Novel cancer antigens and uses thereof

Abstract

Breast cancer is now the most common cancer in the world, and despite treatment progression in decades in the past, metastatic breast cancer is still a disease which cannot be cured. Novel tumor specific antigens (TSA) and tumor associated antigens (TAA) expressed by breast tumor cells are described herein. Synthetic long peptides, nucleic acids, compositions, cells, TCRs, antibodies, and vaccines derived from these TSAs and TAAs are described. The use of the TSA / TAAs, nucleic acids, compositions, antibodies, cells and vaccines for the prevention or treatment of breast cancer, including triple negative breast cancer (TNBC), is also described.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 370,697, filed on August 8, 2022, which is incorporated herein by reference.

[0003] Sequence Listing

[0004] The Sequence Listing is submitted herewith as an XML file named 17971-00066-AD.xml, which was created on August 2, 2023, and is ~181,000 bytes in size. The contents of the above-mentioned file are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates generally to the field of cancer, and more particularly to the treatment of cancer, such as breast cancer. Background Art

[0006] According to recent global statistics, breast cancer is now the most prevalent cancer worldwide, surpassing lung cancer (1). Despite therapeutic advances over the past few decades, metastatic breast cancer remains an incurable disease. From an immunological point of view, breast cancer tumors are divided into two major categories (2). Hormone receptor-positive breast cancer (HR-positive) + ) are considered an immunologically “cold” cancer and have not benefited from recent advances in immunotherapy (3). In contrast, the triple-negative breast cancer subtype (TNBC) is immunologically “hot,” as demonstrated by high levels of leukocyte infiltration and responsiveness to immune checkpoint blockade (CB) (4). In fact, following the IMPassion130 trial, the addition of ICB to chemotherapy is the new standard of care for TNBC tumors with PD-L1 expression (5). High tumor mutational burden (TMB) has previously been used as a biomarker for response to ICB in a variety of cancers (6). However, it is present in only 5% of breast cancer tumors and is not associated with CD8 + T cell infiltration is not involved (6, 7). + The reasons for the different immunogenicity of TNBC and TNBC tumors remain elusive.

[0007] Therefore, there is a need to develop new methods for preventing or treating breast cancer.

[0008] Throughout this specification, a number of documents are referenced, the contents of which are incorporated herein by reference in their entireties.

[0009] Overview

[0010] In various aspects and embodiments, the present disclosure provides the following items 1 to 67:

[0011] 1. A tumor antigen peptide (TAP) comprising or consisting of one of the following amino acid sequences:

[0012] Peptide sequence SEQ ID NO: Peptide sequence SEQ ID NO: SVLGPILIIPK 1 KLKQDTTQK 19 DMFGIRFKR 2 LELFQHVSL 20 EYNAEFGRR 3 MAVNIVLQ 21 FPEDFSILF 4 MLWKLVENV 22 GEAVGFQPP 5 QAWGAGLAY 23 IYLIPEPVLF 6 SEADLQAEV 24 LEISFISP L 7 SLPDNVFSNL 25 RSMRVGVTR 8 SQGQIFSKF 26 SMRSTVVSR 9 SRLELFQHV 27 SVADLLSSF 10 SSQGQIFSK 28 AETDITETSI 11 SVKERESER 29 DIIEYDYEY 12 VEEIFTEEY 30 EEIFTEEY 13 VHDPDYAKILL 31 EEQYIAQF 14 VPWEDKISI 32 ELFQHVSLM 15 VTTLALTF 33 GAYDKARYL 16 WPMHTPLL 34 GTHVWVGLYK 17 WPMHTPLLL 35 HQFTEKVI 18

[0013] Or a nucleic acid encoding said TAP.

[0014] 2. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-A*02:01 molecule and comprises or consists of the sequence of SEQ ID NO:22.

[0015] 3. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-A*03:01 molecule and comprises or consists of the sequence of SEQ ID NO:19.

[0016] 4. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-A*11:01 molecule and comprises or consists of the sequence of SEQ ID NO:1, 17 or 28.

[0017] 5. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-A*24:02 molecule and comprises or consists of the sequence of SEQ ID NO: 6 or 30.

[0018] 6. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-A*25:01 molecule and comprises or consists of the sequence of SEQ ID NO:10.

[0019] 7. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-A*26:01 molecule and comprises or consists of the sequence of SEQ ID NO:15.

[0020] 8. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-A*31:01 molecule and comprises or consists of the sequence of SEQ ID NO:8, 9 or 29.

[0021] 9. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-A*33:01 molecule and comprises or consists of the sequence of SEQ ID NO: 2 or 3.

[0022] 10. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-B*15:01 molecule and comprises or consists of the sequence of SEQ ID NO:26.

[0023] 11. The TAP or nucleic acid of claim 1, wherein the TAP binds to the HLA-B*18:01 molecule and comprises or consists of the sequence of SEQ ID NO: 13, 14 or 33.

[0024] 12. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-B*27:05 molecule and comprises or consists of the sequence of SEQ ID NO:27.

[0025] 13. The TAP or nucleic acid of claim 1, wherein the TAP binds to the HLA-B*35:01 molecule and comprises or consists of the sequence of SEQ ID NO:4, 12 or 23.

[0026] 14. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-B*35:03 molecule and comprises or consists of the sequence of SEQ ID NO:38.

[0027] 15. The TAP or nucleic acid of claim 1, wherein the TAP binds to the HLA-B*38:01 molecule and comprises or consists of the sequence of SEQ ID NO:34.

[0028] 16. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-B*40:01 molecule and comprises or consists of the sequence of SEQ ID NO:20.

[0029] 17. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-B*49:01 molecule and comprises or consists of the sequence of SEQ ID NO: 11 or 24.

[0030] 18. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-B*50:01 molecule and comprises or consists of the sequence of SEQ ID NO:5 or 7.

[0031] 19. The TAP or nucleic acid of claim 1, wherein the TAP binds to the HLA-B*51:01 molecule and comprises or consists of the sequence of SEQ ID NO:35 or 37.

[0032] 20. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-B*52:01 molecule and comprises or consists of the sequence of SEQ ID NO:18.

[0033] 21. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-B*58:01 molecule and comprises or consists of the sequence of SEQ ID NO:36.

[0034] 22. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-C*01:02 molecule and comprises or consists of the sequence of SEQ ID NO:25.

[0035] 23. The TAP or nucleic acid of item 1, wherein the TAP binds to the HLA-C*12:03 molecule and comprises or consists of the sequence of SEQ ID NO:16 or 21.

[0036] 24. The TAP or nucleic acid of any one of items 1 to 23, which is encoded by a sequence located in a non-protein coding region of the genome.

[0037] 25. The TAP or nucleic acid of claim 24, wherein the non-protein coding region of the genome is an intergenic region.

[0038] 26. The TAP or nucleic acid of claim 24, wherein the non-protein coding region of the genome is a long non-coding RNA.

[0039] 27. A combination comprising at least two of the TAPs or nucleic acids defined in any one of items 1 to 26.

[0040] 28. The TAP or nucleic acid of any one of items 1 to 26, or the combination of item 27, wherein the nucleic acid is mRNA.

[0041] 29. The TAP or nucleic acid of any one of items 1 to 26, or the combination of item 27, wherein the nucleic acid is DNA.

[0042] 30. The TAP or nucleic acid of any one of items 1 to 26, or the combination of item 27, wherein the nucleic acid is a component of a viral vector.

[0043] 31. A synthetic long peptide (SLP) comprising at least one of the amino acid sequences defined in item 1, or a nucleic acid encoding SLP.

[0044] 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.

[0045] 33. A vesicle or particle comprising the TAP, nucleic acid, combination or SLP of any one of items 1 to 32.

[0046] 34. The vesicle or particle of claim 33, wherein the vesicle is a lipid nanoparticle (LNP).

[0047] 35. The vesicle or particle of item 33 or 34, which comprises a cationic lipid.

[0048] 36. A composition comprising the TAP, nucleic acid, combination or SLP of any one of items 1 to 32, or the vesicle or particle of any one of items 33-35, and a pharmaceutically acceptable carrier.

[0049] 37. A vaccine comprising the TAP, nucleic acid, combination or SLP of any one of items 1 to 32, the vesicle or particle of any one of items 33-35, or the composition of item 36, and an adjuvant.

[0050] 38. An isolated major histocompatibility complex (MHC) class I molecule comprising the TAP of any one of items 1 to 26 in its peptide binding groove.

[0051] 39. The isolated MHC class I molecule of claim 38, which is in the form of a multimer.

[0052] 40. The isolated MHC class I molecule of claim 39, wherein the multimer is a tetramer.

[0053] 41. An isolated cell comprising (i) a TAP of any one of items 1-26, (ii) a combination of item 27; (iii) an SLP of item 31 or 32; or (iv) a vector comprising a nucleotide sequence encoding a TAP of any one of items 1-26, a combination of item 27, or an SLP of item 31 or 32.

[0054] 42. An isolated cell expressing a major histocompatibility complex (MHC) class I molecule on its surface, said MHC class I molecule comprising a TAP or a combination of any one of items 1 to 30 in its peptide binding groove.

[0055] 43. The cell of item 41 or 42, which is an antigen presenting cell (APC).

[0056] 44. The cell of item 43, wherein the APC is a dendritic cell.

[0057] 45. A T cell receptor (TCR) that specifically recognizes the isolated MHC class I molecule of any one of items 38-40 and / or the MHC class I molecule expressed on the cell surface of any one of items 42-44.

[0058] 46. ​​The TCR of item 45, which is a soluble TCR.

[0059] 47. An antibody or an antigen-binding fragment thereof, which specifically binds to the isolated MHC class I molecule of any one of items 38-40 and / or the MHC class I molecule expressed on the cell surface of any one of items 42-44.

[0060] 48. The TCR of item 45 or 46, or the antibody or its antigen-binding fragment according to item 47, which is a bispecific TCR or a bispecific antibody or its antigen-binding fragment.

[0061] 49. A TCR, antibody or antigen-binding fragment thereof according to item 48, wherein the bispecific antibody or antigen-binding fragment thereof is a single-chain diabody (scDb).

[0062] 50. A 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 signaling molecule.

[0063] 51. A TCR, antibody or antigen-binding fragment thereof according to item 50, wherein the T cell signaling molecule is the CD3 chain.

[0064] 52. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof of item 47 or a nucleic acid encoding the CAR.

[0065] 53. An isolated cell expressing the TCR of item 45 on its cell surface.

[0066] 54. The isolated cells of item 53, which are CD8+T lymphocytes.

[0067] 55. A cell population comprising at least 0.5% or 1% of the isolated cells defined in item 53 or 54.

[0068] 56. A method of treating breast cancer in a subject, comprising administering to the subject an effective amount of:

[0069] (a) a TAP comprising any one of the sequences shown in SEQ ID NOs: 1-61 or any combination thereof or consisting thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences shown in SEQ ID NOs: 1-61;

[0070] (b) at least one nucleic acid encoding a TAP, a combination thereof or a SLP as defined in (a);

[0071] (c) a vesicle or particle comprising a TAP, a combination thereof or a SLP as defined in (a) or at least one nucleic acid as defined in (b);

[0072] (d) a composition comprising a TAP, a combination thereof or a 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;

[0073] (e) a vaccine comprising a TAP, a combination thereof or a 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;

[0074] (f) a cell expressing a major histocompatibility complex (MHC) class I molecule on its surface, wherein the MHC class I molecule comprises a TAP as defined in (a) or a combination thereof in its peptide binding groove;

[0075] (g) a cell expressing on its cell surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of a cell as defined in (f); or

[0076] (h) a soluble TCR, an antibody or an antigen-binding fragment thereof, which specifically binds to an MHC class I molecule expressed on the surface of a cell as defined in (f).

[0077] 57. The method of claim 56, wherein the breast cancer is hormone receptor positive breast cancer (HR+) or triple negative breast cancer (TNBC).

[0078] 58. The method of item 56 or 57, further comprising administering to the subject at least one additional anti-tumor agent or therapy.

[0079] 59. The method of claim 58, wherein at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, immunotherapy, an immune checkpoint inhibitor, radiation therapy, or surgery.

[0080] 60. The following use for treating breast cancer in a subject, or for preparing a medicament for treating breast cancer in a subject:

[0081] (a) a TAP comprising any one of the sequences shown in SEQ ID NOs: 1-61 or any combination thereof or consisting thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences shown in SEQ ID NOs: 1-61;

[0082] (b) at least one nucleic acid encoding a TAP, a combination thereof or a SLP as defined in (a);

[0083] (c) a vesicle or particle comprising a TAP, a combination thereof or a SLP as defined in (a) or at least one nucleic acid as defined in (b);

[0084] (d) a composition comprising a TAP, a combination thereof or a 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;

[0085] (e) a vaccine comprising a TAP, a combination thereof or a 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;

[0086] (f) a cell expressing a major histocompatibility complex (MHC) class I molecule on its surface, wherein the MHC class I molecule comprises a TAP as defined in (a) or a combination thereof in its peptide binding groove;

[0087] (g) a cell expressing on its cell surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of a cell as defined in (f); or

[0088] (h) a soluble TCR, an antibody or an antigen-binding fragment thereof, which specifically binds to an MHC class I molecule expressed on the surface of a cell as defined in (f).

[0089] 61. The use of item 60, wherein the breast cancer is hormone receptor positive breast cancer (HR+) or triple negative breast cancer (TNBC).

[0090] 62. The use of item 60 or 61, further comprising administering at least one additional anti-tumor agent or therapy to the subject.

[0091] 63. The use of claim 62, wherein at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, immunotherapy, an immune checkpoint inhibitor, radiotherapy, or surgery.

[0092] 64. An agent for treating breast cancer in a subject, wherein the agent is:

[0093] (a) a TAP comprising any one of the sequences shown in SEQ ID NOs: 1-61 or any combination thereof or consisting thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences shown in SEQ ID NOs: 1-61;

[0094] (b) at least one nucleic acid encoding a TAP, a combination thereof or a SLP as defined in (a);

[0095] (c) a vesicle or particle comprising a TAP, a combination thereof or a SLP as defined in (a) or at least one nucleic acid as defined in (b);

[0096] (d) a composition comprising a TAP, a combination thereof or a 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;

[0097] (e) a vaccine comprising a TAP, a combination thereof or a 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;

[0098] (f) a cell expressing a major histocompatibility complex (MHC) class I molecule on its surface, wherein the MHC class I molecule comprises a TAP as defined in (a) or a combination thereof in its peptide binding groove;

[0099] (g) a cell expressing on its cell surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of a cell as defined in (f); or

[0100] (h) a soluble TCR, an antibody or an antigen-binding fragment thereof, which specifically binds to an MHC class I molecule expressed on the surface of a cell as defined in (f).

[0101] 65. The agent for use in item 64, wherein the breast cancer is hormone receptor positive breast cancer (HR+) or triple negative breast cancer (TNBC).

[0102] 66. The medicament for use in item 64 or 65, further comprising administering at least one additional anti-tumor agent or therapy to the subject.

[0103] 67. The agent for use in item 66, wherein at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, immunotherapy, an immune checkpoint inhibitor, radiotherapy or surgery.

[0104] Other objects, advantages and features of the present disclosure will become more apparent upon reading the following non-limiting description of certain embodiments of the disclosure, which are given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] In the attached picture:

[0107] Figure 1A -E depicts HR + and the typical immunopeptidome of TNBC tumors. Figure 1A : The number of unique MAPs identified per sample. Figure 1B : HR from HLA ligand ATLAS + and Venn diagram of MAP source genes in TNBC breast cancer tumors and normal tissues. Figure 1C : The total number of MAPs per source gene for the top 1% generators of MAPs. Figure 1D: PANTHER analysis of the top 1% producers of MAPs (n=242) showed enrichment in cytoskeletal, transcriptional and extracellular matrix protein classes. Figure 1E : Gene expression of non-MAPs sources, MAPs sources and the top 1% of MAPs producers (ANOVA, p<0.001).

[0108] Figure 2A -D shows ERE derived MAPs populating HR + and the immunopeptidome of TNBC tumors. Figure 2A : In HR + Similar numbers of ERE-derived MAPs were identified in WT (n=14) and TNBC samples (n=12). Figure 2B : The number of ERE-derived MAPs for each ERE category. Figure 2C :HR + and the number of MAPs identified for each ERE family in TNBC samples. Figure 2D : In tumors from the TCGA cohort, ERE families that lead to MAP generation were more highly expressed at the transcriptomic level than non-source families (t-test, p<0.001, n=741).

[0109] Figure 3A -C shows the identification of target tumor antigens. Figure 3A : Classification workflow of target MAP. Figure 3B : In HR + Number of TSAs and TAAs identified in samples, TNBC samples, or both. Most TSAs were identified only in TNBC samples (n=18 / 25). Figure 3C : Most of the TSAs and TAAs identified using the triage workflow were new.

[0110] Figure 4A -D shows the identification of TSA. Figure 4A : Expression heatmap of TSA coding sequences in normal tissues (GTEX, mTEC and bone marrow). Figure 4B : Genomic origins of TSAs. The identified aeTSAs were mainly derived from exonic regions. Figure 4C : HR of individual TAA expression >2rphm + and percentage of TNBC tumors,. Figure 4D : GSEA analysis of the TCGA breast cancer cohort showed enrichment of tumor-infiltrating leukocyte gene signatures in tumors with high levels (> median) of predictive TSA.

[0111] Figure 5A -C shows the identification of TAA. Figure 5A: Expression heatmap of TAA coding sequences in normal tissues (GTEX, mTEC, and bone marrow) reconstituted by the relevant functions of the source genes. Figure 5B : HR of individual TAA expression >2rphm + and percentage of TNBC tumors. Figure 5C :HR + GSEA analysis in the breast cancer TCGA cohort showed enrichment of both immune suppressive and immune activating pathways in tumors with high levels (> median) of predicted TAAs (as defined by expression > 2 rphm and sufficient HLA alleles for presentation).

[0112] Figure 6 Shown is the expression of CAF-derived TAAs COL11A1, COL10A1, and LRRC15 in different cells from the microenvironment of breast cancer samples.

[0113] Fig. 7A -E shows the survival analysis of predicted TSAs and TSAs in the TCGA cohort. Fig. 7A :HR + Survival analysis of predTSA, which is highly expressed in breast cancer tumors, showed no survival effect. Figure 7B -C: TNBC and HR + Survival analysis of predTAAs that are highly expressed in breast cancer tumors showed no survival effect. Fig.7D -E: Survival analysis in the TNBC cohort of TCGA showed a significant survival benefit in patients with high levels of highly expressed predTSAs derived from noncoding regions and CTAs, whereas high expression of TSAs from noncoding regions or CTAs without potential presentation was associated with decreased survival.

[0114] Figure 8 Schematic diagram of the database construction for MAPs identification, including canonical proteome, ERE proteome, small RNA proteome (smRNA) and cancer-specific proteome.

[0115] Fig. 9 The number of MAPs identified for each HLA allele (n=53) in the data set (n=26) is shown.

[0116] Fig. 10A The percentage of the transcriptome represented at the immunopeptidomic level for each cumulative sample is shown.

[0117] Fig. 10B is a histogram of the total number of MAPs generated by each source gene in the dataset.

[0118] Fig.11A -B is the PANTHER enrichment analysis of source genes specific to cancer immunity peptidome. Fig.11A :HR + Figure 11B: Source genes shared between TNBC and TNBC samples (n=277). 11B: Source genes unique to TNBC tumors (n=259).

[0119] Fig. 12A It is a schematic diagram of the workflow for small RNA database construction. Fig. 12B is a schematic diagram of the filtering steps used for peptide annotation and validation.

[0120] Fig.13 is the genomic map of the identified ERE-derived MAPs.

[0121] Fig.14 is the Spearman correlation of the mutation frequency of each gene in the breast cancer TCGA cohort with the total number of MAPs identified for each gene in the dataset.

[0122] Fig.15 GSEA analysis of breast cancer tumors from TCGA (n=1109) is shown. GSEA analysis shows enrichment of mitotic and immune activation pathways in tumors with high levels (> median) of predicted TSAs (as defined by: expression > 2 rphm and sufficient HLA alleles for presentation). Enrichment of PI3K signaling genes can be observed in tumors with low levels of predicted TSAs.

[0123] Fig.16 GSEA analysis in the TNBC cohort of TCGA is shown. GSEA analysis shows enrichment of immune activation and immune suppression pathways in tumors with high levels (> median) of predicted TAAs (as defined by: expression > 2 rphm and sufficient HLA alleles for presentation).

[0124] Fig.17 Survival analysis in the TNBC cohort of TCGA is shown, which shows a significant survival benefit in patients with high levels of highly expressed preDTSA.

[0125] Fig.18A and 18B Depicted are the results of a functional expansion of specific T cells (FEST) assay showing that after 20 days of stimulation with autologous T cell-depleted PBMCs pulsed with peptide alone, donor D26 ( Fig.18A ) and D27( Fig.18B ) for selected TSAs and TAAs. 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 peptides = MelanA, ELAGIGILTV (SEQ ID NO: 212).

[0126] Detailed disclosure

[0127] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0128] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (ie, meaning "including, but not limited to,") unless otherwise noted.

[0129] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0130] The use of any and all examples, or exemplary language ("such as," "eg," "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.

[0131] 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.

[0132] In this document, the term "about" has its ordinary meaning. The term "about" is used to indicate a value that includes the inherent error variation of the device or method being used to determine the value, or includes values ​​close to the stated value, such as within 10% of the stated value (or range of values).

[0133] Unless otherwise indicated herein, recitation of ranges of values ​​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 into the specification as if it were individually recited herein. All subsets of values ​​within the stated range are also incorporated into the specification as if they were individually recited herein.

[0134] Where features or aspects of the disclosure are described in terms of Markush groups or substituted lists, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or substituted list.

[0135] Unless specifically defined otherwise, all technical and scientific terms used herein should be construed to have the same meanings as commonly understood by one of ordinary skill in the art (e.g., stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0136] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in the following sources: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates up to the present), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JE Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0137] In the studies described herein, the inventors have used a proteogenomic approach to identify TSA and TAA candidates from breast cancer samples. The novel TSA and TAA candidates identified herein can be used, for example, in immunotherapy and vaccines against cancers expressing TSA and TAA candidates, such as breast cancer.

[0138] The present disclosure relates to tumor antigen peptides (TAPs) (or tumor-specific peptides), such as breast cancer (BC) TAPs, comprising or consisting of one of the following amino acid sequences:

[0139] Peptide sequence SEQ ID NO: Peptide sequence SEQ ID NO: SVLGPILIIPK 1 KLKQDTTQK 19 DMFGIRFKR 2 LELFQHVSL 20 EYNAEFGRR 3 MAVNIVLQ 21 FPEDFSILF 4 MLWKLVENV 22 GEAVGFQPP 5 QAWGAGLAY 23 IYLIPEPVLF 6 SEADLQAEV 24 LEISFISP L 7 SLPDNVFSNL 25 RSMRVGVTR 8 SQGQIFSKF 26 SMRSTVVSR 9 SRLELFQHV 27 SVADLLSSF 10 SSQGQIFSK 28 AETDITETSI 11 SVKERESER 29 DIIEYDYEY 12 VEEIFTEEY 30 EEIFTEEY 13 VHDPDYAKILL 31 EEQYIAQF 14 VPWEDKISI 32 ELFQHVSLM 15 VTTLALTF 33 GAYDKARYL 16 WPMHTPLL 34 GTHVWVGLYK 17 WPMHTPLLL 35 HQFTEKVI 18

[0140] In another aspect, the present disclosure also relates to the use of TAP comprising or consisting of one of the following amino acid sequences for treating breast cancer:

[0141]

[0142]

[0143] Typically, the length of the peptide (such as tumor antigen peptide (TAP)) presented in the context of HLA class I varies from about 7 or 8 to about 15, or preferably 8 to 14 amino acid residues. In some embodiments of the method disclosed herein, a longer peptide comprising a TAP sequence defined herein is artificially loaded into a cell (such as an antigen presenting cell (APC)), processed by the cell, and TAP is presented by the MHC class I molecules on the surface of the APC. In this method, a peptide / polypeptide longer than 15 amino acid residues can be loaded into the APC, processed by the protease in the APC cytosol, and the corresponding TAP for presentation as defined herein is provided. In some embodiments, the precursor peptide / polypeptide for producing the TAP defined herein is, for example, 1000, 500, 400, 300, 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20 or 15 amino acids or less. Therefore, all methods and processes using TAP described herein include the use of longer peptides or polypeptides (including natural proteins), i.e., tumor antigen precursor peptides / polypeptides, to induce the presentation of "final" 8-14TAP after cell (APC) processing. In some embodiments, the TAP mentioned herein is about 8 to 14, 8 to 13 or 8 to 12 amino acids long (e.g., 8, 9, 10, 11, 12 or 13 amino acids long), small enough to be directly suitable for HLA class I molecules. In one embodiment, TAP comprises 20 amino acids or less, preferably 15 amino acids or less, more preferably 14 amino acids or less. In one embodiment, TAP comprises at least 7 amino acids, preferably at least 8 amino acids or less, more preferably at least 9 amino acids.

[0144] The term "amino acid" as used herein includes L- and D-isomers of naturally occurring amino acids and other amino acids used in peptide chemistry to prepare synthetic analogs of TAP (e.g., naturally occurring amino acids, non-naturally occurring amino acids, amino acids not encoded by nucleic acid sequences, etc.). 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 of L-amino acids. Naturally occurring non-genetically encoded amino acids and amino acid analogs include, for example, β-alanine, 3-amino-propionic acid, 2,3-diaminopropionic acid, α-aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, tert-butylalanine, tert-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-naphthylalanine, pyridylalanine, 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine , 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, β-2-thienylalanine, methionine sulfoxide, L-homoarginine (Hoarg), N-acetyllysine, 2-aminobutyric acid, 2-aminobutyric acid, 2,4-diaminobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine, homoserine (HoSer), cysteic acid, ε-aminocaproic acid, δ-aminovaleric acid, benzyloxytyrosine, β-phenylalanine or 2,3-diaminobutyric acid (D- or L-) etc. These amino acids are well known in the field of biochemistry / peptide chemistry. Therefore, one or more amino acids in TAP (SEQ ID NO: 1-61 or SEQ ID NO: 1-35) described herein can be substituted by non-genetically encoded amino acids and / or amino acid analogs. TAP may also be modified to improve the proteolytic stability of the peptide, for example by incorporation of methyl amino acids, β-amino acids or peptoids. In one embodiment, TAP comprises only naturally occurring amino acids.

[0145] In some embodiments, TAP described herein includes peptides with altered sequences relative to the sequences mentioned herein, wherein the altered sequences contain substitutions of functionally equivalent amino acid residues. For example, one or more amino acid residues in the sequence can be substituted by another amino acid with similar polarity (having similar physicochemical properties) acting as a functional equivalent, resulting in silent changes. The substitution of amino acids in the 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). Non-polar (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 the non-polar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions carried out in an amino acid family are generally understood to be conservative substitutions. The TAP referred to herein may comprise all L-amino acids, all D-amino acids or a mixture of L- and D-amino acids. In one embodiment, the BC TAP referred to herein comprises all L-amino acids.

[0146] In one embodiment, in the sequence of a TAP comprising or consisting of one of the sequences of SEQ ID NOs: 1-61 or SEQ ID NOs: 1-35, amino acid residues that do not substantially contribute to interaction with a T cell receptor can be modified by replacing them with other amino acids, the incorporation of which does not substantially affect T cell reactivity and does not abolish binding to the relevant MHC.

[0147] TAP can also be modified by replacing one or more amide bonds of the peptide, which can improve chemical stability and / or enhance biological / pharmacological properties (e.g., half-life, absorption, efficacy, efficiency, etc.). Typical peptide bond replacements include esters, polyamines and their derivatives, and substituted alkanes and alkenes, such as aminomethyl and ketomethylene. For example, one or more amide bonds of the above TAP can be replaced by a -CH 2 NH-, -CH 2 S-、-CH 2 -CH 2 -, -CH=CH- (cis or trans), -CH 2 SO-, -CH(OH)CH 2 -or-COCH 2 -key replacement.

[0148] TAP may also be N- and / or C-terminally capped or modified to prevent degradation, increase stability, affinity and / or uptake. Thus, in another aspect, the present disclosure provides a TAP of formula Z1 -XZ 2 The invention relates to a modified TAP of the invention, wherein X is TAP comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 1-61 or SEQ ID NOs: 1-35.

[0149] In one embodiment, the amino terminal residue of TAP (ie, the free amino group at the N-terminus) is modified (eg, to prevent degradation), for example, by covalently attaching a moiety / chemical group (Z 1 ). 1 It can be a straight or branched chain alkyl group of 1 to 8 carbon atoms, or an acyl group (R-CO-), where R is a hydrophobic moiety (e.g., acetyl, propionyl, butyl, isopropionyl or isobutyl), or an aromatic acyl group (Ar-CO-), where Ar is an aromatic group. In one embodiment, the acyl group is C 1 -C 16 or C 3 -C 16 Acyl (straight or branched chain, saturated or unsaturated), in another embodiment, is a saturated C 1 -C 6 Acyl (straight or branched chain) or unsaturated C 3 -C 6 Acyl (straight or branched chain), such as acetyl (CH 3 In one embodiment, Z 1 The carboxyl terminal residue of TAP (i.e., the free carboxyl group at the C-terminus of TAP) may be modified (e.g., to prevent degradation), for example, by amidation (with NH 2 group replaces the OH group), so in this case, Z 2 Yes NH 2 In one embodiment, Z 2 It can be a hydroxamic acid group, a nitrile group, an amide (primary, secondary or tertiary) group, an aliphatic amine of 1 to 10 carbon atoms (such as methylamine, isobutylamine, isovaleramide or cyclohexylamine), an aromatic or aralkylamine (such as aniline, naphthylamine, benzylamine, cinnamylamine or phenylethylamine), an alcohol or CH 2 OH. In one embodiment, Z 2 In one embodiment, TAP comprises one of the amino acid sequences of SEQ ID NO: 1-61 or SEQ ID NO: 1-35. In one embodiment, TAP consists of one of the amino acid sequences of SEQ ID NO: 1-61 or SEQ ID NO: 1-35, i.e., wherein Z 1 and Z 2 Does not exist.

[0150] In another aspect, the present 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 show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-A*02:05, HLA-A*02:06 and / or HLA-A*02:07 molecules.

[0151] In another aspect, the present 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 show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-A*03:02 or HLA-A*30:01 molecules.

[0152] In another aspect, the present invention provides a TAP that binds to an HLA-A*11:01 molecule, comprising or consisting of a sequence of SEQ ID NO: 10, 17 or 28. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-A*03:01, HLA-A*31:01 and / or HLA-A*68:01 molecules.

[0153] In another aspect, the present invention provides a TAP that binds to an HLA-A*24:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 6. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to an HLA-A*23:01 molecule.

[0154] In another aspect, the present invention 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 show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-A*26:01, HLA-A*66:01 or HLA-B*15:02 molecules.

[0155] In another aspect, the present invention 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 show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to an HLA-A*25:01 or HLA-A*66:01 molecule.

[0156] In another aspect, the present invention provides a TAP that binds to an HLA-A*31:01 molecule, comprising or consisting of a sequence of SEQ ID NO: 8, 9 or 29. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-A*11:01, HLA-A*33:01, HLA-A*33:03 or HLA-A*68:01 molecules.

[0157] In another aspect, the present invention provides a TAP that binds to an HLA-A*33:01 molecule, comprising or consisting of a sequence of SEQ ID NO: 2 or 3. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-A*31:01, HLA-A*33:03 or HLA-A*68:01 molecules.

[0158] In another aspect, the present invention 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 show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further 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.

[0159] In another aspect, the present invention provides a TAP that binds to an HLA-B*18:01 molecule, comprising or consisting of a sequence of SEQ ID NO: 13, 14, or 30. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-B*40:01, HLA-B*44:02, HLA-B*44:03, and / or HLA-B*45:01 molecules.

[0160] In another aspect, the present invention provides a TAP that binds to an HLA-B*27:05 molecule, comprising or consisting of the sequence of SEQ ID NO: 27. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-B*27:02.

[0161] In another aspect, the present invention provides a TAP that binds to an HLA-B*35:01 molecule, comprising or consisting of a sequence of SEQ ID NO: 4, 12, 23. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-B*15:02, HLA-B*35:02, HLA-B*35:03 or HLA-B*53:01 molecules.

[0162] In another aspect, the present invention 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 show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further 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.

[0163] In another aspect, the present disclosure provides a TAP that binds to an HLA-B*35:08 molecule, comprising or consisting of the sequence of SEQ ID NO: 35.

[0164] In another aspect, the present invention provides a TAP that binds to an HLA-B*38:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 31. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to an HLA-B*39:01 molecule.

[0165] In another aspect, the present invention provides a TAP that binds to an HLA-B*40:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 20. Since HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-molecules.

[0166] In another aspect, the present invention provides a TAP that binds to an HLA-B*49:01 molecule, comprising or consisting of a sequence of SEQ ID NO: 11 or 24. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further 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.

[0167] In another aspect, the present invention provides a TAP that binds to an HLA-B*50:01 molecule, comprising or consisting of a sequence of SEQ ID NO: 5 or 7.

[0168] In another aspect, the present invention provides a TAP that binds to an HLA-B*51:01 molecule, comprising or consisting of a sequence of SEQ ID NO: 32 or 34. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to an HLA-B*52:01 molecule.

[0169] In another aspect, the present invention 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 show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to an HLA-B*51:01 molecule.

[0170] In another aspect, the present invention 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 show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-A*32:01 or HLA-B*57:01 molecules.

[0171] In another aspect, the present disclosure provides a TAP that binds to an HLA-C*01:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 25.

[0172] In another aspect, the present disclosure provides a TAP that binds to an HLA-C*12:03 molecule, comprising or consisting of a sequence of SEQ ID NO: 16 or 21. Because HLA alleles show promiscuity (certain HLA alleles present similar epitopes), the TAP identified above can further bind to HLA-B*46:01, HLA-C*03:02, HLA-C*03:03, HLA-C*03:04, HLA-C*08:01, HLA-0*12:03, HLA-C*15:02 or HLA-C*16:01 molecules.

[0173] The TAP of the present disclosure can be produced by expression (recombinant expression) in a host cell comprising a nucleic acid encoding TAP or by chemical synthesis (e.g., solid phase peptide synthesis). The peptide can be easily synthesized by manual and / or automatic solid phase procedures well known in the art. Suitable synthesis can be performed, for example, by utilizing a "T-boc" or "Fmoc" procedure. The techniques and procedures for solid phase synthesis are described in, for example, Solid Phase Peptide Synthesis: A Practical Approach, E. Atherton and RC Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, TAP can be prepared by segment condensation, as described, for example, in Liu et al., Tetrahedron Lett. 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. 116:4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91:6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 45:209-216, 1995; Schnolzer and Kent, Science 256:221-225, 1992; Liu and Tam, J. Am. Chem. Soc. 116:4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91:6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 45:209-216, 1995; Res.31:322-334, 1988). Other methods that can be used to synthesize TAP are described in Nakagawa et al., J.Am.Chem.Soc.107:7087-7092, 1985. In one embodiment, TAP is chemically synthesized (synthetic peptide). Another embodiment of the present disclosure relates to non-naturally occurring peptides, wherein the peptide consists of or essentially consists of an amino acid sequence as defined herein, and has been synthetically produced (e.g., synthesized) as a pharmaceutically acceptable salt. The salt of TAP according to the present disclosure is different from a peptide that is essentially derived from an in vivo state, because the peptide produced in vivo is not a salt. The non-natural salt form of the peptide can adjust the solubility of the peptide, particularly in the case of a pharmaceutical composition comprising the peptide, such as a peptide vaccine disclosed herein. Preferably, the salt is a pharmaceutically acceptable salt of the peptide.

[0174] In one embodiment, the TAP mentioned herein is substantially pure. When the compound is separated from the components that naturally accompany it, it is "substantially pure". Typically, when the compound is at least 60% by weight of the total material in the sample, more typically 75% by weight, 80% by weight or 85% by weight, preferably more than 90% by weight, more preferably more than 95% by weight, the compound is substantially pure. Therefore, for example, a polypeptide produced by chemical synthesis or recombinant technology is generally substantially free of its naturally associated components, such as components of its source macromolecules. When a nucleic acid molecule is not directly adjacent to a coding sequence (i.e., covalently linked), the nucleic acid molecule is substantially pure, and the coding sequence is generally adjacent to the coding sequence in the naturally occurring genome of the organism from which the nucleic acid is derived. Substantially pure compounds can be obtained, for example, by extraction from natural sources; by expressing recombinant nucleic acid molecules encoding peptide compounds; or by chemical synthesis. Purity can be measured using any suitable method, such as column chromatography, gel electrophoresis, HPLC, etc. In one embodiment, TAP is in solution. In another embodiment, TAP is in solid form, such as lyophilized.

[0175] 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).

[0176] In another aspect, the disclosure also provides a synthetic long peptide (SLP) comprising at least one TAP as described herein. In one embodiment, SLP includes at least two TAPs, wherein at least one TAP is a TAP as described herein. In one embodiment, SLP includes at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 of the TAPs as described herein. In one embodiment, SLP includes at least one TAP as described herein, which is connected to one or more amino acid sequences or domains that confer the desired properties of SLP, such as stabilizing SLP and / or improving the processing and presentation of MHC molecules, for example, comprising a sequence of a motif that can be cut by a cell protease (such as a cathepsin). In another embodiment, SLP includes at least one TAP as described herein and a TAP in conjunction with an MHC class II molecule. TAPs can be directly attached to each other, or can be indirectly attached by a linker (such as a short amino acid linker). In an embodiment, the joint comprises about 4 to about 20 amino acids, or about 4 to about 15 amino acids, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. In one embodiment, the joint comprises a glycine residue, a serine residue, a proline residue, a threonine residue or a mixture thereof. The joint may include a sequence that promotes SLP processing to release TAP, such as a cathepsin-sensitive joint (for example, a joint comprising 4-6 amino acids of sequence LVGS, ASLG, PIVG, LLSV, VLSVG or LLSVGG, see Rabu et al., Oncoimmunology.2019; 8 (4): e1560919). In one embodiment, SLP has a length of 500, 400, 300, 200, 150, 100, 90, 80, 70, 60 or 50 amino acids or less. In another embodiment, the SLP is 20 to 50, 45 or 40 amino acids in length, such as 20 or 25 amino acids to 30, 35 or 40 amino acids in length.

[0177] In another aspect, the disclosure also provides nucleic acids (isolated) encoding TAP or tumor antigen precursor peptides or SLPs mentioned herein. In one embodiment, the nucleic acid comprises about 24 nucleotides to about 1200 nucleotides, about 24 to about 1000, 900, 800, 700, 600, 500, 400, 300 or 200 nucleotides, for example, about 24 to about 150 or 100 nucleotides, for example, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 53, 66, 69 or 72 nucleotides. As used herein, "isolated" refers to peptides or nucleic acid molecules separated from other components present in the natural environment of the molecule or naturally occurring source macromolecules (e.g., including other nucleic acids, proteins, lipids, sugars, etc.). As used herein, "synthetic" refers to peptides or nucleic acid molecules that are not separated from their natural sources, such as peptides or nucleic acid molecules produced by recombinant technology or using chemical synthesis. In one embodiment, the nucleic acid (DNA, RNA) encoding TAP or SLP of the present disclosure includes any sequence listed in the following table or a corresponding RNA sequence. In one embodiment, the nucleic acid encoding TAP or SLP is an mRNA molecule. In other embodiments, the nucleic acid encoding TAP or SLP is self-amplification mRNA (saRNA), trans-amplification mRNA (taRNA) or circular mRNA (circRNA) (see, e.g., Liu et al., Nature Reviews Cancer, Vol. 23, August 2023, pp. 526-543).

[0178]

[0179]

[0180]

[0181] Of course, due to the degeneracy of the genetic code, the TAP described herein may be encoded by variants of the above sequences.

[0182] The nucleic acid of the present disclosure can be used for recombinant expression of the TAP or SLP of the present disclosure, and can be contained in a vector or plasmid, such as a cloning vector or an expression vector, which can be transfected into a host cell. In one embodiment, the present disclosure provides a clone, expression or viral vector or plasmid comprising a nucleic acid sequence encoding the TAP of the present disclosure. Alternatively, the nucleic acid encoding the TAP of the present disclosure can be incorporated into the genome of the 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 a specific test cell, but also to the offspring or potential offspring of such a cell. The host cell can be any prokaryotic (e.g., Escherichia coli) or eukaryotic cell (e.g., insect cell, yeast cell, plant cell, or mammalian cell) capable of expressing the TAP described herein. The vector or plasmid contains the elements necessary for transcription and translation of the inserted coding sequence, and may contain other components, such as resistance genes, cloning sites, etc. Methods well known to those skilled in the art can be used to construct an expression vector containing a sequence encoding a peptide or polypeptide and an appropriate transcription and translation control / regulatory element operably connected thereto. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. These 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 nucleotide sequences) so that the normal functions of the components can be performed. Thus, a coding sequence operably linked to a regulatory sequence refers to a configuration of nucleotide sequences 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 that participates in regulating the expression of a coding nucleic acid. Thus, the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like. The vector (e.g., expression vector) may have necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences, such as CMV, PGK and EF-1α promoters, ribosome recognition and binding TATA box and 3'UTR AAUAAA transcription termination sequence, for efficient gene transcription and translation in its respective host cell.Other suitable promoters include constitutive promoters of 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 Rous sarcoma virus promoter. Human gene promoters can also be used, including but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. In certain embodiments, inducible promoters are also considered to be part of the vector expressing TAP. This provides a molecular switch that can turn on the expression of the target polynucleotide sequence or turn off the expression. Examples of inducible promoters include but are not limited to metallothionein promoters, glucocorticoid promoters, progesterone promoters or tetracycline promoters. Examples of vectors are plasmids, autonomous replication sequences and transposable elements. Other exemplary vectors include but are not limited to plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC)), phages (such as lambda phages or M13 phages) and animal viruses. Examples of animal virus classes that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). An example of an expression vector is Lenti-X for expression in mammalian cells. TM Bicistronic Expression System (Neo) vector (Contech), pCIneo vector (Promega); pLenti4 / V5-DEST for lentivirus-mediated gene transfer and expression in mammalian cells TM 、pLenti6 / V5-DEST TM and pLenti6.2N5-GW / lacZ (Invitrogen). The coding sequence of TAP disclosed herein can be connected to such expression vectors for expressing TAP in mammalian cells.

[0183] In certain embodiments, a nucleic acid encoding a TAP of the present disclosure is provided in a viral vector. The viral vector may be those derived from adenovirus, vaccinia virus, retrovirus, slow virus or foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into viral vector particles. The viral vector may contain the coding sequence of various TAPs or SLPs described herein to replace non-essential viral genes. In another embodiment, the nucleic acid encoding a TAP or SLP of the present disclosure is provided in a self-amplification or self-replicating RNA (saRNA or srRNA) vector. srRNA is derived from a positive-strand RNA virus, in which the structural protein has been removed and replaced by a heterologous target gene. srRNA has been successfully derived from flaviviruses, nodaviviruses, nidoviruses, and alphaviruses, where the therapeutic version of the technology provides trans-structural proteins to produce single-cycle viral replicon particles (VRP) (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). Vectors and / or particles can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells in vitro or in vivo. Many forms of viral vectors are known in the art.

[0184] In an embodiment, a nucleic acid (DNA, RNA) encoding a TAP or SLP of the present disclosure is contained in a vesicle or nanoparticle, such as a lipid vesicle (e.g., liposome) or lipid nanoparticle (LNP), or any other suitable carrier (e.g., mRNA packaging system). Therefore, in another aspect, the present disclosure provides a vesicle or nanoparticle, such as a lipid vesicle or nanoparticle, comprising a nucleic acid encoding one or more of the TAP or SLP described herein, such as mRNA.

[0185] The term liposome as used herein, according to its ordinary meaning, refers to a microscopic lipid vesicle composed of a bilayer of phospholipids or any similar amphipathic lipids (eg, sphingolipids) enclosing an internal aqueous medium.

[0186] The term "lipid nanoparticle" refers to a liposome-like structure that can include one or more lipid bilayer rings around an internal aqueous medium, or a micellar-like structure that encapsulates a molecule (e.g., nucleic acid) in a non-aqueous core. Lipid nanoparticles typically contain cationic lipids, such as ionizable cationic lipids. Examples of cationic lipids that can be used for LNPs include DOTMA, DOSPA, DOTAP, DOPE, ePC, DLin-MC3-DMA, C12-200, ALC-0315, cKK-E12, lipid H (SM-102), OF-Deg-Lin, A2-lso5-2DC18, 306. i10 , BAME-O16B, TT3, 9A1P9, FTT5, SS-E, SS-EC, SS-OC and SS-OP (see, for example, Hou et al., Nature Reviews Materials, Vol. 6, pp. 1078-1094 (2021); Tenchov et al., ACS Nano, 15, 16982-17015 (2021)).

[0187] Liposomes and lipid nanoparticles often contain other lipid components, such as lipids, lipid-like materials, and polymers, which can improve the properties of the liposome or nanoparticle, 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 cholesterol derivatives), PEGylated lipids (PEG-lipids), such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG 2000 -DMG) and 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol-2000 (PEG 2000 -DSG).

[0188] In one embodiment, the lipid nanoparticles according to the present disclosure include one or more cationic lipids, such as ionizable cationic lipids. Examples of ionizable cationic lipids include those listed in PCT Publication Nos. WO 2017 / 061150 and WO 2019 / 188867, including those sold under the trade names SS-E, SS-EC, SS-OC and SS-OP is a commercially available ionizable cationic lipid.

[0189] The nucleic acid (e.g., mRNA) encoding one or more TAPs can be modified, for example, to increase 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 US10519189 and US10494399). One or more nucleosides of mRNA can be modified or substituted with 1-methyl pseudouridine, pseudouridine (ψ), N6-methyladenosine, 5-methylcytidine and / or 5-methyluridine to increase the stability of the molecule, improve translation efficiency and / or reduce the recognition of the molecule by the innate immune system. The form of modified nucleosides is described in US9371511. Other types of modifications that can be made to mRNA include incorporation of anti-reverse cap analogs (ARCA), 5'-methyl-cytidine triphosphate (m5CTP), N6-methyl-adenosine-5'-triphosphate (m6ATP), 2-thio-uridine triphosphate (s2UTP), pseudouridine triphosphate, N 1 Methyl pseudouridine triphosphate or 5-methoxyuridine triphosphate (5moUTP). mRNA may also include additional modifications to the 5'- and / or 3'-untranslated region (UTR) polyadenylation (polyA) tail (see, e.g., Kim et al., Molecular & Cellular Toxicology Vol. 18, 1 (2022): 1-8). The poly (A) tail preferably comprises 100-200 nucleotides, and more preferably 120-150 nucleotides, and may include modified adenosine. The present disclosure encompasses all of these and other modifications to nucleic acids encoding TAP (e.g., mRNA).

[0190] In another aspect, the present disclosure provides an MHC class I molecule comprising (ie, presenting or binding) one or more BC TAPs comprising or consisting of a sequence of SEQ ID NO: 1-61 or SEQ ID NO: 1-35 as defined herein.

[0191] 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.

[0192] In one embodiment, TAP (e.g., comprising or consisting of a sequence of SEQ ID NO: 1-61 or SEQ ID NO: 1-35 as defined herein) is non-covalently bound to an MHC class I molecule (i.e., TAP is loaded into a peptide binding groove / pocket of an MHC class I molecule or non-covalently bound to a peptide binding groove / pocket of an MHC class I molecule). In another embodiment, TAP is covalently linked / bound to an MHC class I molecule (α chain). In such a construct, TAP and MHC class I molecule (α chain) are produced as synthetic fusion proteins, typically with a short (e.g., 5 to 20 residues, preferably about 8-12, such as 10) flexible linker or spacer (e.g., polyglycine linker). In another aspect, the disclosure provides a nucleic acid encoding a fusion protein comprising a TAP defined herein fused to an MHC class I molecule (α chain). In one embodiment, the MHC class I molecule (α chain)-peptide complex is multimerized. Therefore, in another aspect, the present disclosure provides a polymer of MHC class I molecules loaded (covalently or non-covalently) with TAP mentioned herein. Such a polymer can be connected to a label, such as a fluorescent label, which allows the detection of the polymer. A large number of strategies have been developed for the production of MHC polymers, including MHC dimers, tetramers, pentamers, octamers, etc. (reviewed in Bakker and Schumacher, Current Opinion in Immunology 2005, 17: 428-433). MHC polymers can be used, for example, to detect and purify antigen-specific T cells. Therefore, in another aspect, the present disclosure provides a method for detecting or purifying (separating, enriching) CD8 TAPs specific to TAP defined herein. + A method for detecting T lymphocytes, the method comprising contacting a cell population with a multimer of MHC class I molecules loaded (covalently or non-covalently) with TAP; and detecting or isolating CD8+ T lymphocytes bound by the MHC class I multimer. + T lymphocytes can be isolated using known methods, such as fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

[0193] In another aspect, the disclosure provides cells (e.g., host cells), in one embodiment, there is provided a cell separated, which comprises the nucleic acid, vector or plasmid of the disclosure mentioned herein, i.e., nucleic acid or vector encoding one or more TAPs or SLPs. In another aspect, the disclosure provides a cell, which expresses on its surface binding to or presenting the MHC class I molecules (e.g., the MHC class I molecules of one of the alleles disclosed above) according to the TAP of the disclosure. In one embodiment, the host cell is a eukaryotic cell, such as a mammalian cell, preferably a human cell, a cell line or an 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, a cell line or an immortalized cell. Nucleic acids and vectors can be introduced into cells by conventional transformation or transfection techniques. The terms "transformation" and "transfection" refer to techniques for introducing exogenous nucleic acids into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and virus-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 acids 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.

[0194] Cells, such as APCs, can be loaded with one or more TAPs using a variety of methods known in the art. As used herein, "loading cells" with TAP means transfecting RNA or DNA or TAP encoding TAP into cells, or transforming APCs with nucleic acids encoding TAP. Cells can also be loaded by contacting cells with exogenous TAPs, which can directly bind to MHC class I molecules (e.g., peptide-pulsed cells) present on the cell surface. TAPs can also be fused to domains or motifs that promote their presentation by MHC class I molecules, such as fusion to endoplasmic reticulum (ER) recycling signals, C-terminal Lys-Asp-Glu-Leu sequences (see Wang et al., Eur J Immunol. December 2004; 34 (12): 3582-94).

[0195] In another aspect, the present disclosure provides a composition or peptide combination / pool comprising any one or any combination of TAPs defined herein (or nucleic acids encoding the peptides). In one embodiment, the composition comprises any combination of TAPs defined herein (any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TAPs), or a combination of nucleic acids encoding the TAPs). The present disclosure encompasses compositions comprising any combination / subcombination of TAPs defined herein. In another embodiment, the combination or pool may comprise one or more known tumor antigens.

[0196] Thus, in another aspect, the present disclosure provides a composition comprising any one or any combination of TAPs defined herein (e.g., comprising or consisting of a sequence of SEQ ID NO: 1-61 or SEQ ID NO: 1-35 as defined herein), and a cell expressing an MHC class I molecule (e.g., an MHC class I molecule of one of the alleles disclosed above). APCs for use in the present disclosure are not limited to a particular type of cell, and include professional APCs such as dendritic cells (DCs), Langerhans cells, macrophages, and B cells, which are known to present protein antigens on their cell surfaces for expression by CD8 + T lymphocyte recognition. For example, DC can be induced from peripheral blood mononuclear cells, and then APC can be obtained by contacting (stimulating) TAP in vitro, in vitro or in vivo. APC can also be activated to present TAP in vivo in which one or more TAPs of the present disclosure are applied to a subject, and APCs that induce and present TAP in the subject are induced. The phrase "inducing APC" or "stimulating APC" includes contacting or loading cells with one or more TAPs or nucleic acids encoding TAPs so that TAP is presented by MHC class I molecules on its surface. As described herein, according to the present disclosure, TAPs can be loaded indirectly, for example, using longer peptides / polypeptides (including native proteins) comprising TAP sequences, and then processed in APC (for example, by proteases) to produce TAP / MHC class I complexes on the cell surface. After APC is loaded with TAP and APC is allowed to present TAP, APC can be administered to a subject as a vaccine. For example, ex vivo administration may include the following steps: (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.

[0197] The first subject and the second subject may be the same subject (e.g., an autologous vaccine), or may be different subjects (e.g., an allogeneic vaccine). Alternatively, according to the present disclosure, there is provided the use of the TAP (or a combination thereof) described herein in the preparation of a composition (e.g., a pharmaceutical composition) for inducing antigen presenting cells. In addition, the present disclosure provides a method or process for preparing a pharmaceutical composition for inducing antigen presenting cells, wherein the method or process comprises a step of mixing or formulating the TAP or a combination thereof with a pharmaceutically acceptable carrier. Cells (e.g., APCs) expressing MHC class I molecules (e.g., any of the above HLA molecules) loaded with any one or any combination of the TAPs defined herein can be used to stimulate / amplify CD8 + T lymphocytes, such as autologous CD8 +T lymphocytes. Therefore, in another aspect, the present disclosure provides a composition comprising any one or any combination of TAPs (or nucleic acids or vectors encoding them) as defined herein; cells expressing MHC class I molecules and T lymphocytes, more specifically CD8 + T lymphocytes (e.g., comprising CD8 + T lymphocyte cell population).

[0198] In one embodiment, the composition further comprises a buffer, an excipient, a carrier, a diluent and / or a medium (e.g., a culture medium). In another embodiment, the buffer, excipient, carrier, diluent and / or medium are one or more pharmaceutically acceptable buffers, excipients, carriers, diluents and / or mediums. As used herein, "pharmaceutically acceptable buffers, excipients, carriers, diluents and / or mediums" include any and all solvents, buffers, adhesives, lubricants, fillers, thickeners, disintegrants, plasticizers, coatings, barrier layer preparations, lubricants, stabilizers, release delay agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, etc., that are physiologically compatible, do not interfere with the effectiveness of the biological activity of the active ingredient and are nontoxic to the subject. These media and reagents are well known in the art for the use of pharmaceutically active substances (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4th edition, Pharmaceutical Press, London UK). Unless any conventional medium or agent is incompatible with the active compound (peptide, cell), its use in the composition of the present disclosure is considered. 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 TAPs defined herein or nucleic acids encoding the one or more TAPs (e.g., mRNA) are contained in or complexed with lipid vesicles or liposomes, such as cationic liposomes (see, e.g., Vitor MT et al., Recent Pat Drug Deliv Formul. 2013 August; 7(2): 99-110) or other suitable carriers.

[0199] In another aspect, the present disclosure provides a composition comprising one or more of any one or any combination of TAPs defined herein (e.g., comprising or consisting of a sequence of SEQ ID NOs: 1-61 or SEQ ID NOs: 1-35 as defined herein) (or a nucleic acid, such as an mRNA encoding the peptide), and a buffer, an excipient, a carrier, a diluent and / or a medium. For compositions comprising cells (e.g., APCs, T lymphocytes), the composition comprises a suitable culture medium that allows the maintenance of living cells. Representative examples of such culture media include salt solutions, Earl's balanced salt solution (Life )or (Baxter ). In one embodiment, the composition (e.g., pharmaceutical composition) is an "immunogenic composition", "vaccine composition" or "vaccine". The terms "immunogenic composition", "vaccine composition" or "vaccine" as used herein refer to a composition or formulation comprising one or more TAPs, nucleic acids or vaccine vectors, and when administered to a subject, it is capable of inducing an immune response to one or more TAPs present therein. Vaccination methods for inducing an immune response in a mammal (e.g., a human) include using a vaccine or vaccine vector administered by any conventional route known in the field of vaccines, such as through a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal or urinary tract) surface, by parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous or intraperitoneal) route, or topical administration (e.g., by a transdermal delivery system, such as a patch). In one embodiment, TAP (or a combination thereof) is conjugated to a carrier protein (conjugated vaccine) to increase the immunogenicity of TAP. Therefore, the present disclosure provides a composition (conjugate) comprising TAP (or a combination thereof) or a nucleic acid encoding TAP or a combination thereof, and a carrier protein. For example, the TAP or nucleic acid can be conjugated or complexed to a Toll-like receptor (TLR) ligand (see, e.g., Zorn 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-linked 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 (TAP, nucleic acid and / or cell according to the present disclosure), non-specifically enhances or strengthens the immune response of the host to the agent when exposed 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, such as oil emulsions and surfactant-based formulations, such as MF59 (microfluidized detergent-stabilized oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + QS-21), (3) particulate adjuvants, such as virosomes (unilamellar liposome vehicles incorporating influenza hemagglutinin), AS04 ([SBAS4] aluminum salt with MPL), ISCOMS (structured complex of saponin and lipids), polylactide-co-glycolide (PLG), (4) microbial derivatives (natural and synthetic), such as monophosphoryl lipids A (MPL), Detox (MPL + Mycobacterium phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipid immunostimulator capable of self-organization into liposomes), OM-174 (lipid A derivative), CpG motif (synthetic oligonucleotide containing immunostimulatory CpG motif), modified cholera toxin (CT) and Escherichia coli enterotoxin (LT) (genetically modified bacterial toxins to provide non-toxic adjuvant effects), (5) endogenous human immunomodulators, such as hGM-CSF or hIL-12 (cytokines that can be administered as encoded proteins or plasmids), Immudaptin (C3d tandem array) and / or (6) inert carriers, such as gold particles, etc. In one embodiment, the vaccine is an RNA vaccine.

[0200] In one embodiment, TAP (e.g., comprising or consisting of a sequence of SEQ ID NO: 1-61 or SEQ ID NO: 1-35) or SLP (or a nucleic acid, such as an mRNA encoding the peptide) or a composition comprising the same is in lyophilized form. In another embodiment, TAP, SLP, nucleic acid, or a composition comprising the same is in a liquid composition. In another embodiment, the concentration of TAP or nucleic acid in the composition is from about 0.01 μg / mL to about 100 μg / mL. In a further embodiment, the concentration of TAP or nucleic acid in the composition is from about 0.2 μg / mL to about 50 μg / mL, from about 0.5 μg / mL to about 10, 20, 30, 40 or 50 μg / mL, from about 1 μg / mL to about 10 μg / mL, or about 2 μg / mL.

[0201] As described herein, cells (such as APCs) expressing MHC class I molecules loaded or bound to any one or any combination of TAPs defined herein can be used to stimulate / expand CD8 +T lymphocytes. Therefore, in another aspect, the present disclosure provides T cell receptor (TCR) molecules that can interact or bind with the MHC class I molecules / TAP complexes mentioned herein, and nucleic acid molecules encoding such TOR molecules, and vectors comprising such nucleic acid molecules. According to the TCR of the present disclosure, it is possible to specifically interact or bind with TAP loaded on or presented by MHC class I molecules in vitro or in vivo, preferably on the surface of living cells.

[0202] The term TCR as used herein refers to a member of the immunoglobulin superfamily having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd edition, Current Biology Publications, p. 4: 33, 1997), which is capable of specifically binding to an antigenic peptide bound to an MHC receptor. TCRs can be found on the surface of cells and are typically composed of heterodimers having α and β chains (also referred to as TCRα and TCRβ, respectively). Like immunoglobulins, the extracellular portion of the TCR chain (e.g., α chain, β chain) contains two immunoglobulin regions, a variable region (e.g., TCR variable α region or Vα and TCR variable β region or Vβ; typically amino acids 1 to 116 based on the Rabat numbering of the N-terminus) and a constant region adjacent to the cell membrane (e.g., TCR constant domain βh or Cβ, typically amino acids 117 to 295 based on Rabat). Furthermore, like immunoglobulins, the variable domains contain complementarity determining regions (CDRs, 3 in each chain) separated by framework regions (FRs).In certain embodiments, the TCR is present on the surface of a T cell (or T lymphocyte) and associates with a CD3 complex.

[0203] TCRs and in particular nucleic acids encoding TCRs of the present disclosure can be used, for example, to genetically transform / modify T lymphocytes (e.g., CD8 + T lymphocytes) or other types of lymphocytes that generate new T lymphocyte clones that specifically recognize MHC class I / TAP complexes. In a specific embodiment, 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 infusion). In a specific embodiment, 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 a recipient (allogeneic cell infusion). In another embodiment, the present disclosure provides a CD8 T lymphocyte transformed / transfected by a vector or plasmid encoding a TAP-specific TCR. + T lymphocytes. In another embodiment, the disclosure provides a method for treating a patient with autologous or allogeneic cells transformed with TAP-specific TCRs. In certain embodiments, TCRs are expressed in primary T cells (e.g., cytotoxic T cells) by replacing endogenous loci (e.g., endogenous TRAC and / or TRBC loci) using, for example, CRISPR, TALEN, zinc finger nucleases, or other targeted destruction systems.

[0204] In another embodiment, the disclosure provides a nucleic acid encoding the above-mentioned TCR. In another embodiment, the nucleic acid is present in a vector, such as the above-mentioned vector.

[0205] In another embodiment, a use of a tumor antigen-specific TCR in the preparation of autologous or allogeneic cells for the treatment of breast cancer is provided.

[0206] In some embodiments, patients treated with the compositions of the present disclosure (e.g., pharmaceutical compositions) are treated before 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 comprising TAP nucleic acid (e.g., mRNA) encoding TAP and allogeneic or autologous T lymphocytes (e.g., CD8 + T lymphocytes) or lymphocytes transformed with tumor antigen-specific TCRs. The method of providing T lymphocyte clones capable of recognizing TAP according to the present disclosure can be produced for tumor cells expressing TAP in a subject (e.g., a transplant recipient) (e.g., an allogeneic T lymphocyte and / or donor lymphocyte infusion (DLI) recipient) and can specifically target the tumor cells. Therefore, the present disclosure provides a CD8 T cell receptor encoding and expressing a T cell receptor that can specifically recognize or bind to a TAP / MHC class I molecule complex. + T lymphocytes. The T lymphocytes (e.g., CD8 + The T lymphocytes) can be recombinant (engineered) or naturally selected T lymphocytes. Therefore, the present specification provides at least two methods for producing the CD8 +The method comprises a step of contacting undifferentiated lymphocytes with a TAP / MHC class I molecule complex (usually expressed on the surface of a cell such as an APC) under conditions conducive to triggering T cell activation and expansion, which can be performed in vitro or in vivo (i.e., in a patient administered an APC vaccine, wherein the APC is loaded with TAP, or in a patient treated with a TAP vaccine). Using a combination or pool of TAPs bound to MHC class I molecules, a CD8 TAP capable of recognizing multiple TAPs can be generated. + Alternatively, tumor antigen-specific or targeted T lymphocytes can be generated in vitro or ex vivo by cloning one or more nucleic acids (genes) encoding TCRs (more specifically α and β chains) that specifically bind to the MHC class I molecule / TAP complex (i.e., engineered or recombinant CD8 + T lymphocytes). Nucleic acids encoding TAP-specific TCRs of the present invention can be obtained from T lymphocytes activated ex vivo for TAP (e.g., activated with APCs loaded with TAP) using methods known in the art; or from individuals that exhibit an immune response to a peptide / MHC molecule complex. TAP-specific TCRs of the present invention can be recombinantly expressed in host cells and / or host lymphocytes obtained from a transplant recipient or transplant donor, and optionally differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). Nucleic acids encoding TCR α and β chains (transgenes) can be introduced into T cells (e.g., from a subject to be treated or 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 TCRs specific for TAP can be amplified in vitro using well-known culture methods. + T lymphocytes.

[0207] The present disclosure provides a method for preparing an immune effector cell expressing TCR as described herein. In one embodiment, the method includes transfection or transduction of immune effector cells, such as immune effector cells separated from a subject (such as a subject with breast cancer), so that the immune effector cells express one or more TCR as described herein. In certain embodiments, immune effector cells are separated from individuals and genetically modified in the absence of further in vitro operation. Such cells can then be directly re-applied to individuals. In a further embodiment, immune effector cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express TCR. In this regard, immune effector cells can be cultivated before or after genetic modification (that is, transduction or transfection to express TCR as described herein).

[0208] Prior to the in vitro manipulation or genetic modification of immune effector cells as described herein, cell sources can be obtained from subjects. In particular, immune effector cells used together with TCR as described herein include T cells. T cells can be obtained from many sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from infection sites, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, any technology known to those skilled in the art (e.g., FICOLL TM Separation) obtains T cells from blood units collected from subjects. In one embodiment, cells from individual circulating blood are obtained by apheresis. Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. In one embodiment, cells collected by apheresis can be washed to remove plasma fractions and the cells are placed in appropriate buffers or culture media for subsequent processing. In one embodiment of the present invention, cells are washed with PBS. In an alternative embodiment, the washing solution does not contain calcium and may not contain magnesium, or may not contain many (if not all) divalent cations. As will be understood by those of ordinary skill in the art, the washing step can be completed by methods known to those skilled in the art, such as by using a semi-automatic circulation centrifuge. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions containing or not containing buffers. In certain embodiments, undesirable components of apheresis samples can be removed in the culture medium in which the cells are directly resuspended. In certain embodiments, by lysing red blood cells and depleting monocytes, for example, by PERCOLL TM Gradient centrifugation to separate T cells from peripheral blood mononuclear cells. Specific subsets of T cells, such as CD28 + 、CD4 + 、CD8 + 、CD45RA + and CD45RO +T cells can be further separated by positive or negative selection techniques.For example, enrichment of T cell populations by negative selection can be achieved with a combination of antibodies for surface markers specific to cells selected by negative selection.A method used herein is to sort cells and / or select by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies for cell surface markers present on cells selected by negative selection.For example, in order to enrich CD8+ cells by negative selection, a mixture of monoclonal antibodies generally includes antibodies for CD14, CD20, CD11b, CD16, HLA-DR and CD4.Flow cytometry and cell sorting can also be used to separate the target cell populations used in the present disclosure.PBMC can be directly used for genetic modification performed with TCR using methods as described herein.In certain embodiments, after separation of PBMC, T lymphocytes are further separated, and in certain embodiments, both cytotoxic T lymphocytes and helper T lymphocytes can be sorted into naive T cell subsets, memory T cell subsets and effector T cell subsets before or after genetic modification and / or amplification.

[0209] The present disclosure provides isolated immune cells, such as T lymphocytes (e.g., CD8+ T lymphocytes), which are specifically induced, activated and / or amplified (expanded) by TAPs (i.e., TAPs that bind to MHC class I molecules expressed on the cell surface), or a combination of TAPs. The present disclosure also provides compositions comprising CD8+ TAPs that can recognize TAPs according to the present disclosure (i.e., one or more TAPs that bind to MHC class I molecules) or a combination thereof. + In another aspect, the present disclosure provides a cell population or cell culture enriched for T lymphocytes (e.g., CD8 + T lymphocyte population), the T lymphocytes specifically recognize one or more MHC class I molecules / TAP complexes as described herein. This enriched population can be obtained by ex vivo expansion of specific T lymphocytes using cells such as APCs expressing MHC class I molecules loaded with (e.g., presenting) one or more TAPs disclosed herein. "Enrichment" as used herein means that tumor antigen-specific T cells (e.g., CD8 + T lymphocytes) are significantly higher than the natural population of cells, that is, cells that have not undergone specific T lymphocytes (e.g., CD8 + In a further embodiment, the TAP-specific T lymphocytes (e.g., CD8 + T lymphocytes) in the cell population is at least about 0.5%, such as at least about 1%, 1.5%, 2% or 3%. In some embodiments, the TAP-specific T lymphocytes (e.g., CD8 +The proportion of T lymphocytes (e.g., CD8 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%. This enrichment specifically recognizes one or more target MHC class I molecules / peptide (TAP) complexes. + T lymphocytes) can be used in cancer immunotherapy based on tumor antigens, as described in detail below. In some embodiments, for example, using an affinity-based system, such as a multimer of MHC class I molecules loaded (covalently or non-covalently) with TAP as defined herein, TAP-specific T lymphocytes (e.g., CD8 + Therefore, the present disclosure provides purified or isolated TAP-specific T lymphocytes (e.g., CD8 + T lymphocyte) population, for example, TAP-specific T lymphocytes (e.g., CD8 + The proportion of T lymphocytes) is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0210] In another aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof (e.g., a TCR mimetic or a TCR-like antibody) or a soluble TCR that specifically binds to a complex comprising a TAP as described herein that is bound to an HLA molecule. The term "antibody or an antigen-binding fragment thereof" as used herein 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, as long as they exhibit the desired antigen specificity / binding activity. Antibody fragments comprise a portion of a full-length antibody, typically its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab'), 2 As well as 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 by antibody fragments, single-chain diabodies (scDb), bispecific T cell engagers (BiTE), dual affinity retargeting molecules (DART), bivalent scFv-Fc and trivalent scFv-Fc. Antibody fragments can also refer to binding portions comprising CDRs or antigen binding domains, including but not limited to, V H Area (V H , V H -V H), anticalins, PepBodies, antibody T cell epitope fusions (Troybody) or peptide bodies. In one embodiment, the antibody or its antigen binding fragment is a single-chain antibody, preferably a single-chain Fv (scFv). In one embodiment, the antibody or its antigen binding fragment comprises at least one constant domain, such as a constant domain of a light chain and / or a heavy chain, or a fragment thereof. In another embodiment, the antibody or its antigen binding fragment comprises a fragment crystallizable (Fc) fragment of the constant heavy chain of the antibody. In one embodiment, the antibody or antigen binding fragment is a scFv (scFv-Fc) comprising an Fc fragment. In one embodiment, the scFv component is connected to the Fc fragment by a joint (e.g., a hinge). The presence of the Fc region can be used to induce complement dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP) or antibody dependent cellular toxicity (ADCC) responses against tumor cells.

[0211] In one embodiment, the antibody or its antigen-binding fragment is a multispecific antibody or its antigen-binding fragment, such as a bispecific antibody or its antigen-binding fragment, wherein at least one antigen-binding domain of the multispecific antibody or antibody fragment recognizes a complex comprising TAP as described herein bound to an HLA molecule. In one embodiment, at least one antigen-binding domain of a multispecific antibody or antibody fragment recognizes an immune cell effector molecule. The term "immune cell effector molecule" refers to a molecule (e.g., protein) expressed by an immune cell, and its engagement with a multispecific antibody or antibody fragment leads to the activation of immune cells. Examples of immune cell effector molecules include CD3 signaling complexes in T cells (e.g., CD8 T cells) and various activation receptors (NKG2D, KIR2DS, NKp44, etc.) on NK cells. In another embodiment, at least one antigen-binding domain of a multispecific antibody or antibody fragment recognizes and engages a CD3 signaling complex (e.g., anti-CD3) in a T cell. In yet another embodiment, a multispecific antibody or an antigen fragment single-chain double antibody (scDb). In another embodiment, scDb comprises a first antibody fragment (scFv) and a second antibody fragment (scFv), the first antibody fragment binds to a complex comprising TAP as described herein bound to an HLA molecule, and the second antibody fragment binds and engages an immune cell effector molecule, such as a CD3 signaling complex in a T cell (e.g., anti-CD3 scFv). Such constructs can be used, for example, to induce cytotoxic T cell-mediated killing of tumor cells, which express tumor antigens / MHC complexes recognized by multispecific antibodies or antibody fragments. Antibodies or their antigen-binding fragments can also be used as chimeric antigen receptors (CARs) to produce CAR T cells, CAR NK cells, etc. CAR will provide a specific ligand binding domain (e.g., antibody or antibody fragment) for a desired antigen (e.g., MHC / TAP complex) combined with an activation intracellular domain (or signal transduction domain) portion (e.g., T cell or NK cell activation domain), thereby providing a primary activation signal. Antigen binding fragments of antibodies capable of binding to molecules expressed by tumor cells, more particularly scFv, are typically used as ligand binding domains in CAR.

[0212] In one embodiment, the soluble TCR is a soluble therapeutic 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).

[0213] In one embodiment, a soluble TCR, an antibody or an antibody fragment (e.g., a TCR mimetic) is linked to an anti-tumor agent to form an antibody-drug conjugate (ADC). Such ADCs allow the anti-tumor agent to be targeted to tumor cells expressing one or more TAPs described herein (see, e.g., Shen et al., Asian J Pharm Sci. 2020 November; 15(6): 777-785).

[0214] The present disclosure also provides nucleic acids encoding soluble TCR, antibodies, antibody fragments or CARs described herein, such as mRNA. Such nucleic acids can be formulated into suitable media, such as lipid nanoparticles as described above, and can be used to treat cancer, such as breast cancer, as described below.

[0215] Thus, in another aspect, the present disclosure provides a host cell, preferably an immune cell, such as a T cell or a NK cell, expressing an antibody or antibody fragment (eg, scFv) described herein.

[0216] The present disclosure also relates to a cell-mediated immune response comprising the above-mentioned immune cells (CD8 + T lymphocytes, CAR T cells) or TAP-specific CD8 + Pharmaceutical compositions or vaccines for the treatment of T lymphocyte populations. Such pharmaceutical compositions or vaccines may comprise one or more pharmaceutically acceptable excipients and / or adjuvants, as described above.

[0217] In another aspect, the disclosure also relates to any one of the TAPs, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, APCs, CAR T cells) and / or compositions comprising SEQ ID NOs: 1-61 or SEQ ID NOs: 1-35 or consisting thereof, or any combination thereof as a drug or in the preparation of a drug for the treatment of breast cancer. The disclosure 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) according to the disclosure, or any combination thereof, for the treatment of breast cancer, for example as a breast cancer vaccine. The TAP sequences identified herein can be used to produce synthetic peptides for i) in vitro initiation and amplification of tumor antigen-specific T cells to be injected into tumor patients and / or ii) as a vaccine to induce or enhance anti-tumor T cell responses in breast cancer patients.

[0218] In another aspect, the present disclosure provides TAP or SLP described herein (e.g., comprising or consisting of any sequence of SEQ ID NO: 1-61 or SEQ ID NO: 1-35) or a combination thereof (e.g., a peptide pool) or one or more nucleic acids encoding TAP or SLP as a vaccine for treating breast cancer in a subject. The present disclosure also provides TAP or SLP described herein or a combination thereof (e.g., a peptide pool), or one or more nucleic acids encoding TAP or SLP, which are used as a vaccine for treating breast cancer in a subject. In one embodiment, the subject is a TAP-specific T lymphocyte (e.g., CD8 + Therefore, in another aspect, the present disclosure provides a method for treating breast cancer (e.g., reducing the number of tumor cells, killing tumor cells), the method comprising administering (infusing) an effective amount of T lymphocytes (e.g., CD8 + T lymphocytes) that recognize one or more MHC class I molecules / TAP complexes (expressed on the surface of cells (such as APCs)) (i.e., express a TCR that binds to one or more MHC class I molecules / TAP complexes). In one embodiment, the method further comprises administering / infusing the CD8 + After T lymphocytes, an effective amount of TAP or SLP or a combination thereof, or one or more nucleic acids encoding TAP, and / or cells (e.g., APCs, such as dendritic cells) expressing MHC class I molecules loaded with TAP or SLP are administered to the subject. In another embodiment, the method includes administering a therapeutically effective amount of dendritic cells loaded with one or more TAPs to a subject in need. In another embodiment, the method includes administering a therapeutically effective amount of allogeneic or autologous cells to a patient in need, the cells expressing a recombinant TCR bound to TAP presented by MHC class I molecules.

[0219] In another aspect, the present disclosure provides T lymphocytes (e.g., CD8 + T lymphocytes) for treating breast cancer in a subject (e.g., reducing the number of tumor cells, killing tumor cells), wherein the T lymphocytes recognize one or more MHC class I molecules loaded with (presenting) TAP, or a combination thereof. In another aspect, the present disclosure provides T lymphocytes (e.g., CD8 +T lymphocytes) for preparing / manufacturing a drug for treating breast cancer (such as lymphoblastic leukemia) in a subject (e.g., reducing the number of tumor cells, killing tumor cells), wherein the T lymphocytes recognize one or more MHC class I molecules loaded with (presenting) TAP, or a combination thereof. In another aspect, the present disclosure provides T lymphocytes (e.g., CD8 + T lymphocytes), for treating breast cancer in a subject (e.g., reducing the number of tumor cells, killing tumor cells). In another embodiment, the use further includes using an effective amount of TAP (or a combination thereof), or one or more nucleic acids encoding TAP, and / or expressing cells (e.g., APCs) loaded with (presenting) TAP, after using the TAP-specific T lymphocytes.

[0220] The present disclosure also provides a method for generating an immune response against tumor cells in a subject, wherein the tumor cells express human class I MHC molecules loaded with any TAP disclosed herein (e.g., comprising or consisting of any sequence of SEQ ID NO: 1-61 or SEQ ID NO: 1-35) or a combination thereof, the method comprising administering cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with TAP or a combination of TAPs. The present disclosure also provides the use of cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with any TAP or a combination of TAPs disclosed herein for generating an immune response against tumor cells, wherein the tumor cells express human class I MHC molecules loaded with TAP or a combination thereof.

[0221] Breast cancer can be ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDS) or invasive lobular carcinoma (ILC).Breast cancer can be hormone receptor positive (HR+) breast cancer, or wherein one or more of estrogen receptor (ER), progesterone receptor (PR) or epidermal growth factor receptor (HER2) do not express or express (undetectable by standard test) breast cancer at very low levels.In one embodiment, breast cancer is triple negative breast cancer (TNBC), wherein all receptors (ER, PR and HER2) do not express or express at very low levels.Breast cancer can be in any stage, i.e. breast cancer in situ (Phase 0), early stage (1A, 1B or 2A), locally advanced breast cancer (2B, 3A, 3B or 3C) or metastatic breast cancer (Phase 4).

[0222] In one embodiment, the methods or uses described herein further comprise determining the HLA class I alleles expressed by the patient prior to treatment / use, 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 determined to express HLA-11*01 and HLA-B35*01, any combination of (i) TAPs of SEQ ID NOs: 1, 17, and / or 28 (binding to HLA-A11*01) and (ii) TAPs of SEQ ID NOs: 4, 12, and / or 23 (binding to HLA-B35*01) may be administered or used in the patient.

[0223] 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 according to the present disclosure or any combination thereof can be used in combination with one or more additional active agents or therapies to treat breast cancer, such as chemotherapy (e.g., vinca alkaloids, agents that disrupt 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 agents, anthracyclines, topoisomerase inhibitors, macrolides, retinoids (e.g., all-trans retinoic acid or its derivatives), geldanamycin or its derivatives (e.g., 17-AAG), CDK4 / 6, TG F-β, WNT-β-catenin, MYC or PI3K inhibitors, 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 T cells, CAR NK cells), and cytokines, such as IL-2, IL-7, IL-21 and IL-15. In one embodiment, according to the TAP, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocyte, APC) and / or composition disclosed herein, it is administered / used in combination with immune checkpoint inhibitors. In one embodiment, according to the TAP disclosed herein, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocyte, APC) and / or composition and CDK4 / 6, TGF-β and / or WNT-β-catenin inhibitor combination administration / use. Several CDK4 / 6 inhibitors are in clinical trials, including palbociclib (PD-0332991, Ibrance), ribociclib (LEE-011, Kisqali), bomacilib (LY2835219, Verzenios), SHR6390 and tracilib (G1T28). TGF-β inhibitors include antisense inhibitors such as AP12009 (Trabedersen) and ISTH0036, antibodies and ligand capture agents such as GC1008 (fusuma monoclonal antibody), LY2382770 and P144, vaccines targeting TGF-β pathways such as Belagenpumatucel-L (Lucanix TM ) and FANGTM or vigil (Gemogenovatucel-T), as well as small molecule inhibitors such as LY2157299 (Galunisertib) and TEW-7197. Inhibitors of the WNT-β-catenin pathway include amino acid starvation agents (asparaginase), GSK3 inhibitors, C2 WNT974, ETC-1922159, RXC004, CGX1321, OTSA101-DTPA-90Y, vandetumab (OMP-18R5), efenacitin (OMP-54F28), PRI-724, SM08502, secreted frizzled-related protein / peptide and tankyrase inhibitors (XAV939, JW-55, RK-287107, and G007-LK).

[0224] The additional therapy may be administered before, simultaneously with, or after administration of the TAPs, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, CAR T or NK cells, APCs), and / or compositions according to the present disclosure. Example

[0225] The present disclosure is further illustrated by the following non-limiting examples.

[0226] Example 1: Materials and Methods

[0227] Primary breast cancer samples. Fresh frozen primary breast tumor samples were purchased from Tissue solutions (https: / / www.tissue-solutions.com / ). Samples had a histological diagnosis of invasive ductal carcinoma (n=24) or invasive carcinoma (n=2). Immunohistochemistry data were available through Tissue solutions and samples were classified as HR + (ER or PR positive and HER2 negative; n = 14) or TNBC (ER / PR / HER2 negative, n = 12). Patients did not receive chemotherapy before resection.

[0228] RNA and miRNA sequencing. RNeasy TM Total RNA was isolated using the Qubit mini kit (Qiagen). TM RNA was quantified using the 2100 Bioanalyzer (Thermo Scientific). TM (Agilent Technologies). The quality was assessed using KAPA RNA HyperPrep TM(Roche), and transcriptome libraries were generated using poly-A selection (ThermoScientific). TM Small RNA libraries were prepared using the miRNA library kit (Qiagen) and sequenced on an Illumina NextSeq500 system.

[0229] Database generation. All RNA sequencing reads were trimmed by Trimmomatic v0.35 and aligned to the GRCh38.99 index using STARv2.5.1b. Transcript expression was quantified in transcripts per million (tpm) using Kallisto v0.43.0 with default parameters.

[0230] Standard proteome. Standard proteomes were constructed as described previously (13). Sample-specific exomes were constructed using pyGeno by inserting single-base variants (quality>20) identified using FreeBayes (https: / / github.com / ekg / freebayes). Annotated open reading frames with tpm>0 were computer translated from sample-specific exomes to generate standard proteomes.

[0231] ERE proteome. ERE proteomes were constructed for each sample as described (23). Ambiguous nucleotides were trimmed from the reads of the ERE dataset and then translated in all possible reading frames. Finally, the resulting ERE amino acid sequences were spliced ​​to remove the sequence after the stop codon. Only sequences of at least 8 amino acids were retained and given unique IDs to generate a theoretical ERE proteome. This database was then linked to the canonical proteome to generate a personalized ERE proteome for MAP identification.

[0232] Small RNA (smRNA) proteome. smRNA sequencing reads were linked in fastq.gz files by Qiagen software. A K-mer database (24 Å long) was generated using Jellyfish v2.2.3 and assembled into contigs as described (11). The contigs were then translated in 3 frames and different polypeptides were linked with JJ linkers to avoid bias towards linking with shorter sequences. This database was linked to the standard proteome for each sample for MAP identification.

[0233] Cancer-specific proteomes. Cancer-specific proteomes were assembled using the k-mer analysis described (11, 13). From each cancer sample database, k-mers (33 nucleotides long) that were present at least once in the mTECs k-mer database were removed and the remaining k-mers were assembled into contigs. Finally, the contigs were 3-frame translated and the different peptides were connected using JJ linkers. This database was connected to the standard proteome of each sample for MAP identification.

[0234] Mass spectrometry. MHC I immunoprecipitation, tandem mass tag (TMT) labeling, and liquid chromatography-MS / MS analysis (LC-MS / MS) were performed as described (24). LC-MS / MS data were searched against relevant databases using PEAKS10.5 or Peaks X Pro (Bioinformatics Solution Inc.). For peptide identification, tolerances for precursor and fragment ions were set to 10 ppm and 0.01 Da, respectively. Oxidation (M), deamidation, and TMT modifications were set as variable modifications. After peptide identification, sample-specific thresholds were applied using a modified target-decoy method built-in peak-to-peak scoring to ensure a 1% false discovery rate (FDR), which was calculated as the ratio between the number of bait hits and the number of target hits above the scoring threshold. The peak score corresponding to 1% FDR was determined for each sample, and peptides that passed the threshold were further filtered to match the following criteria: peptide length between 8 and 11 amino acids, affinity classification based on NetMHCpan-4.1b as sample HLA alleles <2% (25). These filtering steps were performed using MAPDP (26).

[0235] Validation of genomic origin and target MAPs (MOI). Tumor antigen candidates were identified as previously described (11, 13, 14). The genomic origin of MAPs was considered exonic when they aligned to exonic sequences with ≥1 read. Otherwise, the genomic origin of the MOI was assigned using the alignment with the highest number of reads. All final alignments of TSAs and TAAs were manually validated with Integrative GenomicsViewer (IGV). MOIs where reads did not match a consistent genomic location or matched a hypervariable region (such as MHC, Ig, or TCR genes) were excluded.

[0236] TSA candidates were classified as mTSAs if they contained variants in their MAP coding sequences that did not match known germline polymorphisms [reported in the Database of Single Nucleotide Polymorphisms (dbSNP) v149, http: / / www.ncbi.nlm.nih.gov / SNP / ]. Leucine and isoleucine variants were indistinguishable by standard MS methods. Therefore, MAPs marking existing variants were discarded as non-MOIs unless they presented higher RNA expression than the variant or mapped in inappropriate regions (in the case of EREMAPs candidates and smRNAMAPs candidates). In addition, all MOI, ERE MAP, and smRNA MAP candidates were validated using COMET software (27) using filters with peaks and an FDR of 5%. For peptides that were not re-identified using COMET, manual spectral validation was performed in-house to further retain or discard MAPs.

[0237] MOI expression in tissues. Expression of MOI coding sequences was assessed in normal tissues from GTEX (n=50 samples / tissue), mTEC (n=11), purified blood and bone marrow samples (n=6), and breast cancer tissues from TCGA (n=1109).

[0238] Predicted TAA and TSA presentation and survival analysis in TCGA breast cancer samples. TAA or TSA was considered to be presented in TCGA samples only if the MAP encoding transcript was expressed (>2rphm) and the patient had an HLA allotype that could present the MAP according to NetMHCPan4.1 (25). The HLA alleles of 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 removed from the analysis to not duplicate their contribution to the results, leaving 915 patients. Kaplan-Meier survival curves and log-rank tests were generated using the TCGAbiolinks package.

[0239] Single-cell analysis. The original single-cell data of 14 breast cancer patients published by Qian et al. (30) were obtained through their website: http: / / scope.lambrechtslab.org / . Visualizations were developed using the Seurat package (31).

[0240] FEST assay. The FEST assay was performed as previously described (Danilova et al., Cancer Immunol Res. 2018 Aug;6(8):888-899. Epub 2018 Jun 122018) with minor modifications. Five million T cells were co-cultured with autologous T cell-depleted PBMCs pulsed with individual peptides during 20 days and restimulated on day 10. After in vitro expansion, CD8 T cells were isolated using the Human CD8+ T Cell Isolation Kit (Miltenyi). DNA extraction was performed with the QIAGEN DNA Blood Mini Kit, followed by TCR VβCDR3 sequencing using the ImmunoSEQ platform. Raw data were processed with the FEST web tool (www.stat-apps.onc.jhmi.edu / FEST). The following parameters were used: FDR 1%, fold change >5, minimum number of templates of 1 and “ignore baseline threshold”. Two negative control groups consisted of PBMCs depleted of autologous T cells that were not pulsed and uncultured CD8 + CD8 T cell co-culture + T cell composition.

[0241] Statistical analysis and data visualization. Analyses and plots were performed using R v4.0.0. The “gplots” and “ggplot2” packages in R were used to generate the different plots. When necessary, tests involving distribution comparisons were performed using t-tests or one-way ANOVAs. Differential gene expression analysis was performed using the limma package (32). GSEA analysis was performed using the “fgsea” package (33).

[0242] Example 2: Global proteomic strategy for identification of MAPs in primary breast cancer samples

[0243] Twenty-six primary breast cancer samples (14 HR+ and 12 TNBC) from untreated patients were analyzed. MAPs were identified by MS analysis using a previously described proteogenomic approach (11, 13, 14). For each sample, a personalized reference database was constructed by computer translation of RNA sequencing data. These databases include four modules: 1) standard proteome (in-frame translation of protein-coding exons), 2) endogenous retrotransposons (ERE) proteome, 3) small-RNA proteome, and 4) cancer-specific proteome. RNA sequencing reads containing cancer-specific proteomes were present in tumor samples but not in the mTECs collection ( Figure 8 ).

[0244] Example 3: Standard immunopeptide panels for HR+ and TNBC tumors are similar

[0245] MAPs encoded by the canonical reading frames of annotated protein-coding genes are collectively referred to as canonical immunopeptidase. A total of 57,094 canonical MAPs from 10,552 protein-coding genes were identified. The average number of MAPs per tumor sample was 4,633, with a mean of 95.3% in HR + There was no difference between the TNBC samples (t test; p>0.05, Figure 1A MAPs are presented by 53 different HLA alleles, of which >20% are presented by two common alleles: HLA-A*02:01 and HLA-B*18:01 ( Fig. 9 Consistent with previous reports (34), 62% of the transcriptome (defined as genes with more than one transcript per kilobase million (TPM)) produced MAPs ( Fig. 10A A high proportion of genes (72.5%, n = 7371) contributed to MAP production in HR+ and TNBC tumors ( Figure 1B ), and most of them (92%) were also reported to be MAP-derived genes in normal tissues from the HLA ligand ATLAS ( 18 ).

[0246] Of the 798 standard MAPs not listed in the HLA ligand ATLAS, 277 were found in HR + 259 were unique to TNBC and 262 were unique to HR + Sample unique ( Figure 1B ). HR + The 277 canonical MAPs shared with TNBC samples were encoded by a gene set enriched in genes encoding extracellular matrix proteins (enrichment fold 7.59, p < 0.05) ( Fig.11A ). 259 canonical MAPs found only in TNBC samples showed enrichment of the glycosyltransferase protein class (enrichment fold 5.58, p<0.05) ( Fig. 11B ). For + No specific enrichment was found for the 262 MAPs found in the samples.

[0247] The contribution of source genes to the immunopeptidome was inconsistent. Although 49% of source genes from the dataset produced ≤5 MAPs, 51% produced >5 MAPs, and a significant proportion of them encoded >100 MAPs ( Figure 1C and Fig. 10B The top 1% of MAP generators are significantly enriched in cytoskeleton and extracellular matrix protein categories ( Figure 1D Together, these results reveal that HR +There was substantial overlap between the standard immunopeptidome of TNBC samples and significant enrichment in MAPs derived from cytoskeletal and extracellular matrix proteins. The immunopeptidome projects a representation of proteins that are actively translated on the cell surface and degraded into the cell (35). Thus, the overrepresentation of MAPs derived from cytoskeletal and extracellular matrix proteins is consistent with the key role of extracellular matrix remodeling in breast cancer tumorigenesis (36-38).

[0248] Example 4: Contribution of ERE and smRNA to the non-classical breast cancer immunopeptidomimetics

[0249] EREs and smRNAs are involved in different steps in neoplastic transformation, including in breast cancer (39, 40). In addition, EREs have been shown to encode immunogenic MAPs in mice and humans (13, 23). Therefore, a personalized proteomic database was specifically investigated for the presence of ERE-encoded MAPs and smRNA-encoded MAPs in breast cancer. ERE-encoded transcripts were retrieved from bulk RNA sequencing, while smRNA sequencing was used to construct the smRNA database ( Fig. 12A -B).

[0250] We identified 75 ERE-derived MAPs, which were evenly distributed across HR + and TNBC samples ( Figure 2A , Table 1). ERE-derived MAPs mapping, intronic regions are similar to intergenic regions ( Fig.13 ). The three main types of ERE contribute similarly to HR + and immunopeptidome of TNBC samples ( Figure 2B MAP-producing EREs are expressed at higher levels than EREs that do not produce MAPs ( Figure 2D Only 9 / 22 ERE families resulted in MAP generation, and the L1 family was the most important contributor ( Figure 2C ). Only three smRNA-derived MAPs were identified (Table 2): one derived from piRNA and two from snRNA. It is concluded that EREs, rather than smRNAs, produce a large number of MAPs in breast cancer tumors. However, EREMAPs can also be found in normal tissues (23). Therefore, further analysis must be performed to evaluate whether EREMAPs can be marked as TAAs or TSAs.

[0251] Table 1: ERE-derived MAPs

[0252]

[0253]

[0254]

[0255]

[0256] Table 2: smRNA-derived MAPs

[0257] Peptide sequence (SEQ ID NO) source Samples for identification Allele ITKVVFPGR(135) snRNA 825,346,1192 HLA-A*31:01 DEDLFTDLY(136) piRNA 825 HLA-B*18:01 WETRLHNLW(137) snRNA 1945 HLA-B*44:03

[0258] Example 5: Identification of potential therapeutic targets: TAA, aeTSA and mTSA

[0259] use Figure 3A MOI was identified and classified as TAA, aeTSA, or mTSA using the workflow outlined in . Overall, 25 TSAs were identified: 1 mTSA and 24 aeTSAs ( Figure 4A ). The only mTSA originated from a non-synonymous mutation in the deubiquitinase OTUB1 gene. This rare mutation is not listed in the COSMIC database (https: / / cancer.sanger.ac.uk / cosmic). The rarity of mTSAs led us to ask whether genes frequently mutated in breast cancer were represented in the immunopeptidomics. A slight positive correlation (p<0.001) was found between the frequency of mutations identified by the TCGA consortium and the generation of MAPs in the dataset ( Fig.14 ). This means that there is no negative bias towards the representation of highly mutated genes in the breast cancer immunopeptidomics. Therefore, highly mutated genes produce MAPs, but these MAPs are not derived from mutated regions. This is consistent with the fact that MAPs preferentially originate from specific regions of MAP-derived proteins (MAP "hotspots") (18, 34, 41). The most parsimonious explanation for the scarcity of mTSA is that breast cancer has relatively few mutations and these mutations are not located in MAP hotspots.

[0260] In the study of TAAs and aeTSAs, only MAPs encoded by transcripts expressed in at least 5% of the TCGA breast cancer cohort samples were considered. It was assumed that antigens expressed in fewer samples were of little interest. The classification of unmutated MAPs as TAAs or aeTSAs was based on a comprehensive transcriptomic analysis of their expression in i) breast cancer samples from TCGA (n=1109), ii) 50 normal tissues from GTEx (@50 samples / tissue), iii) mTEC (n=11), and iv) purified blood and bone marrow samples (n=6). Blood and bone marrow cells were used as surrogates for rapidly proliferating cells, as 90% of the cells produced by humans every day are hematopoietic cells (42). It must be emphasized that the expression analysis only considered MAPs coding sequences, not entire genes or genomic regions. Therefore, aberrations in RNA splicing often result in protein isoforms present only in cancer cells. MAPs derived from such cancer-specific isoforms are labeled as aeTSAs, even if other isoforms are expressed in normal cells.

[0261] MAPs expressed below 8.55 reads per 100 million (rphm) in all normal tissues except testis were labeled as aeTSAs. As reported (11), 8.55 rphm was used as the threshold because expression below this level was associated with a very low probability of MAP generation. Otherwise, MAPs with an MOI above the threshold expression in one or more normal tissues were labeled as TAAs if they were overexpressed in tumors or highly proliferative tissues compared with normal non-hematopoietic tissues. This strategy led to the identification of 24 aeTSAs and 49 TAAs, most of which were novel because they were not listed in the Immune Epitope Database (IEDB) ( Figure 3B -C, Tables 3 and 4).

[0262] Table 3: TSAs identified in this study

[0263]

[0264]

[0265] Table 4: TAAs identified in this study

[0266]

[0267]

[0268]

[0269] Example 6: TSA in TNBC is more effective than in HR + More abundant in breast cancer

[0270] Of the 24 aeTSAs, 17 were encoded by canonical exons: 14 belonged to the MAGE family of CTAs, 2 belonged to genes encoding extracellular matrix components (COL11A1, ITH6), and 1 belonged to a transmembrane protein encoding gene (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).

[0271] Most aeTSA were found in TNBC samples ( Figure 3B We next assessed whether this enrichment in TNBC samples correlated with TSA expression in the TCGA cohort. The proportion of tumors expressing individual aeTSAs of the CTA category was significantly higher in TNBC relative to HR + Tumors were superior (19% vs. 8%, p=0.004) ( Figure 4C ). The distribution of other categories of TSA did not differ significantly.

[0272] Next, it was assessed whether aeTSA presentation was associated with immune infiltration. An aeTSA was considered to be present in a TCGA sample only if the MAP encoding transcript was expressed and the patient had an HLA allotype that could present the MAP (11). The TCGA samples were then classified into two groups presenting high (above the median) and low (below the median) aeTSA numbers. Then, differential gene expression analysis between the two groups was performed. Gene set enrichment analysis using the gene signature of leukocyte infiltration described by Danaher et al. (43) showed that these genes were enriched in the group of tumors with high TSA numbers. This suggests that at least some TSAs are immunogenic in vivo. In addition, it was assessed whether oncogenic pathways involved in immune escape (44) were enriched in samples with high or low levels of TSAs. To this end, the corresponding marker gene sets from the Molecular Signatures Database (45) were selected, and enrichment in the PI3K pathway was found in tumors with low numbers of predicted TSAs (NES = -1.48, p < 0.05; Fig.15 ). The PI3K pathway is involved in tumorigenesis, progression, and resistance to therapy in breast cancer (46).

[0273] Example 7: TAAs are highly shared in breast cancer

[0274] 49 TAAs were identified, 48 of which were derived from canonical protein coding regions ( Figure 5A and Table 4). These antigens are + and TNBC tumors ( Figure 5B The largest group of TAAs (n=14) were derived from genes (COL11A1, COL10A1, LRRC15) reported as markers of cancer-associated fibroblasts (47). These genes are associated with extracellular matrix production and cell migration. To confirm their most likely cellular origin, their expression levels were evaluated in a single-cell dataset from Qian et al. (30) that included 14 breast cancer tumors ( Figure 6 A). The expression of all three genes in tumor fibroblasts was significantly higher than that in other cell subsets, including cancer cells ( Figure 6 B; ANOVA; p < 0.05). Two additional groups of TAAs were identified. Thirteen TAAs were derived from CYP4Z1, which is implicated in many cancer types and elicits autoantibodies against CYP4Z1 in breast cancer patients (48). Eleven TAAs were associated with cell proliferation ( Figure 5B ); they are expressed at low levels in mature epithelial and blood cells but at higher levels in myeloid progenitor cells ( Figure 5A ).

[0275] The number of TAAs per tumor in the TCGA dataset was predicted using the same criteria as for aeTSA: expression of MAP coding sequences and the presence of relevant HLA allotypes ( Figure 5C , Fig.16 ). HR with high level of predictive TAA + TNBC and TNBC tumors showed enrichment of immune activation and immunosuppressive pathways (i.e., PI3K / mTOR, Wnt / B-catenin, and MAPK pathways). In addition, tumors with many TAAs showed enrichment of fibroblast proliferation markers. These findings suggest that in the presence of multiple TAAs, antitumor immune responses can be mitigated by activation of immunosuppressive pathways and accumulation of cancer-associated fibroblasts.

[0276] Example 8: Presentation of multiple TSAs improves overall survival in TNBC

[0277] We next evaluated whether the number of TAAs and aeTSAs presented correlated with overall survival of TCGA patients. As in the previous examples, antigen is considered to be presented in a tumor when both MAP encoding transcripts and the appropriate HLA allotype are expressed (11). Patients were divided into two categories: high numbers of presented antigens (first quartile) and low numbers of presented antigens (second to fourth quartiles).

[0278] The number of TAAs presented has an impact on HR + or TNBC tumors had no effect on survival ( Figure 7B , C). Similarly, the number of aeTSAs submitted is + There was no effect in patients with tumors ( Fig. 7A ), which is a group expressing little aeTSA ( Figure 4C However, more aeTSA presentation was associated with better overall survival in the TNBC group ( Fig.17 This benefit was observed with aeTSAs derived from both CTA and noncoding regions ( Fig.7D , E). Of note, when only TSA expression was considered (without regard to HLA allotype), no differences were found between high and low expressers ( Fig.7D , E). This means that the beneficial effect of aeTSA presentation is HLA-restricted. It is therefore due to MHC I presentation of the aeTSA peptide and not due to expression of the aeTSA encoding transcript itself.

[0279] It can be concluded that aeTSA reported here confers an MHC class I-restricted survival advantage in TNBC patients ( Fig.7D , E) HR + This is not the case for cancer patients ( Fig. 7A), possibly because they present less aeTSA than TNBC tumors ( Figure 4C ). TAA presentation does not confer a similar survival advantage, possibly because TAA expression is associated with activation of immunosuppressive pathways ( Figure 5C ).

[0280] Example 9: TSAs and TAAs induce expansion of specific CD8 T cells

[0281] Next, we evaluated whether the TAAs and TSAs identified in this article could induce CD8 T cell expansion ex vivo. This was evaluated using the FEST (Functional Expansion of Specific T Cells) assay, which integrates T cell receptor sequencing of short-term peptide-stimulated cultures with a bioinformatics platform to identify antigen-specific clonal expansion (Danilova et al., Cancer Immunol Res. 2018 August; 6(8): 888-899. Epub 2018 June 12, 2018).

[0282] like Fig.18A and 18B As shown, significant antigen-specific CD8 T cell clonotype expansions were obtained for selected TSAs and TAAs in two different donors, providing convincing evidence that TSAs and TAAs are immunogenic and can be used to stimulate anti-tumor T cell responses in subjects with cancers expressing these TSAs and / or TAAs.

[0283] Although the present invention has been described above by specific embodiments thereof, modifications may be made thereto without departing from the spirit and nature of the invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including but not limited to". The singular forms "a", "an", and "the" include corresponding plural references unless the context clearly dictates otherwise.

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Claims

1. A tumor antigen peptide (TAP) comprising or consisting of one of the following amino acid sequences: Or a nucleic acid encoding said TAP.

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

22.

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

19.

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

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

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

10.

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

15.

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

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

10. 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 or nucleic acid of claim 1, wherein the TAP binds to an HLA-B*18:01 molecule and comprises or consists of a sequence of SEQ ID NO: 13, 14 or 33.

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

27.

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

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

18.

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

36.

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

25.

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

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

26. 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 of the TAPs or nucleic acids defined in any one of claims 1 to 26.

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. 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. The TAP or nucleic acid of any one of claims 1 to 26, or the combination of 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 the SLP.

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

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

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

35. The vesicle or particle of claim 33 or 34, comprising a cationic lipid.

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

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

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

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

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

41. An isolated cell comprising (i) a TAP of any one of claims 1-26, (ii) a combination of claim 27; (iii) an SLP of claim 31 or 32; or (iv) a vector comprising a nucleotide sequence encoding a TAP of any one of claims 1-26, a combination of claim 27, or an SLP of claim 31 or 32.

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

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) which specifically recognizes the isolated MHC class I molecule of any one of claims 38 to 40 and / or the MHC class I molecule expressed on the cell surface of 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, which specifically binds to the isolated MHC class I molecule of any one of claims 37 to 39 and / or the MHC class I molecule expressed on the surface of a cell of any one of claims 42 to 44.

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

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

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

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

52. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof of claim 47, or a nucleic acid encoding the CAR.

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

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

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

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

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

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

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

60. The following use for treating breast cancer in a subject, or for preparing a medicament for treating breast cancer in a subject: (a) a TAP comprising any one of the sequences shown in SEQ ID NOs: 1-61 or any combination thereof or consisting thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences shown in SEQ ID NOs: 1-61; (b) at least one nucleic acid encoding a TAP, a combination thereof or a SLP as defined in (a); (c) a vesicle or particle comprising a TAP, a combination thereof or a SLP as defined in (a) or at least one nucleic acid as defined in (b); (d) a composition comprising a TAP, a combination thereof or a 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, a combination thereof or a 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 a major histocompatibility complex (MHC) class I molecule on its surface, wherein the MHC class I molecule comprises a TAP as defined in (a) or a combination thereof in its peptide binding groove; (g) a cell expressing on its cell surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of a cell as defined in (f); or (h) a soluble TCR, an antibody or an antigen-binding fragment thereof, which specifically binds to an MHC class I molecule expressed on the surface of a cell as defined in (f).

61. The method of claim 60, wherein the breast cancer is hormone receptor positive breast cancer (HR + ) or triple-negative breast cancer (TNBC).

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

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

64. An agent for treating breast cancer in a subject, wherein the agent is: (a) a TAP comprising any one of the sequences shown in SEQ ID NOs: 1-61 or any combination thereof or consisting thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences shown in SEQ ID NOs: 1-61; (b) at least one nucleic acid encoding a TAP, a combination thereof or a SLP as defined in (a); (c) a vesicle or particle comprising a TAP, a combination thereof or a SLP as defined in (a) or at least one nucleic acid as defined in (b); (d) a composition comprising a TAP, a combination thereof or a 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, a combination thereof or a 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 a major histocompatibility complex (MHC) class I molecule on its surface, wherein the MHC class I molecule comprises a TAP as defined in (a) or a combination thereof in its peptide binding groove; (g) a cell expressing on its cell surface a T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of a cell as defined in (f); or (h) a soluble TCR, an antibody or an antigen-binding fragment thereof, which specifically binds to an MHC class I molecule expressed on the surface of a cell as defined in (f).

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

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

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

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