T cell receptor with improved affinity, nucleic acid molecule, carrier, cell and medicine containing T cell receptor, nucleic acid molecule, carrier and cell for treating tumor

Abstract

The invention relates to the field of immunity, in particular to a T cell receptor with improved affinity, a nucleic acid molecule, a carrier, a cell and a medicine containing the T cell receptor, the nucleic acid molecule, the carrier and the cell for treating tumors. The T cell receptor includes a TCR [alpha] chain variable region and a TCR [beta] chain variable region, which have activity to bind to a KRAS G12V epitope and an HLA-A * 11 complex, and the TCR [alpha] chain variable region includes CDR1 [alpha], CDR2 [alpha] and CDR3 [alpha] complementarity determining regions, and the TCR [beta] chain variable region includes CDR1 [beta], CDR2 [beta] and CDR3 [beta] complementarity determining regions. The TCR provided by the invention has high-affinity target antigen peptide binding capacity, remarkably improves the target cell recognition capacity, shows good activation and killing activity, and shows high technical value and clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of immunity, and specifically, to a T cell receptor with enhanced affinity, a nucleic acid molecule encoding the T cell receptor, a vector containing the nucleic acid molecule, a cell expressing the T cell receptor, or a cell carrying the nucleic acid molecule, or containing the vector, and their uses in detecting, diagnosing, preventing, alleviating or treating KRAS G12V mutation-positive diseases and / or conditions, for example, preparing a drug for treating KRAS G12V-positive tumors. Background Art

[0002] Some cancers may have very limited treatment options, especially when the cancer becomes metastatic and unresectable. Although treatments such as, for example, surgery, chemotherapy, and radiation therapy have advanced, the prognosis for many cancers, such as, for example, pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer, may be poor.

[0003] KVA11-N04 is a natural TCR targeting KRAS G12V isolated by the applicant of this application from the infiltrating lymphocytes (TIL) of the postoperative tumor tissue of a patient with KRAS G12V mutation-positive colorectal cancer (A*11). It has been verified that the TCR has high affinity, high membrane stability, excellent tumor killing activity and specificity for the target antigen peptide. A Chinese patent application was submitted on January 13, 2023, with publication number CN118344478A, and a PCT patent application was submitted on January 12, 2024, with publication number WO2024149347A1.

[0004] However, there is still a need in the art to develop TCR products with high affinity that specifically target KRAS G12V. Summary of the invention

[0005] In order to obtain a TCR with higher affinity for KRAS G12V, the present invention uses KVA11-N04 as a template, uses in vitro mutagenesis to mutate the complementary determining region (CDR) region of the TCR, and obtains mutant clones with high affinity and excellent killing activity by performing in vitro functional positive screening on the mutated TCR, thereby completing the present invention.

[0006] Accordingly, in a first aspect, the present disclosure provides a T cell receptor with enhanced affinity, the T cell receptor comprising a TCR α chain variable region and a TCR β chain variable region, which has the activity of binding to the KRAS G12V epitope and the HLA-A*11 complex, and the TCR α chain variable region comprises CDR1α, CDR2α and CDR3α complementarity determining regions, and the TCR β chain variable region comprises CDR1β, CDR2β and CDR3β complementarity determining regions: wherein,

[0007] The amino acid sequence of CDR1α is NSASDY as shown in SEQ ID NO: 1;

[0008] The amino acid sequence of CDR2α is LYSTMDK as shown in SEQ ID NO: 2;

[0009] The amino acid sequence of CDR3α is AENSDGTSYGKLT as shown in SEQ ID NO:3;

[0010] The amino acid sequence of CDR1β is SGHAT as shown in SEQ ID NO:4;

[0011] The amino acid sequence of CDR2β is FQNNGV as shown in SEQ ID NO:5;

[0012] The amino acid sequence of CDR3β is ASSLVGSPDYEQY as shown in SEQ ID NO:6.

[0013] In a second aspect, the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding a T cell receptor as described above or a complementary sequence thereof.

[0014] In a third aspect, the present disclosure provides a vector comprising the nucleic acid molecule as described above.

[0015] In a fourth aspect, the present disclosure provides a cell, wherein the cell expresses the TCR or antigen-binding fragment thereof targeting KRAS G12V as described above, or carries the nucleic acid molecule as described above, or contains the vector as described above.

[0016] In a fifth aspect, the present disclosure provides a drug for treating KRAS G12V-positive tumors, the drug comprising the TCR or antigen-binding fragment thereof targeting KRAS G12V as described above, or the nucleic acid molecule as described above, or the vector as described above, or the cell as described above.

[0017] The TCR provided by the present invention has the ability to bind to target antigen peptides with high affinity, significantly improving the target cell recognition ability, while showing good activation and killing activity, demonstrating strong technical value and clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The affinity of the TCR (KVA11-M04-178) provided by the present invention is shown.

[0019] Figure 2 The results show that the TCR (KVA11-M04-178) provided by the present invention has a highly efficient and specific killing effect on antigen-positive tumor cells.

[0020] Figure 3 The results of specific INF-γ secretion of the TCR (KVA11-M04-178) provided by the present invention on antigen-positive tumor cells are shown. DETAILED DESCRIPTION

[0021] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. For example, the terms used herein are as defined in Janeway CA Jr, Travers P, Walport M, et al., Immunobiology, Fifth Edition, New York: Garland Science (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations), Leuenberger, HGW, Nagel, B. and Kölbl, H., eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0022] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, the terms "a," "an," "one or more," and "at least one" may be used interchangeably. Similarly, the terms "comprising," "including," and "having" may be used interchangeably.

[0023] When the term "comprising" or "including" is used herein and in the appended claims, it does not exclude other elements. For the purpose of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising or including at least a certain number of embodiments, it should also be understood to disclose a group that preferably consists of only these embodiments.

[0024] T cell receptor (TCR)

[0025] The present invention provides a T cell receptor with enhanced affinity, wherein the T cell receptor comprises a TCR α chain variable region and a TCR β chain variable region, which has the activity of binding to the KRAS G12V epitope and the HLA-A*11 complex, and the TCR α chain variable region comprises CDR1α, CDR2α and CDR3α complementary determining regions, and the TCR β chain variable region comprises CDR1β, CDR2β and CDR3β complementary determining regions: wherein,

[0026] The amino acid sequence of CDR1α is NSASDY as shown in SEQ ID NO: 1;

[0027] The amino acid sequence of CDR2α is LYSTMDK as shown in SEQ ID NO: 2;

[0028] The amino acid sequence of CDR3α is AENSDGTSYGKLT as shown in SEQ ID NO:3;

[0029] The amino acid sequence of CDR1β is SGHAT as shown in SEQ ID NO:4;

[0030] The amino acid sequence of CDR2β is FQNNGV as shown in SEQ ID NO:5;

[0031] The amino acid sequence of CDR3β is ASSLVGSPDYEQY as shown in SEQ ID NO:6.

[0032] The inventors of the present invention found in their research that by introducing amino acid substitutions L, Y, and T mutations at the first position I, the second position R, and the fourth position N of the parental TCR α chain CDR2 (CDR2α), respectively, the modified TCR of the present invention was obtained, which has improved binding affinity to the KRAS G12V mutant antigen.

[0033] The term "parent TCR" as used herein refers to an HLA-A1101-restricted human TCR targeting the KRAS G12V mutant antigen that was previously developed and patented by the applicant of the present invention (KVN04, patent application number: CN118344478A), which is incorporated herein by reference in its entirety.

[0034] According to the different components of TCR, T cells can be divided into αβT cells and γδT cells. At present, αβT cells are the most studied in the field of TCR-T cells. The TCR of αβT cells is composed of α chain and β chain, and recognizes pMHC in the form of heterodimers. The extramembrane parts of α chain and β chain each contain two Ig-like domains, one is the constant region (C region) at the proximal end of the membrane, and the other is the variable region (V region) at the distal end of the membrane. The V region of α chain and β chain together constitute the antigen binding site of TCR. The V region can be further divided into complementarity-determining regions 1 (CDR1), CDR2 and CDR3, namely CDR1α / β, CDR2α / β and CDR3α / β. When TCR recognizes ligands, CDR1 and CDR2 with lower diversity contact the α helices on both sides of pMHC, while CDR3 contacts the central peptide. CDR3 is the most critical sequence that determines the specificity of TCR recognition of antigens. In order to achieve recognition of different pMHC molecules, the CDR3 region has high diversity. TCR diversity is concentrated in the high variability of the CDR3 region.

[0035] The term "avidity" refers to the ability of T cells expressing the TCR of the present invention to respond to a given concentration of ligand in vitro, and is believed to be related to the in vivo effector capacity of cells expressing TCR. According to definition, cells expressing TCR with high binding affinity respond to very low antigen doses in in vitro tests, and such cells with lower binding affinity require higher amounts of antigen before reaching an immune response similar to that of cells expressing TCR with high affinity. Therefore, binding affinity can be regarded as a quantitative determinant of the activation threshold of cells expressing TCR. This is measured by exposing such cells to different amounts of cognate antigens in vitro. Cells with high binding affinity TCR respond to low antigen doses. In some embodiments, EC50 is used as a criterion for judging the affinity of a given TCR.

[0036] As used herein, the term "T cell receptor" or "TCR" can be used interchangeably to refer to a natural TCR or its variants, fragments and constructs. The term thus includes heterodimers, multimers and single-chain constructs comprising a TCR α chain and a TCR β chain; optionally comprising other domains and / or portions, as long as the TCR retains its ability to recognize an antigen target (preferably in a complex with HLA-A*11).

[0037] Both variable regions of the TCR α and β chains contain three hypervariable or complementary determining regions surrounded by a framework (FR) region, as described above. CDR3 is the main determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with specific antigens), while CDR1 and CDR2 mainly interact with MHC molecules presenting antigenic peptides, i.e., the characteristics of TCR are conferred by the six CDR regions.

[0038] In some embodiments, the framework sequence of the variable domain of the TCR of the present invention can be of mouse or human origin, preferably of human origin. In some preferred embodiments, the framework region is derived from the framework region of the TCR for the mutant KRAS. In some more preferred embodiments, the framework region is derived from the framework region of the TCR for the mutant KRAS G12V. In some further preferred embodiments, the framework region is derived from the framework region of the HLA-A*11 restricted TCR for the mutant KRAS G12V. In the most preferred embodiment, the framework region is derived from the framework region of the KVA11-N04 TCR.

[0039] Therefore, after determining the 6 CDR domains of the TCR of the present invention, the TCR α chain variable region and the TCR β chain variable region are not unique. In some embodiments of the present invention, the TCR α chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 7. In some embodiments of the present invention, the TCR β chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 8.

[0040] The term "sequence identity" as used herein refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions or substitutions may have a lower degree of identity. Those skilled in the art will recognize that some algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and ClustalW.

[0041] Therefore, the amino acid sequence of SEQ ID NO: 7 or 8 can be used as a "subject sequence" or "reference sequence", while the amino acid sequence of the variable region of the TCR α chain or β chain different therefrom can be used as a "query sequence".

[0042] In some embodiments, the TCR α chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the TCR β chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the TCR α chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the TCR β chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8.

[0043] SEQ ID NO:7:

[0044] GESVGLHLPTLGVQEGDNSIINCAYSNSASDYFIWYKQESGKGPQFIIDLYSTMDKRQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFCAENSDGTSYGKLTFGQGTILTVHPN

[0045] SEQ ID NO:8

[0046] EAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLVGSPDYEQYFGPGTRLTVT

[0047] In some embodiments, the TCR α chain variable region is contained on a first polypeptide, and the TCR β chain variable region is contained on a different second polypeptide, that is, the TCR is an αβ heterodimer. In some embodiments, the TCR α chain variable region and the TCR β chain variable region are contained on a single polypeptide, that is, the TCR is a scTCR. In a preferred embodiment, the TCR is an αβ heterodimer.

[0048] In some embodiments, the TCR further comprises a TCR constant region or a fragment thereof. In the IMGT system, the α chain constant domain has the following symbol: TRAC*01, where "TR" represents the T cell receptor gene; "A" represents the α chain gene; C represents the constant region; "*01" represents allele 1. The β chain constant domain has the following symbol: TRBC1*01 or TRBC2*01, where "TR" represents the T cell receptor gene; "B" represents the β chain gene; C represents the constant region; "*01" represents allele 1. The constant region of the α chain is uniquely determined, and in the form of the β chain, there are two possible constant region genes "C1" and "C2". Those skilled in the art can obtain the constant region gene sequences of the TCR α and β chains through the public IMGT database.

[0049] As used herein, the term "constant region" may be a human constant region or derived from another species, thereby creating a "chimeric" TCR. For example, the human α and / or β chains may be replaced ("murinization") with their murine counterparts, which have been found to enhance surface expression of the human TCR by supporting preferential pairing of the TCR α and β chains and to enhance more stable binding to the CD3 co-receptor.

[0050] In some embodiments, the TCR constant region is a mouse constant region or a human constant region. In some preferred embodiments, the TCR constant region is a mouse constant region.

[0051] It is reported that adding disulfide bonds to TCRα and β chains can promote the correct pairing of TCRα and β chains. Therefore, in some embodiments of the present invention, an artificial interchain disulfide bond is introduced between the α chain and the β chain. In some embodiments, one or more cysteine ​​modifications are added to the constant region to form a disulfide bond between the TCR α chain and the TCR β chain. In some embodiments, the TCR constant region includes a TCR α chain constant region and a TCR β chain constant region; preferably, the TCR α chain constant region and the TCR β chain constant region respectively contain at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between the TCR α chain and the TCR β chain. In some embodiments, the cysteine ​​mutation is at one or more of the following positions: the 48th position of the wild-type human TCR α chain constant region, the 48th position of the wild-type mouse TCR α chain constant region, the 57th position of the wild-type human TCR β chain constant region, and the 57th position of the wild-type mouse TCR β chain constant region. Other sites for introducing cysteine ​​residues for disulfide bond formation in the constant region may be:

[0052] Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01;

[0053] Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01;

[0054] Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01;

[0055] Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01;

[0056] Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01;

[0057] Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01;

[0058] or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

[0059] In addition, regarding stability, patent document PCT / CN2016 / 077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Therefore, the α chain variable region and the β chain constant region of the high-affinity TCR of the present invention may also contain an artificial interchain disulfide bond. Specifically, the cysteine ​​residue that forms the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR is replaced by:

[0060] amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01;

[0061] amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0062] amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

[0063] or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.

[0064] To facilitate the description of the position of the cysteine ​​mutation, the position of the amino acid sequence of the wild-type TCR constant region in the present invention is numbered according to the naming rules of the International Immunogenetics Information System (IMGT). For example, an amino acid in the TCR α chain constant region (TRAC) is numbered 48 in the position listed in IMGT, and it is described herein as the 48th amino acid in the TCR α chain constant region (TRAC); an amino acid in the TCR β chain constant region (TRBC) is numbered 57 in the position listed in IMGT, and it is described herein as the 57th amino acid in the TCR β chain constant region (TRBC), and so on. Herein, the position numbering of the amino acid sequence of the variable region TRAV and TRBV follows the position numbering listed in IMGT. For example, an amino acid in TRAV is numbered 46 in the position listed in IMGT, and it is described herein as the 46th amino acid in TRAV, and so on. In the present invention, if the sequence position numbering of other amino acids is specially specified, it is in accordance with the special instructions.

[0065] In some embodiments, the TCR α chain constant region further comprises an LVL mutation or a LIV mutation, such that the constant region (and / or the transmembrane region) comprises the amino acid sequence LLVIVLRIL. For example, when the TCR α chain comprises a human constant region, the human constant region may comprise an LVL mutation, such that the constant region (and / or the transmembrane region) comprises the amino acid sequence LLVIVLRIL. When the TCR α chain comprises a mouse constant region, the mouse constant region may comprise an LIV mutation, such that the constant region (and / or the transmembrane region) comprises the amino acid sequence LLVIVLRIL.

[0066] In some embodiments, the TCR alpha chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17. In some embodiments, the TCR beta chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 18. In some embodiments, the TCR alpha chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 17, and the TCR beta chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 18.

[0067] SEQ ID NO:17

[0068] IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS

[0069] SEQ ID NO:18

[0070] EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS

[0071] In some embodiments, the TCR is a TCR comprising an α chain and a β chain. In some cases, the TCR α chain and / or the β chain may comprise a leader sequence. For example, the leader sequence of the TCR α chain may have an amino acid sequence (MAGIRALFMYLWLQLDWVSR) as shown in SEQ ID NO: 19. The leader sequence of the TCR β chain may have an amino acid sequence (MSTRLLCWAALCLLGAELT) as shown in SEQ ID NO: 20.

[0072] In some embodiments, the TCR further comprises a transmembrane region and / or a cytoplasmic region.

[0073] In some embodiments, the TCR comprises a TCR alpha chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one selected from SEQ ID NO: 9. In some embodiments, the TCR comprises a TCR beta chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one selected from SEQ ID NO: 10. In some embodiments, the TCR comprises a TCR alpha chain as described in SEQ ID NO: 9 and a TCR beta chain as described in SEQ ID NO: 10.

[0074] SEQ ID NO:9

[0075] GESVGLHLPTLGVQEGDNSIINCAYSNSASDYFIWYKQESGKGPQFIIDLYSTMDKRQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFCAENSDGTSYGKLTFGQGTILTVHPNIQNPEPAVYQ LKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS

[0076] SEQ ID NO:10

[0077] EAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLVGSPDYEQYFPGGTRLTVTEDLRNVTPPKVSLFEPSKAEIANKQKATL VCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS

[0078] TCR recognizes antigenic peptides that are bound to major histocompatibility complex (MHC) molecules at the surface of antigen presenting cells ("presented / displayed on MHC molecules"). Antigenic peptides presented on MHC molecules are also referred to herein as "complexes of epitopes with MHC molecules", "epitope-MHC complexes" or "target antigen peptide-MHC complexes". There are two different classes of MHC molecules: MHC I and MHC II, which present peptides from different cell compartments. MHC class I molecules are expressed on the surface of all nucleated cells in the human body and display peptides or protein fragments from intracellular compartments to cytotoxic T cells. In humans, MHC is also known as human leukocyte antigen (HLA). There are three main types of MHC class I: HLA-A, HLA-B and HLA-C. Once TCR binds to its specific epitope-MHC complex, the T cell is activated and exerts biological effector functions. In the present invention, "MHC" and "HLA" can be used interchangeably.

[0079] The TCR provided herein can advantageously (specifically) recognize the KRAS G12V epitope or a complex of the epitope and the HLA-A*11 molecule, such as a complex of the KRAS G12V epitope and HLA-A*1101, and has a significantly improved binding affinity compared to its parental TCR.

[0080] In some embodiments of the present invention, the TCR provided herein is capable of binding to a complex of an epitope comprising the amino acid sequence VVVGAVGVGK (SEQ ID NO: 11) and an HLA-A*11 molecule.

[0081] The term "epitope" generally refers to a site on an antigen, typically a (poly)peptide recognized by a binding domain. The term "binding domain" refers to an "antigen binding site" in its broadest sense, i.e., a domain that characterizes a molecule that binds / interacts with a specific epitope on an antigen target. An antigen target may contain a single epitope, but typically contains at least two epitopes, and depending on the size, conformation, and type of the antigen, the antigen target may include any number of epitopes. The term "epitope" generally includes linear epitopes and conformational epitopes. A linear epitope is a continuous epitope contained in an amino acid primary sequence, and it typically includes at least 2 amino acids or more amino acids. A conformational epitope is formed by non-continuous amino acids that are arranged side by side by the folding of a target antigen, and particularly a target (poly)peptide.

[0082] In some embodiments, the TCR is soluble or membrane-bound.

[0083] The TCR of the present invention can be provided in a soluble form, for example, in the form of a soluble TCR. Soluble TCR (sTCR) can be used as a diagnostic tool and a carrier or "adapter" for specifically targeting therapeutic agents or effector cells to, for example, cancer cells expressing an antigen target recognized by a soluble TCR. Soluble TCRs are generally fragments or constructs comprising TCR α chains and / or β chains or their variable regions or CDRs, and optionally they are stabilized by disulfide bonds or covalently linked by suitable linkers. Typically, soluble TCRs do not include, for example, a transmembrane region.

[0084] In some embodiments, the TCR further comprises an intracellular signaling region. In some embodiments, the TCR further comprises one or more antigen binding regions that bind to other antigens or epitopes.

[0085] Nucleic Acids

[0086] The present invention provides a nucleic acid molecule, which comprises a nucleic acid sequence encoding the T cell receptor of the present invention or a complementary sequence thereof.

[0087] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13, which encodes a TCR alpha chain variable region.

[0088] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15, which encodes a TCR β chain variable region.

[0089] In some embodiments, the nucleic acid further comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 13, encoding a TCR α chain variable region, and comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15, encoding a TCR β chain variable region. In some preferred embodiments, the nucleic acid further comprises a nucleotide sequence as shown in SEQ ID NO: 13, encoding a TCR α chain variable region, and comprises a nucleotide sequence as shown in SEQ ID NO: 15, encoding a TCR β chain variable region.

[0090] SEQ ID NO:13

[0091] GGAGAAAGTGTTGGGCTGCATTTGCCTACGTTGGGAGTACAGGAAGGGGACAACAGCATTATCAATTGCGCCTATTCTAACAGTGCTTCCGATTACTTCATCTGGTACAAGCAGGAGTCTGGAAAAGGTCCCCAGTTTATCATTGATCTGTACTCCACAATGGACAAGAGAC AGGGGCAGCGAGTGACTGTCCTGCTCAACAAAACCGTAAAGCACCTTAGCCTGCAGATAGCTGCTACCCAACCCGGAGACAGTGCTGTATACTTCTGCGCGGAGAACTCTGACGGTACGAGTTACGGCAAGCTTACATTTGGACAGGGTACAATACTCACAGTTCACCCCAAT

[0092] SEQ ID NO:15

[0093] GAAGCTGGCGTTGCACAAAGCCCAAGATACAAAATTATCGAGAAGAGGCAGAGCGTCGCCTTCTGGTGTAACCCTATTTCAGGCCACGCAACGCTCTACTGGTATCAGCAGATTCTTGGACAGGGACCAAAGCTCCTCATCCAGTTCCAGAATAACGGAGTGGTTGACGACT CACAACTCCCTAAGGACCGGTTTTCTGCTGAACGCCTCAAAGGTGTGGACAGCACGCTGAAGATACAGCCCGCTAAGCTGGAAGATAGTGCTGTGTATTTGTGTGCTTCTTCTCTGGTCGGCTCACCCGACTACGAGCAGTATTTCGGCCCAGGAACCAGGCTGACTGTGACA

[0094] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21, which encodes a TCR alpha chain constant region.

[0095] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:22, which encodes a TCR β chain constant region.

[0096] In some embodiments, the nucleic acid further comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 21, encoding a TCR α chain constant region, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 22, encoding a TCR β chain constant region. In some embodiments, the nucleic acid further comprises a nucleotide sequence as shown in SEQ ID NO: 21, encoding a TCR α chain constant region, and a nucleotide sequence as shown in SEQ ID NO: 22, encoding a TCR β chain constant region.

[0097] SEQ ID NO:21

[0098] ATTCAGAACCCCGAGCCTGCCGTGTACCAGCTGAAGGACCCCAGAAGCCAGGACTCTACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAGAGCGGCACCTTCATCACCGACAAGTGCGTGCTGGACATGAAGGCCATGGACAGCAAGAGCAACGGCGCCATCGCCTGGAGCAACCAGACC AGCTTCACCTGTCAGGACATCTTCAAGGAGACCAACGCCACCTAACCCCAGCAGCGACGTTCCCTGTGATGCCACACTGACCGAGAAGAGCTTCGAGACCGACATGAACCTGAACTTCCAGAACCTGCTGGTGATCGTGCTGAGGATCTTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGGCTGTGGAGCAGC

[0099] SEQ ID NO:22

[0100] GAGGACCTGAGGAACGTGACCCCTCCCAAGGTGAGCCTGTTCGAGCCCTCTAAGGCCGAGATCGCCAACAAGCAGAAAGCTACCCTGGTGTGCTTGGCCAGGGGCTTCTTCCCCGACCACGTGGAACTT AGCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGAGTGTGCACAGATCCCCAAGCCTACAAGGAGAGCAACTACAGCTACTGCCTGAGCAGCAGGCTGAGGGTGAGCGCCACCTTCTGGCATAATCCTA GGAACCACTTCAGGTGCCAGGTGCAGTTCCATGGCTTGAGCGAGGAAGACAAGTGGCCCGAGGGCAGCCCCAAACCTGTGACCCAAAACATTAGCGCCGAGGCTTGGGGCAGAGCCGACTGCGGCATTAC AAGCGCTTCTTACCAGCAAGGCGTGCTGAGCGCCACCATCCTGTACGAGATTCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTACACTGGTGGTGATGGCCATGGTGAAGAGGAAGAACAGC

[0101] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14, which encodes a TCR alpha chain.

[0102] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16, which encodes a TCR β chain.

[0103] In some embodiments, the nucleic acid further comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 14, encoding a TCR α chain, and comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16, encoding a TCR β chain. In some preferred embodiments, the nucleic acid further comprises a nucleotide sequence as shown in SEQ ID NO: 14, encoding a TCR α chain, and comprises a nucleotide sequence as shown in SEQ ID NO: 16, encoding a TCR β chain.

[0104] Carrier

[0105] The present disclosure provides vectors comprising a nucleic acid of the present disclosure.

[0106] The term "vector" as used herein is a nucleic acid molecule used as a medium for transferring (exogenous) genetic material into a host cell, in which the nucleic acid molecule as a vector can, for example, be replicated and / or expressed. The term "vector" encompasses, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors and adenovirus-associated vectors (AAV)), bacteriophages, phagemids, cosmids and artificial chromosomes (including BACs and YACs). The vector itself is typically a nucleotide sequence, typically a DNA sequence comprising an insert (transgene) and a larger sequence as a vector "skeleton". Engineered vectors are typically included in the starting point of autonomous replication in the host cell (if stable expression of polynucleotides is required), a selection marker and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector may additionally include a promoter, a genetic marker, a reporter gene, a targeting sequence and / or a protein purification tag. As known to those skilled in the art, a large number of suitable vectors are known to those skilled in the art, and many are commercially available. Examples of suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), which is incorporated herein by reference in its entirety.

[0107] In some embodiments, the vector is preferably selected from the group consisting of a lentiviral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

[0108] cell

[0109] The present disclosure provides a cell, which expresses the TCR or antigen-binding fragment thereof targeting KRAS G12V provided by the present invention, or carries the nucleic acid molecule provided by the present invention, or contains the vector provided by the present invention.

[0110] The term "cell" as used herein refers to any type of cell capable of expressing the TCR of the present disclosure. The cell can be a eukaryotic cell, for example, a plant (without the potential to develop into a plant), an animal, a fungus or an algae, or can be a prokaryotic cell, for example, a bacterium or a protozoan. The cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism, such as a human. The cell can be an adherent cell or a suspension cell, i.e., a cell grown in suspension. Suitable host cells are known in the art and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of producing the TCR of the present disclosure, the cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.

[0111] In some embodiments, the cell is selected from lymphocytes (e.g., T cells, NK cells), monocytes (e.g., PBMCs), and stem cells. The term "stem cell" as used herein is a stem cell for expressing the TCR (particularly TCR) of the present disclosure. For example, the stem cell may be a lymphoid progenitor cell, an induced pluripotent stem cell (iPSC), or a hematopoietic stem cell (HSC). In some embodiments, stem cells do not include embryonic stem cells obtained by destroying human embryos, and / or do not include omnipotent stem cells for development and formation of animal individuals. Gene transfer to stem cells generally does not result in expression of TCR on the cell surface because CD3 molecules are not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, the expression of CD3 molecules will initiate expression of the introduced TCR molecules on the surface of thymocytes.

[0112] In some embodiments, the stem cell is a lymphoid progenitor cell or an induced pluripotent stem cell (iPSC).

[0113] In some embodiments, the cell is a T cell. The T cell can be any T cell, such as a cultured T cell, such as a primary T cell or a T cell from a cultured T cell line, such as Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or liquids. The T cell can also be enriched or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell separated from a person. The T cell can be any type of T cell and can be a T cell at any stage of development, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells, such as Th1 and Th2 cells, CD4+T cells, CD8+T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc. In some embodiments, the T cell does not express endogenous TCR.

[0114] In some embodiments, the method of preparing the cells of the present disclosure comprises the step of transducing or transfecting the cells with the vector of the present disclosure.

[0115] As used herein, the term "transfection" is a process in which nucleic acid molecules or polynucleotides (including vectors) are intentionally introduced into target cells. An example is RNA transfection, which is the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into host cells. The term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of viral-mediated nucleic acid molecules or polynucleotides. The transfection of animal cells generally involves opening a transient hole or "hole" in the cell membrane to allow the uptake of materials. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to produce liposomes that fuse with the cell membrane and deposit their cargo into the interior. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation.

[0116] In some embodiments, the method further comprises the step of expanding and / or activating the cells either before or after the transduction or transfection.

[0117] use

[0118] The present disclosure provides use of a T cell receptor or a fragment thereof, a nucleic acid, or a vector provided by the present disclosure in preparing a product for detecting, diagnosing, preventing, alleviating or treating a KRAS G12V-positive disease or condition.

[0119] Specifically, the present invention provides a drug for treating KRAS G12V-positive tumors, wherein the drug comprises the TCR or its antigen-binding fragment targeting KRAS G12V provided by the present invention, or the nucleic acid molecule provided by the present invention, or the vector provided by the present invention, or the cell provided by the present invention.

[0120] In some embodiments, the drug is in the form of a conjugate, which includes a TCR of the present disclosure and an active agent coupled or conjugated to the TCR.

[0121] In some embodiments, the active agent is selected from a detectable label, an immunostimulatory molecule and a therapeutic agent. Preferably, the detectable label is selected from biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent or chemiluminescent molecule. Preferably, the immunostimulatory molecule is selected from a cytokine (such as IL-2 and IFN-γ), a chemokine (such as IL-8), a platelet factor (such as platelet factor 4) and a complement initiator. Preferably, the therapeutic agent is selected from an immunomodulator, a radioactive compound, an enzyme, a chemotherapeutic agent and a toxin. Other suitable therapeutic agents include small molecule cytotoxic agents, i.e. compounds having the ability to kill mammalian cells with a molecular weight of less than 700 Daltons. Such compounds may also contain toxic metals that can have cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e. compounds that decay or are converted to release cytotoxic agents under physiological conditions. Examples of such agents include cisplatin, maytansine derivatives, rachlombycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, pyroximate sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronide, auristatin E, vincristine, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells; for example, ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA e and RNase; radionuclides, i.e. unstable isotopes of elements that emit one or more alpha or beta particles or gamma rays while decaying, for example, iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225 and astatine-213; chelating agents, which can be used to promote the binding of these radionuclides to molecules or their polymers; or heterologous protein domains, allogeneic protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0122] In some embodiments, the drug is in the form of a composition, which comprises the TCR, nucleic acid, vector, or cell of the present disclosure. Preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0123] The term "composition" refers in particular to a composition suitable for administration to humans. However, the term also generally encompasses compositions suitable for administration to non-human animals. The composition and its components (i.e., active agent and optional carrier or excipient) are preferably pharmaceutically acceptable, i.e., capable of inducing the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects. The pharmaceutically acceptable composition of the present invention may be, for example, sterile. Specifically, the term "pharmaceutically acceptable" may mean approved by a regulatory agency or other recognized pharmacopoeia for use in animals, more particularly in humans.

[0124] The term "excipient" includes fillers, binders, disintegrants, coating agents, adsorbents, antiadherents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tension regulators. It is known to those skilled in the art to select suitable excipients to prepare the compositions of the present invention. Exemplary carriers for the compositions of the present invention include saline, buffered saline, glucose and water. Typically, the selection of suitable excipients depends particularly on the desired dosage form of the active agent used, the disease to be treated and the compositions.

[0125] Depending on the active agent used (e.g., soluble TCR), the compositions of the present disclosure may be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration. The processes known to the invention for producing drugs are shown in the 22nd edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa., 2012), and may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. Compositions comprising, for example, host cells or soluble TCRs as described herein are typically provided in liquid form and preferably contain a pharmaceutically acceptable buffer.

[0126] In some embodiments, the compositions of the present disclosure further comprise a second therapeutic agent, preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

[0127] Preferred examples of the second therapeutic agent include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, E), vincristine and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes and RNA enzymes; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as IL-2, chemokines such as IL-8, platelet factor 4; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides.

[0128] In some embodiments, the product is a kit comprising the TCR or conjugate of the present disclosure, and the kit is used to detect the presence of a positive epitope in a sample to be tested, wherein the epitope comprises a KRAS G12V mutation epitope.

[0129] In some embodiments, the kit is a kit for detecting (e.g., diagnosing) a KRAS G12V mutation-positive disease and / or condition in a subject, comprising a TCR or conjugate of the present disclosure.

[0130] In some embodiments, the conjugate comprises a detectable label. Examples of detectable labels include, but are not limited to, biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent or chemiluminescent molecule; preferably an enzyme or a catalytically active fragment thereof, a radionuclide, a fluorescent, phosphorescent or chemiluminescent molecule. In some embodiments, the kit may further comprise instructions for how to use the kit.

[0131] In an embodiment of the present disclosure, the disease and / or condition may include a KRAS G12V mutation-positive disease and / or condition. In some embodiments, the KRAS G12V mutation may be an epitope of the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the KRAS G12V mutation-positive disease may include a tumor. In some embodiments, the tumor may include a solid tumor and / or a hematological tumor. In some embodiments, the tumor includes: at least one of colorectal cancer, pancreatic cancer, lung adenocarcinoma, and endometrial cancer.

[0132] The present invention is further described by the following specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are carried out according to the conventional conditions in the art, for example, the conditions described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press, or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0133] Example

[0134] Example 1 Screening of modified TCRs with enhanced affinity

[0135] The inventors used the human KVA11-N04 sequence (CN118344478A) as a template, substituted the complementary determining region (CDR) of KVA11-N04 by in vitro substitution, and obtained high-affinity substitution clones by in vitro positive screening of the substituted TCR. The sequence information of the preferred TCR sequence screened by this method is as follows:

[0136] The amino acid sequence of CDR1α is shown in SEQ ID NO: 1;

[0137] The amino acid sequence of CDR2α is shown in SEQ ID NO:2;

[0138] The amino acid sequence of CDR3α is shown in SEQ ID NO: 3;

[0139] The amino acid sequence of CDR1β is shown in SEQ ID NO:4;

[0140] The amino acid sequence of CDR2β is shown in SEQ ID NO:5;

[0141] The amino acid sequence of CDR3β is shown in SEQ ID NO:6;

[0142] The amino acid sequence of the TCR α chain variable region is shown in SEQ ID NO:7, and the coding sequence is shown in SEQ ID NO:13;

[0143] The amino acid sequence of the TCR β chain variable region is shown in SEQ ID NO:8, and the coding sequence is shown in SEQ ID NO:15;

[0144] The TCR with improved affinity obtained in this way was named KVA11-M04-178.

[0145] In order to increase the correct pairing and expression of exogenous TCR on the surface of human T cells, the variable regions of the α chain and β chain of TCR were fused to the mouse TCR constant region, and the classic α chain C region "LIV" substitution was introduced into the mouse TCR constant region (i.e., amino acid substitutions were introduced into the mouse TCR α chain constant region so that the constant region contained the amino acid sequence LLVIVLRIL; shown in bold) and the classic α chain T48C and β chain S57C substitutions were introduced into the mouse TCR constant region. The amino acid sequence of the constant region of the TCR α chain is shown in SEQ ID NO:17, and the coding sequence is shown in SEQ ID NO:21; the amino acid sequence of the TCR β chain constant region is shown in SEQ ID NO:18, and the coding sequence is shown in SEQ ID NO:22.

[0146] Through the 2A self-cleavage peptide SEQ ID NO: 12, the α chain and β chain of TCR (the N-termini of the α chain and the β chain respectively contain the signal peptide sequences SEQ ID NO: 19 and SEQ ID NO: 20) are constructed in series into a lentiviral expression vector in the order of β-P2A-α.

[0147] The affinity of the TCR provided by the present invention is determined by the following method:

[0148] The parent TCR and the TCR sequences provided by the present invention were cloned into TCR tandem expression lentiviral vectors, packaged with lentivirus, transduced into reporter cells JKR9, and then sorted for TCR expression-positive cells. HLA-A*1101-positive T2 cells were loaded with different concentrations of KRAS G12V target antigen peptide (SEQ ID NO: 11), and then co-incubated and activated with JKR9 reporter cells transduced with parent TCR and TCR of the present invention, respectively. After 16 hours of activation, the reporter gene was stained for flow cytometry analysis, and the EC50 value was calculated based on the flow cytometry analysis. The EC50 value of KVA11-N04 was 1.592E-08, and the EC50 value of the TCR of the present invention was 3.245E-09. Compared with the parent TCR, the affinity of the TCR provided by the present invention has been increased by 5 times, which has been significantly improved ( Figure 1 ).

[0149] Example 2. Efficient and specific killing of antigen-positive tumor cells by affinity-optimized TCR-T cells

[0150] T cells expressing affinity-optimized TCR or KVA11-N04 were used as effector cells, and PBMCs without TCR transduction were expanded and cultured in parallel as negative control of effector cells. HELA-A1101-G12V-EL (KRAS G12V positive, HLA-A*11:01 positive), PANC1-A1101-G12V-EL cells (KRAS G12V positive, HLA-A*11:01 positive), T2KO-A1101 loaded with KRAS G12V 7-16 The peptide (T2+KV2) is used as the positive target cell matched with HLA-antigen peptide. All target cells stably express the luciferase gene. The effector cells are incubated with different target cells at a 1:1 effector-target ratio (E:T) for 16-20 hours, and the luciferase substrate is added to detect the surviving target cells. The proportion of target cells killed is calculated based on the remaining target cells. The results are as follows: Figure 2 shown.

[0151] Depend on Figure 2 It can be seen that affinity-optimized TCR-T cells have a strong affinity for HELA-A1101-G12V-EL, PANC1-A1101-G12V-EL, and T2KO-A1101 loaded with KRAS G12V 7-16 The peptide-positive target cells have obvious killing activity, and their killing activity is significantly higher than that of KVA11-N04 TCR-T cells. HELA-A1101-EL cells as target antigen-negative target cells, T2 cells as target antigens, and HLA double-negative target cells cannot be killed by TCR-T expressing KVA11-N04 and KVA11-M04-178 in the experiment. These results show that affinity-optimized TCR-T cells have the ability to efficiently and specifically kill HLA-antigen peptide-matched target cells, and their killing activity increases with the increase in affinity.

[0152] Example 3. Specific INF-γ secretion by affinity-optimized TCR-T cells against antigen-positive tumor cells

[0153] T cells expressing affinity-optimized TCR or KVA11-N04 were used as effector cells, and PBMCs without TCR transduction were expanded and cultured in parallel as negative control of effector cells. HELA-A1101-G12V-EL (KRAS G12V positive, HLA-A*11:01 positive), PANC1-A1101-G12V-EL cells (KRAS G12V positive, HLA-A*11:01 positive), T2KO-A1101 loaded with KRAS G12V 7-16 The peptides were used as HLA-antigen peptide-matched positive target cells; effector cells were incubated with different target cells at a 1:1 effector-target ratio (E:T) for 16-20 h, and the IFN-γ secretion in the supernatant was detected using an IFN-γ ELISA kit (ExCell, Cat# EH008-96). The results are shown in Figure 3 shown.

[0154] The results showed that affinity-optimized TCR-T cells and positive target cells HELA-A1101-G12V-EL, PANC1-A1101-G12V-EL, T2KO-A1101 loaded with KRAS G12V 7-16 After co-incubation with peptides, significant IFN-γ secretion can be detected, and the amount of IFN-γ secretion is significantly higher than that of KVA11-N04 TCR-T cells. HELA-A1101-EL cells as target antigen-negative target cells, T2 cells as target antigens, and HLA double-negative target cells were unable to activate KVA11-N04 and KVA11-M04-178 TCR-T to produce IFN-γ secretion in the experiment. These results show that affinity-optimized TCR-T cells have good specific killing effects on HLA-antigen peptide-matched target cells, and this specific killing effect is enhanced with the increase of affinity.

[0155] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications. Including the combination of various specific technical features in any suitable manner. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations. However, these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A T cell receptor with enhanced affinity, the T cell receptor comprising a TCR α chain variable region and a TCR β chain variable region, characterized in that: It has the activity of binding to the KRAS G12V epitope and the HLA-A*11 complex, and the TCR α chain variable region includes CDR1α, CDR2α and CDR3α complementarity determining regions, and the TCR β chain variable region includes CDR1β, CDR2β and CDR3β complementarity determining regions: wherein, The amino acid sequence of CDR1α is NSASDY as shown in SEQ ID NO: 1; The amino acid sequence of CDR2α is LYSTMDK as shown in SEQ ID NO: 2; The amino acid sequence of CDR3α is AENSDGTSYGKLT as shown in SEQ ID NO:3; The amino acid sequence of CDR1β is SGHAT as shown in SEQ ID NO:4; The amino acid sequence of CDR2β is FQNNGV as shown in SEQ ID NO:5; The amino acid sequence of CDR3β is ASSLVGSPDYEQY as shown in SEQ ID NO:

6.

2. The T cell receptor with enhanced affinity according to claim 1, wherein The TCR α chain variable region comprises the amino acid sequence shown in SEQ ID NO:7; and / or the TCR β chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

3. The T cell receptor with enhanced affinity according to claim 2, wherein: The TCR α chain comprises the amino acid sequence shown in SEQ ID NO:9; and / or the TCR β chain comprises the amino acid sequence shown in SEQ ID NO:

10.

4. The T cell receptor with enhanced affinity according to claim 1, wherein The TCR is an αβ heterodimer, and contains an artificial interchain disulfide bond between the α chain and the β chain.

5. The T cell receptor with enhanced affinity according to any one of claims 1 to 4, wherein The KRAS G12V epitope comprises the amino acid sequence VVVGAVGVGK (SEQ ID NO: 11).

6. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid sequence encoding the T cell receptor with enhanced affinity as described in any one of claims 1 to 5 or a complementary sequence thereof.

7. The nucleic acid molecule according to claim 6, wherein The nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 13 encoding the variable region of the TCR α chain, or the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 14 encoding the TCR α chain; and / or The nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 15 encoding the variable region of the TCR β chain, or the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 16 encoding the TCR β chain.

8. A carrier, characterized in that The vector contains the nucleic acid molecule according to claim 6 or 7.

9. A cell, characterized in that The cell expresses the T cell receptor with enhanced affinity as described in any one of claims 1 to 5, or carries the nucleic acid molecule as described in claim 6 or 7, or contains the vector as described in claim 8.

10. A drug for treating KRAS G12V-positive tumors, the drug comprising the affinity-enhanced T cell receptor according to any one of claims 1 to 5, or the nucleic acid molecule according to claim 6 or 7, or the vector according to claim 8, or the cell according to claim 9.

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