Novel tumor-specific antigens against acute myeloid leukemia (AML) and uses thereof

Abstract

Acute myeloid leukemia (AML) has not benefited from innovative immunotherapy, primarily because of the lack of actionable immune targets. To provide a new tumor-specific antigen (TSA) shared by most AML cells.SOLUTION: Most of the TSAs described herein are derived from aberrantly expressed non-mutated genomic sequences that are not expressed in normal tissues, such as intronic and intergenic sequences. Nucleic acids, compositions, cells, and vaccines derived from these TSAs are described. Uses of the TSAs, nucleic acids, compositions, cells, and vaccines for the treatment of leukemia, such as AML, are also described.SELECTED DRAWING: Figure 5A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 63 / 009,853, filed April 14, 2020. No. 6,239,999, filed on Oct. 1, 2003, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Not applicable.

[0003] The present invention relates generally to cancer, and more specifically to T cell-based cancer immunotherapy. This invention relates to tumor antigens specific to acute myeloid leukemia. [Background technology]

[0004] Acute myeloid leukemia (AML), the most aggressive hematologic malignancy, is caused by abnormal epigenetic impaired mitochondrial patterning, impaired mitochondrial proteostasis, and a relatively small number of mutations It is a heterogeneous disease characterized by tos et al.,2016, Ishizawa et al.,2019, Fen nell et al., 2019). Of note, genetic and epigenetic differences in AML Genetic changes may occur years before diagnosis (Abelson et al., 2018, Desai et al., 2018). Not only is it necessary to eliminate tumor cells, but also leukemia stem cells (Shlush et al. al., 2017, Boyd et al., 2018). Currently, most patients The 5-year overall survival rate for patients who relapsed after chemotherapy was 40% for those under 60 years of age and 40% for those over 60 years of age. In patients with AML (which account for the majority of cases), only 10-20% t al.,2018).

[0005] In recent years, enthusiasm for cancer immunotherapy has been driven primarily by two major breakthroughs: i) melanoma and Immune checkpoint therapy for the treatment of selected types of solid tumors, and ii) phosphorylation Therapy for pancreatic malignancies has been facilitated by chimeric antigen receptors. However, AML has not benefited from such innovations, primarily due to the lack of actionable immune targets. Major histocompatibility complex MHC-associated peptides (MAPs) recognized by T cells are involved in anti-cancer responses. In line with the idea that evidence is at the core of the answer (Coulie et al., 2014), suggested that AML cells must present immunogenic MAP to CD8 T cells. i) AML cells express high density of MHC class I molecules (Berlin et al., 2015), and ii) the bone marrow of AML patients contains CD8 T cells and is depleted. possessing the phenotypic and transcriptional characteristics of thirst (and therefore antigen recognition) (Knaus et al. However, AML antigens that can induce protective immune responses are The nature of the remains unknown.

[0006] The first class of MAPs that attracted the attention of cancer immunologists were those found in tumor cells compared with normal cells. High-affinity T cells that recognize self-antigens are overexpressed tumor-associated antigens (TAA). TAAs are inherently low-affinity because they are eliminated by the central immune tolerance process of thymic selection. Therefore, TAA-based vaccines may be effective in preventing the progression of AML. Most studied AML TAA: Wilms tumor Tumor 1 (WT1) produced particularly disappointing results (Di Stasi et al. ,2015, Maslak et al.,2018, Rashidi and Wal Importantly, a recent report showed that WT1-derived peptides targeting TCR gene therapy (in which a gene is engineered to express a high-affinity TCR against a selected antigen) T cell-engineered T cells provide durable protection against relapse in recipients of allogeneic hematopoietic stem cell transplants. It has been shown that this can be achieved (Chapuis et al., 2019). These studies have demonstrated that WT1-derived peptides have low immunogenicity and fully demonstrate their therapeutic potential. This suggests that targeting with engineered T cells is necessary to achieve this.

[0007] In contrast to TAAs, tumor-specific antigens (TSAs) are presented exclusively by tumor cells To date, mutated TSA (mTSA), also known as neoantigens, has been reported to be a type of MAP. The search for vaccines against tumors has recently attracted considerable attention. A is not found in medullary thymic cells (mTECs), which induce central immune tolerance, and therefore does not induce hyperimmune responses. However, mTSA presents two caveats. First, these are generally unique to each patient's tumor (individual neoantigens). Second, they are initially AML is less common than previously predicted (Knaus et al., 2018). Consistent with the low mutation burden in primary AML cells, only one mTSA was detected by mass spectrometry (MS spectrometry) in primary AML cells. ) analysis (van der Lee et al., 2019). The therapeutic potential of this mTSA, which originates from a frameshift in the NPM1 gene, has yet to be evaluated. Although not yet available, available evidence suggests that it does not induce a spontaneous immune response in patients with AML. (van der Lee et al., 2019).

[0008] Considering this, identifying antigens that can induce therapeutic immune responses against AML is crucial. Such antigens are expected to be useful as vaccines (± immune checkpoint inhibitors). or as targets for T cell receptor-based approaches (cell therapy, bispecific biologics) It can be used as follows.

[0009] This description refers to several documents, the contents of which are incorporated herein by reference in their entirety. It is incorporated into the detailed text. Summary of the Invention

[0010] The present disclosure provides the following items 1 to 67. 1. A leukemia tumor antigen peptide (TAP) comprising one of the following amino acid sequences: [Table 1-1] [Table 1-2]

[0011] 2. The method according to item 1, comprising one of the amino acid sequences shown in SEQ ID NOs: 97 to 154. Leukemia TAP.

[0012] 3. The leukemia TAP binds to the HLA-A*01:01 molecule and has the amino acid sequence NTS HLPLIY (SEQ ID NO: 48), HTDDIENAKY (SEQ ID NO: 67), YSHHSG LEY (SEQ ID NO: 89), ILDLESRY (SEQ ID NO: 134), VTDLLALTV( SEQ ID NO: 151), or LSDRQLSL (SEQ ID NO: 164), preferably ILDLES Item 1, comprising RY (SEQ ID NO: 134) or VTDLLALTV (SEQ ID NO: 151) 2. Leukemia TAP according to claim 2.

[0013] 4. The leukemia TAP binds to the HLA-A*02:01 molecule and has the amino acid sequence FLL EFKPVS (SEQ ID NO: 7), LLSRGLLFRI (SEQ ID NO: 11), LLDNILQ SI (SEQ ID NO: 27), FLASFVEKTVL (SEQ ID NO: 32), ILASHNLTV (SEQ ID NO: 33), IQLTSVHLL (SEQ ID NO: 34), LELISFLPVL (SEQ ID NO: 35), No. 35), LLLPESPSI (SEQ ID NO: 43), ALASHLIEA (SEQ ID NO: 51 ), ALDDITIQL (SEQ ID NO: 52), ALGNTVPAV (SEQ ID NO: 53), AL LPAVPSL (SEQ ID NO: 54), GLYYKLHNV (SEQ ID NO: 61), HLLSET PQL (SEQ ID NO: 65), KLLEKAFSI (SEQ ID NO: 72), SLWGQPAEA ( SEQ ID NO: 77), SVFAGVVGV (SEQ ID NO: 82), VLVPYEPPQV (SEQ ID NO: No. 86), VLFGGKVSGA (SEQ ID NO: 104), KLQDKEIGL (SEQ ID NO: 1 08), TLNQGINVYI (SEQ ID NO: 119), ALPVALPSL (SEQ ID NO: 12 3), ALDPLLLRI (SEQ ID NO: 130), KILDVNLRI (SEQ ID NO: 132) , SLLSGLLRA (SEQ ID NO: 146), SLDLLPLSI (SEQ ID NO: 150), I LLEEQSLI (SEQ ID NO: 167), LTSISIRPV (SEQ ID NO: 168), TIS ECPLLI (SEQ ID NO: 169), ILLSNFSSL (SEQ ID NO: 171), RMVAY LQQL (SEQ ID NO: 183), or KLNQAFLVL (SEQ ID NO: 188), preferably VLFGGKVSGA (SEQ ID NO: 104), KLQDKEIGL (SEQ ID NO: 108), T LNQGINVYI (SEQ ID NO: 119), ALPVALPSL (SEQ ID NO: 123), AL DPLLLRI (SEQ ID NO: 130), KILDVNLRI (SEQ ID NO: 132), SLLS GLLRA (SEQ ID NO: 146), or SLDLLPLSI (SEQ ID NO: 150). Leukemia TAP according to items 1 or 2.

[0014] 5. The leukemia TAP binds to the HLA-A*03:01 molecule and has the amino acid sequence RSA SSATQVHK (SEQ ID NO: 5), IVATGSLLK (SEQ ID NO: 18), KIKNKT KNK (SEQ ID NO: 19), KLLSLTIYK (SEQ ID NO: 20), ITSSAVTTAL K (SEQ ID NO: 42), VILIPLPPK (SEQ ID NO: 44), NVNRPLTMK (SEQ ID NO: No. 74), SVYKYLKAK (SEQ ID NO: 91), VVFPFPVNK (SEQ ID NO: 10 5), ILFQNSALK (SEQ ID NO: 113), TVIRIAIVNK (SEQ ID NO: 126 ), ISLIVTGGLK (SEQ ID NO: 131), HVSDGSTALK (SEQ ID NO: 159) , IAYSVRALR (SEQ ID NO: 160), LSSRLPLGK (SEQ ID NO: 180), or is RLVSSTLLQK (SEQ ID NO: 189), preferably VVFPFPVNK (SEQ ID NO: 105), ILFQNSALK (SEQ ID NO: 113), TVIRIAIVNK (SEQ ID NO: 1 26), or ISLIVTGLK (SEQ ID NO: 131). Disease TAP.

[0015] 6. The leukemia TAP binds to the HLA-A*11:01 molecule and has the amino acid sequence SAS SATQVHK (SEQ ID NO: 6), AVLLPKPPK (SEQ ID NO: 45), ATQNTII GK (SEQ ID NO: 96), SLLIIPKKK (SEQ ID NO: 106), SVQLLEQAIH K (SEQ ID NO: 121), STFSLYLKK (SEQ ID NO: 149), or RTQITKVS LKK (SEQ ID NO: 152), preferably SLLIIPKKK (SEQ ID NO: 106), SVQ LLEQAIHK (SEQ ID NO: 121), STFSLYLKK (SEQ ID NO: 149), or R 3. The leukemia TAP of item 1 or 2, comprising TQITKVSLKK (SEQ ID NO: 152).

[0016] 7. The leukemia TAP binds to the HLA-A*24:02 molecule and has the amino acid sequence LYF LGHGSI (SEQ ID NO: 13), NFCMLHQSI (SEQ ID NO: 36), KFSNVTM LF (SEQ ID NO: 71), IYQFIMDRF (SEQ ID NO: 92), LYPSKLTHF (SEQ ID NO: sequence number 95), or RYLANKIHI (SEQ ID NO: 145), preferably RYLANKI 3. The leukemia TAP of item 1 or 2, comprising HI (SEQ ID NO: 145).

[0017] 8. The leukemia TAP binds to the HLA-A*26:01 molecule and has the amino acid sequence ETT Item 1, including SQVRKY (SEQ ID NO: 59) or TVPGIQRY (SEQ ID NO: 185). Or the leukemia TAP according to 2.

[0018] 9. The leukemia TAP binds to the HLA-A*29:02 molecule and has the amino acid sequence VVF DKSDLAKY (SEQ ID NO: 88), FNVALNARY (SEQ ID NO: 99), or LGI SLTLKY (SEQ ID NO: 138), preferably FNVALNARY (SEQ ID NO: 99) or 3. The leukemia virus according to item 1 or 2, comprising one of the following: LGISLTLKY (SEQ ID NO: 138). Disease TAP.

[0019] 10. The leukemia TAP binds to the HLA-A*30:01 molecule and has the amino acid sequence TS RLPKIQK (SEQ ID NO: 26), LSWGYFLFK (SEQ ID NO: 29), or LSHP 3. The leukemia TAP of item 1 or 2, comprising APSSL (SEQ ID NO: 165).

[0020] 11. The leukemia TAP binds to the HLA-A*68:02 molecule and has the amino acid sequence NV SSHVHTV (SEQ ID NO: 50) or SSSPVRGPSV (SEQ ID NO: 148), preferably 3. The leukemia TA according to item 1 or 2, comprising SSSPVRGPSV (SEQ ID NO: 148). P.

[0021] 12. The leukemia TAP binds to the HLA-B*07:02 molecule and has the amino acid sequence GP QVRGSI (SEQ ID NO: 8), SPQSGPAL (SEQ ID NO: 25), VPAPAQAI ( SEQ ID NO: 40), APAPPPVAV (SEQ ID NO: 55), APDKKITL (SEQ ID NO: 5 6), KPMPTKVVF (SEQ ID NO: 73), SPADHRGYASL (SEQ ID NO: 78) , SPQSAAAEL (SEQ ID NO: 79), SPVVHQSL (SEQ ID NO: 80), SPYR TPVL (SEQ ID NO: 81), PPRPLGAQV (SEQ ID NO: 98), GPGSRESTL (SEQ ID NO: 100), APGAAGQRL (SEQ ID NO: 107), TPGRSTQAI (SEQ ID NO: 108) Sequence number 110), APRGTAAL (SEQ ID NO: 111), SPVVRVGL (SEQ ID NO: 1 18), RPRGPRTAP (SEQ ID NO: 120), TLRSPGSSL (SEQ ID NO: 128 ), TVRGDVSSL (SEQ ID NO: 129), LPSFSHFLLL (SEQ ID NO: 157) , PRGFLSAL (SEQ ID NO: 161), IPLNPFSSL (SEQ ID NO: 163), LP SFSRPSGII (SEQ ID NO: 179), or SPARALPSL (SEQ ID NO: 184), Preferably, PPRPLGAQV (SEQ ID NO: 98), GPGSRESTL (SEQ ID NO: 100 ), APGAAGQRL (SEQ ID NO: 107), TPGRSTQAI (SEQ ID NO: 110), APRGTAAL (SEQ ID NO: 111), SPVVRVGL (SEQ ID NO: 118), RPRG PRTAP (SEQ ID NO: 120), TLRSPGSSL (SEQ ID NO: 128), or TVRG 3. The leukemia TAP of item 1 or 2, comprising DVSSL (SEQ ID NO: 129).

[0022] 13. The leukemia TAP binds to the HLA-B*08:01 molecule and has the amino acid sequence SG KLRVAL (SEQ ID NO: 4), NPLQLSLSI (SEQ ID NO: 14), DLMLRESL (SEQ ID NO: 15), IALYKQVL (SEQ ID NO: 17), NILKKTVL (SEQ ID NO: 2 1), NPKLKDIL (SEQ ID NO: 22), NQKKVRIL (SEQ ID NO: 23), RLE VRKVIL (SEQ ID NO: 28), EGKIKRNI (SEQ ID NO: 31), LNHLRTSI (SEQ ID NO: 47), SIQRNLSL (SEQ ID NO: 49), IPHQRSSL (SEQ ID NO: 1 01), NLKEKKALF (SEQ ID NO: 103), ILKKNISI (SEQ ID NO: 114) , VLKEKNASL (SEQ ID NO: 137), DLLPKKLL (SEQ ID NO: 139), SR IHLVVL (SEQ ID NO: 147), QIKTKLLGSL (SEQ ID NO: 156), TLKL KKIFF (SEQ ID NO: 170), MIGIKRLL (SEQ ID NO: 181), or NLKKR EIL (SEQ ID NO: 182), preferably IPHQRSSL (SEQ ID NO: 101), NLKE KKALF (SEQ ID NO: 103), ILKKNISI (SEQ ID NO: 114), VLKEKNA SL (SEQ ID NO: 137), DLLPKKLL (SEQ ID NO: 139), or SRIHLVVL (SEQ ID NO: 147).

[0023] 14. The leukemia TAP binds to the HLA-B*14:01 molecule and has the amino acid sequence DR ELRNLEL (SEQ ID NO: 2), SNLIRTGSH (SEQ ID NO: 39), DQVIRLA GL (SEQ ID NO: 58), HQLYRASAL (SEQ ID NO: 66), SLQILVSSL (SEQ ID NO: Sequence number 124), ERVYIRASL (SEQ ID NO: 133), LYIKSLPAL (SEQ ID NO: No. 136), IAGALRSVL (SEQ ID NO: 141), ISSWLISSL (SEQ ID NO: 1 62), DRGILRNLL (SEQ ID NO: 175), GLRLIHVSL (SEQ ID NO: 176 ), or GLRLLHVSL (SEQ ID NO: 177), preferably SLQILVSSL (SEQ ID NO: SEQ ID NO: 124), ERVYIRASL (SEQ ID NO: 133), LYIKSLPAL (SEQ ID NO: 136), or IAGALRSVL (SEQ ID NO: 141). Blood disease TAP.

[0024] 15. The leukemia TAP binds to the HLA-B*15:01 molecule and has the amino acid sequence KI KVFSKVY (SEQ ID NO: 10), AQMNLLQKY (SEQ ID NO: 57), GQKPVI Item 1 or 2, including LTY (SEQ ID NO: 62), or AQKVSVGQAA (SEQ ID NO: 94) 2. Leukemia TAP.

[0025] 16. The leukemia TAP binds to the HLA-B*27:05 molecule and has the amino acid sequence RQ ISVQASL (SEQ ID NO: 1) or LRSQILSY (SEQ ID NO: 144), preferably L 3. The leukemia TAP of item 1 or 2, comprising RSQILSY (SEQ ID NO: 144).

[0026] 17. The leukemia TAP binds to the HLA-B*38:01 molecule and has the amino acid sequence TQ VSMAESI (SEQ ID NO: 46), HHLVETLKF (SEQ ID NO: 64), or THGS 3. The leukemia TAP of item 1 or 2, comprising EQLHL (SEQ ID NO: 84).

[0027] 18. The leukemia TAP binds to the HLA-B*40:01 molecule and has the amino acid sequence RE Contains PYELTVPAL (SEQ ID NO: 75) or SEAEAAKNAL (SEQ ID NO: 76) , leukemia TAP according to item 1 or 2).

[0028] 19. The leukemia TAP binds to the HLA-B*44:03 molecule and has the amino acid sequence KE 3. The leukemia TAP of item 1 or 2, comprising IFLELRL (SEQ ID NO: 127).

[0029] 20. The leukemia TAP binds to the HLA-B*51:01 molecule and has the amino acid sequence LP IASASLL (SEQ ID NO: 12), PFPLVQVEPV (SEQ ID NO: 24), PLPIV PAL (SEQ ID NO: 38), IAAPILHV (SEQ ID NO: 68), IPLAVRTI (SEQ ID NO: SEQ ID NO: 115), LPRNKPLL (SEQ ID NO: 116), or LPSHSLLI (SEQ ID NO: 190), preferably IPLAVRTI (SEQ ID NO: 115) or LPRNKPLL (SEQ ID NO: The leukemia TAP according to item 1 or 2, comprising the TAP of item 116).

[0030] 21. The leukemia TAP binds to the HLA-B*57:01 molecule and has the amino acid sequence GA RQQIHSW (SEQ ID NO: 3), VTFKLSLF (SEQ ID NO: 16), KGHGGPRS W (SEQ ID NO: 41), GSLDFQRGW (SEQ ID NO: 63), KAFPFHIIF (SEQ ID NO: No. 69), GTLQGIRAW (SEQ ID NO: 93), RTPKNYQHW (SEQ ID NO: 12 2), ISNKVPKLF (SEQ ID NO: 125), KTFVQQKTL (SEQ ID NO: 135) , ILRSPLKW (SEQ ID NO: 153), or LTVPLSVFW (SEQ ID NO: 183), Preferably, RTPKNYQHW (SEQ ID NO: 122), ISNKVPKLF (SEQ ID NO: 12 5), KTFVQQKTL (SEQ ID NO: 135), or ILRSPLKW (SEQ ID NO: 153 3. The leukemia TAP according to item 1 or 2, comprising:

[0031] 22. The leukemia TAP binds to the HLA-B*57:03 molecule and has the amino acid sequence GG SLIHPQW (SEQ ID NO: 60) or LGGAWKAVF (SEQ ID NO: 172) Leukemia TAP described in items 1 or 2).

[0032] 23. The leukemia TAP binds to the HLA-C*03:03 molecule and has the amino acid sequence PA RPAGPL (SEQ ID NO: 37), IASPIALL (SEQ ID NO: 112), or HSLIS IVYL (SEQ ID NO: 140), preferably IASPIALL (SEQ ID NO: 112) or HS 3. The leukemia TAP of item 1 or 2, comprising LISIVYL (SEQ ID NO: 140).

[0033] 24. The leukemia TAP binds to the HLA-C*05:01 molecule and has the amino acid sequence SL 3. The leukemia TAP of item 1 or 2, comprising DLLPLSI (SEQ ID NO: 150).

[0034] 25. The leukemia TAP binds to the HLA-C*06:02 molecule and has the amino acid sequence IR MKAQAL (SEQ ID NO: 9), KATEYVHSL (SEQ ID NO: 70), VSFPDVRK V (SEQ ID NO: 87), IGNPILRVL (SEQ ID NO: 142), LSTGHLSTV (SEQ ID NO: SEQ ID NO: 154), or LRKAVDPIL (SEQ ID NO: 166), preferably IGNPIL Item 1 or 2, including RVL (SEQ ID NO: 142) or LSTGHLSTV (SEQ ID NO: 154) 2. Leukemia TAP.

[0035] 26. The leukemia TAP binds to the HLA-C*07:01 molecule and has the amino acid sequence IG NPILRVL (SEQ ID NO: 142), IYAPHIRLS (SEQ ID NO: 143), TVEE YLVNI (SEQ ID NO: 155), LHNEKGLSL (SEQ ID NO: 178), or VSRN YVLLI (SEQ ID NO: 186), preferably IGNPILRVL (SEQ ID NO: 142) or 3. The leukemia TAP of item 1 or 2, comprising IYAPHIRLS (SEQ ID NO: 143).

[0036] 27. The leukemia TAP binds to the HLA-C*07:02 molecule and has the amino acid sequence TI LPRILTL (SEQ ID NO: 30), SYSPAHARL (SEQ ID NO: 83), TQAPPN VVL (SEQ ID NO: 85), YYLDWIHHY (SEQ ID NO: 90), SLREPQPAL( SEQ ID NO: 109), PAPPHPAAL (SEQ ID NO: 117), or CLRIGPVTL ( SEQ ID NO: 158), preferably SLREPQPAL (SEQ ID NO: 109) or PAPPHP 3. The leukemia TAP of item 1 or 2, comprising AAL (SEQ ID NO: 117).

[0037] 28. The leukemia TAP binds to the HLA-C*08:02 molecule and has the amino acid sequence AQ DIILQAV (SEQ ID NO: 97), LTDRIYLTL (SEQ ID NO: 102), or AGD IIARLI (SEQ ID NO: 174), preferably AQDIILQAV (SEQ ID NO: 97) or 3. The leukemia TAP of item 1 or 2, comprising LTDRIYLTL (SEQ ID NO: 102).

[0038] 29. The leukemia TAP binds to the HLA-C*12:03 molecule and has the amino acid sequence LS 3. The leukemia TAP of item 1 or 2, comprising ASHLSSL (SEQ ID NO: 173).

[0039] 30. Item 1, encoded by sequences located in non-protein-coding regions of the genome The leukemia TAP of any one of claims 1 to 29.

[0040] 31. The non-protein-coding region of the genome is an untranslated transcribed region (UTR). Leukemia TAP described in eyes 30.

[0041] 32. The method according to item 30, wherein the non-protein-coding region of the genome is an intron. Leukemia TAP.

[0042] 33. The method according to item 30, wherein the non-protein-coding region of the genome is an intergenic region. Leukemia TAP.

[0043] 34. At least two of the leukemia TAPs defined in any one of items 1 to 33 combinations, including

[0044] 35. The leukemia TAP according to any one of items 1 to 33, or the combination according to item 34. A nucleic acid encoding the combination.

[0045] 36. The nucleic acid according to item 35, which is an mRNA or a viral vector.

[0046] 37. A leukemia TAP according to any one of items 1 to 33, and a combination according to item 34. 37. A liposome comprising the nucleic acid according to Item 35 or 36.

[0047] 38. A leukemia TAP according to any one of items 1 to 33 and a combination according to item 34. 36. The nucleic acid according to Item 35 or 36, or the liposome according to Item 37, and an acceptable carrier.

[0048] 39. A leukemia TAP according to any one of items 1 to 33, and a combination according to item 34. the nucleic acid according to Item 35 or 36, the liposome according to Item 37, or the liposome according to Item 38 A vaccine comprising the composition described above and an adjuvant.

[0049] 40. An isolated major histocompatibility complex (MHC) class I molecule, comprising a peptide thereof. 34. An isolated M protein comprising, in its docking groove, a leukemia TAP according to any one of items 1 to 33. HC class I molecules.

[0050] 41. The isolated MHC class I molecule according to item 40, in the form of a multimer.

[0051] 42. The isolated MHC class I molecule according to item 41, wherein the multimer is a tetramer. .

[0052] 43. (i) Leukemia TAP according to any one of items 1 to 33, (ii) according to item 34 or (iii) a TAP or a combination thereof according to any one of items 1 to 33. a vector comprising a nucleotide sequence encoding the combination according to item 34, Isolated cells.

[0053] 44. An isolated cell, having on its surface a major histocompatibility complex (MHC) clone. MHC class I molecules are expressed, and MHC class I molecules are inserted into their peptide-binding grooves as described in items 1 to 3. 34. An isolated leukemia TAP comprising the leukemia TAP of any one of items 3 and 35 or the combination of items 34 and 35. Cells.

[0054] 45. The cell according to item 44, which is an antigen-presenting cell (APC).

[0055] 46. ​​The cell according to item 45, wherein the APC is a dendritic cell.

[0056] 47. An isolated MHC class I molecule and / or or an MHC class I molecule expressed on the surface of the cell according to any one of items 44 to 46. T cell receptor (TCR) specifically recognizes molecules.

[0057] 48. The TCR comprises one of the amino acid sequences shown in SEQ ID NOs: 191 to 219. Item 47, comprising a TCR beta (TCRβ) chain containing a complementarity-determining region 3 (CDR3) The TCR described in

[0058] 49. An isolated cell, having on its cell surface a T according to item 47 or 48. Isolated cells expressing CR.

[0059] 50.CD8 + 50. The isolated cell of item 49, which is a T lymphocyte.

[0060] 51. Cells containing at least 0.5% isolated cells as defined in items 49 or 50. Group.

[0061] 52. A method for treating leukemia in a subject, comprising administering to the subject an effective amount of (i) items 1 to 5. 33, (ii) a combination according to item 34, (i (ii) the nucleic acid according to Item 35 or 36, (iv) the liposome according to Item 37, (v) (vi) the composition according to item 38, (vi) the vaccine according to item 39, (vii) items 43 to 49 (viii) the cell according to any one of items 46, 49, and 50, or (viii) the cell according to item 51 A method comprising administering a cell population.

[0062] 53. The method according to item 52, wherein the leukemia is myeloid leukemia.

[0063] 54. The patient according to item 53, wherein the myeloid leukemia is acute myeloid leukemia (AML). Law.

[0064] 55. The method further comprises administering to the subject at least one additional anti-tumor agent or therapy. 55. The method according to any one of Items 52 to 54.

[0065] 56. The at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, an immunotherapy, or an immunotherapy. 56. The method of item 55, wherein the treatment is a checkpoint inhibitor, radiation therapy, or surgery.

[0066] 57. (i) a method according to any one of items 1 to 33 for treating leukemia in a subject; (ii) the combination according to item 34, (iii) the combination according to item 35 or 36. The nucleic acid according to Item 36, (iv) the liposome according to Item 37, and (v) the composition according to Item 38. (vi) the vaccine according to item 39; (vii) the vaccine according to items 43 to 46, 49, and 50. (viii) Use of the cell according to any one of the preceding claims, or the cell population according to item 51.

[0067] 58. For the manufacture of a medicine for treating leukemia in a subject, (i) items 1 to 3 3, (ii) a combination according to item 34, (ii) (i) the nucleic acid according to Item 35 or 36, (iv) the liposome according to Item 37, (v) (vi) the composition according to item 38, (vii) the vaccine according to item 39, (vii) items 43 to 44 (viii) the cell according to any one of items 6, 49, and 50, or (viii) the cell according to item 51. The use of cell populations.

[0068] 59. The use according to item 57 or 58, wherein the leukemia is myeloid leukemia.

[0069] 60. The method according to item 59, wherein the myeloid leukemia is acute myeloid leukemia (AML). For.

[0070] 61. The method of any one of items 57 to 60, further comprising the use of at least one additional anti-tumor agent or therapy. 10. The use according to any one of claims 1 to 9.

[0071] 62. The at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, an immunotherapy, or an immunotherapy. 62. The use according to item 61, wherein the treatment is a checkpoint inhibitor, radiation therapy, or surgery.

[0072] 63. (i) a method according to any one of items 1 to 33 for treating leukemia in a subject; (ii) the combination according to item 34, (iii) the combination according to item 35 or 36. The nucleic acid according to Item 36, (iv) the liposome according to Item 37, and (v) the composition according to Item 38. (vi) the vaccine according to item 39; (vii) the vaccine according to items 43 to 46, 49, and 50. (viii) the cell population of item 51;

[0073] 64. The method for treating leukemia T according to item 63, wherein the leukemia is myeloid leukemia. AP, combination, nucleic acid, liposome, composition, vaccine, cell, or cell population.

[0074] 65. The method according to item 64, wherein the myeloid leukemia is acute myeloid leukemia (AML). Leukemia TAP, combinations, nucleic acids, liposomes, compositions, vaccines, cells, or cell populations.

[0075] 66. For use in combination with at least one additional anti-tumor agent or therapy 66. A leukemia TAP combination for use according to any one of items 63 to 65. , a nucleic acid, a liposome, a composition, a vaccine, a cell, or a cell population.

[0076] 67. The at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, an immunotherapy, or an immunotherapy. 67. For the use according to item 66, the therapeutic agent is a checkpoint inhibitor, radiotherapy, or surgery. , leukemia TAP, combination, nucleic acid, liposome, composition, vaccine, cell, or cell collection Group.

[0077] Other objects, advantages and features of the present invention will be apparent from and will be described by way of example only with reference to the accompanying drawings, in which: This will become more apparent upon reading the following non-limiting description of specific embodiments. [Brief explanation of the drawings]

[0078] In the accompanying drawings: [Figure 1]These graphs demonstrate that hematopoietic progenitor cells are a better control than mTECs for detecting TSAs in AML. Figure 1A: Comparison of the effectiveness of k-mer depletion from each k-mer set of 19 AML specimens with either the combined k-mers from six mTEC samples or six MPC samples. For this comparison, the jellyfish database was generated in canonical mode, ignoring the occurrence of k-mers less than 2. Figure 1B: Overlap between the combined k-mers of all AML specimens and the k-mers from the six mTEC and six MPC samples used in Figure 1A. The parameters for database construction used in Figure 1A were reapplied here. Figure 1C: t-distributed stochastic neighbor embedding (t-SNE) analysis of expressed protein-coding genes (TPM ≥ 1) in purified cell populations from the indicated tissues. Figure 1D: Comparison of the total number of expressed protein-coding genes (TPM ≥ 1) in the indicated tissues and cell populations used to plot panel C. Pluri_stem: pluripotent stem cells, Ery: erythrocytes, Precu: progenitor cells, Lympho: lymphocytes, Granulo: granulocytes, Mono: monocytes. mTECs were compared with other tissues using the Mann-Whitney U test (****p<0.0001). Bars indicate mean and standard deviation. [Figure 2] Schematic diagrams of the MPC-based TSA discovery approach are shown. (A) Schematic diagram of the workflow for TSA discovery based on mTEC k-mer depletion. (B) Schematic diagram of the workflow for ERE-derived MAP discovery. (C) Schematic diagram of the workflow for the mTEC+MPC k-mer depletion TSA discovery approach. (D) Schematic diagram of the workflow for the DKE approach. The workflow for the AML#1 sample is shown here. A fold change of 10 was used as the minimum to consider k-mers as overexpressed (other filters were also applied; see Methods). For the other three approaches, the resulting database of full-frame translated contigs was concatenated with the individualized canonical proteome, followed by MS identification of MAPs eluted from the same AML sample used for RNA sequencing. [Figure 3]Figure 3A shows that the MPC-based approach identifies the majority of TSA-high genes in AML. The σ of the distribution (black plot) is given. Figure 3A: For each AML specimen (n = 19), the proportion of MAPs derived from transcripts separated into 10 distinct groups (deciles) based on their TPM expression. Decile 10 has the most highly expressed transcripts, and decile 1 has the least expressed transcripts. The boxes indicate the median, 25th, and 75th percentiles of the distribution, with whiskers extending to the minimum and maximum values. Figure 3B: Normal distribution of the cumulative frequency of MAPs (dots) as a function of the logarithm of the total number of RNA-seq reads (rphm) that can encode them in AML specimens identified by MS. The mean (μ) and standard deviation (σ) of the distribution (black plot) are given. Figure 3C: Probabilities were calculated based on the normal distribution parameters in Figure 3B for RNA-seq to generate MAPs after the different indicated fold changes (FC, original rphm × FC). Figure 3D: A decision tree was used to separate the MAPs of interest (MOI) into TAA, HSA, and TSA high. "Normal tissue" refers to all tissues (GTEx, purified hematopoietic cells, and mTEC), while blood / BM refers to purified hematopoietic cells only. Figure 3E: Comparison of MOI counts obtained by each indicated proteogenomic approach. Figure 3F: Venn diagram comparing TSA high identities between the indicated approaches. Figure 3G: Pearson correlation between observed retention time and predicted retention time (left) or hydrophobicity index (right). Figure 3H: Median and interquartile range frequencies of successful re-identification of the indicated MAPs by Comet. [Figure 4]This indicates that TSA-highs are primarily derived from intron translation and are shared among many patients. Figure 4A: Heatmap showing the mean RNA expression (logarithm of rphm+1) of each identified TSA-high in either total normal tissue from GTEx (n = 12–50 depending on available samples), normal sorted hematopoietic cell populations (n ​​= 3–16 depending on available samples), or mTECs (n = 11). TAAs evaluated as safe in clinical trials have also been reported. Prec: Progenitor cells. Figure 4B: Comparison of the fold change in TSA-high between mean rphm expression in 19 AML specimens and MPCs (n = 16). Dots indicate each MOI, boxes indicate the median, 25th, and 75th percentiles of the distribution, and whiskers extend to the minimum and maximum values. Figure 4C: Distribution of biotypes (genomic regions or events) that generated the indicated MOIs. Exon-intron: peptides overlapping exon-intron junctions (retention introns), ncRNA: non-coding RNA, OoF translation: out-of-frame translation. Figure 4D: TSA-high RNA expression in 19 AML samples and 437 Leucegene patients. Figure 4E: Population coverage by HLA allotypes capable of presenting TSA-high (19 AML sample alleles presenting TSA-high + promiscuous binders calculated by MHC cluster). This was calculated using the IEDB Population Coverage Tool (www.iedb.org). Bars indicate the frequency of individuals within the global population carrying up to six allotypes (x-axis), and the cumulative percentage of population coverage is shown as dots. Figure 4F: Distribution of HLA-TSA-high complexes in the Leucegene cohort based on TSA-high RNA expression (considered expressed if rphm ≥ 2), patient HLA alleles (OptiType), and promiscuous binders. Figure 4G: Number of predHLA-TSA high complexes in Leucegene patients at diagnosis and relapse. Figure 4H: RNA expression of TSA high that can be presented by HLA alleles in paired purified AML blasts from 15 patients at diagnosis and relapse (data from (Toffalori et al., 2019)).Comparisons were performed using the Wilcoxon paired signed-rank test. Figure 4I: Comparison of shared genes (considered expressed if rphm>0) among TSA-high samples in sorted blast cells (n=12) or leukemia stem cells (LSC, n=8) as reported elsewhere (Corces et al., 2016). Figure 4J: RNA expression of HLA-ABC molecules in the samples shown in Figure 4I. Mean + SD is shown. Figure 4K: GSEA analysis comparing Leucegene patients expressing above the median (rphm>0) TSA-high (n=207) with other patients (n=230) for the indicated LSC signature gene set (Eppert et al., 2011). NES: Normalized Enrichment Score. [Figure 5] The presence of multiple TSA-high mutations correlates with better survival. Figure 5A: Kaplan-Meier survival analysis between Leucegene patients expressing high (n = 98, upper quartile of Figure 5B) versus low (n = 275, all other patients) levels of the HLA-TSA-high complex. Statistical significance was determined by the log-rank test. Figure 5B: Forest plot for multivariate analysis of 5-year overall survival. HR: adjusted hazard ratio, CI: confidence interval, adv: adverse, fav: favorable, int: moderate. NPM1 / FLT3 interaction = presence of both NPM1 variants and FLT3-ITD. Figure 5C: Log-rank p-values ​​calculated after removing the indicated number of TSA-high mutations from the analysis performed in (A). 1000 permutations were performed for each number, and the mean + SD is reported. Figure 5D: Percentage of significant p-values ​​obtained in Figure 5C. Figure 5E: Comparison of log-rank p-values ​​recalculated after alternative removal of each TSA-high mutation from the analysis in Figure 5A. Figure 5F: Comparison of log-rank p-values ​​recalculated after alternative removal of each HLA allele from the analysis in Figure 5A. [Figure 6]Figure 6A: Comparison of immunogenicity scores (Repitope) among MOIs, thymic stromal cell-derived MAPs, and HIV MAPs. Figure 6B: Median and interquartile range of mean RNA expression across 11 available mTEC samples for MOIs, 5112 non-immunogenic MAPs, and 1411 immunogenic MAPs (curated from the IEDB (Ogishi and Yotsuyanagi, 2019)). Figure 6C: IFN-γ ELISpot assay of healthy PBMCs after stimulation of DCs pulsed with the indicated peptides. Results from two independent experiments are combined. Figure 6D: ELISpot assay of the indicated TSA-high (single donor). Figure 6E: Flow cytometry analysis of cytokine secretion of T cells expanded in the presence of the indicated peptides. Figure 6F: Representative flow cytometry plot of the indicated dextramer frequency among T cells expanded in the presence of the indicated peptides. Figure 6G: FEST assay: Significant T cell clonotype expansion after 10 days of stimulation with three different TSA-high pools (5 peptides / pool). Figure 6H: TCR CDR3s per 1,000 TCR reads (CPK, as a measure of clonotype diversity) in Leucegene patients with high versus low counts of the indicated predHLA-MOI (related to Figures 4F and 11D). Figure 6I: Frequency of TSA-high-responsive clonotypes (n = 66-164 / group) in Leucegene, as predicted by ERGO. Figure 6J: Frequency of TAA-responsive clonotypes (n = 74-207 / group) in Leucegene, as predicted by ERGO. Figure 6K: Frequency of clonotypes recognizing the pred MOI presented in the examined samples among all anti-MOI clonotypes (normalized by the number of pred-presented MOIs) (related to I and J). Patients with an anti-presMOI clonotype count of 0 were ignored. Figure 6L: Correlation between RNA expression of CD8A and CD8B genes and the number of TSA highs expressed above 2 rphm in Leucegene. Figure 6M: Correlation between RNA expression of CD8A and CD8B genes and the number of pre dHLA-TSA highs in Leucegene.Figure 6N: Volcano plot of differential gene expression analysis comparing patients with normalized TSA high predilection above the median vs. below the median. Dots indicate genes upregulated in patients above the median. Figure 6O: GO term analysis of the upregulated genes in Figure 6N. [Figure 7] High TSA expression is associated with immune editing, AML driver mutations, and epigenetic abnormalities. Figure 7A: Pearson correlation between the number of HE-TSA highs and the expression of the indicated genes across the complete Leucegene cohort (n=437). In the first panel, HLA-A, -B, and -C expression values ​​were summed. Figure 7B: Comparison of PD-L1 (CD274) gene expression between Leucegene patients expressing above the median HE-TSA high and other patients (stratified as a function of NPM1 mutation status). Figure 7C: Network analysis of GO term enrichment between genes was inversely correlated with the number of HE-TSA highs. Node size is proportional to the size of the gene set. Figure 7D: Network analysis of GO term enrichment between genes was positively correlated with the number of HE-TSA highs. Figure 7E: Comparison of the number of patients expressing above the median HE-TSA high with the number of other patients between wild-type and mutant patients for the indicated genes. Statistical significance established by Fisher's exact test (**p<0.01, ***p<0.0001). Figure 7F: Comparison of HE-TSA counts among patients with 0 to 3 mutations in either NPM1, FLT3, or DNMT3A. Figure 7G: Unsupervised consensus clustering of intron retention ratios for Leucegene patients (n=437, columns) as determined by IRFinder. Rows represent the 1211 top introns with the highest variability and significance for the consensus clustering, clustered hierarchically. Patient FAB types are indicated below the heatmap, along with p-values ​​indicating significant association with the indicated consensus cluster (Fisher's exact test, *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001). [Figure 8A] FIG. 1 is a diagram of the concept of k-mer occurrence. [Figure 8B]10 is a graph showing an example of k-mer frequency distribution in the occurrence function for sample 05H143. [Figure 8C] FIG. 1 is a graph illustrating a comparison of occurrence thresholds used between mTEC-only and mTEC+MPC k-mer depletion approaches (each dot is a different AML sample). [Figure 8D] Graph showing the overlap of k-mer identities between unique k-mer combinations obtained from all 19 AML specimens obtained after depletion of either mTEC or mTEC+MPC. [Figure 9A] FIG. 1 is a schematic diagram providing details of differential k-mer expression analysis and MS database construction. As an example, the construction of the MS database for sample AML#1 is presented. FC: fold change. FIG. 2 is a diagram providing details of differential k-mer expression analysis and MS database construction. As an example, the construction of the MS database for sample AML#1 is presented. FC: fold change. [Figure 9B] Graph showing the average database size (line) versus the cumulative number of personalized canonical peptide identifications (peptides derived from personalized canonical proteomes, either alone (Canon.) or concatenated with contig sequences in the four indicated approaches). [Figure 9C] Venn diagrams comparing the identity overlap of canonical peptides identified based on each approach with peptides identified based on the individualized canonical proteome alone are shown. [Figure 10A] Graph showing a comparison of the percentage of MHC-I associated peptides (MAPs) of interest (MOI) identified by each TSA identification approach. [Figure 10B] Graph showing a comparison of the total number of AML samples (out of 19 samples used to identify TSA in this study) expressing TSA high (rphm>0) identified by either mTEC+MPC k-mer depletion or differential k-mer expression approaches. [Figure 11A] Graph showing distribution of number of TSA highs with RNA expression ≧2 rphm in the Leucegene cohort (n=437). [Figure 11B] Graph showing survival comparison between patients in the Leucegene cohort (n=372, patients sequenced at diagnosis and for whom survival data was available) presenting with a high number of TSA expressed at levels of 2 rphm or greater (top quartile of distribution in left panel) versus patients presenting with low levels (remaining cohort). [Figure 11C] Graphs showing the distribution of HLA-MOI complexes across the entire Leucegene cohort based on RNA expression (considered expressed if rphm≧2), each patient's HLA allele, and promiscuous binder predictions (optitypes and MHC clusters) for HSA (FIG. 11C), TAA (FIG. 11D), and TSA low (FIG. 11E). [Figure 11D] Graphs showing the distribution of HLA-MOI complexes across the entire Leucegene cohort based on RNA expression (considered expressed if rphm≧2), each patient's HLA allele, and promiscuous binder predictions (optitypes and MHC clusters) for HSA (FIG. 11C), TAA (FIG. 11D), and TSA low (FIG. 11E). [Figure 11E] Graphs showing the distribution of HLA-MOI complexes across the entire Leucegene cohort based on RNA expression (considered expressed if rphm≧2), each patient's HLA allele, and promiscuous binder predictions (optitypes and MHC clusters) for HSA (FIG. 11C), TAA (FIG. 11D), and TSA low (FIG. 11E). [Figure 11F] Graphs showing survival comparisons between patients (n=372, patients sequenced at diagnosis and for whom survival data was available) from the Leucegene cohort presenting high levels of HLA-MOI complexes (top quartile of distribution in top panel) versus patients presenting low levels (remainder of cohort) for HSA (FIG. 11F), TAA (FIG. 11G), and TSA low (FIG. 11H). [Figure 11G]Graphs showing survival comparisons between patients (n=372, patients sequenced at diagnosis and for whom survival data was available) from the Leucegene cohort presenting high levels of HLA-MOI complexes (top quartile of distribution in top panel) versus patients presenting low levels (remainder of cohort) for HSA (FIG. 11F), TAA (FIG. 11G), and TSA low (FIG. 11H). [Figure 11H] Graphs showing survival comparisons between patients (n=372, patients sequenced at diagnosis and for whom survival data was available) from the Leucegene cohort presenting high levels of HLA-MOI complexes (top quartile of distribution in top panel) versus patients presenting low levels (remainder of cohort) for HSA (FIG. 11F), TAA (FIG. 11G), and TSA low (FIG. 11H). [Figure 12A] Pearson correlation between the number of HE-TSA highs and the expression of the indicated genes across the complete Leucegene cohort (n=437) is shown. [Figure 12B] A graph showing a comparison of the expression of the indicated genes in patients with high pred presentation levels of TSA high versus the remaining patients is shown (related to Figure 4F). [Figure 12C] Pearson correlation between ZNF445 expression and the number of retained introns in the Leucegene cohort (analysis by IRFinder, defined as retained if >10% of transcripts were retained) is shown. [Figure 12D] Graph showing comparison of patients expressing above median HE-TSA high with other patients between wild-type and mutant patients for the indicated genes. Statistical significance established by Fisher's exact test. [Figure 12E] Graph showing a comparison of patients expressing above median HE-TSA elevation with other patients, among patients who did or did not undergo allogeneic HSCT. Statistical significance established by Fisher's exact test. [Figure 12F]Graph showing the distribution of FAB types in patients with high HE-TSA counts above or below the median high HE-TSA count across the entire Leucegene cohort. [Figure 12G] Graph showing the distribution of 2008 WHO classification of patients with high HE-TSA counts above or below the median high HE-TSA count across the entire Leucegene cohort. [Figure 12H] Graph showing the distribution of cytogenetic profiles of patients with high HE-TSA counts above or below the median high HE-TSA count across the Leucegene cohort. DETAILED DESCRIPTION OF THE INVENTION

[0079] The genetic, molecular biology, biochemistry, and nucleic acid terms and symbols used herein are those of the art. Standard articles and texts in the field, e.g., Kornberg and Bak er,DNA Replication,Second Edition(WHFr eeman, New York, 1992), Lehninger, Biochemis try,Second Edition(Worth Publishers,New York, 1975), Strachan and Read, Human Molec ular Genetics,Second Edition(Wiley-Liss, New York, 1999), Eckstein, editor, Oligonucl. eotides and analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis:A Practical Approach(IRL Press,Oxford,1984 ) etc. All terms should be understood in the typical sense established in the relevant art. It is.

[0080] The terms "a" and "an" refer to one or more of the grammatical objects of an item. is used herein to refer to more than one (i.e., at least one). For example, "An element" means one element or more than one element. Unless the context otherwise requires, The words "including" and "comprising" refer to the steps stated. A step or element or group of steps or elements may be included without including any other step or element. It is understood that the exclusion of any element or step or group of elements is implicit. There will be.

[0081] The recitation of ranges of values ​​herein is merely a range unless otherwise indicated herein. It is intended to serve as a shorthand way of individually referencing each individual value contained within a , each individual value is incorporated herein as if individually listed herein. All subsets of values ​​within ranges are also contemplated herein as if individually recited. It will be incorporated into the specification.

[0082] All methods described herein are intended to be illustrative unless otherwise indicated herein or by context. The steps may be performed in any suitable order unless clearly contradicted by the principles of the invention.

[0083] Any and all examples or exemplary language (e.g., "such as") provided herein The uses are intended only to better illustrate the invention and, unless otherwise stated, No limitation is intended to the scope of the invention.

[0084] No language in the specification should be construed as indicating any element not claimed as essential to the practice of the invention. should not be construed as indicating

[0085] As used herein, the term "about" has its ordinary meaning. Values ​​include the inherent variation of error for the device or method used to determine the value. used to indicate that a value is, or is close to, an enumerated value, e.g., an enumerated value (or The range of values ​​includes values ​​within 10% or 5% of the range of values.

[0086] In the studies described herein, the inventors used a proteogenomics-based approach. Using the technique, we identified candidate TSAs in 19 AML specimens. The majority of these TSAs were Non-exonic sequences (e.g., intronic and intergenic sequences) are expressed in normal tissues. These AML TSA candidates are derived from aberrantly expressed, non-mutated genomic sequences that are not expressed in the AML TSA candidates. Expression of complement is related to mutations in epigenetic modifiers (e.g., DNMT3A) and genomic inactivation. This correlates with the expression of ZNF445, a regulator of imprinting. L TSA candidates were highly shared between patients and expressed in both blasts and leukemic stem cells, These HLA presentations were also shown to be associated with markers of immunoediting and better overall survival. Therefore, the novel AML TSA candidates identified herein are useful for the treatment of leukemia T It may be useful in cell-based immunotherapy.

[0087] Thus, in one aspect, the disclosure includes one of the following amino acid sequences: Regarding the leukemia TAP (or leukemia tumor-specific peptide), it consists of: [Table 2-1] [Table 2-2]

[0088] Generally, peptides (e.g., TAP) presented in the context of HLA class I are expressed in approximately 7 or 8 The length varies from about 8 to about 15, or preferably from 8 to 14, amino acid residues. In some embodiments of the disclosed methods, longer peptides containing the TAP sequences defined herein are used. The peptide is artificially delivered to cells such as antigen-presenting cells (APCs) where it is processed by the cells. TAP is loaded onto the APC and presented by MHC class I molecules on the surface of the APC. This method allows loading of peptides / polypeptides longer than 15 amino acid residues into APCs. and APC cells that provide the corresponding TAPs as defined herein for presentation. In some embodiments, the protein is processed by proteases in the cytoplasm. The precursor peptides / polypeptides used to generate the defined TAPs are, for example: 1000, 500, 400, 300, 200, 150, 100, 75, 50, 45, 40 , 35, 30, 25, 20, or 15 or fewer amino acids. All of the methods and processes using TAP described are "top-down" after treatment with cells (APCs). Longer peptides or polypeptides to induce the final presentation of 8-14 TAPs Use of tumor antigen precursor peptides / polypeptides (including naturally occurring proteins) In some embodiments, the leukemia TAPs described herein include about 8-14, 8-13 , or 8 to 12 amino acids in length (e.g., 8, 9, 10, 11, 12, or 13 amino acids in length) and is small enough to fit directly onto an HLA class I molecule. In an embodiment, the TAP is 20 or fewer amino acids, preferably 15 or fewer amino acids, more preferably 14 or fewer amino acids In an embodiment, the TAP comprises at least 7 amino acids, preferably at least It contains at least eight amino acids or less, more preferably at least nine amino acids.

[0089] As used herein, the term "amino acid" refers to an amino acid that is an amino acid that is used to prepare synthetic analogs of TAP. Naturally occurring amino acids as well as other amino acids used in peptide chemistry for For example, naturally occurring amino acids, non-naturally occurring amino acids, and amino acids encoded by nucleic acid sequences. Examples of naturally occurring amino acids include both L- and D-forms of amino acids (e.g., amino acids that are not naturally occurring). are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc. Other amino acids include, for example, non-genetically encoded forms of amino acids, as well as L-amino acids. Conservative substitutions for amino acids include: For example, β-alanine, 3-aminopropionic acid, 2,3-diaminopropionic acid, α-amino Isobutyric acid (Aib), 4-aminobutyric acid, N-methylglycine (sarcosine), hydrochloride Hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine glycine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexyl Alanine, norleucine (Nle), norvaline, 2-naphthylalanine, pyridylalanine 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine Fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, Penicilla amine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, β-2-thienyl L-Homoarginine (Hoarg), N-Acetyl Lurisin, 2-aminobutyric acid, 2-aminobutyric acid, 2,4-diaminobutyric acid (D- or L- ), p-aminophenylalanine, N-methylvaline, homocysteine, homoserine (H oSer), cysteic acid, ε-aminohexanoic acid, δ-aminovaleric acid, or 2,3-diamino These amino acids are used in biochemistry / peptides. In embodiments, TAP is a naturally occurring amino acid. Contains only.

[0090] In embodiments, the TAPs described herein have functionally enhanced activity compared to the sequences described herein. These include peptides with altered sequences that involve substitutions of potentially equivalent amino acid residues. One or more amino acid residues within the sequence may have a similar polarity (similar amino acids) that act as functional equivalents. The amino acid sequence can be substituted with another amino acid (which has the same physicochemical properties) resulting in a silent change. Substitutes for an amino acid within a string may be selected from other members of the class to which the amino acid belongs. For example, positively charged (basic) amino acids include arginine, lysine, and histidine ( and homoarginine and ornithine). Nonpolar (hydrophobic) amino acids include Isoleucine, alanine, phenylalanine, valine, proline, tryptophan Uncharged polar amino acids include serine, threonine, cysteine, and methionine. Negatively charged (acidic) amino acids include riboflavin, tyrosine, asparagine, and glutamine. The amino acid glycine is a nonpolar amino acid. Amino acids may be included in either the uncharged (neutral) polar amino acid family. Substitutions made within a family of acids are generally understood to be conservative substitutions. The TAPs described herein may be any L-amino acid, any D-amino acid, or any L-amino acid. In embodiments, the TAPs described herein may comprise a mixture of D-amino acids and D-amino acids. Contains all L-amino acids.

[0091] In an embodiment, the sequences of SEQ ID NOs: 1 to 190, preferably SEQ ID NOs: 97 to 154 The sequence of TAP, which comprises or consists of one of the following, is involved in the interaction with the T cell receptor: The amino acid residues that do not substantially contribute do not substantially affect T cell reactivity and are not relevant. It can be modified by substituting other amino acids that do not eliminate binding to the corresponding MHC. Cut.

[0092] TAP also prevents degradation and improves stability, affinity, and / or uptake. , N-terminal and / or C-terminal capping or modification. Now, the present disclosure provides a method for producing a compound of formula Z 1 -XZ 2 wherein X is selected from the group consisting of SEQ ID NO: 1 to 190, preferably one of the amino acid sequences of SEQ ID NOs: 97 to 154, or is a TAP consisting of it.

[0093] In embodiments, the amino terminal residue of TAP (i.e., the free amino group at the N-terminus) is, for example, For example, moieties / chemical groups (Z 1) by covalent attachment of for). Z 1 is a straight or branched chain alkyl group of 1 to 8 carbons, or an acyl group (R- CO-), where R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, isopropionyl, or isobutanyl), or an aroyl group (Ar—CO -), where Ar is an aryl group. In embodiments, the acyl group is C1-C1 6 or C3-C 16 acyl groups (linear or branched, saturated or unsaturated); further embodiments In this case, saturated C1-C6 acyl groups (linear or branched) or unsaturated C3-C6 acyl groups (linear or branched) is branched), for example, an acetyl group (CH3-CO-, Ac). 1 teeth The carboxy-terminal residue of TAP (i.e., the free carboxyl group at the C-terminus of TAP) is absent. The hydroxyl group) can be modified, for example, by amidation (replacement of an OH group by an NH group) (e.g., For example, for protection against decomposition), therefore, in such cases, Z 2 is an NH2 group In an embodiment, Z 2 is a hydroxamate group, a nitrile group, an amide (primary, secondary, or tertiary) group, methylamine, iso-butylamine, iso-valerylamine, or cyclo Aliphatic amines with 1 to 10 carbon atoms, such as hexylamine, aniline, naphthylamine, aromatic or aromatic amines such as benzylamine, cinnamylamine, or phenylethylamine; It may be an arylalkylamine, an alcohol, or CHOH. 2 In an embodiment, TAP is selected from the group consisting of SEQ ID NOs: 1 to 190, preferably SEQ ID NO: 97. In embodiments, TAP comprises one of the amino acid sequences of SEQ ID NOs: 1 to 154. 90, preferably consisting of one of the amino acid sequences of SEQ ID NOs: 97 to 154, i.e. , Z 1 and Z 2 does not exist.

[0094] In another aspect, the present disclosure provides a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 48, 67, 89, 134, 151, or 164, HLA-A*01:01, comprising or consisting of the sequence of SEQ ID NO: 134 or 151 leukemia TAP (or tumor-specific peptide), preferably AML TAP, which binds to a molecule to provide.

[0095] In another aspect, the present disclosure provides SEQ ID NOs: 7, 11, 27, 32, 33, 34, 35, 435 1, 52, 53, 54, 61, 65, 72, 77, 82, 86, 104, 108, 119 , 123, 130, 132, 146, 150, 167, 168, 169, 171, 183 , or 188, preferably SEQ ID NOs: 104, 108, 119, 123, 130, 132, 146 or 150 sequences of the HLA-A*02:01 molecule. providing a leukemia TAP (or tumor-specific peptide), preferably AML TAP, that binds to Because HLA alleles are promiscuous (certain HLA alleles present similar epitopes), (See Table 4). The above identified TAPs are HLA-A*02:05, HLA-A It may further bind to HLA-A*02:06, and / or HLA-A*02:07 molecules.

[0096] In another aspect, the present disclosure provides SEQ ID NOs: 5, 18, 19, 20, 42, 44, 74, 91, 105, 113, 126, 131, 159, 160, 180, or 189, preferably HLA comprising or consisting of the sequence of sequence numbers 105, 113, 126, or 131 - A*03: Leukemia TAP (or tumor-specific peptide), preferably binding to the O1 molecule provides AML TAP. Because HLA alleles are promiscuous (specific HLA alleles are The above identified TAPs exhibit similar epitopes (see Table 4), HLA-A* It can further bind to the 11:01 molecule.

[0097] In another aspect, the present disclosure provides a method for the preparation of a nucleic acid sequence of SEQ ID NO: 6, 45, 96, 106, 121, 149, or 1 52, preferably comprising or including the sequence of SEQ ID NO: 106, 121, 149 or 152. The leukemia TAP (or tumor-specific peptide) that binds to the HLA-A*11:01 molecule consists of Because HLA alleles are promiscuous (especially Certain HLA alleles present similar epitopes (see Table 4), and the above identified TA P is HLA-A*03:01, HLA-A*31:01, and / or HLA-A*68 :01 molecule.

[0098] In another aspect, the present disclosure provides SEQ ID NOs: 13, 36, 71, 92, 95, or 145, preferably or an HLA-A*24:02 molecule comprising or consisting of the sequence of SEQ ID NO: 145. and providing a leukemia TAP (or tumor-specific peptide), preferably an AML TAP, that binds to the Because HLA alleles are promiscuous (certain HLA alleles provide similar epitopes), (See Table 4) The above identified TAPs further bind to the HLA-A*23:01 molecule. Can be combined.

[0099] In another aspect, the disclosure provides a method for the detection of a mutated or mutated nucleotide sequence comprising or consisting of the sequence of SEQ ID NO: 59 or 185. , leukemia TAP (or tumor-specific peptide) that binds to the HLA-A*26:01 molecule; Preferably, AML TAP is provided. Because HLA alleles are promiscuous (specific HLA The TAPs identified above are related to the HL It may further bind to AA*25:01 and / or HLA-A*66:01 molecules.

[0100] In another aspect, the present disclosure provides a method for the preparation of a nucleic acid sequence similar to SEQ ID NO: 88, 99, or 138, preferably SEQ ID NO: 99. or 138, which bind to the HLA-A*29:02 molecule, comprising or consisting of the sequence , a leukemia TAP (or tumor-specific peptide), preferably an AML TAP, is provided. Because HLA alleles exhibit promiscuity (specific HLA alleles present similar epitopes, see Table 4), the above identified TAPs are HLA-A*30:02 and / or HLA-B *15:02 It can further bind to the molecule.

[0101] In another aspect, the disclosure provides a method for the preparation of a nucleic acid sequence comprising or containing the sequence of SEQ ID NO: 26, 29, or 165. The leukemia TAP (or tumor-specific peptide) that binds to the HLA-A*30:01 molecule Tide), preferably AML TAP.

[0102] In another aspect, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising SEQ ID NO: 50 or SEQ ID NO: 148, preferably SEQ ID NO: 14 leukemia, which binds to the HLA-A*68:02 molecule and comprises or consists of the sequence of A TAP (or tumor-specific peptide), preferably AML TAP, is provided.

[0103] In another aspect, the present disclosure provides SEQ ID NOs: 8, 25, 40, 55, 56, 73, 78, 79, 80, 81, 98, 100, 107, 110, 111, 118, 120, 128, 129 , 157, 161, 163, 179, or 184, preferably SEQ ID NOs: 98, 100, 1 07, 110, 111, 118, 120, 128, or 129. The leukemia TAP (or tumor-specific peptide) that binds to the HLA-B*07:02 molecule HLA alleles are promiscuous (specific HLA alleles present similar epitopes (see Table 4), and the above identified TAPs are HLA-B*35:02, HLA-B*35:03, HLA-B*55:01, and and / or may further bind to HLA-B*56:01 molecules.

[0104] In another aspect, the present disclosure provides SEQ ID NOs: 4, 14, 15, 17, 21, 22, 23 , 28, 31, 47, 49, 101, 103, 114, 137, 139, 147, 156 , 170, 181, or 182, preferably SEQ ID NOs: 101, 103, 114, 137, 139 or 147 sequences, or consisting of the HLA-B*08:01 molecule. providing a leukemia TAP (or tumor-specific peptide), preferably AML TAP, that binds to do.

[0105] In another aspect, the present disclosure provides SEQ ID NOs: 2, 39, 58, 66, 124, 133, 136, 141, 162, 175, 176, or 177, preferably SEQ ID NOs: 124, 133, 1 36 or 141 sequences, or consisting of the sequence, binds to the HLA-B*14:01 molecule. and providing a leukemia TAP (or tumor-specific peptide), preferably an AML TAP, that is combined with the do.

[0106] In another aspect, the disclosure comprises the sequence of SEQ ID NO: 10, 57, 62, or 94; or It consists of leukemia TAP (or tumor-specific) that binds to the HLA-B*15:01 molecule. peptide), preferably AML TAP. Certain HLA alleles present similar epitopes (see Table 4), and the above identified T AP is HLA-B*15:02, HLA-B*15:03, and / or HLA-B*4 It can further bind to the 6:01 molecule.

[0107] In another aspect, the present disclosure provides a method for the preparation of a nucleic acid comprising the sequence of SEQ ID NO: 1 or 144, preferably SEQ ID NO: 144. Leukemia TAP (or TAP-like peptide) that binds to the HLA-B*27:05 molecule, comprising or consisting of is a tumor-specific peptide), preferably AML TAP. (specific HLA alleles present similar epitopes, see Table 4) The identified TAP can further bind to the HLA-B*27:02 molecule.

[0108] In another aspect, the disclosure provides a method for the preparation of a medicament comprising or derived from the sequence of SEQ ID NO: 4, 64, or 84. Leukemia TAP (or tumor-specific peptide) that binds to the HLA-B*38:01 molecule. ), preferably AML TAP is provided. Because HLA alleles are promiscuous (specific H LA alleles present similar epitopes, see Table 4 ), the above identified TAPs It may further bind to the HLA-B*39:01 molecule.

[0109] In another aspect, the present disclosure provides a method for the detection of a HIV-1-associated ... Leukemia TAP (or tumor-specific peptide), which binds to the HLA-B*40:01 molecule, Preferably, AML TAP is provided. Because HLA alleles are promiscuous (specific HLA alleles are not included), The above identified TAPs are related to HLA-associated antigens (see Table 4). -B*18:01, HLA-B*40:02, HLA-B*41:02, HLA-B*4 4:02, HLA-B*44:03, and / or HLA-B*45:01 molecules. possible.

[0110] In another aspect, the present disclosure provides an HLA antibody comprising or consisting of the sequence of SEQ ID NO: 127. - leukemia TAP (or tumor-specific peptide), preferably binding to the B*44:03 molecule provides AML TAP. Because HLA alleles are promiscuous (specific HLA alleles are The above identified TAPs exhibit similar epitopes (see Table 4), HLA-B* 18:01, HLA-B*40:01, HLA-B*40:02, HLA-B*41:0 2. Can further bind to HLA-B*44:02 and / or HLA-B*45:01 molecules .

[0111] In another aspect, the present disclosure provides SEQ ID NOs: 12, 24, 38, 68, 115, 116, or 1 90, preferably comprising or consisting of the sequence of SEQ ID NO: 115 or 116 - leukemia TAP (or tumor-specific peptide), preferably binding to the B*51:01 molecule provides AML TAP. Because HLA alleles are promiscuous (specific HLA alleles are The above identified TAPs exhibit similar epitopes (see Table 4), HLA-B* 35:02, HLA-B*35:03, HLA-B*52:01, HLA-B*53:0 1, and can further bind to HLA-B*55:01 and / or HLA-B*56:01 molecules. .

[0112] In another aspect, the present disclosure provides SEQ ID NOs: 3, 16, 41, 63, 69, 93, 122, 12 5, 135, 153, or 183, preferably 122, 125, 135, or 153 Leukemia TAP comprising or consisting of a sequence that binds to the HLA-B*57:01 molecule (or tumor-specific peptide), preferably AML TAP, is provided. To demonstrate heterogeneity (certain HLA alleles present similar epitopes, see Table 4), The identified TAPs are HLA-A*32:01 and / or HLA-B*58:01 mutants. It can further bind to a child.

[0113] In another aspect, the disclosure provides a method for the detection of a mutated or mutated nucleotide sequence comprising or consisting of the sequence of SEQ ID NO: 60 or 172. , leukemia TAP (or tumor-specific peptide) that binds to the HLA-B*57:03 molecule; Preferably, an AML TAP is provided.

[0114] In another aspect, the present disclosure provides a method for the preparation of a nucleic acid sequence similar to SEQ ID NO: 37, 112, or 140, preferably SEQ ID NO: 1 Binds to HLA-C*03:03 molecules, comprising or consisting of the sequences 12 or 140 and providing a leukemia TAP (or tumor-specific peptide), preferably an AML TAP, that Because HLA alleles are promiscuous (particular HLA alleles present similar epitopes), , see Table 4 ), the above identified TAPs are HLA-B*46:01, HLA-C*0 3:02, HLA-C*03:04, HLA-C*08:01, HLA-C*08:02 , HLA-C*12:02, HLA-C*12:03, HLA-C*15:02, and / Or it may further bind to the HLA-C*16:01 molecule.

[0115] In another aspect, the present disclosure provides an HLA antibody comprising or consisting of the sequence of SEQ ID NO: 150. - Leukemia TAP (or tumor-specific peptide), preferably binding to the C*05:01 molecule provides AML TAP. Because HLA alleles are promiscuous (specific HLA alleles are The above identified TAPs exhibit similar epitopes (see Table 4), HLA-C* It may further bind to 08:01 and / or HLA-C*08:02 molecules.

[0116] In another aspect, the present disclosure provides a method for the preparation of a nucleic acid sequence of SEQ ID NO: 9, 70, 87, 142, 154, or 166, preferably Preferably, HLA-C*0 comprises or consists of the sequence of SEQ ID NO: 142 or 154. Leukemia TAP (or tumor-specific peptide), preferably AML, that binds to the 6:02 molecule TAP is provided. Because HLA alleles exhibit promiscuity (a particular HLA allele may have similar endogenous genes), The TAPs identified above represent HLA-B*27:0 2, which can further bind to HLA-C*07:01 and / or HLA-C*07:02 molecules .

[0117] In another aspect, the present disclosure provides SEQ ID NO: 142, 143, 155, 178, or 186, preferably Preferably, HLA-C*0 comprises or consists of the sequence of SEQ ID NO: 142 or 143. Leukemia TAP (or tumor-specific peptide), preferably AML, that binds to the 7:01 molecule TAP is provided. Because HLA alleles exhibit promiscuity (a particular HLA allele may have similar endogenous genes), The TAPs identified above represent HLA-B*27:0 2. HLA-C*07:01, HLA-C*07:02, and / or HLA-C*14: It can further bind to the O2 molecule.

[0118] In another aspect, the present disclosure provides a method for the preparation of a nucleic acid sequence of SEQ ID NO: 30, 83, 85, 90, 109, 117, or 1 58, preferably comprising or consisting of the sequence of SEQ ID NO: 109 or 117 - Leukemia TAP (or tumor-specific peptide), preferably binding to the C*07:02 molecule provides AML TAP. Because HLA alleles are promiscuous (specific HLA alleles are The above identified TAPs exhibit similar epitopes (see Table 4), HLA-B* 27:02, HLA-C*07:01, HLA-C*07:02, and / or HLA-C *14:02 It can further bind to the molecule.

[0119] In another aspect, the present disclosure provides a method for the preparation of a nucleic acid sequence similar to SEQ ID NO: 97, 102, or 174, preferably SEQ ID NO: 9 7 or 102 sequences, which bind to HLA-C*08:02 molecules. The present invention provides a leukemia TAP (or tumor-specific peptide), preferably an AML TAP. Because HLA alleles are promiscuous (specific HLA alleles present similar epitopes, (See Table 4). The above identified TAPs are HLA-C*03:03, HLA-C*03 :04, HLA-C*05:01, HLA-C*08:01, and / or HLA-C*1 5:02 molecule.

[0120] In another aspect, the present disclosure provides an HLA antibody comprising or consisting of the sequence of SEQ ID NO: 173. - Leukemia TAP (or tumor-specific peptide), preferably binding to the C*12:03 molecule provides AML TAP. Because HLA alleles are promiscuous (specific HLA alleles are The above identified TAPs exhibit similar epitopes (see Table 4), HLA-B* 46:01, HLA-C*03:02, HLA-C*03:03, HLA-C*03:0 4, HLA-C*08:01, HLA-C*12:03, HLA-C*15:02, and and / or may further bind to HLA-C*16:01 molecules.

[0121] In embodiments, the TAP is located in the untranslated transcribed region (UTR), i.e., the 3′-UTR or 5′-UTR. In another embodiment, TAP is encoded by a sequence located in the 1′-UTR region. In another embodiment, TAP is encoded by a sequence located in an intergenic In another embodiment, TAP is encoded by a sequence located in an exon. It is encoded by the adjacent sequence and results from a frameshift.

[0122] TAP of the present disclosure can be expressed in a host cell containing a nucleic acid encoding TAP (recombinant expression). Peptides can be produced by synthesis or by chemical synthesis (e.g., solid phase peptide synthesis). The compounds can be readily synthesized by manual and / or automated solid-phase procedures well known in the art. Suitable syntheses can be carried out, for example, using "T-boc" or "Fmoc" procedures. Techniques and procedures for solid phase synthesis are described, for example, in Solid Phase Synthesis. hase Peptide Synthesis:A Practical Appro ach, by E. Atherton and R.C. Sheppard, publi shed by IRL,Oxford University Press,1989 Alternatively, the MiHA peptide can be prepared, for example, as described below. Alternatively, they can be prepared by segment condensation (Liu et al., Tetra hedron Lett.37:933-936,1996, Baca et al., J. Am. Chem. Soc. 117:1881-1887, 1995, Tam et al. al.,Int.J.Peptide Protein Res.45:209-216 ,1995,Schnolzer and Kent,Science 256:221 -225,1992, Liu and Tam, J.Am.Chem.Soc.116: 4149-4153, 1994, Liu and Tam, Proc. Natl. Aca. d.Sci.USA 91:6584-6588,1994, and Yamashiro and Li, Int. J. Peptide Protein Res. 31:322- 334, 1988). Another method useful for the synthesis of TAP is that of Nakagawa et al. ., J. Am. Chem. Soc. 107:7087-7092, 1985 In an embodiment, the TAP is chemically synthesized (synthetic peptide). An embodiment is a non-naturally occurring peptide, wherein the peptide is consisting of or consisting essentially of an amino acid sequence and synthetically produced as a pharmaceutically acceptable salt The salts of TAP according to the present disclosure relate to peptides that are synthesized (e.g., synthesized) in vivo. Since the peptides produced by the method have no salt, the peptides in their in vivo state The non-natural salt forms of peptides are particularly useful in pharmaceutical compositions containing peptides, e.g. For example, in the context of the peptide vaccines disclosed herein, the solubility of the peptide can be modulated. Preferably, the salt is a pharmaceutically acceptable salt of the peptide.

[0123] In embodiments, the TAP described herein is substantially pure. A compound is "substantially pure" when it is separated from compounds that naturally accompany it. At least 60%, more commonly 75%, 80%, or 80% by weight of the total ingredients in the recipe. Substantially pure when the solubility is 5%, preferably greater than 90%, and more preferably greater than 95%. Thus, for example, polynucleotides that are chemically synthesized or produced by recombinant technology Peptides generally have the same structure as their naturally associated components, e.g., the components of the macromolecule from which they are derived. A nucleic acid molecule will be substantially free of naturally occurring genomic DNA of the organism from which the nucleic acid is derived. It is not immediately contiguous (i.e., covalently linked) with the coding sequence that is normally contiguous in the A substantially pure compound is one that is free from, for example, a natural source. The peptide compounds may be synthesized by extraction, by expression of recombinant nucleic acid molecules encoding the peptide compounds, or by chemical synthesis. Purity can be confirmed by column chromatography, gel electrophoresis, HPLC, etc. In embodiments, TAP can be measured using any suitable method. In another embodiment, the TAP is in solid form, for example, lyophilized.

[0124] In another aspect, the present disclosure provides a method for producing a tumor antigen precursor peptide comprising administering to a subject a tumor antigen precursor peptide as described herein. In an embodiment, the nucleic acid is about 21 nucleotides long. to about 45 nucleotides, about 24 to about 45 nucleotides, for example, 24, 27, 30, 33 "Isolated" as used herein refers to a sequence containing 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 3 If present, other components present in the molecule's natural environment or macromolecules of naturally occurring origin (e.g. peptide or nucleic acid molecule separated from other nucleic acids, proteins, lipids, sugars, etc. "Synthetic" as used herein refers to the production of a substance, e.g., through recombinant techniques or chemical synthesis. A peptide or nucleic acid molecule that is produced using chemical synthesis and has not been isolated from its natural source. The nucleic acids of the present disclosure can be used for recombinant expression of the TAP of the present disclosure, and can be used in host A vector, such as a cloning vector or an expression vector, that can be transfected into a cell In embodiments, the present disclosure provides a vector encoding the TAP of the present disclosure. a cloning vector, expression vector, or viral vector containing the nucleic acid sequence; or Alternatively, the nucleic acid encoding the TAP of the present disclosure can be incorporated into the genome of a host cell. In either case, the host cell may be adapted to express the TAP or As used herein, the term "host cell" refers to a specific A host cell refers not only to the subject cell of the present invention but also to the progeny or potential progeny of such a cell. Any prokaryotic cell (e.g., E. coli) capable of expressing the TAP described herein, or The cell may be a eukaryotic cell (e.g., an insect cell, a yeast cell, or a mammalian cell). The plasmid contains the elements necessary for transcription and translation of the inserted coding sequence and encodes resistance genes, clones, and The peptide may contain other components such as a cloning site. or polypeptide coding sequence, and appropriate transcription and transcription factors operably linked thereto. Expression vectors containing translational control / regulatory elements can be constructed. These methods include: These include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al. (1989) Molecular Cloning,A Laboratory Manual,Cold Spring Harbor Press, Plainview, NY, and Ausubel, F. M.et al. (1989)Current Protocols in Molec ular Biology,John Wiley&Sons,New York,N. "Operably linked" refers to components, particularly nucleotide sequences. It refers to the parallel arrangement of components that allows the normal functioning of a column. A coding sequence operably linked to a sequence is one in which the coding sequence is under the regulatory control of the regulatory sequence, i.e. refers to the configuration of a nucleotide sequence that can be expressed under transcriptional and / or translational control. As used herein, "regulatory / control region" or "regulatory / control sequence" refers to a region that regulates the expression of a coding nucleic acid. The term regulatory region therefore refers to a non-coding nucleotide sequence involved in the regulation of a gene. It contains a promoter sequence, a regulatory protein binding site, an upstream activator sequence, etc. Vector (e.g., expression vectors) for efficient gene transcription and translation in the respective host cells. promoter sequences (e.g., CMV, PGK, and EFla promoters) for Ribosome recognition and binding TATA box and 3'UTR AAUAAA transcription termination sequence Other suitable promoters may include the necessary 5' upstream and 3' downstream regulatory elements, such as the promoter sequence. Examples include the Simian virus 40 (SV40) early promoter and mouse mammary tumor virus (MMT) promoter. (MMTV) promoter, HIV LTR promoter, MoMuLV promoter, Avian leukosis virus promoter, EBV immediate early promoter, and Rous sarcoma Vims Constitutive promoters of the promoter may also be used. Human gene promoters may also be used. , actin promoter, myosin promoter, hemoglobin promoter, and Cre promoter In certain embodiments, the promoters include, but are not limited to, acetylcholinesterase promoters. Inducible promoters are also contemplated as part of vectors expressing TAP. , which can turn on or turn off expression of a polynucleotide sequence of interest A molecular switch is provided. An example of an inducible promoter is a metallothionine promoter. -, glucocorticoid promoter, progesterone promoter, or tetracycline Examples of vectors include, but are not limited to, promoters of the pluripotent stem cells. Additional exemplary vectors include: Plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs) ), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophage phage (e.g., λ phage or M13 phage), and animal viruses, Examples of categories of animal viruses useful as vectors include, but are not limited to: Although not widely used, retroviruses (including lentiviruses), adenoviruses, and adeno-associated viruses viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, and bacilli Examples include the flu, papilloma, and papova viruses (e.g., SV40). An example of an expression vector is Lenti-X (trademark) for expression in mammalian cells. ) Bicistronic expression system (Neo) vector (Clontrch), pClneo vector ctor (Promega); lentivirus-mediated gene transfer in mammalian cells and pLenti4 / V5-DEST™, pLenti6 / V5-DES for expression T™, and pLenti6.2N5-GW / lacZ (Invitrogen) The coding sequence for TAP disclosed herein is useful for the expression of TAP in mammalian cells. The vector can be ligated into such an expression vector for

[0125] In certain embodiments, the nucleic acid encoding the TAP of the present disclosure is in a viral vector. The viral vector may be a retrovirus, a lentivirus, or a foamy virus. As used herein, the term "viral vector" refers to a vector that is derived from a viral vector. The term refers to a vector that contains at least one element of viral origin and is packaged into a viral vector particle. A viral vector refers to a nucleic acid vector construct that has the ability to be transduced into a non-essential virus. Instead of the gene, the vector may contain coding sequences for various proteins described herein. The vectors and / or particles can be expressed in various forms, including DNA, RNA, or other nucleic acids, either in vitro or in vivo. A, or other nucleic acids can be used to introduce them into cells. Spectroscopy vectors are known in the art.

[0126] In embodiments, the nucleic acid (DNA, RNA) encoding the TAP of the present disclosure is delivered in a liposome or is contained in any other suitable vehicle.

[0127] In another aspect, the present disclosure provides T MHC class I antigens containing (i.e., presenting or being bound by) one or more of the APs In an embodiment, the MHC class I molecule is an HLA-A1 molecule, and further In one embodiment, the MHC class is HLA-A*01:01. The I molecule is an HLA-A2 molecule, and in a further embodiment, an HLA-A*02:01 molecule. In another embodiment, the MHC class I molecule is an HLA-A3 molecule, and In one embodiment, it is an HLA-A*03:01 molecule. In another embodiment, it is an MHC class I molecule. The molecule is an HLA-A11 molecule, and in a further embodiment, an HLA-A*11:01 molecule. In another embodiment, the MHC class I molecule is an HLA-A24 molecule, and In one embodiment, it is an HLA-A*24:02 molecule. The molecule is an HLA-A26 molecule, and in a further embodiment, an HLA-A*26:01 molecule. In another embodiment, the MHC class I molecule is an HLA-A29 molecule. In one embodiment, it is an HLA-A*29:02 molecule. The molecule is an HLA-A30 molecule, and in a further embodiment, an HLA-A*30:01 molecule. In another embodiment, the MHC class I molecule is an HLA-A68 molecule. In one embodiment, it is an HLA-A*68:02 molecule. The molecule is an HLA-B07 molecule, and in a further embodiment, an HLA-B*07:02 molecule. In another embodiment, the MHC class I molecule is an HLA-B08 molecule, and In one embodiment, it is an HLA-B*08:01 molecule. The molecule is an HLA-B14 molecule, and in a further embodiment, an HLA-B*14:01 molecule. In another embodiment, the MHC class I molecule is an HLA-B15 molecule, and In one embodiment, it is an HLA-B*15:01 molecule. In another embodiment, it is an MHC class I molecule. The molecule is an HLA-B27 molecule, and in a further embodiment, an HLA-B*27:05 molecule. In another embodiment, the MHC class I molecule is an HLA-B38 molecule, and In one embodiment, it is an HLA-B*38:01 molecule. In another embodiment, it is an MHC class I molecule. The molecule is an HLA-B40 molecule, and in a further embodiment, an HLA-B*40:01 molecule. In another embodiment, the MHC class I molecule is an HLA-B44 molecule, and In one embodiment, the HLA-B*44:02 molecule or HLA-B*44:03. In one embodiment, the MHC class I molecule is an HLA-B57 molecule, and in a further embodiment, H In another embodiment, the HLA-B*57:01 or HLA-B*57:03 molecule. The C class I molecule is an HLA-C03 molecule, and in a further embodiment, HLA-C*03 In another embodiment, the MHC class I molecule is an HLA-C04 molecule. In a further embodiment, it is an HLA-C*04:01 molecule. The C class I molecule is an HLA-C05 molecule, and in a further embodiment, HLA-C*05 In another embodiment, the MHC class I molecule is an HLA-C06 molecule. In a further embodiment, it is an HLA-C*06:02 molecule. The C class I molecule is an HLA-C07 molecule, and in a further embodiment, HLA-C*07 In another embodiment, the MHC class I molecule is an HLA-C*07:01 or HLA-C*07:02 molecule. is an HLA-C08 molecule, and in a further embodiment, an HLA-C*08:02 molecule. In another embodiment, the MHC class I molecule is an HLA-C12 molecule, and in a further embodiment In terms of morphology, it is the HLA-C*12:03 molecule.

[0128] In embodiments, TAP is non-covalently bound to an MHC class I molecule (i.e., TAP The peptides are loaded into the peptide-binding groove / pocket of MHC class I molecules or are non-covalently bound. In another embodiment, TAP is covalently bound to an MHC class I molecule (alpha chain). In such a construct, TAP and an MHC class I molecule (alpha chain) are combined. are typically short (e.g., 5-20 residues, preferably about 8-12, e.g., 1 0) synthetic amines with flexible linkers or spacers (e.g., polyglycine linkers) In another aspect, the present disclosure provides a method for producing a fusion protein comprising administering to a mammalian subject the fusion protein .... a nucleic acid encoding a fusion protein comprising a TAP as defined herein fused to a TAP chain (the FA chain); In embodiments, the MHC class I molecule (alpha chain)-peptide complex comprises: Thus, in another aspect, the present disclosure provides a method for preparing a medicament for the treatment of a medicament comprising: The present invention provides multimers of MHC class I molecules bound (covalently or non-covalently) to one another. The multimers may be attached to a tag, such as a fluorescent tag, that allows for detection of the multimer. Numerous strategies have been developed for the production of MHC multimers, including monomers, tetramers, pentamers, octamers, etc. (Bakker and Schumacher, Current Opinion ion in Immunology 2005, 17:428-433 MHC multimers are useful, for example, for the detection and purification of antigen-specific T cells. In another aspect, the present disclosure provides a CD8 specific for TAP as defined herein. + T lymphocytes and methods for detecting or purifying (isolating, enriching) spheres loaded with TAP (covalently linked). contacting the cell population with a multimer of MHC class I molecules (covalently or noncovalently); CD8 bound by class I multimers +detecting or isolating T lymphocytes. CD8 bound by MHC class I multimers + T lymphocytes can be identified by known methods, e.g. For example, fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) can be used. It may also be isolated.

[0129] In another aspect, the present disclosure provides a cell (e.g., a host cell), and in embodiments, the present disclosure provides a cell (e.g., a host cell) A nucleic acid, vector, or plasmid of the present disclosure (i.e., one or more TAP In another aspect, the present invention provides an isolated cell comprising a nucleic acid or vector encoding the The illustration shows an MHC class I molecule (e.g., A cell expressing an MHC class I molecule of one of the alleles disclosed above is provided. In one embodiment, the host cell is a eukaryotic cell, e.g., a mammalian cell, preferably a human cell, In another embodiment, the cell is an antigen-presenting cell (APC). In one embodiment, the host cell is a primary cell, a cell line, or an immortalized cell. In this embodiment, the cells are antigen-presenting cells (APCs). The nucleic acids and vectors can be used in conventional transfection. Alternatively, the gene can be introduced into a cell via transfection techniques. The term "transfection" refers to calcium phosphate or calcium chloride co-precipitation, D EAE-dextran-mediated transfection, lipofection, electroporation, myotube Introducing foreign nucleic acid into host cells, including chromosomal injection and viral-mediated transfection Refers to a technique suitable for transforming or transfecting a host cell. Methods are described, for example, in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known, and include the use of nucleic acids in mammalian cells. can be used to deliver the vectors or plasmids of the present disclosure to a subject for gene therapy .

[0130] Cells such as APCs can be transfected with one or more TAPs using a variety of methods known in the art. As used herein, "loading cells" with TAP refers to loading cells with TAP. RNA or DNA encoding TAP is transfected into the cell, Or alternatively, it means that the APCs are transformed with a nucleic acid encoding TAP. Alternatively, cells may be subjected to a denaturation assay to detect MHC class I receptors present on the cell surface (e.g., peptide-pulsed cells). By contacting cells with exogenous TAP, which can directly bind to the IL-1 molecule, cells can be negatively affected. TAP also contains domains or domains that facilitate its presentation by MHC class I molecules. motif (e.g., endoplasmic reticulum (ER) retrieval signal, C-terminal Lys-Asp-Glu-L eu sequence (Wang et al.,Eur J Immunol.2004 Dec; 34(12):3582-94).

[0131] In another aspect, the present disclosure provides a method for producing a TAP (or a polypeptide encoding the peptide) as defined herein. A composition or a peptide combination comprising any one or any combination of the above nucleic acids. In embodiments, the composition comprises any of the TAPs defined herein. Any combination (2, 3, 4, 5, 6, 7, 8, 9, 10 or more taps) any combination of nucleic acids encoding the TAPs), or any combination of nucleic acids encoding the TAPs. Compositions comprising any combination / subcombination of TAPs as defined herein are disclosed herein. In another embodiment, the combination or pool is comprised of one or more known tumor antigens. may include:

[0132] Thus, in another aspect, the present disclosure provides a composition, comprising a TAP as defined herein. and any one or any combination of MHC class I molecules (e.g., and a cell expressing one of the selected alleles of an MHC class I molecule. APCs for use in this study are not limited to any particular cell type and may be CD8 + T lymphocytes As recognized by the The profiles of dendritic cells (DCs), Langerhans cells, macrophages, and B cells are For example, APCs can be derived from peripheral blood monocytes to form DCs, which can then be transformed into DCs. Contacting (stimulating) TAP either in vitro, ex vivo, or in vivo APCs can also be activated to present TAP in vivo. (one or more of the TAPs of the present disclosure are administered to the subject), and PCs are induced in the subject's body. "Inducing APCs" or "stimulating APCs" The phrase refers to contacting or administering to a cell one or more TAPs or nucleic acids encoding TAPs. These are loaded, resulting in TAP being presented on the cell surface by MHC class I molecules. As described herein, according to the present disclosure, a TAP may be, for example, Use longer peptides / polypeptides (including natural proteins) containing the sequence of TAP. can be indirectly loaded via the APC and then processed (e.g., proteases) inside the APC. TAP / MHC class I complexes are generated on the surface of the cells by the enzyme TAP. After loading the APCs with P and allowing them to present TAP, the APCs are administered to the subject as a vaccine. For example, ex vivo administration can include the steps of: (a) collecting APCs from a first subject; (b) contacting / loading the APCs of step (a) with the TAP to form a surface of the APCs; (c) forming an MHC class I / TAP complex in a subject requiring treatment; administering the peptide-loaded APCs to a second subject.

[0133] The first subject and the second subject may be the same subject (e.g., an autologous vaccine), or Alternatively, according to the present disclosure, the vaccine may be administered to a different subject (e.g., an allogeneic vaccine). The present invention provides a method for producing a composition (e.g., a pharmaceutical composition) for inducing primary presentation cells. Uses of the TAPs (or combinations thereof) described herein are provided. In addition, the present disclosure provides methods for the detection of antigens. A method or process for producing a pharmaceutical composition for inducing cells is provided, and the method or process The process involves mixing or blending TAP, or a combination thereof, with a pharmaceutically acceptable carrier. Any one or any combination of the TAPs defined herein may be used. Combined loaded MHC class I molecules (e.g., HLA-A1, HLA-A2, HLA -A3, HLA-A11, HLA-A24, HLA-A25, HLA-A29, HLA- A32, HLA-B07, HLA-B08, HLA-B14, HLA-B15, HLA- B18, HLA-B39, HLA-B40, HLA-B44, HLA-C03, HLA- C04, HLA-C05, HLA-C06, HLA-C07, HLA-C12, or HL A-C14 molecules) expressing cells such as APCs are expressed by CD8 + T lymphocytes (e.g., autologous C D8 + It can be used to stimulate / expand T lymphocytes. In this regard, the present disclosure provides a method for producing a TAP (or a nucleic acid or vector encoding same) as defined herein. cells expressing MHC class I molecules, and T lymphocytes, more specifically CD8 + T lymphocytes (e.g., CD8 + a cell population containing T lymphocytes), or any combination thereof.

[0134] In embodiments, the composition may contain a buffer, excipient, carrier, diluent, and / or medium (e.g., In further embodiments, the culture medium further comprises a buffer, excipient, carrier, diluent, and / or The medium may be a pharmaceutically acceptable buffer, excipient, carrier, diluent and / or medium. As used herein, "pharmaceutically acceptable buffers, excipients, carriers, diluents, and / or The "medium" or "culture medium" is physiologically compatible, does not interfere with the effectiveness of the biological activity of the active ingredient, and is compatible with Any and all solvents, buffers, binders, lubricants, fillers, thickeners, disintegrants that are not toxic to elephants agents, plasticizers, coatings, barrier layer formulations, lubricants, stabilizers, release retardants, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, etc. The use of such media and agents is well known in the art (Rowe et al., H andbook of pharmaceutical excipients,200 3,4 th edition,Pharmaceutical Press,Londo n UK). Any conventional media or agents are incompatible with the active compound (peptide, cells). Their use in the compositions of the present disclosure is contemplated, except as limited thereto. The liquid, excipient, carrier, and / or medium may contain non-naturally occurring buffers, excipients, carriers, and / or In embodiments, a TAP, or one or more TAPs as defined herein, is used in a culture medium. One or more of the encoding nucleic acids (e.g., mRNA) may be delivered to a liposome (e.g., a cationic contained within or complexed with (aquatic liposomes) or other suitable carriers. For example, Vitor MT et al., Recent Pat Drug Deli v Formul.2013 Aug;7(2):99-110).

[0135] In another aspect, the present disclosure provides a method for producing a TAP (or a polypeptide encoding the peptide) as defined herein. any one or any combination of the above (nucleic acids), as well as buffers, excipients, carriers, diluents, The present invention provides a composition comprising one or more of the following: a cell (e.g., APC), a diluting agent, and / or a medium. For compositions containing cells (e.g., T lymphocytes), the composition should be in a suitable culture medium that allows for the maintenance of viable cells. Representative examples of such media include physiological saline and Earl's buffered salt solution. (Life Technologies®), or PlasmaLyte® Baxter International (registered trademark). In embodiments, the compositions (e.g., pharmaceutical compositions) are referred to as "immunogenic compositions," "vaccine compositions," "vaccine compositions," "immunogenic ... " or "vaccine." As used herein, "immunogenic composition," "vaccine" The term "vaccine composition" or "vaccine" refers to a compound containing one or more TAP or vaccine vectors. and when administered to a subject, induces an immune response against one or more TAPs present therein. refers to a composition or formulation capable of inducing an immune response in a mammal. The use of the vaccine or vaccine vector may be carried out in any conventional manner known in the vaccine field. By route, for example, mucous membranes (e.g., ophthalmic, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary) via the surface of the body; parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) routes; or by topical administration (e.g., via a transdermal delivery system such as a patch) In embodiments, the vaccine or vaccine vector is a TAP (or a combination thereof). The TAP is conjugated to a carrier protein to increase its immunogenicity. conjugate vaccine). Thus, the present disclosure provides a method for administering TAP (or a combination thereof) or a nucleic acid encoding TAP (or a combination thereof) and a carrier protein. For example, TAP binds to Toll-like receptors (TLRs). Ligands (e.g., Zom et al., Adv Immunol. 2012, 114 :177-201) or polymers / dendrimers (see, for example, Liu et al. ,Biomacromolecules.2013 Aug12;14(8):2798 In embodiments, the immunogenic composition may be conjugated or complexed with a The composition or vaccine further comprises an adjuvant. An "adjuvant" is an adjuvant that is used to adjuvantize an antigen (as defined in this disclosure). When added to immunogenic agents such as TAP, nucleic acids, and / or cells, exposure to the mixture It refers to a substance that nonspecifically enhances or strengthens the immune response to a drug in a host upon exposure. Examples of adjuvants currently used in the field of vaccines include: (1) mineral salts (phosphate aluminum and aluminum salts such as aluminum hydroxide, calcium phosphate gel), Squalene, (2) oil-based adjuvants (oil emulsions and surfactant-based formulations) MF59 (microfluidized detergent-stabilized oil-in-water emulsion), Q S21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + Q S-21), (3) particulate adjuvants, such as virosomes (influenza hemagglutinin), unilamellar liposomal vesicles incorporating thymine), AS04 (containing MPL [SBAS4] Aluminum salts), ISCOMS (structural complexes of saponins and lipids), polylactide co-glucan (4) Microbial derivatives (natural and synthetic), e.g., monophosphoryl Pido A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP [RC- 529] (synthetic acylated monosaccharide), DC_Chol (which can self-assemble into liposomes) Lipid immunostimulatory substance that can be used), OM-174 (lipid A derivative), CpG motif (immunostimulatory substance) Synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (non-toxic (5) A genetically engineered bacterial toxin immunoglobulin to provide a potent adjuvant effect. human immunomodulatory agents, such as hGM-CSF or hIL-12 (protein or coding region) Immudatapeptin (a cytokine that can be administered either as a plasmid or (C3d tandem array), and / or (6) an inert vehicle such as gold particles. can be.

[0136] In an embodiment, the TAP or a composition comprising it is in lyophilized form. In some embodiments, the TAP or a composition comprising it is a liquid composition. , in a concentration of about 0.01 μg / mL to about 100 μg / mL in the composition. In the present invention, TAP is present in the composition at a concentration of about 0.2 μg / mL to about 50 μg / mL, about 0.5 μg / mL. L to about 10, 20, 30, 40, or 50 μg / mL, about 1 μg / mL to about 10 μg / mL L, or a concentration of about 2 μg / mL.

[0137] As described herein, any one of the TAPs defined herein, or MHC class I molecules loaded with or bound to any combination thereof Cells such as APCs expressing CD8 + Stimulates / increases T lymphocytes Thus, in another aspect, the present disclosure provides a method for the propagation of M T cell receptors (TCRs) that can interact with or bind to the HC class I molecule / TAP complex TCR) molecules, and nucleic acid molecules encoding such TCR molecules, and such nucleic acid molecules The TCR according to the present disclosure is loaded onto an MHC class I molecule or a vector comprising the and the TAP displayed thereby, preferably on the surface of living cells in vitro or in vivo. can specifically interact or bind at

[0138] In embodiments, the anti-leukemia (e.g., anti-AML) TCR according to the present disclosure is SEQ ID NO: 191 Complementarity determining region 3 (CDR3) containing one of the amino acid sequences shown in It contains the TCR beta (β) chain.

[0139] In embodiments, the TCR is specific for one or more of the following TAPs: SLLSG LLRA, ALPVALPSL, ALDPLLLRI, IASPIALL, and / or S LDLLPLSI, and one of the amino acid sequences shown in SEQ ID NOs: 191 to 199 In embodiments, the TCR comprises a TCR beta chain comprising a CDR3 comprising TAP SLLSG LLRA-specific, one of the amino acid sequences set forth in SEQ ID NOs: 191 to 199 In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising TAP ALPVA LPSL-specific, one of the amino acid sequences shown in SEQ ID NOs: 191 to 199 In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising TAP ALDPL LLRI-specific, one of the amino acid sequences shown in SEQ ID NOs: 191 to 199 In embodiments, the TCR comprises a TCR beta chain comprising a CDR3 comprising TAP IASPI ALL-specific, and one of the amino acid sequences shown in SEQ ID NOs: 191 to 199. In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising TAP SLDLLP It is specific to LSI and has one of the amino acid sequences shown in SEQ ID NOs: 191 to 199. It comprises a TCR β chain comprising a CDR3 comprising

[0140] In another embodiment, the TCR is specific for one or more of the following TAPs: LTD RIYLTL, VLFGGKVSGA, LGISLTLKY, FNVALNARY, and / or TLNQGINVYI is an amino acid sequence represented by SEQ ID NOs: 200 to 209 In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising one of: Among the amino acid sequences shown in SEQ ID NOs: 200 to 209, which are specific to DRIYLTL In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising one of: FGGKVSGA specific, and the amino acid sequence shown in SEQ ID NOs: 200 to 209 In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising one of the following: GISLTLKY-specific, and the amino acid sequence shown in SEQ ID NOs: 200 to 209 In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising one of the following: UNIVERSAL NATIONAL GENE specific, and the amino acid sequence shown in SEQ ID NOs: 200 to 209 In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising one of the following: LNQGINVYI specific, and the amino acid sequence shown in SEQ ID NOs: 200 to 209 It contains a TCR β chain containing a CDR3 containing one of them.

[0141] In another embodiment, the TCR is specific for one or more of the following TAPs: LRS QILSY, KILDVNLRI, HSLISIVYL, KLQDKEIGL, and / or AQDIILQAV, and the amino acid sequence shown in SEQ ID NOs: 210 to 219 In embodiments, the TCR comprises a TCR β chain, including a CDR3 comprising one TAP LR. Among the amino acid sequences shown in SEQ ID NOs: 210 to 219, which are specific to SQILSY In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising one Among the amino acid sequences shown in SEQ ID NOs: 210 to 219, which are specific to DVNLRI In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising one TAP HSL Among the amino acid sequences shown in SEQ ID NOs: 210 to 219, which are specific to ISIVYL In an embodiment, the TCR comprises a TCR β chain comprising a CDR3 comprising one of TAP KLQ Among the amino acid sequences shown in SEQ ID NOs: 210 to 219, which are specific to DKEIGL In embodiments, the TCR comprises a TCR β chain comprising a CDR3 comprising one TAP AQD IILQAV-specific, and among the amino acid sequences shown in SEQ ID NOs: 210 to 219 It contains a TCR β chain containing one CDR3.

[0142] In embodiments, the TCR according to the present disclosure is HLA-A*02:01, HLA-A*29: 02, HLA-B*15:01, HLA-B27:05, HLA-C*01:02, and and / or recognize one or more of the above-mentioned TAPs bound to the HLA-C*03:04 molecule. In embodiments, the TCR according to the present disclosure comprises the above-mentioned TCR bound to the HLA-A*02:01 molecule. In embodiments, the TCRs of the present disclosure recognize one or more of the following TAPs: HLA-A *Recognizes one or more of the above-mentioned TAPs bound to a 29:02 molecule. In an embodiment, The TCR according to the present disclosure comprises one of the above-mentioned TAPs bound to an HLA-B*15:01 molecule. In embodiments, the TCR according to the present disclosure recognizes one or more HLA-B27:05 molecules. In embodiments, the TCR according to the present disclosure recognizes one or more of the above-mentioned TAPs bound thereto. recognizes one or more of the above-mentioned TAPs bound to HLA-C*01:02 molecules. In embodiments, the TCR according to the present disclosure is a TCR as described above bound to an HLA-C*03:04 molecule. Recognize one or more of the APs.

[0143] As used herein, the term TCR refers to a variable binding domain, a constant domain, a transmembrane domain, and a It refers to members of the immunoglobulin superfamily that have a cytoplasmic tail and a long cytoplasmic tail ( For example, Janeway et al, Immunobiology: The Immu ne System in Health and Disease,3rd Ed., Current Biology Publications,p.4:33,1997 TC) can specifically bind to antigenic peptides bound to MHC receptors. TCRs can be found on the surface of cells and are generally made up of an α chain and a β chain (TCRα and TCRβ, respectively). Similar to immunoglobulins, TCR chains are heterodimers with The extracellular portion of the immunoglobulin (e.g., α chain, β chain) is made up of two immunoglobulin domains, the variable domain (e.g., , the TCR variable alpha region or Vα, and the TCR variable beta region or Vβ; typically at the N-terminus (amino acids 1-116 according to the Rabat numbering in the IL-1 domain) and one constant region adjacent to the cell membrane. regions (e.g., TCR constant domains α or Cα, and typically amino acids 117-259, TCR constant domain β or Cβ, typically based on Rabat It also contains a variable domain (amino acids 117 to 295), similar to immunoglobulins. The complementarity determining regions (CDs) in each chain are separated by framework regions (FRs). In certain embodiments, the TCR comprises a TCR found on the surface of a T cell (or T lymphocyte). and associates with the CD3 complex.

[0144] TCRs, and specifically nucleic acids encoding TCRs of the present disclosure, can be applied to, for example, T lymphocytes. spheres (e.g., CD8 + T lymphocytes) or MHC class I / TAP complexes Other types of lymphocytes can be genetically transformed / transformed to generate novel T lymphocyte clones that In certain embodiments, T lymphocytes obtained from a patient (e.g., C D8 + T lymphocytes) are transformed to express one or more TCRs that recognize TAP. The transformed cells are then administered to the patient (autologous cell transfusion). T lymphocytes (e.g., CD8 + T lymphocytes) recognize TAP. The cells are transformed to express one or more TCRs, and the transformed cells are then administered to the recipient. In another embodiment, the present disclosure provides a method for the treatment of rhesus mast cells by administering TAP-specific TCRs to rhesus mast cells. T lymphocytes transformed / transfected with vectors or plasmids ( For example, CD8 + In a further embodiment, the present disclosure provides a TAP-specific The present invention provides a method for treating a patient with autologous or allogeneic cells transformed with a specific TCR. In this embodiment, the TCR is a TCR that ... Other targeted disruption systems have been used to target endogenous loci (e.g., endogenous TRAC and / or or TRBC locus), thereby generating primary T cells (e.g., cytotoxic T It is expressed in the nucleus of the thymus gland (thymus gland cells).

[0145] In another embodiment, the present disclosure provides a nucleic acid encoding the TCR described above. In the form, the nucleic acid is present in a vector, such as the vectors described above.

[0146] In still further embodiments, autologous or cytogenetic alterations for the treatment of cancer (leukemia, e.g., AML) are used. The present invention provides the use of tumor antigen-specific TCRs in the production of allogeneic cells.

[0147] In some embodiments, the patient treated with the compositions (e.g., pharmaceutical compositions) of the present disclosure Before or after treatment with allogeneic stem cell transplantation (ASCL), allogeneic lymphocyte infusion, or autologous lymphocyte infusion The compositions of the present disclosure include an allogeneic antibody that is activated ex vivo against TAP. T lymphocytes (e.g., CD8 + T lymphocytes), allogeneic or autologous AP loaded with TAP C vaccine, TAP vaccine, and allogeneic or autologous T lymphocytes (e.g., CD8 + T lymphocytes), or lymphocytes transformed with tumor antigen-specific TCRs. The method for providing a T lymphocyte clone capable of recognizing a TAP in a subject (e.g., transplant recipients), e.g., ASCT and / or donor lymphocyte infusion (DLI) ) in the recipient, can be generated for tumor cells expressing TAP, and specifically Thus, the present disclosure provides a method for specifically targeting TAP / MHC class I molecule complexes. CD8, which encodes and expresses a T cell receptor that can recognize or bind to + Providing T lymphocytes. The T lymphocytes (e.g., CD8 + T lymphocytes) are recombinant ( The present invention relates to T lymphocytes, which may be either engineered or naturally selected. Indication of CD8 + Provide at least two methods for producing T lymphocytes and T cell activation and undifferentiated lymphocytes (typically APCs, etc.) under conditions conducive to the induction of T cell proliferation. The step of contacting the TAP / MHC class I molecule complex (expressed on the surface of the cell) The TAPs are used in the preparation of TAP-containing APCs, which can be used in vitro or in vivo (i.e., APCs are loaded with TAPs). In patients receiving the APC vaccine or in patients treated with the TAP vaccine A combination or pool of TAP bound to MHC class I molecules can be used. CD8 can recognize multiple TAPs using + To generate a population of T lymphocytes Alternatively, the tumor antigen-specific T lymphocytes or target T lymphocytes may be MHC class I T lymphocytes. RasI molecule / TAP complex (i.e., engineered or recombinant CD8 + T lymphocytes) specifically One or more nuclei encoding the TCR (more specifically, the alpha and beta chains) that bind Produced in vitro or ex vivo by cloning the acid (gene) Nucleic acids encoding the TAP-specific TCRs of the present disclosure can be produced by methods known in the art. from T lymphocytes activated against TAP ex vivo using methods known in For example, by TAP-loaded APCs) or by immunization against peptide / MHC molecule complexes. The TAP-specific TCRs of the present disclosure can be obtained from individuals who show an immune response. Recombination in host cells and / or host lymphocytes obtained from the recipient or graft donor and optionally differentiated in vitro to generate cytotoxic T lymphocytes (CTLs). Nucleic acids (transgenes) encoding the TCR alpha and beta chains can be provided. can be produced by transfection (e.g., electroporation) or transduction (e.g., Use of viral vectors) (e.g., calcium phosphate-DNA co-precipitation, DEAE-Dex transfection, polybrene-mediated transfection, electroporation troporation, microinjection, liposome fusion, lipofection, using any suitable method (e.g., rotoplast fusion, retroviral infection, biolistics, etc.) , can be introduced into T cells (e.g., from the subject to be treated or another individual). Engineered CD8 expressing a novel TCR + T lymphocytes can be cultured in vitro using well-known culture methods. can be propagated in

[0148] The present disclosure provides methods for generating immune effector cells that express the TCRs described herein. In one embodiment, the method comprises administering to a subject the immune effector cells of any of the invention as described herein. Immune effector cells (e.g., leukemia ( For example, immune effector cells isolated from a subject, such as a subject with AML, are transduced into the In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. The cells can then be directly readministered to the individual. Vector cells are first activated and stimulated to proliferate in vitro, after which they express TCR In this regard, immune effector cells are genetically modified to express The cells may be cultured before or after being genetically modified (i.e., the TCs described herein). transduced or transfected to express R).

[0149] Prior to the in vitro manipulation or genetic modification of immune effector cells described herein, the cells The source can be obtained from a subject, particularly for use with the TCRs described herein. Immune effector cells for this include T cells. T cells are derived from peripheral blood mononuclear cells (PBMCs). , bone marrow, lymph node tissue, umbilical cord blood, thymus issues, tissue from infected areas, ascites, pleural effusion, spleen tissue In certain embodiments, T cells can be obtained from a number of sources, including T cells, T cells, and tumors. The cells may be isolated using any number of techniques known to those skilled in the art, such as FICOLL™ separation. In one embodiment, the antibody can be obtained from a unit of blood drawn from an individual's circulating blood. These cells can be obtained by apheresis. The apheresis product is typically These include lymphocytes, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and In one embodiment, the cells collected by apheresis are washed and The plasma fraction is removed by centrifugation and the cells are placed in an appropriate buffer or medium for further processing. In one embodiment of the present invention, the cells are washed with PBS. In an alternative embodiment, The wash solution may be calcium-free, magnesium-free, or may contain many, but not all, of the As will be appreciated by those skilled in the art, the washing step may be by methods known to those skilled in the art, for example, by using semi-automated flow-through centrifugation. After washing, the cells are then washed in various biocompatible buffers or buffer-containing solutions. In certain embodiments, the apheresis sample may be resuspended in saline or other saline solutions that do not contain erythrocytes. Undesirable components of the feed can be removed by resuspending the cells directly in the medium. In embodiments, T cells are activated by lysing red blood cells and depleting monocytes (e.g., PE). from peripheral blood mononuclear cells (PBMCs) by centrifugation through a RCOLL™ gradient. CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells are isolated. Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be achieved by isolating negatively selected T cells. This can be achieved by combining antibodies directed against cell-specific surface markers. One method for use herein is negative magnetic immunoadhesion or flow cytometry. cell sorting and / or selection by the presence of Use a cocktail of monoclonal antibodies directed against cell surface markers. To enrich for CD8+ cells by reactive selection, monoclonal antibody cocktails are typically used. Typically, CD14, CD20, CD11b, CD16, HLA-DR, and CD4 are involved. For use in the present disclosure, antibodies that PBMCs can also be isolated using the methods described herein. In certain embodiments, the TCR can be used directly for genetic modification. After isolation of PBMCs, T lymphocytes are further isolated and, in certain embodiments, genetically modified and and / or proliferation of cytotoxic T lymphocytes and helper T lymphocytes either before or after Both can be sorted into subpopulations of naive, memory, and effector T cells. This can be done.

[0150] The present disclosure relates to TAP (i.e., binding to MHC class I molecules expressed on the surface of cells). specifically induced, activated, and / or Isolated immune cells (e.g., CD8 + This provides T lymphocytes. The disclosure also provides a TAP or combination thereof according to the disclosure (i.e., a TAP or combination thereof that targets an MHC class I molecule). and CD8 that can recognize the TAPs. + T lymphocytes In another aspect, the present disclosure provides a composition comprising one or more of the M CD8 specifically recognizes the HC class I molecule / TAP complex + Enriched in T lymphocytes Selected cell populations or cell cultures (e.g., CD8 + Such a The enriched population is challenged with one or more of the TAPs disclosed herein (e.g., , these) using cells such as APCs that express MHC class I molecules, These can be obtained by ex vivo expansion of target T lymphocytes. The "enrichment" refers to the number of tumor antigen-specific CD8 + The proportion of T lymphocytes is Active populations, i.e., populations that have not been subjected to the step of ex vivo expansion of specific T lymphocytes. In a further embodiment, the TAP in the cell population is significantly higher than that in the population. specific CD8 + The proportion of T lymphocytes is at least about 0.5%, for example, at least about 1% In some embodiments, the TAP-specific expression level in the cell population is 1.5%, 2%, or 3%. target CD8 + The proportion of T lymphocytes is approximately 0.5 to 10%, approximately 0.5 to 8%, and approximately 0.5 to 10%. 5%, about 0.5 to about 4%, about 0.5 to about 3%, about 1 to about 5%, about 1 to about 4%, about 1% about 3%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, or about 3% to approximately 4% of one or more MHC class I molecule / peptide (TAP) complexes of interest CD8 specifically recognizes + Such cell populations or cultures (e.g., For example, CD8 + T lymphocyte populations) are targeted by tumor antigen-based immunoglobulins, as described in detail below. In some embodiments, TAP-specific CD8 + T lymphocytes The population of spheres may be loaded (covalently or non-covalently) with, for example, a TAP as defined herein. Further enrichment can be achieved using affinity-based systems such as multimers of MHC class I molecules. Thus, the present disclosure provides a method for the production of TAP-specific CD8 + Purified or isolated collection of T lymphocytes For example, TAP-specific CD8 + The proportion of T lymphocytes is at least about 50%. 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, Or 100%.

[0151] The present disclosure further provides the above-mentioned immune cells (CD8 + T lymphocytes) or TAP-specific CD8 + T The present invention relates to a pharmaceutical composition or vaccine comprising a population of lymphocytes. , as described above, may contain one or more pharmaceutically acceptable excipients and / or adjuvants. do.

[0152] The present disclosure further provides any TAP, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocytes, APCs), and / or a composition according to the present disclosure, or any combination thereof. In an embodiment, the present invention relates to the use of the compound as a pharmaceutical agent or in the manufacture of a pharmaceutical agent. The product is for the treatment of cancer, for example, a cancer vaccine. Any TAP, nucleic acid, or expression vector for use in therapy, e.g., as a cancer vaccine. vectors, T cell receptors, cells (e.g., T lymphocytes, APCs), and / or The present invention relates to a composition (e.g., a vaccine composition) according to the present invention, or any combination thereof. The TAP sequences specified in the specification are used to: i) identify tumor antigen-specific T cells that are injected into tumor patients; ii) for in vitro stimulation and proliferation, and / or ii) for anti-tumor T cells in cancer patients. Production of synthetic peptides for use as vaccines to induce or enhance cellular responses It can be used for:

[0153] In another aspect, the present disclosure provides a method for treating cancer in a subject, comprising administering to a subject a therapeutically effective amount of the present invention. TAP (SEQ ID NOs: 1 to 190, preferably SEQ ID NOs: 97 to 154) described in the specification, or The present disclosure also provides for the use of combinations (e.g., peptide pools) of peptides in a subject. a TAP as described herein for use as a vaccine for treating cancer; In an embodiment, the subject is provided with a combination (e.g., a peptide pool) of TAP specific CD8 + Thus, in another aspect, the present disclosure provides a method for the treatment of T lymphocytes comprising administering to a subject a subject in need thereof, the method ... and methods for treating cancer (e.g., reducing the number of tumor cells, killing tumor cells). and administering to a subject in need thereof an effective amount of one of the These MHC class I molecule / TAP complexes are recognized by TCs that bind to them. R-expressing)CD8 + In an embodiment, this method includes administering (injecting) T lymphocytes. The law is + After administration / infusion of T lymphocytes, the subject is administered an effective amount of TAP or its equivalent. and / or cells expressing MHC class I molecules loaded with TAP (e.g., In yet a further embodiment, the method further comprises administering APCs, such as dendritic cells. The method comprises administering to a subject in need thereof a therapeutically effective amount of dendritic cells loaded with one or more TAPs. In still further embodiments, the method comprises administering to a patient in need thereof A therapeutically effective amount of a recombinant TCR that binds to TAP presented by an MHC class I molecule is produced. This includes administering allogeneic or autologous cells that express the tumor.

[0154] In another aspect, the present disclosure provides a method for treating cancer in a subject (e.g., reducing the number of tumor cells). and (providing) TAP or a combination thereof for (killing tumor cells). CD8 recognizes one or more MHC class I molecules + Provides use of T lymphocytes In another aspect, the present disclosure provides a method for treating cancer in a subject (e.g., reducing the number of tumor cells). for the preparation / manufacture of a drug (for reducing the risk of cancer and killing tumor cells) recognize one or more MHC class I molecules loaded with (presenting) a combination of CD8 + In another aspect, the present disclosure provides a method for treating cancer in a subject, the method comprising administering a T lymphocyte to a subject. for use in therapy (e.g., to reduce the number of tumor cells, kill tumor cells); One or more MHC class I molecules loaded with (presenting) TAP or a combination thereof CD8 recognizes the child + T lymphocytes (cytotoxic T lymphocytes) are provided. Therefore, this use is directed to the TAP-specific CD8 + After the use of T lymphocytes, an effective amount of TAP (if or a combination thereof), and / or one or more M loaded with (presenting) TAP Further included is the use of cells (eg, APCs) that express HC class I molecules.

[0155] The present disclosure also provides a method for administering to a subject any of the TAPs disclosed herein or combinations thereof. Tumor cells (leukemia cells, AM) expressing human class I MHC molecules loaded with the combination and methods for generating an immune response against TAP or a combination of TAPs. administering cytotoxic T lymphocytes that specifically recognize the loaded class I MHC molecules; The present disclosure also provides a method for the preparation of human class I MHC molecules loaded with TAP or a combination thereof. TAs disclosed herein for generating an immune response against tumor cells expressing the TAs. Specific for class I MHC molecules loaded with either P or a combination of TAP. The present invention provides the use of cytotoxic T lymphocytes that recognize the antigen.

[0156] In embodiments, the methods or uses described herein may comprise administering to the patient a steroid hormone (SEQ ID NO: 1) or a combination thereof (SEQ ID NO: 2) prior to treatment / use. To determine the HLA class I alleles expressed by the patient and and administering or using a TAP that binds to one or more of the I alleles. For example, if a patient is determined to express HLA-A1*01 and HLA-C05*01 , (i) SEQ ID NOs: 48, 67, 89, 134, 151, and / or 164 (HLA-A1 *01), and / or SEQ ID NO: 150 (which binds to HLA-C05*01 Any combination of TAPs (e.g., steroids, anti-inflammatory drugs ...

[0157] In embodiments, the cancer is a hematological cancer, preferably a leukemia, e.g., acute lymphocytic leukemia. (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia In embodiments, the leukemia is A, B, C, D, E, F, F, G, H, HCL, HCC ... The AML treated by the methods and uses described herein is any type of AML. or subtype of AML (e.g., low-risk, intermediate-risk, or high-risk AML), e.g. For example, AML with a translocation between chromosome 8 and chromosome 21 [t(8;21)] AML with abnormalities, translocation or inversion on chromosome 16 [t(16;16) or inv( 16)], AML with PML-RARA fusion gene, and AML staining with chromosome 9 AML with a translocation between chromosome 6 and chromosome 9 [t(9;11)]; AML with locus [t(6:9)], translocation or inversion on chromosome 3 [t(3;3) or inv(3)], AML with a translocation between chromosome 1 and chromosome 22 [t(1:22)] AML (megakaryoblastic), with BCR-ABL1 (BCR-ABL) fusion gene AML, AML with a mutated NPM1 gene, and biallelic mutations in the CEBPA gene AML, AML with mutated RUNX1 gene, AML with mutated ASX1 gene, AML with mutated IDH1 and / or IDH2 genes, AM with mutated FLT3 gene L, AML with myelodysplasia-related changes and A associated with previous chemotherapy or radiation It can be ML.

[0158] In embodiments, the TAPs, nucleic acids, expression vectors, T cell receptors, cells (e.g., For example, T lymphocytes, APCs), and / or compositions, or any combination thereof, To treat cancer, one or more additional active agents or therapies, such as chemotherapy (e.g., Vinca alkaloids, drugs that interfere with microtubule formation (e.g., colchicine and its derivatives), Anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents (e.g., tyrosine kinase kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (e.g., Nucleoside analogues), alkylating agents, platinum-based drugs, anthracycline antibiotics, topoisomerase inhibitors, Isomerase inhibitors, macrolides, retinoids (e.g., all-trans retinoic acid or its derivatives), geldanamycin or its derivatives (17-AAG), surgery, radiation therapy method, immune checkpoint inhibitor (immunotherapeutic agent), immune checkpoint inhibitor (immunotherapeutic agent) Anti-PD-1 / PD-L1 inhibitors such as anti-PD-1 / PD-L1 antibodies, anti-PD-L1 antibodies, CTLA-4 inhibitors such as TLA-4 antibodies, B7-1 / B7-2 antibodies such as anti-B7-1 / B7-2 antibodies, B7-2 inhibitors, TIM3 inhibitors such as anti-TIM3 antibodies, BTLA such as anti-BTLA antibodies inhibitors, CD47 inhibitors such as anti-CD47 antibodies, GITR inhibitors such as anti-GITR antibodies) , antibodies against tumor antigens, cell-based therapies (e.g., CAR T cells, CAR NK cells), cells), cytokines (e.g., IL-2, IL-7, IL-21, IL-15) In embodiments, the TAP, nucleic acid, and expression vector of the present disclosure can be used in combination. The cells, T cell receptors, cells (e.g., T lymphocytes, APCs), and / or compositions are immunocompetent. In embodiments, the TCR-1 inhibitors according to the present disclosure are administered / used in combination with a checkpoint inhibitor. AP, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocyte, APC), and and / or the composition is administered in combination with one or more chemotherapeutic agents used in the treatment of AML; It is given / used in combination with other AML therapies (e.g., stem cell / bone marrow transplant).

[0159] Additional therapies include those comprising TAP, nucleic acids, expression vectors, T cell receptors, cells (e.g., For example, T lymphocytes, APCs), and / or administered before, simultaneously with, or after administration of the composition. can be given.

[0160] Modes for Carrying Out the Invention The present invention is illustrated in further detail by the following non-limiting examples.

[0161] Example 1: Materials and Methods AML specimens Diagnostic AML samples (cryovials of DMSO-frozen leukemic blast cells) were obtained from Banque de cellules leucemiques du Quebec program ( The samples were obtained from the BCLQ (bclq.org). Table 1 provides the technical and clinical characteristics of the samples. 100 million cells of each AML sample (except 14H124, see section below) were thawed (1 minute in a 37°C water bath) and resuspended in 48 ml of 4°C PBS. Cells (1 ml) were pelleted and resuspended in 1 ml of Trizol for RNA sequencing. The remaining 98 million cells were pelleted and flash-frozen in liquid nitrogen for mass spectrometry analysis. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] NC: Unclassifiable by FAB criteria. HLA was determined based on the RNA-Seq data of each sample. , determined by Optitype. Clinical data are available from the Banque de cellul es leucemiques du Quebec program (BC LQ (bclq.org).

[0162] Other Data Sources Human mTEC samples were prepared and sequenced for the needs of our team's previous research. Has it been decided? (#GSE127825 and #GSE127826) (Larouch e et al., 2020, Laumont et al., 2018), or other The paper is published by the University of California, San Diego (E-MTAB-7383) (Fergusson et al. al., 2018), which was previously used by our group for TSA discovery. Only six mTEC samples have been used in the k-mer depletion approach (Laumo nt et al., 2018). The 11 MPC samples used as primary normal controls were Sequenced by the IRIC genome platform and assigned to the Leucegene group These have been published previously (#GSE98310, #GSE51984). All other normal samples used were collected from dbGap (www.ncbi.nlm.nih.gov) v / gap / ), Arrayexpress(www.ebi.ac.uk / array express / ) or GEO (www.ncbi.nlm.nih.gov / geo Downloaded from http: / / www.ncbi.nlm.nih.gov / pubmed / 100237. Leucocytes from 437 RNA-seq AML samples Using the entire egene cohort, the discovered TSA 高 The clinical significance of RN was investigated. A sequencing data was previously published and is available separately (#GSE49642 , #GSE52656, #GSE62190, #GSE66917, #GSE67039 )(Lavallee et al., 2015, Macrae et al., 201 3, Pabst et al., 2016). Selected LSC and BLAST RNA The -Seq data have been published elsewhere and are available in #GSE74246 (Corces et al. RN of pre- and post-relapse (matched samples) of AML blasts was obtained from ( ). A-seq data have been published elsewhere (Toffalori et al., 2019 ), HLA typing of these samples was kindly provided by Dr. Luca Vago. All data obtained from external sources was analyzed using GRC using STAR v2.5.1b. Aligned onto the h38 genome.

[0163] Growth of 14H124 AML cells in NSG mice Only 20 million cells were available for this patient, so the blastocysts from patient 14H124 were used. The cells were thawed, washed with PBS, and then subjected to sublethal whole-body irradiation (2.5 Gy, 137Cs-γ source). ), 24 hours after the injection, 10 NOD-scid IL-2Rγ null (NSG) mice (2 × 10 6 The engraftment of human AML cells was assessed by flow cytometry. Therefore, peripheral blood was assessed on day 122. Briefly, 100 μl of blood was bled via tail vein. and deplete red blood cells using RBC lysis buffer (eBioscience). The cells were then washed with staining buffer (PBS + 3% FBS) and incubated with anti-human CD45-PacificB Anti-mouse CD45-PECy5 (30-F The cells were stained with FACS Canto I (BD 11) at 4°C for 20 minutes and washed with PBS. Data were acquired on a flow cytometer (Becton Dickinson) and analyzed by Fl owjo® Software 7.0 (Tree Star Inc., Ashla nd, OR).

[0164] In 8 / 10 mice, chimerism of more than 1% of human cells was found. Signs of disease (anemia, >20% weight loss, or visible tumor) occurring 188-264 days after transplant The animals were sacrificed within 24 hours and bone marrow, spleen, and solid tumors (interscapular region, neck and hip region, or kidney) were removed. The tumors were analyzed for future processing by mass spectrometry. AML cells were extracted from crushed spleens and explanted from femurs and tibias ( Bone marrow was harvested by flushing with 4°C PBS. The cells were depleted, filtered (100 μm) to remove debris, counted, and filtered as detailed above. Flow cytometry (5 × 10 5 cells) to assess their purity, or All remaining cells) were incubated in Trizol® (Inv) for future RNA sequencing. The samples were lysed in PBS (ribonucleotides) and stored frozen. , size >1cm 3 1 with 6 million bone marrow-derived blast cells with purity >99% Tumors (Figures 14A-B) were selected and processed for identification of MAPs by mass spectrometry. In contrast, two mice with no humanized grafts in the peripheral blood (graft failure) showed no signs of disease. All animals were sacrificed at the end of the experiment (day 264). All sacrifices were performed by CO2 asphyxiation followed by thawing. , was performed humanely by cervical dislocation. Mice were evaluated three times weekly for signs of disease. Daily monitoring was carried out during the experiment.

[0165] RNA extraction, library preparation, and sequencing RNA extraction was performed using RNeasy® Mini extraction columns (Qiagen). Extraction and purification was performed using Rizol® / chloroform. RNA was used for library preparation. The quality of total RNA was confirmed by BioAnalyzer Na All samples were evaluated using a KAPA mR assay (Agilent) and had a RIN greater than 8. Using the NAseq Hyperprep Kit (KAPA, Catalog No. KK8581) Ligation was performed using an Illumina Truseq interface. The final concentration of dextran was 51 nM. Sample 14H124 was prepared using 4 million cells, 1 ug of total DNA. The amplification was performed using 10 PCR cycles instead of 12. Library preparation was performed as for the previous samples, except that QuBit and BioA Libraries were quantified using the analyzer DNA1000. Dilute to 10 nM and use the KAPA Library Quantification Kit (KAPA; catalog number KK Libraries were pooled and normalized to equimolar amounts using the 4973 (Figure 4). A concentration of 2.8 pM of the pooled library was used for Illumina N Nextseq High Output Kit 150 cycles ( Sequencing was performed using approximately 120-200M paired sequences per sample. Library preparation and sequencing were performed by the Immunology and Cancer's Genomics Platform (IRIC) .

[0166] Database generation for shotgun mass spectrometry identification 1) Generation of personalized canonical proteomes, which was performed as previously detailed (Laumont et al., 2018). Briefly, RNA-Seq reads are Cropped using immomatic v0.35 and STAR v2.5.1b Aligned to GRCh38.88 using (Dobin et al., 2013) and - -alignSJoverhangMin, --alignMatesGapMax, - --alignIntronMax and --alignSJstitchMismatc Run with default parameters except for the hNmax parameter to generate a bam file. The default values ​​are 10, 200,000, 200,000, and 5 -155". A single base mutation with a minimum alternative count setting of 5 was eBayes 1.0.2-16-gd466dde(arXiv:1207.3907 ) were used to identify transcript expression. v0.43.0 (Bray et al., 2016) per million Finally, high-quality sample characteristics were analyzed using pyGeno. Alternative single nucleotide variants (freeBayes quality >20) were inserted into the reference exome and expressed Extract sample-specific sequences of known proteins produced by transcripts (tpm>0). The individualized canonical proteome fasta files were generated.

[0167] 2) Generation of an AML-specific proteome by mTEC k-mer depletion (Figure 2A). This was performed as previously detailed ( Laumont et al., 2018 ). Briefly, the R1 and R2 fastq files of each sample were trimmed as reported above. and R1 lead, fastx_rev of FASTX-Toolkit v0.0.14 The k-mer database was created by using the erse_complement function. (24 or 33 length) with Jellyfish v2.2.3 (Marcais and For each AML sample, a single database was generated. The data was generated by concatenating the fastq files of six mTEC samples. The duration of the k-mer assembly (as shown below) was determined by combining the k-mers in a unique database. (see below) exponentially grows over 30 million k-mers, so A 3-nucleotide long k-mer database is used for the assembly step, with up to 30 million k To reach a k-mer, a sample-specific threshold is set at the occurrence (the threshold at which a given k-mer is found in the database). After this filtering, the number of occurrences of m For each sample database, a k-mer that exists at least once in the TEC k-mer database is The remaining k-mers were then extracted from the source and analyzed using NEKT software developed in our facility. The contigs were assembled using AR. Briefly, the presented 33 nucleotide long k One of the k-mers is replaced by a contiguous k-mer that overlaps by 32 nucleotides on the same strand. Randomly select a seed to extend from both ends (with the -r option disabled, The assembly process used the k-mers as a concatenated set of k-mers. Stops if either the match cannot be made or multiple k-mers match (-a1 option for linear assembly). In such cases, a new seed is selected, The assembly process is repeated until all k-mers from the presented list have been used once. Finally, the contigs are split into three frames using a Python script at our facility. The amino acid sequence is then translated, split at internal stop codons, and the resulting subsequences are then split into individual subsequences. The differentiated canonical proteome was linked to each sample.

[0168] 3) Generation of ERE-specific proteomes (Figure 2B). For each sample, RNA-Seq was performed. The code was STAR (Dobin et al., 2013) with default parameters. The sequences were aligned to the human reference genome (GRCh38.88) using BEDtools (PMI D20110278) to intersect the reads with the ERE sequence or Reads mapping perfectly to either of the canonical genes were separated into two datasets. Reads in the ERE read dataset are those whose sequences are also present in the canonical read dataset. If so, they were discarded. Then, unmapped reads, secondary alignments, and low-quality Reads were discarded from the ERE read dataset in samtools view (PM The remaining ERE reads were then analyzed for all possible reading frames. The ERE polypeptide was in silico translated into the ERE polypeptide in the Splice at the stop codon, discard downstream sequences, and retain upstream sequences of 8 or more amino acids. Only the smallest length of MAPs was retained. The resulting ERE proteome was then , concatenated with the individualized canonical proteome of each sample.

[0169] 4) Generation of AML-specific proteomes by mTEC+MPC k-mer depletion (Figure 2 C). To implement this approach, mTEC k-mer depletion was described. The same method was used with the following modifications: (i) 11 used as k-mer control; An additional normal k-mer database was created by combining the fastq files of the MPC samples. These samples were sequenced in stranded mode. Therefore, the k-mer database was generated with the -C option and R1 fas The tq files were not reverse complemented. (ii) The mTEC or MPC k-mer database This step removes AML k-mers present in any of the sequences. The number of filtered k-mers is the same as in the mTEC k-mer depletion approach. (iii) The effectiveness of k-mer depletion in normal samples was higher than that in normal samples. Therefore, it is possible to pre-filter AML k-mers using a lower occurrence threshold. This is possible (Table 1 and Figure 7C), and these data were significantly higher than those obtained with mTEC k-mer depletion alone. This dramatically altered the identity of k-mers present in the database (Figure 7D). , an occurrence threshold of less than 3 was used to exclude possible sequencing errors. We report in the section "Generation of an AML-specific proteome by mTEC k-mer depletion." All other procedures were performed as described above.

[0170] 5) Generation of AML-specific proteomes by differential k-mer expression (Figure 2D). k-mer analysis was performed using a customized version of DE-kupl from fastq files. A computational pipeline for generating a k-mer database, normalizing k-mer abundances, filtering k-mers based on their occurrence and their sharing among samples; statistical Comparison of k-mer abundances between samples under two different conditions by using the test, Assembly of expressed k-mers into contigs, alignment of contigs on the genome, and based on the annotation of contigs based on their genome alignment ( Figure 8) (Audoux et al., 2017). Specifically, first, the following parameters were diff_method Ttest, kmer_length33, gene_di ff_method limma-voom, data_type WGS, lib_t ype unstranded, min_recurrence6, min_recur rence_abundance3, pvalue_threshold0.05, and DE-kupl was run using log2fc_threshold 0.1, and A ML specimens were compared to 11 MPC controls, which revealed significant differences between AML and MPC samples. The differentially expressed 33-nucleotide k-mer (FDR<0.05) sequences and positive The diff-counts.tsv file containing the normalized counts is returned, and there are at least six k-mers were present in a minimum of three occurrences in each sample (either MPC or AML). Custom filtering rules were desired, so we used k-mers in DE-kupl. No restriction is applied to the fold change (log2fc_threshold0.1), and diff-c Manually filter the list of k-mers provided in the counts.tsv file. (i) completely absent (count = 0) in all MPC samples (hence (ii) present in at least six AML samples; or (iii) present in at least six AML samples. (>30% of samples) with a fold change of 10-fold or more, or (iii) a single MP C samples and at a lower abundance than the lowest abundance in AML samples, or (iv) at least were also present in six AML samples, with a fold change of ≥5-fold and an FDR of ≥0.000001. Based on these rules, we kept all k-mers that were below 41×10 6 of Generate a new diff-counts.tsv file containing the k-mers and DE-ku Used to perform k-mer assembly by pL, ~2.1 x 10 6 Contigs Finally, we get the merged-diff-counts.tsv file containing the Contigs generated on the GRCh38 human genome using the annot function in upl was mapped and annotated.

[0171] To obtain individualized contig sequences for each AML sample, DE-kupl anno Use the DiffContigsInfos.tsv output of t to find the All continuations with a length of 34 nucleotides or more (derived from a 1-mer assembly) A bed file of the sequences was constructed and aligned without gaps, insertions, or deletions (N (CIGAR without / D / I). Next, use this bed file and bedtools, sam Using the tools and bcftools suites, each AML sample (sam tools mpileup-C50-uf ref_genome.fasta sa mple.bam|bcftools call-c|vcfutils.pl vcf 2fq-d 8-D 100|awk' / ^@chr.$|^chr..$|^@GL. .......$|^@KI........$ / , / ^+$ / '|sed' / ^+ / d '|tr”@””>”>consensus.fasta) bam file (STAR Reads mapped to GRCh38 using the "individualized canonical proteome" The individualized contigs were generated from the consensus genome generated from the Array (bedtools getfasta-fi consensus.fasta -bed contigs.bed-name>>output.fasta) Ta.

[0172] The part of the contig not covered by the reads (N) is extracted using sed (sed-E s / NNN+ / Λn / g”) and write all contigs to a fasta file. Gaps, insertions, or deletions (and sequences not available from the consensus genome) ) and the associated sample in DiffContigsInfos.tsv The sequences of contigs reported to be expressed by Finally, we used our Python scripts (previously published or pyGen) o(Daouda et al.,2016;Laumont et al.,2018 )) to translate the contigs in six frames. The amino acid sequence is then divided into all possible sequences (single base mutations and overlapping contigs are included in multiple different sequences). The amino acid sequence is converted into a sequence containing an internal termination code (which can encode an amino acid sequence containing an internal termination code). The resulting subsequences are then partitioned into individualized canonical proteomes for each sample. and linked.

[0173] 6) Database size verification - Figure 9B-C. The four proteogens used in this study The MS database used in the omics approach was the canonical (individualized) proteome database. Considering the variable expansion size presented compared to the database, how We investigated whether these larger sizes affected MS identification. The cumulative number of peptides detected was compared across 19 AML samples (Figure 9B). Despite significant differences in database size between approaches, the canonical proteome and showed that the number of identified peptides changed slightly compared to the ERE approach. Next, each database is used to generate its own individualized canonical set for each sample. Concatenated with the proteome, a database of appropriate size allows for the canonical proteome We reasoned that it should be possible to distinguish peptides derived from canonical proteins of similar identity from those derived from single proteins. As shown in Figure 9C, the proteins identified by each approach across all AML samples were The majority of peptides annotated as protein-coding peptides (88.2% - 96%) 0.2%) were common to those identified based on the canonical proteome alone. Based on the findings, various database sizes are suitable for reliable MS identification. The conclusion was reached.

[0174] Isolation of MHC-associated peptides W6 / 32 antibody (BioXcell) at a ratio of 1 mg of antibody per mL of slurry PureProteome Protein A magnetic beads (Millipore) were used in PBS. The antibody was magnetically coupled to the antibody using dimethylpimeridate as described above. The beads were covalently crosslinked. The beads were then resuspended in PBS (pH 7.2) and 0.02% Na Stored in N3 at 4°C. For frozen cell pellet samples (98 million cells / pellet), Thaw the cells and resuspend them in 1 mL of PBS (pH 7.2) with a protease inhibitor cocktail. (Sigma) supplemented with PBS (pH 7.2), 1% (w / v) CHAPS (Sigma) The tumor samples were solubilized by adding 1 mL of detergent buffer containing 1 mL of HCl. , the sample was cut into small pieces (cubes, approximately 3 mm in size) and diluted with a protein inhibitor cocktail 5 ml of ice-cold PBS was added. The tissue pieces were first spun at a speed of 20,000 rpm. Ultra Turrax T25 homogenizer (IKA-Labortechnik ) and homogenize twice for 20 seconds, then set the speed to 25,000 rpm. Ultra Turrax T8 homogenizer (IKA-Labortechnik ) for 20 seconds. Then, 550 μl of ice-cold 10x lysis buffer was added. CHAPS (5% w / v) was added to the samples. The cell pellets and tumor samples were incubated at 4°C. Incubate for 60 minutes with rotation, then centrifuge at 10,000 g for 20 minutes at 4°C. The supernatant was transferred to a magnetic beads containing 1 mg of W6 / 32 antibody covalently linked to Protein A per sample. The mixture was transferred to a new tube containing 100 ml of PBS and incubated at 4°C with rotation for 180 minutes. The sample was placed on a magnet to collect the MHC I complexes bound to the magnetic beads. Dilute the solution first with 8 x 1 mL of PBS, then with 1 x 1 mL of 0.1X PBS, and finally with 1 The MHC fragments were then washed with 1 mL of water. The I complex was eluted from the magnetic beads. The exudate was filtered through a 2.0 mL Costar Spin-X centrifuge tube filter (0.4 mL). The mixture was transferred to a 21 mm diameter octadecane filter (Corning, 5 μm filter) and centrifuged at 855 g for 2 minutes. A homemade stage chip filled with Sil (C-18) solid-phase extraction disks (EMPORE) Using a filter, the peptide-containing filtrate was purified by ELISA to separate the MHC I subunits (HLA molecules and β- The stage tip was first washed with methanol and then with 80% acetonitrile (ACN) in 0.2% trifluoroacetic acid (TFA) The sample was loaded onto the stage tip and washed with 0.2% FA. The peptides were eluted with 30% ACN in 0.1% TFA and washed with vacuum centrifugation. The gel was dried using a PBS and then stored at -20°C until MS analysis.

[0175] mass spectrometry The dried peptide extract was resuspended in 4% formic acid and loaded onto a homemade C18 analytical column (C18 J The tube was loaded onto a 15cm x 150µm inner diameter tube filled with Phenomenex. On an EasynLC II system, acetonitrile was varied from 0% to 30% (0.2% acetonitrile). acid) with a 56-minute gradient (10H005) or a 106-minute gradient (all other samples) and 6 A flow rate of 100 nL / min was used. Samples were then injected into a positive electrode with a Nanospray 2 source at 1.6 kV. A Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) was used in ion mode. Each complete MS spectrum was acquired at a resolution of 60,000. After the analysis, 20 MS / MS spectra were performed, and the most abundant multiply charged ions were identified at a resolution of 30. ,000, 5×10 4 (10H005) or 2 x 10 4 Auto Gain (for all other samples) Control target: 100ms (10H005) or 500ms (15H023, 15H063, 15H080, 05H149) or 800 ms (all other samples) injection time, and It was selected for MS / MS sequencing using a collision energy of 25%.

[0176] Synthetic peptides If sufficient material is available, TSA 高 The amino acid sequence of This was further validated in the presence of thiazol-2-one (Zhao et al., Cancer Immunol Res .2020 Feb11 doi:10.1158 / 2326-6066.CIR-19 -0541.[Epublished ahead of print]).

[0177] Bioinformatics analysis All analyses were performed on trimmed data and were performed as described in the previous section. All alignments were performed using STAR, and unless otherwise stated, all alignments were performed using GRCh38 It was done on .88.

[0178] All liquid chromatography (LC)-MS / MS (LC-MS / MS) data were PEAKS X (Bioinformatics Solution Inc.) used The peptides were identified using precursor ions and phenotypes. The tolerances for the fragment ions were set at 10 ppm and 0.01 Da, respectively. ) and the occurrence of deamidation (NQ) were set as variable modifications.

[0179] 1) Identification of MAPs. After peptide identification, a list of unique peptides is obtained for each sample, A 5% false discovery rate (FDR) was applied to the peptide scores. Binding affinity was predicted by NetMHC4.0 (Andreatta and Nielsen n, 2016), and only peptides with a percentile rank of 8 to 11 amino acids or less were included. , was used for further annotation.

[0180] 2) Identification and validation of the desired MAP (MOI). For both k-mer depletion approaches, This was carried out using a similar approach as previously described (Lau et al. (mont et al., 2018). Briefly, each MAP and its coding sequence are and associated AML and normal canonical proteomes (as detailed above, all mTECs and constructed for MPC), or cancer and normal 24 nucleotide-long km er database (combined mTEC or combined M as detailed above) The normal canonical proteome was queried against the genomic DNA (constructed from either the genomic DNA or the genomic DNA). MAPs were excluded regardless of their coding sequence detection status. MAP, normal canonical process MAPs that were not detected in either the teome or normal k-mers were considered as MOI candidates. Flagged. Absent in both canonical proteomes but not in both k-mer databases. MAP present in the source is overexpressed at least 10-fold in AML compared to normal samples. These RNAs are required to have their coding sequences expressed and are flagged as MOIs. Finally, MA corresponding to several RNA sequences (derived from different proteins) are P if their respective coding sequences are consistently flagged as MOI can only be flagged as MOI.

[0181] Regarding the ERE approach, we investigated the ERE and its application in personalized canonical proteomes. Based on the presence of the amino acid sequence, the ERE states "yes," "maybe," or "no." A status was given to each individual MAP. For "maybe" candidates, the ERE lead and the canonical The expression level of the peptide coding sequence in the read dataset (i.e., peptide The minimum occurrence of a 24-nucleotide k-mer set was calculated. Only "likely" candidates with at least 10-fold higher expression in the set were selected for ER The remaining ERE MAP candidates were then manually verified using IGV. (Robinson et al.,Nat Biotechnol.2011 Ja n;29(1):24-6), the coding sequence of the peptide contains a germline polymorphism, and the ERE sequence Compare the sequence and canonical annotated sequence (if applicable) to determine whether it has the correct orientation. Make a decision.

[0182] For the differential k-mer approach, the complete list of MAPs is used as MOI candidates. Next, we analyzed the RNA expression of each MAP (see below). (following the procedure described in section 3.1.) in 19 AML specimens and DE-kup The best correlation between normal and cancer samples was evaluated in 11 MPCs used as controls in the All MAPs with a small fold change of 5 were flagged as MOIs. Current assessment procedures rely on a reference genome for quantification, and therefore, the risk of mutations is low. We were unable to adequately quantify candidate MOIs and systematically flagged them as MOI candidates. The presence of each MOI in each AML sample in which they were identified was clearly verified at the RNA level. To do this, we extract the MO from the DiffContigsInfo.tsv output of DE-kupl. I extracted the coding sequence and related fastq files (forward R2 fastq The sequence was compared with the reverse complement of the corresponding sequence in the reverse direction (R1 fastq). The MOI was discarded.

[0183] For the entire list of MOI candidates (four different approaches), leucine and isol Because the isin variants cannot be distinguished by standard MS approaches, each list was examined. The MOI at which the existing variant was flagged as a non-MOI was higher than the variant. Unless they showed high RNA expression, they were discarded. MS / MS spectra of all MOIs were obtained manually. Finally, their coding sequences on the reference genome were compared to the original genome. The reads containing the columns were mapped using BLAT (a tool from the UCSC genome browser). Genomic locations were assigned to all MOIs by matching the matched genomic locations. Reads that did not match or matched in hypervariable regions (e.g., MHC, Ig, or TCR genes) The MOI of the matched reads was excluded. For those with matched genomic locations, IGV was used. Using the MOI (dbSNP14) with coding sequences overlapping with known germline polymorphisms, 9) was excluded.

[0184] 3) Quantification of MAP coding sequences in RNA-Seq data. RNA expression of each MAP. To clearly assess the MAP amino acid sequence, all MAP amino acid sequences were compared to all possible nucleotide sequences. Next, the genome was back-translated using GSNAP (Wu et al., 2016). Use the -n1000000 option to map all these possible sequences and All genomic regions capable of encoding a given MAP were located. To confidently capture the MAP encoded by the site sequence, possible MAP codes were The reference sequences are mapped to the transcriptome (cDNA and non-coding RNA). Extract the majority of the transcriptome sequences (80 nucleotides) (samtools faidx --length80 option), then matched to the reference genome. Pinged (GSNAP, --use-splicing and --novelsplic ing=1 option). For OIs, we also performed genome alignment of all reads containing their coding sequences. Filter the output of P to keep only exact matches between the array and the reference given Generate a BED file containing all possible genomic regions amenable to MAP coding samtools view (-F256 option), grep, and wc (- l option) for each desired RNA-Seq sample (e.g., AML) MAP coding sequence at each genomic location in the genomic region (e.g., GTEX, or normal sample). The number of reads containing the sequence was counted and aligned to the reference genome using STAR (bam file). Finally, all read counts (from different regions and coding sequences) for a given MAP were calculated. The total number of reads sequenced in each evaluated sample was summed and normalized to the total number of reads sequenced per 100 million. Reads per minute (RPHM) counts were obtained.

[0185] 4) Evaluation of immunogenicity. The immunogenicity prediction of MOI was performed using Repitope (Ogishi a The predefined MHCI_ Use the Human_MinimumFeatureSet variable to perform feature calculations and The Mendeley repository for the package (https: / / data.mendeley .com / datasets / sydw5xnxpt / 1) Updated reDF_MHCI and FragmentLibrary files (2019 (July 12).

[0186] 5) MOI presentation and expression by AML patients. Given MAP (promiscuous Identify all possible HLA alleles that can present miscuous binders To do this, we used the MHCcluster online tool (http: / / www.cbs.d tu.dk / services / MHCcluster / )(Thomsen et a HLA alleles with a clustering value of 0.4 or less were used. Those who presented the same MAP were considered to be able to do so. To assess MOI presentation by ML patients, we first identified their HLA types using Optit To maximize the probability that the expression at the RNA level is If the patient is more than 2 rphm (not rphm), and if the patient presents with an MOI When the LA allele was expressed (NetM for the original identification of the presented molecule at each MOI found) MHC classifiers predicted by HC4.0 and for identification of promiscuous binders A given MOI was considered to have been presented. If two different HLA alleles that can be expressed are expressed, the MOI is considered to be presented twice. It was done.

[0187] High TSA 高 To assess molecular features associated with expression, TSA 高 In this patient Its expression in the cohort is the median of its expression across the entire cohort (calculated based only on non-null values). A TS was considered to be expressed in a given patient if its expression level was higher than that of the TS expressed by the TS gene. A 高 Total number of (#HE-TSA高 ) were counted for each patient and correlated with gene expression. The following analyses were used to perform correlation analyses of associations with mutations or other clinical features: section).

[0188] 6) Survival analysis. The survival data for 374 patients in the Leucegene cohort were Kindly donated by the Leucegene team (https: / / leucegene.ca) High count HLA-TSA calculated as above 高 Complex (HLA-restricted TSA 高 Survival analysis was performed to assess the association between the presentation of sarcoma and clinical outcome (overall survival). They can present it to the TSA 高 Depending on the total number of markers, two groups were identified: high expressers ( HLA-TSA 高 were divided into low expressers (top quartile of counts) and low expressers (all other patients) Survival rates were compared between the two groups using Kaplan-Meier curves, and significance was The results were evaluated using the log-rank test in GraphPad Prism v7.0. The analysis was carried out using the R package survivalAnalysis v0.1.1. performed, incorporating age as a continuous variable and coding mutations as present / absent (1 / 0). However, cytogenetic risk assessments were treated as individual groups, with intermediate risk versus favorable risk and favorable risk assessed. The risk of harm versus favorable risk was assessed.

[0189] 7) Mutation analysis: NPM1, FLT3-ITD, FLT3-TKD, IDH1 (R132 ) and biallelic CEBPA mutation data were compared with previously published Leucegene cohort The data was taken from the database (Audemard et al., 2019, Lavall ee et al., 2016). ASXL1, TP53, DNMT3A, IDH2(R 140 and R172 only), WT1, RUNX1 and TET2 mutations were identified using Fr The following mutations were detected and filtered using eebayes: (i) mutations less than 20%; (ii) a mutation with a variant allele frequency (VAF) of less than (https: / / cancer.sanger.ac.uk / cosmic) (iii) mutations with low predicted effect (5' UTR premature start codon gain variants, splice region variants, and synonymous variants , stop-retaining variants, synonymous variants), (iv) FATHMM-XF (http: / / fathmm.biocompute.org.uk / fathmm-xf / missense SNPs (Ro) predicted to have mild effects on protein structure and function gers et al., 2018), (iv) insertions with AAAAA+ or TTTTT+ (v) only germline insertions and deletions flagged in the COSMIC database mutations that have been reported (Tate et al., 2018).

[0190] 8) Gene expression analysis. Quantification of the expression of all transcripts was performed with default parameters, k Kallisto v0.43.0 was used. Kallisto transcript levels Count estimates were converted to gene-level counts using the R package tximport. Counts were normalized using the TMM algorithm to counts per million. edgeR was used to output count (cpm) values. Only protein-coding genes were retained (using Ensembl's BioMart tool). As reported at biomart.ensembl.org / biomart). Child expression and HE-TSA 高 Exhaustive Pearson correlation between counts, using cor.tes in R Correlations were performed using the t function. Correlations were performed for all non-NPM1 / FLT3-ITD / DNMT3A The results were compared for mutated and non-FAB-M1 patients. p values ​​were calculated using the Benjamini-Ho Corrected for multiple comparisons with the chberg method (p.adjust in R), three correlation analyses FDR<0.00001 in at least one of the analyses, and FDR< 0.001, a consistent correlation coefficient (positive or negative) in three analyses, and at least one Only genes with a correlation coefficient >0.3 or <-0.3 in the analysis were retained for downstream processing. I held it.

[0191] t-SNE analysis is performed using tximport to estimate gene abundance. Kalli to abundance estimation (expression = 1 if tpm ≥ 1, expression = 0 if tpm < 1) The identities of expressed genes obtained from the aggregation of transcript-level abundance estimates in sto were analyzed using R This analysis was carried out using the tsne package. Only the most susceptible to the formation of

[0192] 9) GO term and enrichment map analysis. Biological process Gene Ontology (GO) terms Overexpression was measured using BiNGO v3.0.3( Maere et al., 2005), using the hypergeometric test, A significance cutoff of FDR-adjusted p-values ​​of 0.5 or less was applied. The output from BiNGO is EnrichmentMap v3.2.1 (Merico) (et al., 2010) to cluster redundant GO terms, and the results The EnrichmentMap was visualized using a Jaccard similarity coefficient of 0.25. A p-value cutoff of 0.001 and an FDR-adjusted cutoff of 0.005 were used. The network was generated using Cyto In scape, the default "Prefuse Force-Directed L Visualization was performed using "ayout." Clusters of similar GO terms were manually circled.

[0193] 10) Intron retention and NMF clustering. Complete Leucogen cohort and intron retention (IR) analysis of 11 primary MPC samples using IRFinder v1. 2.5 (Middleton et al., 2017). o ≥ 10% (introns with ≥ 10% transcript retention) and a minimum coverage of 3 reads Introns with the nucleotide sequence were considered to be retained. and retained in at least two AML samples and not retained in any MPC samples Only the 10% most variable introns (IRa across the complete cohort) were retained. tio) were selected for further analysis (6988 introns). NMF v0.21.0 (Gauj) in R for the IRatio of selected introns oux and Seoighe, 2010) package, and the default Bru net algorithm, and 200 iterations for rank search and clustering. Unsupervised consensus clustering was performed using the 3-15 clusters. For a clustering solution with The cluster results are selected based on the profile of the netic score and the average silhouette width. Ta.

[0194] Top 2% ranking in NMF metagene (W matrix) output file An abundance heatmap was generated by identifying introns in the After removal, a list of 1211 introns was obtained. Generate a matrix of these intron I Ratios and cluster them using NMF. Using the heatmap.3 package in R, sort the output to match the rings. Hierarchical clustering of introns with a central correlation distance metric and complete linkage was performed. Ta.

[0195] ELISPOT assay 1) Generation of monocyte-derived dendritic cells. Monocyte-derived dendritic cells were generated from frozen PBMCs as previously described. The dendritic cells were generated from the dendritic cells (Vincent et al., Biology of Blood od and Marrow Transplantation: Journal of the American Society for Blood and Marr ow Transplantation,22 Oct 2013,20(1):37- 45;Laumont et al.,Nat Commun.2016 Jan5;7 Briefly, DCs were cultured in 5% human serum (Sigma-Aldrich), pi medium (Philips et al., 2014). sodium benzoate (1 mM), IL-4 (100 ng / mL, Peprotech), and X-VIVO ( by culturing in Lonza Biosciences 15 medium for 8 days. DCs were prepared from the adherent PBMC fraction. After 7 days of culture, DCs were treated with IFN-γ (1000 IU / mL, Gibco) and LPS (100ng / mL, Sigma Aldrich) After 2 hours of the maturation process, DCs were loaded with 2 μg / mL of peptide. They were then irradiated (40 Gy) and used as APCs in T-DC cultures. For the control group, DCs were injected with Melan-A, NS3, and Gag-A2 peptides (all three HLA- A*02:01) was pulsed with a mixture containing 1000kJ / mL of ATP.

[0196] 2) In vitro peptide-specific T cell proliferation. Thawed PBMCs were first transfected with human CD8 + CD8 T cell isolation kit (Miltenyi Biotech) was used. + T cells DCs were enriched and co-incubated with DCs pulsed with autologous peptides at an APC:T cell ratio of 1:10. The expanded T cells were cultured in 8% human serum (Sigma-Aldrich), L -Advanced RPMI supplemented with glutamine (Gibco) and cytokines The DCs were cultured in 100% PBS medium (Gibco) for 4 weeks (with restimulation with pulsed DCs every 7 days). During the first week of co-culture, IL-12 (10 ng / mL) and IL-21 (30 ng / mL) were administered. Two days later, IL-2 (100 UI / mL) and a cytokine mixture were added to the medium. In the second week, IL-2 (100 UI / mL), IL-7 (10 ng / mL), IL-15 (5 ng / mL) and IL-21 (30 ng / mL) were added to the medium. During the final 2 weeks of culture, IL-2 (100 UI / mL), IL-7 (10 ng / mL), and The medium was supplemented with the appropriate cytokine mixture. , were added to the co-culture every 2 days. At the end of the fourth week of co-culture, ELISPOT assays were performed. The cells were harvested to perform

[0197] 3) IFNγ ELISPOT assay. To perform the experiment, follow the manufacturer's recommendations. The ELISpot human IFNγ (R&D Systems, USA) kit was used. Then, the collected CD8 + T cells were seeded and irradiated penicillin was used as stimulator cells. The cells were incubated for 24 hours at 37°C in the presence of peptide-pulsed PBMCs (40 Gy). As a gender control, CD8 selected + T cells were incubated with irradiated, non-pulsed PBMCs. The spots were prepared by Imm as described in the protocol of the reagent set manufacturer. unoSpot S5 UV Analyzer(Cellular Technolo IFN was counted using a IFN counting kit (Gy Ltd, Shaker Heights, OH). -γ production was calculated by subtracting spot counts from negative control wells. 6 CDs 8 + It was expressed as the number of peptide-specific spot-forming cells (SFC) per T cell.

[0198] Immunogenicity prediction The immunogenicity prediction of MOI was performed using Repitope (Ogishi and Yotsuya This was performed using the predefined MHCI_Human_Mini Use the mumFeatureSet variable to perform feature calculations and use the package Mende ley repository (https: / / data.mendeley.com / datas ets / sydw5xnxpt / 1) The FragmentLibrary file was updated (July 12, 2019).

[0199] TCR and cytotoxic T cell signature analysis TRUST4 software (Li et al., 2017) and default parameters Using this data, we analyzed the TCR Repertoire analysis was performed. T cell clonotype diversity was measured per kilo TCR read. The number of TCR CDR3 (complete and partial) was estimated by normalizing (CPK) The amino acid sequence of the complete TCRβ CDR3 detected by TRUST4 and the MOI ERGO (Springer et al., 2020) predictions of interactions between Aut Free use of oencoder-based models and VDJdb as training database Through the web portal (http: / / tcr.cs.biu.ac.il / ) went.

[0200] For cytotoxic T cell signature analysis, predicted HLA-T per patient SA 高 The number of pairs was calculated by dividing the number of TSAs with rphm expression ≥ 2. 高 Normalize by dividing by the counts of TSA 高 The presentation level of HLA-TSA was obtained. 高 Patient samples that were not counted or Patient samples not collected at the time of diagnosis were discarded from analysis. These regularized TSA 高 Grouped according to presentation level, patients above the median of the distribution Patients were compared with other patients (below the median) through differential gene expression analysis. R3.6. Analysis was performed at 1. Raw read counts were normalized to library size. Converted to counts per million (cpm) and analyzed using edgeR 3.26.8 (Robin son et al., 2010) and limma3.40.6 (Ritchie et al. al., 2015) to identify genes with cpm > 1 in at least two samples. By retaining the nucleotides, low-expressing genes were filtered out. We performed a Voom transformation and linear modeling using lmfit in MA. Finally, we adjusted the t Statistics were calculated using eBayes. p-value ≤ 0.01 and -0.3 ≥ log2(FC) ≥ 0. .3 genes were considered as significantly differentially expressed.

[0201] Cytokine secretion assay and dextramers Peptide-loaded monocyte-derived dendritic cells based on (Janelle et al., 2015) and 1.0 × 10 after three rounds of stimulation with cytokines. 6 7.5μ cells g / ml brefeldin A (Sigma-Aldrich, Oakville, ON) In the presence of dimethyl sulfoxide (DMSO), 5 μg / ml of the peptide of interest, 5 μg / ml of control peptide (negative control), or 50 ng / ml of phorbol 12-myristate 13-Propanol acetate (PMA), and 500 ng / ml ionomycin (positive control, S The cells were then incubated with either IgG1 or IgG2 (Igma-Aldrich) for 4 hours. Stain with cell surface antibodies and use Cytofix / for intracellular staining according to the manufacturer's instructions. Cytoperm buffer was used for fixation and permeabilization (BD Biosciences) es, Mississauga, ON). Permeabilized cells were incubated with IFNγ, IL-2, and TNFα (BD Biosciences) for 20 min at 4°C. Incubated and supplemented with 2% fetal bovine serum (FBS; ThermoFisher, Waltham After resuspension in phosphate-buffered saline (PBS) supplemented with HCl (H2O, HCl ... This acquisition was performed using an LSRII flow cytometer (BD Biosciences). The analysis was performed using FlowJo™ V10 software (BD Bioscience Data were analyzed using a 1.0 × 10 6 cells, Custom-made fluorescent dextramer (Immudex, Copenhagen, Denmark) The cells were stained with CD8 monoclonal antibody (eBioSc) for 45 minutes at 4°C, and then stained with CD8 monoclonal antibody (eBioSc). The cells were stained with PBS (BioSciences, San Diego, CA) for 30 minutes at 4°C. After washing with 2% FBS, the cells were then placed on an LSRII cytometer (BD Biosciences) Data were acquired using FlowJo™ V10 software (BD Biosciences). The analysis was carried out using the NIMS Sciences.

[0202] FEST assay For the FEST assay, T cells were cultured as previously described with minor modifications (D Briefly, on day 0, healthy donors (BioI Thawed PBMCs from VT were isolated using a human pan T cell isolation kit (Miltenyi). T cells were enriched by 50 μg / mL gentamicin (ThermoFi AIM V medium supplemented with 1% HEPES (Sher Scientific) 2×10 6 The T cell negative fraction was irradiated with 30 G γ, washed, and resuspended at 50 μL / mL. AIM V medium supplemented with 2.0× g / mL gentamicin and 1% HEPES was used. 10 6 1 mL of T cells and irradiated T cell depleted cells per well were resuspended at 1 mL / mL. and both TSA 高 Pool (5 TSA per pool) 高 , 1μ for each TSA in a 12-well plate with or without peptide (final concentration of 100 M). The cells were cultured at 37°C and 5% CO2 for 10 days. On the third and seventh days, the culture medium was Half of the culture medium was supplemented with 100 IU / mL IL-2, 50 ng / mL IL-7, and 50 nM IL-1. g / mL IL-15 (day 3), and 200 IU / mL IL-2, 50 ng / mL Replace with fresh culture medium containing 100 mg / mL IL-7, 50 mg / mL IL-15, and 50 ng / mL IL-15 (day 7). On day 10, human CD8 + A T cell isolation kit (Miltenyi) was used to isolate cells. The cells were collected and CD8 + The cells were further isolated. As a negative control, CD8 + T cells also DNA was isolated from freshly thawed, uncultured PBMCs of the same healthy donor. CD8 was isolated using a liquid mini kit (Qiagen). + DNA was extracted from T cells. munoSEQ platform (Adaptive Biotechnologies TCRVβ CDR3 sequencing was performed using a survey resolution of 1000 kJ / s. The raw data exported from the Q Portal can be accessed using the FEST web tool (www.sta t-apps.onc.jhmi.edu / FEST) with a minimum number of templates and Processed without the "Ignore baseline threshold" parameter .

[0203] Quantification and statistical analysis Unless explicitly mentioned in the figure legends, all statistical tests comparing two conditions were performed using the The Whitney U test was used. All correlations were assessed using the Pearson correlation coefficient. Unless otherwise specified, all boxes in the box plot represent the median, 25th percentile, and and 75th percentiles, and whiskers indicate the 10th and 90th percentiles. Unless otherwise stated, all bar graphs show the mean and standard deviation (SD). The analysis and statistical tests were mainly performed using GraphPad Prism v7.00. For all statistical tests, **** indicates p<0.0001 and *** indicates p<0. 0.01, ** indicates p<0.01, * indicates p<0.05.

[0204] Example 2: Purified hematopoietic progenitor cells are a useful control for detecting TSA in AML is. MS is the only available technique that can directly identify MAPs (Ehx and d Perreault, 2019, Shao et al., 2018). Typically MS-based identification of MAPs is performed by providing acquired tandem MS spectra by the user. Through the use of software tools that match the protein sequences to databases provided However, the reference protein database only contains canonical protein sequences. They contain, and are therefore, a major source of mutations and aberrantly expressed non-canonical genomic regions (aeTSAs). MAPs derived from the MAPs cannot be identified (Laumont et al., 2018 ) Proteomic analysis to build a tailored MS database for global TSA identification. The genomics strategy has been described previously. The adjusted database was generated for each tumor sample. It must be constructed to include all potential TSAs and meet two criteria: comprehensive enough to ensure accurate detection and the extended reference database reduces the risk of false discoveries. The size of the cells is limited from increasing (Nesvizhskii et al. l.,2014, Chong et al.,2020). Database construction involves: (i) (ii) RNA sequencing of tumor samples, which is the core of the data; and (iii) 33-nucleotide-long subsequences. In silico slicing of RNA-seq reads into (k-mers), and (iii) Subtraction of normal k-mers to create modules containing only non-specific k-mers As with many aspects of cancer research, the challenge is determining whether a negative control (here, a normal control) is present. Previous studies have used k-mers from mTECs as a source of normal versus normal k-mers. However, in the case of AML, another type of negative control, i.e., Selected bone marrow progenitor cells (including granulocytic / monocytic progenitors and various types of granulocytic progenitors) MPC) was tested.

[0205] First, 19 target AMs were used to compare the values ​​of mTECs and MPCs as negative controls. L specimens (see Table 1 for characteristics), six mTEC samples, and high-coverage RNA Six MPC samples for which .ALPHA.-seq has previously been performed (Maiga et al., 201 6) were compared. In particular, MPCs were found to be 16 times more likely to be derived from AML than mTECs. 0.4% more k-mers were depleted, resulting in better correlation between MPC and AML than between mTEC and AML. Therefore, mTECs and MTECs showed a greater overlap in their transcriptomes than mTECs (Fig. 1A). PC (approx. 8.7×10 8 for approximately 9.9 × 10 8 ) for both Although a large number of mTECs (approximately 1.9 × 10 8 , approximately 22%) than AML (approx. 3.3×10 8 , approximately 33%) and shared more exclusive k-mers (Figure 1B). To confirm the origin of this higher similarity, we analyzed the expressed proteins. Selected epithelia downloaded from various sources based on the identity of quality-coding genes t-SNE clusters of AML samples along with RNA-seq arrays of AML and hematopoietic cells Clustering was performed (see Methods), which demonstrates that AML samples cluster with hematopoietic cells. The results showed that mTECs clustered with epithelial cells, whereas mTECs clustered with epithelial cells (Fig. 1C). Importantly, mTECs harbor the highest diversity of genes, consistent with their biological functions. (Figure 1D). Collectively, these results demonstrate the transcriptome diversity of mTECs. Despite its usefulness, MPC has advantages over mTEC for detecting TSA in AML. As a corollary, instead of mTEC k-mers, When subtracting MPC k-mers, the size of the database of AML-specific k-mers is It becomes smaller.

[0206] Example 3: Development of an MPC-based TSA discovery approach In addition to capturing the entire AML TSA landscape, the construction of the reference database Four strategies for the construction of endothelial cells were evaluated. The first two strategies have been previously reported (Figure 2A, B), and the other two are novel (Fig. 2C, D). Importantly, MS analysis of AML specimens Each of the four different TSA discovery approaches was performed only once for each AML test. The first strategy was mTEC subtraction (Figure 2). This results in A) (Laumont et al., 2018). The second strategy is to We focus specifically on MAPs encoded by EREs, which can be an abundant source (Figure 2B )(Larouche et al., 2020).

[0207] The third strategy depleted k-mers from both mTECs and MPCs (Figure 2C). Notably, before the depletion step, the final k-mers for contig assembly were limit the number of k-mers to about 30 million (assembling more k-mers requires more computation time). Filtering k-mers based on their occurrences (too strict from a statistical point of view) (The number of times k-mers exist in the same sample, Figure 8A, B). PC k-mer depletion yielded more k-mers from AML samples than mTEC alone. This reduces the threshold for emergence by approximately 2-3 fold, thereby enhancing the mTEC k-mer depletion approach. This allowed us to discover MAPs that had been missed in the Loach database (Figure 8C , D).

[0208] The fourth strategy is to address the main caveat of the k-mer depletion strategy (the difference between k-mers in normal and cancer samples). The aim was to avoid the lack of comparison between k-mer abundances. In the depletion strategy, the presence of k-mers in the normal control indicates that their frequency is higher than that of the normal control. Even at 100-fold higher levels, this results in k-mer filtering in cancer samples. Briefly, differential k-mer expression (DKE) analysis was performed using the DE-kupl computational protocol (Au Doux et al., 2017) and performed with some adjustments at our institution. The results can be summarized as follows (Figure 2D and Figure 9A): (i) at least 30 Pre-filtering of k-mers present in % of AML samples (occurrence ≥ 3 times), (ii ) normalization of k-mer abundances, (iii) k-mer abundances by a user-defined algorithm Statistical comparison of abundance, (iv) significantly differentially overexpressed k-mers (minimum fold change 1 (v) assembly of the contigs to establish the origin region. Genome alignment. In this study, MPs were selected because they were the most closely related normal samples. C was selected and used as normal controls, and 19 AML samples were compared with the 11 available high-coverage The differentially expressed contigs were then compared with the MPC samples. Individualized for each AML sample (based on read coverage and SNP calling) Contig sequences are generated and translated into all possible read frames, and individualized canonical pro- cesses are performed. In combination with the teome, MAPs were identified (Fig. 2).

[0209] Example 4: MPC-based approach improves TSA in AML 高 Identify the majority of Each of the four TSA discovery approaches generated thousands of MAPs across 19 AML samples. To be considered an effective TSA, MAP must be expressed by AML cells. The epitope density must be high enough to be presented abundantly on target cells by CD8 T cells. Because they play a key role in eradication, they must be present in a way that is not presented by normal cells or that does not induce T cell recognition. The signal must be presented at a low enough level that it does not disturb the signal (Cosma and Eisenl ohr, 2019), because MAPs are preferentially derived from highly abundant transcripts (Figure 3A and Pearson et al., 2016), two critical thresholds were established: (i) normal group; (ii) RNA expression levels that can be considered to be low in tissues that are likely to produce MAP; and Establish the fold change (FC) in RNA expression required to significantly increase the probability of presenting a To achieve this, we performed a randomized multicenter study of all identified MAPs in each AML sample. RNA expression was assessed and found to follow a normal distribution plotted as a cumulative frequency distribution. This resulted in an incidence of less than 8.55 reads per hundred million (RPHM) (Figure 3B). Currently, it has been demonstrated that the probability of MAP formation is less than 5%. They express similar levels of MHC molecules compared to granulocytes, with granulocytes having the highest expression levels among normal tissues Considering that they express MHC-I at high levels (Berlin et al., 2015 , Boegel et al., 2018), as the first threshold for all organizations. Based on the same distribution, the difference in the probability of generating MAP was The effect of different FCs can also be evaluated (Figure 3C). This indicates that FCs of 2 to 5 have a larger effect. showed that the minimum FC tends to have a greater effect on the probability than the minimum FC. An FC threshold of 5 was adopted.

[0210] Based on these two thresholds, we identified AML, MPC, other normal hematopoietic cells, and mTEC. We separate MAPs based on their RNA expression in a wide range of normal adult tissues, including A decision tree was established for this purpose (Figure 3D). All MAPs expressed in AML and at higher levels in MPCs The detection of these proteins is highly dependent on the surface of AML cells, while their probability of being presented by normal tissues is low. This was flagged as evidence of their presentation in the TSA. Therefore, there is little FC between AML and MPC. At least 5 TSA 高 Hematopoietic cells compared to other tissues were flagged as Other MAPs that were overexpressed in cells but did not meet these criteria were identified as TAA or hematopoietic-specific MAPs. The antigen was classified as a specific antigen (HSA) (Fig. 3D).

[0211] Pre-filtering steps (see Methods) and decision tree-based classification for each pipeline After this, four lists of desired MAP (MOI) were obtained (Table 2). The roach yielded the highest percentage of HSA, while the TSA 高 Most of the The DKE approach was the first to identify the markers (Figs. 3E, F, and 10A). To pre-filter the k-mers with the smallest occurrence in the smallest number of patients, both MPs The overlap between C-based approaches was low. Most TSA 高 compared with those identified by the DKE approach Overall, these results suggest that the DKE approach is effective in detecting AML. At TSA 高It is most suitable for identifying MPC-based k-mer depletion Complemented by the thirst approach, additional less shared TSA 高 Identifying This shows that it is possible to do this. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] Biotypes were manually assigned upon inspection of the peptide-coding sequences at the indicated genomic locations. The immunogenicity score was calculated using Repitope. HLA alleles were netMHC4. 0 to the most likely presenting peptide in a given sample. The corresponding synthetic peptides were validated using TSA. 高 This was carried out only.

[0212] To assess the robustness of MOI identification, the observed mean retention time (R) of a given peptide was calculated. T) demonstrates two best-in-class methods for validation of MAPs identified by high-throughput MS. Trick: RT (Bouwmeester) calculated by the DeepLC algorithm et al., 2020) and the hydrophobicity index evaluated by SSRcalc (Krokhi n, 2006), both of which were predicted based on peptide sequences. RT distributions of non-canonical MOIs correlate well with predictions and support correct identification of canonical proteolytic sequences. The distribution of the peptides derived from the IL-11 gene was not significantly different from that of the peptides derived from the IL-11 gene (F test) (Fig. 3G). MS database search (initially performed with PEAKS software) The re-identification percentage was calculated using the t algorithm. There was no significant difference between the MOI of 58 and the TSA (Figure 3H, left panel). 高 of Of these, 52 (90%) were re-identified (Figure 3H, right panel). This large overlap between the MAPs identified thus further supports the robustness of non-canonical MOI identification. do.

[0213] Example 5: TSA 高 is an immunogenic MAP that is primarily derived from the translation of an intron. The combined results of the four TSA discovery approaches yielded a total of 47 HSAs and 49 TAs. A, 36 TSA 低 , and 58 T.S.A. 高 (No redundancy) was obtained. Table 2 shows all M The main characteristics of OI are listed. By definition, TSA is responsible for all (from GTEx) It was expressed below threshold in organs, as well as in mTECs and normal hematopoietic cells (Figure 4A). Importantly, TSA in normal tissues 高 Expression of coding RNA is associated with off-target toxicity The results were systematically inferior to those of previously used TAAs in clinical trials without the use of Chapus albicans. is et al.,2019, He et al.,2020, Legat et a l., 2016, Qazilbash et al., 2017). The HLA ligand atlas, which includes human MAPs identified in 9 non-malignant tissues, does not include any T SA 高 does not exist. https: / / www.biorxiv.org / conten t / 10.1101 / 778944v1). This is TSA 低 and 58 TSA 高 (redundant TAAs support the safety of targeting at least one normal tissue. Although HSA expression was elevated, it was restricted to the hematopoietic compartment. Comparison of FC between L specimens and MPC revealed that TSA 高 was most highly overexpressed along with TAA (median 22-fold), whereas HSA was expressed at the highest level in healthy cells ( The median value was 0.6-fold (Figure 4B). Overall, these results suggest that TSA 高 but, This demonstrates that it combines the advantages of both TSA specificity / safety and TAA overexpression.

[0214] TSAs are mostly derived from what are considered non-coding regions of the genome; Of these, only 13% were derived from canonical protein exons, and Among these, 58% were identified as being derived from introns (Fig. 4C). None of these were found to be mutations consistent with the low mutational burden of AML (Lawrence et al. , 2013). TAAs are primarily derived from protein-coding exons, but the origin of HSA is , consistent with previous studies reporting tissue-specific intron retention and ERE expression patterns. It was also dominated by non-coding regions (Middleton et al., 2017 , Larouche et al., 2020). 8 TSAs 高 is a canonical protein core Although derived from the TAAs, their low expression in normal tissues compared to safe TAAs Considering this, they can be considered safe targets (Figure 4A). In support, three of them were associated with known AML biomarkers (LTBP1 , MYCN, and PLPPR3), while the other five have unknown functions or are amplified. They are involved in proliferation, differentiation, or drug resistance (Table 3). [Table 5]

[0215] The therapeutic value of TSA depends in part on the extent to which it is shared by the patient. TSA between sexually transmitted leukemia and atherosclerosis 高 To assess sharing, purified AM for 437 patients was used. Leucine cohort containing RNA-seq data from L-blast cells (Lavall ee et al., 2015, Macrae et al., 2013, Pabst We analyzed the MAPs of different HLA allotypes. The identified TSA may be presented as 高 The presentation begins with the promiscuous binder. The evaluation was carried out by taking into account HLA alleles that present similar epitopes together. Clustering using the MHC cluster tool (Thomsen et al., 2013 ) to allow individual TSA 高 HLA allotypes that can present The complete set can be estimated (Table 4). Based on these data, the world population Among them, 99.92% of individuals have one TSA 高 One or more HLs can be presented The TSA-encoding transcript was then expressed and Only when a patient has an HLA allotype capable of presenting with this TSA can an individual TSA 高 was considered to be present in a given AML sample. Based on these criteria, TSA per patient in the Leucegene cohort 高 The median number of 93.6% of patients had at least one TSA 高 It can be predicted that the F). [Table 6-1] [Table 6-2] [Table 6-3]

[0216] TSA in AML samples analyzed at initial diagnosis 高 When the number of samples was compared with that at relapse (mismatched samples), In another study (Toffalori et al. al., 2019) reported that AML blasts obtained at diagnosis and at relapse after allogeneic hematopoietic cell transplantation TSA that can be presented by patient HLA alleles in matched cell samples 高 RNA expression No difference was observed when comparing the two (Fig. 4H). Because it is a medium for TSA (Shlush et al., 2017), 高 and HLA R NA expression was measured using LSCs obtained from another study ( Corces et al., 2016 ). We also evaluated the RNA-seq data of the blast cells sorted from the blastocysts and found no differences between the two cell populations. However, gene set enrichment analysis (GSEA) revealed no significant differences (Figure 4I-J). ) allows many TSA 高 Patients who develop HIV also have higher levels of were found to express the established LSC gene signature (Eppert et al. al., 2011) (Figure 4K). Overall, these results suggest that TSA 高 High immunogenicity Further supporting this, they are present in almost all AML patients, either at diagnosis or relapse. Therefore, TSA 高 Immune targeting of It can be assumed that there is a possibility of eliminating the LSC.

[0217] Example 6: Multiple TSAs 高 Presentation of IL-1 correlates with better survival. Second, TSA at diagnosis relative to patient survival 高 We investigated the effect of presentation. The most numerous (top quartile) TSAs 高 Patients expressing The survival rate was good (Figure 5A). 高 The survival benefit associated with presenting with age , and other known prognostic factors such as cytogenetic risk, NPM1 and FLT3-ITD mutations. Both remained significant in multivariate analysis (Figure 5B). pre d The same comparison, made independently of presentation, showed no differences between high and low expressers. (Fig. 11A, B). 高 This means that the protective effect of TAA, HSA, or TSA 低 The same analysis performed on These data were consistent with TSA 高 spontaneous anti-AML immune response This suggests that the antibody is sufficiently immunogenic to induce

[0218] TSA 高 The survival advantage provided by their cumulative HLA pred Due to presentation To demonstrate this, high TSA 高 Patients and low TSA 高 The log-rank p-values ​​for the patients were calculated as From the analysis of the increasing TSA 高 Calculated after randomly removing the number of (1 to 29 out of 58) (1000 random permutations / number). Increasing numbers of TSA 高 The probabilistic removal of low expressers (other High expressers (HLA-TSA) compared with all patients 高 Significance of (upper quartile of counts) The survival advantage of individual TSAs was rapidly lost (Fig. 5C-D). 高 The survival advantage associated with subtracting This gradual decrease in 高 This suggests that a large proportion of the phenotype contributes to this survival advantage. TSA presented by a larger proportion of patients 高 had the greatest effect on the p-value (Figure 5E). Similarly, log-rank analysis revealed that common HLA alleles (>5% of patients) Removing the shared alleles had a greater effect on the p-value than removing the low-frequency alleles (Figure 5 F). Collectively, these data suggest the impact of TSA on patient survival. 高 The benefits of presentation are HLA-restricted In the next experiment, we demonstrate that TSA 高 Survival advantages associated with presentation We considered the simplest explanation. TSA 高 teeth 、 Induce a spontaneous anti-AML protective immune response.

[0219] Example 7: TSA 高 Presentation elicits a cytotoxic T cell response TSA 高 and the immunogenicity (i.e., their ability to induce an immune response) of other MOIs. As a prerequisite for evaluation, we rely on public TCR databases to predict the probability of a T cell response. We used Repitope, a machine learning algorithm (Ogishi and Y otsuyanagi, 2019). MAP (Adam) presented by thymic epithelial cells opoulou et al., 2013) or HIV-derived MAP, respectively. When used as a control and positive control, TAA was mostly Although it is non-immunogenic, the other three MOIs are immunogenic similar to the HIV peptides. Therefore, TAA significantly improved the survival rate compared with the other three groups and in the IEDB. Compared with a set of 1411 MAPs reported to be immunogenic in mTECs, All non-TAA MOIs showed expression (approximately 12.1 rphm) compared with other immunogens (Figure 6B). showed very low RNA expression in mTECs compared with the soluble peptides, supporting their immunogenicity. To validate the Repitope predictions, an in vitro T cell assay was performed. , HLA-A*02:01-presenting TSA predicted to be most immunogenic 高 :ALPVAL We started with PSL. [Sequence Table 1] JPEG2026009916000028.jpg945 is one of the most immunogenic human MAPs, and therefore is a candidate for IFN-γ ELISpo It was used as a positive control in ard et al., 2016). The immunogenicity of ALPVALPSL is [Sequence Table 2] The results were similar to those of JPEG2026009916000029.jpg944 (Figure 6C). 高 ELISpot is also These TSAs were confirmed to be immunogenic (Fig. 6D). 高 Regarding cytokines Secretion assays and dextramer staining were also performed, which confirmed the ELISpot results. This supported the specificity of the immune response (Fig. 6E-F). 高 But spontaneous and specific T To further demonstrate that TSA can induce proliferation of cell clonotypes, 高 of Short-term cultures of peripheral blood T cells stimulated with different pools are analyzed by TCR sequencing , functional proliferation of specific T cells (FEST) assay was performed (Danilova et al. al., 2018). The five TSAs tested 高 Each pool has 9-10 different chrono These results support their spontaneous immunogenicity, as demonstrated by the induction of type-specific proliferation of IgG1 and IgG2a (Figure 6G and Table 5). . [Table 7] HLA-A*02:01, HLA-A*29:02, HLA-B*15:01, HLA- HLA of healthy donors: B27:05, HLA-C*01:02, HLA-C*03:04 All TSAs that can be represented by the allele 高 TCR-seq is a method for identifying and characterizing adaptive B The raw data was generated by the FEST analysis tool (h http: / / www.stat-apps.onc.jhmi.edu / FEST Here, the number of pools, the TSAs present in each pool, 高 , significantly expanded in each pool The sequence of each clonotype and the FDR and variances provided by the FEST analysis tool Odds ratios are reported.

[0220] Next, because it is "in vivo veritas," 437 Leucegen eA detailed analysis of transcriptome data from patients was performed to identify T cell-mediated TSA 高 In vivo recognition of First, we used the TRUST4 algorithm to identify TCR receptors in T cells. The diversity of the trees was evaluated (Zhang et al., 2019). (Non-immunogenic control Elevated TSAs, as opposed to TAAs (used herein as 高 number of pred presentation is anti-TSA 高 Associated with a decline in TCR repertoire diversity, suggesting clonotype proliferation To demonstrate the specificity of this proliferation, we used the ERGO algorithm. predicted MOI-TCR interactions (Springer et al., 2020). To identify anti-MOI clonotypes, multiple ERGO probabilities were used. TSA 高 Patients with anti-TSA also had the highest CDR3 count among all detected CDR3s. 高 Kuronota The frequency of the type was higher (Fig. 6I). A similar correlation was not observed for TAA (Fig. 6J). Next, the MOI presented by each AML sample was pred Recognizing Proportion of anti-MOI clonotypes capable of clonal replication (i.e., frequency of cognate TCR-MOI interactions) This ratio was calculated as pred Normalized according to the number of MOIs (not The higher the MOI presented, the higher the percentage of resistance, of course. pred proposal This is because the MOI clonotypes are detected. 高 pred showed that presentation was associated with a dramatically higher frequency of specific T cell recognition than TAAs ( Figure 6K).

[0221] Anti-TSA 高 In light of T cell recognition, TSA 高 pred Presentation of activated CD8 T cells It was speculated that this must be related to the infiltration of TSA. 高 Transcript diversity Gender was inversely correlated with CD8A and CD8B expression in AML samples, whereas their pre d This was not correlated with the diversity of presentation (Fig. 6L-M). 高 High transcript abundance The heterogeneity was due to a slightly higher blast purity in the AML samples (as expected for TSA). To avoid this possible bias, we also investigated the HLA -TSA高 The number of expressed TSA 高 Because it is mathematically related to the number of pred presented TSA 高 The number of expressed TSAs 高 Normalized to the number of transcripts and normalized pred proposal We analyzed differential gene expression in patients with levels above or below the median. In addition, TSA 高 pred Some of the 123 genes positively associated with presentation were C Contains D8A, CD8B, GZMA, GZMB, IL2RB, PRF1, and ZAP70 , and were associated with T cell activation and cytolysis (Fig. 6N). Notably, these 123 genes The associated GO terms were exclusively related to T cell activation and differentiation (Figure 6O). Four genes were not differentially expressed and none of the GO terms were significantly associated with the downregulated genes. Therefore, TSA 高 pred Presenting activated CD8 It was concluded that this was associated with a higher abundance of T cells.

[0222] Example 8: TSA 高 RNA expression of AML-related genes was associated with immunoediting signatures, AML driver mutations, and epigenetic changes. associated with genetic abnormalities TSA 高 Given the potential therapeutic value of In this analysis, it is desirable to obtain highly expressed TSA 高 (HE-TSA 高 ) counts , for each Leucegene patient (i.e., a given TSA 高 All patients with non-null expression of Across the board, those TSA 高 counts at levels higher than their median expression The expression of each protein-coding gene and HE-TSA were then compared. 高 With Count Compare the expression of specific genes by performing pairwise Pearson correlations between TSA 高 The release of We evaluated whether genes involved in MAP presentation could be linked to the (HLA-A, HLA-B, HLA-C, B2M, and NLRC5) expression and HE-TS A 高 showed a consistent inverse correlation with the number of TSA 高 Emergence of immunoediting in response to increased expression These results suggest that immunoediting is related to the inhibition of CD47 (dendritic cell phagocytosis) by dendritic cells (Figs. 7A and 12A-B). It is an immune checkpoint molecule involved in the ), and CD84 (which promotes PD-L1 expression by leukemia cells) (Lewinsky This finding was also supported by a positive correlation with PD-1 (Kuhn et al., 2018). It can regulate the expression of L1 (CD274) (Greiner et al., 2014). 017) for NPM1 変異型 and NPM1 野生型 AML patients were analyzed separately. The analysis showed that HE-TSA 高 NPM1 with count 野生型 Patients had a median Below HE-TSA 高 express significantly higher levels of PD-L1 than patients with This revealed that the ATP-dependent ATPase activity was significantly increased (Figure 7B).

[0223] Next, we analyzed the gene pathways correlated with HE-TSA counts (Figure 7C and Table 6). Pathways correlated with biological processes involved in cell growth (including transport and cell organization) (including mitochondrial OXPHOS and proteasome-mediated protein catabolism) Interestingly, inhibition of mitochondrial activity has been shown to decrease MHC-I expression. and can be used as an immune evasion mechanism by cancer cells (Charni Similarly, inhibition of proteolysis is also associated with the inhibition of MHC-I molecules. The amount of peptide for the assay may be less, and therefore the TSA 高 Present (Tripathi et al., 2016). The reduction in the associated processes is related to MHC- This may be a side effect of downregulation of I (Wang et al., 2019). Overall, these The data is provided by TSA 高 Expression of α-glucan in AML cells may contribute to various responses that may function as an immunoediting mechanism. Indicates that it is related to.

[0224] In contrast to negatively correlated pathways, positively correlated pathways were restricted to regulatory processes. (Figure 7D). Thus, 16.1% of the positively correlated genes (vs. 16.1% of the negatively correlated genes) 2.5% of children are TSA 高 It was a transcription factor that could directly mediate the transcription of Among them, the most correlated genes were those affected by genomic imprinting (i.e., DNA methylation). The most important factor was ZNF445 (Figure 7A), a regulator of the mitochondrial endothelial cell membrane-associated epigenetic processes. (Takahashi et al., 2019). ZNF445 functions by regulating DNA methylation. Typically, TSA 高 AML mutations associated with aberrant DNA methylation and expression We investigated possible associations between the three most frequent AML driver mutations (NPM 1変異型 , FLT3-ITD, and DNMT3A 変異型 ) were tested first, and all three were High (above median) HE-TSA 高 significantly enriched in patients expressing In addition, patients with two or three coexisting mutations were found to have than patients with one or zero HE-TSA 高 The number of β-glucan-1-phosphate dehydrogenase (β-glucan-1-phosphate dehydrogenase) was high (Figure 7F). MS analysis Twelve of the 19 AML specimens used in this study were found to have FLT3-ITD or NPM1 mutations. Regarding other frequent AML mutations, IDH2 and biallelic CEBPA mutations were The difference is also HE-TSA 高 positively associated with increased counts, while ASXL1 and SRSF2 , and U2AF1 mutations were negatively associated with FLT3-TKD, IDH1, RUNX1, and TET 2, TP53, and WT1 were not found to be associated with NPM (Figure 12D). 1, DNMT3A, IDH2, and CEBPA 両変異型 Mutations result in abnormal methylation profiles Because it is associated with IL (Figueroa et al., 2010a, Figueroa et al., 2010b, Figueroa et al., 2010c, Figueroa et al., 2010d, Figueroa et al., 2010e, Figueroa et al., 2010f ...g roa et al., 2010b, Ley et al., 2013), HE-TSA 高 The correlation with the rise in counts is that TSA 高 The significance of epigenetic dysregulation in expression Support. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6]

[0225] Finally, TSA 高 Expression of these genes allows us to predict their presence in AML patients. Other clinical features associated with the French-American-British (FAB) subtype, such as We investigated whether this could be achieved (Fig. 12E-H). TSA 高 Patients expressing AML are over- and under-represented in M1 and M5 AML, respectively. Therefore, patients with differentiated AML without a normal karyotype were evaluated at the highest level. Bell TSA 高 Therefore, this is the test used to discover TSA. This is due to an overestimation of FAB M1 AML in the sample (9 of 19). , and TSA 高 Most of the ERs were located in intron regions (37 / 58, ERs located in introns). E-origin TSA 高 This is due to the different intron conservation between FAB types. It was hypothesized that this could be explained by the presence of specific patterns in AML. Unsupervised consensus clustering performed on the locally retained introns revealed The patients showed a clear clustering according to their FAB type (Figure 7G). The data is provided by TSA 高 Expression is associated with AML subtype-specific intron retention patterns Indicates that it can be attached.

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Claims

1. A leukemia tumor antigen peptide (TAP) comprising one of the following amino acid sequences: Table 1-1 Table 1-2

2. The antibody of claim 1, comprising one of the amino acid sequences set forth in SEQ ID NOs: 97 to 154. Leukemia TAP.

3. The leukemia TAP binds to the HLA-A*01:01 molecule and has the amino acid sequence NTSHL PLIY (SEQ ID NO: 48), HTDDIENAKY (SEQ ID NO: 67), YSHHSGLE Y (SEQ ID NO: 89), ILDLESRY (SEQ ID NO: 134), VTDLLALTV (SEQ ID NO: No. 151), or LSDRQLSL (SEQ ID NO: 164), preferably ILDLESRY (SEQ ID NO: 134) or VTDLLALTV (SEQ ID NO: 151), The leukemia TAP described in

4. The leukemia TAP binds to the HLA-A*02:01 molecule and has the amino acid sequence FLLEF KPVS (SEQ ID NO: 7), LLSRGLLFRI (SEQ ID NO: 11), LLDNILQSI (SEQ ID NO: 27), FLASFVEKTVL (SEQ ID NO: 32), ILASHNLTV (SEQ ID NO: 33), Sequence number 33), IQLTSVHLL (SEQ ID NO: 34), LELISFLPVL (SEQ ID NO: 35), LLLPESPSI (SEQ ID NO: 43), ALASHLIEA (SEQ ID NO: 51), ALDDITIQL (SEQ ID NO: 52), ALGNTVPAV (SEQ ID NO: 53), ALLP AVPSL (SEQ ID NO: 54), GLYYKLHNV (SEQ ID NO: 61), HLLSETPQ L (SEQ ID NO: 65), KLLEKAFSI (SEQ ID NO: 72), SLWGQPAEA (SEQ ID NO: No. 77), SVFAGVVGV (SEQ ID NO: 82), VLVPYEPPQV (SEQ ID NO: 8 6), VLFGGKVSGA (SEQ ID NO: 104), KLQDKEIGL (SEQ ID NO: 108 ), TLNQGINVYI (SEQ ID NO: 119), ALPVALPSL (SEQ ID NO: 123) , ALDPLLLRI (SEQ ID NO: 130), KILDVNLRI (SEQ ID NO: 132), S LLSGLLRA (SEQ ID NO: 146), SLDLLPLSI (SEQ ID NO: 150), ILL EEQSLI (SEQ ID NO: 167), LTSISIRPV (SEQ ID NO: 168), TISEC PLLI (SEQ ID NO: 169), ILLSNFSSL (SEQ ID NO: 171), RMVAYLQ QL (SEQ ID NO: 183), or KLNQAFLVL (SEQ ID NO: 188), preferably VL FGGKVSGA (SEQ ID NO: 104), KLQDKEIGL (SEQ ID NO: 108), TLN QGINVYI (SEQ ID NO: 119), ALPVALPSL (SEQ ID NO: 123), ALDP LLLRI (SEQ ID NO: 130), KILDVNLRI (SEQ ID NO: 132), SLLSGL LRA (SEQ ID NO: 146), or SLDLLPLSI (SEQ ID NO: 150).

3. The leukemia TAP according to 1 or 2.

5. The leukemia TAP binds to the HLA-A*03:01 molecule and has the amino acid sequence RSASS ATQVHK (SEQ ID NO: 5), IVATGSLLK (SEQ ID NO: 18), KIKNKTKN K (SEQ ID NO: 19), KLLSLTIYK (SEQ ID NO: 20), ITSSAVTTALK ( SEQ ID NO: 42), VILIPLPPK (SEQ ID NO: 44), NVNRPLTMK (SEQ ID NO: 74), SVYKYLKAK (SEQ ID NO: 91), VVFPFPVNK (SEQ ID NO: 105) , ILFQNSALK (SEQ ID NO: 113), TVIRIAIVNK (SEQ ID NO: 126), ISLIVTGLK (SEQ ID NO: 131), HVSDGSTALK (SEQ ID NO: 159), I AYSVRALR (SEQ ID NO: 160), LSSRLPLGK (SEQ ID NO: 180), or R LVSSTLLQK (SEQ ID NO: 189), preferably VVFPFPVNK (SEQ ID NO: 10 5), ILFQNSALK (SEQ ID NO: 113), TVIRIAIVNK (SEQ ID NO: 126 3. The leukemia of claim 1 or 2, comprising the gene encoding the leukemia vector, TAP.

6. The leukemia TAP binds to the HLA-A*11:01 molecule and has the amino acid sequence SASSA TQVHK (SEQ ID NO: 6), AVLLPKPPK (SEQ ID NO: 45), ATQNTIIGK (SEQ ID NO: 96), SLLIIPKKK (SEQ ID NO: 106), SVQLLEQAIHK ( SEQ ID NO: 121), STFSLYLKK (SEQ ID NO: 149), or RTQITKVSLK K (SEQ ID NO: 152), preferably SLLIIPKKK (SEQ ID NO: 106), SVQLL EQAIHK (SEQ ID NO: 121), STFSLYLKK (SEQ ID NO: 149), or RTQ 3. The leukemia TAP of claim 1 or 2, comprising ITKVSLKK (SEQ ID NO: 152).

7. The leukemia TAP binds to the HLA-A*24:02 molecule and has the amino acid sequence LYFLG HGSI (SEQ ID NO: 13), NFCMLHQSI (SEQ ID NO: 36), KFSNVTMLF (SEQ ID NO: 71), IYQFIMDRF (SEQ ID NO: 92), LYPSKLTHF (SEQ ID NO: No. 95), or RYLANKIHI (SEQ ID NO: 145), preferably RYLANKIHI 3. The leukemia TAP of claim 1 or 2, comprising: (SEQ ID NO: 145).

8. The leukemia TAP binds to the HLA-A*26:01 molecule and has the amino acid sequence ETTSQ VRKY (SEQ ID NO: 59) or TVPGIQRY (SEQ ID NO: 185). 2 is a leukemia TAP described in 2.

9. The leukemia TAP binds to the HLA-A*29:02 molecule and has the amino acid sequence VVFDK SDLAKY (SEQ ID NO: 88), FNVALNARY (SEQ ID NO: 99), or LGISL TLKY (SEQ ID NO: 138), preferably FNVALNARY (SEQ ID NO: 99) or LG 3. The leukemia of claim 1 or 2, comprising one of the following: ISLTLKY (SEQ ID NO: 138). TAP.

10. The leukemia TAP binds to the HLA-A*30:01 molecule and has the amino acid sequence TSRLP KIQK (SEQ ID NO: 26), LSWGYFLFK (SEQ ID NO: 29), or LSHPAPS 3. The leukemia TAP of claim 1 or 2, comprising SL (SEQ ID NO: 165).

11. The leukemia TAP binds to the HLA-A*68:02 molecule and has the amino acid sequence NVSSH VHTV (SEQ ID NO: 50) or SSSPVRGPSV (SEQ ID NO: 148), preferably S The leukemia TAP of claim 1 or 2, comprising SSPV RGPSV (SEQ ID NO: 148).

12. The leukemia TAP binds to the HLA-B*07:02 molecule and has the amino acid sequence GPQVR GSI (SEQ ID NO: 8), SPQSGPAL (SEQ ID NO: 25), VPAPAQAI (SEQ ID NO: No. 40), APAPPPVAV (SEQ ID NO: 55), APDKKITL (SEQ ID NO: 56), KPMPTKVVF (SEQ ID NO: 73), SPADHRGYASL (SEQ ID NO: 78), SP QSAAAEL (SEQ ID NO: 79), SPVVHQSL (SEQ ID NO: 80), SPYRTPV L (SEQ ID NO: 81), PPRPLGAQV (SEQ ID NO: 98), GPGSRESTL (SEQ ID NO: No. 100), APGAAGQRL (SEQ ID NO: 107), TPGRSTQAI (SEQ ID NO: 110), APRGTAAL (SEQ ID NO: 111), SPVVRVGL (SEQ ID NO: 118) , RPRGPRTAP (SEQ ID NO: 120), TLRSPGSSL (SEQ ID NO: 128), T VRGDVSSL (SEQ ID NO: 129), LPSFSHFLLL (SEQ ID NO: 157), PR GFLSAL (SEQ ID NO: 161), IPLNPFSSL (SEQ ID NO: 163), LPSFS RPSGII (SEQ ID NO: 179), or SPARALPSL (SEQ ID NO: 184), preferably PPRPLGAQV (SEQ ID NO: 98), GPGSRESTL (SEQ ID NO: 100), A PGAAGQRL (SEQ ID NO: 107), TPGRSTQAI (SEQ ID NO: 110), APR GTAAL (SEQ ID NO: 111), SPVVRVGL (SEQ ID NO: 118), RPRGPRT AP (SEQ ID NO: 120), TLRSPGSSL (SEQ ID NO: 128), or TVRGDVS 3. The leukemia TAP of claim 1 or 2, comprising SL (SEQ ID NO: 129).

13. The leukemia TAP binds to the HLA-B*08:01 molecule and has the amino acid sequence SGKLR VAL (SEQ ID NO: 4), NPLQLSLSI (SEQ ID NO: 14), DLMLRESL (SEQ ID NO: No. 15), IALYKQVL (SEQ ID NO: 17), NILKKTVL (SEQ ID NO: 21), NPKLKDIL (SEQ ID NO: 22), NQKKVRIL (SEQ ID NO: 23), RLEVRK VIL (SEQ ID NO: 28), EGKIKRNI (SEQ ID NO: 31), LNHLRTSI (SEQ ID NO: No. 47), SIQRNLSL (SEQ ID NO: 49), IPHQRSSL (SEQ ID NO: 101) , NLKEKKALF (SEQ ID NO: 103), ILKKNISI (SEQ ID NO: 114), VL KEKNASL (SEQ ID NO: 137), DLLPKKLL (SEQ ID NO: 139), SRIHL VVL (SEQ ID NO: 147), QIKTKLLGSL (SEQ ID NO: 156), TLKLKKI FF (SEQ ID NO: 170), MIGIKRLL (SEQ ID NO: 181), or NLKKREIL (SEQ ID NO: 182), preferably IPHQRSSL (SEQ ID NO: 101), NLKEKKA LF (SEQ ID NO: 103), ILKKNISI (SEQ ID NO: 114), VLKEKNASL ( SEQ ID NO: 137), DLLPKKLL (SEQ ID NO: 139), or SRIHLVVL (SEQ ID NO: 140). The leukemia TAP of claim 1 or 2, comprising:

14. The leukemia TAP binds to the HLA-B*14:01 molecule and has the amino acid sequence DRELR NLEL (SEQ ID NO: 2), SNLIRTGSH (SEQ ID NO: 39), DQVIRLAGL ( SEQ ID NO: 58), HQLYRASAL (SEQ ID NO: 66), SLQILVSSL (SEQ ID NO: 124), ERVYIRASL (SEQ ID NO: 133), LYIKSLPAL (SEQ ID NO: 13 6), IAGALRSVL (SEQ ID NO: 141), ISSWLISSL (SEQ ID NO: 162) , DRGILRNLL (SEQ ID NO: 175), GLRLIHVSL (SEQ ID NO: 176), or is GLRLLHVSL (SEQ ID NO: 177), preferably SLQILVSSL (SEQ ID NO: 1 24), ERVYIRASL (SEQ ID NO: 133), LYIKSLPAL (SEQ ID NO: 136 3. The leukemia of claim 1 or 2, comprising the sequence: TAP.

15. The leukemia TAP binds to the HLA-B*15:01 molecule and has the amino acid sequence KIKVF SKVY (SEQ ID NO: 10), AQMNLLQKY (SEQ ID NO: 57), GQKPVILTY (SEQ ID NO: 62), or AQKVSVGQAA (SEQ ID NO: 94). The leukemia TAP described in

16. The leukemia TAP binds to the HLA-B*27:05 molecule and has the amino acid sequence RQISV QASL (SEQ ID NO: 1) or LRSQILSY (SEQ ID NO: 144), preferably LRSQ The leukemia TAP of claim 1 or 2, comprising ILSY (SEQ ID NO: 144).

17. The leukemia TAP binds to the HLA-B*38:01 molecule and has the amino acid sequence TQVSM AESI (SEQ ID NO: 46), HHLVETLKF (SEQ ID NO: 64), or THGSEQL 3. The leukemia TAP of claim 1 or 2, comprising HL (SEQ ID NO: 84).

18. The leukemia TAP binds to the HLA-B*40:01 molecule and has the amino acid sequence REPYE LTVPAL (SEQ ID NO: 75) or SEAEAAKNAL (SEQ ID NO: 76). Item 3. The leukemia TAP according to Item 1 or 2.

19. The leukemia TAP binds to the HLA-B*44:03 molecule and has the amino acid sequence KEIFL 3. The leukemia TAP of claim 1 or 2, comprising ELRL (SEQ ID NO: 127).

20. The leukemia TAP binds to the HLA-B*51:01 molecule and has the amino acid sequence LPIAS ASLL (SEQ ID NO: 12), PFPLVQVEPV (SEQ ID NO: 24), PLPIVPAL (SEQ ID NO: 38), IAAPILHV (SEQ ID NO: 68), IPLAVRTI (SEQ ID NO: 1 15), LPRNKPLL (SEQ ID NO: 116), or LPSSHSLLI (SEQ ID NO: 190 ), preferably IPLAVRTI (SEQ ID NO: 115) or LPRNKPLL (SEQ ID NO: 1 16). The leukemia TAP of claim 1 or 2, comprising:

21. The leukemia TAP binds to the HLA-B*57:01 molecule and has the amino acid sequence GARQQ IHSW (SEQ ID NO: 3), VTFKLSLF (SEQ ID NO: 16), KGHGGPRSW (SEQ ID NO: Sequence number 41), GSLDFQRGW (SEQ ID NO: 63), KAFPFHIIF (SEQ ID NO: 6 9), GTLQGIRAW (SEQ ID NO: 93), RTPKNYQHW (SEQ ID NO: 122), ISNKVPKLF (SEQ ID NO: 125), KTFVQQKTL (SEQ ID NO: 135), IL RSPLKW (SEQ ID NO: 153), or LTVPLSVFW (SEQ ID NO: 183), preferably RTPKNYQHW (SEQ ID NO: 122), ISNKVPKLF (SEQ ID NO: 125), KTFVQQKTL (SEQ ID NO: 135), or ILRSPLKW (SEQ ID NO: 153). The leukemia TAP of claim 1 or 2.

22. The leukemia TAP binds to the HLA-B*57:03 molecule and has the amino acid sequence GGSLI 1. The method of claim 1, comprising administering to a subject a sequence selected from the group consisting of: HPQW (SEQ ID NO: 60) or LGGAWKAVF (SEQ ID NO: 172). or leukemia TAP according to 2.

23. The leukemia TAP binds to the HLA-C*03:03 molecule and has the amino acid sequence PARPA GPL (SEQ ID NO: 37), IASPIALL (SEQ ID NO: 112), or HSLISIVY L (SEQ ID NO: 140), preferably IASPIALL (SEQ ID NO: 112) or HSLIS 3. The leukemia TAP of claim 1 or 2, comprising IVYL (SEQ ID NO: 140).

24. The leukemia TAP binds to the HLA-C*05:01 molecule and has the amino acid sequence SLDLL 3. The leukemia TAP of claim 1 or 2, comprising PLSI (SEQ ID NO: 150).

25. The leukemia TAP binds to the HLA-C*06:02 molecule and has the amino acid sequence IRMKA QAL (SEQ ID NO: 9), KATEYVHSL (SEQ ID NO: 70), VSFPDVRKV (SEQ ID NO: Sequence number 87), IGNPILRVL (SEQ ID NO: 142), LSTGHLSTV (SEQ ID NO: 154), or LRKAVDPIL (SEQ ID NO: 166), preferably IGNPILRVL (SEQ ID NO: 142) or LSTGHLSTV (SEQ ID NO: 154), The leukemia TAP described in

26. The leukemia TAP binds to the HLA-C*07:01 molecule and has the amino acid sequence IGNPI LRVL (SEQ ID NO: 142), IYAPHIRLS (SEQ ID NO: 143), TVEEYLV NI (SEQ ID NO: 155), LHNEKGLSL (SEQ ID NO: 178), or VSRNYVL LI (SEQ ID NO: 186), preferably IGNPILRVL (SEQ ID NO: 142) or IYA 3. The leukemia TAP of claim 1 or 2, comprising PHIRLS (SEQ ID NO: 143).

27. The leukemia TAP binds to the HLA-C*07:02 molecule and has the amino acid sequence TILPR ILTL (SEQ ID NO: 30), SYSPAHARL (SEQ ID NO: 83), TQAPPNVVL (SEQ ID NO: 85), YYLDWIHHY (SEQ ID NO: 90), SLREPQPAL (SEQ ID NO: No. 109), PAPPPHPAAL (SEQ ID NO: 117), or CLRIGPVTL (SEQ ID NO: No. 158), preferably SLREPQPAL (SEQ ID NO: 109) or PAPPPHPAAL 3. The leukemia TAP of claim 1 or 2, comprising: (SEQ ID NO: 117).

28. The leukemia TAP binds to the HLA-C*08:02 molecule and has the amino acid sequence AQDII LQAV (SEQ ID NO: 97), LTDRIYLTL (SEQ ID NO: 102), or AGDIIA RLI (SEQ ID NO: 174), preferably AQDIILQAV (SEQ ID NO: 97) or LTD 3. The leukemia TAP of claim 1 or 2, comprising RIYLTL (SEQ ID NO: 102).

29. The leukemia TAP binds to the HLA-C*12:03 molecule and has the amino acid sequence LSASH 3. The leukemia TAP of claim 1 or 2, comprising LSSL (SEQ ID NO: 173).

30. 1 to 2, which are encoded by sequences located in non-protein-coding regions of the genome 10. The leukemia TAP of any one of claims 9.

31. 3. The non-protein-coding region of the genome is a transcribed untranslated region (UTR). The leukemia TAP according to claim 0.

32. 31. The leukemia virus of claim 30, wherein the non-protein coding region of the genome is an intron. Disease TAP.

33. 31. The method of claim 30, wherein the non-protein-coding region of the genome is an intergenic region. Blood disease TAP.

34. A method comprising administering to a subject a leukemia comprising administering to said subject at least two of the leukemia TAPs defined in any one of claims 1 to 33. Hmm, combination

35. Leukemia TAP according to any one of claims 1 to 33 or the combination according to claim 34. A nucleic acid that encodes a combination.

36. 36. The nucleic acid of claim 35, which is an mRNA or a viral vector.

37. Leukemia TAP according to any one of claims 1 to 33, and the combination according to claim 34 37. A liposome comprising the nucleic acid of claim 35 or 36.

38. Leukemia TAP according to any one of claims 1 to 33, and the combination according to claim 34 35 or 36, or the liposome of claim 37, and a pharmaceutical and a carrier acceptable to the subject.

39. Leukemia TAP according to any one of claims 1 to 33, and the combination according to claim 34 , the nucleic acid according to claim 35 or 36, the liposome according to claim 37, or claim 3 9. A vaccine comprising the composition according to claim 8 and an adjuvant.

40. An isolated major histocompatibility complex (MHC) class I molecule, comprising: An isolated MHC comprising in its groove a leukemia TAP according to any one of claims 1 to 33. Class I molecules.

41. 41. The isolated MHC class I molecule of claim 40, in the form of a multimer.

42. 42. The isolated MHC class I molecule of claim 41, wherein the multimer is a tetramer.

43. (i) the leukemia TAP according to any one of claims 1 to 33, (ii) the leukemia TAP according to claim 34 or (iii) a combination of the above A TAP according to any one of claims 1 to 33 or A single vector comprising a nucleotide sequence encoding the combination of claim 34. Isolated cells.

44. An isolated cell, comprising at its surface a major histocompatibility complex (MHC) class I The MHC class I molecules express a molecule according to claims 1 to 3 in their peptide-binding groove.

35. An isolated leukemia comprising the leukemia TAP of any one of claims 3 to 34 or the combination of claim 34. The cells.

45. 45. The cell of claim 44, which is an antigen-presenting cell (APC).

46. The cell of claim 45, wherein the APC is a dendritic cell.

47. The isolated MHC class I molecule and / or the isolated MHC class I molecule according to any one of claims 40 to 42.

47. An MHC class I molecule expressed on the surface of the cell according to any one of claims 44 to 46. T cell receptor (TCR) specifically recognizes the antigen.

48. The TCR comprises one of the amino acid sequences set forth in SEQ ID NOs: 191 to 219.

48. The method of claim 47, comprising a TCR beta (TCRβ) chain comprising complementarity determining region 3 (CDR3). TCR shown.

49. An isolated cell, comprising at its cell surface a TCR according to claim 47 or 48. An isolated cell expressing

50. CD8 ＋ 50. The isolated cell of claim 49, which is a T lymphocyte.

51. A cell population comprising at least 0.5% of the isolated cells defined in claim 49 or 50. 。

52. A method of treating leukemia in a subject, comprising administering to the subject an effective amount of (i) a compound according to any one of claims 1 to 5. 33, (ii) a combination according to claim 34, ( iii) the nucleic acid according to claim 35 or 36, (iv) the liposome according to claim 37 (v) the composition of claim 38; (vi) the vaccine of claim 39; (vii) A cell according to any one of claims 43 to 46, 49 and 50, or (viii) a cell according to claim 43 52. A method comprising administering the cell population of claim 51.

53. 53. The method of claim 52, wherein the leukemia is myeloid leukemia.

54. 54. The method of claim 53, wherein the myeloid leukemia is acute myeloid leukemia (AML).

55. further comprising administering to said subject at least one additional anti-tumor agent or therapy.

55. The method of any one of claims 52 to 54.

56. The at least one additional anti-tumor agent or therapy may be a chemotherapeutic agent, an immunotherapy, an immune checkpoint inhibitor, or a combination thereof.

56. The method of claim 55, wherein the treatment is a cross-linking agent, a cross-linking inhibitor, radiation therapy, or surgery.

57. (i) a method according to any one of claims 1 to 33 for treating leukemia in a subject Leukemia TAP, (ii) the combination of claim 34, (iii) claim 35 or 36, (iv) the liposome of claim 37, (v) the liposome of claim 38 (vi) a vaccine according to claim 39; (vii) a vaccine according to claims 43 to 46, 49, and and 50, or (viii) a cell population according to claim 51, use.

58. (i) for the manufacture of a medicament for treating leukemia in a subject; Claims 1 to 33 (ii) a combination according to claim 34; (iii) ) the nucleic acid according to claim 35 or 36, (iv) the liposome according to claim 37, (v) (vi) the composition of claim 38, (vi) the vaccine of claim 39, (vii) claim A cell according to any one of claims 43 to 46, 49 and 50, or (viii) claim 51.

2. Use of a cell population according to claim 1.

59. 59. The use of claim 57 or 58, wherein the leukemia is myeloid leukemia.

60. 60. The use of claim 59, wherein the myeloid leukemia is acute myeloid leukemia (AML).

61. 61. The method of any of claims 57 to 60, further comprising the use of at least one additional anti-tumor agent or therapy. The use according to any one of claims 1 to 4.

62. The at least one additional anti-tumor agent or therapy may be a chemotherapeutic agent, an immunotherapy, an immune checkpoint inhibitor, or a combination thereof.

62. The use of claim 61, wherein the treatment is a cross-linking agent, a cross-linking inhibitor, radiation therapy, or surgery.

63. (i) for use in treating leukemia in a subject; Any one of claims 1 to 33 (ii) a combination according to claim 34; (iii) Claim 35 or 36, (iv) the liposome according to claim 37, (v) the liposome according to claim 38 (vi) a vaccine according to claim 39; (vii) Claims 43 to 46, 4 9 and 50, or (viii) a cell collection according to claim 51. Group.

64. 64. The leukemia TAP for use according to claim 63, wherein the leukemia is myeloid leukemia. , combination, nucleic acid, liposome, composition, vaccine, cell, or cell population.

65. 65. The use of claim 64, wherein the myeloid leukemia is acute myeloid leukemia (AML). Leukemia TAP, combinations, nucleic acids, liposomes, compositions, vaccines, cells, or Cell population.

66. For use in combination with at least one additional anti-tumor agent or therapy, Leukemia TAP, combination, nuclear for use according to any one of claims 63 to 65 An acid, a liposome, a composition, a vaccine, a cell, or a cell population.

67. The at least one additional anti-tumor agent or therapy may be a chemotherapeutic agent, an immunotherapy, an immune checkpoint inhibitor, or a combination thereof.

67. The method of claim 66, wherein the therapeutic agent is a cross-linking agent, ... Hematologic TAP, combination, nucleic acid, liposome, composition, vaccine, cell, or cell population.

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