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US-12624084-B2 - T cell receptor recognising KRAS mutation and encoding sequence thereof

US12624084B2US 12624084 B2US12624084 B2US 12624084B2US-12624084-B2

Abstract

Provided is a T cell receptor (TCR) capable of specifically binding to KRAS G12V mutant antigens; the mutant antigen short peptide VVGAVGVGK is capable of forming a complex with HLA A1101, and the TCR specifically binds to said complex. Also provided in the present invention are a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule. Also provided are TCR-transduced cells.

Inventors

  • Jing Hu
  • Hanli SUN

Assignees

  • XLIFESC, LTD.

Dates

Publication Date
20260512
Application Date
20201010
Priority Date
20191010

Claims (19)

  1. 1 . A T-cell receptor (TCR), wherein the TCR is capable of specifically binding to a VVGAVGVGK-HLA A1101 complex, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, wherein, the three complementarity determining regions (CDRs) of the TCRα chain variable domain are: (SEQ ID NO: 10) α CDR1-DRVSQS; (SEQ ID NO: 11) α CDR2-IYSNGD; and (SEQ ID NO: 12) α CDR3- ASLKGNNDMR; and the three CDRs of the TCRβ chain variable domain are: (SEQ ID NO: 13) β CDR1- SGHVS; (SEQ ID NO: 14) β CDR2- FQNEAQ; and (SEQ ID NO: 15) β CDR3- ASSSSRWEQQF, wherein cysteine residues form an artificial disulfide bond between the α and β chain constant domains of the TCR; and wherein the cysteine residues forming the artificial disulfide bond in the TCR are substituted for one or more groups of amino acids selected from the following: Thr48 of TRAC*01 exon 1 and Ser57 of TRBC1*01 or TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; Tyr10 of TRAC*01 exon 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; Ser15 of TRAC*01 exon 1 and Glu15 of TRBC1*01 or TRBC2*01 exon 1; Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1; Pro89 of TRAC*01 exon 1 and Ala19 of TRBC1*01 or TRBC2*01 exon 1; and Tyr10 of TRAC*01 exon 1 and Glu20 of TRBC1*01 or TRBC2*01 exon 1.
  2. 2 . The TCR of claim 1 , comprising a TCRα chain variable domain and a TCRβ chain variable domain, wherein the TCRα chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; and the TCRβ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5, provided that the three complementarity determining regions (CDRs) of the TCRα chain variable domain are: (SEQ ID NO: 10) α CDR1-DRVSQS; (SEQ ID NO: 11) α CDR2-IYSNGD; and (SEQ ID NO: 12) α CDR3- ASLKGNNDMR; and the three CDRs of the TCRβ chain variable domain are: (SEQ ID NO: 13) β CDR1- SGHVS; (SEQ ID NO: 14) β CDR2- FQNEAQ; and (SEQ ID NO: 15) β CDR3- ASSSSRWEQQF.
  3. 3 . The TCR of claim 1 , wherein the TCR comprises an α chain variable domain amino acid sequence SEQ ID NO: 1; and, the TCR comprises a β chain variable domain amino acid sequence SEQ ID NO: 5.
  4. 4 . The TCR of claim 1 , wherein the α chain amino acid sequence of the TCR is SEQ ID NO: 3 and the β chain amino acid sequence of the TCR is SEQ ID NO: 7; or the α chain amino acid sequence of the TCR is SEQ ID NO: 26 and the β chain amino acid sequence of the TCR is SEQ ID NO: 28.
  5. 5 . The TCR of claim 1 , wherein the TCR is soluble; and the TCR is a single chain.
  6. 6 . The TCR of claim 1 , wherein the α chain variable domain amino acid sequence of the TCR comprises SEQ ID NO: 32 and the β chain variable domain amino acid sequence of the TCR comprises SEQ ID NO: 34.
  7. 7 . The TCR of claim 1 , wherein an anti-CD3 antibody is bound to the α chain and/or β chain of the TCR at C- or N-terminal.
  8. 8 . A multivalent TCR complex, comprising at least two TCR molecules, wherein at least one TCR molecule is the TCR of claim 1 .
  9. 9 . A nucleic acid molecule, comprising a nucleic acid sequence encoding the TCR molecule of claim 1 .
  10. 10 . The nucleic acid molecule of claim 9 , wherein the nucleic acid molecule comprises a nucleotide sequence SEQ ID NO: 4 encoding the TCRα chain and comprises a nucleotide sequence SEQ ID NO: 8 encoding the TCRβ chain.
  11. 11 . The nucleic acid molecule of claim 9 , wherein the nucleic acid molecule comprises a nucleotide sequence SEQ ID NO: 2 encoding the TCRα chain variable domain, and a nucleotide sequence SEQ ID NO: 6 encoding the TCRβ chain variable domain; or the nucleic acid molecule comprises a nucleotide sequence SEQ ID NO: 33 encoding the TCRα chain variable domain and a nucleotide sequence SEQ ID NO: 35 encoding the TCRβ chain variable domain.
  12. 12 . A vector, comprising the nucleic acid molecule of claim 9 .
  13. 13 . An isolated host cell, having the nucleic acid molecule of claim 9 integrated into its genome or having a vector comprising the nucleic acid molecule of claim 9 , wherein the nucleic acid molecule is exogenous with respect to the isolated host cell.
  14. 14 . A pharmaceutical composition, comprising a pharmaceutically acceptable carrier, and the TCR of claim 1 .
  15. 15 . A pharmaceutical composition, comprising a pharmaceutically acceptable carrier and a cell, wherein the cell is transduced with the nucleic acid molecule of claim 9 or the cell is transduced with a vector comprising the nucleic acid molecule of claim 9 .
  16. 16 . A method for treating a disease, comprising administering an appropriate amount of the TCR of claim 1 , wherein the disease is KRAS G12V-positive cancer.
  17. 17 . The method of claim 16 , wherein the disease is lung cancer, colorectal cancer, pancreatic cancer or gastric cancer.
  18. 18 . A method for treating a disease, comprising administering an appropriate amount of a cell, wherein the cell is transduced with the nucleic acid molecule of claim 9 or the cell is transduced with a vector comprising the nucleic acid molecule of claim 9 , wherein the disease is KRAS G12V-positive cancer.
  19. 19 . The method of claim 18 , wherein the disease is lung cancer, colorectal cancer, pancreatic cancer or gastric cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a National Stage Application, filed under 35 U.S.C. § 371, of International Application No. PCT/CN2020/120191, filed Oct. 10, 2020, which claims the benefit of priority under 35 U.S.C. § 119 to CN application No. 201910960523.3, filed Oct. 10, 2019, each of which is incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 6, 2022 is named 51766-505N01US_ST25_Sequence.txt and is 34 KB in size. TECHNICAL FIELD The present disclosure relates to a T-cell receptor (TCR) capable of recognising a short peptide derived from a KRAS G12V antigen and an encoding sequence thereof. The present disclosure also relates to KRAS G12V-specific T cells obtained by transducing the TCR and use thereof in the prevention and treatment of diseases related to KRAS G12V. BACKGROUND KRAS gene (p21 gene) is a rat sarcoma virus oncogene, which is about 35 kb long and located on chromosome 12. It is a member of ras gene family and encodes the KRAS protein. The KRAS gene can be divided into the wild-type KRAS gene or the mutant-type KRAS gene. Under normal conditions, the wild-type KRAS can control the pathway of regulating the cell growth. Once the wild-type KRAS mutates, it will continuously stimulate the cell growth and cause tumor. The common mutation sites of the KRAS gene are located at codons 12 and 13 of exon 2 of the KRAS gene, among which G12V is one of the seven common mutation sites (Chin J Lab Med. November 2012, Vol. 35, No. 11). The KRAS protein encoded by the KRAS mutant (mKRAS) will be continuously activated, and then the cell proliferation will be out of control, resulting in the formation of tumors. Studies have shown that KRAS-G12V is related to a variety of human malignant tumors, comprising but not limited to lung cancer and colorectal cancer (Tumour Biol. 2016 May; 37 (5): 6823-30), pancreatic cancer, gastric cancer (J Cancer 2019; 10 (4): 821-828), etc., and tumor patients with KRAS-G12V mutation have the shortest survival time (Br J Cancer. 2015 Oct. 20; 113(8): 1206-1215). VVGAVGVGK (SEQ ID NO: 9) or VVVGAVGVGK (SEQ ID NO: 38) is a short peptide derived from the KRAS G12V antigen and is a target for the treatment of KRAS G12V-related diseases. T cell adoptive immunotherapy is to transfer reactive T cells with specificity for target cell antigen into patients to make theses reactive T cells act on target cells. T-cell receptor (TCR) is a membrane protein on the surface of T cells, which can recognise the corresponding antigenic peptides on the surface of target cells. In the immune system, once antigen-specific TCRs bind to peptide-Major Histocompatibility Complexes (pMHC complexes), it causes direct physical contact of a T cell and an antigen-presenting cell (APC). Then, the interaction of other membrane molecules in both of the T cell and APC occurs and the subsequent cell signaling and other physiological responses are initiated so that a range of different antigen-specific T cells exert immune effects on their target cells. Therefore, those skilled in the art are committed to isolating TCRs that are specific for KRAS G12V antigen mutation short peptides and transducing T cells with such TCRs to obtain T cells having specificity for KRAS G12V antigen short peptides, so that they function in cellular immunotherapy. SUMMARY The object of the present disclosure is to provide a T-cell receptor capable of recognising a KRAS G12V antigen short peptide. In a first aspect of the present disclosure, a T-cell receptor (TCR) is provided, which is capable of specifically binding to a VVGAVGVGK-HLA A1101 complex. In another preferred embodiment, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, an amino acid sequence of CDR3 of the TCRα chain variable domain is ASLKGNNDMR (SEQ ID NO: 12); and/or an amino acid sequence of CDR3 of the TCRβ chain variable domain is ASSSSRWEQQF (SEQ ID NO: 15). In another preferred embodiment, three complementarity determining regions (CDRs) of the TCRα chain variable domain are: (SEQ ID NO: 10)α CDR1-DRVSQS; (SEQ ID NO: 11)α CDR2-IYSNGD;and (SEQ ID NO: 12)α CDR3- ASLKGNNDMR; and/or three CDRs of the TCRβ chain variable domain are: (SEQ ID NO: 13)β CDR1- SGHVS; (SEQ ID NO: 14)β CDR2- FQNEAQ;and (SEQ ID NO: 15)β CDR3- ASSSSRWEQQF. In another preferred embodiment, the TCR is also capable of specifically binding to a VVVGAVGVGK-HLA A1101 complex. In another preferred embodiment, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, where the TCRα chain variable domain is an amino acid sequence having at least 90% of sequence identity with SEQ ID NO: 1; and/or the TCRβ chain variable domain is an amino acid sequence having at least 90% of sequence identity with SEQ ID NO: 5. In