T-cell receptor (TCR) and use thereof

Abstract

Provided are a T-cell receptor (TCR) and the use thereof, wherein the TCR contains an α chain containing a variable region and / or a β chain containing a variable region. The TCR is capable of specifically recognizing an A1101-restricted KRASG12V mutation and an A1101-restricted KRASG12D mutation. A T cell transducing the TCR (TCR-T) can bind to an antigen short peptide-HLA-A1101 complex, and can be used for treating malignant tumors carrying the KRASG12V mutation and KRASG12D mutation.

Description

A T cell receptor (TCR) and its use Technical Field

[0001] The present application relates to the field of medical technology, in particular to a method for specifically recognizing A1101-restricted KRAS G12V Mutated, A1101-restricted KRAS G12D Mutated TCRs and their uses. Background Art

[0002] KRAS mutations are common in patients with metastatic colorectal cancer, with G12D mutations accounting for 45% and G12V mutations for 30%. KRAS, a member of the RAS family, is a GTPase protein encoded by genes that participate in epidermal growth factor receptor (EGFR) signaling, regulating cell growth, differentiation, proliferation, and survival. RAS mutations result in defective GTPase activity, leading to overactivation of the RAF-MAPK cell signaling cascade. Tumors with KRAS mutations are generally more aggressive and have a worse prognosis. Studies have shown that patients with KRAS colorectal cancer are resistant to EGFR monoclonal antibodies (cetuximab or panitumumab). Therefore, the NCCN guidelines recommend RAS genetic testing for all patients with metastatic colorectal cancer, and EGFR monoclonal antibodies should only be used in patients with wild-type RAS. The development of targeted drugs targeting KRAS has been fraught with difficulties, and KRAS was once considered an "undruggable target." While molecular biology has made significant advances in the study of RAS protein mutations and signaling pathways in recent years, the development of targeted drugs remains challenging. In the development of chemical drugs, due to the smooth structure of RAS protein, the hydrophobic pockets on its surface for binding small molecules are not obvious; in the development of biological drugs, antibody drugs need to penetrate the cell membrane to target RAS protein, so the drug delivery efficiency is very low. G12C AMG510, a small molecule targeted drug developed by FDA, was approved for marketing in the United States on May 29, 2021. However, the G12C mutation is more common in lung cancer patients (approximately 50%), but only accounts for about 10% of colorectal cancer patients. Therefore, new drugs targeting KRAS mutations in colorectal cancer are urgently needed.

[0003] Specific T cell immunotherapy utilizes T cells specific for tumor antigens to kill tumor cells. It is a highly personalized approach to tumor immunotherapy. Due to the local immunosuppressive microenvironment of the tumor, the patient's own T cells have limited ability to kill tumors. Therefore, efforts are underway to genetically modify T cells to enhance their tumor-killing capacity. Both TCR-T and CAR-T are genetically modified cell therapies. Upon binding to their target, the transferred T cell receptor (TCR) or chimeric antigen receptor (CAR) gene activates T cells and eliminates tumor cells through the release of granzymes, perforins, and cytokines. However, a significant difference between TCR-T and CAR-T is that CAR-T targets cell surface membrane proteins, while TCR-T targets antigenic peptide-major histocompatibility complexes (pMHC).

[0004] TCRs are restricted by the major histocompatibility complex (MHC). The frequency of human MHC (also known as human leukocyte antigen, HLA) has been found to vary significantly across different populations. The most common HLA types in the Chinese population are A1101 and A2402, while the most common HLA type in Caucasians is A0201. Known TCR targets are limited, and most are presented by the A0201 target, which is more prevalent in Western populations. For example, NY-ESO-1 and HPV E6 / E7, currently sought after by domestic and international TCR-T companies, can only target patients with the A0201 genotype. Therefore, developing TCR-T drugs targeting the more common HLA-A1101 genotype in the Chinese population will benefit more Chinese patients.

[0005] Therefore, those skilled in the art are committed to screening for KRAS that can specifically recognize A1101 restriction. G12V Mutated TCR, A1101-restricted KRAS G12D Mutated TCRs, thus enabling them to play a role in T cell immunotherapy.

[0006] Summary of the Invention

[0007] In order to solve the defect of the above-mentioned prior art that there is no KRAS mutant TCR-T drug developed for HLA-A1101 typing, the present application provides a T cell receptor (TCR) that can specifically recognize A1101-restricted-KRAS G12VMutation, T cells transduced with the TCR of the present application (TCR-T) can bind to the antigen short peptide KRAS G12V -HLA-A1101 complex binding, targeting tumor antigen-specific killing of tumor cells, can be used to treat KRAS G12V The present application provides a T cell receptor (TCR) that can specifically recognize A1101-restricted-KRAS G12D Mutation, T cells transduced with the TCR of the present application (TCR-T) can bind to the antigen short peptide KRAS G12D -HLA-A1101 complex binding, targeting tumor antigen-specific killing of tumor cells, can be used to treat KRAS G12D At the same time, the present application also provides the use of the T cell receptor.

[0008] Specifically, this application relates to the following aspects:

[0009] 1. A T cell receptor (TCR), wherein the TCR comprises an α chain containing a variable region and / or a β chain containing a variable region,

[0010] The variable region of the α chain comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; and / or

[0011] The amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 3 or SEQ ID NO: 4; and / or

[0012] The amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO: 5 or SEQ ID NO: 6, and the T cell receptor (TCR) binds to KRAS G12V mutation.

[0013] 2. The T cell receptor (TCR) according to item 1, wherein

[0014] The variable region of the β chain comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 8; and / or

[0015] The amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 9 or SEQ ID NO: 10; and / or

[0016] The amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO: 11 or SEQ ID NO: 12.

[0017] 3. The T cell receptor (TCR) according to item 1 or 2, wherein

[0018] The variable region of the α chain further comprises a first leader sequence; and / or

[0019] The variable region of the β chain further includes a second leader sequence,

[0020] Preferably, the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14, and / or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 16,

[0021] Preferably, the α chain further comprises an α constant region, and / or the β chain further comprises a β constant region. Preferably, the constant region is a mouse constant region or a human constant region.

[0022] 4. The T cell receptor (TCR) of any one of items 1 to 3, wherein the TCR is isolated or purified or recombinant;

[0023] Preferably, the TCR is human;

[0024] Preferably, the TCR is monoclonal;

[0025] Preferably, the TCR is single chain;

[0026] Preferably, the TCR comprises two chains;

[0027] Preferably, the TCR is in a cell-bound form or a soluble form, preferably a soluble form;

[0028] Preferably, the TCR binds to the antigen short peptide-HLA-A1101 complex, and preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO:1.

[0029] 5. A nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the TCR according to any one of items 1 to 4, or the α chain or β chain of the TCR.

[0030] 6. The nucleic acid molecule according to item 5, wherein the nucleotide sequence encoding the α chain is the nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18; and / or

[0031] The nucleotide sequence encoding the β chain is the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0032] 7. A vector, wherein the vector comprises the nucleic acid molecule according to item 5 or 6.

[0033] 8. The vector according to item 7, wherein the vector is an expression vector;

[0034] Preferably, the vector is a viral vector, preferably a retroviral vector;

[0035] Preferably, the viral vector is a lentiviral vector.

[0036] 9. An engineered cell comprising the TCR of any one of items 1-4, the nucleic acid molecule of any one of items 5-6, or the vector of any one of items 7-8.

[0037] 10. The engineered cell of claim 9, wherein the TCR is heterologous to the cell;

[0038] Preferably, the engineered cell is a cell line;

[0039] Preferably, the engineered cells are primary cells obtained from a subject, preferably a mammalian subject, preferably a human;

[0040] Preferably, the engineered cells are T cells or NK cells, preferably, the T cells are T cells isolated from peripheral blood;

[0041] Preferably, the T cells are CD8+ or CD4+.

[0042] 11. A method for producing the engineered cell according to any one of items 9 to 10, comprising introducing the nucleic acid molecule according to any one of items 5 to 6 or the vector according to any one of items 7 to 8 into a cell in vitro or ex vivo.

[0043] 12. The method according to item 11, wherein the vector is a viral vector and the introduction is performed by transduction.

[0044] 13. A pharmaceutical composition comprising the T cell receptor (TCR) of any one of items 1 to 4, the nucleic acid molecule of any one of items 5 to 6, the vector of any one of items 7 to 8, or the engineered cell of any one of items 9 to 10;

[0045] Preferably, it further comprises a pharmaceutically acceptable carrier or adjuvant.

[0046] 14. Use of the T cell receptor (TCR) of any one of items 1 to 4, the nucleic acid molecule of any one of items 5 to 6, the vector of any one of items 7 to 8, the engineered cell of any one of items 9 to 10, or the pharmaceutical composition of item 13 in the preparation of a medicament for treating a malignant tumor;

[0047] Preferably, the malignant tumor is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, or prostate cancer.

[0048] 15. A T cell receptor (TCR), wherein the TCR comprises an α chain containing a variable region and / or a β chain containing a variable region,

[0049] The variable region of the α chain comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 33; and / or

[0050] The amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 34 or SEQ ID NO: 35; and / or

[0051] The amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO: 36 or SEQ ID NO: 37, and the T cell receptor (TCR) binds to KRAS G12D mutation.

[0052] 16. The T cell receptor (TCR) according to item 15, wherein

[0053] The variable region of the β chain comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 38 or SEQ ID NO: 39; and / or

[0054] The amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 40 or SEQ ID NO: 41; and / or

[0055] The amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO: 42 or SEQ ID NO: 43.

[0056] 17. The T cell receptor (TCR) according to item 15 or 16, wherein

[0057] The variable region of the α chain further comprises a first leader sequence; and / or

[0058] The variable region of the β chain further includes a second leader sequence,

[0059] Preferably, the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 44 or SEQ ID NO: 45, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 45, and / or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 46 or SEQ ID NO: 47, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 46 or SEQ ID NO: 47,

[0060] Preferably, the α chain further comprises an α constant region, and / or the β chain further comprises a β constant region. Preferably, the constant region is a mouse constant region or a human constant region.

[0061] 18. The T cell receptor (TCR) of any one of items 15 to 17, wherein the TCR is isolated or purified or recombinant;

[0062] Preferably, the TCR is human;

[0063] Preferably, the TCR is monoclonal;

[0064] Preferably, the TCR is single chain;

[0065] Preferably, the TCR comprises two chains;

[0066] Preferably, the TCR is in a cell-bound form or a soluble form, preferably a soluble form;

[0067] Preferably, the TCR binds to the antigen short peptide-HLA-A1101 complex, and preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO:32.

[0068] 19. A nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the TCR according to any one of items 15 to 18, or the α chain or β chain of the TCR.

[0069] 20. The nucleic acid molecule according to item 19, wherein the nucleotide sequence encoding the α chain is the nucleotide sequence shown in SEQ ID NO: 48 or SEQ ID NO: 49; and / or

[0070] The nucleotide sequence encoding the β chain is the nucleotide sequence shown in SEQ ID NO: 50 or SEQ ID NO: 51.

[0071] 21. A vector, wherein the vector comprises the nucleic acid molecule according to item 19 or 20.

[0072] 22. The vector according to item 21, wherein the vector is an expression vector;

[0073] Preferably, the vector is a viral vector, preferably a retroviral vector;

[0074] Preferably, the viral vector is a lentiviral vector.

[0075] 23. An engineered cell comprising the TCR of any one of items 15-18, the nucleic acid molecule of any one of items 19-20, or the vector of any one of items 21-22.

[0076] 24. The engineered cell of item 23, wherein the TCR is heterologous to the cell;

[0077] Preferably, the engineered cell is a cell line;

[0078] Preferably, the engineered cells are primary cells obtained from a subject, preferably a mammalian subject, preferably a human;

[0079] Preferably, the engineered cells are T cells or NK cells, preferably, the T cells are T cells isolated from peripheral blood;

[0080] Preferably, the T cells are CD8+ or CD4+.

[0081] 25. A method for producing the engineered cell of any one of items 23-24, comprising introducing the nucleic acid molecule of any one of items 19-20 or the vector of any one of items 7-8 into the cell in vitro or ex vivo.

[0082] 26. The method of claim 25, wherein the vector is a viral vector and the introducing is performed by transduction.

[0083] 27. A pharmaceutical composition comprising the T cell receptor (TCR) of any one of items 15-18, the nucleic acid molecule of any one of items 19-20, the vector of any one of items 21-22, or the engineered cell of any one of items 23-24;

[0084] Preferably, it further comprises a pharmaceutically acceptable carrier or adjuvant.

[0085] 28. Use of the T cell receptor (TCR) of any one of items 15 to 18, the nucleic acid molecule of any one of items 19 to 20, the vector of any one of items 21 to 22, the engineered cell of any one of items 23 to 24, or the pharmaceutical composition of item 27 in the preparation of a medicament for treating a malignant tumor;

[0086] Preferably, the malignant tumor is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, or prostate cancer.

[0087] Effects of the Invention

[0088] The T cell receptor (TCR) of the present application can specifically recognize A1101-restricted-KRAS G12V Mutation, T cells transduced with the TCR of the present application (TCR-T) can bind to the antigen short peptide KRAS G12V -HLA-A1101 complex binding, targeting tumor antigen-specific killing of tumor cells, can be used to treat KRAS G12V The T cell receptor (TCR) of the present application can specifically recognize A1101-restricted-KRAS G12D Mutation, T cells transduced with the TCR of the present application (TCR-T) can bind to the antigen short peptide KRAS G12D -HLA-A1101 complex binding, targeting tumor antigen-specific killing of tumor cells, can be used to treat KRAS G12D Mutated malignant tumors.

[0089] And the T cells transduced with the TCR of the present application can express A11 and KRAS G12V Mutated tumor cells are specifically activated and have good specificity, recognizing only KRAS G12V mutations, but not wild-type KRAS and KRAS G12D Mutation. T cells transduced with the TCR of the present application can express A11 and KRAS G12D Mutated tumor cells are specifically activated and have good specificity, recognizing only KRAS G12D mutations, but not wild-type KRAS and KRAS G12V mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] FIG1 shows that the TCR described in the present application can be correctly expressed in the Jurkat T cell line.

[0091] FIG2 shows that T cells transduced with the TCR described in this application can be loaded with KRAS G12V Target cells react to short antigenic peptides.

[0092] FIG3 shows that the TCR described in the present application can be correctly expressed in primary T cells.

[0093] FIG4 shows T cells transduced with the TCR described in this application and expressing A11 and KRAS G12V Release of interferon-γ after co-incubation of mutant tumor cells.

[0094] Figure 5 shows that the TCR of the present application has good specificity and only recognizes KRAS G12V mutations, but not wild-type KRAS and KRAS G12D mutation.

[0095] FIG6 shows that the TCR described in the present application can be correctly expressed in the Jurkat T cell line.

[0096] FIG7 shows that T cells transduced with the TCR described in this application can be loaded with KRAS G12D Target cells react to short antigenic peptides.

[0097] FIG8 shows that the TCR described in the present application can be correctly expressed in primary T cells.

[0098] FIG9 shows T cells transduced with the TCR described in this application and expressing A11 and KRAS G12D Release of interferon-gamma after co-incubation of mutant tumor cells.

[0099] Figure 10 shows that the TCR of the present application has good specificity and only recognizes KRAS G12D mutations, but not wild-type KRAS and KRAS G12V mutation. DETAILED DESCRIPTION

[0100] The present application is described in detail below with reference to the embodiments described in the accompanying drawings, wherein like numbers in all figures represent like features. Although specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. Instead, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0101] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of this application shall be as defined by the attached claims.

[0102] The present application provides a T cell receptor (TCR), wherein the TCR comprises an α chain containing a variable region and / or a β chain containing a variable region, the variable region of the α chain comprises a complementarity determining region 1 (CDR1) with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; and / or a complementarity determining region 2 (CDR2) with an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4; and / or a complementarity determining region 3 (CDR3) with an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 6,

[0103] The amino acid sequence shown in SEQ ID NO:1 is: DRVSQS; the amino acid sequence shown in SEQ ID NO:2 is: TSENNYY; the amino acid sequence shown in SEQ ID NO:3 is: IYSNGD; the amino acid sequence shown in SEQ ID NO:4 is: QEAYKQQN; the amino acid sequence shown in SEQ ID NO:5 is: AAVSGGSYIPT; the amino acid sequence shown in SEQ ID NO:6 is: AFMNGETSGSRLT.

[0104] In one embodiment, the variable region of the beta chain comprises a complementarity determining region 1 (CDR1) with an amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 8; and / or a complementarity determining region 2 (CDR2) with an amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 10; and / or a complementarity determining region 3 (CDR3) with an amino acid sequence as shown in SEQ ID NO: 11 or SEQ ID NO: 12,

[0105] The amino acid sequence shown in SEQ ID NO:7 is: SGDLS; the amino acid sequence shown in SEQ ID NO:8 is: SQVTM; the amino acid sequence shown in SEQ ID NO:9 is: YYNGEE;

[0106] The amino acid sequence shown in SEQ ID NO: 10 is: ANQGSEA; the amino acid sequence shown in SEQ ID NO: 11 is: ASSVGGLAGELLETQY; the amino acid sequence shown in SEQ ID NO: 12 is: SVIPHGLYEQY.

[0107] In one embodiment, the variable region of the α chain further comprises a first leader sequence; and / or the variable region of the β chain further comprises a second leader sequence. The first leader sequence of the variable region of the α chain and the second leader sequence of the variable region of the β chain are well known to those skilled in the art. For example, the first leader sequence of the variable region of the α chain can use an amino acid sequence such as the leader sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22, and the second leader sequence of the variable region of the β chain can use an amino acid sequence such as the leader sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24.

[0108] The amino acid sequence shown in SEQ ID NO:21 is: MKSLRVLLVILWLQLSWVWSQ; the amino acid sequence shown in SEQ ID NO:22 is: MTRVSLLWAVVVSTCLESGM; the amino acid sequence shown in SEQ ID NO:23 is: MGFRLLCCVAFCLLGAGPV; the amino acid sequence shown in SEQ ID NO:24 is: MLSLLLLLLGLGSVF;

[0109] In one embodiment, the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14, and / or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 16,

[0110] The amino acid sequence shown in SEQ ID NO: 13 is:

[0111] The amino acid sequence shown in SEQ ID NO: 14 is:

[0112] The amino acid sequence shown in SEQ ID NO: 15 is:

[0113] The amino acid sequence shown in SEQ ID NO: 16 is:

[0114] The amino acid sequence of the α chain variable region has at least 90% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14, and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14. The amino acid sequence of the β chain variable region has at least 90% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16, and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16.

[0115] In one embodiment, the α chain further comprises an α constant region, and / or the β chain further comprises a β constant region, preferably, the constant region is a mouse constant region or a human constant region. For example, the amino acid sequence of the mouse α constant region is as shown in SEQ ID NO: 25; and / or the amino acid sequence of the mouse β constant region is as shown in SEQ ID NO: 26. That is, the constant regions of the α chains of the above-mentioned TCRs can all have the same constant region, and similarly, the constant regions of the β chains of all TCRs can also have the same constant region.

[0116] The amino acids shown in SEQ ID NO: 25 are:

[0117] The amino acids shown in SEQ ID NO: 26 are:

[0118] The constant region of the TCR can contain a short linker sequence in which cysteine ​​residues form a disulfide bond, thereby connecting the two chains of the TCR. The TCR can have additional cysteine ​​residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant region.

[0119] In one embodiment, the amino acid sequence of the α chain variable region of the TCR is as shown in SEQ ID NO: 13 and the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 15, or the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 14 and the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 16.

[0120] In one embodiment, an artificial disulfide bond is introduced between the residues of the constant regions of the α chain and the β chain of the TCR. The positions of the disulfide bonds that can be introduced are well known to those skilled in the art.

[0121] In one embodiment, the TCR is isolated or purified or recombinant.

[0122] In one embodiment, the TCR is human.

[0123] In one embodiment, the TCR is monoclonal.

[0124] In one embodiment, the TCR is single chain.

[0125] In one embodiment, the TCR comprises two chains.

[0126] TCRs can be obtained from biological sources, such as from cells (e.g., from T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available resources, for example, TCRs can be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals, such as typically from human.

[0127] In some embodiments, the TCR may be in a cell-bound form or a soluble form, preferably a soluble form. The soluble form of the TCR refers to a TCR that has mutations in its hydrophobic core region, and these mutations in the hydrophobic core region are preferably mutations that can improve the stability of the soluble TCR of the present application.

[0128] The present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the TCR or the α chain or β chain of the TCR.

[0129] In one embodiment, the nucleotide sequence encoding the α chain is the nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18; and / or the nucleotide sequence encoding the β chain is the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0130] The nucleotide sequence shown in SEQ ID NO: 17 is:

[0131] The nucleotide sequence shown in SEQ ID NO: 18 is:

[0132] The nucleotide sequence shown in SEQ ID NO: 19 is:

[0133] The nucleotide sequence shown in SEQ ID NO:20 is:

[0134] In one embodiment, the nucleotide sequence encoding the alpha chain and / or the nucleotide sequence encoding the beta chain are codon-optimized. Typically, codon optimization involves balancing the percentage of selected codons with the abundance of disclosed human transfer RNAs so that none of them is overloaded or restricted. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Codon usage differences between transfected genes and host cells may affect protein expression and immunogenicity of nucleic acid constructs. Typically, for codon optimization, codons are selected to select those codons that are balanced with human usage frequency. Typically, the redundancy of amino acid codons is such that different codons encode a single amino acid. In some embodiments, when selecting a codon for replacement, the resulting mutation may be a silent mutation so that the codon change does not affect the amino acid sequence. Typically, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence.

[0135] The present application provides a vector comprising the nucleic acid molecule described above.

[0136] For example, one or more nucleic acids encoding one or both chains of the TCR described above are cloned into one or more suitable expression vectors, which can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses.

[0137] The vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific to the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), taking into account whether the vector is DNA-based or RNA-based. The vector may also contain a non-natural promoter operably linked to the nucleotide sequence encoding the TCR. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat sequence of the mouse stem cell virus, and other promoters known to those skilled in the art are also contemplated.

[0138] The vector is an expression vector, preferably a viral vector, preferably a retroviral vector, and more preferably a lentiviral vector.

[0139] The application also provides a host cell comprising the nucleic acid molecules described above, and in order to recombinantly produce TCR, the nucleic acid encoding TCR can be separated and inserted into one or more vectors to further clone and / or express in the host cell. Conventional techniques can be used (for example, by using oligonucleotide probes that can bind specifically to the genes encoding the α chain and β chain of the TCR) to easily separate and sequence such nucleic acids. In some embodiments, a method for preparing TCR is provided, wherein the method includes culturing a host cell comprising a nucleic acid encoding TCR as provided above under conditions suitable for expressing the TCR molecule, and optionally recovering the TCR from the host cell (or host cell culture medium).

[0140] The term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations.

[0141] The present application also provides an engineered cell comprising the T cell receptor (TCR), the nucleic acid molecule or the vector.

[0142] In one embodiment, the TCR is allogeneic to the cell.

[0143] In one embodiment, the engineered cell is a cell line.

[0144] In one embodiment, the engineered cell is a primary cell obtained from a subject, preferably a mammalian subject, preferably a human.

[0145] In one embodiment, the engineered cells are T cells, preferably T cells isolated from peripheral blood.

[0146] In one embodiment, the T cells are CD8+ or CD4+.

[0147] In some embodiments, the engineered cell is a cell that is derived from blood, bone marrow, lymph or lymphoid organs, and is a cell of the immune system, such as a cell of innate immunity or adaptive immunity, such as bone marrow or lymphoid cells (including lymphocytes, typically T cells and / or NK cells). Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSCs). Cell is typically primary cells, such as directly separated from a subject and / or separated and frozen from a subject. In some embodiments, cell includes one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells and subgroups thereof.

[0148] Subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, etc.

[0149] In some embodiments, the engineered cells are natural killer (NK) cells, preferably, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils.

[0150] The present application provides a method for producing the engineered cells described above, which comprises introducing the nucleic acid molecule or the vector into cells in vitro or ex vivo.

[0151] The vector is a viral vector, and the introduction is performed by transduction.

[0152] The present application provides a pharmaceutical composition comprising the above-mentioned T cell receptor (TCR), the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned engineered cell.

[0153] In one embodiment, it further comprises a pharmaceutically acceptable carrier or adjuvant.

[0154] The pharmaceutically acceptable carrier or adjuvant refers to a component in a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers or adjuvants include but are not limited to buffers, excipients, stabilizers or preservatives.

[0155] The pharmaceutical composition can utilize timed release, delayed release, and sustained release delivery systems so that delivery of the composition occurs before sensitization of the treated area and has sufficient time to cause sensitization. Many types of release delivery systems are available and known. Such systems can be used to avoid repeated administration of the composition, thereby increasing the convenience of the subject and the physician.

[0156] The present application provides the use of the aforementioned T cell receptor (TCR), the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned engineered cell or the aforementioned pharmaceutical composition in the preparation of a drug for treating malignant tumors.

[0157] In one embodiment, the malignant tumor is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, or prostate cancer.

[0158] In one embodiment, the colorectal cancer is metastatic colorectal cancer.

[0159] The T cell receptor (TCR) of the present application can specifically recognize A1101-restricted-KRAS G12V Mutation, T cells transduced with the TCR of the present application (TCR-T) can bind to the antigen short peptide KRAS G12V -HLA-A1101 complex binding, targeting tumor antigen-specific killing of tumor cells, can be used to treat KRAS G12V Mutated malignant tumors.

[0160] And the T cells transduced with the TCR of the present application can express A11 and KRAS G12V Mutated tumor cells are specifically activated and have good specificity, recognizing only KRAS G12V mutations, but not wild-type KRAS and KRAS G12D mutation.

[0161] The present application provides a T cell receptor (TCR), wherein the TCR comprises an α chain containing a variable region and / or a β chain containing a variable region, the variable region of the α chain comprises a complementarity determining region 1 (CDR1) with an amino acid sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 33; and / or a complementarity determining region 2 (CDR2) with an amino acid sequence as shown in SEQ ID NO: 34 or SEQ ID NO: 35; and / or a complementarity determining region 3 (CDR3) with an amino acid sequence as shown in SEQ ID NO: 36 or SEQ ID NO: 37,

[0162] The amino acid sequence shown in SEQ ID NO:32 is: TRDTTYY; the amino acid sequence shown in SEQ ID NO:33 is: SSVVY; the amino acid sequence shown in SEQ ID NO:34 is: RNSFDEQN; the amino acid sequence shown in SEQ ID NO:35 is: YLSGSTLV; the amino acid sequence shown in SEQ ID NO:36 is: ALSEAAPGGSYIPT; and the amino acid sequence shown in SEQ ID NO:37 is: AVIGNDYKLS.

[0163] In one embodiment, the variable region of the beta chain comprises a complementarity determining region 1 (CDR1) with an amino acid sequence as shown in SEQ ID NO: 38 or SEQ ID NO: 39; and / or a complementarity determining region 2 (CDR2) with an amino acid sequence as shown in SEQ ID NO: 40 or SEQ ID NO: 41; and / or a complementarity determining region 3 (CDR3) with an amino acid sequence as shown in SEQ ID NO: 42 or SEQ ID NO: 43,

[0164] The amino acid sequence shown in SEQ ID NO:38 is: MDHEN; the amino acid sequence shown in SEQ ID NO:39 is: SNHLY; the amino acid sequence shown in SEQ ID NO:40 is: SYDVKM; the amino acid sequence shown in SEQ ID NO:41 is: FYNNEI;

[0165] The amino acid sequence shown in SEQ ID NO:42 is: ASSLGPGQHNSPLH; the amino acid sequence shown in SEQ ID NO:43 is: ASSGTGGIEAF.

[0166] In one embodiment, the variable region of the α chain further comprises a first leader sequence; and / or the variable region of the β chain further comprises a second leader sequence. The first leader sequence of the variable region of the α chain and the second leader sequence of the variable region of the β chain are well known to those skilled in the art. For example, the first leader sequence of the variable region of the α chain can use an amino acid sequence such as a leader sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53, and the second leader sequence of the variable region of the β chain can use an amino acid sequence such as a leader sequence shown in SEQ ID NO: 54 or SEQ ID NO: 55.

[0167] The amino acid sequence shown in SEQ ID NO:52 is: MLTASLLRAVIASICVVSSM; the amino acid sequence shown in SEQ ID NO:53 is: MLLLLVPAFQVIFTLGGTR; the amino acid sequence shown in SEQ ID NO:54 is: MGIRLLCRVAFCFLAVGLV; the amino acid sequence shown in SEQ ID NO:55 is: MDTWLVCWAIFSLLKAGLT;

[0168] In one embodiment, the amino acid sequence of the α chain variable region is as shown in SEQ ID NO:44 or SEQ ID NO:45, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:44 or SEQ ID NO:45, and / or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO:46 or SEQ ID NO:47, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:46 or SEQ ID NO:47,

[0169] The amino acid sequence shown in SEQ ID NO:44 is:

[0170] The amino acid sequence shown in SEQ ID NO:45 is:

[0171] The amino acid sequence shown in SEQ ID NO:46 is:

[0172] The amino acid sequence shown in SEQ ID NO:47 is:

[0173] The amino acid sequence of the α chain variable region has at least 90% sequence identity with SEQ ID NO: 44 or SEQ ID NO: 45, and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 44 or SEQ ID NO: 45. The amino acid sequence of the β chain variable region has at least 90% sequence identity with SEQ ID NO: 46 or SEQ ID NO: 47, and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 46 or SEQ ID NO: 47.

[0174] In one embodiment, the α chain further comprises an α constant region, and / or the β chain further comprises a β constant region, preferably, the constant region is a mouse constant region or a human constant region. For example, the amino acid sequence of the mouse α constant region is as shown in SEQ ID NO: 56; and / or the amino acid sequence of the mouse β constant region is as shown in SEQ ID NO: 57. That is, the constant regions of the α chains of the above-mentioned TCRs can all have the same constant region, and similarly, the constant regions of the β chains of all TCRs can also have the same constant region.

[0175] The amino acids shown in SEQ ID NO:56 are:

[0176] The amino acids shown in SEQ ID NO:57 are:

[0177] The constant region of the TCR can contain a short linker sequence in which cysteine ​​residues form a disulfide bond, thereby connecting the two chains of the TCR. The TCR can have additional cysteine ​​residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant region.

[0178] In one embodiment, the amino acid sequence of the α chain variable region of the TCR is as shown in SEQ ID NO:44 and the amino acid sequence of the β chain variable region is as shown in SEQ ID NO:46, or the amino acid sequence of the α chain variable region is as shown in SEQ ID NO:45 and the amino acid sequence of the β chain variable region is as shown in SEQ ID NO:47.

[0179] In one embodiment, an artificial disulfide bond is introduced between the residues of the constant regions of the α chain and the β chain of the TCR. The positions of the disulfide bonds that can be introduced are well known to those skilled in the art.

[0180] In one embodiment, the TCR is isolated or purified or recombinant.

[0181] In one embodiment, the TCR is human.

[0182] In one embodiment, the TCR is monoclonal.

[0183] In one embodiment, the TCR is single chain.

[0184] In one embodiment, the TCR comprises two chains.

[0185] TCRs can be obtained from biological sources, such as from cells (e.g., from T cells (e.g., cytotoxic T cells)), T cell hybridomas, or other publicly available resources, for example, TCRs can be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals, such as typically from human.

[0186] In some embodiments, the TCR may be in a cell-bound form or a soluble form, preferably a soluble form. The soluble form of the TCR refers to a TCR that has mutations in its hydrophobic core region, and these mutations in the hydrophobic core region are preferably mutations that can improve the stability of the soluble TCR of the present application.

[0187] The present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the TCR or the α chain or β chain of the TCR.

[0188] In one embodiment, the nucleotide sequence encoding the α chain is the nucleotide sequence shown in SEQ ID NO:48 or SEQ ID NO:49; and / or the nucleotide sequence encoding the β chain is the nucleotide sequence shown in SEQ ID NO:50 or SEQ ID NO:51.

[0189] The nucleotide sequence shown in SEQ ID NO:48 is:

[0190] The nucleotide sequence shown in SEQ ID NO:49 is:

[0191] The nucleotide sequence shown in SEQ ID NO:50 is:

[0192] The nucleotide sequence shown in SEQ ID NO:51 is:

[0193] In one embodiment, the nucleotide sequence encoding the alpha chain and / or the nucleotide sequence encoding the beta chain are codon-optimized. Typically, codon optimization involves balancing the percentage of selected codons with the abundance of disclosed human transfer RNAs so that none of them is overloaded or restricted. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Codon usage differences between transfected genes and host cells may affect protein expression and immunogenicity of nucleic acid constructs. Typically, for codon optimization, codons are selected to select those codons that are balanced with human usage frequency. Typically, the redundancy of amino acid codons is such that different codons encode a single amino acid. In some embodiments, when selecting a codon for replacement, the resulting mutation may be a silent mutation so that the codon change does not affect the amino acid sequence. Typically, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence.

[0194] The present application provides a vector comprising the nucleic acid molecule described above.

[0195] For example, one or more nucleic acids encoding one or both chains of the TCR described above are cloned into one or more suitable expression vectors, which can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses.

[0196] The vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific to the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), taking into account whether the vector is DNA-based or RNA-based. The vector may also contain a non-natural promoter operably linked to the nucleotide sequence encoding the TCR. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat sequence of the mouse stem cell virus, and other promoters known to those skilled in the art are also contemplated.

[0197] The vector is an expression vector, preferably a viral vector, preferably a retroviral vector, and more preferably a lentiviral vector.

[0198] The application also provides a host cell comprising the nucleic acid molecules described above, and in order to recombinantly produce TCR, the nucleic acid encoding TCR can be separated and inserted into one or more vectors to further clone and / or express in the host cell. Conventional techniques can be used (for example, by using oligonucleotide probes that can bind specifically to the genes encoding the α chain and β chain of the TCR) to easily separate and sequence such nucleic acids. In some embodiments, a method for preparing TCR is provided, wherein the method includes culturing a host cell comprising a nucleic acid encoding TCR as provided above under conditions suitable for expressing the TCR molecule, and optionally recovering the TCR from the host cell (or host cell culture medium).

[0199] The term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations.

[0200] The present application also provides an engineered cell comprising the T cell receptor (TCR), the nucleic acid molecule or the vector.

[0201] In one embodiment, the TCR is allogeneic to the cell.

[0202] In one embodiment, the engineered cell is a cell line.

[0203] In one embodiment, the engineered cell is a primary cell obtained from a subject, preferably a mammalian subject, preferably a human.

[0204] In one embodiment, the engineered cells are T cells, preferably T cells isolated from peripheral blood.

[0205] In one embodiment, the T cells are CD8+ or CD4+.

[0206] In some embodiments, the engineered cell is a cell that is derived from blood, bone marrow, lymph or lymphoid organs, and is a cell of the immune system, such as a cell of innate immunity or adaptive immunity, such as bone marrow or lymphoid cells (including lymphocytes, typically T cells and / or NK cells). Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSCs). Cell is typically primary cells, such as directly separated from a subject and / or separated and frozen from a subject. In some embodiments, cell includes one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells and subgroups thereof.

[0207] Subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, etc.

[0208] In some embodiments, the engineered cells are natural killer (NK) cells, preferably, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils.

[0209] The present application provides a method for producing the engineered cells described above, which comprises introducing the nucleic acid molecule or the vector into cells in vitro or ex vivo.

[0210] The vector is a viral vector, and the introduction is performed by transduction.

[0211] The present application provides a pharmaceutical composition comprising the above-mentioned T cell receptor (TCR), the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned engineered cell.

[0212] In one embodiment, it further comprises a pharmaceutically acceptable carrier or adjuvant.

[0213] The pharmaceutically acceptable carrier or adjuvant refers to a component in a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers or adjuvants include but are not limited to buffers, excipients, stabilizers or preservatives.

[0214] The pharmaceutical composition can utilize timed release, delayed release, and sustained release delivery systems so that delivery of the composition occurs before sensitization of the treated area and has sufficient time to cause sensitization. Many types of release delivery systems are available and known. Such systems can be used to avoid repeated administration of the composition, thereby increasing the convenience of the subject and the physician.

[0215] The present application provides the use of the aforementioned T cell receptor (TCR), the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned engineered cell or the aforementioned pharmaceutical composition in the preparation of a drug for treating malignant tumors.

[0216] In one embodiment, the malignant tumor is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, prostate cancer, etc.

[0217] In one embodiment, the colorectal cancer is metastatic colorectal cancer.

[0218] The T cell receptor (TCR) of the present application can specifically recognize A1101-restricted-KRAS G12D Mutation, T cells transduced with the TCR of the present application (TCR-T) can bind to the antigen short peptide KRAS G12D -HLA-A1101 complex binding, targeting tumor antigen-specific killing of tumor cells, can be used to treat KRAS G12D Mutated malignant tumors.

[0219] And the T cells transduced with the TCR of the present application can express A11 and KRAS G12D Mutated tumor cells are specifically activated and have good specificity, recognizing only KRAS G12D mutations, but not wild-type KRAS and KRAS G12V mutation.

[0220] Example

[0221] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0222] Example 1: Cloning of KRAS G12V Acquisition of antigen short peptide-specific T cells and TCR genes

[0223] Peripheral blood lymphocytes from healthy volunteers with the HLA-A1101 genotype were stimulated with a synthetic peptide (SEQ ID NO: 27: VVVGAVGVGK) (Jiangsu GenScript Biotechnology Co., Ltd.). The VVVGAVGVGK peptide was annealed with biotin-labeled HLA-A1101 to prepare pHLA (peptide-human leukocyte antigen complex) monomers. These monomers were combined with PE-labeled streptavidin (BD Company) to form PE (phycoerythrin)-labeled tetramers (the preparation method of pHLA monomers and tetramers referred to the protocol published by the NIH Tetramer Core Facility, and the specific steps are shown on the website https: / / tetramer.yerkes.emory.edu / support / protocols#1). The tetramers and anti-CD8-FITC (fluorescein isothiocyanate) double-positive cells were enriched, and the obtained double-positive cells were flow cytometry sorted to obtain single cells, which are antigen-specific T cells. The sorted single cells were used to amplify the TCR α chain and β chain respectively using a one-step RT-PCR kit (QIAGEN, catalog number 210212), and the PCR products were sequenced. By comparing the sequencing results with the sequences in the public database of IMGT (International Immunogenetics Information System), the nucleotide sequences of the α chain variable region and the β chain variable region of TCR (TCR059 and TCR076) and their CDR1, CDR2, and CDR3 information can be obtained.

[0224] The nucleotide sequence of the α chain variable region of TCR059 is: SEQ ID NO: 28:

[0225] The nucleotide sequence of the β chain variable region of TCR059 is: SEQ ID NO: 29:

[0226] The amino acid sequence of the complementarity determining region 1 (CDR1) of the α chain of TCR059 is: SEQ ID NO: 1: DRVSQS;

[0227] The amino acid sequence of the complementarity determining region 2 (CDR2) of the α chain of TCR059 is: SEQ ID NO: 3: IYSNGD;

[0228] The amino acid sequence of the complementarity determining region 3 (CDR3) of the α chain of TCR059 is: SEQ ID NO: 5: AAVSGGSYIPT;

[0229] The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain of TCR059 is: SEQ ID NO: 7: SGDLS;

[0230] The amino acid sequence of the complementarity determining region 2 (CDR2) of the β chain of TCR059 is: SEQ ID NO: 9: YYNGEE;

[0231] The amino acid sequence of the complementarity determining region 3 (CDR3) of the β chain of TCR059 is: SEQ ID NO: 11: ASSVGGLAGELLETQY;

[0232] The nucleotide sequence of the α chain variable region of TCR076 is: SEQ ID NO: 30:

[0233] The nucleotide sequence of the β chain variable region of TCR076 is: SEQ ID NO: 31:

[0234] The amino acid sequence of the complementarity determining region 1 (CDR1) of the α chain of TCR076 is: SEQ ID NO: 2: TSENNYY;

[0235] The amino acid sequence of the complementarity determining region 2 (CDR2) of the α chain of TCR076 is: SEQ ID NO: 4: QEAYKQQN;

[0236] The amino acid sequence of the complementarity determining region 3 (CDR3) of the α chain of TCR076 is: SEQ ID NO: 6: AFMNGETSGSRLT;

[0237] The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain of TCR076 is: SEQ ID NO: 8: SQVTM;

[0238] The amino acid sequence of the complementarity determining region 2 (CDR2) of the β chain of TCR076 is: SEQ ID NO: 10: ANQGSEA;

[0239] The amino acid sequence of the complementarity determining region 3 (CDR3) of the β chain of TCR076 is: SEQ ID NO: 12: SVIPHGLYEQY.

[0240] Example 2: Construction of VVVGAVGVGK antigen short peptide-specific TCR lentiviral vector and lentiviral packaging

[0241] (1) Construction of TCR lentiviral vector

[0242] The VVVGAVGVGK TCR α and β chain variable region sequences were cloned into a pLKO-based expression plasmid (Addgene). The α or β variable domains were cloned into a pLKO-based expression plasmid containing the murine α or β constant region using standard methods using the Multi-Fragment Recombination Cloning Kit (Novozymes Biotech, catalog number C113). The ligated plasmids were transformed into competent Escherichia coli strain Stbl3 cells (Shanghai Weidi Biotechnology Co., Ltd.) and plated on LB / agar plates containing 100 μg / ml ampicillin. After overnight incubation at 37°C, individual colonies were picked and grown overnight at 37°C in 10 ml of LB containing 100 μg / ml ampicillin with shaking. The cloned plasmids were purified using a miniprep kit (TIANGEN Biotech, catalog number DP118-02) and sequenced to obtain the VVVGAVGVGK TCR (i.e., TCR059, TCR076) plasmids.

[0243] (2) Lentiviral packaging

[0244] Assay culture medium: 10% FBS (Lonsera, catalog number S711-001), DMEM (ThermoFisher, catalog number C11995500BT).

[0245] Prepare 293T cells (American Type Culture Collection, ATCC, catalog number CRL-1573) in a 10cm dish and begin plasmid transfection when no more than 80% confluency is reached. Use a 1:1 ratio of viral packaging plasmid to VVVGAVGVGK TCR plasmid, for a total of 10 μg. Mix the plasmids in serum-free DMEM medium with PEI (polyethylenimine). Add this mixture to the 293T cells and incubate at 37°C. After 72 hours, concentrate the supernatant using a 100 kD ultrafiltration tube to collect the viral vector.

[0246] Example 3: Construction and functional characterization of a Jurkat cell line expressing a VVVGAVGVGK antigen peptide-specific TCR

[0247] Nuclear Factor of Activated T Cells (NFAT) Reporter Gene Expression Method

[0248] The following experiment was performed to demonstrate the specific activation response of TCR-transduced T cells to target cells. NFAT expression was measured using flow cytometry as a readout of T cell activation.

[0249] (1) Reagents

[0250] Assay medium: 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT)

[0251] (2) Method

[0252] Target cell preparation

[0253] The target cells used in this experiment were T2-A11 cells (T2 cells were deposited in ATCC with the deposit catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). Target cells were prepared in experimental culture medium and the target cell concentration was adjusted to 1.6×10 6 50 μl was taken per well to obtain 80,000 cells / well.

[0254] Effector cell preparation

[0255] The effector cells in this experiment were Jurkat-CD8-NFAT (JK8NF) cells transduced with the TCR of the present application, and JK8NF cells not transfected with the TCR of the present application were used as a control group.

[0256] JK8NF cells (Jurkat cells were deposited in ATCC with the deposit catalog number TIB-152. JK8NF cells were constructed based on Jurkat cells with reference to Cancer Res 2006; 66(23):11455-61, Front. Immunol. 11:633) were added with the lentivirus carrying the TCR gene of the present application obtained in Example 2 at an MOI (multiplicity of infection) of 10. After 72 hours, the transfection positive rate was determined by flow cytometry to be approximately 100% (the results are shown in FIG1 ). The concentration of the effector cells after expansion culture was adjusted to 1.6×10 6 50 μl was taken per well to obtain 80,000 cells / well.

[0257] Preparation of short peptide solution

[0258] The original concentration of 5 mg / ml short peptide (VVVGAVGVGK) was diluted to 400 μg / ml, and then diluted down in 10-fold ratio to 40 μg / ml, 4 μg / ml, 0.4 μg / ml, 0.04 μg / ml, 0.004 μg / ml, 0.0004 μg / ml, and 0.00004 μg / ml.

[0259] 50 μl was taken from each well to make the final concentration of the short peptide in the 96-well plate 100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0.001 μg / ml, 0.0001 μg / ml, and 0.00001 μg / ml, respectively.

[0260] Finally, add 50 μl of target cells, 50 μl of effector cells, 50 μl of short peptide dilution of corresponding concentration and 50 ul of culture medium to each well of a 96-well flat-bottom plate and incubate in a cell culture incubator at 37 degrees for 12 hours.

[0261] (3) Results

[0262] The expression of NFAT in the TCR-transduced T cells of the present application in response to target cells loaded with the VVVGAVGVGK antigen peptide was tested by the above method. The expression level curve of NFAT was plotted using Graphpad Prism 8, and the results are shown in FIG2 .

[0263] As can be seen from FIG2 , T cells transduced with the TCR of the present application have a good activation response to target cells loaded with their specific short peptides.

[0264] Example 4: In vitro functional characterization of primary T cells expressing VVVGAVGVGK antigen peptide-specific TCR using IFN gamma ELISPOT method

[0265] (1) Reagents

[0266] Assay medium: ELISPOT kit (BD, catalog number 551849), 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT)

[0267] (2) Method

[0268] Effector T cell preparation

[0269] The effector cells (T cells) of this experiment were T cells transduced with the TCR of the present application, and T cells of the same volunteer that were not transfected with the TCR of the present application were used as control T cells (TCR negative control group).

[0270] The peripheral blood of volunteers was subjected to density gradient centrifugation to obtain peripheral blood mononuclear cells, and the peripheral blood mononuclear cells were plated at 5.0×10 5 / 500μl was placed in the well, and a total of 1x10 6T cells were stimulated with anti-CD3 / CD28 magnetic beads and then cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cell clumping was observed and the TCR lentivirus obtained in Example 3 was added at an MOI (multiplicity of infection) of 2 for transduction. The cells were then expanded in 1640 medium containing 10% FBS and 200 IU / ml IL-2 until 3-4 days after transduction. The TCR transfection efficiency was determined by flow cytometry (the results are shown in Figure 3). The concentration of the effector cells after expansion was adjusted to 5.0×10 4 positive cells / ml, 100 μl was taken from each well to obtain 5000 positive cells / well.

[0271] In the T2-A11 target cell positive control group, T2-A11+KRAS G12V , the number of TCR-T cells was 2000 / well.

[0272] Target cell preparation

[0273] The target cells used in this experiment, SW620 (purchased from ATCC, catalog number CCL-227), inherently express the G12V mutation and lack A11 expression. Therefore, SW620-A11 cells were constructed using a lentiviral vector to overexpress the A11 gene. SW620-A11 cells served as one target cell group, and SW620 cells served as a negative control group. The cell count was 50,000 cells / well. Both groups were treated with an HLA class I neutralizing antibody (anti-HLA I, clone W6 / 32, purchased from Biolegend, catalog number 311402) as a control group. The T2-A11 cells used in this experiment were the target cell positive control group (T2 cells were deposited in ATCC with the deposit catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). The number of cells used was 20,000 cells / well.

[0274] Preparation of short peptide solution

[0275] The original concentration of 5 mg / ml of the short peptide (VVVGAVGVGK) was diluted to 4 μg / ml, and 50 μl was taken to make the final concentration of the short peptide in the 96-well plate 1 μg / ml.

[0276] ELISPOT assay

[0277] Prepare the plates according to the manufacturer's instructions (BD, catalog number 551849) as follows: Dilute anti-human IFN-γ capture antibody 1:400 in 5 ml of sterile PBS per plate, then aliquot 50 μl of the diluted capture antibody into each well. Incubate the plates overnight at 4°C. After incubation, wash the plates to remove excess capture antibody. Block the plates by adding 200 μl of PBS containing 10% FBS and incubate at room temperature for 2 hours. Discard the blocking solution and flick and tap the ELISPOT plates to remove any residual blocking solution.

[0278] The corresponding target and effector cells and the corresponding peptide were then added, and the plates were incubated overnight (37°C / 5% CO2). The next day, the medium was discarded, and the plates were washed twice with double-distilled water and three times with wash buffer, patting gently on a paper towel to remove any residual wash buffer. The detection antibody was then diluted 1:400 in PBS containing 10% FBS, and 100 μl / well was added to each well. The plates were incubated at room temperature for 2 hours, then washed three times with wash buffer, patting gently on a paper towel to remove any excess wash buffer.

[0279] Dilute streptavidin-alkaline phosphatase 1:200 in PBS containing 10% FBS. Add 100 μL of the diluted streptavidin-alkaline phosphatase to each well and incubate the plate at room temperature for 1 hour. Then, wash three times with wash buffer and three times with PBS. Tap the plate gently on a paper towel to remove excess wash buffer and PBS. After washing, add 100 μL of the BCIP / NBT solution provided in the kit per well for development. Cover the plate with aluminum foil to protect it from light during development and let it stand for 5-15 minutes. During this time, routinely inspect the spots on the developed plate to determine the optimal time to terminate the reaction. Remove the BCIP / NBT solution and rinse the plate with deionized water to terminate the development reaction. Shake dry, then remove the bottom of the plate and dry it at room temperature until each well is completely dry. Count the spots formed on the inner membrane of the plate using an immunospot plate counter. The function of TCR-transduced T cells of the present invention was tested using an ELISPOT assay (described above). The number of ELISPOT spots observed in each well was plotted using GraphPad Prism 6.

[0280] (3) Results

[0281] As shown in Figure 4, T cells TCR059 and TCR076, which express the VVVGAVGVGK-A1101 antigen peptide-specific TCR, strongly activated the SW620-A11 cell line, while control T cells showed no activation response. Addition of HLA class I neutralizing antibodies inhibited TCR059 and TCR076 recognition of the SW620-A11 cell line.

[0282] Example 5: Identification of cross-reactivity of VVVGAVGVGK antigen short peptide-specific TCR

[0283] Nuclear Factor of Activated T Cells (NFAT) Reporter Gene Expression Method

[0284] The following experiments were performed to demonstrate that the VVVGAVGVGK antigen peptide-specific TCR is resistant to wild-type KRAS and KRAS G12D The mutations were not cross-reactive.

[0285] Flow cytometry was used to detect NFAT expression as a readout of T cell activation.

[0286] (1) Reagents

[0287] Assay culture medium: 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT).

[0288] (2) Method

[0289] Target cell preparation

[0290] The target cells used in this experiment were T2-A11 cells (T2 cells were deposited in ATCC with the deposit catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). Target cells were prepared in experimental culture medium and the target cell concentration was adjusted to 1.6×10 6 50 μl was taken per well to obtain 80,000 cells / well.

[0291] Effector cell preparation

[0292] The effector cells in this experiment were Jurkat-CD8-NFAT (JK8NF) cells transduced with the TCR of the present application, and JK8NF cells not transfected with the TCR of the present application were used as a control group.

[0293] JK8NF cells (Jurkat cells were deposited in ATCC with the deposit catalog number TIB-152. JK8NF cells were constructed based on Jurkat cells with reference to Cancer Res 2006; 66(23):11455-61, Front. Immunol. 11:633) were added with the lentivirus carrying the TCR gene of the present application obtained in Example 2 at an MOI (multiplicity of infection) of 10. After 72 hours, the transfection positive rate was determined by flow cytometry to be approximately 100% (the results are shown in FIG1 ). The concentration of the effector cells after expansion culture was adjusted to 1.6×106 50 μl was taken per well to obtain 80,000 cells / well.

[0294] Preparation of short peptide solution

[0295] The original concentration of 5 mg / ml short peptide (VVVGAVGVGK(G12V), VVVGAGGVGK(WT)VVVGADGVGK(G12D)) was diluted to 400 μg / ml, and then diluted down in 10-fold ratio to 40 μg / ml, 4 μg / ml, 0.4 μg / ml, 0.04 μg / ml, 0.004 μg / ml, 0.0004 μg / ml, and 0.00004 μg / ml.

[0296] 50 μl was taken from each well to make the final concentration of the short peptide in the 96-well plate 100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0.001 μg / ml, 0.0001 μg / ml, and 0.00001 μg / ml, respectively.

[0297] Finally, add 50 μl of target cells, 50 μl of effector cells, 50 μl of short peptide dilution of corresponding concentration and 50 ul of culture medium to each well of a 96-well flat-bottom plate and incubate in a cell culture incubator at 37 degrees for 12 hours.

[0298] (3) Results

[0299] The TCR-transduced T cells of the present application were tested for KRAS-loaded T cells by the above method. G12V , KRAS WT and KRAS G12D The expression of NFAT in target cells responsive to the three antigenic peptides was plotted using Graphpad Prism8, and the results are shown in FIG5 .

[0300] As can be seen from Figure 5, the TCR of this application has good specificity and only recognizes KRAS G12V mutations, but not wild-type KRAS and KRAS G12D mutation.

[0301] In summary, the T cells transduced with the TCR in this application can express A11+KRAS G12V The mutant complex is activated specifically in tumor cells and has good specificity, recognizing only KRAS G12V mutations, but not wild-type KRAS and KRAS G12D mutation.

[0302] Example 6: Cloning of KRAS G12DAcquisition of antigen short peptide-specific T cells and TCR genes

[0303] Peripheral blood lymphocytes from healthy volunteers with the HLA-A1101 genotype were stimulated with a synthetic peptide (SEQ ID NO: 58: VVVGADGVGK) (Jiangsu GenScript Biotechnology Co., Ltd.). The VVVGADGVGK peptide was annealed with biotin-labeled HLA-A1101 to prepare pHLA (peptide-human leukocyte antigen complex) monomers. These monomers were combined with PE-labeled streptavidin (BD Company) to form PE (phycoerythrin)-labeled tetramers (the preparation method of pHLA monomers and tetramers referred to the protocol published by the NIH Tetramer Core Facility, and the specific steps are shown on the website https: / / tetramer.yerkes.emory.edu / support / protocols#1). The tetramers and anti-CD8-FITC (fluorescein isothiocyanate) double-positive cells were enriched, and the obtained double-positive cells were flow cytometry sorted to obtain single cells, which are antigen-specific T cells. The sorted single cells were used to amplify the TCR α chain and β chain respectively using a one-step RT-PCR kit (QIAGEN, catalog number 210212), and the PCR products were sequenced. By comparing the sequencing results with the sequences in the public database of IMGT (International Immunogenetics Information System), the nucleotide sequences of the α chain variable region and the β chain variable region of TCR (TCR104 and TCR106) and their CDR1, CDR2, and CDR3 information can be obtained.

[0304] The nucleotide sequence of the α chain variable region of TCR104 is: SEQ ID NO: 59:

[0305] The nucleotide sequence of the β chain variable region of TCR104 is: SEQ ID NO: 60:

[0306] The amino acid sequence of the complementarity determining region 1 (CDR1) of the α chain of TCR104 is: SEQ ID NO: 32: TRDTTYY;

[0307] The amino acid sequence of the complementarity determining region 2 (CDR2) of the α chain of TCR104 is: SEQ ID NO: 34: RNSFDEQN;

[0308] The amino acid sequence of the complementarity determining region 3 (CDR3) of the α chain of TCR104 is: SEQ ID NO: 36: ALSEAAPGGSYIPT;

[0309] The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain of TCR104 is: SEQ ID NO: 38: MDHEN;

[0310] The amino acid sequence of the complementarity determining region 2 (CDR2) of the β chain of TCR104 is: SEQ ID NO: 40: SYDVKM;

[0311] The amino acid sequence of the complementarity determining region 3 (CDR3) of the β chain of TCR104 is: SEQ ID NO: 42: ASSLGPGQHNSPLH;

[0312] The nucleotide sequence of the α chain variable region of TCR106 is: SEQ ID NO: 61:

[0313] The nucleotide sequence of the β chain variable region of TCR106 is: SEQ ID NO: 62:

[0314] The amino acid sequence of the complementarity determining region 1 (CDR1) of the α chain of TCR106 is: SEQ ID NO: 33: SSVSVY;

[0315] The amino acid sequence of the complementarity determining region 2 (CDR2) of the α chain of TCR106 is: SEQ ID NO: 35: YLSGSTLV;

[0316] The amino acid sequence of the complementarity determining region 3 (CDR3) of the α chain of TCR106 is: SEQ ID NO: 37: AVIGNDYKLS;

[0317] The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain of TCR106 is: SEQ ID NO: 39: SNHLY;

[0318] The amino acid sequence of the complementarity determining region 2 (CDR2) of the β chain of TCR106 is: SEQ ID NO: 41: FYNNEI;

[0319] The amino acid sequence of the complementarity determining region 3 (CDR3) of the β chain of TCR106 is: SEQ ID NO: 43: ASSGTGGIEAF.

[0320] Example 7: Construction of VVVGADGVGK antigen short peptide-specific TCR lentiviral vector and lentiviral packaging

[0321] (1) Construction of TCR lentiviral vector

[0322] The VVVGADGVGK TCR α and β chain variable region sequences were cloned into a pLKO-based expression plasmid (Addgene). The α or β variable domains were cloned into a pLKO-based expression plasmid containing the murine α or β constant region using standard methods using the Multi-Fragment Recombination Cloning Kit (Novozymes Biotech, catalog number C113). The ligated plasmids were transformed into competent Escherichia coli strain Stbl3 cells (Shanghai Weidi Biotechnology Co., Ltd.) and plated on LB / agar plates containing 100 μg / ml ampicillin. After overnight incubation at 37°C, individual colonies were picked and grown overnight at 37°C in 10 ml of LB containing 100 μg / ml ampicillin with shaking. The cloned plasmids were purified using a miniprep kit (TIANGEN Biotech, catalog number DP118-02) and sequenced to obtain the VVVGADGVGK TCR (i.e., TCR104, TCR106) plasmids.

[0323] (2) Lentiviral packaging

[0324] Assay culture medium: 10% FBS (Lonsera, catalog number S711-001), DMEM (ThermoFisher, catalog number C11995500BT).

[0325] Prepare 293T cells (American Type Culture Collection, ATCC, catalog number CRL-1573) in a 10cm dish and begin plasmid transfection when no more than 80% confluency is reached. Use a 1:1 ratio of viral packaging plasmid to VVVGAVGVGK TCR plasmid, for a total of 10 μg. Mix the plasmids in serum-free DMEM medium with PEI (polyethylenimine). Add this mixture to the 293T cells and incubate at 37°C. After 72 hours, concentrate the supernatant using a 100 kD ultrafiltration tube to collect the viral vector.

[0326] Example 8: Construction and functional characterization of a Jurkat cell line expressing a VVVGADGVGK antigen peptide-specific TCR

[0327] Nuclear Factor of Activated T Cells (NFAT) Reporter Gene Expression Method

[0328] The following experiment was performed to demonstrate the specific activation response of TCR-transduced T cells to target cells. NFAT expression was measured using flow cytometry as a readout of T cell activation.

[0329] (1) Reagents

[0330] Assay culture medium: 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT).

[0331] (2) Method

[0332] Target cell preparation

[0333] The target cells used in this experiment were T2-A11 cells (T2 cells were deposited in ATCC with the deposit catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). Target cells were prepared in experimental culture medium and the target cell concentration was adjusted to 1.6×10 6 50 μl was taken per well to obtain 80,000 cells / well.

[0334] Effector cell preparation

[0335] The effector cells in this experiment were Jurkat-CD8-NFAT (JK8NF) cells transduced with the TCR of the present application, and JK8NF cells not transfected with the TCR of the present application were used as a control group.

[0336] JK8NF cells (Jurkat cells were deposited in ATCC with the deposit catalog number TIB-152. JK8NF cells were constructed based on Jurkat cells with reference to Cancer Res 2006; 66(23):11455-61, Front. Immunol. 11:633) were added with the lentivirus carrying the TCR gene of the present application obtained in Example 2 at an MOI (multiplicity of infection) of 10. After 72 hours, the transfection positive rate was determined by flow cytometry to be approximately 100% (the results are shown in FIG1 ). The concentration of the effector cells after expansion culture was adjusted to 1.6×10 6 50 μl was taken per well to obtain 80,000 cells / well.

[0337] Preparation of short peptide solution

[0338] The original concentration of 5 mg / ml short peptide (VVVGADGVGK) was diluted to 400 μg / ml, and then diluted down in 10-fold ratio to 40 μg / ml, 4 μg / ml, 0.4 μg / ml, 0.04 μg / ml, 0.004 μg / ml, 0.0004 μg / ml, and 0.00004 μg / ml.

[0339] 50 μl was taken from each well to make the final concentration of the short peptide in the 96-well plate 100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0.001 μg / ml, 0.0001 μg / ml, and 0.00001 μg / ml, respectively.

[0340] Finally, add 50 μl of target cells, 50 μl of effector cells, 50 μl of short peptide dilution of corresponding concentration and 50 ul of culture medium to each well of a 96-well flat-bottom plate and incubate in a cell culture incubator at 37 degrees for 12 hours.

[0341] (3) Results

[0342] The expression of NFAT in the TCR-transduced T cells of the present application in response to target cells loaded with the VVVGADGVGK antigen peptide was tested by the above method. The expression level curve of NFAT was plotted using Graphpad Prism 8, and the results are shown in FIG2 .

[0343] As can be seen from FIG2 , T cells transduced with the TCR of the present application have a good activation response to target cells loaded with their specific short peptides.

[0344] Example 9: In vitro functional characterization of primary T cells expressing VVVGADGVGK antigen peptide-specific TCR using IFN gamma ELISPOT method

[0345] (1) Reagents

[0346] Assay medium: ELISPOT kit (BD, catalog number 551849), 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT).

[0347] (2) Method

[0348] Effector T cell preparation

[0349] The effector cells (T cells) of this experiment were T cells transduced with the TCR of the present application, and T cells of the same volunteer that were not transfected with the TCR of the present application were used as control T cells (TCR negative control group).

[0350] The peripheral blood of volunteers was subjected to density gradient centrifugation to obtain peripheral blood mononuclear cells, and the peripheral blood mononuclear cells were plated at 5.0×10 5 / 500μl was placed in the well, and a total of 1x10 6T cells were stimulated with anti-CD3 / CD28 magnetic beads and then cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cell clumping was observed. After transduction with the TCR lentivirus obtained in Example 2 at an MOI (multiplicity of infection) of 2, the cells were expanded in 1640 medium containing 10% FBS and 200 IU / ml IL-2 until 3-4 days after transduction. The TCR transfection efficiency was determined by flow cytometry (the results are shown in Figure 3). The concentration of the effector cells after expansion culture was adjusted to 5.0×10 4 positive cells / ml, 100 μl was taken from each well to obtain 5000 positive cells / well.

[0351] In the T2-A11 target cell positive control group, T2-A11+KRAS G12D , the number of TCR-T cells was 2000 / well.

[0352] Target cell preparation

[0353] The target cells used in this experiment, panc0813 (purchased from ATCC, catalog number CRL-2551), inherently express the G12D mutation and do not express A11. Therefore, panc0813-A11 cells were constructed by overexpressing the A11 gene via a lentiviral vector. Panc0813-A11 cells served as one target cell group, and panc0813 cells served as a negative control group for target cells. The cell count was 50,000 cells / well. Both groups were treated with an HLA class I neutralizing antibody (anti-HLA I, clone W6 / 32, purchased from Biolegend, catalog number 311402) as a control group. The T2-A11 cells used in this experiment were the target cell positive control group (T2 cells were deposited in ATCC with the deposit catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). The number of cells used was 20,000 cells / well.

[0354] Preparation of short peptide solution

[0355] The original concentration of 5 mg / ml of the short peptide (VVVGADGVGK) was diluted to 4 μg / ml, and 50 μl was taken to make the final concentration of the short peptide in the 96-well plate 1 μg / ml.

[0356] ELISPOT assay

[0357] Prepare the plates according to the manufacturer's instructions (BD, catalog number 551849) as follows: Dilute anti-human IFN-γ capture antibody 1:400 in 5 ml of sterile PBS per plate, then aliquot 50 μl of the diluted capture antibody into each well. Incubate the plates overnight at 4°C. After incubation, wash the plates to remove excess capture antibody. Block the plates by adding 200 μl of PBS containing 10% FBS and incubate at room temperature for 2 hours. Discard the blocking solution and flick and tap the ELISPOT plates to remove any residual blocking solution.

[0358] The corresponding target and effector cells and the corresponding peptide were then added, and the plates were incubated overnight (37°C / 5% CO2). The next day, the medium was discarded, and the plates were washed twice with double-distilled water and three times with wash buffer, patting gently on a paper towel to remove any residual wash buffer. The detection antibody was then diluted 1:400 in PBS containing 10% FBS, and 100 μl / well was added to each well. The plates were incubated at room temperature for 2 hours, then washed three times with wash buffer, patting gently on a paper towel to remove any excess wash buffer.

[0359] Dilute streptavidin-alkaline phosphatase 1:200 in PBS containing 10% FBS. Add 100 μL of the diluted streptavidin-alkaline phosphatase to each well and incubate the plate at room temperature for 1 hour. Then, wash three times with wash buffer and three times with PBS. Tap the plate gently on a paper towel to remove excess wash buffer and PBS. After washing, add 100 μL of the BCIP / NBT solution provided in the kit per well for development. Cover the plate with aluminum foil to protect it from light during development and let it stand for 5-15 minutes. During this time, routinely inspect the spots on the developed plate to determine the optimal time to terminate the reaction. Remove the BCIP / NBT solution and rinse the plate with deionized water to terminate the development reaction. Shake dry, then remove the bottom of the plate and dry it at room temperature until each well is completely dry. Count the spots formed on the inner membrane of the plate using an immunospot plate counter. The function of TCR-transduced T cells of the present invention was tested using an ELISPOT assay (described above). The number of ELISPOT spots observed in each well was plotted using GraphPad Prism 6.

[0360] (3) Results

[0361] As shown in Figure 4, TCR104 and TCR106, which express TCRs specific for the VVVGADGVGK-A1101 antigen peptide, strongly activated the panc0813-A11 cell line, while control T cells showed no activation. Addition of HLA class I neutralizing antibodies inhibited TCR104 and TCR106 recognition of the panc0813-A11 cell line.

[0362] Example 10: Identification of cross-reactivity of TCR specific for VVVGADGVGK antigen short peptide

[0363] Nuclear Factor of Activated T Cells (NFAT) Reporter Gene Expression Method

[0364] The following experiments were performed to demonstrate that the VVVGADGVGK antigen peptide-specific TCR is resistant to wild-type KRAS and KRAS G12D The mutations were not cross-reactive.

[0365] Flow cytometry was used to detect NFAT expression as a readout of T cell activation.

[0366] (1) Reagents

[0367] Assay culture medium: 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT).

[0368] (2) Method

[0369] Target cell preparation

[0370] The target cells used in this experiment were T2-A11 cells (T2 cells were deposited in ATCC with the deposit catalog number CRL-1992. T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032). Target cells were prepared in experimental culture medium and the target cell concentration was adjusted to 1.6×10 6 50 μl was taken per well to obtain 80,000 cells / well.

[0371] Effector cell preparation

[0372] The effector cells in this experiment were Jurkat-CD8-NFAT (JK8NF) cells transduced with the TCR of the present application, and JK8NF cells not transfected with the TCR of the present application were used as a control group.

[0373] JK8NF cells (Jurkat cells were deposited in ATCC with the deposit catalog number TIB-152. JK8NF cells were constructed based on Jurkat cells with reference to Cancer Res 2006; 66(23):11455-61, Front. Immunol. 11:633) were added with the lentivirus carrying the TCR gene of the present application obtained in Example 2 at an MOI (multiplicity of infection) of 10. After 72 hours, the transfection positive rate was determined by flow cytometry to be approximately 100% (the results are shown in FIG1 ). The concentration of the effector cells after expansion culture was adjusted to 1.6×106 50 μl was taken per well to obtain 80,000 cells / well.

[0374] Preparation of short peptide solution

[0375] The original concentration of 5 mg / ml short peptides (VVVGADGVGK(G12D), VVVGAGGVGK(WT), VVVGAVGVGK(G12V)) was diluted to 400 μg / ml, and then diluted down in 10-fold ratio to 40 μg / ml, 4 μg / ml, 0.4 μg / ml, 0.04 μg / ml, 0.004 μg / ml, 0.0004 μg / ml, and 0.00004 μg / ml.

[0376] 50 μl was taken from each well to make the final concentration of the short peptide in the 96-well plate 100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0.001 μg / ml, 0.0001 μg / ml, and 0.00001 μg / ml, respectively.

[0377] Finally, add 50 μl of target cells, 50 μl of effector cells, 50 μl of short peptide dilution of corresponding concentration and 50 ul of culture medium to each well of a 96-well flat-bottom plate and incubate in a cell culture incubator at 37 degrees for 12 hours.

[0378] (3) Results

[0379] The TCR-transduced T cells of the present application were tested for KRAS-loaded T cells by the above method. G12D , KRAS WT and KRAS G12D The expression of NFAT in target cells responsive to the three antigenic peptides was plotted using Graphpad Prism8, and the results are shown in FIG5 .

[0380] As can be seen from Figure 5, the TCR of this application has good specificity and only recognizes KRAS G12D mutations, but not wild-type KRAS and KRAS G12D mutation.

[0381] In summary, the T cells transduced with the TCR in this application can express A11+KRAS G12D The mutant complex is activated specifically in tumor cells and has good specificity, recognizing only KRAS G12D mutations, but not wild-type KRAS and KRAS G12V mutation.

[0382] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

[0383] Sequence Listing

Claims

1. A T cell receptor (TCR) that binds to a KRAS mutation, wherein: The TCR comprises an α chain containing a variable region and / or a β chain containing a variable region, The variable region of the α chain comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 32 or SEQ ID NO: 2 or SEQ ID NO: 33; and / or The amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 3 or SEQ ID NO: 34 or SEQ ID NO: 4 or SEQ ID NO: 35; and / or The amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO: 5 or SEQ ID NO: 36 or SEQ ID NO: 6 or SEQ ID NO:

37.

2. The T cell receptor (TCR) according to claim 1, wherein The variable region of the beta chain comprises a complementarity determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 38 or SEQ ID NO: 8 or SEQ ID NO: 39; and / or The amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 9 or SEQ ID NO: 40 or SEQ ID NO: 10 or SEQ ID NO: 41; and / or The amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO: 11 or SEQ ID NO: 42 or SEQ ID NO: 12 or SEQ ID NO:

43.

3. The T cell receptor (TCR) according to claim 1 or 2, wherein The variable region of the α chain further comprises a first leader sequence; and / or The variable region of the β chain further includes a second leader sequence, Preferably, the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 13 or SEQ ID NO: 44 or SEQ ID NO: 14 or SEQ ID NO: 45, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 44 or SEQ ID NO: 14 or SEQ ID NO: 45, and / or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 15 or SEQ ID NO: 46 or SEQ ID NO: 16 or SEQ ID NO: 47, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 46 or SEQ ID NO: 16 or SEQ ID NO: 47, Preferably, the α chain further comprises an α constant region, and / or the β chain further comprises a β constant region. Preferably, the constant region is a mouse constant region or a human constant region.

4. The T cell receptor (TCR) according to any one of claims 1 to 3, wherein The TCR is isolated or purified or recombinant; Preferably, the TCR is human; Preferably, the TCR is monoclonal; Preferably, the TCR is single chain; Preferably, the TCR comprises two chains; Preferably, the TCR is in a cell-bound form or a soluble form, preferably a soluble form; Preferably, the TCR binds to the antigen short peptide-HLA-A1101 complex. Preferably, the amino acid sequence of the antigen short peptide is as shown in SEQ ID NO: 1 or SEQ ID NO:

32.

5. A nucleic acid molecule, wherein The nucleic acid molecule comprises a nucleotide sequence encoding the TCR according to any one of claims 1 to 4, or the α chain or β chain of the TCR.

6. The nucleic acid molecule according to claim 5, wherein the nucleotide sequence encoding the α chain is the nucleotide sequence shown in SEQ ID NO: 17 or SEQ ID NO: 48 or SEQ ID NO: 18 or SEQ ID NO: 49; and / or The nucleotide sequence encoding the β chain is the nucleotide sequence shown in SEQ ID NO: 19, SEQ ID NO: 50, SEQ ID NO: 20, or SEQ ID NO:

51.

7. A vector, wherein The vector comprises the nucleic acid molecule according to claim 5 or 6.

8. The carrier according to claim 7, wherein The vector is an expression vector; Preferably, the vector is a viral vector, preferably a retroviral vector; Preferably, the viral vector is a lentiviral vector.

9. An engineered cell comprising the TCR of any one of claims 1-4, the nucleic acid molecule of any one of claims 5-6, or the vector of any one of claims 7-8.

10. The engineered cell according to claim 9, wherein The TCR is heterologous to the cell; Preferably, the engineered cell is a cell line; Preferably, the engineered cells are primary cells obtained from a subject, preferably a mammalian subject, preferably a human; Preferably, the engineered cells are T cells or NK cells, preferably, the T cells are T cells isolated from peripheral blood; Preferably, the T cells are CD8+ or CD4+.

11. A method for producing the engineered cell according to any one of claims 9-10, comprising introducing the nucleic acid molecule according to any one of claims 5-6 or the vector according to any one of claims 7-8 into a cell in vitro or ex vivo.

12. The method according to claim 11, wherein The vector is a viral vector, and the introduction is performed by transduction.

13. A pharmaceutical composition comprising the T cell receptor (TCR) of any one of claims 1-4, the nucleic acid molecule of any one of claims 5-6, the vector of any one of claims 7-8, or the engineered cell of any one of claims 9-10; Preferably, it further comprises a pharmaceutically acceptable carrier or adjuvant.

14. Use of the T cell receptor (TCR) of any one of claims 1-4, the nucleic acid molecule of any one of claims 5-6, the vector of any one of claims 7-8, the engineered cell of any one of claims 9-10, or the pharmaceutical composition of claim 13 in the preparation of a medicament for treating malignant tumors; Preferably, the malignant tumor is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, or prostate cancer.

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