Novel T-cell activated immunotherapy for treating human cancer expressing mucoprotein 1 protein

Abstract

Provided herein are multi-epitope peptides comprising at least one mucoprotein 1 (MUC1) peptide having an MHC affinity for at least one HLA serotype and recognized by a CD4 + T cell receptor and / or a CD8 + T cell receptor. Also provided herein are compositions, including vaccine compositions, comprising the multi-epitope peptides and cationic lipids. In various aspects, the cationic lipid is R-DOTAP. The invention also provides methods of using the multi-epitope peptides and the compositions and vaccine compositions. Methods of use include methods of treating cancer in a subject and methods of inducing a MUC-specific multifunction cytolytic T cell response.

Description

[0001] Cross-reference to related applications This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 417,640, filed on October 19, 2022. The disclosure of the prior application is considered part of the disclosure of the present application and is incorporated herein by reference in its entirety.

[0002] Incorporation of Sequence Listing The material in the accompanying sequence listing is hereby incorporated into this application by reference. The accompanying sequence listing xml file (named ST26.xml) was created on and is 1 kb. Background of the Invention Field of the Invention

[0003] The present invention relates generally to anti-cancer vaccines, and more particularly to T cell activating immunotherapeutic compositions comprising cationic lipids.

[0004] Background Information A key component of cancer immunotherapy is the activation, expansion, and targeting of cancer antigen-specific cytotoxic T cells; these therapies play a vital role in modulating the host immune system to recognize tumor-associated antigens and triggering cellular anti-tumor immune responses to eliminate targeted cancer cells. There are several approaches being developed for new T-cell activation cancer-targeted immunotherapies: 1) whole tumor cell-based immunotherapies, combining tumor cell components with immunostimulatory adjuvant compounds; 2) immunotherapies based on similar recombinant tumor antigen proteins / peptides; and 3) immunotherapies based on DNA / RNA encoding target tumor antigens. In all other previous examples developed to date, the various immunostimulatory adjuvants used in these therapies, including oil-in-water emulsions, alum particles, and various polysaccharide and lipid components, have failed to induce strong and effective yet safe multifunctional cytotoxic T cell responses in patients. This type of therapeutic cancer immunotherapy must overcome several important obstacles to induce anti-tumor immunity. The first obstacle is the correct selection of tumor antigens. Ideal candidates for this are antigens that are specifically 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 obstacle to overcome is the ability to effectively present these antigens to dendritic cells so that they can be presented more effectively via both MHC I (CD8+ T cells) and MHC II (CD4 T cells). Currently used methods and immunoadjuvants have been ineffective in this regard. The third obstacle to effective immunotherapy is the activation of T cell immune responses in a broadly targeted patient population expressing a wide range of HLA subtypes.

[0005] The present invention describes novel modified multi-epitope peptide formulations derived from MUC1, consisting of short and long peptides, including epitope-enhanced peptide agonist sequences, associated with R-DOTAP immunostimulatory nanoparticles, which can induce and generate high levels of MUC1 antigen-specific cytotoxic killer T-cells that recognize and kill MUC1-expressing cancer cells. The unique modified peptide antigens include long multi-epitope peptides derived from MUC1, as well as specific HLA A2, A11, and A23 antigen sequences from MUC1, whose MHC binding has been increased by amino acid substitutions and further modified by lipidation to form multi-epitope high-molecular-weight micellar structures that are effectively associated with R-DOTAP immunostimulatory nanoparticles for efficient delivery to dendritic cells for antigen processing, thereby generating a strong class I immune response. SUMMARY OF THE INVENTION The present invention is based on the profound discovery of using mucin-1 (also referred to herein as MUCl)-derived antigenic peptides and polyepitopic MUCl peptides thereof in vaccine compositions with cationic lipids as adjuvants to induce multifunctional anti-cancer specific T cell responses.

[0007] In one embodiment, the present invention provides a polyepitopic peptide having at least 80%, 85%, 90% or 95% sequence identity to the amino acid sequence comprising SEQ ID NO:1.

[0008] In one aspect, the polyepitope peptide has at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 9-14 and 20-37. In one aspect, the epitope peptide comprises at least one mucin 1 (MUC1) peptide. In one aspect, the polyepitope peptide has an MHC affinity for at least one of HLA-A2, HLA-A3, HLA-A11, and / or HLA-A24. In one aspect, the polyepitope peptide is recognized by a CD4+ T cell receptor and / or a CD8+ T cell receptor. In one aspect, the polyepitope peptide has the sequence of SEQ ID NO: 1. In one aspect, the polyepitope peptide is oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated. In one aspect, the polyepitope peptide is palmitoylated. In one aspect, the polyepitope peptide is a cleavable anionic N-terminal sequence. In one aspect, the cleavable anionic N-terminal sequence is the amino acid sequence SSEEDE (SEQ ID NO: 38) or SSEEDEE (SEQ ID NO: 39).

[0009] In another embodiment, the present invention provides a composition having a polyepitopic peptide and a cationic lipid, wherein the polyepitopic peptide comprises at least one MUC-1 peptide.

[0010] In one aspect, the multi-epitope peptide has at least 80%, 85%, 90% or 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 1. In one aspect, at least one MUC-1 peptide has at least 80%, 85%, 90% or 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 9-14 and 20-37. In one aspect, 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 aspect, the cationic lipid is R-DOTAP. In one aspect, the multi-epitope peptide is oxidized, cross-linked, PEGylated, glycosylated, phosphorylated, palmitoylated, methylated or biotinylated. In one aspect, the polyepitope peptide has an MHC affinity for at least one of HLA-A2, HLA-A3, HLA-A11, and / or HLA-A24. In one aspect, the polyepitope peptide is recognized by the CD4+ T cell receptor and / or the CD8+ T cell receptor. In one aspect, the polyepitope peptide comprises a cleavable anionic N-terminal sequence. In one aspect, the cleavable anionic N-terminal sequence comprises the amino acid sequence SSEEDE (SEQ ID NO: 38) or SSEEDEE (SEQ ID NO: 39). In one aspect, the polyepitope peptide is encapsulated in a liposome comprising a cationic lipid. In one aspect, one or more polyepitope peptides are mixed with preformed cationic lipid nanoparticles in a 1:1 ratio. In one aspect, one or more polyepitope peptides are mixed with preformed cationic lipid nanoparticles as separate micelles. In one aspect, the composition further comprises an enhancer agonist epitope and / or an analog thereof.

[0011] In additional embodiments, the present application provides a vaccine composition having a multi-epitope peptide, wherein the multi-epitope has at least one mucin 1 (MUC1) peptide: and a cationic lipid.

[0012] In one aspect, 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 aspect, the cationic lipid is R-DOTAP. In one aspect, the polyepitope peptide has a sequence comprising at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one aspect, at least one MUC-1 peptide has a sequence comprising at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 9-14 and 20-37. In one aspect, the polyepitope peptide has an MHC affinity for at least one of HLA-A2, HLA-A3, HLA-A11, and / or HLA-A24. In one aspect, the polyepitope peptide is oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated. In one aspect, the polyepitope peptide is palmitoylated. In one aspect, the polyepitope peptide has a cleavable anionic N-terminal sequence. In one aspect, the cleavable anionic N-terminal sequence has the amino acid sequence SSEEDE (SEQ ID NO:38) or SSEEDEE (SEQ ID NO:39). In one aspect, the polyepitope peptide has a sequence comprising SEQ ID NO:1. In one aspect, the polyepitope peptide has an amino acid sequence that is at least 80% identical to SEQ ID NOs:18, 19, 42, or 43. In one aspect, the polyepitope peptide is encapsulated in liposomes comprising cationic lipids. In one aspect, the polyepitope peptide and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one aspect, the polyepitope peptide is mixed as separate micelles with preformed cationic lipid nanoparticles. In one aspect, the polyepitope peptide comprises a sequence having the amino acid sequence of SEQ ID NO:1.

[0013] In a further embodiment, the present invention provides a method of treating cancer in a subject, comprising administering to the subject a vaccine composition having a polyepitope peptide, wherein the polyepitope peptide has at least one mucin 1 (MUC1) peptide; and a cationic lipid.

[0014] In one aspect, the multi-epitope peptide has a sequence comprising at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 1. In one aspect, at least one MUCl peptide has a sequence comprising at least 80%, 85%, 90%, or 95% identity to the amino acid sequence comprising any one of SEQ ID NOs: 9-14 and 20-37. In one aspect, 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 aspect, the cationic lipid is R-DOTAP. In one aspect, the multi-epitope peptide is encapsulated in cationic lipid nanoparticles. In one aspect, the multi-epitope peptide and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one aspect, the multi-epitope peptide is mixed as separate micelles with preformed cationic lipid nanoparticles. In one aspect, one or more MUCl peptides induce presentation of non-HLA restricted peptides to CD4 + and CD8 + T cells. In one aspect, treating cancer includes preventing the progression of cancer in a subject. In one aspect, the cancer has cancer cells that express MUC1. In one aspect, the cancer is prostate cancer, breast cancer, or acute myeloid leukemia (AML). In one aspect, the method further includes administering an anticancer treatment to the subject. In one aspect, the anticancer treatment includes immune checkpoint inhibitor therapy. In one aspect, treating cancer includes inducing a MUC-specific multifunctional cytolytic T cell response in the subject.

[0015] In one embodiment, the present invention provides a method of inducing a MUC-specific multifunctional cytolytic T cell response in a subject by administering to the subject a composition comprising a polyepitope peptide, wherein the polyepitope peptide has one or more mucin 1 (MUC1) peptides: and a cationic lipid.

[0016] In one aspect, 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 aspect, the cationic lipid is R-DOTAP. In one aspect, the multi-epitope peptide is encapsulated in cationic lipid nanoparticles. In one aspect, the multi-epitope peptide and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one aspect, the multi-epitope peptide is mixed as a separate micelle with preformed cationic lipid nanoparticles. In one aspect, the multi-epitope peptide induces presentation of non-HLA restricted peptides to CD4 by antigen presenting cells. + and CD8 + T cells. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph illustrating the induction of HLA-A2-specific CD8 T cells in response to a peptide antigen derived from MUCl protein incorporated into R-DOTAP nanoparticles.

[0018] Figure 2 is a graph illustrating the induction of HLA-A2-specific CD8 T cells in response to a peptide antigen derived from MUCl protein incorporated into R-DOTAP nanoparticles.

[0019] Figure 3 is a graph illustrating HLA-A2-specific CD8 T cell responses to formulations comprising peptide antigens C1A (SEQ ID NO: 9) and C2A (SEQ ID NO: 10) incorporated into the lipid bilayer of R-DOTAP nanoparticles.

[0020] Figure 4 is a graph illustrating HLA-A2-specific CD8 T cell responses to a formulation comprising peptide antigens SSEEDE-C1A (SEQ ID NO: 18) and SSEEDE-C2A (SEQ ID NO: 19) formulated with R-DOTAP nanoparticles and a micellar mixture having six lipidated peptide agonist antigens pC3A: (SEQ ID NO: 15), pV1A: (SEQ ID NO: SEQ ID NO: 2), pV2A: (SEQ ID NO: SEQ ID NO: 3), pC5A: (SEQ ID NO: SEQ ID NO: 4), pC6A: (SEQ ID NO: SEQ ID NO: 5), and pP93L: (SEQ ID NO: 16).

[0021] Figure 5is a graph illustrating HLA-A2-specific CD8 T cell responses to a formulation comprising peptide antigens SSEEDE-C1A (SEQ ID NO:9) and SSEEDE-C2A (SEQ ID NO:10) formulated with R-DOTAP nanoparticles and a micellar mixture having six lipidated peptide agonist antigens pC3A: (SEQ ID NO:15), pV1A: (SEQ ID NO:2), pV2A: (SEQ ID NO:3), pC5A: (SEQ ID NO:4), pC6A: (SEQ ID NO:5), pP93L: (SEQ ID NO:16).

[0022] Figure 6 is a graph illustrating HLA-A2-specific CD8 T cell responses to a MUC1 / RDOTAP vaccine formulation containing the long MUC1 peptide antigen YL-40 (containing C1A (SEQ ID NO: 2) and C2A (SEQ ID NO: 3) antigens).

[0023] Figure 7 is a graph illustrating HLA-A2-specific CD8 T cell responses to formulations comprising peptide antigens C1A (SEQ ID NO: 9) and C2A (SEQ ID NO: 10) incorporated into the lipid bilayer of R-DOTAP nanoparticles.

[0024] Figure 8 is a graph illustrating HLA-A2-specific CD8 T cell responses to formulations containing peptide antigens C1A (SEQ ID NO: 9) and C2A (SEQ ID NO: 10) mixed or encapsulated within the lipid bilayer of R-DOTAP nanoparticles.

[0025] Details The present invention is based on the highly influential discovery of using mucin-1-derived antigenic peptides and polyepitope peptides thereof in vaccine compositions with cationic lipids as adjuvants to induce multifunctional anti-cancer specific T cell responses.

[0026] Before describing the compositions and methods of the present invention, it should be understood that the invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0027] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the 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 and so forth.

[0028] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0029] As used herein, the term "about" in relation to a numerical value is intended to include any additional numerical values ​​that are reasonably close to the numerical value shown. For example, and based on the context, a value may vary up or down by 5-10%. For example, for a value of about 100, 90 to 110 (or any value between 90 and 110) is intended.

[0030] 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.

[0031] 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 the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the present disclosure. Preferred methods and materials are now described.

[0032] Mucin 1 (also known as CD227, episialin, PEM, H23Ag, EMA, CA15-3, and MCA) is a transmembrane glycoprotein that is aberrantly expressed on many epithelial cancer cells and varies in its cellular distribution, function, and glycosylation. The protein is a heterodimer in which a large extracellular domain is covalently bound to a small intracellular domain. The extracellular domain consists of a large tandem repeat region (VNTR) and a non-tandem region. The C-terminal domain of MUC1 consists of interaction sites for several signaling molecules and has been shown to have oncogenic potential.

[0033] Human clinical trials testing mucin 1 (MUC1) as a tumor-associated antigen therapeutic target have used several approaches to generate cytotoxic T cells capable of killing MUC1-expressing cancer cells. These clinical trials include the use of MUC1 polypeptides, DNA sequences, or viral vectors composed of T cell epitopes derived from the N-terminal immunogenic VNTR region and non-VNTR regions of MUC1 to generate cytotoxic T cells. None of these approaches met the criteria for clinical benefit because they failed to generate sufficient T cells capable of killing MUC1-expressing cancer cells. Current approaches target sequences and peptide modifications from the C-terminus of MUC1 to generate epitope-enhanced polypeptides that could improve vaccine immunogenicity.

[0034] Cross presentation refers to that soluble protein or peptide enters cell from the outside and enters the immune pathway of MHC I class processing pathway. This can occur in two ways, via cytoplasmic pathway or endosomal pathway. In these two pathways, peptide / protein is initially taken in endosome / phagosome. In the cytoplasmic pathway, a part of the endosomal protein of one or more partial degradations finally enters cytoplasm, where they are processed by proteasome, and the resulting peptide is transported to the endoplasmic reticulum or other endosomes for being combined with MHC I class by antigen processing related transporter (TAP). On the other hand, protein can be degraded in endosome, and peptide can be combined with the MHC I class present in endosome. The latter approach does not rely on proteasome and is inefficient, because it relies on endosomal protease to accidentally produce correct peptide. Protein entering the early endosome containing limited proteolytic activity is conducive to cross presentation, and the late endosome containing higher level proteolytic activity can suppress cross presentation.

[0035] Certain specific enantiomeric cationic lipids are unique in their ability to rapidly bind to antigen-presenting dendritic cells in a receptor-independent manner and be taken up into early endosomes along with the relevant peptide or protein antigen. We demonstrate that these specific cationic lipids can facilitate the entry of orders of magnitude more protein and peptide antigens into the MHC class I and MHC class II pathways than other current approaches to induce dendritic cell maturation, such as using alum or oil-in-water adjuvants. Furthermore, vaccination with these peptide-antigen-associated cationic lipid nanoparticles induces superior T cell immune responses in vivo compared to either the peptide alone or the peptide formulated with traditional adjuvants.

[0036] In one embodiment, the present invention provides a polyepitopic peptide having at least 80%, 85%, 90% or 95% sequence identity to the amino acid sequence comprising SEQ ID NO:1.

[0037] For example, a polyepitope peptide has at least 80% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. A polyepitope peptide has at least 85% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. A polyepitope peptide has at least 90% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. A polyepitope peptide has at least 91% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. A polyepitope peptide has at least 92% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. A polyepitope peptide has at least 93% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. A polyepitope peptide has at least 94% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. A polyepitope peptide has at least 95% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. A polyepitope peptide has at least 96% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. A polyepitope peptide has at least 97% sequence identity with an amino acid sequence comprising SEQ ID NO: 1. The polyepitope peptide has at least 98% sequence identity to the amino acid sequence comprising SEQ ID NO: 1. The polyepitope peptide has at least 99% sequence identity to the amino acid sequence comprising SEQ ID NO: 1. The polyepitope peptide has at least 99.5% sequence identity to the amino acid sequence comprising SEQ ID NO: 1. The polyepitope peptide has at least 99.9% sequence identity to the amino acid sequence comprising SEQ ID NO: 1.

[0038] 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 (with respect to DNA) and translated (with respect to mRNA) into a polypeptide 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. The coding sequence may 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. The transcription termination sequence will typically be located 3' of the coding sequence.

[0039] The terms "sequence identity" or "percent identity" are used interchangeably herein. In order to determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison (e.g., a gap can be introduced into the sequence of the first polypeptide or polynucleotide for optimal comparison with the second polypeptide or polynucleotide sequence). The amino acids or nucleotides at the 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 the position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e., overlapping positions) x 100). In some embodiments, the length of the reference sequence (e.g., SEQ ID NOs: 1-37) aligned for comparison is at least 80% of the length of the comparison sequence, and in some embodiments, at least 90% or 100%. In one embodiment, the two sequences are identical in length.

[0040] The range of the degree of sequence identity desired is about 80%-100% and integer values ​​in between. The percentage identity between the disclosed sequence and the claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9%. In general, an exact match represents 100% identity over the length of the reference sequence (e.g., SEQ ID NOs: 1-43).

[0041] Polypeptides and polynucleotides having about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 9999.5% or more identity to the polypeptides and polynucleotides described herein are encompassed by the present disclosure. For example, a polypeptide can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs: 1-43.

[0042] Variants of the disclosed sequences also include peptides or full-length proteins that contain substitutions, deletions, or insertions in the protein backbone that retain at least about 70% homology to the original protein within the corresponding portion. A greater degree of homology departure is permitted if similar amino acids, i.e., conservative amino acid substitutions, are not considered changes in the sequence. Examples of conservative substitutions involve amino acids with identical or similar properties. Illustrative 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 to leucine.

[0043] As used herein, the terms "polyepitope peptide," "multi-epitope peptide," and the like refer to a peptide or polypeptide comprising at least two epitopes as described herein. For example, the multi-epitope peptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more epitopes of the invention.

[0044] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., a part or 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 part of the protein. The terms "epitope", "antigenic peptide", "antigenic epitope", "immunogenic peptide", "antigenic fragment" and "MHC binding peptide" are used interchangeably herein and preferably relate to a representative of an antigen that is capable of inducing an immune response against the antigen or a cell that expresses or contains and preferably presents the antigen. "Antigen" according to the present invention includes any substance that will induce 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). According to the present invention, the term "antigen" includes any molecule that comprises at least one epitope. Preferably, an antigen in the context of the present invention is a molecule that optionally induces an immune response after processing. According to the present invention, any suitable antigen can be used, which is a candidate for an immune response, wherein the immune response is preferably a cellular immune response. In the context of embodiments of the present invention, antigens are preferably presented by cells, preferably by antigen presenting cells (which include diseased cells, particularly cancer cells) in the context of MHC molecules, which results in an immune response against the antigen. The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens include tumor antigens.

[0045] The epitopes referred to in this application include any epitope that can be derived from the MUC1 peptide. This includes, for example, an epitope having the amino acid sequence of SEQ ID NO: 1.

[0046] In another aspect, the polyepitopic peptide comprises a mucin 1 (MUC1) peptide.

[0047] In one aspect, the MUCl peptide has a sequence that is at least 80%, 85%, 90% or 95% identical to the sequence of any one of SEQ ID NOs: 9-14 and 20-37.

[0048] For example, the MUCl peptide has a sequence that is at least 80% identical to the sequence of any one of SEQ ID NOs: 9-14 and 20-37.

[0049] The MUC1 peptide has a sequence that has at least 85% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 91% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 92% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 93% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 94% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 95% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 96% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 97% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 98% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 99% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUC1 peptide has a sequence that has at least 99.5% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37. The MUCl peptide has a sequence that has at least 99.9% sequence identity to the sequence of any one of SEQ ID NOs: 9-14 and 20-37.

[0050] As noted above, peptide epitopes, such as MUCl epitopes, can have affinities for different MHC molecules expressed by antigen presenting cells (APCs), which in turn dictates which immune cell receptors can present those peptide epitopes, which immune cell receptors recognize them, and therefore which type of immune response can be induced by the peptide epitope. As detailed in the Examples section, the MUCl epitopes described herein have affinities for several HLA molecules and are therefore capable of inducing several types of immune responses.

[0051] In some aspects, the MUCl epitope has MHC affinity for HLA-A2, A3, Al 1, and A24. In various aspects, the MUCl epitope is recognized by the CD4+ T cell receptor and / or the CD8+ T cell receptor.

[0052] As used herein, a peptide epitope that is "recognized" by an immune cell receptor may be referred to interchangeably as an "immune cell receptor epitope." That is, a MUCl epitope that is recognized by a CD4+ T cell receptor may be referred to as a CD4+ T cell receptor epitope or a MUCl epitope, depending on the emphasis placed on the peptide from which the epitope is derived (e.g., MUCl) or the immune cell receptor (e.g., CD4+ T cell receptor and / or CD8+ T cell receptor) with which it is recognized (or interacts based on affinity).

[0053] In one aspect, the polyepitopic peptide comprises an amino acid sequence comprising at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO:1.

[0054] In another aspect, the one or more epitope peptides are oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated or biotinylated. In various aspects, the one or more epitope peptides are palmitoylated.

[0055] In one aspect, the one or more epitope peptides comprise a cleavable anionic N-terminal extension. In some aspects, the cleavable anionic N-terminal extension comprises the amino acid sequence SSEEDE (SEQ ID NO: 38).

[0056] In another aspect, the cleavable anionic N-terminal sequence is the amino acid sequence SSEEDEE (SEQ ID NO:39). SEQ ID NO:38 and SEQ ID NO:39 are variations of anionic tags that can be used alternatively. It is noteworthy that any sequence disclosed herein as comprising an anionic tag is disclosed as comprising a variation of the anionic tag. For example, disclosure of SEQ ID NO:18, SSEEDE-YLAIVYLIAL, comprising the anionic tag of SEQ ID NO:38 refers to disclosure of a modified peptide comprising the anionic tag of SEQ ID NO:39. Non-limiting examples of such alternatively modified peptides include SEQ ID NO:42 and SEQ ID NO:43.

[0057] In another embodiment, the present invention provides a composition comprising one or more of the multi-epitope peptides described herein and a cationic lipid.

[0058] As used herein, the term composition is intended to include pharmaceutical compositions that may also contain other therapeutic agents and can be formulated, for example, using conventional pharmaceutically acceptable vehicles or diluents and pharmaceutical additives (e.g., excipients, preservatives, etc.) of a type suitable for the desired mode of administration according to techniques known in the art of pharmaceutical formulation. In certain embodiments, the compositions disclosed herein are formulated with additional agents that promote entry into the desired cells or tissues. Such additional agents include micelles, liposomes, and dendrimers.

[0059] The term "pharmaceutically acceptable" refers to the fact 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 a combination thereof, can be administered to a subject with the conjugate of the present invention without causing any undesirable biological effects or interacting in an undesirable manner with any other components of the pharmaceutical composition in which it is contained.

[0060] Pharmaceutical compositions comprising the peptides or compositions described herein can be administered by any suitable means, e.g., parenteral, such as by subcutaneous, intravenous, intramuscular, intrathecal or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions) in dosage formulations containing a non-toxic, pharmaceutically acceptable vehicle or diluent. Depending on the condition being treated, these pharmaceutical compositions can be formulated and administered systemically or topically. Techniques for formulation and administration are well known in the art. Suitable routes can be, for example, parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous or intraperitoneal. For injection, the pharmaceutical compositions of the present invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as water, Hanks solution, Ringer's solution or physiologically buffered saline.

[0061] Adjuvants are often used to modify or enhance the effectiveness of vaccines by stimulating the immune system to make a more powerful response to vaccines and thereby provide increased immunity to specific diseases. Adjuvants accomplish this task by mimicking a specific evolutionarily conserved group of molecules (so-called pathogen-associated molecular patterns), including liposomes, lipopolysaccharides, molecular cages for antigens, components of bacterial cell walls, and endocytic nucleic acids, such as RNA, double-stranded RNA, single-stranded DNA, and DNA containing unmethylated CpG dinucleotides. Because the immune system has evolved to recognize these specific antigenic moieties, the presence of adjuvants in conjunction with vaccines can greatly increase the innate immune response to antigens by enhancing the activity of dendritic cells, lymphocytes, and macrophages by mimicking natural infection.

[0062] The compositions described herein can be formulated with lipid nanoparticles as adjuvants to enhance the presentation of antigens to antigen-presenting cells and thereby increase the immune response induced by the antigen.

[0063] In some aspects 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 have a protonatable group and are positively charged at a pH below the pKa.

[0064] Suitable cationic lipids according to the present disclosure include, but are not limited to: 3-β[4N1N,8-biguanidinospermidine)-carbamoyl]cholesterol (BGSC); 3-β[N,N-biguanidinoethyl-aminoethane)-carbamoyl]cholesterol (BGTC); N,N.1N2N3 tetramethyltetrapalmitylspermine (cellfectin); N-tert-butyl-N'-tetradecyl-3-tetradecyl-aminopropionamidine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-dioleoyloxy-N-[2(sperminamido)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propylamine salt)(2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-p-ropanaminium trifluorocetate))(DOSPA); 1,3-dioleoyloxy-2-(6-carboxycetyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-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); Nl-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) or other N-(N,Nl-dioxane) oxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; 1,2-dioleoyl-3-(4'-trimethylammonium)butanol-sn-glycerol (DOBT) or cholesterol (4'trimethylammonium) butyrate (ChOTB), wherein the trimethylammonium group is linked to the double chain (for DOTB) or cholesterol group (for ChOTB) via a butanol spacer; DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) (DORIE) or their analogs, such as WO 93 / 03709; 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC); cholesterol hemisuccinate (ChOSC); lipopolyamines, such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidylethanolamylspermine (DPPES), cholesterol-3β-carboxamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesterol carboxylate iodide, cholesterol-3-O-carboxamidoethyleneamine, cholesterol-3-β-oxysuccinyl-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesterol-3-β-oxysuccinate iodide, 2-(2- trimethylammonium)-ethylmethylaminoethyl-cholesterol-3-β-oxysuccinate iodide, 3-β-N-(N',N'-dimethylaminoethane)carbamoylcholesterol (DC-chol), and 3-β-N-(polyethyleneimine)carbamoylcholesterol; O,O'-dimyristyl-N-lysyl-aspartic acid (DMKE); O,O'-dimyristyl-N-lysyl-glutamate (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-trimethylammonium propane (DOTAP); dioleoyldimethylaminopropane (DODAP); 1,2-palmitoyl-3-trimethylammonium propane (DPTAP); 1,2-distearoyl-3-trimethylammonium propane (DSTAP), 1,2-myristoyl-3-trimethylammonium propane (DMTAP); and sodium dodecyl sulfate (SDS). In addition, structural variants and derivatives of any of the cationic lipids are also contemplated. ;

[0065] In some aspects, the cationic lipid is selected from DOTAP, DOTMA, DOEPC and combinations thereof. In other aspects, the cationic lipid is DOTAP. In yet other aspects, the cationic lipid is DOTMA. In other aspects, the cationic lipid is DOEPC. In some aspects, the cationic lipid is purified.

[0066] In some embodiments, the cationic lipid is an enantiomer of a cationic lipid. The term "enantiomer" refers to a stereoisomer of a cationic lipid that is a non-superimposable mirror image of a stereoisomer of its counterpart, such as an R and S enantiomer. In each example, 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 aspects, the enantiomer is purified.

[0067] In one aspect, the cationic lipid is selected from DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations thereof, and analogs thereof.

[0068] In another aspect, the polyepitope peptide is oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated.

[0069] In one aspect, the epitope peptide is a mucin 1 (MUC1) epitope.

[0070] In one aspect, the MUCl epitope has MHC affinity for HLA-A2, A3, Al 1, and A24.

[0071] In another aspect, the MUCl epitope is recognized by the CD4+ T cell receptor and / or the CD8+ T cell receptor.

[0072] In one aspect, the one or more epitope peptides comprise a cleavable anionic N-terminal extension. In some aspects, the cleavable anionic N-terminal extension comprises the amino acid sequence SSEEDE (SEQ ID NO: 38) or SSEEDEE (SEQ ID NO: 39).

[0073] In another aspect, one or more polyepitope peptides are encapsulated in liposomes comprising cationic lipids or mixed as micelles with preformed cationic lipid nanoparticles. In some aspects, one or more polyepitope peptides and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

[0074] In one aspect, the composition further comprises an enhancer agonist epitope and / or an analog thereof.

[0075] In additional embodiments, the present invention provides a vaccine composition comprising: (a) one or more polyepitope peptides comprising at least two peptides that are targeted by CD4 + and / or CD8 + An epitope peptide recognized by a T cell receptor, wherein the epitope peptide is a mucin 1 (MUC1) epitope; and (b) a cationic lipid.

[0076] According to the present invention, the term "vaccine" refers to a pharmaceutical preparation (pharmaceutical composition) or product that, when administered, induces an immune response, particularly a cellular immune response, that recognizes and attacks pathogens or diseased cells, such as cancer cells. Vaccines can be used to prevent or treat disease. The term "personalized cancer vaccine" refers to a specific cancer patient and means that the cancer vaccine is tailored to the needs or special circumstances of the individual cancer patient.

[0077] In one aspect, the cationic lipid is selected from DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations thereof, and analogs thereof. In some aspects, the cationic lipid is R-DOTAP.

[0078] In another aspect, at least two epitope peptides recognized by the CD4+ and / or CD8+ T cell receptors comprise a sequence comprising at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 2-37 and 42-43. In one aspect, the MUC1 epitope has MHC affinity for HLA-A2, A3, A11, and A24. In another aspect, one or more epitope peptides are oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated. In some aspects, one or more epitope peptides are palmitoylated. In one aspect, one or more epitope peptides comprise a cleavable anionic N-terminal extension. In some aspects, the cleavable anionic N-terminal extension comprises the amino acid sequence SSEEDE (SEQ ID NO: 38) or SSEEDEE (SEQ ID NO: 39). In another aspect, at least two epitope peptides recognized by CD4+ and / or CD8+ T cell receptors comprise a sequence comprising at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 9, 10, 13, 14, 18, 19, 42, or 43. In one aspect, the polyepitope peptide comprises a sequence comprising at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 1. In another aspect, at least two epitope peptides recognized by CD4+ and / or CD8+ T cell receptors are covalently modified to improve association with cationic lipids. In some aspects, the covalent modification comprises palmitoylation or the addition of an anionic sequence. In various aspects, the modified peptide comprises an amino acid sequence comprising at least 80% identity to SEQ ID NOs: 18, 19, 42, or 43. In another aspect, one or more polyepitope peptides are encapsulated in liposomes comprising cationic lipids or mixed as micelles with preformed cationic lipid nanoparticles. In some aspects, at least two polyepitopic peptides and preformed cationic lipid nanoparticles are mixed in a 1: 1 ratio. In one aspect, one or more polyepitopic peptides comprise a sequence comprising at least 80% identity to the amino acid sequence of SEQ ID NO:1.

[0079] In a further embodiment, the present invention provides a method of treating cancer in a subject, comprising administering to the subject a vaccine composition comprising: (a) a multi-epitope peptide comprising one or more mucin 1 (MUC1)-derived epitopes; and (b) a cationic lipid, thereby treating the cancer in the subject.

[0080] As used herein, the term "subject" refers to any individual or patient to whom the subject method is administered. Typically, the subject is a human, although as will be appreciated by those skilled in the art, the subject can be an animal. Thus, other animals, including vertebrates such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals including cattle, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of subject.

[0081] The term "treatment" is used interchangeably herein with the term "therapeutic method" and refers to 1) therapeutic treatment or measures that cure, slow down, alleviate the symptoms of, and / or prevent the progression of a diagnosed pathological condition or disorder, and 2) preventative / prophylactic measures. Those in need of treatment may include individuals already suffering from a particular medical condition as well as those who may eventually acquire the condition (i.e., those in need of preventative measures).

[0082] The terms "therapeutically effective amount," "effective dose," "therapeutically effective dose," "effective amount," and the like refer to the amount of the subject compound that will elicit the biological or medical response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or other clinician. Generally, the response is an improvement in symptoms or a desired biological outcome (e.g., treatment of cancer) in a patient.

[0083] The term "administration of" and or "administering" should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount 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, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual, rectal, vaginal, naso-ocular administration, as well as infusion, inhalation and nebulization.

[0084] In one aspect, the one or more MUCl-derived epitopes comprise a sequence comprising at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 2-37 and 42- 43. In another aspect, the polyepitope peptide comprises a sequence comprising at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 1.

[0085] In one aspect, the cationic lipid is selected from DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations thereof, and analogs thereof. In some aspects, the cationic lipid is R-DOTAP.

[0086] In another aspect, the multi-epitope peptides are encapsulated in cationic lipid nanoparticles or mixed as micelles with preformed cationic lipid nanoparticles.

[0087] In some aspects, the multi-epitope peptide and pre-formed cationic lipid nanoparticles are mixed in a 1:1 ratio.

[0088] In one aspect, one or more MUCl-derived epitopes induce presentation of non-HLA restricted peptides to CD4 by antigen presenting cells. + and CD8 + T cells.

[0089] The epitopes and polyepitopic peptides described herein are HLA class I and / or HLA class II non-restricted epitopes.

[0090] The human leukocyte antigen (HLA) system or complex is a gene complex located on chromosome 6 in humans and encodes cell surface proteins responsible for 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 they have many different alleles, allowing them to fine-tune the adaptive immune system. The HLAs (A, B, and C), which correspond to MHC class I, are all HLA class 1 groups and present peptides from the inside of the cell. These peptides are produced by digested proteins that are broken down in the proteasome. In general, these specific peptides are small polymers, 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-positive or cytotoxic T-cells), which destroy cells. MHC class I proteins are associated with β2-microglobulin, which, unlike HLA proteins, is encoded by genes on chromosome 15. The HLAs (DP, DM, DO, DQ and DR) corresponding to MHC class II present antigens from the extracellular space to T-lymphocytes. These specific antigens stimulate the proliferation of T-helper cells (also known as CD4-positive T cells), which in turn stimulate antibody-producing B-cells to produce antibodies against the specific antigens. Self-antigens are suppressed by regulatory T cells.

[0091] MHC-restricted antigen recognition, MHC restriction, or HLA-restriction refers to the fact that T cells can interact with self-major histocompatibility complex molecules and foreign peptides bound to them, but will only respond to an antigen when it is bound to a specific MHC molecule. When foreign proteins enter cells, they are broken down into peptides. These peptides, or antigens, can be derived from pathogens such as viruses or intracellular bacteria. The foreign peptides are brought to the cell surface and presented to the T cell by proteins called the major histocompatibility complex (MHC). During T cell development, the T cell undergoes a selection process in the thymus to ensure that the T cell receptor (TCR) will not recognize MHC molecules that present self-antigens, that is, its affinity is not too high. High affinity means that it will be self-reactive, but no affinity means that it will not bind to the MHC strongly enough. This selection process results in the development of T cells with specific TCRs, which may only respond to certain MHC molecules and not others. The fact that the TCR will only recognize some MHC molecules but not others contributes to "MHC restriction." The biological reason for MHC restriction is to prevent the production of extra wandering lymphocytes, thereby saving energy and cell building materials. T-cells are a type of lymphocyte that is important in the immune system to activate other immune cells. T-cells will recognize foreign peptides through T-cell receptors (TCRs) on the surface of the T cell, and then perform different actions depending on the type of T cell to which they belong, to protect the host from foreign peptides that may have come from pathogens such as bacteria, viruses or parasites. By enforcing the restriction that T cells are only activated by peptide antigens when the antigen is bound to self-MHC molecules, MHC restriction adds another dimension to the specificity of the T cell receptor, so that the antigen is only recognized as a peptide-MHC complex. MHC restriction in T cells occurs during their development in the thymus, specifically positive selection. Only thymocytes (T cells developing in the thymus) that can bind to MHC molecules with appropriate affinity can receive survival signals and enter the next level of selection. MHC restriction is important for T cells to function properly once they leave the thymus because it allows the T cell receptor to bind to MHC and detect cells infected with intracellular pathogens, viral proteins, and cells with genetic defects.

[0092] In another aspect, treating cancer includes preventing the progression of cancer in a subject.

[0093] Cancer is a group of diseases involving abnormal cell growth that has the potential to infect or spread to other parts of the body. In 2015, approximately 90.5 million people were living with cancer, with approximately 14.1 million new cases occurring each year, and it causing approximately 8.8 million deaths (15.7% of deaths). The most common types of cancer in men are lung cancer, prostate cancer, colorectal cancer, and stomach cancer. In women, the most common types are breast cancer, colorectal cancer, lung cancer, and cervical cancer.

[0094] The term "cancer" refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation that begins in one site (primary site) with the potential to infect and spread to other sites (secondary sites, metastases), which distinguishes cancer (malignant tumors) from benign tumors. Almost all organs can be affected, resulting in more than 100 types of cancer that can affect humans. Cancer can be caused by many causes, including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, smoking and / or alcohol consumption, obesity, poor diet, lack of physical activity, or any combination thereof.

[0095] As used herein, "neoplasm" or "tumor," including grammatical variations thereof, refers to a new and abnormal growth of tissue, which may be benign or cancerous. In related aspects, a neoplasm is indicative of a neoplastic disease or condition, 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.

[0096] Exemplary cancers described by the National Cancer Institute include: acute lymphoblastic leukemia in adults; acute lymphoblastic leukemia in children; acute myeloid leukemia in adults; adrenocortical carcinoma; adrenocortical carcinoma in children; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; cerebellar astrocytoma in children; cerebral astrocytoma in children; extrahepatic bile duct cancer; bladder cancer; bladder cancer in children; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brain stem glioma in children; brain tumors in adults; brain tumors, brain stem glioma in children; brain tumors, cerebellar astrocytoma in children; brain tumors, cerebral astrocytoma / malignant glioma in children; brain tumors, ependymoma in children; medulloblastoma in children Childhood Brain Tumors, Supratentorial Primary Neuroectodermal Tumors; Childhood Brain Tumors, Visual Pathway and Hypothalamic Gliomas; Childhood Brain Tumors (Other); Breast Cancer; Breast Cancer and Pregnancy; Childhood Breast Cancer; Male Breast Cancer; Childhood Bronchial Adenoma / Carcinoid Tumors: Childhood Carcinoid Tumors; Gastrointestinal Carcinoid Tumors; Adrenal Cortical Carcinoma; Islet Cell Carcinoma; Carcinoma of Unknown Primary; Primary Central Nervous System Lymphoma; Childhood Cerebellar Astrocytoma; Childhood Cerebral Astrocytoma / Malignant Glioma; Cervical Cancer; Childhood Cancer; Chronic Lymphocytic Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of the Tenosynovium; Colon Cancer; Childhood Colorectal Cancer; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Childhood Ependymoma; Epithelial Ovarian Cancer; Esophageal cancer; esophageal cancer in children; Ewing family of tumors; extracranial ectodermal tumors in children; gonadal ectodermal tumors; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; stomach (gastric) cancer; stomach (gastric) cancer in children; gastrointestinal carcinoid tumors; extracranial ectodermal tumors in children; gonadal ectodermal tumors; ovarian ectodermal tumors; gestational trophoblastic tumors; brainstem gliomas in children; optic pathway and hypothalamic gliomas in children; hairy cell leukemia; head and neck cancer; primary hepatocellular carcinoma in adults; primary hepatocellular carcinoma in children; Hodgkin lymphoma in adults; Hodgkin lymphoma in children; Hodgkin lymphoma during pregnancy; hypopharyngeal cancer; hypothalamic and optic pathway gliomas in children; intraocular melanoma; pancreatic islet cell carcinoma Cancer (endocrine pancreas); Kaposi's sarcoma; kidney cancer; laryngeal cancer; childhood laryngeal cancer; acute lymphoblastic leukemia (adults); acute lymphoblastic leukemia (children); acute myeloid leukemia (adults); acute myeloid leukemia (children); chronic lymphocytic leukemia; chronic myeloid leukemia; hairy cell leukemia; lip and oral cavity cancer; liver cancer (primary) in adults; liver cancer (primary) in children; non-small cell lung cancer; small cell lung cancer; acute lymphoblastic leukemia (adults); acute lymphoblastic leukemia (children); chronic lymphocytic leukemia; AIDS-related lymphoma; central nervous system (primary) lymphoma; cutaneous T-cell lymphoma; Hodgkin lymphoma (adults); Hodgkin lymphoma (children); 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; Melanoma; Intraocular Melanoma; Merkel Cell Carcinoma; Malignant Mesothelioma; Metastatic Squamous Neck Cancer with Occult Primary Primary); Multiple Endocrine Neoplasia Syndrome (Childhood); Multiple Myeloma / Plasma Cell Neoplasms; Mycosis Fungoides; Myelodysplasia Syndrome; Chronic Myelogenous Leukemia; Acute Myelogenous Leukemia (Childhood); Multiple Myeloma; Chronic Myeloproliferative Disorders; Nasal and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer (Childhood); Neuroblastoma; Adult Non-Hodgkin Lymphoma; Childhood Non-Hodgkin Lymphoma; Non-Hodgkin Lymphoma (Pregnancy); Non-Small Cell Lung Cancer; Oral Cancer (Childhood); Oral and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer (Childhood); Epithelial Ovarian Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor Tumor); pancreatic cancer; pancreatic cancer, islet cell pancreatic cancer in children; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pheochromocytoma; primary neuroectodermal tumors of the pineal gland and supratentorial gland in children; pituitary tumors; plasma cell neoplasms / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; pregnancy and Hodgkin lymphoma; pregnancy and non-Hodgkin lymphoma; primary central nervous system lymphoma; primary liver cancer in adults; primary liver cancer in children; prostate cancer; rectal cancer; renal cell (kidney) cancer; renal cell cancer in children; renal pelvis and ureter, transitional cell cancer; retinoblastoma; rhabdomyosarcoma in children; salivary gland cancer; salivary gland cancer in children; Ewing family of tumors; Kaposi's sarcoma; sarcomas (osteosarcoma, malignant fibrous histiocytoma of bone; sarcomas, rhabdomyosarcomas in children; soft tissue cancer in adults) Sarcoma; childhood soft tissue sarcoma; Sézary syndrome; skin cancer; childhood skin cancer; skin cancer (melanoma); Merkel cell skin cancer; small cell lung cancer; small bowel cancer; adult soft tissue sarcoma; childhood soft tissue sarcoma; occult primary metastatic squamous neck cancer; stomach (gastric) cancer; childhood stomach (gastric) cancer; childhood primary neuroectodermal tumor (supratentorial) of the kidneys; cutaneous T-cell lymphoma; testicular cancer; childhood thymoma; malignant thymoma; thyroid cancer; childhood thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic tumor; cancer of unknown primary site in children; rare childhood cancers; ureteral and renal pelvis, transitional cell carcinoma; urethral cancer; uterine sarcoma; vaginal cancer; optic pathway and hypothalamic glioma in children; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0097] In one aspect, the cancer comprises cancer cells that express MUCl.

[0098] MUC1 is expressed in a variety of tissues and organs, including the nasopharynx, bronchi, stomach, colon, rectum, gallbladder, fallopian tube, endometrium, cervix, placenta, lung, esophagus, duodenum, small intestine, pancreas, kidney, bladder, testis, epididymis, seminal vesicle, breast, appendix, adrenal gland, oral mucosa, salivary gland, prostate, skin, lymph nodes, tonsils and bone marrow. Thus, non-limiting examples of cancers comprising cells expressing MUC1 include nasopharyngeal carcinoma, lung cancer, gastric cancer, colorectal cancer, gallbladder cancer, fallopian tube cancer, endometrial cancer, cervical cancer, esophageal cancer, duodenum cancer, small intestine cancer, pancreatic cancer, kidney cancer, bladder cancer, testicular cancer, epididymal cancer, seminal vesicle cancer, breast cancer, adrenal cancer, oral mucosa cancer, salivary gland cancer, prostate cancer, skin cancer, melanoma, lymphoma, tonsil cancer, myeloma and multiple myeloma.

[0099] In some aspects, the cancer is prostate cancer, breast cancer, or acute myeloid leukemia (AML).

[0100] In another aspect, the method further comprises administering an anti-cancer therapy to the subject.

[0101] As used herein, the term "anti-cancer therapy" refers to any therapeutic method that can be used to treat cancer, including, but not limited to, surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, and any combination thereof.

[0102] In some aspects, administration can be combined with one or more additional therapeutic agents. The phrases "combination therapy", "combined with..." and the like refer to the simultaneous use of more than one drug therapy or treatment to increase the response. The composition of the present invention can be used in combination with other drugs or treatments in use 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 therapy such as an immune checkpoint inhibitor therapy. This therapy can be administered before, simultaneously with, or after the administration of the composition of the present invention.

[0103] In some aspects, the anti-cancer treatment comprises immune checkpoint inhibitor therapy.

[0104] Checkpoint inhibitor therapy is a current form of cancer treatment that uses immune checkpoints, which affect how the immune system functions. Immune checkpoints can be either stimulatory or inhibitory. Tumors can use these checkpoints to protect themselves from immune system attack. Checkpoint therapy blocks inhibitory checkpoints, thereby restoring immune system function. 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-associated protein 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 inhibitor of T cell activation).

[0105] Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a cell surface receptor that plays an important role in downregulating the immune system and promoting self-tolerance by inhibiting T cell inflammatory activity. PD-1 is an immune checkpoint and prevents autoimmunity through a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells).

[0106] PD-1 has two ligands, PD-L1 and PD-L2, both members of the B7 family. PD-L1 protein is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment and on T and B cells during TCR and B cell receptor signaling, while PD-L1 mRNA can be detected in the heart, lung, thymus, spleen, and kidney of resting mice. PD-L1 is expressed on nearly all murine tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma, upon treatment with IFN-γ. PD-L2 expression is more restricted and is primarily expressed by DCs and a few tumor lines.

[0107] CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that downregulates the immune response when it functions as an immune checkpoint. CTLA4 is constitutively expressed in regulatory T cells, but is upregulated in conventional T cells only after activation - a phenomenon that is particularly noteworthy 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 co-stimulatory 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 it to outcompete CD28 for its ligands. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. CTLA4 is also found in regulatory T cells and contributes to their inhibitory function. Activation of T cells through the T cell receptor and CD28 results in increased CTLA-4 expression.

[0108] There are several checkpoint inhibitors currently 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 iplimumab (Yervoy). There are several other checkpoint inhibitors in development, including an anti-B7-H3 antibody (MGA271), an anti-KIR antibody (lirilumab), and an anti-LAG3 antibody (BMS-986016).

[0109] So-called "directed therapy" refers to any molecular directed therapy that blocks cancer cell growth by interfering with specific targeted molecules required for carcinogenesis and tumor growth, rather than by simply interfering with all rapidly dividing cells (e.g., using traditional chemotherapy). Biomarkers are often needed to help select patients who are likely to respond to a given directed therapy. Non-limiting examples of directed therapy include: tyrosine kinase inhibitors, small molecule drug conjugates, serine / threonine kinase inhibitors, monoclonal antibodies, and histone deacetylase (HDAC) inhibitors.

[0110] In other aspects, targeted therapies include HDAC inhibitors.

[0111] Histone deacetylase (HDAC) is a class of enzymes that remove acetyl groups (O=C-CH3) from the ε-N-acetyllysine amino acids on histone and non-histone proteins. HDACs allow histones to wrap around DNA more tightly. This is important because DNA is wrapped around histones and DNA expression is regulated by acetylation and deacetylation. The effect of HDAC is opposite to that of histone acetyltransferase. HDAC proteins are now also referred to as lysine deacetylases (KDACs) to describe their function rather than their target, which also includes non-histone proteins. In general, they inhibit gene expression.

[0112] Histone deacetylase inhibitors (HDAC inhibitors, HDACi, HDIs) are compounds that inhibit histone deacetylases. "Classic" HDIs specifically target class I, II, and IV HDACs by binding to their zinc-containing catalytic domains. These classic HDIs can be categorized into groups named after 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, in descending order of typical zinc-binding affinity, include: 1. hydroxamic acids (or hydroxamates), such as trichostatin A; 2. cyclic tetrapeptides (such as trapoxin B) and ester peptides; 3. benzamides; 4. electrophilic ketones; and 5. aliphatic acid compounds, such as phenylbutyric acid and valproic acid.

[0113] "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). Sirtuin III class HDACs are NAD+ dependent and are therefore inhibited by nicotinamide and derivatives of NAD, dihydrocoumarins, naphthopyranone, and 2-hydroxynaphthaldehyde. Non-limiting examples of HDAC inhibitors include Istodax (romidepsin), Zolinza (vorinostat), Farydak (panobinostat), and Belodaq (belinostat).

[0114] In one aspect, treating cancer comprises inducing a MUCl -specific multifunctional cytolytic T cell response in a subject.

[0115] The term "immune response" refers to the overall body response to an antigen, and preferably refers to a cellular immune response or a cellular and humoral immune response. The immune response may be protective / prophylactic / preventative and / or therapeutic.

[0116] 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 protects the body from pathogens and other foreign substances, destroys infected and malignant cells, and removes cellular debris. This system 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 immunity of the adaptive immune system and are important for immune surveillance. T cells, or T lymphocytes, are a type of lymphocyte that plays a major 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 play a role in regulating immune responses. Killer T cells only recognize antigens coupled to class I MHC molecules, while helper T cells only recognize antigens coupled to class II MHC molecules. These two antigen presentation mechanisms reflect the different roles of the two types of T cells. The third, minor subtype is the γδ T cell, which recognizes intact antigens not bound to MHC receptors. In contrast, B cell antigen-specific receptors are antibody molecules on the surface of B cells and recognize entire pathogens without any need for antigen processing. Each B cell lineage expresses different antibodies, so the complete B cell antigen receptor represents the full range of antibodies that the body can produce.

[0117] By "cellular immune response," "cellular response," "cellular response to an antigen," or similar terms is meant a cellular response that includes responses to cells characterized by antigen presentation using class I or class II MHC. The cellular response involves cells called T cells or T-lymphocytes, which act as "helper cells" or "killer cells." Helper T cells (also known as CD4+ T cells) play a major role by regulating the immune response, and killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill diseased cells, such as cancer cells, thereby preventing the production of more diseased cells. In a preferred embodiment, the present invention is directed to stimulating an anti-tumor CTL response against tumor cells that express one or more tumor-expressed antigens and that preferably present such tumor-expressed antigens using class I MHC.

[0118] The terms "immunoreactive cell", "immune cell" or "immune effector cell" in the context of the present invention relate to cells that perform effector functions during an immune response. An "immunoreactive cell" is preferably capable of binding to an antigen or is a cell characterized by presenting an antigen or an antigenic peptide derived from an antigen and mediating an immune response. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancerous cells and optionally eliminate such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages and dendritic cells.

[0119] "Inducing an immune response" may mean that there is no immune response to a specific antigen before induction, but it may also mean that there is a certain level of immune response to a specific antigen before induction and that the immune response is enhanced after induction. Therefore, "inducing an immune response" also includes "enhancing an immune response". Preferably, after inducing an immune response in a subject, the subject is protected from developing a disease, such as a cancer disease, or the disease condition is improved by inducing the immune response. For example, an immune response against an antigen expressed by a tumor can be induced in a patient suffering from a cancer disease or in a subject at risk of developing a cancer disease. Inducing an immune response in such a case may mean that the subject's disease condition is improved, the subject does not develop metastasis, or a subject at risk of developing a cancer disease does not develop the cancer disease.

[0120] In the human mucin 1 oncoprotein, multiple high-affinity MHC-binding peptide antigens specific for HLA-A2, A3, A11, and A24 have been identified (Table 1). The MHC-binding peptide antigens specific for HLA-A2 have been identified: YLAIVYLIAL (SEQ ID NO: 9, C1A), YLIALAVCQV (SEQ ID NO: 10, C2A), YLAPPAHGV (SEQ ID NO: 13, V1A), and YLDTRPAPV (SEQ ID NO: 14, V2A).

[0121] Table 1. Shows the sequences of native and modified agonist peptide antigens derived from the human oncoprotein MUCl, where the amino acid substitutions shown in bold underline indicate modifications used to generate agonist antigens with enhanced MHC binding affinity.

[0122] Specifically, all four peptides, C1A (SEQ ID NO: 9) and C2A (SEQ ID NO: 10), V1A (SEQ ID NO: 13) and V2A (SEQ ID NO: 14), have been modified by amino acid substitutions in their native sequences to increase MHC binding affinity, thereby increasing their cellular display on antigen-presenting human dendritic cells and their potential effectiveness in inducing high levels of antigen-specific cytotoxic T cells (see Table 1). However, the agonist amino acid modifications have the negative consequence of leaving the already hydrophobic peptides C1A and C2A without polar residues to promote aqueous solubility. These highly hydrophobic peptide antigens, which have very high MHC binding but limited aqueous solubility, are ineffective molecules for use in traditional vaccine compositions in vivo due to the difficulty in successfully synthesizing and delivering highly purified antigens to the antigen-presenting cells of the immune system in standard vaccine compositions such as CFA or other oil-in-water emulsions or when combined with any water-compatible formulation. Three successful approaches to address this problem have been developed: 1. By synthesizing these hydrophobic peptides with an anionically charged, cleavable N-terminal extension on the peptide antigen, which allows both high-purity peptide synthesis and peptide solubility and compatibility with cationic R-DOTAP immunostimulatory nanoparticles. The association of the anionic domain of the antigen with the cationic surface of the nanoparticle results in high levels of antigen delivery and immune cell activation ( Figure 4 and 5 ).

[0123] 2. These two highly hydrophobic antigens are delivered by inserting them into the nonpolar inner membrane bilayer of cationic surface-charged liposomal nanoparticles formed by the unique enantiomeric immunostimulatory lipid R-DOTAP. This unique delivery vehicle effectively captures water-insoluble peptide antigens and quantitatively delivers them to the dendritic cells of the immune system in highly charged water-compatible immunostimulatory nanoparticles, where the antigens are displayed on the surface MHC of activated dendritic cells to induce high levels of antigen-specific cytotoxic T-cells ( Figure 1 、 2 and 3). This entrapment and delivery of nonpolar peptide antigens within the lipid bilayer of enantiomeric cationic lipid nanoparticles resulted in very high levels of induction of antigen-specific T-cells in ELISpot assays in murine humanized HLA-A2 transgenic mice ( Figure 1 、 2 and 3).

[0124] 3. A third approach to addressing the delivery of hydrophobic peptide antigens C1A and C2A was also developed, involving the incorporation of an amino acid sequence into a much larger peptide derived from the sequence of the MUC1 oncoprotein, which can be efficiently associated with and delivered using R-DOTAP immunostimulatory nanoparticles in an aqueous environment. The large peptide, YL40 (SEQ ID NO: 1), contains two hydrophobic agonist peptides at the N-terminus of the molecule, where they can be efficiently processed by intracellular proteases in dendritic cells to generate separate agonist peptide antigens for display on cell surface MHC. This approach was found to produce high-level detection of peptide agonist C1A-specific T-cell responses following vaccination and ELISpot analysis of humanized transgenic HLA-A mice. Figure 6 and Figure 7 ).

[0125] In the design of peptide-based immunotherapies designed to induce cytotoxic T cells that can recognize and kill tumor cells expressing target antigens, the peptide antigens must be delivered in combination with an effective immune stimulus that can promote efficient antigen uptake by dendritic cells, direct peptide antigen processing into the class I (CD8) pathway, and provide the correct cytokine activation and signaling to induce large amounts of effector T-cells that produce multiple cytokines. The inventors herein have identified peptide sequences that uniquely and effectively associate with R-DOTAP immunostimulatory nanoparticles, which are taken up and processed by dendritic cells in this context and produce high levels of multi-cytokine-producing MUCl-specific killer CD8 T-cells. Many other combinations of MUCl peptide sequences, including short specific peptide antigens from the MUCl protein and other long overlapping peptide sequences, have been shown to be ineffective when combined with or incorporated into R-DOTAP immunostimulatory nanoparticles (see Example 5 below). Therefore, the unique combination of short lipidated agonist peptide epitopes and long MUCl peptide sequences that form high molecular weight micellar structures reported herein results in a strong antigen-specific immune response when delivered in combination with R-DOTAP immunostimulatory nanoparticles. The peptides can be incorporated into immunogenic compositions, such as vaccines. The peptide sequences used in the resulting compositions have the benefit of inducing potent cytotoxic T cell responses.

[0126] Disclosed herein are methods for designing and using unique peptide sequences comprising human cytotoxic T lymphocyte (CTL) epitopes encoded in the MUCl protein. The sequences shown in Table 1, including YL40 (SEQ ID NO: 1), pV1A (SEQ ID NO: 2), pV2A (SEQ ID NO: 3), pC5A (SEQ ID NO: 4), pC6A (SEQ ID NO: 5), and pC7A (SEQ ID NO: 6), are unique epitope-enhancing peptides selected to promote the processing and presentation of T cell antigens encoded in the MUCl protein when delivered in combination with R-DOTAP nanoparticles.

[0127] Table 1: Peptide sequences: Example

[0128] Example 1: Design of MUC1 protein-derived peptides Disclosed herein are methods for the design and use of unique peptide sequences (SEQ ID NOs: 1-6) derived from the MUCl protein that are designed to be efficiently processed and presented to T cells when delivered in conjunction with R-DOTAP immunostimulatory nanoparticles.

[0129] Here, the limited aqueous solubility of the highly hydrophobic peptides C1A and C2A was overcome by developing three successful approaches to address the problem: (1) synthesis of these hydrophobic peptides with an anionically charged, cleavable N-terminal extension on the peptide antigen, (2) delivery of these two highly hydrophobic antigens by inserting them into the nonpolar inner membrane bilayer of cationic surface-charged liposomal nanoparticles formed from the unique enantiomeric immunostimulatory lipid R-DOTAP, and (3) incorporation of the amino acid sequences of individual epitope peptides into larger (polyepitope) peptides.

[0130] Example 2 Effect of adding anionically charged cleavable N-terminal extensions Synthesis of these hydrophobic peptides with an anionically charged, cleavable N-terminal extension on the peptide antigen allows both high-purity synthesis of the peptide and peptide solubility and compatibility with cationic R-DOTAP immunostimulatory nanoparticles. Association of the anionic domain of the antigen with the cationic surface of the nanoparticle results in high levels of antigen delivery and immune cell activation ( Figure 4 and 5 ).

[0131] like Figure 4As exemplified in , HLA-A2-specific CD8 T cell responses were evaluated against a formulation comprising peptide antigens SSEEDE-C1A (SEQ ID NO: 18) and SSEEDE-C2A (SEQ ID NO: 19) formulated with R-DOTAP nanoparticles and a micellar mixture of six lipidated peptide agonist antigens pC3A: (SEQ ID NO: 15), pV1A: (SEQ ID NO: 2), pV2A: (SEQ ID NO: 3), pC5A: (SEQ ID NO: 4), pC6A: (SEQ ID NO: 5), and pP93L: (SEQ ID NO: 16). Due to low peptide solubility in aqueous systems, the highly hydrophobic peptide antigens C1A and C2A are generally inactive in generating an immune response when included in an antigen mixture mixed with R-DOTAP immunostimulatory nanoparticles. Adding an anionic amino acid sequence (SSEEDE, SEQ ID NO: 38) to the N-terminus of the hydrophobic peptide not only allows the synthesis of high-purity antigens, but also ensures that otherwise insoluble antigens are effectively delivered to the dendritic cells of the immune system through strong interactions with cationic nanoparticles in the suspension. These results were confirmed for four replicate preparations of the antigen mixture / R-DOTAP preparation. The efficacy of these vaccine preparations in generating T cell responses was measured by administering two 0.1 ml subcutaneous injections of the vaccine to transgenic mice expressing HLA-A2 (AAD mice) on day 0 and day 7. Negative control mice were vaccinated with the MUC1 peptide formulated in sucrose buffer. By counting MUC1 antigen-specific T cells in triplicate wells in the spleens obtained from vaccinated mice, the specific immune response to the vaccine preparation was measured in an ELISPOT assay. Each bar in the chart represents the average SFU per 250,000 splenocytes of a group of vaccinated mice. Error bars represent the mean ± SEM of five mice in each group. To identify MUCl-specific T cells, splenocytes were stimulated with the peptide sequences C1A (SEQ ID NO:9: YLAIVYLIAL), C2A (SEQ ID NO:10), V1A (SEQ ID NO:13), V2A (SEQ ID NO:14:) C3A (SEQ ID NO:12), and p93L (SEQ ID NO:11).

[0132] like Figure 5As shown in , HLA-A2-specific CD8 T cell responses were evaluated against a formulation comprising peptide antigens SSEEDE-C1A (SEQ ID NO: 9) and SSEEDE-C2A (SEQ ID NO: 10) formulated with R-DOTAP nanoparticles and a micellar mixture of six lipidated peptide agonist antigens pC3A: (SEQ ID NO: 15), pV1A: (SEQ ID NO: 2), pV2A: (SEQ ID NO: 3), pC5A: (SEQ ID NO: 4), pC6A: (SEQ ID NO: 5), and pP93L: (SEQ ID NO: 16). Due to low peptide solubility in aqueous systems, the highly hydrophobic peptide antigens C1A and C2A are generally inactive in generating an immune response when included in an antigen mixture mixed with R-DOTAP immunostimulatory nanoparticles. Adding an anionic amino acid sequence (SSEEDE, SEQ ID NO: 38) to the N-terminus of the hydrophobic peptide not only allows the synthesis of high-purity antigens, but also ensures that otherwise insoluble antigens are effectively delivered to the dendritic cells of the immune system through strong interactions with cationic nanoparticles in the suspension. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml subcutaneous injections of the vaccine to transgenic mice expressing HLA-A2 (AAD mice) on days 0 and 7. Negative control mice were vaccinated with the MUC1 peptide formulated in sucrose buffer. By counting MUC1 antigen-specific T cells in triplicate wells in the spleens obtained from vaccinated mice, the specific immune response to the vaccine formulation was measured in an ELISPOT assay. Each bar in the chart represents the average SFU per 250,000 splenocytes of a group of vaccinated mice. Error bars represent the mean ± SEM of five mice in each group. To identify MUCl-specific T cells, splenocytes were stimulated with the peptide sequences C1A (SEQ ID NO:9: YLAIVYLIAL), C2A (SEQ ID NO:10), V1A (SEQ ID NO:13), V2A (SEQ ID NO:14) C3A (SEQ ID NO:12), and p93L (SEQ ID NO:11).

[0133] Example 3 Effect of formulation of R-DOTAP cationic surface charged liposomal nanoparticles Two highly hydrophobic antigens were inserted into the nonpolar inner membrane bilayer of cationic surface-charged liposomal nanoparticles formed by the unique enantiomeric immunostimulatory lipid R-DOTAP. This unique delivery vehicle efficiently captured water-insoluble peptide antigens and quantitatively delivered them to the dendritic cells of the immune system within the highly charged, water-compatible immunostimulatory nanoparticles, where the antigens were displayed on the surface MHC of activated dendritic cells to induce high levels of antigen-specific cytotoxic T-cells ( Figure 1 、 2 and 3).

[0134] like Figure 1 As shown in , HLA-A2-specific CD8 T cell peptide antigens derived from MUC1 protein incorporated into R-DOTAP nanoparticles exhibited effective immunoactivity of insoluble C1A (SEQ ID NO: 9) when inserted into the lipid bilayer of liposome nanoparticles, thereby delivering low water-soluble antigens to immune-activated cells using R-DOTAP immunostimulants. Control peptide antigen preparations were prepared with 0.5 ml Montanide plus CpG or an "NCI adjuvant" formulation consisting of 50 μg / ml GM-CSF, 20 μg / ml IL-12, and 0.8 mg / ml HBV core (128-140) peptide in incomplete Freund's adjuvant. The efficacy of these vaccine preparations in generating T cell responses was measured by administering two 0.1 ml subcutaneously delivered vaccine injections to transgenic mice expressing HLA-A2 (AAD mice) on days 0 and 7. Control mice were vaccinated with a single MUC1 peptide formulated in sucrose buffer. The immune response specific to the vaccine formulation was measured in an ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in spleens obtained from vaccinated mice. Each data point in the graph represents the mean SFU per 250,000 splenocytes in vaccinated mice. Error bars represent the mean ± SEM of five mice in each group. To identify MUC1-specific T cells, splenocytes were stimulated with the peptide sequence C1A (SEQ ID NO: 9: YLAIVYLIAL).

[0135] like Figure 2As exemplified in the present invention, HLA-A2-specific CD8 T cell peptide antigens derived from the MUC1 protein incorporated into R-DOTAP nanoparticles exhibited potent immunoreactivity of insoluble C2A (SEQ ID NO: 10) when incorporated into the lipid bilayer of liposome nanoparticles, thereby delivering low water-soluble antigens to immune-activated cells using R-DOTAP immunostimulants. Control peptide antigen preparations were prepared with 0.5 ml of Montanide plus CpG or an "NCI adjuvant" formulation consisting of 50 μg / ml GM-CSF, 20 μg / ml IL-12, and 0.8 mg / ml HBV core (128-140) peptide in incomplete Freund's adjuvant. The efficacy of these vaccine preparations in generating T cell responses was measured by administering two 0.1 ml subcutaneously delivered injections of the vaccine to transgenic mice expressing HLA-A2 (AAD mice) on days 0 and 7. A control group of mice was vaccinated with the MUC1 peptide alone formulated in sucrose buffer. The immune response specific to the vaccine formulation was measured in an ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in spleens obtained from vaccinated mice. Each data point in the graph represents the mean SFU per 250,000 splenocytes in vaccinated mice. Error bars represent the mean ± SEM of five mice in each group. To identify MUC1-specific T cells, splenocytes were stimulated with the peptide sequence C2A (SEQ ID NO: 10: YLIALAVCQV).

[0136] like Figure 3As exemplified in , HLA-A2-specific CD8 T cell responses were evaluated against formulations containing peptide antigens C1A (SEQ ID NO: 9) and C2A (SEQ ID NO: 10) incorporated into the lipid bilayer of R-DOTAP nanoparticles. The highly hydrophobic peptide antigens C1A and C2A are typically inactive in generating an immune response when included in an antigen mixture formulated with R-DOTAP immunostimulatory nanoparticles. Insertion of the hydrophobic peptides into the nanoparticle lipid bilayer ensures that otherwise insoluble antigens are efficiently delivered to the dendritic cells of the immune system, generating a strong immune response comparable to the potent antigens V1A (SEQ ID NO: 13) and V2A (SEQ ID NO: 14) in the micellar peptide antigen component of the vaccine. Lipidated peptide agonist antigens V1A and V2A (0.2-1.0 mg / peptide) forming a micellar mixture and agonist antigens C1A and C2A incorporated into the liposome bilayer of R-DOTAP were formulated in 1 ml of formulation using 3 mg of R-DOTAP. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml subcutaneous injections of the vaccine to transgenic mice expressing HLA-A2 (AAD mice) on days 0 and 7. Negative control mice were vaccinated with the MUC1 peptide formulated in sucrose buffer. The immune response specific to the vaccine formulation was measured in an ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in the spleens obtained from vaccinated mice. Each data point in the chart represents the average SFU per million splenocytes in the vaccinated mice. The error bars represent the mean ± SEM of five mice in each group. To identify MUC1-specific T cells, splenocytes were stimulated with a mixture of peptide sequences C1A (SEQ ID NO: 9: YLAIVYLIAL), C2A (SEQ ID NO: 10: YLIALAVCQV), V1A (SEQ ID NO: 13YLAPPAHGV), and V2A (SEQ ID NO: 14: YLDTRPAPV).

[0137] This entrapment and delivery of nonpolar peptide antigens within the lipid bilayer of enantiomeric cationic lipid nanoparticles resulted in very high levels of induction of antigen-specific T-cells in ELISpot assays in murine humanized HLA-A2 transgenic mice ( Figure 1 、 2 and 3).

[0138] Example 4 Effects of incorporating epitope peptides into polyepitope peptides Incorporating the C1A and C2A amino acid sequences into a much larger peptide derived from the sequence of the MUC1 oncoprotein allows for efficient association and delivery with R-DOTAP immunostimulatory nanoparticles in an aqueous environment. The large peptide, YL40 (SEQ ID NO: 1), contains two hydrophobic agonist peptides at the N-terminus of the molecule, where they can be efficiently processed by intracellular proteases in dendritic cells to generate separate agonist peptide antigens for display on cell surface MHC. The method was found to produce high-level detection of peptide agonist C1A-specific T-cell responses following vaccination and ELISpot analysis of humanized transgenic HLA-A mice. Figure 6 and Figure 7 ).

[0139] like Figure 6 As shown in , HLA-A2-specific CD8 T cell responses to a MUC1 / R-DOTAP vaccine formulation containing the long MUC1 peptide antigen YL-40 (containing C1A (SEQ ID NO: 2) and C2A (SEQ ID NO: 3) antigens) were evaluated. The inclusion of peptide YL40 (SEQ ID NO: 1) resulted in a strong antigen-specific response to antigens C1A and C2A. Lipidated peptide antigen agonists derived from MUC1 (0.2-1.0 mg / peptide) were formulated as peptide micelles along with the long peptide antigen YL-40 in a 1 ml formulation with 3 mg of R-DOTAP. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml subcutaneously delivered injections of the vaccine to transgenic mice expressing HLA-A2 (AAD mice) on days 0 and 7. A control group of mice was vaccinated with the MUC1 peptide formulated in a sucrose / water solution. The immune response specific to the vaccine formulation was measured in an ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in spleens obtained from vaccinated mice. Each data point in the graph represents the mean SFU per million splenocytes in vaccinated mice. Error bars represent the mean ± SEM of five mice in each group. To identify MUC1-specific T cells, splenocytes were stimulated with a mixture of peptide sequences C1A (SEQ ID NO: 9: YLAIVYLIAL), C2A (SEQ ID NO: 10: YLIALAVCQV), V1A (SEQ ID NO: 13 YLAPPAHGV), V2A (SEQ ID NO: 14: YLDTRPAPV).

[0140] like Figure 7As exemplified in , HLA-A2-specific CD8 T cell responses were evaluated to formulations containing peptide antigens C1A (SEQ ID NO: 9) and C2A (SEQ ID NO: 10) incorporated into the lipid bilayer of R-DOTAP nanoparticles. The highly hydrophobic peptide antigens C1A and C2A are generally inactive in generating an immune response when included in an antigen mixture formulated with R-DOTAP immunostimulatory nanoparticles. Insertion of the hydrophobic peptides into the nanoparticle lipid bilayer ensures that otherwise insoluble antigens are efficiently delivered to the dendritic cells of the immune system, thereby generating a strong immune response comparable to the potent antigens pV1A (SEQ ID NO: 2) and pV2A (SEQ ID NO: 3) in the micellar peptide antigen component of the vaccine. Lipidated peptide agonist antigens (0.2-1.0 mg / peptide) forming a micellar mixture and agonist antigens C1A and C2A incorporated into the liposomal bilayer of R-DOTAP were formulated in 1 ml of formulation using 3 mg of R-DOTAP. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml subcutaneous injections of the vaccine to transgenic mice expressing HLA-A2 (AAD mice) on days 0 and 7. Negative control mice were vaccinated with the MUC1 peptide formulated in sucrose buffer. The immune response specific to the vaccine formulation was measured in an ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in the spleens obtained from vaccinated mice. Each data point in the chart represents the average SFU per million splenocytes in the vaccinated mice. The error bars represent the mean ± SEM of five mice in each group. To identify MUC1-specific T cells, splenocytes were stimulated with a mixture of peptide sequences C1A (SEQ ID NO: 9: YLAIVYLIAL), C2A (SEQ ID NO: 10: YLIALAVCQV), V1A (SEQ ID NO: 13YLAPPAHGV), and V2A (SEQ ID NO: 14: YLDTRPAPV).

[0141] Example 5 Effect of mixture and encapsulation on inducing immune response Many combinations of MUCl peptide sequences, including short specific peptide antigens from the MUCl protein and other long overlapping peptide sequences, have been generated and proven to be ineffective when combined with or incorporated into R-DOTAP immunostimulatory nanoparticles.

[0142] For example, and if Figure 8As exemplified in Figure 1, while the long peptide YL40 (SEQ ID NO: 1) induced a significant C1A response (activity), the peptide YL35 (SEQ ID NO: 40) did not produce a significant C2A response (using the same method as YL40); YL35 did induce an immune response to C2A, but it was weak. The long peptide sequence YL40, when mixed with R-DOTAP, enabled a strong immune response to the C-terminus, particularly C1A. The long peptide YL35, which contains the C2A sequence, did not enable a strong immune response when mixed with R-DOTAP. This suggests that not all long peptides have the ability to promote a strong immune response to the incorporated sequence, even when mixed with R-DOTAP.

[0143] Furthermore, it was demonstrated that the short C1A and C2A peptides mixed with R-DOTAP did not produce a significant response, but encapsulation of the short C1A and C2A peptides did produce a significant response. The short peptides C1A and C2A induced an immune response only when incorporated into the R-DOTAP bilayer, but not when mixed with R-DOTAP.

[0144] Figure 8 It was also shown that, as expected, the short peptide in the absence of R-DOTAP was inactive.

[0145] Although the present invention has been described with reference to the above embodiments, it will be understood that modifications and variations are encompassed within the spirit and scope of the present invention. Accordingly, the present invention is limited only by the following claims.

Claims

1. A polyepitopic peptide comprising at least 80%, 85%, 90% or 95% sequence identity to the amino acid sequence comprising SEQ ID NO:

1.

2. The multi-epitope peptide of claim 1, wherein the multi-epitope peptide comprises at least 80%, 85%, 90% or 95% sequence identity to an amino acid sequence comprising any one of SEQ ID NOs: 9-14 and 20-37. 3 . The multi-epitope peptide according to claim 1 , wherein the epitope peptide comprises at least one mucin 1 (MUC1) peptide. 4 . The multi-epitope peptide according to claim 1 , wherein the multi-epitope peptide has MHC affinity for at least one of HLA-A2, HLA-A3, HLA-A11 and / or HLA-A24. The multi-epitope peptide according to claim 3 , wherein the multi-epitope peptide is recognized by CD4+ T cell receptor and / or CD8+ T cell receptor. The multi-epitope peptide according to claim 2 , wherein the multi-epitope peptide comprises a sequence comprising SEQ ID NO:

1.

7. The multi-epitope peptide according to claim 1, wherein the multi-epitope peptide is oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated or biotinylated. The multi-epitope peptide according to claim 7 , wherein the multi-epitope peptide is palmitoylated.

9. The multi-epitope peptide of claim 1, wherein the multi-epitope peptide comprises a cleavable anionic N-terminal sequence.

10. The multi-epitope peptide of claim 9, wherein the cleavable anionic N-terminal sequence comprises the amino acid sequence SSEEDE (SEQ ID NO: 38) or SSEEDEE (SEQ ID NO: 39).

11. A composition comprising a polyepitopic peptide and a cationic lipid, wherein the polyepitopic peptide comprises at least one MUC-1 peptide.

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

1.

13. The composition of claim 12, wherein the at least one MUC-1 peptide comprises at least 80%, 85%, 90% or 95% sequence identity to an amino acid sequence comprising any one of SEQ ID NOs: 9-14 and 20-37.

14. The composition of claim 11, 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, variations thereof, or analogs thereof.

15. The composition of claim 14, wherein the cationic lipid is R-DOTAP.

16. The composition of claim 11, wherein the multi-epitope peptide is oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated.

17. The composition of claim 11, wherein the multi-epitope peptide has MHC affinity for at least one of HLA-A2, HLA-A3, HLA-A11 and / or HLA-A24.

18. The composition according to claim 11, wherein the multi-epitope peptide is recognized by CD4+ T cell receptor and / or CD8+ T cell receptor.

19. The composition of claim 11, wherein the polyepitopic peptide comprises a cleavable anionic N-terminal sequence.

20. The composition of claim 19, wherein the cleavable anionic N-terminal sequence comprises the amino acid sequence SSEEDE (SEQ ID NO: 38) or SSEEDEE (SEQ ID NO: 39).

21. The composition of claim 11, wherein the multi-epitope peptide is encapsulated in liposomes comprising cationic lipids.

22. The composition of claim 21, wherein the multi-epitope peptide and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

23. The composition of claim 11, wherein the multi-epitope peptide is mixed as micelles with preformed cationic lipid nanoparticles.

24. The composition of claim 11, further comprising an enhancer agonist epitope and / or an analog thereof.

25. A vaccine composition comprising: (a) a polyepitope peptide, wherein the polyepitope comprises at least one mucin 1 (MUC1) peptide: and (b) Cationic lipids.

26. The vaccine of claim 25, 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, variations thereof, or analogs thereof.

27. The vaccine of claim 25, wherein the cationic lipid is R-DOTAP.

28. The vaccine of claim 25, wherein the multi-epitope peptide comprises a sequence comprising at least 80%, 85%, 90% or 95% sequence identity to an amino acid sequence comprising SEQ ID NO:

1.

29. The vaccine of claim 28, wherein the at least one MUC-1 peptide comprises a sequence comprising at least 80%, 85%, 90% or 95% sequence identity to an amino acid sequence comprising any one of SEQ ID NOs: 9-14 and 20-37.

30. The vaccine of claim 25, wherein the multi-epitope peptide has MHC affinity for at least one of HLA-A2, HLA-A3, HLA-A11 and / or HLA-A24.

31. The vaccine of claim 25, wherein the multi-epitope peptide is oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated.

32. The vaccine of claim 31 , wherein the multi-epitope peptide is palmitoylated.

33. The vaccine of claim 25, wherein the multi-epitope peptide comprises a cleavable anionic N-terminal sequence.

34. The vaccine of claim 33, wherein the cleavable anionic N-terminal sequence comprises the amino acid sequence SSEEDE (SEQ ID NO: 38) or SSEEDEE (SEQ ID NO: 39).

35. The vaccine of claim 25, wherein the multi-epitope peptide comprises a sequence comprising SEQ ID NO:

1.

36. The vaccine of claim 35, wherein the multi-epitope peptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NOs: 18, 19, 42, or 43.

37. The vaccine of claim 25, wherein the multi-epitope peptide is encapsulated in liposomes comprising cationic lipids.

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

39. The vaccine of claim 25, wherein the multi-epitope peptides are mixed as individual micelles with preformed cationic lipid nanoparticles.

40. The vaccine of claim 25, wherein the multi-epitope peptide comprises a sequence comprising the amino acid sequence of SEQ ID NO:

1.

41. A method of treating cancer in a subject, comprising administering to the subject a vaccine composition comprising: (a) a polyepitopic peptide, wherein the polyepitopic peptide comprises at least one mucin 1 (MUC1) peptide; and (b) cationic lipids, Cancer is thereby treated in the subject.

42. The method of claim 41, wherein the polyepitopic peptide comprises a sequence comprising at least 80%, 85%, 90% or 95% identity to an amino acid sequence comprising SEQ ID NO:

1.

43. The method of claim 41, wherein the at least one MUCl peptide comprises a sequence comprising at least 80%, 85%, 90% or 95% identity to an amino acid sequence comprising any one of SEQ ID NOs: 9-14 and 20-37.

44. The method of claim 41, 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, variations thereof, or analogs thereof.

45. The method of claim 41, wherein the cationic lipid is R-DOTAP.

46. ​​The method of claim 41, wherein the multi-epitope peptide is encapsulated in cationic lipid nanoparticles.

47. The method of claim 46, wherein the multi-epitope peptide and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

48. The method of claim 41, wherein the multi-epitope peptide is mixed as micelles with preformed cationic lipid nanoparticles.

49. The method of claim 41, wherein the one or more MUCl peptides induce presentation of non-HLA restricted peptides to CD4 + and CD8 + T cells.

50. The method of claim 41, wherein treating cancer comprises preventing progression of cancer in the subject.

51. The method of claim 41, wherein the cancer comprises cancer cells expressing MUCl.

52. The method of claim 41, wherein the cancer is prostate cancer, breast cancer, or acute myeloid leukemia (AML).

53. The method of claim 41, further comprising administering an anti-cancer therapy to the subject.

54. The method of claim 53, wherein the anti-cancer treatment comprises immune checkpoint inhibitor therapy.

55. The method of claim 41, wherein treating cancer comprises inducing a MUC-specific multifunctional cytolytic T cell response in the subject.

56. A method of inducing a MUC-specific multifunctional cytolytic T cell response in a subject, comprising administering to the subject a composition comprising: (a) a polyepitopic peptide, wherein the polyepitopic peptide comprises one or more mucin 1 (MUC1) peptides: and (b) cationic lipids, A MUC-specific multifunctional cytolytic T cell response is thereby induced in the subject.

57. The method of claim 56, wherein the polyepitopic peptide comprises a sequence comprising at least 80%, 85%, 90% or 95% identity to an amino acid sequence comprising SEQ ID NO:

1.

58. The method of claim 57, wherein the at least one MUC peptide comprises a sequence comprising at least 80%, 85%, 90% or 95% identity to an amino acid sequence comprising any one of SEQ ID NOs: 9-14 and 20-37.

59. The method of claim 58, 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, variations thereof, or analogs thereof.

60. The method of claim 59, wherein the cationic lipid is R-DOTAP.

61. The method of claim 56, wherein the multi-epitope peptide is encapsulated in cationic lipid nanoparticles.

62. The method of claim 61, wherein the multi-epitope peptide and preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

63. The method of claim 56, wherein the multi-epitope peptide is mixed as micelles with preformed cationic lipid nanoparticles.

64. The method of claim 56, wherein the multi-epitope peptide induces presentation of non-HLA restricted peptides to CD4 by antigen presenting cells. + and CD8 + T cells.

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