Prostate-specific antigen peptides and uses thereof

Abstract

The present disclosure provides isolated peptides derived from prostate-specific antigen, peptide-based molecules (e.g., peptide-MHC (pMHC) complexes), polynucleotides and vectors encoding the peptides or peptide-based molecules, pharmaceutical compositions (e.g., vaccine compositions), and their use for treatment or prevention of prostate cancer. The present disclosure also provides binding moieties that bind to the peptides or peptide-based molecules disclosed herein, and their use for treatment or prevention of prostate cancer.

Description

PROSTATE-SPECIFIC ANTIGEN PEPTIDES AND USES THEREOFSEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 18, 2025, is named 67000-1481_WO_SL.xml and is 54,474 bytes in size.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] The present application claims priority to US Provisional Application No. 63 / 678,314, filed 01 -August-2024, which is hereby incorporated in its entirety.TECHNICAL FIELD

[0003] The present disclosure relates to methods and compositions that involve isolated peptides derived from prostate tissue-specific antigens, and the use of such methods and compositions for treatment or prevention of prostate cancer.BACKGROUND

[0004] Prostate cancer is a commonly diagnosed cancer in men and a leading cause of cancer death. If detected at an early and treatable stage, prostate cancer is curable. Unfortunately, a majority of cases are diagnosed at later stages when metastasis of the primary tumor has already occurred. Even early diagnosis is controversial because not all individuals who test positive in prostate cancer screens develop cancer.

[0005] Prostate-specific antigen (PSA), is a protein produced by normal, as well as malignant, cells of the prostate gland. PSA is a serum glycoprotein member of the glandular kallikrein gene family. Like all other members of the kallikrein family, PSA is a serine protease that is synthesized in an inactive form as a zymogen which is composed of a pre-peptide (also known as signal peptide) and a pro-peptide (which maintains the enzyme in the latent form). PSA has a chymotrypsin-like substrate specificity (i.e., it cleaves on the carboxyl side of a hydrophobic amino acid residue, namely Tyr, Phe, Trp, and Leu). The physiological function of PSA is degradation of the gel-forming proteins semenogelins I and II in semen after ejaculation.

[0006] The blood level of PSA is often elevated in people with prostate cancer. The tissue specificity of PSA makes PSA useful as both a diagnostic target and as a potential therapeutic target for active specific immunotherapy.SUMMARY

[0007] As specified in the Background section above, there is a great need in the art for development of a therapeutic treatment related to elevated levels of PSA in prostate cancer patients for specific immunotherapy. The present application addresses these and other needs.

[0008] In one aspect, provided herein is a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11 in the form of a pharmaceutically acceptable salt.

[0009] In some embodiments, the peptide has a sequence of any one of SEQ ID NOs: 1-6 or any one of SEQ ID NOs: 7-11. In some embodiments, the peptide has a sequence of SEQ ID NO: 2.

[0010] In some embodiments, the salt is an acetate salt, a sodium salt, or an HCL salt. In some embodiments, the acetate salt is a trifluoroacetate (TFA) salt.

[0011] In another aspect, provided herein is a composition comprising a peptide having a sequence selected from the group consisting of SEQ ID NOs: 1-11, wherein the composition comprises an adjuvant and a pharmaceutically acceptable carrier.

[0012] In another aspect, provided herein is a polynucleotide encoding a peptide and a regulatory sequence, wherein: a) the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 1-4 and 7-8; and b) the polynucleotide is not identical to a KLK3 gene or a portion thereof. In some embodiments, the polynucleotide encodes a peptide having SEQ ID NO: 2.

[0013] Regulatory elements are specific nucleotide sequences that play a role in controlling gene expression. These elements do not code for proteins but insteadregulate the transcription of genes. The main types of DNA regulatory elements include promoters, enhancers, silencers, insulators, and operators.

[0014] In another aspect, provided herein is a polynucleotide encoding a peptide and a regulatory sequence, wherein: a) the peptide has a sequence as set forth in SEQ ID NO:5 or SEQ ID NO: 9; and b) the polynucleotide is not identical to a KLK4 gene or a portion thereof.

[0015] In another aspect, provided herein is a polynucleotide encoding a peptide and a regulatory sequence, wherein: a) the peptide has a sequence as set forth in SEQ ID NO:6 or SEQ ID NO: 10; and b) the polynucleotide is not identical to a KLK2 gene or a portion thereof.

[0016] In another aspect, provided herein is a polynucleotide encoding a peptide and a regulatory sequence, wherein: a) the peptide has a sequence as set forth in SEQ ID Nos: 1-11; and b) the polynucleotide is not identical to an ACP3 gene or a portion thereof.

[0017] In another aspect, provided herein is a vector comprising a polynucleotide encoding a peptide having a sequence of any one of SEQ ID NOs: 1-11. In some embodiments, the vector is an expression vector. In other embodiments, the vector is a viral vector.

[0018] In another aspect, provided herein is a host cell comprising a polynucleotide encoding a peptide having a sequence of any one of SEQ ID NOs: 1 -11. In some embodiments, the host cell is a prokaryotic or a eukaryotic cell. In other embodiments, the host cell is an APC.

[0019] In another aspect, provided herein is a modified peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11.

[0020] In some embodiments, the modified peptide is pegylated or has at least one amino acid of the peptide a D-amino acid.

[0021] In some embodiments, the modified peptide has at least one residue-specific modification selected from the group consisting of: phosphorylation, acetylation, methylation, ubiquitination, glycosylation, SUMOylation, oxidation, nitrosylation, and palmitoylation.

[0022] In some embodiments, the modified peptide has a sequence of any one of SEQ ID Nos: 1-6 or SEQ IDs NO: 7-11. In some embodiments, the peptide has a sequence of SEQ ID NO: 2.

[0023] In another aspect, provided herein is a polypeptide comprising: a) a peptide sequence selected from the group consisting of: SEQ ID NOs: 1-11; b) a linker; and c) a 02-microglobulin (02M) polypeptide, or a portion thereof.

[0024] In some embodiments, the modified peptide has a sequence of any one of SEQ ID Nos: 1-6 or SEQ IDs NO: 7-11. In some embodiments, the peptide has a sequence of SEQ ID NO: 2.

[0025] In another aspect, provided herein is an isolated protein complex comprising: a) at least one part of a Major Histocompatibility Complex (MHC) molecule; and b) a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11.

[0026] In some embodiments, the HLA in the isolated protein complex is HLA-A. In some embodiments, the Major Histocompatibility Complex (MHC) molecule in the isolated protein complex is an HLA heavy chain, a truncated HLA heavy chain or 0-2 macroglobulin.

[0027] In some embodiments, the peptide in the isolated protein complex has a sequence of any one of SEQ ID Nos: 1-6 or SEQ IDs NO: 7-11. In some embodiments, the peptide has a sequence of SEQ ID NO: 2.

[0028] In some embodiments, the MHC molecule in the isolated protein complex is selected from the group consisting of class I, class II, and class III.

[0029] In some embodiments, the complex between the peptide and the MHC molecule in the isolated protein complex is a covalently bound complex. In some embodiments, the covalently bound complex is a fusion protein of the MHC molecule with the peptide.

[0030] In some embodiments, there is a non-native linkage between the peptide, and a binding groove of the MHC in the isolated protein complex. In some embodiments, the non-native linkage is between the C-terminal anchor residue of the peptide and an amino acid residue in the F pocket of the binding groove of the MHC molecule.

[0031] In another aspect, provided herein is an isolated molecule that binds the peptide, the modified peptide, the polypeptide, or the isolated protein complex of the present disclosure.

[0032] In some embodiments, the isolated molecule of is an antibody or an antigenbinding fragment thereof. In some embodiments, the antibody is a bispecific antibody.

[0033] In some embodiments, isolated molecule is an alternative scaffold, a chimeric antigen receptor (CAR), or a T cell receptor (TCR).

[0034] In another aspect, provided herein is an isolated cell comprising the CAR of the present disclosure.

[0035] In some embodiments, the isolated cell is an immune cell. In some embodiments, the immune cell is a T cell, an NK cell, or a macrophage.

[0036] In another aspect, provided herein is an isolated cell comprising the TCR of the present disclosure.

[0037] In some embodiments, the isolated cell an immune cell. In some embodiments, the immune cell is a T cell, an NK cell, or a macrophage.

[0038] In another aspect, provided herein is a nanoparticle complex comprising a nanoparticle core and the isolated protein complex of the present disclosure covalently coupled to the nanoparticle core.

[0039] In another aspect, provided herein is a modified single chain TCR comprising an alpha chain and a beta chain, wherein the TCR specifically binds to the isolated protein complex of the present disclosure.

[0040] In another aspect, provided herein is an artificial antigen presenting cell (aAPC) comprising a liposome comprising a phospholipid and the isolated protein complex of the present disclosure.

[0041] In another aspect, provided herein is a vaccine composition capable of rising a specific T-cell response comprising: a) a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11; b) one or more polynucleotides encoding a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11; c) a population of autologous dendritic cells or antigen presenting cells presenting at its surface the prostate-specific antigen epitope having a sequence selected from the group consisting of: SEQ ID NOs: 1-11; or d) one or more MHC molecules or parts thereof loaded with the prostate-specific antigen epitope having a sequence selected from the group consisting of: SEQ ID NOs: 1-11, and a pharmaceutically acceptable carrier other than water.

[0042] In some embodiments, the peptide in the vaccine or immunogenic composition of the present disclosure has a sequence of any one of SEQ ID Nos: 1-6 or SEQ IDs NO: 7-11. In some embodiments, the peptide has a sequence of SEQ ID NO: 2.

[0043] In another aspect, provided herein is a pharmaceutical composition comprising a first peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11 and a pharmaceutically acceptable carrier other than water.

[0044] In some embodiments, the first peptide in the pharmaceutical composition of the present disclosure is not covalently bound to a second peptide or a molecule. In some embodiments, first peptide is bound by hydrogen bonds to the second peptide or molecule such as an HLA molecule or part thereof. In some embodiments, HLA molecule is selected from the group consisting of class I, class II and class III. In some embodiments, the HLA molecule is a class I molecule encoded by an HLA gene.

[0045] In some embodiments, the first peptide in the pharmaceutical composition of the present disclosure is part of a complex that comprises (i) an HLA-A heavy chain or a part thereof; and (ii) a P-2 macroglobulin or a part thereof.

[0046] In another aspect, provided herein is a pharmaceutical composition comprising the peptide salt of the present disclosure and a pharmaceutically acceptable carrier other than water.

[0047] In another aspect, provided herein is a pharmaceutical composition comprising the polynucleotide of the present disclosure and a pharmaceutically acceptable carrier other than water.

[0048] In another aspect, provided herein is a pharmaceutical composition comprising the modified of the present disclosure and a pharmaceutically acceptable carrier other than water.

[0049] In another aspect, provided herein is a pharmaceutical composition comprising the polypeptide of the present disclosure and a pharmaceutically acceptable carrier other than water.

[0050] In another aspect, provided herein is a pharmaceutical composition comprising the isolated protein complex of the present disclosure and a pharmaceutically acceptable carrier other than water.

[0051] In another aspect, provided herein is a pharmaceutical composition comprising the nanoparticle complex of the present disclosure and a pharmaceutically acceptable carrier other than water.

[0052] In another aspect, provided herein is a pharmaceutical composition comprising the single chain TCR of the present disclosure and a pharmaceutically acceptable carrier other than water.

[0053] In another aspect, provided herein is a pharmaceutical composition comprising the artificial antigen presenting cell (aAPC) of the present disclosure and a pharmaceutically acceptable carrier other than water.

[0054] In another aspect, provided herein is a method for isolating an antigen specific T cell, the method comprising the steps of: a) providing a composition comprising a particle attached to an MHC display moiety comprising at least one antigenic peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11; b)providing a sample comprising one or more T cells; c) contacting the composition with the sample, wherein the contacting comprises providing conditions sufficient for a single T cell to bind the MHC display moiety attached to the at least one particle; and d) isolating a single antigen specific T cell associated with the at least one particle.

[0055] In some embodiments, the MHC display moiety is attached to the particle by a linker.

[0056] In another aspect, provided herein is a method of inducing an immune response in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11 or any of the pharmaceutical compositions of the present disclosure.

[0057] In another aspect, provided herein is a method of treating or reducing the likelihood of prostate cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11 or any of the pharmaceutical compositions of the present disclosure.

[0058] In another aspect, provided herein is a method of inducing a CD8+ cytotoxic T cell response in a subject having prostate cancer overexpressing prostate-specific antigen comprising a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11, the method comprising administering to the subject a therapeutically effective amount of the peptide or any of the pharmaceutical compositions of the present disclosure.

[0059] In some embodiments, the patient is HLA-Al l:01+ or HLA-A03:01+ and the peptide has SEQ ID NO: 2.

[0060] In another aspect, provided herein is a kit comprising: (i) a) one or more peptides of any one of claims 1-7; b) one or more polynucleotides of the present disclosure; c) one or more vectors of the present disclosure; d) one or more modified peptides of the present disclosure; e) one or more polypeptides of the present disclosure; f) one or more isolated protein complexes of the present disclosure; g) one or more nanoparticle complexes of the present disclosure; h) one or more modified single chain TCRs of thepresent disclosure; i) one or more artificial antigen presenting cell (aAPC) of the present disclosure; j) one or more vaccines of the present disclosure; or k) one or more pharmaceutical compositions of the present disclosure; and (ii) packaging and / or instructions for use for the same.

[0061] In another aspect, provided herein is a method of generating a peptide binding moiety that recognizes one of SEQ ID NOs: 1-11, the method comprising: a) immunizing a non-human animal with a composition comprising: (i) one or more polynucleotides of the present disclosure; (ii) one or more vectors of the present disclosure; (iii) one or more polypeptides of the present disclosure; (iv) one or more isolated protein complexes of the present disclosure; (v) one or more nanoparticle complexes of the present disclosure; (vi) one or more artificial antigen presenting cell (aAPC) of the present disclosure; (vii) one or more vaccines of the present disclosure; or (viii) one or more pharmaceutical compositions of the present; and b) isolating one or more T cells, B cells, TCRs, and / or antibodies that bind one of SEQ ID NOs: 1-11 from the non-human animal.

[0062] In some embodiments, the non-human animal is genetically modified such as for example a genetically modified rodent or mouse.

[0063] In some embodiments, the genetically modified non-human animal is genetically modified to express: i) a human or humanized immunoglobulin, comprising a human or humanized immunoglobulin heavy chain and / or a human or humanized immunoglobulin light chain; ii) a human or humanized TCR; and / or ii) a human or humanized MHC class I molecule, and optionally a human or humanized 2m.

[0064] These and other aspects of the present invention will be apparent to those of ordinary skill in the art in the following description and claims.DETAILED DESCRIPTION

[0065] PSA is widely recognized as the serum biomarker utilized for screening for the presence, aggressiveness and / or recurrence of prostate cancer. PSA was cloned and characterized as a product of the human prostate gland; however, PSA is produced by many organs in the human, in both males and females. In females, PSA is produced inbenign breast, endometrial and ovarian tissues, and malignancies of these tissues, as well as in non-hormonally regulated tissues and their cancers, such as the parotid gland. Across all these malignancies, including prostate cancer, tissue levels of PSA in the cancer tissue are inversely correlated with prognosis.

[0066] PSA undergoes proteolytic processing from a full length protein that initially contains a signal peptide sequence (residues 1-17 of the lull length protein), a sequence corresponding to an activation peptide (residues 18-24), and a sequence corresponding to the processed PSA protein, which ordinarily consists of 237 amino acids spanning residues 25-261 of the full length protein. The 237 amino acid protein (25-261) was used in the studies that include PSA as described herein.

[0067] The full length (i.e., unprocessed) PSA protein consists of the amino acid sequence:MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCG GVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDM SLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYAS GWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGK STCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVA NP (SEQ ID NO: 12).

[0068] The clinical effects of immune checkpoint blockade in patients have demonstrated increased overall survival indicating that T cells of cancer patients can potentially respond to and eliminate cancer cells. Cytotoxic CD8+ T cells of the adaptive immune system protect a host against intracellular pathogens and tumors. As such, cytotoxic CD8+ T cells play a pivotal role in controlling tumor growth. Because of the critical role of CD8+ T-cells in defending against tumors, harnessing CD8+ T- cells has become an important aspect of immunotherapies. Cytotoxic T-cells mediate adaptive immunity by recognizing HLA-peptide complexes presented on the surface of infected cells via interactions with the T cell receptors (TCRs) expressed on their surface. These HLA-peptide complexes can be used as targets for developing different immunotherapeutics, including, for example, bispecific antibodies and CAR-T therapies. Accordingly, there is a strong interest in CD8+ T-cell-based immunotherapystudies and the identification of antigens presented by the human leukocyte antigen (HLA) class I of cancer cells.

[0069] Peptides presented by HLA molecules on the cell surface play a crucial role in adaptive immunity, mediating the communication between T cells and antigen presenting cells. Such tumor-specific HLA-peptide complexes can potentially serve as antigens to generate antibodies or TCR’s for use as or in therapeutic agents for cancer immunotherapy, especially when there are no traditional cell-surface protein targets available.

[0070] These antigens can be targeted by cytotoxic CD8+ T cells, which are responsible for the immune-mechanized eradication of cancer cells. There is mounting evidence that immune recognition of tumor-derived, cancer-specific HLA class-I restricted epitopes is a crucial mechanism in successfully triggering immune-mediated tumor rejection. HLA class I presents a repertoire of endogenously derived peptides for immune surveillance by cytotoxic T lymphocytes.

[0071] While virtually all cellular proteins undergo proteolysis, only a subset is selected for presentation by HLA. Therapeutic targeting of these antigens with chimeric antigen receptor or engineered TCR T cell therapies, bispecific monoclonal antibodies, or vaccines is a promising, personalized treatment for many tumor types including prostate adenocarcinomas.

[0072] To generate precise immunotherapeutics, the amino acid sequence of the peptide presented in the HLA-peptide complex needs to be known. Prostate lineage antigens are attractive immunotherapy targets because of their tissue specificity and the specific nature of T cell receptors in T cells to mount an immune response against tumor cells. Mass spectrometry-based HLA-peptide sequencing provides means to detect the HLA-presented peptides present on the surface of a tumor cell. A synthetic analog spike-in method can be used to estimate copy number of peptides of interest and help determine which immunotherapeutic approach (like CART, bi-specific antibodies, etc.) is most appropriate.

[0073] The present disclosure provides, among other things, isolated peptides derived from PSA or other prostate lineage antigens, and fragments or derivatives thereof. Specifically, the present disclosure provides peptides identified from the prostate lineage antigens, Prostate Specific Antigen (KLK3), Kallikrein-4 (KLK4), Kallikrein- 2 (KLK2), and Prostatic acid phosphatase (ACP3). Various peptide-based molecules including complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, and conjugates comprising the peptides are also provided. Further provided herein are polynucleotides and vectors encoding the peptides or peptide-based molecules described herein. Binding moieties (e.g., antibodies, alternative scaffolds, T-cell receptors (TCRs) or chimeric antigen receptors (CARs)) that bind to the peptides or peptide-based molecules are also provided. The compositions of the present disclosure can be used to induce an immune response and / or for treatment or prevention of prostate cancer.

[0074] Kallikreins are a subgroup of serine proteases, enzymes capable of cleaving peptide bonds in proteins. In humans, tissue kallikrein-related peptidases (KLKs) encode a family of fifteen closely related serine proteases. These genes are localized to chromosome 19ql3, forming the largest contiguous cluster of proteases within the human genome. Kallikreins are responsible for the coordination of various physiological functions including blood pressure, semen liquefaction and skin desquamation.

[0075] The protein encoded by the human KLK2 gene (genomic location: chrl9:50, 861, 568-50, 880, 567) is a highly active trypsin-like serine protease that selectively cleaves at arginine residues. This protein is primarily expressed in prostatic tissue and is responsible for cleaving pro-prostate-specific antigen into its enzymatically active form.

[0076] The protein encoded by the human KLK3 gene (genomic location: chrl9:50, 854, 915-50, 860, 764) functions in the process of liquefaction of seminal coagulum by hydrolysis of the high molecular mass seminal vesicle protein.

[0077] The protein encoded by the human KLK4 gene (genomic location: chrl9:50, 819, 146-50, 823, 787) possesses serine-type endopeptidase activity andfunctions in collagen chain trimerization and regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs).

[0078] The protein encoded by the human ACP3 gene (genomic location: chr3: 132, 317, 369-132, 368, 302) catalyzes the conversion of orthophosphoric monoester to alcohol and orthophosphate. It is synthesized under androgen regulation and is secreted by the epithelial cells of the prostate gland.

[0079] Lymphocytes, such as T cells, play important roles in adaptive anti-infection, antitumor, autoimmune, and transplant rejection responses. Generally, a T cell mediated immune response involves close contact, e.g., an immunological synapse, between a T cell and an antigen presenting cell (APC). The pairing of several molecules is involved in the formation of the immunological synapse, including, but not limited to: (a) a T-cell receptor (TCR) on a T cell, which specifically binds to a peptide presented in the peptide binding groove of a major histocompatibility complex (MHC) molecule on an APC; and (b) CD28 (on the T cell), which pairs with a B7 molecule on the APC. A TCR, together with CD3 molecules, form a TCR complex, and upon pairing of the TCR to the peptide-MHC (pMHC) complex, a signal is sent through CD3. Signaling through both the TCR complex and CD28 on the T cell results in activation of the T cell.

[0080] T cell receptors are heterodimeric structures composed of two types of chains (an a (alpha) and p (beta) chain, or a y (gamma) and 5 (delta) chain). The a chain is encoded by the nucleic acid sequence located within the a locus (on human or mouse chromosome 14), which also encompasses the entire 8 locus that encodes the 8 chain, and the chain is encoded by the nucleic acid sequence located within the P locus (on mouse chromosome 6 or human chromosome 7). Most T cells have an ap TCR, while a minority of T cells bear a y8 TCR. T cell receptor a and p polypeptides (and similarly y and 8 polypeptides) are linked to each other via a disulfide bond. Each of the two polypeptides that make up the TCR contains an extracellular domain comprising constant and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and the cytoplasmic tail also being a part of the constant region).

[0081] The variable region of each TCR comprises a unique and characteristic structure, i.e., an idiotope that determines the specificity of the TCR. Generally, a TCR will bind to a pMHC complex only if the TCR comprises an idiotype that recognizes the peptide being presented in the context of MHC, e.g., the unique conformation of a particular pMHC complex.

[0082] Immunotherapeutic approaches to treating disease work to regulate T cell activity in vivo, e.g., to enhance anti-infection and antitumor responses, or, for example, downregulate autoimmune and transplant rejection responses. However, such methods can lack specificity since immunotherapies can target signaling by the TCR complex by binding CD3 and / or the pairing of costimulatory molecules. Such approaches can result in undesirable side effects, e.g., a hyperactive immune response or generalized immune suppression. Accordingly, therapies that take advantage of the uniquely specific interaction between a TCR and pMHC complex may provide the ability to specifically modulate the activity of specific T cells in vivo, and provide treatments based on T cell modulation.

[0083] The presentation of prostate lineage antigen-derived peptides by MHC molecules on the surface of an infected cell and the recognition of these pMHC complexes by, and subsequent activation of, CD8+ cytotoxic T cells provides an important mechanism for immunity-based protection against tumors. Prostate cancer cells can express various prostate lineage antigen-derived antigens. Peptides derived from these antigens may be displayed on the tumor cell surface in complex with MHC molecules. Detection of an MHC-presented prostate lineage antigen-derived peptide by a T cell bearing the corresponding TCR, leads to targeted killing of the tumor cell. However, because of the selection processes which occur during T cell maturation in the thymus, there is often a scarcity of T cells in the circulating repertoire, which recognize prostate lineage antigen-derived peptides with a sufficiently high level of affinity. As a result, tumor cells often escape elimination by the immune system.

[0084] The identification of prostate lineage antigen-derived peptides presented on prostate cancer cells can allow for the development of immunotherapeutic reagentsdesigned to specifically target and destroy the prostate cancer cells. Such reagents may be moieties that bind to the prostate lineage antigen-derived peptide and / or pMHC complexes and the reagents, e.g., moieties that can function by inducing a T cell response. For example, such reagents may be based on antibodies, TCRs, and / or CARs.

[0085] The present disclosure, in part, is based on a proteogenomic approach that detects MHC-associated prostate lineage antigen-derived peptides from prostate cancer cells. The repertoire of HLA-I-prostate lineage antigen-derived peptides that are expressed in prostate cancer cells characterized herein can provide an accurate representation of prostate lineage antigen epitopes in a population.

[0086] HLA-restricted prostate lineage antigen-derived peptides as potential targets may be leveraged for delivering immunotherapeutics (such as antibodies (e.g., bispecific antibodies), engineered TCR- or CAR- based cellular therapies) to prostate cancer cells. However, differences in patient HLA alleles, can be a challenge in developing therapeutics against these peptide targets. To address this challenge, in one aspect the present disclosure provides a proteogenomics approach for generating patient-specific databases that allows for the comprehensive identification of prostate lineage antigen-derived peptides. The HLA-associated prostate lineage antigen- derived peptides disclosed herein may be used in the development of immunotherapeutics (such as antibodies (e.g., bispecific antibodies), engineered TCR- or CAR- based cellular therapies) for the treatment of prostate cancer and to inform vaccine development. The proteogenomic discovery platform described herein provides a method for identifying prostate lineage antigen-derived peptides as targets for anti-tumor related immunotherapy.Definitions

[0087] Unless defined otherwise, 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 disclosure belongs.

[0088] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes oneor more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0089] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0090] The term “antigen” encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that, when introduced into an immunocompetent host (directly or upon expression as in, e.g., DNA or RNA vaccines) is recognized by the immune system of the host and can elicit an immune response by the host. The T-cell receptor (TCR) recognizes a peptide presented in the context of a major histocompatibility complex (MHC) as part of an immunological synapse. The peptide-MHC (pMHC) complex is recognized by TCR, with the peptide (antigenic determinant) and the TCR idiotype providing the specificity of the interaction. Accordingly, the term “antigen” encompasses peptides presented in the context of MHCs, e.g., pMHC complexes. The peptide displayed on MHC may also be referred to as an “epitope” or an “antigenic determinant”. The terms “peptide,” “antigenic determinant,” “epitopes,” etc., encompass not only those presented naturally by antigen-presenting cells (APCs), but may be any desired peptide so long as it is recognized by an immune cell, e.g., when presented appropriately to the cells of an immune system. For example, a peptide having an artificially prepared amino acid sequence may also be used as the epitope.

[0091] A single antigen (such as an antigenic polypeptide) may have more than one epitope. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and are defined as those residues that directly contribute to the affinity of the interaction between an MHC molecule and the antigen. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically activesurface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three- dimensional structural characteristics, and / or specific charge characteristics. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.

[0092] The terms “major histocompatibility complex,” and “MHC” encompass the terms “human leukocyte antigen” or “HLA” (the latter two of which are generally reserved for human MHC molecules), naturally occurring MHC molecules (e.g., MHC class I molecule comprising MHC class I a (heavy) chain and 02 microglobulin; MHC class II molecule comprising MHC class II a chain and MHC class II 0 chain), individual chains of MHC molecules (e.g., MHC class I a (heavy) chain, MHC class II a chain, and MHC class II P chain), individual subunits of such chains of MHC molecules (e.g., al, a2, and / or a3 subunits of MHC class I a chain, al-a2 subunits of MHC class II a chain, P1-P2 subunits of MHC class II P chain) as well as portions (e.g., the peptide-binding portions, e.g., the peptide-binding grooves), mutants and various derivatives thereof (including fusions proteins), wherein such portion, mutants and derivatives retain the ability to display an antigenic peptide for recognition by a TCR, e.g., an antigen-specific TCR. An MHC class I molecule comprises a peptide binding groove formed by the al and a2 domains of the heavy a chain that can stow a peptide of around 8-10 amino acids. Despite the fact that both classes of MHC bind a core of about 9 amino acids (e.g., 5 to 17 amino acids) within peptides, the open-ended nature of MHC class II peptide binding groove (the al domain of a class II MHC a polypeptide in association with the 1 domain of a class II MHC 0 polypeptide) allows for a wider range of peptide lengths. Peptides binding MHC class II usually vary between 13 and 17 amino acids in length, though shorter or longer lengths are not uncommon. As a result, peptides may shift within the MHC class II peptide binding groove, changing which 9-mer sits directly within the groove at any given time. In some embodiments, the peptide-MHC complex described herein may be a peptide-MHC complex from a non-human animal. In other embodiments, the peptide-MHC complex described herein may include an peptide-HLA complex, i.e., a peptide-MHC complex from a human. Conventional identifications of particular MHC variants are used herein. For example,HLA-A11 refers to a human leucocyte antigen from the A gene group (hence a class I type MHC) gene position (known as a gene locus) number 11; gene HLA-DR11, refers to a human leucocyte antigen coded by a gene from the DR region (hence a class II type MHC) locus number 11.

[0093] “MHC-peptide complex,” “peptide-MHC complex,” “pMHC complex,” “peptide-in-groove,” and the like include (i) an MHC molecule, e.g., a human and / or non-human animal MHC molecule, or portion thereof (e.g., the peptide-binding groove thereof, and e.g., the extracellular portion thereof, and (ii) an antigenic peptide (e.g., a prostate lineage antigen-derived peptide), where the MHC molecule and the antigenic peptide are complexed in such a manner that the pMHC complex can specifically bind a T-cell receptor. A pMHC complex encompasses cell surface expressed pMHC complexes and soluble pMHC complexes.

[0094] “HLA-peptide complex,” “peptide-HLA complex,” “pHLA complex,” and the like refer to an MHC-peptide complex wherein the MHC molecule is a Human Leukocyte Antigen (HLA) molecule.

[0095] The term “T cell” or “T lymphocyte” is used herein in its broadest sense to refer to all types of immune cells expressing CD3, including, but not limited to, T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), tumor infiltrating cytotoxic T cells (TIL; CD8+ T cell), CD4+CD8+ T cells, T-regulatory cells (Treg), and NK-T cells. T cells can include thymocytes, naive T cells, memory T cells, immature T cells, mature T cells, resting T cells, or activated T cells. T cells may also include “gamma-delta T cells (y8 T cells),” which refer to a specialized population that to a small subset of T cells possessing a distinct TCR on their surface, and unlike the majority of T cells in which the TCR is composed of two glycoprotein chains designated a- and 0-TCR chains, the TCR in y8 T cells is made up of a y-chain and a 8-chain.

[0096] The term “antigen presenting cell” or “APC” refers to any cell that presents on the surface of the cell an antigen in association with a major histocompatibility complex molecule, either MHC class I or MHC class II molecule, or both.

[0097] The terms “antibody,” “antibodies,” “immunoglobulin”, and the like refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced. The terms include monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antigen-specific TCR), and epitope-binding fragments of any of the above. The terms “antibody” and “antibodies” also refer to covalent diabodies such as those disclosed in U.S. Pat. Appl. Pub. 2007 / 0004909, incorporated herein by reference in its entirety, and Ig-DARTS such as those disclosed in U.S. Pat. Appl. Pub. 2009 / 0060910, incorporated herein by reference in its entirety. Antibodies useful in the present disclosure include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

[0098] The term “specifically binds,” “binds in a specific manner,” “antigen-specific” or the like, indicates that the molecules involved in the specific binding are able to form a complex with each other that is relatively stable under physiological conditions, and are unable to form stable complexes non-specifically with other molecules outside the specified binding pair. Accordingly, a peptide binding moiety (e.g., an antibody, an alternative scaffold, a CAR, or a TCR) that binds in a specific manner to a prostate lineage antigen-derived peptide, or a peptide-based molecule (such as a complex (e.g., a pMHC complex), fusion protein, or conjugate comprising the described peptide) indicates that the peptide binding moiety forms a stable intermolecular non-covalent bonds with the prostate lineage antigen-derived peptide or peptide-based molecule (such as a complex (e.g., a pMHC complex), fusion protein, or conjugate comprising the described peptide). Specific binding can be characterized by an equilibrium dissociation constant (KD) in the low micromolar to picomolar range (i.e., a smaller KD denotes a tighter binding). High specificity may be in the low nanomolar range, with very high specificity being in the picomolar range. For example, a peptide bindingmoiety may exhibit binding to a prostate lineage antigen-derived peptide or peptide- based molecule (such as a complex (e.g., a pMHC complex), fusion protein, or conjugate comprising the described peptide) with a KD of about 3000 nM or less, about 2000 nM or less, about 1000 nM or less, about 500 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 1 nM or less, or about 0.5 nM or less. Methods for determining whether two molecules specifically bind to one another are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0099] The terms “protein” and “polypeptide”, used interchangeably herein, encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP- ribosylation, PEGylation, biotinylation, etc.). Small polypeptides of less than 100 amino acids, preferably less than 50 amino acids, may be referred to as “peptides”.

[0100] The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0101] The term “operably linked” or the like refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. For example, a control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences include both expression control sequences that are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control a gene ofinterest (or sequence of interest). The term “expression control sequence” includes polynucleotide sequences, which are necessary to affect the expression and processing of coding sequences to which they are ligated. “Expression control sequences” include: appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance polypeptide stability; and when desired, sequences that enhance polypeptide secretion. The nature of such control sequences differs depending upon the host organism. For example, in prokaryotes, such control sequences generally include promoter, ribosomal binding site and transcription termination sequence, while in eukaryotes typically such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0102] The term “isolated” refers to a homogenous population of molecules (such as polynucleotides or polypeptides) which have been substantially separated and / or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. “Isolated” refers to a molecule that is substantially free of other cellular material and / or chemicals and encompasses molecules that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0103] The term “derivative” as used herein refers to a peptide, polypeptide, or polynucleotide, or a variant or analog thereof, comprising one or more mutations and / or chemical modifications as compared to a reference peptide, polypeptide or polynucleotide. Mutations and / or chemical modifications are further detailed below and can include, for example, insertions, substitutions, deletions, transversions, and / or inversions at one or more locations in the amino acid or nucleotide sequence.

[0104] The terms “treat” or “treatment” of a state, disorder, disease, or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder, disease, or condition developing in a subject that may be afflicted with or predisposed to the state, disorder, disease, or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder, disease, or condition; or (2) inhibiting the state, disorder, disease, or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the state, disorder, disease, or condition, i.e., causing regression of the state, disorder, disease, or condition or at least one of the clinical or sub-clinical symptoms of the state, disorder, disease, or condition. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0105] An “individual” or “subject” or “animal” refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of diseases (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0106] The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0107] The phrase “pharmaceutically acceptable”, as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federalor a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0108] The term “administration” and the like refer to and includes the administration of a composition to a subject or system (e.g., to a cell, organ, tissue, organism, or relevant component or set of components thereof). The skilled artisan will appreciate that route of administration may vary depending, for example, on the subject or system to which the composition is being administered, the nature of the composition, the purpose of the administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., to a human or a rodent) may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and / or vitreal. In some embodiments, administration may involve intermittent dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0109] In accordance with the disclosure herein, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, F.M. et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include site directed mutagenesis as described in Kunkel, Proc. Natl. Acad. Sci. USA 82: 488- 492 (1985), U. S. Patent No. 5,071, 743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360 (1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh andGuengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech. 13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang and Malcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patents Nos. 5,789, 166 and 5,932, 419, Hogrefe, Strategies 14. 3: 74-75 (2001), U. S. Patents Nos. 5,702,931, 5,780,270, and 6,242,222, Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson, Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996), Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch and Joly, Nucl. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178: 3346- 3349 (1996), Boles and Miogsa, Curr. Genet. 28: 197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al., Meth. Molec. Biol. 67: 209-218.Peptides Disclosed Herein

[0110] In one aspect, the present disclosure provides isolated peptides comprising an amino acid sequence derived from PSA or other prostate lineage antigens.

[0111] Amino acids sequences derived from prostate lineage antigens disclosed herein may include naturally occurring proteogenic amino acids as well as non- proteogenic amino acids and non-naturally occurring amino acids such as amino acid analogs. In some embodiments, the amino acids that may be used in the practice of the present disclosure may include, for example, without limitation, naturally occurring proteogenic (L)-amino acids, their optical (D)-isomers, chemically modified amino acids, including, e.g., amino acid analogs such as, e.g., selenocysteine (Sec), penicillamine (3-mercapto-D-valine), pyroglutamic acid (5 -oxoproline), etc., naturally occurring non-proteogenic amino acids such as norleucine, and chemically synthesized amino acids that have properties known in the art to be characteristic of an amino acid, and amino acid equivalents.

[0112] In some embodiments, an isolated peptide of the present disclosure comprises an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence ofany one of SEQ ID NOs: 1-11, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, an isolated peptide of the present disclosure comprises an amino acid sequence of any one of SEQ ID NOs: 1 -11. In some embodiments, an isolated peptide of the present disclosure consists essentially of an amino acid sequence of any one of SEQ ID NOs: 1-11. In some embodiments, the isolated peptide comprises two or more sequences selected from any one or SEQ ID NOs: 1-11, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0113] A list of non-limiting examples of prostate lineage antigen-derived peptides are provided in Table 1 below.Table 1. Examples of Prostate Lineage Antigen-derived Peptides

[0114] A peptide of the disclosure may be synthetically produced or produced by hydrolysis. Synthetically produced peptides can include randomly generated peptides, specifically designed peptides, and peptides where at least some of the amino acid positions are conserved among several peptides and the remaining positions are random. Alternatively, a peptide of the present disclosure may be produced by expression in a heterologous host cell.

[0115] In nature, peptides that are produced by hydrolysis undergo hydrolysis prior to binding of the antigen to an MHC molecule. Class I MHC typically present peptides derived from proteins actively synthesized in the cytoplasm of the cell. In contrast, class II MHC typically present peptides derived either from exogenous proteins that enter a cell’s endocytic pathway or from proteins synthesized in the endoplasmic reticulum (ER). Intracellular trafficking permits a peptide to become associated with an MHC molecule.

[0116] The binding groove of MHC complexes can be considered to be divided into six pockets or subsites, designated pockets A to F. The pockets at each end of the binding groove (A and F) are highly conserved and are responsible for binding the N and C terminal anchor residues of peptide through extensive hydrogen bonding networks. The other pockets are polymorphic and therefore play a role in determining the specificity of peptide interaction. The binding of a peptide to an MHC peptide binding groove can control the spatial arrangement of MHC and / or peptide amino acid residues recognized by a TCR. Such spatial control is due in part to hydrogen bonds formed between a peptide and an MHC molecule. Based on the knowledge on how peptides bind to various MHC molecules, the major MHC anchor amino acids and the surface exposed amino acids that are varied among different peptides can be determined.

[0117] Preferably, the length of an MHC-binding peptide is from about 5 to about 40 amino acid residues, more preferably from about 6 to about 30 amino acid residues, and even more preferably from about 8 to about 20 amino acid residues, and even more preferably between about 9 and 11 amino acid residues, including any size peptide between 5 and 40 amino acids in length, in whole integer increments (i.e., 5, 6,7, 8, 9 . . . 40). While naturally-occurring MHC class Il-bound peptides vary from about 9-40 amino acids, in nearly all cases the peptide can be truncated to an about 9-11 amino acid core without loss of MHC binding activity or T cell recognition.

[0118] In some embodiments, the isolated peptides of the disclosure may be about 8-12 amino acids in length. For example, a peptide disclosed herein may be 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids in length. In some embodiments, the isolated peptides of the disclosure may be 8-11 amino acids, 8-10 amino acids, or 8-9 amino acids in length. In some embodiments, the isolated peptides of the disclosure may be 9-12 amino acids, 9-11 amino acids, or 9-10 amino acids in length. In some embodiments, the isolated peptides of the disclosure may be 10-12 amino acids or 10-11 amino acids in length. In some embodiments, the isolated peptides of the disclosure may be 11-12 amino acids in length.

[0119] A peptide of the disclosure may comprise one or more reverse peptide bonds, one or more non-peptide bonds, one or more chemical modifications, one or more D-isomers of amino acids, or any combination thereof.

[0120] In some embodiments, a peptide disclosed herein may be modified to comprise one or more reverse peptide bonds or non-peptide bonds. Such modifications may improve stability and / or binding of the peptide to MHC molecules to elicit a stronger immune response. In a reverse peptide bond, amino acid residues are not j oined by peptide ( — CO — NH — ) linkages but the peptide bond is reversed. Such retro- inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al., 1997 (Meziere C., et al. J Immunol 1997). This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Such pseudopeptides may be useful, for example, for desired MHC binding and / or T helper cell responses. Retro-inverse peptides, which contain NH — CO bonds instead of CO — NH peptide bonds, are much more resistant to proteolysis. Additional non-peptide bond that may be used are, for example, — CH2— NH, — CH2S— , — CH2CH2— , — CH=CH— , — COCH2— , — CH(OH)CH2— , and — CH2SO— .

[0121] The amino acid residues comprising the peptides of the disclosure may be chemically modified. Non-limiting examples of chemical modifications include, for example, phosphorylation, acetylation, deamidation acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide modification of carboxyl groups and sulphydryl modification by performic acid oxidation of cysteine to cysteic acid, formation of mercurial derivatives, formation of mixed disulfides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide and carbamoylation with cyanate at alkaline pH. Chemical modifications may not correspond to those that may be present in vivo.

[0122] For example, modification of arginyl residues in proteins may be based on the reaction of vicinal dicarbonyl compounds such as phenylglyoxal, 2,3- butanedione, and 1,2-cyclohexanedione to form an adduct. Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without concomitant modification of other nucleophilic sites such as lysine and histidine. Selective reduction of disulfide bonds in proteins can also be performed. Disulfide bonds can be formed and oxidized during the heat treatment of biopharmaceuticals. Woodward’s Reagent K may be used to modify specific glutamic acid residues. N-(3- (dimethylamino)propyl)-N'-ethylcarbodiimide can be used to form intra-molecular crosslinks between a lysine residue and a glutamic acid residue. For example, diethylpyrocarbonate and 4-hydroxy-2-nonenal can be used to modify histidyl residues in proteins. The reaction of lysine residues and other a-amino groups is, for example, useful in binding of peptides to surfaces or the cross-linking of proteins / peptides. Lysine is the site of attachment of poly(ethylene)glycol and the major site of modification in the glycosylation of proteins. Methionine residues in proteins can be modified with e.g. iodoacetamide, bromoethylamine, and chloramine T. Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosyl residues. Cross-linking via the formation of dityrosine can be accomplished with hydrogen peroxide / copper ions. N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole) have been used in recent studies for the modification of tryptophan. Successful modification of therapeutic proteins and peptides with PEG can lead to an extension ofcirculatory half-life while cross-linking of proteins / peptides with glutaraldehyde, polyethylene glycol diacrylate and formaldehyde can be used for the preparation of hydrogels. Chemical modification of allergens for immunotherapy can be achieved by carbamylation with potassium cyanate.

[0123] Peptides of the present disclosure may also be synthesized with additional chemical groups present at their N- and / or C-termini, to enhance the stability, bioavailability, and / or affinity of the peptides.

[0124] N-terminal modifications can include methylation (e.g., — NHCH3 or —N(CH3)2), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as a-chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO — or sulfonyl functionality defined by R — SO2 — , where R is selected from alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N-terminal amino group) to decrease susceptibility to proteases or to restrict the conformation of the peptide. Additionally, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to the N-terminus. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the N-terminus.

[0125] C-terminal modifications can include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. One can also cyclize the peptides of the disclosure, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide. C-terminal functional groups of the compounds of the present disclosure include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof. Additionally, the hydrophobic group, t- butyloxycarbonyl, or an amido group may be added to the C-terminus.

[0126] Further examples of non-natural modifications include incorporation of non-encoded a-amino acids, photoreactive cross-linking amino acids, N-methylated amino acids, and -amino acids, backbone reduction, retroinversion by using D-amino acids, and C-terminal amidation and PEGylation.

[0127] Peptides described herein may comprise one or more (e.g., 1, 2, 3, or 4) amino acid substitutions and / or insertions and / or deletions. Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position. Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue. One or more (e.g., 1, 2, 3 or 4) amino acids may be substituted and / or inserted and / or deleted from the sequence of any one of SEQ ID NOs: 1-11. Each substitution and / or insertion and / or deletion can take place at any position of any one of SEQ ID NOs: 1-11.

[0128] In some embodiments, the peptides of the disclosure may comprise additional amino acids (e.g., 1, 2, 3 or 4) at the C-terminal end and / or at the N-terminal end of the sequence of any one SEQ ID NOs: 1-11. A peptide of the disclosure may comprise the amino acid sequence of any one of SEQ ID NOs: 1-11 except for one or more (e.g., 1, 2, 3, or 4) amino acid substitutions, insertions or deletions.

[0129] Inserted amino acids and substituted amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain, and / or be linked together via non-native peptide bonds. Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007; 50(7): 1598-609 and Hoppes et al., J. Immunol 2014; 193(10):4803- 13 and references therein. If more than one amino acid residue is substituted and / or inserted, the replacement / inserted amino acid residues may be the same as each other or different from one another. Each replacement amino acid may have a different side chain to the amino acid being replaced.

[0130] D-amino acids may be substituted for the L-amino acids in the antigenic peptides of the disclosure. In addition, non-standard amino acids (i.e., other than the common naturally occurring proteinogenic amino acids such as P-y-5-amino acids, as well as many derivatives of L-a-amino acids) may also be used for substitutions or additions to produce peptides of the present disclosure.

[0131] Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. For example, the following groups of amino acids share similar chemical properties such as size, charge, and polarity: Group 1 - Ala, Ser, Thr, Pro, Gly; Group 2 - Asp, Asn, Glu, Gin; Group 3 - His, Arg, Lys; Group 4 - Met, Leu, He, Vai, Cys; Group 5 - Phe, Thy, Trp.

[0132] Substantial changes in function (e.g., affinity for MHC molecules and / orTCRs) can be made by selecting substitutions that are less conservative than those described above, in other words, selecting residues that differ more significantly in their effect on maintaining the structure of the peptide backbone in the area of the substitution (e.g., as a sheet or helical conformation), the bulk of the side chain, or the charge or hydrophobicity of the peptide at the positions involved for MHC or TCR binding. The substitutions which in general are expected to produce the greatest changes in peptide properties will be those in which (a) a hydrophilic residue, e.g. Ser, is substituted for (or by) a hydrophobic residue, e.g. Leu, He, Phe, Vai or Ala; (b) a residue having an electropositive side chain, e.g., Lys, Arg, or His, is substituted for (or by) an electronegative residue, e.g. Glu or Asp; or (c) a residue having a bulky side chain, e.g. Phe, is substituted for (or by) a residue not having a side chain, e.g., Gly.

[0133] One can also replace the naturally occurring side chains of the 20 genetically encoded amino acids (or the stereoisomeric D-amino acids) with other side chains, for instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7- membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7- membered heterocyclic. For example, proline analogues in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed.Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1 -piperidyl, piperidine), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

[0134] Other examples of amino acid replacements include stereoisomers (e.g., D-amino acids) and unnatural amino acids such as, for example, L-omithine, L- homocysteine, L-homoserine, L-citrulline, 3-sulfino-L-alanine, N-(L- arginino)succinate, 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, 3,5-diiodo-L- tyrosine, triiodothyronine, L-thyroxine, L-selenocysteine, N-(L-arginino)taurine, 4- aminobutylate, (R,S)-3-amino-2-methylpropanoate, a,a-disubstituted amino acids, N- alkyl amino acids, lactic acid, P-alanine, 3 -pyridylalanine, 4-hydroxyproline, O- phosphoserine, N-methylglycine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5 -hydroxylysine, nor-leucine, and other similar amino acids and imino acids.

[0135] The amino acid residues that do not substantially contribute to interactions with the T-cell receptor can be modified by replacement with other amino acid whose incorporation does not substantially affect T-cell reactivity and does not eliminate binding to the relevant MHC.

[0136] The peptides may also comprise isosteres of two or more residues. An “isostere” as used here refers to a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Suchmodifications include modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks.

[0137] Combinations of several substitutions / additions / deletions at more than one position can be developed and tested to determine if the combination results in an additive or synergistic effects on the immunogenicity of the peptide. In some embodiments, no more than 4 positions within the peptide are simultaneously altered.

[0138] Preferably, peptides of the disclosure bind to an MHC molecule in the peptide binding groove of the MHC molecule. Generally, the amino acid modifications described above will not impair the ability of the peptide to bind to the MHC molecule. In some embodiments, the amino acid modifications improve the ability of the peptide to bind to the MHC molecule. For example, mutations may be made at positions which anchor the peptide to the MHC molecule. Such anchor positions and the preferred residues at these locations for peptides which bind, in particular, HLA-A*02 may comprise, e.g., amino acids residues at position 2, and / or at the C-terminus of the peptide, which may be considered primary anchor positions. Preferred anchor residues may be different for each HLA type. As a non-limiting example, the preferred amino acids in position 2 for HLA-A*02 are Leu, lie, Vai, or Met and at the C-terminus are Vai or Leu. Multiple positions may be important for stable peptide binding to HLA- A*02, including positions 2, 3, 5-7, and 9. The anchor residues at position 2 and 9 may be of prime importance for peptide binding to HLA-A2. However, other peptide side chains, e.g., at position 3, may contribute to the stability of the interaction. In certain cases, the optimal length for peptide binding can be longer than 9 residues.

[0139] The immunologic properties of peptides can be described as a function of binding to MHC molecules (Kon and Koff) and TCR (affinity of interaction between TCR and MHC-peptide complexes). Modifications of primary MHC anchor residues exhibit a significant degree of predictability about overall impact on binding to MHC molecules. Modifications of secondary MHC anchor residues can impact the affinity of interaction of the MHC-peptide complex to TCR as well as with the Kon and Koff relative to peptide-MHC interaction.

[0140] When the prostate lineage antigen-derived peptide is a mutant peptide, T cell lines against a natural (non-mutated) epitope are generated, and an immunization strategy potent enough to generate a useful response in transgenic mice carrying human MHC (such as the A2 allele) is used. Prostate lineage antigen-derived peptides are interrogated ex vivo in the presence of competent APCs and the functional impact of T cells specific for natural (non-mutated) epitopes is measured. The evaluation is done at various concentrations of peptide, because the expected effect is biphasic in the instance of cross-reactive peptides (activating at limited concentrations and inhibiting at higher concentrations, due to antigen-induced cell death [AICD]). Measurement of the following three parameters is used to define basic and useful characteristics of prostate lineage antigen-derived peptides:1. Minimal required concentration of peptide metrics to induce effects indicative of T cell activation (e.g., cytokine [e.g., IFN-y] production);2. Maximal (peak value) effect (e.g., cytokine [e.g., IFN-y] production) at any peptide concentration; and3. Peptide concentration at peak value of activating effect (e.g., cytokine [e.g., IFN-y] concentration).

[0141] By way of a non-limiting example, prostate lineage antigen-derived peptides that result in reduced values associated with parameters number 1 and number 3, but increased number 2, can be useful. Use of natural epitopes and / or unrelated non- cross-reactive peptides as references is valuable for identifying classes of peptides of possible value. Peptides possessing properties quantitatively comparable to or even moderately attenuated from those of natural epitopes are still considered useful since, while they retain cross-reactivity, they may exhibit immunologic properties that are distinct from those of the natural peptide, e.g., reduced capacity to break tolerance or reestablish responsiveness in vivo or lower propensity to induce AICD.

[0142] In addition to practicality and rapidity, additional advantages of this screening approach include, but are not limited to, use of more relevant polyclonal T cell lines instead of potentially biased T cell clones as a read out, and the composite value, integrating parameters such as Kon, Kotr and TCR affinity that can translate intocross-reactivity and functional avidity of peptide-MHC complexes relative to TCR. These parameters can be predictive of the in vivo immunologic properties and thus can define useful panels of peptides eligible for further evaluation, optimization and practical applications. Peptides that bind to MHC and retain cross-reactivity against TCR specific for the nominal wild-type peptide are predicted to elicit a measurable effect in this assay.

[0143] A peptide of the disclosure, or pharmaceutically acceptable salt thereof, or fragment or derivative thereof, may be used to induce an immune response. If this is the case, it is important that the immune response is specific to the intended target (e.g., prostate cancer cells) to avoid the risk of unwanted side effects that may be associated with an “off target” immune response. Therefore, it is preferred that the amino acid sequence of a peptide of the disclosure does not match the amino acid sequence of a peptide from any other endogenous protein(s), particularly that of another human protein. Also, the amino acid modifications described herein should not impair the ability of the peptide inducing an antigen-specific immune response when presented in a complex with an MHC molecule on the surface of an antigen presenting cell (APC).

[0144] The peptides may be also modified to improve half-life and / or bioavailability, for example, by PEGylation, glycosylation, polysialylation, HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticulate encapsulation, cholesterol fusion, iron fusion, or acylation.

[0145] The peptides of the disclosure can also serve as structural models for non-peptidic compounds with similar biological activity. A variety of techniques can be used to construct compounds with the same or similar desired biological activity as the lead peptide compound, but with more favorable activity than the lead with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis. These techniques include replacing the peptide backbone with a backbone composed of amidates, phosphonates, carbamates, sulfonamides, secondary amines, and N- methylamino acids.

[0146] Multiple peptides described herein may be operably linked together. Accordingly, in one aspect, the present disclosure provides an isolated peptide or polypeptide comprising two or more amino acid sequences selected from SEQ ID NO: 1 - 11 , or a derivative thereof, or a pharmaceutically acceptable salt thereof. For example, such a multi-epitope peptide or polypeptide may comprise 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 single-epitope peptides as described herein (e.g., SEQ ID NO: 1-11). The single-epitope peptides (e.g., SEQ ID NO: 1-11) may be arranged in any order, and may be identical or different.

[0147] In some embodiments, the present disclosure provides an isolated peptide or polypeptide comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 amino acid sequences selected from SEQ ID NO: 1-11, or a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0148] The single-epitope peptides may be linked via a linker. The linker may comprise of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The linker can be selected from, e.g., Table 2, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present linker need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the linker will usually be at least one or two residues, more usually three to six residues.

[0149] Peptides of the disclosure can be synthesized by e.g., solid phase synthesis. As such, the peptides may be immobilized, for example to a solid support such as a bead. Peptides of the disclosure may be synthesized by the Fmoc-polyamide mode of solid-phase peptide synthesis. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is done using 20% piperidine in N, N- dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acidand aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6- trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalizing agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives except for asparagine and glutamine, which are added using a reversed N, N-dicyclohexyl-carbodiimide / 1 -hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used include ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesized. Also, a combination of solid phase and solution phase methodologies for the synthesis of peptides is possible.

[0150] Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilization of the aqueous phase affords the crude peptide free of scavengers.

[0151] Purification may be performed by techniques such as re-crystallization, ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography and reverse-phase high performance liquid chromatography using e.g. acetonitrile / water gradient separation, or a combination thereof.

[0152] Peptides may be analyzed using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF mass spectrometric analysis.

[0153] Alternatively, the peptide may be produced by recombinant expression in a heterologous host cell. Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the peptide to be expressed, to express the polypeptide in vivo-, for example, in bacteria, yeast, insect or mammalian cells.

[0154] In further embodiments, in vitro cell-free systems may be used. The peptides may be isolated and / or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other peptides or proteins.Peptide-MHC (pMHC) Complexes Disclosed Herein

[0155] In another aspect, the disclosure provides a complex of a peptide of the disclosure and an MHC molecule. Preferably, the peptide is bound to the peptide binding groove of the MHC molecule. In some embodiments, the peptide and the MHC molecule form a non-covalent complex. In other embodiments, the peptide and the MHC molecule may be covalently linked, for example, via a linker.

[0156] MHC molecules are generally classified into two categories: class I and class II MHC molecules. An MHC class I molecule is an integral membrane protein comprising a glycoprotein heavy chain, also referred to herein as the a chain, which has three extracellular domains (i.e., al, a2 and a3) and two intracellular domains (i.e., a transmembrane domain (TM) and a cytoplasmic domain (CYT)). The heavy chain is noncovalently associated with a soluble subunit called 02 microglobulin (02m or 02M). An MHC class II molecule or MHC class II protein is a heterodimeric integral membrane protein comprising one a chain and one 0 chain in noncovalent association. The a chain has two extracellular domains (al and a2), and two intracellular domains (a TM domain and a CYT domain). The 0 chain contains two extracellular domains (01 and 02), and two intracellular domains (a TM domain and CYT domain).

[0157] The domain organization of class I and class II MHC molecules forms the antigenic determinant binding site, e.g., the peptide-binding portion or peptide binding groove, of the MHC molecule. A peptide binding groove refers to a portion of an MHC molecule that forms a cavity in which a peptide, e.g., antigenic determinant, can bind. The conformation of a peptide binding groove is capable of being altered upon binding of a peptide to enable proper alignment of amino acid residues important for TCR binding to the peptide-MHC (pMHC) complex.

[0158] In some embodiments, MHC molecules include fragments of MHC chains that are sufficient to form a peptide binding groove. For example, a peptide binding groove of a class I protein can comprise portions of the al and a2 domains of the heavy chain capable of forming two 0-pleated sheets and two a helices. Inclusion of a portion of the 02 microglobulin chain stabilizes the MHC class I molecule. While for most versions of MHC class II molecules, interaction of the a and 0 chains can occur in the absence of a peptide, the two-chain molecule of MHC class II is unstable until the binding groove is filled with a peptide. A peptide binding groove of a class II protein can comprise portions of the al and 01 domains capable of forming two 0-pleated sheets and two a helices. A first portion of the al domain forms a first 0-pleated sheet and a second portion of the al domain forms a first a helix. A first portion of the 01 domain forms a second 0-pleated sheet and a second portion of the 01 domain forms a second a helix. The X-ray crystallographic structure of class II protein with a peptide engaged in the binding groove of the protein shows that one or both ends of the engaged peptide can project beyond the MHC protein. Thus, the ends of the al and 01 a helices of class II form an open cavity such that the ends of the peptide bound to the binding groove are not buried in the cavity. Moreover, the X-ray crystallographic structure of class II proteins shows that the N-terminal end of the MHC 0 chain apparently projects from the side of the MHC protein in an unstructured manner since the first 4 amino acid residues of the 0 chain could not be assigned by X-ray crystallography.

[0159] The peptides of the present disclosure can bind to an MHC molecule in a manner such that the pMHC complex can bind to a TCR, preferably in a specificmanner. In certain embodiments, binding of the pMHC complex to the TCR may induce a T cell response.

[0160] Whether or not a given peptide will form a complex with an MHC molecule can be determined by assessing whether the MHC can be refolded in the presence of the peptide using the process described in, for example, PCT Application W02018 / 083505, which is incorporated herein in its entirety for all purposes. If the peptide does not form a complex with MHC, then MHC will not refold. Refolding can be confirmed using an antibody that recognizes MHC in a folded state only. Alternatively, the ability of a peptide to stabilize MHC on the surface of transporter associated with antigen processing (TAP)-deficient cell lines such as T2 cells, which lack the capacity for TAP-mediated translocation of cytosolic peptides into the endoplasmic reticulum (ER) for peptide loading onto MHC class I molecules, or other biophysical methods to determine interaction parameters can be determined.

[0161] The peptides according to the present disclosure may be provided as an MHC groove-binding peptide. In some embodiments, the MHC groove-binding peptide can be designed such that the peptide may vary in some or all the positions involved in MHC binding. For example, the MHCBN is a comprehensive database of MHC binding and non-binding peptides compiled from published literature and existing databases. The latest version of the database has 25,860 entries including 20,717 MHC binders and 4,022 MHC non-binders for more than 450 MHCs. The database has sequence and structure data of (a) source proteins of peptides and (b) MHCs. MHCBN has a number of web tools that include: (i) mapping of peptide on query sequence; (ii) search on any field; (iii) creation of data sets; and (iv) online data submission.

[0162] In some cases, a peptide binding tool for prediction of binding to MHC- I or MHC-II can be, for example, Antibody Epitope Prediction, ANTIGENIC, BepiPred, CTLPred, DiscoTope, EPIPREDICT, Epitope Cluster Analysis, Epitope Conservancy Analysis, EUiPro, HLA Peptide Binding Predictions, HLABinding, MAPPP, MHCBench, MHC-I processing predictions, Mosaic Vaccine Tool Suite, NetChop, NetCTL, NetMHC, NetMHCII, NetMHCpan, nHLAPred-I, OptiTope, PAProC, POPI, PREDEP, Prediction of Antigenic Determinants, ProPred, ProPred-1,RankPep, SMM, SVMHC, TAPPred, VaxiJen, or combinations thereof. Additional example programs are used such as BIMAS, SYFPEITHI, or Rankpep.

[0163] In one specific embodiment, a library of altered peptides is produced by genetically engineering the library using polymerase chain reaction (PCR) or any other suitable technique to construct a DNA fragment encoding the peptide. With PCR techniques, by using oligonucleotides that are randomly mutated within particular triplet codons, the resultant fragment pool encodes all possible combination of codons at these positions. Preferably, certain of the amino acid positions are maintained constant, which are the conserved amino acids that are required for binding to the MHC peptide binding groove, and which do not contact the T cell receptor (TCR).

[0164] In some embodiments, when a library of altered peptides is produced by genetically engineering the library using polymerase chain reaction (PCR) or any other suitable technique to construct a DNA fragment encoding the peptide, the target TCR is a TCR for which it is desired to identify the peptide epitope recognized by the receptor. In some embodiments, the target TCR is from a patient with prostate cancer. In some embodiments, the TCR includes an a-chain and a 0-chain.

[0165] MHC molecules used in pMHC complexes described herein include naturally occurring full-length MHC molecules as well as individual chains of MHC molecules (e.g., MHC class I a (heavy) chain, 02-microglobulin, MHC class II a chain, and MHC class II 0 chain), individual subunits of such chains of MHCs (e.g., al, a2 and / or a3 subunits of MHC class I a chain, al and / or a2 subunits of MHC class II a chain, 01 and / or 02 subunits of MHC class II 0 chain) as well as fragments, mutants, and various derivatives thereof (including fusion proteins, e.g., fusions with viral envelope proteins or fusogens), wherein such fragments, mutants, and derivatives retain the ability to display an antigenic determinant for recognition by an antigen-specific TCR. In one specific embodiment, the MHC comprises a transmembrane domain embedded in the lipid envelope of a liposome, a recombinant viral particle, or a viruslike particle (VLP).

[0166] Naturally-occurring MHC molecules are encoded by a cluster of genes on human chromosome 6 or mouse chromosome 17. MHCs are also referred to as H-2 in mice and Human Leucocyte Antigen (HLA) in humans. MHC class I molecules specifically bind CD8 molecules expressed on cytotoxic T lymphocytes (CD8+ T cells), whereas MHC class II molecules specifically bind CD4 molecules expressed on helper T lymphocytes (CD4+ T cells). MHCs include, but are not limited to, HLA specificities such as A (e.g. A1-A74), B (e.g., B 1-B77), C (e.g., Cl-Cl l), D (e.g., D1-D26), E, G, DR (e.g., DR1-DR8), DQ (e.g., DQ1-DQ9) and DP (e.g. DP1-DP6). More preferably, HLA specificities include Al, A2, A3, Al l, A23, A24, A28, A30, A33, B7, B8, B35, B44, B53, B60, B62, DR1, DR2, DR3, DR4, DR7, DR8, and DR-11.

[0167] In some embodiments, the MHC molecule in a pMHC complex of the present disclosure is a human leukocyte antigen (HLA) molecule. The MHC molecule may be a human HLA molecule selected from the group consisting of HLA-A, HLA- B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the MHC class I or MHC II polypeptides may be derived from any functional human HLA-A, B, C, DR, or DQ molecules. Non-limiting examples of HLA-A alleles comprise, without limitation, A*0101, A*0201, A*0202, A*0301, A* 1101, A*2301, A*2402, A*2501, A*2601, A*2901 , A*2902, A*3101, A*3201, A*3301, A*3401, A*3601, A*4301, A*6601, A*6801, A*6901, A*7401, and A*8001. Non-limiting examples of HLA-B alleles comprise, without limitation, B*0702. B*0801, B*1301, B*1401, B*1402, B*1501, B*1801, B*1802, B*2701, B*2702, B*3501, B*3502, B*3701, B*3801, B*3901, B*4001, B*4101, B*4201, B*4402, B*4501, B*4601, B*4701, B*4801, B*4901, B*5001, B*5101, B*5201, B*5301, B*5401, B*5501, B*5502, B*5601, B*5701, B*5801, B*5901, B*6701, B*7301, B*1517, B*8101, B*8201, and B*8301. Non-limiting examples of HLA-C alleles comprise, without limitation, Cw*0101, Cw*0202, Cw*0303, Cw*0401, Cw*0501, Cw*0602, Cw*0701, Cw*0702, Cw*0802, Cw*1203, Cw*1401, Cw*1502, Cw*1601, Cw*1701, and. Cw*1801. Non-limiting examples of HLA-DR alleles comprise, without limitation, DRBl*0101, DRBl*0103, DRB1*15O1, DRB1*15O2, DRB1*16O1, DRB1*16O2, DRBl*0301, DRBl*0401, DRBl*0404, DRBl*1101, DRB1*12O1, DRBl*1301, DRB1*13O2, DRBl*1401, DRB1*14O2, DRBl*0701, DRBl*0801, DRBl*0802, DRB1*O8O3, DRBl*0901, and DRBl*1001.

[0168] In some embodiments, the MHC class I molecule may be selected from HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*24, HLA- B*07, HLA-B*08, HLA-B*40, HLA-B*44, HLA-B*15, HLA-C*04, HLA*C*03 HLA-C*07. There are also allelic variants of the above HLA types, all of which are encompassed by the present disclosure. In some embodiments, the MHC molecule may be HLA-A*02 or HLA-A*11.

[0169] The MHC molecules used herein may also be from any other mammalian or avian species, for example, non-human primates, rodents (e.g., mice), rabbits, equines, bovines, canines, felines, pigs, etc.

[0170] Naturally occurring MHC class I molecules bind peptides derived from proteolytically degraded proteins, especially endogenously synthesized proteins, by a cell. Small peptides obtained accordingly are transported into the endoplasmic reticulum where they associate with nascent MHC class I molecules before being routed through the Golgi apparatus and displayed on the cell surface for recognition by cytotoxic T lymphocytes.

[0171] Naturally occurring MHC class I molecules consist of an a (heavy) chain associated with 02-microglobulin. The heavy chain consists of subunits al-a3. The 02- microglobulin protein and a3 subunit of the heavy chain are associated. In certain embodiments, 02-microglobulin and a3 subunit are covalently bound. In certain embodiments, 02-microglobulin and a3 subunit are non-covalently bound. The al and a2 subunits of the heavy chain fold to form a groove for a peptide, e.g., antigenic determinant, to be displayed and recognized by TCR.

[0172] Class I molecules can bind peptides of about 8-10 amino acids in length.All humans have between three and six different class I molecules, which can each bind many different types of peptides.

[0173] In some embodiments, the MHC contained in the pMHC complexes of the disclosure comprises (i) a class I MHC polypeptide or a fragment, mutant or derivative thereof, and, optionally, (ii) a 02 microglobulin polypeptide or a fragment,mutant or derivative thereof. In one specific embodiment, the class I MHC polypeptide is linked to the 02 microglobulin polypeptide by a peptide linker.

[0174] In one specific embodiment, the class I MHC polypeptide is a human class I MHC polypeptide selected from the group consisting of HLA-A, HLA-B, HLA- C, HLA-E, HLA-F, and HLA-G. In another specific embodiment, the class I MHC polypeptide is a murine class I MHC polypeptide selected from the group consisting of H-2K, H-2D, H-2L, H2-IA, H2-IB, H2-IJ, H2-IE, and H2-IC.

[0175] In some embodiments, the peptide disclosed herein forms a complex with one or more MHC class I a heavy chains. In some embodiments, the MHC class I a heavy chain is fully human. In some embodiments, the MHC class I a heavy chain is humanized. Humanized MHC class I a heavy chains are described, e.g., in U.S. Pat. Pub. Nos. 2013 / 0111617, 2013 / 0185819 and 2014 / 0245467. In some embodiments, the MHC class I a heavy chain comprises a human extracellular domain (human al, a2, and / or a3 domains) and a cytoplasmic domain of another species. In some embodiments, the class I a heavy chain polypeptide is HLA-A, HLA-B, HLA-C, HLA- E, HLA-F, HLA-G, HLA-K, or HLA-L. In some embodiments, the HLA-A sequence can be an HLA-A*0201 sequence. In various aspects, the peptide-MHC can include all the domains of an MHC class I heavy chain.

[0176] In some embodiments, the MHC molecule comprises a 02- microglobulin. In some embodiments, the 02-microglobulin is fully human. In some embodiments, the 02-microglobulin is humanized.

[0177] In some embodiments, the MHC class I molecule comprises a mutation in a 02-microglobulin (02m or B2M) polypeptide and in the Heavy Chain sequence to effect a disulfide bond between the B2M and the Heavy Chain. In some cases, the Heavy Chain is an HLA and wherein the disulfide bond links one of the following pairs of residues: B2M residue 12, HLA residue 236; B2M residue 12, HLA residue 237; B2M residue 8, HLA residue 234; B2M residue 10, HLA residue 235; B2M residue 24, HLA residue 236; B2M residue 28, HLA residue 232; B2M residue 98, HLA residue 192; B2M residue 99, HLA residue 234; B2M residue 3, HLA residue 120; B2Mresidue 31, HLA residue 96; B2M residue 53, HLA residue 35; B2M residue 60, HLA residue 96; B2M residue 60, HLA residue 122; B2M residue 63, HLA residue 27; B2M residue Arg3, HLA residue Glyl20; B2M residue His31, HLA residue Gln96; B2M residue Asp53, HLA residue Arg35; B2M residue Trp60, HLA residue Gln96; B2M residue Trp60, HLA residue Aspl22; B2M residue Tyr63, HLA residue Tyr27; B2M residue Lys6, HLA residue Glu232; B2M residue Gln8, HLA residue Arg234; B2M residue TyrlO, HLA residue Pro235; B2M residue Serl l, HLA residue Gln242; B2M residue Asn24, HLA residue Ala236; B2M residue Ser28, HLA residue Glu232; B2M residue Asp98, HLA residue Hisl92; and B2M residue Met99, HLA residue Arg234, first linker position Gly2, Heavy Chain (HLA) position Tyr84; Light Chain (B2M) position Argl2, HLA Ala236; and / or B2M residue Argl2, HLA residue Gly237.

[0178] In some embodiments, the antigenic determinant amino acid sequence can be that of a peptide described herein, which can be presented by an MHC class I molecule. In certain embodiments, the sequence can comprise from about 8 to about 15 contiguous amino acids. In certain embodiments, the sequence can comprise from about 8 to about 12 contiguous amino acids.

[0179] In some embodiments, at least one chain of the MHC and the peptide are comprised within a fusion protein. In one specific embodiment, the MHC and the peptide are separated by a linker sequence. For example, the single chain molecule can comprise, from amino to carboxy terminal, an antigenic determinant, a p2- microglobulin sequence, and a class I a (heavy) chain sequence. Alternatively, the single chain molecule can comprise, from amino to carboxy terminal, an antigenic determinant, a class I a (heavy) chain sequence, and a p2-microglobulin sequence. The single-chain molecule can further comprise a signal peptide sequence at the amino terminal. In certain embodiments, there can be a linker sequence between the peptide sequence and the p2-microglobulin sequence. In certain embodiments, there can be a linker sequence between the p2-microglobulin sequence and the class I a (heavy) chain sequence. A single-chain molecule can further comprise a signal peptide sequence at the amino terminal, as well as first linker sequence extending between the peptide sequence and the p2-microglobulin sequence, and / or a second linker sequence extending between the p2-microglobulin sequence and the class I heavy chainsequence. In certain embodiments, the 02-microglobulin and the class I a (heavy) chain sequences can be human, murine, or porcine.

[0180] In some embodiments, a single-chain molecule can comprise a first flexible linker between the peptide ligand segment and the 02-microglobulin segment. For example, linkers can extend from and connect the carboxy terminal of the peptide ligand segment to the amino terminal of the 02-microglobulin segment. Preferably, the linkers are structured to allow the linked peptide ligand to fold into the binding groove resulting in a functional MHC-antigen peptide. In some embodiments, this linker can comprise at least about 10 amino acids, up to about 15 amino acids. In some embodiments, a single-chain molecule can comprise a second flexible linker inserted between the 02-microglobulin and heavy chain segments. For example, linkers can extend from and connect the carboxy terminal of the 02-microglobulin segment to the amino terminal of the heavy chain segment. In certain embodiments, the 02- microglobulin and the heavy chain can fold into the binding groove resulting in a molecule which can function in promoting T cell expansion.

[0181] Suitable linkers used in the MHCs can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Non-limiting examples of linkers include, e.g., glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 15) and (GGGS)n (SEQ ID NO: 13), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains). Exemplary linkers can comprise amino acid sequences including, but not limited to, those listed in Table 2. In some embodiments, a linker peptide includes a cysteine residue that can form a disulfide bond with a cysteine residue present in a second polypeptide.Table 2. Examples of Linker Sequences

[0182] In certain embodiments, the single-chain molecule can comprise a peptide covalently attached to an MHC class I a (heavy) chain via a disulfide bridge (i.e., a disulfide bond between two cystines). In certain embodiments, the disulfide bond comprises a first cysteine, comprising a linker extending from the carboxy terminal of an antigen peptide, and a second cysteine comprising an MHC class I heavy chain (e.g., an MHC class I a (heavy) chain which has a non-covalent binding site for the antigen peptide). In certain embodiments, the second cysteine can be a mutation (addition or substitution) in the MHC class I a (heavy) chain. In certain embodiments, the single-chain molecule can comprise one contiguous polypeptide chain as well as a disulfide bridge. In certain embodiments, the single-chain molecule can comprise two contiguous polypeptide chains which are attached via the disulfide bridge as the only covalent linkage. In some embodiments, the linking sequences can comprise at least one amino acid in addition to the Cys residues, including one or more Gly residues, one or more Ala residues, and / or one or more Ser residues.

[0183] In certain embodiments, the disulfide bridge can link an antigen peptide described herein in the class I groove of the pMHC complex if the pMHC complex comprises a first cysteine in a Gly-Ser linker extending between the C-terminus of the peptide and the p2-microglobulin, and a second cysteine in a proximal heavy chain position.

[0184] Attaching the peptide to the MHC class I or MHC class II molecule via a flexible linker has the can help ensure that the peptide will occupy and stay associated with the MHC molecule during biosynthesis, transport, and display. However, there may be situations in which this linker can interfere with peptide binding to the MHC molecule or with TCR recognition of the complex. As an alternate approach, in some embodiments, the MHC molecule and the peptide are expressed separately.

[0185] In some embodiments, the p2-microglobulin sequence can comprise a full-length p2-microglobulin sequence. In certain embodiments, the p2-microglobulin sequence lacks the leader peptide sequence. As such, in some configurations, the p2- microglobulin sequence can comprise about 99 amino acids, and can be a mouse P2- microglobulin sequence (e.g., GenBank Accession No. X01838). In some otherconfigurations, the 02-microglobulin sequence can comprise about 99 amino acids, and can be a human 02-microglobulin sequence (e.g., GenBank Accession No. AF072097.1).

[0186] In some embodiments, the pMHC complex can contain MHC sequences as disclosed in U.S. Patent Nos. 4,478,823; 6,011,146; 8,518,697; 8,895,020; 8,992,937; WO 96 / 04314; Mottez et al. J. Exp. Med. 181: 493-502, 1995; Madden et al. Cell 70: 1035-1048, 1992; Matsumura et al., Science 257: 927-934, 1992; Mage et al., Proc. Natl. Acad. Sci. USA 89: 10658-10662, 1992; Toshitani et al, Proc. Nat’l Acad. Sci. 93: 236-240, 1996; Chung et al, J. Immunol. 163:3699-3708, 1999; Uger and Barber, J. Immunol. 160: 1598-1605, 1998; Uger et al., J. Immunol. 162, pp. 6024- 6028, 1999; White et al., J. Immunol. 162: 2671-2676, 1999; Yu et al., J. Immunol. 168:3145-3149, 2002; Truscott et al., J. Immunol. 178: 6280-6289, 2007, all of which are incorporated by reference in their entireties.

[0187] In some embodiments, the MHC comprises a class II MHC polypeptide or a fragment, mutant or derivative thereof. In one specific embodiment, the MHC comprises a and polypeptides of a class II MHC complex or a fragment, mutant or derivative thereof. In one specific embodiment, the a and 0 polypeptides are linked by a peptide linker. In one specific embodiment, the MHC comprises a and 0 polypeptides of a human class II MHC complex selected from the group consisting of HLA-DP, HLA-DR, HLA-DQ, HLA-DM and HLA-DO. In another specific embodiment, the MHC comprises a and 0 polypeptides of a murine H-2A or H-2E class II MHC complex.

[0188] Naturally occurring MHC class II molecules can contain two polypeptide chains, a and 0. The chains may come from the DP, DQ, or DR gene groups. There are about 40 known different human MHC class II molecules. All have the same basic structure but can vary subtly in their molecular structure. MHC class II molecules can bind peptides of 13-18 amino acids in length.

[0189] In some embodiments, the MHC class II a chain is fully human. In some embodiments, the MHC class II a chain is humanized. Humanized MHC class II achains are described, e.g., in U.S. Pat. Nos. 8,847,005, 9,043,996, and 10,154,658, which are incorporated herein by reference in their entireties. In some embodiments, the humanized MHC class II a chain polypeptide comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II a chain is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA or HLA-DRA. In some embodiments, the class II a chain polypeptide is humanized HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA and / or HLA-DRA.

[0190] In some embodiments, the peptide of the present disclosure forms a complex with one or more MHC class II 0 chains. In some embodiments, the MHC class II 0 chain is fully human. In some embodiments, the MHC class II 0 chain polypeptide is humanized. Humanized MHC class II 0 chain polypeptides are described, e.g., in U.S. Pat. Nos. 8,847,005, 9,043,996, and 10,154,658, which are incorporated herein by reference in their entireties. In some embodiments, the humanized MHC class II 0 chain comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II 0 chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB or HLA-DRB. In some embodiments, the class II 0 chain is humanized HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB and / or HLA-DRB.

[0191] The pMHC complexes of the disclosure may be isolated and / or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other peptides or proteins. MHC molecules as disclosed herein can include recombinant MHC molecules, non-naturally occurring MHC molecules, and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained. For example, MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, attached to a tag, biotinylated and / or in multimeric form. A peptide disclosed herein may be covalently attached to the MHC.

[0192] Methods to produce soluble recombinant MHC molecules with which peptides disclosed herein can form a complex include, but are not limited to, expressionand purification from E. coli cells or insect cells. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

[0193] The peptides disclosed herein may be presented on the surface of a cell in complex with MHC. Thus, the present disclosure also provides a cell presenting on its surface a pMHC complex disclosed herein. Such a cell may be a mammalian cell, preferably a cell of the immune system, and a specialized antigen-presenting cell (APC) such as a dendritic cell or a B cell. Other preferred cells include T2 cells. Cells presenting the peptide or pMHC complex of the disclosure may be isolated, preferably in the form of a homogenous population, or provided in a substantially pure form. Such cells may not naturally present the complex of the disclosure, or alternatively said cells may present the complex at a level higher than they would in nature. Such cells may be obtained by pulsing said cells with one or more peptides (e.g., 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides) of the disclosure, or genetically modifying the cells (via DNA or RNA transfer) to express one or more peptides (e.g., 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides) of the disclosure. Pulsing involves incubating the cells with the peptide for several hours using peptide concentrations typically ranging from 10-5to 10-12M. Such cells may additionally be transduced with HLA molecules, such as HLA-A*02 to further induce presentation of the peptide(s). Cells may be produced recombinantly. Cells presenting peptides of the disclosure may be used to isolate T cells and TCRs which are activated by, or bind to, the cells.Fusion Proteins, Conjugates and Oligomeric Complexes Disclosed Herein

[0194] Peptides or pMHC complexes disclosed herein may be fused or conjugated to one or more heterologous molecules. Peptides or pMHC complexes disclosed herein may also be in multimeric form. Accordingly, the present disclosure also provides fusion proteins, conjugates, and oligomeric complexes comprising a peptide or a pMHC complex of the disclosure.

[0195] In some embodiments, a peptide disclosed herein is fused or conjugated to one or more heterologous molecules which include an MHC molecule (or fragments thereof).

[0196] Heterologous molecules suitable for genetical fusion and / or chemical conjugation with the peptides or the pMHC complexes of the disclosure include, but are not limited to, peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, sugars, etc. The heterologous molecule(s) may be fused at the N- and / or C- terminus of the peptide and / or another polypeptide chain in the pMHC complex.

[0197] Heterologous peptides and polypeptides include, but are not limited to, an epitope (e.g., FLAG) or a tag sequence (e.g., Hise (SEQ ID NO: 61), and the like) to allow for the detection and / or isolation of a fusion protein; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region (e.g., an Fc domain); a half-life-extending sequence comprising a combination of two or more (e.g., 2, 5, 10, 15, 20, 25, etc.) naturally occurring or non- naturally occurring charged and / or uncharged amino acids (e.g., Ser, Gly, Glu or Asp) designed to form a predominantly hydrophilic or predominantly hydrophobic fusion partner for a fusion protein; a functional or non-functional antibody (e.g., an antibody that is specific for dendritic cells), or a heavy or light chain thereof; and a polypeptide which has an activity, such as a therapeutic activity, different from fusion proteins of the present disclosure. In some embodiments, the one or more heterologous molecules enhances a peptide-specific immune response in a subject. In some embodiments, the one or more heterologous molecules mediates peptide delivery to a specific site within a subject.

[0198] In some embodiments, fusion proteins of the disclosure may comprise one or more affinity tags, e.g., to allow for affinity purification or coupling to another molecule. Examples of affinity tags include, but are not limited to, a Hise tag (SEQ ID NO: 61), an Avi-tag, a biotin, a hemagglutinin (HA) tag, a FLAG tag, a Myc tag, a GST tag, a MBP tag, a chitin binding protein tag, a calmodulin tag, a V5 tag, a streptavidin binding tag, a green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, SUMO tag, and Ubiquitin tag.

[0199] In some embodiments, fusion proteins of the disclosure may comprise one or more epitopes that is not present in the prostate lineage antigen. One such example is the use of fusion peptides where a promiscuous T helper epitope is covalently linked (e.g., via a polypeptide linker or spacer) to the peptide sequence. Nonlimiting examples of promiscuous T helper epitopes include the PADRE peptide, tetanus toxoid peptide (830-843), or influenza haemagglutinin, HA(307-319).

[0200] Peptides or pMHC complexes of the disclosure may be conjugated to additional moieties such as carrier molecules or adjuvants for use as vaccines. Examples of adjuvants used in vaccines include microbes, such as the bacterium Bacillus Calmette-Guerin (BCG), and / or substances produced by bacteria, such as Detox B (an oil droplet emulsion of monophosphoryl lipid A and mycobacterial cell wall skeleton). KLH (keyhole limpet hemocyanin), bovine serum albumin (BSA), the E2 core protein of the pyruvate dehydrogenase complex are examples of suitable carrier proteins used in vaccine compositions. Additional examples of carrier proteins suitable for use in the compositions of the present disclosure include, but are not limited to, ovalbumin (OVA), blue carrier protein (BCP), thyroglobulin (THY), a soybean trypsin inhibitor (STI), and multiple attachment peptide (MAP), albumin, serum albumin, c- reactive protein, conalbumin, lactalbumin, ion carrier protein, acyl carrier protein, signal transduction adapter protein, androgen binding protein, calcium binding protein, calmodulin binding protein, ceruloplasmin, cholesterol Ester transfer protein, f box protein, fatty acid binding protein, follistatin, follistatin related protein, GTP binding protein, insulin-like growth factor binding protein, iron binding protein, latent TGF beta binding protein, light-harvesting protein complex, lymph Sphere antigen, membrane transport protein, neurophysin, periplasmic binding protein, phosphate binding protein, phosphatidylethanolamine binding protein, phospholipid transport protein, retinol binding protein, RNA binding protein, s-phase kinase related protein, sex hormone binding globulin, Thyroxine binding protein, transcobalamin, transcortin, transferrin binding protein, and / or vitamin D binding protein.

[0201] As a further example, a peptide or pMHC complex of the present disclosure may be fused to, for example, the 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (p33 or li) as derived from the NCBI, GenBank Accession-number X00497). The li fragment may facilitate an efficient introduction of the peptide or pMHC complex into the cells.

[0202] Peptides or pMHC complexes of the present disclosure may also be attached, covalently (e.g., via a linker) or non-covalently, to a moiety capable of eliciting a therapeutic effect, such as antibodies, or cytokines, such as interleukin 2, interferon-a, and granulocyte-macrophage colony-stimulating factor. Alternatively, or additionally, the peptides or pMHC complexes may be encapsulated into liposomes.

[0203] Other suitable heterologous molecules include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes, and contrast reagents, antibodies, or enzymes that produce a detectable product. Methods for detecting heterologous molecules may include flow cytometry, microscopy, electrophoresis, or scintillation counting.

[0204] In some embodiments, peptides or pMHC complexes of the disclosure may be conjugated with fluorocarbon to increase cellular immunogenicity. Where the peptide or another polypeptide chain of the pMHC complex is linked to a fluorocarbon, the terminus of the peptide or polypeptide chain, such as the terminus that is not conjugated to the fluorocarbon, or other attachment, can be altered, for example to promote solubility of the fluorocarbon-peptide / polypeptide construct via the formation of micelles. To facilitate large-scale synthesis of the construct, the N- or C-terminal amino acid residues of the peptide or another polypeptide chain of the pMHC complex can be modified. When the desired peptide or another polypeptide chain of the pMHC complex is particularly sensitive to cleavage by peptidases, the normal peptide bond can be replaced by a non-cleavable peptide mimetic. Such bonds and methods of synthesis are well known in the art.

[0205] Peptides or pMHC complexes of the disclosure may be provided in soluble form, or may be immobilized by attachment to a suitable solid support. Examples of solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, and a column. pMHC complexes may beattached to an ELISA plate, a magnetic bead, or a surface plasmon resonance biosensor chip. Methods of attaching peptides or pMHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens. In some embodiments, peptides or pMHC complexes are labeled with biotin and attached to streptavidin-coated surfaces.

[0206] Peptides or pMHC complexes of the disclosure may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octameric, or greater. Accordingly, in some aspects, the present disclosure provides oligomeric complexes comprising the peptides or pMHC complexes of the present disclosure. As used herein, the terms “oligomer”, “oligomeric”, “oligomerize” and “oligomerization” or the like encompass a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or higher species of polymerized monomers that comprise the peptide or pMHC complex. Having multiple copies of the peptides or pMHC complexes in a large complex may enhance their biological activity, e.g., immunogenic activity.

[0207] For example, the peptides of the disclosure may be oligomerized using the biotin / streptavidin system. Biotinylated analogs of peptide monomers may be synthesized by standard techniques. For example, the peptide may be C-terminally biotinylated. These biotinylated peptide monomers are then oligomerized by incubation with streptavidin [e.g., at a 4: 1 molar ratio at room temperature in phosphate buffered saline (PBS) or HEPES-buffered RPMI medium for 1 hour]. In a variation of this embodiment, biotinylated peptide monomers may be oligomerized by incubation with anti-biotin antibodies [e.g., goat anti-biotin IgG].

[0208] In general, oligomeric pMHC complexes may be produced using pMHC tagged with a biotin residue and complexed through fluorescently labeled streptavidin. A biotinylation site may be introduced to the pMHC complex to which biotin can be added, for example, using the BirA enzyme. Alternatively, oligomeric pMHC complexes may be formed by using immunoglobulin as a molecular scaffold. In this system, the extracellular domains of MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker. Oligomeric pMHC complexes have also been produced using carrier molecules such as dextran.Oligomeric pMHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.

[0209] In other embodiments, the peptides or pMHC complexes of the disclosure can be oligomerized by covalent attachment to at least one linker. The linker moiety can be a peptide linker, such as those described herein (e.g., in Table 2). In some embodiments, polyethylene glycol (PEG) may serve as the linker that oligomerizes the peptide monomers. For example, a single PEG moiety may be simultaneously attached to the N-termini of both peptide chains of a peptide dimer.

[0210] Alternatively, oligomeric peptide or pMHC complexes may also contain one or more intramolecular disulfide bonds between cysteine residues of the peptide or pMHC monomers. Preferably, the two monomers contain at least one intramolecular disulfide bond. Most preferably, both monomers contain an intramolecular disulfide bond, such that each monomer contains a cyclic group. Such disulfide bonds may be formed by oxidation of the cysteine residues of the peptide core sequence. In one embodiment the control of cysteine bond formation is exercised by choosing an oxidizing agent of the type and concentration effective to optimize formation of the desired isomer. For example, oxidation of a peptide dimer to form two intramolecular disulfide bonds (one on each peptide chain) is preferentially achieved (over formation of intermolecular disulfide bonds) when the oxidizing agent is DMSO. The formation of cysteine bonds can be controlled by the selective use of thiol-protecting groups during peptide synthesis.

[0211] In some embodiments, peptides or pMHC complexes described herein may be fused or conjugated to a dimerization moiety. The dimerization moiety may contain, for example, an immunoglobulin domain, such as from an IgG antibody (e.g., human IgG), which connects two monomers generating a homodimer or heterodimer molecule. As a non-limiting example, the dimerization motif in the proteins according to the present disclosure may be constructed to include a hinge region and an immunoglobulin domain (e.g. Cy3 domain), e.g., carboxyterminal C domain (CH3 domain), or a sequence that is substantially identical to the C domain. The hinge region may be Ig derived and contributes to the dimerization through the formation of aninterchain covalent bond(s), e.g. disulfide bridge(s). In addition, such homodimer or heterodimer molecules may further comprise one or more targeting moieties that bind to target molecules present on, for example, antigen-presenting cells (APCs) such as dendritic cells or B cells. In such instances, the hinge region may function as a flexible spacer between the domains allowing the two targeting units to bind simultaneously to two target molecules on the APC expressed with variable distances. The immunoglobulin domains contribute to dimerization through non-covalent interactions, e.g. hydrophobic interactions. In a preferred embodiment the CH3 domain is derived from IgG. These dimerization moieties may be exchanged with other multimerization moieties from e.g., other Ig isotypes / subclasses. Preferably the dimerization motif is derived from native human proteins, such as human IgG. Examples of such homodimer protein construct are described in US 10,590,195, which is incorporated herein by reference in its entirety.Nucleic Acids and Vectors

[0212] In another aspect, the disclosure provides an isolated polynucleotide comprising a nucleic acid sequence encoding one or more peptide(s) and / or peptide- based molecules (such as complexes (e.g., pMHC complexes), fusion proteins, or conjugates comprising the described peptides) of the disclosure. The polynucleotide may be, for example, DNA, cDNA, PNA, RNA or a combination thereof, either single- and / or double-stranded. The polynucleotide may be in a native or stabilized form, such as, for example, a polynucleotide with a phosphorothioate backbone. The polynucleotide may or may not contain introns so long as the polynucleotide codes for the peptide.

[0213] In some embodiments, the polynucleotide described herein encodes a peptide comprising an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1 -11 , or a fragment or derivative thereof. In some embodiments, the polynucleotide described herein encodes a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-11, or a fragment or derivative thereof.

[0214] In some embodiments, the polynucleotide described herein encodes more than one peptide selected from any one of SEQ ID NOs: 1-11 or fragments thereof. For example, the polynucleotide described herein may encode 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides as described herein (e.g., SEQ ID NO: 1- 11), or fragments thereof. The peptides may be arranged in any order and may be identical or different.

[0215] In some embodiments, the polynucleotide described herein encodes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 amino acid sequences selected from SEQ ID NO: 1-11, or fragments thereof.

[0216] In some embodiments, the polynucleotide described herein is a DNA molecule.

[0217] Methods to deliver DNA to a subject include, for example, direct delivery, as naked DNA. Delivery may also be achieved by nanoparticles; gene gun, microneedle array and in situ electroporation. The nucleic acids can also be administered using ballistic delivery. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles.

[0218] In some embodiments, the polynucleotide described herein is an RNA molecule. For example, the RNA molecule may be mRNA or a self-replicating RNA.

[0219] A polynucleotide encoding RNA disclosed herein can be used to make a vaccine. RNA cannot integrate into the genome and has therefore little oncogenic potential; thus, RNA can be usefid for making a vaccine. Also, RNA only needs to enter the cytoplasm, contrary to DNA which needs to enter the nucleus. An RNA molecule disclosed herein may be chemically modified and / or incorporate modified nucleosides to overcome susceptibility to degradation. RNA vaccines may comprise mRNA and / or self-replicating RNA (also known as RNA replicons). Delivery techniques for RNAvaccines may also encompass, for example, condensation with protamine and encapsulation into liposomes or nanoparticles.

[0220] The nucleic acids (either DNA or RNA) can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in WO 91 / 06309; WO 93 / 24640; WO 96 / 18372; U.S. Pat No. 5,279,833, which are incorporated herein by reference in their entireties.

[0221] A nucleic acid molecule described herein may be generated synthetically. One method is the phosphoramidite method. Without wishing to be bound by theory, in this chemistry, a phosphoramidite (a nucleoside with side protecting groups that preserve the integrity of the sugar, the phosphodiester linkage, and the base during chain extension steps) is coupled through its reactive 3' phosphorous group to the 5' hydroxyl group of a nucleoside immobilized on a solid support column. The steps of oligonucleotide synthesis can include the following: (1) Detritylation, in which the dimethoxytrityl (DMT or trityl) group on the 5' hydroxyl of the support nucleoside is removed by treatment with trichloroacetic acid (TCA). (2) In the coupling step, a phosphoramidite, made reactive by tetrazole (a weak acid), is chemically coupled to the last base added to the column support material. (3) In the capping step, any free 5' hydroxyl groups of unreacted column nucleotides are acetylated by treatment with acetic anhydride and N-methylimidazole. (4) In the oxidation step, the unstable intemucleotide phosphate linkage between the previously coupled base and the most recently added base is oxidized by treatment with iodine and water to a more stable phosphotriester linkage. Following coupling of all bases in the oligonucleotide's sequence, the completed nucleic acid chain may be cleaved from the column by treatment with ammonium hydroxide, and the base protecting groups are removed by heating in the ammonium hydroxide solution.

[0222] By way of a non-limiting example, a synthesis cycle may comprise growth of the nucleotide chain from an initial protected nucleoside derivatized via its terminal 3' hydroxyl to a solid support. Reagents and solvents can be pumped through the support to induce the consecutive removal and addition of sugar protecting groupsin order to isolate the reactivity of a specific chemical moiety on the monomer and effect its stepwise addition to the growing oligonucleotide chain. Assembly of the protected oligonucleotide chain can be carried out in chemical steps, for example, without limitation, deblocking, activation / coupling, oxidation, and capping. Cleavage and deprotection then reveal the single-stranded nucleic acid.

[0223] Nucleic acid synthesis methods disclosed herein can comprise, for example, oligonucleotide synthesis, column-based oligonucleotide synthesis, microarray-based oligonucleotide synthesis, gene synthesis from oligonucleotides, gene synthesis from array-derived oligonucleotide pools, and any of various error correction and sequence validation steps, or any combination thereof.

[0224] RNA chemical synthesis may be similar to that used for DNA. In some embodiments, RNA chemical synthesis methods may comprise an additional protecting group at the 2' hydroxyl of ribose. The 2' hydroxyl of ribose position may be protected with tert-butyldimethyl silyl groups, which can be stable throughout the synthesis, and can be removed at the final deprotection step by addition of a basic fluoride ion such as tetrabutylammonium fluoride (TBAF). The remaining positions on both the sugar and the bases can be protected in the same fashion as for DNA. By adjusting several parameters in the DNA synthesis protocol such as, but not limited to the coupling times, monomer delivery rate, frequency of washing steps, and types of capping reagents, stepwise coupling efficiencies of up to 99% can be obtained.

[0225] Viral nucleic acid synthesis may be catalyzed by both viral and host enzymes, the relative contribution of which can be determined by the type of virus and the specific molecule. Viruses with RNA genomes, except for the retroviruses, synthesize mRNA and replicate their genomes using virus-encoded RNA-dependent RNA polymerases. In contrast, retroviruses synthesize a double-stranded complementary DNA (cDNA) copy of their single-stranded RNA genome using a virion-encoded RNA-dependent DNA polymerase (reverse transcriptase). In subsequent steps, the retroviral cDNA may be integrated into the host chromosome and transcribed by host-encoded DNA-dependent RNA polymerase II (pol II) to yield viral messages and genomic RNA. DNA viruses, except for poxviruses, also use host-encoded pol II to transcribe their messages. Poxviruses, because they replicate in the cytoplasm and do not have access to pol II, assemble a novel transcriptase composed of multiple poxvirus-specific (and possibly one or more host-derived) subunits. Most DNA virus families (e.g. Poxviridae, Iridoviridae, Herpesviridae, Adenoviridae) synthesize a virus-encoded DNA-dependent, DNA polymerase. However, two families (i.e., Parvoviridae and Papovaviridae) utilize host DNA polymerase, and the Hepadnaviridae replicate viral DNA through an RNA intermediate using a virus- encoded reverse transcriptase.

[0226] Due to the degeneracy of the genetic code, nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence. For expression in various hosts, the polynucleotides may be codon-optimized.

[0227] In a further aspect, the disclosure provides a vector comprising a nucleic acid sequence according to the third aspect of the disclosure. The vector may include, in addition to a nucleic acid sequence encoding only a peptide of the disclosure, one or more additional nucleic acid sequences encoding one or more additional peptides. Such additional peptides may, once expressed, be fused to the N-terminus or the C-terminus of the peptide of the disclosure. Examples of such additional peptides are detailed in the sections above. In one embodiment, the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG- tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.

[0228] The vector utilized in the context of the present disclosure desirably comprises sequences appropriate for introduction into cells. For instance, the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.

[0229] In the context of the present disclosure, the term “vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the disclosure, under the control of afunctional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.

[0230] The following vectors are provided by way of example: bacteriophages such as lambda (X) bacteriophage, EMBL bacteriophage; bacterial vectors such as pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a; pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5; eukaryotic vectors such as pWLneo, pSV2cat, pOG44, PXR1, pSG, pSVK3, pBPV, pMSG and pSVL; and transposons such as Sleeping Beauty transposon and PiggyBac transposon.

[0231] In some embodiments, the vector is a viral vector. Viral vectors can be derived from naturally occurring virus genomes, which typically are modified to be replication incompetent, e.g. non-replicating. Non-replicating viruses require the provision of proteins in trans for replication. Typically, those proteins are stably or transiently expressed in a viral producer cell line, thereby allowing replication of the virus. The viral vectors are, thus, typically infectious and non-replicating. Viral vectors may be adenovirus vectors, adeno-associated virus (AAV) vectors (e.g., AAV type 5 and type 2), alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), Sindbis virus (SIN), Semliki forest virus (SFV), and VEE-SIN chimeras), herpes virus vectors (e.g., vectors derived from cytomegaloviruses, like rhesus cytomegalovirus (RhCMV)), arena virus vectors (e.g. lymphocytic choriomeningitis virus (LCMV) vectors), measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors (canarypox (ALVAC) and fowlpox (FPV) vectors), vesicular stomatitis virus (VSV) vectors, retrovirus vectors, lentivirus vectors, simian virus 40 (SV40), bovine papilloma viruses, Epstein-Barr viruses, Moloney murine leukemia viruses, Harvey murine sarcoma viruses, murine mammary tumor viruses, Rous sarcoma viruses, poxvirus viral like particles, baculoviral vectors and bacterial spores.

[0232] As further examples, adenovirus vectors may be derived from human adenovirus (Ad) but also from adenoviruses that infect other species, such as bovine adenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g. CAdV2), aporcine adenovirus (e.g. PAdV3 or 5), or great apes, such as Chimpanzee (Pan), Gorilla (Gorilla), Orangutan (Pongo), Bonobo (Pan paniscus) and common chimpanzee (Pan troglodytes). Poxvirus (Poxviridae) vectors may be derived from smallpox virus (variola), vaccinia virus, cowpox virus or monkeypox virus. Exemplary vaccinia viruses are the Copenhagen vaccinia virus (W), New York Attenuated Vaccinia Virus (NYVAC), ALVAC, TROVAC and Modified Vaccinia Ankara (MVA).

[0233] Many expression systems are known in the art, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus spec.), plant cells, animal cells (e.g., mammalian cells), and insect cells.

[0234] In yet another aspect, the disclosure provides a host cell comprising the vector of the disclosure. The host cell can be either prokaryotic or eukaryotic. Bacterial cells may be preferred prokaryotic host cells in some circumstances and typically are a strain of E. coli such as, for example, the E. coli strains DH5 and RR1. Non-limiting examples of eukaryotic host cells include yeast, insect, and mammalian cells (e.g., from a mouse, rat, monkey, or human cell lines). Non-limiting examples of yeast host cells include, e.g., YPH499, YPH500, and YPH501. Non-limiting examples of mammalian host cells include Chinese hamster ovary (CHO) cells, NIH Swiss mouse embryo cells NIH / 3T3, monkey kidney-derived COS-1 cells, and 293 cells which are human embryonic kidney cells. Examples of insect cells include Sf9 cells, which can be transfected with baculovirus expression vectors.

[0235] Transformation of appropriate cell hosts with a DNA construct of the present disclosure is accomplished by well-known methods that typically depend on the type of vector used. Successfully transformed cells, i.e. cells that contain a DNA construct of the present disclosure, can be identified by, for example, PCR. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

[0236] It will be appreciated that certain host cells of the disclosure are useful in the preparation of the peptides or peptide-based molecules of the disclosure, forexample bacterial, yeast, and insect cells. However, other host cells may be useful in certain therapeutic methods. For example, antigen-presenting cells (APCs), such as dendritic cells or B cells, may be used to express the peptides of the disclosure such that the peptides may be loaded into appropriate MHC molecules.

[0237] A further aspect of the disclosure provides a method of producing peptides or peptide-based molecules of the disclosure, the method comprising culturing a host cell and isolating the peptide or peptide-based molecule from the host cell or its culture medium.Peptide and pMHC Binding Moieties

[0238] Peptides, pMHC complex, or other peptide-based molecules (such as a complex, fusion protein, or conjugate comprising a peptide disclosed herein) of the present disclosure can be used to identify and / or isolate binding moieties that bind specifically to a peptide, pMHC complex, or other peptide-based molecule of the disclosure. Such binding moieties may be used as immunotherapeutic reagents and may include, e.g., antibodies (or antigen-binding fragments thereof), alternative scaffolds, TCRs, and CARs.

[0239] In one aspect, the disclosure provides a peptide binding moiety that binds a peptide of the disclosure. Preferably the peptide binding moiety binds a peptide when the peptide is in complex with MHC. In the latter instance, the peptide binding moiety may bind partially to the MHC, provided that the peptide binding moiety also binds to the peptide. The peptide binding moiety may bind only the peptide, and that binding may be specific. The peptide binding moiety may bind only the pMHC complex and that binding may be specific.

[0240] The disclosure also provides a method of identifying a peptide binding moiety that binds a pMHC complex of the disclosure, the method comprising contacting a candidate peptide binding moiety with the pMHC complex and determining whether the candidate peptide binding moiety binds the complex. Methods to determine binding to pMHC complexes include, for example, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in whicha biological response is detected upon binding, for example, cytokine release or cell apoptosis.

[0241] The candidate peptide binding moiety may be a peptide binding moiety of the type already described, such as an antibody or a TCR.

[0242] For example, antibodies and TCRs may be obtained from display libraries in which the pMHC complex of the disclosure is used to pan the library. TCRs can be displayed on the surface of phage particles and yeast particles, for example, and such libraries have been used for the isolation of high affinity variants of TCR derived from T cell clones. TCR phage libraries can be used to isolate TCRs with novel antigen specificity. Such libraries can be constructed with a- and 0- chain sequences corresponding to those found in a natural repertoire. However, the random combination of these a- and 0- chain sequences, which occurs during library creation, can produce a repertoire of TCRs that may not be naturally occurring.

[0243] In some embodiments, the pMHC complex of the disclosure may be used to screen a library of diverse TCRs displayed on the surface of phage particles. The TCRs displayed by said library may not correspond to those contained in a natural repertoire, for example, they may contain a- and 0- chain pairing that would not be present in vivo, and / or the TCRs may contain non-natural mutations and / or the TCRs may be in soluble form. Screening may involve panning the phage library with pMHC complexes of the disclosure and subsequently isolating bound phage particles. For this purpose, pMHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound pMHC complexes isolated, with a magnet, or by chromatography, respectively. The panning steps may be repeated several times. Isolated phage may be further expanded in E. coli cells. Isolated phage particles may be tested for specific binding to pMHC complexes of the disclosure. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument. The DNA sequence of the T cell receptor displayed by pMHC binding phage can be further identified by PCR methods.

[0244] Various methods for generating and / or isolating peptide binding moieties, such as antibodies and TCRs, including those that target peptides presented as part of pMHC complexes are well known in the art. Peptide binding moieties may be generated by immunizing immunocompetent host animals, such as non-human host animals (e.g., rodents such as mice or rats) with antigens such as peptides or pMHC complexes. In certain implementations, the host animal may be genetically modified such that it generates human or humanized peptide binding moieties in response to the immunization. For instance, TCRs may be generated by immunizing with antigenic peptide (e.g., a prostate lineage antigen-derived peptide such as any one SEQ ID NOs: 1-11) a rodent genetically modified to express human or humanized TCRs and, optionally, to further express human or humanized MHC (e.g., MHC I a, MHC II a, and / or MHC II ), human or humanized 02 microglobulin (02M), and / or human or humanized T Cell co receptor (e.g., CD4, CD8 a, and / or CD8 0). See, e.g., U.S. Pat. No. 11,259,510; U.S. Pub. No. 2022 / 0322648; Moore et al, Sci Immunol. 2021 Dec 17; 6(66):eabj4026, which are each herein incorporated by reference in their entirety. Likewise, antibodies, or more specifically TCR-mimetic antibodies, may be generated by immunizing with antigenic pMHC complexes (e.g, a pMHC complex, such as an MHC-prostate lineage antigen-derived peptide complex) a rodent genetically modified to express human or humanized immunoglobulins (e.g, IgGs) and, optionally, further express human or humanized MHC (e.g, MHC I a, MHC II a, and / or MHC II 0) and / or human or humanized 02 microglobulin (02M). See, e.g, U.S. Pat No. 8,791,323; U.S. Pat. No. 8,642,835; U.S. Pat. No. 8,754,287; U.S. Pat. No. 10,143,186; U.S. Pat. Pub. No. 2019 / 0292263; Proc Natl Acad Sci U S A. 2014 Apr 8;1 ll(14):5153-8, each of which is herein incorporated by reference in its entirety. Methods of peptide binding moieties (e.g, cells expressing peptide binding moieties such as T cells and B cells) and isolating and cloning sequences, such as variable domain coding sequences, are well known in the art.

[0245] The ability to generate antibodies has been harnessed in genetically modified animals, which are able to generate therapeutic antibodies or antibody fragments against human targets. Exemplary genetically modified mice, comprising human V(D)J gene segments, for generation of therapeutic antibodies are those described in U.S. Pat. Nos. 5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598,6,150,584, 6,998,514, 7,795,494, 7,910,798, 8,232,449, 8,703,485, 8,907,157, and 9,145,588, each of which is hereby incorporated by reference in its entirety, as well as in U.S. Pat. Pub. Nos. 2008 / 0098490, 2010 / 0146647, 2013 / 0145484, 2012 / 0167237, 2013 / 0167256, 2013 / 0219535, 2012 / 0207278, 2015 / 0113668, 2019 / 0127757, and 2021 / 0059229 each of which is hereby incorporated by reference in its entirety, and in PCT Pub. Nos. W02007117410, W02008151081, W02009157771, W02010039900, WO2011004192, WO2011123708, WO2013045916, WO2015049517, W02014093908, W02014093908, W02006008548, W02010109165, W02016062990, W02018039180, WO2011158009, WO2013041844, WO2013041846, WO2013079953, W02013061098, WO2013144567, WO2013144566, W02013171505, W02012018610, WO2022126113, WO2020132557, WO2017035274, WO2019236670, WO2019236671, W02019008123, WO2021123090, W02020169022, and WO2021244522, each of which are hereby incorporated by reference in its entirety. Other exemplary genetically modified mice, comprising human V(D)J gene segments, for generation of therapeutic antibodies are those described in U.S. Pat. Nos.6,596,541, 6,586,251, 8,642,835, 9,706,759, 10,238,093, 8,754,287, 10,143,186, 9,796,788, 10,130,081, 9,226,484, 9,012,717, 10,246,509, 9,204,624, and 9,686,970, each of which is hereby incorporated by reference in its entirety, as well as in U.S. Pat. Pub. Nos. 2013 / 0212719, 2015 / 0289489, 2017 / 0347633, 2019 / 0223418, 2018 / 0125043, 2019 / 0261612, and 2019 / 0380316, each of which is hereby incorporated by reference in its entirety, in PCT Pub. Nos. WO2013138680, WO2013138712, WO2013138681, W02015042250, WO2012148873, WO2013134263, WO2013184761, W02014160179, WO2017214089, WO2016149678, and WO2017123808, WO 2012018764, WO2019241692, WO2017123804, WO2022140219 and Murphy, A., “Veloclmmune: Immunoglobulin Variable Region Humanized Mouse,” in Recombinant Antibodies for Immunotherapy, New York, NY, Cambridge University Press, 101-107 (2009), each of which are hereby incorporated by reference in its entirety. Additional detailed embodiments of certain exemplary genetically engineered non-human animals, e.g., rodents, e.g., rats or mice, are described below.

[0246] Thus, in some embodiments, an immunized non-human animal host asdescribed herein is a rodent, e.g., a rat or mouse. In some embodiments, an immunized non-human animal host as described herein is a genetically modified nonhuman animal host, e.g., a genetically modified rodent. Various embodiments of the genetically modified non-human animals, e.g., rodents, e.g., rats or mice, are described in more detail herein below.

[0247] In some embodiments, the immunized non-human animal host is a rodent such as a rat or mouse. In some embodiments, the host is a genetically modified rodent that comprises in its genome an immunoglobulin heavy chain variable region comprising one or more human heavy chain V gene segments, one or more human D gene segments, and one or more human heavy chain J gene segments, wherein the immunoglobulin heavy chain variable region is operably linked to a constant region, and an immunoglobulin light chain variable region comprising one or more human light chain V gene segments and one or more human light chain J gene segments, wherein the light chain is operably linked to a constant region.

[0248] In some embodiments, the host is a genetically modified mouse that comprises in its genome an immunoglobulin heavy chain variable region comprising one or more human heavy chain V gene segments, one or more human D gene segments, and one or more human heavy chain J gene segments, wherein the heavy chain variable region is operably linked to a murine (e.g., a rat or mouse) constant region, and an immunoglobulin light chain variable region comprising one or more human light chain V gene segments and one or more human light chain J gene segments, wherein the light chain is operably linked to a murine constant region.

[0249] In one aspect, the immunoglobulin heavy chain variable region is operably linked to a mouse heavy chain constant region, and the immunoglobulin light chain variable region is operably linked to a mouse light chain constant region. In a further aspect, the immunoglobulin heavy chain variable region operably linked to a mouse heavy chain constant region resides at the endogenous mouse heavy chain locus, and the immunoglobulin light chain variable region operably linked to a mouse light chain constant region resides at the endogenous mouse light chain locus. One exemplary embodiment is described in Macdonald et al, Proc. Natl. Acad. Sci. USA111:5147-52 and supporting information (www.pnas.org / cgi / content / short / 1323896111), which is hereby incorporated by reference in its entirety.

[0250] In some embodiments, the host is a genetically modified rat that comprises in its genome an immunoglobulin heavy chain variable region comprising one or more human heavy chain V gene segments, one or more human D gene segments, and one or more human heavy chain J gene segments, wherein the human heavy chain variable region is operably linked to a human or murine (e.g., a rat or mouse) constant region. In some embodiments, a host is a genetically modified rat that comprises in its genome an immunoglobulin light chain variable region comprising one or more human light chain V gene segments and one or more human light chain J gene segments, wherein the light chain is operably linked to a human or murine constant region.

[0251] In one aspect, the immunoglobulin heavy chain variable region is operably linked to a rat heavy chain constant region. In some embodiments, the immunoglobulin heavy chain variable region operably linked to a rat heavy chain constant region resides at the endogenous rat heavy chain locus. In a further aspect, the immunoglobulin light chain variable region is operably linked to a rat light chain constant region. In some embodiment, the immunoglobulin light chain variable region operably linked to a rat light chain constant region resides at the endogenous rat light chain locus.

[0252] In another aspect, the immunoglobulin heavy chain variable region is operably linked to a rat heavy chain constant region and the immunoglobulin light chain variable region is operably linked to a human light chain constant region. In some embodiments, the immunoglobulin heavy chain variable region operably linked to a rat heavy chain constant region resides at the endogenous rat heavy chain locus. In some embodiments, the immunoglobulin light chain variable region operably linked to a human light chain constant region resides at the endogenous rat light chain locus. In a further aspect, an engineered immunoglobulin heavy chain locus comprises a human Ep enhancer. In some embodiments, an engineered immunoglobulin heavy chain locuscomprises a rat 3 ’ enhancer. In some embodiments, an engineered immunoglobulin light chain locus (e.g., a kappa light chain locus) comprises a human kappa deleting element (KDE). In some embodiments, an engineered immunoglobulin light chain locus (e.g., a lambda light chain locus) comprises a human 3’ enhancer. Exemplary embodiments are described in W02014093908 and Osborn, et al., Journal of Immunology 190.4 (2013): 1481-1490, which are herein incorporated by reference in their entirety.

[0253] According to certain aspects of the disclosure, immunoglobulins that are specific for pMHC complexes (e.g., comprising prostate lineage antigen-derived peptides such as one of SEQ ID NOs: 1-11) may be generated in a genetically modified non-human animal (e.g., a rodent such as a mouse) described in PCT Pub. No. WO2019190922, which is hereby incorporated by reference in its entirety, and / or generated according to the methods described therein.

[0254] According to certain aspects of the disclosure, T cells and / or TCRs that are specific for pMHC complexes (e.g., comprising prostate lineage antigen-derived peptides such as one of SEQ ID NOs: 1-11) may be generated in a genetically modified non-human animal (e.g., a rodent such as a mouse) described in PCT Pub. No. WO2016164492, WO2022212582, and / or W02024064860, each of which is hereby incorporated by reference in its entirety, and / or generated according to the methods described therein.

[0255] Alternatively, antigen binding T cells and TCRs can be isolated from fresh blood obtained from patients or healthy donors. Such a method involves stimulating T cells using autologous dendritic cells (DCs), followed by autologous B cells, and then pulsed with a peptide disclosed herein. Several rounds of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the disclosure (for example using an IFNy ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g. IFNy), or expression of a cell surface marker (such as CD 137). Sorted cells may be expanded and further validated,for example, by ELISpot assay and / or cytotoxicity against target cells and / or staining by peptide-MHC tetramer. The TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.

[0256] A peptide binding moiety disclosed herein can include, for example, without limitation, an antibody, a TCR, or a CAR.

[0257] Peptide binding moieties may be screened for binding to one or more peptides (e.g., prostate lineage antigen-derived peptides such as SEQ ID NOs: 1-11) according to various methods known in the art. The peptide binding moieties (e.g., antibodies, TCRs, CARs) and / or the one or more peptides may be presented in solution, presented on a cell surface (e.g., a reporter cell), or immobilized on a solid support, including the specific cells or solid supports described elsewhere herein. For example, the peptide binding moiety may be presented on a cell surface (e.g., on a heterologous host cell) or immobilized on a solid support and the one or more peptides may be presented in solution (e.g., as soluble pMHC complexes), presented on a cell surface (e.g., loaded onto MHC of a heterologous cell), or immobilized on a solid support. Similarly, the one or more peptides may be presented on a cell surface (e.g., loaded onto MHC of a heterologous cell) or immobilized on a solid support (e.g., as a pMHC complex) and the peptide binding moiety may be presented in solution (e.g., as soluble pMHC complexes), presented on a cell surface (e.g., on a heterologous host cell), or immobilized on a solid support. In some instances, peptide binding moiety binding to a peptide presented on a cell surface may be detected using a genetically modified reporter cell that emits a signal in response to the binding, including in cell-to-cell screening systems in which the antigen-recognition molecule and one or more peptides are presented on different cell surfaces.

[0258] For example, TCR screening can be performed using TCR activation assays. For example, JRT3-T3.5 cells (ATCC TIB-153), a Jurkat subline lacking endogenous TCR surface expression may be utilized as described in Moore et al., Sci. Immunol. 6, eabj4026 (2021), which is herein incorporated by reference in its entirety. T cell receptor alpha (TCRA) and T cell receptor beta (TCRB) sequences of interest can be introduced into the cells by lentiviral transduction, and surface TCR+ cells can be sorted. Antigen-presenting cells (e.g., 293T cells) that are pulsed with a targetpeptide or off-target peptide can be incubated with the TCR-transduced or parental JRT3 cells. A readout (e.g., luciferase activity) is then measured as an indication of TCR-mediated activation.

[0259] In some embodiments, monoclonal antibody screening can be performed with cells isolated from the spleens and lymphoid tissue harvested from mice with optimal titers using hybridoma and B cell sorting (BST) platforms. Counterscreening approaches using one or more off-target peptides of the prostate lineage antigen-derived peptides can help to identify and eliminate B-cells and hybridomas that show cross-reactivity with peptides that form pHLA complexes resembling the targeted complex. For example, antigen positive (Ag+) clones that have cross-reactivity to off- target peptides can be identified by examining cell supernatants for antibody binding to cells (e.g., T2 cells) pulsed with the target peptide or off-target peptides using a cell binding assay. As another example, Ag+ B-cells can be captured using a biotin-labeled HLA-target peptide complex in the presence of high concentrations of one or more unlabeled, HLA-off-target peptide complexes to enrich for antibodies specific for the HLA-target peptide complex. The antibody variable domains of Ag+ B-cells can be subsequently cloned as full-length mAbs and expressed (e.g., in CHO cells) for further screening.

[0260] Antibody binding to target / off-target peptides can be determined using ELISA. For example, MHC-target peptide complexes or MHC-off-target peptide complexes can be coated onto a plate (e.g., 96-well microtiter plate). A sample comprising test antibodies can be added to the plate and the reaction can be incubated under a condition to allow binding to occur. The plate is then washed, and a secondary antibody can be then added to the plate to detect the antibodies bound to an MHC- peptide complex. Typically, the secondary antibody can produce a signal that is indicative of the amount of the antibodies bound to the MHC-peptide complex.

[0261] In various embodiments of the methods described herein, pMHC complexes may be provided in soluble form, or may be immobilized by attachment to a suitable solid support. Examples of solid supports include, but are not limited to, a bead (e.g., a magnetic bead), a membrane, sepharose, a plate, a tube, a column. pMHCcomplexes may be atached to an ELISA plate, a magnetic bead, or a surface plasmon resonance biosensor chip. Methods of ataching pMHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g., biotin and streptavidin, or antibodies and antigens. In some embodiments, pMHC complexes are labeled with biotin and atached to streptavidin-coated surfaces.

[0262] In various embodiments of the methods described herein, pMHC complexes may be present on a cell (e.g., on a surface of a cell). Such a cell may be a mammalian cell, preferably a cell of the immune system, and a specialized antigen- presenting cell (APC) such as a dendritic cell or a B cell. Other preferred cells include T2 cells. Cells presenting the peptide or pMHC complex of the disclosure may be isolated, preferably in the form of a homogenous population, or provided in a substantially pure form. Such cells may be obtained by pulsing said cells with one or more peptides (e.g., 2 to 10, 2 to 20, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides) of the disclosure, or genetically modifying the cells (via DNA or RNA transfer) to express one or more peptides (e.g., 2 to 10, 2 to 20, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides) of the disclosure. Pulsing involves incubating the cells with the peptide for several hours using peptide concentrations typically ranging from 10-5 to 10-12 M. Such cells may additionally be transduced with HLA molecules to further induce presentation of the peptide(s). Cells may be produced recombinantly.

[0263] In various embodiments of the methods described herein, the method is performed in a high-throughput format (e.g., a 96-well plate).

[0264] In yet another aspect, provided herein is a method of enriching a sample for peptide binding moieties that specifically bind a target peptide, comprising (a) contacting a sample comprising a plurality of peptide binding moieties with the target peptide in the presence of one or more off-target peptides associated with said target peptide, wherein each of said target peptide and said one or more off-target peptides is presented in a complex with a major histocompatibility complex (MHC) molecule (MHC-target peptide complex or MHC-off-target peptide complex); and (b) enriching the sample by isolating the peptide binding moieties that are bound to the MHC-target peptide complex. The method may further comprise repeating steps (a)-(b) to furtherenrich the sample. In some embodiments, the target prostate lineage antigen-derived peptide or MHC-peptide complex is labeled and said off-target peptides or proteins or fragments thereof comprising said off-target peptides or MHC-off-target peptide complexes are not labeled or are labeled differently.

[0265] There are various ways that can allow isolation of the peptide binding moieties that are bound to the MHC-target peptide complex. For example, the MHC- target peptide complex may be present on antigen-presenting cells while the MHC-off- target peptide complexes are not present on antigen-presenting cells (e.g., in a soluble form). Alternatively, the MHC-target peptide complex may be immobilized on a solid support while the MHC-off-target peptide complexes may be soluble or immobilized to a different solid support. The MHC-target peptide complex and the MHC-off-target peptide complexes may also be differentially labeled such that specific detection and isolation of the MHC-target peptide complex and the peptide binding moieties bound thereon can be achieved. The peptide binding moieties may be eluted from the MHC- target peptide complex after isolation.

[0266] In some embodiments, the peptide binding moiety of the disclosure may be an antibody or antigen-binding fragment thereof. Antibodies or antigen-binding fragments thereof encompass derivatives, functional equivalents, and homologues of antibodies, humanized antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. A humanized antibody may be a modified antibody having the variable regions of a nonhuman, e.g. murine, antibody, and the constant region of a human antibody. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; or fragments that comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as “mAb”.

[0267] In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody. The bispecific antibody may comprise a second targeting moiety that targets the desired cell or tissue, e.g., prostate, or to another desired antigen associated with the same or similar disease or disorder (e.g., a prostate lineage antigen, such as PSA).

[0268] It is possible to take an antibody, for example a monoclonal antibody, and use recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. A hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

[0269] It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers, (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion, and (x) VHH or VNAR antibodies, also known as single-domain antibodies or nanobodies (Nb), which may be derived from heavy-chain antibodies from e.g., dromedaries, camels, llamas, alpacas, or sharks.

[0270] Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with eachother to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94 / 13804). Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways, e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain “Janusins”. Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against an antigen of interest, then a library can be made where the other arm is varied, and an antibody of appropriate specificity selected. An “antigen binding domain” is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0271] In some embodiments, the peptide binding moiety may be an antibodylike molecule that has been designed to specifically bind a peptide or peptide-MHC complex of the disclosure. In some embodiments the peptide binding moiety may comprise a TCR-mimic antibody. In some embodiments, such TCR-mimic antibodies can comprise high-affinity soluble antibody molecules endowed with a TCR-like specificity towards tumor epitopes that can target tumor cells and mediate their specific killing.

[0272] Also encompassed within the present disclosure are binding moieties based on engineered protein scaffolds or “alternative scaffolds”. Alternative scaffoldsare derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest. Examples of alternative scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices; anticalins, derived from lipocalins, that incorporate binding sites for small ligands at the open end of a P-barrel fold; monobodies, designed to incorporate the fibronectin type III domain (Fn3) of fibronectin or tenascin as a protein scaffold or synthetic FN3 domains (e.g., tencon); nanobodies, and DARPins. Additional alternative scaffolds include Adnectin™, iMab, EETI-II / AGRP, Kunitz domain, thioredoxin peptide aptamer, Affilin, Tetranectin, Fynomer, and Avimer. Alternative scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo. Short peptides may also be used to bind a target protein. Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit / disrupt protein-protein interactions in vivo.

[0273] Alternative scaffolds are typically single chain polypeptidic frameworks that contain a highly structured core associated with variable domains of high conformational tolerance allowing insertions, deletions, or other substitutions within the variable domains. Libraries introducing diversity to one or more variable domains, and in some cases to the structured core, may be generated using known protocols and the resulting libraries may be screened for binding to the peptide and / or the pMHC complex of the disclosure, and the identified binders may be further characterized for their specificity using known methods. Alternative scaffolds may be derived from Protein A, in particular, the Z-domain thereof (affibodies), ImmE7 (immunity proteins), BPTI / APPI (Kunitz domains), CTLA-4, charybdotoxin (Scorpion toxin), Min-23 (knottins), lipocalins (anticalins), Ras-binding protein AF-6 (PDZ-domains), neokarzinostatin, a fibronectin domain, an ankyrin consensus repeat domain, or thioredoxin.

[0274] In some embodiments, the antibodies or alternative scaffolds described herein can be immobilized on viral vectors. Such modified recombinant viral vectorscan be usefill for the targeted introduction of genetic materials encoded by the viral vectors into cells and / or tissues (e.g., prostate cells and / or prostate tissues). Various means can be used to mobilize the antibodies or alternative scaffolds to the viral vectors, for example, by using an affinity binding pair, such as c-Myc / anti-Myc antibody, streptavidin / biotin, or via spy-tag / spy-catcher system. Exemplary vectors that may be modified with the antibodies or alternative scaffolds described herein include, but are not limited to, adeno-associated virus (AAV) vectors (e.g., AAV1, AAV2, AAV6, AAV9, or AAV9.PHP), retroviral vectors, lentiviral vectors, and targeted oncolytic viruses (e.g., herpes simplex virus (HSV)).

[0275] In some embodiments, the peptide binding moiety may be a TCR. TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and the IMGT public database of TCR sequences.

[0276] The TCRs of the present disclosure may be in any format. For example, the TCRs may be a0 heterodimers, or aa or 00 homodimers.

[0277] a / 0 heterodimeric TCRs have an a-chain and a 0-chain. Broadly, each chain comprises variable, joining and constant region, and the 0-chain also usually contains a short diversity region between the variable and joining regions, but this diversity region is often considered as part of the joining region. Each variable region comprises three hypervariable CDRs (Complementarity Determining Regions) embedded in a framework sequence; CDR3 is believed to be the main mediator of antigen recognition. There are several types of a- chain variable (Va) regions and several types of 0-chain variable (V0) regions distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence.

[0278] The TCRs of the disclosure may not correspond to TCRs as they exist in nature. For example, they may comprise a- and 0- chain combinations that are not present in a natural repertoire. Alternatively or additionally, a TCR described herein may be soluble, and / or the a- and / or 0- chain constant domain may be truncated relative to the native / naturally occurring TRAC / TRBC sequences such that, for example, the C-terminal transmembrane domain and intracellular regions are not present. Suchtruncation may result in removal of the cysteine residues from TRAC / TRBC that form the native interchain disulfide bond.

[0279] In addition, the TRAC / TRBC domains may contain modifications. For example, the a-chain extracellular sequence may include a modification relative to the native / naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48. Likewise, the 0-chain extracellular sequence may include a modification relative to the native / naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57. These cysteine substitutions relative to the native a- and 0- chain extracellular sequences enable the formation of a non-native interchain disulfide bond which stabilizes the refolded soluble TCR, i.e. the TCR formed by refolding extracellular eland - chains. This non-native disulfide bond facilitates the display of correctly folded TCRs on phage. In addition, the use of the stable disulfide linked soluble TCR enables more convenient assessment of binding affinity and binding half-life. Alternative positions for the formation of a non-native disulfide include, for example, Thr 45 of exon 1 of TRAC*01 and Ser 77 of exon 1 of TRBCl*01 or TRBC2*01; Tyr 10 of exon 1 of TRAC*01 and Ser 17 of exon 1 of TRBCl*01 or TRBC2*01; Thr 45 of exon 1 of TRAC*01 and Asp 59 of exon 1 of TRBCl*01 or TRBC2*01; and Ser 15 of exon 1 of TRAC*01 and Glu 15 of exon 1 of TRBCl*01 or TRBC2*01. TCRs with a non-native disulfide bond may be full length or may be truncated.

[0280] TCRs of the disclosure may be in single chain format. Single chain TCRs include a TCR polypeptides of the type: Va-L-V0, V0-L-Va, Va-Ca-L-V0, Va-L-V0-C0 or Va-Ca-L-V0-C0, optionally in the reverse orientation, wherein Va and V0 are TCR a and 0 variable regions respectively, Ca and C0 are TCR a and 0 constant regions respectively, and L is a linker sequence. Single chain TCRs may contain a non- native disulfide bond. The TCR may be in a soluble form (i.e. having no transmembrane or cytoplasmic domains) or may contain full length a- and 0- chains. The TCR may be provided on the surface of a cell, such as a T cell.

[0281] TCRs of the disclosure may be engineered to include mutations. Methods for producing mutated high affinity TCR variants such as phage display andsite directed mutagenesis. Preferably, mutations to improve affinity are made within the variable regions of a- and / or 0- chains. More preferably mutations to improve affinity are made within the CDRs. There may be between 1 and 15 mutations in the a- and or 0- chain variable regions.

[0282] TCRs of the disclosure may also be labeled with an imaging compound, for example a label that is suitable for diagnostic purposes. Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD 1 -antigen complexes, bacterial superantigens, and MHC-peptide / superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand. In multimeric high affinity TCR complexes (formed, for example, using biotinylated heterodimers) fluorescent streptavidin can be used to provide a detectable label. A fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.

[0283] A TCR of the present disclosure (or multivalent complex thereof) may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine. A multivalent high affinity TCR complex of the present disclosure may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer. Thus, the multivalent high affinity TCR complexes according to the disclosure are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses. The high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.

[0284] High affinity TCRs of the disclosure may be used in the production of soluble bi-specific reagents. A preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti-CD3 specific antibody fragment.

[0285] In a further aspect, the disclosure provides nucleic acid encoding the TCR of the disclosure, a TCR expression vector comprising nucleic acid encoding a TCR of the disclosure, as well as a cell harboring such a vector. The TCR may be encoded either in a single open reading frame or two distinct open reading frames. Also included in the scope of the disclosure is a cell harboring a first expression vector which comprises nucleic acid encoding an a- chain of a TCR of the disclosure, and a second expression vector which comprises nucleic acid encoding a P-chain of a TCR of the disclosure. Alternatively, one vector may encode both an a- and a - chain of a TCR of the disclosure.

[0286] A further aspect of the present disclosure provides a cell displaying on its surface a TCR of the disclosure. The cell may be a T cell, or other immune cell. The T cell may be modified such that it does not correspond to a T cell as it exists in nature. For example, the cell may be transfected with a vector encoding a TCR of the disclosure such that the T cell expresses a further TCR in addition to the native TCR. Additionally or alternatively, the T cell may be modified such that it is not able to present the native TCR. There are a number of methods suitable for the transfection of T cells with DNA or RNA encoding the TCRs of the disclosure. As a non-limiting example, the transfection method may comprise a rapid RNA-based transfection system. T cells expressing the TCRs of the disclosure are suitable for use in adoptive therapy-based treatment of diseases such as cancers. There are a number of suitable methods by which adoptive therapy can be carried out. For example, adoptive cell therapy (ACT) may comprise use of autologous tumor-infiltrating lymphocytes and may include a lymphodepletion preparative regimen prior to ACT. In some embodiments, viruses, e.g., retroviruses, that encode TCRs may be used for genetic modification of lymphocytes to convert normal lymphocytes into lymphocytes with anti-cancer activity. The adoptive transfer of lymphocytes with anti-cancer activity into patients requiring treatment of, e.g., metastatic melanoma, can mediate tumor regression. In some embodiments, ACT may comprise treatment of patients with cancers expressing viral or alloantigens, treatment of patients with cancers expressing viral antigens, and / or ACT using gene-modified lymphocytes. In some embodiments, ACT methods may include, for example, genetic modification of lymphocytes to introduce new recognitionspecificities using, e.g., aPTCR(s) and / or chimeric TCR(s); genetic modification of lymphocytes to alter function of T cells using, e.g., co-stimulatory molecules (e.g., CD28, 41BB), cytokines (e.g., IL2, IL15), homing molecules (e.g., CD62L, CCR7), and / or molecules capable of preventing apoptosis (BCL2); modification of host lymphodepletion using, e.g., selective depletion of CD4+ cells or T regulatory cells; blocking of inhibitory signals on reactive lymphocytes using, e.g., antibodies to CTLA4 and / or PD-1; administration of vaccines to stimulate transferred cells using, e.g., recombinant virus encoding antigen(s); administration of alternative cytokines to support cell growth using, e.g., IL15 and / or IL21; stimulation of APCs using, e.g., tolllike receptor agonists; generation of less differentiated lymphocytes using, e.g., alternate culture conditions and growth promoting cytokines in vitro-, and, overcoming antigen escape variants using, e.g., natural killer cells.

[0287] The TCRs of the disclosure intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells. The glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene.

[0288] In some embodiments, the peptide binding moiety may be a chimeric antigen receptor (CAR). CARs are genetically engineered receptors. CARs may be generated that bind the peptides or pMHC complexes of the present disclosure by incorporating an antigen binding domain that specifically binds the peptide or pMHC complex to the extracellular domain of the CAR. CARs may be introduced into and expressed by immune cells, such as T cells, NK cells, or macrophages. CARs can be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell presenting that antigen. When these antigens exist on tumor cells, an immune cell that expresses the CAR can target and kill the tumor cell.

[0289] The general structure of a CAR typically comprises an extracellular domain that binds the antigen (e.g. the peptides or pMHC complexes of the present disclosure), a hinge, a transmembrane domain, and an intercellular domain comprising a signaling domain and optionally one or more co-stimulatory domains.

[0290] Extracellular domains of the CAR may contain any polypeptide that specifically binds the desired antigen (e.g. the peptides or pMHC complexes of the present disclosure). For example, the extracellular domain may comprise an antibody fragment such as scFv or VHH. The CARs may also be engineered to bind two or more desired antigens that may be arranged in tandem and separated by linker sequences. For example, one or more domain antibodies, scFvs, llama VHH antibodies or other VH only antibody fragments may be organized in tandem via a linker to provide bispecificity or multispecificity to the CAR.

[0291] A hinge domain may be present between the extracellular domain and the transmembrane domain of the CAR, e.g., to provide flexibility to allow effective binding of the extracellular domain to its intended target. The hinge domain may be a polypeptide of about 2 to 100 amino acids in length. The hinge may include or be composed of flexible residues such as Gly and Ser so that the adjacent protein domains are free to move relative to one another. Longer hinges may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. The hinge may be derived from a hinge region or portion of the hinge region of any immunoglobulin. Non-limiting examples of linkers include a part of human CD8a chain, extracellular domain of CD28, an Ig hinge from IgG, IgM, IgA, IgD, or IgE, FcyRllla receptor, or a functional fragment thereof.

[0292] Transmembrane domains of the CAR may be derived transmembrane proteins, such as an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD2, CD4, CD5, CD8, CD9, CD16, CD18, CD19, CD22, CD27, CD29, CD33, CD37, CD40, CD45, CD49a, CD64, CD80, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134, CD154, CD160 (BY55), KIRDS2, 0X40, LFA-1 (CDl la, CD18), CDl lb, CDl lc, CDl ld, ICOS (CD278), 4-1 BB (CD137), 4-1 BBL, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, ITGAL, LFA-1, ITGAM, ITGAX, ITGB1, ITGB2, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CEACAM1, CRT AM, Ly9 (CD229), PSGL1, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp30, NKp44, NKp46, NKG2D, and NKG2C, or functional fragment thereof.

[0293] The intracellular signaling domain of a CAR participates in transducing the signal of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit an effector cell function, e.g., activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular CAR domain. Non-limiting examples of intracellular signaling domains of the CAR include those derived from CD3 , CD3E, CD38, CD3y, CD5, CD22, CD39, CD79A, CD79B, CD66d, CD226, DAP10, DAP12, Fc epsilon receptor I gamma chain (FCER1G), or FcR .

[0294] Intracellular co-stimulatory domains of the CAR can provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Such co-stimulatory domains may be derived from one or more co-stimulatory molecules, such as, but not limited to, 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP 10, LAT, NKD2C SLP76, TRIM, BTLA, GITR, CD226, HVEM, and ZAP70.

[0295] The CARs can be generated by standard molecular biology techniques. The extracellular domain that binds the desired antigen may be derived from the antibodies or their antigen binding fragments described herein.

[0296] In another aspect, the disclosure further provides cells that comprise a peptide binding moiety (e.g., TCRs and CARs) of the present disclosure. In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is T cell, NK cell, or a macrophage. The host cell may be autologous or allogeneic with respective to the subject receiving the cell (as treatment).

[0297] In some embodiments, TCRs of the present disclosure are provided as TCR-T cells. In some embodiments, CARs of the present disclosure are provided asCAR-T cells. Any methods known in the art for modifying T cells to express a TCR or CAR can be employed to generate the TCR-T or CAR-T cells of the present disclosure.

[0298] The cells expressing a peptide binding moiety (e.g., TCRs and CARs) of the present disclosure may also contain one or more additional genes. The additional genes can be used to increase the effector function of the cells expressing the peptide binding moiety (e.g., TCRs and CARs). Non-limiting examples of classes of additional genes include (a) a second targeting moiety, such as antibodies, including fragments thereof and bispecific antibodies (e.g., bispecific T cell engagers (BiTEs)), (b) secretable cytokines (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18), (c) membrane bound cytokines (e.g., IL-15), (d) chimeric cytokine receptors (e.g., IL-2 / IL-7, IL-4 / IL-7), (e) constitutive active cytokine receptors (e.g., C7R), (f) dominant negative receptors (DNR; e.g., TGFRII DNR), (g) ligands of co-stimulatory molecules (e.g., CD80, 4- 1BBL), (h) nuclear factor of activated T cells (NFATs) (e.g., NFATcl, NFATc2, NFATc3, NFATc4, and NFAT5), or (j) suicide genes (e.g., CD20, truncated EGFR or HER2, inducible caspase 9 molecules). In some embodiments, the cells expressing a peptide binding moiety (e.g., TCRs and CARs) of the present disclosure may express a second targeting moiety that targets to the liver or to another known liver cancer antigen.Pharmaceutical Compositions, Dosage Forms, and Administration

[0299] In a further aspect, the disclosure provides a pharmaceutical composition comprising a peptide, a peptide-based molecule (such as a complex (e.g., peptide-MHC (pMHC) complex), fusion protein, or conjugate comprising the peptide), a nucleic acid molecule, a vector, a cell, or a peptide binding moiety of the disclosure together with a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical compositions of the disclosure may be in any suitable form (depending upon the desired method of administering to a patient). Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et al., Blood. 2009; 114(3):535-46.

[0300] The pharmaceutical compositions may comprise the peptides or peptide- based molecules of the disclosure either in the free form or in the form of apharmaceutically acceptable salt. The term “pharmaceutically acceptable salt” as used herein refers to a derivative of the disclosed peptides wherein the peptide is modified by making acid or base salts of the agent. For example, acid salts are prepared from the free base (typically wherein the neutral form of the drug has a neutral — NH2 group) involving reaction with a suitable acid. Suitable acids for preparing acid salts include both organic acids, e.g., acetic acid, benzoic acid, citric acid, propionic acid, glycolic acid, trifluoroacetic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and the like. Conversely, preparation of basic salts of acid moieties which may be present on a peptide are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like.

[0301] Compositions of the disclosure may comprise multiple peptides, e.g., 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides as described herein (e.g., SEQ ID NO: 1-11). In some embodiments, the compositions of the disclosure may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 amino acid sequences selected from SEQ ID NO: 1-11, or a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0302] In some embodiments, the peptides or peptide-based molecules may be present in a solution at a concentration of about 1 pg / rnL to 50 mg / mL, for example, about 0.1 mg / mL to 10 mg / mL, about 0.2 mg / mL to 5 mg / mL, about 0.5 mg / mL to 8 mg / mL, about 0.8 mg / mL to 12 mg / mL, about 1 mg / mL to 15 mg / mL, about 2 mg / mL to 20 mg / mL, or about 5 mg / mL to 25 mg / mL, or about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 1.75 mg / mL, 2 mg / mL, 2.25 mg / mL, 2. 5 mg / mL, 2.75 mg / mL, 3 mg / mL, 3.25 mg / mL, 3. 5 mg / mL, 3.75 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, or 20 mg / mL

[0303] The pharmaceutical composition may be adapted for administration by any appropriate route such as, e.g., parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes.

[0304] Such compositions may be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0305] In addition, disclosed herein are pharmaceutical dosage forms comprising the peptides, peptide-based molecules (such as complexes (e.g., peptide- MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide(s)), nucleic acid molecules, vectors, cells, or binding moieties of the disclosure.

[0306] Pharmaceutical compositions based on the peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide(s)), nucleic acid molecules, vectors, cells, or binding moieties disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. The peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide(s)), nucleic acid molecules, vectors, cells, or binding moieties may be formulated for administration by, for example, injection, inhalation, or insulation (either through the mouth or the nose) or by oral, buccal, parenteral or rectal administration, or by administration directly to an organ or tissue.

[0307] The pharmaceutical compositions can be formulated for a variety of modes of administration, including systemic, topical, or localized administration. Techniques and formulations can be found in, for example, Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For the purposes of injection, the pharmaceutical compositions can be formulated in liquid solutions, preferably in physiologicallycompatible buffers, such as Hank’s solution or Ringer’s solution. In addition, the pharmaceutical compositions may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms of the pharmaceutical composition are also suitable.

[0308] In some embodiments, the pharmaceutical compositions of the present disclosure may be lyophilized. As a non-limiting example, the obtained lyophilizate can be reconstituted into a hydrous composition by adding a hydrous solvent. In some embodiments, the hydrous composition may be able to be directly administered parenterally to a patient. Therefore, a further embodiment of the present disclosure is a hydrous pharmaceutical composition, obtainable via reconstitution of the lyophilizate with a hydrous solvent.

[0309] In some embodiments, the pharmaceutical composition disclosed herein may comprise a lyophilized formulation. As a non-limiting example, the lyophilization formulation may comprise peptides of the disclosure, mannitol, and / or TWEEN 80®. As another non-limiting example, the lyophilization formulation may comprise the peptides disclosed herein, mannitol and poloxamer 188. In some embodiments, the pharmaceutical composition may comprise a lyophilization formulation comprising a reconstituted-liquid composition.

[0310] In some embodiments, pharmaceutical compositions of the present disclosure may provide a formulation with an enhanced solubility and / or moistening of the lyophilizate over previously known compositions. As a non-limiting example, enhanced solubility and / or moistening of the lyophilizate may be achieved using an appropriate composition of excipients. In this way, pharmaceutical compositions of the present disclosure comprising peptides of SEQ ID NO: 1 to 54 and variants thereof may be developed to show a desired shelf stability at (e.g., at -20° C, +5° C, or +25° C) and can be easily resolubilized such that the lyophilizate can be completely dissolved through the use of a buffer or other excipients from seconds up to two or more minutes, with or without the use of an of ultrasonic homogenizer. Furthermore, the composition can be easily provided to a patient in need of treatment via any appropriate delivery route disclosed herein, e.g., parenteral (including subcutaneous, intramuscular, orintravenous), enteral (including oral or rectal), inhalation, or intranasal routes. As a nonlimiting example, the pH-value of the resulting solution may be between pH 2.7 and pH 9.

[0311] For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g. sodium lauryl sulfate). The tablets can also be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

[0312] The pharmaceutical compositions can be formulated for parenteral administration by injection, e.g. by bolus injection or continuous infusion. Formulations for injection can be presented in a unit dosage form, e.g. in ampoules or in multi-dose containers, with an optionally added preservative. The pharmaceutical compositions can further be formulated as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain other agents including suspending, stabilizing and / or dispersing agents.

[0313] Additionally, the pharmaceutical compositions can also be formulated as a depot preparation. These long-acting formulations can be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection.Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g. as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Other suitable delivery systems include microspheres, which offer the possibility of local noninvasive delivery of drugs over an extended period of time. This technology can include microspheres having a precapillary size, which can be injected via a coronary catheter into any selected part of an organ without causing inflammation or ischemia. The administered therapeutic is men slowly released from the microspheres and absorbed by the surrounding cells present in the selected tissue.

[0314] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts, and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration can occur using nasal sprays or suppositories. For topical administration, the vector particles described herein can be formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can also be used locally to treat an injury or inflammation in order to accelerate healing.

[0315] Pharmaceutical forms suitable for injectable use can include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid. It must be stable under the conditions of manufacture and certain storage parameters (e.g. refrigeration and freezing) and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0316] If formulations disclosed herein are used as a therapeutic to boost an immune response in a subject, a therapeutic agent can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0317] A carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0318] Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

[0319] Upon formulation, solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but slow-release capsules or microparticles and microspheres and the like can also be employed.

[0320] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intratumorally, intramuscular, subcutaneous and intraperitoneal administration. In this context, sterile aqueous media that can beemployed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion.

[0321] The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. For example, a subject may be administered peptides, peptide-based molecules (such as complexes (e.g., peptide- MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide(s)), nucleic acid molecules, vectors, cells, or binding moieties described herein on a daily or weekly basis for a time period or on a monthly, bi-yearly or yearly basis depending on need or a condition in the subject (e.g. cancer).

[0322] In addition to the compounds formulated for parenteral administration, such as intravenous, intratumorally, intradermal or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; biodegradable and any other form currently used.

[0323] One may also use intranasal or inhalable solutions or sprays, aerosols or inhalants. Nasal solutions can be aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions can be prepared so that they are similar in many respects to nasal secretions. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation. Various commercial nasal preparations are known and can include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.

[0324] Oral formulations can include excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. In certain defined embodiments, oral pharmaceutical compositions willinclude an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0325] The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of Wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.

[0326] Further embodiments disclosed herein can concern kits for use with methods and compositions. Kits can also include a suitable container, for example, vials, tubes, mini- or microfiige tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide(s)), nucleic acid molecules, vectors, cells, or binding moieties and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Optionally, one or more additional active agents may be needed for compositions described.

[0327] Dose ranges and frequency of administration can vary depending on the nature of the composition and the medical condition as well as parameters of a specific patient and the route of administration used. A dose can also depend on the subject in which it is being administered. For example, a lower dose may be required if the subject is juvenile, and a higher dose may be required if the subject is an adult human subject. In certain embodiments, a more accurate dose can depend on the weight of the subject. A suitable, non-limiting example of a dosage of a pharmaceutical composition containing the same disclosed herein may vary depending upon the age and the size of a subject to be administered, target disease, the purpose of the treatment, conditions, route of administration, and the like. Non-limiting examples of suitable dosages include, e.g., 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0328] Compositions may include administration to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, via a catheter, via a lavage, in a cream, or in a lipid composition.

[0329] Certain additional agents used in the combination therapies can be formulated and administered by any means known in the art.

[0330] Compositions as disclosed herein can also include adjuvants such as aluminum salts and other mineral adjuvants, tensoactive agents, bacterial derivatives, vehicles and cytokines. Adjuvants can also have antagonizing immunomodulating properties. For example, adjuvants can stimulate Thl or Th2 immunity. Compositions and methods as disclosed herein can also include adjuvant therapy.

[0331] The peptides or peptides-based molecules of the disclosure may be provided in the form of a vaccine composition. The vaccine composition may be useful for the treatment or prevention of prostate cancer. As will be appreciated, vaccines may take several forms (see, e.g., Schlom, J Natl Cancer Inst. 2012; 104(8): 599-613; Salgaller, Cancer Res. 1996; 56(20):4749-57 and Marchand, Int J Cancer. 1999; 80(2):219-30). The vaccine composition may include additional peptides or peptides- based molecules such that the peptide or peptides-based molecule of the disclosure is one of a mixture of peptides or peptides-based molecules. Adjuvants may be added to the vaccine composition to augment the immune response. In particular for peptide- containing vaccines compositions of the disclosure, pharmaceutically acceptable adjuvants include, but are not limited to, aluminum salts, Amplivax, AS 15, Aquila’s QS21 stimulon, AsA404 (DMXAA), beta-glucan, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, hniquimod, hnuFactEVlP321, IS Patch, ISS, 1018 ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, poly-ICLC, PepTel®, Pam3Cys, PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF- 17D, VEGF trap, R848, and / or vadimezan.

[0332] Alternatively, the vaccine composition may take the form of an APC displaying the peptide of the disclosure in complex with MHC. Preferably the APC is an immune cell, more preferably a dendritic cell or a B cell. The peptide may be pulsed onto the surface of the cell (Thumer, J Exp Med. 1999; 190(11): 1669-78), or nucleic acid encoding for the peptide of the disclosure may be introduced into dendritic cells or B cells (e.g., by electroporation. Van Tendeloo, Blood. 2001; 98(l):49-56).

[0333] The pharmaceutical compositions of the disclosure may be administered directly into the patient, into the affected organ or systemically i.d., i.m., s.c., i.p. and i.v., or applied ex vivo to cells derived from the patient or a human cell line which are subsequently administered to the patient, or used in vitro to select a subpopulation of immune cells derived from the patient, which are then re-administered to the patient. If the nucleic acid is administered to cells in vitro, it may be useful for the cells to be transfected so as to co-express immune-stimulating cytokines, such as interleukin-2. The peptide or peptide-based molecule may be substantially pure, or combined with an immune-stimulating adjuvant or used in combination with immune-stimulatory cytokines, or be administered with a suitable delivery system, e.g., liposomes, viral particles, VLPs. The peptide or peptide-based molecule may also be conjugated to a suitable carrier such as keyhole limpet haemocyanin (KLH) or mannan (see, e.g., WO 95 / 18145 and Longenecker et al., 1993).

[0334] In some embodiments, the peptide-containing compositions described herein further comprise an accessory molecule which can modulate a survival or an activity of TCR-expressing cells.

[0335] Non-limiting examples of useful accessory molecules include, e.g., an anti-CD28 antibody, an anti-CD80 (B7.1) antibody, an anti-CD86 (B7.2) antibody, an anti-anti-CD3 antibody, an anti-CD2 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD47 antibody, and functional derivatives, mutants and fragments thereof.

[0336] Accessory molecules used in the peptide-containing compositions described herein include molecules that provide a signal which, in addition to the primary signal provided by, for instance, binding of a TCR / CD3 complex with a pMHC complex, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.

[0337] The accessory molecule can be, for example, an inhibitory or stimulatory antibody, a peptide ligand, a costimulatory peptide, a cytokine, etc. Nonlimiting examples of accessory molecules that can be used in the peptide-containingcompositions described herein include, e.g., CD7, B7.1 (CD80), B7.2 (CD86), PD-L1 , PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible co stimulatory ligand (ICOS- L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, FIVEM, lymphotoxin 0 receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds to B7-H3 as well as antibodies that specifically bind to CD27, CD28, B7.1 (CD80), B7.2 (CD86), 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD3, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0338] Additional non-limiting examples of accessory molecules include, e.g., TNF / TNF family members (e.g., OX40L, ICOSL, FASL, LTA, LTB TRAIL, CD153, TNFSF9, RANKL, TWEAK, TNFSF13, TNFSF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG, CD70); members of the Immunoglobulin superfamily (e.g., VISTA, PD1, PD-L1 , PD-L2, B71 , B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T cell receptor chains, ICOS, ICOS ligand, HHLA2, butyrophilms, BTLA, B7-H3, B7-H4, CD3, CD79a, CD79b, IgSF CAMS (including CD2, CD58, CD48, CD150, CD229, CD244, ICAM-1), Leukocyte immunoglobulin like receptors (LILR), killer cell immunoglobulin like receptors (KIR)), lectin superfamily members, selectins, cytokines / chemokine and cytokine / chemokine receptors, growth factors and growth factor receptors), adhesion molecules (integrins, fibronectins, cadherins), or ectodomains of multi-span integral membrane proteins, or antibodies directed to any of these molecules.

[0339] In some embodiments, the peptide-containing compositions described herein further comprise a cytotoxic agent. In one specific embodiment, the cytotoxic agent is a toxin or a radioactive isotope (e.g., a radioconjugate) or a suicide gene. Nonlimiting examples of toxins which can be used in the peptide-containing compositions described herein include, e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments, mutants or derivatives thereof. Enzymatically active toxins and fragments thereof that can be used include, for example, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, a-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Non-limiting examples of suicide genes include, e.g., thymidine kinase, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, P-galactosidase, hepatic cytochrome P450- 2B1, linamarase, horseradish peroxidase, and carboxypeptidase.

[0340] Methods for introducing polypeptide or polynucleotides of the present disclosure into a cell or subject can include, for example, vector delivery, particle- mediated delivery, exosome-mediated delivery, lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediated delivery, or implantable-device-mediated delivery. In some embodiments, a nucleic acid or protein can be introduced into a cell or subject in a carrier such as a poly(lactic acid) (PLA) microsphere, a poly(D,L-lactic-coglycolic- acid) (PLGA) microsphere, a liposome, a micelle, an inverse micelle, a lipid cochleate, or a lipid microtubule.

[0341] The use of nanoparticles to deliver the polypeptide or polynucleotides compositions of the disclosure is contemplated herein. Exemplary nanoparticles include, but are not limited to, polymeric nanoparticles, inorganic nanoparticles, liposomes, lipid nanoparticles (LNP), an immune stimulating complex (ISCOM), a virus-like particle (VLP), or a self-assembling protein. The nanoparticles may be calcium phosphate nanoparticles, silicon nanoparticles or gold nanoparticles. For examples, the polymeric nanoparticles may comprise one or more synthetic polymers, such as poly(d,l-lactide-co-glycolide) (PLG), poly(d,l-lactic-coglycolic acid) (PLGA), poly(g-glutamic acid) (g-PGA), poly(ethylene glycol) (PEG), or polystyrene or one or more natural polymers such as a polysaccharide, for example pullulan, alginate, inulin, and chitosan. The use of a polymeric nanoparticles may be advantageous due to the properties of the polymers that may be include in the nanoparticle. For instance, the natural and synthetic polymers recited above may have good biocompatibility and biodegradability, a non-toxic nature and / or the ability to be manipulated into desired shapes and sizes. The polymeric nanoparticle may also form hydrogel nanoparticles, hydrophilic three-dimensional polymer networks with favorable properties including flexible mesh size, large surface area for multivalent conjugation, high water content,and high loading capacity for antigens. Polymers such as Poly(L-lactic acid) (PLA), PLGA, PEG, and polysaccharides are suitable for forming hydrogel nanoparticles. Inorganic nanoparticles typically have a rigid structure and comprise a shell in which an antigen is encapsulated or a core to which the antigen may be covalently attached. The core may comprise one or more atoms such as gold (Au), silver (Ag), copper (Cu) atoms, Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Pd or Au / Ag / Cu / Pd or calcium phosphate (CaP).

[0342] Other molecules suitable for complexing with the polypeptide or polynucleotides of the disclosure include cationic molecules, such as, polyamidoamine , dendritic polylysine, polyethylene irinine or polypropylene imine, polylysine, chitosan, DNA-gelatin coarcervates, DEAE dextran, dendrimers, or polyethylenimine (PEI).

[0343] In some embodiments, antibodies of the present disclosure can be conjugated to nanoparticles. Nanoparticles that may be used for conjugation with antibodies of the present disclosure include but not are limited to PEGylated liposomes, poly(d,l-lactide-co-glycolide) / montmorillonite nanoparticles (PLGA / MMT NPs), poly(lactide-co-glycolide) (PLGA) nanoparticles, poly-(malic acid)-based nanoparticles, chitosan-shelled nanoparticles, carbon nanotubes, and other inorganic nanoparticles (such as nanoparticles made of magnesium-aluminium layered double hydroxides with disuccinimidyl carbonate (DSC), and TiC nanoparticles). Nanoparticles can be developed and conjugated to an antibody contained in a pharmaceutical composition for targeting cancer cells.Treatment Methods

[0344] Compositions of the present disclosure, including the peptides, peptide- based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide(s)), nucleic acid molecules, vectors, cells, or binding moieties of the disclosure, may be used in the prophylaxis and / or treatment of prostate cancer.

[0345] In one aspect, disclosed herein is a method for modulating an activity, proliferation or survival of a cell comprising a TCR, comprising contacting the cell with a composition (e.g., peptide, complex (e.g., pMHC complex), fusion protein, or conjugate) of the disclosure.

[0346] In some embodiments, the cell is a lymphocyte such as, e.g., a T-cell (e.g., a CD4+ T-cell or a CD8+ T-cell). In some embodiments, the target T cell is a CD4+ T cell such as, e.g., a helper T cell (e.g., a Thl, Th2, or Thl7 cell) or a CD4+ / CD25+ / FOXP3+ regulatory T (Treg) cell. In some cases, the target T cell is a CD8+ T cell such as, e.g., a cytotoxic T cell. In some cases, the target T cell is a memory T cell, which can be a CD4+ T cell or a CD8+ T cell, where memory T cells are generally CD45RO+. In some cases, the target T cell is an NK-T cell.

[0347] In some embodiments, the contacting is ex vivo. In some embodiments, the contacting is in vivo in a subject (e.g., human).

[0348] In some embodiments, the cell is a mammalian cell (e.g., a human cell).

[0349] In some embodiments, e.g., where the target T cell is a CD8+ T cell, the peptide is presented by a class I MHC polypeptide. In some embodiments, e.g., where the target T cell is a CD4+ T cell, the peptide is presented by class II MHC polypeptides.

[0350] The interaction of a T cell with the peptides described herein can result in, e.g., activation, induction of anergy, or death of a T cell that occurs when the TCR of the T cell is bound by a TCR-binding molecule (e.g., pMHC complex). “Activation of a T cell” refers to induction of signal transduction pathways in the T cell resulting in production of cellular products (e.g., interleukin-2) by that T cell. “Anergy” refers to the diminished reactivity by a T cell to an antigen. Activation and anergy can be measured by, for example, measuring the amount of IL-2 produced by a T cell after a pMHC complex has bound to the TCR. Anergic cells will have decreased IL-2 production when compared with stimulated T cells. Another method for measuring the diminished activity of anergic T cells includes measuring intracellular and / orextracellular calcium mobilization by a T cell upon engagement of its TCR’s. “T cell death” refers to the permanent cessation of substantially all functions of the T cell.

[0351] In another aspect, provided herein is a method of inducing an immune response against a prostate cancer cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition (e.g., one or more peptides, complexes (e.g., pMHC complex), fusion proteins, conjugates, nucleic acid molecules, vectors, cells, or binding moieties) of the present disclosure.

[0352] In certain embodiments, generating an immune response comprises an increase in target antigen-specific cytotoxic T lymphocytes (CTL) activity of about 1.5- fold to 20-fold, or more fold in a subject administered a composition of the disclosure as compared to a control. In certain embodiments, generating an immune response comprises an increase in target-specific CTL activity of about 1 ,5-fold to 20-fold, or more fold in a subject administered the composition of the disclosure as compared to a control. In a further embodiment, generating an immune response that comprises an increase in target antigen-specific cell-mediated immunity activity as measured by ELISpot assays measuring cytokine secretion, such as interferon-gamma (IFN-y), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-a), or other cytokines, of about 1.5-fold to 20-fold, or more fold as compared to a control.

[0353] In a further embodiment, generating an immune response comprises an increase in target- specific antibody production of between 1.5-fold and 5-fold in a subject administered the composition of the disclosure as compared to an appropriate control. In another embodiment, generating an immune response comprises an increase in target- specific antibody production of about 1.5-fold to 20-fold, or more fold in a subject administered the composition of the disclosure as compared to a control.

[0354] T cell activation may be determined, e.g., by measuring changes in the level of expression of cytokines and / or T cell activation markers, and / or the induction of antigen-specific proliferating cells. Techniques known to those of skill in the art, including, but not limited to, immunoprecipitation followed by western blot analysis,ELISAs, flow cytometry, northern blot analysis, and RT-PCR can be used to measure the expression cytokines and T cell activation markers. Cytokine release may be measured by measuring secretion of cytokines including but not limited to Interleukin- 2 (IL-2), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin- 12 (IL-12), Interleukin- 16 (IL-16), PDGF, TGF-a, TGF-0, TNF-a, TNF- , GCSF, GM-CSF, MCSF, IFN-a, IFN-P, IFN-y, TFN-y, IGF-I, and IGF-II.

[0355] T cell modulation may also be evaluated by measuring, e.g., proliferation by, e.g.,3H-thymidine incorporation, trypan blue cell counts, and fluorescence activated cell sorting (FACS).

[0356] The anti-tumor responses of T cells may be determined in xenograft tumor models. Tumors may be established using any human cancer cell line expressing the relevant tumor-associated antigen. To establish xenograft tumor models, about 5xl06viable cells, may be injected, e.g., subcutaneously into nude athymic mice using for example Matrigel (Becton Dickinson). The endpoint of the xenograft tumor models can be determined based on the size of the tumors, weight of animals, survival time and histochemical and histopathological examination of the cancer, using methods known to one skilled in the art.

[0357] In a related aspect, disclosed herein is a method of treating prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition (e.g., one or more peptides, complexes (e.g., pMHC complex), fusion proteins, conjugates, nucleic acid molecules, vectors, cells, binding moieties) of the present disclosure.

[0358] In a related aspect, disclosed herein is a method of reducing the likelihood of developing prostate cancer disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition (e.g., one or more peptides, complexes (e.g., pMHC complex), fusion proteins, conjugates, nucleic acid molecules, vectors, a cells, binding moieties) of the present disclosure.

[0359] It is contemplated that when used to treat various diseases, the compositions and methods can be combined with other therapeutic agents suitable for the same or similar diseases. Also, two or more embodiments described herein may be also co-administered to generate additive or synergistic effects. When co-administered with a second therapeutic agent, the embodiment described herein and the second therapeutic agent may be simultaneously or sequentially (in any order). Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0360] The compositions and methods described herein can be combined with other immunomodulatory treatments such as, e.g., therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 41BB, 0X40, etc.). The inhibitory treatments described herein can be also combined with other treatments that possess the ability to modulate NKT function or stability, including but not limited to CD Id, CD ld- fusion proteins, CD Id dimers or larger polymers of CD Id either unloaded or loaded with antigens, CD 1 d-chimeric antigen receptors (CDld-CAR), or any other of the five known CD1 isomers existing in humans (CD la, CD lb, CDlc, CDle), in any of the aforementioned forms or formulations, alone or in combination with each other or other agents.

[0361] Therapeutic methods described herein can be combined with additional immunotherapies and therapies. For example, when used for treating cancer, NKT cells described herein can be used in combination with cancer therapies, such as, e.g., surgery, radiotherapy, chemotherapy or combinations thereof, depending on type of the tumor, patient condition, other health issues, and a variety of factors. In certain aspects, other therapeutic agents useful for combination cancer therapy with the inhibitors described herein include anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including, e.g., TNP-470, platelet factor 4, thrombospondin-1 , tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor P, interferon-a, soluble KDR and FLT-1receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000). In some embodiments, the inhibitors described herein can be used in combination with a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).

[0362] Non-limiting examples of chemotherapeutic compounds which can be used in combination treatments include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0363] These chemotherapeutic compounds may be categorized by their mechanism of action into, for example, following groups: anti-metabolites / anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK- 506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.Kits

[0364] The present disclosure further comprises a kit which may comprise any of various compositions of the present disclosure, including the peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide(s)), nucleic acid molecules, vectors, cells, or binding moieties of the disclosure.

[0365] In one aspect, the present disclosure may include a kit comprising, for example: (a) a container that contains a pharmaceutical composition disclosed herein, for example, a pharmaceutical composition in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; and / or (c) optionally, instructions for (i) use of the solution or (ii) reconstitution and / or use of the lyophilized formulation.

[0366] In some embodiments, the kit may further comprise, for example, without limitation, one or more of (i) a buffer, (ii) a diluent, (iii) a filter, (iv) a needle, and / or (v) a syringe. As a non-limiting example, the container may be a bottle, a vial, a syringe or test tube. In some embodiments, the container may be a multi-use container. In some the pharmaceutical composition may be lyophilized.

[0367] Kits of the present disclosure may comprise a lyophilized formulation of the present disclosure in a suitable container and instructions for its reconstitution and / or use. Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The kit and / or container may contain instructions on or associated with the container that indicate directions for reconstitution of the lyophilized formulation and / or use of the kit. For example, the label may indicate that the lyophilized formulation is to be reconstituted to an appropriate peptide concentration. The label may indicate that the formulation is useful or intended for any route of administration disclosed herein, e.g., parenteral administration routes disclosed herein.

[0368] The container holding the formulation may be a multi-use vial, which may allow for repeat administrations (e.g., from 2-6 administrations) of the reconstituted formulation. The kit may further comprise a second container comprising a suitable diluent (e.g., sodium bicarbonate solution).

[0369] Upon mixing of the diluent and the lyophilized formulation, the final peptide concentration in the reconstituted formulation is reached. The kit may further include other materials desirable from a commercial and / or user standpoint, including, for example, without limitation, other buffers, diluents, filters, needles, syringes, and / or package inserts which may comprise, e.g., instructions for use.

[0370] Kits of the present disclosure may have a single container that contains the formulation of the pharmaceutical compositions according to the present disclosure with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds) or may have a distinct container for each component.

[0371] In some embodiments, kits of the disclosure may include a formulation of the disclosure packaged for use in combination with the co-administration of a second compound (such as adjuvants (e.g., GM-CSF, a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenesis agent or inhibitor, an apoptosis-inducing agent or a chelator) or a pharmaceutical composition thereof. The components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient. The components of the kit may be provided in one or more liquid solutions. A liquid solutions described herein may be an aqueous solution, for example, a sterile aqueous solution. The components of the kit may also be provided as solids, which may be converted into liquids such as by addition of suitable solvents, which may be provided in another distinct container.

[0372] The container of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid. When there is more than one component, the kit may contain a second vial or other container, which may allow for separate dosing. The kit may also contain another container for a pharmaceuticallyacceptable liquid. In some embodiments, a kit may contain an apparatus (e.g., one or more needles, syringes, eye droppers, pipettes, etc.), which may allow for administration of the agents of the disclosure that are components of the present kit.EXAMPLES

[0373] The present disclosure is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the disclosure may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the disclosure in spirit or in scope. The disclosure is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.Example 1. Generation of RNAseq databases from prostate cancer samples.RNA-Seq / WES Database GenerationGermline mutation calling / Whole-exome sequencing database generation

[0374] Germline mutations were called using the Sentieon Germline FASTQ to VCF (V.4.1.1) applet with the mark duplicates option. The Haplotyper algorithm was selected for mutation calling with the following inputs: extra BWA option -K 10000000, and extra GVCFtyper options — genotype model multinomial to match GATK4.1's default behavior.

[0375] For all called germline mutations, variant consequence was annotated using SnpEff (version 4.3t). SnpEff was also used to output wild-type and mutated version of each protein. Using a custom bash / PERL scripts the final database of 27mer peptides centered around the mutation position was created. For frameshifted peptides, the custom script included all sections of the frameshifted peptide that did not also occur in the wild-type version of the protein.RNA-sequencing database generation - RNAseq modulesCT and CT-like genes detection:

[0376] Expression of all genes in a sample was first quantified using kallisto and RNA-seq sequencing reads (FASTQ or BAM). A custom bash / PERL script was used to annotate CT and CT-like genes that were expressed in the sample. Protein sequences of the expressed CT and CT-like genes were then included in the final database by the same annotation script.Alternative splicing module:

[0377] The alternative splicing module of the workflow was a three-step process: first the raw RNAseq reads were aligned to the transcriptome reference using STAR aligner. Next rMATS software was run to detect all potential splicing aberrations (skipped exons, retained introns, alternative 3' and 5' splice sites, mutually exclusive exons). No filtering of events was performed based on FDR. Next, a SPLICIFY script was run to translate all splicing events detected in three frames considering adjacent upstream and downstream exons around each event in question. Three frame translation was used since it was not known if the frame was shifted by another splicing event that could potentially occur upstream of the event in question. Due to the splicing module and to accounting for upstream and downstream exons, peptides were detected that were encoded by some UTR sequences (if the splicing aberration occurred in one of the external exons, second exon or second to last exon).Gene fusion detection:

[0378] Gene fusion detection was performed by STAR-fusion algorithm with default parameters. After all gene fusions were identified, a custom bash / PERL script translated gene fusion nucleotide sequences into peptides in three frames using transeq software.HERVs / retrotransposon detection

[0379] Retrotransposon / HERV detection module was a two-step process. First the raw sequencing reads were mapped to repetitive sequences using SalmonTE software, default parameters, and the default database of consensus sequences that came with the software. Second, a custom script checked for expressed repetitive sequencesfrom the default SalmonTE database and those sequences were translated in six frames using transeq software.Noncoding / out-of-frame translation events / non-human peptide detection / RNA editing module:

[0380] This module is the most comprehensive of all. As a first step, because all sequencing was performed using stranded protocol, reverse strand FASTQ RNAseq reads were reversed using seqtk software (this step is optional for unstranded RNAseq sequencing protocol). Next, a kmer database of nucleotide sequences was created using KMC software with the following parameters -k33 -m64 -cil -tl6 -b -cslOOOOOOOOO and forward and reversed FASTQ files. Next, to keep the database size of this module small, any kmers that could be found in the published database of mTEC kmers (DOI: 10.1126 / scitranslmed.aau5516) were filtered. Next, kmers that survive filtering were put into contigs using abyss software and the following parameters: k=33 n=l c=l e=l E=0 1=33 m=32 q=0 t=0 d=0 j=l 6 s=34, allowing kmers to be put together only if 32 out of 33 nucleotides overlapped between a kmer and another kmer / contig. As the final step, all contigs and unitigs (kmers that did not overlap with any other kmers) were translated in three frames using transeq software.Final database creation:

[0381] The final database for a sample was created using a custom PERL / bash script that concatenates individual databases from each module and creates a custom header for each peptide / protein entry in the database. This allowed for the identification of relevant hits quickly and efficiently.Example 2. Characterization of the immunopeptidome of prostate cancer samples.

[0382] 173 primary prostate tumor tissue samples were de-identified prior to purchase from commercial vendors and processed as described below. In brief, primary prostate tumor samples were pulverized and lysed, prior to HLA affinity enrichment. Isolated HLA-peptides were analyzed by LC-MS / MS and raw mass spectrometry results were searched against a consolidated database of human UniProtKB (homo sapiens) with PEAKS DB search engine. On average, around 3000-5000 9mer length peptides were detected per sample.Example 3. Identification of peptides predicted to bind HLA using NetMHC pan 4.0 analysis.

[0383] The polymorphic nature of HLA-I has a profound effect on the epitopes presented on the tissue surface. Hence, DNA sequencing of prostate cancer samples was performed to determine the HLA genotype (Table 3), which was used to predict the binding of detected peptides by NetMHC pan 4.1.

[0384] The HLA-eluted peptides, which were isolated by the HLA affinity enrichments, were mixed with synthetic peptides (50 finol) containing heavy containing heavy Leucine (13C(6)15N(1), +7.0172) or heavy Valine (13C(5)15N(1), +6.0138) and analyzed by LC-MS / MS. The identity of the endogenous peptides was confirmed by comparing the elution and fragmentation profile with the corresponding heavy peptides. Furthermore, the abundance of endogenous peptides was estimated based on the intensity of heavy and endogenous peptides, using Avogadro’s number. For this calculation, a 50% sample loss was assumed during the sample preparation.

[0385] PEAKS Online (Peaks Online 11, Bioinformatics Solutions Inc.) was used for de novo-assisted database search with precursor mass tolerance of 8 ppm, and fragment ion tolerance of 0.02 Da. Enzyme selection was set to none, methionine oxidation, and cysteine carbamidomethylation were set as variable modifications with 3 maximum allowed modifications per peptide. The search was performed with a 5% false-discovery rate (FDR) at the peptide level, and the peptides were further filtered based on a -logP score of 20 (corresponding to 1% FDR).Table 3. Prostate lineage antigen target discovery by mass spec

[0386] Below are the methods used in the Examples described above.

[0387] Prostate Tissue Procurement. 173 primary prostate tumor tissues were cryopreserved until sample preparation.

[0388] Tissue Lysis, and HLA Affinity Enrichment. The tissues were pulverized using SPEX SamplePrep Freezer / Mill Dual-Chamber Cryogenix Grinder in liquid nitrogen, and lysed in ice-cold lysis buffer (1% NP-40, 150mM NaCl, 50mM Tris-HCl pH 8.0 and lO M EDTA pH 8.0) supplemented with HALT protease and phosphatase inhibitors and 0.2 mM iodoacetamide per 10 ml lysis buffer (Sigma-Aldrich, cat. A3221). The volume of lysis buffer was determined by the tissue weight (for example,5 ml lysis buffer for 0.5 g tissue). The pulverized tissue and lysis buffer were rotated for a period of time, for example, 1 hour at 4 °C followed by freezing the sample prior to the affinity enrichment. The tissue lysate was further disrupted by a brief period of sonication on ice and then cleared during a 30-minute centrifugation, for example, at 20,913g at 4 °C.

[0389] Anti-HLA Class I (W6 / 32) was conjugated to NHS-sepharose beads by overnight incubation in coupling buffer (0.2 M NaHCCh + 0.5 M NaCl pH 8.3) in 4 °C. The reaction was quenched with 0.1 M Tris-HCl pH 8.5, and the beads were washed with the same Tris-HCl solution and 0.1 M acetate buffer.

[0390] The pre-cleared tissue lysate was passed through a column packed with 1 ml of HLA Class-I beads bed under gravity. The column was subsequently washed with Seppro Dilution Buffer and 20mM Tris-HCl pH 8, and HLA-peptide complex was eluted with 0.1M glycine pH 2.7.

[0391] Sample Preparation for Mass Spectrometry. The glycine eluate was loaded onto the C18 Sep-Pak, followed by selective elution of peptides by 30% ACN / 0.1% TFA. The peptides were further cleaned up, and analyzed by nano-LC- MS / MS.

[0392] Liquid Chromatography with tandem mass spectrometry (LC-MS / MS). HLA peptides as described above were loaded onto a nanoViper Acclaim PepMaplOO C18 trap column (75 pm i.d. x 2 cm, 3 pm, 100 A) and were separated using a nanoViper Acclaim PepMap RSLC Cl 8 column (75 pm i.d. x 25 cm, 2 pm, 100 A) heated to 40 °C and retrofitted with a New Objective SilicaTip (7 cm) with a distal conductive coating at the inlet end of the emitter. The gradient was delivered by an EASY-nLC 1200 HPLC system at 300 nL / min. The following 120-minute elution gradient with mobile phase A (Water / 0.1% formic acid) and B (80% Acetonitrile / 0.1% formic acid) was used: 3% B at 3 min, linear to 35% B at 100 min, and linear to 45% B at 123 min. The peptides eluted from the column were ionized via Flex ion source at 1.9 kV and analyzed by the Thermo Fusion Lumos Tribrid mass spectrometer using Xcalibur 4.1.31.9. The data acquisition was performed in data-dependent mode, wheresurvey scans were carried out in the high field Orbitrap analyzer (range of m / z 300- 1500 at a resolution of 60,000) with the automatic gain control target of 4.0E5 and maximal ion fill time of 100 ms. The MS / MS analyses were performed by 1.2 m / z precursor ion isolation with the quadrupole, applying normalized HCD (higher-energy collisional dissociation) collision energy of 32%, and analysis of fragment ions in the Orbitrap at a resolution of 15,000. The dynamic exclusion window was set to 6 seconds, monoisotopic precursor selection (MIPS) to peptide, maximum injection time to 100 ms, and charge states unknown. +1-+4 charge states were included and the advanced peak determination was toggled on.

[0393] For FAIMS-enabled experiments, the settings were identical except the FAIMS device was placed between the nanoelectrospray source and the mass spectrometer. FAIMS separations were performed with the following settings: inner and outer electrode temperature was set to 100 °C (except where noted), FAIMS carrier gas flow of 5.0 L / min, asymmetric waveform with DV -5000 V, entrance plate voltage 250 V, and CV settling time of 25 ms. The FAIMS carrier gas was N2, and the ion separation gap is 1.5 mm. The noted CVs were applied to the FAIMS electrodes. For external stepping or single CV experiments, the selected CV was applied to all scans throughout the analysis. For internal CV stepping experiments, each of the selected CVs was applied to sequential survey scans and MS / MS cycles (1 s); the MS / MS CV was always paired with the appropriate CV from the corresponding survey scan.

[0394] HLA Genotyping for Patient Samples. For sample preparation and sequencing, DNA sample quantity was determined by fluorescence and quality was assessed by running 25 ng of sample on a 1% pre-cast agarose gel. Samples with high molecular weight gDNA with a majority of the DNA fragments greater than 20 kb and a concentration of no less than 10 ng / ul passed the quality assessment. The DNA samples were normalized to 10 ng / ul and 50 ng was used to amplify the full-length HLA amplicons. The targets were amplified in three pools of variant tolerant primers optimized for similar binding temperatures and PCR conditions with LA Taq DNA Polymerase. The resulting amplicon pools were combined equimolarly as determined by automated capillary electrophoresis and fluorescence. DNA libraries were prepared for Illumina-based sequencing with a custom NEB kit. The amplicons wereenzymatically fragmented to a mean insert size of 250 bp and universal adapters were ligated onto the DNA fragments. Unique 10 base pair barcode sequences were added to the DNA fragments during PCR with NEBNext Ultra II Q5 Master Mix to facilitate highly multiplexed sequencing. The samples were pooled and sequenced using 150 base pair paired-end sequencing on an Illumina Nextseq 500.

[0395] For data analysis, upon completion of sequencing, raw data from each Illumina Nextseq run was gathered in local buffer storage and uploaded to a local high- performance computing platform for automated analysis. The FASTQ-formatted reads were converted from the BCL files and assigned to samples identified by specific barcodes using the bcl2fastq conversion software (Illumina Inc., San Diego, CA). All the reads in sample-specific FASTQ files were subjected to HLA typing analysis using an updated version of PHLAT program with the reference sequences consisting of GRCh38 genomic sequences and HLA type reference sequences in the IPD- IMGT / HLA database v3.30.0.

[0396] RNA sequencing. Total RNA was extracted from prostate cancer tissue using MagMAX kit. Strand-specific RNA-seq libraries were prepared from 1 pg RNA using KAPA stranded mRNA-Seq Kit (KAPA Biosystems). Twelve-cycle PCR was performed to amplify libraries. The amplified libraries were size-selected at 400-600 bp using PippinHT. Sequencing was performed on Illumina HiSeq®2500 (Illumina) by multiplexed paired-read run with 2X100 cycles.Example 4. Confirmation of HLA Peptide Loading by Cell Pulsing

[0397] Select KLK3-derived peptides were tested for loading to relevant HLAs identified in Table 3. KLK3159-169 (SEQ ID NO: 1), KLK348-56 (SEQ ID NO: 2), KLK3241-249 (SEQ ID NO: 4), and KLK3?9-89 (SEQ ID NO: 7) peptides were assessed for loading on 3T3 cells engineered to express human 2 microglobulin (B2M) and HLA-A11 (3T3 / B2M / HLA-All:01), HLA-A03 (3T3 / B2M / HLA-A03:01) or HLA- B15 (3T3 / B2M / HLA-B 15:01) and / or on HLA-A02 expressing T2 cells. Cells were pulsed by resuspending cells in AIM V medium (Gibco. Cat#31035-025) at a density of 1x106cells / ml followed by the addition of 10 pg / ml human b2 microglobulin (hB2M; EMD Millipore Cat#475828) and 100 pg / ml peptide. Cells were thenincubated overnight at 37°C. Peptide pulsed cells were then harvested and plated in staining buffer (PBS, without Calcium and Magnesium (Coming, Ref # 21-031-CV) + 2% FBS (Seradigm, Lot#238B15) at a density of 200,000 cells per well in a 96 well V- Bottom plate. Cells were incubated with three-fold serial dilutions (1.7 pM - 100 nM) with a pan HLA Class I antibody (Novus, Cat# DDX0250P-100) or an HLA-A2 specific antibody (REGN4262) for 30 mins at 4°C, washed once in staining buffer, and incubated with an Alexa-647 conjugated secondary antibody (Jackson ImmunoResearch, Cat # 115-606-071) at 5 ug / ml for 30 mins at 4°C. Cells were again washed once in staining buffer and stained with viability dye (CellTrace™ Violet Cell Proliferation Kit, Thermo-Fisher Cat# C34557) for 30 mins at 4°C. Lastly cells were washed once in staining buffer and fixed using a 50% solution of BD Cytofix (BD, Cat # 554655). Samples were run and analyzed on an intellicyt iQue flow cytometer to calculate mean fluorescence intensity (MFI) on live cells. MFI values were plotted in Graphpad Prism. The secondary antibody alone (i.e. no primary antibody) for each dose-response curve is also included in the analysis as a continuation of the three-fold serial dilution and was represented as the lowest dose. The signal to noise (S / N) was calculated by dividing maximum MFI on the pulsed cells by the maximum MFI on unpulsed cells (Table 4). Peptides were determined to load if the S / N from pulsed cells / unpulsed cells was greater than 1.3. The data demonstrates that KLK3159-169 (SEQ ID NO: 1) loaded on HLA-A11:01 expressing cells; KLK348-56 (SEQ ID NO: 2) loaded on HLA-Al l:01 and HLA-A03:01 expressing cells; KLK3241-249 (SEQ ID NO: 4) loaded on HLA-A03:01 and HLA-B15:01 expressing cells; and KLK3?9-89 (SEQ ID NO: 7) loaded on HLA-A2:01 expressing cells.Table 4: Cell binding S / N for peptide pulsed onto 3T3 / B2M / HLA-A11:O1, 3T3 / B2M / HLA-A03:01, 3T3 / B2M / HLA-B 15:01 or T2 cells.Example 5. Determination of MHC binding capacity by UV-ligand exchange

[0398] Candidate peptides according to the present disclosure are tested for their MHC binding capacity (affinity). The individual peptide-MHC (pMHC) complexes are produced by UV-ligand exchange. A UV-sensitive peptide is cleaved upon UV-irradiation and exchanged with the peptide of interest. Peptide candidates that effectively bind and stabilize the peptide-receptive MHC molecules prevent dissociation of the MHC complexes. To determine the yield of the exchange reaction, an ELISA is performed based on the detection of the light chain (02m) of stabilized MHC complexes. Briefly, 96-well plates are coated with streptavidin, washed, and blocked. Refolded HLA-A monomers serve as standards, covering a pre-determined concentration range. Peptide-MHC monomers of the UV-exchange reaction are diluted in blocking buffer. Samples are incubated, washed, incubated with HRP conjugated anti- 02m, washed again and detected with a chromogenic substrate solution that is stopped per the manufacturer’s protocol. Absorption is measured, for example, at 450 nm. Candidate peptides that show a high exchange yield are preferred for generation and production of antibodies or fragments thereof, and / or T cell receptors or fragments thereof. Candidate peptides demonstrate avidity to the MHC molecules and prevent dissociation of the MHC complexes.Example 6. Preparation of peptide-MHC (pMHC) complexes.

[0399] This example relates to a method for the preparation of soluble recombinant HLA loaded with a prostate lineage antigen-derived peptide.

[0400] Class I HLA molecules (HLA-heavy chain and HLA light-chain (02m)) are expressed separately in E. coli as inclusion bodies using suitable expression vectors. HLA-heavy chain additionally comprises a C-terminal biotinylation tag which replaces, for example, the transmembrane and / or cytoplasmic domains. E. coli cells are lysed and inclusion bodies processed to approximately 80% purity.

[0401] Inclusion bodies of 02m and heavy chain are denatured separately in denaturation buffer. Refolding buffer is prepared. Synthetic peptides are dissolved to a final concentration and added to the refold buffer. Then 2m followed by heavy chain are added. Refolding is performed to completion.

[0402] The refold mixture is then dialyzed. The protein solution is subsequently filtered through a filter and loaded onto an exchange column (pre-equilibrated). Protein is eluted such as by way of a linear salt gradient using additional purifier. HLA-peptide complex is eluted, and peak fractions are collected. A cocktail of protease inhibitors is added and the fractions are chilled on ice.

[0403] Biotin-tagged pHLA molecules are buffer exchanged into a buffer using a fast desalting column equilibrated in the same buffer. Upon elution, the proteincontaining fractions are chilled on ice and protease inhibitor cocktail is added. Biotinylation reagents are then added. The mixture is then allowed to incubate.

[0404] The biotinylated pHLA molecules are further purified, for example, by gel filtration chromatography using purifier with a column pre-equilibrated with filtered PBS. The biotinylated pHLA mixture is concentrated to a final volume, loaded onto the column and developed. Biotinylated pHLA molecules elute, e.g., as a single peak. Fractions containing protein are pooled, chilled on ice, and protease inhibitor cocktail is added. Protein concentration is determined and aliquots of biotinylated pHLA molecules are stored frozen.

[0405] Such peptide-MHC (pMHC) complexes are used in soluble form or immobilized through their C-terminal biotin moiety on to a solid support, to be used forthe detection of T cells and T cell receptors which bind the peptide-MHC complex. For example, such complexes are used in panning phage libraries, performing ELISA assays and / or preparing sensor chips for measurements of affinity and binding kinetics.Example 7. Identification of T cell receptors (TCRs) that bind to pMHC complexes.

[0406] Antigen binding T cell receptors (TCRs) are obtained using peptides disclosed herein to pan a TCR phage library. The library is constructed using a- and p- chain sequences obtained from a natural repertoire. The random combination of these a- and - chain sequences occurs during library creation, thereby producing a nonnatural repertoire of a / p chain combinations.

[0407] TCRs obtained from the library are assessed by enzyme-linked immunoassay (ELISA) to confirm specific antigen recognition. ELISA plates are coated with streptavidin and incubated with the biotinylated peptide-HLA complex. TCR-bearing phage clones are added to each well and detection is carried out using an HRP antibody conjugate. Bound antibody is detected using a peroxidase Substrate System. An absence of binding to alternative peptide-HLA complexes indicated that the TCR is not highly cross reactive.

[0408] Further confirmation that TCRs can bind a peptide-HLA complex of the disclosure is obtained by surface plasmon resonance (SPR) using isolated TCRs. In this case a- and - chain sequences are expressed in E. coli as soluble TCRs. Binding of the soluble TCRs to the complexes is analyzed by surface plasmon resonance. Biotinylated peptide-HLA monomers are prepared and immobilized on a streptavidin-coupled sensor chip. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-HLAs and the response values at equilibrium are determined for each concentration. Data are analyzed, for example, by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 : 1 interaction.

[0409] TCRs that specifically recognize peptide-HLA complexes of the disclosure are obtained from the library. Data generated according to the above- described experiments confirm that antigen specific TCRs can be isolated.Example 8. Further characterization of binding to MHC and stability of pMHC complex.

[0410] T2 cell-based peptide binding assay. T2 cells which do not express the transporter associated with antigen processing (TAP), and as such do not assemble stable MHC class I on the cell surface, are pulsed with different concentrations of peptides (controls or prostate lineage antigen-derived peptide of interest [POI]), washed, detected with fluorescently-tagged antibody recognizing MHC class I (e.g., A2 allele), and run through a FACS Scan analyzer. The difference between the MFI (mean fluorescence intensity) corresponding to a given concentration of POI and the negative control (non-MHC binder) is a function of the number of stabilized pMHC complexes displayed on the cell surface. Therefore, at limiting concentrations of the peptide, it is largely a measurement of Kon, and at saturation levels of the peptide it is a measurement of both Kon and Kotr. The binding is quantified by two related factors: relative affinity (1 / RA) and half maximal binding (the peptide concentration responsible for 50% of the signal corresponding to saturation). Relative affinity, RA, is binding normalized to a reference (e.g., a wild-type peptide in instances where a mutant POI is being tested), e.g., the ratio between half maximal binding of control relative to POI. The higher the 1 / RA index and the lower the half maximal binding, the higher the Konof the interaction between the POI and the MHC.

[0411] Characterization of binding and stability by ELISA. Avidin-coated microtiter plates containing class I monomer loaded with a placeholder peptide are used to evaluate peptide binding, affinity, and off-rate. The monomer-coated plates are supplied as part of a kit, e.g., the iTopia Epitope Discovery System Kit. Assay buffers, anti-MHC-FITC mAb and p2-microglobulin and control peptides are also supplied with the kits.

[0412] Binding assay: POIs are first evaluated for their ability to bind each MHC molecule by binding assay. This assay measures the ability of individual peptidesto bind HLA molecules under optimal standardized binding conditions. Monomer- coated plates are first stripped, releasing the placeholder peptide and leaving only the MHC heavy chain bound to the plate. Test peptides are then introduced under optimal folding conditions, along with the anti-MHC-FITC mAb. Plates were incubated. The anti-MHC-FITC mAb binds preferentially to a refolded MHC complex. Thus, the fluorescence intensity resultant from each peptide is related to the peptide's capacity to complex with MHC molecule. Each peptide's binding is evaluated relative to a positive control peptide, and the results are expressed, for example, as percent (%) binding.

[0413] Affinity assay: For the affinity assay, after the initial stripping of the placeholder peptide, increasing concentrations of POI are added to a series of wells and incubated under the conditions described previously. Plates are read on the fluorometer. Dose response curves are generated. The amount of peptide required to achieve 50% of the maximum is recorded as ED50 value.

[0414] Off-Rate assay: Plates are washed after incubation under conditions to remove excess peptide. The plates are then incubated on allele-specific monomer plates. The plates are measured at multiple time points (e.g., 0, 0.5, 1, 1.5, 2, 4, 6 and 8 hrs) for relative fluorescence intensity. The time required for 50% of the peptide to dissociate from the MHC monomer is defined as the T1 value (hrs).

[0415] iScore calculation: an iScore is a multi-parameter calculation provided within the iTopia software. Its value is calculated based on the binding, affinity, and stability data.Example 9. Determination of responses against tumor cells.

[0416] A suitable number of groups of mice are immunized with a plasmid expressing prostate lineage antigen-derived peptides disclosed herein by direct inoculation. By way of a non-limiting example, mice are inoculated into lymph nodes, e.g., inguinal lymph nodes, with plasmids at an appropriate concentration at day 0, and at subsequent days over the time course of the experiment, e.g., at days 3, 14, and 17. In certain cases, this is followed by one or more additional peptide boost(s) on following days, e.g., days 28 and 31, using a negative control peptide and POI. Splenocytes arestimulated ex vivo with POI and tested against Chromium-51 (51Cr) -labeled tumor cells at various E:T ratios.Example 10. In vivo assessment of enhanced immunity against prostate lineage antigen-derived peptides.

[0417] A suitable number of groups of mice are immunized with a plasmid expressing prostate lineage antigen-derived peptides disclosed herein by direct inoculation. By way of a non-limiting example, mice are inoculated into lymph nodes, e.g., inguinal lymph nodes, with plasmids at an appropriate concentration at day 0, and at subsequent days over the time course of the experiment, e.g., at days 3, 14, and 17. In certain cases, this is followed by one or more additional peptide boost(s) on following days, e.g., days 28 and 31, using a negative control peptide and POI.

[0418] To evaluate the in vivo response against prostate lineage antigen-derived peptides, splenocytes were isolated from littermate control mice and incubated with one or more appropriate concentrations of POI for a pre-determined period of time. These cells were then stained with CFSEhi fluorescence and intravenously co-injected into immunized mice with an equal number of control splenocytes stained with CFSElo fluorescence. After a pre-determined period of time, the specific elimination of target cells was measured by removing spleen and peripheral blood mononuclear cells (PBMCs) from challenged animals and measuring CFSE fluorescence by flow cytometry. The relative depletion of the populations corresponding to peptide loaded splenocytes was calculated relative to the control (unloaded) population and expressed as percent (%) specific lysis.Example 11. Testing of POI’s increased immunogenicity and ability to overcome tolerization

[0419] POIs are used in in vitro for immunization of blood to generate cytotoxic T lymphocytes (CTLs).

[0420] PBMCs from normal donors are purified from buffy coats by centrifugation in standard sterile medium designed for isolating lymphocytes. Cultures are carried out using autologous plasma (AP). For in vitro generation of peptide-specificCTL, autologous dendritic cells (DCs) are used as antigen presenting cells (APCs). DCs are generated and CTLs are induced with DCs and peptides from PBMCs. Monocyte- enriched cell fractions are cultured to induce maturation. Specific numbers of CD8+- enriched T lymphocytes and peptide-pulsed DCs are co-cultured. Cultures are restimulated on various days with autologous irradiated peptide-pulsed DCs. Immunogenicity is assayed using in vitro cytotoxicity and cytokine production assays.Example 12. CD8+ T cell responses against peptides.

[0421] Based on the predicted or experimentally verified HLA-binding peptides, whether T cells can be generated to recognize the tumor-specific peptides is determined. Peptides with appropriate binding scores are synthesized. To generate T cells of desired specificity, T cells are stimulated with peptide-pulsed (individual peptide or peptide pool) autologous APCs such as dendritic cells and / or CD40L- expanded autologous B cells on a predetermined schedule, for example, in the presence of IL-2 and IL-7. After several rounds of stimulation, the expanded CD8+ cells are tested on ELISpot for evidence of reactivity against the peptide based on IFNy secretion.Example 13. Cytokine production assay.

[0422] For cytokine (e.g., IL-2 and IFNy) production assays, T cells are harvested after contact with the peptide-pulsed APC, centrifuged, and both cell pellets and supernatants are collected. Cytokine production is measured by ELISA from the supernatant.Example 14. Enhancement of a peptide-specific CD8+ T cell immunity in a peptide-presenting tumor model.

[0423] For in vivo studies, the ability of the prostate lineage antigen-derived peptide of interest in the context of MHC I molecules to enhance a CD8+ T cell response against peptide-presenting tumors is tested in a general purpose strain mice, e.g., C57B1 / 6 mice, grafted with a peptide-expressing tumor cell line.

[0424] The peptide-expressing tumor cell line is first grown in vitro, then injected (e.g., subcutaneously) into the mice. After tumor cell injection, when tumorsare of palpable size, the prostate lineage antigen-derived peptide, for example, in a specific groove of the MHC I or control peptide (e.g., ovalbumin [OVA] peptide) in an alternate groove of the MHC I molecule, are injected intratumorally. Tumor cell volume is measured. Tumor, blood, and / or spleen is collected, and homogenized in single cell suspensions, when applicable. The level of CD8+ T cells in the homogenized samples is determined at all suitable time points.

[0425] Below are additional example methods that may be used in accordance with the disclosure.

[0426] Splenocytes and antigen presenting cell primary cultures. Spleens of adult general purpose strain mice, e.g., C57B1 / 6 mice, are excised and placed in cold buffer. Tissues are then homogenized to break apart the spleens. Dissociated cells are centrifuged. After centrifugation, the cell pellet is resuspended in a suitable volume of lysis buffer designed to remove red blood cells, e.g., ACK (Ammonium-Chloride- Potassium) lysis buffer, and incubated in the buffer. After incubation, the cell suspension is added to buffer, and centrifuged. The cell pellet is then resuspended in buffer and the cell solution is filtered. The filtered cell suspension is centrifuged again, and the subsequent pellet is resuspended in a suitable volume of buffer. Total spleen cells are counted, and global antigen-presenting cells (APC) are sorted using anti-MHC class II Microbeads and appropriate sorting technology. After elution, the MHC class Il-positive cell fraction is collected and resuspended at a given concentration in APC cell medium.

[0427] Pulsing of APCs with peptides. APC are incubated with prostate lineage antigen-derived or control peptides. Peptide-pulsed APC are harvested and washed prior to addition to any of various suitable T cell lines (e.g., J.RT3-T3.5 derived cells), after infection with the viral particles. The T cells are co-cultured with the pulsed APC and subsequently used in proliferation, luciferase and / or cytokines production assays.

[0428] T cell activation post transduction. Alternatively, to co-culture with peptides-pulsed APCs, suitable T cell lines (e.g., J.RT3-T3.5-derived cell lines) areactivated after transduction with either phytohemagglutinin (PHA,), phorbol 12- myristate 13-acetate (PMA), or a combination of PHA and PMA.

[0429] Another alternative method to activate T cells after transduction is to use soluble or immobilized antibodies such as, but not limited to anti-CD3 and / or anti- CD28 monoclonal antibodies. Soluble antibodies are added at an appropriate concentration. In some experiments, plates pre-coated with anti-CD3 antibodies, for example, are used in combination with soluble anti-CD28 antibody.

[0430] Another alternative method to activate T cell lines is activating beads coupled with anti-CD3 / anti-CD28 antibodies. After transduction, infected cells are counted, and anti-CD3 / anti-CD28 beads are added to the culture medium at a given ratio.

[0431] Cells staining and FACS analysis. Fluorescence-activated cell sorting (FACS) is performed after the transduction. Transduced cells are counted and seeded into a cell culture plate. Cells are spun, washed, and then spun again any number of times. Cells are incubated with a dye for determining viability of cells, e.g., Live / Dead™ Fixable Near-IR stain, washed, and incubated with Fc block in buffer, e.g., FACS stain buffer. After being washed any number of times again with FACS stain buffer, cells are subsequently incubated with antibodies such as, but not limited to, fluorescent-labeled antibodies targeting TCR and control. Cells are washed with FACS stain buffer, fixed, washed again, and resuspended in FACS stain buffer. Samples are run for analysis with a FACS analyzer.

[0432] Proliferation and cytokine production assays. To measure T cell proliferation, 3H-thymidine is added to assay cultures following contact with the peptide-pulsed APC. Following incubation, cultures are harvested onto filter bottom microplates. MicroScint 20 scintillation fluid or the like is added to each well, and plates are counted on a Scintillation Counter.

[0433] For transcription factor activity analysis and cytokines production assay, cells are harvested after contact with the peptide-pulsed APC, centrifuged, and cell pellets and supernatants are collected.

[0434] Cell pellets are processed for RNA extraction for transcriptomics analysis via qPCR. Cytokine production is measured by ELISA from the supernatant. Cell pellets are processed for luciferase detection assay to measure programmed cell death, e.g., API activity.

[0435] Splenocytes isolation and CD8+T cell culture. Spleens of adult general purpose strain mice, e.g., C57B1 / 6 mice, are excised and placed in cold buffer. Tissues are then homogenized to break apart the spleens. Dissociated cells are centrifuged. After centrifugation, the cell pellet is resuspended in a suitable volume of lysis buffer designed to remove red blood cells, e.g., ACK (Ammonium-Chloride-Potassium) lysis buffer, and incubated in the buffer. After incubation, the cell suspension is added to buffer, and centrifuged. The cell pellet is then resuspended in buffer and the cell solution is filtered. The filtered cell suspension is centrifuged again and the subsequent pellet is resuspended in a suitable volume of buffer. CD8+ T cells are then isolated such as, for example, by using a CD8a+ T cell Isolation Kit.

[0436] Peptide immunization of mice. For immunization, a suitable amount of prostate lineage antigen-derived peptide or control peptide is diluted in buffer and emulsified at a given ratio with an adjuvant such as, but not limited to, complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (IF A) using, for example, a double syringes system, to reach a final desired volume of peptide / adjuvant emulsion. A measured volume (e.g., 200 pl) of the peptide / adjuvant emulsion is then injected by a given route of administration, for example, subcutaneously, into mice (e.g., C57B1 / 6 mice) under appropriate anesthesia in appropriate location(s).

[0437] Quantification of cytotoxicity activity by flow cytometry. Preparation of target cells: For preparation of target cells (e.g., autologous B cells), the target cells are isolated from splenocytes of OT-1 or P14 mice by using a Mouse B cell Kit (or the like, according to the manufacturer's instruction), and are subsequently stimulated for apredetermined duration in the presence of a set concentration of IFN-y. Targets cells are then counted, divided into tubes, and washed in buffer. A proportion of the target cells are stained with a high concentration (e.g., 0.2 pM) of CFSE (CFSEHigh) and a separate proportion with a low concentration (e.g, 0.02 pM) of CFSE (CFSELow) in buffer. After the incubation, cells are pelleted and resuspended in an appropriate media to quench the labeling reaction. Target cells stained with the low concentration of CFSE are pulsed by adding the prostate lineage antigen-derived peptides or control OVA peptides at a suitable final concentration under appropriate culture conditions. Both target cells stained with different concentration of CFSE are then washed, resuspended at a measured concentration in media and mixed with an appropriate ratio, such as a 1 : 1 ratio (CFSEHigh: CFSELow).

[0438] Effector cells: Total CD8+ T cells containing the effector cells are enriched from splenocytes of P14 and OT-1 mice using a Negative Selection Human CD8 T cell isolation Kit. CD8+ T cells are counted, resuspended in complete T cell medium, and serially diluted volume: volume in a given volume of complete T cell medium. From each dilution, a given volume of cells are seeded in duplicate to multiwell cell culture plate. The mixed target cells are added to each dilution of effector cells. To measure basal apoptosis, a number of wells are seeded with target cells alone. Cell mixtures are incubated under standard cell culture conditions.

[0439] Flow cytometry staining and acquisition: Cells are transferred to multiwell cell culture plate, washed in FACS staining buffer and stained with, for example, iTag Tetramer / APC-H-2Kb OVA9® (MBL), iTag Tetramer / APC-H-2Db HBV®Alexa Fluor® and Live / Dead® Fixable Near-IR stain (ThermoFisher) under appropriate conditions. Cells are then washed in FACS stain buffer before staining with, for example, Fluorescently-labeled monoclonal antibody that specifically binds to CD8 alpha such as, but not limited to, BV421 aCD8a. Cells are washed with FACS stain buffer and re-suspended fixative. Acquisition is performed and all cells are acquired. Post-acquisition data analysis performed.

[0440] Preparation of vectors encoding peptides. A therapeutic DNA or RNA vaccine comprising polynucleotides or vectors encoding polynucleotides to be used isprepared by GMP manufacturing of the plasmid vaccine according to regulatory authorities' guidelines. The vaccine is appropriately formulated, for example, by dissolving in a saline solution, at a suitable concentration. The vaccine may be administered either intradermal or intramuscular with or without following electroporation or alternatively with a jet injector.★ A w

[0441] The claimed subject matter is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the claimed subject matter in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0442] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Claims

CLAIMS1. A polynucleotide encoding a peptide and a regulatory sequence, wherein: a) the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 7, 1, 2, 8, 3, and 4; and b) the polynucleotide is not identical to a KLK3 gene or a portion thereof.

2. The polynucleotide of claim 1, wherein the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 1-4.

3. The polynucleotide of claim 2, wherein the peptide has the sequence as set forth in SEQ ID NO: 2.

4. The polynucleotide of claim I, wherein the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 7-8.

5. A polynucleotide encoding a peptide and a regulatory sequence, wherein: a) the peptide has a sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 9; and b) the polynucleotide is not identical to a KLK4 gene or a portion thereof.

6. The polynucleotide of claim 5, wherein the peptide has SEQ ID NO: 5.

7. A polynucleotide encoding a peptide and a regulatory sequence, wherein: a) the peptide has a sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 10; and b) the polynucleotide is not identical to a KLK2 gene or a portion thereof.

8. A polynucleotide encoding a peptide and a regulatory sequence, wherein: a) the peptide has a sequence as set forth in SEQ ID NO: 11 ; and b) the polynucleotide is not identical to an ACP3 gene or a portion thereof.

9. A vector comprising the polynucleotide of any one of claims 1-8.

10. The vector of claim 9, wherein the vector is an expression vector.

11. The vector of claim 9 or claim 10, wherein the vector is a viral vector.

12. A host cell comprising the polynucleotide of any one of claims 1-8 or the vector of any one of claims 9-11.

13. The host cell of claim 12, wherein the host cell is a prokaryotic cell.

14. The host cell of claim 12, wherein the host cell is a eukaryotic cell.

15. The host cell of claim 14, wherein the host cell is an APC.

16. A polypeptide comprising: a) a peptide sequence selected from the group consisting of: SEQ ID NOs: 1- 11; b) a linker; and c) a p2-microglobulin (02M) polypeptide or a part thereof.

17. The polypeptide of claim 16, wherein the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 1-6.

18. The polypeptide of claim 16, wherein the peptide has sequence SEQ ID NO: 2.

19. The polypeptide of claim 16, wherein the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 7-11.

20. An isolated protein complex comprising:a) at least one part of a Major Histocompatibility Complex (MHC) molecule; and b) a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11.

21. The isolated protein complex of claim 20, wherein the Major Histocompatibility Complex (MHC) molecule is HLA.

22. The isolated protein complex of claim 20, wherein the Major Histocompatibility Complex (MHC) molecule is a truncated HLA heavy chain.

23. The isolated protein complex of claim 20, wherein the MHC complex molecule is an HLA heavy chain or p-2 microglobulin.

24. The isolated protein complex of claim 20, wherein the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 1-6.

25. The isolated protein complex of claim 20, wherein the peptide has sequence SEQ ID NO: 2.

26. The isolated protein complex of claim 20, wherein the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 7-11.

27. The isolated protein complex of claim 20 comprising: a) an HLA heavy chain; b) P-2 microglobulin; and c) a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11.

28. The isolated protein complex of claim 20, wherein the MHC molecule is a class I, class II, or class III MHC molecule.

29. The isolated protein complex of claim 20, wherein the complex between the peptide and said MHC molecule is a covalently bound complex.

30. The isolated protein complex of claim 29, wherein the covalently bound complex is a fusion protein of the MHC molecule with the peptide.

31. The isolated protein complex of claim 20, wherein there is a non-native linkage between the peptide, and a binding groove of the MHC molecule.

32. The isolated protein complex of claim 31 , wherein the non-native linkage is between the C-terminal anchor residue of the peptide and an amino acid residue in the F pocket of the binding groove of the MHC molecule.

33. An isolated molecule that binds the polypeptide of any one of claims 16-19, or the isolated protein complex of any one of claims 20-32.

34. The isolated molecule of claim 33, wherein the molecule is an antibody or an antigen-binding fragment thereof.

35. The isolated molecule of claim 34, wherein the antibody is a bispecific antibody.

36. The isolated molecule of claim 33, wherein the molecule is an alternative scaffold.

37. The isolated molecule of claim 33, wherein the molecule is a chimeric antigen receptor (CAR).

38. The isolated molecule of claim 33, wherein the molecule is a T cell receptor (TCR).

39. An isolated cell comprising the CAR of claim 37.

40. The isolated cell of claim 39, wherein the isolated cell is an immune cell.

41. The isolated cell of claim 40, wherein the immune cell is a T cell, an NK cell, or a macrophage.

42. An isolated cell comprising the TCR of claim 38.

43. The isolated cell of claim 42, wherein the isolated cell is an immune cell.

44. The isolated cell of claim 43, wherein the immune cell is a T cell, an NK cell, or a macrophage.

45. A nanoparticle complex comprising a nanoparticle core and the isolated protein complex of any one of claims 20-32 covalently coupled to the nanoparticle core.

46. A modified single chain TCR comprising an alpha chain and a beta chain, wherein the TCR specifically binds to the isolated protein complex of any one of claims 20-32.

47. An artificial antigen presenting cell (aAPC) comprising a liposome comprising a phospholipid and the isolated protein complex of any one of claims 20-32.

48. A vaccine composition capable of rising a specific T-cell response comprising: a) a peptide having a sequence selected from the group consisting of: SEQ IDNOs: 1-11; b) one or more polynucleotides encoding a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11; c) a population of autologous dendritic cells or antigen presenting cells presenting at its surface the prostate-specific antigen epitope having a sequence selected from the group consisting of: SEQ ID NOs: 1-11 ; ord) one or more MHC molecules or parts thereof loaded with the prostatespecific antigen epitope having a sequence selected from the group consisting of: SEQ ID NOs: 1-11, and a pharmaceutically acceptable carrier other than water.

49. The vaccine of claim 48, wherein the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 1-6.

50. The vaccine of claim 48, wherein the peptide has sequence SEQ ID NO: 2.

51. The vaccine of claim 48, wherein the peptide has a sequence selected from the group consisting of: SEQ ID NOs: 7-11.

52. A pharmaceutical composition comprising a first peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11 and a pharmaceutically acceptable carrier other than water.

53. The pharmaceutical composition of claim 52, wherein the first peptide is not covalently bound to a second peptide or a molecule.

54. The pharmaceutical composition of claim 53, wherein the first peptide is bound by hydrogen bonds to the second peptide or molecule.

55. The pharmaceutical composition of claim 54, wherein the molecule is an HLA molecule or part thereof.

56. The pharmaceutical composition of claim 55, wherein the HLA molecule is selected from the group consisting of class I, class II and class III.

57. The pharmaceutical composition of claim 56, wherein the HLA molecule is a class I HLA molecule.

58. The pharmaceutical composition of claim 56, wherein the HLA molecule or part thereof is encoded by an HLA-A gene.

59. The pharmaceutical composition of claim 58, wherein the first peptide is part of a complex that comprises (i) an HLA-A heavy chain or a part thereof; and (ii) a P-2 macroglobulin or a part thereof.

60. A pharmaceutical composition comprising the polynucleotide of any of claims 1-8 and a pharmaceutically acceptable carrier other than water.

61. A pharmaceutical composition comprising the vector of any of claims 9-11 and a pharmaceutically acceptable carrier other than water.

62. A pharmaceutical composition comprising the polypeptide of any one of claims 16-19 and a pharmaceutically acceptable carrier other than water.

63. A pharmaceutical composition comprising the isolated protein complex of any one of claims 20-32 a pharmaceutically acceptable carrier other than water.

64. A pharmaceutical composition comprising the nanoparticle complex of claim 45 and a pharmaceutically acceptable carrier other than water.

65. A pharmaceutical composition comprising the single chain TCR of claim 46 and a pharmaceutically acceptable carrier other than water.

66. A pharmaceutical composition comprising the artificial antigen presenting cell (aAPC) of claim 47 and a pharmaceutically acceptable carrier other than water.

67. A method for isolating an antigen specific T cell, the method comprising the steps of: a) providing a composition comprising a particle attached to an MHC display moiety comprising at least one antigenic peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11;b) providing a sample comprising one or more T cells; c) contacting the composition with the sample, wherein the contacting comprises providing conditions sufficient for a single T cell to bind the MHC display moiety attached to the particle; and d) isolating a single antigen specific T cell associated with the particle.

68. The method of claim 67, wherein the MHC display moiety is attached to the particle by a linker.

69. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11 or the pharmaceutical composition of any one of claims 67-83.

70. A method of treating or reducing the likelihood of prostate cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11 or the pharmaceutical composition of any one of claims 67-83.

71. A method of inducing a CD8+ cytotoxic T cell response in a subject having prostate cancer overexpressing prostate-specific antigen comprising a peptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-11, the method comprising administering to the subject a therapeutically effective amount of the peptide or a pharmaceutical composition of any one of claims 52-66.

72. The method of claim 71, wherein the patient is HLA-A11 :01+ or HLA- A03:01+ and the peptide has SEQ ID NO: 2.

73. A kit comprising:(i) a) one or more polynucleotides of any one of claims 1-8; b) one or more vectors of any one of claims 9-11; c) one or more polypeptides of any one of claims 16-19; d) one or more isolated protein complexes of any one of claims 20-32;e) one or more nanoparticle complexes of claim 45; f) one or more modified single chain TCRs of claim 46; g) one or more artificial antigen presenting cell (aAPC) of claim 47; h) one or more vaccines of any one of claims 48-51 ; or i) one or more pharmaceutical compositions of any one of claims 52-66; and(ii) packaging and / or instructions for use for the same.

74. A method of generating a peptide binding moiety that recognizes one of SEQ ID NOs: 1-11, the method comprising: a) immunizing a non-human animal with a composition comprising:(i) one or more polynucleotides of any one of claims 1-8;(ii) one or more vectors of any one of claims 9-11;(iii) one or more polypeptides of any one of claims 16-19;(iv) one or more isolated protein complexes of any one of claims 20- 32;(v) one or more nanoparticle complexes of claim 45;(vi) one or more artificial antigen presenting cell (aAPC) of claim 47;(vii) one or more vaccines of any one of claims 48-51 ; or(viii) one or more pharmaceutical compositions of any one of claims52-64 or 66; and b) isolating one or more T cells, B cells, TCRs, and / or antibodies that bind one of SEQ ID NOs: 1-11 from the non-human animal.

75. The method of claim 74, wherein the non-human animal is genetically modified.

76. The method of claim 75, wherein the genetically modified non-human animal is a rodent, optionally a mouse.

77. The method of claim 75 or 76, wherein the genetically modified non-human animal is genetically modified to express:i) a human or humanized immunoglobulin, comprising a human or humanized immunoglobulin heavy chain and / or a human or humanized immunoglobulin light chain; ii) a human or humanized TCR; and / or ii) a human or humanized MHC class I molecule, and optionally a human or humanized 2m.

78. The method of any one of claims 74-77, wherein the peptide binding moiety comprises any one of the isolated molecules of claims 33-34 or 38.