Novel tumor antigens for lung cancer and their use

Abstract

Lung cancer remains the leading cause of cancer-related death worldwide. Despite the significant advancements in lung cancer treatment brought about by the introduction of immune checkpoint inhibitors (ICIs), the prognosis for the disease remains poor, with a considerable proportion of patients failing to respond to such treatments. Cancer vaccines may offer a complementary approach that enhances anti-tumor immunity and acts synergistically with ICIs. This specification describes novel tumor antigens common to the majority of lung tumor cells. Some of the tumor antigens described herein are derived from abnormally expressed non-mutant genomic sequences, such as intronic and intergenetic sequences, that are not expressed in normal tissues. Nucleic acids, compositions, cells, and vaccines derived from these tumor antigens are described. The use of tumor antigens, nucleic acids, compositions, cells, and vaccines for the treatment of lung cancer is also described.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 489,855, filed on 13 March 2023, and U.S. Provisional Patent Application No. 63 / 515,149, filed on 24 July 2023, all of which are incorporated herein by reference.

[0002] Sequence List The sequence listing was created on March 11, 2024, and is submitted herein as an XML file named G17971-00077_Seq Listing.xml, with a size of approximately 183,462 bytes. The contents of the aforementioned file are incorporated herein by full citation.

[0003] This invention generally relates to the field of cancer, and more specifically to the treatment of cancer such as lung cancer. [Background technology]

[0004] Lung cancer remains the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) accounts for approximately 80-85% of all lung cancers, with 30% of patients presenting with stage III disease. NSCLC comprises several histopathological subtypes, the most common being adenocarcinoma (40-60%) and squamous cell carcinoma (30%). Many patients with NSCLC often have advanced cancer at the time of diagnosis. The standard treatment for patients with good performance status (PS) and unresectable stage III NSCLC has been platinum-based dual chemotherapy and radiotherapy (cCRT) administered concurrently with the aim of curative treatment.

[0005] Various immune checkpoint inhibitors (ICIs), such as pembrolizumab, atezolizumab, and durvalumab, have been successively introduced into clinical medicine and have shown remarkable efficacy in the treatment of lung cancer. Thus, the introduction of ICIs has brought about significant progress in lung cancer treatment, but the prognosis of the disease remains poor. The median survival time for first-line patients with metastatic NSCLC is approximately 22 months in the non-squamous cell carcinoma group and 15.9 months in the squamous cell carcinoma group (Paz-Ares L, et al., Gadgeel S, et al., J. Clin. Oncol. 2020;38(14):1505-1517). In addition, 40% to 60% of the subjects do not respond to ICI-based treatment. Cancer vaccines may offer a complementary approach that enhances anti-tumor immunity and acts synergistically with ICIs. Identification of tumor-specific antigens is necessary for such cancer vaccines.

[0006] In light of this, there is a need to identify tumor antigens that can induce a therapeutic immune response against lung cancer. Such antigens can be used as vaccines (± immune checkpoint inhibitors) or targets in T cell receptor-based approaches (cell therapy, bispecific biological agents).

[0007] These descriptions cite numerous sources, the contents of which are incorporated in their entirety by reference in this specification. [Overview of the project]

[0008] In various aspects and embodiments, this disclosure provides the following items 1 to 77.

[0009] 1. A tumor antigen peptide (TAP) comprising or consisting of one of the amino acid sequences described in any one of SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74.

[0010] 2. TAP is a TAP or nucleic acid as described in item 1, comprising or consisting of one of the amino acid sequences of sequence numbers 15, 22, 28, 203, 1-14, 16-21, 23-27, 29-38, and 74.

[0011] 3. TAP is a TAP as described in item 2, which contains or consists of one of the amino acid sequences of sequence numbers 15, 22, 28, 203, 1-14, 16-21, 23-27, 29-35, and 74.

[0012] 4. TAP is HLA-A * 01:01 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of sequence numbers 14, 33, 40, 42-45, 48, or 60.

[0013] 5. TAP is HLA-A * 02:01 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of sequence numbers 12, 39, 50, 53, 56, or 70.

[0014] 6. TAP is HLA-A * 02:03 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs. 6, 12, 16, 39, 50, 53, 56, 61, or 70.

[0015] 7. TAP is HLA-A * 03:01 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs: 11, 14, 17, 19-22, 26, 29, 36, 40, 47-49, 51, 59, 63, 65, 67, or 68.

[0016] 8. TAP is HLA-A * A TAP as described in any one of items 1 to 3, which is bound to the 11:01 molecule and contains or consists of the sequence of sequence numbers 11, 14, 17, 19-22, 26, 29, 36, 40, 44, 45, 47-49, 51, 59, 63, 65, 67, 68, or 74.

[0017] 9. TAP binds to the HLA-A * 24:02 molecule and is the TAP according to any one of items 1 to 3, comprising or consisting of the sequence of SEQ ID NO: 12, 15, 18, 25, 35, 46, 54, or 58. [[ID=⑦]]

[0018] [[ID=⑧]] [[ID=⑨]]10. TAP binds to the HLA-A[[ID=⑩]] * [[ID=⑪]]33:03 molecule and is the TAP according to any one of items 1 to 3, comprising or consisting of the sequence of SEQ ID NO: 5, 7, 34, 36, 63, 65, 67 or 68. [[ID=⑫]] [[ID=⑬]]

[0019] [[ID=⑭]] [[ID=⑮]]11. TAP binds to the HLA-A[[ID=⑯]] * [[ID=⑰]]68:01 molecule and is the TAP according to any one of items 1 to 3, comprising or consisting of the sequence of SEQ ID NO: 5, 7, 14, 26, 29, 36, 42, 44, 45, 47, 49, 51, 63, 65, 67, 68, or 71. [[ID=⑱]] [[ID=⑲]]

[0020] [[ID=⑳]] [[ID=㉑]]12. TAP binds to the HLA-B[[ID=㉒]] * [[ID=㉓]]The 07:02 molecule and is the TAP according to any one of items 1 to 3, comprising or consisting of the sequence of SEQ ID NO: 13, 16, 23, 24, 27, 28, 32, 38, 42, 52, 60, 61, 66 or 73. [[ID=㉔]] [[ID=㉕]]

[0021] [[ID=㉖]] [[ID=㉗]]13. TAP binds to the HLA-B[[ID=㉘]] * [[ID=㉙]]The 07:05 molecule and is the TAP according to any one of items 1 to 3, comprising or consisting of the sequence of SEQ ID NO: 13, 16, 23, 24, 27, 28, 32, 38, 42, 52, 60, 61, 66, or 73. [[ID=㉚]] [[ID=㉛]]

[0022] [[ID=㉜]] [[ID=㉝]]14. TAP binds to the HLA-B[[ID=㉞]] * [[ID=㉟]]The 08:01 molecule and is the TAP according to any one of items 1 to 3, comprising or consisting of the sequence of SEQ ID NO: 15, 16, 23, 24, 27, 31, 37-39, 42, 45, 50, 52, 54-57, 61, 66, 70, or 73. [[ID=㊱]] [[ID=㊲]]

[0023] [[ID=㊳]] It should be noted that in the above translation, for the convenience of distinction, the numbers in the original text are marked with Chinese characters in the translation. In actual use, it should be adjusted according to specific requirements. And the specific meaning of these contents needs to be understood in combination with relevant professional knowledge.15. TAP is HLA-B * 15:01 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of sequence numbers 12, 16, 39, 40, 42-46, 48, 58, 60-62, 72, or 176.

[0024] 16. TAP is HLA-B * 15:25 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs: 12, 16, 38-40, 42, 44-46, 48, 58, 60, 61, or 62.

[0025] 17. TAP is HLA-B * 18:01 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs: 1, 2, 9, 10, 13, 30, 33, 35, 41-46, 54, 55, 58, 60, 64, 69, 72, or 73.

[0026] 18. TAP is HLA-B * 27:06 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs: 8, 12, 15, 25, 46, 54, 55, 58, 61, 64, 66, or 70.

[0027] 19. TAP is HLA-B * 35:01 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs: 13, 23, 24, 27, 35, 38, 42-45, 52, 54, 60, 66, or 73.

[0028] 20. TAP is HLA-B * 39:01 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs: 1, 8, 15, 25, 27, 38, 42, 45, 46, 52, 54, 55, 58, 61, 64, 70, or 73.

[0029] 21. TAP is HLA-B *A TAP as described in any one of items 1 to 3, which is bound to a 40:01 molecule and contains or consists of the sequence of SEQ ID NOs: 1-4, 9, 30, 33, 43, 46, 55, 58, 61, 64, 69, or 72.

[0030] 22. TAP is HLA-B * A TAP as described in any one of items 1 to 3, which is bound to a 44:02 molecule and contains or consists of the sequence of SEQ ID NOs: 1-4, 9, 10, 33, 41-46, 55, 58, 61, 64, 69, or 72.

[0031] 23. TAP is HLA-B * A TAP as described in any one of items 1 to 3, which is bound to a 44:03 molecule and contains or consists of the sequence of SEQ ID NOs: 1-4, 9, 10, 33, 41-46, 55, 58, 60, 62, 64, 69, or 72.

[0032] 24. TAP is HLA-B * 54:01 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of sequence numbers 24, 27, 32, 38, 42, 52, 60, or 73.

[0033] 25. TAP is HLA-C * 03:04 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs: 12, 24, 27, 28, 38, 39, 42, 44, 45, 50, 52, 70, or 73.

[0034] 26. TAP is HLA-C * 07:04 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs: 8, 12, 15, 16, 24, 25, 27, 35, 38, 39, 42, 44, 45, 50, 52, 54, 55, 61, 64, 70, or 73.

[0035] 27. TAP is HLA-C *15:05 A TAP as described in any one of items 1 to 3, which is bound to a molecule and contains or consists of the sequence of SEQ ID NOs: 12, 28, 31, 38, 39, 42, 44, 45, 61, or 70.

[0036] 28. A TAP described in any one of items 1-27, encoded by a sequence located in a non-protein-coding region of the genome.

[0037] 29. Non-protein-coding regions of the genome are inter-gene regions, as described in item 28 (TAP).

[0038] 30. Non-protein-coding regions of the genome are long non-coding RNAs, as described in item 28 (TAP).

[0039] 31. Non-protein-coding regions of the genome are introns, as described in item 28 (TAP).

[0040] 32. A combination comprising at least two of the TAPs or nucleic acids defined in any one of items 1 through 31.

[0041] 33. A synthetic long-chain peptide (SLP) containing at least one of the amino acid sequences defined in item 1.

[0042] 34. A nucleic acid that codes for one or more of the following: a TAP as described in any one of items 1-31, a combination as described in item 32, or an SLP as described in item 33.

[0043] 35. The nucleic acid is mRNA, and the mRNA optionally contains one or more 5' terminal modifications, 3' terminal modifications, and / or modified nucleosides, thereby increasing mRNA stability, improving translation, and / or reducing immunogenicity, as described in item 34.

[0044] 36. Nucleic acids are components of viral vectors, as described in item 34.

[0045] 37. Vesicles or particles containing any TAP, combination, SLP, or nucleic acid as described in any one of items 1 through 36.

[0046] 38. A vesicle is a lipid nanoparticle (LNP), as described in item 37.

[0047] 39. Vesicles or particles as described in item 37 or 38, containing cationic lipids.

[0048] 40. A composition comprising a TAP, combination, SLP, or nucleic acid as described in any one of items 1 to 36, or a vesicle or particle as described in any one of items 37 to 39, and a pharmaceutically acceptable carrier.

[0049] 41. A vaccine comprising a TAP, combination, SLP, or nucleic acid as described in any one of items 1 to 36, a vesicle or particle as described in any one of items 37 to 39, or a composition as described in item 40, and an adjuvant.

[0050] 42. A molecule comprising an isolated major histocompatibility complex (MHC) class I molecule containing a TAP described in any one of items 1 to 31 within its peptide bond groove.

[0051] 43. An isolated MHC class I molecule described in item 42, in the form of a polymer.

[0052] 44. The polymer is a tetramer, as described in item 43, for isolated MHC class I molecules.

[0053] 45. Isolated cells comprising (i) a TAP as described in any one of items 1 to 31, (ii) a combination as described in item 32, (iii) an SLP as described in item 33, (iv) a nucleic acid as described in any one of items 34 to 36, or (v) a vector comprising a nucleic acid as described in any one of items 34 to 36.

[0054] 46. ​​Isolated cells expressing a major histocompatibility complex (MHC) class I molecule on its surface containing a TAP or combination described in any one of items 1 to 32 within a peptide bond groove.

[0055] 47. Antigen-presenting cells (APCs), as described in item 45 or 46.

[0056] 48. APCs are dendritic cells, as described in item 47.

[0057] 49. A T cell receptor (TCR) that specifically recognizes an isolated MHC class I molecule as described in any one of items 42-44, and / or an MHC class I molecule expressed on the surface of a cell as described in any one of items 46-48.

[0058] 50. A soluble TCR, as described in item 49.

[0059] 51. An antibody or its antigen-binding fragment that specifically binds to an isolated MHC class I molecule as described in any one of items 42-44, and / or an MHC class I molecule expressed on the surface of a cell as described in any one of items 46-48.

[0060] 52. A bispecific TCR or a bispecific antibody or its antigen-binding fragment, as described in item 49 or 50, or an antibody or its antigen-binding fragment as described in item 51.

[0061] 53. A bispecific antibody or its antigen-binding fragment is a single-chain diabody (scDb), a TCR as described in item 52, or an antibody or its antigen-binding fragment.

[0062] 54. A bispecific TCR, or a bispecific antibody or its antigen-binding fragment, as described in item 52 or 53, which also specifically binds to a T cell signaling molecule.

[0063] 55. T cell signaling molecules are the CD3 chain, the TCR as described in item 54, or an antibody or its antigen-binding fragment.

[0064] 56. A chimeric antigen receptor (CAR) comprising an antibody or its antigen-binding fragment as described in any one of items 51-55, or a nucleic acid encoding a CAR.

[0065] 57. Isolated cells expressing the TCR described in item 49 or the CAR described in item 56 on their cell surface.

[0066] 58. CD8 + T lymphocytes (e.g., tumor-infiltrating CD8) + Isolated cells (T lymphocytes) as described in item 57.

[0067] 59. A cell population containing at least 0.5% isolated cells as defined in item 57 or 58.

[0068] 60. A method for treating cancer in a subject, wherein an effective amount of the subject (a) A TAP or any thereof containing or consisting of any one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-35 A combination of the above, or a synthetic long-chain peptide (SLP) comprising at least one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 176, 1-14, 16-21, 23-27, and 29-35, (b) The TAPs as defined in (a), a combination thereof, or at least one nucleic acid encoding an SLP, (c) The TAP as defined in (a), a combination thereof or SLP, or a vesicle or particle containing at least one nucleic acid as defined in (b), (d) A composition comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), or the vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), the vesicle or particle as defined in (c), or the composition as defined in (d), and an adjuvant. (f) Cells expressing a major histocompatibility complex (MHC) class I molecule containing the TAP defined in (a) or a combination thereof within a peptide bond groove on its surface, (g) A cell which expresses a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes the MHC class I molecule expressed on the surface of the cell as defined in (f), or A method comprising administering a soluble TCR, or an antibody or its antigen-binding fragment, that specifically binds to an MHC class I molecule expressed on the surface of the cell as defined in (h)(f).

[0069] 61. Cancer is lung cancer, as described in item 60.

[0070] 62. Lung cancer is non-small cell lung cancer (NSCLC), as described in item 61.

[0071] 63. Lung cancer is adenocarcinoma, large cell carcinoma, or squamous cell carcinoma, as described in item 61 or 62.

[0072] 64. The method according to any one of items 60 to 63, further comprising administering at least one additional antitumor agent to the subject or performing at least one additional therapy.

[0073] 65. At least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery, as described in item 64.

[0074] 66. For the manufacture of a drug for treating cancer in a subject, or for the manufacture of a drug for treating cancer in a subject. (a) A TAP or any thereof containing or consisting of any one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-35 A combination of the above, or a synthetic long-chain peptide (SLP) comprising at least one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-35, (b) The TAPs as defined in (a), a combination thereof, or at least one nucleic acid encoding an SLP, (c) The TAP as defined in (a), a combination thereof or SLP, or a vesicle or particle containing at least one nucleic acid as defined in (b), (d) A composition comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), or the vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), the vesicle or particle as defined in (c), or the composition as defined in (d), and an adjuvant. (f) Cells expressing a major histocompatibility complex (MHC) class I molecule containing the TAP defined in (a) or a combination thereof within a peptide bond groove on its surface, (g) A cell which expresses a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes the MHC class I molecule expressed on the surface of the cell as defined in (f), or A soluble TCR, antibody, or antigen-binding fragment thereof that specifically binds to MHC class I molecules expressed on the surface of cells as defined in (h)(f). Use.

[0075] 67. Cancer is lung cancer, use as described in item 66.

[0076] 68. Lung cancer is non-small cell lung cancer (NSCLC), as described in item 67.

[0077] 69. Lung cancer is adenocarcinoma, large cell carcinoma, or squamous cell carcinoma, as described in item 67 or 68.

[0078] 70. Uses described in any one of items 66-69, further comprising the use of at least one additional antitumor agent or therapy to the subject.

[0079] 71. At least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery, as described in item 70.

[0080] 72. A drug used to treat cancer in the subject, (a) A TAP or any thereof containing or consisting of any one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-35 A combination of the above, or a synthetic long-chain peptide (SLP) comprising at least one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-35, (b) The TAPs as defined in (a), a combination thereof, or at least one nucleic acid encoding an SLP, (c) The TAP as defined in (a), a combination thereof or SLP, or a vesicle or particle containing at least one nucleic acid as defined in (b), (d) A composition comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), or the vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), the vesicle or particle as defined in (c), or the composition as defined in (d), and an adjuvant. (f) Cells expressing a major histocompatibility complex (MHC) class I molecule containing the TAP defined in (a) or a combination thereof within a peptide bond groove on its surface, (g) A cell which expresses a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes the MHC class I molecule expressed on the surface of the cell as defined in (f), or A method comprising administering a soluble TCR, or an antibody or its antigen-binding fragment, that specifically binds to an MHC class I molecule expressed on the surface of the cell as defined in (h)(f).

[0081] 73. Cancer is lung cancer, the drug for use as described in item 72.

[0082] 74. Lung cancer is non-small cell lung cancer (NSCLC), and the drugs are for use as described in item 73.

[0083] 75. Lung cancer is adenocarcinoma, large cell carcinoma, or squamous cell carcinoma, and the drugs for use as described in item 73 or 74.

[0084] 76. A drug for use as described in any one of items 72-75, further comprising the use of at least one additional antitumor agent or therapy to the subject.

[0085] 77. At least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery, as described in item 76.

[0086] Other purposes, advantages, and features of this disclosure will become more apparent upon reading the following non-restrictive description of specific embodiments, which are given only as examples with reference to the accompanying drawings. [Brief explanation of the drawing]

[0087] [Figure 1A] This shows the sharing and regulation of TA expression across cancer samples. The stacked bar graph shows the proportion (and absolute number of TAs) of shared TA types among different numbers of analyzed NSCLC samples. [Figure 1B]This shows the sharing and regulation of TA expression across cancer samples. Each TA (gray dot) is shown in a box plot representing the percentage of TCGA samples that express at least twice as much TA as the 95th percentile value of that TA in normal bronchial brushing samples and GTEx samples excluding testes. The box plot shows the median and interquartile range (IQR), with the whisker extending to the maximum value within 1.5*IQR from the box hinge. [Figure 1C] This demonstrates the sharing and regulation of TA expression across cancer samples. Spearman correlations (Figure 1C) between RPHM expression of each NSCLC TA and corresponding omics values ​​(FPKM expression, copy number variation, methylation β value, and TM) across LUSC and LUAD samples analyzed from TCGA. [Figure 1D] This shows the sharing and regulation of TA expression across the entire cancer sample. The proportion of TAs with significant correlations (p-adj < 0.05, asterisked cells in Figure 1C) among TAs for which omics data is available (non-blank cells in Figure 1C) (Figure 1D, TSA = left bar, TAA = middle bar, LSA = right bar) is shown. [Figure 2] This bar graph shows the proportion (and absolute number) of NSCLC TAA and aeTSA (read count greater than 1) expressed only in cancer cells, as well as the proportion of NSCLC LSA expressed only in cancer cells and normal alveolar cells, based on NSCLC scRNA-seq data from Lambrechts et al. 6. [Figure 3A] This shows the sharing of non-mutant TA at the immunopeptide level and RNA level. The box plots show stem cell scores obtained using ssGSEA in TCGA samples studied herein, across LUAD, LUSC, and SKCM cohorts. P-values ​​of the two-sided Wilcoxon test are also shown. [Figure 3B] This shows the sharing of non-mutant TAs at the immunopeptide and RNA levels. It also shows the number of TAs identified from samples of each NSCLC subtype. P-values ​​for comparing adenocarcinoma and squamous cell carcinoma samples using a two-sided, uncorresponding t-test are provided. [Figure 3C]This shows the sharing of non-mutant TAs at the immunopeptide level and RNA level. It also shows the non-synonymous mutation rates (obtained from Firebrowse) in samples analyzed from TCGA, depending on the NSCLC subtype and smoking history from cBioPortal. P-values ​​for comparing smokers and non-smokers using a two-tailed, unpaired t-test are also provided. [Figure 3D] This shows the sharing of non-mutant TA at the immunopeptidomic and RNA levels. It also shows RNA expression of non-mutant TA in TCGA samples analyzed according to NSCLC subtype and smoking history. P-values ​​for comparing smokers and non-smokers using the two-sided Wilcoxon test are provided. [Figure 3E] This shows the sharing of non-mutant TAs at the immunopeptide level and RNA level. It shows the number of TAs with non-null RNA expression across TCGA samples analyzed according to NSCLC subtype and smoking history. P-values ​​for comparing smokers and non-smokers using two-sided unpaired t-tests are shown. Box plots show the median and interquartile range (IQR), with whiskers extending to the maximum value within 1.5*IQR from the box hinge. No adjustment was made for p-values ​​in multiple tests. [Figure 4A] This shows annotations for scRNA-seq data from previous NSCLC studies. It is a balloon plot showing the mean expression and percentage of cells expressing cluster annotation indicator genes within each cluster identified across NSCLC samples from Lambrechts et al. 6. The cluster annotation genes were obtained from the original research paper. [Figure 4B] Annotations of scRNA-seq data from previous studies of NSCLCs are shown. A UMAP is shown from Lambrechts et al. 6, showing the identified clusters (top) and their cell type annotations (bottom) according to the genes (c) across NSCLC samples. [Figure 5A] This shows the expression of non-mutant TAs in NSCLC scRNA-seq data. Figure 2 is a box plot showing read counts of cancer cell-specific NSCLC TAs or NSCLC LSAs specific to cancer cells and alveolar cells across cell types in NSCLC samples from Lambrechts et al. 6. Each gray dot represents one TA per cell. [Figure 5B] This shows the expression of non-mutant TA in scRNA-seq data from NSCLC. The box plot shows the read count of NSCLC TA expressed in non-cancer cell types from NSCLC samples by Lambrechts et al. 6. Each gray dot represents one TA per cell. The box plot shows the median and interquartile range (IQR), with the whisker extending to the maximum value within 1.5*IQR from the box hinge. [Modes for carrying out the invention]

[0088] In the context of describing the technology (particularly in the context of the following claims), the use of the terms “a,” “an,” and “the,” as well as similar referents, is to be interpreted as encompassing both singular and plural forms, unless otherwise indicated herein or unless it is clearly inconsistent with the context.

[0089] The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including, but not limited to”) unless otherwise noted.

[0090] All methods described herein may be carried out in any preferred order, unless otherwise indicated herein or unless the context clearly contradicts it.

[0091] Any and all examples provided herein, or any use of illustrative expressions (such as "for example," "etc.") are intended merely to better illustrate embodiments of the claimed technology and, unless otherwise claimed, do not constitute a limitation of scope.

[0092] Nothing expressed herein should be construed as indicating that any non-claimed element is essential to the implementation of the claimed embodiment of the technology.

[0093] In this specification, the term “about” has its usual meaning. The term “about” is used to indicate that a value includes inherent variability due to errors in the instruments or methods used to determine the value, or that it includes values ​​close to the enumerated values, for example, values ​​within 10% of the enumerated values ​​(or range of values).

[0094] References to ranges of values ​​in this specification are intended simply as a convenient way to refer individually to each individual value within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually referred to herein. All subsets of values ​​within the range are also incorporated herein as if they were individually enumerated herein.

[0095] If any feature or aspect of the present disclosure is described in terms of the Markush group or list of substitutes, a person skilled in the art will recognize that the present disclosure is also described in terms of any individual component or subgroup of components of the Markush group or list of substitutes.

[0096] Unless otherwise specifically defined, all technical and scientific terms used herein should be interpreted as having the same meaning as that generally understood by those skilled in the art (for example, in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0097] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in the following sources: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present); Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0098] In the studies described herein, the inventors identified candidate tumor antigens from lung cancer specimens using proteogenomics-based methods. The novel tumor antigens identified herein, including tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), may be useful in immunotherapy and vaccines for cancers that express tumor antigens, such as lung cancer (e.g., NSCLC).

[0099] This disclosure relates to tumor antigen peptides (TAPs) (or tumor-specific peptides), such as lung cancer TAPs, comprising or consisting of one of the following amino acid sequences: [Table 1-1] [Table 1-2] [Table 1-3]

[0100] In one embodiment, TAP includes or consists of one of the sequences of sequence numbers 1-38, 74, and 203. In a further embodiment, TAP includes or consists of one of the sequences of sequence numbers 1-35, 74, and 203.

[0101] In another embodiment, TAP is an abnormally expressed tumor-specific antigen (aeTSA). In a further embodiment, TAP comprises or consists of one of the sequences of SEQ ID NOs: 6, 7, 10, 12, 13, 15-23, 28, 31, 32, 34, 35, 57 and 203. In a further embodiment, TAP comprises or consists of one of the sequences of SEQ ID NOs: 6, 7, 10, 12, 13, 15-22, 28, 31, 32, 34, 35, 57 and 203.

[0102] Generally, peptides such as tumor antigen peptides (TAPs) presented in the context of HLA class I are amino acid residues whose length varies in the range of about 7 or 8 to about 15, or preferably 8 to 14. In some embodiments of the methods of this disclosure, longer peptides containing the TAP sequence as defined herein are artificially loaded onto cells such as antigen-presenting cells (APCs) that are processed by cells, and the TAP is presented on the surface of the APC by an MHC class I molecule. In this method, peptides / polypeptides longer than 15 amino acid residues can be loaded onto the APC and processed by a protease in the cytoplasm of the APC that provides the corresponding TAP as defined herein for presentation. In some embodiments, precursor peptides / polypeptides used to generate the TAP as defined herein are, for example, 1000, 500, 400, 300, 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, or 15 or fewer amino acids. Accordingly, all methods and processes using TAP described herein involve the use of longer peptides or polypeptides (including native proteins), i.e., tumor antigen precursor peptides / polypeptides, to induce the presentation of a “final” 8-14 amino acid TAP after treatment with cells (APCs). In some embodiments, the TAP described herein has an amino acid length of about 8-14, 8-13, or 8-12 (e.g., 8, 9, 10, 11, 12, or 13 amino acids) and is small enough to directly fit into an HLA class I molecule. In one embodiment, the TAP contains 20 or fewer amino acids, preferably 15 or fewer, more preferably 14 or fewer. In one embodiment, the TAP contains at least 7 amino acids, preferably at least 8 or fewer, more preferably at least 9 amino acids.

[0103] As used herein, the term “amino acid” includes not only both L-isomers and D-isomers of naturally occurring amino acids, but also other amino acids used in peptide chemistry to prepare synthetic analogs of TAP (e.g., naturally occurring amino acids, naturally occurring amino acids, amino acids not encoded by nucleic acid sequences, etc.). Examples of naturally occurring amino acids include glycine, alanine, valine, leucine, isoleucine, serine, and threonine. Other amino acids include, for example, non-gene-coding amino acids, amino acid analogs, and conserved substitutions of L-amino acids. Naturally occurring amino acids and amino acid analogs that are not encoded by genes include, for example, β-alanine, 3-aminopropionic acid, 2,3-diaminopropionic acid, α-aminoisobutyric acid (Aib), 4-aminobutyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-naphthylalanine, pyridylalanine, 3-benzothienylalanine, 4-chlorophenylalanine, and 2-fluorophenylalanine. These include nin, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, beta-2-thienylalanine, methionine sulfoxide, L-homoarginine (HoArg), N-acetyllysine, 2-aminobutyric acid, 2,4-diaminobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine, homoserine (HoSer), cysteic acid, epsilon-aminohexanoic acid, delta-aminovaleric acid, benzyloxytyrosine, β-phenylalanine, or 2,3-diaminobutyric acid (D- or L-). These amino acids are well known in the field of biochemistry / peptide chemistry. Therefore, one or more of the amino acids in the TAPs described herein (SEQ ID NOs: 1-74, 149-175, and 203) may be substituted with amino acids and / or amino acid analogs that are not encoded by genes.TAP can also be modified to improve the protease resistance of peptides by incorporating, for example, methyl amino acids, β-amino acids, or peptoids. In one embodiment, TAP contains only naturally occurring amino acids.

[0104] In several embodiments, the TAPs described herein include peptides having altered sequences that include functionally equivalent amino acid residue substitutions compared to the sequences described herein. For example, one or more amino acid residues in a sequence may be substituted with another amino acid of similar polarity (having similar physicochemical properties) that acts as a functional equivalent, resulting in a silent mutation. Amino acid substitutions in a sequence 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 (as well as homoarginine and ornithine). Nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan, and methionine. Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Negatively charged (acidic) amino acids include glutamic acid and aspartic acid. The amino acid glycine may belong to either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within an amino acid family are generally understood to be conservative substitutions. The TAPs described herein may comprise any L-amino acid, any D-amino acid, or a mixture of L-amino acids and D-amino acids. In one embodiment, the TAPs described herein comprise all L-amino acids. In one embodiment, in a TAP sequence containing or consisting of one of the sequences of SEQ ID NOs: 1-74, 149-175, and 203, amino acid residues that do not substantially contribute to interaction with the T cell receptor may be modified by substituting them with other amino acids whose incorporation does not substantially affect T cell reactivity and does not eliminate binding to the relevant MHC.

[0105] TAPs can also be modified by replacing one or more amide bonds of a peptide, which can improve chemical stability and / or enhance biological / pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.). Typical peptide bond substitutions include esters, polyamines and their derivatives, as well as substituted alkanes and alkenes such as aminomethyl and ketomethylenes. For example, in the above TAPs, one or more amide bonds may be replaced by bonds such as -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH-(cis or trans), -CH2SO-, -CH(OH)CH2-, or -COCH2-.

[0106] TAP may also be capped or modified at its N-terminus and / or C-terminus to prevent degradation and improve stability, affinity, and / or uptake. Thus, in another embodiment, the present disclosure relates to formula Z 1 -XZ 2 The formula provides a modified TAP, where X is a TAP containing or consisting of one of the amino acid sequences of SEQ ID NOs: 1-74, 149-175, and 203.

[0107] In one embodiment, the amino-terminal residue of TAP (i.e., the free amino group at the N-terminus) is modified (e.g., to prevent degradation) by, for example, a covalent partial / chemical group (Z 1 Modified by the addition of Z. 1 C1-C14 is a linear or branched alkyl group of 1 to 8 carbon atoms, or an acyl group (R-CO-), where R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, isopropionyl, or isobutanyl) or an alloyl group (Ar-CO-), where Ar is an aryl group. In one embodiment, the acyl group is C1-C14 16 Or C3-C 16 The acyl group (linear or branched, saturated or unsaturated) is, in further embodiments, a saturated C1-C6 acyl group (linear or branched) or an unsaturated C3-C6 acyl group (linear or branched), for example, an acetyl group (CH3-CO-, Ac). In one embodiment, Z1 It does not exist. The carboxyl-terminal residue of TAP (i.e., the free carboxyl group at the C-terminus of TAP) may be modified, for example, by amidation (substitution of the OH group with an NH2 group) (e.g., to prevent degradation), and in such a case Z2 is an NH2 group. In one embodiment, Z 2 This may be a hydroxysamate group, a nitrile group, an amide (primary, secondary, or tertiary) group, an aliphatic amine with 1 to 10 carbon atoms such as methylamine, isobutylamine, isovalerylamine, or cyclohexylamine, an aromatic or arylalkylamine such as aniline, naphthylamine, benzylamine, cinnamylamine, or phenylethylamine, an alcohol, or CH2OH. In one embodiment, Z 2 It does not exist. In one embodiment, TAP includes or consists of one of the amino acid sequences of SEQ ID NOs: 1-74, 149-175 and 203, for example, SEQ ID NOs: 1-38, 74 and 203 or SEQ ID NOs: 1-35, 74 and 203. In one embodiment, TAP consists of one of the amino acid sequences of SEQ ID NOs: 1-74, 149-175 and 203 (for example, SEQ ID NOs: 1-38, 74 and 203 or SEQ ID NOs: 1-35, 74 and 203), i.e., Z 1 and Z 2 It does not exist.

[0108] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 14, 33, 40, 42-45, 48, or 60, which is an HLA-A sequence. * This provides TAPs that bind to the 01:01 molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0109] In another aspect, the Disclosure relates to an HLA-A sequence comprising or consisting of sequence number 12, 39, 50, 53, 56, or 70. *02:01 provides TAPs that bind to molecules. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0110] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 6, 12, 16, 39, 50, 53, 56, 61, or 70, which is an HLA-A sequence. * 02:03 provides TAPs that bind to molecules. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0111] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 11, 14, 17, 19-22, 26, 29, 36, 40, 47-49, 51, 59, 63, 65, 67, or 68, which is an HLA-A sequence. * 03:01 provides TAPs that bind to molecules. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0112] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 11, 14, 17, 19-22, 26, 29, 36, 40, 44, 45, 47-49, 51, 59, 63, 65, 67, 68, or 74, which is an HLA-A sequence. * This provides a TAP that binds to the 11:01 molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0113] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 12, 15, 18, 25, 35, 46, 54, or 58, which is an HLA-A sequence. *This provides TAPs that bind to the 24:02 molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0114] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs. 5, 7, 34, 36, 63, 65, 67, or 68, which is an HLA-A sequence. * 33:03 provides a TAP that binds to the molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0115] In another aspect, the Disclosure relates to an HLA-A sequence comprising or consisting of sequence numbers 5, 7, 14, 26, 29, 36, 42, 44, 45, 47, 49, 51, 63, 65, 67, 68, or 71. * This provides a TAP that binds to the 68:01 molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0116] In another embodiment, the Disclosure relates to an HLA-B sequence comprising or consisting of sequence numbers 13, 16, 23, 24, 27, 28, 32, 38, 42, 52, 60, 61, 66, or 73. * This provides TAPs that bind to the 07:02 molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0117] In another embodiment, the Disclosure relates to an HLA-B sequence comprising or consisting of sequence numbers 13, 16, 23, 24, 27, 28, 32, 38, 42, 52, 60, 61, 66, or 73. *07:05 provides TAPs that bind to molecules. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0118] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 15, 16, 23, 24, 27, 31, 37-39, 42, 45, 50, 52, 54-57, 61, 66, 70, or 73, which is an HLA-B * 08:01 provides a TAP that binds to the molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0119] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 12, 16, 39, 40, 42-46, 48, 58, 60-62, 72, or 203, which is an HLA-B * 15:01 provides a TAP that binds to the molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0120] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 12, 16, 38-40, 42, 44-46, 48, 58, 60, 61, or 62, which is an HLA-B * 15:25 provides TAPs that bind to molecules. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0121] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 1, 2, 9, 10, 13, 30, 33, 35, 41-46, 54, 55, 58, 60, 64, 69, 72, or 73, which is an HLA-B *18:01 provides a TAP that binds to the molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0122] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 8, 12, 15, 25, 46, 54, 55, 58, 61, 64, 66, or 70, which is an HLA-B * 27:06 provides TAPs that bind to molecules. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0123] In another embodiment, the present disclosure includes or comprises the sequence of SEQ ID NOs: 13, 23, 24, 27, 35, 38, 42-45, 52, 54, 60, 66, or 73, which is an HLA-B * This provides a TAP that binds to the 35:01 molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0124] In another embodiment, the present disclosure includes or comprises the sequence of SEQ ID NOs: 1, 8, 15, 25, 27, 38, 42, 45, 46, 52, 54, 55, 58, 61, 64, 70, or 73, which is an HLA-B * 39:01 provides a TAP that binds to the molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0125] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 1-4, 9, 30, 33, 43, 46, 55, 58, 61, 64, 69, or 72, which is an HLA-B *This provides a TAP that binds to the 40:01 molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0126] In another embodiment, the Disclosure provides TAPs that bind to the HLA-B*44:02 molecule, comprising or consisting of the sequences SEQ ID NOs: 1-4, 9, 10, 33, 41-46, 55, 58, 61, 64, 69, or 72. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0127] In another aspect, the present disclosure includes or comprises the sequence of SEQ ID NOs: 1-4, 9, 10, 33, 41-46, 55, 58, 60, 62, 64, 69, or 72, which is an HLA-B * This provides a TAP that binds to the 44:03 molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0128] In another embodiment, the present disclosure includes or comprises the sequence of SEQ ID NOs: 24, 27, 32, 38, 42, 52, 60, or 73, which is an HLA-B * This provides a TAP that binds to the 54:01 molecule. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0129] In another aspect, the Disclosure relates to an HLA-C sequence comprising or consisting of sequence numbers 12, 24, 27, 28, 38, 39, 42, 44, 45, 50, 52, 70, or 73. *03:04 provides TAPs that bind to molecules. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0130] In another embodiment, the Disclosure provides TAPs that bind to HLA-C*07:04 molecules, comprising or consisting of sequences SEQ ID NOs: 8, 12, 15, 16, 24, 25, 27, 35, 38, 39, 42, 44, 45, 50, 52, 54, 55, 61, 64, 70, or 73. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0131] In another aspect, the Disclosure relates to an HLA-C sequence comprising or consisting of sequence numbers 12, 28, 31, 38, 39, 42, 44, 45, 61, or 70. * 15:05 provides TAPs that bind to molecules. Because HLA alleles exhibit cross-reactivity (certain HLA alleles present similar epitopes), the TAPs identified above can further bind to other HLA alleles, as shown in Table 3A.

[0132] The TAPs of this disclosure can be produced by expression in host cells containing the nucleic acid encoding the TAP (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis). The peptides can be readily synthesized by manual and / or automated solid-phase procedures widely known in the art. Preferred synthesis can be carried out, for example, by utilizing the "T-boc" or "Fmoc" procedures. Techniques and procedures for solid-phase synthesis are described, for example, in Solid Phase Peptide Synthesis: A Practical Approach, E. Atherton and RC Sheppard, IRL, Oxford University Press, 1989. Alternatively, TAP can be used as described in, for example, Liu et al., Tetrahedron Lett. 37:933-936, 1996; Baca et al., J. Am. Chem. Soc. 117: 1881-1887, 1995; Tam et al., Int. 256:221-225,1992;Liu and Tam,J.Am.Chem.Soc.116:4149-4153,1994;Liu and Tam,Proc.Natl.Acad.Sci.USA 91:6584-6588,1994;and Yamashiro and Li,Int.J.Peptide Protein TAP may be prepared by segment condensation, as described in Res.31:322-334, 1988. Other methods useful for TAP synthesis are described in Nakagawa et al., J.Am.Chem.Soc.107:7087-7092, 1985. In one embodiment, TAP is chemically synthesized (synthetic peptide). Another embodiment of the present disclosure relates to a peptide that does not exist in nature, which consists of or is essentially composed of an amino acid sequence as defined herein, and is produced by synthesis (e.g., synthesized) as a pharmaceutically acceptable salt.The TAP salts described herein differ significantly from the peptide(s) in vivo, as peptides produced in vivo do not take the form of salts. These non-natural peptide salts can be used to adjust the solubility of peptides in peptide-containing pharmaceutical compositions, particularly in peptide vaccines disclosed herein. These salts are preferably pharmaceutically acceptable peptide salts.

[0133] In one embodiment, the TAP described herein is substantially pure. A compound is considered "substantially pure" if it is separated from its naturally occurring components. Typically, a compound is considered substantially pure if it is present in a weight ratio of at least 60%, more commonly 75%, 80%, or 85%, preferably more than 90%, and even more preferably more than 95%, relative to the total amount of substance in the sample. Therefore, for example, a polypeptide produced by chemical synthesis or recombinant technology is usually substantially separated from its naturally occurring components, such as macromolecular components of its source. A nucleic acid molecule is considered substantially pure if it is not immediately adjacent (i.e., not covalently bonded) to a coding sequence that is normally adjacent within the naturally occurring genome of the organism from which it originates. Substantially pure compounds can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid molecule encoding a peptide compound, or by chemical synthesis. Purity can be measured using any suitable method, such as column chromatography, gel electrophoresis, or HPLC. In one embodiment, the TAP is in solution. In another embodiment, the TAP exists in a solid form, such as in a freeze-dried state.

[0134] In one embodiment, TAP is encoded by a sequence located in a non-protein-coding region of the genome. In another embodiment, TAP is encoded by a sequence located in an intergeneric region. In yet another embodiment, TAP is encoded by non-coding RNA (ncRNA). In yet another embodiment, TAP is encoded by a sequence located in an intron. In yet another embodiment, TAP is encoded by a sequence located in an untranslated region (UTR), for example, the 5'UTR. In yet another embodiment, TAP is encoded by a sequence located in a non-coding exon.

[0135] In another embodiment, the disclosure further provides synthetic long-chain peptides (SLPs) comprising at least one of the TAPs described herein. In one embodiment, the SLP comprises at least two TAPs, at least one of which is a TAP as described herein. In one embodiment, the SLP comprises at least two, three, four, or five of the TAPs described herein. In one embodiment, the SLP comprises at least ten, fifteen, twenty, twenty-five, thirty, thirty-five, or forty of the TAPs described herein. In one embodiment, the SLP comprises at least one TAP bound to one or more amino acid sequences or domains described herein that confer desired properties to the SLP, for example, a sequence or domain that stabilizes the SLP and / or improves processing and presentation by MHC molecules, for example, a sequence containing a motif that can be cleaved by an intracellular protease such as a cathepsin. In another embodiment, the SLP comprises at least one of the TAPs described herein and a TAP that binds to an MHC class II molecule. TAPs may be directly linked to each other, or they may be indirectly linked via linkers such as short-chain amino acid linkers. In several embodiments, the linker contains about 4 to 20 amino acids, or about 4 to 15 amino acids, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In one embodiment, the linker contains a glycine residue, a serine residue, a proline residue, a threonine residue, or a mixture thereof. The linker may contain a sequence that promotes SLP processing and releases TAP, for example, a cathepsin-sensitive linker (e.g., a 4-6 amino acid linker containing the sequences LVGS (SEQ ID NO: 205), ASLG (SEQ ID NO: 206), PIVG (SEQ ID NO: 207), LLSV (SEQ ID NO: 208), VLSVG (SEQ ID NO: 209), or LLSVGG (SEQ ID NO: 210). See Rabu et al., Oncoimmunology. 2019;8(4):e1560919). In one embodiment, the length of the SLP is 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, or 50 amino acids or less.In further embodiments, the length of the SLP is 20 to 50, 45, or 40 amino acids, for example, 20 or 25 amino acids to 30, 35, or 40 amino acids.

[0136] In another aspect, the disclosure further provides (isolated) nucleic acids encoding TAP or tumor antigen precursor peptides or SLPs as described herein. In one embodiment, the nucleic acids include about 24 to 1200 nucleotides, about 24 to 1000, 900, 800, 700, 600, 500, 400, 300 or 200 nucleotides, for example, about 24 to 150 or 100 nucleotides, for example, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69 or 72 nucleotides. As used herein, “isolated” means a peptide or nucleic acid molecule that has been separated from other components present in the natural environment of the molecule or from macromolecules of naturally occurring sources (e.g., including other nucleic acids, proteins, lipids, sugars, etc.). As used herein, “synthesized” means a peptide or nucleic acid molecule that has not been isolated from its natural source, produced, for example, through recombinant technology or using chemical synthesis. In one embodiment, the nucleic acid (DNA, RNA) encoding the TAP or SLP of this disclosure comprises one of the sequences listed in Table 1 below, or a corresponding RNA sequence. In one embodiment, the nucleic acid encoding the TAP or SLP is an mRNA molecule. In other embodiments, the nucleic acid encoding the TAP or SLP is self-amplified mRNA (saRNA), transcriptionally amplified mRNA (taRNA), or circular mRNA (circRNA) (see, for example, Liu et al., Nature Reviews Cancer, Vol. 23, August 2023, pp. 526-543). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0137] Of course, because the genetic code is degenerate, the TAPs described herein may be encoded by variants of the sequences mentioned above.

[0138] In one embodiment, the nucleic acid (DNA, RNA) encoding the TAP or SLP of the Disclosure comprises or consists of one or more of the sequences of SEQ ID NOs. 23-28, 31, 32, 34, 35, 39, 42, or 44, or the corresponding RNA sequence.

[0139] In one embodiment, the nucleic acid (DNA, RNA) encoding the TAP or SLP of the Disclosure comprises or consists of one or more sequences from sequence numbers 75-112, 148, or 204, or the corresponding RNA sequence. In one embodiment, the nucleic acid (DNA, RNA) encoding the TAP of the Disclosure comprises or consists of one of sequences from sequence numbers 75-109, 148, or 204, or the corresponding RNA sequence.

[0140] In one embodiment, a nucleic acid (DNA, RNA) encodes one or more aeTSAs and comprises or consists of one or more sequences, for example, sequence numbers 6, 7, 10, 12, 13, 15-23, 28, 31, 32, 34, 35, 57 and 203. In one embodiment, a nucleic acid encoding one or more aeTSA includes or consists of one or more of the sequences of sequence numbers 80, 81, 84, 86, 87, 89-97, 102, 105, 106, 108, 109, 131, and 204, for example, one or more of the sequences of sequence numbers 80, 81, 84, 86, 87, 89-96, 102, 105, 106, 108, 109, 131, and 204.

[0141] The nucleic acids of this disclosure can be used for recombinant expression of the TAP or SLP of this disclosure and may be included in vectors or plasmids that can be transfected into host cells, such as cloning vectors or expression vectors. In one embodiment, this disclosure provides a cloning vector, expression vector, viral vector or plasmid containing a nucleic acid sequence encoding the TAP of this disclosure. Alternatively, the nucleic acid encoding the TAP of this disclosure may be incorporated into the genome of a host cell. In either case, the host cell expresses the TAP or protein encoded by the nucleic acid. The term “host cell” as used herein refers not only to a specific target cell but also to a progeny or potential progeny of such a cell. The host cell may be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., insect cell, yeast cell, plant cell, or mammalian cell) capable of expressing the TAP described herein. The vector or plasmid may contain elements necessary for the transcription and translation of the inserted coding sequence and may contain other components such as resistance genes or cloning sites. Expression vectors containing a sequence encoding a peptide or polypeptide and appropriate transcriptional and translational regulatory / modulatory elements operably linked thereto may be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, and Ausubel, FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY. "Operatably linked" refers to the parallel arrangement of components, particularly nucleotide sequences, that enable them to perform their normal function.Therefore, a coding sequence operably linked to a regulatory sequence refers to a nucleotide sequence configuration in which the coding sequence can be expressed under the regulatory control of the regulatory sequence, i.e., under transcriptional and / or translational control. As used herein, “regulatory / controllable region” or “regulatory / controllable sequence” refers to a non-coding nucleotide sequence involved in the regulation of coding nucleic acid expression. Therefore, the term regulatory region includes promoter sequences, regulatory protein binding sites, and upstream activator sequences, etc. A vector (e.g., an expression vector) may have promoter sequences such as CMV, PGK, and EF-1α promoters, ribosome recognition and binding TATA boxes, and necessary 6' upstream and 3'' downstream regulatory elements such as a 3'UTR AAUAAA transcription termination sequence for efficient gene transcription and translation in the respective host cell. Other suitable promoters include the constitutive promoters of Simiambimus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV) promoter, HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV very early promoter, and Rous sarcoma vims promoter. Human gene promoters may also be used, including, but are not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. In certain embodiments, the inducible promoter is also intended to be part of a vector expressing TAP. This provides a molecular switch that can turn on or off the expression of a target polynucleotide sequence. Examples of inducible promoters include, but are not limited to, metallothione promoters, glucocorticoid promoters, progesterone promoters, or tetracycline promoters. Examples of vectors include plasmids, autonomous replication sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or Pl-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the Lenti-X® Bicistronic Expression System (Neo) vector (Contech) and the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST®, pLenti6 / V5-DEST®, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of TAP disclosed herein can be ligated into such expression vectors for the expression of TAP in mammalian cells.

[0142] In certain embodiments, the nucleic acid encoding the TAP of this disclosure is provided in a viral vector. The viral vector may be derived from an adenovirus, vaccinia virus, retrovirus, lentivirus, or formyvirus. As used herein, the term “viral vector” refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. The viral vector may contain coding sequences for various proteins described herein instead of non-essential viral genes. In another embodiment, the nucleic acid encoding the TAP of this disclosure is provided incorporated into a self-amplifying RNA or self-replicating RNA (srRNA) vector. The srRNA is derived from a forward-chain RNA virus from which structural proteins have been removed and replaced with the heterologous gene of interest. srRNA has been successfully derived from flaviviruses, nodamuraviruses, nidoviruses, and alphaviruses, and in a therapeutic version of this technology, single-cycle viral replicon particles (VRPs) are produced by supplying structural proteins in a trans state (see, e.g., Aliahmad et al. Next generation self-replicating RNA vectors for vaccines and immunotherapies). Cancer Gene Ther (2022). See https: / / doi.org / 10.1038 / s41417-022-00435-8. The vectors and / or particles can be used to introduce DNA, RNA, or other nucleic acids into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0143] In embodiments, the nucleic acids (DNA, RNA) encoding the TAP or SLP of the Disclosure are contained within vesicles or nanoparticles such as lipid vesicles (e.g., liposomes) or lipid nanoparticles (LNPs), or any other suitable vehicle. Thus, in another embodiment, the Disclosure provides vesicles or nanoparticles, e.g., lipid vesicles or lipid nanoparticles, containing nucleic acids encoding one or more of the TAP or SLPs, e.g., mRNA.

[0144] As used herein, the term liposome refers, in its usual sense, to a tiny lipid vesicle consisting of a phospholipid bilayer or any similar amphiphilic lipid (e.g., sphingolipid) enclosing an internal aqueous medium.

[0145] The term "lipid nanoparticles" refers to liposome-like structures that may include one or more lipid bilayer rings surrounding an internal aqueous medium similar to that of liposomes, or micelle-like structures that encapsulate molecules (e.g., nucleic acids) within a non-aqueous core. Lipid nanoparticles typically contain cationic lipids, such as ionizable cationic lipids. Examples of cationic lipids that can be used in LNPs include DOTMA, DOSPA, DOTAP, ePC, DLin-MC3-DMA, C12-200, ALC-0315, cKK-E12, Lipid H(SM-102), OF-Deg-Lin, A2-Iso5-2DC18, 306Oi10, BAME-O16B, TT3, 9A1P9, FTT5, COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC, and COATSOME® SS-OP (see, for example, Hou et al., Nature Reviews Materials, volume 6, pages 1078-1094 (2021); Tenchov et al., ACS Nano, 15, 16982-17015 (2021)).

[0146] Liposomes and lipid nanoparticles typically contain lipids, lipid-like materials, and other lipid components such as polymers that can improve the properties of liposomes or nanoparticles, including stability, delivery effectiveness, tolerability, and biodistribution. These include phospholipids such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and DOPE (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol), sterols (such as cholesterol and its derivatives), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG). 2000 -DMG) and 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG 2000 It contains PEGylated lipids (PEG-lipids) such as DSG.

[0147] In one embodiment, the lipid nanoparticles described herein include one or more cationic lipids, such as ionizable cationic lipids. Examples of ionizable cationic lipids include those described in PCT published patents WO2017 / 061150 and WO2019 / 188867, which encompass ionizable cationic lipids commercially available under the trademark names COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC, and COATSOME® SS-OP.

[0148] Nucleic acids encoding one or more of TAP or SLP (e.g., mRNA) can be modified, for example, to increase stability and / or reduce immunogenicity. For example, the 5' end can be capped to stabilize the molecule and reduce immunogenicity (as described, for example, in US10519189 and US10494399). One or more nucleosides of mRNA may be modified or substituted with 1-methylpseudouridine to either increase the stability of the molecule or reduce recognition of the molecule by the innate immune system. Forms of modified nucleosides are described in US9371511. Other types of modifications that can be performed on mRNA include anti-reverse cap analogs (ARCA), 5'-methylcytidine triphosphate (m5CTP), N6-methyladenosine-5'-triphosphate (m6ATP), 2-thiouridine triphosphate (s2UTP), pseudouridine triphosphate, N 1 Examples include the incorporation of methylpsoiduridine triphosphate or 5-methoxyuridine triphosphate (5moUTP). The mRNA may also include additional modifications to the 5′ and / or 3′ untranslated region (UTR) and the polyadenylated (polyA) tail (see, e.g., Kim et al., Molecular & Cellular Toxicology, Vol. 18, I (2022): 1–8). The poly(A) tail preferably comprises 100–200 nucleotides, more preferably 120–150 nucleotides, and may contain modified adenosine. All of these modifications, and other modifications to nucleic acids encoding TAP (e.g., mRNA), are encompassed by this disclosure.

[0149] In another aspect, the present disclosure provides an MHC class I molecule comprising (i.e., presenting or binding to) one or more TAPs comprising or consisting of the sequences of SEQ ID NOs: 1-74, 149-175 and 203 as defined herein, preferably SEQ ID NOs: 1-38, 74 and 203, more preferably SEQ ID NOs: 1-35, 74 and 203.

[0150] In one embodiment, the MHC class I molecule is the HLA-A*01:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A*02:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A*02:03 molecule. In one embodiment, the MHC class I molecule is the HLA-A*03:01 molecule. In one embodiment, the MHC class I molecule is the HLA-A * It is 11:01. In one embodiment, the MHC class I molecule is the HLA-A*24:02 molecule. In one embodiment, the MHC class I molecule is the HLA-A * 33:03 molecule. In one embodiment, the MHC class I molecule is HLA-A * In one embodiment, the MHC class I molecule is an HLA-B*07:02 molecule. In one embodiment, the MHC class I molecule is an HLA-B*07:05 molecule. In one embodiment, the MHC class I molecule is an HLA-B*08:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*15:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*15:25 molecule. In one embodiment, the MHC class I molecule is an HLA-B*18:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*27:06 molecule. In one embodiment, the MHC class I molecule is an HLA-B*35:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*39:01 molecule. In one embodiment, the MHC class I molecule is an HLA-B*40:01 molecule. In one embodiment, the MHC class I molecule is HLA-B*44:02. In one embodiment, the MHC class I molecule is HLA-B*44:03. In one embodiment, the MHC class I molecule is HLA-B*54:01. In one embodiment, the MHC class I molecule is HLA-C*03:04. In one embodiment, the MHC class I molecule is the HLA-C*07:04 molecule. In one embodiment, the MHC class I molecule is the HLA-C*15:05 molecule.

[0151] In one embodiment, TAP (for example, including or consisting of sequences SEQ ID NOs: 1-74, 149-175 and 203, SEQ ID NOs: 1-38, 74 and 203, or 1-35, 74 and 203 as defined herein) is non-covalently bonded to an MHC class I molecule (i.e., TAP is loaded into or non-covalently bonded to the peptide bond groove / pocket of the MHC class I molecule). In another embodiment, TAP is covalently bonded to an MHC class I molecule (alpha chain). In such a configuration, TAP and the MHC class I molecule (alpha chain) are produced as a synthetic fusion protein having a short (e.g., 5-20 residues, preferably about 8-12, e.g., 10) highly mobile linker or spacer (e.g., polyglycine linker). In another aspect, the disclosure provides a nucleic acid encoding a fusion protein in which TAP as defined herein is fused to an MHC class I molecule (alpha chain). In one embodiment, an MHC class I molecule (alpha chain)-peptide complex is multimerized. Therefore, in another embodiment, this disclosure provides a multimer of an MHC class I molecule loaded with TAP (covalently or otherwise) as described herein. Such a multimer may be attached to a tag, such as a fluorescent tag, enabling the detection of the multimer. Regarding the production of MHC multimers, many strategies have been developed, including MHC dimers, tetramers, pentamers, octamers, etc. (as outlined in Bakker and Schumacher, "Current Opinion in Immunology 2005, 17:428-433"). MHC multimers are useful, for example, for the detection and purification of antigen-specific T cells. Therefore, in another embodiment, this disclosure provides a TAP-specific CD8 as defined herein. + The present invention provides a method for detecting or purifying (isolating, concentrating) T lymphocytes, which involves contacting a cell population with a multimer of MHC class I molecules loaded with TAP (covalently or noncovalently) and CD8 conjugated by the MHC class I multimer. + This includes detecting or isolating T lymphocytes. CD8 to which MHC class I multimers bind. +T lymphocytes can be isolated using known methods, such as fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS).

[0152] In another embodiment, the Disclosure provides cells (e.g., host cells), and in one embodiment, provides isolated cells containing the nucleic acids, vectors, or plasmids described herein (i.e., nucleic acids or vectors encoding one or more TAPs). In another embodiment, the Disclosure provides cells expressing on their surface an MHC class I molecule (e.g., any one of the MHC class I molecules of the alleles disclosed above) bound to or presenting the TAP described herein. In one embodiment, the host cell is a eukaryotic cell, e.g., a mammalian cell, preferably a human cell, cell line, or immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC). In one embodiment, the host cell is a primary cell, cell line, or immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC). The nucleic acids and vectors can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing exogenous nucleic acids into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, and virus-mediated transfection. Appropriate methods for transforming or transfecting host cells can be found, for example, in Sambrook et al. (mentioned above) and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known and can be used to deliver the vectors or plasmids of this disclosure to subjects for gene therapy.

[0153] Cells such as APCs can be loaded with one or more TAPs using various methods known in the field. As used herein, “loading cells with TAP” means transfecting cells with RNA or DNA encoding TAP, or TAP itself, or transforming APCs with nucleic acids encoding TAP. The cells can also be loaded by contacting exogenous TAPs that can directly bind to MHC class I molecules present on the cell surface (e.g., peptide pulsed cells). TAPs can also be fused to domains or motifs that facilitate their presentation by MHC class I molecules, such as endoplasmic reticulum (ER) retrieval signals or C-terminal Lys-Asp-Glu-Leu sequences (see Wang et al., Eur J Immunol. 2004 Dec;34(12):3582-94).

[0154] In another embodiment, the Disclosure provides compositions or combinations / pools of peptides comprising any one or any combination of TAPs (or nucleic acids encoding the peptide(s)) as defined herein. In one embodiment, the composition comprises any combination of TAPs as defined herein (any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TAPs) or a combination of nucleic acids encoding the TAPs. Compositions comprising any combination / partial combination of TAPs as defined herein are encompassed by the Disclosure. In another embodiment, the combination or pool may comprise one or more known tumor antigens.

[0155] Accordingly, in another aspect, the Disclosure provides a composition comprising one or any combination of any of the TAPs defined herein (for example, comprising or consisting of sequences 1-74, 149-175 and 203, 1-38, 74 and 203, or 1-35, 74 and 203 as defined herein). The APCs for use in the Disclosure are not limited to any particular type of cell, including CD8+ This includes professional APCs such as dendritic cells (DCs), Langerhans cells, macrophages, and B cells, which are known to present proteinaceous antigens on their cell surface so as to be recognized by T lymphocytes. For example, APCs can be obtained either in vitro, ex vivo, or in vivo by inducing DCs from peripheral blood monocytes and then contacting (stimulating) them with TAPs. APCs can also be activated to present TAPs in vivo, where one or more TAPs of this disclosure are administered to a subject, and APCs that present TAPs are induced in the subject's body. The phrases “inducing APCs” or “stimulating APCs” include contacting or loading cells with one or more TAPs or nucleic acids encoding TAPs, resulting in the presentation of TAPs on the cell surface by MHC class I molecules. As described herein, according to this disclosure, TAP can be indirectly loaded using, for example, a longer peptide / polypeptide (including a native protein) containing the sequence of TAP, which is then processed internally within the APC (e.g., by a protease) to generate a TAP / MHC class I complex on the cell surface. After loading the APC with TAP and causing the APC to present TAP, the APC can be administered to a subject as a vaccine. For example, ex vivo administration may include the steps of: (a) collecting an APC from a first subject; (b) contacting / loading the APC from step (a) with TAP to form an MHC class I / TAP complex on the surface of the APC; and (c) administering the peptide-loaded APC to a second subject in need of treatment.

[0156] The first and second subjects may be the same subject (e.g., autovaccines) or different subjects (e.g., allovaccines). Alternatively, the Disclosure provides the use of the TAPs described herein (or combinations thereof) for manufacturing compositions (e.g., pharmaceutical compositions) for inducing antigen-presenting cells. In addition, the Disclosure provides a method or process for manufacturing pharmaceutical compositions for inducing antigen-presenting cells, the method or process comprising the step of mixing or compounding the TAPs, or combinations thereof, with a pharmaceutically acceptable carrier. Cells such as APCs expressing MHC class I molecules (e.g., any of the HLA molecules listed above) and loaded with any one or any combination of the TAPs defined herein can be used to stimulate / amplify CD8+ T lymphocytes, e.g., autologous CD8+ T lymphocytes. Thus, in another aspect, the Disclosure provides any one or any combination of the TAPs defined herein (or nucleic acids or vectors encoding them), cells expressing MHC class I molecules, and T lymphocytes, more specifically CD8+ + The present invention provides a composition containing T lymphocytes (e.g., a cell population including CD8+ T lymphocytes).

[0157] In one embodiment, the composition further comprises a buffer, excipients, carriers, diluents, and / or a culture medium (e.g., a culture medium). In further embodiments, the buffer, excipients, carriers, diluents, and / or a culture medium are pharmaceutically acceptable buffers, excipients, carriers, diluents, and / or a culture medium. As used herein, “pharmaceutically acceptable buffers, excipients, carriers, diluents, and / or a culture medium” includes all solvents, buffers, binders, lubricants, fillers, thickeners, disintegrants, plasticizers, coatings, barrier layer formulations, stabilizers, release retarders, dispersion media, coatings, antimicrobial and antifungal agents, and isotonic agents, etc., that are physiologically compatible, do not impede the efficacy of the biological activity of the active ingredient(s), and are non-toxic to the subject. The use of culture media and drugs in this manner for pharmaceutically active substances is well known in the art (Rowe et al., "Handbook of pharmaceutical excipients, 2003, 4th edition," Pharmaceutical Press, London UK). Unless any conventional culture media or drug is incompatible with the active compound (peptide, cell), their use in the compositions of the present disclosure is intended. In one embodiment, the buffer, excipient, carrier, and / or culture medium is a buffer, excipient, carrier, and / or culture medium that does not exist naturally. In one embodiment, one or more of the TAPs defined herein, or nucleic acids (e.g., mRNA) encoding the one or more TAPs, are contained in or complexed with lipid vesicles or liposomes, e.g., cationic liposomes (see, e.g., Vitor MT et al., Recent Pat Drug Deliv Formul. 2013 Aug;7(2):99-110) or other suitable carriers.

[0158] In another embodiment, the Disclosure provides a composition comprising any one or any combination thereof of TAPs as defined herein (e.g., comprising or consisting of sequences SEQ ID NOs: 1-74, 149-175 and 203, SEQ ID NOs: 1-38, 74 and 203, or 1-35, 74 and 203 as defined herein), or SLPs, (or nucleic acids such as mRNA encoding the above peptides), as well as a buffer, excipients, carriers, diluents and / or culture media. For compositions comprising cells (e.g., APCs, T lymphocytes), the composition comprises a suitable culture medium that enables the maintenance of viable cells. Typical examples of such culture media include physiological saline, Earl's buffered salt solutions (Life Technologies®), or PlasmaLyte® (Baxter International®). In one embodiment, the composition (e.g., a pharmaceutical composition) is an "immunogenic composition," a "vaccine composition," or a "vaccine." As used herein, the terms “immunogenic composition,” “vaccine composition,” or “vaccine” refer to a composition or formulation comprising one or more TAPs, nucleic acids, or vaccine vectors that, when administered to a subject, has the ability to induce an immune response to one or more TAPs present therein. Methods of vaccination for inducing an immune response in mammals (e.g., humans) include the use of vaccines or vaccine vectors administered via any conventional route known in the vaccine field, e.g., via mucosal surfaces (eyes, nasal cavity, lungs, oral cavity, stomach, intestine, rectum, vagina, or urinary tract), via parenteral routes (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal), or by topical administration (e.g., transdermal delivery systems such as patches). In one embodiment, a TAP or SLP (or a combination thereof) is conjugated to a carrier protein to increase the immunogenicity of the TAP(s) (conjugated vaccine). Accordingly, this disclosure provides compositions (complexes) comprising a TAP (or a combination thereof) or nucleic acid encoding a TAP, or a combination thereof, and a carrier protein.For example, TAP(or TAP) or nucleic acid(or nucleic acid) may be a Toll-like receptor (TLR) ligand (see, e.g., Zom et al., Adv Immunol. 2012, 114:177-201), or a polymer / dendrimer (see, e.g., Liu et al., Biomacromolecules. 2013 Aug 12;14(8):2798-806), such as a polymer-conjugated TLR agonist (see, e.g., Lynn et al., Nature Biotechnology 33:1201-1210(2015); Lynn et al., Nature Biotechnology 38:320-332(2020)). In one embodiment, the immunogenic composition or vaccine further comprises an adjuvant. "Adjuvant" means a substance that, when added to an immunogenic agent such as an antigen (AP, nucleic acid, and / or cell as described herein), nonspecifically enhances or strengthens the immune response to the drug in a host upon exposure to the mixture.Examples of adjuvants currently used in the vaccine field include: (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gel), squalene, (2) oil-based adjuvants (oil-based emulsions and surfactant-based formulations, e.g., MF59 (microfluidized detergent-stabilized oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (water-in-oil emulsion + MPL + QS-21)), (3) particle adjuvants, e.g., virosomes (monolayer liposome vehicle incorporating influenza hemagglutinin), AS04 [SBAS4] (aluminum salt and MPL), ISCOMS (structured complex of saponin and lipid), polylactic acid coglycolic acid (PLG), and (4) microbial-derived (natural and synthetic) adjuvants. Examples include monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP[RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoid immunostimulant capable of self-assembling to form liposomes), OM-174 (lipid A derivative), CpG motif (synthetic oligonucleotide containing an immunostimulatory CpG motif), genetically modified cholera toxin (CT) and Escherichia coli enterotoxin (LT) (bacterial toxins genetically modified to provide a non-toxic adjuvant effect), (5) endogenous human immunomodulators, such as hGM-CSF or hIL-12 (cytokines administered as either a protein or an encoded plasmid), Immudaptin (C3d tandem array), and / or (6) inert vehicles such as gold particles.

[0159] In one embodiment, the TAP, SLP, (or nucleic acids such as mRNA encoding the peptide(s)), or composition containing them (for example, sequences 1-74, 149-175 and 203, 1-38, 74 and 203, or 1-35, 74 and 203) is in a lyophilized form. In another embodiment, the TAP(s), nucleic acids(s), or composition containing them is a liquid composition. In a further embodiment, the TAP(s) or nucleic acids(s) are present in the composition at a concentration of about 0.01 μg / mL to about 100 μg / mL. In several further embodiments, TAP(or nucleic acid(or nucleic acid)) is present at concentrations of approximately 0.2 μg / mL to approximately 50 μg / mL, approximately 0.5 μg / mL to approximately 10, 20, 30, 40 or 50 μg / mL, approximately 1 μg / mL to approximately 10 μg / mL, or approximately 2 μg / mL.

[0160] As described herein, cells such as APCs expressing MHC class I molecules loaded with or bound to any one or any combination of TAPs may undergo CD8 in vivo or ex vivo. + It can be used to stimulate / amplify T lymphocytes. In another aspect, the present disclosure provides a T cell receptor (TCR) molecule that can interact with or bind to the MHC class I molecule / TAP complex described herein, a nucleic acid molecule encoding such a TCR molecule, and a vector comprising such a nucleic acid molecule. The TCR described herein can specifically interact with or bind to TAP loaded on or presented on an MHC class I molecule, preferably on the surface of a living cell in vitro or in vivo. As used herein, the term TCR refers to immunoglobulin superfamily members having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail (see, for example, Janeway et al, Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p.4:33, 1997), and capable of specifically binding to antigen peptides bound to MHC receptors. TCRs may reside on the cell surface and generally consist of heterodimers having α and β chains (also known as TCRα and TCRβ, respectively). Similar to immunoglobulins, the extracellular portion of the TCR chain (e.g., α chain, β chain) contains two immunoglobulin regions: a variable region (e.g., TCR variable α region or Vα, and TCR variable β region or Vβ; typically amino acids 1-116 based on Rabat numbering at the N-terminus) and a constant region adjacent to the cell membrane (e.g., TCR constant domain α or Cα, typically amino acids 117-259 based on Rabat, TCR constant domain β or Cβ, typically amino acids 117-295 based on Rabat). Also similar to immunoglobulins, the variable domain contains complementarity-determining regions (CDRs, three per chain) separated by a framework region (FR). In certain embodiments, the TCR is present on the surface of T cells (or T lymphocytes) and associates with the CD3 complex. In other embodiments, the TCR is a soluble TCR.

[0161] By applying TCRs and, more specifically, nucleic acids encoding the TCRs of this disclosure, T lymphocytes (e.g., CD8+ T lymphocytes) or other types of lymphocytes that generate novel T lymphocyte clones that specifically recognize the MHC class I / TAP complex can be genetically transformed / modified. In certain embodiments, T lymphocytes obtained from a patient (e.g., CD8+ T lymphocytes) are transformed to express one or more TCRs that recognize TAP, and the transformed cells are administered to the patient (autologous cell transfusion). In certain embodiments, T lymphocytes obtained from a donor (e.g., CD8+ T lymphocytes) are transformed to express one or more TCRs that recognize TAP, and these transformed cells are administered to the recipient (allogeneic cell transfusion). In another embodiment, this disclosure provides T lymphocytes, e.g., CD8+ T lymphocytes transformed / transfected with a vector or plasmid encoding a TAP-specific TCR. In further embodiments, the present disclosure provides a method for treating a patient with autologous or allogeneic cells transformed with a TAP-specific TCR. In certain embodiments, the TCR is expressed in primary T cells (e.g., cytotoxic T cells) by replacing endogenous loci, such as the endogenous TRAC and / or TRBC loci, using, for example, CRISPR, TALEN, zinc finger, or other targeted disruption systems.

[0162] In yet another embodiment, the use of tumor antigen-specific TCRs in the production of autologous or allogeneic cells for the treatment of cancer such as lung cancer (e.g., NSCLC) is provided.

[0163] In some embodiments, patients receiving treatment with the compositions of the Disclosure (e.g., pharmaceutical compositions) receive treatment before or after treatment with antitumor agents and / or immunotherapies (e.g., CAR therapy, immune checkpoint inhibitor therapy). The compositions of the Disclosure include allogeneic or autologous T lymphocytes (e.g., CD8+ T lymphocytes) ex vivo activated against TAP, allogeneic or autologous APC vaccines loaded with TAP, vaccines containing nucleic acids (e.g., mRNA) encoding TAP, and allogeneic or autologous T lymphocytes (e.g., CD8+ T lymphocytes). + This includes T lymphocytes, or lymphocytes transformed with a tumor antigen-specific TCR. The method for providing T lymphocyte clones capable of recognizing TAP as described in this disclosure allows for the production of T lymphocyte clones for tumor cells expressing TAP in subjects (e.g., graft recipients), such as allogeneic or autologous T lymphocytes and / or donor lymphocyte infusion (DLI) recipients, and for the specific targeting of these tumor cells. Accordingly, this disclosure relates to a CD8 that encodes and expresses a T cell receptor capable of specifically recognizing or binding to the TAP / MHC class I molecular complex. + This specification provides T lymphocytes. The above T lymphocytes (e.g., CD8+ T lymphocytes) may be recombinant (engineered) T lymphocytes or naturally selected T lymphocytes. Therefore, this specification provides for the CD8 described herein. + The present invention provides at least two methods for producing T lymphocytes, comprising the step of contacting undifferentiated lymphocytes with a TAP / MHC class I molecule complex (typically expressed on the surface of cells such as APCs) under conditions that induce T cell activation and T cell proliferation, which can be performed in vitro or in vivo (i.e., in patients administered an APC vaccine in which APCs are loaded with TAP, or in patients treated with a TAP vaccine). By using a combination or pool of TAPs bound to MHC class I molecules, CD8 can recognize multiple TAPs. + It is possible to produce a population of T lymphocytes. Alternatively, an MHC class I molecule / TAP complex (i.e., an engineered modified CD8) can be produced.+ By cloning one or more nucleic acids (genes) encoding TCRs (more specifically, alpha and beta chains) that specifically bind to T lymphocytes or recombinant CD8+ T lymphocytes, tumor antigen-specific or targeted T lymphocytes can be produced in vitro or ex vivo. The nucleic acids encoding the TAP-specific TCRs of this disclosure can be obtained ex vivo from T lymphocytes activated against TAP (e.g., using TAP-loaded APCs) or from individuals exhibiting an immune response to peptide / MHC molecular complexes using methods known in the art. The TAP-specific TCRs described herein can be recombinantly expressed in host cells and / or host lymphocytes obtained from graft recipients or graft donors and, optionally, differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). Nucleic acids (or transgenes) encoding the alpha and beta chains of the TCR can be introduced into T cells (e.g., from a target or another individual) using any preferred method such as transfection (e.g., electroporation) or transduction (e.g., using a viral vector). Engineered CD8 expressing a TCR specific to TAP. + T lymphocytes can be proliferated in vitro using well-known culture methods.

[0164] This disclosure provides a method for producing immunoeffector cells expressing TCRs as described herein. In one embodiment, the method comprises transfecting or transduction immunoeffector cells, e.g., immunoeffector cells isolated from a subject such as a subject having lung cancer, so that the immunoeffector cells express one or more TCRs. In a particular embodiment, the immunoeffector cells are isolated from an organism and genetically modified in vitro without further manipulation. Such cells can then be directly re-administered to the organism. In a further embodiment, the immunoeffector cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express TCRs. In this regard, the immunoeffector cells can be cultured before or after genetic modification (i.e., transduction or transfection to express TCRs as described herein).

[0165] Prior to the in vitro manipulation or genetic modification of immunoeffector cells described herein, cell sources can be obtained from a subject. In particular, immunoeffector cells for use with TCRs described herein include T cells. T cells can be obtained from several sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic problems, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL® isolation. In one embodiment, cells derived from the circulating blood of an individual can be obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in a suitable buffer or culture medium for further processing. In one embodiment of the present invention, cells are washed with PBS. In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or many but not all divalent cations. As will be understood by those skilled in the art, the washing step can be achieved by methods known to those skilled in the art, for example, by using a semi-automatic flow-through centrifuge. After washing, these cells can be resuspended in various biocompatible buffers or other salines, with or without buffers. In certain embodiments, unwanted components in the apheresis sample can be removed intracellularly, and the cells can be resuspended directly in culture medium. In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing erythrocytes and depleting monocytes, for example, by centrifugation via a PERCOLL® gradient. CD28 + CD4 + CD8 + CD45RA + , and CD45RO +Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies that target surface markers specific to negatively selected cells. One method for use herein is the sorting and / or selection of cells via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies that target cell surface markers present on negatively selected cells. For example, to enrich CD8+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD4. The desired cell population for use in this disclosure can also be isolated using flow cytometry and cell sorting. PBMCs can be used directly for gene modification by TCR using the methods described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic T lymphocytes and helper T lymphocytes can be sorted into subpopulations of naive T cells, memory T cells, and effector T cells, either before or after gene modification and / or proliferation.

[0166] This disclosure provides isolated immune cells (e.g., CD8+ T lymphocytes) that are specifically induced, activated, and / or amplified (proliferated) by TAP (i.e., TAP bound to MHC class I molecules expressed on the surface of cells), or a combination of TAPs. + T lymphocytes are tumor-infiltrating lymphocytes (TILs) obtained from a subject and may be proliferated ex vivo using one or more of the described TAPs before being administered to a patient. The disclosure also describes the TAPs or combinations thereof described herein (i.e., one or more TAPs bound to an MHC class I molecule) and CD8 capable of recognizing such TAP(s). + A composition containing T lymphocytes is provided.

[0167] In another aspect, the disclosure relates to a cell population or cell culture (e.g., CD8+ T lymphocytes) enriched in T lymphocytes (e.g., CD8+ T lymphocytes) that specifically recognize one or more MHC class I molecule / TAP complexes described herein. + The present invention provides a population of T lymphocytes. Such a concentrated population can be obtained by ex vivo proliferation of specific T lymphocytes using cells such as APCs expressing MHC class I molecules loaded with (e.g., presenting) one or more of the TAPs disclosed herein. As used herein, “concentration” refers to tumor antigen-specific T lymphocytes (e.g., CD8) in the population. + This means that the proportion of T lymphocytes is significantly higher compared to the innate cell population, i.e., the population not subjected to the step of ex vivo proliferation of specific T lymphocytes. In one embodiment, the cell population is a TIL population or derived from TILs (e.g., TILs isolated from a patient and proliferated ex vivo). In a further embodiment, the proportion of TAP-specific T lymphocytes (e.g., CD8+ T lymphocytes) in the cell population is at least about 0.5%, e.g., at least about 1%, 1.5%, 2%, or 3%. In some embodiments, the percentage of TAP-specific T lymphocytes (e.g., CD8+ T lymphocytes) within a cell population is approximately 0.5–10%, 0.5–8%, 0.5–5%, 0.5–4%, 0.5–3%, 1–5%, 1–4%, 1–3%, 2–5%, 2–4%, 2–3%, 3–5%, or 3–4%. Such a cell population or culture (e.g., CD8+ T lymphocyte population) is characterized by T lymphocytes (e.g., CD8+ T lymphocytes) that specifically recognize one or more MHC class I molecule / peptide (TAP) complexes of interest. +The TAP-specific CD8+ T lymphocytes (T lymphocytes) enriched with TAP can be used in tumor antigen-based cancer immunotherapy, as detailed below. In some embodiments, populations of TAP-specific CD8+ T lymphocytes are further enriched using affinity-based systems, such as multimers of MHC class I molecules (commonly or non-commonly) loaded with TAP(plural) as defined herein. Thus, this disclosure relates to purified or isolated populations of TAP-specific T lymphocytes (e.g., CD8+ T lymphocytes), for example, TAP-specific CD8 + The present invention provides purified or isolated populations of TAP-specific CD8+ T lymphocytes, where the proportion of T lymphocytes is at least approximately 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0168] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to a complex comprising a TAP as described herein that binds to an HLA molecule, such as an HLA molecule defined herein, or a soluble TCR (e.g., a TCR mimetic). Such antibodies are generally referred to as TCR-like antibodies. As used herein, the term "antibody or antigen-binding fragment thereof" refers to any type of antibody / antibody fragment, including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired antigen specificity / binding activity. Antibody fragments include a portion of a full-length antibody, generally including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain Fv, scFv), single-domain antibodies (e.g., from camelids), shark NAR single-domain antibodies, as well as antibody fragments, single-chain diabodies (scDb), bispecific T cell engagers (BiTE), dual-affinity retargeting molecules (DART), bivalent scFv-Fc, and multispecific antibodies formed from trivalent scFv-Fc. Antibody fragments also include binding portions that contain a CDR or antigen-binding domain, which includes V H region (V H , V H -V HThis includes, but is not limited to, antikalin, peptobodies, antibody-T cell epitope fusions (troybodies), or peptibodies. In one embodiment, the antibody or its antigen-binding fragment is a single-chain antibody, preferably a single-chain Fv (scFv). In one embodiment, the antibody or its antigen-binding fragment comprises at least one constant domain, e.g., a constant domain of the light chain and / or heavy chain, or a fragment thereof. In a further embodiment, the antibody or its antigen-binding fragment comprises a crystallizable fragment (Fc) fragment derived from the constant heavy chain of the antibody. In one embodiment, the antibody or antigen-binding fragment is an scFv comprising an Fc fragment (scFV-Fc). In one embodiment, the scFv component is connected to the Fc fragment by a linker, e.g., a hinge. The presence of the Fc region is useful for inducing a response to tumor cells by complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated phagocytosis (ADCP), or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0169] In one embodiment, the antibody or its antigen-binding fragment is a multispecific antibody or its antigen-binding fragment, such as a bispecific antibody or its antigen-binding fragment, where at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognizes a complex containing TAP as described herein, bound to an HLA molecule. In one embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognizes an immune cell effector molecule. The term “immune cell effector molecule” refers to a molecule (e.g., a protein) expressed by an immune cell whose engagement by its multispecific antibody or antibody fragment results in activation of the immune cell. Examples of immune cell effector molecules include the CD3 signaling complex in T cells, such as CD8 T cells, and various activating receptors on NK cells (e.g., NKG2D, KIR2DS, NKp44). In a further embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognizes and engages with the CD3 signaling complex in T cells (e.g., anti-CD3). In a further embodiment, the multispecific antibody or antibody fragment is a single-stranded diabody (scDb). In further embodiments, scDb comprises a first antibody fragment (e.g., scFv) that binds to a complex containing TAP as described herein, conjugated to an HLA molecule, and a second antibody fragment (e.g., scFv) that binds to and engages with an immune cell effector molecule, such as an intracellular CD3 signaling complex (e.g., anti-CD3scFv). Such constructs can be used, for example, to induce cytotoxic T cell-mediated killing of tumor cells expressing a tumor antigen / MHC complex recognized by a multispecific antibody or antibody fragment. The antibody or its antigen-binding fragment may also be used as a chimeric antigen receptor (CAR) for producing CAR T cells, CAR NK cells, etc. A CAR combines a ligand-binding domain (e.g., antibody or antibody fragment) that provides specificity to a desired antigen (e.g., an MHC / TAP complex) with an activated intracellular domain (or signaling domain) moiety, such as a T cell or NK cell activation domain, to provide a primary activation signal.Antibodies that can bind to molecules expressed by tumor cells, more specifically, antigen-binding fragments of scFvs, are commonly used as ligand-binding domains in CARs.

[0170] In one embodiment, the soluble TCR is a therapeutically soluble bispecific TCR (see, for example, Robinson et al., FEBS J. 2021 Nov. 288(21):6159-6173; Dilchert et al., Antibodies (Basel). 2022 May 10;11(2):34).

[0171] In one embodiment, a soluble TCR (e.g., a TCR mime), an antibody, or an antibody fragment conjugates with an antitumor agent to form an antibody-drug conjugate (ADC). Such an ADC enables the delivery of an antitumor agent to tumor cells expressing one or more TAPs as described herein (see, for example, Shen et al., Asian J Pharm Sci. 2020 Nov;15(6):777-785).

[0172] In another embodiment, the disclosure provides nucleic acids (e.g., DNA, mRNA) encoding the above-mentioned TCR, TCR mime, antibody, or antibody fragment. In a further embodiment, this nucleic acid resides within a vector, such as the vector described above.

[0173] Therefore, in another aspect, the present disclosure provides host cells, preferably immune cells such as T cells or NK cells, that express antibodies or antibody fragments (e.g., scFv) described herein.

[0174] This disclosure further describes the above immune cells (CD8 + T lymphocytes, CAR T cells, or TAP-specific CD8 + The present invention relates to a pharmaceutical composition or vaccine comprising a population of T lymphocytes. Such a pharmaceutical composition or vaccine may contain one or more pharmaceutically acceptable excipients and / or adjuvants as described above.

[0175] In another aspect, the Disclosure further relates to the use of any one of the TAPs, SLPs, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, APCs, CAR T cells), and / or compositions, or any combination thereof, as a pharmaceutical product for the treatment of cancer (e.g., lung cancer such as NSCLC) or in the manufacture of such pharmaceutical product. The Disclosure also relates to any one of the TAPs, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, APCs, CAR T cells), and / or compositions (e.g., vaccine compositions), or any combination thereof, for use in the treatment of cancer (e.g., lung cancer such as NSCLC) (e.g., as a lung cancer vaccine). The TAP sequences identified herein can be used to produce synthetic peptides that are used i) for in vitro priming and proliferation of tumor antigen-specific T cells injected into tumor patients, and / or ii) as vaccines to induce or enhance the antitumor T cell response in cancer patients (e.g., lung cancer such as NSCLC).

[0176] In another embodiment, the Disclosure provides the use of one or more nucleic acids encoding TAP or SLP as described herein (e.g., comprising or consisting of any one of the sequences of, for example, SEQ ID NOs: 1-74, 149-175, and 203, e.g., SEQ ID NOs: 1-38, 74, and 203, or 1-35, 74, and 203) or a combination thereof (e.g., a peptide pool) or one or more nucleic acids encoding TAP as a vaccine for treating cancer in a subject (e.g., lung cancer such as NSCLC). The Disclosure also provides one or more nucleic acids encoding TAP as described herein for use as a vaccine for treating cancer in a subject (e.g., lung cancer such as NSCLC). In one embodiment, the subject is TAP-specific T lymphocytes (e.g., CD8 +a recipient of T lymphocytes). Thus, in another aspect, the present disclosure provides a method of treating cancer, more specifically lung cancer such as NSCLC (e.g., reducing the number of tumor cells, killing tumor cells), the method comprising administering (infusing) to a subject in need of such treatment an effective amount of T lymphocytes (e.g., CD8 + T lymphocytes) that recognize (i.e., express a TCR that binds to) one or more MHC class I molecule / TAP complexes expressed on the surface of cells such as APCs. In one embodiment, the method further comprises administering to the subject, after administration / infusion of the CD8 + T lymphocytes, an effective amount of TAP or SLP, or a combination thereof, or one or more nucleic acids encoding TAP(s) or SLP(s), and / or cells (e.g., APCs such as dendritic cells) that express MHC class I molecule(s) loaded with TAP(s). In a further embodiment, the method comprises administering to a subject in need thereof dendritic cells loaded with a therapeutically effective amount of one or more TAPs. In a further embodiment, the method comprises administering to a patient in need thereof allogeneic or autologous cells that express a recombinant TCR that binds to TAP presented by MHC class I molecules, at a therapeutically effective amount.

[0177] In another aspect, the present disclosure provides the use of T lymphocytes (e.g., CD8 + T lymphocytes) that recognize one or more MHC class I molecules loaded with (presenting) TAP or a combination thereof, for treating cancer, more specifically lung cancer such as NSCLC (e.g., reducing the number of tumor cells, killing tumor cells) in a subject. In another aspect, the present disclosure provides the preparation / manufacture of a medicament for treating cancer, more specifically lung cancer such as NSCLC (e.g., reducing the number of tumor cells, killing tumor cells) in a subject, using T lymphocytes (e.g., CD8 +In another aspect, the disclosure presents the use of T lymphocytes that recognize one or more MHC class I molecules (e.g., CD8) loaded with TAP or a combination thereof for use in the treatment of cancer in a subject, more specifically lung cancer such as NSCLC (e.g., to reduce the number of tumor cells, to kill tumor cells). + The present invention provides T lymphocytes. In a further embodiment, the use further comprises the use of cells (e.g., APCs) expressing an effective amount of TAP (or a combination thereof), one or more nucleic acids encoding TAP, and / or one or more MHC class I molecules loaded (presented) with TAP, after the use of the TAP-specific T lymphocytes.

[0178] This disclosure also provides a method for inducing an immune response against tumor cells expressing human class I MHC molecules loaded with any one of the TAPs or SLPs disclosed herein (e.g., sequences 1-74, 149-175, and 203, e.g., sequences 1-38, 74, and 203, or 1-35, 74, and 203). This disclosure also provides the use of cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with any one of the TAPs or combinations of TAPs disclosed herein for generating an immune response against tumor cells expressing human class I MHC molecules loaded with TAPs or a combination of TAPs.

[0179] The TAPs, combinations thereof (e.g., peptide pools), SLPs, TAPs(or more) or nucleic acids(or more) encoding SLPs(or more), as well as antibodies, TCRs, cells (e.g., CD8 T cells, APCs), vaccines, and compositions disclosed herein can be used for the prevention or treatment of any cancer expressing TAP.

[0180] The TAPs, combinations thereof (e.g., peptide pools), SLPs, TAPs or SLPs, nucleic acids (or multiples) encoding them, as well as antibodies / antibody fragments, TCRs, cells (e.g., T lymphocytes, CAR T cells or NK cells, APCs), vaccines, and compositions disclosed herein can be used to induce or stimulate an immune response against cancer cells expressing TAPs.

[0181] In one embodiment, the cancer is lung cancer such as NSCLC. In one embodiment, the lung cancer is adenocarcinoma. In one embodiment, the lung cancer is squamous cell carcinoma. In one embodiment, the lung cancer is large cell carcinoma. In one embodiment, the lung cancer is stage 1 lung cancer. In one embodiment, the lung cancer is stage 2 lung cancer. In one embodiment, the lung cancer is stage 3 lung cancer. In one embodiment, the lung cancer is stage 4 lung cancer. In one embodiment, the lung cancer is recurrent lung cancer. In one embodiment, the lung cancer is chemotherapy-resistant lung cancer. In one embodiment, the lung cancer is NSCLC.

[0182] In one embodiment, the methods or uses described herein further include identifying the HLA class I alleles expressed by the patient prior to treatment / use, and administering or using TAPs that bind to one or more HLA class I alleles expressed by the patient. For example, if the patient is determined to express HLA-A03*01 and HLA-B40*01, any combination of (i) TAPs of sequence numbers 11, 14, 17, 19-22, 26, and / or 29 (which bind to HLA-A03*01) and (ii) TAPs of sequence numbers 1-4, and / or 9 (which bind to HLA-B40*01) may be administered or used to the patient.

[0183] In some embodiments, patients treated with the compositions of the Disclosure (e.g., pharmaceutical compositions) are treated before or after treatment with allogeneic stem cell transplantation (ASCL), allogeneic lymphocyte infusion, or autologous lymphocyte infusion.

[0184] In one embodiment, the TAP, SLP, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, CAR T cells or NK cells, APCs), and / or compositions, or any combination thereof, according to the Disclosure, may be used to treat cancer (e.g., lung cancer such as NSCLC) as one or more additional activators or therapies, e.g., chemotherapy (e.g., vinca alkaloids), agents that inhibit microtubule formation (e.g., colchicine and its derivatives, monomethyl auristatin E (MMAE)), anti-angiogenic agents, therapeutic antibodies, EGFR targeters, tyrosine kinase targeters (e.g., tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (e.g., nucleoside analogs), alkylating agents, platinum-based drugs, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (e.g., all thrombocytosis inhibitors), Inhibitors of thretinoic acid or its derivatives, geldanamycin or its derivatives (17-AAG), CDK4 / 6, TGF-β, WNT-β-catenin, MYC, or PI3K, surgery, immune checkpoint inhibitors or immunotherapies (e.g., PD-1 / PD-L1 inhibitors such as anti-PD-1 / PD-L1 antibodies, CTLA-4 inhibitors such as anti-CTLA-4 antibodies, B7-1 / B7-2 inhibitors such as anti-B7-1 / B7-2 antibodies, TIM3 inhibitors such as anti-TIM3 antibodies, BTLA inhibitors such as anti-BTLA antibodies, CD47 inhibitors such as anti-CD47 antibodies, GITR inhibitors such as anti-GITR antibodies), antibodies against tumor antigens (e.g., anti-CD19, anti-CD22 antibodies), cell-based therapies (e.g., CAR It can be used in combination with T cells (CAR NK cells), and cytokines such as IL-2, IL-7, IL-21, and IL-15. In one embodiment, the TAP, nucleic acid, expression vector, T cell receptor, cells (e.g., T lymphocytes, APCs), and / or compositions described herein are administered / used in combination with immune checkpoint inhibitors. In one embodiment, the TAP, nucleic acid, expression vector, T cell receptor, cells (e.g., T lymphocytes, APCs), and / or compositions described herein are administered / used in combination with inhibitors of CDK4 / 6, TGF-β, and / or WNT-β-catenin.Several CDK4 / 6 inhibitors, including Palbociclib (PD-0332991, Ibrance), Ribociclib (LEE-011, Kisqali), Abemaciclib (LY2835219, Verzenios), SHR6390, and Trilaciclib (G1T28), are in clinical trials. TGF-β inhibitors include antisense inhibitors such as AP12009 (Trabedersen) and ISTH0036, antibody and ligand traps such as GC1008 (Fresolimumab), LY2382770, and P144, vaccines targeting the TGF-β pathway such as Belagenpumatucel-L (Lucanix®), and small molecule inhibitors such as LY2157299 (Galunisertib) and TEW-7197. Inhibitors of the WNT-β-catenin pathway include amino acid starvation factors (asparaginase), GSK3 inhibitors, C2 (see Chemical Formula 1), WNT974, ETC-1922159, RXC004, CGX1321, OTSA101-DTPA-90Y, Vantictumab (OMP-18R5), Ipafricept (OMP-54F28), PRI-724, SM08502, secreted frizzled-related proteins / peptides, and tankyrase inhibitors (XAV939, JW-55, RK-287107, and G007-LK). [ka] In one embodiment, the TAP, SLP, nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, CAR T cells or NK cells, APCs), and / or compositions according to the Disclosure, or any combination thereof according to the Disclosure, are administered / used in combination with one or more chemotherapeutic agents used for the treatment of NSCLC, or in combination with other NSCLC therapies. Additional therapies may involve administering TAP, TAP nucleic acids, expression vectors, T cell receptors, antibodies / antibody fragments, cells (e.g., T lymphocytes, CAR T or NK cells, APCs), and / or compositions described in this disclosure before, concurrently with, or after administration. [Examples]

[0185] This disclosure is illustrated in further detail by the following non-limiting embodiments.

[0186] Example 1: Identification of lung cancer tumor antigens Sample Acquisition: The 26 rapidly cryopreserved NSCLC samples used in this study were purchased from Tissue Solutions, BioIVT, and Reprocell. The NSCLC subtypes included in this study were lung adenocarcinoma (n=14), squamous cell lung carcinoma (n=11), and large cell carcinoma (n=1) (Table 2). This project was approved by the Research Ethics Committee of the University of Montreal. 500 mg to 1.3 g per tumor were used for mass spectrometry, and 30 mg was used for RNA sequencing. [Table 3-1] [Table 3-2]

[0187] Immunoprecipitation of MHC I. W6 / 32 antibody (BioXcell) was incubated for 60 minutes at room temperature in PBS using PureProteome protein A magnetic beads (Millipore) at a ratio of 1 mg antibody per 1 mL slurry. The antibody was covalently crosslinked to the magnetic beads using the aforementioned dimethyl pimelidate. The beads were stored at 4°C in pH 7.2 PBS and 0.02% NaN3. Biological replicas of the cell pellet were resuspended in 1 mL of pH 7.2 PBS and solubilized by adding 1 mL of washing buffer containing pH 7.2 PBS and 1% (w / v) CHAPS (Sigma) supplemented with a protease inhibitor cocktail (Sigma). After incubation by tumbling at 4°C for 60 minutes, the sample was rotated at 16,000 g at 4°C for 30 minutes. 1 × 10 6The supernatant was transferred to a new tube containing magnetic beads conjugated with W6 / 32 antibody at a ratio of 10 μg of W6 / 32 antibody per cell. The sample was incubated by tumbling at 4°C for 180 minutes and placed on a magnet to recover the MHC I complexes conjugated to the magnetic beads. The magnetic beads were washed first with 8 × 1 mL of PBS, then with 1 × 1 mL of 0.1X PBS, and finally with 1 × 1 mL of water. The MHC I complexes were eluted from the magnetic beads by acid treatment with 0.2% formic acid (FA). To remove any remaining magnetic beads, the eluate was transferred to a 2 mL Costar mL Spin-X centrifuge filter (0.45 μm, Corning) and rotated at 855 g for 2 minutes. The peptide-containing filtrate was separated from the MHC I subunits (HLA molecules and β-2 macroglobulin) using a homemade stage tip packed with a 21 mm diameter octadecyl (C-18) solid-phase extraction disk (EMPORE). The stage tip was pre-washed first with methanol, then with 80% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA), and finally with 0.1% FA. The sample was loaded onto the stage tip, and the peptide was retained on the stage tip while HLA molecules and β-2 macroglobulin were found in the flow-through. The stage tip was washed with 0.1% FA, and the peptide was eluted in 30% ACN in 0.1% TFA. The peptide was dried using vacuum centrifugation and then stored at -20°C until MS analysis.

[0188] TMT labeling. In this study, one sample was TMT labeled (715-18T). The sample was reconstituted in 100 μL of 200 mM HEPES buffer (pH 8.2). The TMT reagent (Thermo Fisher Scientific) was dissolved in 40 μL of anhydrous ACN (Sigma-Aldrich) and added to the peptide. The solution was gently mixed and incubated at room temperature for 90 minutes without agitation, after which the reaction was stopped with hydroxylamine (Thermo Fisher Scientific). The sample was desalted using a silica C18 UltraMicroSpin column (The Nest Group), dried, and reconstituted in 4% FA (EMD Millipore).

[0189] Liquid chromatography-tandem MS analysis. Dried peptide extracts were resuspended in 4% FA and loaded onto a custom-made C18 analytical column (20 cm × 150 μm inner diameter, packed with C18 Jupiter Phenomenex) on an EASY-nLC II system with a 106-minute gradient of 0% to 30% ACN (0.2% FA) and a flow rate of 600 nL / min. Twelve samples were analyzed with a Q-Exactive HF and fourteen samples with an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). For the Q-Exactive HF, analysis was performed in positive ion mode at 1.6 kV using a Nanospray 2 source. Twenty MS / MS spectra followed each complete MS spectrum acquired at a resolution of 60,000, with the most abundant polyvalent ions being 2 × 10⁶ at a resolution of 30,000. 4An automated gain-controlled target, an 800 ms injection time, and a 25% collision energy were selected for MS / MS sequencing. Analysis was performed using an Orbitrap Exploris 480 mass spectrometer in positive ion mode at 2.8 kV with a Nanoflex source. Twenty MS / MS spectra followed each complete MS spectrum acquired at a resolution of 240,000, with the most abundant polyvalent ions selected for MS / MS sequencing with a resolution of 30,000, a 100% automated gain-controlled target, a 700 ms injection time, and a 40% collision energy.

[0190] Construction of an MS database. Cancer-specific proteomes were assembled using k-mer profiling as described above (1, 2). k-mers (33-nucleotide-length) that appeared multiple times in the mTEC k-mer database were removed from each cancer sample database, and the remaining k-mers were assembled into contigs. Finally, the contigs were translated using three frames, and different polypeptides were linked using a JJ linker. This database was linked to the standard proteome of the corresponding sample and used for TA identification.

[0191] MAP identification. LC-MS / MS data were searched for in relevant databases using Peaks X Pro (Bioinformatics Solution Inc.). For peptide identification, the tolerances for precursor and fragment ions were set to 10 ppm and 0.01 Da, respectively. Oxidation (M) and deamidation were set as variable modifications. After peptide identification, we applied a sample-specific threshold to the PEAKS score using the modified target-decoy approach built into PEAKS to ensure a 5% false discovery rate (FDR), calculated as the ratio between the number of decoy hits and the number of target hits exceeding the score threshold. A PEAKS score corresponding to a 5% FDR was determined for each sample, and peptides exceeding the threshold were further screened to meet the following criteria: a peptide length of 8–11 amino acids, and an eluting ligand likelihood prediction rank of less than 2% based on NetMHCpan-4.1b(3) for any one of the sample's HLA alleles. These filtering steps were performed using MAPDP(4).

[0192] Selection of TSAs and TAAs. NSCLC TA candidates were selected based on source RNA expression (FC≧10) in originating cancer samples and mTECs, as described above (1,2). Next, the expression of TA candidate coding sequences was evaluated using BamQuery(2,5) in normal GTEX tissues (n=50 samples per tissue, 50 tissues), bronchial brushing samples (n=82), mTECs (n=11), purified blood and bone marrow samples (n=115), and NSCLC samples from the TCGA cohort (n=385). The TAs listed herein are peptides that meet the following criteria: 1. Source RNAs for TSA and TAA are expressed at levels greater than twice the 95th percentile expression level of GTEx / mTEC samples in at least 5% of the samples from the TCGA_LUAD or TCGA_LUSC cohorts. 2. A) In the case of TSA: The source RNA of TSA is expressed at less than 8.55 reads per 100 million (rphm) in more than 90% of normal samples from each GTEx tissue except mTEC, blood and bone marrow cells and testes, and is overexpressed in TCGA (LUAD or LUSC) compared to normal (GTEx lung samples or bronchial brushing), with mean expression TCGA / normal ≥ 2. B) In the case of TAA: The source RNA of TAA can be expressed in more than 10% of samples from any normal tissue (GTEx, bronchial brushing, mTEC, and / or blood and bone marrow cells) at a read-to-read ratio (rphm) greater than 8.55 per 100 million, but is overexpressed in TCGA (LUAD or LUSC) compared to all normal tissues except the testes (mean expression TCGA / normal ≥ 2). C) In the case of LSA: The source RNA of LSA is expressed at an rphm of less than 8.55 in more than 90% of normal samples from mTECs, blood, and bone marrow cells, as well as from each GTEx tissue (excluding testicular and progenitor tumor tissue). In the case of NSCLC, LSA is expressed at an rphm of greater than 8.55 in at least 10% of GTEx lung or bronchial brushing samples, and the mean expression in these samples is higher than in all other GTEx tissues.

[0193] Single-cell RNA-seq analysis. Previously published single-cell RNA-seq data from NSCLC were downloaded from Array Express (accession number E-MTAB-6653). Read alignment to the human reference genome (GRCh38), filtering, barcode counting, and UMI counting were performed using the Cell Ranger counting function in version 6.1.2 with default parameters. Cell size coefficients were normalized using scran version 1.18.7, and read counts were log-normalized using scuttle with default parameters. Large-variance features were selected using scran based on mean-variance trends derived from a Poisson distribution of noise. Inter-batch sequencing depth adjustment and nearest-nearest-neighbor (MNN) correction were calculated using batchelor version 1.6.3. Cell clustering was performed using scran, with the Jaccard index used for edge weighting and the Louvain method for community detection. (Lambrechts et al.) 6 The gene list was used to annotate the cell population.

[0194] TA expression in scRNA-seq data. BamQuery 5 Using Lambrechts et al. 6 The expression of non-mutant TAs (in read counts) in single cells from NSCLC samples was quantified. For each TA type, the proportion of cancer cell-specific TAs (or LSAs specific to cancer cells and alveolar cells in NSCLC) was defined as the number of TAs specifically expressed in cancer cells (or cancer cells and alveolar cells of NSCLC-derived LSAs) (read count > 1) divided by the number of TAs expressed in at least one cell (read count > 1). Cell doublet analysis was performed for each sample using the computeDoubletDensity function from the scDblFinder package in R.

[0195] The characteristics of TSA and TAA identified in this specification are described in Tables 3A to 3D. 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 Table 4-15 Table 4-16 Table 4-17 Table 4-18 Table 4-19 Table 4-20 Table 4-21 Table 4-22 Table 4-23 Table 4-24 Table 4-25 Table 4-26 Table 4-27 Table 4-28 Table 4-29 Table 4-30 Table 4-31 Table 4-32 Table 4-33 Table 4-34 Table 4-35 Table 4-36 Table 4-37 Table 4-38 Table 4-39 Table 4-40 Table 4-41 Table 4-42 Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 5-9 [Table 5-10] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7]

[0196] Example 2: Evidence that aeTSA is a good candidate for immunotherapy A therapeutically attractive feature of non-mutant TA is its sharing among patients. In contrast to patient-specific mTSA, aeTSA from NSCLC was abundant and shared at the peptide and RNA levels (Figures 1A-1B). Nevertheless, TA showed expression patterns and regulation specific to TA type, cancer type, and cancer subtype. Specifically, aeTSA encoded by carcinoembryonic (or oncogenic germline) genes and TAA encoded by cell cycle genes were found to be abundant in TCGA's pulmonary squamous cell carcinoma (LUSC) samples, consistent with the increased stem cell characteristics of these tumors (Figures 1B and 3A). In contrast, NSCLC-derived LSAs were primarily derived from ROS1, extracellular matrix-related genes (i.e., COL6A5, ADAMTS12), or genes involved in surfactant production (i.e., SFTPA1), and were preferentially detected in the lung adenocarcinoma (LUAD) subtype, where the main originating cells are type 2 alveolar cells (Figures 1B and 3B).

[0197] Using TCGA multi-omics data, a significant correlation was found between TA expression and its corresponding source gene, suggesting gene-level regulation (Figures 1C-1D). aeTSA expression was often correlated with hypomethylation of the source gene promoter in NSCLCs. In LUSCs, CNVs contributed most significantly to TAA expression, while LSA was underexpressed in this subtype (Figures 1C-1D and 3B). In LUADs, TAA and LSA expression were associated with smoking status and TMB. Smoking history correlated with higher TMB, higher TAA (and aeTSA) expression, and lower LSA expression (Figures 1C-1D and 3C-3D), consistent with reports indicating increased TMB in LUAD samples with poor translocation and differentiation. Consequently, LUAD nonsmokers had a higher number of LSAs expressed at the RNA level, which was often correlated with increased CNVs and hypomethylation of the source gene promoter (Figures 1D and 3E). In summary, these results, consistent with the role of MAP in reflecting the internal cellular state, indicate that TA expression reflects cancer cell programs that are shared among patients and regulated at least partially mutational and / or epigenetic levels. The association with cancer (sub)type, smoking history, and degree of dedifferentiation suggests that prioritizing non-mutant TA is possible and likely important for effective treatment.

[0198] MAPs obtained from MHC I immunoprecipitation of bulk tumor lysates contain tumor-infiltrating immune cells and the tumor microenvironment. 8 Peptides from other stromal cells within the cell are "contaminating" it. Therefore, the published NSCLC 6Using a single-cell RNA-seq (scRNA-seq) dataset, we aimed to verify that non-mutant TA expression is associated with malignant cells (or LSA originating cell lineages) (Figures 4A-4B). TA was found to be highly and primarily expressed by NSCLC cancer cells (Figures 5A-5B). Most of the detected aeTSA was expressed only in cancer cells (88% in NSCLC), while most NSCLC LSAs were specific to cancer cells and alveolar cells (Figure 2). Therefore, aeTSA is specific to cancer cells, and its detection in other cell populations was attributed to technical limitations in single-cell sample preparation.

[0199] Overall, the results presented in this study strongly support the immunotargeting of non-mutant TAs across cancers with varying TMB levels. aeTSA is a particularly attractive target for immunotherapy given its cancer specificity, immunogenicity, high abundance, and patient-to-patient sharing.

[0200] While the present invention has been described by the specific embodiments described above, it may be modified without departing from the spirit and essence of the invention as defined in the appended claims. In the claims, the term “including” is used as an open-ended term and is substantially synonymous with the expression “including, but not limited to.” The singular forms “a,” “an,” and “the” also include their corresponding plural forms unless the context clearly indicates a different meaning.

[0201] References 1.Laumont CM, Vincent K, Hesnard L, Audemard E, Bonneil E, Laverdure JP, et al.Noncoding regions are the main source of targetable tumor-specific antigens.Science Translational Medicine 2018;10:eaau5516. 2.Ehx G,Larouche J-D,Durette C,Laverdure J-P,Hesnard L,Vincent K,et al.Atypical acute myeloid leukemia-specific transcripts generate shared and immunogenic MHC class-I-associated epitopes.Immunity 2021;54:737-52.e10. 3.Reynisson B,Alvarez B,Paul S,Peters B,Nielsen M.NetMHCpan-4.1 and NetMHCIIpan-4.0:improved predictions of MHC antigen presentation by concur-rent motif deconvolution and integration of MS MHC eluted ligand data.Nucleic Acids Res 2020;48:W449-W54. 4.Courcelles M,Durette C,Daouda T,Laverdure JP,Vincent K,Lemieux S,et al.MAPDP:A Cloud-Based Computational Platform for Immunopeptidomics Analyses.J Proteome Res 2020;19:1873-81. 5.Ruiz Cuevas MV et al.BamQuery:a new proteogenomic tool to explore the immunopeptidome.BioRxiv,October 8,2022,doi:https: / / doi.org / 10.1101 / 2022.10.07.510944. 6.Lambrechts,D.et al.Phenotype molding of stromal cells in the lung tumor microenvironment.Nat.Med.24,1277-1289(2018). 7.Stein,M.K.et al.Tumor Mutational Burden Is Site Specific in Non-Small-Cell Lung Cancer and Is Highest in Lung Adenocarcinoma Brain Metastases.JCO Precis.Oncol.3,1-13(2019). 8.Jaeger,A.M.et al.Deciphering the immunopeptidome in vivo reveals new tumour antigens.Nature 607,149-155(2022).

Claims

1. A tumor antigen peptide (TAP) comprising or consisting of one of the amino acid sequences described in any one of SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74.

2. The TAP or nucleic acid according to claim 1, wherein the TAP comprises or consists of one of the amino acid sequences of SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, 29-38, and 74.

3. The TAP or nucleic acid according to claim 2, wherein the TAP comprises or consists of one of the amino acid sequences of SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, 29-35, and 74.

4. The aforementioned TAP is HLA-A ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 01:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 14, 33, 40, 42-45, 48, or 60.

5. The aforementioned TAP is HLA-A ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 02:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 12, 39, 50, 53, 56, or 70.

6. The aforementioned TAP is HLA-A ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 02:03 molecule and comprises or consists of the sequence of SEQ ID NOs: 6, 12, 16, 39, 50, 53, 56, 61, or 70.

7. The aforementioned TAP is HLA-A ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 03:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 11, 14, 17, 19-22, 26, 29, 36, 40, 47-49, 51, 59, 63, 65, 67, or 68.

8. The aforementioned TAP is HLA-A ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to an 11:01 molecule and comprises or consists of the sequences of SEQ ID NOs: 11, 14, 17, 19-22, 26, 29, 36, 40, 44, 45, 47-49, 51, 59, 63, 65, 67, 68, or 74.

9. The aforementioned TAP is HLA-A ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 24:02 molecule and comprises or consists of the sequence of sequence numbers 12, 15, 18, 25, 35, 46, 54, or 58.

10. The aforementioned TAP is HLA-A ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 33:03 molecule and comprises or consists of the sequence of SEQ ID NOs: 5, 7, 34, 36, 63, 65, 67, or 68.

11. The aforementioned TAP is HLA-A ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 68:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 5, 7, 14, 26, 29, 36, 42, 44, 45, 47, 49, 51, 63, 65, 67, 68, or 71.

12. The TAP binds to the HLA-B ＊ 07:02 molecule and comprises or consists of a sequence of SEQ ID NO: 13, 16, 23, 24, 27, 28, 32, 38, 42, 52, 60, 61, 66 or 73, the TAP or nucleic acid according to any one of claims 1 to 3.

13. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 07:05 molecule and comprises or consists of the sequence of SEQ ID NOs: 13, 16, 23, 24, 27, 28, 32, 38, 42, 52, 60, 61, 66, or 73.

14. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to the 08:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 15, 16, 23, 24, 27, 31, 37-39, 42, 45, 50, 52, 54-57, 61, 66, 70, or 73.

15. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 15:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 12, 16, 39, 40, 42-46, 48, 58, 60-62, 72, or 203.

16. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 15:25 molecule and comprises or consists of the sequence of SEQ ID NOs: 12, 16, 38-40, 42, 44-46, 48, 58, 60, 61, or 62.

17. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a molecule and comprises or consists of the sequence of SEQ ID NOs: 1, 2, 9, 10, 13, 30, 33, 35, 41-46, 54, 55, 58, 60, 64, 69, 72, or 73.

18. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 27:06 molecule and comprises or consists of the sequence of SEQ ID NOs: 8, 12, 15, 25, 46, 54, 55, 58, 61, 64, 66, or 70.

19. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 35:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 13, 23, 24, 27, 35, 38, 42-45, 52, 54, 60, 66, or 73.

20. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 39:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 1, 8, 15, 25, 27, 38, 42, 45, 46, 52, 54, 55, 58, 61, 64, 70, or 73.

21. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 40:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 1-4, 9, 30, 33, 43, 46, 55, 58, 61, 64, 69, or 72.

22. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 44:02 molecule and comprises or consists of the sequence of SEQ ID NOs: 1-4, 9, 10, 33, 41-46, 55, 58, 61, 64, 69, or 72.

23. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 44:03 molecule and comprises or consists of the sequence of SEQ ID NOs: 1-4, 9, 10, 33, 41-46, 55, 58, 60, 62, 64, 69, or 72.

24. The aforementioned TAP is HLA-B ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 54:01 molecule and comprises or consists of the sequence of SEQ ID NOs: 24, 27, 32, 38, 42, 52, 60, or 73.

25. The aforementioned TAP is HLA-C ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 03:04 molecule and comprises or consists of the sequence of SEQ ID NOs: 12, 24, 27, 28, 38, 39, 42, 44, 45, 50, 52, 70, or 73.

26. The aforementioned TAP is HLA-C ＊ A TAP or nucleic acid according to any one of claims 1 to 3, which is bound to a 07:04 molecule and comprises or consists of the sequence of SEQ ID NOs: 8, 12, 15, 16, 24, 25, 27, 35, 38, 39, 42, 44, 45, 50, 52, 54, 55, 61, 64, 70, or 73.

27. The aforementioned TAP is HLA-C ＊ A TAP or nucleic acid according to any one of claims 1 to 3, comprising or consisting of the sequence of sequence numbers 12, 28, 31, 38, 39, 42, 44, 45, 61, or 70, bound to a 15:05 molecule.

28. A TAP or nucleic acid according to any one of claims 1 to 27, encoded by a sequence located in a non-protein coding region of the genome.

29. The TAP or nucleic acid according to claim 28, wherein the non-protein coding region of the genome is an intergenetic region.

30. The TAP or nucleic acid according to claim 28, wherein the non-protein coding region of the genome is a long non-coding RNA.

31. The TAP or nucleic acid according to claim 28, wherein the non-protein coding region of the genome is an intron.

32. A combination comprising at least two of the TAPs or nucleic acids defined in any one of claims 1 to 31.

33. A synthetic long-chain peptide (SLP) comprising at least one of the amino acid sequences defined in claim 1.

34. A nucleic acid encoding one or more of the TAPs described in any one of claims 1 to 31, the combination described in claim 32, or the SLPs described in claim 33.

35. The nucleic acid according to claim 34, wherein the nucleic acid is mRNA, and the mRNA optionally comprises one or more 5'-terminal modifications, 3'-terminal modifications, and / or modified nucleosides to improve the stability of the mRNA, improve translation, and / or reduce immunogenicity.

36. The nucleic acid according to claim 34, wherein the nucleic acid is a component of a viral vector.

37. A vesicle or particle comprising a TAP, combination, SLP, or nucleic acid according to any one of claims 1 to 36.

38. The vesicle or particle according to claim 37, wherein the vesicle is a lipid nanoparticle (LNP).

39. A vesicle or particle according to claim 37 or 38, comprising a cationic lipid.

40. A composition comprising a TAP, combination, SLP, or nucleic acid according to any one of claims 1 to 36, or a vesicle or particle according to any one of claims 37 to 39, and a pharmaceutically acceptable carrier.

41. A vaccine comprising a TAP, combination, SLP, or nucleic acid according to any one of claims 1 to 36, a vesicle or particle according to any one of claims 37 to 39, or a composition according to claim 40, and an adjuvant.

42. A molecule comprising an isolated major histocompatibility complex (MHC) class I molecule containing a TAP according to any one of claims 1 to 31 within its peptide bond groove.

43. An isolated MHC class I molecule according to claim 42, which is in the form of a polymer.

44. The isolated MHC class I molecule according to claim 43, wherein the polymer is a tetramer.

45. Isolated cells comprising (i) a TAP according to any one of claims 1 to 31, (ii) a combination according to claim 32, (iii) an SLP according to claim 33, (iv) a nucleic acid according to any one of claims 34 to 36, or (v) a vector comprising a nucleic acid according to any one of claims 34 to 36.

46. An isolated cell expressing, on its surface, a major histocompatibility complex (MHC) class I molecule containing the TAP or combination described in any one of claims 1 to 31 within a peptide bond groove.

47. The cell according to claim 45 or 46, which is an antigen-presenting cell (APC).

48. The cell according to claim 47, wherein the APC is a dendritic cell.

49. A T cell receptor (TCR) that specifically recognizes an isolated MHC class I molecule according to any one of claims 42 to 44, and / or an MHC class I molecule expressed on the surface of a cell according to any one of claims 46 to 48.

50. The TCR according to claim 49, which is a soluble TCR.

51. An antibody or its antigen-binding fragment that specifically binds to an isolated MHC class I molecule according to any one of claims 42 to 44, and / or an MHC class I molecule expressed on the surface of a cell according to any one of claims 46 to 48.

52. A TCR according to claim 49 or 50, or an antibody or antigen-binding fragment according to claim 51, which is a bispecific TCR or a bispecific antibody or an antigen-binding fragment thereof.

53. The TCR according to claim 52, or an antibody or its antigen-binding fragment, wherein the bispecific antibody or its antigen-binding fragment is a single-chain diabody (scDb).

54. The TCR, antibody, or antigen-binding fragment according to claim 52 or 53, wherein the bispecific TCR, or the bispecific antibody or its antigen-binding fragment, also specifically binds to T cell signaling molecules.

55. The TCR according to claim 54, or an antibody or its antigen-binding fragment, wherein the T cell signaling molecule is a CD3 chain.

56. A chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof according to any one of claims 51 to 55, or a nucleic acid encoding the CAR.

57. An isolated cell expressing the TCR described in claim 49 or the CAR described in claim 56 on its cell surface.

58. CD8 ＋ The isolated cells according to claim 57, which are T lymphocytes.

59. A cell population comprising at least 0.5% of isolated cells as defined in claim 57 or 58.

60. A method for treating cancer in a subject, wherein an effective amount of the subject (a) A TAP or any thereof containing or consisting of any one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-35 A combination of the above, or a synthetic long-chain peptide (SLP) comprising at least one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 176, 1-14, 16-21, 23-27, and 29-35, (b) At least one nucleic acid that codes for the TAP, combination thereof, or SLP as defined in (a), (c) A vesicle or particle containing the TAP as defined in (a), a combination thereof, or an SLP, or the at least one nucleic acid as defined in (b), (d) A composition comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), or the vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), the vesicles or particles as defined in (c), or the composition as defined in (d), and an adjuvant. (f) Cells expressing a major histocompatibility complex (MHC) class I molecule on its surface that contains the TAP or a combination thereof as defined in (a) within a peptide bond groove, (g) A cell which expresses a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes the MHC class I molecule expressed on the surface of the cell as defined in (f), or A method comprising administering a soluble TCR, or an antibody or its antigen-binding fragment, that specifically binds to the MHC class I molecule expressed on the surface of the cell as defined in (h)(f).

61. The method according to claim 60, wherein the cancer is lung cancer.

62. The method according to claim 61, wherein the lung cancer is non-small cell lung cancer (NSCLC).

63. The method according to claim 61 or 62, wherein the lung cancer is adenocarcinoma, large cell carcinoma, or squamous cell carcinoma.

64. The method according to any one of claims 60 to 63, further comprising administering at least one additional antitumor agent to the subject or performing at least one additional therapy.

65. The method according to claim 64, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery.

66. For the manufacture of pharmaceuticals for treating cancer in a subject, or for the manufacture of pharmaceuticals for treating cancer in a subject, (a) A TAP or any thereof containing or consisting of any one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-35 A combination of the above, or a synthetic long-chain peptide (SLP) comprising at least one of the sequences described in SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-74, preferably SEQ ID NOs: 15, 22, 28, 203, 1-14, 16-21, 23-27, and 29-38, more preferably SEQ ID NOs: 15, 22, 28, 176, 1-14, 16-21, 23-27, and 29-35, (b) At least one nucleic acid encoding the TAP, combination thereof, or SLP as defined in (a), (c) A vesicle or particle containing a TAP as defined in (a), a combination thereof, or an SLP, or the at least one nucleic acid as defined in (b), (d) A composition comprising a TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), or a vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), the vesicle or particle as defined in (c), or the composition as defined in (d), and an adjuvant. (f) Cells expressing a major histocompatibility complex (MHC) class I molecule on its surface that contains the TAP or a combination thereof as defined in (a) within a peptide bond groove, (g) A cell which expresses a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes the MHC class I molecule expressed on the surface of the cell as defined in (f), or A soluble TCR, antibody, or antigen-binding fragment thereof that specifically binds to the MHC class I molecule expressed on the surface of the cell as defined in (h) and (f). Use.

67. The use according to claim 66, wherein the cancer is lung cancer.

68. The use according to claim 67, wherein the lung cancer is non-small cell lung cancer (NSCLC).

69. The use according to claim 67 or 68, wherein the lung cancer is adenocarcinoma, large cell carcinoma, or squamous cell carcinoma.

70. The use according to any one of claims 66 to 69, further comprising the use of at least one additional antitumor agent or therapy to the subject.

71. The use according to claim 70, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery.

72. A drug for use in the treatment of cancer in the subject, wherein the drug is (a) A TAP comprising or consisting of any one of the sequences described in SEQ ID NOs: 1 to 74, preferably SEQ ID NOs: 1 to 38 and 74, more preferably SEQ ID NOs: 1 to 35 and 74, or any combination thereof, or a synthetic long-chain peptide (SLP) comprising at least one of the sequences described in SEQ ID NOs: 1 to 74, preferably SEQ ID NOs: 1 to 38 and 74, more preferably SEQ ID NOs: 1 to 35 and 74, (b) At least one nucleic acid that codes for the TAP, combination thereof, or SLP as defined in (a), (c) A vesicle or particle containing the TAP as defined in (a), a combination thereof, or an SLP, or the at least one nucleic acid as defined in (b), (d) A composition comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), or the vesicle or particle as defined in (c), and a pharmaceutically acceptable carrier. (e) A vaccine comprising the TAP as defined in (a), a combination thereof or SLP, the at least one nucleic acid as defined in (b), the vesicles or particles as defined in (c), or the composition as defined in (d), and an adjuvant. (f) Cells expressing a major histocompatibility complex (MHC) class I molecule on its surface that contains the TAP or a combination thereof as defined in (a) within a peptide bond groove, (g) A cell which expresses a T cell receptor (TCR) or chimeric antigen receptor (CAR) on its cell surface that specifically recognizes the MHC class I molecule expressed on the surface of the cell as defined in (f), or A drug that is a soluble TCR, or an antibody or its antigen-binding fragment, that specifically binds to the MHC class I molecule expressed on the surface of the cell as defined in (h) and (f).

73. The drug for use according to claim 72, wherein the cancer is lung cancer.

74. The agent for use according to claim 73, wherein the lung cancer is non-small cell lung cancer (NSCLC).

75. The agent for use according to claim 73 or 74, wherein the lung cancer is adenocarcinoma, large cell carcinoma, or squamous cell carcinoma.

76. The agent for use according to any one of claims 72 to 75, further comprising the use of at least one additional antitumor agent or therapy on the subject.

77. The agent for use according to claim 76, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiotherapy, or surgery.

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