Novel antigens for cancer and their uses

Abstract

Breast cancer is currently the most prevalent cancer in the world, and despite therapeutic advances over the past few decades, metastatic breast cancer remains an incurable disease. This specification describes novel tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) expressed by breast cancer cells. Synthetic long peptides, nucleic acids, compositions, cells, TCRs, antibodies, and vaccines derived from these TSAs and TAAs are described. Furthermore, the use of the TSAs / TAAs, nucleic acids, compositions, antibodies, cells, and vaccines for the prevention or treatment of breast cancer, including triple-negative breast cancer (TNBC), is described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 370,697, filed August 8, 2022, the contents of which are incorporated herein by reference.

[0002] Sequence Listing A Sequence Listing is submitted herewith in XML format under the file name 17971-00066-AD.xml, with a file size of approximately 181 kilobytes, created on August 2, 2023. The contents of the foregoing file are incorporated herein by reference in their entirety.

[0003] The present invention relates generally to the field of cancer, and more particularly to the treatment of cancer, such as breast cancer. [Background technology]

[0004] According to recent global statistics, breast cancer has now surpassed lung cancer as the most common cancer worldwide (1). Despite advances in treatment over the past few decades, metastatic breast cancer remains an incurable disease. From an immunological perspective, breast cancer tumors are broadly classified into two categories (2): hormone receptor-positive breast cancer (HR-positive) and hormone receptor-positive breast cancer (HR-positive). + ) are considered immunologically "cold" cancers and have not benefited from recent advances in immunotherapy (3). In contrast, the triple-negative breast cancer (TNBC) subtype is immunologically "hot," as evidenced by its high levels of leukocyte infiltration and its responsiveness to immune checkpoint blockade (ICB) (4). Indeed, following the IMPassion130 trial, the addition of ICB to chemotherapy has become the new standard of care for TNBC tumors with PD-L1 expression (5). High tumor mutation burden (TMB) has previously been used as a biomarker for responsiveness to ICB in multiple cancers (6). However, only 5% of breast cancer tumors have high TMB, and CD8 + There is no correlation with T cell infiltration (6, 7).+ The factors behind the differences in immunogenicity between tumors and TNBC tumors remain unclear.

[0005] Therefore, there is a need to develop new methods for preventing and treating breast cancer.

[0006] These descriptions cite numerous documents, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention

[0007] In various aspects and embodiments, the present disclosure provides items 1-67 below.

[0008] 1. The following amino acid sequence: [Table 1] A tumor antigen peptide (TAP) comprising or consisting of one of the following, or a nucleic acid encoding said TAP.

[0009] 2. A TAP or nucleic acid according to item 1, wherein said TAP binds to an HLA-A*02:01 molecule and comprises or consists of the sequence of SEQ ID NO: 22.

[0010] 3. A TAP or nucleic acid according to item 1, wherein said TAP binds to an HLA-A*03:01 molecule and comprises or consists of the sequence of SEQ ID NO: 19.

[0011] 4. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-A*11:01 molecule and comprises or consists of the sequence of SEQ ID NO: 1, 17 or 28.

[0012] 5. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-A*24:02 molecule and comprises or consists of the sequence of SEQ ID NO: 6 or 30.

[0013] 6. A TAP or nucleic acid according to item 1, wherein said TAP binds to an HLA-A*25:01 molecule and comprises or consists of the sequence of SEQ ID NO: 10.

[0014] 7. A TAP or nucleic acid according to item 1, wherein said TAP binds to an HLA-A*26:01 molecule and comprises or consists of the sequence of SEQ ID NO: 15.

[0015] 8. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-A*31:01 molecule and comprises or consists of the sequence of SEQ ID NO: 8, 9 or 29.

[0016] 9. A TAP or nucleic acid according to item 1, wherein said TAP binds to an HLA-A*33:01 molecule and comprises or consists of the sequence of SEQ ID NO: 2 or 3.

[0017] 10. A TAP or nucleic acid according to item 1, wherein said TAP binds to an HLA-B*15:01 molecule and comprises or consists of the sequence of SEQ ID NO: 26.

[0018] 11. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-B*18:01 molecule and comprises or consists of the sequence of SEQ ID NO: 13, 14 or 33.

[0019] 12. A TAP or nucleic acid according to item 1, wherein said TAP binds to an HLA-B*27:05 molecule and comprises or consists of the sequence of SEQ ID NO: 27.

[0020] 13. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-B*35:01 molecule and comprises or consists of the sequence of SEQ ID NO: 4, 12 or 23.

[0021] 14. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-B*35:03 molecule and comprises or consists of the sequence of SEQ ID NO: 38.

[0022] 15. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-B*38:01 molecule and comprises or consists of the sequence of SEQ ID NO: 34.

[0023] 16. A TAP or nucleic acid according to item 1, wherein said TAP binds to an HLA-B*40:01 molecule and comprises or consists of the sequence of SEQ ID NO: 20.

[0024] 17. A TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-B*49:01 molecule and comprises or consists of the sequence of SEQ ID NO: 11 or 24.

[0025] 18. A TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-B*50:01 molecule and comprises or consists of the sequence of SEQ ID NO: 5 or 7.

[0026] 19. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-B*51:01 molecule and comprises or consists of the sequence of SEQ ID NO: 35 or 37.

[0027] 20. A TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-B*52:01 molecule and comprises or consists of the sequence of SEQ ID NO: 18.

[0028] 21. A TAP or nucleic acid according to item 1, wherein said TAP binds to an HLA-B*58:01 molecule and comprises or consists of the sequence of SEQ ID NO: 36.

[0029] 22. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-C*01:02 molecule and comprises or consists of the sequence of SEQ ID NO: 25.

[0030] 23. The TAP or nucleic acid according to item 1, wherein the TAP binds to an HLA-C*12:03 molecule and comprises or consists of the sequence of SEQ ID NO: 16 or 21.

[0031] 24. The TAP or nucleic acid according to any one of items 1 to 23, which is encoded by a sequence located in a non-protein-coding region of the genome.

[0032] 25. The TAP or nucleic acid according to item 24, wherein the non-protein-coding region is an intergenic region.

[0033] 26. The TAP or nucleic acid according to item 24, wherein the non-protein-coding region of the genome is a long non-coding RNA.

[0034] 27. A combination comprising at least two of the TAPs or nucleic acids defined in any one of items 1 to 26.

[0035] 28. The TAP or nucleic acid according to any one of items 1 to 26, or the combination according to item 27, wherein the nucleic acid is mRNA.

[0036] 29. The TAP or nucleic acid according to any one of items 1 to 26, or the combination according to item 27, wherein the nucleic acid is DNA.

[0037] 30. The TAP or nucleic acid according to any one of items 1 to 26, or the combination according to item 27, wherein the nucleic acid is a component of a viral vector.

[0038] 31. A synthetic long peptide (SLP) comprising at least one of the amino acid sequences defined in item 1, or a nucleic acid encoding said SLP.

[0039] 32. The SLP or nucleic acid according to item 31, wherein the SLP comprises at least 5, 10, 15 or 20 of the amino acid sequences defined in item 1.

[0040] 33. A vesicle or particle comprising a TAP, a nucleic acid, a combination or an SLP according to any one of items 1 to 32.

[0041] 34. The vesicle or particle according to item 33, wherein the vesicle is a lipid nanoparticle (LNP).

[0042] 35. A vesicle or particle according to item 33 or 34, comprising a cationic lipid.

[0043] 36. A composition comprising a TAP, nucleic acid, combination or SLP according to any one of items 1 to 32, or a vesicle or particle according to any one of items 33 to 35, and a pharmaceutically acceptable carrier.

[0044] 37. A vaccine comprising a TAP, nucleic acid, combination or SLP according to any one of items 1 to 32, a vesicle or particle according to any one of items 33 to 35, or a composition according to item 36, and an adjuvant.

[0045] 38. An isolated major histocompatibility complex (MHC) class I molecule comprising within its peptide-binding site a TAP according to any one of items 1 to 26.

[0046] 39. The isolated MHC class I molecule according to item 38, which is in the form of a multimer.

[0047] 40. The isolated MHC class I molecule according to item 39, wherein the multimer is a tetramer.

[0048] 41. An isolated cell comprising (i) a TAP according to any one of items 1 to 26, (ii) the combination according to item 27, (iii) an SLP according to item 31 or 32, or (iv) a vector comprising a nucleotide sequence encoding a TAP according to any one of items 1 to 26, the combination according to item 27 or an SLP according to item 31 or 32.

[0049] 42. An isolated cell expressing on its surface a major histocompatibility complex (MHC) class I molecule, said molecule comprising within its peptide-binding site a TAP or combination according to any one of items 1 to 30.

[0050] 43. The cell according to item 41 or 42, which is an antigen-presenting cell (APC).

[0051] 44. The cell according to item 43, wherein the APC is a dendritic cell.

[0052] 45. A T cell receptor (TCR) that specifically recognizes the isolated MHC class I molecule according to any one of items 38 to 40 and / or the MHC class I molecule expressed on the surface of the cell according to any one of items 42 to 44.

[0053] 46. The TCR according to item 45, which is a soluble TCR.

[0054] 47. An antibody or antigen-binding fragment thereof that specifically binds to the isolated MHC class I molecule according to any one of items 38 to 40 and / or the MHC class I molecule expressed on the surface of the cell according to any one of items 42 to 44.

[0055] 48. A TCR according to item 45 or 46, or an antibody or antigen-binding fragment according to item 47, which is a bispecific TCR or a bispecific antibody or bispecific antigen-binding fragment.

[0056] 49. The TCR, antibody or antigen-binding fragment according to item 48, wherein the bispecific antibody or antigen-binding fragment is a single-chain diabody (scDb).

[0057] 50. The TCR, antibody or antigen-binding fragment thereof according to item 48 or 49, wherein the bispecific TCR, antibody or antigen-binding fragment thereof also specifically binds to a T cell signalling molecule.

[0058] 51. The TCR, antibody or antigen-binding fragment according to item 50, wherein the T cell signaling molecule is a CD3 chain.

[0059] 52. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof according to item 47, or a nucleic acid encoding the CAR.

[0060] 53. An isolated cell, which expresses on its surface the TCR according to item 45.

[0061] 54. The isolated cell according to item 53, which is a CD8+ T lymphocyte.

[0062] 55. A cell population comprising at least 0.5% or 1% of the isolated cells defined in item 53 or 54.

[0063] 56. A method of treating breast cancer in a subject, comprising administering to the subject an effective amount of any of the following: (a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1 to 61, or a combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1 to 61; (b) at least one nucleic acid encoding a TAP, combination of TAPs or SLP as defined in (a); (c) a TAP, combination of TAPs, or SLP as defined in (a), or a vesicle or particle comprising at least one nucleic acid as defined in (b); (d) a composition comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier; (e) a vaccine comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant; (f) a cell expressing on its surface a major histocompatibility complex (MHC) class I molecule containing within its peptide-binding groove a TAP or combination of TAPs as defined in (a); (g) A cell expressing on its surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of a cell defined in (f), or (h) A method comprising administering a soluble TCR, antibody or antigen-binding fragment thereof that specifically binds to an MHC class I molecule expressed on the surface of a cell defined in (f).

[0064] 57. The method of item 56, wherein the breast cancer is hormone receptor positive breast cancer (HR+) or triple negative breast cancer (TNBC).

[0065] 58. The method according to item 56 or 57, further comprising administering or treating the subject with at least one additional anti-tumor agent.

[0066] 59. The method according to item 58, wherein the at least one additional anti-tumor agent or treatment is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy or surgery.

[0067] 60. (a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1 to 61, or a combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1 to 61; (b) at least one nucleic acid encoding a TAP, combination of TAPs or SLP as defined in (a); (c) a TAP, combination of TAPs, or SLP as defined in (a), or a vesicle or particle comprising at least one nucleic acid as defined in (b); (d) a composition comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier; (e) a vaccine comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant; (f) a cell expressing on its surface a major histocompatibility complex (MHC) class I molecule containing within its peptide-binding groove a TAP or combination of TAPs as defined in (a); (g) A cell expressing on its surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of a cell defined in (f), or (h) A soluble TCR, antibody, or antigen-binding fragment thereof that specifically binds to an MHC class I molecule expressed on the surface of a cell defined in (f). Use of a compound of claim 1 for treating breast cancer in a subject or for manufacturing a medicament for treating breast cancer in a subject.

[0068] 61. The use according to item 60, wherein the breast cancer is hormone receptor positive breast cancer (HR+) or triple negative breast cancer (TNBC).

[0069] 62. The use according to item 60 or 61, further comprising administering to the subject at least one additional anti-tumor agent or treatment.

[0070] 63. The use according to item 62, wherein the at least one additional anti-tumor agent or treatment is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy or surgery.

[0071] 64. A drug for use in treating breast cancer in a subject, the drug comprising: (a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1 to 61, or a combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1 to 61; (b) at least one nucleic acid encoding a TAP, combination of TAPs or SLP as defined in (a); (c) a TAP, combination of TAPs, or SLP as defined in (a), or a vesicle or particle comprising at least one nucleic acid as defined in (b); (d) a composition comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier; (e) a vaccine comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant; (f) a cell expressing on its surface a major histocompatibility complex (MHC) class I molecule containing within its peptide-binding groove a TAP or combination of TAPs as defined in (a); (g) A cell expressing on its surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of a cell defined in (f), or (h) A drug that is a soluble TCR, antibody, or antigen-binding fragment thereof that specifically binds to an MHC class I molecule expressed on the surface of a cell defined in (f).

[0072] 65. The agent for use according to item 64, wherein the breast cancer is hormone receptor positive breast cancer (HR+) or triple negative breast cancer (TNBC).

[0073] 66. The agent for use according to item or 65, further comprising administering to the subject at least one additional anti-tumor agent or treatment.

[0074] 67. The agent for use according to item 66, wherein the at least one additional antitumor agent or treatment is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy or surgery.

[0075] Other objects, advantages and features of the present disclosure will become more apparent upon reading the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. [Brief explanation of the drawings]

[0076] [Figure 1A] The number of unique MAPs identified per sample. [Figure 1B]Canonical immunopeptidome of HR+ and TNBC breast cancer tumors. Venn diagram of MAP-derived genes in HR+ and TNBC breast cancer tumors and in normal tissues from the HLA ligand ATLAS. [Figure 1C] Canonical immunopeptidome of HR+ and TNBC breast cancer tumors. Total number of MAPs per gene contributing the top 1% of MAPs. [Figure 1D] Canonical immunopeptidome of HR+ and TNBC breast cancer tumors. PANTHER analysis of the top 1% of MAP-producing genes (n=242) showing enrichment in cytoskeletal, transcriptional, and extracellular matrix protein classes. [Figure 1E] Canonical immunopeptidome of HR+ and TNBC breast cancer tumors. Expression of MAP non-contributing genes, MAP-contributing genes, and top 1% MAP-producing genes (ANOVA: p<0.001). [Figure 2A] Figure 1 shows that ERE-derived MAPs are enriched in the immunopeptidome of HR+ and TNBC tumors. Similar numbers of ERE-derived MAPs were identified in HR+ (n=14) and TNBC (n=12) samples. [Figure 2B] Figure 1 shows that ERE-derived MAPs are enriched in the immunopeptidome of HR+ and TNBC tumors. Number of ERE-derived MAPs per ERE class. [Figure 2C] Figure 1 shows that ERE-derived MAPs are enriched in the immunopeptidome of HR+ and TNBC tumors. Number of MAPs identified per ERE family in HR+ and TNBC samples. [Figure 2D] We demonstrate that ERE-derived MAPs are enriched in the immunopeptidome of HR+ and TNBC tumors, demonstrating that ERE families leading to MAP production are more highly expressed at the transcriptional level than non-receiving families in tumors from the TCGA cohort (t-test: p<0.001, n=741). [Figure 3A] Identification of tumor antigens of interest. Classification workflow of MAPs of interest. [Figure 3B]Identification of tumor antigens of interest is shown. Number of TSAs and TAAs identified in HR+ samples, TNBC samples, or both. Most TSAs were identified only in TNBC samples (n=18 / 25). [Figure 3C] Identification of tumor antigens of interest. The majority of TSAs and TAAs identified using the classification workflow are novel. [Figure 4A] Figure 1 shows the identification of TSA. Expression heatmap of the coding sequence of TSA in normal tissues (GTEX, mTEC, and bone marrow). [Figure 4B] Identification of TSAs. Genomic origin of TSAs. Identified aeTSAs primarily derived from exon regions. [Figure 4C] Identification of TSAs is shown. Percentage of HR+ and TNBC tumors with individual expression of TAA >2 rphm. [Figure 4D] Figure 1 shows the identification of TSA. GSEA analysis of the TCGA breast cancer cohort demonstrates that tumor-infiltrating leukocyte gene markers are enriched in tumors with high (above the median) levels of predicted TSA. [Figure 5A] Figure 1 shows the identification of TAAs. Expression heatmap of coding sequences of TAAs in normal tissues (GTEX, mTEC and bone marrow) reclassified by the associated function of the contributing gene. [Figure 5B] Identification of TAAs is shown. Percentage of HR+ and TNBC tumors with individual TAA expression >2 rphm. [Figure 5C] Identification of TAAs: GSEA analysis in the TCGA cohort of HR+ breast cancers shows that both immunosuppressive and immunostimulatory pathways are enriched in tumors with high (above median) levels of predicted TAAs (defined by expression >2 rphm and appropriate HLA alleles for presentation). [Figure 6] CAF-derived TAA are shown. Expression of COL11A1, COL10A1, and LRRC15 in different cells of the microenvironment of breast cancer samples. [Figure 7A]Figure 1 shows survival analysis of predicted TSA. Survival analysis of highly expressed predTSA in HR+ breast cancer tumors shows no impact on survival. [Figure 7B] Figure 1 shows survival analysis of predicted TSAs. Survival analysis of highly expressed predTAAs in TNBC and HR+ breast cancer tumors shows no impact on survival. [Figure 7C] Figure 1 shows survival analysis of predicted TSAs. Survival analysis of highly expressed predTAAs in TNBC and HR+ breast cancer tumors shows no impact on survival. [Figure 7D] Figure 1 shows survival analysis of TSA in the TCGA cohort. Survival analysis in the TCGA TNBC cohort demonstrates a significant survival benefit in patients with high predTSA expression from non-coding regions and CTAs, whereas high expression of TSA from non-coding regions or CTAs and not likely to present is associated with decreased survival. [Figure 7E] Figure 1 shows survival analysis of TSA in the TCGA cohort. Survival analysis in the TCGA TNBC cohort demonstrates a significant survival benefit in patients with high predTSA expression from non-coding regions and CTAs, whereas high expression of TSA from non-coding regions or CTAs and not likely to present is associated with decreased survival. [Figure 8] FIG. 1 is a schematic diagram of database construction for the identification of MAPs, including the canonical proteome, the ERE proteome, the small RNA proteome (smRNA) and the cancer-specific proteome. [Figure 9] The number of MAPs identified for each HLA allele (n=53) in the dataset (n=26) is shown. [Figure 10A] The percentage of transcripts expressed at the immunopeptidome level per cumulative sample is shown. [Figure 10B] 1 is a histogram of the total number of MAPs produced per fed gene in the dataset. [Figure 11A]PANTHER enrichment analysis of contributing genes specific to the cancer immunopeptidome. Shared contributing genes between HR+ and TNBC samples (n=277). [Figure 11B] PANTHER enrichment analysis of contributing genes specific to the cancer immunopeptidome. Contributing genes specific to TNBC tumors (n=259). [Figure 12A] Schematic diagram of the small RNA database construction workflow. [Figure 12B] FIG. 1 is a schematic diagram of the filtering procedure for peptide characterization and validation. [Figure 13] Genomic mapping of the identified ERE-derived MAPs. [Figure 14] Spearman correlation between mutation frequency per gene in the TCGA breast cancer cohort and the total number of MAPs identified per gene in the dataset. [Figure 15] GSEA analysis of breast cancer tumors from TCGA (n=1109). This GSEA analysis shows enrichment of mitotic and immune activation pathways in tumors with high (above median) levels of predicted TSA (defined by expression >2 rphm and appropriate HLA alleles). In tumors with low levels of predicted TSA, enrichment of PI3K signaling genes can be observed. [Figure 16] GSEA analysis of the TCGA TNBC cohort shows that immune stimulatory and immune suppressive pathways are enriched in tumors with high (above median) levels of predictive TAAs (defined as expression >2 rphm and appropriate HLA alleles for presentation). [Figure 17] Survival analysis in the TCGA TNBC cohort is shown, demonstrating a significant survival benefit in patients with high predTSA expression levels. [Figure 18A]

[0039] Figure 1 shows the results of a functional expansion of specific T cells (FEST) assay demonstrating significant expansion of antigen-specific clonotypes to select TSAs and TAAs in donor D26 after 20 days of stimulation with autologous T cell-depleted PBMCs pulsed with individual peptides. For each peptide, left circle = CD8+ T cells cultured with peptide, middle circle = CD8+ T cells cultured without peptide; right circle = CD8+ T cells not cultured. Control peptide = MelanA, ELAGIGILTV (SEQ ID NO: 212). [Figure 18B] Figure 1 shows the results of a functional expansion of specific T cells (FEST) assay demonstrating significant expansion of antigen-specific clonotypes to selected TSAs and TAAs after 20 days of stimulation with autologous T cell-depleted PBMCs pulsed with individual peptides at D27. For each peptide, left circle = CD8+ T cells cultured with peptide, middle circle = CD8+ T cells cultured without peptide; right circle = uncultured CD8+ T cells. Control peptide = MelanA, ELAGIGILTV (SEQ ID NO: 212). DETAILED DESCRIPTION OF THE INVENTION

[0077] In the context of describing the technology (particularly in the context of the claims which follow), the use of the terms "a," "an," and "the," and similar referents, are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0078] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0079] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0080] Any and all examples provided herein, or the use of exemplary language ("for example," "e.g.," "etc.") are intended merely to better illustrate embodiments of the claimed technology and do not pose a limitation on scope unless otherwise claimed.

[0081] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.

[0082] As used herein, the term "about" has its ordinary meaning. The term "about" is used to indicate that a value includes the inherent variation for error of the device or method being used to determine the value, or encompasses a value near the recited value, e.g., within 10% of the recited value (or range of values).

[0083] Recitation of ranges of values herein, unless otherwise stated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually referred to herein. Every subset of values within a range is also incorporated herein as if it were individually listed herein.

[0084] Where features or aspects of the present disclosure are described in terms of a Markush group or list of alternatives, those skilled in the art will recognize that the present disclosure is also thereby described in terms of any individual component or subgroup of components of the Markush group or list of alternatives.

[0085] Unless specifically defined otherwise, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0086] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, "A Practical Guide to Molecular Cloning" (John Wiley and Sons, 1984), J. Sambrook et al., "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 1989), T.A. Brown (ed.), "Essential Molecular Biology: Practical Approach, Volumes 1 and 2" (IRL Press, 1991), D.M.G.lover and B.D.Hames (eds.), "DNA Cloning: A Practical Approach, Volumes 1-4" (IRL Press, 1995 and 1996), F.M.A.usubel et al. (eds.), "Current Protocols in Molecular Biology" (Greene Pub: Associates and Wiley-Interscience, 1988, including the latest updates), Ed. Harlow and David Lane (eds.), "Antibodies: A Laboratory Manual" (Cold Spring Harbor Laboratory, 1988), and J.E. Coligan et al. (eds.), "Current Protocols in The use of chemotherapeutic agents has been described and explained throughout the literature, including The Journal of Immunology (John Wiley & Sons, including the latest updates).

[0087] In the studies described herein, the inventors used a proteogenomics-based approach to identify candidate TSAs and TAAs from breast cancer specimens. The novel candidate TSAs and TAAs identified herein may be useful for immunotherapy and vaccines against cancers that express these candidate TSAs and TAAs, such as breast cancer.

[0088] The present disclosure relates to tumor antigen peptides (TAPs) (or tumor-specific peptides), for example, breast cancer (BC) TAP, that comprise or consist of one of the following amino acid sequences: [Table 2]

[0089] In another aspect, the present disclosure further relates to the use of TAP comprising or consisting of any one of the following amino acid sequences for the treatment of breast cancer: [Table 3]

[0090] Generally, peptides such as tumor antigen peptides (TAPs) presented in the context of HLA class I are variable in length, ranging from about 7 or 8 to about 15, and preferably 8 to 14, amino acid residues. In some embodiments of the methods of the present disclosure, longer peptides comprising a TAP sequence as defined herein are artificially loaded into cells, such as antigen-presenting cells (APCs), where they are processed by the cells, and the TAP is presented by MHC class I molecules on the surface of the APCs. In this method, peptides / polypeptides longer than 15 amino acid residues can be loaded into APCs and processed by proteases in the APC cytoplasm to provide the corresponding TAP as defined herein for presentation. In some embodiments, the precursor peptides / polypeptides used to generate the TAPs defined herein are, for example, 1000, 500, 400, 300, 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, or 15 amino acids or less. Accordingly, all methods and processes using TAP described herein involve the use of longer peptides or polypeptides (including naturally occurring proteins), i.e., tumor antigen precursor peptides / polypeptides, to induce presentation of the "final" 8-14 amino acid TAP after processing by cells (APCs). In some embodiments, the TAPs described herein are approximately 8-14, 8-13, or 8-12 amino acids in length (e.g., 8, 9, 10, 11, 12, or 13 amino acids in length), small enough to fit directly onto an HLA class I molecule. In one embodiment, the TAP contains 20 or fewer amino acids, preferably 15 or fewer amino acids, and more preferably 14 or fewer amino acids. In one embodiment, the TAP contains at least 7 amino acids, preferably at least 8 or fewer amino acids, and more preferably at least 9 amino acids.

[0091] As used herein, the term "amino acid" includes both L- and D-isomers of naturally occurring amino acids, as well as other amino acids (e.g., naturally occurring amino acids, non-naturally occurring amino acids, amino acids not encoded by nucleic acid sequences, etc.) used in peptide chemistry to prepare synthetic analogs of TAP. Examples of naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc. Other amino acids include, for example, non-genetically encoded forms of amino acids, amino acid analogs, and conservative substitutions for L-amino acids. Naturally occurring non-genetically encoded amino acids and amino acid analogs Examples of amino acids include β-alanine, 3-aminopropionic acid, 2,3-diaminopropionic acid, α-aminoisobutyric acid (Aib), 4-aminobutyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-naphthylalanine, pyridylalanine, 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, Examples of amino acids include methylparaben, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, beta-2-thienylalanine, methionine sulfoxide, L-homoarginine (HoArg), N-acetyllysine, 2-aminobutyric acid, 2,4-diaminobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine, homoserine (HoSer), cysteic acid, epsilon-aminohexanoic acid, delta-aminovaleric acid, benzyloxytyrosine, β-phenylalanine, and 2,3-diaminobutyric acid (D- or L-). These amino acids are widely known in the fields of biochemistry / peptide chemistry. Therefore, one or more amino acids contained in the TAPs (SEQ ID NOS: 1-61 or 1-35) described herein may be substituted with non-genetically encoded amino acids and / or amino acid analogs.TAP can also be modified to improve the protease resistance of the peptide, for example, by incorporating methyl amino acids, β-amino acids, or peptoids. In one embodiment, TAP contains only naturally occurring amino acids.

[0092] In some embodiments, the TAPs described herein include peptides with altered sequences containing functionally equivalent amino acid residue substitutions compared to the sequences described herein. For example, one or more amino acid residues within a sequence can be substituted with another amino acid of similar polarity (having similar physicochemical properties) that acts as a functional equivalent, resulting in a silent mutation. Substitutes for amino acids within a sequence can be selected from other members of the class to which the amino acid belongs. For example, positively charged (basic) amino acids include arginine, lysine, and histidine (as well as homoarginine and ornithine). Nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan, and methionine. Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Negatively charged (acidic) amino acids include glutamic acid and aspartic acid. The amino acid glycine can be included in either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions. The TAPs described herein can contain any L-amino acid, any D-amino acid, or a mixture of L- and D-amino acids. In one embodiment, the BC TAPs described herein contain all L-amino acids.

[0093] In one embodiment, in the sequence of TAP comprising or consisting of any one of SEQ ID NOs: 1 to 61 or 1 to 35, amino acid residues that do not substantially contribute to interaction with the T cell receptor may be modified by substituting them with other amino acids that do not substantially affect T cell responsiveness and do not impair binding to the relevant MHC.

[0094] TAPs can also be modified by replacing one or more of the peptide's amide bonds, which may improve chemical stability and / or enhance biological / pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.). Typical peptide bond replacements include esters, polyamines and their derivatives, and substituted alkanes and alkenes such as aminomethyl and ketomethylene. For example, in the above TAP, one or more amide bonds may be replaced with bonds such as -CHNH-, -CHS-, -CH-CH-, -CH=CH- (cis or trans), -CHSO-, -CH(OH)CH-, or -COCH-.

[0095] TAP may also be N-terminally and / or C-terminally capped or modified to prevent degradation and improve stability, affinity and / or uptake. Thus, in another aspect, the present disclosure provides a compound of formula Z 1 -XZ 2 In the formula, X is a TAP comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 61 or 1 to 35.

[0096] In one embodiment, the amino terminal residue of TAP (i.e., the N-terminal free amino group) is modified (e.g., to prevent degradation), e.g., by covalent attachment of a moiety / chemical group (Z 1 ) is modified by the addition of Z 1 may be a linear or branched alkyl group of 1 to 8 carbons, or an acyl group (R—CO—), where R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, iso-propionyl, or iso-butanyl), or an aroyl group (Ar—CO—), where Ar is an aryl group. In one embodiment, the acyl group is a C1-C 16 or C3-C 16 In a further embodiment, Z is a saturated C1-C6 acyl group (linear or branched, saturated or unsaturated), or an unsaturated C3-C6 acyl group (linear or branched), such as an acetyl group (CH3-CO-, Ac). 1The carboxy-terminal residue of TAP (i.e., the free carboxy group at the C-terminus of TAP) may be modified (e.g., to prevent degradation), for example, by amidation (substitution of an OH group with an NH2 group), and in such cases, Z 2 is an NH group. In one embodiment, Z 2 may be a hydroxamate group, a nitrile group, an amide (primary, secondary, or tertiary) group, an aliphatic amine of 1 to 10 carbons such as methylamine, iso-butylamine, iso-valerylamine, or cyclohexylamine, an aromatic or arylalkyl amine such as aniline, naphthylamine, benzylamine, cinnamylamine, or phenylethylamine, an alcohol, or CHOH. 1 In one embodiment, TAP comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 61 or 1 to 35. In one embodiment, TAP consists of the amino acid sequence of any one of SEQ ID NOs: 1 to 61 or 1 to 35, i.e., Z 1 and Z 2 does not exist.

[0097] In another aspect, the disclosure provides a TAP that binds to an HLA-A*02:01 molecule comprising or consisting of the sequence of SEQ ID NO: 22. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-A*02:05, HLA-A*02:06, and / or HLA-A*02:07 molecules.

[0098] In another aspect, the disclosure provides a TAP that binds to an HLA-A*03:01 molecule comprising or consisting of the sequence of SEQ ID NO: 19. Because HLA alleles are cross-reactive (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-A*03:02 or HLA-A*30:01 molecules.

[0099] In another aspect, the disclosure provides a TAP that binds to an HLA-A*11:01 molecule comprising or consisting of the sequence of SEQ ID NO: 10, 17, or 28. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-A*03:01, HLA-A*31:01, and / or HLA-A*68:01 molecules.

[0100] In another aspect, the disclosure provides a TAP that binds to HLA-A*24:02 molecules, comprising or consisting of the sequence of SEQ ID NO: 6. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAP can also bind to HLA-A*23:01 molecules.

[0101] In another aspect, the disclosure provides a TAP that binds to an HLA-A*25:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 1. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to an HLA-A*23:01 molecule.

[0102] In another aspect, the disclosure provides a TAP that binds to an HLA-A*26:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 15. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-A*25:01 or HLA-A*66:01 molecules.

[0103] In another aspect, the disclosure provides a TAP that binds to an HLA-A*31:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 8, 9, or 29. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-A*11:01, HLA-A*33:01, HLA-A*33:03, or HLA-A*68:01 molecules.

[0104] In another aspect, the disclosure provides a TAP that binds to an HLA-A*33:01 molecule comprising or consisting of the sequence of SEQ ID NO: 2 or 3. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-A*31:01, HLA-A3*3:03, or HLA-A*68:01 molecules.

[0105] In another aspect, the disclosure provides a TAP that binds to an HLA-B*15:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 26. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-A*25:01, HLA-A*29:02, HLA-B*15:01, HLA-B*15:03, HLA-B*15:18, HLA-B*35:01, or HLA-B*46:01 molecules.

[0106] In another aspect, the disclosure provides a TAP that binds to HLA-B*18:01 molecules, comprising or consisting of the sequence of SEQ ID NO: 13, 14, or 30. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-B*40:01, HLA-B*44:02, HLA-B*44:03, and / or HLA-B*45:01 molecules.

[0107] In another aspect, the disclosure provides a TAP that binds to HLA-B*27:05 molecules, comprising or consisting of the sequence of SEQ ID NO: 27. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAP can also bind to HLA-B*27:02 molecules.

[0108] In another aspect, the disclosure provides a TAP that binds to an HLA-B*35:01 molecule comprising or consisting of the sequence of SEQ ID NO: 4, 12, or 23. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-B*15:02, HLA-B*35:02, HLA-B*35:03, or HLA-B*53:01 molecules.

[0109] In another aspect, the disclosure provides a TAP that binds to an HLA-B*35:03 molecule comprising or consisting of the sequence of SEQ ID NO: 35. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-B*07:02, HLA-B*35:01, HLA-B*35:03, HLA-B*51:01, HLA-B*53:01, HLA-B*55:01, or HLA-B*56:01 molecules.

[0110] In another aspect, the disclosure provides a TAP that binds to an HLA-B*35:08 molecule, comprising or consisting of the sequence of SEQ ID NO:35.

[0111] In another aspect, the disclosure provides a TAP that binds to HLA-B*38:01 molecules, comprising or consisting of the sequence of SEQ ID NO: 31. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-B*39:01 molecules.

[0112] In another aspect, the present disclosure provides a TAP that binds to an HLA-B*40:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 20. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-molecules.

[0113] In another aspect, the disclosure provides a TAP that binds to an HLA-B*49:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 11 or 24. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-B*18:01, HLA-B*40:02, HLA-B*41:02, HLA-B*44:02, HLA-B*44:03, or HLA-B*45:01 molecules.

[0114] In another aspect, the disclosure provides a TAP that binds to an HLA-B*50:01 molecule, comprising or consisting of the sequence of SEQ ID NO:5 or SEQ ID NO:7.

[0115] In another aspect, the disclosure provides a TAP that binds to HLA-B*51:01 molecules, comprising or consisting of the sequence of SEQ ID NO: 32 or 34. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-B*52:01 molecules.

[0116] In another aspect, the disclosure provides a TAP that binds to an HLA-B*52:01 molecule comprising or consisting of the sequence of SEQ ID NO: 18. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the TAPs identified above can also bind to HLA-B*51:01 molecules.

[0117] In another aspect, the disclosure provides a TAP that binds to an HLA-B*58:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 33. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-A*32:01 or HLA-B*57:01 molecules.

[0118] In another aspect, the disclosure provides a TAP that binds to an HLA-C*01:02 molecule, comprising or consisting of the sequence of SEQ ID NO:25.

[0119] In another aspect, the disclosure provides a TAP that binds to an HLA-C*12:03 molecule, comprising or consisting of the sequence of SEQ ID NO: 16 or 21. Because HLA alleles exhibit cross-reactivity (particular HLA alleles present similar epitopes), the above-identified TAPs can also bind to HLA-B*46:01, HLA-C*03:02, HLA-C*03:03, HLA-C*03:04, HLA-C*08:01, HLA-C*12:03, HLA-C*15:02, or HLA-C*16:01 molecules.

[0120] The TAPs of the present disclosure can be produced by expression in a host cell containing a nucleic acid encoding the TAP (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis). Peptides can be readily synthesized by manual and / or automated solid-phase procedures well known in the art. Suitable synthesis can be carried out, for example, by utilizing the "T-boc" or "Fmoc" procedures. Techniques and procedures for solid-phase synthesis are described, for example, in E. Atherton and R.C. Sheppard, "Solid Phase Peptide Synthesis: A Practical Approach" (IRL, Oxford University Press, 1989). Alternatively, TAP can be prepared by the methods described in Liu et al., Tetrahedron Lett. 37:37:933-936, 1996; Baca et al., J. Am. Chem. Soc. 117:1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res. 45:209-216, 1995; Schnolzer and Kent, Science 256:221-225, 1992; Liu and Tam, J. Am. Chem. Soc. 116:116:4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91:91:6584-6588, 1994, and Yamashiro and Li, Int. J. Peptide Protein Res. 31:31:322-334, 1988. Other methods useful for synthesizing TAP are described in Nakagawa et al., J. Am. Chem. Soc. 107:107:7087-7092, 1985. In one embodiment, TAP is chemically synthesized (synthetic peptide). Another embodiment of the present disclosure relates to a non-naturally occurring peptide, which consists of or consists essentially of an amino acid sequence defined herein and is synthetically produced (e.g., synthesized) as a pharmaceutically acceptable salt. The salts of TAP described in the present disclosure differ significantly from the in vivo state(s) of the peptide, since peptides produced in vivo do not take the salt form.The non-natural peptide salts can adjust the solubility of the peptides, particularly in pharmaceutical compositions containing the peptides, such as the peptide vaccines disclosed herein. These salts are preferably pharmaceutically acceptable salts of the peptides. In one embodiment, the TAP described herein is substantially pure. A compound being "substantially pure" refers to the state in which the compound has been separated from components that naturally accompany it. Typically, a compound is considered substantially pure when it is present in a weight ratio of at least 60%, more usually 75%, 80%, or 85%, preferably greater than 90%, and even more preferably greater than 95%, of the total amount of material in a sample. Thus, for example, a chemically synthesized or recombinantly produced polypeptide is generally substantially separated from its naturally accompanyable components, e.g., components of its source macromolecule. A nucleic acid molecule is considered substantially pure when it is not immediately adjacent to (i.e., covalently linked to) coding sequences with which it is normally adjacent in the naturally occurring genome of the organism from which it is derived. A substantially pure compound can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid molecule encoding the peptide compound, or by chemical synthesis. Purity can be measured using any suitable method, such as column chromatography, gel electrophoresis, or HPLC. In one embodiment, the TAP is in solution. In another embodiment, the TAP is in a solid form, for example, lyophilized.

[0121] In one embodiment, TAP is encoded by a sequence located in a non-protein-coding region of the genome. In one embodiment, TAP is encoded by a sequence located in an intergenic region. In another embodiment, TAP is encoded by a non-coding RNA (ncRNA).

[0122] In another aspect, the present disclosure further provides synthetic long peptides (SLPs) comprising at least one of the TAPs described herein. In one embodiment, the SLP comprises at least two TAPs, wherein at least one TAP is as described herein. In one embodiment, the SLP comprises at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 of the TAPs described herein. In one embodiment, the SLP comprises at least one TAP linked to one or more amino acid sequences or domains described herein that confer a desired property to the SLP, e.g., a sequence or domain that stabilizes the SLP and / or improves processing and presentation by MHC molecules, e.g., a sequence containing a motif cleavable by an intracellular protease, such as a cathepsin. In another embodiment, the SLP comprises at least one TAP described herein and a TAP that binds to an MHC class II molecule. The TAPs may be linked directly to each other or indirectly via a linker, such as a short amino acid linker. In some embodiments, the linker comprises about 4-20 amino acids, or about 4-15 amino acids, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In one embodiment, the linker comprises glycine, serine, proline, or threonine residues, or a mixture thereof. The linker can include a sequence that facilitates processing of the SLP to release TAP, such as a cathepsin-sensitive linker (e.g., a 4-6 amino acid linker including the sequence LVGS, ASLG, PIVG, LLSV, VLSVG, or LLSVGG; see Rabu et al., Oncoimmunology. 2019;8(4):e1560919). In one embodiment, the SLP is no longer than 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, or 50 amino acids in length. In a further embodiment, the SLP is from 20 to 50, 45 or 40 amino acids in length, for example, from 20 or 25 amino acids to 30, 35 or 40 amino acids in length.

[0123] In another aspect, the present disclosure further provides (isolated) nucleic acids encoding the TAP or tumor antigen precursor peptides or SLPs described herein. In one embodiment, the nucleic acids comprise about 24-1200 nucleotides, about 24-1000, 900, 800, 700, 600, 500, 400, 300, or 200 nucleotides, e.g., about 24-150 or 100 nucleotides, e.g., 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, or 72 nucleotides. As used herein, "isolated" refers to a peptide or nucleic acid molecule that is separated from other components present in the molecule's natural environment or macromolecules of its naturally occurring source (e.g., including other nucleic acids, proteins, lipids, sugars, etc.). As used herein, "synthetic" refers to a peptide or nucleic acid molecule that has not been isolated from its natural source, e.g., produced through recombinant technology or using chemical synthesis. In one embodiment, a nucleic acid (DNA, RNA) encoding a TAP or SLP of the present disclosure comprises any one of the sequences set forth in the table below or the corresponding RNA sequences. In one embodiment, the nucleic acid encoding the TAP or SLP is an mRNA molecule. In other embodiments, the nucleic acid encoding the TAP or SLP is a self-amplifying mRNA (saRNA), a transcription-amplified mRNA (taRNA), or a circular mRNA (circRNA) (see, e.g., Liu et al., Nature Reviews Cancer, 23:8, 2023, 526-543). [Table 4-1] [Table 4-2]

[0124] Of course, due to the degeneracy of the genetic code, the TAPs described herein may be encoded by variants of the above sequences.

[0125] The nucleic acids of the present disclosure can be used for recombinant expression of the TAPs or SLPs of the present disclosure and can be included in vectors or plasmids, such as cloning or expression vectors, that can be transfected into host cells. In one embodiment, the present disclosure provides a cloning vector, expression vector, viral vector, or plasmid containing a nucleic acid sequence encoding a TAP of the present disclosure. Alternatively, a nucleic acid encoding a TAP of the present disclosure can be integrated into the genome of a host cell. In either case, the host cell expresses the TAP or protein encoded by the nucleic acid. The term "host cell," as used herein, refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. A host cell can be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., insect, yeast, plant, or mammalian cell) capable of expressing a TAP described herein. A vector or plasmid contains the necessary elements for the transcription and translation of an inserted coding sequence and may include other components, such as resistance genes, cloning sites, etc. Methods well known to those skilled in the art can be used to construct expression vectors in which a peptide or polypeptide coding sequence and appropriate transcriptional and translational control / regulatory elements are operably linked thereto. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al. (1989) "Molecular Cloning, A Laboratory Manual" (Cold Spring Harbor Press), Plainview, NY, and Ausubel, FM et al. (1989) "Current Protocols in Molecular Biology" (John Wiley & Sons), New York, NY. "Operably linked" refers to the juxtaposition of components, particularly components that enable the normal function of the nucleotide sequence to be carried out. Thus, a coding sequence operably linked to a regulatory sequence refers to a nucleotide sequence configuration in which the coding sequence can be expressed under the regulatory control, i.e., transcriptional and / or translational control, of the regulatory sequence.As used herein, "regulatory / control region" or "regulatory / control sequence" refers to a non-coding nucleotide sequence involved in regulating the expression of an encoding nucleic acid. Thus, the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like. A vector (e.g., an expression vector) may have necessary 6' upstream and 3" downstream regulatory elements for efficient gene transcription and translation in a respective host cell, such as a promoter sequence, such as the CMV, PGK, and EF-1α promoters, a ribosome recognition and binding TATA box, and a 3' UTR AAUAAA transcription termination sequence. Other suitable promoters include the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukosis virus promoter, EBV immediate early promoter, and constitutive promoter of the Rous sarcoma Vims promoter. Human gene promoters may also be used, including, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In certain embodiments, an inducible promoter is also contemplated as part of a vector expressing TAP. This provides a molecular switch that can turn on or off expression of a polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, or tetracycline promoters. Examples of vectors include plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.Examples of categories of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the Lenti-X™ Bicistronic Expression System (Neo) vector (Contech) for expression in mammalian cells, the pClneo vector (Promega), and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequence for TAP disclosed herein can be ligated into such expression vectors for expression of TAP in mammalian cells.

[0126] In certain embodiments, nucleic acids encoding the TAPs of the present disclosure are provided in a viral vector. The viral vector may be derived from adenovirus, vaccinia virus, retrovirus, lentivirus, or foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and is capable of being packaged into a viral vector particle. The viral vector may contain coding sequences for various TAPs or SLPs described herein in place of non-essential viral genes. In another embodiment, nucleic acids encoding the TAPs or SLPs of the present disclosure are provided in a self-amplifying or self-replicating RNA (saRNA or srRNA) vector. The srRNA is derived from a positive-stranded RNA virus from which structural proteins have been removed and replaced with a heterologous gene of interest. srRNA has been successfully derived from flaviviruses, nodamuraviruses, nidoviruses, and alphaviruses, and therapeutic versions of this technology use structural proteins delivered in trans to generate single-cycle viral replicon particles (VRPs) (see, e.g., Aliahmad et al., "Next generation self-replicating RNA vectors for vaccines and immunotherapies." Cancer Gene Ther (2022). https: / / doi.org / 10.1038 / s41417-022-00435-8). The vectors and / or particles can be used to introduce DNA, RNA, or other nucleic acids into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0127] In one embodiment, a nucleic acid (DNA, RNA) encoding a TAP or SLP of the disclosure is contained within a vesicle or nanoparticle, such as a lipid vesicle (e.g., liposome) or lipid nanoparticle (LNP), or other suitable vehicle (e.g., mRNA packaging system). Thus, in another aspect, the disclosure provides a vesicle or nanoparticle, such as a lipid vesicle or lipid nanoparticle, comprising a nucleic acid, e.g., mRNA, encoding one or more TAPs or SLPs.

[0128] The term liposome, as used herein, refers according to its ordinary meaning to a microscopic lipid vesicle composed of a bilayer of phospholipids or any similar amphiphilic lipid (e.g., sphingolipid) encapsulating an internal aqueous medium.

[0129] The term "lipid nanoparticle" refers to a liposome-like structure that can include one or more lipid bilayer rings surrounding an internal aqueous medium, similar to liposomes, or a micelle-like structure that encapsulates molecules (e.g., nucleic acids) within a non-aqueous core. Lipid nanoparticles typically contain a cationic lipid, such as an ionizable cationic lipid. Examples of cationic lipids that can be used in LNPs include DOTMA, DOSPA, DOTAP, DOPE, ePC, DLin-MC3-DMA, C12-200, ALC-0315, cKK-E12, Lipid H (SM-102), OF-Deg-Lin, A2-Iso5-2DC18, 306O i10 , BAME-O16B, TT3, 9A1P9, FTT5, COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC, and COATSOME® SS-OP (see, e.g., Hou et al., Nature Reviews Materials, Vol. 6, pp. 1078-1094 (2021); Tenchov et al., ACS Nano, 15, pp. 16982-17015 (2021)).

[0130] Liposomes and lipid nanoparticles typically contain lipids, lipid-like materials, and other lipid components, such as polymers, which can improve liposome or nanoparticle properties such as stability, delivery efficacy, tolerability, and biodistribution. These include phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol), such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and DOPE, sterols (e.g., cholesterol and its derivatives), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG), and other lipid-containing polymers. 2000 -DMG) and 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG 2000 PEGylated lipids (PEG-lipids) such as PEG-DSG.

[0131] In one embodiment, the lipid nanoparticles described herein comprise one or more cationic lipids, such as ionizable cationic lipids. Examples of ionizable cationic lipids include those described in PCT Publication Nos. WO2017 / 061150 and WO2019 / 188867, including those commercially available under the trade names COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC, and COATSOME® SS-OP.

[0132] Nucleic acids (e.g., mRNA) encoding one or more of the TAPs may be modified to improve stability and / or reduce immunogenicity. For example, the 5' end can be capped to stabilize the molecule and reduce immunogenicity (e.g., as described in US 10,519,189 and US 10,494,399). One or more nucleosides in the mRNA may be substituted or modified with 1-methylpseudouridine, pseudouridine (ψ), N6-methyladenosine, 5-methylcytidine, and / or 5-methyluridine, which improves the molecule's stability, improves translation efficiency, and / or reduces recognition by the innate immune system. Modified nucleoside forms are described in US 9,371,511. Other examples of modifications that can be made to mRNA include the incorporation of anti-reverse cap analog (ARCA), 5'-methyl-cytidine triphosphate (m5CTP), N6-methyl-adenosine-5'-triphosphate (m6ATP), 2-thio-uridine triphosphate (s2UTP), pseudouridine triphosphate, N1-methylpseudouridine triphosphate, or 5-methoxyuridine triphosphate (5moUTP). The mRNA may also contain additional modifications to the 5' and / or 3' untranslated regions (UTRs) and polyadenylation (polyA) tails (see, e.g., Kim et al., Molecular & Cellular Toxicology, Vol. 18(1) (2022):1-8). The poly(A) tail preferably contains 100-200 nucleotides, more preferably 120-150 nucleotides, and may contain modified adenosines. All of these modifications, and other modifications to nucleic acids (eg, mRNA) encoding TAP, are encompassed by the present disclosure.

[0133] In another aspect, the present disclosure provides an MHC class I molecule comprising (i.e., presenting or bound to) one or more of the BC TAPs comprising or consisting of the sequences of SEQ ID NOs: 1-61 or 1-35.

[0134] In one embodiment, the MHC class I molecule is an HLA-A*02:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A*03:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A*11:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A*24:02 molecule. In one embodiment, the MHC class I molecule is an HLA-A*25:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A*26:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A*31:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A*33:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*15:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*18:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*27:05 molecule. In one embodiment, the MHC class I molecule is an HLA-B*35:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*35:03 molecule. In one embodiment, the MHC class I molecule is an HLA-B*38:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*40:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*49:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*50:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*51:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*52:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*58:01 molecule. In one embodiment, the MHC class I molecule is an HLA-C*01:02 molecule. In one embodiment, the MHC class I molecule is an HLA-C*12:03 molecule.

[0135] In one embodiment, TAP (e.g., comprising or consisting of a sequence of SEQ ID NOS: 1-61 or 1-35 as defined herein) is non-covalently linked to an MHC class I molecule (i.e., TAP is loaded into or non-covalently bound to the peptide-binding groove / pocket of the MHC class I molecule). In another embodiment, TAP is covalently linked / bound to an MHC class I molecule (alpha chain). In such a configuration, TAP and the MHC class I molecule (alpha chain) are typically produced as a synthetic fusion protein with a short (e.g., 5-20 residues, preferably about 8-12, e.g., 10) flexible linker or spacer (e.g., a polyglycine linker). In another aspect, the present disclosure provides a nucleic acid encoding a fusion protein in which TAP as defined herein is fused to an MHC class I molecule (alpha chain). In one embodiment, the MHC class I molecule (alpha chain)-peptide complex is multimerized. Thus, in another aspect, the present disclosure provides multimers of MHC class I molecules loaded (covalently or otherwise) with a TAP as described herein. Such multimers may be attached to a tag, e.g., a fluorescent tag, that allows for detection of the multimer. Many strategies have been developed for producing MHC multimers, including MHC dimers, tetramers, pentamers, octamers, etc. (reviewed in Bakker and Schumacher, Current Opinion in Immunology 2005, 17:428-433). MHC multimers are useful, for example, for the detection and purification of antigen-specific T cells. Thus, in another aspect, the present disclosure provides multimers of CD8 specific for a TAP as defined herein. + A method for detecting or purifying (isolating, enriching) T lymphocytes is provided, the method comprising contacting a cell population with a multimer of MHC class I molecules loaded (covalently or non-covalently) with TAP, and detecting CD8 + and detecting or isolating T lymphocytes. +T lymphocytes can be isolated using known methods, for example, fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

[0136] In yet another aspect, the disclosure provides cells (e.g., host cells), and in one embodiment, an isolated cell comprising a nucleic acid, vector, or plasmid described herein (i.e., a nucleic acid or vector encoding one or more TAPs or SLPs). In another aspect, the disclosure provides a cell that expresses on its surface an MHC class I molecule (e.g., an MHC class I molecule of any one of the alleles disclosed above) bound to or presenting a TAP described in this disclosure. In one embodiment, the host cell is a eukaryotic cell, e.g., a mammalian cell, preferably a human cell, cell line, or immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC), such as a dendritic cell. In one embodiment, the host cell is a primary cell, cell line, or immortalized cell. Nucleic acids and vectors can be introduced into cells via conventional transformation or transfection techniques. The terms "transformation" and "transfection" refer to techniques for introducing foreign nucleic acid into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can be found, for example, in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing nucleic acid into mammalian cells in vivo are also known and can be used to deliver the vectors or plasmids of the present disclosure to a subject for gene therapy.

[0137] Cells, such as APCs, can be loaded with one or more TAPs using various methods known in the art. As used herein, "loading cells with TAP" refers to transfecting cells with RNA or DNA encoding TAP, or TAP itself, or transforming APCs with a nucleic acid encoding TAP. Cells can also be loaded by contacting them with exogenous TAP capable of directly binding to MHC class I molecules present on the cell surface (e.g., peptide-pulsed cells). TAP can also be fused to a domain or motif that promotes its presentation by MHC class I molecules, such as an endoplasmic reticulum (ER) retrieval signal, a C-terminal Lys-Asp-Glu-Leu sequence (see Wang et al., Eur J Immunol. 2004 Dec;34(12):3582-94).

[0138] In another aspect, the disclosure provides compositions or combinations / pools of peptides comprising any one or any combination of the TAPs (or nucleic acids encoding said peptide(s)) defined herein. In one embodiment, the composition comprises any combination of the TAPs defined herein (any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TAPs) or nucleic acids encoding said TAPs. Compositions comprising any combination / subcombination of the TAPs defined herein are encompassed by the disclosure. In another embodiment, the combination or pool can comprise one or more known tumor antigens.

[0139] Thus, in another aspect, the present disclosure provides compositions comprising one or a combination of a TAP as defined herein (e.g., comprising or consisting of a sequence of SEQ ID NOs: 1-61 or 1-35) and a cell expressing an MHC class I molecule (e.g., an MHC class I molecule derived from any one of the alleles disclosed above). APCs for use in the present disclosure are not limited to a particular cell type, and may be CD8 +These include professional APCs, such as dendritic cells (DCs), Langerhans cells, macrophages, and B cells, which are known to present protein antigens on their cell surface for recognition by T lymphocytes. For example, APCs can be obtained either in vitro, ex vivo, or in vivo by inducing DCs from peripheral blood monocytes and then contacting (stimulating) them with TAPs. APCs can also be activated to present TAPs in vivo, where one or more of the TAPs disclosed herein are administered to a subject, and APCs that present TAPs are induced within the subject. The phrases "inducing APCs" or "stimulating APCs" include contacting or loading cells with one or more TAPs or nucleic acids encoding TAPs, resulting in presentation of the TAPs on the cell surface by MHC class I molecules. As described herein, according to the present disclosure, TAP can be indirectly loaded using, for example, a longer peptide / polypeptide (including a natural protein) containing the sequence of TAP, which is then processed (e.g., by a protease) inside the APC to generate a TAP / MHC class I complex on the surface of the cell. After loading the APC with TAP and allowing the APC to present the TAP, the APC can be administered to a subject as a vaccine. For example, ex vivo administration can include the steps of: (a) collecting APCs from a first subject; (b) contacting / loading the APCs of step (a) with TAP to form an MHC class I / TAP complex on the surface of the APCs; and (c) administering the peptide-loaded APCs to a second subject in need of treatment.

[0140] The first subject and the second subject may be the same subject (e.g., an autologous vaccine) or different subjects (e.g., an allogeneic vaccine). Alternatively, the present disclosure provides use of a TAP (or a combination thereof) described herein for manufacturing a composition (e.g., a pharmaceutical composition) for inducing antigen-presenting cells. In addition, the present disclosure provides a method or process for manufacturing a pharmaceutical composition for inducing antigen-presenting cells, the method or process comprising mixing or formulating a TAP, or a combination thereof, with a pharmaceutically acceptable carrier. Cells such as APCs that express MHC class I molecules (e.g., any of the HLA molecules listed above) and are loaded with any one or any combination of the TAPs defined herein can express CD8 + T lymphocytes, e.g., autologous CD8 + Thus, in another aspect, the present disclosure provides a method for the stimulation / expansion of T lymphocytes, comprising administering to a subject a therapeutic agent comprising any one or any combination of TAPs as defined herein (or a nucleic acid or vector encoding the same), a cell expressing an MHC class I molecule, and a T lymphocyte, more specifically a CD8 + T lymphocytes (e.g., CD8 + The present invention provides a composition comprising a cell population comprising T lymphocytes.

[0141] In one embodiment, the composition further comprises a buffer, excipient, carrier, diluent, and / or medium (e.g., culture medium). In a further embodiment, the buffer, excipient, carrier, diluent, and / or medium is a pharmaceutically acceptable buffer(s), excipient(s), carrier(s), diluent(s), and / or medium. As used herein, "pharmaceutically acceptable buffer, excipient, carrier, diluent, and / or medium" includes all solvents, buffers, binders, lubricants, fillers, thickeners, disintegrants, plasticizers, coatings, barrier layer formulations, stabilizers, release retardants, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and the like that are physiologically compatible, do not interfere with the effectiveness of the biological activity of the active ingredient(s), and are non-toxic to the subject. The use of such media and agents for pharmaceutically active substances is well known in the art (Rowe et al., Handbook of Pharmaceutical Excipients, 2003, 4th Edition, Pharmaceutical Press, London UK). Use of any conventional media or agent in the compositions of the present disclosure is contemplated, except insofar as it is incompatible with the active compound (peptide, cells). In one embodiment, the buffer, excipient, carrier, and / or medium is a non-naturally occurring buffer, excipient, carrier, and / or medium. In one embodiment, one or more of the TAPs defined herein, or a nucleic acid (e.g., mRNA) encoding said one or more TAPs, is contained within or complexed with a lipid vesicle or liposome, e.g., a cationic liposome (see, e.g., Vitor MT et al., Recent Pat Drug Deliv Formul, 2013 Aug;7(2):99-110), or other suitable carrier.

[0142] In another aspect, the present disclosure provides a composition comprising one or more of any one of the TAPs defined herein (e.g., a TAP comprising or consisting of a sequence of SEQ ID NOs: 1-61 or 1-35) (or a nucleic acid, such as an mRNA, encoding said peptide(s)), or any combination thereof, and a buffer, excipient, carrier, diluent, and / or vehicle. For compositions comprising cells (e.g., APCs, T lymphocytes), the composition comprises a suitable medium that allows for the maintenance of viable cells. Representative examples of such media include saline, Earl's Buffered Saline Solution (Life Technologies®), or PlasmaLyte® (Baxter International®). In one embodiment, the composition (e.g., pharmaceutical composition) is an "immunogenic composition," "vaccine composition," or "vaccine." As used herein, the terms "immunogenic composition," "vaccine composition," or "vaccine" refer to a composition or formulation that comprises one or more TAPs, nucleic acids, or vaccine vectors and that, when administered to a subject, is capable of eliciting an immune response against one or more TAPs present therein. Vaccination methods for inducing an immune response in a mammal (e.g., a human) include the use of vaccines or vaccine vectors administered via any conventional route known in the vaccine art, for example, via mucosal (ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary) surfaces, via parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) routes, or by topical administration (e.g., transdermal delivery systems such as patches). In one embodiment, a TAP (or combination thereof) is conjugated to a carrier protein (conjugate vaccine) to increase the immunogenicity of the TAP(s). Accordingly, the present disclosure provides compositions (conjugates) comprising a TAP (or combination thereof) or a nucleic acid encoding a TAP, or a combination thereof, and a carrier protein.For example, the TAP(s) or nucleic acid(s) can be conjugated or complexed to a Toll-like receptor (TLR) ligand (see, e.g., Zom et al., Adv Immunol. 2012;114:177-201) or a polymer / dendrimer (see, e.g., Liu et al., Biomacromolecules. 2013 Aug. 12;14(8):2798-806), such as a polymer-conjugated TLR agonist (see, e.g., Lynn et al., Nature Biotechnology 33:1201-1210 (2015); Lynn et al., Nature Biotechnology 38:320-332 (2020)). In one embodiment, the immunogenic composition or vaccine further comprises an adjuvant. "Adjuvant" refers to a substance that, when added to an immunogenic agent such as an antigen (an AP, nucleic acid, and / or cell as described in this disclosure), non-specifically enhances or potentiates the immune response to the agent in a host upon exposure to the mixture.Examples of adjuvants currently used in the vaccine field include: (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gel), squalene; (2) oil-based adjuvants (oil emulsions and surfactant-based formulations, e.g., MF59 (microfluidized detergent-stabilized oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (water-in-oil emulsion + MPL + QS-21)); (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza hemagglutinin), AS04 [SBAS4] (aluminum salts and MPL), ISCOMS (structured complexes of saponin and lipids), polylactic-co-glycolic acid (PLG); and (4) those derived from microorganisms (natural and synthetic), e.g., monophosphoryl Examples of suitable adjuvant vaccines include lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipidic immunostimulatory substance capable of self-assembly to form liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), genetically modified cholera toxin (CT) and Escherichia coli enterotoxin (LT) (bacterial toxins genetically modified to provide a non-toxic adjuvant effect), (5) endogenous human immunomodulatory factors, such as hGM-CSF or hIL-12 (cytokines administered either as proteins or as encoded plasmids), Immudaptin (C3d tandem array), and / or (6) an inert vehicle such as gold particles. In one embodiment, the vaccine is an RNA vaccine.

[0143] In one embodiment, the TAP(s) (e.g., comprising or consisting of a sequence of SEQ ID NOs: 1-61 or 1-35) or SLP (or nucleic acid, such as mRNA, encoding said peptide(s)), or composition comprising them, is in lyophilized form. In another embodiment, the TAP(s), SLP(s), nucleic acid(s), or composition comprising them, is a liquid composition. In a further embodiment, the TAP(s) or nucleic acid(s) are present in the composition at a concentration of about 0.01 μg / mL to about 100 μg / mL. In further embodiments, the TAP(s) or nucleic acid(s) are present at a concentration of about 0.2 μg / mL to about 50 μg / mL, about 0.5 μg / mL to about 10, 20, 30, 40, or 50 μg / mL, about 1 μg / mL to about 10 μg / mL, or about 2 μg / mL.

[0144] As described herein, cells such as APCs expressing MHC class I molecules loaded with or bound to any one or any combination of TAPs can be used to induce CD8 + can be used to stimulate / expand T lymphocytes. Thus, in another aspect, the present disclosure provides T cell receptor (TCR) molecules capable of interacting with or binding to the MHC class I molecule / TAP complexes described herein, as well as nucleic acid molecules encoding such TCR molecules, and vectors comprising such nucleic acid molecules. The TCRs described in the present disclosure can specifically interact with or bind to TAP loaded on or presented by MHC class I molecules, preferably on the surface of living cells in vitro or in vivo.

[0145] As used herein, the term TCR refers to a member of the immunoglobulin superfamily that has a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail (e.g., as described in Janeway et al., "Immunobiology: The Immune System in Health and Disease," 3rd ed., Current Biology Publications, p. 4:33 (1997)), and is capable of specifically binding an antigenic peptide bound to an MHC receptor. TCRs can be present on the surface of cells and generally consist of a heterodimer having an α chain and a β chain (also known as TCRα and TCRβ, respectively). Similar to immunoglobulins, the extracellular portion of a TCR chain (e.g., α chain, β chain) contains two immunoglobulin regions: a variable region (e.g., a TCR variable α region or Vα, and a TCR variable β region or Vβ; typically, amino acids 1-116 according to Rabat numbering at the N-terminus) and one constant region adjacent to the cell membrane (e.g., a TCR constant domain α or Cα, typically, amino acids 117-259 according to Rabat; a TCR constant domain β or Cβ, typically, amino acids 117-295 according to Rabat). Also similar to immunoglobulins, the variable domains contain complementarity-determining regions (CDRs, three in each chain) separated by framework regions (FRs). In certain embodiments, TCRs are present on the surface of T cells (or T lymphocytes) and associate with the CD3 complex.

[0146] TCRs, and specifically nucleic acids encoding TCRs of the present disclosure, can be applied to, for example, T lymphocytes (e.g., CD8 + T lymphocytes), or other types of lymphocytes, can be genetically transformed / modified to generate novel T lymphocyte clones that specifically recognize the MHC class I / TAP complex. In certain embodiments, T lymphocytes (e.g., CD8 + T lymphocytes (e.g., CD8 T lymphocytes) are transformed to express one or more TCRs that recognize TAP, and the transformed cells are administered to the patient (autologous cell transfusion). In certain embodiments, T lymphocytes (e.g., CD8 T lymphocytes) obtained from a donor are transfected to express one or more TCRs that recognize TAP, and the transfected cells are administered to the patient (autologous cell transfusion). +In another embodiment, the present disclosure provides a method for the transfection of T lymphocytes, e.g., CD8 T lymphocytes, transformed / transfected with a vector or plasmid encoding a TAP-specific TCR. + T lymphocytes are provided. In further embodiments, the present disclosure provides methods of treating patients with autologous or allogeneic cells transformed with a TAP-specific TCR. In certain embodiments, the TCR is expressed in primary T cells (e.g., cytotoxic T cells) by replacing an endogenous locus, e.g., the endogenous TRAC and / or TRBC locus, using, e.g., CRISPR, TALEN, zinc finger, or other targeted disruption systems.

[0147] In another embodiment, the present disclosure provides a nucleic acid encoding the TCR described above. In a further embodiment, the nucleic acid is present in a vector, such as the vector described above.

[0148] In yet a further embodiment, there is provided the production and use of autologous or allogeneic cells using tumor antigen-specific TCRs for the treatment of breast cancer.

[0149] In some embodiments, patients treated with compositions (e.g., pharmaceutical compositions) of the present disclosure are treated prior to or after treatment with anti-tumor agents and / or immunotherapy (e.g., CAR therapy, immune checkpoint inhibitor therapy). The compositions of the present disclosure include allogeneic T lymphocytes (e.g., CD8 + T lymphocytes), allogeneic or autologous APC vaccines loaded with TAP, vaccines containing nucleic acid (e.g., mRNA) encoding TAP and allogeneic or autologous T lymphocytes (e.g., CD8 +These include lymphocytes transformed with tumor antigen-specific TCRs, or tumor antigen-specific TCRs. By providing a T lymphocyte clone capable of recognizing TAP as described herein, T lymphocyte clones can be generated in a subject (e.g., a transplant recipient), such as an allogeneic T lymphocyte and / or donor lymphocyte infusion (DLI) recipient, for tumor cells expressing TAP and capable of specifically targeting the tumor cells. Thus, the present disclosure provides a method for generating a CD8 T cell clone that encodes and expresses a T cell receptor capable of specifically recognizing or binding to a TAP / MHC class I molecule complex. + Provide T lymphocytes. The above T lymphocytes (e.g., CD8 + The CD8 T lymphocytes described herein may be recombinant (engineered) T lymphocytes or naturally selected T lymphocytes. + At least two methods for producing T lymphocytes are provided, including contacting undifferentiated lymphocytes with TAP / MHC class I molecule complexes (typically expressed on the surface of cells such as APCs) under conditions that induce T cell activation and T cell proliferation, which can be done in vitro or in vivo (i.e., in patients who have received an APC vaccine in which APCs have been loaded with TAP, or in patients who have been treated with a TAP vaccine). By using combinations or pools of TAPs bound to MHC class I molecules, CD8 T cells that can recognize multiple TAPs can be generated. + Alternatively, it is possible to generate a T lymphocyte population that is specifically engineered to contain the MHC class I molecule / TAP complex (i.e., engineered CD8 + T lymphocytes or recombinant CD8 +Tumor antigen-specific or targeted T lymphocytes can be produced / produced in vitro or ex vivo by cloning one or more nucleic acids (genes) encoding TCRs (more specifically, alpha and beta chains) that specifically bind to TAP (T lymphocytes). Nucleic acids encoding the TAP-specific TCRs of the present disclosure can be obtained from T lymphocytes activated against TAP ex vivo (e.g., using APCs loaded with TAP) or from individuals that have an immune response to a peptide / MHC molecule complex, using methods known in the art. The TAP-specific TCRs described in the present disclosure can be recombinantly expressed in host cells and / or host lymphocytes obtained from the graft recipient or graft donor and, optionally, differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). Nucleic acid(s) (transgene(s)) encoding the TCR alpha and beta chains can be introduced into T cells (e.g., from the subject to be treated or from another individual) using any suitable method, such as transfection (e.g., electroporation) or transduction (e.g., using a viral vector). Engineered CD8 T cells expressing a TCR specific for TAP can be used to induce T cell proliferation and differentiation. + T lymphocytes can be expanded in vitro using well-known culture methods.

[0150] The present disclosure provides methods for generating immune effector cells that express a TCR described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells, e.g., immune effector cells isolated from a subject, such as a subject with breast cancer, so that the immune effector cells express one or more TCRs. In certain embodiments, immune effector cells are isolated from an individual and genetically modified in vitro without further manipulation. Such cells can then be directly readministered to the individual. In a further embodiment, immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a TCR. In this regard, the immune effector cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express a TCR described herein).

[0151] Prior to in vitro manipulation or genetic modification of immune effector cells described herein, a cell source can be obtained from a subject. In particular, immune effector cells for use with the TCRs described herein include T cells. T cells can be obtained from several sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation. In one embodiment, cells from an individual's circulating blood can be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing. In one embodiment of the present invention, the cells are washed with PBS. In alternative embodiments, the wash solution may lack calcium, magnesium, or many, but not all, divalent cations. As will be appreciated by those skilled in the art, the wash step can be accomplished by methods known to those skilled in the art, for example, by using a semi-automated flow-through centrifuge. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions, with or without buffers. In certain embodiments, unwanted components in the apheresis sample can be removed intracellularly and resuspended directly in culture medium. In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO +Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies targeting surface markers unique to the negatively selected cells. One method for use herein includes cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich for CD8+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD4. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure. PBMCs can be used directly for TCR-mediated genetic modification using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated and, in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into subpopulations of naive, memory, and effector T cells, either before or after genetic modification and / or expansion.

[0152] The present disclosure provides methods for the preparation of isolated immune cells (e.g., CD8 + The present disclosure also provides a TAP or combination thereof according to the present disclosure (i.e., one or more TAPs bound to an MHC class I molecule) and a CD8 T lymphocyte capable of recognizing the TAP(s). + In another aspect, the present disclosure provides compositions comprising T lymphocytes (e.g., CD8 T lymphocytes) that specifically recognize one or more MHC class I molecule / TAP complex(es) described herein. + T lymphocytes) or cell cultures (e.g., CD8 +Such enriched populations can be obtained by ex vivo expansion of specific T lymphocytes using cells, such as APCs, that express MHC class I molecules loaded with (e.g., presenting) one or more of the TAPs disclosed herein. As used herein, "enriched" refers to the number of tumor antigen-specific T lymphocytes (e.g., CD8 T lymphocytes) in a population. + In a further embodiment, the percentage of TAP-specific T lymphocytes (e.g., CD8 T lymphocytes) in the cell population is much higher than in an untreated cell population, i.e., one that has not undergone a step of ex vivo expansion of the specific T lymphocytes. + In some embodiments, the percentage of TAP-specific T lymphocytes (e.g., CD8 T lymphocytes) in the cell population is at least about 0.5%, e.g., at least about 1%, 1.5%, 2%, or 3%. + T lymphocytes) is about 0.5 to about 10%, about 0.5 to about 8%, about 0.5 to about 5%, about 0.5 to about 4%, about 0.5 to about 3%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, or about 3% to about 4%. Such cell populations or cultures (e.g., CD8 + T lymphocyte population) that specifically recognize one or more MHC class I molecule / peptide (TAP) complex(es) of interest (e.g., CD8 + The TAP-specific CD8 T lymphocytes can be used for tumor antigen-based cancer immunotherapy, as described in more detail below. + The population of T lymphocytes may be further enriched using affinity-based systems, such as multimers of MHC class I molecules (covalently or non-covalently) loaded with TAP(s) as defined herein. Thus, the present disclosure provides methods for the identification of TAP-specific T lymphocytes (e.g., CD8 + purified or isolated populations of T lymphocytes, e.g., TAP-specific CD8 +A purified or isolated population of TAP-specific CD8+ T lymphocytes is provided, wherein the percentage of T lymphocytes is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0153] In another aspect, the present disclosure provides an antibody or antigen-binding fragment (e.g., a TCR mimetic antibody or TCR-like antibody), or a soluble TCR, that specifically binds to a complex in which a TAP as described herein is bound to an HLA molecule, such as a plurality of HLA molecules as defined herein. As used herein, the term "antibody or antigen-binding fragment thereof" refers to any type of antibody / antibody fragment, including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies, and antibody fragments, so long as it exhibits the desired antigen specificity / binding activity. An antibody fragment comprises a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain Fv, scFv), single-domain antibodies (e.g., from camelids), shark NAR single-domain antibodies, and multispecific antibodies formed from antibody fragments, single-chain diabodies (scDbs), bispecific T cell engagers (BiTEs), dual affinity retargeting molecules (DARTs), bivalent scFv-Fc, and trivalent scFv-Fc. Antibody fragments also include binding portions comprising the CDRs or antigen-binding domains, including V H Area (V H , V H -V H), anticalins, pepbodies, antibody-T cell epitope fusions (troibodies), or peptibodies. In one embodiment, the antibody or antigen-binding fragment thereof is a single-chain antibody, preferably a single-chain Fv (scFv). In one embodiment, the antibody or antigen-binding fragment thereof comprises at least one constant domain, e.g., a light chain and / or heavy chain constant domain, or a fragment thereof. In a further embodiment, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable (Fc) fragment derived from the constant heavy chain of the antibody. In one embodiment, the antibody or antigen-binding fragment is an scFv comprising an Fc fragment (scFV-Fc). In one embodiment, the scFv component is connected to the Fc fragment by a linker, e.g., a hinge. The presence of an Fc region is useful for inducing responses to tumor cells via complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), or antibody-dependent cellular cytotoxicity (ADCC).

[0154] In one embodiment, the antibody or antigen-binding fragment thereof is a multispecific antibody or antigen-binding fragment thereof, such as a bispecific antibody or antigen-binding fragment thereof, wherein at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognizes a complex comprising a TAP described herein bound to an HLA molecule. In one embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognizes an immune cell effector molecule. The term "immune cell effector molecule" refers to a molecule (e.g., a protein) expressed by an immune cell, the engagement of which by the multispecific antibody or antibody fragment results in the activation of the immune cell. Examples of immune cell effector molecules include the CD3 signaling complex in T cells, such as CD8 T cells, and various activating receptors on NK cells (e.g., NKG2D, KIR2DS, NKp44, etc.). In a further embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognizes and engages the CD3 signaling complex in T cells (e.g., anti-CD3). In a further embodiment, the multispecific antibody or antibody fragment is a single-chain diabody (scDb). In further embodiments, the scDb comprises a first antibody fragment (e.g., scFv) that binds to a complex containing a TAP described herein bound to an HLA molecule, and a second antibody fragment (e.g., scFv) that binds to and engages an immune cell effector molecule, such as a CD3 signaling complex (e.g., anti-CD3 scFv) within a T cell. Such constructs can be used, for example, to induce cytotoxic T cell-mediated killing of tumor cells expressing a tumor antigen / MHC complex recognized by the multispecific antibody or antibody fragment. The antibody or antigen-binding fragment thereof can also be used as a chimeric antigen receptor (CAR) for the production of CAR T cells, CAR NK cells, and the like. CARs combine a ligand-binding domain (e.g., antibody or antibody fragment) that provides specificity for a desired antigen (e.g., an MHC / TAP complex) with an intracellular activation domain (or signaling domain), such as a T cell or NK cell activation domain, to provide the primary activation signal.Antigen-binding fragments of antibodies, more specifically scFvs, that can bind to molecules expressed by tumor cells are commonly used as the ligand-binding domain in CARs.

[0155] In one embodiment, the soluble TCR is a therapeutic soluble bispecific TCR (see, e.g., Robinson et al., FEBS J. 2021 Nov;288(21):6159-6173; Dilchert et al., Antibodies (Basel). 2022 May 10;11(2):34)).

[0156] In one embodiment, a soluble TCR, antibody, or antibody fragment (e.g., a TCR mimetic antibody) is conjugated with an anti-tumor agent to form an antibody-drug conjugate (ADC). Such an ADC allows for targeted delivery of the anti-tumor agent to tumor cells expressing one or more of the TAPs described herein (see, e.g., Shen et al., Asian J Pharm Sci. 2020 Nov;15(6):777-785).

[0157] The present disclosure also provides nucleic acids, such as mRNAs, encoding the soluble TCRs, antibodies, antibody fragments, or CARs described herein. Such nucleic acids can be formulated in a suitable vehicle, such as lipid nanoparticles, as described above, and used to treat cancers, such as breast cancer, as described below.

[0158] Thus, in another aspect, the present disclosure provides a host cell, preferably an immune cell such as a T cell or an NK cell, that expresses an antibody or antibody fragment (e.g., scFv) described herein.

[0159] The present disclosure further provides a method for producing the above-mentioned immune cells (CD8 + T lymphocytes, CAR T cells) or TAP-specific CD8 +The present invention relates to a pharmaceutical composition or vaccine comprising a population of T lymphocytes. Such a pharmaceutical composition or vaccine may comprise one or more pharmaceutically acceptable excipients and / or adjuvants as described above.

[0160] In another aspect, the present disclosure further relates to the use of a TAP, nucleic acid, expression vector, T cell receptor, antibody / antibody fragment, cell (e.g., T lymphocyte, APC, CAR T cell), and / or composition described herein, or any combination thereof, comprising or consisting of any of the sequences of SEQ ID NOs: 1-61 or 1-35, as a medicament or in the manufacture of a medicament for the treatment of breast cancer. The present disclosure also relates to any TAP, nucleic acid, expression vector, T cell receptor, antibody / antibody fragment, cell (e.g., T lymphocyte, APC), and / or composition (e.g., vaccine composition), or any combination thereof, for use in the treatment of breast cancer (e.g., as a breast cancer vaccine) according to the present disclosure. The TAP sequences identified herein can be used to generate synthetic peptides for use i) in vitro priming and expansion of tumor antigen-specific T cells for administration to tumor patients and / or ii) as vaccines to induce or enhance anti-tumor T cell responses in breast cancer patients.

[0161] In another aspect, the disclosure provides for the use of a TAP or SLP described herein (e.g., comprising or consisting of any of the sequences of SEQ ID NOS: 1-61 or 1-35), or a combination thereof (e.g., a peptide pool), or one or more nucleic acid(s) encoding the TAP(s) or SLP(s), as a vaccine for treating breast cancer in a subject. The disclosure also provides for the use of a TAP or SLP described herein, or a combination thereof (e.g., a peptide pool), or one or more nucleic acid(s) encoding the TAP(s) or SLP(s), as a vaccine for treating breast cancer in a subject. In one embodiment, the subject is provided with TAP-specific T lymphocytes (e.g., CD8 +Thus, in another aspect, the disclosure provides a method of treating breast cancer (e.g., reducing the number of tumor cells, killing tumor cells), the method comprising targeting T lymphocytes (e.g., CD8 T lymphocytes) that recognize (i.e., express a TCR that binds to) one or more MHC class I molecule / TAP complexes (expressed on the surface of cells such as APCs). + In one embodiment, the method comprises administering (infusing) an effective amount of TAPs or SLPs, or a combination thereof, one or more nucleic acid(s) encoding the TAP(s), and / or cells (e.g., APCs such as dendritic cells) expressing MHC class I molecule(s) loaded with the TAP(s) or SLP(s) to a subject in need thereof. + and administering T lymphocytes to said subject after administration / infusion. In yet a further embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of dendritic cells loaded with one or more TAPs. In yet a further embodiment, the method comprises administering to a patient in need thereof a therapeutically effective amount of allogeneic or autologous cells expressing a recombinant TCR that binds to a TAP presented by an MHC class I molecule.

[0162] In another aspect, the present disclosure provides a method for the production of T lymphocytes (e.g., CD8 + In another aspect, the present disclosure provides for the use of T lymphocytes (e.g., CD8 T lymphocytes) that recognize one or more MHC class I molecules, or combinations thereof, loaded (presented) with TAP. +In another aspect, the present disclosure provides for the use of T lymphocytes (e.g., CD8 T lymphocytes) that recognize one or more MHC class I molecule(s) loaded (presented) with TAP, or a combination thereof, for use in the treatment of breast cancer (e.g., to reduce the number of tumor cells, kill tumor cells), e.g., lymphoblastic leukemia. In another aspect, the present disclosure provides for the use of T lymphocytes (e.g., CD8 T lymphocytes) that recognize one or more MHC class I molecule(s) loaded (presented) with TAP, or a combination thereof, for use in the treatment of breast cancer in a subject (e.g., to reduce the number of tumor cells, kill tumor cells). + In a further embodiment, the use further comprises, after use of said TAP-specific T lymphocytes, use of cells (e.g., APCs) expressing an effective amount of TAP (or a combination thereof), one or more nucleic acid(s) encoding TAP(s), and / or one or more MHC class I molecule(s) loaded (presented) with TAP.

[0163] The present disclosure also provides a method of generating an immune response in a subject against tumor cells expressing human class I MHC molecules loaded with any of the TAPs disclosed herein (e.g., comprising or consisting of any of the sequences of SEQ ID NOS: 1-61 or 1-35), or combinations thereof, the method comprising administering cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with the TAP or combinations of TAPs. The present disclosure also provides the use of cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with any of the TAPs or combinations of TAPs disclosed herein to generate an immune response against tumor cells expressing human class I MHC molecules loaded with the TAP or combinations thereof.

[0164] The breast cancer may be ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), or invasive lobular carcinoma (ILC). The breast cancer may be hormone receptor positive (HR) or hormone receptor negative (HRP). +The breast cancer may be a breast cancer characterized by a cytoplasmic ...

[0165] In one embodiment, the methods or uses described herein further comprise, prior to treatment / use, identifying the HLA class I alleles expressed by the patient and administering or using a TAP that binds to one or more of the HLA class I alleles expressed by the patient. For example, if the patient is identified as expressing HLA-11*01 and HLA-B35*01, any combination of (i) TAPs of SEQ ID NOs: 1, 17, and / or 28 (which bind to HLA-A11*01), and (ii) TAPs of SEQ ID NOs: 4, 12, and / or 23 (which bind to HLA-B35*01) may be administered or used to the patient.

[0166] In one embodiment, the TAP, nucleic acid, expression vector, T cell receptor, antibody / antibody fragment, cell (e.g., T lymphocyte, CAR T or NK cell, APC), and / or composition described herein, or any combination thereof, is administered in combination with one or more additional active agents or therapies, for example, chemotherapy (e.g., vinca alkaloids, agents that interfere with microtubule formation (e.g., colchicine and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR-targeting agents, tyrosine kinase-targeting agents (e.g., tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (e.g., nucleoside analogs), alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (e.g., all-trans retinoic acid or its derivatives), geldrugs, or the like, for the treatment of breast cancer. Namycin or its derivatives (17-AAG), inhibitors of CDK4 / 6, TGF-β, WNT-β-catenin, MYC, or PI3K, surgery, immune checkpoint inhibitors or immunotherapeutics (e.g., PD-1 / PD-L1 inhibitors such as anti-PD-1 / PD-L1 antibodies, CTLA-4 inhibitors such as anti-CTLA-4 antibodies, B7-1 / B7-2 inhibitors such as anti-B7-1 / B7-2 antibodies, TIM3 inhibitors such as anti-TIM3 antibodies, BTLA inhibitors such as anti-BTLA antibodies, CD47 inhibitors such as anti-CD47 antibodies, GITR inhibitors such as anti-GITR antibodies), antibodies against tumor antigens (e.g., anti-CD19, anti-CD22 antibodies), cell-based therapies (e.g., CAR TAPs may be used in combination with immune checkpoint inhibitors, such as T cells, CAR NK cells, and cytokines such as IL-2, IL-7, IL-21, and IL-15. In one embodiment, the TAPs, nucleic acids, expression vectors, T cell receptors, cells (e.g., T lymphocytes, APCs), and / or compositions described herein are administered / used in combination with immune checkpoint inhibitors. In one embodiment, the TAPs, nucleic acids, expression vectors, T cell receptors, cells (e.g., T lymphocytes, APCs), and / or compositions described herein are administered / used in combination with inhibitors of CDK4 / 6, TGF-β, and / or WNT-β-catenin.Several CDK4 / 6 inhibitors are in clinical trials, including Palbociclib (PD-0332991, Ibrance), Ribociclib (LEE-011, Kisqali), Abemaciclib (LY2835219, Verzenios), SHR6390, and Trilaciclib (G1T28). Inhibitors of TGF-β include antisense inhibitors such as AP12009 (Trabedersen) and ISTH0036, antibodies and ligand traps such as GC1008 (Fresolimumab), LY2382770, and P144, vaccines targeting the TGF-β pathway such as Belagenpumatucel-L (Lucanix™), and small molecule inhibitors such as LY2157299 (Galunisertib) and TEW-7197. Inhibitors of the WNT-β-catenin pathway include amino acid starvation factor (asparaginase), GSK3 inhibitors, and C2(. [ka] ), WNT974, ETC-1922159, RXC004, CGX1321, OTSA101-DTPA-90Y, Vantictumab (OMP-18R5), Ipafricept (OMP-54F28), PRI-724, SM08502, secreted frizzled-related proteins / peptides, and tankyrase inhibitors (XAV939, JW-55, RK-287107, and G007-LK).

[0167] The additional therapy can be administered before, simultaneously with, or after administration of the TAP, nucleic acid, expression vector, T cell receptor, antibody / antibody fragment, cell (e.g., T lymphocyte, CAR T or NK cell, APC), and / or composition described in this disclosure. [Example]

[0168] The present disclosure is illustrated in further detail by the following non-limiting examples.

[0169] Example 1: Materials and Methods Primary breast cancer samples. Fresh-frozen primary breast tumor samples were purchased from Tissue Solutions (https: / / www.tissue-solutions.com / ). Samples were histologically diagnosed as invasive ductal carcinoma (n=24) or invasive carcinoma (n=2). Immunohistochemistry data were available through Tissue Solutions, and samples were analyzed by HR. + Patients were classified as either TNBC (ER or PR positive, HER2 negative, n=14) or TNBC (ER / PR / HER2 all negative, n=12). Patients had not received chemotherapy before resection.

[0170] RNA and miRNA sequencing. Total RNA was extracted using the RNeasy™ mini kit (Qiagen) according to the manufacturer's instructions. RNA quantification was performed using Qubit™ (Thermo Scientific), and quality assessment was performed using the 2100 Bioanalyzer™ (Agilent Technologies). Transcriptome libraries were generated using KAPA RNA HyperPrep™ (Roche) with poly-A selection (Thermo Scientific). Small RNA libraries were prepared using the QIAseq™ miRNA Library Kit (Qiagen). Sequencing was performed using an Illumina NextSeq500 system.

[0171] Database generation. All RNA-seq reads were trimmed using Trimmomatic v0.35 and aligned to the GRCh38.99 index using STAR v2.5.1b. Transcript expression levels were quantified in transcripts per million (tpm) using Kallisto v0.43.0 with default parameters.

[0172] Canonical proteome. A canonical proteome was constructed according to previous studies (13). Single-nucleotide variants (quality >20) identified using FreeBayes (https: / / github.com / ekg / freebayes) were inserted, and sample-specific exomes were constructed using pyGeno. Open reading frames annotated with tpm >0 were translated in silico from the sample-specific exomes to generate the canonical proteome.

[0173] ERE Proteome. Following previous studies, an ERE proteome was constructed for each individual sample (23). After trimming ambiguous nucleotides from the reads of the ERE dataset, translation was performed in all possible reading frames. Finally, the resulting ERE amino acid sequences were spliced, and sequences following the stop codon were removed. Only sequences longer than 8 amino acids were retained and assigned unique IDs to create a theoretical ERE proteome. This database was concatenated with the canonical proteome to generate a personalized ERE proteome for MAP identification.

[0174] Small RNA (smRNA) proteome. smRNA sequencing reads were concatenated within fastq.gz files using Qiagen software. K-mer databases (24 bases long) were generated using Jellyfish v2.2.3 and assembled into contigs according to previous studies (11). Contigs were then translated in three frames, and different polypeptides were linked with JJ linkers to avoid bias due to shorter sequences. This database was concatenated with the canonical proteome of each sample, and MAP identification was performed.

[0175] Cancer-specific proteome. Following previous studies, we assembled cancer-specific proteomes using k-mer profiling (11, 13). From each cancer sample database, k-mers (33 nucleotides long) that occurred at least once in the mTECs k-mer database were removed, and the remaining k-mers were assembled into contigs. Finally, the contigs were translated in three frames, and distinct polypeptides were linked using JJ linkers. This database was then concatenated with the canonical proteome of each sample, and MAP identification was performed.

[0176] Mass spectrometry analysis. MHC class I immunoprecipitation, tandem mass tag (TMT) labeling, and liquid chromatography-MS / MS analysis (LC-MS / MS) were performed according to previous studies (24). LC-MS / MS data were searched against relevant databases using PEAKS 10.5 or Peaks X Pro (Bioinformatics Solution Inc.). Peptide identification was performed using precursor and fragment ion tolerances set to 10 ppm and 0.01 Da, respectively. Variable modifications included oxidation (M), deamidation, and TMT modifications. After peptide identification, a modified target-decoy approach was implemented in PEAKS, applying sample-specific thresholds to the PEAKS score to ensure a false discovery rate (FDR) of 1%, calculated as the ratio between the number of decoy hits and the number of target hits exceeding the score threshold. The PEAKS score corresponding to a 1% FDR was determined for each sample, and peptides exceeding the threshold were further selected if they met the following criteria: The peptides were filtered based on the following criteria: peptide length was 8–11 amino acids, and the binding affinity ranking for the HLA alleles of the sample based on NetMHCpan-4.1b (25) was <2%. These filtering steps were performed using MAPDP (26).

[0177] Genomic origin and validation of the MAP of interest (MOI). Candidate tumor antigens were identified according to previous studies (11, 13, 14). If a MAP aligned to an exon sequence with at least one read, the genomic origin of the MAP was considered to be from the exon sequence. Otherwise, the alignment with the highest number of reads was used to compare the genomic origin of the MOI. The final alignment of the TSA and TAA was manually verified using Integrative Genomics Viewer (IGV). MOIs that did not match the genomic location with matching reads or that matched hypervariable regions (e.g., MHC, Ig, TCR genes) were excluded.

[0178] Candidate TSAs were classified as mTSAs if they contained mutations within the MAP coding sequence that did not match known germline polymorphisms reported in the Single Nucleotide Polymorphism (SNP) Database (dbSNP) v149 (http: / / www.ncbi.nlm.nih.gov / SNP / ). Standard MS methods cannot distinguish between leucine and isoleucine mutations. Therefore, MAPs flagged with pre-existing mutations were discarded as non-MOIs unless they exhibited higher RNA expression than the mutation or mapped to an inappropriate region (as in the case of ERE and smRNA MAP candidates). Furthermore, all MOI, ERE, and smRNA MAP candidates were validated using the COMET software (27) with the same filters as in Peaks and a 5% FDR. For peptides not re-identified by COMET, manual spectral validation was performed in-house to determine whether to retain or discard the MAPs.

[0179] Expression of MOI in tissues. Expression of the MOI coding sequence was assessed in normal tissues from GTEX (n = 50 per tissue), mTECs (n = 11), purified blood and bone marrow samples (n = 6), and breast cancer tissues from TCGA (n = 1109).

[0180] Predictive presentation and survival analysis of TAA and TSA in TCGA breast cancer samples. TCGA samples were considered to be present for a TAA or TSA only if the MAP-encoding transcript was expressed (>2 rphm) and the patient had an HLA allotype that allowed for MAP presentation using NetMHCPan4.1 (25). HLA alleles for TCGA patients were determined using Optitype software (28). TCGA survival data were obtained using the "TCGAbiolinks" package (29). Patients with more than one biopsy were excluded from the analysis to avoid overlapping contributions to the results, leaving 915 patients. Kaplan-Meier survival curves and log-rank tests were generated using the TCGAbiolinks package.

[0181] Single-cell analysis. Raw single-cell data from 14 breast cancer patients published by Qian et al. (30) were obtained from their website (http: / / scope.lambrechtslab.org / ). Visualization graphs were generated using the Seurat package (31).

[0182] FEST assay. The FEST assay was performed according to a previous study (Danilova et al., Cancer Immunol Res. 2018 Aug;6(8):888-899. Epub 2018 Jun 12, 2018) with minor modifications. Five million T cells were co-cultured with autologous T cell-depleted PBMCs pulsed with each peptide for 20 days, with restimulation on day 10. After in vitro expansion, CD8+ T cells were cultured as human CD8 +T Cells were isolated using a T Cell Isolation Kit (Miltenyi). DNA extraction was performed using the QIAGEN DNA blood mini kit, followed by TCR Vβ CDR3 sequencing using the ImmunoSEQ platform. Raw data were processed using the FEST web tool (www.stat-apps.onc.jhmi.edu / FEST). The following parameters were used: FDR 1%, fold change >5, minimum template number 1, and "Ignore baseline threshold." Two negative control groups consisted of CD8 T cells co-cultured with unpulsed autologous T cell-depleted PBMCs. + T cells and uncultured CD8 + It consisted of T cells.

[0183] Statistical analysis and data visualization. Analyses and figures were performed using R v4.0.0. Various graphs were created using the "gplots" and "ggplot2" packages in R. Tests involving comparison of distributions were performed using t-tests or one-way ANOVA tests as appropriate. Differential gene expression analysis was performed using the limma package (32). GSEA analysis was performed using the "fgsea" package (33).

[0184] Example 2: Global proteogenomics strategy for MAP identification in primary breast cancer samples We analyzed 26 primary breast cancer samples (14 HR+ and 12 TNBC) from treatment-naive patients. MAPs were identified by MS analysis using a proteogenomics approach described in previous studies (11, 13, 14). For each sample, we constructed individual reference databases by in silico translation of RNA-seq data. These databases included four modules: 1) the canonical proteome (in-frame translation of protein-coding regions), 2) the endogenous retroelement (ERE) proteome, 3) the small RNA proteome, and 4) the cancer-specific proteome. The cancer-specific proteome incorporated RNA-seq reads present in tumor samples but absent from the mTEC collection (Figure 8).

[0185] Example 3: The canonical immunopeptidomes of HR+ and TNBC tumors are similar The MAPs encoded by the canonical reading frames of annotated protein-coding genes are collectively referred to as the canonical immunopeptidome. A total of 57,094 canonical MAPs derived from 10,552 protein-coding genes were identified. The average number of MAPs per tumor sample was 4,633, with HR + No differences were observed between HR+ and TNBC samples (t-test; p>0.05, Figure 1A). MAPs were presented by 53 distinct HLA alleles, of which over 20% were presented by two common alleles, namely, HLA-A02:01 and HLA-B18:01 (Figure 9). Consistent with previous studies (34), 62% of the transcriptome, i.e., genes with ≥1 transcript per kilobase million (TPM), produced MAPs (Figure 10A). Many genes (72.5%, n = 7371) produced MAPs in both HR+ and TNBC tumors (Figure 1B), the majority of which (92%) were also reported as MAP-donating genes in normal tissues derived from the HLA ligand ATLAS (18).

[0186] Of the 798 canonical MAPs not listed in the HLA ligand ATLAS, 277 were HR + were detected in both HR and TNBC samples, 259 were specific for TNBC, and 262 were HR + The results were specific to the sample (Figure 1B). + The 277 canonical MAPs commonly detected in both HR and TNBC samples were enriched in the gene set encoding extracellular matrix proteins (enrichment factor 7.59, p<0.05) (Figure 11A). The 259 canonical MAPs found exclusively in TNBC samples were enriched in the glycosyltransferase protein class (enrichment factor 5.58, p<0.05) (Figure 11B). + No specific enrichment was observed for the 262 MAPs found only in the sample.

[0187] Contributing genes did not contribute uniformly to the immunopeptidome. While 49% of contributing genes in the dataset generated 5 or fewer MAPs, 51% generated more than 5 MAPs, and among them, a significant number of genes encoded more than 100 MAPs (Fig. 1C and Fig. S10B). The top 1% of genes generating the most MAPs were significantly enriched in the cytoskeletal and extracellular matrix protein classes (Fig. 1D). Taken together, these results suggest that HR + We found significant overlap between the canonical immunopeptidomes of breast cancer and TNBC samples, and furthermore, a significant enrichment of MAPs derived from cytoskeletal and extracellular matrix proteins. The immunopeptidome reflects the phenotype of proteins that are actively translated at the cell surface and degraded intracellularly (35). Thus, the overexpression of MAPs derived from cytoskeletal and extracellular matrix proteins is consistent with the important role of extracellular matrix remodeling in breast cancer tumorigenesis (36-38).

[0188] Example 4: Contribution of EREs and smRNAs to the non-canonical breast cancer immunopeptidome EREs and smRNAs are involved in various stages of neoplastic transformation, including breast cancer (39, 40). Furthermore, EREs have been shown to encode immunogenic MAPs in mice and humans (13, 23). Therefore, we specifically searched personalized proteogenomics databases for the presence of EREs and smRNA-encoded MAPs in breast cancer. ERE-encoding transcripts were obtained from bulk RNA sequencing data, and smRNA sequencing was used to construct the smRNA database (Figure 12A-B).

[0189] HR + We identified 75 ERE-derived MAPs that were evenly distributed between HR and TNBC samples (Figure 2A, Table 1). ERE-derived MAPs were similarly mapped to intronic and intergenic regions (Figure 13). Three major classes of EREs were identified: + EREs contributed equally to the immunopeptidome of breast cancer and TNBC samples (Figure 2B). MAP-producing EREs were expressed at higher levels than non-MAP-producing EREs (Figure 2D). Only 9 / 22 ERE families produced MAPs, with the L1 family being the most significant contributor (Figure 2C). Only three smRNA-derived MAPs were identified (Table 2), one derived from piRNA and the other two from snRNA. From these results, we concluded that EREs generate a significant number of MAPs in breast cancer tumors, whereas smRNAs do not. However, ERE-derived MAPs can also be present in normal tissues (23). Therefore, further analysis was required to assess whether ERE-derived MAPs could be classified as TAAs or TSAs.

[0190] [Table 5-1] [Table 5-2] [Table 5-3]

[0191] [Table 6]

[0192] Example 5: Identification of potential therapeutic targets: TAAs, aeTSAs, and mTSAs MOIs were identified and classified as TAA, aeTSA, or mTSA using the workflow outlined in Figure 3A. Overall, 25 TSAs were identified: one mTSA and 24 aeTSA (Figure 4A). The only mTSA was derived from a nonsynonymous mutation in the deubiquitinase OTUB1 gene. This rare mutation is not listed in the COSMIC database (https: / / cancer.sanger.ac.uk / cosmic). Given the rarity of mTSAs, we investigated whether frequently mutated genes in breast cancer are reflected within the immunopeptidome. We found a slight positive correlation between mutation frequency identified by the TCGA consortium and MAP generation in our dataset (p<0.001) (Figure 14). This indicates that there is no negative bias in the breast cancer immunopeptidome, which suppresses the representation of hypermutated genes. Thus, although hypermutated genes generate MAPs, these MAPs are not derived from mutated regions. This is consistent with the fact that MAPs arise preferentially from specific regions of MAP-supplying proteins (MAP “hotspots”). (18, 34, 41) The most plausible explanation for the paucity of mTSAs is that there are relatively few mutations in breast cancer, and these mutations are not located in MAP hotspots.

[0193] In our study of TAAs and aeTSAs, we considered only MAPs encoded by transcripts that were expressed in at least 5% of the TCGA breast cancer cohort samples. Antigens with low expression levels were considered of little interest. Classification of unmutated MAPs as TAAs or aeTSAs was based on comprehensive transcriptome analysis of their expression in i) breast cancer samples from TCGA (n = 1109), ii) 50 normal tissues from GTEX (50 samples per tissue), iii) mTECs (n = 11), and iv) purified blood and bone marrow samples (n = 6). Because hematopoietic cells account for 90% of the cells produced daily in humans, blood and bone marrow cells were used as a surrogate for rapidly proliferating cells (42). It is important to emphasize that expression profiling only considers the sequences encoding the MAPs, not the entire gene or genomic region. Therefore, aberrant RNA splicing typically results in protein isoforms being present only in cancer cells. MAPs derived from such cancer-specific isoforms are classified as aeTSAs even if other isoforms are expressed in normal cells.

[0194] MAPs with expression levels below 8.55 reads per 100 million reads (rphm) in all normal tissues except testis were classified as aeTSAs. As previously reported (11), 8.55 rphm was set as the threshold because expression levels below this value are associated with a very low probability of MAP formation. On the other hand, MOIs with expression levels above the threshold in at least one normal tissue were classified as TAA if they were overexpressed in tumor or hyperproliferative tissues compared with normal nonhematopoietic tissues. This strategy identified 24 aeTSAs and 49 TAAs, the majority of which are novel because they are not registered in the Immune Epitope Database (IEDB) (Figure 3B-C, Tables 3 and 4).

[0195] [Table 7]

[0196] [Table 8-1] [Table 8-2]

[0197] Example 6: TSA is HR + More common in TNBC than in breast cancer Of the 24 aeTSAs, 17 were encoded in canonical exons: 14 belonged to the MAGE family of CTAs, two to genes encoding extracellular matrix components (COL11A1, ITH6), and one to a gene encoding a transmembrane protein (ABCC11) (Fig. 4 and Table 3). Seven aeTSAs were derived from non-protein-coding regions, two of which overlapped with EREs and could be classified as ERE-derived MAPs (Fig. 4 and Table 3).

[0198] We found the majority of aeTSAs within TNBC samples (Figure 3B). We next assessed whether this enriched identification of TSAs in TNBC samples correlated with TSA expression in the TCGA cohort. The proportion of tumors expressing individual aeTSAs of the CTA class was significantly higher in TNBC than in the control group. + The distribution of TSA was significantly higher in patients with glaucoma compared with those with tumors (19% vs. 8%, p = 0.004) (Figure 4C). No significant differences were observed in the distribution of other TSA categories.

[0199] Next, we evaluated whether aeTSA presentation correlates with immune infiltration. AeTSA was considered present in a TCGA sample only if the transcript encoding the MAP was expressed and the patient possessed an HLA allotype capable of presenting this MAP (11). TCGA samples were then classified into two groups: those with high (above the median) and those with low (below the median) aeTSA presentations. Differential gene expression analysis was then performed between the two groups. Gene set enrichment analysis using gene markers of leukocyte infiltration, as described by Danaher et al. (43), revealed enrichment of these genes in tumors with high TSA numbers. This result suggests that at least some TSAs are immunogenic in vivo. Furthermore, we evaluated whether oncogenic pathways involved in immune escape (44) were enriched in samples from high or low TSA groups. To this end, we selected a set of corresponding hallmark genes from the Molecular Signature Database (45) and found that the PI3K pathway was enriched in tumors predicted to have low TSA counts (NES = -1.48, p < 0.05, Figure 15). The PI3K pathway is involved in breast cancer tumorigenesis, progression, and therapeutic resistance (46).

[0200] Example 7: TAAs are widely shared in breast cancer We identified 49 TAAs, 48 of which were derived from canonical protein-coding regions (Figure 5A and Table 4). These antigens were involved in HR +These genes were widely shared in both breast and TNBC tumors (Figure 5B). The largest group of TAAs (n = 14) derived from genes (COL11A1, COL10A1, and LRRC15) has been reported as markers of cancer-associated fibroblasts (47). These genes are involved in extracellular matrix production and cell migration. To identify their most likely cell of origin, we assessed their expression levels using a single-cell dataset from Qian et al. (30) containing 14 breast cancer tumors (Figure 6A). All three genes showed significantly higher expression in tumor fibroblasts compared with other cell subpopulations, including cancer cells (Figure 6B, ANOVA, p < 0.05). Two other large groups of TAAs were identified. Thirteen TAAs derived from CYP4Z1 are involved in many cancer types and are known to induce autoantibodies against CYP4Z1 in breast cancer patients (48). Eleven TAAs were associated with cell proliferation (Fig. 5B) and showed low expression in mature epithelial cells and blood cells, but high expression in myeloid progenitor cells (Fig. 5A).

[0201] The same criteria as for aeTSA (i.e., expression of MAP-encoding sequences and presence of relevant HLA alleles) were used to predict the number of TAA for each tumor in the TCGA dataset (Figure 5C, Figure 16). HR + In HBsAg and TNBC tumors, those with high predicted TAA levels showed enrichment in immune activation and immune suppression pathways, namely, the PI3K / mTOR pathway, the Wnt / B-catenin pathway, and the MAPK pathway. Furthermore, tumors with high numbers of TAAs also showed enrichment in markers of fibroblast proliferation. These findings suggest that in the presence of high numbers of TAAs, the antitumor immune response is mitigated by the activation of immune suppression pathways and the accumulation of cancer-associated fibroblasts.

[0202] Example 8: Presentation of multiple TSAs improves overall survival in TNBC Next, we evaluated whether the number of presented TAAs and aeTSAs correlated with overall survival in TCGA patients. As in previous studies, an antigen was considered to be presented in the tumor if both the transcript encoding the MAP and the appropriate HLA allotype were expressed (11). Patients were divided into two categories: those with a high number of presented antigens (1st quartile) and those with a low number of presented antigens (2nd-4th quartile).

[0203] The number of presented TAAs was HR + The number of aesthesia-associated tumors (aesthesia-associated tumors) did not affect the survival rate of patients with TNBC tumors (Figures 7B, C). Similarly, the ... + In tumor-bearing patients, aesthesia had no effect (Figure 7A, Figure 4C). However, in the TNBC cohort, aesthesia presentation correlated with improved overall survival (Figure 17). This benefit was observed for both aesthesia derived from CTAs and non-coding regions (Figure 7D, E). Notably, when considering TSA expression alone (and not HLA allotype), no difference was observed between the high- and low-expression groups (Figure 7D, E). This indicates that the beneficial effect of aesthesia presentation is HLA-restricted. Therefore, this is due to presentation of aesthesia peptides by MHC I and not to the expression of the aesthesia-encoding transcript itself.

[0204] It can be concluded that the aeTSA reported herein confers an MHC class I-restricted survival benefit in patients with TNBC (Fig. 7D, E). This is likely due to the HR + Patients with tumors presented fewer aesthesia-associated steroids compared with TNBC tumors (Figure 4C). + This was not the case for tumor-bearing patients (Figure 7A). TAA presentation did not show a similar survival benefit, as TAA expression tends to be associated with activation of immunosuppressive pathways (Figure 5C).

[0205] Example 9: TSAs and TAAs induce specific CD8 T cell proliferation We next assessed whether the TAAs and TSAs identified herein could induce CD8+ T cell expansion ex vivo using the Functional Expansion of Specific T cells (FEST) assay, which integrates T cell receptor sequencing in short-term peptide-stimulated cultures with a bioinformatics platform to identify antigen-specific clonotype expansion (Danilova et al., Cancer Immunol Res. 2018 Aug;6(8):888-899. Epub 2018 Jun 12, 2018).

[0206] As shown in Figures 18A and 18B, significant antigen-specific CD8 T cell clonotype expansion was obtained in two different donors for selected TSAs and TAAs, providing compelling evidence that the TSAs and TAAs are immunogenic and will be useful for stimulating anti-tumor T cell responses in subjects afflicted with cancers expressing these TSAs and / or TAAs.

[0207] While the present invention has been described with reference to specific embodiments above, modifications can be made without departing from the spirit and essence of the invention as defined in the appended claims. In the claims, the term "comprising" is used as an open-ended term and is substantially equivalent to the phrase "including, but not limited to." The singular forms "a," "an," and "the" also include the corresponding plural forms unless the context clearly dictates otherwise.

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Claims

1. The following amino acid sequence: 【Table 1】 A tumor antigen peptide (TAP) comprising or consisting of one of the following, or a nucleic acid encoding said TAP.

2. 2. The TAP or nucleic acid of claim 1, wherein said TAP binds to an HLA-A*02:01 molecule and comprises or consists of the sequence of SEQ ID NO:

22.

3. 2. The TAP or nucleic acid of claim 1, wherein said TAP binds to an HLA-A*03:01 molecule and comprises or consists of the sequence of SEQ ID NO:

19.

4. The TAP binds to an HLA-A*11:01 molecule and comprises or consists of the sequence of SEQ ID NO: 1, 17 or 28.

5. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-A*24:02 molecule and comprises or consists of the sequence of SEQ ID NO: 6 or 30.

6. 2. The TAP or nucleic acid of claim 1, wherein said TAP binds to an HLA-A*25:01 molecule and comprises or consists of the sequence of SEQ ID NO:

10.

7. 2. The TAP or nucleic acid of claim 1, wherein said TAP binds to an HLA-A*26:01 molecule and comprises or consists of the sequence of SEQ ID NO:

15.

8. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-A*31:01 molecule and comprises or consists of the sequence of SEQ ID NO: 8, 9 or 29.

9. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-A*33:01 molecule and comprises or consists of the sequence of SEQ ID NO: 2 or 3.

10. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*15:01 molecule and comprises or consists of the sequence of SEQ ID NO:

26.

11. The TAP binds to an HLA-B*18:01 molecule and comprises or consists of the sequence of SEQ ID NO: 13, 14 or 33.

12. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*27:05 molecule and comprises or consists of the sequence of SEQ ID NO:

27.

13. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*35:01 molecule and comprises or consists of the sequence of SEQ ID NO: 4, 12, or 23.

14. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*35:03 molecule and comprises or consists of the sequence of SEQ ID NO:

38.

15. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*38:01 molecule and comprises or consists of the sequence of SEQ ID NO:

34.

16. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*40:01 molecule and comprises or consists of the sequence of SEQ ID NO:

20.

17. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*49:01 molecule and comprises or consists of the sequence of SEQ ID NO: 11 or 24.

18. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*50:01 molecule and comprises or consists of the sequence of SEQ ID NO: 5 or 7.

19. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*51:01 molecule and comprises or consists of the sequence of SEQ ID NO: 35 or 37.

20. 2. The TAP or nucleic acid of claim 1, wherein said TAP binds to an HLA-B*52:01 molecule and comprises or consists of the sequence of SEQ ID NO:

18.

21. 2. The TAP or nucleic acid of claim 1, wherein said TAP binds to an HLA-B*58:01 molecule and comprises or consists of the sequence of SEQ ID NO:

36.

22. 2. The TAP or nucleic acid of claim 1, wherein said TAP binds to an HLA-C*01:02 molecule and comprises or consists of the sequence of SEQ ID NO:

25.

23. 2. The TAP or nucleic acid of claim 1, wherein the TAP binds to an HLA-C*12:03 molecule and comprises or consists of the sequence of SEQ ID NO: 16 or 21.

24. 24. The TAP or nucleic acid of any one of claims 1 to 23, encoded by a sequence located in a non-protein coding region of the genome.

25. 25. The TAP or nucleic acid of claim 24, wherein the non-protein coding region of the genome is an intergenic region.

26. 25. The TAP or nucleic acid of claim 24, wherein the non-protein coding region of the genome is a long non-coding RNA.

27. A combination comprising at least two TAPs or nucleic acids as defined in any one of claims 1 to 26.

28. 28. The TAP or nucleic acid of any one of claims 1 to 26, or the combination of claim 27, wherein the nucleic acid is mRNA.

29. 28. The TAP or nucleic acid of any one of claims 1 to 26, or the combination of claim 27, wherein the nucleic acid is DNA.

30. A TAP or nucleic acid according to any one of claims 1 to 26, or a combination according to claim 27, wherein the nucleic acid is a component of a viral vector.

31. A synthetic long peptide (SLP) comprising at least one of the amino acid sequences defined in claim 1, or a nucleic acid encoding said SLP.

32. 32. The SLP or nucleic acid of claim 31, wherein the SLP comprises at least 5, 10, 15 or 20 of the amino acid sequences defined in claim 1.

33. A vesicle or particle comprising a TAP, a nucleic acid, a combination or an SLP according to any one of claims 1 to 32.

34. 34. The vesicle or particle of claim 33, which is a lipid nanoparticle (LNP).

35. 35. A vesicle or particle according to claim 33 or 34, comprising a cationic lipid.

36. 36. A composition comprising a TAP, nucleic acid, combination or SLP according to any one of claims 1 to 32, or a vesicle or particle according to any one of claims 33 to 35, and a pharmaceutically acceptable carrier.

37. 37. A vaccine comprising a TAP, nucleic acid, combination or SLP according to any one of claims 1 to 32, a vesicle or particle according to any one of claims 33 to 35, or a composition according to claim 36, and an adjuvant.

38. 27. An isolated major histocompatibility complex (MHC) class I molecule comprising a TAP according to any one of claims 1 to 26 within its peptide-binding groove.

39. 39. The isolated MHC class I molecule of claim 38, in the form of a multimer.

40. 40. The isolated MHC class I molecule of claim 39, wherein the multimer is a tetramer.

41. (i) a TAP according to any one of claims 1 to 26, (ii) a combination according to claim 27, (iii) an SLP according to claim 31 or 32, or (iv).

32. An isolated cell comprising a vector comprising a nucleotide sequence encoding a TAP according to any one of claims 1 to 26, a combination according to claim 27, or an SLP according to claim 31 or 32.

42. 31. An isolated cell expressing a major histocompatibility complex (MHC) class I molecule comprising, on its surface, within its peptide-binding groove, a TAP according to any one of claims 1 to 30, or a combination thereof.

43. 43. The cell of claim 41 or 42, which is an antigen-presenting cell (APC).

44. The cell of claim 43, wherein the APC is a dendritic cell.

45. A T cell receptor (TCR) that specifically recognizes an isolated MHC class I molecule according to any one of claims 38 to 40 and / or an MHC class I molecule expressed on the surface of a cell according to any one of claims 42 to 44.

46. The TCR of claim 45, which is a soluble TCR.

47. An antibody or an antigen-binding fragment thereof that specifically binds to an isolated MHC class I molecule described in any one of claims 37 to 39 and / or an MHC class I molecule expressed on the surface of a cell described in any one of claims 42 to 44.

48. 48. The TCR of claim 45 or 46, or the antibody or antigen-binding fragment thereof of claim 47, which is a bispecific TCR, a bispecific antibody, or an antigen-binding fragment thereof.

49. 49. The TCR, antibody or antigen-binding fragment thereof of claim 48, wherein the bispecific antibody or antigen-binding fragment thereof is a single-chain diabody (scDb).

50. 50. The TCR, antibody or antigen-binding fragment thereof of claim 48 or 49, wherein the bispecific TCR, antibody or antigen-binding fragment thereof also specifically binds to a T cell signaling molecule.

51. 51. The TCR, antibody or antigen-binding fragment thereof of claim 50, wherein the T cell signaling molecule is a CD3 chain.

52. 48. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof of claim 47, or a nucleotide sequence encoding said CAR.

53. An isolated cell, wherein the cell expresses the TCR of claim 45 or the CAR of claim 52 on its cell surface.

54. CD8 ＋ 54. The isolated cell of claim 53, which is a T lymphocyte.

55. 55. A cell population comprising at least 0.5% or 1% of isolated cells as defined in claim 53 or 54.

56. 1. A method of treating breast cancer in a subject, comprising administering to said subject an effective amount of any of the following: (a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1 to 61, or a combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1 to 61; (b) at least one nucleic acid encoding a TAP, combination of TAPs or SLP as defined in (a); (c) a TAP, a combination of TAPs or an SLP as defined in (a), or a vesicle or particle comprising at least one nucleic acid as defined in (b); (d) a composition comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier; (e) a vaccine comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant; (f) a cell expressing on its surface a major histocompatibility complex (MHC) class I molecule containing within its peptide-binding groove a TAP or combination of TAPs as defined in (a); (g) A cell expressing on its surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of the cell defined in (f), or (h) A method comprising administering a soluble TCR, antibody, or antigen-binding fragment thereof that specifically binds to an MHC class I molecule expressed on the surface of a cell defined in (f).

57. The breast cancer is hormone receptor positive breast cancer (HR ＋ 57. The method of claim 56, wherein the cancer is triple-negative breast cancer (TNBC).

58. 58. The method of claim 56 or 57, further comprising administering to the subject at least one additional anti-tumor agent or at least one additional treatment.

59. 59. The method of claim 58, wherein the at least one additional anti-tumor agent or treatment is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy, or surgery.

60. (a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1 to 61, or a combination thereof, or a synthetic long peptide (SLP) comprising at least one sequence set forth in SEQ ID NOs: 1 to 61; (b) at least one nucleic acid encoding a TAP, combination of TAPs or SLP as defined in (a); (c) a TAP, a combination of TAPs or an SLP as defined in (a), or a vesicle or particle comprising at least one nucleic acid as defined in (b); (d) a composition comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier; (e) a vaccine comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant; (f) a cell expressing on its surface a major histocompatibility complex (MHC) class I molecule containing within its peptide-binding groove a TAP or combination of TAPs as defined in (a); (g) A cell expressing on its surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of the cell defined in (f), or (h) A soluble TCR, antibody, or antigen-binding fragment thereof that specifically binds to an MHC class I molecule expressed on the surface of a cell defined in (f). Use of a compound of claim 1 for treating breast cancer in a subject or for manufacturing a medicament for treating breast cancer in a subject.

61. The breast cancer is hormone receptor positive breast cancer (HR ＋ ) or triple-negative breast cancer (TNBC).

62. 62. The use of claim 60 or 61, further comprising the use or administration of at least one additional anti-tumor agent or treatment to said subject.

63. 63. The use of claim 62, wherein the at least one additional anti-tumor agent or treatment is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy, or surgery.

64. 1. A medicament for use in treating breast cancer in a subject, said medicament comprising: (a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1 to 61, or a combination thereof, or a synthetic long peptide (SLP) comprising at least one sequence set forth in SEQ ID NOs: 1 to 61; (b) at least one nucleic acid encoding a TAP, combination of TAPs or SLP as defined in (a); (c) a TAP, a combination of TAPs or an SLP as defined in (a), or a vesicle or particle comprising at least one nucleic acid as defined in (b); (d) a composition comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier; (e) a vaccine comprising a TAP, combination of TAPs or SLP as defined in (a), at least one nucleic acid as defined in (b), a vesicle or particle as defined in (c), or a composition as defined in (d), and an adjuvant; (f) a cell expressing on its surface a major histocompatibility complex (MHC) class I molecule containing within its peptide-binding groove a TAP or combination of TAPs as defined in (a); (g) A cell expressing on its surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of the cell defined in (f), or (h) A drug which is a soluble TCR, antibody or antigen-binding fragment thereof that specifically binds to an MHC class I molecule expressed on the surface of a cell defined in (f).

65. The breast cancer is hormone receptor positive breast cancer (HR ＋ 65. The method of claim 64, wherein the cancer is a triple-negative breast cancer (TNBC) or triple-negative breast cancer (TNBC).

66. 66. The method of claim 64 or 65, further comprising the use of at least one additional anti-tumor agent or administration of said therapy to said subject.

67. 67. The method of claim 66, wherein the at least one additional anti-tumor agent or treatment is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy, or surgery.

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