Novel T cell-activating immunotherapeutic agents for treating human cancers expressing mucin 1 protein

Abstract

Provided herein are multiepitope peptides comprising at least one mucin 1 (MUC1) peptide, having MHC affinity for at least one HLA serotype, and recognized by CD4+ T cell receptors and / or CD8+ T cell receptors. Also provided herein are compositions comprising the multiepitope peptides and a cationic lipid, including vaccine compositions. In various embodiments, the cationic lipid is R-DOTAP. The present invention also provides methods of using the multiepitope peptides, as well as compositions and vaccine compositions. These methods of use include methods for treating cancer in a subject and methods for inducing MUC-specific polyfunctional and cytolytic T cell responses in a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 417,640, filed October 19, 2022. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated herein by reference in its entirety in the disclosure of this application.

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

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

[0004] Background information A key element of cancer immunotherapy treatment is the activation, expansion, and targeting of cancer antigen-specific cytotoxic T cells; these treatments play a crucial role in modulating the host immune system to recognize tumor-associated antigens, trigger a cellular antitumor immune response, and eliminate targeted cancer cells. Several approaches are currently under development for novel immunotherapeutic agents that activate T cells and target cancer: 1) whole-tumor cell-based immunotherapeutics, which combine tumor cell components with immunostimulatory adjuvant compounds; 2) immunotherapeutics based on similar recombinant tumor antigen proteins / peptides; and 3) immunotherapeutics based on DNA / RNA encoding the target tumor antigen. In all other previous studies developed to date, the various immunostimulatory adjuvants utilized in these therapeutics, including oil-in-water emulsions, alum particles, and various polysaccharide and lipid components, have failed to induce potent, effective, safe, and polyfunctional cytotoxic T cell responses in patients. There are several significant hurdles that must be overcome for such therapeutic cancer immunotherapeutics to induce antitumor immunity. The first hurdle is the correct selection of tumor antigens. Ideal candidates are antigens expressed exclusively in tumors, such as tumor-specific antigens ("TSAs"), or antigens that are uniquely altered in tumor cells, such as neoantigens or tumor-associated antigens ("TAAs"). The second hurdle to overcome is the ability to effectively present these antigens to dendritic cells, thereby enabling more effective presentation via both MHC I (CD8+ T cells) and MHC II (CD4 T cells). Currently available approaches and immune adjuvants have been ineffective in this regard. The third hurdle for an effective immunotherapeutic agent is activating a T cell immune response in a broad target patient population expressing a wide range of HLA subtypes.

[0005] The present invention describes a novel MUC1-derived modified multiepitope peptide formulation consisting of short and long peptides containing epitope-enhanced peptide agonist sequences conjugated to the immunostimulatory nanoparticle R-DOTAP, which can induce and generate high levels of MUC1 antigen-specific cytotoxic killer T cells that recognize and kill MUC1-expressing cancer cells. This unique modified peptide antigen contains a MUC1-derived long multiepitope peptide and specific MUC1-derived HLA A2, A11, and A23 antigenic sequences, which have increased MHC binding properties through amino acid substitutions and are further modified by lipidation to form multiepitope high-molecular-weight micellar structures that are effectively conjugated to the immunostimulatory nanoparticle R-DOTAP for delivery to dendritic cells for antigen processing and potent class I immune responses. Summary of the Invention

[0006] The present invention is based on the breakthrough discovery that the use of antigenic peptides derived from mucin-1 (also referred to herein as MUC1) and multi-epitope MUC1 peptides thereof in vaccine compositions, together with cationic lipids as adjuvants, induces polyfunctional, cancer-specific T cell responses.

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

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

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

[0011] In a further embodiment, the present application provides a vaccine composition comprising a multi-epitope peptide comprising at least one mucin 1 (MUC1) peptide; and a cationic lipid.

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

[0013] In another embodiment, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a vaccine composition comprising: a multi-epitope peptide having at least one mucin 1 (MUC1) peptide; and a cationic lipid.

[0014] In one embodiment, the multi-epitope peptide has a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 1. In one embodiment, at least one MUC1 peptide has a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to an amino acid sequence comprising any of SEQ ID NOs: 9-14 and 20-37. In one embodiment, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant thereof, or an analog thereof. In one embodiment, the cationic lipid is R-DOTAP. In one embodiment, the multi-epitope peptide is encapsulated within a cationic lipid nanoparticle. In one embodiment, the multi-epitope peptide and the preformed cationic lipid nanoparticle are mixed in a 1:1 ratio. In one embodiment, the multi-epitope peptide is mixed with a preformed cationic lipid nanoparticle as separate micelles. In one embodiment, one or more MUC1 peptides are associated with CD4 expression by antigen-presenting cells. + T cells and CD8 +Inducing presentation of non-HLA-restricted peptides to T cells. In one embodiment, treating cancer comprises inhibiting progression of cancer in a subject. In one embodiment, the cancer has cancer cells that express MUC1. In one embodiment, the cancer is prostate cancer, breast cancer, or acute myeloid leukemia (AML). In one embodiment, the method further comprises administering an anti-cancer treatment to the subject. In one embodiment, the anti-cancer treatment comprises immune checkpoint inhibitor therapy. In one embodiment, treating cancer comprises inducing a MUC-specific polyfunctional cytolytic T cell response in a subject.

[0015] In one embodiment, the present invention provides a method for inducing a mucin 1 (MUC1)-specific polyfunctional and cytolytic T cell response in a subject by administering to the subject a composition comprising a multi-epitope peptide having one or more mucin 1 (MUC1) peptides; and a cationic lipid.

[0016] In one embodiment, the cationic lipid is DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, a variant or analog thereof. In one embodiment, the cationic lipid is R-DOTAP. In one embodiment, the multi-epitope peptide is encapsulated within a cationic lipid nanoparticle. In one embodiment, the multi-epitope peptide and pre-formed cationic lipid nanoparticles are mixed in a 1:1 ratio. In one embodiment, the multi-epitope peptide is mixed with pre-formed cationic lipid nanoparticles as separate micelles. In one embodiment, the multi-epitope peptide is used to induce CD4 expression by antigen-presenting cells. + T cells and CD8 + Induce presentation of non-HLA-restricted peptides to T cells. [Brief explanation of the drawings]

[0017] [Figure 1]FIG. 1 is a graph showing the induction of HLA-A2-specific CD8 T cells in response to a peptide antigen derived from MUC1 protein incorporated into R-DOTAP nanoparticles.

[0018] [Figure 2] FIG. 2 is a graph showing the induction of HLA-A2-specific CD8 T cells in response to a peptide antigen derived from MUC1 protein incorporated into R-DOTAP nanoparticles.

[0019] [Figure 3] FIG. 3 is a graph showing HLA-A2-specific CD8 T cell responses to a formulation containing 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] FIG. 4 is a graph showing HLA-A2-specific CD8 T cell responses to a formulation containing peptide antigens SSEEDE-C1A (SEQ ID NO: 18) and SSEEDE-C2A (SEQ ID NO: 19) combined 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).

[0021] [Figure 5] Figure 5 is a graph showing HLA-A2-specific CD8 T cell responses to a formulation containing peptide antigens SSEEDE-C1A (SEQ ID NO: 9) and SSEEDE-C2A (SEQ ID NO: 10) combined 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).

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

[0023] [Figure 7] FIG. 7 is a graph showing HLA-A2-specific CD8 T cell responses to a formulation containing 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] Figure 8 is a graph showing 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 incorporated into the lipid bilayer of R-DOTAP nanoparticles. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention is based on the groundbreaking discovery that the use of mucin-1-derived antigenic peptides and their multi-epitope peptides in vaccine compositions with cationic lipids as adjuvants induces polyfunctional, cancer-specific T cell responses.

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

[0027] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure.

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

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

[0032] Mucin 1 (also known as CD227, episialin, PEM, H23Ag, EMA, CA15-3, and MCA) is a transmembrane glycoprotein aberrantly expressed on many epithelial cancer cells, with variable cellular distribution, function, and glycosylation. The protein is a heterodimer consisting of a large extracellular domain covalently linked to a small intracellular domain. The extracellular domain consists of multiple tandem repeat regions (VNTRs) and non-tandem repeat regions. The C-terminal domain of MUC1 contains interaction sites with several signaling molecules and has been shown to have oncogenic potential.

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

[0034] Cross-presentation refers to the immunological pathway in which soluble proteins or peptides enter cells from the outside and enter the MHC class I processing pathway. Cross-presentation can occur in two ways: via the cytoplasmic pathway or the endosomal pathway. In both pathways, peptides / proteins are first internalized into endosomes / phagosomes. In the cytoplasmic pathway, partially degraded endosomal proteins eventually enter the cytoplasm, where they are processed through the proteasome. The resulting peptides are transported by TAP (transporter associated with Ag-processing) to the endoplasmic reticulum or other endosomes and bind to MHC class I. Alternatively, proteins can be degraded in endosomes, and the peptides can bind to MHC class I present in endosomes. This latter pathway is proteasome-independent and inefficient because it relies on the serendipitous generation of the correct peptide by endosomal proteases. Entry of proteins into early endosomes, where proteolytic activity is limited, is favorable for cross-presentation, whereas late endosomes, where proteolytic activity levels are high, can inhibit cross-presentation.

[0035] Certain enantiomeric cationic lipids are unique in their ability to rapidly bind to antigen-presenting dendritic cells in a receptor-independent manner and be internalized into early endosomes along with bound peptide or protein antigens. These specific cationic lipids have been shown to promote the migration of orders of magnitude more protein and peptide antigens into the MHC class I and MHC class II pathways than other current approaches, such as the use of alum or oil-in-water adjuvants to induce dendritic cell maturation. Furthermore, vaccination with these peptide antigen-conjugated cationic lipid nanoparticles induces superior T cell immune responses in vivo compared with peptides alone or peptides formulated with conventional adjuvants.

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

[0037] For example, the multi-epitope peptide has at least 80% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 85% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 90% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 91% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 92% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 93% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 94% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 96% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 97% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 98% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 99% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 99.5% sequence identity to an amino acid sequence comprising SEQ ID NO: 1. The multi-epitope peptide has at least 99.9% sequence identity to an 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 (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, either in vitro or in vivo, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will typically be located 3' to the coding sequence.

[0039] The terms "sequence identity" and "percent identity" are used interchangeably herein. To determine percent identity between two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into a first polypeptide or polynucleotide sequence to optimally align it with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). In some embodiments, the length of a reference sequence (e.g., SEQ ID NOS: 1-37) aligned for comparison purposes is at least 80% of the length of the sequence being compared, and in some embodiments, at least 90% or 100%. In one embodiment, the two sequences are the same length.

[0040] Desirable ranges of sequence identity are approximately 80% to 100% and integer values ​​therebetween. The percent identity between the disclosed and claimed sequences 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%. Generally, a perfect match indicates 100% identity over the entire length of the reference sequence (e.g., SEQ ID NOS: 1-43).

[0041] Also encompassed by the present disclosure are polypeptides and polynucleotides that have about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity to the polypeptides and polynucleotides described herein. For example, a polypeptide may have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NOs: 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 but still retain at least about 70% homology to the original protein over the corresponding portions. Greater degrees of deviation from homology are permitted, provided that similar amino acids, i.e., conservative amino acid substitutions, are not counted as changes in the sequence. Examples of conservative substitutions involve amino acids with identical or similar properties. Exemplary conservative amino acid substitutions include the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamic acid; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine to leucine.

[0043] As used herein, the terms "polyepitopic peptide," "multiepitope peptide," and the like refer to a peptide or polypeptide that comprises at least two epitopes as described herein. For example, a polyepitopic peptide may comprise two, three, four, five, six, seven, eight, nine, ten, or more of the epitopes of the invention.

[0044] The term "epitope" refers to an antigenic determinant within a molecule such as an antigen, i.e., a portion within a molecule or a fragment of a molecule that is recognized by the immune system. An epitope of a protein, such as a tumor antigen, preferably comprises a continuous or discontinuous portion of the protein. The terms "epitope," "antigenic peptide," "antigenic epitope," "immunogenic peptide," "antigenic fragment," and "MHC-binding peptide" may be used interchangeably herein and preferably relate to the presentation of an antigen capable of eliciting an immune response against the antigen or against a cell expressing, containing, or preferably presenting the antigen. An "antigen" according to the present invention encompasses any substance that elicits an immune response. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with an antibody or T lymphocyte (T cell). In the present invention, the term "antigen" includes any molecule that contains at least one epitope. Preferably, an antigen in the context of the present invention is a molecule that elicits an immune response, optionally after processing. Any suitable antigen that is a candidate for an immune response, preferably a cellular immune response, may be used in the present invention. In the context of the present embodiment, the antigen is preferably presented by cells, preferably antigen-presenting cells including abnormal cells, particularly cancer cells, in the context of MHC molecules, leading to an immune response against the antigen. Preferably, the antigen corresponds to a naturally occurring antigen or is a product derived from a naturally occurring antigen. Such naturally occurring antigens include tumor antigens.

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

[0046] In another embodiment, the multi-epitope peptide comprises a mucin 1 (MUC1) peptide.

[0047] In one embodiment, the MUC1 peptide has a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% identity to any of the sequences of SEQ ID NOs: 9-14 and 20-37.

[0048] For example, the MUC1 peptide has a sequence that has at least 80% identity with any of the sequences of SEQ ID NOs: 9-14 and 20-37.

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

[0050] As mentioned above, peptide epitopes, such as the MUC1 epitope, can have affinity for various MHC molecules expressed by antigen-presenting cells (APCs), thereby dictating which immune cell receptors can present and recognize these peptide epitopes, and consequently, which types of immune responses can be induced by these peptide epitopes. As detailed in the Examples section, the MUC1 epitopes described herein have affinity for several HLA molecules and are therefore capable of inducing several types of immune responses.

[0051] In some embodiments, the MUC1 epitope has MHC affinity for HLA-A2, HLA-A3, HLA-A11, and HLA-A24. In various embodiments, the MUC1 epitope is recognized by the CD4+ T cell receptor and / or the CD8+ T cell receptor.

[0052] As used herein, a peptide epitope "recognized by" an immune cell receptor may be referred to interchangeably as an "immune cell receptor epitope." That is, a MUC1 epitope recognized by a CD4+ T cell receptor may be referred to as a CD4+ T cell receptor epitope or a MUC1 epitope, depending on whether emphasis is placed on the peptide from which the epitope is derived (e.g., MUC1) or on the immune cell receptor (e.g., CD4+ T cell receptor and / or CD8+ T cell receptor) with which the epitope may be recognized (or may interact with based on affinity).

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

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

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

[0056] In another embodiment, 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 the anion tag and may be used alternatively. It should be noted that any sequence provided herein that includes an anion tag is intended to be disclosed as encompassing variations of the anion tag. For example, the disclosure of SSEEDE-YLAIVYLIAL of SEQ ID NO:18, which includes the anion tag of SEQ ID NO:38, is intended to encompass the disclosure of modified peptides that include the anion 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 encompass pharmaceutical compositions that may also contain other therapeutic agents and may be formulated, for example, according to techniques known in the pharmaceutical formulation art, by using conventional pharmaceutically acceptable solvents or diluents and types of pharmaceutical additives (e.g., excipients, preservatives, etc.) appropriate for the desired mode of administration. In certain embodiments, the compositions disclosed herein are formulated with additional agents that facilitate entry into desired cells or tissues. Such additional agents include micelles, liposomes, and dendrimers.

[0059] The term "pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. For example, the carrier, diluent, or excipient, or composition thereof, could be administered to a subject, together with a conjugate of the present invention, without causing any undesired biological effects or interacting in an undesired manner with any of the other ingredients of the pharmaceutical composition contained therein.

[0060] Pharmaceutical compositions containing the peptides or compositions described herein may be administered by any suitable means, for example, parenterally, e.g., by subcutaneous, intravenous, intramuscular, intrathecal, or intracisternal injection or infusion (e.g., as a sterile injectable aqueous or non-aqueous solution or suspension) in a dosage formulation containing a non-toxic pharmaceutically acceptable solvent or diluent. Depending on the condition being treated, these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration are generally known in the art. A suitable route may be, for example, parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, or intraperitoneal delivery. For injection, the pharmaceutical compositions of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as water, Hank's solution, Ringer's solution, or physiologically buffered saline.

[0061] Adjuvants are often used to modulate or enhance the efficacy of vaccines by stimulating the immune system to respond more vigorously to the vaccine, thereby providing enhanced immunity against specific diseases. Adjuvants accomplish this task by mimicking a specific set of evolutionarily conserved molecules, so-called pathogen-associated molecular patterns (PAMs). This set includes liposomes, lipopolysaccharides, molecular cages for antigens, components of bacterial cell walls, and endocytosed nucleic acids such as RNA, double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. Because the immune system has evolved to recognize these specific antigenic moieties, the presence of adjuvants with vaccines can significantly enhance the innate immune response to antigens by mimicking natural infection and enhancing the activity of dendritic cells, lymphocytes, and macrophages.

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

[0063] In some embodiments described herein, the adjuvant is a cationic lipid. As used herein, the term "cationic lipid" refers to any of a number of lipid species that have a net positive charge at physiological pH or that have a protonatable group and have a positive charge at a pH lower than the pKa.

[0064] Suitable cationic lipids according to the present disclosure include, but are not limited to: 3-β [4NlN,8-diguanidinospermidine)-carbamoyl] cholesterol (BGSC); 3-β [N,N-Diguanidinoethyl-aminoethane)-carbamoyl]cholesterol (BGTC); N,N,1N2N3 tetra-methyltetrapalmitylspermine (Cellfectin); Nt-butyl-N'-tetradecyl-3-tetradecyl-aminopropionamidine (CLONfectin); dimethyldioctadecylammonium bromide (DDAB); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate trifluorocetate) (DOSPA); 1,3-dioleoyloxy-2-(6-carboxyspermyl)-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); N-1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), or other N-(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; 1,2 dioleoyl-3-(4'-trimethylammonio)butanol- sn -glycerol (DOBT) or cholesteryl (4'-trimethylammonium) butanoate (ChOTB) (the trimethylammonium group is connected to the duplex (in the case of DOTB) or cholesteryl group (in the case of ChOTB) via a butanol spacer arm);DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) (DORIE) or analogs thereof as disclosed in WO 93 / 03709; 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate (ChOSC); lipopolyamines, such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidylethanolamylspermine (DPPES), cholesteryl-3 β-Carboxyl-amido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterylcarboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine, cholesteryl-3-β-oxysuccinamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxysuccinate iodide, 2-(2-trimethylammonio)-ethylmethylaminoethyl-cholesteryl-3-β-oxysuccinate iodide, 3-β-N-(N',N'-dimethylaminoethane)carbamoylcholesterol (DC-chol), and 3-β-N-(polyethyleneimine)-carbamoylcholesterol. O,O'-Dimyristyl-N-lysyl aspartate (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-trimethylammoniumpropane (DPTAP); 1,2-distearoyl-3-trimethylammoniumpropane (DSTAP), 1,2-myristoyl-3-trimethylammoniumpropane (DMTAP); and sodium dodecyl sulfate (SDS). Additionally, structural variants and derivatives of any of the above cationic lipids are contemplated.

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

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

[0067] In one embodiment, the cationic lipid is selected from the group consisting of DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variants thereof, and analogs thereof.

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

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

[0070] In one embodiment, the MUC1 epitope has MHC affinity for HLA-A2, HLA-A3, HLA-A11, and HLA-A24.

[0071] In another embodiment, the MUC1 epitope is recognized by the CD4+ T cell receptor and / or by the CD8+ T cell receptor.

[0072] In one embodiment, one or more epitope peptides comprise a cleavable anionic N-terminal extension. In some embodiments, 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 embodiment, the one or more multiepitope peptides are encapsulated in liposomes containing cationic lipids or mixed with preformed cationic lipid nanoparticles as micelles. In some embodiments, the one or more multiepitope peptides and the preformed cationic lipid nanoparticles are mixed in a 1:1 ratio.

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

[0075] In a further embodiment, the present invention provides a method for detecting a CD4 + T cell receptor and / or CD8 + The present invention provides a vaccine composition comprising: (a) one or more multi-epitope peptides, each of which contains at least two epitope peptides recognized by a T cell receptor, the epitope peptides being mucin 1 (MUC1) epitopes; and (b) a cationic lipid.

[0076] In the present invention, the term "vaccine" relates to a pharmaceutical preparation (pharmaceutical composition) or a drug that, after administration, induces an immune response, in particular a cellular immune response, that recognizes and attacks pathogens or abnormal cells such as cancer cells. Vaccines can be used for the prevention or treatment of diseases. The term "personalized cancer vaccine" relates to a specific cancer patient and means that the cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.

[0077] In one embodiment, the cationic lipid is selected from the group consisting of DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations and analogs thereof. In some embodiments, the cationic lipid is R-DOTAP.

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

[0079] In another embodiment, the present invention provides a method for 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 cancer in the subject.

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

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

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

[0083] The terms "administration of" and / or "administering" should be understood to mean providing a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. The route of administration can be enteral, topical, or parenteral. Thus, routes of administration include, but are not limited to, intradermal, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, intrathecal, intraspinal, and intrasternal administration, oral, sublingual, buccal, rectal, vaginal, nasal, and ocular administration, as well as infusion, inhalation, and nebulization.

[0084] In one embodiment, the one or more MUC1-derived epitopes comprise a sequence that comprises at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 2 to 37 and SEQ ID NOs: 42 to 43. In another embodiment, the multi-epitope peptide comprises a sequence that comprises at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 1.

[0085] In one embodiment, the cationic lipid is selected from the group consisting of DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations and analogs thereof. In some embodiments, the cationic lipid is R-DOTAP.

[0086] In another embodiment, the multi-epitope peptide is encapsulated within the cationic lipid nanoparticles or mixed with pre-formed cationic lipid nanoparticles as micelles.

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

[0088] In one embodiment, one or more MUC1-derived epitopes are expressed by antigen-presenting cells (e.g., CD4 + T cells and CD8 + Induce presentation of non-HLA-restricted peptides to T cells.

[0089] The epitopes and multi-epitope 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 complex of genes located on chromosome 6 in humans. These genes encode cell surface proteins involved in regulating the immune system. The HLA system is also known as the human version of the major histocompatibility complex (MHC), found in many animals. HLA genes are highly polymorphic, meaning that there are many different alleles of HLA genes, allowing for fine-tuning of the adaptive immune system. HLAs (A, B, and C) corresponding to MHC class I are all part of the HLA class 1 group and present peptides from within cells. These peptides are generated from proteins after digestion in the proteasome. These specific peptides are usually small polymers of approximately 8–10 amino acids in length. Foreign antigens presented by MHC class I attract T lymphocytes called killer T cells (also known as CD8+ T cells or cytotoxic T cells), which destroy the cells. MHC class I proteins bind to β2-microglobulin, which, unlike HLA proteins, is encoded by a gene on chromosome 15. HLA (DP, DM, DO, DQ, and DR), which correspond to MHC class II, present extracellular antigens to T lymphocytes. These specific antigens stimulate the proliferation of helper T cells (also called CD4-positive T cells), which in turn stimulate antibody-producing B cells to produce antibodies against the specific antigen. Self-antigens are suppressed by regulatory T cells.

[0091] MHC-restricted antigen recognition, MHC restriction, or HLA restriction refers to the fact that T cells can interact with self-specific major histocompatibility complex molecules and foreign peptides bound to them, but will only respond to the antigen if the antigen is bound to a specific MHC molecule. When foreign proteins enter cells, they are degraded into peptides. These peptides, or antigens, can be derived from pathogens such as viruses or intracellular bacteria. The foreign peptides are delivered to the cell surface and presented to T cells by proteins called major histocompatibility complexes (MHCs). During T cell development, T cells undergo a selection process in the thymus to prevent their T cell receptors (TCRs) from recognizing MHC molecules that present self-antigens, i.e., to prevent the TCR from having too high an affinity. High affinity means autoreactivity, while no affinity means that the TCR does not bind strongly enough to the MHC. This selection process results in the development of T cells with specific TCRs that may respond only to certain MHC molecules but not to other MHC molecules. This fact that TCRs recognize only some MHC molecules but not others contributes to "MHC restriction." The biological reason for MHC restriction is to conserve energy and cellular components by preventing the generation of redundant, wandering lymphocytes. T cells are a type of lymphocyte important in the immune system, activating other immune cells. Upon recognizing foreign peptides via their T cell receptors (TCRs) on their surface, T cells play different roles in defending the host against foreign peptides, which may be derived from pathogens such as bacteria, viruses, or parasites. MHC restriction adds another dimension to T cell receptor specificity by enforcing the restriction that T cells are activated by peptide antigens only when bound to self-MHC molecules, ensuring that antigens are recognized only as peptide-MHC complexes. MHC restriction in T cells occurs during T cell development in the thymus, particularly during positive selection.Only thymocytes (developing T cells in the thymus) that can bind MHC molecules with the appropriate affinity can receive survival signals and progress to the next level of selection. MHC restriction is important for proper functioning of T cells upon exiting the thymus, as it allows the T cell receptor to bind to MHC and detect cells infected by intracellular pathogens, viral proteins, and cells with genetic abnormalities.

[0092] In another embodiment, treating cancer comprises inhibiting the progression of cancer in a subject.

[0093] Cancer is a group of diseases involving the growth of abnormal cells that can invade other parts of the body and metastasize. In 2015, approximately 90.5 million people had cancer, with approximately 14.1 million new cases occurring annually, resulting in approximately 8.8 million deaths (15.7% of deaths). The most common types of cancer in men are lung, prostate, colorectal, and stomach cancer. In women, the most common types are breast, colorectal, lung, and cervical cancer.

[0094] The term "cancer" refers to a group of diseases characterized by abnormal and uncontrolled cell growth that begins at one site (primary site) and may invade and metastasize to other sites (secondary sites, metastases); this characteristic distinguishes cancer (malignant tumors) from benign tumors. Virtually every organ can be affected, and there are over 100 types of cancer that can affect humans. Cancer can arise as a result of many causes, including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, tobacco and / or alcohol use, obesity, poor diet, physical inactivity, or any combination of these.

[0095] As used herein, "neoplasm" or "tumor," including grammatical variations thereof, refers to a new, abnormal growth of tissue, which may be benign or cancerous. In a related aspect, neoplasm refers to a neoplastic disease or disorder, including, but not limited to, various cancers. For example, such cancers may include prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, pancreatic cancer, brain cancer, and head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0096] Exemplary cancers reported by the National Cancer Institute include: adult acute lymphoblastic leukemia; childhood acute lymphoblastic leukemia; adult acute myeloid leukemia; adrenocortical carcinoma; childhood adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; childhood cerebellar astrocytoma; childhood cerebral astrocytoma; extrahepatic bile duct cancer; bladder cancer; childhood bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma; childhood brain stem glioma; adult brain tumor; childhood brain tumor, brain stem glioma; childhood brain tumor, cerebellar astrocytoma; childhood brain tumor, cerebral astrocytoma / malignant glioma; childhood brain tumor, ependymoma; childhood brain tumor, medulloblastoma; childhood brain tumor, supratentorial primitive neuroectodermal tumor; childhood brain tumor, visual pathway and hypothalamic glioma; childhood brain tumor (other); breast cancer; pregnancy-associated breast cancer Pregnancy); Pediatric Breast Cancer; Male Breast Cancer; Childhood Bronchial Adenoma / Carcinoid; Childhood Carcinoid Tumors; Gastrointestinal Carcinoid Tumors; Adrenocortical Carcinoma; Islet Cell Carcinoma; Cancer of Unknown Primary Site; Primary Central Nervous System Lymphoma; Childhood Cerebellar Astrocytoma; Childhood Cerebral Astrocytoma / Malignant Glioma; Cervical Cancer; Childhood Cancer; Chronic Lymphocytic Leukemia; Chronic Myeloid Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of the Tendon Sheath; Colon Cancer; Childhood Colorectal Cancer; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Childhood Ependymoma; Epithelial Ovarian Cancer; Esophageal Cancer; Childhood Esophageal Cancer; Ewing's Sarcoma Family Tumors; Childhood Extracranial Germ Cell Tumors; Extragonadal Germ Cell Tumors; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric Cancer; Childhood Gastric Cancer; Gastrointestinal Carcinoid Tumors; Childhood Extracranial Germ Cell Tumors; Sexuality Extraglandular germ cell tumor; Ovarian germ cell tumor; Gestational trophoblastic tumor; Pediatric brain stem glioma; Pediatric visual pathway and hypothalamic glioma; Hairy cell leukemia; Head and neck cancer; Adult (primary) hepatocellular (liver) cancer; Pediatric (primary) hepatocellular (liver) cancer; Adult Hodgkin's lymphoma; Pediatric Hodgkin's lymphoma; Hodgkin's lymphoma during pregnancy; Hypopharyngeal cancer; Pediatric hypothalamic and visual pathway glioma; Intraocular melanoma Chromoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Renal cancer; Laryngeal cancer; Childhood laryngeal cancer; Adult acute lymphoblastic leukemia; Childhood acute lymphoblastic leukemia; Adult acute myeloid leukemia; Childhood acute myeloid leukemia; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Hairy cell leukemia; Lip and oral cavity cancer; Adult (primary) liver cancer; Childhood (primary) liver cancer; Non-small cell lung cancer; Small cell lung cancer;Adult acute lymphoblastic leukemia;Childhood acute lymphoblastic leukemia;Chronic lymphocytic leukemia;AIDS-related lymphoma;Central nervous system (primary) lymphoma;Cutaneous T-cell lymphoma;Adult Hodgkin lymphoma;Childhood Hodgkin lymphoma;Hodgkin lymphoma during pregnancy;Adult non-Hodgkin lymphoma;Childhood non-Hodgkin lymphoma;Non-Hodgkin lymphoma during pregnancy;Primary central nervous system lymphoma;Waldenstrom's macroglobulinemia;Male breast cancer;Adult malignant mesothelioma;Childhood malignant mesothelioma;Malignant thymoma;Childhood medulloblastoma tumor; melanoma; intraocular melanoma; Merkel cell carcinoma; malignant mesothelioma; metastatic squamous cell neck cancer of unknown primary; childhood multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndrome; chronic myeloid leukemia; childhood acute myeloid leukemia; multiple myeloma; chronic myeloproliferative disorders; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; pediatric nasopharyngeal cancer; neuroblastoma; adult non-Hodgkin's lymphoma; childhood non-Hodgkin's lymphoma; non-Hodgkin's lymphoma during pregnancy; non-small cell lung cancer; oral cancer, Pediatric; Oral cavity and lip cancer; Oropharyngeal cancer; Osteosarcoma / Malignant fibrous histiocytoma of bone; Pediatric ovarian cancer; Ovarian epithelial cancer; Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pediatric pancreatic cancer, pancreatic islet cell cancer; Cancer of the paranasal sinuses and nasal cavity; Parathyroid cancer; Penile cancer; Pheochromocytoma; Pediatric pineal and supratentorial primitive neuroectodermal tumor; Pituitary tumor; Plasma cell neoplasm / multiple myeloma; Pleuropulmonary blastoma; Pregnancy-associated breast cancer; Pregnancy-associated Hodgkin lymphoma; Pregnancy-associated non-Hodgkin lymphoma; Primary central nervous system lymphoma; Adult primary liver cancer; Pediatric primary liver cancer; Prostate cancer; Rectal cancer; Renal cell (kidney) cancer; Pediatric renal cell carcinoma; Transitional cell carcinoma of the renal pelvis and ureter; Retinoblastoma; Pediatric rhabdomyosarcoma; Salivary gland cancer; Pediatric salivary gland cancer; Ewing's sarcoma family of tumors; Kaposi's sarcoma; Sarcoma (osteosarcoma) Malignant fibrous histiocytoma of bone;Pediatric rhabdomyosarcoma;Adult soft tissue sarcoma;Pediatric soft tissue sarcoma;Sezary syndrome;Skin cancer;Pediatric skin cancer;Skin cancer (melanoma);Merkel cell skin cancer;Small cell lung cancer;Small intestine cancer;Adult soft tissue sarcoma;Pediatric soft tissue sarcoma;Metastatic squamous cell neck cancer of unknown primary site;Gastric cancer;Pediatric gastric cancer;Pediatric supratentorial primitive neuroectodermal tumor;Cutaneous T-cell lymphoma;Testicular cancer;Pediatric thymoma;Malignant thymoma;Thyroid cancer;Pediatric thyroid cancer;Transitional cell carcinoma of the renal pelvis and ureter;Gestational trophoblastic tumor;Pediatric cancer of unknown primary site;Pediatric rare cancers;Transitional cell carcinoma of the ureter and renal pelvis;Urethral cancer; uterine sarcoma; vaginal cancer; childhood visual pathway and hypothalamic glioma; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0097] In one embodiment, the cancer comprises cancer cells that express MUC1.

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

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

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

[0101] As used herein, the term "anti-cancer therapy" is intended to refer to any therapeutic approach that can be used to treat cancer. This term includes, but is not limited to, surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, and any combination thereof.

[0102] In some embodiments, administration can be combined with one or more additional therapeutic agents. The terms "combination therapy," "combined with," and the like refer to the simultaneous use of two or more agents or therapies to enhance response. For example, the compositions of the present invention may be used in combination with other agents or therapies used to treat cancer. Specifically, administration of the peptides, multi-epitope peptides, or vaccines described herein to a subject can be combined with another anti-cancer treatment, such as immune checkpoint inhibitor therapy. Such treatments can be administered before, simultaneously with, or after administration of the compositions of the present invention.

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

[0104] "Checkpoint inhibitor therapy" is a current form of cancer treatment that exploits immune checkpoints that affect immune system function. Immune checkpoints can be either stimulatory or inhibitory. Tumors can exploit these checkpoints to protect themselves from immune system attack. Checkpoint therapy can restore immune system function by blocking inhibitory checkpoints. Checkpoint proteins include programmed cell death protein 1 (PDCD1, PD-1; also known as CD279) and its ligand, PD-1 ligand 1 (PD-L1, CD274), cytotoxic T lymphocyte antigen 4 (CTLA-4), A2AR (adenosine A2A receptor), B7-H3 (or CD276), B7-H4 (or VTCN1), BTLA (B and T lymphocyte attenuator, or CD272), IDO (indoleamine 2,3-dioxygenase), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin domain and mucin domain 3), and VISTA (V domain Ig suppressor of T cell activation).

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

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

[0107] CTLA4 or CTLA-4 (cytotoxic T lymphocyte antigen 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint, downregulating immune responses. CTLA4 is constitutively expressed in regulatory T cells, but its expression increases in normal T cells only after activation, a phenomenon particularly pronounced in cancer. CTLA4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits inhibitory signals to T cells. CTLA4 is homologous to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 binds to CD80 and CD86 with greater affinity and avidity than CD28, allowing CTLA-4 to outcompete CD28 for its ligand. CTLA-4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. CTLA-4 is also present on regulatory T cells and contributes to their suppressive function. T cell activation via the T cell receptor and CD28 results in increased expression of CTLA-4.

[0108] There are several checkpoint inhibitors currently being used to treat cancer. PD-1 inhibitors include pembrolizumab (Keytruda) and nivolumab (Opdivo). PD-L1 inhibitors include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). CTLA-4 inhibitors include ipilimumab (Yervoy). Several other checkpoint inhibitors are in development, including the anti-B7-H3 antibody (MGA271), the anti-KIR antibody (lirilumab), and the anti-LAG3 antibody (BMS-986016).

[0109] "Targeted therapy" refers to any molecularly targeted therapy used to block the growth of cancer cells by interfering with specific target molecules required for carcinogenesis and tumor growth, rather than simply interfering with all rapidly dividing cells (e.g., as in conventional chemotherapy). Biomarkers are typically needed to help select patients likely to respond to a given targeted therapy. Non-limiting examples of targeted therapies include: tyrosine-kinase inhibitors, small molecule-drug conjugates, serine-threonine kinase inhibitors, monoclonal antibodies, and histone deacetylase (HDAC) inhibitors.

[0110] In other embodiments, the targeted therapy comprises an HDAC inhibitor.

[0111] Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups (O=C-CH3) from ε-N-acetyllysine amino acids on both histone and nonhistone proteins. HDACs allow histones to wrap more tightly around DNA. This is important because DNA wraps around histones and DNA expression is controlled by acetylation and deacetylation. The action of HDACs is opposite to that of histone acetyltransferases. HDAC proteins are now also called lysine deacetylases (KDACs) to describe their function, rather than their target, which also includes nonhistone proteins. Normally, HDAC proteins repress gene expression.

[0112] Histone deacetylase inhibitors (HDAC inhibitors, HDACi, HDI) are chemical compounds that inhibit histone deacetylases. "Classical" HDIs act exclusively on class I, class II, and class IV HDACs by binding to the zinc-containing catalytic domain of HDACs. These classical HDIs can be classified into several groups named according to the chemical moiety that binds to the zinc ion (excluding cyclic tetrapeptides, which bind to the zinc ion via a thiol group). Some examples of typical HDIs, in descending order of zinc-binding affinity, are: 1. hydroxamic acids (or hydroxamates), such as trichostatin A; 2. cyclic tetrapeptides (such as trapoxin B) and depsipeptides; 3. benzamides; 4. electrophilic ketones; and 5. aliphatic acid compounds, such as phenylbutyrate and valproic acid.

[0113] "Second-generation" HDAs include the hydroxamic acids vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589); and the benzamides entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103). Sirtuins, class III HDACs, are NAD+-dependent and are therefore inhibited by nicotinamide, as well as the NAD derivatives dihydrocoumarin, naphthopyranone, and 2-hydroxynaphthaldehyde. Non-limiting examples of HDAC inhibitors include Istodax (romidepsin), Zolinza (vorinostat), Farydak (panobinostat), and Belodaq (belinostat).

[0114] In one embodiment, treating cancer comprises inducing a MUC1-specific polyfunctional and cytolytic T cell response in a subject.

[0115] The term "immune response" refers to the body's integrated reaction to an antigen, preferably a cellular immune response, or a cellular and humoral immune response. The immune response can be protective / preventive / prophylactic and / or therapeutic.

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

[0117] The terms "cellular immune response," "cellular response," "cellular response to antigen," or similar terms are intended to encompass cellular responses directed against cells characterized by antigen presentation by class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes, which act as "helpers" or "killers." Helper T cells (also known as CD4+ T cells) play a central role by regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill abnormal cells, such as cancer cells, and prevent the production of more abnormal cells. In a preferred embodiment, the invention involves stimulating an antitumor CTL response against tumor cells expressing one or more tumor-expressed antigens, preferably presenting such tumor-expressed antigens by class I MHC.

[0118] In the context of the present invention, the terms "immune response cell," "immune cell," or "immune effector cell" refer to cells that perform effector functions during an immune response. "Immune response cells" are preferably cells characterized by binding to an antigen or antigen-presenting antigen, or antigenic peptides derived from an antigen, and are capable of mediating an immune response. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancerous cells, and, if necessary, eliminate such cancerous cells. For example, immune response cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.

[0119] "Inducing an immune response" may refer to the absence of an immune response against a specific antigen before induction, or may refer to the presence of a certain level of immune response against a specific antigen before induction, and the enhancement of the immune response after induction. That is, "inducing an immune response" also encompasses "enhancing an immune response." After inducing an immune response in a subject, it is preferable that the subject is protected from developing a disease such as a cancer, or that the induction of an immune response improves the condition of the subject. For example, an immune response against a tumor-expressed antigen can be induced in a patient with cancer or in a subject at risk of developing a cancer. In this case, the induction of an immune response may be intended to improve the subject's condition, prevent metastasis from occurring in the subject, or prevent a subject at risk of developing a cancer from developing the disease.

[0120] Multiple high-affinity MHC-binding peptide antigens specific for HLA-A2, HLA-A3, HLA-A11, and HLA-A24 were identified within the human mucin 1 oncoprotein (Table 1). MHC-binding peptide antigens specific for HLA-A2 were 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). [Table 1]

[0121] Table 1. Native and modified sequences of agonist peptide antigens derived from the human oncoprotein MUC1 are shown, with the amino acid substitutions shown in bold and underlined text indicating the modifications utilized to create agonist antigens with enhanced MHC binding affinity.

[0122] Specifically, four peptides, C1A (SEQ ID NO: 9), C2A (SEQ ID NO: 10), V1A (SEQ ID NO: 13), and V2A (SEQ ID NO: 14), were all modified by amino acid substitutions in their native sequences to increase MHC binding affinity, thereby enhancing their cell presentation on antigen-presenting human dendritic cells and increasing their potential effectiveness in inducing high levels of antigen-specific cytotoxic T cells (see Table 1). However, the amino acid modifications of the agonists have the negative consequence that the inherently hydrophobic peptides C1A and C2A lack polar residues that promote water solubility. Therefore, these highly hydrophobic peptide antigens, which have very high MHC binding affinity but limited water solubility, are ineffective molecules in vivo for use in conventional vaccine compositions due to the difficulty in successfully synthesizing the antigens in high purity and the difficulty in delivering the antigens to 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 aqueous-compatible formulation. To solve this problem, three effective approaches have been developed: 1. Synthesizing these hydrophobic peptides with an anionic, cleavable N-terminal extension on the peptide antigen allows for the synthesis of high-purity peptides and confers peptide solubility and compatibility with cationic R-DOTAP immunostimulatory nanoparticles. Linking the anionic domain of the antigen to the cationic surface of the nanoparticles results in high levels of antigen delivery and immune cell activation (Figures 4 and 5). 2. Delivery of two highly hydrophobic antigens by inserting them into the nonpolar interior of the membrane bilayer of cationic, surface-charged liposomal nanoparticles formed from a unique chiral immunostimulatory lipid, R-DOTAP. This unique delivery vehicle effectively encapsulates and quantitatively delivers water-insoluble peptide antigens within highly charged, water-compatible immunostimulatory nanoparticles to dendritic cells of the immune system, where they are presented on the MHC surface of activated dendritic cells, inducing high levels of antigen-specific cytotoxic T cells (Figures 1, 2, and 3). Encapsulation and delivery of this nonpolar peptide antigen into the bilayer of chiral cationic lipid nanoparticles resulted in the induction of very high levels of antigen-specific T cells in ELISpot assays using mouse-humanized HLA-A2 transgenic mice (Figures 1, 2, and 3). 3. A third approach to solving the problem of delivering the hydrophobic peptide antigens C1A and C2A has also been developed. This approach involves incorporating these amino acid sequences into a much larger peptide derived from the oncoprotein MUC1, which can be effectively bound to and delivered by the immunostimulatory nanoparticle R-DOTAP in an aqueous environment. This large peptide, YL40 (SEQ ID NO: 1), contains a hydrophobic agonist peptide at the N-terminus of the molecule, which can be efficiently processed by intracellular proteases in dendritic cells to generate individual agonist peptide antigens for presentation on cell surface MHC. This approach was found to detect high levels of peptide agonist C1A-specific T cell responses after vaccination of humanized transgenic HLA-A mice and ELISpot analysis (Figures 6 and 7).

[0123] In designing peptide-based immunotherapeutics designed to elicit cytotoxic T cells capable of recognizing and killing tumor cells expressing a target antigen, peptide antigens must be delivered in conjunction with potent immunostimuli capable of promoting effective antigen uptake by dendritic cells, directing peptide antigen processing down the class I (CD8) pathway, and demonstrating correct cytokine activation and signaling, thereby inducing large numbers of multicytokine-producing effector T cells. Herein, we have identified peptide sequences that uniquely and effectively associate with the immunostimulatory nanoparticle R-DOTAP, which, in this context, is taken up and processed by dendritic cells, resulting in the generation of high levels of multicytokine-producing MUC1-specific CD8 killer T cells. Numerous other combinations of MUC1 peptide sequences, including short specific peptide antigens and other long overlapping peptide sequences derived from the MUC1 protein, have been shown to be ineffective when combined with or incorporated into the immunostimulatory nanoparticle R-DOTAP (see Example 5, below). Thus, the unique combination of a short, lipidated agonist peptide epitope and a long MUC1 peptide sequence reported herein, which forms a high molecular weight micellar structure, when delivered in association with the immunostimulatory nanoparticle R-DOTAP, results in a potent antigen-specific immune response. These peptides can be incorporated into immunogenic compositions, such as vaccines. The peptide sequences utilized in the resulting compositions have the advantage of inducing a potent cytotoxic T cell response.

[0124] Disclosed herein are methods for the design and use of unique peptide sequences containing human cytotoxic T lymphocyte (CTL) epitopes encoded by the MUC1 protein. The sequences shown in Table 1 include 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), which are unique epitope-enhancing peptides selected to enhance processing and presentation of the MUC1 protein-encoded T cell antigen when delivered in association with R-DOTAP nanoparticles.

[0125] [Table 2] [Example]

[0126] Example 1: Design of peptides derived from MUC1 protein Disclosed herein are methods for the design and use of unique peptide sequences (SEQ ID NOS: 1-6) derived from the MUC1 protein that are designed to be efficiently processed and presented to T cells when delivered in association with R-DOTAP immunostimulatory nanoparticles.

[0127] Here, the water solubility limitations of the highly hydrophobic peptides C1A and C2A were overcome by developing three effective approaches to solve this problem: (1) synthesizing these hydrophobic peptides with an anionic charged cleavable N-terminal extension on the peptide antigen, (2) delivering these two highly hydrophobic antigens by inserting them into the nonpolar interior of the membrane bilayer of cationic surface-charged liposomal nanoparticles formed from the unique enantiomeric immunostimulatory lipid R-DOTAP, and (3) incorporating the amino acid sequences of individual epitope peptides into larger (multiepitope) peptides.

[0128] Example 2 Effect of adding an anionic charged cleavable N-terminal extension The synthesis of these hydrophobic peptides with an anionic, cleavable N-terminal extension on the peptide antigen enabled the synthesis of high-purity peptides and resulted in peptide solubility and compatibility with cationic R-DOTAP immunostimulatory nanoparticles. Linking the anionic domain of the antigen to the cationic surface of the nanoparticles resulted in high levels of antigen delivery and immune cell activation (Figures 4 and 5).

[0129] HLA-A2-specific CD8 T cell responses were evaluated against formulations containing peptide antigens SSEEDE-C1A (SEQ ID NO: 18) and SSEEDE-C2A (SEQ ID NO: 19) combined 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), as shown in Figure 4. The highly hydrophobic peptide antigens C1A and C2A are typically inactive in generating an immune response when included in antigen mixtures mixed with R-DOTAP immunostimulatory nanoparticles due to their poor solubility in aqueous systems. The addition of an anionic amino acid sequence (SSEEDE, SEQ ID NO: 38) to the N-terminus of the hydrophobic peptide enabled the synthesis of highly purified antigens and ensured effective delivery of the otherwise insoluble antigen to dendritic cells of the immune system through strong interactions with cationic nanoparticles in suspension. These results were confirmed in four replicate preparations of the antigen mixture / R-DOTAP formulation. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml vaccine injections subcutaneously to HLA-A2-expressing transgenic mice (AAD mice) on days 0 and 7. Negative control mice were inoculated with MUC1 peptide formulated in sucrose buffer. Vaccine formulation-specific immune responses were measured in ELISPOT assays by counting MUC1 antigen-specific T cells in triplicate wells in spleens from vaccinated mice. Each bar in the graph represents the average SFU per 250,000 splenocytes in the vaccinated mice group. Error bars represent the mean ± SEM of five mice per group. To identify MUC1-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).

[0130] HLA-A2-specific CD8 T cell responses were evaluated against formulations containing peptide antigens SSEEDE-C1A (SEQ ID NO: 9) and SSEEDE-C2A (SEQ ID NO: 10) combined 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), as shown in Figure 5. The highly hydrophobic peptide antigens C1A and C2A are typically inactive in generating an immune response when included in antigen mixtures mixed with R-DOTAP immunostimulatory nanoparticles due to their poor solubility in aqueous systems. The addition of an anionic amino acid sequence (SSEEDE, SEQ ID NO: 38) to the N-terminus of the hydrophobic peptide allows for the synthesis of highly purified antigens and ensures effective delivery of otherwise insoluble antigens to dendritic cells of the immune system through strong interactions with cationic nanoparticles in suspension. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml vaccine injections subcutaneously to HLA-A2-expressing transgenic mice (AAD mice) on days 0 and 7. Negative control mice were inoculated with MUC1 peptide formulated in sucrose buffer. Vaccine formulation-specific immune responses were measured in an ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in spleens from vaccinated mice. Each bar on the graph represents the average SFU per 250,000 splenocytes in the vaccinated mouse group. Error bars represent the mean ± SEM of five mice per group. To identify MUC1-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).

[0131] Example 3 Effect of formulation within R-DOTAP cationic surface-charged liposomal nanoparticles Two highly hydrophobic antigens were inserted into the nonpolar interior of the membrane bilayer of cationic, surface-charged liposomal nanoparticles formed from the unique chiral immunostimulatory lipid R-DOTAP. This unique delivery vehicle effectively encapsulates and quantitatively delivers water-insoluble peptide antigens within highly charged, water-compatible immunostimulatory nanoparticles to dendritic cells of the immune system, where they are presented on the surface MHC of activated dendritic cells and induce high levels of antigen-specific cytotoxic T cells (Figures 1, 2, and 3).

[0132] As shown in Figure 1, the HLA-A2-specific CD8 T cell peptide antigen derived from the MUC1 protein incorporated into R-DOTAP nanoparticles exhibited the effective immunoreactivity of insoluble C1A (SEQ ID NO: 9) when inserted into the lipid bilayer of liposomal nanoparticles, thereby delivering the poorly water-soluble antigen to immunoreactive cells along with the immunostimulatory activity of R-DOTAP. Control peptide antigen formulations were prepared with 0.5 ml of Montanide + 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 Freud's adjuvant. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml vaccine injections subcutaneously to HLA-A2-expressing transgenic mice (AAD mice) on days 0 and 7. Control mice were inoculated with MUC1 peptide formulated in sucrose buffer alone. Vaccine formulation-specific immune responses were measured by ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in spleens from vaccinated mice. Each data point on the graph represents the average SFU per 250,000 splenocytes of vaccinated mice. Error bars represent the mean ± SEM of five mice per group. To identify MUC1-specific T cells, splenocytes were stimulated with the peptide sequence C1A (SEQ ID NO: 9: YLAIVYLIAL).

[0133] As shown in Figure 2, the HLA-A2-specific CD8 T cell peptide antigen derived from the MUC1 protein incorporated into R-DOTAP nanoparticles exhibited effective immunoreactivity of insoluble C2A (SEQ ID NO: 10) when inserted into the lipid bilayer of liposomal nanoparticles, thereby delivering the poorly water-soluble antigen to immunoreactive cells together with the immunostimulant R-DOTAP. Control peptide antigen formulations were prepared with 0.5 ml of Montanide + CpG or the "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 Freud's adjuvant. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml vaccine injections subcutaneously to HLA-A2-expressing transgenic mice (AAD mice) on days 0 and 7. Control mice were inoculated with MUC1 peptide formulated in sucrose buffer alone. Vaccine formulation-specific immune responses were measured by ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in spleens from vaccinated mice. Each data point on the graph represents the average SFU per 250,000 splenocytes of vaccinated mice. Error bars represent the mean ± SEM of five mice per group. To identify MUC1-specific T cells, splenocytes were stimulated with the peptide sequence C2A (SEQ ID NO: 10: YLIALAVCQV).

[0134] As shown in Figure 3, HLA-A2-specific CD8 T cell responses were evaluated against a formulation 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 normally inactive in generating immune responses when included in an antigen mixture formulated with R-DOTAP immunostimulatory nanoparticles. Insertion of the hydrophobic peptide into the nanoparticle lipid bilayer effectively delivered the otherwise insoluble antigen to dendritic cells of the immune system, generating a potent 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 liposomal bilayer of R-DOTAP were formulated with 3 mg of R-DOTAP in a 1 ml formulation. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml vaccine injections subcutaneously to HLA-A2-expressing transgenic mice (AAD mice) on days 0 and 7. Negative control mice were inoculated with MUC1 peptide formulated in sucrose buffer. Vaccine formulation-specific immune responses were measured in an ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in spleens from vaccinated mice. Each data point on the graph represents the average SFU per million splenocytes from vaccinated mice. Error bars represent the mean ± SEM of five mice per group. To identify 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), and V2A (SEQ ID NO: 14: YLDTRPAPV).

[0135] Encapsulation and delivery of this nonpolar peptide antigen into the bilayer of chiral cationic lipid nanoparticles resulted in the induction of very high levels of antigen-specific T cells in mouse humanized HLA-A2 transgenic mouse ELISpot assays (Figures 1, 2, and 3).

[0136] Example 4 Effect of incorporating epitope peptides into multi-epitope peptides Incorporating the C1A and C2A amino acid sequences into a much larger peptide derived from the oncoprotein MUC1 enabled efficient binding and delivery to the immunostimulatory nanoparticle R-DOTAP in an aqueous environment. This large peptide, YL40 (SEQ ID NO: 1), contains a hydrophobic agonist peptide at the N-terminus of the molecule, which can be efficiently processed by intracellular proteases in dendritic cells to generate individual agonist peptide antigens for presentation on cell surface MHC. This approach demonstrated the detection of high levels of peptide agonist C1A-specific T cell responses following vaccination of humanized transgenic HLA-A mice and ELISpot analysis (Figures 6 and 7).

[0137] As shown in Figure 6, HLA-A2-specific CD8 T cell responses were evaluated against a MUC1 / R-DOTAP vaccine formulation containing the long MUC1 peptide antigen YL-40, which contains the C1A antigen (SEQ ID NO: 2) and the C2A antigen (SEQ ID NO: 3). The inclusion of peptide YL40 (SEQ ID NO: 1) resulted in strong antigen-specific responses to the C1A and C2A antigens. MUC1-derived lipidated peptide antigen agonists (0.2–1.0 mg / peptide) were formulated as peptide micelles with 3 mg of R-DOTAP and the long peptide antigen YL-40 in a 1 ml formulation. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml vaccine injections subcutaneously to HLA-A2-expressing transgenic mice (AAD mice) on days 0 and 7. Control mice were vaccinated with MUC1 peptides formulated in a sucrose / water solution. Vaccine formulation-specific immune responses were measured by ELISPOT assay by counting MUC1 antigen-specific T cells in triplicate wells in spleens from vaccinated mice. Each data point on the graph represents the average SFU per million splenocytes from vaccinated mice. Error bars represent the mean ± SEM of five mice per group. To identify 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), and V2A (SEQ ID NO: 14: YLDTRPAPV).

[0138] As shown in Figure 7, HLA-A2-specific CD8 T cell responses were evaluated against a formulation 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 normally inactive in generating immune responses when included in antigen mixtures formulated with R-DOTAP immunostimulatory nanoparticles. Insertion of the hydrophobic peptide into the nanoparticle lipid bilayer effectively delivers otherwise insoluble antigens to dendritic cells of the immune system, generating potent immune responses 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 micellar mixtures and agonist antigens C1A and C2A incorporated into the liposomal bilayer of R-DOTAP were formulated with 3 mg of R-DOTAP in 1 ml formulations. The efficacy of these vaccine formulations in generating T cell responses was measured by administering two 0.1 ml vaccine injections subcutaneously to HLA-A2-expressing transgenic mice (AAD mice) on days 0 and 7. Negative control mice were inoculated with MUC1 peptides formulated in sucrose buffer. Vaccine formulation-specific immune responses were measured in ELISPOT assays by counting MUC1 antigen-specific T cells in triplicate wells in spleens from vaccinated mice. Each data point on the graph represents the average SFU per million splenocytes of vaccinated mice. Error bars represent the mean ± SEM of five mice per group. To identify 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), and V2A (SEQ ID NO: 14: YLDTRPAPV).

[0139] Example 5 Comparison of the effects of mixtures and encapsulation on the induction of immune responses Numerous combinations of MUC1 peptide sequences, including short specific peptide antigens and other long overlapping peptide sequences derived from the MUC1 protein, have been generated and shown to be ineffective when combined with or incorporated into the immunostimulatory nanoparticle R-DOTAP.

[0140] For example, as shown in Figure 8, the YL40 (SEQ ID NO: 1) long peptide induced a significant C1A response (activity), while the YL35 (SEQ ID NO: 40) peptide did not produce a significant C2A response (using the same approach as YL40); YL35 induced 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, specifically C1A. The long peptide YL35, which contains the C2A sequence, did not enable a strong immune response even when mixed with R-DOTAP. This indicates that not all long peptides have the ability to promote a strong immune response to the incorporated sequence, even when mixed with R-DOTAP.

[0141] Furthermore, we demonstrated that short C1A and C2A peptides induced an immune response only when incorporated into the R-DOTAP bilayer, but not when mixed with R-DOTAP, whereas no significant responses were observed when the short C1A and C2A peptides were encapsulated.

[0142] FIG. 8 also shows that, as expected, short peptides without R-DOTAP have no activity.

[0143] Although the invention has been described with reference to the above examples, it should be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. A multi-epitope peptide comprising at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO:

1.

2. The multi-epitope peptide of claim 1, which has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to an amino acid sequence comprising any one of SEQ ID NOs: 9 to 14 and SEQ ID NOs: 20 to 37.

3. The multi-epitope peptide of claim 1 , comprising at least one mucin 1 (MUC1) peptide.

4. The multi-epitope peptide of claim 1, which has MHC affinity for at least one of HLA-A2, HLA-A3, HLA-A11, and / or HLA-A24.

5. The multi-epitope peptide according to claim 3 , which is recognized by the CD4+ T cell receptor and / or the CD8+ T cell receptor.

6. The multi-epitope peptide of claim 2, comprising a sequence comprising SEQ ID NO:

1.

7. The multi-epitope peptide of claim 1, which is oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated.

8. The multi-epitope peptide of claim 7, which is palmitoylated.

9. The multi-epitope peptide of claim 1 , comprising 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 multiepitope peptide and a cationic lipid, wherein the multiepitope peptide comprises at least one MUC-1 peptide.

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

1.

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

14. 12. 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, a variant thereof, or an analog 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 of claim 11 , wherein the multi-epitope peptide is recognized by a CD4+ T cell receptor and / or a CD8+ T cell receptor.

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

20. 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 a liposome comprising a cationic lipid.

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

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

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

25. (a) a multi-epitope peptide comprising at least one mucin 1 (MUC1) peptide; and (b) a cationic lipid; 10. A vaccine composition comprising:

26. 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, a variant thereof, or an analog thereof.

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

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

1.

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

30. 26. 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. 26. The vaccine of claim 25, wherein the multi-epitope peptide is oxidized, cross-linked, pegylated, glycosylated, phosphorylated, palmitoylated, methylated, or biotinylated.

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

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

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

1.

36. 36. The vaccine of claim 35, wherein the multi-epitope peptide comprises an amino acid sequence comprising at least 80% identity to SEQ ID NO: 18, 19, 42, or 43.

37. 26. The vaccine of claim 25, wherein the multi-epitope peptide is encapsulated in a liposome comprising a cationic lipid.

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

39. 26. The vaccine of claim 25, wherein the multi-epitope peptide is mixed with preformed cationic lipid nanoparticles as separate micelles.

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

1.

41. 1. A method of treating cancer in a subject, comprising: (a) a multi-epitope peptide comprising at least one mucin 1 (MUC1) peptide; and (b) a cationic lipid; treating cancer in a subject by administering to said subject a vaccine composition comprising: A method comprising:

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

1.

43. 42. The method of claim 41, wherein the at least one MUC1 peptide comprises a sequence comprising at least 80%, at least 85%, at least 90%, or at least 95% identity to an amino acid sequence comprising any of SEQ ID NOs: 9-14 and 20-37.

44. 42. 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, a variant thereof, or an analog thereof.

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

46. 42. The method of claim 41, wherein the multi-epitope peptide is encapsulated within a cationic lipid nanoparticle.

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

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

49. The one or more MUC1 peptides are expressed as CD4 by antigen-presenting cells. ＋ T cells and CD8 ＋ 42. The method of claim 41, which induces presentation of non-HLA-restricted peptides to T cells.

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

51. 42. The method of claim 41, wherein the cancer comprises cancer cells that express MUC1.

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

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

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

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

56. 1. A method for inducing a MUC-specific polyfunctional and cytolytic T cell response in a subject, comprising: (a) a multi-epitope peptide comprising one or more mucin 1 (MUC1) peptides; and (b) a cationic lipid; inducing a MUC-specific polyfunctional and cytolytic T cell response in the subject by administering to the subject a composition comprising: A method comprising:

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

1.

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

59. 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, a variant thereof, or an analog thereof.

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

61. 57. The method of claim 56, wherein the multi-epitope peptide is encapsulated within a cationic lipid nanoparticle.

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

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

64. The multi-epitope peptide binds to CD4 by antigen-presenting cells. ＋ T cells and CD8 ＋ 57. The method of claim 56, which induces presentation of non-HLA-restricted peptides to T cells.

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