Tissue-specific antigens for cancer immunotherapy

Abstract

The present invention provides compositions related to tissue-specific antigens and methods for identifying tissue-specific antigens.The present invention also provides pharmaceutical compositions and treatment methods related to tissue-specific antigens.The present invention provides methods and compositions that solve these problems, including tissue-specific antigens that have not been previously considered, such as tissue-specific antigens that are specific to non-essential tissues.Tissue-specific epitope sequences can be predicted to be presented on tumor cells or non-essential normal cells derived from the same lineage of non-essential tissues, and are predicted to have zero or low expression levels in essential tissues.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 125,269, filed December 14, 2020, which is incorporated by reference herein in its entirety. [Background technology]

[0002] background Personalized immunotherapy using tumor-specific peptides has been described (Ott et al., Hematol. Oncol. Clin. N. Am. 28 (2014) 559-569). Prior to this disclosure, the majority of cancer immunotherapies focused on epitopes that are believed to display "tumor-specific" or "tumor-associated" expression patterns. Examples of such epitopes include MAGEA3, NY-ESO-1, and MSLN. In general, these genes suffer from either low expression in tumors or non-negligible expression in essential normal tissues. These issues are likely to interfere with efficacy. However, focusing on tissue-specific antigens can change the scope of possible targets. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Ott et al., Hematol. Oncol. Clin. N. Am. 28 (2014) 559-569 Summary of the Invention [Means for solving the problem]

[0004] overview The present invention provides methods and compositions that solve these problems, including tissue-specific antigens that have not been previously considered, such as tissue-specific antigens that are specific to non-essential tissues.Tissue-specific epitope sequences can be predicted to be presented on tumor cells or non-essential normal cells derived from the same lineage of non-essential tissues, and are predicted to have zero or low expression levels in essential tissues.Therefore, epitope sequence information of tissue-specific antigens, such as antigens that are specific to tumors derived from specific tissues, can be translated into therapeutic methods and compositions for diseases or conditions, such as cancer.In some embodiments, tissue-specific antigens are tumor antigens. ANKRD30A, COL10A1, CTCFL, PPIAL4G, POTEE, DLL3, MMP13, SSX1, DCAF4L2, MAGEA4, MAGEA11, MAGEC2, MAGEA12, PRAME, CLDN6, EPYC, KLK3, KLK2, KLK4, TGM4, POTEG, RLN1, POTEH, SLC45A2, TSPAN10, PAGE5, CSAG1, PRDM7, TG, TSHR, RSPH6A, SCXB, HIST1H4K, ALPPL2, PRM2, PRM1, TNP1, LELP1, HMGB4, AKAP4, CETN1, UBQLN3, ACTL7A, ACTL9, ACTRT2, PGK2, C2orf 53, KIF2B, ADAD1, SPATA8, CCDC70, TPD52L3, ACTL7B, DMRTB1, SYCN, CELA2A, CELA2B, PNLIPRP1, CTRC, AMY2A, SERPINI2 The present invention provides a composition comprising: a tissue-specific antigenic peptide comprising an epitope sequence of a protein encoded by a gene selected from the group consisting of: RBPJL, AQP12A, IAPP, KIRREL2, G6PC2, AQP12B, CYP11B1, CYP11B2, STAR, CYP11A1, and MC2R, wherein the protein is expressed by a cancer; a polynucleotide encoding the tissue-specific antigenic peptide; one or more antigen presenting cells (APCs) comprising the tissue-specific antigenic peptide; a T cell receptor (TCR) or antibody or functional portion thereof specific for an MHC:peptide complex, wherein the MHC:peptide complex comprises the tissue-specific antigenic peptide; or a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a TCR.

[0005] In some embodiments, the tumor antigen epitope may include an epitope derived from any one of the proteins TSHR, TG, RSPH6A, SCXB, SSX1, or any combination thereof, and the cancer comprises thyroid cancer.

[0006] Also provided herein is a population of T cells for cancer therapy to a human subject in need thereof, wherein the population of T cells is selected from the group consisting of ANKRD30A, COL10A1, CTCFL, PPIAL4G, POTEE, DLL3, MMP13, SSX1, DCAF4L2, MAGEA4, MAGEA11, MAGEC2, MAGEA12, PRAME, CLDN6, EPYC, KLK3, KLK2, KLK4, TGM4, POTEG, RLN1, POTEH, SLC45A2, TSPAN10, PAGE5, CSAG1, PRDM7, TG, TSHR, RSPH6A, SCXB, HIST1H4K, ALPPL2, PRM2, PRM1, TNP1, LELP1, HMGB4, AKAP4, C and T cells that specifically recognize one of the epitope sequences of a protein encoded by a gene selected from the group consisting of ETN1, UBQLN3, ACTL7A, ACTL9, ACTRT2, PGK2, C2orf53, KIF2B, ADAD1, SPATA8, CCDC70, TPD52L3, ACTL7B, DMRTB1, SYCN, CELA2A, CELA2B, PNLIPRP1, CTRC, AMY2A, SERPINI2, RBPJL, AQP12A, IAPP, KIRREL2, G6PC2, AQP12B, CYP11B1, CYP11B2, STAR, CYP11A1, and MC2R, wherein the epitope is expressed by a cancer cell of the human subject.

[0007] Improved ex vivo method for preparing tumor antigen-specific T cells, comprising the steps of: depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells, wherein the population of immune cells is derived from a biological sample from a human subject; incubating the population of CD14- and / or CD25-depleted immune cells comprising APCs and the first population of T cells in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L) and (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject having cancer, or (B) a polynucleotide encoding the polypeptide, for a first period of time, thereby forming a population of cells comprising stimulated T cells; and expanding the population of cells comprising stimulated T cells, thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells (i) express at least one tumor antigen epitope sequence. and (ii) a T cell specific for a complex comprising an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii); and administering to the human subject the expanded population of cells comprising the tumor antigen-specific T cells, wherein the tumor antigen epitope is selected from the group consisting of ANKRD30A, COL10A1, CTCFL, PPIAL4G, POTEE, DLL3, MMP13, SSX1, DCAF4L2, MAGEA4, MAGEA11, MAGEC2, MAGEA12, PRAME, CLDN6, EPYC, KLK3, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, KLK15, KLK16, KLK17, KLK18, KLK19, KLK20, KLK21, KLK19, KLK22, KLK30, KLK19, KLK21, KLK18, KLK19, KLK22, KLK19, KLK23, KLK30, KLK19, KLK20, KLK19, KLK21, KLK19, KLK20, KLK3 ... LK4, TGM4, POTEG, RLN1, POTEH, SLC45A2, TSPAN10, PAGE5, CSAG1, PRDM7, TG, TSHR, RSPH6A, SCXB, HIST1H4K, ALPPL2, PRM2, PRM1, TNP1, LELP1, HMGB 4, AKAP4, CETN1, UBQLN3, ACTL7A, ACTL9, ACTRT2, PGK2, C2orf53, KIF2B, ADAD1, SPATA8, CCDC70, TPD52L3, ACTL7B, DMRTB1, SYCN, CELA2A, CELA2B,The method provided herein may be one or more of PNLIPRP1, CTRC, AMY2A, SERPINI2, RBPJL, AQP12A, IAPP, KIRREL2, G6PC2, AQP12B, CYP11B1, CYP11B2, STAR, CYP11A1, and MC2R, and the epitope is expressed by a cancer cell of a human subject. In some embodiments, the tumor antigen epitope may include an epitope derived from any one of the proteins TSHR, TG, RSPH6A, SCXB, SSX1, or any combination thereof, and the cancer comprises thyroid cancer.

[0008] The present invention provides a tissue-specific antigenic peptide comprising an epitope sequence of a protein, wherein the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs:1 to 8962, and the protein is expressed by a cancer; a polynucleotide encoding the tissue-specific antigenic peptide; one or more antigen-presenting cells (APCs) that present the tissue-specific antigenic peptide; a T cell receptor (TCR) or antibody or a functional portion thereof specific to an MHC:peptide complex, wherein the MHC:peptide complex comprises the tissue-specific antigenic peptide; or a composition comprising a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a TCR.

[0009] Provided herein is a composition comprising: a tissue-specific antigenic peptide comprising an epitope sequence of a protein, the protein being expressed by a tumor in a target tissue; a polynucleotide encoding the tissue-specific antigenic peptide; one or more antigen-presenting cells (APCs) presenting the tissue-specific antigenic peptide; a T cell receptor (TCR) or antibody or functional portion thereof specific for an MHC:peptide complex, the MHC:peptide complex comprising the tissue-specific antigenic peptide; or a population of immune cells from a biological sample comprising at least one antigen-specific T cell comprising a TCR, the epitope sequence binding or predicted to bind to a protein encoded by an MHC allele expressed by a human subject, the protein being encoded by a tissue-specific antigen epitope gene whose expression level in the target tissue is at least twice the expression level of the tissue-specific antigen gene in each of a plurality of non-target tissues different from the target tissue.

[0010] In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 6846-7061, 7359-7448, 7629-8099, and 8619-8744, and the cancer comprises thyroid cancer.

[0011] In some embodiments, the protein comprises RBPJL, AQP12A, AQP12B, IAPP, CELA2A, CELA2B, AMY2A, CTRC, G6PC2, KIRREL2, PNLIPRP1, SERPINI2, SYNC, or any combination thereof, and the cancer comprises pancreatic cancer.

[0012] In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 720-814, 989-1182, 1373-1565, 2120-2211, 2920-3009, 3101-3196, 3320-3440, 5193-5284, 6487-6579, 7062-7150, and 7539-7628, and the cancer comprises pancreatic cancer.

[0013] In some embodiments, the protein comprises CYP11A1, CYP11B1, CYP11B2, MC2R, STAR, or any combination thereof, and the cancer comprises adrenal cancer.

[0014] In some embodiments, the epitope sequence has 70%-100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 22122523, 4817-4915, and 7449-7538, and the cancer comprises adrenal cancer.

[0015] In some embodiments, the protein comprises ALPPL2, POTEE, PRAME, or any combination thereof, and the cancer comprises uterine cancer.

[0016] In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 627-719, 5285-5431, and 6085-6183, and the cancer comprises uterine cancer.

[0017] In some embodiments, the protein comprises KLK2, KLK3, KLK4, POTEH, POTEG, TGM4, RLN1, POTEE, PPIAL4G, or any combination thereof, and the cancer comprises prostate cancer.

[0018] In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 3441-4274, 5285-6084, 6580-6845, and 8100-8434, and the cancer comprises prostate cancer.

[0019] In some embodiments, the protein comprises ANKRD30A, COL10A1, or a combination thereof, and the cancer comprises breast cancer.

[0020] In some embodiments, the epitope sequence has 70%-100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 815-988, and 1749-1867, and the cancer comprises breast cancer.

[0021] In some embodiments, the protein comprises CTCFL, PRAME, CLDN6, EPYC, or any combination thereof, and the cancer comprises ovarian cancer.

[0022] In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1659-1748, 1964-2119, 2827-2919, and 6085-6183, and the cancer comprises ovarian cancer.

[0023] In some embodiments, the protein comprises CTCFL and the cancer comprises cervical cancer.

[0024] In some embodiments, the epitope sequence has 70%-100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1964-2119, and the cancer comprises cervical cancer.

[0025] In some embodiments, the protein comprises POTEE, PPIAL4G, or a combination thereof, and the cancer comprises colorectal cancer.

[0026] In some embodiments, the epitope sequence has 70%-100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 5285-5431, and 5996-6084, and the cancer comprises colorectal cancer.

[0027] In some embodiments, the protein comprises DLL3 and the cancer comprises glioma.

[0028] In some embodiments, the epitope sequence has 70%-100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2619-2736, and the cancer comprises glioma.

[0029] In some embodiments, the protein comprises MMP13 and the cancer comprises head and neck cancer.

[0030] In some embodiments, the epitope sequence has 70%-100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 4916-5010, and the cancer comprises head and neck cancer.

[0031] In some embodiments, the protein comprises DCAF4L2, SSX1, or a combination thereof, and the cancer comprises liver cancer.

[0032] In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2524-2618 and 7359-7448, and the cancer comprises liver cancer.

[0033] In some embodiments, the protein comprises SSX1, MAGEA4, PRAME, CSAG1, MAGEA12, MAGEA2, MAGEC2, PAGE5, PRDM7, SLC45A2, TSPAN10, or any combination thereof, and the cancer comprises melanoma.

[0034] In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1868-1963, 4458-4550, 4551-4637, 4638-4728, 4729-4816, 5011-5100, 6085-6183, 6184-6307, 7151-7264, 7359-7448, and 8745-8835, and the cancer comprises melanoma.

[0035] In some embodiments, the protein comprises MAGEA11, MAGEA4, PRAME, or any combination thereof, and the cancer comprises lung squamous cell carcinoma.

[0036] In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 4368-4457, 4638-4728, and 6085-6183, and the cancer comprises lung squamous cell carcinoma.

[0037] In some embodiments, the protein comprises ACTL7A, ACTL7B, ACTL9, ACTRT2, ADAD1, AKAP4, C2orf53, CCDC70, CETN1, DMRTB1, HMGB4, KIF2B, LELP1, PGK2, PRM1, PRM2, SPATA8, TNP1, TPD52L3, UBQLN3, or any combination thereof, and the cancer comprises testicular cancer.

[0038] In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1-626, 1183-1372, 1566-1658, 2737-2826, 3010-3100, 3197-3319, 4275-4367, 5101-5192, 6308-6486, 7265-7358, 8435-8618, and 8836-8962, and the cancer comprises testis cancer.

[0039] In some embodiments, the protein comprises KLK2, KLK3, KLK4, ANKRD30A, PRAME, MAGE4, ​​or a combination thereof.

[0040] In some embodiments, the protein comprises KLK2, KLK3 or KLK4, and the cancer comprises prostate cancer. In some embodiments, the epitope sequence is selected from the group consisting of AYSEKVTEF (SEQ ID NO: 3534), GLWTGGKDTCGV (SEQ ID NO: 3468), HPEDTGQVF (SEQ ID NO: 3988), HPEYNRPLL (SEQ ID NO: 4143), QRVPVSHSF (SEQ ID NO: 3544), SESDTIRSI (SEQ ID NO: 4176), SLFHPEDTGQV (SEQ ID NO: 3775), SLQCVSLHL (SEQ ID NO: 3456), VILLGRHSL (SEQ ID NO: 3891), VLVHPQWVL (SEQ ID NO: 3757), LFHPEDTGQVF (SEQ ID NO: 3827), RPRSLQCVSL (SEQ ID NO: 357 8), GYLQGLVSF (SEQ ID NO: 4094), IRNKSVILL (SEQ ID NO: 3974), KLQCVDLHV (SEQ ID NO: 3740), LLANGRMPTV (SEQ ID NO: 4029), LRPGDDSTL (SEQ ID NO: 3767), MPALPMVL (SEQ ID NO: 3874), NRPLLANDL (SEQ ID NO: 4216), SLQCVSLHL (SEQ ID NO: 3456), TWIAPPLQV (SEQ ID NO: 3784), VFQVSHSF (SEQ ID NO: 3828) and YSEKVTEFML (SEQ ID NO: 3454). In some embodiments, the epitope sequence has between 70% and 100% sequence identity to a peptide sequence selected from the group consisting of AYSEKVTEF (SEQ ID NO: 3534), HPEDTGQVF (SEQ ID NO: 3988), HPEYNRPLL (SEQ ID NO: 4143), QRVPVSHSF (SEQ ID NO: 3544), LFHPEDTGQVF (SEQ ID NO: 3827), GYLQGLVSF (SEQ ID NO: 4094), IRNKSVILL (SEQ ID NO: 3974), KLQCVDLHV (SEQ ID NO: 3740), LLANGRMPTV (SEQ ID NO: 4029), LRPGDDSTL (SEQ ID NO: 3767), MPALPMVL (SEQ ID NO: 3874), NRPLLANDL (SEQ ID NO: 4216), SLQCVSLHL (SEQ ID NO: 3456), TWIAPPLQV (SEQ ID NO: 3784), VFQVSHSF (SEQ ID NO: 3828) and YSEKVTEFML (SEQ ID NO: 3454).

[0041] In some embodiments, the protein comprises ANKRD30A and the cancer comprises breast cancer. In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of LLSHGAVIEV (SEQ ID NO: 831), SIPTKALEL (SEQ ID NO: 942), SQYSGQLKV (SEQ ID NO: 927), SVPNKALEL (SEQ ID NO: 941), SLSKILDTV (SEQ ID NO: 826) and SLDQKLFQL (SEQ ID NO: 827). In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of LLSHGAVIEV (SEQ ID NO: 831), SIPTKALEL (SEQ ID NO: 942), SVPNKALEL (SEQ ID NO: 941), SLSKILDTV (SEQ ID NO: 826) and SLDQKLFQL (SEQ ID NO: 827).

[0042] In some embodiments, the protein comprises PRAME and the cancer comprises lung squamous cell carcinoma; melanoma; ovarian cancer, uterine cancer, or any combination thereof. In some embodiments, the epitope sequence is selected from the group consisting of DSLFFLRGR (SEQ ID NO: 6132), ELFSYLIEK (SEQ ID NO: 6108), FYDPEPILC (SEQ ID NO: 6166), ISISALQSL (SEQ ID NO: 6161), ITDDQLLAL (SEQ ID NO: 6158), KRKKNVLRL (SEQ ID NO: 6173), LQSLLQHLI (SEQ ID NO: 6146), LSHIHASSY (SEQ ID NO: 6152), PYLGQMINL (SEQ ID NO: 6120), QLLALLPSL (SEQ ID NO: 6093), SFY The epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of GNSISI (SEQ ID NO: 6174), SLLQHLIGL (SEQ ID NO: 6095), SPSVSQLSVL (SEQ ID NO: 6139), SPYLGQMINL (SEQ ID NO: 6138), TSPRRLVEL (SEQ ID NO: 6159), VLYPVPLESY (SEQ ID NO: 6154), VSPEPLQAL (SEQ ID NO: 6156), YLHARLREL (SEQ ID NO: 6157) and RLDQLLRHV (SEQ ID NO: 6104). In some embodiments, the epitope sequence has 70% to 100% sequence identity to the peptide sequence of SLLQHLIGL (SEQ ID NO: 6095).

[0043] In some embodiments, the protein comprises MAGE4 and the cancer comprises lung squamous cell carcinoma. In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of EVDPASNTY (SEQ ID NO: 4638), GVYDGREHTV (SEQ ID NO: 4653), KEVDPASNTY (SEQ ID NO: 4640), KVDELAHFL (SEQ ID NO: 4648), QIFPKTGL (SEQ ID NO: 4692), QSPQGASAL (SEQ ID NO: 4707), SALPTTISF (SEQ ID NO: 4699), TVYGEPRKL (SEQ ID NO: 4722), VYGEPRKL (SEQ ID NO: 4727), YPSLREAAL (SEQ ID NO: 4689), ALLEEEEGV (SEQ ID NO: 4698) and KVLEHVVRV (SEQ ID NO: 4697). In some embodiments, the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of EVDPASNTY (sequence number 4638), GVYDGREHTV (sequence number 4653), KVDELAHFL (sequence number 4648) and KVLEHVVRV (sequence number 4697).

[0044] In some embodiments, the target tissue is a non-essential tissue.

[0045] In some embodiments, each non-target tissue is an essential tissue.

[0046] In some embodiments, the tissue-specific antigenic peptide is an isolated, purified and / or synthetic peptide.

[0047] In some embodiments, the tissue-specific antigenic peptide further comprises accessory sequences adjacent to the epitope sequence.

[0048] In some embodiments, the polynucleotide comprises deoxyribonucleic acid (DNA).

[0049] In some embodiments, the polynucleotide comprises ribonucleic acid (RNA).

[0050] In some embodiments, the composition comprises a viral vector containing the polynucleotide.

[0051] In some embodiments, the viral vector is an adenovirus viral vector, an adeno-associated virus (AAV) viral vector, a herpes simplex virus (HSV) viral vector, a Semliki Forest virus (SFV) viral vector, a lentivirus viral vector, a retrovirus viral vector, a poxvirus viral vector, an alphavirus viral vector, a vaccinia virus viral vector, a hepatitis B virus (HBV) viral vector, a human papillomavirus viral vector, or a pseudotype thereof, or any combination thereof.

[0052] In some embodiments, the tissue-specific antigenic peptide activates CD8+ T cells, CD4+ T cells, or both.

[0053] The present invention provides a composition for autologous T cell therapy for cancer in a subject in need thereof, comprising a population of T cells expressing an antigen-specific TCR, wherein the antigen is a cancer antigen as disclosed herein. A population of immune cells from a biological sample comprising at least one antigen-specific T cell comprising a TCR, wherein the epitope sequence binds or is predicted to bind to a protein encoded by an MHC allele expressed by a human subject in need of autologous T cell therapy, the TCR binds to the epitope when the epitope is present in a complex with a protein encoded by an MHC allele expressed by the human subject, and the epitope is a tissue-specific epitope encoded by a tissue-specific antigen epitope gene whose expression level in the target tissue is at least twice that of each of a plurality of non-target tissues different from the target tissue, is contemplated. In some embodiments, the T cells are non-engineered cells. In some embodiments, the T cells are autologous to the subject. In some embodiments, the T cells are modified ex vivo.

[0054] In some embodiments, the TCR is specific for a tissue-specific antigenic peptide complexed with a class I or class II MHC protein.

[0055] In some embodiments, at least one antigen-specific T cell expresses CD8 or CD4.

[0056] In some embodiments, at least one antigen-specific T cell comprises an exogenous polynucleotide encoding a TCR.

[0057] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 6846-7061, 7359-7448, 7629-8099, and 8619-8744, and the cancer comprises thyroid cancer.

[0058] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from protein RBPJL, AQP12A, AQP12B, IAPP, CELA2A, CELA2B, AMY2A, CTRC, G6PC2, KIRREL2, PNLIPRP1, SERPINI2, SYNC, or any combination thereof, and the cancer comprises pancreatic cancer.

[0059] In some embodiments, the at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 720-814, 989-1182, 1373-1565, 2120-2211, 2920-3009, 3101-3196, 3320-3440, 5193-5284, 6487-6579, 7062-7150, and 7539-7628, and the cancer comprises pancreatic cancer.

[0060] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the proteins: CYP11A1, CYP11B1, CYP11B2, MC2R, STAR, or any combination thereof, and the cancer comprises adrenal cancer.

[0061] In some embodiments, the at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70%-100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 22122523, 4817-4915, and 7449-7538, and the cancer comprises adrenal cancer.

[0062] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the proteins: ALPPL2, POTEE, PRAME, or any combination thereof, and the cancer comprises uterine cancer.

[0063] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 627-719, 5285-5431, and 6085-6183, and the cancer comprises uterine cancer.

[0064] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the following proteins: KLK2, KLK3, KLK4, POTEH, POTEG, TGM4, RLN1, POTEE, PPIAL4G, or any combination thereof, and the cancer comprises prostate cancer.

[0065] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 3441-4274, 5285-6084, 6580-6845, and 8100-8434, and the cancer comprises prostate cancer.

[0066] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the proteins: ANKRD30A, COL10A1, or a combination thereof, and the cancer comprises breast cancer.

[0067] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 815-988, and 1749-1867, and the cancer comprises breast cancer.

[0068] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the proteins: CTCFL, PRAME, CLDN6, EPYC, or any combination thereof, and the cancer comprises ovarian cancer.

[0069] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1659-1748, 1964-2119, 2827-2919, and 6085-6183, and the cancer comprises ovarian cancer.

[0070] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the protein: CTCFL, and the cancer comprises cervical cancer.

[0071] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1964-2119, and the cancer comprises cervical cancer.

[0072] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the proteins: POTEE, PPIAL4G, or a combination thereof, and the cancer comprises colorectal cancer.

[0073] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 5285-5431, and 5996-6084, and the cancer comprises colorectal cancer.

[0074] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the protein, DLL3, and the cancer comprises glioma.

[0075] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2619-2736, and the cancer comprises glioma.

[0076] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the protein, MMP13, and the cancer comprises head and neck cancer.

[0077] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 4916-5010, and the cancer comprises head and neck cancer.

[0078] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the protein, DCAF4L2, or SSX1, or a combination thereof, and the cancer comprises liver cancer.

[0079] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2524-2618 and 7359-7448, and the cancer comprises liver cancer.

[0080] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the following proteins: SSX1, MAGEA4, PRAME, CSAG1, MAGEA12, MAGEA2, MAGEC2, PAGE5, PRDM7, SLC45A2, TSPAN10, or any combination thereof, and the cancer comprises melanoma.

[0081] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1868-1963, 4458-4550, 4551-4637, 4638-4728, 4729-4816, 5011-5100, 6085-6183, 6184-6307, 7151-7264, 7359-7448, and 8745-8835, and the cancer comprises melanoma.

[0082] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the proteins: MAGEA11, MAGEA4, PRAME, or any combination thereof, and the cancer comprises lung squamous cell carcinoma.

[0083] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 4368-4457, 4638-4728, and 6085-6183, and the cancer comprises lung squamous cell carcinoma.

[0084] In some embodiments, at least one antigen-specific T cell comprises a TCR specific for an epitope sequence derived from the following proteins: ACTL7A, ACTL7B, ACTL9, ACTRT2, ADAD1, AKAP4, C2orf53, CCDC70, CETN1, DMRTB1, HMGB4, KIF2B, LELP1, PGK2, PRM1, PRM2, SPATA8, TNP1, TPD52L3, UBQLN3, or any combination thereof, and the cancer comprises testicular cancer.

[0085] In some embodiments, the at least one antigen-specific T cell comprises a TCR specific for an epitope sequence having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1-626, 1183-1372, 1566-1658, 2737-2826, 3010-3100, 3197-3319, 4275-4367, 5101-5192, 6308-6486, 7265-7358, 8435-8618, and 8836-8962, and the cancer comprises testis cancer.

[0086] In some embodiments, the composition comprises at least one antigen-specific T cell, and the tissue-specific antigen peptide comprises an epitope sequence of a protein encoded by a gene selected from the group consisting of ANKRD30A, DLL3, PRAME, CLDN6, EPYC, SLC45A2, TSPAN10, TSHR, LELP1, AQP12A, KIRREL2, G6PC2, AQP12B, and MC2R.

[0087] In some embodiments, the biological sample is from a subject with cancer or a donor other than the subject with cancer.

[0088] In some embodiments, the donor has a natural immune response to the tissue-specific antigenic peptide.

[0089] In some embodiments, the cancer comprises prostate cancer and the donor is female.

[0090] In some embodiments, the cancer comprises breast cancer or ovarian cancer and the donor is male.

[0091] In some embodiments, the protein is encoded by a tissue-specific antigen epitope gene having an mRNA expression level in each of a plurality of non-target tissues that are different from the tumor target tissue of at most about 5 mRNA transcripts per million total mRNA transcripts in each non-target tissue.

[0092] In some embodiments, the protein is encoded by a tissue-specific antigen epitope gene that has an mRNA expression level in the target tissue of at least about 100 mRNA transcripts per million total mRNA transcripts in the target tissue.

[0093] Provided herein is a pharmaceutical composition comprising a composition described herein and a pharma- ceutically acceptable carrier.

[0094] Provided herein is a method comprising a step of identifying an epitope sequence that binds or is predicted to bind to a protein encoded by an MHC allele expressed by a human subject, and that is encoded by a tissue-specific antigen epitope gene whose expression level in a tumor from a target tissue is at least twice the expression level of the tissue-specific antigen epitope gene in each of a plurality of non-target tissues distinct from the target tissue.

[0095] Provided herein is a method for preparing a T cell comprising a T cell receptor (TCR) specific for a complex of (i) an epitope sequence of a tissue-specific antigenic peptide of a protein and (ii) a protein encoded by an HLA allele of a human subject, the method comprising incubating the T cell in the presence of an antigen-presenting cell (APC) comprising the epitope sequence, wherein the APC expresses the protein encoded by the HLA allele of the human subject.

[0096] In some embodiments, the APC comprises a polypeptide comprising an epitope sequence or a polynucleotide encoding a polypeptide comprising an epitope sequence. In some embodiments, the APC is an APC from a human subject. In some embodiments, the T cell is a T cell from a human subject. In some embodiments, the method further comprises administering the T cell to a human subject in need thereof.

[0097] Provided herein is a method of treatment comprising administering a composition to a human subject in need thereof, the composition comprising an epitope sequence of a protein, the epitope sequence comprising a tissue-specific antigenic peptide expressed by a tumor; a polynucleotide encoding the tissue-specific antigenic peptide; one or more antigen-presenting cells (APCs) presenting the tissue-specific antigenic peptide; a T cell receptor (TCR) specific for the tissue-specific antigenic peptide; or a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a TCR, the epitope sequence binding or predicted to bind to a protein encoded by an MHC allele expressed by the human subject, the protein being encoded by a tissue-specific antigen epitope gene whose expression level in the tumor is at least twice the expression level of the tissue-specific antigen gene in each of a plurality of non-target tissues distinct from the target tissue.

[0098] In some embodiments, each organization of the plurality of organizations is a required organization.

[0099] In some embodiments, the plurality of tissues comprises skeletal muscle, coronary arteries, heart, adipose, uterus, vagina, skin, salivary glands, brain, lung, esophagus, stomach, colon, small intestine, nerve, or any combination thereof.

[0100] In some embodiments, each non-target tissue of the plurality of non-target tissues is a non-essential tissue.

[0101] In some embodiments, the MHC allele is a class I MHC allele or a class II MHC allele.

[0102] Provided herein is a method of treating cancer comprising administering to a subject in need thereof a composition described herein.

[0103] In some embodiments, the cancer comprises adrenal gland cancer, breast cancer, cervical cancer, colorectal cancer, fallopian tube cancer, glioma, head and neck cancer, liver cancer, lung squamous cell carcinoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, or any combination thereof.

[0104] In some embodiments, the protein comprises KLK2, KLK3, KLK4, ANKRD30A, PRAME, MAGE4, ​​or a combination thereof. In some embodiments, the protein comprises KLK2, KLK3, or KLK4, and the cancer comprises prostate cancer. In some embodiments, the epitope sequence is AYSEKVTEF (SEQ ID NO: 3534), and the human subject expresses a protein encoded by an HLA-C06:02 or HLA-A24:02 allele; the epitope sequence is GLWTGGKDTCGV (SEQ ID NO: 3468), and the human subject expresses a protein encoded by an HLA-A02:01 allele; the epitope sequence is HPEDTGQVF (SEQ ID NO: 3988), and the human subject expresses a protein encoded by an HLA-C*04:01 or or expressing a protein encoded by the HLA-C07:01 allele; the epitope sequence is HPEYNRPLL (SEQ ID NO: 4143), and the human subject expresses a protein encoded by the HLA-C*07:01 or HLA-B07:02 allele; the epitope sequence is QRVPVSHSF (SEQ ID NO: 3544), and the human subject expresses a protein encoded by the HLA-C*07:01, HLA-C*07:02, or HLA-A24:02 allele. the epitope sequence is SESDTIRSI (SEQ ID NO: 4176) and the human subject expresses a protein encoded by the HLA-B13:02 allele; the epitope sequence is SLFHPEDTGQV (SEQ ID NO: 3775) and the human subject expresses a protein encoded by the HLA-A02:01 allele; the epitope sequence is SLQCVSLHL (SEQ ID NO: 3456) and the human subject expresses a protein encoded by the HLA-A02:01 allele. the epitope sequence is VILLGRHSL (SEQ ID NO: 3891) and the human subject expresses a protein encoded by the HLA-B08:01 allele; the epitope sequence is VLVHPQWVL (SEQ ID NO: 3757) and the human subject expresses a protein encoded by the HLA-A02:01 allele; the epitope sequence is LFHPEDTGQVF (SEQ ID NO: 3827) and the human subject expresses a protein encoded by the HLA-A24:02 allele;The epitope sequence is RPRSLQCVSL (SEQ ID NO: 3578) and the human subject expresses a protein encoded by the HLA-B07:02 allele; the epitope sequence is GYLQGLVSF (SEQ ID NO: 4094) and the human subject expresses a protein encoded by the HLA-A24:02 allele; the epitope sequence is IRNKSVILL (SEQ ID NO: 3974) and the human subject expresses a protein encoded by the HLA-C*06:02, HLA-C*07:02 or HLA-C07:01 allele. the epitope sequence is KLQCVDLHV (SEQ ID NO: 3740), and the human subject expresses a protein encoded by the HLA-A02:01 allele; the epitope sequence is LLANGRMPTV (SEQ ID NO: 4029), and the human subject expresses a protein encoded by the HLA-A02:01 allele; the epitope sequence is LRPGDDSTL (SEQ ID NO: 3767), and the human subject expresses a protein encoded by the HLA-C07:02 allele; the epitope sequence is MPALPMV L (SEQ ID NO: 3874), and the human subject expresses a protein encoded by the HLA-B07:02 allele; the epitope sequence is NRPLLANDL (SEQ ID NO: 4216), and the human subject expresses a protein encoded by the HLA-C*06:02, HLA-C*07:02 or HLA-C01:02 allele; the epitope sequence is SLQCVSLHL (SEQ ID NO: 3456), and the human subject expresses a protein encoded by the HLA-A02:01 allele; the epitope sequence is TWI APPLQV (SEQ ID NO: 3784), and the human subject expresses a protein encoded by the HLA-C*04:01 or HLA-A02:01 allele; the epitope sequence is VFQVSHSF (SEQ ID NO: 3828), and the human subject expresses a protein encoded by the HLA-C*07:02 or HLA-A24:02 allele; or the epitope sequence is YSEKVTEFML (SEQ ID NO: 3454), and the human subject expresses a protein encoded by the HLA-A01:01 allele.

[0105] In some embodiments, the protein comprises ANKRD30A and the cancer comprises breast cancer. In some embodiments, the epitope sequence is LLSHGAVIEV (SEQ ID NO: 831) and the human subject expresses a protein encoded by an HLA-A02:01 allele, the epitope sequence is SQYSGQLKV (SEQ ID NO: 927) and the human subject expresses a protein encoded by an HLA-B13:02 allele, the epitope sequence is SVPNKALEL (SEQ ID NO: 941) and the human subject expresses a protein encoded by an HLA-C*04:01 or HLA-C01:02 allele. In one embodiment, the epitope sequence is SLSKILDTV (SEQ ID NO: 826) and the human subject expresses a protein encoded by the HLA-A02:01 allele, the epitope sequence is SIPTKALEL (SEQ ID NO: 942) and the human subject expresses a protein encoded by the HLA-C*04:01 or HLA-C01:02 allele, or the epitope sequence is SLDQKLFQL (SEQ ID NO: 827) and the human subject expresses a protein encoded by the HLA-A02:01 allele.

[0106] In some embodiments, the protein comprises PRAME, and the cancer comprises lung squamous cell carcinoma; melanoma; ovarian cancer, uterine cancer, or any combination thereof. In some embodiments, the epitope sequence is DSLFFLRGR (SEQ ID NO: 6132), and the human subject expresses a protein encoded by the HLA-A33:03 allele; the epitope sequence is ELFSYLIEK (SEQ ID NO: 6108), and the human subject expresses a protein encoded by the HLA-A03:01 allele; the epitope sequence is FYDPEPILC (SEQ ID NO: 6166), and the human subject expresses a protein encoded by the HLA-C04:01 allele; The epitope sequence is ISISALQSL (SEQ ID NO: 6161) and the human subject expresses a protein encoded by the HLA-C03:04 allele; the epitope sequence is ITDDQLLAL (SEQ ID NO: 6158) and the human subject expresses a protein encoded by the HLA-A01:01 allele; the epitope sequence is KRKKNVLRL (SEQ ID NO: 6173) and the human subject expresses a protein encoded by the HLA-C07:01 allele; the epitope sequence is LQS The epitope sequence is LLQHLI (SEQ ID NO: 6146), and the human subject expresses a protein encoded by the HLA-B13:02 allele; the epitope sequence is LSHIHASSY (SEQ ID NO: 6152), and the human subject expresses a protein encoded by the HLA-B46:01 allele; the epitope sequence is PYLGQMINL (SEQ ID NO: 6120), and the human subject expresses a protein encoded by the HLA-A24:02 allele; the epitope sequence is QLLALLPSL (SEQ ID NO: No. 6093), the human subject expresses a protein encoded by the HLA-A02:01 allele, the epitope sequence is SFYGNSISI (SEQ ID NO: 6174), the human subject expresses a protein encoded by the HLA-C07:01 allele, the epitope sequence is SLLQHLIGL (SEQ ID NO: 6095), the human subject expresses a protein encoded by the HLA-A02:01 allele, the epitope sequence is SPSVSQLSVL (SEQ ID NO: 6139),The human subject expresses a protein encoded by the HLA-B07:02 allele, the epitope sequence is SPYLGQMINL (SEQ ID NO: 6138), the human subject expresses a protein encoded by the HLA-B07:02 allele, the epitope sequence is TSPRRLVEL (SEQ ID NO: 6159), the human subject expresses a protein encoded by the HLA-C01:02 allele, the epitope sequence is VLYPVPLESY (SEQ ID NO: 6154), and the human subject expresses a protein encoded by the HLA-A03:01 allele. the epitope sequence is VSPEPLQAL (SEQ ID NO: 6156) and the human subject expresses a protein encoded by the HLA-C01:02 allele; the epitope sequence is YLHARLREL (SEQ ID NO: 6157) and the human subject expresses a protein encoded by the HLA-B08:01 allele; or the epitope sequence is RLDQLLRHV (SEQ ID NO: 6104) and the human subject expresses a protein encoded by the HLA-A02:01 allele.

[0107] In some embodiments, the protein comprises MAGE4 and the cancer comprises lung squamous cell carcinoma. In some embodiments, the epitope sequence is EVDPASNTY (SEQ ID NO: 4638) and the human subject expresses a protein encoded by the HLA-A01:01 allele, the epitope sequence is GVYDGREHTV (SEQ ID NO: 4653) and the human subject expresses a protein encoded by the HLA-A02:01 allele, the epitope sequence is KEVDPASNTY (SEQ ID NO: 4640) and the human subject expresses a protein encoded by the HLA-A01:01 allele. The epitope sequence is KVDELAHFL (SEQ ID NO: 4648), and the human subject expresses a protein encoded by the HLA-A02:01 allele; the epitope sequence is QIFPKTGL (SEQ ID NO: 4692), and the human subject expresses a protein encoded by the HLA-B08:01 allele; the epitope sequence is QSPQGASAL (SEQ ID NO: 4707), and the human subject expresses a protein encoded by the HLA-C01:02 allele. the epitope sequence is SALPTTISF (SEQ ID NO: 4699) and the human subject expresses a protein encoded by the HLA-B46:01 allele; the epitope sequence is TVYGEPRKL (SEQ ID NO: 4722) and the human subject expresses a protein encoded by the HLA-C07:01 allele; the epitope sequence is VYGEPRKL (SEQ ID NO: 4727) and the human subject expresses a protein encoded by the HLA-C07:02 allele. , the epitope sequence is YPSLREAAL (sequence number 4689) and the human subject expresses a protein encoded by the HLA-B07:02 allele, the epitope sequence is ALLEEEEGV (sequence number 4698) and the human subject expresses a protein encoded by the HLA-A02:01 allele, or the epitope sequence is KVLEHVVRV (sequence number 4697) and the human subject expresses a protein encoded by the HLA-A02:01 allele.

[0108] The present invention provides a method comprising: (a) contacting a T cell with an antigenic peptide complexed with HLA of an APC; and (b) determining the sequence of a TCR of the T cell that recognizes the antigenic peptide complexed with HLA, the T cell being suspected of having no or reduced immune tolerance to the tissue of origin of the antigenic peptide. In some embodiments, the T cell is derived from a female subject, and the antigenic peptide is specific for a tissue selected from the group consisting of bulbourethral gland, epididymis, penis, prostate, scrotum, seminal vesicle, and testis. In some embodiments, the T cell is derived from a female subject, and the antigenic peptide is specific for a tissue selected from the group consisting of Bartholin's gland, fallopian tube, ovary, Skene's gland, uterus, cervix, vagina, and any combination thereof. In some embodiments, the T cell is derived from a male subject, and the antigenic peptide is specific for an ovary. In some embodiments, the T cell is derived from a type 1 diabetes patient, and the antigenic peptide is specific for a pancreas. In some embodiments, the T cell is derived from a subject with thyroid autoimmune condition, and the antigenic peptide is specific for thyroid.In some embodiments, the T cell is derived from a subject who is negative for HLA allele.In some embodiments, the T cell is derived from a subject who is negative for HLA allele, and the antigenic peptide binds to the HLA encoded by the HLA allele. Incorporation by Reference

[0109] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0110] [Figure 1] FIG. 1 is a box plot illustrating the expression levels of the gene ANKRD30A in several different normal tissues and tumors.

[0111] [Diagram 2]FIG. 2 is a box plot illustrating the expression levels of the gene COL10A1 in several different normal tissues and tumors.

[0112] [Diagram 3] FIG. 3 is a box plot illustrating the expression levels of the gene CTCFL in several different normal tissues and tumors.

[0113] [Figure 4] FIG. 4 is a box plot illustrating the expression levels of the gene PPIAL4G in several different normal tissues and tumors.

[0114] [Diagram 5] FIG. 5 is a box plot illustrating the expression levels of the gene POTEE in several different normal tissues and tumors.

[0115] [Figure 6] FIG. 6 is a box plot illustrating the expression levels of the gene DLL3 in several different normal tissues and tumors.

[0116] [Figure 7] FIG. 7 is a box plot illustrating the expression levels of the gene MMP13 in several different normal tissues and tumors.

[0117] [Figure 8] FIG. 8 is a box plot illustrating the expression levels of the gene SSX1 in several different normal tissues and tumors.

[0118] [Figure 9] FIG. 9 is a box plot illustrating the expression levels of the gene DCAF4L2 in several different normal tissues and tumors.

[0119] [Figure 10]FIG. 10 is a box plot illustrating the expression levels of the gene MAGEA4 in several different normal tissues and tumors.

[0120] [Figure 11] FIG. 11 is a box plot illustrating the expression levels of the gene MAGEA11 in several different normal tissues and tumors.

[0121] [Figure 12] FIG. 12 is a box plot illustrating the expression levels of the gene MAGEC2 in several different normal tissues and tumors.

[0122] [Figure 13] FIG. 13 is a box plot illustrating the expression levels of the gene MAGEA12 in several different normal tissues and tumors.

[0123] [Figure 14] FIG. 14 is a box plot illustrating the expression levels of the gene PRAME in several different normal tissues and tumors.

[0124] [Figure 15] FIG. 15 is a box plot illustrating the expression levels of the gene CLDN6 in several different normal tissues and tumors.

[0125] [Figure 16] FIG. 16 is a box plot illustrating the expression levels of the gene EPYC in several different normal tissues and tumors.

[0126] [Figure 17] FIG. 17 is a box plot illustrating the expression levels of the gene KLK3 in several different normal tissues and tumors.

[0127] [Figure 18]FIG. 18 is a box plot illustrating the expression levels of the gene KLK2 in several different normal tissues and tumors.

[0128] [Figure 19] FIG. 19 is a box plot illustrating the expression levels of the gene KLK4 in several different normal tissues and tumors.

[0129] [Figure 20] FIG. 20 is a box plot illustrating the expression levels of the gene TGM4 in several different normal tissues and tumors.

[0130] [Figure 21] FIG. 21 is a box plot illustrating the expression levels of the gene POTEG in several different normal tissues and tumors.

[0131] [Figure 22] FIG. 22 is a box plot illustrating the expression levels of gene RLN1 in several different normal tissues and tumors.

[0132] [Figure 23] FIG. 23 is a box plot illustrating the expression levels of the gene POTEH in several different normal tissues and tumors.

[0133] [Figure 24] FIG. 24 is a box plot illustrating the expression levels of the gene SLC45A2 in several different normal tissues and tumors.

[0134] [Diagram 25] FIG. 25 is a box plot illustrating the expression levels of the gene TSPAN10 in several different normal tissues and tumors.

[0135] [Figure 26]FIG. 26 is a box plot illustrating the expression levels of the gene PAGE5 in several different normal tissues and tumors.

[0136] [Figure 27] FIG. 27 is a box plot illustrating the expression levels of the gene CSAG1 in several different normal tissues and tumors.

[0137] [Figure 28] FIG. 28 is a box plot illustrating the expression levels of the gene PRDM7 in several different normal tissues and tumors.

[0138] [Figure 29] FIG. 29 is a box plot illustrating the expression levels of gene TG in several different normal tissues and tumors.

[0139] [Diagram 30] FIG. 30 is a box plot illustrating the expression levels of the gene TSHR in several different normal tissues and tumors.

[0140] [Diagram 31] FIG. 31 is a box plot illustrating the expression levels of the gene RSPH6A in several different normal tissues and tumors.

[0141] [Diagram 32] FIG. 32 is a box plot illustrating the expression levels of the gene SCXB in several different normal tissues and tumors.

[0142] [Diagram 33] FIG. 33 is a box plot illustrating the expression levels of the gene HIST1H4K in several different normal tissues and tumors.

[0143] [Diagram 34]FIG. 34 is a box plot illustrating the expression levels of the gene ALPPL2 in several different normal tissues and tumors.

[0144] [Diagram 35] FIG. 35 is a box plot illustrating the expression levels of the gene PRM2 in several different normal tissues and tumors.

[0145] [Diagram 36] FIG. 36 is a box plot illustrating the expression levels of the gene PRM1 in several different normal tissues and tumors.

[0146] [Figure 37] FIG. 37 is a box plot illustrating the expression levels of the gene TNP1 in several different normal tissues and tumors.

[0147] [Figure 38] FIG. 38 is a box plot illustrating the expression levels of the gene LELP1 in several different normal tissues and tumors.

[0148] [Figure 39] FIG. 39 is a box plot illustrating the expression levels of the gene HMGB4 in several different normal tissues and tumors.

[0149] [Diagram 40] FIG. 40 is a box plot illustrating the expression levels of the gene AKAP4 in several different normal tissues and tumors.

[0150] [Diagram 41] FIG. 41 is a box plot illustrating the expression levels of the gene CETN1 in several different normal tissues and tumors.

[0151] [Diagram 42]FIG. 42 is a box plot illustrating the expression levels of the gene UBQLN3 in several different normal tissues and tumors.

[0152] [Diagram 43] FIG. 43 is a box plot illustrating the expression levels of the gene ACTL7A in several different normal tissues and tumors.

[0153] [Diagram 44] FIG. 44 is a box plot illustrating the expression levels of the gene ACTL9 in several different normal tissues and tumors.

[0154] [Diagram 45] FIG. 45 is a box plot illustrating the expression levels of the gene ACTRT2 in several different normal tissues and tumors.

[0155] [Diagram 46] FIG. 46 is a box plot illustrating the expression levels of the gene PGK2 in several different normal tissues and tumors.

[0156] [Figure 47] FIG. 47 is a box plot illustrating the expression levels of the gene C2orf53 in several different normal tissues and tumors.

[0157] [Figure 48] FIG. 48 is a box plot illustrating the expression levels of the gene KIF2B in several different normal tissues and tumors.

[0158] [Figure 49] FIG. 49 is a box plot illustrating the expression levels of the gene ADAD1 in several different normal tissues and tumors.

[0159] [Figure 50]FIG. 50 is a box plot illustrating the expression levels of the gene SPATA8 in several different normal tissues and tumors.

[0160] [Figure 51] FIG. 51 is a box plot illustrating the expression levels of the gene CCDC70 in several different normal tissues and tumors.

[0161] [Figure 52] FIG. 52 is a box plot illustrating the expression levels of the gene TPD52L3 in several different normal tissues and tumors.

[0162] [Figure 53] FIG. 53 is a box plot illustrating the expression levels of the gene ACTL7B in several different normal tissues and tumors.

[0163] [Figure 54] FIG. 54 is a box plot illustrating the expression levels of the gene DMRTB1 in several different normal tissues and tumors.

[0164] [Figure 55] FIG. 55 is a box plot illustrating the expression levels of the gene SYCN in several different normal tissues and tumors.

[0165] [Figure 56] FIG. 56 is a box plot illustrating the expression levels of the gene CELA2A in several different normal tissues and tumors.

[0166] [Figure 57] FIG. 57 is a box plot illustrating the expression levels of the gene CELA2B in several different normal tissues and tumors.

[0167] [Figure 58]FIG. 58 is a box plot illustrating the expression levels of the gene PNLIPRP1 in several different normal tissues and tumors.

[0168] [Figure 59] FIG. 59 is a box plot illustrating the expression levels of the gene CTRC in several different normal tissues and tumors.

[0169] [Figure 60] FIG. 60 is a box plot illustrating the expression levels of the gene AMY2A in several different normal tissues and tumors.

[0170] [Figure 61] FIG. 61 is a box plot illustrating the expression levels of the gene SERPINI2 in several different normal tissues and tumors.

[0171] [Figure 62] FIG. 62 is a box plot illustrating the expression levels of the gene RBPJL in several different normal tissues and tumors.

[0172] [Figure 63] FIG. 63 is a box plot illustrating the expression levels of the gene AQP12A in several different normal tissues and tumors.

[0173] [Figure 64] FIG. 64 is a box plot illustrating the expression levels of the gene IAPP in several different normal tissues and tumors.

[0174] [Figure 65] FIG. 65 is a box plot illustrating the expression levels of the gene KIRREL2 in several different normal tissues and tumors.

[0175] [Figure 66]FIG. 66 is a box plot illustrating the expression levels of the gene G6PC2 in several different normal tissues and tumors.

[0176] [Figure 67] FIG. 67 is a box plot illustrating the expression levels of the gene AQP12B in several different normal tissues and tumors.

[0177] [Figure 68] FIG. 68 is a box plot illustrating the expression levels of the gene CYP11B1 in several different normal tissues and tumors.

[0178] [Figure 69] FIG. 69 is a box plot illustrating the expression levels of the gene CYP11B2 in several different normal tissues and tumors.

[0179] [Figure 70] FIG. 70 is a box plot illustrating the expression levels of the gene STAR in several different normal tissues and tumors.

[0180] [Figure 71] FIG. 71 is a box plot illustrating the expression levels of the gene CYP11A1 in several different normal tissues and tumors.

[0181] [Figure 72] FIG. 72 is a box plot illustrating the expression levels of the gene MC2R in several different normal tissues and tumors.

[0182] [Figure 73] FIG. 73 shows a schematic of an exemplary workflow for epitope mapping using targeted proteomics.

[0183] [Figure 74]Figure 74 shows an exemplary graph demonstrating the spectral validation of class I HLA epitopes by mass analysis of endogenous peptides using targeted proteomics. Chromatographs of six characteristic fragment ions for the light (endogenous) and heavy chain isotope-labeled synthetic peptide sequence "HPEYNRPLL" from KLK4 (HLA-B*07:02, endogenous peptide identified in human prostate specimens). Matched chromatographic retention times of the peptide fragment ions and a high dot product similarity score (0.992 calculated using Skyline software) provide validation that this epitope is processed and presented on the HLA-B*07:02 molecule.

[0184] [Figure 75] Figure 75 shows two exemplary spectra illustrating the spectral validation of endogenous peptides using targeted proteomics. Spectra are shown for the light chain (endogenous) HPEYNRPLL epitope identified in a human prostate specimen (left) and the corresponding heavy chain isotopically labeled synthetic peptide (right). B and Y fragment ions are shown, showing high spectral similarity and confirming the detection of the endogenous epitope. For each peptide, the top 200 more intense ions are plotted, and the corresponding mass errors of the highlighted b and y ions are plotted below the spectral plot.

[0185] [Figure 76] Figure 76 shows exemplary flow cytometry plots of naive T cells from healthy donors stained with peptide-MHC multimers for target epitopes after induction with the indicated HLA-I molecules. The multimer positive population and percentage of multimer positive cells are shown. The top panel shows the identification of positive samples using combinatorial multimer analysis. The bottom panel shows the results of a confirmatory combinatorial analysis performed on frozen samples after the initial identification in the top panel. Multimer positive cells from the analysis in the bottom panel are selected for downstream TCR identification.

[0186] [Figure 77]Figure 77 shows a graph showing exemplary TCR clonotypes identified from the 10-round genomic pipeline. Each graph originates from a single selected multimer positive population. All samples in this case contained two unique TCR clonotypes identified by paired alpha and beta sequences. In cases where the 10-round genomic pipeline identified clonotypes that contained multiples of either alpha or beta sequences, all possible combinations were synthesized for antigen specificity and avidity.

[0187] [Figure 78] FIG. 78 shows an exemplary plot depicting the avidity of an exemplary TCR. The plot reflects CD69 expression on transduced Jurkat cells (identified by simultaneous expression of murine TCR, CD8, and CD3) after overnight co-culture with target cell lines presenting HLA and loaded with variable amounts of peptide. For the seven TCRs tested, five of these show a peptide-dependent increase in CD69 expression. The concentration required to achieve 50% activation (EC50) was calculated from these plots and the results are shown on the plots. Target cells were pre-transduced to overexpress the allele of interest. A375 were plated at 50K / well for 2-5 hours and then pulsed with peptide for 1 hour before adding effector cells. T2 were plated at 10K / well and then pulsed with peptide for 1 hour before adding effector cells. Peptides were pulsed at a final concentration between 10e3-10e-1 nM. Cells were co-cultured overnight, then harvested and stained for CD69 expression by flow using CD8, CD3, and mouse TCR constant antibodies as lineage markers for effector cells.

[0188] [Figure 79]Figure 79 shows an exemplary plot showing the intrinsic activity of two different exemplary TCRs. Exemplary TCR avidity. The plot reflects the activation of two different TCR sequences (hereafter designated mTCR21-033 and mTCR-034) after co-culture with the cell line MDA-PCa-2b, which is endogenous for both HLA-B07 and KLK4. These plots show an increase in the activation of mTCR21-033, but not mTCR21-034, after 24 hours of treatment with a cocktail of interferons (IFNs). IFN treatment increases surface HLA expression on the cell lines, and increased surface HLA expression may result in greater expression of HLA-B07 bound to the KLK4 epitope. MDA-PCa-2b cells were plated at 50K / well in F12K medium. The next day, the cultures were treated with a cocktail of interferon alpha, beta, and gamma at a final concentration of 1 U / μL. The next day, the cells were washed with RPMI supplemented with 10% FBS and Glutamax. The cultures were then pulsed with peptides at a final concentration of 2 μM for 1 hour before adding effector cells. The cells were co-cultured overnight, then harvested and stained for CD69 expression by flow using CD8, CD3, and mouse TCR constant antibodies as lineage markers for effector cells and HLA-B07 as lineage marker for target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0189] Detailed Description definition The terminology used herein is merely for the purpose of describing a particular instance and is not limiting. In this application, the use of the singular includes the plural unless otherwise specified. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0190] In this application, the use of "or" means "and / or" unless otherwise specified. The terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably when used herein. These terms may convey that any combination is specifically intended. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C." The term "or" may be used conjunctively or disjunctively unless the context specifically indicates a disjunctive use.

[0191] The term "about" or "approximately" may mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" may mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% for a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, within 5-fold, or more preferably within 2-fold of a value. When a particular value is described in this application and claims, the term "about" should be assumed to mean within an acceptable error range for the particular value, unless otherwise specified.

[0192] As used in the specification and claim(s), the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is intended that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to realize the methods of the disclosure.

[0193] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" may mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, certain terms and phrases are defined below.

[0194] "Major histocompatibility complex" or "MHC" may refer to a cluster of genes that play a role in controlling cellular interactions involved in physiological immune responses. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of MHC and HLA complexes, see Paul, Fundamental Immunology, 3rd Ed., Raven Press, New York (1993). "Major histocompatibility complex (MHC) proteins or molecules," "MHC molecules," "MHC proteins," or "HLA proteins" should be understood to mean proteins capable of binding peptides resulting from proteolytic cleavage of protein antigens and transporting those peptides to the cell surface where they are presented to specific cells, in particular cytotoxic T-lymphocytes, helper T cells, or B cells. The major histocompatibility complex in the genome contains gene regions whose gene products expressed on the cell surface are important for binding and presenting endogenous and / or foreign antigens, and thus for regulating immunological processes. The major histocompatibility complex is divided into two groups of genes that code for different proteins: MHC class I molecules and MHC class II molecules. The cell biology and expression patterns of the two MHC classes are compatible with their distinct roles.

[0195] "Human leukocyte antigen" or "HLA" can refer to human class I or class II major histocompatibility complex (MHC) proteins (see, e.g., Stites, et al., Immunology, 8th Ed., Lange Publishing, Los Altos, Calif. (1994)).

[0196] "Polypeptide" and "peptide" are used interchangeably and may refer to a polymer of amino acid residues as used herein. A "mature protein" is a protein that is full length and optionally includes glycosylation or other modifications typical of proteins in a given cellular environment. The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may include synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indomethacin, cyclohexylglycine ... Examples of suitable lysine-2-carboxylic acids include lysine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. It is further contemplated by the present disclosure that expression of the polypeptides described herein in engineered cells may be accompanied by post-translational modification of one or more amino acids of the polypeptide construct.Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glycosylphosphatidylinositol glypiation, lipoylation and iodination. The nomenclature used to describe peptides or proteins follows the conventional convention of showing the amino group on the left (amino-terminus or N-terminus) and the carboxyl group on the right (carboxy-terminus or C-terminus) of each amino acid residue. When referring to the position of amino acid residues in a peptide epitope, the amino acid residues are numbered in the amino to carboxyl direction, with the residue located at the amino terminus of the epitope or the peptide or protein of which it is a part being numbered 1. In the formulas representing selected particular embodiments of the present disclosure, the amino and carboxyl terminal groups are not specifically shown, but are in the form they assume at physiological pH values ​​unless otherwise specified. In amino acid structural formulas, each residue is generally represented by a standard three-letter or one-letter name. The L-form of an amino acid residue is represented by a single capital letter or the first letter of the three-letter symbol with a capital letter, and the D-form of those amino acid residues having a D-form is represented by a single lower-case letter or a three-letter symbol with a lower-case letter. However, when the three-letter symbol or the full name is used without capital letters, it may refer to an L-amino acid residue. Glycine does not have an asymmetric carbon atom and is simply referred to as "Gly" or "G". The amino acid sequences of the peptides described herein are generally designated using the standard one-letter symbols (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine).

[0197] An "immunogenic" peptide or "immunogenic" epitope can refer to an epitope-containing peptide or peptides that contain an allele-specific motif, such that the peptide binds to an HLA molecule and elicits a cell-mediated or humoral response, such as cytotoxic T lymphocytes (CTLs (e.g., CD8 + )), helper T lymphocytes (Th (e.g., CD4 + )) and / or B lymphocyte responses. Thus, the immunogenic peptides described herein are capable of binding to the appropriate HLA molecule and subsequently inducing a CTL (cytotoxic) or HTL (and humoral) response against the peptide.

[0198] "Reference" can be used to correlate and compare the results obtained from tumor specimen in the method of the present disclosure.Generally, "reference" can be obtained based on one or more normal specimens obtained from either a patient or one or more different individuals, for example healthy individuals, particularly individuals of the same species, particularly specimens that are not affected by cancer disease."Reference" can be empirically determined by testing a sufficiently large number of normal specimens.

[0199] An "epitope" may be a collective feature of a molecule, such as the primary, secondary and tertiary peptide structure and charge, which together form the site recognized by, for example, an immunoglobulin, a T cell receptor, an HLA molecule, or a chimeric antigen receptor. Alternatively, an epitope may be defined as the set of amino acid residues involved in recognition by a particular immunoglobulin, or, for T cells, the set of residues required for recognition by a T cell receptor protein, a chimeric antigen receptor, and / or a major histocompatibility complex (MHC) receptor. Epitopes may be prepared by isolation from natural sources or may be synthesized according to standard protocols in the art. Synthetic epitopes may include artificial amino acid residues, "amino acid mimetics," e.g., D isomers of naturally occurring L amino acid residues, or non-naturally occurring amino acid residues such as cyclohexylalanine. Throughout this disclosure, epitopes may in some cases be referred to as peptides or peptide epitopes. It should be understood that proteins or peptides that contain the epitopes or analogs described herein as well as additional amino acid(s) still fall within the scope of the present disclosure. In certain embodiments, the peptides include fragments of antigens. In certain embodiments, the peptides of the present disclosure are limited in length. A limited-length embodiment occurs when a protein or peptide that contains an epitope described herein includes a region (i.e., a continuous series of amino acid residues) that has 100% identity to a native sequence. For example, to avoid reading the entire natural molecule to define an epitope, the length of any region that has 100% identity to the native peptide sequence is limited.Thus, for peptides comprising the epitopes described herein and regions having 100% identity to a native peptide sequence, the length of the region having 100% identity to the native sequence is generally less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues. In certain embodiments, an "epitope" as described herein is comprised of a peptide having a region of less than 51 amino acid residues, e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues, with 100% identity to the native peptide sequence.

[0200] A "T cell epitope" should be understood to mean a peptide sequence which can be bound by an MHC molecule of class I or II and in which the MHC molecule or MHC complex presents the peptide, which can then be recognised and bound by a T cell, such as a T-lymphocyte or a helper T cell.

[0201] As used herein, the term "affinity" may refer to a measure of the strength of binding between two members of a binding pair, for example, an HLA-binding peptide and class I or II HLA. Dis the dissociation constant and has units of molarity. The affinity constant is the inverse of the dissociation constant. Affinity constant is sometimes used as a general term to describe this chemical entity. It is a direct measure of the energy of binding. Affinity can be determined experimentally, for example, by surface plasmon resonance (SPR) using a commercially available Biacore SPR unit. Affinity can also be expressed as the inhibitory concentration 50 (IC50), the concentration at which 50% of the peptide is displaced. 50 ) can also be expressed as ln(IC 50 ) is an IC 50 It refers to the natural logarithm of K off refers to, for example, the dissociation rate constant for the dissociation of an HLA-binding peptide with class I or II HLA. Throughout this disclosure, "binding data" results are referred to as "IC 50 ". IC 50 is the concentration at which 50% inhibition of binding of the tested peptide to the labeled reference peptide in the binding assay is observed. Given the conditions under which the assay is performed (i.e., the limiting HLA protein and labeled reference peptide concentrations), these values ​​are D The binding is close to the value. Assays for determining binding are well known in the art and are described in detail in, for example, PCT Publication Nos. WO94 / 20127 and WO94 / 03205, and other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154: 247 (1995); and Sette, et al., Mol. Immunol. 31: 813 (1994). Alternatively, binding can be expressed relative to the binding by a reference standard peptide. For example, its IC 50 IC of the reference standard peptide based on 50may be relative to Other assay systems can also be used to determine binding, including those using live cells (e.g., Ceppellini et al., Nature 339: 392 (1989); Christnick et al., Nature 352: 67 (1991); Busch et al., Int. Immunol. 2: 443 (1990); Hill et al., J. Immunol. 147: 189 (1991); del Guercio et al., J. Immunol. 154: 685 (1995)), cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21: 2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152: 4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11: 2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268: 15425 (1993)); high flow soluble phase assays (Hammer et al., J. Exp. Med. 180: 2353 (1994)), and measurements of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346: 476 (1990); Schumacher et al., Cell 62: 563 (1990); Townsend et al., Cell 62: 285 (1990); Parker et al., J. Immunol. 149: 1896 (1992)). "Cross-reactive binding" indicates that a peptide binds to more than one HLA molecule; a synonym is degenerate binding.

[0202] A "synthetic peptide" may refer to a peptide obtained from a non-natural source, e.g., man-made. Such peptides may be made using methods such as chemical synthesis or recombinant DNA technology. In some embodiments, a "synthetic peptide" may include a "fusion protein."

[0203] The term "motif" can refer to a pattern of residues in a peptide of a defined length that is recognized by a particular HLA molecule, for example, less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, for example, about 8 amino acid residues to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13) for class I HLA motifs, and about 6 amino acid residues to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for class II HLA motifs. Motifs are generally different for each HLA protein encoded by a given human HLA allele. These motifs differ in the pattern of major and minor anchor residues. In some embodiments, MHC class I motifs are identified as peptides of 9, 10, or 11 amino acid residues in length.

[0204] According to the present disclosure, the term "vaccine" may refer to a pharmaceutical preparation (composition) or product that, when administered, induces an immune response, e.g., a cellular or humoral immune response, that recognizes and attacks a pathogen or diseased cell, such as a cancer cell. Vaccines can be used to prevent or treat disease. The term "individualized cancer vaccine" or "personalized cancer vaccine" refers to a specific cancer patient and to adapting the cancer vaccine to the needs or special circumstances of the individual cancer patient.

[0205] A "protective immune response" or a "therapeutic immune response" can refer to a CTL and / or HTL response to an antigen derived from a pathogenic antigen (e.g., a tissue-specific antigen) that in some way prevents or at least partially arrests disease symptoms, side effects, or exacerbation. The immune response can include an antibody response promoted by stimulation of helper T cells.

[0206] The term "antibody," as used herein, may refer to an immunoglobulin protein that comprises two heavy chains bound to one another, and each heavy chain may also be paired with a light chain.

[0207] A "functional portion of an antibody" as used herein may refer to a portion having at least one property shared with said antibody in kind, but not necessarily in amount. A functional portion is capable of binding to the same antigen as the antibody, although not necessarily to the same extent. A functional portion of an antibody comprises at least a heavy chain variable domain (V H ) and the light chain variable domain (V L In some embodiments, a functional portion of an antibody preferably comprises at least a heavy chain variable domain (V H Non-limiting examples of functional portions of antibodies include single domain antibodies, single chain antibodies, nanobodies, unibodies, single chain variable fragments (scFv), bispecific T cell-inducing antibodies (BiTEs), Fab fragments, and F(ab') 2 It may be a fragment.

[0208] "Antigen processing" or "processing" and its grammatical equivalents can refer to the degradation of a polypeptide or antigen into process products that are fragments of said polypeptide or antigen (e.g., degradation of a polypeptide into peptides), and the association (e.g., by binding) of one or more of these fragments to an MHC molecule for presentation by a cell, e.g., an antigen-presenting cell, to a specific T cell.

[0209] An "antigen-presenting cell" (APC) can be a cell that presents peptide fragments of protein antigens associated with MHC molecules on the cell surface. Some APCs can activate antigen-specific T cells. Professional antigen-presenting cells are highly efficient at internalizing antigens, either by phagocytosis or receptor-mediated endocytosis, and then displaying fragments of antigens bound to class II MHC molecules on their membranes. T cells recognize and interact with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. Additional costimulatory signals are then generated by the antigen-presenting cell, leading to T cell activation. The expression of costimulatory molecules is a defining feature of professional antigen-presenting cells. The major types of professional antigen-presenting cells are dendritic cells, macrophages, B cells, and certain activated epithelial cells, which have the broadest range of antigen presentation and are perhaps the most important antigen-presenting cells. Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via both MHC class II and I antigen presentation pathways. It is well known that dendritic cells are potent inducers of immune responses and activation of these cells is a crucial step for inducing antitumor immunity. Dendritic cells are conveniently categorized into "immature" and "mature" cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with a high capacity for antigen uptake and processing, which correlates with high expression of Fc receptors (FcR) and mannose receptors. The mature phenotype is generally characterized by lower expression of these markers but high expression of cell surface molecules involved in T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1 BB).

[0210] As used herein, the term "identical" and its grammatical equivalents or "sequence identity" in reference to the amino acid sequences of two nucleic acid sequences or polypeptides may refer to the residues in the two sequences being the same when aligned for maximum correspondence over a specified comparison window. A "comparison window," as used herein, may refer to a segment of at least about 20, typically about 50 to about 200, more typically about 100 to about 150 contiguous positions that a sequence can be compared to a reference sequence having the same number of contiguous positions after optimally aligning the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2: 482 (1981); by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. USA, 85: 2444 (1988); computerized implementations of these algorithms (including, but not limited to, CLUSTAL in the PC / Gene program by Intelligentics, Mountain View Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package of the Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA).The CLUSTAL program is well described in Higgins and Sharp, Gene, 73: 237-244 (1988) and Higgins and Sharp, CABIOS, 5: 151-153 (1989);Corpet et al., Nucleic Acids Res., 16: 10881-10890 (1988);Huang et al., Computer Applications in the Biosciences, 8: 155-165 (1992);and Pearson et al., Methods in Molecular Biology, 24: 307-331 (1994). Alignment is also often performed by visual inspection and manual alignment. In one class of embodiments, the polypeptides of the invention have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference polypeptide, or a fragment thereof, as measured, for example, by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, a nucleic acid can be described with reference to a starting nucleic acid, e.g., having 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% sequence identity to a reference nucleic acid or a fragment thereof, as measured, e.g., by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have a certain percentage of sequence identity to a larger molecule, it means that, when the two molecules are optimally aligned, said percentage of residues in the smaller molecule match with the residues of the larger molecule according to the order in which the two molecules are optimally aligned.

[0211] The term "substantially identical" and its grammatical equivalents, when applied to a nucleic acid or amino acid sequence, can mean that the nucleic acid or amino acid sequence has a sequence with at least 90% or more sequence identity, at least 95%, at least 98% and at least 99% sequence identity, when compared to a reference sequence using the above programs, such as BLAST, and using standard parameters. For example, the BLASTN program (nucleotide sequences) uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1992)). The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide sequence in the comparison window can include additions or deletions (i.e., gaps) compared to the reference sequence (without additions or deletions) in order to optimally align the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. In some embodiments, substantial identity exists over a region that is at least about 50 residues long of the sequence, over a region of at least about 100 residues, and in some embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, the sequences are substantially identical over the entire length of the coding region.

[0212] The term "vector" as used herein can refer to a construct that can deliver one or more genes or sequences of interest to host cells and usually express them in host cells.Examples of vectors include, but are not limited to, virus vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors that are combined with cationic condensing agents, and DNA or RNA expression vectors that are encapsulated in liposomes.

[0213] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition may be a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. An isolated polypeptide, antibody, polynucleotide, vector, cell, or composition includes those that have been purified to the extent that they are no longer in a form found in nature. In some embodiments, an isolated polypeptide, antibody, polynucleotide, vector, cell, or composition is substantially pure. For example, isolated peptides do not contain some or all of the materials that normally accompany the peptide in their in situ environment. For example, naturally occurring polynucleotides or peptides present in living animals are not isolated, whereas the same polynucleotides or peptides separated from some or all of the coexisting materials in a natural system are isolated. Such polynucleotides may be part of a vector, and / or such polynucleotides or peptides may be part of a composition, and still be "isolated" in that such vectors or compositions are not part of their natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and further include such molecules made synthetically.

[0214] The terms "polynucleotide", "nucleotide", "nucleic acid", "polynucleic acid" or "oligonucleotide" and their grammatical equivalents are used interchangeably herein and can refer to polymers of nucleotides of any length, including DNA and RNA, such as mRNA. Thus, these terms include double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA. Modified and unmodified forms of polynucleotides, for example, by methylation and / or capping, are also included. These terms are also intended to include molecules that include non-naturally occurring or synthetic nucleotides and nucleotide analogs. The nucleic acid sequences and vectors disclosed or contemplated herein can be introduced into cells, for example, by transfection, transformation, or transduction. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, the polynucleotides and nucleic acids can be in vitro transcribed mRNA. In some embodiments, the polynucleotide administered using the methods of the present disclosure is mRNA.

[0215] "Transfection", "transformation" or "transduction" as used herein may refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.Many transfection techniques are known in the art, including, for example, calcium phosphate DNA coprecipitation (see, for example, Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-assisted microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells by growing the infectious particles in suitable packaging cells, many of which are commercially available.

[0216] The term "subject" may refer to any animal (e.g., mammal), including but not limited to, humans, non-human primates, canines, felines, rodents, etc., that will be the recipient of a particular treatment. In general, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0217] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" may refer to an amount of a therapeutic agent effective to "treat" a disease or disorder in a subject or mammal. A therapeutically effective amount of a drug has a therapeutic effect and may thus prevent the onset of a disease or disorder, slow the onset of a disease or disorder, slow the progression of a disease or disorder, relieve to some extent one or more of the symptoms associated with a disease or disorder, reduce morbidity and mortality, improve quality of life, or a combination of such effects.

[0218] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" can refer to both (1) therapeutic measures that cure, slow, alleviate the symptoms, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to having the disorder; and those in whom the disorder is to be prevented.

[0219] "Pharmaceutically acceptable" may generally refer to a composition or component of a composition that is non-toxic, inert, and / or physiologically compatible.

[0220] A "pharmaceutical excipient" or "excipient" can include materials such as, for example, adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, etc. A "pharmaceutical excipient" is a pharmaceutical acceptable excipient.

[0221] A "tissue-specific" antigen may refer to an epitope sequence encoded by a gene that has a higher level of expression in target tissue than in non-target tissue. tissue specific antigen

[0222] Tissue-specific antigens may have great potential as targets for immunotherapy. In particular, tissue-specific antigens, compositions containing or producing tissue-specific antigens, and methods for identifying tissue-specific antigens are provided in the present invention. One advantage of targeting tissue-specific antigens for immunotherapy is that tissue-specific genes are generally very highly expressed in their given tissues, thereby enhancing the possibility that they are robustly presented. Such an approach may eliminate both tumors and corresponding healthy tissues of the same lineage. However, in many cases, this may be an acceptable trade-off. For example, CAR-T therapy that targets CD19 surface marker eliminates both healthy and leukemic B cells. Although the loss of normal B cells may impair immune function, patients can tolerate B cell loss.

[0223] In some embodiments, tissue-specific antigens are specific to non-essential tissues. Tissue-specific epitope sequences can be predicted to be presented on tumor cells or non-essential normal cells derived from the same lineage of non-essential tissues, and can be predicted to have zero or low expression levels in essential tissues. Thus, epitope sequence information of tissue-specific antigens, such as antigens specific to tumors derived from specific tissues, can be translated into therapeutic methods and compositions for diseases or conditions, such as cancer. In some embodiments, tissue-specific antigens presented herein can be expressed at high levels in tumor tissues originating from or present in non-essential tissues. In some embodiments, tissue-specific antigens may or may not be expressed in normal non-essential tissues, but can be expressed at relatively very low levels in essential tissues.

[0224] As provided herein, a tissue-specific antigen may refer to an epitope sequence encoded by a gene that has a higher expression level in a target tissue than in a non-target tissue, in which case the tissue-specific antigen may be referred to as "specific for the target tissue." In some embodiments, the target tissue specific antigen has an expression level in the target tissue that is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.1 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times, at least 3.2 times, at least 3.4 times, at least 3.5 times, at least 3.6 times, at least 3.8 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times , at least 15x, at least 16x, at least 17x, at least 18x, at least 19x, at least 20x, at least 22x, at least 24x, at least 25x, at least 26x, at least 28x, at least 30x, at least 35x, at least 40x, at least 45x, at least 50x, at least 55x, at least 60x, at least 70x, at least 80x, at least 90x, at least 100x, at least 120x, at least 140x, at least 150x, at least 160x, at least 180x, at least 200x, at least 250x, at least 300x, at least 400x, at least 500x, at least 600x, at least 700x, at least 800x, at least 900x, at least 1000x, at least 2000x, at least 4000x, at least 5000x, at least 10 4 times at least 10 5 times, or at least 10 6The epitope gene is derived from the epitope gene that is double-double. In some embodiments, the tissue-specific antigen can be specific to one particular type of tissue, for example, the tissue-specific antigen can be specific only to pancreatic tissue, cardiac tissue, prostate tissue, or epithelial tissue. In some embodiments, the tissue-specific antigen can be specific to more than one type of tissue, for example, the tissue-specific antigen can be specific to two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more different types of tissue. The criteria for setting "tissue specificity" can vary depending on the purpose of application of the subject matter presented herein. As will be discussed in detail, the subject matter presented herein can be applied to various situations in which different criteria for the selection of tissue-specific antigen can be utilized.

[0225] In some embodiments, the tissue-specific antigen is specific for a target tissue other than an essential tissue. In some embodiments, the target tissue is a non-essential tissue. As provided herein, an essential tissue may refer to a tissue in a living body whose vital functions cannot be replaced by internal or external support. As provided herein, a non-essential tissue may refer to a tissue in a living body whose vital functions can be replaced (e.g., the function of the tissue can be performed, at least in part, by some other tissue in the body or by a tissue transplant or artificial device) or can be dispensed with (e.g., the function of the tissue is not necessary for the survival of the body). In some embodiments, the essential tissue comprises brain or colon tissue. In some embodiments, the essential tissue comprises bone marrow. In some embodiments, the non-essential tissue comprises thyroid, pancreas, adrenal gland, fallopian tube, prostate, breast, ovary, or cervical tissue.

[0226] In some aspects, the present disclosure provides tissue-specific antigens, such as tissue-specific antigenic peptides.The tissue-specific antigens presented herein can comprise tumor epitope sequences.The tissue-specific antigens presented herein can comprise tumor epitope sequences derived from the tumor-expressed proteins presented herein.In some embodiments, the tumor-expressed proteins presented herein are specific to tumors derived from a certain type of tissue, for example, the tumor-expressed protein TSHR can be specific to thyroid cancer from thyroid tissue.

[0227] In some embodiments, the proteins expressed in the tumors presented herein are ACTL7A, ACTL7B, ACTL9, ACTRT2, ADAD1, AKAP4, ALPPL2, AMY2A, ANKRD30A, AQP12A, AQP12B, C2orf53, CCDC70, CELA2A, CELA2B, CETN1, CLDN6, COL10A1, CSAG1, CTCFL, CTRC, CYP11A1, CYP11B1, CYP11B2, DCAF4L2, DLL3, DMRTB1, EPYC, G6PC2, HMGB4, IAPP, KIF2B, KIRREL2, KLK2, KLK3, KLK4, LELP1, MAGEA11, MAGEA12, MAGEA2, MAGEA4, MAGEC2, MC2R, MMP13, PAGE5, PGK2, PNLIPRP1, POTEE, POTEG, POTEH, PPIAL4G, PRAME, PRDM7, PRM1, PRM2, RBPJL, RLN1, RSPH6A, SCXB, SERPINI2, SLC45A2, SPATA8, SSX1, STAR, SYCN, TG, TGM4, TNP1, TPD52L3, TSHR, TSPAN10, UBQLN3, or any combination thereof.

[0228] The tumor-expressed proteins presented herein may include TSHR, TG, RSPH6A, SCXB, SSX1, or any combination thereof, each of which may be specific for thyroid cancer. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 6846-7061, 7359-7448, 7629-8099, and 8619-8744, each of which may be specific for thyroid cancer. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 6846-7061, 7359-7448, 7629-8099, and 8619-8744, each of which may be specific for thyroid cancer.

[0229] The tumor-expressed proteins presented herein may include RBPJL, AQP12A, AQP12B, IAPP, CELA2A, CELA2B, AMY2A, CTRC, G6PC2, KIRREL2, PNLIPRP1, SERPINI2, SYNC, or any combination thereof, each of which may be specific to pancreatic cancer. The epitope sequences provided by the present invention may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 720-814, 989-1182, 1373-1565, 2120-2211, 2920-3009, 3101-3196, 3320-3440, 5193-5284, 6487-6579, 7062-7150, and 7539-7628, each of which may be specific to pancreatic cancer. The epitope sequences provided by the present invention may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 720-814, 989-1182, 1373-1565, 2120-2211, 2920-3009, 3101-3196, 3320-3440, 5193-5284, 6487-6579, 7062-7150, and 7539-7628, each of which may be specific to pancreatic cancer.

[0230] The tumor-expressed proteins presented herein may include CYP11A1, CYP11B1, CYP11B2, MC2R, STAR, or any combination thereof, each of which may be specific for adrenal cancer. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2212-2523, 4817-4915, and 7449-7538, each of which may be specific for adrenal cancer. The epitope sequences provided by the present invention may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2212-2523, 4817-4915, and 7449-7538, each of which may be specific to adrenal cancer.

[0231] The tumor-expressed proteins presented herein may include ALPPL2, POTEE, PRAME, or any combination thereof, each of which may be specific to uterine cancer. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 627-719, 5285-5431, and 6085-6183, each of which may be specific to uterine cancer. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 627-719, 5285-5431, and 6085-6183, each of which may be specific to uterine cancer.

[0232] The tumor-expressed proteins presented herein may include KLK2, KLK3, KLK4, POTEH, POTEG, TGM4, RLN1, POTEE, PPIAL4G, or any combination thereof, each of which may be specific to prostate cancer.The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to peptide sequences selected from the group consisting of SEQ ID NOs: 3441-4274, 5285-6084, 6580-6845, and 8100-8434, each of which may be specific to prostate cancer. The epitope sequences provided by the present invention may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 3441-4274, 5285-6084, 6580-6845, and 8100-8434, each of which may be specific to prostate cancer.

[0233] The tumor-expressed proteins presented herein may include ANKRD30A, COL10A1, or a combination, each of which may be specific to breast cancer. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 815-988, and 1749-1867, each of which may be specific to breast cancer. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 815-988, and 1749-1867, each of which may be specific to breast cancer.

[0234] The tumor-expressed proteins presented herein may include CTCFL, PRAME, CLDN6, EPYC, or any combination thereof, each of which may be specific to ovarian cancer. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1659-1748, 1964-2119, 2827-2919, and 6085-6183, each of which may be specific to ovarian cancer. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1659-1748, 1964-2119, 2827-2919, and 6085-6183, each of which may be specific to ovarian cancer.

[0235] The tumor-expressed proteins presented herein may include CTCFL, each of which may be specific to cervical cancer. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1964-2119, each of which may be specific to cervical cancer. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1964-2119, each of which may be specific to cervical cancer.

[0236] The tumor-expressed proteins presented herein may include POTEE, PPIAL4G, or a combination thereof, each of which may be specific to colorectal cancer. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 5285-5431, and 5996-6084, each of which may be specific to colorectal cancer. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 5285-5431, and 5996-6084, each of which may be specific to colorectal cancer.

[0237] The tumor-expressed proteins presented herein may include DLL3, each of which may be specific to glioma. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2619-2736, each of which may be specific to glioma. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2619-2736, each of which may be specific to glioma.

[0238] The tumor-expressed proteins presented herein may include MMP13, each of which may be specific to head and neck cancer. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 4916-5010, each of which may be specific to head and neck cancer. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 4916-5010, each of which may be specific to head and neck cancer.

[0239] The tumor-expressed proteins presented herein may include DCAF4L2, SSX1, or a combination thereof, each of which may be specific to liver cancer. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2524-2618, and 7359-7448, each of which may be specific to liver cancer. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 2524-2618, and 7359-7448, each of which may be specific to liver cancer.

[0240] The tumor-expressed proteins presented herein may include SSX1, MAGEA4, PRAME, CSAG1, MAGEA12, MAGEA2, MAGEC2, PAGE5, PRDM7, SLC45A2, TSPAN10, or any combination thereof, each of which may be specific to melanoma. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1868-1963, 4458-4550, 4551-4637, 4638-4728, 4729-4816, 5011-5100, 6085-6183, 6184-6307, 7151-7264, 7359-7448, and 8745-8835, each of which may be specific to melanoma. The epitope sequences provided by the present invention may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1868-1963, 4458-4550, 4551-4637, 4638-4728, 4729-4816, 5011-5100, 6085-6183, 6184-6307, 7151-7264, 7359-7448, and 8745-8835, each of which may be specific to melanoma.

[0241] The tumor-expressed proteins presented herein may include MAGEA11, MAGEA4, PRAME, or any combination thereof, each of which may be specific to lung squamous cell carcinoma. The epitope sequences provided herein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 4368-4457, 4638-4728, and 6085-6183, each of which may be specific to lung squamous cell carcinoma. The epitope sequences provided herein may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 4368-4457, 4638-4728, and 6085-6183, each of which may be specific to lung squamous cell carcinoma.

[0242] Tumor-expressed proteins presented herein may include ACTL7A, ACTL7B, ACTL9, ACTRT2, ADAD1, AKAP4, C2orf53, CCDC70, CETN1, DMRTB1, HMGB4, KIF2B, LELP1, PGK2, PRM1, PRM2, SPATA8, TNP1, TPD52L3, UBQLN3, or any combination thereof, each of which may be specific to testis cancer. The epitope sequences provided by the present invention may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1-626, 1183-1372, 1566-1658, 2737-2826, 3010-3100, 3197-3319, 4275-4367, 5101-5192, 6308-6486, 7265-7358, 8435-8618, and 8836-8962, each of which may be specific to testis cancer. The epitope sequences provided by the present invention may have at least 70% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1-626, 1183-1372, 1566-1658, 2737-2826, 3010-3100, 3197-3319, 4275-4367, 5101-5192, 6308-6486, 7265-7358, 8435-8618, and 8836-8962, each of which may be specific to testis cancer.

[0243] Table 1A provides a summary of a number of peptide sequences that are potential tissue-specific antigens, and also lists the HLA alleles that each peptide sequence is predicted to bind, as well as the cancer type for which each peptide sequence is specific.

[0244] Table 1B provides a summary of exemplary peptide sequences that may be tissue-specific antigens, and also lists the HLA alleles that each peptide sequence is predicted to bind, as well as the cancer type for which each peptide sequence is specific.

[0245] Table 1C provides a summary of exemplary peptide sequences from Table 1B that were verified by mass spectrometry to be presented by antigen-presenting cells. [Table 1A-1] [Table 1A-2] [Table 1A-3] [Table 1A-4] [Table 1A-5] [Table 1A-6] [Table 1A-7] [Table 1A-8] [Table 1A-9] [Table 1A-10] [Table 1A-11] [Table 1A-12] [Table 1A-13] [Table 1A-14] [Table 1A-15]

Table 1A-16

Table 1A-17

Table 1A-18

Table 1A-19

Table 1A-20

Table 1A-21

Table 1A-22

Table 1A-23

Table 1A-24

Table 1A-25

Table 1A-26

Table 1A-27

Table 1A-28

Table 1A-29

Table 1A-30

Table 1A-31

Table 1A-32

Table 1A-33

Table 1A-34

Table 1A-35

Table 1A-36

Table 1A-37

Table 1A-38

Table 1A-39

Table 1A-40

Table 1A-41

Table 1A-42

Table 1A-43

Table 1A-44

Table 1A-45

Table 1A-46

Table 1A-47

Table 1A-48

Table 1A-49

Table 1A-50

Table 1A-51

Table 1A-52

Table 1A-53

Table 1A-54

Table 1A-55

Table 1A-56

Table 1A-57

Table 1A-58

Table 1A-59

Table 1A-60

Table 1A-61

Table 1A-62

Table 1A-63

Table 1A-64

Table 1A-65

Table 1A-66

Table 1A-67

Table 1A-68

Table 1A-69

Table 1A-70

Table 1A-71

Table 1A-72

Table 1A-73

Table 1A-74

Table 1A-75

Table 1A-76

Table 1A-77

Table 1A-78

Table 1A-79

Table 1A-80

Table 1A-81

Table 1A-82

Table 1A-83

Table 1A-84

Table 1A-85

Table 1A-86

Table 1A-87

Table 1A-88

Table 1A-89

Table 1A-90

Table 1A-91

Table 1A-92

Table 1A-93

Table 1A-94

Table 1A-95

Table 1A-96

Table 1A-97

Table 1A-98

Table 1A-99

Table 1A-100

Table 1A-101

Table 1A-102

Table 1A-103

Table 1A-104

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Table 1A-114

Table 1A-115

[0246] In some aspects, the present invention provides compositions comprising tissue-specific antigens. In some embodiments, the compositions comprise antigenic peptides comprising tissue-specific antigens. In some embodiments, the tissue-specific antigen comprises tumor epitope sequence(s) presented herein. In some embodiments, the present invention also provides compositions comprising polynucleotides encoding tissue-specific antigens.

[0247] In some embodiments, the sizes of the antigenic peptides presented herein include, but are not limited to, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid molecule residues, and any range derivable therein.

[0248] In some embodiments, the antigenic peptide is equal to or less than 50 amino acids. In some embodiments, the antigenic peptide is equal to about 20 to about 30 amino acids. Longer peptides can be designed in several ways. For example, where the HLA binding regions are predicted or known, the longer peptides can consist of individual binding peptides with 0-10 amino acid extensions toward the N-terminus and C-terminus of each of the corresponding gene products. The longer peptides can also consist of a partial or full concatenation of binding peptides, each with a sequence extension.

[0249] Antigenic peptides and polypeptides can bind or be expected to bind to HLA protein.Antigenic peptides can have or be expected to have IC50 of less than about 1000nM, less than about 500nM, less than about 250nM, less than about 200nM, less than about 150nM, less than about 100nM, or less than about 50nM.In some embodiments, antigenic peptides do not induce autoimmune response and / or cause epidemiological tolerance when administered to a subject. Identification of tissue-specific antigens

[0250] In some aspects, the present disclosure provides a method for identifying a tissue-specific antigen. In some embodiments, the tissue-specific antigen may be a tumor tissue-specific epitope sequence.

[0251] In some embodiments, the methods provided herein include identifying an epitope sequence encoded by a tissue-specific antigen epitope gene that binds or is predicted to bind to a protein encoded by an MHC allele expressed by a human subject, and whose expression level in a tumor from a target tissue is at least twice the expression level of the tissue-specific antigen epitope gene in each of a plurality of non-target tissues distinct from the target tissue.

[0252] In some embodiments, the method presented herein comprises identifying an epitope gene whose expression level in target tissue is higher than that in non-target tissue.For example, the method can comprise identifying an epitope gene whose expression level in human pancreatic tissue is higher than that in human breast tissue, human lung tissue, or other human essential tissue.In some cases, the expression level in human pancreatic tissue can be at least twice as high as that in human breast tissue.In some embodiments, identifying an epitope gene whose expression level in target tissue is higher than that in non-target tissue comprises comparing the expression level of the epitope gene in target tissue with the expression level of the epitope gene in non-target tissue. Comparisons were made with TCGA (portal.gdc.cancer.gov / , last accessed September 2018), GTEX (gtexportal.org / home / , last accessed September 2018), GENT (medicalgenome.kribb.re.kr / GENT / , last accessed September 2018), The Human Protein Atlas (proteinatlas.org / , last accessed September 2018), Expression Atlas (ebi.ac.uk / gxa / home, last accessed September 2018), BioXpress (hive.biochemistry.gwu.edu / tools / bioxpress, last accessed September 2018), MERAV (merav.wi.mit.edu, last accessed September 2018), and Global Cancer This can be done by examining the expression levels of epitope genes profiled in accumulated datasets such as Global Cancer Map (globalcancermap.com / , last accessed September 2018), and CGAP (cgap.nci.nih.gov / , last accessed September 2018), at either the mRNA transcript or protein level or both.Alternatively, the comparison can be made by experimental methods for assessing gene expression levels, such as, for example, but not limited to, techniques for assessing mRNA transcript levels, such as real-time RT-PCR (real-time polymerase chain reaction), microarrays, Northern blots, ISH (in situ hybridization), and RNA-seq (RNA sequencing), as well as techniques for assessing protein expression levels, such as mass spectrometry, protein arrays, peptide arrays, immunostaining, and Western blots. Alternatively, the comparison can be made by 1) first examining the profiled expression levels in a pooled data set, such as those described above, and then experimentally verifying the expression levels in the tissue of interest.

[0253] In some embodiments, the method provided herein comprises identifying a tumor epitope gene whose expression level in a tumor derived from a target tissue is higher than the expression level in each of a plurality of non-target tissues different from the target tissue.For example, a prostate tumor is derived from prostate tissue, and the method provided herein can comprise identifying a tumor epitope gene whose expression level in a prostate tumor is higher than the expression level in each of a plurality of non-target tissues different from the prostate, such as, but not limited to, the brain, colon, lung, heart and bone marrow.

[0254] In some embodiments, the method presented herein comprises identifying a tumor epitope gene whose expression level in tumors derived from target tissue is higher than that in essential tissue.In some embodiments, the target tissue is a non-essential tissue.In some embodiments, the essential tissue comprises brain, colon, heart, bone marrow, or lung.In some embodiments, the non-essential tissue comprises thyroid, pancreas, adrenal, fallopian tube, prostate, breast, ovary, or cervix.

[0255] As presented herein, the tissue from which the tumor originates may be referred to as the target tissue, and other tissues, or in some cases essential tissues, may be referred to as off-target tissues. In some embodiments, the methods presented herein include identifying tissue-specific antigens based on absolute expression levels in target and off-target tissues. Expression levels may be evaluated in some cases by RNA-seq readings. In some cases, expression levels may be expressed in units such as "transcripts per million" (TPM), which may mean that a gene of interest has a certain number of mRNA transcripts over a million total mRNA transcripts in the tissue of interest. In some embodiments, TPM allows for partitioning of protein-coding mRNA transcripts and removing non-protein-coding genes from consideration. In some embodiments, the methods presented herein include identifying epitope sequences encoded by tumor epitope genes that have expression levels in target tissues of at least about 100 TPM and expression levels in off-target tissues of at most about 5 TPM. In some embodiments, the expression level of the epitope gene in the target tissue is at least 10 TPM, at least 20 TPM, at least 30 TPM, at least 40 TPM, at least 50 TPM, at least 60 TPM, at least 70 TPM, at least 80 TPM, at least 90 TPM, at least 100 TPM, at least 110 TPM, at least 120 TPM, at least 130 TPM, at least 140 TPM, at least 150 TPM, at least 200 TPM, at least 300 TPM, at least 400 TPM, at least 500 TPM, at least 600 TPM, at least 700 TPM, at least 800 TPM, at least 1000 TPM, at least 2000 TPM, at least 3000 TPM, at least 5000 TPM, at least 10 4In some embodiments, the expression level of the epitope gene in the off-target tissue may be at most 1000 TPM, at most 500 TPM, at most 100 TPM, at most 50 TPM, at most 20 TPM, at most 10 TPM, at most 9 TPM, at most 8 TPM, at most 7 TPM, at most 6 TPM, at most 5 TPM, at most 4 TPM, at most 3 TPM, at most 2 TPM, at most 1 TPM, at most 1 TPM, at most 2 ... up to 0.9 TPM, up to 0.8 TPM, up to 0.7 TPM, up to 0.6 TPM, up to 0.5 TPM, up to 0.4 TPM, up to 0.3 TPM, up to 0.2 TPM, up to 0.1 TPM, up to 0.050 TPM, up to 0.02 TPM, up to 0.010 TPM, up to 0.005 TPM, up to 0.002 TPM, up to 0.001 TPM, or less.

[0256] In some embodiments, the method comprises using a computer algorithm to screen the tissue-specific epitope genes presented herein. The computer algorithm can be constructed to access and search available databases that contain expression data of several genes in different types of tissues. The computer algorithm can also be constructed to extract and compare expression data provided from various databases to identify genes of interest, such as tissue-specific genes, such as tissue-specific tumor epitope genes. In some embodiments, the computer algorithm can be constructed to report and display the screening results so that they can be viewed, extracted, and / or further processed by other computer algorithms. For example, the computer algorithm presented herein can include different modules, among which there are one or more modules for identifying the tissue-specific genes presented herein, and there are also one or more modules for identifying epitope sequences from the identified tissue-specific genes.

[0257] In some embodiments, the method presented herein comprises identifying epitope sequences that may bind or be predicted to bind to proteins encoded by MHC alleles. In some embodiments, the MHC alleles are expressed by a human subject. In some embodiments, the identification of epitope sequences that may bind or be predicted to bind to proteins encoded by MHC alleles expressed by a human subject is based on MHC binding affinity prediction, for example, by one or more predictive algorithms. In some embodiments, the identification is based on experimental validation, as discussed below. In some embodiments, the identification is based on both algorithmic prediction and experimental validation. In some embodiments, computer algorithms applicable to the subject matter include, but are not limited to, evolutionary algorithms, algorithms based on artificial neural networks, algorithms involving ant colonies, hidden Markov models, support vector machines, and motif searches, and any combination thereof. The computer algorithms may be based on convolutional neural networks (artificial intelligence or deep learning). The algorithms applicable to the subject matter may be based on any suitable predictive model.Non-limiting exemplary affinity prediction programs, tools, or online resources can include NetMHC, NetMHCIIpan, SVRMHC, DeepMHC, BiodMHC, sNebula, MHCPred, EpiToolKit, FRED, NNAlign, ProPred, HLA-DR4Pred, EpiTOP, CTLPred, TEPITOPEpan, SMM-align, ICES, GPS-MBA, EpiJen, PREDIVAC, EpicCapo, Epitopemap, ARB, EpiDOCK, HLArestrictor, MULTIPRED, MHCcluster, IMS (Immunogenetic Management Software), PAAQD, MHC2Pred, TEpredict, TepiTool, MMBPred, MHCMIR, HLAV3D, MHCBench, FDR4, LIGAP, MHC, HLAPred, HLA, POPISK, BiodMHC, MultiRTA, and MHC-BPS.

[0258] In some embodiments, the method provided herein comprises identifying an epitope sequence that can bind or can be predicted to bind to a protein encoded by an MHC allele, and can be presented or can be predicted to be presented by an antigen presenting cell. In some embodiments, the MHC allele is expressed by a human subject. In some embodiments, the antigen presenting cell is a human antigen presenting cell. The identification of affinity binding with the MHC allele and presentation by APC can be based on a prediction algorithm, experimental validation, or both. Therapeutic Methods and Compositions

[0259] The present invention provides therapeutic compositions comprising peptides identified according to the methods disclosed herein or presented herein. The present invention also provides a method for producing anti-tumor immunity in a mammal, comprising administering to the mammal a polynucleic acid comprising a sequence encoding a peptide identified according to the methods described herein. The present invention also provides a method for producing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a peptide having a sequence of a peptide identified according to the methods described herein. The present invention also provides a method for producing anti-tumor immunity in a mammal, comprising administering to the mammal a cell comprising a peptide comprising a sequence of a peptide identified according to the methods described herein. The present invention also provides a method for producing anti-tumor immunity in a mammal, comprising administering to the mammal a cell comprising a polynucleic acid comprising a sequence encoding a peptide comprising a sequence of a peptide identified according to the methods described herein. In some embodiments, the cell presents the peptide as an HLA-peptide complex.

[0260] Provided herein is a therapeutic composition comprising a polynucleotide comprising a sequence encoding a peptide identified according to the methods disclosed herein or a peptide presented herein.Also provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject a polynucleic acid comprising a sequence encoding a peptide identified according to the methods disclosed herein or a peptide presented herein.

[0261] Provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of a peptide comprising a peptide sequence identified according to the methods described herein or a peptide sequence presented herein. Provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject a cell comprising a peptide comprising a peptide sequence identified according to the methods described herein or a peptide sequence presented herein. Provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject a cell comprising a polynucleic acid comprising a sequence encoding a peptide comprising a peptide sequence identified according to the methods described herein or a peptide sequence presented herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the method further comprises administering to the subject an immune checkpoint inhibitor.

[0262] In some embodiments, the present invention relates to therapeutic or pharmaceutical compositions, e.g., vaccine compositions, capable of generating a tissue-specific antigen response (e.g., a humoral or cell-mediated immune response). In some embodiments, the pharmaceutical composition comprises an antigen therapeutic agent described herein (e.g., a peptide, a polynucleotide, a TCR, a CAR, a cell containing a TCR or a CAR, a dendritic cell containing a polypeptide, a dendritic cell containing a polynucleotide, an antibody, etc.) that corresponds to a tissue-specific antigen identified herein.

[0263] In some embodiments, the pharmaceutical compositions provided herein comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one tissue-specific antigenic peptide sequence provided herein. In some embodiments, the T cell is prepared by incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample, and incubating at least one T cell of the biological sample with an APC that presents at least one tissue-specific antigenic peptide sequence.

[0264] One skilled in the art can select antigenic therapeutics, for example, by testing in vitro generation of T cells and their efficiency and overall presence, proliferation, affinity and expansion of a particular T cell for a particular peptide, and functionality of the T cells, for example, by analyzing IFN-γ production or tumor killing by the T cells. The most efficient peptides can then be combined into an immunogenic composition.

[0265] In some embodiments of the present invention, different antigenic peptides and / or polypeptides are selected, such that one pharmaceutical composition comprises antigenic peptides and / or polypeptides capable of associating with different MHC molecules, such as different MHC class I molecules. In some embodiments, the pharmaceutical composition comprises antigenic peptides and / or polypeptides capable of associating with the most frequently occurring MHC class I molecule. Thus, the immunogenic compositions described herein comprise different peptides capable of associating with at least two, at least three, or at least four MHC class I or class II molecules.

[0266] In some embodiments, the pharmaceutical compositions described herein are capable of generating a specific cytotoxic T cell response, a specific helper T cell response, or a B cell response.

[0267] In some embodiments, the pharmaceutical composition described herein may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided herein below. The polypeptide and / or polynucleotide in the composition may be associated with a carrier, such as a protein, or an antigen-presenting cell, such as a dendritic cell (DC), capable of presenting peptides to T cells or B cells. In further embodiments, DC-binding peptides are used as carriers to target antigenic peptides and polynucleotides encoding tissue-specific antigenic peptides to dendritic cells (Sioud et al. FASEB J 27: 3272-3283 (2013)).

[0268] In some embodiments, the antigenic polypeptides or polynucleotides of the present disclosure may be provided as antigen-presenting cells (e.g., dendritic cells) containing such polypeptides or polynucleotides. In other embodiments, such antigen-presenting cells are used to stimulate T cells for use in patients.

[0269] In some embodiments, the antigen-presenting cells are dendritic cells. In related embodiments, the dendritic cells are autologous dendritic cells pulsed with antigenic peptides or nucleic acids. The antigenic peptides can be any suitable peptides that generate appropriate T cell responses. T cell therapy using autologous dendritic cells pulsed with peptides derived from tumor-associated antigens is disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278. In some embodiments, the T cells are CTLs. In some embodiments, the T cells are HTLs.

[0270] Thus, one embodiment of the present invention provides a pharmaceutical composition comprising at least one antigen-presenting cell (e.g., dendritic cell) pulsed or loaded with one or more antigenic polypeptides or polynucleotides described herein. In some embodiments, such APCs are autologous (e.g., autologous dendritic cells). Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient can be loaded with antigenic peptides or polynucleotides ex vivo. In related embodiments, such APCs or PBMCs are injected back into the patient.

[0271] The polynucleotide of the present disclosure may be any suitable polynucleotide capable of transducing dendritic cells and thus resulting in the presentation of tissue-specific antigenic peptides and the induction of immunity. In some embodiments, the polynucleotide may be naked DNA that is taken up by passive loading into cells. In another embodiment, the polynucleotide is part of a delivery vehicle, such as a liposome, a virus-like particle, a plasmid, or an expression vector. In another embodiment, the polynucleotide is delivered by a vector-free delivery system, such as high-performance electroporation and high-speed cell transformation. In several embodiments, such antigen-presenting cells (APCs) (e.g., dendritic cells) or peripheral blood mononuclear cells (PBMCs) are used to stimulate T cells (e.g., autologous T cells). In related embodiments, the T cells are CTLs. In other related embodiments, the T cells are HTLs. Such T cells are then injected into a patient. In some embodiments, the CTLs are injected into a patient. In some embodiments, the HTLs are injected into a patient. In some embodiments, both the CTLs and the HTLs are injected into a patient. Administration of any of the therapeutic agents may be carried out simultaneously or sequentially in any order.

[0272] In some aspects, the present disclosure provides therapeutic compositions comprising immune cells, such as T cells that target tissue-specific antigens as presented herein, and methods for producing the compositions. In some embodiments, T cells are stimulated ex vivo with one or more of the antigens described herein. In some embodiments, T cells that are ex vivo induced to recognize and target tissue-specific antigens are infused into a patient. In some embodiments, the infused T cells are derived from the patient himself. In some embodiments, the infused T cells are derived from another subject.

[0273] In some aspects, the present disclosure provides therapeutic compositions comprising TCRs targeting tissue-specific antigens as presented herein, and methods of generating the compositions. The TCRs presented herein can recognize one or more specific antigens. For example, in some cases, TCRs can be engineered to be bispecific. In some cases, TCRs can specifically recognize one specific antigen. In some cases, TCRs can specifically recognize one specific antigen. In some embodiments, TCRs that recognize one or more tissue-specific antigens are identified a priori, for example, from healthy donors. In some embodiments, TCR(s) are knocked into T cells from a patient or other subject, for example, T cells are genetically modified to express TCR(s) that have been identified to recognize one or more tissue-specific antigens. In some embodiments, genetically modified T cells are infused into a patient.

[0274] In some aspects, the disclosure provides a method for discovering an epitope, e.g., a TCR that recognizes a tissue-specific antigen. In some embodiments, the method includes obtaining T cells from a donor and contacting the T cells with an antigenic peptide complexed with HLA of an APC from the donor. In some embodiments, the contacting step can induce proliferation of the T cells. In some embodiments, the method further includes determining the sequence of the TCR that recognizes the antigenic peptide. In some embodiments, the donor is known to have no or reduced immune tolerance to the tissue of origin of the antigenic peptide. Without wishing to be bound by any particular theory, a subject, e.g., a human, may normally develop immune tolerance to a protein or peptide encoded by almost all of the subject's normal genes (e.g., wild-type genes) in healthy somatic tissues. However, in some cases, when tissues of the same species are xenogeneic to the subject, the subject may have no or reduced immune tolerance to proteins or peptides normally expressed in such tissues. For example, human females may have low to no immune tolerance to human prostate-specific peptides (e.g., peptides specifically expressed in human prostates), and human males may have low to no immune tolerance to human ovarian-specific peptides (e.g., peptides specifically expressed in human ovaries).In some other cases, when the subject's immune system lacks the development of immune tolerance to one or more of its own tissues, the subject may also have low to no immune tolerance to peptides specifically expressed in one or more tissues.For example, a type I diabetes subject may have autoimmunity to pancreatic-specific peptides.

[0275] In some embodiments of the TCR discovery method provided herein, the donor is a female subject, and the antigenic peptide is specific to a tissue selected from the group consisting of bulbourethral gland, epididymis, penis, prostate, scrotum, seminal vesicle, and testis. In some embodiments, the donor is a female subject, and the antigenic peptide is specific to the prostate. In some embodiments, the donor is a male subject, and the antigenic peptide is specific to a tissue selected from the group consisting of Bartholin's gland, fallopian tube, ovary, Skene's gland, uterus, cervix, vagina, and any combination thereof. In some embodiments, the donor is a male subject, and the antigenic peptide is specific to the ovary. In some embodiments, the TCR discovered by contacting the prostate-specific antigenic peptide with T cells from a female subject can be used to treat prostate cancer. In some embodiments, the TCR discovered by contacting the ovary-specific antigenic peptide with T cells from a male subject can be used to treat ovarian cancer.

[0276] In some embodiments, the donor is a type 1 diabetic patient, and the antigen peptide is specific to pancreas. In some embodiments, the TCR discovered by contacting pancreas-specific antigen peptide with T cells from a type I diabetic subject can be used to treat pancreatic cancer. In some embodiments, the donor has a thyroid autoimmune condition, and the antigen peptide is specific to thyroid. In some embodiments, the TCR discovered by contacting thyroid-specific antigen peptide with T cells from a subject with a thyroid autoimmune condition can be used to treat thyroid cancer.

[0277] In some aspects, the present disclosure provides therapeutic compositions comprising antibodies or functional portions thereof targeting tissue-specific antigens presented herein, and methods of producing the compositions. The antibodies presented herein may recognize one or more specific antigens. In some cases, the antibodies described herein may specifically recognize one specific antigen. In some embodiments, the antibodies presented herein may be specifically used for specific binding to tissue-specific antigens expressed on the cell surface. In some embodiments, the antibodies presented herein may be specifically used for specific binding to tissue-specific antigens secreted outside of cells. In some embodiments, the antibodies may be isolated, recombinant, or purified for therapeutic compositions. The production of the antibodies or functional portions thereof may be performed by techniques available to those skilled in the art. In some embodiments, the antibodies may be produced by hybridomas or by such B-cell cultures. The antibodies may be collected and used, for example, for anti-cancer therapy. In some embodiments, the antibodies may be humanized before use to reduce side effects.

[0278] The pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment are intended for parenteral, topical, nasal, oral or local administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the compositions can be administered intratumorally. To induce a local immune response against the tumor, the compositions can be administered at the site of surgical resection. In some embodiments, compositions for parenteral administration are described herein that include a solution of antigenic peptides, and the immunogenic compositions are dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques or can be sterile filtered. The resulting aqueous solutions can be packaged as is for use, or lyophilized, and the lyophilized preparations can be combined with a sterile solution prior to administration. The compositions may contain, as necessary, pharma- ceutically acceptable auxiliary substances, such as, for example, pH adjusting and buffering agents, isotonicity adjusting agents, wetting agents, and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like, to approximate physiological conditions.

[0279] The concentration of the antigenic peptides and polynucleotides described herein in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1% by weight, usually at or about 2%, up to as much as 20%-50% or more, and is selected depending on fluid volumes, viscosities, etc., depending on the particular mode of administration selected.

[0280] The antigenic peptides and polynucleotides described herein can also be administered via liposomes, which target the peptide to specific cellular tissues, such as lymphatic tissues. Liposomes are also useful for increasing the half-life of peptides. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the peptide to be delivered is incorporated as part of the liposome, either alone or in conjunction with, for example, a molecule that binds to a receptor that is widespread among lymphatic cells, such as a monoclonal antibody that binds to the DEC205 antigen, or other therapeutic or immunogenic compositions. Thus, liposomes filled with the desired peptide or polynucleotide described herein can be directed to the site of lymphatic cells, where the selected therapeutic / immunogenic polypeptide / polynucleotide composition is then delivered by the liposome. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral, negatively charged phospholipids and sterols, such as cholesterol. The selection of lipid is generally guided by considering, for example, liposome size, acid lability and stability of liposome in bloodstream.A variety of methods for preparing liposome are available, as described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), U.S. Patent No. 4,235,871, U.S. Patent No. 4,501,728, U.S. Patent No. 4,837,028 and U.S. Patent No. 5,019,369.

[0281] For targeting to immune cells, antigenic polypeptides or polynucleotides are incorporated into liposomes directed against cell surface determinants of desired immune system cells. Liposomal suspensions containing peptides can be administered intravenously, topically, locally, etc., in doses that vary depending, among other things, on the mode of administration, the polypeptide or polynucleotide being delivered, and the stage of the disease being treated.

[0282] In some embodiments, antigenic polypeptides and polynucleotides are targeted to dendritic cells.In some embodiments, antigenic polypeptides and polynucleotides are targeted to dendritic cells using markers DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, TSLP receptor, Clec9a or CD1a.

[0283] For solid compositions, conventional or nanoparticulate non-toxic solid carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. For oral administration, a pharma- ceutically acceptable non-toxic composition is formed by incorporating any of the commonly used excipients, such as those previously listed carriers, and generally 10-95% of the active ingredient, i.e., one or more antigenic polypeptides or polynucleotides described herein, at a concentration of 25%-75%.

[0284] For aerosol administration, the antigenic polypeptide or polynucleotide can be supplied in finely divided form together with a surfactant and a propellant. Representative of such agents are esters or partial esters of fatty acids containing 6 to 22 carbon atoms, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, oleic acid, and the like, with aliphatic polyhydric alcohols or their cyclic anhydrides. Mixed esters, such as mixed or natural glycerides, can be used. The surfactant can comprise 0.1% to 20% by weight of the composition, or 0.25 to 5%. The remainder of the composition can be a propellant. A carrier can also be included as desired, as in the case of, for example, lecithin for intranasal delivery.

[0285] Additional methods for delivering the antigenic polynucleotides described herein are also known in the art. For example, nucleic acid can be delivered directly as "naked DNA". This approach is described, for example, in Wolff et al., Science 247: 1465-1468 (1990) and U.S. Patent Nos. 5,580,859 and 5,589,466. Nucleic acid can also be administered using ballistic delivery, for example, as described in U.S. Patent No. 5,204,253. Particles consisting of DNA alone can be administered. Alternatively, DNA can be attached to particles, such as gold particles.

[0286] For treatment or immunization, mRNA encoding antigenic peptide or peptide-binding agent can also be administered to the patient. In some embodiments, the mRNA is a self-amplifying RNA. In further embodiments, the self-amplifying RNA is part of a synthetic lipid nanoparticle formulation (Geall et al., Proc Natl Acad Sci US A. 109: 14604-14609 (2012)).

[0287] Nucleic acid can also be delivered in a complex with cationic compounds, such as cationic lipids.Lipid-mediated gene delivery methods are described, for example, in WO96 / 18372, WO93 / 24640;Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988);U.S. Patent No. 5,279,833;WO91 / 06309;and Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).

[0288] The antigenic peptides and polypeptides described herein can also be expressed by attenuated viruses such as vaccinia or fowlpox. This approach involves using vaccinia virus as a vector to express nucleotide sequences encoding the peptides described herein. When introduced into an acutely or chronically infected or uninfected host, the recombinant vaccinia virus expresses the immunogenic peptides, thereby eliciting a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351: 456-460 (1991)). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides described herein will be apparent to those skilled in the art from the description herein.

[0289] Adjuvants are any substances that are mixed into pharmaceutical compositions to enhance or otherwise modify the immune response to therapeutic agents. Carriers are scaffolds, such as polypeptides or polysaccharides, that can bind tissue-specific antigenic polypeptides or polynucleotides. Optionally, adjuvants are covalently or non-covalently conjugated to the polypeptides or polynucleotides described herein.

[0290] The ability of adjuvants to increase immune response to antigens is generally manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms.For example, the increase in humoral immunity can be manifested by a significant increase in the titer of antibodies against antigens, and the increase in T cell activity can be manifested by an increase in cell proliferation, or cytotoxicity, or cytokine secretion.Adjuvants can also modify immune response, for example, by changing a predominantly humoral or T helper 2 response to a predominantly cellular or T helper 1 response.

[0291] Suitable adjuvants are known in the art (see WO2015 / 095811) and include, but are not limited to, poly(I:C), poly-ICLC, STING agonist, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide Adjuvants include ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulon derived from saponin (Aquila Biotech, Worcester, Mass., USA), mycobacterial extracts and synthetic bacterial cell wall mimics, as well as other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Adjuvants also include incomplete Freund's or GM-CSF. Some immunological adjuvants specific for dendritic cells (e.g., MF59) and their preparation have been previously described (Dupuis M, et al., Cell Immunol. 1998; 186 (1): 18-27; Allison AC; Dev Biol Stand. 1998; 92: 3-11) (Mosca et al. Frontiers in Bioscience, 2007; 12: 4050-4060) (Gamvrellis et al. Immunol & Cell Biol. 2004; 82: 506-516). Cytokines can also be used.Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1b, IL-4, IL-6, and CD40L) (U.S. Pat. No. 5,849,589, incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996 (6): 414-418).

[0292] It has also been reported that CpG immunostimulatory oligonucleotides enhance the effect of adjuvants in the context of vaccines. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cell immunogenic pharmaceutical compositions, and polysaccharide conjugates in both prophylactic and therapeutic immunogenic pharmaceutical compositions. Importantly, this enhances dendritic cell maturation and differentiation, resulting in enhanced activation of TH1 cells and potent cytotoxic T lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides show much greater adjuvant activity when formulated or co-administered with other adjuvants or as preparations such as microparticles, nanoparticles, lipid emulsions or similar preparations, which is necessary to induce strong responses, especially when antigens are relatively weak.CpG oligonucleotides can also accelerate immune responses, reduce the dose of antigen, and in some experiments, accompany antibody responses equivalent to the full dose of immunogenic pharmaceutical compositions without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484).US Patent No. 6,406,705 B1 describes the use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens in combination to induce antigen-specific immune responses. A commercially available CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) from Mologen (Berlin, Germany), which is a component of the pharmaceutical compositions described herein.Other TLR binding molecules, for example RNA binding TLR7, TLR8 and / or TLR9, can also be used.

[0293] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly ICLC, poly (I:C) (e.g., poly i:CI2U), non-CpG bacterial DNA or RNA, ssRNA40 against TLR8, and immunologically active small molecules and antibodies that may act therapeutically and / or as adjuvants, such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, AZD2171, ipilimumab, tremelimumab, and SC58175. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of skill in the art without undue experimentation. Additional adjuvants include colony stimulating factors such as granulocyte-macrophage colony stimulating factor (GM-CSF, sargramostim).

[0294] In some embodiments, the pharmaceutical composition according to the present invention comprises more than one different adjuvant. Moreover, the present invention encompasses therapeutic compositions comprising any adjuvant material, including any of the above or a combination thereof. Antigenic therapeutics (e.g., humoral or cell-mediated immune response) are also contemplated. In some embodiments, the pharmaceutical composition comprises a tissue-specific antigen therapeutic (e.g., peptides, polynucleotides, TCRs, CARs, cells containing TCRs or CARs, dendritic cells containing polypeptides, dendritic cells containing polynucleotides, antibodies, etc.), and the adjuvant can be administered separately and in any suitable order.

[0295] Carriers may exist independent of adjuvants. The function of a carrier may be to increase the molecular weight of certain mutants, for example, to increase their activity or immunogenicity, to confer stability, to increase biological activity, or to extend serum half-life. In addition, a carrier may assist in presenting peptides to T cells. A carrier may be any suitable carrier known to those skilled in the art, for example, a protein or an antigen-presenting cell. Carrier proteins may be, but are not limited to, keyhole limpet hemocyanin, serum proteins, such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin, or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. In some embodiments, the carrier comprises a human fibronectin type III domain (Koide et al. Methods Enzymol. 2012; 503: 135-56). For human immunization, the carrier must be a physiologically acceptable carrier that is acceptable and safe for humans. However, in some embodiments of the invention, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier may be a dextran, such as sepharose.

[0296] In some embodiments, instead of coupling the polypeptide to a carrier to increase immunogenicity, the polypeptide can be synthesized as a multiply linked peptide, such molecules are also known as multiple antigenic peptides (MAPS).

[0297] The tissue-specific antigens described herein that induce an immune response can be combined with an acceptable carrier or excipient and used as compositions that are useful for in vitro or in vivo analysis or for administration to a subject in vivo or ex vivo to treat the subject with a disease.

[0298] Thus, in addition to the active ingredient, a pharmaceutical composition may contain pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials may depend on the route of administration.

[0299] Pharmaceutical formulations containing a protein of interest, e.g., a tissue-specific antigen as described herein, can be prepared for storage by mixing the antigen having the desired degree of purity with physiologically acceptable carriers, excipients or stabilizers as required, and providing it in the form of a lyophilized formulation or aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Oslo, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers, such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin. hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0300] Acceptable carriers are physiologically acceptable to the patient receiving the administration and retain the therapeutic properties of the compound administered with / in the carrier. Acceptable carriers and their formulations are generally described, for example, in Remington's Pharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA 1990). One exemplary carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is involved in carrying or transporting the subject compound from the administration site in one organ or body part to another organ or body part, or in an in vitro assay system. Acceptable carriers are compatible with other ingredients of the formulation and are not harmful to the subject receiving the administration. Acceptable carriers should also not alter the specific activity of tissue-specific antigens.

[0301] In one aspect, the present invention provides pharma- ceutically or physiologically acceptable compositions, including solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic agents, and absorption enhancers or retarders that are suitable for pharmaceutical administration. Thus, pharmaceutical compositions or pharmaceutical preparations refer to compositions that are suitable for use as medicines in subjects. Pharmaceutical compositions and preparations include an amount of tissue-specific antigens (or polynucleotides encoding tissue-specific antigens) presented herein and a pharma-ceutically or physiologically acceptable carrier. The compositions can be formulated to be compatible with a particular route of administration (i.e., systemic or local). Thus, the compositions include carriers, diluents, or excipients that are suitable for administration by various routes.

[0302] In some embodiments, the composition further comprises an acceptable additive to improve the stability of the tissue-specific antigen in the composition and / or to control the release rate of the composition. The acceptable additive does not modify the specific activity of the tissue-specific antigen. Exemplary acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. The acceptable additive can be combined with an acceptable carrier and / or excipient, such as dextrose. Alternatively, exemplary acceptable additives include, but are not limited to, surfactants, such as polysorbate 20 or polysorbate 80, to increase the stability of the peptide and to reduce gelling of the solution. The surfactant can be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0303] The pharmaceutical composition can be administered, for example, by injection. Compositions for injection include aqueous solutions (if water soluble) or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, polyalcohols such as sugars, mannitol, sorbitol, and sodium chloride, can be included in the composition. The resulting solution can be packaged as is for use, or can be lyophilized. The lyophilized preparation can later be combined with a sterile solution before administration. For intravenous injection or injection into the painful area, the active ingredient is made into the form of parenterally acceptable aqueous solution, which is pyrogen-free and has appropriate pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions well, for example, using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as necessary. Sterile injections can be prepared by incorporating the required amount of active ingredient into a suitable solvent with one or a combination of ingredients listed above, and then sterilizing by filtration as required. In general, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders for preparing sterile injections, the preferred preparation method is vacuum drying and freeze-drying, which obtains a powder of the active ingredient and any additional desired ingredients from its solution that has been previously sterilized and filtered.

[0304] The composition can be conventionally administered intravenously, for example, by injection of a unit dose.For injection, the active ingredient can be in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has suitable pH, isotonicity and stability.For example, suitable solutions can be prepared using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as necessary.Furthermore, the composition can be administered by aerosolization.

[0305] In some embodiments, the composition is freeze-dried, for example, to extend shelf life under storage.When considering the composition for use in medicine or any of the methods presented herein, it is intended that the composition is substantially free of pyrogens, and therefore, when administered to human patients, the composition does not cause inflammatory reaction or unsafe allergic reaction.Testing the composition for pyrogens and preparing the composition that is substantially free of pyrogens are well understood by those skilled in the art, and can be achieved using commercially available kits.

[0306] Acceptable carriers may contain compounds that stabilize, increase or delay absorption, or increase or delay clearance. Such compounds include, for example, carbohydrates, such as glucose, sucrose, or dextran; low molecular weight proteins; compositions that reduce peptide clearance or hydrolysis; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Surfactants can also be used to stabilize or increase or decrease the absorption of pharmaceutical compositions, including liposomal carriers. To protect compounds from digestion, compounds can be complexed with compositions that provide resistance to acid and enzyme hydrolysis, or compounds can be complexed with suitable resistant carriers, such as liposomes.

[0307] The composition can be administered in a therapeutically effective amount in a manner compatible with the dosage form.The amount administered can depend on the subject to be treated, the ability of the subject's immune system to utilize the active ingredient, and the degree of binding capacity desired.The exact amount of active ingredient that needs to be administered depends on the judgment of the practitioner and is specific to each individual.The appropriate regime for initial administration and booster vaccination can also vary, but is typified by an initial administration, followed by repeated doses by subsequent injections or other administrations at one or more time intervals.Alternatively, continuous intravenous infusion sufficient to maintain blood concentration is contemplated.

[0308] The peptide-based immunogenic pharmaceutical composition can be formulated using any of the well-known techniques, carriers, and excipients, as appropriate and understood in the art.The polypeptide can be a cocktail of multiple polypeptides containing the same sequence, or a cocktail of multiple copies of different polypeptides.The peptide can be modified, for example, by lipidation or attachment to a carrier protein.Lipidation can be the covalent attachment of lipid groups to the polypeptide.Lipidated peptides or lipidated polypeptides can stabilize the structure and enhance the efficacy of treatment.

[0309] Lipid addition can be classified into several different types, such as N-myristoylation, palmitoylation, GPI-anchor addition, prenylation, and several additional types of modification. N-myristoylation is the covalent attachment of myristic acid, a C14 saturated acid, to glycine residues. Palmitoylation is the thioester linkage of a long-chain fatty acid (C16) to a cysteine ​​residue. GPI-anchor addition is the glycosyl-phosphatidylinositol (GPI) linkage via an amide bond. Prenylation is the thioether linkage of an isoprenoid lipid (e.g., farnesyl (C-15), geranylgeranyl (C-20)) to a cysteine ​​residue. Additional types of modification can include the attachment of S-diacylglycerol through the sulfur atom of cysteine, O-octanoyl conjugation through serine or threonine residues, S-archeol conjugation to cysteine ​​residues, and cholesterol attachment.

[0310] Fatty acids for generating lipidated peptides can include C2-C30 saturated, monounsaturated, or polyunsaturated fatty acyl groups. Exemplary fatty acids can include palmitoyl, myristoyl, stearoyl, and decanoyl groups. In some cases, lipid moieties with adjuvant properties are attached to the polypeptide of interest to elicit or enhance immunogenicity in the absence of exogenous adjuvants. The lipidated peptide or lipopeptide can be referred to as a self-adjuvanting lipopeptide. Any of the fatty acids described above and elsewhere herein can elicit or enhance the immunogenicity of the polypeptide of interest. Fatty acids that can elicit or enhance immunogenicity can include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, and decanoyl groups.

[0311] A polypeptide, such as a naked peptide or a lipidated peptide, can be incorporated into a liposome. Sometimes, a lipidated peptide can be incorporated into a liposome. For example, the lipid portion of the lipidated peptide can be spontaneously incorporated into the lipid bilayer of the liposome. Thus, the lipopeptide can be presented on the "surface" of the liposome.

[0312] Exemplary liposomes suitable for incorporation into the formulation include, but are not limited to, multilamellar vesicles (MLVs), oligolamellar vesicles (OLVs), unilamellar vesicles (UVs), small unilamellar vesicles (SUVs), medium unilamellar vesicles (MUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), multivesicular vesicles (MVVs), single or oligolamellar vesicles produced by reverse phase evaporation (REVs), multilamellar vesicles produced by reverse phase evaporation (MLV-REVs), stable plurilamellar vesicles (SPLVs), freeze-thawed MLVs (FATMLVs), vesicles prepared by extrusion (VETs), vesicles prepared by French press (FPVs), vesicles prepared by fusion (FUVs), dehydrated-rehydrated vesicles (DRVs), and bubblesomes (BSVs).

[0313] Depending on the method of preparation, liposomes can be unilamellar or multilamellar and can vary in size with diameters ranging from about 0.02 μm to more than about 10 μm. Many types of cells can be adsorbed to the liposomes, which can then release the incorporated agent (e.g., peptides described herein). In some cases, the liposomes fuse with the target cell, which then allows the contents of the liposome to enter the target cell. The liposomes can be taken up by endocytosis by phagocytic cells. After endocytosis, the liposomal lipids are degraded intralysosomally, releasing the encapsulated agent.

[0314] The liposomes presented herein may also include a carrier lipid. In some embodiments, the carrier lipid is a phospholipid. Carrier lipids capable of forming liposomes include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Other suitable phospholipids include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidyglycerol (DPPA); dimyristoylphosphatidylic acid (DMPA), distearoylphosphatidylcholine (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylic acid (DPPA); dimyristoylphosphatidylic acid (DMPA), distearoylphosphatidylcholine (DSPG), dimyristoylphosphatidylcholine ... The liposome further comprises phosphatidylserine (DSPA), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidyethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), or the like, or combinations thereof. In some embodiments, the liposome further comprises a sterol (e.g., cholesterol) that modulates liposome formation. The carrier lipid can be any known non-phosphorylated polar lipid.

[0315] The pharmaceutical composition can be encapsulated in liposomes using well-known techniques.Biodegradable microspheres can also be used as carriers for the pharmaceutical composition of the present invention.

[0316] Pharmaceutical compositions can be administered as liposomes or microspheres (or microparticles).The methods for preparing liposomes and microspheres for administration to patients are well known to those skilled in the art.Essentially, material is dissolved in an aqueous solution, appropriate phospholipids and lipids are added, together with surfactants if necessary, and the material is dialyzed or sonicated as necessary.

[0317] Microspheres formed of polymers or proteins are well known to those of skill in the art and are adapted to pass through the gastrointestinal tract directly into the bloodstream. Alternatively, compounds can be incorporated into microspheres or composites of microspheres and implanted for slow release over a period of days to months.

[0318] Polypeptides can also be attached to carrier proteins for delivery. Carrier proteins can be immunogenic carrier elements and can be attached by any recombinant technique. Exemplary carrier proteins include marine cultured keyhole limpet hemocyanin (mcKLH), PEGylated mcKLH, Blue Carrier*Protein, bovine serum albumin (BSA), cationized BSA, ovalbumin, and bacterial proteins such as tetanus toxoid (TT).

[0319] Polypeptides can also be prepared as multiple antigenic peptides (MAPs). Peptides can be attached to the N-terminus or C-terminus of a small non-immunogenic core. Building peptides onto the core in this way can result in a highly localized peptide density. The core can be a dendritic core residue or a matrix composed of bifunctional units. Core molecules suitable for constructing MAPs can include ammonia, ethylenediamine, aspartic acid, glutamic acid, and lysine. For example, a lysine core molecule can have two additional lysines attached by peptide bonds through each of the amino groups.

[0320] Polypeptides can be chemically synthesized or recombinantly expressed in cellular or cell-free systems. Peptides can be synthesized, for example, by liquid phase synthesis, solid phase synthesis, or microwave-assisted peptide synthesis. Polypeptides can be modified by, for example, acylation, alkylation, amidation, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation, glycosylation, malonylation, hydroxylation, iodination, nucleotide addition (e.g., ADP-ribosylation), oxidation, phosphorylation, adenylylation, propionylation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, glycosylation, palmitoylation, myristoylation, isoprenylation, or prenylation (e.g., farnesylation or The polypeptide can be modified by, for example, geranylgeranylation), glycosylphosphatidylinositol addition, lipoylation, attachment of a flavin moiety (e.g., FMN or FAD), attachment of heme C, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol attachment, hypusine formation, biotinylation, PEGylation, ISGylation, SUMUylation, ubiquitinylation, NEDDylation, Pupylation, citrullination, deamidation, eliminylation, carbamylation, or a combination thereof.

[0321] After the polypeptide is produced, it can be subjected to one or more rounds of purification steps to remove impurities. The purification step can be, for example, a chromatography step using a separation method such as affinity-based, size-exclusion-based, ion-exchange-based, etc. In some cases, the polypeptide is up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or free of impurities. In some cases, the polypeptide is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or free of impurities.

[0322] Polypeptides may contain natural amino acids, unnatural amino acids, or combinations thereof. Amino acid residues may refer to molecules that contain both amino and carboxyl groups. Suitable amino acids include, without limitation, both D- and L-isomers of naturally occurring amino acids, as well as unnatural amino acids prepared by organic synthesis or other metabolic pathways. The term amino acid, as used herein, includes, without limitation, α-amino acids, natural amino acids, unnatural amino acids, and amino acid analogs.

[0323] The term "α-amino acid" can refer to a molecule that contains both an amino group and a carboxyl group bonded to a carbon referred to as the α-carbon.

[0324] The term "β-amino acid" may refer to a molecule that contains both an amino group and a carboxyl group in the β configuration.

[0325] A "naturally occurring amino acid" can refer to any one of the twenty amino acids commonly found in naturally synthesized peptides and are known by the one-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. A table summarizing the properties of the natural amino acids can be found, for example, in U.S. Patent Application Publication No. 20130123169, which is incorporated herein by reference.

[0326] The peptides presented herein may contain one or more hydrophobic, polar, or charged amino acids. "Hydrophobic amino acids" include small hydrophobic amino acids and large hydrophobic amino acids. "Small hydrophobic amino acids" may be glycine, alanine, proline, and analogs thereof. "Large hydrophobic amino acids" may be valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof. "Polar amino acids" may be serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof. "Charged amino acids" may be lysine, arginine, histidine, aspartic acid, glutamic acid, and analogs thereof.

[0327] The peptides presented herein may contain one or more amino acid analogs. An "amino acid analog" may be a molecule that is structurally similar to an amino acid and can replace the amino acid in forming a peptidomimetic macrocycle. Amino acid analogs include, but are not limited to, β-amino acids and amino acids in which amino or carboxy groups are replaced with similarly reactive groups (e.g., primary amines are replaced with secondary or tertiary amines, or carboxy groups are replaced with esters).

[0328] The peptides presented herein may contain one or more unnatural amino acids. An "unnatural amino acid" may be an amino acid that is not one of the 20 amino acids commonly found in naturally synthesized peptides and known by the one-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. Unnatural amino acids or amino acid analogs include, for example, the structures disclosed in US Patent Publication No. 20130123169, which is incorporated herein by reference.

[0329] The amino acid analogs may include β-amino acid analogs. Examples of β-amino acid analogs and analogs of alanine, valine, glycine, leucine, arginine, lysine, aspartic acid, glutamic acid, cysteine, methionine, phenylalanine, tyrosine, proline, serine, threonine, and tryptophan may include, for example, structures disclosed in U.S. Patent Publication No. 20130123169, which is incorporated herein by reference.

[0330] The amino acid analogs may be racemic. In some cases, the D-isomers of the amino acid analogs are used. In some cases, the L-isomers of the amino acid analogs are used. In some cases, the amino acid analogs contain a chiral center in the R or S configuration. Sometimes, the amino group(s) of the β-amino acid analogs are replaced by a protecting group, such as tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, etc. Sometimes, the carboxylic acid function of the β-amino acid analogs is protected, for example, as its ester derivative. In some cases, a salt of the amino acid analogs is used.

[0331] A "non-essential" amino acid residue can be a residue that can be altered from the wild-type sequence of a polypeptide without eliminating or substantially altering an essential biological or biochemical activity of the polypeptide (e.g., receptor binding or activation). An "essential" amino acid residue can be a residue that, when altered from the wild-type sequence of the polypeptide, results in eliminating or substantially eliminating an essential biological or biochemical activity of the polypeptide.

[0332] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families can include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), non-polar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H). Thus, for example, a predicted non-essential amino acid residue in a polypeptide can be replaced with another amino acid residue from the same side chain family. Other examples of permissible substitutions can be based on isosteric considerations (e.g., replacement of methionine with norleucine) or on other properties (e.g., replacement of phenylalanine with 2-thienylalanine, or replacement of tryptophan with 6-Cl-tryptophan).

[0333] Nucleic acid-based immunogenic pharmaceutical compositions can also be administered to a subject. Nucleic acid-based immunogenic pharmaceutical compositions can be formulated using any of the well-known techniques, carriers, and excipients, as appropriate and understood in the art. The nucleic acid can be DNA, genomic DNA or cDNA, RNA, or hybrid, and the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acid can be obtained by chemical synthesis methods or by recombinant methods. The immunogenic pharmaceutical composition can be a DNA-based immunogenic pharmaceutical composition, an RNA-based immunogenic pharmaceutical composition, a hybrid DNA / RNA-based immunogenic pharmaceutical composition, or a hybrid nucleic acid / peptide-based immunogenic pharmaceutical composition. The peptide can be a peptide derived from a peptide of Table 1A, Table IB, Table 1C or Table 2, a peptide having a sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more identical to a peptide of Table 1A, Table IB, Table 1C or Table 2, or a peptide having a sequence that is at most 40%, 50%, 60%, 70%, 80%, 90%, 95% or less identical to a peptide of Table 1A, Table IB, Table 1C or Table 2.

[0334] The nucleic acids described herein may contain phosphodiester bonds, but in some cases, as outlined below (e.g., for the construction of primers and probes, such as labeled probes), include nucleic acid analogs that may have alternative backbones, including, for example, phosphoramide, phosphorothioate, O-methyl phosphoramidite linkages, and peptide nucleic acid backbones and linkages. Other nucleic acid analogs include locked nucleic acids, which contain bicyclic structures with positive backbones and non-ribose backbones. Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids. Locked nucleic acids (LNAs) are also included in the definition of nucleic acid analogs. LNAs are a class of nucleic acid analogs in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4'-C atom. These modifications of the ribose phosphate backbone can be made to increase the stability and half-life of such molecules in physiological environments. For example, PNA:DNA and LNA-DNA hybrids may exhibit higher stability and therefore can be used in some embodiments. Nucleic acids can be single-stranded or double-stranded as specified, or contain portions of double-stranded or single-stranded sequences. Depending on the application, nucleic acids can be DNA (including, for example, genomic DNA, mitochondrial DNA, and cDNA), RNA (including, for example, mRNA and rRNA), or hybrids in which the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthan gum, hypoxanthan gum, isocytosine, isoguanine, and the like.

[0335] The nucleic acid-based immunogenic pharmaceutical composition may be in the form of a vector. The vector may be a circular plasmid or a linear nucleic acid. The circular plasmid or linear nucleic acid may be capable of directing the expression of a particular nucleotide sequence in the appropriate subject's cells. The vector may have a promoter operably linked to the nucleotide sequence encoding the polypeptide, which may be operably linked to a termination signal. The vector may contain sequences necessary for the proper translation of the nucleotide sequence. The vector containing the nucleotide sequence of interest may be chimeric, that is, at least one of its components may be heterologous to at least one of the other components. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter that may initiate transcription only when the host cell is exposed to some specific internal or external stimulus.

[0336] The vector may be a plasmid, which may be useful for transfecting cells with a nucleic acid encoding a polypeptide, and the transformed host cells may be cultured and maintained under conditions where expression of the polypeptide occurs.

[0337] Plasmids may contain nucleic acid sequences that code for one or more of the various polypeptides disclosed herein.A single plasmid may contain the coding sequence for a single polypeptide, or the coding sequence for more than one polypeptide.Sometimes, plasmids may further contain coding sequences that code for adjuvants, such as immunostimulatory molecules, such as cytokines.

[0338] The plasmid may further comprise a start codon that may be upstream of the coding sequence, and a stop codon that may be downstream of the coding sequence. The start and stop codons may be in frame with the coding sequence. The plasmid may also comprise a promoter operably linked to the coding sequence, and an enhancer upstream of the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as that derived from CMV, FMDV, RSV or EBV.

[0339] The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and generate multiple copies of the plasmid in the cell. The plasmid may also contain a regulatory sequence that can be fully adapted for gene expression in the cell to which the plasmid is administered. The coding sequence may contain codons that can allow more efficient transcription of the coding sequence in the host cell.

[0340] The nucleic acid-based immunogenic pharmaceutical composition may be a linear nucleic acid immunogenic pharmaceutical composition or a linear expression cassette that can be efficiently delivered to a subject by electroporation and express one or more polypeptides disclosed herein.

[0341] Cell-based immunogenic pharmaceutical compositions can also be administered to subjects.For example, antigen-presenting cell (APC)-based immunogenic pharmaceutical compositions can be formulated using any of well-known techniques, carriers and excipients, as appropriate and understood in the art.APCs include monocytes, monocyte-derived cells, macrophages and dendritic cells.Sometimes, APC-based immunogenic pharmaceutical compositions can be dendritic cell-based immunogenic pharmaceutical compositions.

[0342] Dendritic cell-based immunogenic pharmaceutical compositions can be prepared by any method known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared by ex vivo or in vivo methods. Ex vivo methods can include using autologous DCs pulsed ex vivo with a polypeptide described herein to activate or load DCs before administration to a patient. In vivo methods can include using an antibody coupled with a polypeptide described herein to target a specific DC receptor. DC-based immunogenic pharmaceutical compositions can further include DC activators, such as TLR3, TLR-7-8, and CD40 agonists. DC-based immunogenic pharmaceutical compositions can further include an adjuvant and a pharmaceutically acceptable carrier.

[0343] Adjuvants can be used to enhance the immune response (humoral and / or cellular) elicited in patients receiving the immunogenic pharmaceutical composition. Sometimes, adjuvants can elicit a Th1-type response. Other times, adjuvants can elicit a Th2-type response. A Th1-type response can be characterized by the production of cytokines such as IFN-γ, whereas a Th2-type response can be characterized by the production of cytokines such as IL-4, IL-5 and IL-10.

[0344] In some embodiments, lipid-based adjuvants, such as MPLA and MDP, can be used with the immunogenic pharmaceutical compositions disclosed herein.For example, monophosphoryl lipid A (MPLA) is an adjuvant that induces the presentation of liposomal antigens to specific T lymphocytes.In addition, muramyl dipeptide (MDP) can also be used as a suitable adjuvant in conjunction with the immunogenic pharmaceutical preparations described herein.

[0345] Adjuvants may also include stimulatory molecules such as cytokines, non-limiting examples of which include CCL20, alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFp, GM-CSF, epidermal growth factor (EGF), cutaneous T-cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1-, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1. , LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant form of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, A po-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, and TAP2.

[0346] Additional adjuvants include MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibronectin, and fibronectin. Fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5 , KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

[0347] In some embodiments, the adjuvant can be a modulator of toll-like receptor. Examples of modulators of toll-like receptor include TLR-9 agonists, and are not limited to small molecule modulators of toll-like receptor, such as imiquimod. Other examples of adjuvants used in combination with the immunogenic pharmaceutical compositions described herein can include, but are not limited to, saponin, CpG ODN, and the like. Sometimes, the adjuvant is selected from bacterial toxoids, polyoxypropylene-polyoxyethylene block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or combinations thereof. Sometimes, the adjuvant is an oil-in-water emulsion. The oil-in-water emulsion can include at least one oil and at least one surfactant, and the oil(s) and surfactant(s) are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion can be less than 5 μm in diameter, or even have submicron diameters, and these small sizes are achieved using a microfluidizer to produce stable emulsions. Droplets less than 220 nm in size can be subjected to filter sterilization.

[0348] In some cases, the immunogenic pharmaceutical compositions may include carriers and excipients (including, but not limited to, buffers, carbohydrates, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents, and / or preservatives), water, oils including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, saline solutions, aqueous dextrose and glycerol solutions, flavorings, coloring agents, adhesion reducers and other acceptable additives, adjuvants, or binders, other pharma- ceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH buffering agents, isotonicity agents, emulsifying agents, wetting agents, and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, etc. In other cases, the pharmaceutical preparation is substantially free of preservatives. In other cases, the pharmaceutical preparation may contain at least one preservative. Any suitable carrier known to those skilled in the art can be used to administer the pharmaceutical composition described herein, and it will be understood that the type of carrier varies depending on the mode of administration.

[0349] The immunogenic pharmaceutical composition may include a preservative, such as thiomersal or 2-phenoxyethanol. In some cases, the immunogenic pharmaceutical composition is substantially free of mercurial materials (e.g., <10 μg / ml), e.g., thiomersal-free. α-Tocopherol succinate can be used as an alternative to mercurial compounds.

[0350] Physiological salts, such as sodium salts, can be included in the immunogenic pharmaceutical composition to control tonicity. Other salts include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride.

[0351] The osmolality of the immunogenic pharmaceutical composition may be between 200 mOsm / kg and 400 mOsm / kg, between 240 mOsm / kg and 360 mOsm / kg, or within the range of 290 to 310 mOsm / kg.

[0352] The immunogenic pharmaceutical composition may include one or more buffers, such as Tris buffer, borate buffer, succinate buffer, histidine buffer (particularly with an aluminum hydroxide adjuvant), or citrate buffer, etc. The buffer is included in some cases in the range of 5-20 mM.

[0353] The pH of the immunogenic pharmaceutical composition can be between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8.

[0354] The immunogenic pharmaceutical composition may be sterile. The immunogenic pharmaceutical composition may be non-pyrogenic, e.g., containing <1 EU (endotoxin unit, standard criteria) per dose, and <0.1 EU per dose. The composition may be gluten-free.

[0355] The immunogenic pharmaceutical composition may include a surfactant, such as a polyoxyethylene sorbitan ester surfactant (known as "Tween"), or an octoxynol (e.g., octoxynol-9 (such as Triton® X-100) or t-octylphenoxypolyethoxyethanol). The surfactant may be present only in trace amounts. The immunogenic pharmaceutical composition may include octoxynol-10 and polysorbate 80 at less than 1 mg / mL each. Other components remaining in trace amounts may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0356] The immunogenic pharmaceutical composition can be formulated as a sterile solution or suspension in a suitable vehicle known in the art.The pharmaceutical composition can be sterilized by conventional well-known sterilization techniques, or can be sterile filtered.The aqueous solution obtained can be packaged directly for use, or can be lyophilized, and the lyophilized preparation can be combined with a sterile solution before administration.

[0357] The immunogenic pharmaceutical composition can be formulated with one or more pharma- ceutically acceptable salts. Pharmaceutically acceptable salts can include salts of inorganic ions, such as sodium, potassium, calcium, magnesium ions, and the like. Such salts can include salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid, or maleic acid. Furthermore, when the agent(s) contains a carboxyl group or other acidic group, it can be converted into a pharma- ceutically acceptable addition salt with an inorganic or organic base. Examples of suitable bases include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine, diethanolamine, triethanolamine, and the like.

[0358] For example, pharmaceutical compositions comprising an active agent, such as a peptide, nucleic acid, antibody or fragment thereof, and / or APC, as described herein, in combination with one or more adjuvants, can be formulated to include a certain molar ratio. For example, a molar ratio of about 99:1 to about 1:99 of an active agent, such as a peptide, nucleic acid, antibody or fragment thereof, as described herein, and / or APC, in combination with one or more adjuvants can be used. In some cases, the range of molar ratios of an active agent, such as a peptide, nucleic acid, antibody or fragment thereof, as described herein, and / or APC, in combination with one or more adjuvants can be selected from about 80:20 to about 20:80; about 75:25 to about 25:75, about 70:30 to about 30:70, about 66:33 to about 33:66, about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90. The molar ratio of the combination of active agents, such as peptides, nucleic acids, antibodies or fragments thereof, and / or APCs described herein, and one or more adjuvants can be about 1:9, and in some cases, about 1:1. Active agents, such as peptides, nucleic acids, antibodies or fragments thereof, and / or APCs described herein, can be combined with one or more adjuvants and formulated together in the same dosage unit, e.g., one vial, suppository, tablet, capsule, aerosol spray, or each agent, form, and / or compound can be formulated as separate units, e.g., two vials, suppositories, tablets, two capsules, a tablet and a vial, aerosol spray, etc.

[0359] In some cases, the immunogenic pharmaceutical composition can be administered with additional agents. The selection of additional agents can depend, at least in part, on the condition to be treated. Additional agents can include any agent that has a therapeutic effect against pathogen infection (e.g., viral infection), including, for example, drugs used to treat inflammatory conditions, such as NSAIDs, for example, ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. As another example, the formulation can further contain one or more supplements, such as vitamin C, vitamin E, or other antioxidants.

[0360] Pharmaceutical compositions comprising an active agent, such as a peptide, a nucleic acid, an antibody or fragment thereof, and / or an APC, as described herein, in combination with one or more adjuvants, can be formulated in a conventional manner using one or more physiologically acceptable carriers, including, for example, excipients, diluents, and / or auxiliary agents that facilitate processing of the active agent into a preparation that can be administered. The appropriate formulation can depend, at least in part, on the route of administration selected. The agent(s) described herein can be delivered to a patient using several routes or forms of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular application, as well as by inhalation.

[0361] The active agent can be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and can be provided in unit dose form in ampoules, prefilled syringes, small injections or multi-dose containers with added preservatives. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, e.g., a solution in aqueous polyethylene glycol.

[0362] For injection formulations, the vehicle can be selected from those known in the art to be suitable, including aqueous solutions or oil suspensions, or emulsions using sesame, corn, cottonseed, or peanut oil, as well as elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles. The formulation can also include biocompatible, biodegradable polymer compositions, such as poly(lactic acid-co-glycolic acid). These materials can be made into microspheres or nanospheres, loaded with drugs, and further coated or derivatized to provide excellent sustained release performance. Vehicles suitable for periocular or intraocular injection include, for example, injection grade water, liposomes, and suspensions of therapeutic agents in vehicles suitable for lipophilic substances. Other vehicles for periocular or intraocular injection are well known in the art.

[0363] In some cases, the pharmaceutical composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous administration to humans.Generally, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.If necessary, the composition may also contain a solubilizing agent such as lidocaine and a local anesthetic to ease pain at the injection site.Generally, the components are supplied separately or mixed in unit dosage form, for example, as a dry, lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the quantity of active agent.When the composition is administered by injection, it can be dispensed into an injection bottle containing sterile pharmaceutical grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline is provided so that the components can be mixed before administration.

[0364] When administration is by injection, the active agent can be formulated in an aqueous solution, specifically in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution can contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active compound can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. In another embodiment, the pharmaceutical composition does not contain an adjuvant or any other added substance for enhancing the immune response stimulated by the peptide. In another embodiment, the pharmaceutical composition contains a substance that inhibits the immune response to the peptide.

[0365] In addition to the above-mentioned formulations, active agent can also be formulated as a depot preparation.Such long-acting formulations can be administered by implantation or transcutaneous delivery (e.g., subcutaneous or intramuscular), intramuscular injection or by using a transdermal patch.Thus, for example, active agent can be formulated with suitable polymer or hydrophobic material (e.g., as an emulsion in acceptable oil) or ion exchange resin, or as sparingly soluble derivative, for example, as a sparingly soluble salt.

[0366] In some cases, pharmaceutical compositions containing one or more agents exert local and regional effects when administered locally or injected at or near a particular site of infection. For example, direct topical application of, for example, viscous liquids, solutions, suspensions, dimethylsulfoxide (DMSO)-based solutions, liposomal formulations, gels, jellies, creams, lotions, ointments, suppositories, foams, or aerosol sprays can be used for local administration to produce local and / or regional effects. Pharmaceutically suitable vehicles for such formulations include, for example, lower aliphatic alcohols, polyglycols (e.g., glycerol or polyethylene glycol), esters of fatty acids, oils, fats, silicones, and the like. Such preparations can also include preservatives (e.g., p-hydroxybenzoic acid esters) and / or antioxidants (e.g., ascorbic acid and tocopherol). See also Dermatological Formulations: Percutaneous absorption, Barry (Ed.), Marcel Dekker Incl, 1983. In another embodiment, topical / local formulations containing transporter, carrier, or ion channel inhibitors are used to treat epidermal or mucosal viral infections.

[0367] The pharmaceutical composition may contain a cosmetically or dermatologically acceptable carrier. Such a carrier is compatible with skin, nails, mucous membranes, tissues and / or hair, and may include any conventionally used cosmetic or dermatological carrier that meets these requirements. Such a carrier can be easily selected by those skilled in the art. For the formulation of a skin ointment, the active agent or combination of active agents can be formulated in an oily hydrocarbon base, an anhydrous absorption base, a water-in-oil absorption base, an oil-in-water water-removable base and / or a water-soluble base. Examples of such carriers and excipients include, but are not limited to, humectants (e.g., urea), glycols (e.g., propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleic acid), surfactants (e.g., isopropyl myristate and sodium lauryl sulfate), pyrrolidone, glycerol monolaurate, sulfoxides, terpenes (e.g., menthol), amines, amides, alkanes, alkanols, water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0368] Ointments and creams can be formulated, for example, with aqueous or oily bases and with the addition of suitable thickening and / or gelling agents.Lotions can be formulated with aqueous or oily bases and generally also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickening agents or coloring agents.The construction and use of transdermal patches for the delivery of pharmaceuticals is well known in the art.Such patches can be constructed for continuous, pulsatile or on-demand delivery of pharmaceuticals.

[0369] Lubricants that can be used to form pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, or mixtures thereof. Additional lubricants include, for example, syloid silica gel, aerosol of synthetic silica, or mixtures thereof. Lubricants can be added, if necessary, in an amount of less than about 1 weight percent of the pharmaceutical composition.

[0370] The pharmaceutical compositions may be in any form suitable for topical application, including aqueous, aqueous-alcoholic or oily solutions, lotions or serum dispersions, aqueous, anhydrous or oily gels, emulsions obtained by dispersing a fatty phase in an aqueous phase (O / W or oil in water) or conversely, emulsions obtained by dispersing an aqueous phase in a fatty phase (W / O or water in oil), microemulsions or alternatively microcapsules, microparticles or lipid vesicle dispersions of ionic and / or non-ionic type. These compositions can be prepared according to conventional methods. The amounts of the various constituents of the compositions are conventionally used in the art. These compositions constitute creams, milks, lotions, gels or foams, in particular for the protection, treatment or care of the face, hands, body and / or mucous membranes, or for cleansing the skin. The compositions may also consist of solid preparations constituting soaps or cleansing bars.

[0371] The pharmaceutical compositions may contain adjuvants such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, sunscreens, odor absorbents and dyes. The amounts of these various adjuvants are those conventionally used in the fields under consideration, for example from about 0.01% to about 20% of the total weight of the composition. Depending on their nature, these adjuvants can be introduced into the fatty phase, into the aqueous phase and / or into lipid vesicles.

[0372] For local / topical application, the pharmaceutical composition may include one or more permeation enhancers. For example, the formulation may include a suitable solid or gel phase carrier or excipient that enhances penetration or aids in delivery of the agent or combination of agents of the present invention across a permeability barrier, such as the skin. Many of these penetration enhancing compounds are known in the topical formulation art and include, for example, water, alcohols (e.g., terpenes such as methanol, ethanol, 2-propanol), sulfoxides (e.g., dimethyl sulfoxide, decyl methyl sulfoxide, tetradecyl methyl sulfoxide), pyrrolidones (e.g., 2-pyrrolidone, N-methyl-2-pyrrolidone, N-(2-hydroxyethyl)pyrrolidone), laurocapram, acetone, dimethylacetamide, dimethylformamide, tetrahydrofurfuryl alcohol, L-α-amino acids, anionic, cationic, amphoteric or nonionic surfactants (e.g., isopropyl myristate and sodium lauryl sulfate), fatty acids, fatty alcohols (e.g., oleic acid), amines, amides, clofibric acid amide, hexamethylene lauramide, proteolytic enzymes, α-bisabolol, d-limonene, urea, and N,N-diethyl-m-toluamide. Additional examples include humectants (e.g., urea), glycols (e.g., propylene glycol and polyethylene glycol), glycerol monolaurate, alkanes, alkanols, ORGELASE, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and / or other polymers. In another embodiment, the pharmaceutical composition includes one or more such permeation enhancers.

[0373] Pharmaceutical compositions for topical / local application may contain one or more antimicrobial preservatives, such as, for example, quaternary ammonium compounds, organic mercurials, p-hydroxybenzoic acid, aromatic alcohols, chlorobutanol, and the like.

[0374] The pharmaceutical composition can be formulated into an aerosol solution, suspension or dry powder. The aerosol can be administered through the respiratory system or nasal cavity. For example, those skilled in the art will understand that the composition of the present invention can be suspended or dissolved in a suitable carrier, such as a pharma- ceutically acceptable propellant, and administered directly to the lungs using a nasal spray or inhalant. For example, the aerosol formulation containing the transporter, carrier, or ion channel inhibitor can be dissolved, suspended, or emulsified in a propellant or a mixture of a solvent and a propellant, for example, to be administered as a nasal spray or inhalant. The aerosol formulation can contain any acceptable propellant under pressure, such as, for example, a cosmetically or dermatologically or pharma- ceutically acceptable propellant that is conventionally used in the art.

[0375] Aerosol formulations for nasal administration are generally aqueous solutions designed to be administered to the nasal cavity as drops or sprays. Nasal solutions can be similar to nasal secretions in that they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values ​​outside this range can also be used. Antimicrobial agents or preservatives can also be included in the formulation.

[0376] Aerosol formulations for inhalation and inhalation can be designed so that the agent or combination of agents is carried into the subject's respiratory tree when administered via nasal or oral respiratory route.Inhalation solutions can be administered, for example, by nebulizers.For example, inhalation or insufflation containing finely powdered or liquid drugs can be delivered to the respiratory system as a medicinal aerosol of a solution or suspension of the agent or combination of agents in a propellant to aid distribution.The propellant can be a liquefied gas, including halocarbons, for example, fluorocarbons, for example, fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.

[0377] Halocarbon propellants may include fluorocarbon propellants in which all hydrogens are replaced by fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced by chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen-containing chlorofluorocarbon propellants. Hydrocarbon propellants useful in the present invention include, for example, propane, isobutane, n-butane, pentane, isopentane, and neopentane. Mixtures of hydrocarbons can also be used as propellants. Ether propellants include, for example, dimethyl ether and ether. Aerosol formulations of the present invention may contain more than one propellant. For example, aerosol formulations may contain more than one propellant of the same class, for example, two or more fluorocarbons; or more than one, more than two, more than three propellants of different classes, for example, fluorohydrocarbons and hydrocarbons. The pharmaceutical compositions of the present invention may also be formulated using compressed gases, for example, inert gases such as carbon dioxide, nitrous oxide, or nitrogen.

[0378] The aerosol formulation may also contain other components, such as ethanol, isopropanol, propylene glycol, and surfactants or oils and surfactants, which may serve to stabilize the formulation and / or lubricate valve components.

[0379] Aerosol formulations can be packaged under pressure, and can be formulated as aerosols using solutions, suspensions, emulsions, powders and semi-solid preparations.For example, solution aerosol formulations can include a solution of the agent of the present invention, such as a transporter, carrier, or ion channel inhibitor, in a (substantially) pure propellant or as a mixture of a propellant and a solvent. Solvents can be used to dissolve the agent and / or retard the evaporation of the propellant. Solvents can include, for example, water, ethanol and glycol. Any combination of suitable solvents can be used, optionally in combination with preservatives, antioxidants, and / or other aerosol components.

[0380] Aerosol formulations can be dispersions or suspensions.Suspension aerosol formulations can include the suspension of the agent or agent combination of the present invention, such as a transporter, carrier, or ion channel inhibitor, and a dispersing agent.Dispersing agents can include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin, and corn oil.Suspension aerosol formulations can also include lubricants, preservatives, antioxidants, and / or other aerosol components.

[0381] Aerosol formulations can be formulated in the same way as emulsions.Emulsion aerosol formulations can include, for example, alcohol such as ethanol, surfactant, water and propellant, and the agent or combination of agents of the present invention, such as transporter, carrier, or ion channel.The surfactant used can be non-ionic, anionic, or cationic.One example of emulsion aerosol formulation includes, for example, ethanol, surfactant, water and propellant.Another example of emulsion aerosol formulation includes, for example, vegetable oil, glycerin monostearate, and propane.

[0382] Pharmaceutical compounds can be formulated for administration as suppositories.First melt a low melting wax such as triglyceride, fatty acid glyceride, Witepsol® S55 (trademark of Dynamite Nobel Chemical, Germany) or a mixture of cocoa butter, and disperse the active ingredient homogeneously, for example by stirring.The homogeneous molten mixture is then poured into convenient size molds, cooled, and solidified.

[0383] Pharmaceutical compositions can be formulated for vaginal administration: pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0384] The pharmaceutical composition can be releasably attached to a biocompatible polymer for use in sustained release formulations on, in, or attached to an insert for local, intraocular, periocular, or systemic administration.Controlled release from biocompatible polymers can also be used with water-soluble polymers to form drop-injectable formulations.For example, controlled release from biocompatible polymers such as PLGA microspheres or nanospheres can also be used in formulations suitable for intraocular implantation or injection for sustained release administration.Any suitable biodegradable and biocompatible polymer can be used. Production of tissue-specific antigens

[0385] The present disclosure is based at least in part on the ability to present a patient's immune system with one or more tissue-specific antigens. From this disclosure and knowledge in the art, the skilled artisan will recognize that there are various methods of producing such tissue-specific antigens. In general, such tissue-specific antigens can be produced either in vitro or in vivo. Tissue-specific antigens, such as peptides or polypeptides, which can then be formulated into a vaccine or pharmaceutical composition and administered to a subject, can be produced in vitro. As described in further detail herein, such in vitro production can occur by various methods known to those skilled in the art, such as, for example, peptide synthesis or expression of a peptide / polypeptide from DNA or RNA molecules in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptide / polypeptide. Alternatively, tissue-specific antigens can be produced in vivo by introducing molecules (e.g., DNA, RNA, viral expression systems, etc.) encoding the tissue-specific antigen into a subject, whereupon the encoded tissue-specific antigen is expressed. Methods of in vitro and in vivo production of antigens are also described further herein with respect to pharmaceutical compositions and therapeutic delivery methods.

[0386] In vitro peptide / polypeptide synthesis

[0387] Proteins or peptides of the disclosure, e.g., tissue-specific antigen peptides, e.g., tissue-specific antigens comprising tumor epitope sequences provided herein, can be made by any technique known to those of skill in the art, including expression of the protein, polypeptide or peptide by standard molecular biology techniques, isolation of the protein or peptide from a natural source, in vitro translation, or chemical synthesis of the protein or peptide.

[0388] The peptides of the present disclosure can be easily chemically synthesized utilizing reagents that are free of bacterial or animal material contamination (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc.85:2149-54, 1963). In some embodiments, the antigenic peptides of the present disclosure are prepared by (1) parallel solid phase synthesis on a multichannel instrument using homogeneous synthesis and cleavage conditions; (2) purification on a RP-HPLC column with column stripping; and re-cleavage (without replacement) between peptides; followed by (3) analysis with a limited set of the most informative assays. Good Manufacturing Practice (GMP) footprints can be defined for individual patient peptide sets, so that a series of switchover procedures are only required during the synthesis of peptides for different patients.

[0389] Alternatively, a nucleic acid (e.g., polynucleotide) encoding the antigenic peptide of the present disclosure can be used to generate the antigenic peptide in vitro. The polynucleotide can be, for example, single-stranded and / or double-stranded DNA, cDNA, PNA, CNA, RNA, or a polynucleotide in a natural or stabilized form, such as a polynucleotide with a phosphorothioate backbone, or a combination thereof, and the polynucleotide can contain an intron so long as it encodes a peptide. In one embodiment, in vitro translation is used to generate the peptide. There are many exemplary systems available to those skilled in the art (e.g., Retic Lysate IVT Kit, Life Technologies, Waltham, MA). An expression vector capable of expressing the polypeptide can also be prepared. Expression vectors for various cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in the appropriate orientation and correct reading frame for expression. If necessary, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such controls are generally available in the expression vector. The vector is then introduced into a host bacterium for cloning using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0390] Also contemplated is an expression vector comprising the isolated polynucleotide, and a host cell containing the expression vector.Antigen peptides can be provided in the form of RNA or cDNA molecules that code desired antigen peptides.One or more antigen peptides of the present disclosure can be encoded by a single expression vector.

[0391] In some embodiments, a polynucleotide may include, for example, a coding sequence for a tissue-specific antigenic peptide fused in the same reading frame as a polynucleotide that aids in the expression and / or secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide out of the cell). A polypeptide having a leader sequence may be a preprotein, with the leader sequence being cleaved by the host cell to form the mature form of the polypeptide.

[0392] In some embodiments, the polynucleotides may include coding sequences for antigenic peptides of the present disclosure fused in the same reading frame with a marker sequence that allows for purification of the encoded polypeptide, which may then be incorporated into a personalized vaccine or immunogenic composition. For example, the marker sequence may be a hexa-histidine tag provided by the pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence may be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. Additional tags include, but are not limited to, Calmodulin tag, FLAG tag, Myc tag, S tag, SBP tag, Softag1, Softag3, V5 tag, Xpress tag, Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tag, GST tag, fluorescent protein tag (e.g., green fluorescent protein tag), maltose binding protein tag, Nus tag, Strep tag, thioredoxin tag, TC tag, Ty tag, and the like.

[0393] In some embodiments, a polynucleotide may contain the coding sequence for one or more of the tissue-specific antigenic peptides fused in the same reading frame to create a single concatemerized antigenic peptide construct capable of producing multiple antigenic peptides.

[0394] In some embodiments, isolated nucleic acid molecules may be provided that have a nucleotide sequence that is at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding a tissue-specific antigenic peptide of the present disclosure.

[0395] The isolated tissue-specific antigenic peptides described herein can be produced in vitro (e.g., in a laboratory) by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding isolated polypeptide sequences and expressing these sequences in a suitable transformed host. In some embodiments, DNA sequences are constructed by isolating or synthesizing DNA sequences encoding wild-type proteins of interest using recombinant techniques. If necessary, the sequences can be mutagenized by site-directed mutagenesis to provide functional analogs thereof. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

[0396] In some embodiments, the DNA sequence encoding the polypeptide provided herein will be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and the selection of those codons that are preferred in the host cell in which the recombinant polypeptide of interest is produced. Standard methods can be applied to synthesize the isolated polynucleotide sequence encoding the isolated polypeptide of interest. For example, the complete amino acid sequence can be used to construct a reverse-translated gene. In addition, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding parts of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complement assembly.

[0397] Once assembled (e.g., by synthesis, site-directed mutagenesis, or other methods), the polynucleotide sequence encoding the particular isolated polypeptide is inserted into an expression vector and, if necessary, operably linked to an appropriate expression control sequence for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of the transfected gene in the host, the gene can be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0398] Recombinant expression vectors can be used to amplify and express DNA encoding the tissue-specific antigenic peptides described herein. A recombinant expression vector is a replicable DNA construct with synthetic or cDNA-derived DNA fragments encoding tissue-specific antigenic peptides, or biologically equivalent analogs operably linked to suitable transcription or translation regulatory elements derived from mammalian, microbial, viral or insect genes. A transcription unit, as described in detail herein, generally includes an assembly of (1) genetic element(s) that have a regulatory role in gene expression, such as a transcription promoter or enhancer, (2) structural or coding sequences that are transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Such regulatory elements may include operator sequences to control transcription. Usually, the ability to replicate in a host, conferred by an origin of replication, and a selection gene that facilitates the recognition of transformants may be further incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor involved in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to allow translation. Generally, operably linked means contiguous, and in the case of a secretory leader, contiguous and in reading frame. Structural elements intended for use in yeast expression systems include leader sequences that allow extracellular secretion of the translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue, which is then cleaved, if necessary, from the expressed recombinant protein to provide the final product.

[0399] Useful expression vectors for producing the polypeptides of the present disclosure in eukaryotic hosts, particularly mammals or humans, include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as plasmids from Escherichia coli, including pCR1, pBR322, pMB9 or their derivatives, broad host range plasmids such as M13 and filamentous single-stranded DNA phages.

[0400] Suitable host cells for expressing the polypeptide of the present disclosure may include prokaryotes, yeast, insect or higher eukaryotic cells under the control of suitable promoters. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or bacillus. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems may also be used. Cloning and expression vectors suitable for use with bacterial, fungal, yeast and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0401] A variety of mammalian or insect cell culture systems can be used to express the recombinant proteins provided herein. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell systems include the COS-7 system of monkey kidney cells described in Gluzman (Cell 23:175, 1981), as well as other cell systems capable of expressing suitable vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa and BHK cell systems. Mammalian expression vectors can include non-transcribed elements such as origins of replication, suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, as well as 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0402] The proteins provided herein produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography, etc.), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence, glutathione-S-transferase, etc. can be added to the protein and passed through a suitable affinity column to allow easy purification. The isolated protein can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography. For example, the supernatant from a system that secretes recombinant proteins into culture medium can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate with pendant diethylaminoethyl (DEAE) groups, can be employed. The matrix may be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step may be employed. Suitable cation exchangers include various insoluble matrices that contain sulfopropyl or carboxymethyl groups. Finally, the cancer stem cell protein-Fc composition may be further purified using one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, for example, silica gel with pendant methyl or other aliphatic groups. Some or all of the above purification steps may also be used in various combinations to provide homogeneous recombinant protein.

[0403] Recombinant proteins as described herein produced in bacterial culture can be isolated, for example, by initial extraction from a cell pellet followed by one or more concentration, salting out, aqueous ion exchange or size exclusion chromatography steps. A final purification step can employ high performance liquid chromatography (HPLC). Microbial cells used for expression of recombinant proteins can be disrupted by any convenient method, including freeze-thaw cycle treatment, sonication, mechanical disruption, or the use of cell lysing agents.

[0404] In vivo peptide / polypeptide synthesis

[0405] The present disclosure also contemplates the use of nucleic acid molecules as vehicles for delivering antigenic peptides / polypeptides in vivo, e.g., in the form of a DNA / RNA vaccine, to a subject in need thereof (see, e.g., WO2012 / 159643, and WO2012 / 159754, which are hereby incorporated by reference in their entireties).

[0406] In some embodiments, antigens can be administered to subjects in need thereof by using plasmids. These are usually plasmids consisting of a strong viral promoter that drives the transcription and translation of the gene of interest (or complementary DNA) in vivo (Mor, et al., (1995). The Journal of Immunology 155 (4): 2039-2046). Intron A can be included to improve mRNA stability and thereby increase protein expression (Leitner, et al. (1997). The Journal of Immunology 159 (12): 6112-6119). The plasmid also contains a strong polyadenylation / transcription termination signal, such as the bovine growth hormone or rabbit beta-globulin polyadenylation sequence (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410;Robinson et al., (2000). Adv. Virus Res. Advances in Virus Research 55: 1-74;Bohmet al., (1996). Journal of Immunological Methods 193 (1): 29-40.). Multicistronic vectors may be constructed to express two or more immunogens, or to express an immunogen and an immunostimulatory protein (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).

[0407] In some embodiments, the plasmid can be introduced into animal tissue by several different methods. Two approaches in particular can be injection of DNA in saline using a standard hypodermic needle and gene gun delivery. Injection in saline is usually performed intramuscularly (IM) or intradermally (ID) in skeletal muscle, and DNA can be delivered to the extracellular space. This can be assisted by electroporation, by temporarily damaging muscle fibers with myotoxins such as bupivacaine; or by using hypertonic solutions of saline or sucrose (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410). The immune response to this delivery method can be influenced by many factors, including needle type, needle alignment, injection speed, injection volume, muscle type, and the age, sex, and physiological condition of the animal receiving the injection (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410).

[0408] Also useful for the present disclosure is gene gun delivery, in which plasmid DNA (pDNA) adsorbed to gold or tungsten microparticles is ballistically accelerated into target cells using compressed helium as an accelerant (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410; Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).

[0409] Alternative delivery methods include aerosol instillation of naked DNA onto mucosal surfaces such as the nasal and pulmonary mucosa (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88) and topical administration of pDNA to the ocular and vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54: 129-88). Mucosal surface delivery can be achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors. DNA or RNA can be delivered to cells after mild mechanical disruption of the cell membrane and transient permeabilization of the cells. Such mild mechanical disruption of the membrane can be accomplished by gently pushing the cells through a small opening (Ex vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells, Sharei et al, PLOS ONE | DOI:10.1371 / journal.pone.0118803 April 13, 2015).

[0410] In some embodiments, a vaccine or pharmaceutical composition comprising a tissue-specific antigen may, for example, comprise separate DNA plasmids encoding one or more antigenic peptides / polypeptides identified by the present disclosure. As discussed herein, the exact choice of expression vector may depend on the peptide / polypeptide to be expressed and is within the skill of the artisan. The predicted persistence of the DNA construct (e.g., in an episomal, non-replicating, non-integrated form within muscle cells) is expected to provide an extended period of protection.

[0411] One or more antigenic peptides of the present disclosure may be encoded and expressed in vivo using a viral-based system (e.g., an adenovirus system, an adeno-associated virus (AAV) vector, a poxvirus, or a lentivirus). In one embodiment, the vaccine or pharmaceutical composition may include a viral-based vector for use in a human patient in need thereof, such as, for example, an adenovirus (see, e.g., Baden et al. First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). J Infect Dis.2013 Jan 15;207(2):240-7, which is hereby incorporated by reference in its entirety). Plasmids that can be used for adeno-associated virus, adenovirus, and lentivirus delivery have been previously described (see, e.g., U.S. Pat. Nos. 6,955,808 and 6,943,019, and U.S. Patent Application No. 20080254008, which are hereby incorporated by reference).

[0412] The peptides and polypeptides of the present disclosure can also be expressed by vectors, such as the nucleic acid molecules discussed herein, such as RNA or DNA plasmids, viral vectors, such as poxviruses, such as orthopoxviruses, avipoxviruses, or adenoviruses, AAV or lentiviruses.This approach involves the use of vectors to express the nucleotide sequence that codes for the peptides of the present disclosure.When introduced into an acutely or chronically infected host or into a non-infected host, the vectors can express immunogenic peptides, thereby inducing host CTL responses.

[0413] Of the vectors that can be used in the practice of the present disclosure, integration in the host genome of cells is possible through retroviral gene transfer methods, often resulting in long-term expression of the inserted transgene. In some embodiments, the retrovirus is a lentivirus. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues. The tropism of the retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Retroviruses can also be engineered to allow conditional expression of the inserted transgene, such that only certain cell types are infected by the lentivirus. Cell type-specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (thus both lentiviral and retroviral vectors can be used in the practice of the present disclosure). Furthermore, lentiviral vectors can transduce or infect non-dividing cells, typically resulting in high viral titers. Thus, the choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors are composed of cis-acting long terminal repeat sequences with packaging capacity for foreign sequences up to 6-10 kb. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate and provide persistent expression of the desired nucleic acid in the target cell.Widely used retroviral vectors that can be used in the practice of the present disclosure include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., (1992) J. Virol. 66:2731-2739; Johann et al., (1992) J. Virol.66:1635-1640; Sommnerfelt et al., (1990) Virol.176:58-59; Wilson et al., (1998) J. Virol.63:2374-2378; Miller et al., (1991) J. Virol.65:2220-2224; PCT / US94 / 05700).

[0414] Also useful in the practice of the present disclosure are minimal non-primate lentiviral vectors, such as lentiviral vectors based on Equine Infectious Anemia Virus (EIAV) (see, e.g., Balagaan, (2006) J Gene Med; 8: 275- 285, Published online 21 November 2005 in Wiley InterScience (interscience.wiley.com). DOI: 10.1002 / jgm.845). The vectors may have a cytomegalovirus (CMV) promoter driving expression of the target gene. Thus, the present disclosure contemplates viral vectors, including retroviral and lentiviral vectors, among vectors useful in the practice of the present disclosure.

[0415] Lentiviral vectors have been disclosed in the treatment of Parkinson's disease.See, for example, US Patent Publication No. 20120295960 and US Patent No. 7303910 and US Patent No. 7351585.Lentiviral vectors have also been disclosed for delivery to the brain.See, for example, US Patent Publication No. US20110293571; US20040013648, US20070025970, US20090111106 and US Patent No. US7259015.In another embodiment, lentiviral vectors are used to deliver vectors to the brain of those who are treated for disease, such as glioma. For lentiviral vector systems useful in the practice of the present disclosure, reference is made to U.S. Patent Nos. 6,428,953, 6,165,782, 6,013,516, 5,994,136, 6,312,682, and 7,198,784, and documents cited therein. In the embodiments herein, delivery is via lentivirus. Zou et al. 9 Recombinant lentivirus with a titer of 10 ... 5 Or about 1 x 10 5 Plaque-forming units (PFU), 5 × 10 5 Or about 5 x 10 5 PFU, 1 × 10 6 Or about 1 x 10 6 PFU, 5 × 10 6 Or about 5 x 10 6 PFU, 1 × 10 7 Or about 1 × 107 PFU, 5 × 10 7 Or about 5 x 10 7PFU, 1 × 10 8 Or about 1 x 10 8 PFU, 5 × 10 8 Or about 5 x 10 8 PFU, 1 × 10 9 Or about 1 x 10 9 PFU, 5 × 10 9 Or about 5 x 10 9 PFU, 1 × 10 10 Or about 1 x 10 10 PFU or 5 × 10 10 Or about 5 x 10 10 The dosage may be PFU or may be adjusted for the weight and size and species of the subject.Those skilled in the art can determine the appropriate dosage.The appropriate dosage of the virus can be empirically determined.

[0416] Adenoviral vectors are also useful in the practice of the present disclosure. One advantage is the ability of recombinant adenoviruses to efficiently transfer and express recombinant genes in various mammalian cells and tissues in vitro and in vivo, resulting in high expression of the transferred nucleic acid. In addition, the ability to infect and grow in quiescent cells expands the usefulness of recombinant adenoviral vectors. Furthermore, high expression levels ensure that the product of the nucleic acid is expressed to a sufficient level to generate an immune response (see, for example, U.S. Patent No. 7,029,848, which is hereby incorporated by reference). Adenoviral vectors useful in the practice of the present disclosure are referred to in U.S. Patent No. 6,955,808. The adenoviral vector used may be selected from the group consisting of Ad5, Ad35, Ad11, C6, and C7 vectors. The sequence of the adenovirus 5 ("Ad5") genome has been published. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285, the contents of which are hereby incorporated by reference.) Ad35 vectors are described in U.S. Patent Nos. 6,974,695, 6,913,922, and 6,869,794. Ad11 vectors are described in U.S. Patent No. 6,913,922. C6 adenoviral vectors are described in U.S. Patent Nos. 6,780,407; 6,537,594; 6,309,647; 6,265,189; 6,156,567; 6,090,393; 5,942,235 and 5,833,975. C7 vectors are described in U.S. Patent No. 6,277,558. E1-deleted or deleted, E3-deleted or deleted, and / or E4-deleted or deleted adenoviral vectors can also be used.Certain adenoviruses with mutations in the E1 region have an improved safety margin because E1-deficient adenovirus mutants are replication-deficient or at least highly attenuated in non-permissive cells. Adenoviruses with mutations in the E3 region can increase immunogenicity by disrupting the mechanism by which adenovirus downregulates MHC class I molecules. Adenoviruses with E4 mutations can reduce the immunogenicity of adenoviral vectors due to suppression of late gene expression. Such vectors may be particularly useful when repeated revaccination utilizing the same vector is desired. Adenoviral vectors that are deleted or mutated in E1, E3, E4, E1 and E3, and E1 and E4 may be used in accordance with the present disclosure. In addition, "gutless" adenoviral vectors in which all viral genes have been deleted may also be used in accordance with the present disclosure. Such vectors require a helper virus for their replication and require a special human 293 cell line that expresses both E1a and Cre, conditions that do not exist in the natural environment. Such "gutless" vectors are non-immunogenic, so the vectors can be administered multiple times as revaccination. "Gutless" adenoviral vectors can be used for insertion of heterologous inserts / genes, such as the transgenes of the present disclosure, and can even be used for simultaneous delivery of multiple heterologous inserts / genes. In some embodiments, delivery is via adenovirus, which can be a single booster dose. In some embodiments, the adenovirus is delivered in multiple doses. For in vivo delivery, AAV is advantageous over other viral vectors because it is less toxic and less likely to cause insertional mutagenesis because it does not integrate into the host genome. AAV has a packaging limit of 4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kb result in significantly reduced virus production. There are many promoters that can be used to drive expression of nucleic acid molecules. The AAV ITRs can serve as promoters, which is advantageous in eliminating the demand for additional promoter elements.For ubiquitous expression, the following promoters can be used: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chain, etc. For expression in the brain, the following promoters can be used: SynapsinI for all neurons, CaMKII alpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters used to drive RNA synthesis can include: Pol III promoters such as U6 or H1. Pol II promoters and intron cassettes can be used to express guide RNAs (gRNAs). For the AAV vectors useful in the practice of the present disclosure, reference is made to U.S. Patent Nos. 5,658,785, 7,115,391, 7,172,893, 6,953,690, 6,936,466, 6,924,128, 6,893,865, 6,793,926, 6,537,540, 6,475,769 and 6,258,595 and documents cited therein.For AAV, AAV can be AAV1, AAV2, AAV5 or any combination thereof.For the cell to be targeted, AAV can be selected; for example, AAV serotype 1, 2, 5 or hybrid capsid AAV1, AAV2, AAV5 or any combination thereof can be selected for targeting brain or neuronal cells; AAV4 can be selected for targeting heart tissue.AAV8 is useful for delivery to liver. In some embodiments, delivery is via AAV. Dosage can be adjusted to balance the therapeutic benefit against any side effects.

[0417] In some embodiments, effective activation of a cellular immune response to a vaccine or pharmaceutical composition can be achieved by expressing the relevant antigen in the vaccine or pharmaceutical composition in a non-pathogenic microorganism. Well-known examples of such microorganisms are Mycobacterium bovis BCG, Salmonella and Pseudomonas (see U.S. Patent No. 6,991,797, which is hereby incorporated by reference in its entirety).

[0418] In some embodiments, poxvirus is used in vaccine or immunogenic composition.These include orthopoxvirus, avipox, vaccinia, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see, for example, Verardi et al., Hum Vaccin Immunother. 2012 July;8(7):961-70; and Moss, Vaccine. 2013;31(39):4220-4222).Poxvirus expression vectors were described in 1982 and quickly became widely used in many fields of research as well as vaccine development.The advantages of vectors include simple construction, ability to accommodate large amounts of foreign DNA and high expression levels. Information regarding poxviruses that can be used in the practice of the present disclosure, such as poxviruses (vertebrate poxviruses) of the Chordopoxvirinae subfamily, e.g., orthopoxviruses and avipoxviruses, e.g., vaccinia viruses (e.g., Wyeth strain, WR strain (e.g., ATCC® VR-1354), Copenhagen strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox viruses (e.g., Wheatley C93 strain, ALVAC), fowlpox viruses (e.g., FP9 strain, Webster strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoonpox, particularly synthetic or non-naturally occurring recombinant forms thereof, their uses, and methods for making and using such recombinants, can be found in the scientific and patent literature.

[0419] In some embodiments, vaccinia viruses are used in vaccines or pharmaceutical compositions to express tissue-specific antigens. (Rolph et al., Recombinant viruses as vaccines and immunological tools. Curr Opin Immunol 9:517-524, 1997). Recombinant vaccinia viruses can replicate in the cytoplasm of infected host cells, and thus the polypeptide of interest can induce an immune response. In addition, poxviruses are widely used as vaccine or pharmaceutical composition vectors due to their ability to self-adjuvant, but also to target encoded antigens for processing by the major histocompatibility complex class I pathway by directly infecting immune cells, particularly antigen-presenting cells.

[0420] In some embodiments, ALVAC is used as a vector in a vaccine or immunogenic composition. ALVAC is a canarypox virus that can be modified to express foreign transgenes and has been used as a method for vaccination against both prokaryotic and eukaryotic antigens. (Horig H, Lee DS, Conkright W, et al. Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule. Cancer Immunol Immunother 2000;49:504-14;von Mehren M, Arlen P, Tsang KY, et al. Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas. Clin Cancer Res 2000;6:2219-28;Musey L, Ding Y, Elizaga M, et al. HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T cell immunity in HIV-1-uninfected individuals. J Immunol 2003;171:1094-101;Paoletti E. Applications of pox virus vectors to vaccination: an update. Proc Natl Acad Sci USA 1996;93:11349-53; U.S. Patent No. 7,255,862).In a phase I clinical trial, ALVAC virus expressing the tissue-specific antigen CEA showed a good safety profile in selected patients and resulted in increased CEA-specific T-cell responses; however, no objective clinical responses were observed (Marshall JL, Hawkins MJ, Tsang KY, et al. Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen. J Clin Oncol 1999;17:332-7).

[0421] In some embodiments, modified vaccinia Ankara (MVA) virus can be used as a viral vector for antigen vaccines or immunogenic compositions. MVA is a member of the orthopoxvirus family and has been generated by serial passage of the Ankara strain of vaccinia virus (CVA) in chicken embryo fibroblast cells for approximately 570 generations (for review, see Mayr, A., et al., Infection 3, 6-14, 1975). As a result of these passages, the resulting MVA virus contains 31 kilobases less genomic information compared to CVA and is highly host cell restricted (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by its extreme attenuation, i.e., reduced virulence or infectivity, while still retaining excellent immunogenicity. When tested in various animal models, MVA has been proven to be avirulent, even in immunosuppressed individuals. Additionally, MVA-BN®-HER2 is a candidate immunotherapy designed for the treatment of HER-2 positive breast cancer, currently undergoing clinical trials. (Mandl et al., Cancer Immunol Immunother. Jan 2012; 61(1): 19-29). Methods for making and using recombinant MVA have been described (see, e.g., U.S. Patent Nos. 8,309,098 and 5,185,146, which are hereby incorporated in their entireties).

[0422] In some embodiments, the recombinant viral particles of the vaccine or pharmaceutical composition are administered to a patient in need thereof. Modifications to peptides / polypeptides

[0423] In some embodiments, the present disclosure includes modified antigen peptides. Modifications may include covalent chemical modifications that do not alter the primary amino acid sequence of the antigen peptide itself. Modifications may result in peptides with desired properties, such as increased in vivo half-life, increased stability, reduced clearance, altered immunogenicity or allergenicity, enabling the production of specific antibodies, cellular targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation. Alterations to antigen peptides that may be performed include, but are not limited to, conjugation to carrier proteins, conjugation to ligands, conjugation to antibodies, PEGylation, polysialylation, HESylation, recombinant PEG mimics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of surfactants, addition of amino acid mimics, or addition of non-natural amino acids.

[0424] In some embodiments, the disclosure also includes various modifications to overcome problems associated with short plasma half-life or susceptibility to protease degradation, including conjugating or linking the polypeptide sequence to any of a variety of non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene (e.g., typically via a linking moiety covalently attached to both the protein and the non-proteinaceous polymer, e.g., PEG). Such PEG-conjugated biomolecules have been shown to have clinically useful properties, including better physical and thermal stability, protection from susceptibility to enzymatic degradation, improved solubility, extended in vivo circulatory half-life and reduced clearance, reduced immunogenicity and antigenicity, and reduced toxicity.

[0425] PEG suitable for conjugation to polypeptide sequences is generally soluble in water at room temperature and has the general formula R(O-CH 2 -CH 2)nO-R, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, R generally has 1 to 8 carbons. The PEG conjugated to the polypeptide sequence may be linear or branched. Branched PEG derivatives, "star PEGs" and multi-arm PEGs are contemplated by the present disclosure.

[0426] The present disclosure also contemplates compositions of conjugates in which PEG has different n values, and thus various different PEGs are present in specific ratios. For example, some compositions contain a mixture of conjugates with n=1, 2, 3, and 4. In some compositions, the percentage of conjugates with n=1 is 18-25%, the percentage of conjugates with n=2 is 50-66%, the percentage of conjugates with n=3 is 12-16%, and the percentage of conjugates with n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. For example, cation exchange chromatography can be used to separate the conjugates, and then fractions containing, for example, conjugates with a desired number of PEGs attached are identified and purified to be free of unmodified protein sequences and conjugates with other numbers of PEGs attached.

[0427] PEG can be attached to the polypeptide of the present disclosure via a terminal reactive group ("spacer"). The spacer is, for example, a terminal reactive group that mediates the bond between one or more free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. PEG with a spacer that can be attached to a free amino group includes N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinic acid ester of polyethylene glycol with N-hydroxysuccinimide. Another activated polyethylene glycol that can be attached to a free amino group is 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride. Activated polyethylene glycols that are attached to free carboxyl groups include polyoxyethylene diamines.

[0428] Conjugation of one or more of the polypeptide sequences of the present disclosure to PEG with a spacer can be carried out by a variety of conventional methods. For example, the conjugation reaction can be carried out in solution at a pH of 5-10, at a temperature of 4°C to room temperature, for 30 minutes to 20 hours, utilizing a molar ratio of reagent to protein of 4:1 to 30:1. Reaction conditions can be selected to produce primarily the desired degree of substitution in the reaction. In general, low temperatures, low pH (e.g., pH=5), and short reaction times tend to reduce the number of PEGs attached, while high temperatures, neutral to high pH (e.g., pH>7), and longer reaction times tend to increase the number of PEGs attached. Various means known in the art can be used to terminate the reaction. In some embodiments, the reaction is terminated by acidifying the reaction mixture and freezing, for example, at -20°C.

[0429] The present disclosure also contemplates the use of PEG mimetics. Recombinant PEG mimetics have been developed that retain the attributes of PEG (e.g., enhanced serum half-life) while imparting some additional advantageous properties. For example, simple polypeptide chains (e.g., containing Ala, Glu, Gly, Pro, Ser and Thr) that can form extended structures similar to PEG can be recombinantly produced (e.g., Amunix's XTEN technology; Mountain View, CA) already fused to the peptide or protein drug of interest. This eliminates the need for additional conjugation steps during the manufacturing process. Furthermore, established molecular biology techniques allow control of the side chain composition of the polypeptide chain, allowing optimization of immunogenicity and manufacturing properties.

[0430] Glycosylation can affect the physical properties of a protein and may also be important in protein stability, secretion, and subcellular localization. The present disclosure also includes compositions comprising polypeptides with glycosylation modifications. Proper glycosylation can be important for biological activity. Indeed, some genes from eukaryotes, when expressed in bacteria (e.g., E. coli) that lack the cellular processes for glycosylation of proteins, result in proteins that are recovered with little or no activity due to the lack of glycosylation. Addition of glycosylation sites can be accomplished by altering the amino acid sequence. Alterations to the polypeptide can be made, for example, by the addition or substitution of one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites). The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type can differ. One type of sugar commonly found in both is N-acetylneuraminic acid (hereafter referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, due to its negative charge, may confer acidic properties to glycoproteins. Embodiments of the present disclosure include the generation and use of N-glycosylation variants.

[0431] The polypeptide sequences of the present disclosure can be modified as necessary by changes at the DNA level, in particular by mutating the DNA encoding the polypeptide at preselected bases so that a codon that translates to the desired amino acid is generated. Another means of increasing the number of carbohydrate moieties of a polypeptide is by chemically or enzymatically coupling glycosides to the polypeptide. Removal of carbohydrates can be accomplished chemically or enzymatically, or by substitution of the codon that codes for the glycosylated amino acid residue. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties of polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases.

[0432] Additional components and molecules suitable for conjugation include, for example, molecules for targeting the lymphatic system, thyroglobulin; albumins such as human serum albumin (HAS); tetanus toxoid; diphtheria toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); VP6 polypeptide of rotavirus; influenza virus hemagglutinin, influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and core protein and surface antigen of Hepatitis B virus; or any combination of the foregoing.

[0433] Fusion of albumin to one or more polypeptides of the present disclosure can be achieved by genetic engineering, for example, such that DNA encoding HSA, or a fragment thereof, is joined to DNA encoding one or more polypeptide sequences. A suitable host can then be transformed or transfected with the fusion nucleotide sequence, for example in the form of a suitable plasmid, to express the fusion polypeptide. Expression can be in vitro, for example from prokaryotic or eukaryotic cells, or in vivo, for example from transgenic organisms. In some embodiments of the present disclosure, expression of the fusion protein is carried out in a mammalian cell system, for example, a CHO cell system. Transformation is used broadly herein to refer to the genetic alteration of a cell that occurs by the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings, as well as uptake across the cell membrane(s). Transformation occurs naturally in some species of bacteria, but can also be achieved by artificial means in other cells. Additionally, albumin itself can be modified to increase its circulating half-life. Fusion of modified albumin to one or more polypeptides can be achieved by the above-mentioned genetic engineering techniques or chemical conjugation; the resulting fusion molecule has a half-life that exceeds that of fusion with unmodified albumin. (See WO2011 / 051489). As an alternative to direct fusion, several albumin binding strategies have been developed, including albumin binding via conjugated fatty acid chains (acylation). Since serum albumin is a transport protein for fatty acids, these natural ligands with albumin binding activity have been used to extend the half-life of small molecule protein therapeutics. For example, insulin detemir (LEVEMIR) is an approved product for diabetes, which contains myristyl chains conjugated to genetically modified insulin, resulting in a long-acting insulin analog.

[0434] Another type of modification provided by the present disclosure is to conjugate (e.g., link) one or more additional components or molecules, such as another protein (e.g., a protein having an amino acid sequence heterologous to the protein of interest) or a carrier molecule, to the N-terminus and / or C-terminus of the polypeptide sequences provided herein. Thus, exemplary polypeptide sequences can be provided as conjugates with another component or molecule.

[0435] In some embodiments, modification of the conjugates provided herein may result in a polypeptide sequence that retains activity with additional or complementary functions or activities of a second molecule.For example, the polypeptide sequence may be conjugated to a molecule to facilitate, for example, solubility, storage, in vivo or storage half-life or stability, reduced immunogenicity, delayed or controlled release in vivo, etc.Other functions or activities include conjugates that reduce toxicity compared to unconjugated polypeptide sequences, conjugates that target cell or organ types more efficiently than unconjugated polypeptide sequences, or drugs to further combat the causes or effects associated with disorders or diseases (e.g., diabetes) as described herein.

[0436] The polypeptides provided herein can also be conjugated to large, slowly metabolic macromolecules such as proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads; polymeric amino acids, such as polyglutamic acid, polylysine; amino acid copolymers; inactivated virus particles; inactivated bacterial toxins, such as diphtheria, tetanus, cholera toxoids, leukotoxin molecules; inactivated bacteria; and dendritic cells.

[0437] Candidates for further components and molecules for conjugation to the polypeptide sequence of the present disclosure can include those suitable for isolation or purification.Specific non-limiting examples include binding molecules such as biotin (biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or molecules that constitute solid supports, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.Purification methods such as cation exchange chromatography can be used to separate conjugates by charge difference, which effectively separates conjugates into various molecular weights.The content of the fraction obtained by cation exchange chromatography can be identified by molecular weight using conventional methods, such as mass spectrometry, SDS-PAGE, or other known methods for separating molecular entities by molecular weight.

[0438] In some embodiments, the amino or carboxyl terminus of the polypeptide sequences of the present disclosure can be fused to an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, and thus biopharmaceutical products may require less frequent administration.

[0439] Fc can bind to the neonatal Fc receptor (FcRn) on endothelial cells lining blood vessels, and upon binding, the Fc fusion molecule is protected from degradation and re-released into the circulation, allowing the molecule to circulate longer. This Fc binding may be the mechanism by which endogenous IgG maintains a long plasma half-life. More recent Fc fusion technology links a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to traditional Fc fusion conjugates.

[0440] The present disclosure also contemplates the use of other modifications of polypeptides, whether known or developed in the future, to improve one or more properties.One such method for extending the circulating half-life of the polypeptides of the present disclosure, increasing stability, reducing clearance, or changing immunogenicity or allergenicity can involve the modification of polypeptide sequence by hesylation, which utilizes hydroxyethyl starch derivatives linked to other molecules to modify molecular characteristics.Various aspects of hesylation are described, for example, in US Patent Application Nos. 2007 / 0134197 and 2006 / 0258607.

[0441] In some embodiments, peptide derivatives such as tissue-specific antigens provided herein may include tissue-specific antigens with enhanced affinity. Such affinity-enhanced tissue-specific antigens may include one or more substitutions or modifications that result in enhanced immunogenicity compared to unmodified versions of tissue-specific antigens.

[0442] For example, an affinity-enhanced tissue-specific antigen may be prepared or derived from a parent peptide, where the affinity-enhanced tissue-specific antigen contains a substituted non-natural amino acid in place of a naturally occurring amino acid residue at one or more primary anchor positions, e.g., one primary anchor position, or two primary anchor positions.

[0443] The parent peptide may be an MHCI-restricted antigen and the peptide derivative may be an MHCI-restricted antigen that binds to at least the same MHCI molecule as the parent peptide, e.g., if the parent peptide binds to HLA-A*0201, then the peptide derivative also binds to HLA-A*0201. Additionally, the peptide derivative may be capable of inducing the expansion of T cells capable of binding to the parent peptide when the parent peptide is complexed with MHCI.

[0444] A peptide derivative may also have increased immunogenicity compared to the parent peptide, hi some embodiments, a peptide derivative exhibits at least one, or at least two, or at least three, or at least four, or all five of the following properties:

[0445] The first property is that the peptide derivative generates a T cell immune response that is greater than that generated by the parent peptide. In one embodiment, the parent peptide generates a detectable T cell immune response, but the peptide derivative generates a T cell immune response that is greater than that generated by the parent peptide. In another embodiment, the parent peptide does not generate a detectable T cell immune response, but the peptide derivative generates a T cell immune response that can be detected. In further embodiments, the immune response may be T cell lysis of the target cell, cytokine release, and / or T cell degranulation.

[0446] The second property is that the peptide derivative binds to MHCI with a higher affinity than the parent peptide binds to MHCI, i.e., the peptide derivative has a lower K D It is to have.

[0447] The third property is that the affinity of the T cell receptor for the complex formed between MHCI and the peptide derivative is higher than the affinity of the T cell receptor for the complex formed between MHCI and the parent peptide. This increased affinity can be determined using a tetramer assay (Laugel, B., et al., 2007, J. Biol. Chem. 282, 23799-23810; Holmberg, K., et al., 2003, J. Immunol. 171, 2427-2434; Yee, C., et al., 1999, J. Immunol. 162, 2227-2234).

[0448] The fourth property is that the complex formed between MHCI and the peptide derivative is more stable (ie, has a slower rate of dissociation) than the complex formed between MHCI and the parent peptide.

[0449] The fifth property is that the peptide derivatives induce the expansion of a broader number of T cell clones that recognize the parent peptide than does the parent peptide.

[0450] Methods for producing antigen-specific T cells for therapeutic use

[0451] Methods for producing antigen-specific T cells are provided herein. Methods for preparing T cell compositions, such as therapeutic T cell compositions, are provided herein. For example, the methods can include expanding or inducing antigen-specific T cells. The step of preparing (e.g., inducing or expanding) T cells can also refer to producing T cells, and in a broad sense, refers to any type of T cell (e.g., CD4 + T cells and CD8 + The present invention encompasses procedures for isolating, stimulating, culturing, inducing, and / or expanding immune cells (T cells) from a biological sample that is depleted of cells expressing CD14 and / or CD25. In one aspect, provided herein is a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, comprising incubating APCs with a population of immune cells from a biological sample that is depleted of cells expressing CD11b and / or CD19. In some embodiments, the method comprises preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, comprising incubating APCs with a population of immune cells from a biological sample that is depleted of cells expressing CD11b and / or CD19. In some embodiments, the method comprises incubating APCs with a population of immune cells from a biological sample that is depleted of cells expressing any CD11b and / or CD19 and / or CD14 and / or CD25 or any combination thereof.

[0452] In a second aspect, provided herein is a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) stimulated APCs with a population of immune cells from a biological sample.

[0453] In a third aspect, provided herein is a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample for a first period of time, and thereafter incubating at least one T cell of the biological sample with an APC.

[0454] In a fourth aspect, provided herein is a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods less than 28 days after incubating the population of immune cells with a first APC preparation of the one or more APC preparations, whereby at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is derived.

[0455] In a fifth aspect, provided herein is a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating a population of immune cells from a biological sample with three or fewer APC preparations for three or fewer separate time periods, whereby at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is derived.

[0456] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate periods of time, thereby stimulating the T cells to become antigen-specific T cells, wherein the percentage of antigen-specific T cells is greater than or equal to the total CD4 + T cells, total CD8 + at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the T cells, total T cells or total immune cells. In some embodiments, a method of preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with three or fewer APC preparations for three or fewer separate periods of time, thereby stimulating the T cells to become antigen-specific T cells. In some embodiments, a method of preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with two or fewer APC preparations for two or fewer separate periods of time, thereby stimulating the T cells to become antigen-specific T cells.

[0457] In some embodiments, a method is provided herein that includes incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate periods of time, thereby stimulating the T cells to become antigen-specific T cells, where the APC preparation is a PBMC cell population that is depleted of cells expressing one or more cell surface markers before antigen loading of the APC population. In some embodiments, CD14+ cells are depleted before antigen loading of the APC population. In some embodiments, CD25+ cells are depleted before antigen loading of the APC population. In some embodiments, CD11b+ cells are depleted before antigen loading of the APC population. In some embodiments, CD19+ cells are depleted before antigen loading of the APC population. In some embodiments, CD3+ cells are depleted before antigen loading of the APC population. In some embodiments, CD25+ cells and CD14+ cells are depleted before antigen loading of the APC population. In some embodiments, CD11b+ and CD25+ cells are depleted before antigen loading of the APC population. In some embodiments, CD11b+ and CD14+ cells are depleted before antigen loading of the APC population. In some embodiments, CD11b+, CD14+ and CD25+ cells are depleted before antigen loading of the APC population. In some embodiments, CD11b+ and CD19+ cells are depleted before antigen loading of the APC population. In some embodiments, CD11b+, CD19+ and CD25+ cells are depleted before antigen loading of the APC population. In some embodiments, CD11b+, CD14+, CD19+ and CD25+ cells are depleted before antigen loading of the APC population. In some embodiments, the method comprises adding a PBMC-derived population of APC-enriched cells depleted of CD3+ cells to any of the depleted APC populations. In some embodiments, the PBMC-derived population of APC-enriched cells is depleted of CD3+ and depleted of any one or more of CD11b+, CD14+, CD19+ or CD25+.

[0458] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the method comprises adding to the PBMC sample a composition comprising one or more antigenic peptides or nucleic acids encoding same, thereby loading APCs within the PBMCs with antigen for antigen presentation to T cells in the PBMCs.

[0459] In some embodiments, the method includes (a) obtaining a biological sample from a subject that contains at least one antigen presenting cell (APC); and (b) enriching cells expressing CD11c from the biological sample, thereby enriching for CD11c. + (c) obtaining a cell enriched sample; and + (d) incubating the cell enriched sample with at least one cytokine or growth factor for a first period of time; and (e) coupling at least one peptide to the CD11c peptide of (c). + (e) incubating the APC peptide-loaded sample with one or more cytokines or growth factors for a third period of time, thereby obtaining an APC peptide-loaded sample; (f) incubating APCs of the mature APC sample with a CD11b and / or CD14 and / or CD25-depleted sample comprising PBMCs for a fourth period of time; (g) incubating the PBMCs with APCs of the mature APC sample for a fifth period of time; (h) incubating the PBMCs with APCs of the mature APC sample for a sixth period of time; and (i) administering at least one T cell of the PBMCs to a subject in need thereof.

[0460] In some embodiments, the method includes (a) obtaining a biological sample from a subject that contains at least one antigen presenting cell (APC); and (b) enriching cells expressing CD14 from the biological sample, thereby enriching for CD14. + (c) obtaining a cell-enriched sample; and + (d) incubating the cell enriched sample with at least one cytokine or growth factor for a first period of time; and (e) incubating the cell enriched sample with at least one peptide to bind to the CD14 +(e) incubating the APC peptide-loaded sample with one or more cytokines or growth factors for a third period of time, thereby obtaining an APC peptide-loaded sample; (f) incubating APCs of the mature APC sample with a CD14 and / or CD25-depleted sample comprising PBMCs for a fourth period of time; (g) incubating the PBMCs with APCs of the mature APC sample for a fifth period of time; (h) incubating the PBMCs with APCs of the mature APC sample for a sixth period of time; and (i) administering at least one T cell of the PBMCs to a subject in need thereof.

[0461] In some embodiments, the method includes the steps of: (a) obtaining a biological sample from a subject comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD11b and / or CD19 from the biological sample, thereby obtaining a CD11b and / or CD19 cell-depleted sample; (c) incubating the CD11b and / or CD19 cell-depleted sample with FLT3L for a first period of time; (d) incubating at least one peptide with the CD11b and / or CD19 cell-depleted sample of (c) for a second period of time, thereby obtaining an APC peptide-loaded sample; and (e) incubating the CD11b and / or CD19 cell-depleted sample of (c) with FLT3L for a second period of time, thereby obtaining an APC peptide-loaded sample. (f) incubating PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; (g) incubating PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and (h) administering at least one T cell of the third stimulated PBMC sample to a subject in need thereof.

[0462] In some embodiments, the method includes (a) obtaining a biological sample from a subject comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD11b and / or CD19 and / or CD14 and / or CD25 from the biological sample, thereby obtaining a CD11b and / or CD19 cell-depleted sample; (c) incubating the CD11b and / or CD19 and / or CD14 and / or CD25 cell-depleted sample with FLT3L for a first period of time; and (d) incubating at least one peptide with the CD11b and / or CD19 and / or CD14 and / or CD25 cell-depleted sample of (c) for a second period of time. (e) incubating the APC peptide-loaded sample with at least one PBMC for a third period of time, thereby obtaining a first stimulated PBMC sample; (f) incubating PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; (g) incubating PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and (h) administering at least one T cell of the third stimulated PBMC sample to a subject in need thereof.

[0463] In some embodiments, the method includes the steps of: (a) obtaining a biological sample from a subject comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD14 and / or CD25 from the biological sample, thereby obtaining a CD14 and / or CD25 cell-depleted sample; (c) incubating the CD14 and / or CD25 cell-depleted sample with FLT3L for a first period of time; (d) incubating at least one peptide with the CD14 and / or CD25 cell-depleted sample of (c) for a second period of time, thereby obtaining an APC peptide-loaded sample; and (e) incubating the CD14 and / or CD25 cell-depleted sample of (c) with FLT3L for a second period of time, thereby obtaining an APC peptide-loaded sample. (f) incubating PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; (g) incubating PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and (h) administering at least one T cell of the third stimulated PBMC sample to a subject in need thereof.

[0464] In some embodiments, a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating APCs with a population of immune cells from a biological sample that has been depleted of cells expressing CD14 and / or CD25.

[0465] In some embodiments, provided herein is a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, comprising incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate periods of less than 28 days after incubating the population of immune cells with a first APC preparation of the one or more APC preparations, whereby at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is derived. In some embodiments, provided herein is a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, comprising incubating a population of immune cells from a biological sample with three or fewer APC preparations for three or fewer separate periods of time, whereby at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is derived.

[0466] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells (e.g., PBMCs) with APCs. In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells (e.g., PBMCs) with APCs for a period of time. In some embodiments, the population of immune cells is derived from a biological sample. In some embodiments, the population of immune cells is derived from a sample (e.g., biological sample) that has been depleted of CD14-expressing cells. In some embodiments, the population of immune cells is derived from a sample (e.g., biological sample) that has been depleted of CD25-expressing cells. In some embodiments, the population of immune cells is derived from a sample (e.g., biological sample) that has been depleted of CD14-expressing cells and CD25-expressing cells.

[0467] In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) stimulated APCs with a population of immune cells from a biological sample. In some embodiments, provided herein is a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample for a first period of time, and then incubating at least one T cell of the biological sample with APCs.

[0468] In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells from a sample (e.g., a biological sample) with FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells from a sample (e.g., a biological sample) with APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a sample (e.g., a biological sample) with APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample (e.g., for a period of time) and then contacting the T cells of the biological sample with APCs. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells from a sample (e.g., biological sample) with one or more APC preparations. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a sample (e.g., biological sample) with one or more APC preparations for one or more separate periods of time.In some embodiments, a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a sample (e.g., a biological sample) with one or more APC preparations for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate time periods. In some embodiments, the one or more separate time periods are less than 28 days calculated from incubating the population of immune cells with a first APC preparation of the one or more APC preparations.

[0469] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells with APCs for a period of time, wherein the population of immune cells is derived from a biological sample comprising PBMCs. In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells with APCs for a period of time, wherein the population of immune cells is derived from a biological sample that has been depleted of CD14 and / or CD25 expressing cells.

[0470] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) for a period of time.

[0471] In some embodiments, a method of preparing a pharmaceutical composition comprising antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample, and then contacting the T cells of the biological sample with APCs.

[0472] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods, thereby inducing or expanding antigen-specific T cells, wherein the one or more separate time periods are less than 28 days calculated from incubating the population of immune cells with a first APC preparation of the one or more APC preparations. In some embodiments, incubating the population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods is performed in a medium containing IL-7, IL-15, or a combination thereof. In some embodiments, the medium further comprises an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof. The IDO inhibitor can be epacadostat, navoximide, 1-methyltryptophan, or a combination thereof. In some embodiments, the IDO inhibitor is an antigen-specific CD8 + In some embodiments, the IDO inhibitor may increase the number of memory CD8 + The functional profile of the T cell response may be maintained. PD-1 antibodies may increase the absolute number of antigen-specific memory CD8+ T cell responses. PD-1 antibodies may increase the proliferation rate of cells treated with such antibodies. The addition of IL-12 may increase the number of antigen-specific cells and / or CD8 + This can result in an increase in the frequency of T cells.

[0473] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate periods of time, thereby expanding or inducing the antigen-specific T cells, the antigen-specific T cells, the antigen-specific CD4 + T cells, or antigen-specific CD8 + The percentage of T cells is: total T cells, total CD4 +T cells, total CD8 + at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the T cells, total immune cells, or total cells.

[0474] In some embodiments, a method of preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with three or fewer APC preparations for three or fewer separate time periods, thereby stimulating the T cells to become antigen-specific T cells.

[0475] In some embodiments, the population of immune cells is derived from a biological sample that is depleted of CD14 and / or CD25 expressing cells. In some embodiments, the APC is an FMS-like tyrosine kinase 3 receptor ligand (FL...

Claims

1. A composition for use in the treatment of a tumor in a target tissue in a human subject, said composition comprising (a) a tissue-specific antigen peptide comprising an epitope sequence of a protein, said epitope sequence being expressed by said tumor of said target tissue, the tissue-specific antigen peptide; (b) a polynucleotide encoding said tissue-specific antigen peptide; (c) one or more antigen-presenting cells (APCs) presenting said epitope sequence; (d) (i) a T cell receptor (TCR) specific for a complex of said epitope sequence and (ii) a protein encoded by an HLA allele of said human subject; or (e) a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising said TCR comprising any one of, said epitope sequence binds or is predicted to bind to a protein encoded by an HLA allele expressed by said human subject, the gene encoding the tissue-specific antigen peptide of (a) or (b) has an expression level in said target tissue that is at least 2-fold higher than the expression level of the gene encoding the tissue-specific antigen peptide in each of a plurality of non-target tissues different from said target tissue of said human subject, the tissue-specific antigen peptide of (a) or (b) is specific for a non-essential tissue, said non-essential tissue being a tissue that is not necessary for the survival of said human subject, composition.

2. The composition for use according to claim 1, wherein the tissue-specific antigen peptide comprising the epitope sequence of the protein is encoded by PRAME or MAGEA4, and the epitope sequence has a sequence identity of 70% to 100% with SEQ ID NO: 6139 (PRAME) or SEQ ID NO: 4653 (MAGEA4). **Claim 3**: The composition for use according to claim 1, wherein the tissue-specific antigen peptide containing the epitope sequence of the protein is encoded by a gene selected from the group consisting of ANKRD30A, COL10A1, CTCFL, PPIAL4G, POTEE, DLL3, MMP13, SSX1, DCAF4L2, MAGEA4, MAGEA11, MAGEC2, MAGEA12, PRAME, CLDN6, EPYC, KLK3, KLK2, KLK4, TGM4, POTEG, RLN1, POTEH, SLC45A2, TSPAN10, PAGE5, CSAG1, PRDM7, TG, TSHR, RSPH6A, SCXB, HIST1H4K, ALPPL2, PRM2, PRM1, TNP1, LELP1, HMGB4, AKAP4, CETN1, UBQLN3, ACTL7A, ACTL9, ACTRT2, PGK2, C2orf53, KIF2B, ADAD1, SPATA8, CCDC70, TPD52L3, ACTL7B, DMRTB1, SYCN, CELA2A, CELA2B, PNIPRP1, CTRC, AMY2A, SERPINI2, RBPJL, AQP12A, IAPP, KIRREL2, G6PC2, AQP12B, CYP11B1, CYP11B2, STAR, CYP11A1, and MC2R. **Claim 4** The composition for use according to any one of claims 1 to 3, wherein the mRNA expression level of the gene encoding the tissue-specific antigen peptide is at most 5 mRNA transcripts per million total mRNA transcripts in each non-target tissue of the human subject, and at most 5 mRNA transcripts in non-target essential tissues. **Claim 5** The composition for use according to claim 4, wherein the mRNA expression level of the gene encoding the tissue-specific antigen peptide is at least 100 mRNA transcripts per million total mRNA transcripts in the target tissue. **Claim 6**: (i) The human subject is a female subject, and the tissue-specific antigen is specific to non-essential tissues selected from the group consisting of the bulbourethral gland, the epididymis, the penis, the prostate, the scrotum, the seminal vesicle, the testis, and any combination thereof; (ii) The human subject is a male subject, and the tissue-specific antigen is specific to non-essential tissues selected from the group consisting of the Bartholin gland, the fallopian tube, the ovary, the Skene gland, the uterus, the cervix, the vagina, and any combination thereof. (iii) the human subject is a type 1 diabetic patient and the tissue-specific antigen is specific to the pancreas, or (iv) the human subject has an autoimmune condition of the thyroid and the tissue-specific antigen is specific to the thyroid, A composition for use according to any one of claims 1 to 5.

7. The composition for use according to any one of claims 1 to 6, wherein the epitope sequence is SPSVSQLSVL (SEQ ID NO: 6139) and the human subject expresses a protein encoded by the HLA-B07:02 allele.

8. The composition for use according to any one of claims 1 to 6, wherein the epitope sequence is GVYDGREHTV (SEQ ID NO: 4653) and the human subject expresses a protein encoded by the HLA-A02:01 allele.

9. The composition for use according to any one of claims 1 to 8, wherein the at least one antigen-specific T cell or the TCR is specific for a complex of (a) the epitope sequence of SPSVSQLSVL (SEQ ID NO: 6139) and a protein encoded by the HLA-B07:02 allele of the human subject, or (b) the epitope sequence of GVYDGREHTV (SEQ ID NO: 4653) and a protein encoded by the HLA-A02:01 allele of the human subject.

10. The composition for use according to any one of claims 1 or 3 to 6, wherein the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 1 to 4652, 4654 to 6138, and 6140 to 8962.

11. The composition for use according to any one of claims 1, 3 to 6 or 10, wherein the epitope sequence has 70% to 100% sequence identity to a peptide sequence selected from the group consisting of SEQ ID NOs: 3441 to 4274, 5285 to 6084, 6580 to 6845, and 8100 to 8434.

12. The composition for use according to any one of claims 1, 3 to 6 or 10 to 11, wherein the epitope sequence of the protein is an epitope sequence of KLK2, KLK3 or KLK4 and the non-essential tissue is the prostate.

13. The composition for use according to claim 12, wherein the subject is female.

14. The composition for use according to any one of claims 1, 3 to 6 or 10 to 13, wherein the epitope sequence of the protein is the epitope sequence of KLK2.

15. The composition for use according to any one of claims 1, 3 to 6 or 10 to 14, having 70% to 100% sequence identity to a peptide sequence selected from the group consisting of AYSEKVTEF (SEQ ID NO: 3534), GLWTGGKDT CGV (SEQ ID NO: 3468), HPEDTGQVF (SEQ ID NO: 3988), HPEYNRPLL (SEQ ID NO: 4143), QRVPVSHSF (SEQ ID NO: 3544), SESDTIRSI (SEQ ID NO: 4176), SLFHPEDTGQV (SEQ ID NO: 3775), SLQCVSLHL (SEQ ID NO: 3456), VILLGRHSL (SEQ ID NO: 3891), VLVHPQWVL (SEQ ID NO: 3757), LFHPEDTGQVF (SEQ ID NO: 3827), RPRSLQCVSL (SEQ ID NO: 3578), GYLQGLVSF (SEQ ID NO: 4094), IRNKSVILL (SEQ ID NO: 3974), KLQCVDLHV (SEQ ID NO: 3740), LLANGRMMPV (SEQ ID NO: 4029), LRPGDDSTL (SEQ ID NO: 3767), MPALPMVL (SEQ ID NO: 3874), NRPLLANDL (SEQ ID NO: 4216), TWIAAPPLQV (SEQ ID NO: 3784), VFQVSHSF (SEQ ID NO: 3828) and YSEKVTEFML (SEQ ID NO: 3454).

16. The at least one antigen-specific T cell or the TCR is (a) a complex of the epitope sequence of AYSEKVTEF (SEQ ID NO: 3534) and a protein encoded by the HLA-C06:02 or HLA-A24:02 allele of the human subject; (b) a complex of the epitope sequence of GLWTGGKDT CGV (SEQ ID NO: 3468) and a protein encoded by the HLA-A02:01 allele of the human subject; (c) a complex of the epitope sequence of HPEDTGQVF (SEQ ID NO: 3988) and a protein encoded by the HLA-C04:01 or HLA-C07:01 allele of the human subject; (d) A complex of the epitope sequence of HPEYNRPLL (SEQ ID NO: 4143) and a protein encoded by the HLA-C07:01 or HLA-B07:02 allele of the human subject, (e) A complex of the epitope sequence of QRVPVSHSF (SEQ ID NO: 3544) and a protein encoded by the HLA-C07:01, HLA-C07:02 or HLA-A24:02 allele of the human subject, (f) A complex of the epitope sequence of SESDTIRSI (SEQ ID NO: 4176) and a protein encoded by the HLA-B13:02 allele of the human subject, (g) A complex of the epitope sequence of SLFHPEDTGQV (SEQ ID NO: 3775) and a protein encoded by the HLA-A02:01 allele of the human subject, (h) A complex of the epitope sequence of SLQCVSLHL (SEQ ID NO: 3456) and a protein encoded by the HLA-A02:01 allele of the human subject, (i) A complex of the epitope sequence of VILLGRHSL (SEQ ID NO: 3891) and a protein encoded by the HLA-B08:01 allele of the human subject, (j) A complex of the epitope sequence of VLVHPQWVL (SEQ ID NO: 3757) and a protein encoded by the HLA-A02:01 allele of the human subject, (k) A complex of the epitope sequence of LFHPEDTGQVF (SEQ ID NO: 3827) and a protein encoded by the HLA-A24:02 allele of the human subject, (l) A complex of the epitope sequence of RPRSLQCVSL (SEQ ID NO: 3578) and a protein encoded by the HLA-B07:02 allele of the human subject, (m) A complex of the epitope sequence of GYLQGLVSF (SEQ ID NO: 4094) and a protein encoded by the HLA-A24:02 allele of the human subject, (n) A complex of the epitope sequence of IRNKSVILL (SEQ ID NO: 3974) and a protein encoded by the HLA-C06:02, HLA-C07:02 or HLA-C07:01 allele of the human subject, (o) A complex of the epitope sequence of KLQCVDLHV (SEQ ID NO: 3740) and a protein encoded by the HLA-A02:01 allele of the human subject, The complex of the epitope sequence of (p) LLANGRMCTV (SEQ ID NO: 4029) and the protein encoded by the HLA-A02:01 allele of the human subject, The complex of the epitope sequence of (q) LRPGDDSTL (SEQ ID NO: 3767) and the protein encoded by the HLA-C07:02 allele of the human subject, The complex of the epitope sequence of (r) MPALPMVL (SEQ ID NO: 3874) and the protein encoded by the HLA-B07:02 allele of the human subject, The complex of the epitope sequence of (s) NRPLLANDL (SEQ ID NO: 4216) and the protein encoded by the HLA-C06:02, HLA-C07:02 or HLA-C01:02 allele of the human subject, The complex of the epitope sequence of (t) TWIAPPQLQV (SEQ ID NO: 3784) and the protein encoded by the HLA-C04:01 or HLA-A02:01 allele of the human subject, The complex of the epitope sequence of (u) VFQVSHSF (SEQ ID NO: 3828) and the protein encoded by the HLA-C07:02 or HLA-A24:02 allele of the human subject, or The complex of the epitope sequence of (v) YSEKVTEFML (SEQ ID NO: 3454) and the protein encoded by the HLA-A01:01 allele of the human subject A composition for use according to any one of claims 1, 3 to 6 or 10 to 15, which is specific for.

17. The at least one antigen-specific T cell or the TCR is The complex of the epitope sequence of (a) AYSEKVTEF (SEQ ID NO: 3534) and the protein encoded by the HLA-C06:02 or HLA-A24:02 allele of the human subject, The complex of the epitope sequence of (b) GLWTGGKDT CGV (SEQ ID NO: 3468) and the protein encoded by the HLA-A02:01 allele of the human subject, The complex of the epitope sequence of (c) QRVPVSHSF (SEQ ID NO: 3544) and the protein encoded by the HLA-C07:01, HLA-C07:02 or HLA-A24:02 allele of the human subject, (d) A complex of the epitope sequence of SLQCVSLHL (SEQ ID NO: 3456) and the protein encoded by the HLA-A02:01 allele of the human subject, (e) A complex of the epitope sequence of RPRSLQCVSL (SEQ ID NO: 3578) and the protein encoded by the HLA-B07:02 allele of the human subject, (f) A complex of the epitope sequence of YSEKVTEFML (SEQ ID NO: 3454) and the protein encoded by the HLA-A01:01 allele of the human subject A composition for use according to claim 16, which is specific for

18. A method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for a complex of (i) an epitope sequence of a tissue-specific antigen peptide and (ii) a protein encoded by an HLA allele of a human subject, (a) Contacting T cells ex vivo with an antigen peptide comprising the epitope sequence complexed with HLA of an antigen-presenting cell (APC), or with a cancer cell, wherein the T cells have reduced tolerance to the antigenic peptide or no tolerance, or the T cells have reduced tolerance to the tissue from which the antigen peptide is derived or no immune tolerance, (b) Determining the sequence of the TCR of the T cells that recognize the antigen peptide complexed with the HLA; (c) (i) Expanding the T cells in the presence of the antigen peptide complexed with the HLA, or (ii) expressing a recombinant polynucleotide encoding the sequence of the TCR in isolated T cells thereby preparing the T cells comprising the TCR, wherein the T cells are derived from a human subject, the tissue-specific antigen peptide is specific for a non-essential tissue, and the non-essential tissue is a tissue that is not necessary for the survival of the human subject.

19. The method according to claim 18, wherein the subject is female, the non-essential tissue is the prostate, and the epitope sequence of the tissue-specific antigen peptide is the epitope sequence of KLK2, KLK3 or KLK4.

20. The TCR is (a) A complex of the epitope sequence of AYSEKVTEF (SEQ ID NO: 3534) and the protein encoded by the HLA-C06:02 or HLA-A24:02 allele of the human subject, (b) A complex of an epitope sequence of GLWTGGKDTCCGV (SEQ ID NO: 3468) and a protein encoded by the HLA-A02:01 allele of the human subject, (c) A complex of an epitope sequence of QRVPVSHSF (SEQ ID NO: 3544) and a protein encoded by the HLA-C07:01, HLA-C07:02 or HLA-A24:02 allele of the human subject, (d) A complex of an epitope sequence of SLQCVSLHL (SEQ ID NO: 3456) and a protein encoded by the HLA-A02:01 allele of the human subject, (e) A complex of an epitope sequence of RPRSLQCVSL (SEQ ID NO: 3578) and a protein encoded by the HLA-B07:02 allele of the human subject, or (f) A complex of an epitope sequence of YSEKVTEFML (SEQ ID NO: 3454) and a protein encoded by the HLA-A01:01 allele of the human subject The method according to claim 18 or 19, which is specific to. **Claim 21** The method according to any one of claims 18 to 20, further comprising the step of depleting CD14+ cells and / or CD25+ cells from a population of immune cells containing APCs prior to the ex vivo contact of the T cells with the antigenic peptide. **Claim 22** The method according to any one of claims 18 to 21, wherein the APC is an APC stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

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