of active agent is administered at least 12 hours prior to the first administration of the T cell activation therapy, preferably at least 2, 4, or 6 days prior to the first administration of the T cell activation therapy. In further embodiments, the first dose of active agent may be provided about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, or 14 days or more prior to the first administration of the T cell activation therapy. In certain embodiments, administration of the first active agent occurs 1-4 days prior to the first administration of the T cell activation therapy. In certain embodiments, administration of the first active agent occurs about 1 week prior to the first administration of the T cell activation therapy.

[0408] After the first dose of active agent, subsequent doses may be administered at any desired time interval between doses, so long as at least two doses of the agent are administered before the first administration of the T cell activating therapy. Administration with the active agent may cease before, during, or after a course of treatment with the T cell activating therapy.

[0409] In one embodiment, the first dose of active agent may be followed by one or more maintenance doses. The term "maintenance dose" as used herein is meant to encompass a dose of active agent given at intervals and / or amounts that maintain a sufficient amount of the agent and / or its active metabolites in the subject's body (e.g., avoiding total systemic clearance of the agent and / or its active metabolites). Providing a maintenance dose may allow the immune modulating effect of the active agent to be extended and / or maintained for an extended period of time before, during, and / or after a course of administration with a T cell activating therapeutic.

[0410] In certain embodiments, to maintain immune modulating effect, active agent may be administered 1, 2, 3, 4 or 5 times or more per day. In certain embodiments, to maintain immune modulating effect, active agent may be administered 1, 2, 3, 4 or 5 times or more per day, as long as low dose administration is maintained (e.g., multiple smaller doses are added up to the desired daily low dose). A single dose (i.e., administration) of active agent may be given at a single time point, such as a pill that is swallowed. Alternatively, a single dose of active agent may be given over a short continuous period, such as by intravenous infusion.

[0411] For embodiments of the invention in which the active agent is cyclophosphamide, it may be appropriate to provide a maintenance dose, for example, every 6 to 18 hours. One of skill in the art would be aware of, or could determine by routine techniques, appropriate intervals for maintenance doses of cyclophosphamide, as well as other active agents encompassed herein.

[0412] In certain embodiments, active agent is administered for at least 2 consecutive days before the first administration of T cell activation therapeutic agent.On these days, active agent can be administered to subject at least 1, 2, 3 or 4 times a day or any desired number of times.In certain embodiments, active agent is administered to subject at least 1, 2, 3 or 4 times a day or any desired number of times to provide the amount of daily low dose of agent.

[0413] In another embodiment, the active agent is administered for a period of about one week before the first administration of the T cell activation therapeutic agent. Multiple doses may be provided during this one week period. In an exemplary embodiment, the active agent may be administered once a day, once every two days, or at any suitable interval to provide the maintenance dose described. For example, in certain embodiments, the method of the present invention includes administering the active agent twice a day for a period of about one week before the administration of the T cell activation therapeutic agent.

[0414] In the method of the present invention, there may be a break in treatment with the active agent before the first administration of the T cell activation therapeutic agent. In such an embodiment, administration of the active agent may be stopped permanently or temporarily before the first administration of the T cell activation therapeutic agent. The period between the last dose of the active agent and the first dose of the T cell activation therapeutic agent may be any suitable period, so long as the subject still obtains immune-modulating benefits from the agent. For example, but not limited to, administration of the active agent may be stopped at the same time as the first dose of the T cell activation therapeutic agent is administered, or at any time up to about one week before the first dose of the T cell activation therapeutic agent. For example, but not limited to, administration of the active agent may be stopped about 6, 12, 18, 24, 36, 48, 60, or 72 hours or more before the first dose of the T cell activation therapeutic agent. In certain embodiments, administration of the active agent is stopped about 2, 4, or 7 days before the first dose of the T cell activation therapeutic agent.

[0415] In an alternative embodiment, the treatment of the subject with the active agent continues throughout the course of treatment with the T cell activation therapeutic agent, with or without interruption in the administration of the agent. In a further embodiment, the treatment with the active agent can continue after the treatment with the T cell activation therapeutic agent is stopped. Thus, in one embodiment, the active agent can be administered during the period before each administration of the T cell activation therapeutic agent. Alternatively, the active agent can be administered only during the period before the first administration of the T cell activation therapeutic agent.

[0416] As described herein, treatment with active agent may continue after the first administration of T cell activation therapeutic agent. In one embodiment, administration of active agent continues daily, with or without intermittent interruptions, throughout the course of treatment with T cell activation therapeutic agent. Thus, in some embodiments, it is expected that the agent will be administered before and during treatment with T cell activation therapeutic agent. In such an example, once administration of T cell activation therapeutic agent begins, the active agent can be administered simultaneously with the T cell activation therapeutic agent, immediately thereafter, or at a different time during the day. When an active agent is administered simultaneously with the T cell activation therapeutic agent, it may be included in the T cell activation therapeutic agent composition of the present invention as a single composition, or may be administered in a separate composition.

[0417] Alternatively, administration of the active agent may be suspended during the days that the T cell activating therapy is administered. Thus, the regimens of the invention may include discontinuing administration of the T cell activating therapy during the period of administration of the T cell activating therapy.

[0418] The embodiments described herein for administering an active agent prior to a first administration of a T cell activating therapy also apply to administration of the agent after the first administration of a T cell activating therapy (e.g., prior to each subsequent administration of a T cell activating therapy).

[0419] In certain embodiments, the method of the present invention comprises metronomic treatment of a subject with an active agent.For the purposes of the present invention, "metronomic treatment", "metronomic regimen", or "metronomic administration", or "low-dose intermittent", etc., are meant to refer to periodically administering an agent that interferes with DNA replication at a lower amount than the normal dose.The term "normal dose amount" as used herein can refer to, for example, but not limited to, either (i) the established maximum tolerated dose (MTD) or standard dose according to conventional administration schedule, or (ii) the low dose amount in the case where a low single bolus amount is established for a particular active agent.

[0420] In metronomic dosing, a cumulative dose may ultimately be administered over a certain period of time that is the same as, lower than, or higher than the dose expected to be administered via a conventional dosing schedule. In particularly preferred embodiments, this is accomplished by extending the time frame over which administration is performed and / or increasing the frequency of administration while decreasing the amount administered compared to the amount of a regular dose. For example, a dose of 300 mg / m of active agent may be administered. 2 Where a low dose of is typically administered (e.g., by a single bolus injection), a metronomic regimen may involve administering the same amount over a period of several days by administering periodic low doses. By this approach, metronomic administration can be used, for example, to provide a maintenance dose as described herein.

[0421] In one embodiment of the method of the present invention, the treatment with metronomic active agent is intended to include a daily low dose of the agent administered for a certain period, for example, 2, 3, 4, 5, 6 or 7 consecutive days, or for a period of longer consecutive days.During these days of metronomic administration, the active agent may be provided at regular intervals or at variable intervals.For example, in one embodiment, the dose of the active agent may be administered every 1, 2, 3, 4, 6, 8, 12 or 24 hours.In another embodiment, the dose of the active agent may be administered every 2, 3 or 4 days.

[0422] In some embodiments of the method of the present invention, there may be interruptions or gaps in the period of metronomic treatment with active agent.In this manner, metronomic treatment with active agent may be performed in a cyclical manner with alternating on-period and off-period of administration.Particularly preferred is an interval in which active agent is administered to subject daily, alternating with weekly intervals.For example, a period of one week of administration of active agent is followed by one week of treatment being stopped, and the cycle is repeated.

[0423] In one embodiment, the methods of the invention comprise administering an active agent to a subject every other week for a period of one week. In a particular aspect of this embodiment, administration of the active agent begins about one week prior to the first administration of the T cell activating therapy.

[0424] In the context of the T cell activation therapeutics of the present invention, in some embodiments, it may be preferred to administer the T cell activation therapeutic composition to the subject at intervals of once a week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, or once every 15 weeks. In certain embodiments, the T cell activation therapeutic composition is administered once every 3 weeks. In certain embodiments, the T cell activation therapeutic composition is administered once every 6 weeks to once every 12 weeks. However, the frequency and duration of administration of the T cell activation therapeutic can be adjusted as required for any given subject, and may be more or less frequent than once a week, once every 2 weeks, or once every 3 weeks. The interval between administrations may also not be constant during the treatment period with the T cell activation therapeutic. In the methods of the present invention, the T cell activation therapeutic may be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. It is expected that the treatment with T cell activation therapeutic agent may continue for an indefinite period of time, depending on how advanced the tumor treatment in the subject is.In the case of the T cell activation therapeutic agent of the present invention, in some embodiments, it may be suitable to administer the T cell activation therapeutic agent to the subject as an adjuvant or neoadjuvant treatment.In the case of the T cell activation therapeutic agent of the present invention, in some embodiments, it may be suitable to administer the T cell activation therapeutic agent to the subject as an adjuvant and neoadjuvant treatment.

[0425] In some embodiments, the method of the present disclosure can be used as adjuvant treatment.As used herein, "adjuvant treatment" refers to any additional cancer treatment administered after primary treatment.In some embodiments, adjuvant treatment is administered to reduce the risk of cancer recurrence.Adjuvant therapy includes, but is not limited to, chemotherapy, radiation therapy, hormone therapy, targeted therapy, biological therapy, or a combination thereof.

[0426] In some embodiments, the method of the present disclosure can be used as neoadjuvant treatment.As used herein, "neoadjuvant treatment" refers to any treatment that is administered as the first step to shrink tumor before the main treatment, which is usually surgery, is administered.Neoadjuvant therapy includes, but is not limited to, chemotherapy, radiation therapy, hormone therapy, or a combination thereof.

[0427] In some embodiments, the method of the present disclosure can be used as consolidation therapy. As used herein, "consolidation therapy" refers to any treatment administered after the initial treatment and after the cancer has disappeared. In some embodiments, consolidation therapy is used to kill any cancer cells that may remain in the body. In some embodiments, consolidation therapy can include, but is not limited to, radiation therapy, stem cell transplantation, treatment with drugs that kill cancer cells, or a combination thereof. Also referred to as intensive therapy and post-remission therapy.

[0428] In some embodiments, the method of the present disclosure can be used as maintenance therapy.As used herein, "maintenance therapy" refers to any therapy that is administered after the initial treatment and the cancer has disappeared, to help prevent the recurrence of cancer.In some embodiments, maintenance therapy can include, but is not limited to, treatment with drugs, vaccines, or antibodies that kill cancer cells, or combinations thereof, and can be administered for a long period of time.

[0429] In certain embodiments, the T cell activating therapeutic agent may be administered to the subject once every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, or every 15 weeks during the neoadjuvant phase. In certain embodiments, the T cell activating therapeutic agent may be administered to the subject once every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks during the neoadjuvant phase. In certain embodiments, the T cell activating therapeutic agent may be administered to the subject once every 3 weeks during the neoadjuvant phase.

[0430] In certain embodiments, it may be suitable to administer the T cell activating therapy to a subject once every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, or every 15 weeks during the adjuvant phase. In certain embodiments, it may be suitable to administer the T cell activating therapy to a subject once every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, or every 15 weeks during the adjuvant phase. In certain embodiments, it may be suitable to administer the T cell activating therapy to a subject at intervals of once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks during the adjuvant phase.

[0431] In certain embodiments, the T cell activation therapeutic agent may be administered to the subject once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks during the neoadjuvant phase. In certain embodiments, the T cell activation therapeutic agent may be administered to the subject once every 3 weeks during the neoadjuvant phase. In certain embodiments, the T cell activation therapeutic agent may be administered to the subject once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, or once every 15 weeks during the adjuvant phase. In certain embodiments, it may be suitable to administer the T cell activating therapy to a subject at intervals of once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks during the adjuvant phase.

[0432] In certain embodiments, it may be suitable to administer the T cell activating therapy to a subject at intervals of once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks during the neoadjuvant phase, and once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, or once every 15 weeks during the adjuvant phase.

[0433] In certain embodiments, it may be suitable to administer the T cell activating therapy to a subject at intervals of once every 3 weeks during the neoadjuvant phase, and once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks during the adjuvant phase.

[0434] In certain embodiments, the T cell activation therapy may be administered in an amount of from about 5 μg to about 1000 μg, from about 10 μg to about 950 μg, from about 15 μg to about 900 μg, from about 20 μg to about 850 μg, from about 25 μg to about 800 μg, from about 30 μg to about 750 μg, from about 35 μg to about 700 μg, from about 40 μg to about 650 μg, from about 45 μg to about 600 μg, from about 50 μg to about 550 μg, from about The antibody is administered in a dose of from about 55 μg to about 500 μg, from about 60 μg to about 450 μg, from about 65 μg to about 400 μg, from about 65 μg to about 350 μg, from about 70 μg to about 300 μg, from about 75 μg to about 275 μg, from about 80 μg to about 250 μg, from about 85 μg to about 225 μg, from about 90 μg to about 200 μg, from about 95 μg to about 175 μg, or from about 100 μg to about 150 μg. In certain embodiments, the T cell activating therapy is administered at a dose of about 50 μg to about 500 μg, about 50 μg to about 100 μg, about 60 μg to about 90 μg, 70 μg to about 80 μg, about 100 μg to about 500 μg, about 120 μg to about 480 μg, about 140 μg to about 460 μg, about 160 μg to about 440 μg, about 180 μg to about 420 μg, about 200 μg to about 400 μg, about 220 μg to about 380 μg, about 240 μg to about 360 μg, about 260 μg to about 340 μg, about 280 μg to about 320 μg, or about 300 μg to about 310 μg.

[0435] In one embodiment of the methods of the invention, the active agent may be administered as a priming agent for intermittent periods prior to each administration of the T cell activating therapy.

[0436] In certain embodiments, methods of the invention involving a combination of an active agent and a T cell activation therapy are expected to include administering the T cell activation therapy to a subject at intervals of once every three weeks (e.g., on days 0, 21, 42, 63, 84, etc.), with a first administration of the active agent beginning about one week prior to administration of the first T cell activation therapy (e.g., on day -7) and continuing daily (e.g., metronomically) alternating weekly intervals. Such a treatment regimen is shown in FIG. 1A.

[0437] As will be understood by those skilled in the art, the frequency and duration of administration of the active agent and the T cell activation therapy can be adjusted as required for any given subject.Factors that may be taken into account include, for example, the nature of the T cell activation antigen or antigens in the T cell activation therapy, the type of cancer, the age, physical condition, weight, sex and diet of the subject; and other clinical factors.

[0438] The active agent can be administered by any suitable delivery means and any suitable administration route.In one embodiment, the active agent is administered orally, for example in the form of a pill, tablet or capsule.In an alternative embodiment, the agent is administered by injection (e.g., intravenously).In a particular embodiment of the method of the present invention, the agent is cyclophosphamide, which is administered orally.

[0439] The T cell activation therapeutic agent of the present invention described herein can be formulated in a form suitable for oral, nasal, rectal or parenteral administration. Parenteral administration includes intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, transepithelial, pulmonary, intrathecal and topical modes of administration. In an embodiment, the T cell activation therapeutic agent is formulated as a composition to achieve a depot effect at the injection site as described above. The T cell activation therapeutic agent and the active agent do not necessarily have to be administered by the same route of administration or at the same time.

[0440] In certain embodiments of the methods of the present invention, the active agent is an alkylating agent, such as cyclophosphamide.

[0441] In certain embodiments, an additional therapeutic agent is administered.

[0442] In certain embodiments, administration of an additional therapeutic agent and a T cell activation therapy to a single patient is intended to include instances where the agent and the T cell activation therapy may not necessarily be administered by the same route of administration or at the same time, e.g., the additional therapeutic agent and the T cell activation therapy may be administered separately, sequentially, or using alternating administration.

[0443] In certain embodiments, the active agent is administered prior to, simultaneously with, or following administration of a T cell activating therapy.

[0444] The additional therapeutic agent is typically administered in an amount sufficient to provide an immune-modulating effect.

[0445] In certain embodiments, the additional therapeutic agent is from about 10 mg to about 1 g, from about 5 mg to about 5 g, from about 10 mg to about 4.5 g, from about 15 mg to about 4 g, from about 20 mg to about 3.5 g, from about 25 mg to about 3 g, from about 30 mg to about 2.5 g, from about 35 mg to about 2 g, from about 40 mg to about 1.5 g, from about 45 mg to about 1 g, from about 50 mg to about 900 mg, from about 55 mg to about 850 mg, from about 60 mg to about 800 mg, from about 65 mg to about 750 mg, from about 70 mg to about 700 mg, from about 75 mg to about 650 mg, from about 80 mg to about 600 mg, about 85 mg to about 550 mg, about 90 mg to about 500 mg, about 95 mg to about 450 mg, about 100 mg to about 400 mg, about 110 mg to about 350 mg, about 120 mg to about 300 mg, about 130 mg to about 290 mg, about 140 mg to about 280 mg, about 150 mg to about 270 mg, about 160 mg to about 260 mg, about 170 mg to about 250 mg, about 180 mg to about 240 mg, about 190 mg to about 230 mg, or about 200 mg to about 220 mg. In certain embodiments, the additional therapeutic agent is administered at a dose of about 50 mg to about 350 mg, about 100 mg to about 300 mg, or about 150 mg to about 250 mg.In certain embodiments, the additional therapeutic agent is at a dose of about 5 mg, or at least about 5 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 125 mg, at least about 150 mg, at least about 175 mg, at least about 200 mg, at least about 225 mg, at least about 250 mg, at least about 275 mg, at least about 300 mg, at least about 325 mg, at least about 350 mg, at least about 375 mg, at least about 400 mg, g, at least about 425 mg, at least about 450 mg, at least about 475 mg, at least about 500 mg, at least about 525 mg, at least about 550 mg, at least about 575 mg, at least about 600 mg, at least about 625 mg, at least about 650 mg, at least about 675 mg, at least about 700 mg, at least about 725 mg, at least about 750 mg, at least about 775 mg, at least about 800 mg, at least about 825 mg, at least about 850 mg, at least about 875 mg, at least about 900 mg, at least about 925 mg, at least about 950 mg, at least about 975 mg, at least about 1 g, at least about 2 g, at least about 3 g, at least about 4 g, or at least about 5 g. In certain embodiments, the additional therapeutic agent is administered at a dose of about 100 mg / dose. In certain embodiments, the additional therapeutic agent is administered at a dose of about 200 mg / dose. In certain embodiments, the additional therapeutic agent is administered at a dose of about 200 mg. In certain embodiments of the methods disclosed herein, the additional therapeutic agent is a checkpoint agent. In certain embodiments, the additional therapeutic agent is an inhibitor of PD-1. In certain embodiments, the inhibitor of PD-1 is an antibody. In certain embodiments, the antibody is pembrolizumab.

[0446] In certain embodiments, the additional therapeutic agent is administered at a dose of less than about 300 mg / dose, less than about 275 mg / dose, less than about 250 mg / dose, less than about 225 mg / dose, less than about 200 mg / dose, less than about 175 mg / dose, less than about 150 mg / dose, less than about 125 mg / dose, or less than about 100 mg / dose. I...

Claims

[Claim 1] 1. A pharmaceutical composition for delivering at least two T cell activating therapeutic agents to a subject, the pharmaceutical composition comprising: i) at least two T cell activating therapeutic agents; ii) one or more lipid-based structures; and iii) a carrier, wherein the at least two T cell activating therapeutic agents comprise at least one survivin antigen and at least one melanoma-associated antigen 9 (MAGE-A9) antigen.

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