Novel TCR gamma alternative reading frame protein (TARP) compositions for non-HLA-restricted T cells and uses thereof

Abstract

Novel compositions combining TCRγ alternative reading frame protein (TARP) peptides with cationic lipids such as DOTAP, particularly R-DOTAP, induce high levels of TARP-specific polyfunctional and cytolytic T cells. The compositions and methods of use are provided. The compositions comprise a pair of overlapping N- and C-terminal peptide sequences that overlap the critical central antigenic region of TARP and encompass the entire protein. These overlapping peptide sequence pairs have been selected and designed to be efficiently presented by antigen-presenting cells and prime cytotoxic T cells specific for TARP-derived T cell peptide antigens when delivered in combination with immunostimulatory nanoparticles composed of the cationic lipid R-DOTAP.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 416,899, filed October 17, 2022. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated herein by reference in its entirety in the disclosure of this application.

[0002] Incorporating a sequence listing The material in the attached Sequence Listing is hereby incorporated by reference into the present application. The attached Sequence Listing xml file, named ST26.xml, was created in and is in KB.

[0003] Technical Field The present invention relates generally to anti-cancer vaccines, and more particularly to HLA-unrestricted multi-epitope TARP vaccine compositions comprising cationic lipids. [Background technology]

[0004] Historically, five categories of tumor antigens have been utilized in immunotherapy: mutated antigens (e.g., p53 or RAS), overexpressed self-antigens (e.g., HER2 / neu), differentiation antigens (e.g., gplOO, tyrosinase), cancer-testis antigens (e.g., MAGE family, BAGE family, or CAGE family, NYESO1), and viral antigens (e.g., HPV16 E6 or E7, EBV). The advantages of therapeutic cancer vaccines using proteins and peptides include their ease of production and the relative lack of major safety and regulatory issues. All cells expressing major histocompatibility complex (MHC) class I molecules can present tumor-associated antigens or short peptides derived from viruses whose chronic infection is associated with malignancies (e.g., human papillomavirus, hepatitis B virus, and hepatitis C virus). However, essential signals for stimulating T cells and inducing sustained, potent, and effective immune responses are often missing, resulting in suboptimal CD8 T cell proliferation due to the lack of appropriate cell-mediated signaling through dendritic cells (DCs). + The use of R-DOTAP as an immune enhancer in vaccines has been shown to solve this problem and result in a strong and effective immune response to these types of peptide antigens.

[0005] TARP (T-cell receptor alternate reading frame) protein is a 58-amino acid protein identified using expressed gene sequence databases. Its mRNA initiates in the Jy1 exon of the T-cell receptor (TCR) gamma sequence, and the expressed protein initiates in an alternate reading frame that differs from the reading frame of the TCR gamma coding sequence. TARP is highly expressed in primary prostate cancer as well as metastatic prostate cancer and other cancers; it is expressed in both hormone-sensitive and castration-resistant prostate cancer. TARP is expressed by both normal and malignant prostate tissue, and is overexpressed in 95% of prostate cancer specimens and approximately 50% of breast cancer specimens, with little or no expression in normal cells. This makes TARP a good target antigen for therapeutic vaccination. TARP is overexpressed in approximately 90% of prostate cancer specimens and approximately 50% of breast cancer specimens, but little or no expression in normal cells. Modified CD8 T cells that recognize TARP-derived T cell epitopes are also expressed. + T cells can kill tumor cells that express TARP proteins in an antigen-dependent manner.

[0006] Combining immunotherapy treatments, such as immune checkpoint inhibitors and therapeutic cancer vaccines, is rapidly gaining interest as an approach to treating cancer. A key component of combination cancer immunotherapy treatments is the activation, expansion, and targeting of cancer antigen-specific cytotoxic T cells; these treatments play a crucial role in modulating the host immune system to recognize tumor-associated antigens, trigger cellular antitumor immune responses, and eliminate targeted cancer cells. Several approaches are currently under development for novel immunotherapeutic agents that activate T cells and target cancer: 1) whole-tumor cell-based immunotherapeutics, which combine tumor cell components with immunostimulatory adjuvant compounds; 2) immunotherapeutics based on analogous recombinant tumor antigen proteins / peptides; and 3) immunotherapeutics based on DNA / RNA encoding target tumor antigens. In all other previous studies developed to date, the various immunostimulatory adjuvants utilized in these treatments, including oil-in-water emulsions, alum particles, and various polysaccharide and lipid components, have failed to induce potent, effective, safe, and polyfunctional cytotoxic T cell responses in patients. There are several significant hurdles that such therapeutic cancer immunotherapeutics must overcome in order to induce antitumor immunity. The first hurdle is the correct selection of tumor antigens. Ideal candidates are antigens that are exclusively expressed in tumors, such as tumor-specific antigens (TSAs), or antigens that are uniquely altered in tumor cells, such as neoantigens or tumor-associated antigens (TAAs). The second hurdle to overcome is the effective presentation of these antigens to dendritic cells, thereby inducing MHC I (CD8 + The key to effective immunotherapeutics is the ability to activate T cell immune responses in a broad target patient population expressing a wide range of HLA subtypes.

[0007] Cationic lipids are unique in their ability to rapidly bind to antigen-presenting dendritic cells in a receptor-independent manner and be internalized into early endosomes along with bound peptide or protein antigens. These particular cationic lipids can promote the migration of orders of magnitude more protein and peptide antigens into the MHC class I and MHC class II pathways than other current approaches, such as the use of alum or oil-in-water adjuvants to induce dendritic cell maturation. Furthermore, vaccination with these peptide-loaded cationic lipid nanoparticles induces superior T cell immune responses in vivo compared to peptides alone or peptides formulated with conventional adjuvants.

[0008] The present invention provides a highly effective and safe cancer immunotherapy for the HLA-nonrestricted treatment of human cancers expressing the tumor-associated antigen TARP. Summary of the Invention

[0009] The present invention is based on the breakthrough discovery that the use of multi-epitope TARP peptides with overlapping amino acid sequences in vaccine compositions, together with cationic lipids as adjuvants, induces non-HLA-restricted, multifunctional, anti-cancer-specific T cell responses.

[0010] In one embodiment, the present invention provides a multi-epitope peptide comprising at least one multi-epitope peptide having an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32.

[0011] In one embodiment, at least one multiepitope peptide is modified. In one embodiment, at least one multiepitope peptide is covalently modified. In one embodiment, the modification comprises palmitoylation or the addition of an anionic sequence.

[0012] In one embodiment, at least one multiepitope peptide is modified. In one embodiment, at least one multiepitope peptide is covalently modified. In one embodiment, the modification comprises palmitoylation or the addition of an anionic sequence.

[0013] In one embodiment, the present invention provides a composition comprising one or more multiepitope peptides having an amino acid sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32, and an adjuvant that is a cationic lipid.

[0014] In one embodiment, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations thereof, or analogs thereof. In one embodiment, the cationic lipid is R-DOTAP.

[0015] In one embodiment, one or more multiepitope peptides are modified by palmitoylation or by the addition of at least one anionic amino acid. In one embodiment, one or more multiepitope peptides are encapsulated in cationic liposomes or mixed with preformed cationic lipid nanoparticles as separate micelles. In one embodiment, one or more multiepitope peptides and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

[0016] In one embodiment, the composition comprises an enhancer agonist epitope and / or an analog thereof.

[0017] In one embodiment, the present invention provides a vaccine composition comprising a multi-epitope peptide comprising at least one TARP peptide and an adjuvant that is a cationic lipid.

[0018] In one aspect, the multi-epitope peptide has at least one multi-epitope peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32.

[0019] In one aspect, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NO:27 and SEQ ID NO:28.

[0020] In one embodiment, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 27. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 28. In one embodiment, at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8.

[0021] In one embodiment, at least one multi-epitope peptide is modified by palmitoylation or by the addition of an anionic amino acid sequence.

[0022] In one embodiment, the at least one multi-epitope peptide comprises the amino acid sequence of a T cell receptor alternative reading frame protein (TARP). In one embodiment, the at least one multi-epitope peptide binds to CD4+ T cells and / or CD8+ T cells.

[0023] In one embodiment, at least one multiepitope peptide is modified. In one embodiment, at least one multiepitope peptide is covalently modified. In one embodiment, the modification comprises palmitoylation or the addition of an anionic sequence.

[0024] In one embodiment, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant or analog thereof, hi one embodiment, the cationic lipid is R-DOTAP.

[0025] In one embodiment, the at least one multi-epitope peptide is encapsulated in a cationic liposome or mixed separately with the pre-formed cationic lipid nanoparticle as a micelle. In one embodiment, the at least one multi-epitope peptide and the pre-formed cationic lipid nanoparticle are mixed in a 1:1 ratio.

[0026] In one embodiment, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a vaccine composition comprising at least one multi-epitope peptide comprising at least one TARP peptide; and an adjuvant that is a cationic lipid.

[0027] It has at least one multi-epitope peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32.

[0028] In one embodiment, at least one multiepitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 27 to 30 and 32. In one embodiment, at least one multiepitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 27 and 28. In one embodiment, at least one multiepitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NOs: 27 and 28. In one embodiment, at least one multiepitope peptide has the amino acid sequence of SEQ ID NO: 27. In one embodiment, at least one multiepitope peptide has the amino acid sequence of SEQ ID NO: 28. In one embodiment, at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8.

[0029] In one embodiment, at least one multi-epitope peptide comprises the amino acid sequence of a T cell receptor alternative reading frame protein (TARP). In one embodiment, at least one multi-epitope peptide is modified. In one embodiment, the modification includes palmitoylation or the addition of an anionic sequence.

[0030] In one embodiment, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant or analog thereof, hi one embodiment, the cationic lipid is R-DOTAP.

[0031] In one embodiment, the at least one multi-epitope peptide is encapsulated by the cationic lipid nanoparticle or mixed with the preformed cationic lipid nanoparticle as a separate micelle. In one embodiment, the at least one multi-epitope peptide and the preformed cationic lipid nanoparticle are mixed in a 1:1 ratio.

[0032] In one embodiment, at least one multi-epitope peptide is capable of inhibiting CD4 expression by antigen-presenting cells. + T cells and CD8 + Inducing presentation of non-HLA-restricted peptides to T cells. In one embodiment, treating cancer comprises inhibiting progression of cancer in a subject. In one embodiment, the cancer comprises cancer cells expressing a TARP. In one embodiment, the cancer is prostate cancer, breast cancer, or acute myeloid leukemia (AML). In one embodiment, the method comprises administering an anti-cancer treatment to the subject. In some embodiments, the anti-cancer treatment comprises surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, or any combination thereof. In various embodiments, the immunotherapy comprises immune checkpoint inhibitor therapy. In many embodiments, the checkpoint inhibitor therapy comprises a programmed cell death 1 protein (PD-1) inhibitor, a PD-1 ligand 1 (PD-L1) inhibitor, and / or a cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor. In other embodiments, the targeted therapy comprises a histone deacetylase (HDAC) inhibitor. In one embodiment, treating cancer comprises inducing a TARP-specific polyfunctional cytolytic T cell response in a subject.

[0033] In one embodiment, the present invention provides a method for inducing a TARP-specific polyfunctional and cytolytic T cell response in a subject, the method comprising administering to the subject a vaccine composition comprising at least one multiepitope peptide, the multiepitope peptide comprising at least one TARP peptide; and an adjuvant that is a cationic lipid. In one aspect, the at least one multiepitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOS: 27-30 and 32. In one aspect, the at least one multiepitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NOS: 27 and 28. In one aspect, the at least one multiepitope peptide comprises the amino acid sequence of SEQ ID NOS: 27 and 28. In one embodiment, at least one multiepitope peptide comprises the amino acid sequence of SEQ ID NO: 28. In one embodiment, at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8. In one embodiment, at least one multiepitope peptide comprises the amino acid sequence of a T cell receptor alternative reading frame protein (TARP). In one embodiment, at least one multiepitope peptide is modified. In one embodiment, the modification comprises palmitoylation or the addition of an anionic sequence. In one embodiment, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variants or analogs thereof. In one embodiment, the cationic lipid is R-DOTAP. In one embodiment, at least one multiepitope peptide is encapsulated by cationic lipid nanoparticles or mixed with preformed cationic lipid nanoparticles as separate micelles.In one embodiment, the at least one multi-epitope peptide and the preformed cationic lipid nanoparticles are mixed in a 1: 1 ratio. In one embodiment, the at least one multi-epitope peptide is capable of targeting CD4 by antigen-presenting cells. + T cells and CD8 + Induce presentation of non-HLA-restricted peptides to T cells. [Brief explanation of the drawings]

[0034] [Figure 1] Figure 1 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. Mice in the TARP.Suc group were inoculated with TARP peptides (SEQ ID NO: 27 and SEQ ID NO: 28) formulated in sucrose solution. Ace: Two long overlapping peptides synthesized with acetate counterions. HCL: Two long overlapping peptides synthesized with HCL counterions.

[0035] [Figure 2] FIG. 2 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ producing cells.

[0036] [Figure 3] Figure 3 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. Anionic TARP: a mixture of SEQ ID NO:29 and SEQ ID NO:30. TARP-Suc: sucrose control mice were vaccinated with TARP peptides (SEQ ID NO:11 and SEQ ID NO:12) formulated in sucrose solution alone. TARP.CFA: mice were vaccinated with TARP peptides (SEQ ID NO:27 and SEQ ID NO:28) formulated in Freund's adjuvant.

[0037] [Figure 4A]Figures 4A-4C show the induction of T cell responses by a mixture of long TARP peptides. Figure 4A is a graph showing the efficacy of the vaccine formulation in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. [Figure 4B] Figures 4A-4C show the induction of T cell responses by a mixture of long TARP peptides, and Figure 4B is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. [Figure 4C] Figures 4A-4C show the induction of T cell responses by a mixture of long TARP peptides, and Figure 4C is a graph showing the efficacy of the vaccine formulation in generating T cell responses, as measured by dosing the number of IFNγ-producing cells.

[0038] [Figure 5] FIG. 5 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells.

[0039] [Figure 6] FIG. 6 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ producing cells.

[0040] [Figure 7] FIG. 7 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells.

[0041] [Figure 8]FIG. 8 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ producing cells.

[0042] [Figure 9] FIG. 9 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ producing cells.

[0043] [Figure 10] Figure 10 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. X-axis: peptide vaccine mixture.

[0044] [Figure 11] Figure 11 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. X-axis: peptide vaccine mixture.

[0045] [Figure 12] Figure 12 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. X-axis: peptide vaccine mixture.

[0046] [Figure 13] Figure 13 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. X-axis: peptide vaccine mixture.

[0047] [Figure 14]Figure 14 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. X-axis: peptide vaccine mixture.

[0048] [Figure 15] Figure 15 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. X-axis: peptide vaccine mixture.

[0049] [Figure 16] Figure 16 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. X-axis: peptide vaccine mixture.

[0050] [Figure 17] Figure 17 is a graph showing the efficacy of vaccine formulations in generating T cell responses, as measured by dosing the number of IFNγ-producing cells. X-axis: peptide vaccine mixture. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention is based on the breakthrough discovery that the use of multi-epitope TARP peptides with overlapping amino acid sequences in vaccine compositions, together with cationic lipids as adjuvants, induces non-HLA-restricted, multifunction, cancer-specific T cell responses.

[0052] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. Also, since the scope of the present invention is defined only by the appended claims, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0053] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure.

[0054] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0055] As used herein, the term "about" in connection with a numerical value is intended to include any additional numerical value reasonably close to the stated numerical value. For example, depending on the context, the value may vary above or below 5-10%. For example, a value of about 100 means 90-110 (or any value between 90-110).

[0056] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0057] Unless otherwise defined, 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. Any methods and materials similar or equivalent to those described herein can also be used in practicing or testing the present invention, but it is understood that modifications and variations are encompassed within the spirit and scope of this disclosure. Preferred methods and materials are described below.

[0058] overview Disclosed herein are methods for designing and using unique long peptide sequences derived from the TARP protein, including (SEQ ID NO:27) and (SEQ ID NO:28). These two novel long peptides consist of an N-terminal TARP peptide and a C-terminal TARP peptide, each consisting of 32-38 amino acids. They share a 12-aa overlap at the C- and N-termini of each peptide, respectively, which encompasses the key immunogenic regions of the TARP protein. As demonstrated using HLA-expressing human transgenic mice and C57BL / 6 mice, these unique sequences, when delivered with cationic lipid nanoparticles, enable efficient processing of antigens into MHC class I and MHC class II epitopes, enabling T cell antigen presentation encoded by the TARP protein.

[0059] An important consideration in the design of peptide-based vaccines designed to elicit CD8 and CD4 T cell-mediated immune responses in humans is the polymorphism of HLA class I and HLA class II molecules within the population. HLA class I can accept short peptides ranging from 8 to 10 aa in length, while HLA class II peptides can accept peptide sequences ranging from 13 to 25 amino acids. Because different HLA alleles bind different peptides, it is important that peptide vaccines contain enough different peptides to be immunogenic in a high proportion of the population. As shown by the data above, neither peptide sequences containing minimal CD8 T cell epitopes nor peptide sequences containing multiple CD8 T cell epitopes, even with modifications to enhance interaction with adjuvants, were sufficient to render the formulation immunogenic. This limitation was overcome by designing longer peptides containing multiple CD4 and CD8 T cell epitopes. The sequences in this unique formulation contain peptide lengths ranging from 32 to 38 amino acids, with 12 amino acid overlaps at the junctions. To maximize the number of peptides that the formulation can present to target populations, this unique peptide design incorporates the entire TARP protein sequence. As detailed in the Examples below, ELISpot assays and mouse models expressing different MHC class I molecules were used to confirm TARP protein presentation, TARP protein processing, and induction of TARP protein-specific CD8 T cells. Furthermore, peptides known to stimulate TARP-specific CD8 T cells in an HLA-A2-dependent manner were selected and confirmed to have the ability to induce human T cell immune responses. As shown in the data provided, these long peptide vaccine formulations were able to process and present validated HLA-A2-specific peptides and stimulate peptide-specific T cell responses in HLA-A2 transgenic mice after vaccination.

[0060] Multi-epitope peptides In one embodiment, the present invention provides a multi-epitope peptide comprising at least one multi-epitope peptide having an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32.

[0061] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to any chain of at least two amino acids linked by a covalent chemical bond. As used herein, a polypeptide may refer to a complete amino acid sequence encoding an entire protein, or a portion thereof. A "protein coding sequence," or a sequence "encoding" a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, either in vitro or in vivo, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will typically be located 3' to the coding sequence.

[0062] The term "long peptide," as used herein, refers to a peptide consisting of at least 30 amino acids. The long peptides of the present invention have immunological properties not found in shorter peptides or pools of shorter peptides covering the same sequence. Examples of such long peptides include, but are not limited to, SEQ ID NOS: 27-30 and 32.

[0063] In one embodiment, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 27. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 28.

[0064] The terms "sequence identity" and "percent identity" are used interchangeably herein. To determine percent identity between two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into a first polypeptide or polynucleotide sequence to optimally align it with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). In some embodiments, the length of a reference sequence (e.g., any of SEQ ID NOS: 1-33) aligned for comparison purposes is at least 80% of the length of the sequence being compared, and in some embodiments, at least 90% or 100%. In one embodiment, the two sequences are the same length.

[0065] Desirable ranges of sequence identity are approximately 80% to 100% and integer values ​​therebetween. The percent identity between the disclosed and claimed sequences may be at least 80%. The percent identity between the disclosed and claimed sequences may be at least 85%. The percent identity between the disclosed and claimed sequences may be at least at least 90%. The percent identity between the disclosed and claimed sequences may be at least at least 95%. The percent identity between the disclosed and claimed sequences may be at least at least 96%. The percent identity between the disclosed and claimed sequences may be at least at least 97%. The percent identity between the disclosed and claimed sequences may be at least at least 98%. The percent identity between the disclosed and claimed sequences may be at least at least 99%. The percent identity between the disclosed and claimed sequences may be at least at least 99.5%. The percent identity between the disclosed and claimed sequences can be at least at least 99.9%. Generally, an exact match indicates 100% identity over the entire length of the reference sequence (e.g., any of SEQ ID NOS: 1-32).

[0066] Also encompassed by the present disclosure are polypeptides and polynucleotides that have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or more identity to the polypeptides and polynucleotides described herein. For example, a polypeptide may have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NOs: 2-26, 27, 28, 29, 30, 31, or 32. In some embodiments, a polypeptide may have 80% identity to SEQ ID NO: 27. In some embodiments, a polypeptide may have 80% identity to SEQ ID NO: 27. In some embodiments, a polypeptide may have 80% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 85% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 90% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 91% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 92% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 93% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 94% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 95% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 96% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 97% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 98% identity to SEQ ID NO:27. In some embodiments, a polypeptide may have 99% identity to SEQ ID NO:27. In some embodiments, the polypeptide may have 80% identity to SEQ ID NO: 28. In some embodiments, the polypeptide may have 85% identity to SEQ ID NO: 28. In some embodiments, the polypeptide may have 90% identity to SEQ ID NO: 28.In some embodiments, a polypeptide may have 91% identity to SEQ ID NO:28. In some embodiments, a polypeptide may have 92% identity to SEQ ID NO:28. In some embodiments, a polypeptide may have 93% identity to SEQ ID NO:28. In some embodiments, a polypeptide may have 94% identity to SEQ ID NO:28. In some embodiments, a polypeptide may have 95% identity to SEQ ID NO:28. In some embodiments, a polypeptide may have 96% identity to SEQ ID NO:28. In some embodiments, a polypeptide may have 97% identity to SEQ ID NO:28. In some embodiments, a polypeptide may have 98% identity to SEQ ID NO:28. In some embodiments, a polypeptide may have 99% identity to SEQ ID NO:28.

[0067] Variants of the disclosed sequences also include peptides or full-length proteins that contain substitutions, deletions, or insertions in the protein backbone but still retain at least about 70% homology to the original protein over the corresponding portions. Greater degrees of deviation from homology are permitted, provided that similar amino acids, i.e., conservative amino acid substitutions, are not counted as changes in the sequence. Examples of conservative substitutions involve amino acids with identical or similar properties. Exemplary conservative amino acid substitutions include the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamic acid; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.

[0068] As used herein, the terms "polyepitopic peptide," "multiepitope peptide," and the like refer to a peptide or polypeptide that comprises at least two epitopes as described herein. For example, a polyepitopic peptide may comprise two, three, four, five, six, seven, eight, nine, ten, or more of the epitopes of the invention.

[0069] The term "epitope" refers to an antigenic determinant within a molecule such as an antigen, i.e., a portion within a molecule or a fragment of a molecule that is recognized by the immune system. An epitope of a protein, such as a tumor antigen, preferably comprises a continuous or discontinuous portion of the protein. The terms "epitope," "antigenic peptide," "antigenic epitope," "immunogenic peptide," "antigenic fragment," and "MHC-binding peptide" may be used interchangeably herein and preferably relate to the presentation of an antigen capable of eliciting an immune response against the antigen or against a cell expressing, containing, or preferably presenting the antigen. An "antigen" according to the present invention encompasses any substance that elicits an immune response. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with an antibody or T lymphocyte (T cell). In the present invention, the term "antigen" includes any molecule that contains at least one epitope. Preferably, an antigen in the context of the present invention is a molecule that elicits an immune response, optionally after processing. Any suitable antigen that is a candidate for an immune response, preferably a cellular immune response, may be used in the present invention. In the context of the present embodiment, the antigen is preferably presented by cells, preferably antigen-presenting cells including abnormal cells, particularly cancer cells, in the context of MHC molecules, leading to an immune response against the antigen. Preferably, the antigen corresponds to a naturally occurring antigen or is a product derived from a naturally occurring antigen. Such naturally occurring antigens include tumor antigens.

[0070] The epitopes referred to in this application include any epitope that can be derived from the TARP peptide having the amino acid sequence of SEQ ID NO:1.

[0071] Non-limiting examples of multi-epitope peptides include SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32.

[0072] The multiepitope peptides of the present invention may comprise at least one multiepitope peptide, at least two multiepitope peptides, at least three multiepitope peptides, at least four multiepitope peptides, at least five multiepitope peptides, at least six multiepitope peptides, at least seven multiepitope peptides, at least eight multiepitope peptides, at least nine multiepitope peptides, or at least ten multiepitope peptides. In one embodiment, the multiepitope peptide comprises at least two multiepitope peptides. The multiepitope peptides of the present invention may comprise one multiepitope peptide, two multiepitope peptides, three multiepitope peptides, four multiepitope peptides, five multiepitope peptides, six multiepitope peptides, seven multiepitope peptides, eight multiepitope peptides, nine multiepitope peptides, or ten multiepitope peptides. In one embodiment, the multi-epitope peptide comprises two multi-epitope peptides.

[0073] The multiepitope peptides of the present invention may be modified. In one embodiment, at least one multiepitope peptide is covalently modified. In one embodiment, the modification comprises palmitoylation or the addition of an anionic sequence. In one embodiment, the anionic sequence is SSEEEDE (SEQ ID NO: 33). In one embodiment, the anionic sequence is SSEEEDEE (SEQ ID NO: 34). In one embodiment, the anionic sequence is SEEEDESS (SEQ ID NO: 35). In one embodiment, the anionic sequence is SEEEDESEED (SEQ ID NO: 36).

[0074] Composition of multi-epitope peptides In one embodiment, the present invention provides a composition comprising one or more multiepitope peptides having an amino acid sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32, and an adjuvant that is a cationic lipid.

[0075] As used herein, the term composition is intended to encompass pharmaceutical compositions that may also contain other therapeutic agents and may be formulated, for example, according to techniques known in the pharmaceutical formulation art, by using conventional pharmaceutically acceptable solvents or diluents and types of pharmaceutical additives (e.g., excipients, preservatives, etc.) appropriate for the desired mode of administration. In certain embodiments, the compositions disclosed herein are formulated with additional agents that facilitate entry into desired cells or tissues. Such additional agents include micelles, liposomes, and dendrimers.

[0076] The term "pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. For example, the carrier, diluent, or excipient, or composition thereof, could be administered to a subject, together with a conjugate of the present invention, without causing any undesired biological effects or interacting in an undesired manner with any of the other ingredients of the pharmaceutical composition contained therein.

[0077] Pharmaceutical compositions containing the peptides or compositions described herein may be administered by any suitable means, for example, parenterally, e.g., by subcutaneous, intravenous, intramuscular, intrathecal, or intracisternal injection or infusion (e.g., as a sterile injectable aqueous or non-aqueous solution or suspension) in a dosage formulation containing a non-toxic pharmaceutically acceptable solvent or diluent. Depending on the condition being treated, these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration are generally known in the art. A suitable route may be, for example, parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, or intraperitoneal delivery. For injection, the pharmaceutical compositions of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as water, Hank's solution, Ringer's solution, or physiologically buffered saline.

[0078] Adjuvants are often used to modulate or enhance the efficacy of vaccines by stimulating the immune system to respond more vigorously to the vaccine, thereby providing enhanced immunity against specific diseases. Adjuvants accomplish this task by mimicking a specific set of evolutionarily conserved molecules, so-called pathogen-associated molecular patterns (PAMs). This set includes liposomes, lipopolysaccharides, molecular cages for antigens, components of bacterial cell walls, and endocytosed nucleic acids such as RNA, double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. Because the immune system has evolved to recognize these specific antigenic moieties, the presence of adjuvants with vaccines can significantly enhance the innate immune response to antigens by mimicking natural infection and enhancing the activity of dendritic cells, lymphocytes, and macrophages.

[0079] The compositions described herein can be formulated with lipid nanoparticles as adjuvants to enhance the presentation of the antigens to antigen-presenting cells and increase the immune response induced by the antigen.

[0080] In some embodiments described herein, the adjuvant is a cationic lipid. As used herein, the term "cationic lipid" refers to any of a number of lipid species that have a net positive charge at physiological pH or that have a protonatable group and have a positive charge at a pH lower than the pKa.

[0081] Suitable cationic lipids according to the present disclosure include, but are not limited to: 3-β [4NlN,8-diguanidinospermidine)-carbamoyl] cholesterol (BGSC); 3-β [N,N-Diguanidinoethyl-aminoethane)-carbamoyl]cholesterol (BGTC); N,N,1N2N3-tetramethyltetrapalmitylspermine (Cellfectin); Nt-butyl-N'-tetradecyl-3-tetradecyl-aminopropionamidine (CLONfectin); dimethyldioctadecylammonium bromide (DDAB); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); 1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxypropyl)-1-methyl-1H-imidazole (DPIM) N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butane-diammonium iodide) (Tfx-50); N-1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), or other N-(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; 1,2 dioleoyl-3-(4'-trimethylammonio)butanol-sn-glycerol (DOBT) or cholesteryl (4'-trimethylammonium) butanoate (ChOTB) (the trimethylammonium group is connected to the duplex (in the case of DOTB) or to the cholesteryl group (in the case of ChOTB) via a butanol spacer arm); DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) (DORIE) or analogs thereof as disclosed in WO 93 / 03709;1,2-Dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidylethanolamylspermine (DPPES), cholesteryl-3 β-carboxyl-amido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterylcarboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine, cholesteryl-3 β-oxysuccinamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3 β-oxysuccinate Iodide, 2-(2-trimethylammonio)-ethylmethylaminoethyl-cholesteryl-3-β-oxysuccinate iodide, 3-β-N-(N',N'-dimethylaminoethane)carbamoylcholesterol (DC-chol), and 3-β-N-(polyethyleneimine)-carbamoylcholesterol; O,O'-Dimyristyl-N-lysylaspartate (DMKE); O,O'-Dimyristyl-N-lysylglutamate (DMKD); 1, 2-Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC); 1, 2-Distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC); 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP); Dioleoyldimethylaminopropane (DODAP); 1,2-Palmitoyl-3-trimethylammoniumpropane (DPTAP); 1,2-Distearoyl-3-trimethylammoniumpropane (DSTAP), 1,2-Myristoyl-3-trimethylammoniumpropane (DMTAP);and sodium dodecyl sulfate (SDS). Additionally, structural variants and derivatives of any of the above cationic lipids are contemplated.

[0082] In some embodiments, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations thereof, analogs thereof, and combinations thereof. In other embodiments, the cationic lipid is DOTAP. In yet other embodiments, the cationic lipid is DOTMA. In other embodiments, the cationic lipid is DOEPC. In some embodiments, the cationic lipid is purified.

[0083] In some embodiments, the cationic lipid is an enantiomer of the cationic lipid. The term "enantiomer" refers to a stereoisomer of the cationic lipid that is a non-superimposable mirror image of its counterpart, such as the R- and S-enantiomers. In various examples, the enantiomer is R-DOTAP or S-DOTAP. In one example, the enantiomer is R-DOTAP. In another example, the enantiomer is S-DOTAP. In some embodiments, the enantiomer is purified.

[0084] In one embodiment, the cationic lipid is selected from the group consisting of DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variants thereof, and analogs thereof.

[0085] In one embodiment, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 27. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 28.

[0086] In another embodiment, one or more multi-epitope peptides are oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated.

[0087] In one embodiment, the one or more multiepitope peptides are modified by palmitoylation or by the addition of at least one anionic amino acid.

[0088] In one embodiment, one or more multi-epitope peptides are encapsulated within liposomes comprising cationic lipid nanoparticles or mixed as separate micelles with preformed cationic lipid nanoparticles.

[0089] The peptides / multiepitope peptides described herein are engineered through lipidation to spontaneously form high molecular weight micellar structures. In some embodiments, the peptides / multiepitope peptides do not need to interact with cationic lipid (e.g., R-DOTAP) nanoparticles to form micelles.

[0090] In one embodiment, the one or more multi-epitope peptides and the pre-formed cationic lipid nanoparticles are mixed in a 1:1 ratio.

[0091] In another embodiment, the composition further comprises an enhancer agonist epitope and / or an analog thereof.

[0092] Vaccine Composition In one embodiment, the present invention provides a vaccine composition comprising at least one multiepitope peptide, the multiepitope peptide comprising at least one TARP peptide, and an adjuvant that is a cationic lipid.

[0093] In the present invention, the term "vaccine" relates to a pharmaceutical preparation (pharmaceutical composition) or a drug that, after administration, induces an immune response, in particular a cellular immune response, that recognizes and attacks pathogens or abnormal cells such as cancer cells. Vaccines can be used for the prevention or treatment of diseases. The term "personalized cancer vaccine" relates to a specific cancer patient and means that the cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.

[0094] By encompassing a "T cell receptor alternative reading frame protein (TARP) amino acid sequence," it is meant that the amino acid sequences of the peptides, when combined, encompass the entire amino acid sequence of a TARP protein, e.g., having the amino acid sequence of SEQ ID NO: 1. This inclusion can include overlap between the amino acid sequences of the peptides.

[0095] In another embodiment, at least one multi-epitope peptide is modified.

[0096] In another embodiment, at least one multi-epitope peptide is covalently modified.

[0097] In some embodiments, the modification comprises palmitoylation or the addition of an anionic sequence.

[0098] In one aspect, at least one multi-epitope peptide has an amino acid sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32.

[0099] In one aspect, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NO:27 and SEQ ID NO:28.

[0100] In one embodiment, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:27 and SEQ ID NO:28.

[0101] In one embodiment, at least one multi-epitope peptide has the amino acid sequences of SEQ ID NO:27 and SEQ ID NO:28.

[0102] In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO:27.

[0103] In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO:28.

[0104] In one embodiment, at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8.

[0105] In some embodiments, the at least one multi-epitope peptide is encapsulated in a liposome comprising a cationic lipid or mixed with a preformed cationic lipid nanoparticle as a separate micelle. In various embodiments, the at least one multi-epitope peptide and the preformed cationic lipid nanoparticle are mixed in a 1:1 ratio.

[0106] In one embodiment, at least one multi-epitope peptide includes the amino acid sequence of a T cell receptor alternative reading frame protein (TARP).

[0107] In one embodiment, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant or analog thereof, hi one embodiment, the cationic lipid is R-DOTAP.

[0108] In one embodiment, at least one multi-epitope peptide binds to CD4+ T cells and / or CD8+ T cells.

[0109] Treatment method In a further embodiment, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a vaccine composition comprising at least one multi-epitope peptide comprising at least one TARP peptide and an adjuvant that is a cationic lipid.

[0110] The term "subject," as used herein, refers to any individual or patient on whom the method is performed. Typically, the subject is a human, although, as will be understood by those skilled in the art, the subject may also be an animal. That is, other animals, including farm animals, including rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, cows, horses, goats, sheep, pigs, chickens, etc., and vertebrates such as primates (including monkeys, chimpanzees, orangutans, and gorillas), are also included within the definition of a subject.

[0111] The term "treatment" is used interchangeably herein with the term "therapy" and refers to both 1) therapeutic procedures or measures that cure, slow, relieve the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and 2) prophylactic / preventative measures. Those in need of treatment can include individuals who already have a particular medical disorder as well as those who may eventually acquire the disorder (i.e., those in need of preventative measures).

[0112] The terms "therapeutically effective amount," "effective amount," "therapeutically effective dose," "effective amount," or like terms refer to that amount of the subject compound that elicits the biological or medical response in a tissue, system, animal, or human that is sought by a researcher, veterinarian, physician, or other clinician. Typically, this response is either an improvement in symptoms in a patient, or a desired biological outcome (e.g., treatment of cancer).

[0113] The terms "administration of" and / or "administering" should be understood to mean providing a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. The route of administration can be enteral, topical, or parenteral. Thus, routes of administration include, but are not limited to, intradermal, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, intrathecal, intraspinal, and intrasternal administration, oral, sublingual, buccal, rectal, vaginal, nasal, and ocular administration, as well as infusion, inhalation, and nebulization.

[0114] In one aspect, at least one multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32.

[0115] In one embodiment, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, at least one multi-epitope peptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 27 and SEQ ID NO: 28. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 27. In one embodiment, at least one multi-epitope peptide has the amino acid sequence of SEQ ID NO: 28.

[0116] In one embodiment, at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8.

[0117] In some embodiments, the at least one multi-epitope peptide comprises a first multi-epitope peptide, a second multi-epitope peptide.

[0118] For example, a vaccine may comprise SEQ ID NO: 27 as the first multiepitope peptide and SEQ ID NO: 28 as the second multiepitope peptide. Alternatively, a vaccine composition may comprise (i) SEQ ID NO: 27 as the first multiepitope peptide and SEQ ID NO: 30 as the second multiepitope peptide; (ii) SEQ ID NO: 29 as the first multiepitope peptide and SEQ ID NO: 28 as the second multiepitope peptide; (iii) SEQ ID NO: 29 as the first multiepitope peptide and SEQ ID NO: 30 as the second multiepitope peptide; (iv) SEQ ID NO: 32 as the first multiepitope peptide and SEQ ID NO: 28 as the second multiepitope peptide; or (v) SEQ ID NO: 32 as the first multiepitope peptide and SEQ ID NO: 30 as the second multiepitope peptide.

[0119] A vaccine may also comprise three or more multi-epitope peptides. Non-limiting examples of such combinations may include: (i) SEQ ID NOs: 27, 28, 29, (ii) SEQ ID NOs: 27, 29, 30, (iii) SEQ ID NOs: 29, 32, 28, (iv) SEQ ID NOs: 29, 32, 30, (v) SEQ ID NOs: 27, 32, 28, (vi) SEQ ID NOs: 27, 32, 30, (vii) SEQ ID NOs: 28, 30, 27, (viii) SEQ ID NOs: 28, 30, 29, (ix) SEQ ID NOs: 28, 30, 32, (x) SEQ ID NOs: 27, 29, 28, 30, (xi) SEQ ID NOs: 27, 32, 28, 30, and (xii) SEQ ID NOs: 27, 29, 32, 28, 30.

[0120] In another embodiment, the cationic lipid is selected from the group consisting of DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variants thereof, and analogs thereof.

[0121] In one embodiment, the cationic lipid is R-DOTAP.

[0122] In another embodiment, the first and second peptides are encapsulated by cationic lipid nanoparticles or mixed as separate micelles with preformed cationic lipid nanoparticles.

[0123] In another embodiment, the first and second multi-epitope peptides and the pre-formed cationic lipid nanoparticles are mixed in a 1:1 ratio.

[0124] In one embodiment, at least one multiepitope peptide comprises the amino acid sequence of a T cell receptor alternative reading frame protein (TARP). In one embodiment, at least one multiepitope peptide is modified. In one embodiment, at least one multiepitope peptide is covalently modified. In one embodiment, the modification includes palmitoylation or the addition of an anionic sequence.

[0125] In one embodiment, the first and second peptides are capable of binding to CD4 by antigen-presenting cells. + T cells and CD8 + This involves the presentation of non-HLA-restricted peptides to T cells.

[0126] The epitopes and multi-epitope peptides described herein are HLA class I and / or HLA class II non-restricted epitopes.

[0127] The human leukocyte antigen (HLA) system or complex is a complex of genes located on chromosome 6 in humans. These genes encode cell surface proteins involved in regulating the immune system. The HLA system is also known as the human version of the major histocompatibility complex (MHC), found in many animals. HLA genes are highly polymorphic, meaning that there are many different alleles of HLA genes, allowing for fine-tuning of the adaptive immune system. HLAs (A, B, and C) corresponding to MHC class I are all part of the HLA class 1 group and present peptides from within cells. These peptides are generated from proteins after digestion in the proteasome. These specific peptides are usually small polymers of approximately 8–10 amino acids in length. Foreign antigens presented by MHC class I attract T lymphocytes called killer T cells (also known as CD8+ T cells or cytotoxic T cells), which destroy the cells. MHC class I proteins bind to β2-microglobulin, which, unlike HLA proteins, is encoded by a gene on chromosome 15. HLA (DP, DM, DO, DQ, and DR), which correspond to MHC class II, present extracellular antigens to T lymphocytes. These specific antigens stimulate the proliferation of helper T cells (also called CD4-positive T cells), which in turn stimulate antibody-producing B cells to produce antibodies against the specific antigen. Self-antigens are suppressed by regulatory T cells.

[0128] MHC-restricted antigen recognition, MHC restriction, or HLA restriction refers to the fact that T cells can interact with self-specific major histocompatibility complex molecules and foreign peptides bound to them, but will only respond to the antigen if the antigen is bound to a specific MHC molecule. When foreign proteins enter cells, they are degraded into peptides. These peptides, or antigens, can be derived from pathogens such as viruses or intracellular bacteria. The foreign peptides are delivered to the cell surface and presented to T cells by proteins called major histocompatibility complexes (MHCs). During T cell development, T cells undergo a selection process in the thymus to prevent their T cell receptors (TCRs) from recognizing MHC molecules that present self-antigens, i.e., to prevent the TCR from having too high an affinity. High affinity means autoreactivity, while no affinity means that the TCR does not bind strongly enough to the MHC. This selection process results in the development of T cells with specific TCRs that may respond only to certain MHC molecules but not to other MHC molecules. This fact that TCRs recognize only some MHC molecules but not others contributes to "MHC restriction." The biological reason for MHC restriction is to conserve energy and cellular components by preventing the generation of excessively wandering lymphocytes. T cells are a type of lymphocyte important in the immune system, activating other immune cells. Upon recognizing foreign peptides via their T cell receptors (TCRs) on their surface, T cells play different roles in defending the host against foreign peptides, which may be derived from pathogens such as bacteria, viruses, or parasites. MHC restriction adds another dimension to T cell receptor specificity by enforcing the restriction that T cells are activated by peptide antigens only when bound to self-MHC molecules, ensuring that antigens are recognized only as peptide-MHC complexes. MHC restriction in T cells occurs during T cell development in the thymus, particularly during positive selection.Only thymocytes (developing T cells in the thymus) that can bind MHC molecules with the appropriate affinity receive survival signals and progress to the next level of selection. MHC restriction is important for the proper functioning of T cells upon exiting the thymus, as it allows the T cell receptor to bind to MHC and detect cells that are infected by intracellular pathogens, carry viral proteins, and have genetic abnormalities.

[0129] In some embodiments, the epitope peptide comprises a TARP epitope. Peptide epitopes, such as TARP epitopes, may have affinity for various MHC molecules expressed by antigen-presenting cells (APCs), thereby determining which immune cell receptors can present and recognize these peptide epitopes, and consequently, which types of immune responses can be induced by these peptide epitopes. As detailed in the Examples section, the TARP epitopes described herein have affinity for several HLA molecules and are therefore capable of inducing several types of immune responses.

[0130] In some embodiments, the MUC1 epitope has MHC affinity for HLA-A2, HLA-A3, HLA-A11, and HLA-A24. In various embodiments, the MUC1 epitope is recognized by the CD4+ T cell receptor and / or the CD8+ T cell receptor.

[0131] As used herein, a peptide epitope "recognized by" an immune cell receptor may be referred to interchangeably as an "immune cell receptor epitope." That is, a TARP epitope recognized by a CD4+ T cell receptor may be referred to as a CD4+ T cell receptor epitope or a TARP epitope, depending on whether emphasis is placed on the peptide (e.g., TARP) from which the epitope is derived or on the immune cell receptor (e.g., CD4+ T cell receptor and / or CD8+ T cell receptor) with which the epitope may be recognized (or may interact with based on affinity).

[0132] By their very nature, the multi-epitope peptides described herein provide not only HLA-A2 antigens, but also HLA-A3 antigens, HLA-A11 antigens, HLA-A24 antigens, and the like.

[0133] In another embodiment, treating cancer comprises inhibiting the progression of cancer in a subject.

[0134] Cancer is a group of diseases involving the growth of abnormal cells that can invade other parts of the body and metastasize. In 2015, approximately 90.5 million people had cancer, with approximately 14.1 million new cases occurring annually, resulting in approximately 8.8 million deaths (15.7% of deaths). The most common types of cancer in men are lung, prostate, colorectal, and stomach cancer. In women, the most common types are breast, colorectal, lung, and cervical cancer.

[0135] The term "cancer" refers to a group of diseases characterized by abnormal and uncontrolled cell growth that begins at one site (primary site) and may invade and metastasize to other sites (secondary sites, metastases); this characteristic distinguishes cancer (malignant tumors) from benign tumors. Virtually every organ can be affected, and there are over 100 types of cancer that can affect humans. Cancer can arise as a result of many causes, including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, tobacco and / or alcohol use, obesity, poor diet, physical inactivity, or any combination of these.

[0136] As used herein, "neoplasm" or "tumor," including grammatical variations thereof, refers to a new, abnormal growth of tissue, which may be benign or cancerous. In a related aspect, neoplasm refers to a neoplastic disease or disorder, including, but not limited to, various cancers. For example, such cancers may include prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, pancreatic cancer, brain cancer, and head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0137] Exemplary cancers reported by the National Cancer Institute include: adult acute lymphoblastic leukemia; childhood acute lymphoblastic leukemia; adult acute myeloid leukemia; adrenocortical carcinoma; childhood adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; childhood cerebellar astrocytoma; childhood cerebral astrocytoma; extrahepatic bile duct cancer; bladder cancer; childhood bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma; childhood brain stem glioma; adult brain tumor; childhood brain tumor, brain stem glioma; childhood brain tumor, cerebellar astrocytoma; childhood brain tumor, cerebral astrocytoma / malignant glioma; childhood brain tumor, ependymoma; childhood brain tumor, medulloblastoma; childhood brain tumor, supratentorial primitive neuroectodermal tumor; childhood brain tumor, visual pathway and hypothalamic glioma; childhood brain tumor (other); breast cancer; pregnancy-associated breast cancer Pregnancy); Pediatric Breast Cancer; Male Breast Cancer; Childhood Bronchial Adenoma / Carcinoid; Childhood Carcinoid Tumors; Gastrointestinal Carcinoid Tumors; Adrenocortical Carcinoma; Islet Cell Carcinoma; Cancer of Unknown Primary Site; Primary Central Nervous System Lymphoma; Childhood Cerebellar Astrocytoma; Childhood Cerebral Astrocytoma / Malignant Glioma; Cervical Cancer; Childhood Cancer; Chronic Lymphocytic Leukemia; Chronic Myeloid Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of the Tendon Sheath; Colon Cancer; Childhood Colorectal Cancer; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Childhood Ependymoma; Epithelial Ovarian Cancer; Esophageal Cancer; Childhood Esophageal Cancer; Ewing's Sarcoma Family Tumors; Childhood Extracranial Germ Cell Tumors; Extragonadal Germ Cell Tumors; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric Cancer; Childhood Gastric Cancer; Gastrointestinal Carcinoid Tumors; Childhood Extracranial Germ Cell Tumors; Sexuality Extraglandular germ cell tumor; Ovarian germ cell tumor; Gestational trophoblastic tumor; Pediatric brain stem glioma; Pediatric visual pathway and hypothalamic glioma; Hairy cell leukemia; Head and neck cancer; Adult (primary) hepatocellular (liver) cancer; Pediatric (primary) hepatocellular (liver) cancer; Adult Hodgkin's lymphoma; Pediatric Hodgkin's lymphoma; Hodgkin's lymphoma during pregnancy; Hypopharyngeal cancer; Pediatric hypothalamic and visual pathway glioma; Intraocular melanoma Chromoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Renal cancer; Laryngeal cancer; Childhood laryngeal cancer; Adult acute lymphoblastic leukemia; Childhood acute lymphoblastic leukemia; Adult acute myeloid leukemia; Childhood acute myeloid leukemia; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Hairy cell leukemia; Lip and oral cavity cancer; Adult (primary) liver cancer; Childhood (primary) liver cancer; Non-small cell lung cancer; Small cell lung cancer;Adult acute lymphoblastic leukemia;Childhood acute lymphoblastic leukemia;Chronic lymphocytic leukemia;AIDS-related lymphoma;Central nervous system (primary) lymphoma;Cutaneous T-cell lymphoma;Adult Hodgkin lymphoma;Childhood Hodgkin lymphoma;Hodgkin lymphoma during pregnancy;Adult non-Hodgkin lymphoma;Childhood non-Hodgkin lymphoma;Non-Hodgkin lymphoma during pregnancy;Primary central nervous system lymphoma;Waldenstrom's macroglobulinemia;Male breast cancer;Adult malignant mesothelioma;Childhood malignant mesothelioma;Malignant thymoma;Childhood medulloblastoma tumor; melanoma; intraocular melanoma; Merkel cell carcinoma; malignant mesothelioma; metastatic squamous cell neck cancer of unknown primary; childhood multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndrome; chronic myeloid leukemia; childhood acute myeloid leukemia; multiple myeloma; chronic myeloproliferative disorders; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; pediatric nasopharyngeal cancer; neuroblastoma; adult non-Hodgkin's lymphoma; childhood non-Hodgkin's lymphoma; non-Hodgkin's lymphoma during pregnancy; non-small cell lung cancer; oral cancer, Pediatric; Oral cavity and lip cancer; Oropharyngeal cancer; Osteosarcoma / Malignant fibrous histiocytoma of bone; Pediatric ovarian cancer; Ovarian epithelial cancer; Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pediatric pancreatic cancer, pancreatic islet cell cancer; Cancer of the paranasal sinuses and nasal cavity; Parathyroid cancer; Penile cancer; Pheochromocytoma; Pediatric pineal and supratentorial primitive neuroectodermal tumor; Pituitary tumor; Plasma cell neoplasm / multiple myeloma; Pleuropulmonary blastoma; Pregnancy-associated breast cancer; Pregnancy-associated Hodgkin lymphoma; Pregnancy-associated non-Hodgkin lymphoma; Primary central nervous system lymphoma; Adult primary liver cancer; Pediatric primary liver cancer; Prostate cancer; Rectal cancer; Renal cell (kidney) cancer; Pediatric renal cell carcinoma; Transitional cell carcinoma of the renal pelvis and ureter; Retinoblastoma; Pediatric rhabdomyosarcoma; Salivary gland cancer; Pediatric salivary gland cancer; Ewing's sarcoma family of tumors; Kaposi's sarcoma; Sarcoma (osteosarcoma) Malignant fibrous histiocytoma of bone;Pediatric rhabdomyosarcoma;Adult soft tissue sarcoma;Pediatric soft tissue sarcoma;Sezary syndrome;Skin cancer;Pediatric skin cancer;Skin cancer (melanoma);Merkel cell skin cancer;Small cell lung cancer;Small intestine cancer;Adult soft tissue sarcoma;Pediatric soft tissue sarcoma;Metastatic squamous cell neck cancer of unknown primary site;Gastric cancer;Pediatric gastric cancer;Pediatric supratentorial primitive neuroectodermal tumor;Cutaneous T-cell lymphoma;Testicular cancer;Pediatric thymoma;Malignant thymoma;Thyroid cancer;Pediatric thyroid cancer;Transitional cell carcinoma of the renal pelvis and ureter;Gestational trophoblastic tumor;Pediatric cancer of unknown primary site;Pediatric rare cancers;Transitional cell carcinoma of the ureter and renal pelvis;Urethral cancer; uterine sarcoma; vaginal cancer; childhood visual pathway and hypothalamic glioma; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0138] In one embodiment, the cancer comprises cancer cells that express TARP.

[0139] By "cancer cells that express TARP" it is meant that the methods described herein can be used to treat any cancer that can be characterized by the presence of cancer cells that express TARP.

[0140] In some embodiments, the cancer is prostate cancer, breast cancer, or acute myeloid leukemia (AML).

[0141] In another aspect, the method further comprises administering to the subject an anti-cancer treatment. As used herein, the term "anti-cancer treatment" is intended to refer to any therapeutic approach that can be used to treat cancer. This term includes, but is not limited to, surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, and any combination thereof.

[0142] In some embodiments, administration can be combined with one or more additional therapeutic agents. The terms "combination therapy," "combined with," and the like refer to the simultaneous use of two or more agents or therapies to enhance response. For example, the compositions of the present invention may be used in combination with other agents or therapies used to treat cancer. Specifically, administration of the peptides, multi-epitope peptides, or vaccines described herein to a subject can be combined with another anti-cancer treatment, such as immune checkpoint inhibitor therapy. Such treatments can be administered before, simultaneously with, or after administration of the compositions of the present invention.

[0143] In various embodiments, the immunotherapy comprises immune checkpoint inhibitor therapy.

[0144] "Checkpoint inhibitor therapy" is a current form of cancer treatment that exploits immune checkpoints that affect immune system function. Immune checkpoints can be either stimulatory or inhibitory. Tumors can exploit these checkpoints to protect themselves from immune system attack. Checkpoint therapy can restore immune system function by blocking inhibitory checkpoints. Checkpoint proteins include programmed cell death 1 protein (PDCD1, PD-1; also known as CD279) and its ligand, PD-1 ligand 1 (PD-L1, CD274), cytotoxic T lymphocyte antigen 4 (CTLA-4), A2AR (adenosine A2A receptor), B7-H3 (or CD276), B7-H4 (or VTCN1), BTLA (B and T lymphocyte attenuator, or CD272), IDO (indoleamine 2,3-dioxygenase), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin domain and mucin domain 3), and VISTA (V domain Ig suppressor of T cell activation).

[0145] Programmed cell death 1 protein, also known as PD-1 and CD279 (cluster of differentiation 279), is a cell surface receptor that plays a key role in downregulating the immune system and promoting self-tolerance by suppressing the inflammatory activity of T cells. PD-1 is an immune checkpoint and sentinel against autoimmunity through a dual mechanism: it promotes apoptosis (programmed cell death) in antigen-specific T cells within lymph nodes while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory suppressor T cells).

[0146] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein expression is increased on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and on T cells and B cells upon TCR and B cell receptor signaling. In resting mice, PD-L1 mRNA can be detected in the heart, lung, thymus, spleen, and kidney. PD-L1 is expressed on nearly all murine cancer cell lines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma, upon IFN-γ treatment. PD-L2 expression is more restricted, primarily expressed by DCs and a few tumor lines.

[0147] CTLA4 or CTLA-4 (cytotoxic T lymphocyte antigen 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint, downregulating immune responses. CTLA4 is constitutively expressed in regulatory T cells, but its expression increases in normal T cells only after activation, a phenomenon particularly pronounced in cancer. CTLA4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits inhibitory signals to T cells. CTLA4 is homologous to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 binds to CD80 and CD86 with greater affinity and avidity than CD28, allowing CTLA-4 to outcompete CD28 for its ligand. CTLA-4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. CTLA-4 is also present on regulatory T cells and contributes to their suppressive function. T cell activation via the T cell receptor and CD28 results in increased expression of CTLA-4.

[0148] There are several checkpoint inhibitors currently being used to treat cancer. PD-1 inhibitors include pembrolizumab (Keytruda) and nivolumab (Opdivo). PD-L1 inhibitors include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). CTLA-4 inhibitors include ipilimumab (Yervoy). Several other checkpoint inhibitors are in development, including the anti-B7-H3 antibody (MGA271), the anti-KIR antibody (lirilumab), and the anti-LAG3 antibody (BMS-986016).

[0149] In many embodiments, the checkpoint inhibitor therapy includes a programmed cell death 1 protein (PD-1) inhibitor, a PD-1 ligand 1 (PD-L1) inhibitor, and / or a cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor.

[0150] "Targeted therapy" refers to any molecularly targeted therapy used to block the growth of cancer cells by interfering with specific target molecules required for carcinogenesis and tumor growth, rather than simply interfering with all rapidly dividing cells (e.g., as in conventional chemotherapy). Biomarkers are typically needed to help select patients likely to respond to a given targeted therapy. Non-limiting examples of targeted therapies include: tyrosine-kinase inhibitors, small molecule-drug conjugates, serine-threonine kinase inhibitors, monoclonal antibodies, and histone deacetylase (HDAC) inhibitors.

[0151] In other embodiments, the targeted therapy comprises an HDAC inhibitor.

[0152] Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups (O=C-CH3) from ε-N-acetyllysine amino acids on both histone and nonhistone proteins. HDACs allow histones to wrap more tightly around DNA. This is important because DNA wraps around histones and DNA expression is controlled by acetylation and deacetylation. The action of HDACs is opposite to that of histone acetyltransferases. HDAC proteins are now also called lysine deacetylases (KDACs) to describe their function, rather than their target, which also includes nonhistone proteins. Normally, HDAC proteins repress gene expression.

[0153] Histone deacetylase inhibitors (HDAC inhibitors, HDACi, HDI) are chemical compounds that inhibit histone deacetylases. "Classical" HDIs act exclusively on class I, class II, and class IV HDACs by binding to the zinc-containing catalytic domain of HDACs. These classical HDIs can be classified into several groups named according to the chemical moiety that binds to the zinc ion (except for cyclic tetrapeptides, which bind to the zinc ion via a thiol group). Some examples of typical zinc-binding affinities in descending order are: 1. Hydroxamic acids (or hydroxamates), such as trichostatin A; 2. Cyclic tetrapeptides (such as trapoxin B) and depsipeptides, 3. Benzamide, 4. Electrophilic ketones, and 5. Aliphatic acid compounds, such as phenylbutyrate and valproic acid.

[0154] "Second-generation" HDIs include the hydroxamic acids vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589); and the benzamides entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103). Class III HDACs, sirtuins, are NAD+-dependent and are therefore inhibited by nicotinamide as well as the NAD derivatives dihydrocoumarin, naphthopyranone, and 2-hydroxynaphthaldehyde.

[0155] Non-limiting examples of HDAC inhibitors include: Istodax (romidepsin), Zolinza (vorinostat), Farydak (panobinostat), and Belodaq (belinostat).

[0156] In one embodiment, treating cancer comprises inducing a TARP-specific polyfunctional and cytolytic T cell response in a subject.

[0157] The term "immune response" refers to the body's integrated reaction to an antigen, preferably a cellular immune response, or a cellular and humoral immune response. The immune response can be protective / preventive / prophylactic and / or therapeutic.

[0158] The immune system is a system of biological structures and processes within an organism that protects against disease. This system is a diffuse, complex network of interacting cells, cell products, and cell-forming tissues that protect the body from foreign substances such as pathogens, destroy infected and malignant cells, and remove necrotic debris. It includes the thymus, spleen, lymph nodes and lymphoid tissues, stem cells, white blood cells, antibodies, and lymphokines. B cells, or B lymphocytes, are a type of lymphocyte in the humoral immune system of the adaptive immune system and are important in immune surveillance. T cells, or T lymphocytes, are a type of lymphocyte that plays a central role in cell-mediated immunity. There are two major subtypes of T cells: killer T cells and helper T cells. In addition, there are suppressor T cells, which are involved in regulating the immune response. Killer T cells recognize only antigens bound to MHC class I molecules, while helper T cells recognize only antigens bound to MHC class II molecules. These two antigen-presenting mechanisms reflect the different roles of these two types of T cells. A third minor subtype, gamma delta T cells, recognize intact antigens not bound to MHC receptors. In contrast, B cell antigen-specific receptors are antibody molecules on the surface of B cells that recognize whole pathogens without requiring antigen processing. Because different lineages of B cells express different antibodies, the complete set of B cell antigen receptors represents all the antibodies the body can produce.

[0159] The terms "cellular immune response," "cellular response," "cellular response to antigen," or similar terms are intended to encompass cellular responses directed against cells characterized by antigen presentation by class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes, which act as "helpers" or "killers." Helper T cells (also known as CD4+ T cells) play a central role by regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill abnormal cells, such as cancer cells, and prevent the production of more abnormal cells. In a preferred embodiment, the invention involves stimulating an antitumor CTL response against tumor cells expressing one or more tumor-expressed antigens, preferably presenting such tumor-expressed antigens by class I MHC.

[0160] In the context of the present invention, the terms "immune response cell," "immune cell," or "immune effector cell" refer to cells that perform effector functions during an immune response. "Immune response cells" are preferably cells characterized by binding to an antigen or antigen-presenting antigen, or antigenic peptides derived from an antigen, and are capable of mediating an immune response. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancerous cells, and, if necessary, eliminate such cancerous cells. For example, immune response cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.

[0161] "Inducing an immune response" may refer to the absence of an immune response against a specific antigen before induction, or may refer to the presence of a certain level of immune response against a specific antigen before induction, and the enhancement of the immune response after induction. That is, "inducing an immune response" also encompasses "enhancing an immune response." After inducing an immune response in a subject, it is preferable that the subject is protected from developing a disease such as a cancer, or that the induction of an immune response improves the condition of the subject. For example, an immune response against a tumor-expressed antigen can be induced in a patient with cancer or in a subject at risk of developing a cancer. In this case, the induction of an immune response may be intended to improve the subject's condition, prevent metastasis from occurring in the subject, or prevent a subject at risk of developing a cancer from developing the disease.

[0162] Numerous combinations of TARP peptide sequences, including specific peptide antigens and long overlapping peptide sequences encompassing the entire TARP protein, have been shown to be ineffective when combined with or incorporated into cationic lipid nanoparticles. Additionally, various TARP peptide antigen modifications designed to promote binding and effective responses when combined with the immunostimulatory nanoparticle R-DOTAP, including peptide lipidation to form high-molecular-weight micelles, and ionic modifications of the peptide sequence (which have been shown to elicit strong antigen responses with numerous other peptide and protein antigens combined with R-DOTAP nanoparticles), have been shown to be ineffective against TARP-derived peptides. Here, we overcome these challenges with TARP tumor antigens by designing two specific long overlapping peptides covering the entire TARP sequence, which uniquely and effectively bind to cationic immunostimulatory nanoparticles, promoting cellular uptake and processing by dendritic cells and promoting the generation of high levels of multicytokine-producing TARP-specific CD8 T cells.

[0163] In one embodiment, the present invention provides a method for inducing a TARP-specific polyfunctional and cytolytic T cell response in a subject, the method comprising administering to the subject a vaccine composition comprising at least one multiepitope peptide, the multiepitope peptide comprising at least one TARP peptide; and an adjuvant that is a cationic lipid. In one aspect, the at least one multiepitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOS: 27-30 and 32. In one aspect, the at least one multiepitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NOS: 27 and 28. In one aspect, the at least one multiepitope peptide comprises the amino acid sequence of SEQ ID NOS: 27 and 28. In one embodiment, at least one multiepitope peptide comprises the amino acid sequence of SEQ ID NO: 28. In one embodiment, at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8. In one embodiment, at least one multiepitope peptide comprises the amino acid sequence of a T cell receptor alternative reading frame protein (TARP). In one embodiment, at least one multiepitope peptide is modified. In one embodiment, the modification comprises palmitoylation or the addition of an anionic sequence. In one embodiment, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variants or analogs thereof. In one embodiment, the cationic lipid is R-DOTAP. In one embodiment, at least one multiepitope peptide is encapsulated by cationic lipid nanoparticles or mixed with preformed cationic lipid nanoparticles as separate micelles.In one embodiment, the at least one multi-epitope peptide and the preformed cationic lipid nanoparticles are mixed in a 1: 1 ratio. In one embodiment, the at least one multi-epitope peptide is capable of targeting CD4 by antigen-presenting cells. + T cells and CD8 + Induce presentation of non-HLA-restricted peptides to T cells.

[0164] An additional important consideration in the design of effective TARP-based immunotherapeutics designed to elicit high levels of both CD4 and CD8 T cell responses in humans is the polymorphism of HLA class I and class II molecules within the population. Because different HLA alleles bind different peptides, it is important that protein / peptide antigen-based immunotherapies contain enough unique, high-affinity HLA-binding epitopes to be immunogenic in a high proportion of the population. In addition, peptide antigens must be delivered in conjunction with potent immune stimuli that promote effective antigen uptake by dendritic cells, direct peptide antigen processing down both the class I (CD8) and class II (CD4) pathways, and provide the correct cytokine activation and signaling to induce large numbers of multicytokine-producing effector T cells. Herein, we overcome these challenges for TARP tumor antigens by designing two specific long overlapping peptides covering the entire TARP sequence. These peptides uniquely and effectively bind to the immunostimulatory nanoparticle R-DOTAP, where they are internalized and processed by dendritic cells, generating high levels of multicytokine-producing TARP-specific CD8 killer T cells. Numerous other combinations of TARP peptide sequences, including short specific peptide antigens and other long overlapping peptide sequences derived from TARP proteins, have been shown to be ineffective when combined with or incorporated into cationic lipid nanoparticles. Additionally, various TARP peptide antigen modifications, including peptide lipidation to form high-molecular-weight micelles and the addition of anionic peptide sequences to promote peptide-cationic nanoparticle association, have been shown to result in strong antigenic responses to other peptide and protein antigens combined with cationic lipid nanoparticles, but not to be effective against TARP peptides. The unique combination of two overlapping TARP peptide sequences reported here results in a potent antigen-specific immune response when delivered in association with cationic lipid nanoparticles.These peptide sequences can be incorporated into immunogenic compositions, such as vaccines, and the resulting compositions utilize these peptide sequences, which have the advantage of inducing T cell immune responses in a non-HLA-restricted manner.

[0165] The TARP amino acid sequence (58 AA residues, isoelectric point approximately 12.3) is as follows: [ka]

[0166] Potential peptide antigens within the TARP protein sequence: SEQ ID NO: 2: FVFLRNFSL = wild-type (WT) HLA-A*0201 binding peptide TARP27-35

[0167] SEQ ID NO: 3: FLRNFSLMV = epitope-enhanced (EE) HLA-A*0201 binding peptide TARP29-37-9V, these two short specific peptides were the original epitopes in the TARP HLA-A2 specific vaccine platform formulated with an oil-in-water adjuvant.

[0168] Additional epitopes used in the second TARP vaccine platform, which includes an oil-in-water adjuvant, are as follows:

[0169] Five 20 amino acid long peptides, overlapping by 10 residues, spanning the entire 58 amino acid TARP protein, as shown below: TARP1-20: MQMFPPSPLFFFLQLLKQSS SEQ ID NO: 4 TARP11-30:FFLQLLKQSSRRLEHTFVFL SEQ ID NO: 5 TARP21-40:RRLEHTFVFLRNFSLMLLRG SEQ ID NO: 6 TARP31-50:RNFSLMLLRGIGKKRRATRF SEQ ID NO: 7 TARP41-58:IGKKRRATRFWDPRRGTP SEQ ID NO: 8

[0170] Set forth below are examples discussing non-HLA-restricted multi-epitope TARP vaccine compositions contemplated for the uses discussed above. The following examples are provided to further illustrate embodiments of the present invention and are not intended to limit the scope of the invention. The following examples are typical of those that might be used, although other procedures, methodologies, or techniques known to those of skill in the art may alternatively be used. [Example]

[0171] Example 1: Evaluation of SEQ ID NOs: 4-8 in a cationic lipid-based T cell vaccine To establish the feasibility of successfully using the immunostimulatory nanoparticle R-DOTAP in a TARP prostate cancer therapeutic vaccine, we engineered a series of TARP peptides corresponding to SEQ ID NOS: 4–8, which possess modifications, including palmitoylation, that have been identified as useful for promoting binding or activity with cationic lipid nanoparticles, as well as the addition of an anionic sequence at the N-terminus to promote charge-mediated binding to cationic lipid nanoparticles. These peptides were formulated with R-DOTAP nanoparticles in two ways: as a 1:1 volumetric mixture of peptide antigen and R-DOTAP nanoparticles, with the peptide not encapsulated within the nanoparticles, and as a mixture with the peptide antigen encapsulated within the R-DOTAP nanoparticles. When the modified peptide formed a high-molecular-weight micellar structure (i.e., palmitoylated peptide), the antigen complex was observed to be internalized by dendritic cells along with the R-DOTAP nanoparticles due to the nanoparticles' large size. Furthermore, highly charged cationic nanoparticles can also bind to the antigen micellar structure, promoting the co-internal internalization of the antigen and immunostimulatory nanoparticles.

[0172] [Table 1]

[0173] [Table 2]

[0174] [Table 3]

[0175] Physiological activity method:

[0176] Randomized mice were divided into groups and vaccinated on days 0 and 7. Each mouse was subcutaneously inoculated with 100 μl of each formulation into the lateral hind leg. One week after the second vaccination, the spleen was removed and processed to generate a single cell suspension. The spleen cells were used to detect antigen-specific T cell activity using an ELISPOT assay to measure IFN-γ. To identify TARP-specific T cells, splenocytes were stimulated with a mixture of long peptide sequences spanning the TARP protein.

[0177] As shown in Figure 5, TARP peptides 29-37 9V (SEQ ID NO: 10), 21-40 (SEQ ID NO: 6), 21-40 modified by the addition of an anionic amino acid group (SEQ ID NO: 21), 21-40 modified by palmitoylation (SEQ ID NO: 13), and HPV positive control peptide YT-10 formulated in CFA failed to elicit antigen-specific ELISPOT responses in HLA-A2 transgenic mice. As seen previously, TARP 27-35 (SEQ ID NO: 2 without the 9V substitution) was apparently inactive in this mouse model, as shown here, and failed to elicit a response when used as a stimulating peptide in ELISPOT. Each marker represents an individual mouse response. Antigen amount in vaccine formulation: 100 micrograms.

[0178] As shown in Figure 6, TARP peptides 29-37 9V (SEQ ID NO: 3), 21-40 (SEQ ID NO: 6), 21-40 modified by the addition of an anionic amino acid group (SEQ ID NO: 21), 21-40 (SEQ ID NO: 13) modified by palmitoylation and formulated with R-DOTAP nanoparticles, and TARP peptide 21-40 (SEQ ID NO: 13) formulated in CFA did not elicit antigen-specific ELISPOT responses in HLA-A2 transgenic mice. As shown here, TARP 27-35 (SEQ ID NO: 3 with the 9V substitution) was apparently inactive in this mouse model and did not elicit a response when used as a stimulatory peptide in ELISPOT. Positive control samples, in which human papillomavirus E7 and E6 oncoprotein peptide antigens were formulated with RDOTAP, elicited responses in the case of two HPV peptide antigens, YT10 and RF9. Each marker represented an individual mouse response. Antigen amount in vaccine formulation: 100 micrograms

[0179] As further shown in Figure 7, TARP peptides 29-37 9V (SEQ ID NO: 3), 21-40 (SEQ ID NO: 13), 21-40 modified by the addition of an anionic amino acid group (SEQ ID NO: 21), 21-40 (SEQ ID NO: 13) modified by palmitoylation and formulated with R-DOTAP nanoparticles, and TARP peptide 21-40 (SEQ ID NO: 13) formulated in CFA did not elicit antigen-specific ELISPOT responses in HLA-A2 transgenic mice, or only very weak responses. TARP peptide antigen 21-40 (SEQ ID NO: 6) showed inactive or very weak activity in this mouse model, as it did not elicit a response when used as a stimulatory peptide in ELISPOT. Peptide 21-40 modified by the addition of an anionic amino acid group (SEQ ID NO: 21) elicited a response to vaccination in only 2 of 6 mice. Each marker represents an individual mouse response. Antigen amount in vaccine formulation: 100 micrograms

[0180] As shown in Figure 8, TARP peptides 29-37 9V (SEQ ID NO: 3), 21-40 (SEQ ID NO: 6), 21-40 modified by the addition of an anionic amino acid group (SEQ ID NO: 21), and 21-40 (SEQ ID NO: 13) modified by palmitoylation and formulated with R-DOTAP nanoparticles elicited no or very weak TARP 21-40 ELISPOT responses in HLA-A2 transgenic mice after two immunizations. In the presence of Freund's complete adjuvant (CFA), a potent immunopotentiator, TARP 21-40(17V) (SEQ ID NO: 6) showed very weak activity when stimulated with TARP 21-40 (SEQ ID NO: 6). X-axis: peptide vaccine mixture. In the ELISPOT assay, the stimulatory peptide TARP 21-40(17V) was solubilized in water / sucrose.

[0181] As shown in Figure 9, TARP 29-37(9V) (SEQ ID NO: 3) and TARP 21-40(17V) (SEQ ID NO: 6), or variants of this peptide, when encapsulated in R-DOTAP nanoparticles, did not appear to elicit any ELISPOT responses to TARP 29-37(9V) in HLA-A2 transgenic mice after two immunizations. When mice were immunized with TARP 29-37(9V) in the presence of the potent adjuvant, Freund's complete adjuvant (CFA), very weak or no ELISPOT responses were observed to TARP 29-37(9V) (SEQ ID NO: 3). X-axis: peptide vaccine mixture. The stimulatory peptide TARP 29-37(9V) solubilized in water / sucrose was used in the ELISPOT assay (see Figure 9).

[0182] Example 2: Development of a long multi-epitope TARP peptide T cell vaccine Development of a novel approach to the TARP program resulted in the development of two novel long peptides, each consisting of 32-38 amino acids, containing an N-terminal TARP peptide and a C-terminal TARP peptide, sharing a 12-aa overlap and containing the immunogenic regions of the TARP protein at the N- and C-termini of each peptide. These two long overlapping peptides encompass the entire TARP antigen and were constructed as follows: one extending from the N-terminal amino acid sequence through the previously identified HLA-A2 antigen, FLRNFSLMV TARP 29-37-9V (TARP2, SEQ ID NO:10) (SEQ ID NO:27), and the other extending from the C-terminal region through the same antigen, FLRNFSLMV TARP 29-37-9V (TARP2, SEQ ID NO:10) (SEQ ID NO:28). We also prepared various versions of the long-chain overlapping peptide to evaluate whether charge modification of the long-chain antigen overlapping peptide by adding anionic amino acid groups to the peptide termini would enhance the binding of the antigen to cationic R-DOTAP nanoparticles and improve immune activity.

[0183] TARP full-length sequence: [ka]

[0184] [ka]

[0185] [ka]

[0186] Anionic tag variants:

[0187] [ka]

[0188] [ka]

[0189] HLA-A2 antigen sequence:

[0190] FVFLRNFSL TARP 27-35 (TARP1) - SEQ ID NO: 2

[0191] FLRNFSLMV TARP 29-37-9V (TARP2) - SEQ ID NO: 10

[0192] Long peptides were formulated with R-DOTAP by mixing solubilized peptides with preformed nanoparticles prior to immunization. The formulations were tested in the same mouse assay as described in Example 1. The long peptides were formulated at 4 mg / mL (FP32 and ML38), and R-DOTAP at 6 mg / mL. These were then combined, resulting in 2 mg / mL TARP peptides and 3 mg / mL R-DOTAP. The human dose was 1 mL, but the mouse dose was 1 / 10 of that for humans. The peptides were not encapsulated but were present separately in a mixture with R-DOTAP. The results are shown in Figures 1 and 4A and discussed below.

[0193] Table 2 summarizes the peptide sequences used in these experiments.

[0194] [Table 4]

[0195] Example 3: Immunological activity of long overlapping TARP peptides in combination with R-DOTAP Two immunogenic vaccine formulations, each consisting of 2 mg of the two long TARP peptide sequences, TARP 1-38 (SEQ ID NO: 27) and TARP 27-58 (SEQ ID NO: 28), were formulated in 1 ml formulations with 3 mg of R-DOTAP nanoparticles. The efficacy of these vaccine formulations in generating T cell responses was measured by subcutaneously injecting 0.1 ml of the vaccine formulation twice, on days 0 and 7, into HLA-A2-expressing transgenic mice (AAD mice) and C57BL / 6 mice (B6 mice). Vaccine formulation-specific immune responses were measured in an ELISPOT assay by counting TARP antigen-specific T cells in triplicate wells in the spleens of vaccinated mice (Figure 1). Each data point on the graph represents the average SFU per million splenocytes of vaccinated mice. Error bars represent the mean ± SEM of five mice per group. To identify TARP-specific T cells, splenocytes were stimulated with a mixed pool of peptide sequences spanning the TARP protein (SEQ ID NOs: 9, 10, 4, 6, 7, 8, and 5). The data indicate that two long overlapping sequences (SEQ ID NOs: 27 and 28) result in efficient antigen processing and presentation, enabling the TARP vaccine to generate TARP-specific T cells in an HLA-unrestricted manner.

[0196] Example 4: TARP antigen processing and various CD8 + and CD4 + Induction of TARP epitopes An immunogenic vaccine formulation was prepared using 2 mg of each of the two long TARP peptide sequences (SEQ ID NO: 27 and SEQ ID NO: 28) combined with 3 mg of R-DOTAP nanoparticles in a 1 ml formulation. The efficacy of this vaccine formulation in generating T cell responses was measured by subcutaneously injecting 0.1 ml of the vaccine formulation twice into HLA-A2-expressing transgenic mice (AAD mice) on days 0 and 7. Immune responses specific to individual regions of the TARP protein were measured in an ELISPOT assay by counting TARP antigen-specific T cells in triplicate wells in the spleens of vaccinated mice (Figure 2). Each data point on the graph represents the average SFU per million splenocytes of vaccinated mice. Error bars represent the mean ± SEM of five mice per group. To identify TARP-specific T cells, splenocytes were stimulated with individual peptide sequences known to be immunogenic in the context of HLA-A2 (TARP1: SEQ ID NO:2; TARP2: SEQ ID NO:3; TARP3: SEQ ID NO:31; TARP4: SEQ ID NO:4; TARP6: SEQ ID NO:6; TARP7: SEQ ID NO:7; and the TARP7 mix (SEQ ID NOs:2, 3, 31, 4, 6, and 7). This data demonstrates that a TARP vaccine composed of SEQ ID NO:27 and SEQ ID NO:28 was efficiently processed and presented in vivo to multiple known HLA-A2 epitopes.

[0197] Example 5: Long TARP peptides combined with R-DOTAP induce enhanced immune responses without sequence modification Two immunogenic vaccine formulations were formulated with 2 mg each of two long-chain modified TARP peptide sequences (SEQ ID NOs: 27 and 28; and SEQ ID NOs: 29 and 30) and 3 mg of R-DOTAP in 1 ml formulations. The efficacy of these vaccine formulations in generating T cell responses was measured by administering 0.1 ml of the vaccine formulations subcutaneously to HLA-A2-expressing transgenic mice (AAD mice) twice on days 0 and 7. SEQ ID NOs: 29 and 30 (containing an additional C-terminal leucine), which are anionic versions of SEQ ID NOs: 27 and 28, respectively, were investigated to determine whether charge modification by the addition of an anionic amino acid group would increase the association of the antigen with cationic R-DOTAP nanoparticles. Additional immunogenic vaccine formulations were prepared with 2 mg each of the two long-chain modified TARP peptide sequences (SEQ ID NOs: 27 and 28) and complete Freund's adjuvant (CFA). The immune response specific to the vaccine formulation was measured by ELISPOT assay by counting TARP antigen-specific T cells in triplicate wells in spleens from vaccinated mice (Figure 3). Each data point on the graph represents the average SFU per million splenocytes of vaccinated mice. Error bars represent the mean ± SEM of five mice per group. To identify TARP-specific T cells, splenocytes were stimulated with a mixture of peptide sequences spanning the TARP protein (SEQ ID NOS: 2, 3, 31, 4, 6, and 7). The data show the activity of TARP peptide sequences (SEQ ID NOS: 27 and 28) when administered in a cationic lipid formulation. Adding an anionic tag to the peptide resulted in an approximately 50% reduction in T cell induction. Furthermore, the peptides (SEQ ID NOS: 27 and 28) showed significantly weaker activity when formulated with the potent adjuvant CFA compared to when formulated with R-DOTAP.

[0198] Example 6: A study demonstrating that long peptides (SEQ ID NO: 27 and SEQ ID NO: 28) induce enhanced immune responses together with R-DOTAP As shown in Figures 4A-4C, a mixture of long TARP peptides and R-DOTAP nanoparticles induced a strong response in an ELISPOT assay. Vaccine formulations were prepared in 1 ml of a vaccine containing 2 mg each of two long-chain modified TARP peptide sequences (SEQ ID NOs: 27 and 28; and SEQ ID NOs: 29 and 30) and 3 mg of R-DOTAP. The efficacy of these vaccine formulations in generating T cell responses was measured by administering 0.1 ml of the vaccine formulation subcutaneously to HLA-A2-expressing transgenic mice (AAD mice) twice on days 0 and 7 (Figure 4A). Figures 4B-4C show graphs depicting exemplary vaccine formulations consisting of modified short TARP peptide sequences (1-2 mg / peptide) with an anion tag (SEED TARP2: SEQ ID NO: 18, SEED TARP4: SEQ ID NO: 19, SEED TARP6: SEQ ID NO: 21) or lipidated (PALM.TARP2: SEQ ID NO: 10, PALM.TARP4: SEQ ID NO: 12, PALM.TARP6: SEQ ID NO: 13) combined with 4-5 mg of R-DOTAP in a 1 ml formulation, or formulations consisting of two long TARP peptide sequences (SEQ ID NO: 27) and (SEQ ID NO: 28), each 2 mg combined with 3 mg of R-DOTAP in a 1 ml formulation. The efficacy of these vaccine formulations in generating T cell responses was measured in HLA-A2-expressing transgenic mice (AAD mice) by administering two 0.1 ml vaccine injections subcutaneously on days 0 and 7. Vaccine formulation-specific immune responses were measured by ELISPOT assay by counting TARP antigen-specific T cells in triplicate wells in spleens from vaccinated mice. TARP246.CFA mice were vaccinated with TARP peptide / complete Freund's adjuvant. Each data point on the graph represents the average SFU per million splenocytes from vaccinated mice. Error bars represent the mean ± SEM of five mice per group. To identify TARP-specific T cells, splenocytes were stimulated with validated HLA-A1-binding epitopes TARP2 (SEQ ID NO: 4), TARP3 (SEQ ID NO: 10), TARP4 (SEQ ID NO: 5), or TARP6 (SEQ ID NO: 6).Encapsulation of peptides within R-DOTAP nanoparticles had little effect on ELISPOT responses. TARP peptides modified with anionic peptide sequences were positive, while unmodified peptides were more potent stimulators. TARP peptides labeled with sucrose or complete Freud's adjuvant (CFA), a potent immunopotentiator, elicited no responses.

[0199] Example 7: Peptide vaccine formulations fail to induce enhanced immune responses with R-DOTAP Vaccine formulations were prepared as shown in Table 4 and evaluated for their ability to induce enhanced immune responses with R-DOTAP, potent NCI adjuvants (GM-CSF, IL-12, HBV core peptide in IFA), or sucrose. Various vaccine formulations were tested to evaluate long peptides that were modified (i.e., palmitoylated or anion-tagged) and mixed with R-DOTAP or encapsulated within R-DOTAP nanoparticles.

[0200] [Table 5] Palmitoylation-modified pTARP1-7 peptide pool mixture (SEQ ID NOs: 9-15 and Unmodified sTARP1-7 peptide pool mixture (SEQ ID NOS: 2-8, and 31) Anion tag-modified nTARP1-7 peptide pool mixture (SEQ ID NOs: 17-24) EN: Encapsulated formulation AD: Mixed formulation

[0201] TARP 20-amino acid overlapping peptide pools, whether unmodified, palmitoylated, anion-tagged, formulated with R-DOTAP, sucrose, or the potent NCI adjuvant, did not induce antigen-specific T cell responses against TARP peptides 1-8 (SEQ ID NOS: 2-8 and 31). TARP 20-amino acid overlapping peptide pools covering the entire sequence of the TARP protein (including palmitoylated and anion-modified versions of these peptides) were encapsulated within R-DOTAP nanoparticles or mixed with preformed R-DOTAP nanoparticles and used to vaccinate HLA-A2-humanized transgenic mice on days 0 and 14. Mouse splenocytes were harvested on day 21 and analyzed for Tarp peptide antigen-specific T cells by ELISPOT (Figures 10-17). Mixtures of SEQ ID NOS: 2-8, and 31, and their anionic tagged (SEQ ID NOS: 17-24) and palmitoylated (SEQ ID NOS: 9-15) derivatives, do not induce any ELISPOT responses to the TARP1-8 peptide antigens (SEQ ID NOS: 2-8 and 31) in HLA-A2 transgenic mice after two immunizations. None of the signals were considered significant compared to the minimal baseline response. Each marker is the mean ELISPOT count for an individual mouse.

[0202] The data show that in HLA-A2 transgenic mice, there is no or very weak immune response to a pool of overlapping TARP peptides spanning the length of the TARP amino acid sequence, in contrast to the immune response generated by two long peptides (SEQ ID NO: 27 and SEQ ID NO: 28). These data indicate that the two long peptides have unexpected immune properties.

[0203] Example 8 Summary Disclosed herein are methods for the design and use of unique long peptide sequences derived from TARP proteins that are designed to be efficiently processed and presented to T cells in an HLA-unrestricted manner when delivered in association with R-DOTAP immunostimulatory nanoparticles, as evidenced by the data in Figures 1-6. This novel combination contains two long peptide sequences, each consisting of 32-38 amino acids. These unique peptide sequences can be incorporated into immunogenic compositions, such as vaccines. As shown in Figures 1-6, these formulations can induce robust TARP protein-specific T cell immune responses.

[0204] Although the invention has been described with reference to the above examples, it should be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. A multi-epitope peptide comprising at least one multi-epitope peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32.

2. The multi-epitope peptide of claim 1, wherein the at least one multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 27 and SEQ ID NO:

28.

3. The multi-epitope peptide of claim 1, wherein the at least one multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27 and SEQ ID NO:

28.

4. The multi-epitope peptide of claim 1 , wherein the at least one multi-epitope peptide comprises an amino acid sequence having SEQ ID NO: 27 and SEQ ID NO:

28.

5. The multi-epitope peptide of claim 1, wherein the at least one multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

27.

6. The multi-epitope peptide of claim 1, wherein the at least one multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

28.

7. The multiepitope peptide according to any one of claims 1 to 6, wherein at least one of the multiepitope peptides is modified.

8. The multi-epitope peptide of claim 7 , wherein the modification comprises palmitoylation or the addition of an anionic sequence.

9. A composition comprising one or more multi-epitope peptides and an adjuvant, wherein the adjuvant is a cationic lipid and the one or more multi-epitope peptides comprise at least one TARP peptide.

10. The composition of claim 9, wherein the one or more multi-epitope peptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 27 to 30 and 32.

11. The composition of claim 9, wherein the at least one multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NO: 27 and SEQ ID NO:

28.

12. The composition of claim 9, wherein the at least one multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27 and SEQ ID NO:

28.

13. The composition of claim 9, wherein the at least one multi-epitope peptide comprises an amino acid sequence having SEQ ID NO: 27 and SEQ ID NO:

28.

14. The composition of claim 9, wherein the at least one multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

27.

15. The composition of claim 9, wherein the at least one multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

28.

16. 10. The composition of claim 9, wherein the at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8.

17. 10. The composition of claim 9, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

18. The composition of claim 10, wherein the cationic lipid is R-DOTAP.

19. The composition of claim 9, wherein the one or more multiepitope peptides are modified by palmitoylation or by the addition of at least one anionic amino acid.

20. 10. The composition of claim 9, wherein the one or more multi-epitope peptides are encapsulated within cationic liposomes or mixed with preformed cationic lipid nanoparticles as separate micelles.

21. 10. The composition of claim 9, wherein the one or more multi-epitope peptides and the preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

22. The composition of claim 9 further comprising an enhancer agonist epitope and / or an analog thereof.

23. (a) a multi-epitope peptide comprising at least one TARP peptide; and (b) an adjuvant that is a cationic lipid; 10. A vaccine composition comprising:

24. The vaccine composition of claim 23, wherein the multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 27-30 and 32.

25. 24. The vaccine composition of claim 23, wherein the multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27 and / or SEQ ID NO:

28.

26. 24. The vaccine composition of claim 23, wherein the multi-epitope peptide comprises the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO:

28.

27. 24. The vaccine composition of claim 23, wherein the multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

27.

28. 24. The vaccine composition of claim 23, wherein the multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

28.

29. 24. The vaccine composition of claim 23, wherein the at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8.

30. The vaccine composition of any one of claims 23 to 29, wherein the multi-epitope peptide is modified by palmitoylation or by the addition of at least one anionic amino acid.

31. 24. The vaccine composition of claim 23, wherein the multi-epitope peptide comprises the amino acid sequence of a T-cell receptor alternative reading frame protein (TARP).

32. The vaccine composition of claim 23, wherein the multi-epitope peptide binds to CD4+ T cells and / or CD8+ T cells.

33. 24. The vaccine composition of claim 23, wherein the multi-epitope peptide is modified.

34. 34. The vaccine composition of claim 33, wherein the modification comprises palmitoylation or the addition of an anionic sequence.

35. 24. The vaccine composition of claim 23, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

36. 36. The vaccine composition of claim 35, wherein the cationic lipid is R-DOTAP.

37. 24. The vaccine composition of claim 23, wherein the multi-epitope peptide is encapsulated within a cationic liposome or mixed with a preformed cationic lipid nanoparticle as a separate micelle.

38. 38. The vaccine composition of claim 37, wherein the multi-epitope peptide and the pre-formed cationic lipid nanoparticles are mixed in a 1:1 ratio.

39. 1. A method of treating cancer in a subject, comprising: (a) at least one multi-epitope peptide comprising at least one TARP peptide; and (b) an adjuvant that is a cationic lipid; treating cancer in a subject by administering to said subject a vaccine composition comprising: A method comprising:

40. 40. The method of claim 39, wherein the at least one multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 27-30 and 32.

41. 40. The method of claim 39, wherein the at least one multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27 and / or SEQ ID NO:

28.

42. 40. The method of claim 39, wherein the at least one multi-epitope peptide comprises the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO:

28.

43. 40. The method of claim 39, wherein the at least one multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

27.

44. 40. The method of claim 39, wherein the at least one multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

28.

45. 40. The method of claim 39, wherein the at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8.

46. 40. The method of claim 39, wherein the at least one multi-epitope peptide comprises the amino acid sequence of T-cell receptor alternative reading frame protein (TARP).

47. 40. The method of claim 39, wherein the at least one multi-epitope peptide is modified.

48. 48. The method of claim 47, wherein the modification comprises palmitoylation or the addition of an anionic sequence.

49. 49. The method of claim 48, wherein the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

50. 40. The method of claim 39, wherein the cationic lipid is R-DOTAP.

51. 40. The method of claim 39, wherein the at least one multi-epitope peptide is encapsulated by a cationic lipid nanoparticle or mixed with a preformed cationic lipid nanoparticle as a separate micelle.

52. 52. The method of claim 51, wherein the at least one multi-epitope peptide and the preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

53. The at least one multi-epitope peptide is capable of binding to CD4 by antigen-presenting cells. ＋ T cells and CD8 ＋ 40. The method of claim 39, which induces presentation of non-HLA-restricted peptides to T cells.

54. 40. The method of claim 39, wherein treating cancer comprises inhibiting the progression of cancer in the subject.

55. 40. The method of claim 39, wherein the cancer comprises cancer cells that express TARP.

56. 40. The method of claim 39, wherein the cancer is prostate cancer, breast cancer, or acute myeloid leukemia (AML).

57. 40. The method of claim 39, further comprising administering to the subject an anti-cancer treatment.

58. 58. The method of claim 57, wherein the anti-cancer treatment comprises surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, or any combination thereof.

59. 59. The method of claim 58, wherein the immunotherapy comprises immune checkpoint inhibitor therapy.

60. 60. The method of claim 59, wherein the checkpoint inhibitor therapy comprises a programmed cell death 1 protein (PD-1) inhibitor, a PD-1 ligand 1 (PD-L1) inhibitor, and / or a cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor.

61. 61. The method of claim 60, wherein the targeted therapy comprises a histone deacetylase (HDAC) inhibitor.

62. 40. The method of claim 39, wherein treating cancer comprises inducing a TARP-specific polyfunctional and cytolytic T cell response in the subject.

63. 1. A method for inducing a TARP-specific polyfunctional and cytolytic T cell response in a subject, comprising: (a) at least one multi-epitope peptide comprising at least one TARP peptide; and (b) an adjuvant that is a cationic lipid; inducing a TARP-specific polyfunctional and cytolytic T cell response in the subject by administering to the subject a composition comprising: A method comprising:

64. 64. The method of claim 63, wherein the at least one multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 27-30 and 32.

65. 64. The method of claim 63, wherein the at least one multi-epitope peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:27 and SEQ ID NO:

28.

66. 64. The method of claim 63, wherein the at least one multi-epitope peptide comprises the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO:

28.

67. 64. The method of claim 63, wherein the at least one multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

27.

68. 64. The method of claim 63, wherein the at least one multi-epitope peptide comprises the amino acid sequence of SEQ ID NO:

28.

69. 64. The method of claim 63, wherein the at least one TARP peptide comprises an amino acid sequence comprising any of SEQ ID NOs: 2-8.

70. 64. The method of claim 63, wherein the at least one multi-epitope peptide comprises the amino acid sequence of T-cell receptor alternative reading frame protein (TARP).

71. 64. The method of claim 63, wherein the at least one multi-epitope peptide is modified.

72. 72. The method of claim 71, wherein the modification comprises palmitoylation or the addition of an anionic sequence.

73. 64. The method of claim 63, wherein the cationic lipid comprises DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof.

74. 64. The method of claim 63, wherein the cationic lipid is R-DOTAP.

75. 64. The method of claim 63, wherein the at least one multi-epitope peptide is encapsulated by a cationic lipid nanoparticle or mixed with a preformed cationic lipid nanoparticle as a separate micelle.

76. 76. The method of claim 75, wherein the at least one multi-epitope peptide and the preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

77. The at least one multi-epitope peptide is capable of binding to CD4 by antigen-presenting cells. ＋ T cells and CD8 ＋ 64. The method of claim 63, which induces presentation of non-HLA-restricted peptides to T cells.

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