RAS MUTANT EPITOPE PEPTIDE AND T CELL RECEPTOR RECOGNIZING RAS MUTANT

Abstract

The present invention relates to the field of immunology and tumor treatment. Specifically, an Ras G12V mutant epitope peptide, an antigen presenting cell expressing the epitope peptide, a tumor vaccine containing same, and a use of the tumor vaccine in preventing or treating a tumor having RAS G12V mutation. The present invention further relates to a T cell receptor (TCR) specifically recognizing an Ras G12V mutant, a conjugate and a fusion protein containing the TCR, an immune cell expressing the TCR, a T cell drug containing same, and a use of the T cell drug in preventing or treating a tumor having RAS G12V mutation.

Claims

1. An isolated epitope peptide or variant thereof, wherein the epitope peptide consists of 11-30 (e.g., 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11) consecutive amino acid residues of RAS G12V mutant, and comprises amino acid residues at positions 3-13 of the RAS G12V mutant; the variant differs from the epitope peptide from which it is derived only in a substitution of one or several (e.g., 1, 2 or 3) amino acid residues, and does not comprise an amino acid substitution at positions corresponding to amino acid positions 4, 5, 6, 9 and 12 of the RAS G12V mutant, and retains a biological function of the epitope peptide from which it is derived; preferably, the epitope peptide consists of 11-25 (e.g., 11-23, 11-13) consecutive amino acid residues of the RAS G12V mutant.

2. The epitope peptide or variant thereof according to claim 1, wherein the epitope peptide or variant thereof is capable of being presented by a MHC-II molecule, and the epitope peptide or variant thereof associated with the MHC-II molecule is capable of being recognized by a T cell, for example recognized by an antigen-specific T cell receptor on the T cell; preferably, the MHC-II molecule is HLA-DP; preferably, the HLA-DP comprises HLA-DPB1*03:01 and/or HLA-DPB1*14:01; preferably, the HLA-DP further comprises one selected from the group consisting of HLA-DPA1*02:02, HLA-DPA1*02:01, HLA-DPA1*01:03.

3. The epitope peptide or variant thereof according to claim 1 or 2, wherein the amino acid residues at positions 3-13 of the RAS G12V mutant have a sequence as set forth in SEQ ID NO: 2.

4. The epitope peptide or variant thereof according to any one of claims 1-3, wherein the epitope peptide comprises amino acid residues at positions 3-13, amino acid residues at positions 2-13, amino acid residues at positions 3-14 or amino acid residues at positions 2-14 of the RAS G12V mutant; preferably, the amino acid residues at positions 3-13, the amino acid residues at positions 2-13, the amino acid residues at positions 3-14 or the amino acid residues at positions 2-14 of the RAS G12V mutant have a sequence as set forth in SEQ ID NOs: 2-5, respectively.

5. The epitope peptide or variant thereof according to claims 1-4, wherein the RAS G12V mutant has a sequence as set forth in SEQ ID NO: 1.

6. The epitope peptide or variant thereof according to any one of claims 1-5, wherein the epitope peptide comprises a sequence set forth in any one of SEQ ID NOs: 2-5; the variant comprises a sequence selected from the group consisting of: (i) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) compared to the sequence set forth in any one of SEQ ID NOs: 2-5; (ii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared to the sequence set forth in any one of SEQ ID NOs: 2-5.

7. An isolated T cell receptor or antigen-binding fragment thereof, which is capable of specifically recognizing the epitope peptide or variant thereof according to any one of claims 1-6; preferably, the epitope peptide or variant thereof is presented by a MHC-II molecule; preferably, the MHC-II molecule is HLA-DP; preferably, the HLA-DP comprises HLA-DPB1*03:01 and/or HLA-DPB1*14:01; preferably, the HLA-DP further comprises one selected from the group consisting of HLA-DPA1*02:02, HLA-DPA1*02:01, HLA-DPA1*01:03; preferably, the TCR is soluble or membrane-bound; preferably, the TCR is a full-length TCR, a soluble TCR or a single-chain TCR.

8. An isolated T cell receptor (TCR) or antigen-binding fragment thereof, which is capable of specifically recognizing RAS G12V mutant, wherein the TCR or antigen-binding fragment thereof comprises an -chain variable region (V) and/or a -chain variable region (V), wherein, (a) the V comprises CDR1, CDR2 and CDR3a, wherein the CDR3a comprises a sequence as set forth in AVRDX.sub.1X.sub.2X.sub.3GGNKLT (SEQ ID NO: 25); wherein: X.sub.1 is selected from the group consisting of G, A, D, L, M, Q, R, S, V, W, Y; preferably G, A, D or R; X.sub.2 is selected from the group consisting of R, A, D, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y; preferably R or T; X.sub.3 is selected from the group consisting of G, A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; preferably G, A, H, N, S or W; and/or, (b) the V comprises CDR1, CDR2 and CDR3, wherein the CDR3 comprises a sequence as set forth in ASSX.sub.4GX.sub.5RDNSPLH (SEQ ID NO: 26); wherein: X.sub.4 is selected from the group consisting of P, A, H, M, S, T, V, Y; preferably P, A or T; X.sub.5 is selected from the group consisting of Q or S; preferably, the TCR is soluble or membrane-bound; preferably, the TCR is a full-length TCR, a soluble TCR or a single-chain TCR.

9. The TCR or antigen-binding fragment thereof according to claim 8, which has one or more of the following characteristics: (i) the CDR1 comprises a sequence as set forth in VSGX.sub.6PY (SEQ ID NO: 27); wherein, X.sub.6 is selected from the group consisting of N, A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y (preferably N, E, P, Q, R or S); (ii) the CDR2 comprises a sequence as set forth in YX.sub.7X.sub.8GDNLV (SEQ ID NO: 28); wherein, X.sub.7 is selected from the group consisting of I, A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y (preferably I, D, E, F, G, H, N, P, R, W or Y); X.sub.8 is selected from the group consisting of T, A, D, E, H, I, L, N, Q, R, S, W, Y (preferably T, A, D, H, I, Q or R); (iii) the CDR1 comprises a sequence set forth in SEQ ID NO: 11; (iv) the CDR2 comprises a sequence as set forth in SX.sub.9X.sub.10VNX.sub.11 (SEQ ID NO: 29); wherein, X.sub.9 is selected from the group consisting of Q, A, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y (preferably Q, A, I, M, N, S, T or Y); X.sub.10 is selected from the group consisting of I, F, H, T, V (preferably I or T); X.sub.11 selected from the group consisting of D, A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y (preferably D, G, L, M, N, T or Y).

10. The TCR or antigen-binding fragment thereof according to claim 8 or 9, wherein, (a) the V comprises FR1, FR2, FR3 and FR4, wherein: the FR1 comprises a sequence set forth in SEQ ID NO: 30; the FR2 comprises a sequence as set forth in LFWYVQYPNRGLQFLLX.sub.12 (SEQ ID NO: 38); wherein, X.sub.12 is selected from the group consisting of K, A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y (preferably K, G, H, N, P, R, S, V, W or Y); the FR3 comprises a sequence set forth in SEQ ID NO: 32; the FR4 comprises a sequence set forth in SEQ ID NO: 33; and/or, (b) the V comprises FR1, FR2, FR3 and FR4, wherein: the FR1 comprises a sequence set forth in SEQ ID NO: 34; the FR2 comprises a sequence as set forth in MYWYRQDPGQGLRLIYX.sub.13 (SEQ ID NO: 39); wherein, X.sub.13 is selected from the group consisting of Y, A, E, F, I, K, L, M, N, P, Q, R, S, T, V, W (preferably Y, A, E, F or K); the FR3 comprises a sequence as set forth in FX.sub.14 KGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLC (SEQ ID NO: 40); wherein, X.sub.14 is selected from the group consisting of Q, A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y (preferably Q, A, D, E, F, H, L, R, T or W); the FR4 comprises a sequence set forth in SEQ ID NO: 37.

11. The TCR or antigen-binding fragment thereof according to any one of claims 8-10, wherein, (a) the V comprises a sequence set forth in SEQ ID NO: 6 or variant thereof, wherein the variant comprises an amino acid substitution at one or more (e.g., 1, 2, 3 or 4) amino acid positions selected from the group consisting of amino acid positions 30, 49, 51, 52, 96, 97, 98 as determined according to the IMGT TCR numbering system; and/or, (b) the V comprises a sequence set forth in SEQ ID NO: 7 or variant thereof, and the variant comprises an amino acid substitution at one or more (e.g., 1, 2, 3 or 4) amino acid positions selected from the group consisting of amino acid positions 48, 50, 51, 54, 56, 95, 97 as determined according to the IMGT TCR numbering system.

12. The TCR or antigen-binding fragment thereof according to claim 11, wherein the V comprises a variant of the sequence set forth in SEQ ID NO: 6, the variant comprises one or more (e.g., 1, 2, 3 or 4) amino acid substitutions selected from the followings, in which the amino acid position is determined according to the IMGT TCR numbering system: substitution of the amino acid at position 30 with A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y (preferably E, P, Q, R or S); substitution of the amino acid at position 49 with A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y (preferably G, H, N, P, R, S, V, W or Y); substitution of the amino acid at position 51 with A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y (preferably D, E, F, G, H, N, P, R, W or Y); substitution of the amino acid at position 52 with A, D, E, H, I, L, N, Q, R, S, W or Y (preferably A, D, H, I, Q or R); substitution of the amino acid at position 96 with A, D, L, M, Q, R, S, V, W or Y (preferably A, D or R); substitution of the amino acid at position 97 with A, D, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y (preferably T); substitution of the amino acid at position 98 with A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y (preferably A, H, N, S or W); preferably, the V comprises a sequence set forth in SEQ ID NO:7.

13. The TCR or antigen-binding fragment thereof according to claim 11 or 12, wherein the V comprises a variant of the sequence set forth in SEQ ID NO: 7, the variant comprises one or more (e.g., 1, 2, 3 or 4) amino acid substitutions selected from the followings, in which the amino acid position is determined according to the IMGT TCR numbering system: substitution of the amino acid at position 48 with A, E, F, I, K, L, M, N, P, Q, R, S, T, V or W (preferably A, E, F or K); substitution of the amino acid at position 50 with A, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y (preferably A, I, M, N, S, T or Y); substitution of the amino acid at position 51 with F, H, T or V (preferably T); substitution of the amino acid at position 54 with A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y (preferably G, L, M, N, T or Y); substitution of the amino acid at position 56 with A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y (preferably A, D, E, F, H, L, R, T or W); substitution of the amino acid at position 95 with A, H, M, S, T, V or Y (preferably A or T); substitution of the amino acid at position 97 with S; preferably, the V comprises a sequence set forth in SEQ ID NO:6.

14. The TCR or antigen-binding fragment thereof according to any one of claims 8-13, wherein the TCR or antigen-binding fragment thereof is capable of specifically recognizing the epitope peptide or variant thereof according to any one of claims 1-6; preferably, the epitope peptide or variant thereof is presented by a MHC-II molecule; preferably, the MHC-II molecule is HLA-DP; preferably, the HLA-DP comprises HLA-DPB1*03:01 and/or HLA-DPB1*14:01; preferably, the HLA-DP further comprises one selected from the group consisting of HLA-DPA1*02:02, HLA-DPA1*02:01, HLA-DPA1*01:03; preferably, a T cell expressing on its surface the TCR or antigen-binding fragment thereof is activated under co-cultivation with a second cell (e.g., APC) displaying the epitope peptide or variant thereof according to any one of claims 1-6.

15. A conjugate, which comprises the TCR or antigen-binding fragment thereof according to any one of claims 7-14 and an effector moiety conjugated thereto; preferably, the TCR or antigen-binding fragment thereof is soluble.

16. A fusion protein, which comprises the TCR or antigen-binding fragment thereof according to any one of claims 7-14 and an additional peptide or protein; preferably, the TCR or antigen-binding fragment thereof is soluble.

17. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the epitope peptide or variant thereof according to any one of claims 1-6, or comprises a nucleotide sequence encoding the TCR or antigen-binding fragment thereof according to any one of claims 7-14 or its -chain variable region and/or -chain variable region, or comprises a nucleotide sequence encoding the fusion protein according to claim 16.

18. A vector, which comprises the isolated nucleic acid molecule according to claim 17; preferably, the vector comprises a nucleotide sequence encoding the epitope peptide or variant thereof according to any one of claims 1-6; preferably, the vector comprises a nucleotide sequence encoding the TCR or antigen-binding fragment thereof according to any one of claims 7-14 or its -chain variable region and/or -chain variable region; preferably, the vector comprises a nucleotide sequence encoding the fusion protein according to claim 16; preferably, the vector is a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a baculoviral vector.

19. A host cell, which comprises the isolated nucleic acid molecule according to claim 17, or the vector according to claim 18; preferably, the host cell comprises a nucleotide sequence encoding the epitope peptide or variant thereof according to any one of claims 1-6; preferably, the host cell comprises a nucleotide sequence encoding the TCR or antigen-binding fragment thereof according to any one of claims 7-14 or its -chain variable region and/or -chain variable region; preferably, the host cell comprises a nucleotide sequence encoding the fusion protein according to claim 16; preferably, the host cell comprises Escherichia coli, yeast, insect cell, or mammalian cell.

20. A method for preparing the epitope peptide or variant thereof according to any one of claims 1-6, or the TCR or antigen-binding fragment thereof according to any one of claims 7-14, or the fusion protein according to claim 16, which comprises culturing the host cell according to claim 19 under conditions that allow protein expression, and recovering the epitope peptide or variant thereof, or the TCR or antigen-binding fragment thereof, or the fusion protein from a culture of the cultured host cell.

21. An engineered antigen-presenting cell (APC), presenting on its surface the epitope peptide or variant thereof according to any one of claims 1-6; preferably, the epitope peptide or variant thereof is presented by a MHC-II molecule; preferably, the MHC-II molecule is HLA-DP; preferably, the HLA-DP comprises HLA-DPB1*03:01 and/or HLA-DPB1*14:01; preferably, the HLA-DP further comprises one selected from the group consisting of HLA-DPA1*02:02, HLA-DPA1*02:01, HLA-DPA1*01:03; preferably, the APC is selected from the group consisting of dendritic cell, monocyte, macrophage, B lymphocyte (e.g., B-lymphoblastoid cell B-LCL), or any combination thereof; preferably, the APC is positive for HLA-DPB1*03:01 or positive for HLA-DPB1*14:01; preferably, the APC is further positive for HLA-DPA1*02:02, positive for HLA-DPA1*02:01, or positive for HLA-DPA1*01:03; preferably, the APC is isolated from a subject positive for HLA-DPB1*03:01 or a subject positive for HLA-DPB1*14:01; preferably, the subject is further positive for HLA-DPA1*02:02, positive for HLA-DPA1*02:01, or positive for HLA-DPA1*01:03.

22. A method for preparing the engineered APC according to claim 21, which comprises: (1) providing an APC from a subject; (2) contacting the APC with the epitope peptide or variant thereof according to any one of claims 1-6 in vitro or introducing an expression vector comprising a nucleotide sequence encoding the epitope peptide or variant thereof according to any one of claims 1-6 into the APC, thereby obtaining an APC presenting on its surface the epitope peptide or variant thereof.

23. An engineered immune cell, expressing on its surface the TCR or antigen-binding fragment thereof according to any one of claims 7-14; preferably, the engineered immune cell comprises a nucleotide sequence encoding the TCR or antigen-binding fragment thereof according to any one of claims 7-14; preferably, the immune cell is a lymphocyte; preferably, the immune cell is selected from the group consisting of T cell (e.g., T cell, T cell or iPSC-derived T cell), tumor infiltrating lymphocyte (TIL), natural killer (NK) cell, natural killer T (NKT) cell, or any combination thereof.

24. A method for preparing the engineered immune cell according to claim 23, which comprises: (1) providing an immune cell from a subject; (2) introducing the isolated nucleic acid molecule according to claim 17 or the vector according to claim 18 into the immune cell of step (1), the nucleic acid molecule or vector comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof according to any one of claims 7-14, thereby obtaining an immune cell expressing the TCR or antigen-binding fragment thereof; preferably, in step (1), the immune cell undergoes pretreatment; the pretreatment comprises sorting, activation and/or proliferation of the immune cell; preferably, the pretreatment comprises contacting the immune cell with one or more selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, IL-2 and IL-15, to stimulate the immune cell and induce its proliferation, thereby producing a pretreated immune cell.

25. A pharmaceutical composition, which comprises the epitope peptide or variant thereof according to any one of claims 1-6, a nucleic acid molecule or carrier or host cell comprising a nucleotide sequence encoding the epitope peptide or variant thereof, or the engineered antigen-presenting cell (APC) according to claim 21; and a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition is a tumor vaccine; preferably, the pharmaceutical composition comprises an adjuvant; preferably, the pharmaceutical composition further comprises an additional therapeutic agent, such as an antitumor agent or an immunopotentiator; preferably, the antitumor agent is selected from the group consisting of alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, anti-angiogenic agent, cytokine, immune checkpoint inhibitor (e.g., PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, or TIM3 antibody); preferably, the immunopotentiator is selected from the group consisting of immunostimulatory antibody (e.g., anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody or any combination thereof), or immunostimulatory cytokine (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-, IL-10, TGF-, GM-CSF, or any combination thereof).

26. A pharmaceutical composition, which comprises the TCR or antigen-binding fragment thereof according to any one of claims 7-14, the conjugate according to claim 15, the fusion protein according to claim 16, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or the fusion protein, or the engineered immune cell according to claim 23; and a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition further comprises an additional therapeutic agent, such as an antitumor agent or an immunopotentiator; preferably, the antitumor agent is selected from the group consisting of alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, anti-angiogenic agent, cytokine, immune checkpoint inhibitor (e.g., PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, or TIM3 antibody); preferably, the immunopotentiator is selected from the group consisting of immunostimulatory antibody (e.g., anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody or any combination thereof) or immunostimulatory cytokine (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-, IL-10, TGF-, GM-CSF, or any combination thereof).

27. Use of the epitope peptide or variant thereof according to any one of claims 1-6, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the epitope peptide or variant thereof, or the engineered antigen-presenting cell (APC) according to claim 21, or the pharmaceutical composition according to claim 25, in the manufacture of a medicament, wherein the medicament is used for inducing an immune response against a tumor with RAS G12V mutation in a subject, and/or preventing or treating a tumor with RAS G12V mutation in a subject; preferably, the tumor with RAS G12V mutation is selected from the group consisting of colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast cancer, head and neck cancer, or acute myeloid leukemia; preferably, the subject is a human; preferably, the subject is positive for HLA-DPB1*03:01 or positive for HLA-DPB1*14:01; preferably, the subject is further positive for HLA-DPA1*02:02, positive for HLA-DPA1*02:01, or positive for HLA-DPA1*01:03; preferably, the epitope peptide or variant thereof, nucleic acid molecule or vector or host cell, engineered antigen presenting cell (APC), or pharmaceutical composition is administered in combination with an additional therapeutic agent, for example, administered simultaneously, separately or sequentially; preferably, the additional therapeutic agent is an immunostimulant or an antitumor agent.

28. Use of the TCR or antigen-binding fragment thereof according to any one of claims 7-14, the conjugate according to claim 15, the fusion protein according to claim 16, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or the fusion protein, or the engineered immune cell according to claim 23, or the pharmaceutical composition according to claim 26, in the manufacture of a medicament, wherein the medicament is used for inducing an immune response against a tumor with RAS G12V mutation in a subject, and/or preventing or treating a tumor with RAS G12V mutation in a subject; wherein the nucleic acid molecule, vector or host cell comprises a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or the fusion protein; preferably, the tumor with RAS G12V mutation is selected from the group consisting of colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast cancer, head and neck cancer, or acute myeloid leukemia; preferably, the subject is a human; preferably, the subject is positive for HLA-DPB1*03:01 or positive for HLA-DPB1*14:01; preferably, the subject is further positive for HLA-DPA1*02:02, positive for HLA-DPA1*02:01, or positive for HLA-DPA1*01:03; preferably, the TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule or vector or host cell, engineered immune cell, or pharmaceutical composition is administered in combination with an additional therapeutic agent, for example, administered simultaneously, separately or sequentially; preferably, the additional therapeutic agent is an immunostimulant or antitumor agent.

29. A method for inducing an immune response against a tumor with RAS G12V mutation in a subject, and/or preventing or treating a tumor with RAS G12V mutation in a subject, wherein the method comprises administering to the subject in need thereof an effective amount of the epitope peptide or variant thereof according to any one of claims 1-6, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the epitope peptide or variant thereof, or the engineered antigen-presenting cell (APC) according to claim 21, or the pharmaceutical composition according to claim 25; preferably, the tumor with RAS G12V mutation is selected from the group consisting of colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast cancer, head and neck cancer, or acute myeloid leukemia; preferably, the subject is a human; preferably, the subject is positive for HLA-DPB1*03:01 or positive for HLA-DPB1*14:01; preferably, the subject is further positive for HLA-DPA1*02:02, positive for HLA-DPA1*02:01, or positive for HLA-DPA1*01:03; preferably, the method further comprises administering to the subject an additional therapeutic agent, such as an immunopotentiator or an antitumor agent.

30. A method for inducing an immune response against a tumor with RAS G12V mutation in a subject, and/or preventing or treating a tumor with RAS G12V mutation in a subject, wherein the method comprises administering to the subject in need thereof an effective amount of the TCR or antigen-binding fragment thereof according to any one of claims 7-14, the conjugate according to claim 15, the fusion protein according to claim 16, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or the fusion protein, or the engineered immune cell according to claim 23, or the pharmaceutical composition according to claim 26; preferably, the tumor with RAS G12V mutation is selected from the group consisting of colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast cancer, head and neck cancer, or acute myeloid leukemia; preferably, the subject is a human; preferably, the subject is positive for HLA-DPB1*03:01 or positive for HLA-DPB1*14:01; preferably, the subject is further positive for HLA-DPA1*02:02, positive for HLA-DPA1*02:01, or positive for HLA-DPA1*01:03; preferably, the method further comprises administering to the subject an additional therapeutic agent, such as an immunopotentiator or an antitumor agent; preferably, the method comprises: (1) providing an immune cell required by the subject; (2) introducing a nucleotide sequence encoding the TCR or antigen-binding fragment thereof according to any one of claims 7-14 into the immune cell of step (1), thereby obtaining an immune cell expressing on its surface the TCR or antigen-binding fragment thereof; (3) administering the immune cell obtained in step (2) to the subject; preferably, the immune cell is a lymphocyte; preferably, the immune cell is selected from the group consisting of T cell (e.g., T cell, T cell or iPSC-derived T cell), tumor infiltrating lymphocyte (TIL), natural killer (NK) cell, natural killer T (NKT) cell, or any combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0206] FIG. 1 shows the results of IFN-specific release against different LCL clones in Example 5.

[0207] FIG. 2 shows the results of IFN release after co-culturing the antigen-presenting cells loaded with different peptides and B13.14.1 TCR-T cells in Example 6.

[0208] FIG. 3 shows the results of IFN release assay of B13.14.1 TCR-T induced by different G12V peptide segments in Example 7.

[0209] FIG. 4 shows the results of IFN release assay of B13.14.1 TCR-T induced by mutation-containing G12V peptide segment in Example 8.

[0210] FIG. 5 shows the results of selective killing of SW620-CIITA-DPA02:02/DPB03:01 cells by B13.14.1 TCR-T in Example 9.

[0211] FIGS. 6A to 6B show the results of affinity assay of B13.14.1 TCR-T to the RAS G12V mutant in Example 10.

[0212] FIG. 7 shows the results of in vivo efficacy assay of B13.14.1 TCR-T in Example 11.

[0213] FIG. 8 shows the determination results of the binding activity of TCR mutant (CDR3 region) to KRAS-G12V antigen peptide in Example 12.

[0214] FIG. 9 shows the determination results of the binding activity of TCR mutant (MHC-II binding region) to KRAS-G12V antigen peptide in Example 12.

SEQUENCE INFORMATION

[0215] Information on the partial sequences involved in the present invention is provided in the table below.

TABLE-US-00001 SEQ ID NO Description Sequenceinformation 1 RASG12Vmutantamino MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYR acidsequence KQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVF AINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRT VDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKH KEKMSKDGKKKKKKSKTKCVIM 2 RASG12Vmutantamino EYKLVVVGAVG acidsequenceaa3- 13/G12V-T9 3 RASG12Vmutantamino TEYKLVVVGAVG acidsequenceaa2- 13/G12V-T3 4 RASG12Vmutantamino EYKLVVVGAVGV acidsequenceaa3- 14/G12V-T16 5 RASG12Vmutantamino TEYKLVVVGAVGV acidsequenceaa2- 14/G12V-T15 6 B13.14.1TCRVamino MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVK acidsequence CTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGF EAEFNKSQTSFHLKKPSALVSDSALYFCAVRDGRGGGNKLT FGTGTQLKVEL 7 B13.14.1TCRVamino MSNQVLCCVVLCLLGANTVDGGITQSPKYLFRKEGQNVTLS acidsequence CEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAE GYSVSREKKESFPLTVTSAQKNPTAFYLCASSPGQRDNSPLH FGNGTRLTVT 8 B13.14.1TCRCDR1 VSGNPY aminoacidsequence 9 B13.14.1TCRCDR2 YITGDNLV aminoacidsequence 10 B13.14.1TCRCDR3 AVRDGRGGGNKLT aminoacidsequence 11 B13.14.1TCRCDR1 LNHDA aminoacidsequence 12 B13.14.1TCRCDR2 SQIVND aminoacidsequence 13 B13.14.1TCRCDR3 ASSPGQRDNSPLH aminoacidsequence 14 RASG12V-mRNA CTTGTTCTTTTTGCAGAAGCTCAGAATAAACGCTCAACTTT nucleotidesequence GGGCCACCATGCCCCGGCAGCTCAGCGCGGCGGCCGCGCT CTTCGCGTCCCTGGCCGTAATTTTGCACGATGGCAGTCAA ATGAGAGCAAAAGCATTTCCAGAAACCAGAGATTATTCTC AACCTACTGCAGCAGCAACAGTACAGGACATAAAAAAAC CTGTCCAGCAACCAGCTAAGCAAGCACCTCACCAAACTTT AGCAGCAAGATTCATGGATGGTCATATCACCTTTCAAACA GCGGCCACAGTAAAAATTCCAACAACTACCCCAGCGACTA CAAAAAACACTGCAACCACCAGCCCAATTACCTACACCCT GGTCACAACCCAGGCCACACCCAACAACTCACACACAGCT CCTCCAGTTACTGAAGTTACAGTCGGCCCTAGCTTAGCCC CTTATTCACTGCCACCCACCATCACCCCACCAGCTCATAC AACTGGAACCAGTTCATCAACCGTCAGCCACACAACTGGG AACACCACTCAACCCAGTAACCAGACCACCCTTCCAGCAA CTTTATCGATAGCACTGCACAAAAGCACAACCGGTCAGAA GCCTGTTCAACCCACCCATGCCCCAGGAACAACGGCAGCT GCCCACAATACCACCCGCACAGCTGCACCTGCCTCCACGG TTCCTGGGCCCACCCTTGCACCTCAGCCATCGTCAGTCAA GACTGGAATTTATCAGGTTCTAAACGGAAGCAGACTCTGT ATAAAAGCAGAGATGGGGATACAGCTGATTGTTCAAGAC AAGGAGTCGGTTTTTTCACCTCGGAGATACTTCAACATCG ACCCCAACGCAACGCAAGCCTCTGGGAACTGTGGCACCCG AAAATCCAACCTTCTGTTGAATTTTCAGGGCGGATTTGTG AATCTCACATTTACCAAGGATGAAGAATCATATTATATCA GTGAAGTGGGAGCCTATTTGACCGTCTCAGATCCAGAGAC AATTTACCAAGGAATCAAACATGCGGTGGTGATGTTCCAG ACAGCAGTCGGGCATTCCTTCAAGTGCGTGAGTGAACAGA GCCTCCAGTTGTCAGCCCACCTGCAGGTGAAAACAACCGA TGTCCAACTTCAAGCCTTTGATTTTGAAGATGACCACTTTG GAAATGTGGATGAGTGCTCGTCTGACTACACAACTGAATA TAAACTTGTGGTAGTTGGAGCTGTTGGCGTAGGCAAGAGT GCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACG AATATGATCCAACAATAGAGGATTCCTACAGGAAGCAAG TAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGA CACAGCAGGTCAAGAGGAGTACAGTGCAATGAGGGACCA GTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCC ATAAATAATACTAAATCATTTGAAGATATTCACCATTATA GAGAACAAATTAAAAGAGTTAAGGACTCTGAAGATGTAC CTATGGTCCTAGTAGGAAATAAATGTGATTTGCCTTCTAG AACAGTAGACACAAAACAGGCTCAGGACTTAGCAAGAAG TTATGGAATTCCTTTTATTGAAACATCAGCAAAGACAAGA CAGGGTGTTGATGATGCCTTCTATACATTAGTTCGAGAAA TTCGAAAACATAAAGAAAAGATGAGCAAAGATGGTAAAA AGAAGAAAAAGAAGTCAAAGACAAAGTGTGTAATTATGA TTGTGCTTCCTGTGATTGGGGCCATCGTGGTTGGTCTCTGC CTTATGGGTATGGGTGTCTATAAAATCCGCCTAAGGTGTC AATCATCTGGATACCAGAGAATCTAATCCTCTAGAGGATC TGGTTACCACTAAACCAGCCTCAAGAACACCCGAATGGAG TCTCTAAGCTACATAATACCAACTTACACTTACAAAATGT TGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAA AAAGAAAGTTTCTTCAC 15 B13.14.1TCRV ATGGCCTCTGCACCCATCTCGATGCTTGCGATGCTCTTCAC nucleotidesequence ATTGAGTGGGCTGAGAGCTCAGTCAGTGGCTCAGCCGGAA GATCAGGTCAACGTTGCTGAAGGGAATCCTCTGACTGTGA AATGCACCTATTCAGTCTCTGGAAACCCTTATCTTTTTTGG TATGTTCAATACCCCAACCGAGGCCTCCAGTTCCTTCTGA AATACATCACAGGGGATAACCTGGTTAAAGGCAGCTATG GCTTTGAAGCTGAATTTAACAAGAGCCAAACCTCCTTCCA CCTGAAGAAACCATCTGCCCTTGTGAGCGACTCCGCTTTG TACTTCTGTGCTGTGAGAGACGGTAGGGGAGGAGGAAAC AAACTCACCTTTGGGACAGGCACTCAGCTAAAAGTGGAAC TC 16 B13.14.1TCRV ATGAGCAACCAGGTGCTCTGCTGTGTGGTCCTTTGTCTCCT nucleotidesequence GGGAGCAAACACCGTGGATGGTGGAATCACTCAGTCCCC AAAGTACCTGTTCAGAAAGGAAGGACAGAATGTGACCCT GAGTTGTGAACAGAATTTGAACCACGATGCCATGTACTGG TACCGACAGGACCCAGGGCAAGGGCTGAGATTGATCTACT ACTCACAGATAGTAAATGACTTTCAGAAAGGAGATATAGC TGAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTT CCTCTCACTGTGACATCGGCCCAAAAGAACCCGACAGCTT TCTATCTCTGTGCCAGTAGCCCCGGACAACGAGATAATTC ACCCCTCCACTTTGGGAACGGGACCAGGCTCACTGTGACA 17 RASWT-T15 TEYKLVVVGAGGV 18 MurineTCRconstant DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTF region ITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATY aminoacidsequence PSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGF NLLMTLRLWSS 19 MurineTCRconstant GACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAG region ATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGA nucleotidesequence CTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCT GGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAG CTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAA CCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACC AACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCA CGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAA CTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGC TGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCT GTGGTCCAGT 20 MurineTCRconstant EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVE region LSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATF aminoacidsequence WHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWG RADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVM AMVKRKNS 21 MurineTCRconstant GAGGACCTGCGCAACGTCACCCCACCAAAGGTCAGTTTGT region TTGAGCCATCAAAGGCGGAGATCGCCAACAAACAGAAAG nucleotidesequence CTACGCTCGTGTGTTTGGCTCGGGGCTTCTTCCCAGACCAC GTAGAACTTTCCTGGTGGGTCAATGGAAAGGAGGTTCATT CCGGAGTGTCCACTGATCCCCAAGCGTACAAGGAATCCAA CTATAGCTACTGTCTCTCATCTCGGCTCCGGGTGAGTGCG ACATTCTGGCATAATCCTCGGAACCACTTTCGATGCCAAG TGCAGTTTCATGGGTTGAGCGAGGAAGACAAGTGGCCCG AGGGCAGTCCTAAACCAGTCACTCAAAACATAAGCGCCG AGGCATGGGGTAGAGCCGATTGTGGGATTACTAGCGCTTC ATACCAACAAGGGGTATTGAGCGCTACAATTCTTTACGAA ATTCTCCTCGGCAAGGCGACGCTCTACGCCGTACTGGTGT CTACTCTCGTGGTTATGGCAATGGTGAAACGGAAAAACAG C 22 P2Anucleotidesequence GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGG AGGAGAACCCCGGCCCC 23 B13.14.1/TRAV+mCa ATGGCCTCTGCACCCATCTCGATGCTTGCGATGCTCTTCAC nucleotidesequence ATTGAGTGGGCTGAGAGCTCAGTCAGTGGCTCAGCCGGAA GATCAGGTCAACGTTGCTGAAGGGAATCCTCTGACTGTGA AATGCACCTATTCAGTCTCTGGAAACCCTTATCTTTTTTGG TATGTTCAATACCCCAACCGAGGCCTCCAGTTCCTTCTGA AATACATCACAGGGGATAACCTGGTTAAAGGCAGCTATG GCTTTGAAGCTGAATTTAACAAGAGCCAAACCTCCTTCCA CCTGAAGAAACCATCTGCCCTTGTGAGCGACTCCGCTTTG TACTTCTGTGCTGTGAGAGACGGTAGGGGAGGAGGAAAC AAACTCACCTTTGGGACAGGCACTCAGCTAAAAGTGGAAC TCGACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAA AGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACC GACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAAT CTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAA AGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGC AACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGA CCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGC CACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTA AACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCC TGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAG GCTGTGGTCCAGT 24 B13.14.1/TRBV+mCb ATGAGCAACCAGGTGCTCTGCTGTGTGGTCCTTTGTCTCCT nucleotidesequence GGGAGCAAACACCGTGGATGGTGGAATCACTCAGTCCCC AAAGTACCTGTTCAGAAAGGAAGGACAGAATGTGACCCT GAGTTGTGAACAGAATTTGAACCACGATGCCATGTACTGG TACCGACAGGACCCAGGGCAAGGGCTGAGATTGATCTACT ACTCACAGATAGTAAATGACTTTCAGAAAGGAGATATAGC TGAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTT CCTCTCACTGTGACATCGGCCCAAAAGAACCCGACAGCTT TCTATCTCTGTGCCAGTAGCCCCGGACAACGAGATAATTC ACCCCTCCACTTTGGGAACGGGACCAGGCTCACTGTGACA GAGGACCTGCGCAACGTCACCCCACCAAAGGTCAGTTTGT TTGAGCCATCAAAGGCGGAGATCGCCAACAAACAGAAAG CTACGCTCGTGTGTTTGGCTCGGGGCTTCTTCCCAGACCAC GTAGAACTTTCCTGGTGGGTCAATGGAAAGGAGGTTCATT CCGGAGTGTCCACTGATCCCCAAGCGTACAAGGAATCCAA CTATAGCTACTGTCTCTCATCTCGGCTCCGGGTGAGTGCG ACATTCTGGCATAATCCTCGGAACCACTTTCGATGCCAAG TGCAGTTTCATGGGTTGAGCGAGGAAGACAAGTGGCCCG AGGGCAGTCCTAAACCAGTCACTCAAAACATAAGCGCCG AGGCATGGGGTAGAGCCGATTGTGGGATTACTAGCGCTTC ATACCAACAAGGGGTATTGAGCGCTACAATTCTTTACGAA ATTCTCCTCGGCAAGGCGACGCTCTACGCCGTACTGGTGT CTACTCTCGTGGTTATGGCAATGGTGAAACGGAAAAACAG C 25 GeneralformulaofTCR AVRDX.sub.1X.sub.2X.sub.3GGNKLT CDR3 26 GeneralformulaofTCR ASSX.sub.4GX.sub.5RDNSPLH CDR3 27 GeneralformulaofTCR VSGX.sub.6PY CDR1 28 GeneralformulaofTCR YX.sub.7X.sub.8GDNLV CDR2 29 TCRCDR2General SX.sub.9X.sub.10VNX.sub.11 formulaof 30 B13.14.1TCRFR1 MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVK CTYS 31 B13.14.1TCRFR2 LFWYVQYPNRGLQFLLK 32 B13.14.1TCRFR3 KGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFC 33 B13.14.1TCRFR4 FGTGTQLKVEL 34 B13.14.1TCRFRI MSNQVLCCVVLCLLGANTVDGGITQSPKYLFRKEGQNVTLS CEQN 35 B13.14.1TCRFR2 MYWYRQDPGQGLRLIYY 36 B13.14.1TCRFR3 FQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLC 37 B13.14.1TCRFR4 FGNGTRLTVT 38 GeneralformulaofTCR LFWYVQYPNRGLQFLLX.sub.12 FR2 39 GeneralformulaofTCR MYWYRQDPGQGLRLIYX.sub.13 FR2 40 GeneralformulaofTCR FX.sub.14KGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLC FR3 41 G12V-T1 TEYKLVVVGAVGVGKSALTIQLI 42 G12V-T2 AVGVGKSALTIQLI 43 G12V-T4 VVVGAVGVGKS 44 G12V-T5 VGAVGVGKSALTIQ 45 G12V-T6 YKLVVVGAVGVG 46 G12V-T7 VVVGAVGVGKSALT 47 G12V-T8 LVVVGAVGVGKS 48 G12V-T10 YKLVVVGAVG 49 G12V-T11 KLVVVGAVG 50 G12V-T12 TEYKLVVVGAV 51 G12V-T13 EYKLVVVGAV 52 G12V-T14 YKLVVVGAV 53 G12V-T17 YKLVVVGAVGV 54 G12V-T18 TEYKLVVVGAVGVG 55 G12V-T19 TEYKLVVVGAVGVGK 56 G12V-T20 TEYKLVVVGAVGVGKS 57 G12V-T21 TEYKLVVVGAVGVGKSA 58 G12V-T15-A1 AEYKLVVVGAVGV 59 G12V-T15-A2 TAYKLVVVGAVGV 60 G12V-T15-A3 TEAKLVVVGAVGV 61 G12V-T15-A4 TEYALVVVGAVGV 62 G12V-T15-A5 TEYKAVVVGAVGV 63 G12V-T15-A6 TEYKLAVVGAVGV 64 G12V-T15-A7 TEYKLVAVGAVGV 65 G12V-T15-A8 TEYKLVVAGAVGV 66 G12V-T15-A9 TEYKLVVVAAVGV 67 G12V-T15-G10 TEYKLVVVAGVGV 68 G12V-T15-A12 TEYKLVVVGAVAV 69 G12V-T15-A13 TEYKLVVVGAVGA

EXAMPLES

[0216] The present invention will now be described with reference to the following examples, which are intended to illustrate the present invention, but not to limit it.

[0217] Unless otherwise specified, the molecular biology experiment methods and immunoassay methods used in the present invention were basically referred to the methods described by J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the restriction enzymes were used in accordance with the conditions recommended by the product manufacturers. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the claimed invention.

Example 1: Culture and Expansion of Tumor-Infiltrating T Cell

[0218] The tumor samples obtained by resection surgery from patients with colorectal cancer with RAS mutation G12V were minced with scalpel to tumor pieces of 2 mm to 4 mm, washed twice with DPBS solution, and cultured in a TIL medium containing IL2 (6000 IU/ml), human AB serum (2%), Hepes (25 mM), and Xvivol5 in a 24-well plate. Half of the medium was replaced every 2 to 3 days, and when the tumor-infiltrating T lymphocytes (TIL) grew to 60-80% confluence (containing about 0.5-3.010.sup.6 TILs), the TILs were harvested and stored in CS10 cryoprotectant solution.

[0219] The TIL cells can be expanded by co-cultured with peripheral blood mononuclear cells derived from different donors (pooled PBMC, donor>3) irradiated with -rays (4000 cGy) at a ratio of 1:30 to 1:200, and cultured in a T175 culture flask with no more than 110.sup.8 cells per flask (the culture medium was TIL medium supplemented with 10 ng/ml OKT3). After 3 days of culture, half of the medium (Xvivol5 medium containing 3000 IU/ml IL2, 2% human AB serum) was replaced; after 7 days of culture, the cells were washed once, the medium was replaced and passaged at 1.010.sup.6/ml; the cells were harvested and cryopreserved after 10-14 days of culture.

Example 2: Preparation of RAS G12V Antigen mRNA

[0220] The vector UTR-LAMP3 Lumenal-KRAS.sup.G12V-LMP3 Sorting-UTR was constructed according to the following design for sequence containing RAS mutation G12V, the sequence was set forth in SEQ ID NO: 14 in Table 1, in which KRAS was marked with a single underline, and the G12V mutation was marked with a double underline. The above sequence was synthesized and cloned into the pcDNA3.1 vector, and the mRNA transcription was prepared in vitro using T7 promoter (mMESSAGE mMACHINE T7 Transcription Kit, Thermofisher), and the mRNA after transcription was sub-packaged and stored at 80 C.

Example 3: Screening by Antigen-Presenting Cell

[0221] Maturation of DC cell (Dendritic cell): The patient's autologous peripheral blood CD14-positive cells were isolated with MACS CD14 Isolation Kit, and cultured in AIM-V medium containing IL4 (1000 IU/ml), GM-CSF (1000 IU/ml) and 1% human AB serum. The culture medium was replaced with fresh medium on the third day, and the cells were cryopreserved in CS10 cryoprotectant solution after culturing for 5-6 days.

[0222] Induction and generation of LCL cell (Lymphoblastoid cell line): The patient's peripheral blood mononuclear cells in an amount of 510.sup.6 were resuspended in RPMI1640 medium containing 10% fetal bovine serum, and added with B95.8 supernatant of cell culture which contains EBV for induction. The induction was generally completed within 14 to 30 days, and half of the medium was replaced every 7 days during the induction period, and the established LCL cell line after induction was expanded and cryopreserved.

[0223] After recovery, the TIL cells were cultured for at least 48 hours for screening. The DC cells or LCL cells, referred to as APC (antigen-presenting cell), were subjected to transfection of KRAS G12V mRNA using a Neon electroporator, in which APC cells were resuspended in the electroporation solution to 110.sup.7/ml, 100 l of the cells was added with 5-8 g mRNA for each electroporation (1500V, 30 ms, 1 pulse). Transfected APCs can be used next day after culturing. 1 to 210.sup.5 of TIL cells and 0.510.sup.5 of electroporated DC cells or 410.sup.5 of electroporated LCL cells were cultured in Xvivol5 medium in a 96-well U-bottom plate; after 16 hours of culture, the cell culture supernatant was collected, and the IFN release in the supernatant was determined using Human IFN Flex Set. The patient numbered as B13 had metastatic colorectal cancer which had KRAS.sup.G12V mutation. The TIL screening results were as follows:

TABLE-US-00002 TABLE 1 TIL screening results TIL Fraction IFN-Ctrl IFN-G12V Relative change B13.1 579 1937 3.3 B13.2 1115 1106 1.0 B13.3 2340 3551 1.5 B13.4 3189 20387 6.39 B13.5 588 744 1.3 B13.8 20304 34676 1.7 B13.10 815 812 1.0 B13.11 1846 3403 1.8 B13.12 1313 2452 1.8 B13.13 676 980 1.4 B13.14 811 3899 4.8 B13.15 7369 18518 2.5 B13.16 8181 788 10.3 [0224] Ctrl: TIL co-cultured with non-electroporated APC [0225] G12V: TIL co-cultured with APC electroporated with G12V mRNA

Example 4: Sorting and Expansion of TIL

[0226] The TIL cells stimulated by APC cells were sorted by flow cytometry. 110.sup.6 TIL cells were resuspended in flow buffer (DPBS solution containing 1% human AB serum, 2 mM EDTA), added with CD3/CD137 antibody and PI (propidium iodide solution), incubated at 4 C. for 1 hour, washed twice with flow buffer, and sorted with BD FACSAiraII flow sorter. The sorted population was a cell population negative for PI, positive for CD3 and positive for CD137. The sorted cells were preserved in RPMI1640 medium containing 10% human AB serum, and placed on ice. The collected sorted cells (CD3 and CD137+) were centrifuged at 300 g for 10 minutes at 4 C. to remove 80% of the preservation solution, washed twice with DPBS solution, resuspended in DPBS, and subjected to 10 Genomics single-cell sequencing.

Example 5: Screening for TCR Recognizing KRAS.SUP.G12V

[0227] The TCR clone obtained by the single-cell sequencing was subjected to gene-synthesis according to the sequence of TRAVmCa-P2A-TRBVmCb, wherein TRAV was the -chain variable region of TCR, mCa was the murine TCR constant region (its amino acid sequence and nucleotide sequence were set forth in SEQ ID NO: 18 and 19, respectively), TRBV was the -chain variable region of TCR, mCb was the murine TCR constant region (its amino acid sequence and nucleotide sequence were set forth in SEQ ID NO: 20 and 21, respectively), and P2A was the self-cleaving peptide (its nucleotide sequence was set forth in SEQ ID NO: 22); the above sequence was cloned into a lentiviral transfer vector (GV401); the transfer and package vectors were transiently transfected into 293T cells according to the standard lentiviral vector packaging method, the culture supernatant was collected which contained the lentiviral vector expressing TCR. T cells from a healthy donor were activated in OKT3/15E8 antibody-coated 6-well plate for 24 hours, then transduced with the TCR-containing lentiviral vector and cultured for 6 to 8 days for TCR screening (the transduced T cells were collected, washed with FACS buffer solution, and 110.sup.6 modified T cells were stained by using an antibody recognizing murine TCR constant region to detect the expression of recombinant TCR), thereby obtaining T cells modified by recombinant TCR.

[0228] The autologous LCL cells of Patient No. B13 were transiently transfected with KRAS.sup.G12V mRNA and cultured overnight according to the electroporation method in Example 3; the T cells modified by recombinant TCR and the electroporated LCL cells at a ratio of 110.sup.5:110.sup.5 were inoculated and co-cultured in a 96-well U-bottom plate, the IFN specific release in the supernatant was detected, and the results were shown in FIG. 1. The screening results showed that the TCR with clone number B13.14.1 could specifically recognize the KRAS G12V point mutation but not wild-type RAS.

[0229] The sequence of B13.14.1 TCR was shown in the table below:

TABLE-US-00003 TABLE 2 TCR sequences B13.14.1-TCR V region B13.14.1-TCR V region TRAV3*01-J10*01 TRBV19*01-D1*01-J1-6*02 Variable SEQ ID NO: 6 SEQ ID NO: 7 region CDR1 SEQ ID NO: 8 SEQ ID NO: 11 CDR2 SEQ ID NO: 9 SEQ ID NO: 12 CDR3 SEQ ID NO: 10 SEQ ID NO: 13

Example 6: HLA Restriction Determination of B13.14.1 TCR

[0230] 6.1 HLA Restriction Determination 1

[0231] T cells expressing recombinant B13.14.1 TCR (hereinafter referred to as B13.14.1 TCR-T) were prepared according to the method described in Example 5, which comprises transducing T cells from a healthy donor with a lentiviral vector containing a nucleotide sequence encoding the recombinant TCR, wherein the nucleotide sequences of the -chain (TRAVmCa) and -chain (TRBVmCb) of the recombinant TCR were set forth in SEQ ID NO: 23 and 24, respectively. The HLA restriction of the B13.14.1 TCR was determined by the method described below.

[0232] SW620 cell line (colorectal cancer lymph-node metastasis) contained KRAS G12V homozygous mutation; CFPAC-1 cell line (pancreatic cancer) contained KRAS G12V heterozygous mutation; and their HLA-DP matched-types were shown in the following table:

TABLE-US-00004 TABLE 3 HLA-DP matched-type of SW620 cell Cell line HLA-DP gene Allelic gene SW620 DPA1 01:03/02:01 DPB1 01:01/04:01 CFPAC-1 DPA1 01:03/01:03 DPB1 04:01/04:01

[0233] The SW620-CIITA or CFPAC1-CIITA cells, SW620/CFPAC1-CIITA-DPB1*03:01 cells and SW620/CFPAC1-CIITA-DPA1*02:02-DPB1*03:01 cells were collected and resuspended in RPMI1640 medium, incubated at 37 C. for 2 hours, and washed twice with DPBS solution. The antigen-presenting cells (the above-mentioned modified SW620 or CFPAC-1 cells) were co-cultured overnight with B13.14.1 TCR-T cells in a ratio of 210.sup.4:210.sup.4 in RPMI1640 medium containing 2% fetal bovine serum, the IFN release in the supernatant was measured, and the results were shown in FIG. 2.

[0234] The results showed that B13.14.1 TCR could recognize the G12V mutation restricted and presented by HLA-DPB1*03:01, in which all three alleles of HLA-DPA1 in combination with DPB1*03:01 could be used for presentation.

[0235] 6.2 HLA Restriction Determination 2

[0236] The LCL cell lines prepared from different donor sources were loaded with KRAS-G12V T15 antigen peptide (10 g/ml), then were co-incubated with Mock T or B13.14.1 TCR-T in RPMI1640 medium containing 2% FBS, and the IFN content in supernatant was determined. The ability of B13.14.1 TCR to recognize KRAS-G12V presented by human high-frequency HLA-DPB1 genes was determined, and the results were shown in the table below.

TABLE-US-00005 TABLE 4 Results of HLA restriction determination IFN release IFN release HLA-DPB1 LCL cell Mock T B13.14.1 TCR DPB1*02:01 D5-LCL 373 212 DPB1*02:02 D12-LCL 620 1156 DPB1*03:01 B13-LCL 398 38765 DPB1*04:01 B8-LCL 543 874 DPB1*04:02 B11-LCL 607 923 DPB1*05:01 B4-LCL 1284 1021 DPB1*09:01 YY-LCL 621 907 DPB1*13:01 B10-LCL 507 444 DPB1*14:01 B1-LCL 473 28375 DPB1*17:01 D350-LCL 418 295 DPB1*38:01 D2-LCL 725 251 DPB1*135:01 WY-LCL 413 324

[0237] The results in Table 4 showed that B13.14.1 TCR could not only recognize the KRAS-G12V antigen peptide presented by HLA-DPB1*03:01, but also recognize the KRAS-G12V antigen peptide presented by HLA-DPB1*14:01.

Example 7: Determination of Presenting Epitope of RAS G12V

[0238] Peptides with length of 9-23 (SEQ ID NOs: 2-5, 18-34) containing G12V mutation site were synthesized according to the following table and subjected to antigen presentation by autologous LCL cells, then the IFN release was determined to screen the RAS G12V epitope recognized by B13.14.1 TCR.

TABLE-US-00006 TABLE5 RasG12Vepitopescreeningtable Peptide SEQID PeptideNo. length Aminoacidsequence NO: G12V-T1 23 TEYKLVVVGAVGVGKSALTIQLI 41 G12V-T2 15 AVGVGKSALTIQLI 42 G12V-T3 12 TEYKLVVVGAVG 3 G12V-T4 11 VVVGAVGVGKS 43 G12V-T5 15 VGAVGVGKSALTIQ 44 G12V-T6 12 YKLVVVGAVGVG 45 G12V-T7 15 VVVGAVGVGKSALT 46 G12V-T8 12 LVVVGAVGVGKS 47 G12V-T9 11 EYKLVVVGAVG 2 G12V-T10 10 YKLVVVGAVG 48 G12V-T11 9 KLVVVGAVG 49 G12V-T12 11 TEYKLVVVGAV 50 G12V-T13 10 EYKLVVVGAV 51 G12V-T14 9 YKLVVVGAV 52 G12V-T15 13 TEYKLVVVGAVGV 5 G12V-T16 12 EYKLVVVGAVGV 4 G12V-T17 11 YKLVVVGAVGV 53 G12V-T18 14 TEYKLVVVGAVGVG 54 G12V-T19 16 TEYKLVVVGAVGVGK 55 G12V-T20 16 TEYKLVVVGAVGVGKS 56 G12V-T21 17 TEYKLVVVGAVGVGKSA 57

[0239] The above peptides were synthesized and then dissolved by adding DMSO; the autologous LCL cells of Patient B13 were resuspended in RPMI1640 medium, added with the above peptides to a final concentration of 1 g/ml, incubated for 2 hours, washed twice with DPBS solution, and resuspended in RPMI1640 medium with % fetal bovine serum to reach 210.sup.5/ml. The antigen-presenting cells loaded with the peptides were co-cultured with B13.14.1 TCR-T cells overnight according to a ratio of 210.sup.4:210.sup.4, the IFN release in supernatant was measured, and the results were shown in FIG. 3.

[0240] The results showed that all G12V-T3, G12V-T9, G12V-T15, and G12V-T16 peptides could effectively induce the IFN release of B13.14.1 TCR-T, indicating that HLA-DPB1*03:01 could present the above peptides. The above results also indicated that the peptides comprising amino acid residues at positions 3-13 of the RAS G12V mutant could effectively activate T lymphocytes, thereby inducing an immune response against tumors with RAS G12V mutation.

Example 8: Determination of Key Amino Acids Presented by RAS G12V Epitope by Alanine Scanning

[0241] By performing one-by-one alanine replacement on RAS G12V epitope peptides, the key amino acids involved in antigen presentation in the RAS G12V epitopes could be screened out. The peptide segments after alanine mutation (the mutated amino acid was underlined) were shown in the following table:

TABLE-US-00007 TABLE6 AlaninescanningofRASG12Vepitope Mutated SEQ amino Aminoacid ID Peptidenumber acid sequence NO: G12V-T15-A1 T1A AEYKLVVVGAVGV 58 G12V-T15-A2 E2A TAYKLVVVGAVGV 59 G12V-T15-A3 Y3A TEAKLVVVGAVGV 60 G12V-T15-A4 K4A TEYALVVVGAVGV 61 G12V-T15-A5 L5A TEYKAVVVGAVGV 62 G12V-T15-A6 V6A TEYKLAVVGAVGV 63 G12V-T15-A7 V7A TEYKLVAVGAVGV 64 G12V-T15-A8 V8A TEYKLVVAGAVGV 65 G12V-T15-A9 G9A TEYKLVVVAAVGV 66 G12V-T15-G10 A10G TEYKLVVVAGVGV 67 G12V-T15-A12 G12A TEYKLVVVGAVAV 68 G12V-T15-A13 V13A TEYKLVVVGAVGA 69

[0242] After the above peptides were synthesized, IFN or IL2 in the supernatant after co-culture of antigen-presenting cells with B13.14.1 TCR-T was measured according to the method in Example 7, and the results were shown in FIG. 4. The above results showed that in the TEYKLVVVGAVGV epitope, the key amino acids involved in antigen presentation were p3Y, p4K, p5L, and p8V, which was manifested in that after these four amino acids were mutated to alanine, the ability of B13.14.1 TCR-T to recognize the RAS G12V epitope was reduced, the most important of these are p3Y, p4K and p5L.

Example 9: Tumor Lysis Determination of B13.14.1 TCR-T

[0243] SW620-CIITA-DPA02:02/DPB03:01 cells were resuspended in RPMI1640 medium containing 2% FBS, inoculated in a 96-well plate according to 10.sup.4/well, and added with Mock-T, B13.14.1 TCR-CD4.sup.+T and B13.14.1 TCR-CD8.sup.+T according to an E:T ratio of 10, 3, 1, 0.3, 0.1, and a control without T cell was set; after 48 hours of co-incubation, the medium was sucked out and 200 l of DPBS solution was added to each well to perform washing once, then 100 l of RPMI1640 medium containing 2% FBS and 10 l of CCK8 detection reagent (Cell Counting Kit-8) were added to each well, incubated at 37 C. for 1 hour, and the light absorption at 450 nm was read.

Killing rate=1(light absorption value/light absorption value of control well)

[0244] The results were shown in FIG. 5. The results showed that B13.14.1 TCR-T could kill tumor cells in a dose-dependent manner, and showed a dependency on CD4.

Example 10: Functional Avidity Determination of B13.14.1 TCR-T

[0245] B13 autologous LCL cells were subjected to antigen loading with different concentrations (10 g/ml, 1 g/ml, 0.1 g/ml, 0.01 g/ml, 0.001 g/ml) of RAS G12V-T15 peptide (SEQ ID NO: 5) and the corresponding wild-type peptide (SEQ ID NO: 17), respectively, cultured at 37 C. for 2 hours, then washed twice with DPBS solution; the antigen-presenting cells were co-cultured overnight with B13.14.1 TCR-T cells according to a ratio of 210.sup.4:210.sup.4 in RPMI1640 medium containing 2% fetal bovine serum, and the release of IL2 and IFN in the supernatant was measured. The results were shown in FIGS. 6A to 6B. The results showed that B13.14.1 TCR-T could specifically recognize the RAS G12V mutant peptide within the tested concentration range, and could release IFN and IL2, and it could still efficiently recognize the RAS G12V mutant peptide when the antigen peptide concentration was 0.1 g/ml, indicating that the T cells expressing this TCR had high affinity and high specificity for the RAS G12V mutant.

Example 11: Determination of In Vivo Efficacy of B13.14.1 TCR-T

[0246] The SW620-DPAB tumor cell line (overexpressing HLA-DPB1*03:01) was inoculated subcutaneously in NSG mice (110.sup.7/mouse), and when the tumor volume reached about 50 mm.sup.3, the blank preparation, MockT (non-transduced T cell control), B13.14.1CD4TCRT (CD4 T cells genetically modified with B13.14.1TCR), B13.14.1CD8TCRT (CD8 T cells genetically modified with B13.14.1TCR), or B13.14.1 MixTCRT (CD4 T cells and CD8 T cells modified with B13.14.1TCR, which were mixed at a ratio of 1:1) were administered via tail vein, respectively, the dose was 110.sup.7 mTCR+ cells/animal, and the tumor growth was measured. The results were shown in FIG. 7.

[0247] The results showed that B13.14.1TCRT could eliminate the tumor of SW620-DPAB cell line, and showed dependence on CD4 co-receptor.

Example 12: Affinity Maturation of B13.14.1 TCR

[0248] According to the table below, point mutations were performed on the V and V of B13.14.1 TCR (including CDR3 region and MEW II binding region), and a lentiviral vector shuttle plasmid encoding the TCR mutant was constructed, packaged with the standard lentiviral vector in 293T cells, and B13.14.1TCR mutant were subjected to function screening.

TABLE-US-00008 TABLE 7 Mutation region Amino acid at the mutated position V-CDR3 95D, 96G, 97R, 98G V-CDR3 95P, 96G, 97Q, 98R, 99D V-MHCII binding region 30N, 32Y, 49K, 51I, 52T V-MHCII binding region 48Y, 50Q, 51I, 54D, 56Q Note: The amino acid positions were determined according to the IMGT TCR numbering system.

[0249] 12.1 TCR CDR3 Mutant Screening

[0250] The TCR mutant (CDR3 region) lentiviral vector was transduced into Jurkat-NFAT-Luc cell line, and B13 LCL cells loaded with KRAS-G12V antigen (RAS G12V-T15 peptide, SEQ ID NO: 5) were used as antigen-presenting cells. 210.sup.4 TCR-T and 210.sup.4 antigen-loaded B13 LCL cells were co-cultured for 16-24 hours, and added with ONE Glo Luciferase to detect the expression of fluorescent signal, and the TCR Mut RLU/WT RLU (ratio of RLU signal value of TCR mutant to RLU signal value of wild-type B13.14.1 TCR) was calculated. The results were as follows.

TABLE-US-00009 TABLE 8 Screening results of TCR CDR3 mutants TCR mutant TCR Mut RLU/WT RLU mTCR % 95D-A 0.5% 89.90% 95D-E 0.9% 85.60% 95D-F 1.0% 59.10% 95D-G 1.5% 79.20% 95D-H 0.8% 92.80% 95D-I 1.6% 83.70% 95D-K 1.4% 57.50% 95D-L 1.4% 84.50% 95D-M 0.7% 90.20% 95D-N 11.9% 85.40% 95D-P 1.2% 80.70% 95D-Q 1.3% 88.70% 95D-R 1.8% 65.50% 95D-S 2.2% 87.80% 95D-T 4.7% 84.90% 95D-V 2.0% 87.80% 95D-W 2.3% 84.80% 95D-Y 2.4% 88.20% 96G-A 45.5% 83.90% 96G-D 30.7% 56.30% 96G-E 1.7% 73.80% 96G-F 2.7% 73.80% 96G-H 2.2% 80.30% 96G-I 2.6% 75.40% 96G-K 5.7% 87.70% 96G-L 123.2% 84.80% 96G-M 126.2% 86.90% 96G-N 19.3% 71.30% 96G-P 9.4% 70.90% 96G-Q 90.0% 81.20% 96G-R 122.9% 89.50% 96G-S 76.1% 78.00% 96G-T 4.5% 84.00% 96G-V 48.1% 54.80% 96G-W 58.8% 49.50% 96G-Y 90.0% 47.60% 97R-A 68.6% 70.90% 97R-D 46.2% 41.40% 97R-E 6.5% 40.00% 97R-F 21.0% 62.10% 97R-G 45.4% 51.90% 97R-H 50.1% 48.40% 97R-I 71.9% 52.80% 97R-K 113.0% 59.00% 97R-L 33.8% 33.20% 97R-M 36.5% 35.10% 97R-N 79.4% 38.70% 97R-P 38.9% 36.60% 97R-Q 37.5% 49.30% 97R-S 44.4% 7.06% 97R-T 245.3% 49.50% 97R-V 40.3% 26.50% 97R-W 35.5% 56.40% 97R-Y 46.9% 42.90% 98G-A 467.6% 48.20% 98G-D 113.1% 32.90% 98G-E 144.2% 38.20% 98G-F 110.3% 17.10% 98G-H 162.2% 32.50% 98G-I 216.3% 58.40% 98G-K 65.4% 31.70% 98G-L 89.5% 23.60% 98G-M 115.8% 48.60% 98G-N 235.3% 42.50% 98G-P 46.4% 62.60% 98G-Q 478.8% 51.10% 98G-R 59.6% 30.60% 98G-S 321.2% 39.30% 98G-T 155.2% 36.20% 98G-V 99.0% 44.80% 98G-W 75.8% 18.60% 98G-Y 58.5% 29.60% 95P-A 226.0% 44.30% 95P-D 11.8% 25.20% 95P-E 12.4% 8.98% 95P-F 20.1% 33.50% 95P-G 12.4% 18.00% 95P-H 31.4% 18.00% 95P-I 18.2% 48.60% 95P-K 11.9% 30.90% 95P-L 4.6% 61.80% 95P-M 72.8% 59.00% 95P-N 25.2% 35.50% 95P-Q 9.0% 21.50% 95P-R 8.7% 30.70% 95P-S 58.5% 25.30% 95P-T 175.3% 40.20% 95P-V 77.8% 42.00% 95P-W 5.4% 0.36% 95P-Y 38.9% 24.40% 96G-A 23.3% 17.40% 96G-D 5.6% 42.80% 96G-E 7.3% 28.80% 96G-F 9.4% 56.40% 96G-H 8.1% 18.20% 96G-I 8.2% 34.10% 96G-K 24.0% 39.10% 96G-L 9.2% 21.40% 96G-M 7.6% 10.60% 96G-N 8.3% 14.90% 96G-P 8.8% 20.20% 96G-Q 7.6% 6.67% 96G-R 5.9% 41.90% 96G-S 17.4% 24.30% 96G-T 10.2% 12.70% 96G-V 6.3% 28.00% 96G-W 8.7% 28.80% 96G-Y 8.8% 20.90% 97Q-A 12.6% 31.50% 97Q-D 10.4% 54.50% 97Q-E 5.9% 55.70% 97Q-F 15.0% 15.10% 97Q-G 6.2% 28.40% 97Q-H 11.9% 29.80% 97Q-I 6.7% 21.10% 97Q-K 7.4% 33.90% 97Q-L 6.4% 40.90% 97Q-M 5.0% 11.00% 97Q-N 5.8% 19.50% 97Q-P 10.3% 20.50% 97Q-R 12.4% 59.80% 97Q-S 175.5% 42.70% 97Q-T 11.6% 22.10% 97Q-V 2.7% 47.40% 97Q-W 3.4% 33.90% 97Q-Y 4.1% 75.70% 98R-A 3.5% 57.40% 98R-D 2.5% 45.50% 98R-E 6.2% 39.90% 98R-F 3.2% 72.40% 98R-G 4.0% 45.30% 98R-H 5.4% 48.40% 98R-I 4.1% 47.80% 98R-K 7.1% 57.60% 98R-L 10.9% 21.30% 98R-M 6.8% 35.50% 98R-N 4.0% 75.50% 98R-P 4.2% 71.20% 98R-Q 5.8% 51.90% 98R-S 1.5% 9.96% 98R-T 3.2% 51.70% 98R-V 3.4% 55.00% 98R-W 6.1% 34.40% 98R-Y 4.3% 53.50% 99D-A 5.0% 31.30% 99D-E 5.8% 28.50% 99D-F 6.3% 50.10% 99D-G 10.3% 29.10% 99D-H 5.6% 44.90% 99D-I 2.2% 65.10% 99D-K 2.6% 74.70% 99D-L 6.4% 36.60% 99D-M 6.2% 51.40% 99D-N 4.6% 57.20% 99D-P 6.3% 53.30% 99D-Q 6.8% 33.90% 99D-R 19.7% 19.30% 99D-S 6.3% 47.40% 99D-T 8.8% 41.30% 99D-V 6.9% 45.50% 99D-W 5.6% 30.20% 99D-Y 6.5% 32.30%

[0251] The above results showed that: [0252] 1) The results of mTCR expression detection showed that all TCR mutants could be expressed on the surface of Jurkat-NFAT-Luc cells; [0253] 2) In the mutations in CDR3 and CDR3 regions of B13.14.1 TCR, the amino acid positions that had an important impact on TCR specificity were CDR3-95D, CDR3-96G, CDR3-98R and CDR3-99D, and after the amino acid were mutated at these positions, all mutants could not effectively recognize KRAS.sup.G12V antigen peptide presented by the antigen-presenting cells;

[0254] The TCR CDR3 and CDR3 mutants with activity (maintaining >30% wild-type B 13.14.1 TCR RLU signal value) were shown in the table below.

TABLE-US-00010 TABLE 9 Activity-maintaining mutation sites TCR Wild-type region amino acid TCR mutant with activity CDR3 95D None 96G A, D, L, M, Q, R, S, V, W, Y 97R A, D, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y 98G A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y CDR3 95P A, H, M, S, T, V, Y 96G None 97Q S 98R None 99D None

[0255] In the above table, those with a higher ratio of TCR Mut RLU/WT RLU were underlined, which were preferred TCR mutants. The Emax and EC50 (Functional Avidity) of these TCR mutants in recognition of the KRAS-G12V antigen peptide were further determined. B13 LCL cells were loaded with different concentrations of G12V antigen peptide (10 g/ml, 10-fold dilution, 5 concentrations), and co-incubated with Jurkat-NFAT-Luc cells expressing the TCR mutants for 16-24 hours, the fluorescence signal expression was detected by adding ONE Glo Luciferase, and the results were shown in FIG. 8 and the table below. The results showed that the preferred TCR mutants could recognize the KRAS-G12V mutant antigen peptide in a concentration-dependent manner, and had stronger antigen recognition ability than the wild-type B13.14.1WT.

TABLE-US-00011 TABLE 10 Recognition activity of TCR mutants to KRAS-G12V antigen peptide Emax relative EC50 g/ml TCR mutant to WT (Functional Avidity) B13.14.1WT 100% 0.288 96G-A 232% 0.142 96G-D 104% 0.212 96G-R 183% 0.312 97R-T 263% 0.272 98G-A 319% 0.245 98G-H 275% 0.520 98G-S 140% 0.075 98G-N 125% 0.255 98G-W 104% 0.064 95P-A 256% 0.204 95P-T 94% 0.325 97Q-S 192% 0.123

[0256] 12.2 Screening of TCR MHC-II Binding Region Mutants

[0257] The TCR mutant (MHC-II binding region) lentiviral vector was transduced into the Jurkat-NFAT-Luc cell line, and B13 LCL cells loaded with KRAS-G12V antigen (RAS G12V-T15 peptide, SEQ ID NO: 5) were used as the antigen-presenting cells. 210.sup.4 TCR-T and 210.sup.4 antigen-loaded B13 LCL cells were co-cultured for 16-24 hours, the fluorescence signal expression was detected by adding ONE Glo Luciferase, and TCR Mut RLU/WT RLU (ratio of RLU signal value of TCR mutant to RLU signal value of wild-type B13.14.1TCR) was calculated. The results were shown in the table below.

TABLE-US-00012 TABLE 11 Screening results of TCR MHC-II binding region mutants TCR mutant TCR Mut RLU/WT RLU mTCR % 30N-A 46.85% 97.20% 30N-D 62.37% 96.70% 30N-E 100.86% 96.10% 30N-F 48.29% 98.10% 30N-G 41.51% 95.30% 30N-H 65.37% 95.50% 30N-I 30.71% 97.60% 30N-K 67.81% 94.30% 30N-L 65.66% 97.30% 30N-M 42.72% 92.70% 30N-P 90.36% 94.70% 30N-Q 90.70% 96.00% 30N-R 88.21% 95.20% 30N-S 80.22% 93.40% 30N-T 76.98% 95.60% 30N-V 63.25% 95.40% 30N-W 65.55% 97.80% 30N-Y 73.45% 94.90% 32Y-A 4.68% 91.30% 32Y-D 4.13% 95.20% 32Y-E 4.59% 93.10% 32Y-F 3.81% 95.70% 32Y-G 4.03% 97.40% 32Y-H 5.60% 94.00% 32Y-I 4.64% 83.80% 32Y-K 4.00% 94.40% 32Y-L 6.35% 89.60% 32Y-M 4.94% 90.20% 32Y-N 4.17% 94.90% 32Y-P 3.98% 96.90% 32Y-Q 3.12% 89.00% 32Y-R 5.60% 82.10% 32Y-S 5.77% 83.70% 32Y-T 3.77% 91.70% 32Y-V 4.71% 88.80% 32Y-W 3.81% 90.30% 49K-A 52.40% 96.30% 49K-D 33.44% 98.70% 49K-E 46.49% 96.10% 49K-F 64.93% 93.70% 49K-G 132.39% 96.30% 49K-H 85.38% 97.70% 49K-I 77.25% 91.90% 49K-L 65.99% 95.50% 49K-M 44.35% 96.10% 49K-N 90.90% 93.00% 49K-P 93.63% 94.70% 49K-Q 66.90% 97.10% 49K-R 91.14% 96.10% 49K-S 112.11% 95.80% 49K-T 63.63% 95.50% 49K-V 100.99% 93.90% 49K-W 94.30% 93.10% 49K-Y 119.44% 94.90% 51I-A 56.93% 95.90% 51I-D 93.65% 93.20% 51I-E 160.66% 93.70% 51I-F 107.59% 94.40% 51I-G 90.09% 90.10% 51I-H 85.85% 94.60% 51I-K 61.76% 94.40% 51I-L 75.40% 95.20% 51I-M 68.25% 92.40% 51I-N 84.17% 91.00% 51I-P 82.47% 86.10% 51I-Q 71.70% 93.10% 51I-R 87.31% 86.70% 51I-S 78.65% 86.40% 51I-T 68.06% 93.20% 51I-V 48.02% 94.40% 51I-W 128.73% 95.20% 51I-Y 161.75% 92.60% 52T-A 139.10% 87.70% 52T-D 101.16% 63.20% 52T-E 43.18% 49.60% 52T-F 14.98% 72.90% 52T-G 9.36% 80.50% 52T-H 114.90% 71.60% 52T-I 113.54% 80.70% 52T-K 11.05% 78.90% 52T-L 79.97% 78.70% 52T-M 16.65% 78.70% 52T-N 72.41% 81.80% 52T-P 12.69% 76.30% 52T-Q 102.55% 75.60% 52T-R 106.98% 73.20% 52T-S 76.99% 77.00% 52T-V 19.70% 74.80% 52T-W 37.02% 78.40% 52T-Y 68.60% 86.30% 48Y-A 110.29% 67.20% 48Y-D 15.07% 61.90% 48Y-E 90.23% 73.80% 48Y-F 125.84% 34.60% 48Y-G 8.49% 76.60% 48Y-H 11.40% 75.40% 48Y-I 34.66% 71.00% 48Y-K 83.49% 75.40% 48Y-L 37.97% 54.30% 48Y-M 66.36% 96.10% 48Y-N 42.82% 50.10% 48Y-P 36.89% 20.80% 48Y-Q 42.56% 74.30% 48Y-R 62.60% 61.70% 48Y-S 34.02% 54.30% 48Y-T 40.26% 46.40% 48Y-V 63.15% 67.10% 48Y-W 32.62% 54.90% 50Q-A 81.49% 84.80% 50Q-D 19.13% 95.10% 50Q-E 2.80% 89.00% 50Q-F 42.98% 73.40% 50Q-G 34.63% 58.60% 50Q-H 64.73% 90.20% 50Q-I 94.65% 95.60% 50Q-K 67.50% 88.60% 50Q-L 62.99% 92.90% 50Q-M 85.81% 77.80% 50Q-N 109.90% 95.10% 50Q-P 78.46% 87.50% 50Q-R 72.99% 73.20% 50Q-S 88.72% 78.10% 50Q-T 101.44% 89.00% 50Q-V 35.26% 86.10% 50Q-W 72.66% 74.20% 50Q-Y 89.49% 84.20% 51I-A 27.57% 72.70% 51I-D 4.56% 81.60% 51I-E 6.28% 80.10% 51I-F 32.79% 81.20% 51I-G 5.49% 80.60% 51I-H 34.95% 82.80% 51I-K 3.82% 91.90% 51I-L 25.46% 78.60% 51I-M 26.84% 85.80% 51I-N 7.61% 89.00% 51I-P 19.95% 39.10% 51I-Q 12.98% 86.60% 51I-R 9.90% 84.20% 51I-S 21.00% 80.30% 51I-T 88.58% 86.00% 51I-V 35.46% 80.30% 51I-W 22.56% 61.00% 51I-Y 19.64% 86.20% 54D-A 63.81% 92.20% 54D-E 53.62% 40.70% 54D-F 49.69% 85.80% 54D-G 92.37% 89.50% 54D-H 71.90% 91.40% 54D-I 67.27% 94.40% 54D-K 61.65% 86.70% 54D-L 85.12% 80.80% 54D-M 91.57% 91.80% 54D-N 82.50% 85.60% 54D-P 40.29% 65.40% 54D-Q 60.43% 90.10% 54D-R 79.21% 72.40% 54D-S 57.33% 26.70% 54D-T 89.58% 85.40% 54D-V 52.11% 79.90% 54D-W 62.51% 85.70% 54D-Y 84.41% 87.90% 56Q-A 100.38% 84.90% 56Q-D 92.36% 77.60% 56Q-E 104.57% 84.90% 56Q-F 82.86% 79.10% 56Q-G 58.76% 61.80% 56Q-H 96.49% 88.50% 56Q-I 57.81% 15.20% 56Q-K 71.30% 87.30% 56Q-L 100.05% 88.10% 56Q-M 72.61% 74.40% 56Q-N 75.67% 73.10% 56Q-P 78.34% 55.00% 56Q-R 96.16% 85.60% 56Q-S 58.09% 77.00% 56Q-T 118.84% 73.60% 56Q-V 56.07% 66.80% 56Q-W 99.67% 83.50% 56Q-Y 66.39% 44.10%

[0258] The above results showed that: [0259] 1) The results of mTCR expression detection showed that all TCR mutants could be expressed on the surface of Jurkat-NFAT-Luc cells; [0260] 2) In the mutations in WWII binding region of B13.14.1 TCR, the amino acid position that had an important impact on TCR specificity was V-32Y. After amino acid was mutated at the above position, the mutant could not effectively recognize the KRAS.sup.G12V antigen peptide presented by the antigen-presenting cells; [0261] 3) The TCR mutants with activity (maintaining 30% wild-type B13.14.1 TCR RLU signal value) were showed in the table below.

TABLE-US-00013 TABLE 12 Activity-maintaining mutation sites TCR Wild-type region amino acid TCR mutant with activity V 30N A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y 32Y None 49K A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y 51I A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y 52T A, D, E, H, I, L, N, Q, R, S, W, Y V 48Y A, E, F, I, K, L, M, N, P, Q, R, S, T, V, W 50Q A, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y 51I F, H, T, V 54D A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y 56Q A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y

[0262] In the above table, those with TCR Mut RLU/WT RLU ratio 80% were underlined, which were the preferred TCR mutants. The Emax and EC50 (Functional Avidity) of the TCR mutants to recognize the KRAS-G12V antigen peptide were further determined. SW620-DPB cells (SW620 expressing HLA-DPB1*03:01) were loaded with different concentrations of G12V antigen peptide (10 g/ml, 10-fold dilution, 5 concentrations), and co-incubated with Jurkat-NFAT-Luc cells expressing the TCR mutants for 16-24 hours, and the fluorescence signal expression was detected by adding ONE Glo Luciferase. The results were shown in FIG. 9 and the table below. The results showed that the preferred TCR mutants could recognize the KRAS-G12V mutant antigen peptide in a concentration-dependent manner, and had stronger antigen recognition ability than the wild-type B13.14.1WT.

TABLE-US-00014 TABLE 13 Recognition activity of TCR mutants to KRAS-G12V antigen peptide Emax relative EC50 g/ml TCR mutant to WT (Functional Avidity) B13.14.1WT 100% 0.868 49K-G 104% 0.809 49K-S 106% 1.208 49K-Y 82% 0.960 51I-E 95% 0.927 51I-W 134% 1.235 51I-Y 94% 0.894 52T-A 131% 1.565 52T-H 111% 1.028 52T-I 95% 1.350 48Y-F 180% 9.508 50Q-N 97% 1.189 50Q-T 78% 1.340 56Q-A 103% 0.859 56Q-E 100% 1.052 56Q-H 97% 1.263 56Q-L 108% 1.412 56Q-T 131% 1.762 56Q-W 53% 0.556

[0263] Although the specific models for carrying out the present invention have been described in detail, those skilled in the art will understand that: according to all the teachings that have been disclosed, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the claims appended hereto and any equivalents thereof.