A T cell receptor (TCR) and its use

Abstract

The present application provides a T cell receptor (TCR) and its use, 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 complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; and / or an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:4. The complementary determining region 2 (CDR2); and / or an amino acid sequence as shown in SEQ ID NO:5 or SEQ ID NO:6. The variable region of the β chain comprises a complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:8; and / or an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:10. The complementary determining region 2 (CDR2); and / or an amino acid sequence as shown in SEQ ID NO:11 or SEQ ID NO:12. The TCR of the present application can specifically recognize A1101-restricted KRAS G12V mutation, T cells (TCR-T) transduced with the TCR of the present application can bind to the antigen short peptide-HLA-A1101 complex and can be used to treat KRAS G12V Mutation of malignant tumors.

Description

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 TCRs and their uses. Background Art

[0002] KRAS mutations are common in patients with metastatic colorectal cancer, of which G12D mutations account for 45% and G12V mutations account for 30%. KRAS is a GTPase protein encoded by genes of RAS family members, which participates in epidermal growth factor receptor EGFR signal transduction and regulates cell growth, differentiation, proliferation and survival. RAS mutations lead to GTPase activity defects, resulting in 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 that all patients with metastatic colorectal cancer undergo RAS gene testing, and EGFR monoclonal antibodies can only be used in patients with wild-type RAS. The development of targeted drugs for KRAS has always been difficult, and KRAS was once considered an "undruggable target." In recent years, molecular biology has made significant progress in the study of mutation forms and signaling pathways of RAS proteins, but the development of related targeted drugs is still challenging. In the development of chemical drugs, the hydrophobic pockets on the surface of RAS protein are not obvious due to its smooth structure; 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, was approved for marketing in the United States on May 29, 2021. However, the G12C mutation is more common in lung cancer patients (about 50%) and only accounts for about 10% in colorectal cancer patients. Therefore, new drugs targeting KRAS mutations in colon cancer are urgently needed in the clinic.

[0003] Specific T cell immunotherapy refers to the use of specific T cells targeting tumor antigens to kill tumor cells. It is a highly personalized tumor immunotherapy method. Due to the presence of the local immunosuppressive microenvironment of the tumor, the patient's own T cells have limited ability to kill tumors. Therefore, people try to improve their ability to kill tumors by genetically modifying T cells. Both TCR-T and CAR-T are genetically modified cell therapy drugs. After the transferred T cell receptor (TCR) or chimeric antigen receptor (CAR) gene binds to the corresponding target, T cells can be activated and tumor cells can be cleared by using granzymes, perforins, cytokines, etc. released by T cells; however, the significant difference between TCR-T and CAR-T is that the target of CAR-T is the membrane protein on the cell surface, while the target of TCR-T is the antigen short peptide-MHC complex (pMHC).

[0004] TCR has major histocompatibility complex (MHC) restriction. It has been found that the frequency of human MHC (also known as human leukocyte antigen, human leukocyte antigen, HLA) in different populations varies greatly. The most common HLA types in the Chinese population are A1101 and A2402, and the most common HLA type in Caucasians is A0201. There are limited known TCR targets, and most of them are A0201 presentation targets that are more distributed in Western populations. For example, NY-ESO-1 and HPV E6 / E7, which are currently being sought after by domestic and international TCR-T companies, can only target patients with A0201 typing. Therefore, developing KRAS mutant TCR-T drugs for more HLA-A1101 typing in the Chinese population will benefit more Chinese patients.

[0005] Therefore, those skilled in the art are committed to screening for specific A1101-restricted KRAS G12V Mutated TCRs, thus making them useful in T cell immunotherapy. Summary of the invention

[0006] 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 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. At the same time, the application also provides the use of the T cell receptor.

[0007] Specifically, this application involves the following aspects:

[0008] 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,

[0009] 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

[0010] The amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 3 or SEQ ID NO: 4; and / or

[0011] The amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO:5 or SEQ ID NO:6.

[0012] 2. The T cell receptor (TCR) according to item 1, wherein

[0013] 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: 8; and / or

[0014] The amino acid sequence is the complementarity determining region 2 (CDR2) shown in SEQ ID NO: 9 or SEQ ID NO: 10; and / or

[0015] The amino acid sequence is the complementarity determining region 3 (CDR3) shown in SEQ ID NO:11 or SEQ ID NO:12.

[0016] 3. The T cell receptor (TCR) according to item 1 or 2, wherein

[0017] The variable region of the α chain further comprises a first leader sequence; and / or

[0018] The variable region of the β chain also includes a second leader sequence,

[0019] 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 with SEQ ID NO: 13 or SEQ ID NO: 14, and / or the amino acid sequence of the variable region of the β chain is as shown in SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16,

[0020] 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.

[0021] 4. A T cell receptor (TCR) according to any one of items 1 to 3, wherein the TCR is isolated or purified or recombinant;

[0022] Preferably, the TCR is human;

[0023] Preferably, the TCR is monoclonal;

[0024] Preferably, the TCR is a single chain;

[0025] Preferably, the TCR comprises two chains;

[0026] Preferably, the TCR is in a cell-bound form or in a soluble form, preferably in a soluble form;

[0027] 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.

[0028] 5. A nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the TCR described in any one of items 1 to 4 or the α chain or β chain of the TCR.

[0029] 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

[0030] The nucleotide sequence encoding the β chain is the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0031] 7. A vector, wherein the vector comprises the nucleic acid molecule described in item 5 or 6.

[0032] 8. The vector according to item 7, wherein the vector is an expression vector;

[0033] Preferably, the vector is a viral vector, preferably a retroviral vector;

[0034] Preferably, the viral vector is a lentiviral vector.

[0035] 9. An engineered cell comprising the TCR described in any one of items 1-4, the nucleic acid molecule described in any one of items 5-6, or the vector described in any one of items 7-8.

[0036] 10. The engineered cell of item 9, wherein the TCR is heterologous to the cell;

[0037] Preferably, the engineered cell is a cell line;

[0038] Preferably, the engineered cell is a primary cell obtained from a subject, preferably, the subject is a mammalian subject, preferably a human;

[0039] Preferably, the engineered cells are T cells or NK cells, preferably, the T cells are T cells isolated from peripheral blood;

[0040] Preferably, the T cells are CD8+ or CD4+.

[0041] 11. A method for producing the engineered cell described in any one of items 9 to 10, comprising introducing the nucleic acid molecule described in any one of items 5 to 6 or the vector described in any one of items 7 to 8 into the cell in vitro or ex vivo.

[0042] 12. The method according to item 11, wherein the vector is a viral vector and the introduction is performed by transduction.

[0043] 13. A pharmaceutical composition comprising the T cell receptor (TCR) of any one of items 1-4, the nucleic acid molecule of any one of items 5-6, the vector of any one of items 7-8, or the engineered cell of any one of items 9-10;

[0044] Preferably, it further comprises a pharmaceutically acceptable carrier or adjuvant.

[0045] 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 drug for treating malignant tumors;

[0046] Preferably, the malignant tumor is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, or prostate cancer.

[0047] Effects of the Invention

[0048] 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 Mutation of malignant tumors.

[0049] 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 it has good specificity, only recognizing KRAS G12V mutations, but not wild-type KRAS and KRAS G12D mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It was shown that the TCR described in the present application can be correctly expressed in the Jurkat T cell line.

[0051] Figure 2 It is shown that T cells transduced with the TCR described in this application can be loaded with KRAS G12V Target cells react to short antigenic peptides.

[0052] Figure 3 It was shown that the TCR described in the present application can be correctly expressed in primary T cells.

[0053] Figure 4 It is shown that T cells transduced with the TCR described in this application express A11 and KRAS G12V Release of interferon-γ after co-incubation of mutant tumor cells.

[0054] Figure 5 It 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. DETAILED DESCRIPTION

[0055] The present application is described in detail below in conjunction with the embodiments described in the accompanying drawings, wherein the same numbers in all the accompanying drawings represent the same features. Although specific embodiments of the present application are shown in the accompanying 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. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to be able to fully convey the scope of the present application to those skilled in the art.

[0056] 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 functional differences of 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 the present application shall be determined by the attached claims.

[0057] 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 complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; and / or a complementary determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4; and / or a complementary determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 6,

[0058] The amino acid sequence shown in SEQ ID NO: 1 is: DRVSQS;

[0059] The amino acid sequence shown in SEQ ID NO:2 is: TSENNYY;

[0060] The amino acid sequence shown in SEQ ID NO:3 is: IYSNGD;

[0061] The amino acid sequence shown in SEQ ID NO:4 is: QEAYKQQN;

[0062] The amino acid sequence shown in SEQ ID NO:5 is: AAVSGGSYIPT;

[0063] The amino acid sequence shown in SEQ ID NO:6 is: AFMNGETSGSRLT.

[0064] 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,

[0065] The amino acid sequence shown in SEQ ID NO:7 is: SGDLS;

[0066] The amino acid sequence shown in SEQ ID NO:8 is: SQVTM;

[0067] The amino acid sequence shown in SEQ ID NO:9 is: YYNGEE;

[0068] The amino acid sequence shown in SEQ ID NO: 10 is: ANQGSEA;

[0069] The amino acid sequence shown in SEQ ID NO: 11 is: ASSVGGLAGELLETQY;

[0070] The amino acid sequence shown in SEQ ID NO: 12 is: SVIPHGLYEQY.

[0071] 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: 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 a leader sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24.

[0072] The amino acid sequence shown in SEQ ID NO:21 is: MKSLRVLLVILWLQLSWVWSQ;

[0073] The amino acid sequence shown in SEQ ID NO:22 is: MTRVSLLWAVVVSTCLESGM;

[0074] The amino acid sequence shown in SEQ ID NO:23 is: MGFRLLCCVAFCLLGAGPV;

[0075] The amino acid sequence shown in SEQ ID NO:24 is: MLSLLLLLLGLGSVF;

[0076] 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 having at least 90% sequence identity to SEQ ID NO:13 or SEQ ID NO:14, and / or the amino acid sequence of the variable region of the β chain 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, and the amino acid sequence shown in SEQ ID NO:13 is: MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRVSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAVSGGSYIPTFGRGTSLIVHPY; The amino acid sequence shown in SEQ ID NO:14 is: MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFMNGETSGSRLTFGEGTQLTVNPD; the amino acid sequence shown in SEQ ID NO:15 is: MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVGGLAGELLETQYFGPGTRLLVL; The amino acid sequence shown in NO:16 is: MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVIPHGLYEQYFGPGTRLTVT.

[0077] 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%, 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%, 98% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 16.

[0078] 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, for the constant region of the α chain of the above-mentioned TCR, they 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.

[0079] The amino acid shown in SEQ ID NO:25 is: IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS;

[0080] The amino acid shown in SEQ ID NO: 26 is: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS.

[0081] The constant region of the TCR may contain a short linker sequence in which cysteine ​​residues form a disulfide bond, thereby connecting the two chains of the TCR. The TCR may have additional cysteine ​​residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant region.

[0082] 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.

[0083] 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, and the positions of the disulfide bonds that can be introduced are well known to those skilled in the art.

[0084] In one embodiment, the TCR is isolated or purified or is recombinant.

[0085] In one embodiment, the TCR is human.

[0086] In one embodiment, the TCR is monoclonal.

[0087] In one embodiment, the TCR is single chain.

[0088] In one embodiment, the TCR comprises two chains.

[0089] The TCR can be obtained from a biological source, such as from a cell (such as from a T cell (e.g., a cytotoxic T cell)), a T cell hybridoma, or other publicly available resource, for example, the TCR can be derived from one of a variety of animal species, such as human, mouse, rat or other mammal, such as typically from human.

[0090] In some embodiments, the TCR may be in a cell-bound form or in a soluble form, preferably in a soluble form. The TCR in a soluble form refers to a TCR that has mutations in its hydrophobic core region, and the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the soluble TCR of the present application.

[0091] 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.

[0092] 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.

[0093] The nucleotide sequence shown in SEQ ID NO:17 is as follows: ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGTTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTCCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGCTGTATCAGGAGGAAGCTACATACCTACATTTGGAAGAGGAACCAGCCTTATTGTTCATCCGTATATCCAGAATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTGCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCCTTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGGCTGTGGAGCTCC

[0094] The nucleotide sequence shown in SEQ ID NO:18 is as follows: ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTTGAATCCGGCATGGCCCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGTGAGAATAATTATTATTTGTTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGACACTGCGATGTATTTCTGTGCTTTCATGAATGGAGAAACCAGTGGCTCTAGGTTGACCTTTGGGGAAGGAACACAGCTCACAGTGAATCCTGATATCCAGAATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTGCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCCTTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGGCTGTGGAGCTCC

[0095] The nucleotide sequence shown in SEQ ID NO:19 is as follows: ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTAGGCGGGCTAGCGGGAGAACTATTGGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTAGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCACATTTTGGCACAACCCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTCTTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC

[0096] The nucleotide sequence shown in SEQ ID NO:20 is as follows: ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGTGCTGTCATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACCTCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACCGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGCAAATCAGGGCTCTGAGGCCACATATGAGAGTGGATTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACTGTGAGCAACATGAGCCCTGAAGACAGCAGCATATATCTCTGCAGCGTTATCCCACACGGACTCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCACATTTTGGCACAACCCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTCTTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC

[0097] In one embodiment, the nucleotide sequence encoding the alpha chain and / or the nucleotide sequence encoding the beta chain are codon optimized. Generally, codon optimization involves balancing the percentage of the selected codon with the abundance of the disclosed human transfer RNA so that none of them is overloaded or limited. 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. The codon usage difference between the transfected gene and the host cell may affect the protein expression and immunogenicity of the nucleic acid construct. Generally, for codon optimization, codons are selected to select those codons that are balanced with the human frequency of use. Generally, the redundancy of amino acid codons makes different codons encode a kind of amino acid. In some embodiments, when selecting a codon for replacement, the resulting mutation may be required to be a silent mutation so that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence.

[0098] The present application provides a vector, which comprises the nucleic acid molecule described above.

[0099] For example, one or more nucleic acids encoding one or both chains of the above-mentioned TCR are cloned into suitable one or more 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.

[0100] The vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, 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 a cytomegalovirus (CMV) promoter, a SV40 promoter, a RSV promoter, and a promoter found in the long terminal repeat sequence of the mouse stem cell virus, and other promoters known to the skilled person are also contemplated.

[0101] The vector is an expression vector, preferably a viral vector, preferably a retroviral vector, and more preferably a lentiviral vector.

[0102] 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 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 TCR molecules, and optionally recovering TCR from the host cell (or host cell culture medium).

[0103] The 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 primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to parent cells in nucleic acid content, but may contain mutations.

[0104] The present application also provides an engineered cell, which comprises the T cell receptor (TCR), the above nucleic acid molecule or the above vector.

[0105] In one embodiment, the TCR is heterologous to the cell.

[0106] In one embodiment, the engineered cell is a cell line.

[0107] In one embodiment, the engineered cell is a primary cell obtained from a subject, preferably a mammalian subject, preferably a human.

[0108] In one embodiment, the engineered cells are T cells, preferably T cells isolated from peripheral blood.

[0109] In one embodiment, the T cells are CD8+ or CD4+.

[0110] The engineered cell can be, for example, a cell colony or a genetically engineered cell expressing TCR, and the cell is generally a eukaryotic cell, such as a mammalian cell, and is generally a human cell. In some embodiments, the cell 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, generally 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 (iPSC). Cells are generally primary cells, such as those directly separated from a subject and / or separated and frozen from a subject. In some embodiments, cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells and subgroups thereof.

[0111] 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, mucosa-associated constant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, etc.

[0112] 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.

[0113] 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.

[0114] The vector is a viral vector, and the introduction is performed by transduction.

[0115] 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.

[0116] In one embodiment, it further comprises a pharmaceutically acceptable carrier or adjuvant.

[0117] The pharmaceutically acceptable carrier or adjuvant refers to a component in a pharmaceutical composition that is non-toxic to a subject except for the active ingredient. Pharmaceutically acceptable carriers or adjuvants include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0118] 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 doctor.

[0119] The present application provides the use of the above-mentioned T cell receptor (TCR), the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned engineered cell or the above-mentioned pharmaceutical composition in the preparation of a drug for treating malignant tumors.

[0120] 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.

[0121] In one embodiment, the colorectal cancer is metastatic colorectal cancer.

[0122] 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 Mutation of malignant tumors.

[0123] 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 it has good specificity, only recognizing KRAS G12V mutations, but not wild-type KRAS and KRAS G12D mutation.

[0124] Example

[0125] The present application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained commercially.

[0126] Example 1: Cloning of KRAS G12V Acquisition of antigen short peptide-specific T cells and TCR genes

[0127] The synthetic short peptide SEQ ID NO: 27: VVVGAVGVGK (Jiangsu GenScript Biotechnology Co., Ltd.) was used to stimulate peripheral blood lymphocytes from healthy volunteers with the genotype of HLA-A1101. The VVVGAVGVGK short peptide was renatured with HLA-A1101 labeled with biotin to prepare pHLA (short peptide-human leukocyte antigen complex) monomer. These monomers were combined with PE-labeled streptavidin (BD Company) to form PE (phycoerythrin)-labeled tetramers (wherein, the method for preparing pHLA monomers and tetramers refers 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), and the tetramers and anti-CD8-FITC (fluorescein isothiocyanate) double-positive cells were enriched. The obtained double-positive cells were flow-sorted to obtain single cells, which are antigen-specific T cells. The single cells obtained by sorting 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 sequence of the α chain variable region and the β chain variable region of TCR (TCR059 and TCR076) and the information of their CDR1, CDR2, and CDR3 can be obtained.

[0128] The nucleotide sequence of the α chain variable region of TCR059 is: SEQ ID NO:28: ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGTTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGA GTTGATAATGTCCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGTCCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGCTGTATCAGGAGGAAGCTACATACCTACATTTGGAAGAGGAACCAGCCTTATTGTTCATCCGTAT;

[0129] The nucleotide sequence of the β chain variable region of TCR059 is: SEQ ID NO:29: ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAG TATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTAGGCGGGCTAGCGGGAGAACTATTGGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTA;

[0130] The amino acid sequence of the complementarity determining region 1 (CDR1) of the α chain of TCR059 is: SEQ ID NO: 1: DRVSQS;

[0131] The amino acid sequence of the complementarity determining region 2 (CDR2) of the α chain of TCR059 is: SEQ ID NO: 3: IYSNGD;

[0132] The amino acid sequence of the complementarity determining region 3 (CDR3) of the α chain of TCR059 is: SEQ ID NO: 5: AAVSGGSYIPT;

[0133] The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain of TCR059 is: SEQ ID NO: 7: SGDLS;

[0134] The amino acid sequence of the complementarity determining region 2 (CDR2) of the β chain of TCR059 is: SEQ ID NO: 9: YYNGEE;

[0135] The amino acid sequence of the complementarity determining region 3 (CDR3) of the β chain of TCR059 is: SEQ ID NO: 11: ASSVGGLAGELLETQY;

[0136] The nucleotide sequence of the α chain variable region of TCR076 is: SEQ ID NO:30: ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTTGAATCCGGCATGGCCCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGTGAGAATAATTATTTGTTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGT TATTCGCCAAGAAGCTTAAGCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGACACTGCGATGTATTTCTGTGCTTTCATGAATGGAGAAACCAGTGGCTCTAGGTTGACCTTTGGGGAAGGAACACAGCTCACAGTGAATCCTGAT;

[0137] The nucleotide sequence of the β chain variable region of TCR076 is: SEQ ID NO:31:ATGCTGAGTCTTCTGCTCCTTCCTGGGACTAGGCTCTGTGTTTCAGTGCTGTCATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACCTCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACCGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGC AAATCAGGGCTCTGAGGCCACATATGAGAGTGGATTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACTGTGAGCAACATGAGCCCTGAAGACAGCAGCATATATCTCTGCAGCGTTATCCCACACGGACTCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACA;

[0138] The amino acid sequence of the complementarity determining region 1 (CDR1) of the α chain of TCR076 is: SEQ ID NO: 2: TSENNYY;

[0139] The amino acid sequence of the complementarity determining region 2 (CDR2) of the α chain of TCR076 is: SEQ ID NO: 4: QEAYKQQN;

[0140] The amino acid sequence of the complementarity determining region 3 (CDR3) of the α chain of TCR076 is: SEQ ID NO: 6: AFMNGETSGSRLT;

[0141] The amino acid sequence of the complementarity determining region 1 (CDR1) of the β chain of TCR076 is: SEQ ID NO: 8: SQVTM;

[0142] The amino acid sequence of the complementarity determining region 2 (CDR2) of the β chain of TCR076 is: SEQ ID NO: 10: ANQGSEA;

[0143] The amino acid sequence of the complementarity determining region 3 (CDR3) of the β chain of TCR076 is: SEQ ID NO: 12: SVIPHGLYEQY.

[0144] Example 2: Construction of VVVGAVGVGK antigen short peptide-specific TCR lentiviral vector and lentiviral packaging

[0145] (1) Construction of TCR lentiviral vector

[0146] The VVVGAVGVGK TCR α and β chain variable region sequences were cloned into pLKO-based expression plasmids (Addgene), and the α or β variable domains were cloned into PLKO-based expression plasmids containing mouse α or β constant regions by standard methods of the multi-fragment recombination cloning kit (Novozyme Biotech, catalog number C113). The ligated plasmids were transformed into competent E. coli strain Stbl3 cells (Shanghai Weidi Biotechnology Co., Ltd.) and inoculated on LB / agar plates containing 100 μg / ml ampicillin. After overnight incubation at 37°C, single 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 mini-prep kit (Tiangen Biochemical Technology Co., Ltd. (TIANGEN) catalog number # DP118-02) and the plasmids were sequenced to obtain VVVGAVGVGK TCR (i.e., TCR059, TCR076) plasmids.

[0147] (2) Lentiviral packaging

[0148] Assay medium: 10% FBS (Lonsera, catalog number S711-001), DMEM (ThermoFisher, catalog number C11995500BT).

[0149] Prepare 293T cells (the depository is the American Type Culture Collection, ATCC, with a deposit catalog number of CRL-1573) and culture them in a 10 cm dish. Start plasmid transfection when the cell is no more than 80% full. The ratio of viral packaging plasmid and VVVGAVGVGK TCR plasmid is 1:1, with a total of 10 μg. Add the above plasmids to serum-free DMEM medium and mix with PEI (polyethylenimine), then add the plasmid mixture to 293T cells and culture at 37 degrees. After 72 hours, concentrate the cell supernatant with a 100 kd ultrafiltration tube to collect the viral vector.

[0150] Example 3: Construction and functional identification of Jurkat cell line expressing VVVGAVGVGK antigen short peptide-specific TCR

[0151] Nuclear factor of activated T cells (NFAT) reporter gene expression method

[0152] The following experiment was performed to demonstrate the activation response of TCR-transduced T cells to target cells. NFAT expression was detected by flow cytometry as a readout of T cell activation.

[0153] (1) Reagents

[0154] Assay medium: 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT)

[0155] (2) Methods

[0156] Target cell preparation

[0157] The target cells used in this experiment were T2-A11 cells (T2 cells were deposited in ATCC with a deposit catalog number of 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 the 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.

[0158] Effector cell preparation

[0159] The effector cells of 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.

[0160] JK8NF cells (Jurkat cells are deposited in ATCC with a deposit catalog number of TIB-152. JK8NF cells are 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 about 100% (the results are shown in FIG. Figure 1 The concentration of effector cells after expansion culture was adjusted to 1.6×10 6 50 μl was taken per well to obtain 80,000 cells / well.

[0161] Preparation of short peptide solution

[0162] The original concentration of 5 mg / ml short peptide (VVVGAVGVGK) was diluted to 400 μg / ml, and then diluted down by 10 times 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.

[0163] 50 μl was taken from each well to make the final concentration of the short peptide in the 96-well plate be 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.

[0164] Finally, 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 were added to each well of a 96-well flat-bottom plate and incubated in a cell culture incubator at 37 degrees for 12 hours.

[0165] (3) Results

[0166] The expression of NFAT in the TCR-transduced T cells of the present application in response to the target cells loaded with the VVVGAVGVGK antigen short peptide was tested by the above method. The expression level curve of NFAT was drawn using Graphpad prism8, and the results are shown in FIG. Figure 2 shown.

[0167] from Figure 2 It can be seen that the T cells transduced with the TCR of the present application have a good activation response to the target cells loaded with their specific short peptides.

[0168] Example 4: In vitro functional identification of primary T cells expressing VVVGAVGVGK antigen short peptide-specific TCR

[0169] IFN gamma ELISPOT method

[0170] (1) Reagents

[0171] Assay medium: ELISPOT kit (BD, catalog number 551849), 10% FBS (ThermoFisher, catalog number 10099-044), RPMI1640 (ThermoFisher, catalog number C11875500BT)

[0172] (2) Methods

[0173] Effector T cell preparation

[0174] 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).

[0175] 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 per well in a 24-well plate. 5 / 500μl was placed in the well, and a total of 1x10 6 The cells were stimulated with anti-CD3 / CD28 magnetic beads and then cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cell clustering was observed, and the TCR lentivirus obtained in Example 3 was added at an MOI (multiplicity of infection) = 2 for transduction. The cells were amplified in a 1640 medium containing 10% FBS and 200 IU / ml IL-2 until 3-4 days after transduction, and the TCR transfection efficiency was identified by flow cytometry (the results are shown in FIG. Figure 3 The concentration of 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.

[0176] In the T2-A11 target cell positive control group, targeting T2-A11+KRAS G12V , the number of TCR-T cells was 2000 / well.

[0177] Target cell preparation

[0178] The target cell SW 620 (purchased from ATCC, catalog number: CCL-227) used in this experiment expresses the G12V mutation itself and does not express A11. Therefore, the SW620-A11 cells were constructed by overexpressing the A11 gene through a lentiviral vector. SW620-A11 cells were a target cell group, and SW620 cells were a target cell negative control group. The number of cells was 50,000 cells / well. HLA class I neutralizing antibodies (anti-HLA I, clone number W6 / 32, purchased from Biolegend, catalog number 311402) were added to the above two groups at the same time as the control groups of the above two groups. The T2-A11 cells used in this experiment were the target cell positive control group (T2 cells were deposited in ATCC with a collection catalog number of CRL-1992, and T2-A11 cells were constructed based on T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032), and the number of cells used was 20,000 cells / well.

[0179] Preparation of short peptide solution

[0180] 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.

[0181] ELISPOT assay

[0182] Prepare the plates as follows according to the instructions provided by the manufacturer (BD, catalog number 551849): dilute the anti-human IFN-γ capture antibody 1:400 in 5 ml sterile PBS per plate, and then add 50 μl of the diluted capture antibody to 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 the plates at room temperature for 2 hours. Pour off the blocking solution and flick and tap the ELISPOT plate to remove any residual blocking solution.

[0183] Then add the corresponding target cells and effector cells and the corresponding short peptides, and incubate the plate overnight (37°C / 5% CO2). The next day, discard the culture medium, wash the plate twice with double distilled water, and then wash it three times with washing buffer, and pat it on a paper towel to remove the residual washing buffer. Then dilute the detection antibody 1:400 with PBS containing 10% FBS, and add 100 μl / well to each well. Incubate the plate at room temperature for 2 hours, then wash it three times with washing buffer, and pat the plate on a paper towel to remove excess washing buffer.

[0184] Dilute streptavidin-alkaline phosphatase 1:200 with PBS containing 10% FBS, add 100 μl of diluted streptavidin-alkaline phosphatase to each well and incubate the well plate at room temperature for 1 hour. Then wash 3 times with wash buffer and 3 times with PBS, and pat the well plate on a paper towel to remove excess wash buffer and PBS. After washing, add 100 μl / well of BCIP / NBT solution provided by the kit for development. Cover the well plate with tin foil to avoid light during development and let it stand for 5-15 minutes. During this period, routinely detect the spots on the developing well plate to determine the best time to terminate the reaction. Remove the BCIP / NBT solution and rinse the well plate with double distilled water to stop the development reaction, spin dry, then remove the bottom of the well plate, dry the well plate at room temperature until each well is completely dry, and then use an immunospot plate counter to count the spots formed on the bottom membrane of the well plate. The function of the TCR-transduced T cells of the present invention is tested by ELISPOT experiment (as described above). The number of ELISPOT spots observed in each well is plotted using graphpad prism6.

[0185] (3) Results

[0186] from Figure 4 It can be seen that TCR059 and TCR076 expressing the VVVGAVGVGK-A1101 antigen short peptide specific TCR have a strong activation response to the SW620-A11 cell line, while the control T cells have no activation response. The addition of HLA class I neutralizing antibodies can inhibit the recognition of TCR059 and TCR076 to the SW620-A11 cell line.

[0187] Example 5: Method for cross-reaction identification of nuclear factor of activated T cells (NFAT) reporter gene expression by VVVGAVGVGK antigen short peptide-specific TCR

[0188] 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.

[0189] Flow cytometry was used to detect the expression of NFAT as a readout of T cell activation.

[0190] (1) Reagents

[0191] Assay medium: 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (ThermoFisher, catalog number C11875500BT).

[0192] (2) Methods

[0193] Target cell preparation

[0194] The target cells used in this experiment were T2-A11 cells (T2 cells were deposited in ATCC with a deposit catalog number of 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 the 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.

[0195] Effector cell preparation

[0196] The effector cells of 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.

[0197] JK8NF cells (Jurkat cells are deposited in ATCC with a deposit catalog number of TIB-152. JK8NF cells are 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 about 100% (the results are shown in FIG. Figure 1 The concentration of effector cells after expansion culture was adjusted to 1.6×10 6 50 μl was taken per well to obtain 80,000 cells / well.

[0198] Preparation of short peptide solution

[0199] 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 by 10 times 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.

[0200] 50 μl was taken from each well to make the final concentration of the short peptide in the 96-well plate be 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.

[0201] Finally, 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 were added to each well of a 96-well flat-bottom plate and incubated in a cell culture incubator at 37 degrees for 12 hours.

[0202] (3) Results

[0203] The TCR-transduced T cells of the present application were tested for KRAS loading by the method described above. G12V , KRAS WT and KRAS G12D The expression of NFAT in target cells that respond to three antigenic peptides. The expression level curve of NFAT was drawn using Graphpad prism8. The results are shown in the figure. Figure 5 shown.

[0204] from Figure 5 It can be seen 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.

[0205] In summary, the T cells transduced with the TCR in the present application can express A11+KRAS G12V The complex is activated specifically in tumor cells with mutations and has good specificity, recognizing only KRAS G12V mutations, but not wild-type KRAS and KRAS G12D mutation.

[0206] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

[0207] Sequence Listing

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

Claims

1. A T cell receptor (TCR), wherein: The TCR comprises an α chain containing a variable region and a β chain containing a variable region, The variable region of the α chain comprises a complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 1, a complementary determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO: 3, and a complementary determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 5, The variable region of the beta chain comprises a complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO:7, a complementary determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO:9, and a complementary determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO:11; or The variable region of the α chain comprises a complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 2, a complementary determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO: 4, and a complementary determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO: 6, The variable region of the β chain comprises a complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO:8, a complementary determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO:10, and a complementary determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO:

12.

2. The T cell receptor (TCR) according to claim 1, wherein The variable region of the α chain further comprises a first leader sequence; and / or The variable region of the beta chain also includes a second leader sequence.

3. The T cell receptor (TCR) according to claim 1, wherein The amino acid sequence of the α chain variable region is shown as SEQ ID NO:13 or SEQ ID NO:14, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:13 or SEQ ID NO:14, and / or the amino acid sequence of the β chain variable region is shown as SEQ ID NO:15 or SEQ ID NO:16, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:15 or SEQ ID NO:

16.

4. The T cell receptor (TCR) according to claim 1, wherein The alpha chain further comprises an alpha constant region, and / or the beta chain further comprises a beta constant region.

5. The T cell receptor (TCR) according to claim 4, wherein The constant region is a mouse constant region or a human constant region.

6. The T cell receptor (TCR) according to any one of claims 1 to 5, wherein The TCR is isolated or purified or recombinant.

7. The T cell receptor (TCR) according to any one of claims 1 to 5, wherein The TCR is human.

8. The T cell receptor (TCR) according to any one of claims 1 to 5, wherein The TCR is monoclonal.

9. The T cell receptor (TCR) according to any one of claims 1 to 5, wherein The TCR is single chain.

10. The T cell receptor (TCR) according to any one of claims 1 to 5, wherein The TCR comprises two chains.

11. The T cell receptor (TCR) according to any one of claims 1 to 5, wherein The TCR is in a cell-bound form or in a soluble form.

12. The T cell receptor (TCR) according to claim 11, wherein The TCR is in soluble form.

13. The T cell receptor (TCR) according to any one of claims 1 to 5, wherein The TCR binds to the antigen short peptide-HLA-A1101 complex, The amino acid sequence of the antigenic short peptide is shown in SEQ ID NO:

1.

14. A nucleic acid molecule, wherein The nucleic acid molecule comprises a nucleic acid encoding the TCR of any one of claims 1-13.

15. The nucleic acid molecule according to claim 14, 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 The nucleotide sequence encoding the β chain is the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO:

20.

16. A vector, wherein The vector comprises the nucleic acid molecule of claim 14 or 15.

17. The carrier according to claim 16, wherein The vector is an expression vector.

18. The carrier according to claim 16, wherein The vector is a viral vector.

19. The carrier according to claim 18, wherein The vector is a retroviral vector.

20. The vector according to claim 18, wherein The viral vector is a lentiviral vector.

21. An engineered cell comprising the TCR of any one of claims 1-13, the nucleic acid molecule of any one of claims 14-15, or the vector of any one of claims 16-20.

22. The engineered cell according to claim 21, wherein The TCR is heterologous to the cell.

23. The engineered cell according to claim 21, wherein The engineered cells are cell lines.

24. The engineered cell according to claim 21, wherein The engineered cells are primary cells obtained from a subject.

25. The engineered cell according to claim 24, wherein The subject is a mammalian subject.

26. The engineered cell according to claim 24, wherein The subject is a human.

27. The engineered cell according to claims 21-26, wherein The engineered cells are T cells or NK cells.

28. The engineered cell according to claim 27, wherein The T cells are T cells isolated from peripheral blood.

29. The engineered cell according to claim 27, wherein The T cells are CD8+ or CD4+.

30. A method for producing the engineered cell of any one of claims 21-29, comprising introducing the nucleic acid molecule of any one of claims 14-15 or the vector of any one of claims 16-20 into a cell in vitro or ex vivo.

31. The method according to claim 30, wherein: The vector is a viral vector, and the introduction is performed by transduction.

32. A pharmaceutical composition comprising the T cell receptor (TCR) of any one of claims 1-13, the nucleic acid molecule of any one of claims 14-15, the vector of any one of claims 16-20, or the engineered cell of any one of claims 21-29.

33. The pharmaceutical composition according to claim 32, further comprising a pharmaceutically acceptable carrier or adjuvant.

34. The T cell receptor (TCR) of any one of claims 1 to 13, the nucleic acid molecule of any one of claims 14 to 15, the vector of any one of claims 16 to 20, the engineered cell of any one of claims 21 to 29, or the pharmaceutical composition of claim 32 or 33 for the preparation of a method for treating KRAS G12V Use of drugs in the treatment of malignant tumors with mutations.

35. The use according to claim 34, wherein The malignant tumors are colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, and prostate cancer.

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