T cell receptors with enhanced affinity, nucleic acid molecules, vectors, cells, and drugs for treating tumors comprising them

Abstract

The present invention relates to the field of immunology, and particularly to a T cell receptor with enhanced affinity, a nucleic acid molecule, a vector, a cell, and a medicament for treating tumors comprising them. The T cell receptor comprises a variable region of the TCR α chain and a variable region of the TCR β chain, which has the activity of binding to the KRAS G12V epitope and the HLA-A*11 complex, and the variable region of the TCR α chain comprises CDR1α, CDR2α, and CDR3α complementarity-determining regions, and the variable region of the TCR β chain comprises CDR1β, CDR2β, and CDR3β complementarity-determining regions. The TCR provided by the present invention has the ability to bind to the target antigen peptide with high affinity, significantly improves the target cell recognition ability, and at the same time exhibits good activation and killing activities, showing strong technical value and clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of immunology. Specifically, it relates 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 use in detecting, diagnosing, preventing, alleviating or treating KRAS G12V mutation-positive diseases and / or disorders. For example, preparing a drug for treating KRAS G12V-positive tumors. Background Art

[0002] Some cancers can have very limited treatment options, especially when the cancer becomes metastatic and inoperable. Despite the progress made in treatments such as surgery, chemotherapy, and radiotherapy, the prognosis of many cancers (such as pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer) may be poor.

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

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

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

[0006] Accordingly, in a first aspect, the present disclosure provides an affinity-enhanced T cell receptor, which includes a variable region of TCR α chain and a variable region of TCR β chain, having the activity of binding to the KRAS G12V epitope and HLA-A*11 complex, and the variable region of TCR α chain includes CDR1α, CDR2α and CDR3α complementarity-determining regions, and the variable region of TCR β chain includes 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, which contains the nucleic acid sequence encoding the T cell receptor as described above or its complementary sequence.

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

[0015] In a fourth aspect, the present disclosure provides a cell, which expresses the TCR targeting KRAS G12V as described above or its antigen-binding fragment, 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, which contains the TCR targeting KRAS G12V as described above or its antigen-binding fragment, 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 the target antigen peptide with high affinity, significantly improves the target cell recognition ability, and at the same time exhibits good activation and killing activities, showing great technical value and clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 Show the high-efficiency specific killing effect of the TCR (KVA11-M04-178) provided by the present invention against antigen-positive tumor cells.

[0020] Figure 3 Show the specific INF-γ secretion results of the TCR (KVA11-M04-178) provided by the present invention against antigen-positive tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, the terms used herein such as those in Janeway CA Jr, Travers P, Walport M et al.'s "Immunobiology", Fifth Edition, New York: Garland Science (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by Leuenberger, H.G.W, Nagel, B. and Kölbl, H. (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 referents 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 terms "comprising" or "including" are used herein and in the appended claims, they do not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined herein as including or comprising at least a certain number of embodiments, it should also be understood that a group consisting only of these embodiments is disclosed.

[0024] T cell receptor (TCR)

[0025] The present invention provides a T cell receptor with enhanced affinity. The T cell receptor comprises a variable region of the TCR α chain and a variable region of the TCR β chain, which has the activity of binding to the KRAS G12V epitope and HLA-A*11 complex, and the variable region of the TCR α chain comprises CDR1α, CDR2α and CDR3α complementary determining regions, and the variable region of the TCR β chain 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 the research that by introducing amino acid substitution mutations of L, Y, and T at the first I, second R, and fourth N positions of the parental TCR α chain CDR2 (CDR2α), the modified TCR of the present invention is obtained, which has improved binding affinity for the KRAS G12V mutant antigen.

[0033] As used herein, the term "parental TCR" refers to the HLA-A*1101-restricted human TCR (KVN04, patent application number: CN118344478A) targeting the KRAS G12V mutant antigen previously developed and patented by the applicant of the present invention, which is incorporated herein by reference in its entirety.

[0034] According to the different TCR components, T cells can be divided into αβ T cells and γδ T cells. Currently, more research has been conducted on αβ T cells in the field of TCR-T cells. The TCR of αβ T cells is composed of an α chain and a β chain, which recognize pMHC in the form of a heterodimer. The extracellular parts of the α chain and the β chain each contain two Ig-like domains, one is the membrane-proximal constant region (C region), and the other is the membrane-distal variable region (V region). The V regions of the α chain and the β chain together form the antigen-binding site of the 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 the TCR recognizes the ligand, CDR1 and CDR2 with lower diversity contact the α helices on both sides of the pMHC, while CDR3 contacts the central peptide. CDR3 is the most critical sequence determining the antigen recognition specificity of the TCR. In order to achieve the recognition of different pMHC molecules, the CDR3 region has a high degree of diversity. The 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 considered to be related to the in vivo effector ability of the cells expressing the TCR. By definition, cells expressing the TCR with high binding avidity respond to very low antigen doses in in vitro assays, while such cells with lower binding avidity require higher amounts of antigen before achieving an immune response similar to that of cells expressing the TCR with high avidity. Therefore, the binding avidity can be regarded as a quantitative determinant of the activation threshold of cells expressing the TCR. This is determined by exposing such cells to different amounts of homologous antigen in vitro. Cells with a TCR having high binding avidity respond to low antigen doses. In some embodiments, EC50 is used as the criterion for judging the affinity of a given TCR.

[0036] As used herein, the terms "T cell receptor" or "TCR" are used interchangeably and refer to a native TCR or its variants, fragments, and constructs. Thus, the term includes heterodimers comprising a TCR α chain and a TCR β chain, as well as multimeric and single-chain constructs; optionally comprising other domains and / or moieties, provided that the TCR retains its ability to recognize an antigen target (preferably in its complex with HLA-A*11).

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

[0038] In some embodiments, the framework sequences of the TCR variable domains of the present invention can be murine or human, preferably human. In some preferred embodiments, the framework region is derived from the framework region of a TCR against mutant KRAS. In some more preferred embodiments, the framework region is derived from the framework region of a TCR against mutant KRAS G12V. In some further preferred embodiments, the framework region is derived from the framework region of an HLA-A*11-restricted TCR against mutant KRAS G12V. In the most preferred embodiments, the framework region is derived from the framework region of KVA11-N04 TCR.

[0039] Therefore, after determining the six CDR domains of the TCR of the present invention, the variable regions of the TCR α-chain and TCR β-chain are not unique. In some embodiments of the present invention, the variable region of the TCR α-chain contains 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 variable region of the TCR β-chain contains 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] As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies with exactly the same sequence have 100% identity, but sequences with lower levels of high conservation and with deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several 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] Thus, the amino acid sequence of SEQ ID NO: 7 or 8 can, for example, serve as the "subject sequence" or "reference sequence", while the amino acid sequence of the variable region of a TCR α chain or β chain that differs therefrom can serve as the "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 variable region of the TCR α chain is included on a first polypeptide, and the variable region of the TCR β chain is included on a different second polypeptide, i.e., the TCR is an αβ heterodimer. In some embodiments, the variable regions of the TCR α chain and the TCR β chain are included on a single polypeptide, i.e., 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 constant domain of the α chain 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 constant domain of the β chain has the following symbols: 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. 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 publicly available IMGT database.

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

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

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

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

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

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

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

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

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

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

[0059] In addition, for stability, patent document PCT / CN2016 / 077680 also discloses that introducing an artificial inter-chain disulfide bond between the variable region of the α-chain and the constant region of the β-chain of the TCR can significantly improve the stability of the TCR. Therefore, an artificial inter-chain disulfide bond may also be included between the variable region of the α-chain and the constant region of the β-chain of the high-affinity TCR of the present invention. Specifically, the cysteine residues forming the artificial inter-chain disulfide bond between the variable region of the α-chain and the constant region of the β-chain of the TCR replace:

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

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

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

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

[0064] For the convenience of describing the positions of cysteine mutations, the positions of the amino acid sequences of the wild-type TCR constant regions in the present invention are numbered according to the nomenclature rules of the International Immunogenetics Information System (IMGT). For example, for a certain amino acid in the constant region of the TCR α-chain (TRAC), if the position number listed in IMGT is 48, it will be described herein as the 48th amino acid of the constant region of the TCR α-chain (TRAC); for a certain amino acid in the constant region of the TCR β-chain (TRBC), if the position number listed in IMGT is 57, it will be described herein as the 57th amino acid of the constant region of the TCR β-chain (TRBC), and so on. In this article, the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are according to the position numbers listed in IMGT. For example, for a certain amino acid in TRAV, if the position number listed in IMGT is 46, it will be described in the present invention as the 46th amino acid of TRAV, and so on. In the present invention, if there are special instructions for the sequence position numbers of other amino acids, they shall be in accordance with the special instructions.

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

[0066] In some embodiments, the TCR α-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 β-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 α-chain constant region comprises the amino acid sequence as shown in SEQ ID NO:17, and the TCR β-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 β-chain may comprise a leader sequence. For example, the leader sequence of the TCR α-chain may have the amino acid sequence shown in SEQ ID NO:19 (MAGIRALFMYLWLQLDWVSR). The leader sequence of the TCR β-chain may have the amino acid sequence shown in SEQ ID NO:20 (MSTRLLCWAALCLLGAELT).

[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 α-chain that 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% identity to any one selected from SEQ ID NO: 9. In some embodiments, the TCR comprises a TCR β-chain that 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 selected from SEQ ID NO: 10. In some embodiments, the TCR comprises the TCR α-chain as described in SEQ ID NO: 9 and the TCR β-chain as described in SEQ ID NO: 10.

[0074] SEQ ID NO:9

[0075] GESVGLHLPTLGVQEGDNSIINCAYSNSASDYFIWYKQESGKGPQFIIDLYSTMDKRQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFCAENSDGTSYGKLTFGQGTILTVHPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS

[0076] SEQ ID NO:10

[0077] EAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLVGSPDYEQYFGPGTRLTVTEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS

[0078] The TCR recognizes an antigenic peptide that binds to a major histocompatibility complex (MHC) molecule at the surface of an antigen-presenting cell (“presented / displayed on the MHC molecule”). The antigenic peptide presented on the MHC molecule is also referred to herein as “epitope-MHC molecule complex”, “epitope-MHC complex” or “target antigenic peptide-MHC complex”. There are two different classes of MHC molecules: MHC I and MHC II, which present peptides from different cellular 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 referred to as human leukocyte antigen (HLA). There are three main types of MHC class I: HLA-A, HLA-B, and HLA-C. Once the TCR binds its specific epitope-MHC complex, the T cell is activated and exerts a biological effector function. In the present invention, “MHC” and “HLA” can be used interchangeably.

[0079] The TCRs provided herein are capable of advantageously (specifically) recognizing the KRAS G12V epitope or the complex of said epitope with an HLA-A*11 molecule, such as the complex of the KRAS G12V epitope with HLA-A*1101, and having a significantly improved binding affinity compared to their parental TCRs.

[0080] In some embodiments of the present invention, the TCRs provided herein are capable of binding the complex of an epitope comprising the amino acid sequence VVVGAVGVGK (SEQ ID NO:11) with 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" in its broadest sense refers to an "antigen-binding site", i.e., a domain of a molecule that characterizes binding / interaction with a specific epitope on an antigen target. The antigen target can 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 can include any number of epitopes. The term "epitope" generally includes linear epitopes and conformational epitopes. A linear epitope is a continuous epitope contained within the primary amino acid sequence, and it typically includes at least 2 amino acids or more. A conformational epitope is formed by non-contiguous amino acids that are juxtaposed through folding of the target antigen, and particularly the 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 as a vector or "adapter" for specifically targeting therapeutic agents or effector cells to, for example, cancer cells expressing an antigen target recognized by the soluble TCR. Soluble TCR generally comprises fragments or constructs of the TCR α-chain and / or β-chain or their variable regions or CDRs, and optionally it is stabilized by disulfide bonds or covalently linked by a suitable linker. Generally, soluble TCR does 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 other antigens or epitopes.

[0085] Nucleic acid

[0086] The present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the T cell receptor of the present invention or its complementary sequence.

[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 the variable region of the TCR α-chain.

[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 the variable region of the TCR β chain.

[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, which encodes the variable region of the 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:15, which encodes the variable region of the TCR β chain. In some preferred embodiments, the nucleic acid further comprises the nucleotide sequence as shown in SEQ ID NO:13, which encodes the variable region of the TCR α chain, and comprises the nucleotide sequence as shown in SEQ ID NO:15, which encodes the variable region of the TCR β chain.

[0090] SEQ ID NO:13

[0091] GGAGAAAGTGTTGGGCTGCATTTGCCTACGTTGGGAGTACAGGAAGGGGACAACAGCATTATCAATTGCGCCTATTCTAACAGTGCTTCCGATTACTTCATCTGGTACAAGCAGGAGTCTGGAAAAGGTCCCCAGTTTATCATTGATCTGTACTCCACAATGGACAAGAGACAGGGGCAGCGAGTGACTGTCCTGCTCAACAAAACCGTAAAGCACCTTAGCCTGCAGATAGCTGCTACCCAACCCGGAGACAGTGCTGTATACTTCTGCGCGGAGAACTCTGACGGTACGAGTTACGGCAAGCTTACATTTGGACAGGGTACAATACTCACAGTTCACCCCAAT

[0092] SEQ ID NO:15

[0093] GAAGCTGGCGTTGCACAAAGCCCAAGATACAAAATTATCGAGAAGAGGCAGAGCGTCGCCTTCTGGTGTAACCCTATTTCAGGCCACGCAACGCTCTACTGGTATCAGCAGATTCTTGGACAGGGACCAAAGCTCCTCATCCAGTTCCAGAATAACGGAGTGGTTGACGACTCACAACTCCCTAAGGACCGGTTTTCTGCTGAACGCCTCAAAGGTGTGGACAGCACGCTGAAGATACAGCCCGCTAAGCTGGAAGATAGTGCTGTGTATTTGTGTGCTTCTTCTCTGGTCGGCTCACCCGACTACGAGCAGTATTTCGGCCCAGGAACCAGGCTGACTGTGACA

[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 the constant region of the TCR α chain.

[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 the constant region of the TCR β chain.

[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, which encodes the TCR α-chain constant 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:22, which encodes the TCR β-chain constant region. In some embodiments, the nucleic acid further comprises the nucleotide sequence as shown in SEQ ID NO:21, which encodes the TCR α-chain constant region, and comprises the nucleotide sequence as shown in SEQ ID NO:22, which encodes the TCR β-chain constant region.

[0097] SEQ ID NO:21

[0098] ATTCAGAACCCCGAGCCTGCCGTGTACCAGCTGAAGGACCCCAGAAGCCAGGACTCTACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAGAGCGGCACCTTCATCACCGACAAGTGCGTGCTGGACATGAAGGCCATGGACAGCAAGAGCAACGGCGCCATCGCCTGGAGCAACCAGACCAGCTTCACCTGTCAGGACATCTTCAAGGAGACCAACGCCACCTACCCCAGCAGCGACGTTCCCTGTGATGCCACACTGACCGAGAAGAGCTTCGAGACCGACATGAACCTGAACTTCCAGAACCTGCTGGTGATCGTGCTGAGGATCTTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGGCTGTGGAGCAGC

[0099] SEQ ID NO:22

[0100] GAGGACCTGAGGAACGTGACCCCTCCCAAGGTGAGCCTGTTCGAGCCCTCTAAGGCCGAGATCGCCAACAAGCAGAAAGCTACCCTGGTGTGCTTGGCCAGGGGCTTCTTCCCCGACCACGTGGAACTTAGCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGAGTGTGCACAGATCCCCAAGCCTACAAGGAGAGCAACTACAGCTACTGCCTGAGCAGCAGGCTGAGGGTGAGCGCCACCTTCTGGCATAATCCTAGGAACCACTTCAGGTGCCAGGTGCAGTTCCATGGCTTGAGCGAGGAAGACAAGTGGCCCGAGGGCAGCCCCAAACCTGTGACCCAAAACATTAGCGCCGAGGCTTGGGGCAGAGCCGACTGCGGCATTACAAGCGCTTCTTACCAGCAAGGCGTGCTGAGCGCCACCATCCTGTACGAGATTCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTACACTGGTGGTGATGGCCATGGTGAAGAGGAAGAACAGC

[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 α 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, which encodes the 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, which encodes the TCR β chain. In some preferred embodiments, the nucleic acid further comprises the nucleotide sequence as shown in SEQ ID NO:14, which encodes the TCR α chain, and comprises the nucleotide sequence as shown in SEQ ID NO:16, which encodes the TCR β chain.

[0104] Vector

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

[0106] As used herein, the term "vector" is a nucleic acid molecule used as a vehicle to transfer (exogenous) genetic material into a host cell, in which the nucleic acid molecule serving as the vector can, for example, replicate and / or be expressed. The term "vector" encompasses, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyomavirus vectors, and adeno-associated vectors (AAV)), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). A vector itself is typically a nucleotide sequence, usually a DNA sequence containing an insert (transgene) and a larger sequence serving as the "backbone" of the vector. Engineered vectors typically contain an origin of replication for autonomous replication in a host cell (if stable expression of the polynucleotide is desired), a selectable marker, and restriction enzyme cleavage sites (such as a multiple cloning site, MCS). Vectors may additionally contain promoters, genetic markers, reporter genes, targeting sequences, and / or protein purification tags. 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 lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes.

[0108] Cell

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

[0110] As used herein, the term "cell" 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 alga, or can be a prokaryotic cell, for example, a bacterium or a protozoan. The cell can be a cultured cell or a primary cell, that is, directly isolated from an organism, such as a human. The cell can be an adherent cell or a suspension cell, that is, a cell that grows 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 (such as T cells, NK cells), monocytes (such as PBMC), and stem cells. As used herein, the term "stem cell" is a stem cell for expressing the TCR of the present disclosure (especially the TCR). For example, the stem cell can be a lymphoid progenitor cell, an induced pluripotent stem cell (iPSC), or a hematopoietic stem cell (HSC). In some embodiments, the stem cell does not include embryonic stem cells obtained by destroying human embryos, and / or does not include totipotent stem cells used for developing and forming an animal individual. Transferring a gene to a stem cell generally does not result in the expression of the TCR on the cell surface because the CD3 molecule is not expressed on the surface of the stem cell. However, when the stem cell differentiates into a lymphoid precursor that migrates to the thymus, the expression of the CD3 molecule will initiate the expression of the introduced TCR molecule 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 cells are T cells. The T cells can be any T cells, such as cultured T cells, e.g., primary T cells or T cells from a cultured T cell line, such as Jurkat, SupTl, etc., or T cells obtained from a mammal. If obtained from a mammal, the T cells can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can also be enriched or purified. Preferably, the T cells are human T cells. More preferably, the T cells are isolated from humans. The T cells can be any type of T cell and can be 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 cells do not express endogenous TCR.

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

[0115] As used herein, the term "transfection" is the process of deliberately introducing a nucleic acid molecule or polynucleotide (including a vector) into a target cell. An example is RNA transfection, i.e., the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into a host cell. This term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells usually involves creating transient pores or "holes" in the cell membrane to allow the uptake of materials. Transfection can be carried out using calcium phosphate, by electroporation, by cell squeezing, or by mixing cationic lipids with the material to produce liposomes that fuse with the cell membrane and deposit their cargo inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection, and electroporation.

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

[0117] Use

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

[0119] Specifically, the present invention provides a drug for treating KRAS G12V-positive tumors, which comprises the TCR targeting KRAS G12V provided by the present invention or an antigen-binding fragment thereof, 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 exists in the form of a conjugate, which comprises the TCR of the present disclosure and an active agent conjugated or linked to the TCR.

[0121] In some embodiments, the active agent is selected from detectable labels, immunostimulatory molecules, and therapeutic agents. 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 cytokines (such as IL-2 and IFN-γ), chemokines (such as IL-8), platelet factors (such as platelet factor 4), and complement initiators. Preferably, the therapeutic agent is selected from immunomodulators, radioactive compounds, enzymes, chemotherapeutic agents, and toxins. Other suitable therapeutic agents include small molecule cytotoxic agents, i.e., compounds capable of killing mammalian cells with a molecular weight less than 700 daltons. Such compounds may also contain toxic metals capable of having a cytotoxic effect. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decay or are converted under physiological conditions to release a cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, reichamycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porfimer sodium photosensitizer II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E, vincristine, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof capable of killing mammalian cells; for example, ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, Dnase, and RNase; radionuclides, i.e., unstable isotopes of elements that emit one or more α or β particles or γ rays while decaying, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225, and astatine-213; chelating agents that can be used to facilitate 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 exists 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" particularly refers to compositions suitable for administration to humans. However, the term generally also encompasses compositions suitable for administration to non-human animals. The compositions and their components (i.e., active agents and optional carriers or excipients) are preferably pharmaceutically acceptable, i.e., capable of eliciting the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects. The pharmaceutically acceptable compositions 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, and more particularly in humans.

[0124] The term "excipient" includes fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesion agents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifying agents, stabilizers, and tonicity regulators. Those skilled in the art know how to select suitable excipients for preparing the compositions of the present invention. Exemplary carriers for use in the compositions of the present invention include saline, buffered saline, glucose, and water. Generally, the selection of suitable excipients depends particularly on the active agent used, the disease to be treated, and the desired dosage form of the composition.

[0125] Depending on the active agent employed (such as soluble TCR), the compositions of the present disclosure can 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 in the art for producing pharmaceuticals 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, entrapping, or lyophilizing processes. Compositions containing, for example, host cells or soluble TCR as described herein are generally 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 antibodies, chemotherapeutic agents, and small molecule drugs.

[0127] Preferred examples of the second therapeutic agent include known anti-cancer 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, vincristine, and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase, and RNase; 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 antibody 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 that includes a TCR or conjugate of the present disclosure for detecting 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 disorder in a subject, and includes a TCR or conjugate of the present disclosure.

[0130] In some embodiments, the conjugate includes a detectable label. Examples of detectable labels include, but are not limited to, biotin, streptavidin, an enzyme or 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 catalytically active fragment thereof, a radionuclide, a fluorescent, phosphorescent, or chemiluminescent molecule. In some embodiments, the kit may further include instructions on how to use the kit.

[0131] In an embodiment of the present disclosure, the disease and / or disorder may include a KRAS G12V mutation-positive disease and / or disorder. 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 will be further illustrated by the following specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are carried out according to the conventional conditions in the art, for example, the conditions described in Molecular Cloning: A Laboratory Manual (Third Edition) (2001), Cold Spring Harbor Laboratory Press, by Sambrook and Russell et al., or according to the conditions recommended by the manufacturer. Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available.

[0133] Example

[0134] Example 1 Screening of Modified TCRs with Enhanced Affinity

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

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

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

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

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

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

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

[0142] The amino acid sequence of the variable region of the TCR α chain 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 variable region of the TCR β chain is shown in SEQ ID NO:8, and the coding sequence is shown in SEQ ID NO:15;

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

[0145] To increase the correct pairing and expression of exogenous TCR on the surface of human T cells, the variable regions of the α and β chains of the TCR were fused to the murine TCR constant region, and classical α-chain C-region "LIV" substitutions (i.e., amino acid substitutions were introduced into the murine TCR α-chain constant region to make the constant region contain the amino acid sequence LLVIVLRIL; shown in bold) and classical α-chain T48C and β-chain S57C substitutions were introduced. 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 constant region of the TCR β chain is shown in SEQ ID NO:18, and the coding sequence is shown in SEQ ID NO:22.

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

[0147] The affinity of the TCR provided by the present invention was measured as follows:

[0148] The parental TCR and the TCR sequence provided by the present invention were respectively cloned into the TCR tandem expression lentiviral vector, the lentivirus was packaged, the reporter cells JKR9 were transduced, and then the TCR-expressing positive cells were sorted. T2 cells positive for HLA-A*1101 were loaded with different concentrations of the KRAS G12V target antigen peptide (SEQ ID NO:11), and then co-incubated and activated with the JKR9 reporter cells transduced with the parental TCR and the TCR of the present invention respectively. After 16 h 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. The TCR provided by the present invention had a 5-fold increase in affinity compared to the parental TCR, showing a significant improvement ( Figure 1 )

[0149] Example 2. Efficient 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 not transduced with TCR were parallelly amplified and cultured as the negative control of effector cells. HELA-A1101-G12V-E-L (KRAS G12V positive, HLA-A*11:01 positive), PANC1-A1101-G12V-E-L cells (KRAS G12V positive, HLA-A*11:01 positive), and T2KO-A1101 loaded with KRAS G12V 7-16 polypeptide (T2+KV2) were used as HLA-antigen peptide-matched positive target cells. All target cells stably expressed the luciferase gene. Effector cells were incubated with different target cells at an effector-to-target ratio (E:T) of 1:1 for 16 - 20 h, and luciferase substrate was added to detect the surviving target cells. The proportion of killed target cells was calculated based on the remaining target cells, and the results are as Figure 2 shown.

[0151] It can be seen from Figure 2 that the affinity-optimized TCR-T cells had obvious killing activity against HELA-A1101-G12V-E-L, PANC1-A1101-G12V-E-L, and T2KO-A1101 loaded with KRAS G12V 7-16 peptide positive target cells, and their killing activity was significantly higher than that of KVA11-N04 TCR-T cells. HELA-A1101-E-L cells as target antigen-negative target cells and T2 cells as target antigen- and HLA-double-negative target cells could not be killed by TCR-T expressing KVA11-N04 and KVA11-M04-178 in the experiment. These results indicate that the 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 improvement of affinity.

[0152] Example 3. Specific INF-γ Secretion of 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 transduced TCR were used as negative controls for effector cells after parallel amplification culture. HELA-A1101-G12V-E-L (KRAS G12V positive, HLA-A*11:01 positive), PANC1-A1101-G12V-E-L cells (KRAS G12V positive, HLA-A*11:01 positive), and T2KO-A1101 loaded with KRAS G12V 7-16 polypeptides were used as HLA-antigen peptide-matched positive target cells; effector cells and different target cells were incubated at an effector-to-target ratio (E:T) of 1:1 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 as Figure 3 shown.

[0154] The results showed that obvious IFN-γ secretion could be detected after co-incubation of affinity-optimized TCR-T cells with positive target cells HELA-A1101-G12V-E-L, PANC1-A1101-G12V-E-L, and T2KO-A1101 loaded with KRAS G12V 7-16 polypeptides, and the amount of IFN-γ secretion was significantly higher than that of KVA11-N04 TCR-T cells. HELA-A1101-E-L cells as target antigen-negative target cells and T2 cells as target antigen- and HLA-double-negative target cells could not activate the IFN-γ secretion of TCR-T of KVA11-N04 and KVA11-M04-178 in the experiment. These results indicate 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 in affinity.

[0155] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. Including any suitable combination of each specific technical feature. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods. But these simple modifications and combinations should also be regarded as the content disclosed by the present invention and all fall within the protection scope of the present invention.

Claims

1. An affinity-enhanced T cell receptor, the T cell receptor comprising a variable region of the TCR α chain and a variable region of the TCR β chain, characterized in that, It has the activity of binding to the KRAS G12V epitope-HLA-A*11 complex, and the variable region of the TCR α chain includes CDR1α, CDR2α and CDR3α complementarity-determining regions, and the variable region of the TCR β chain includes CDR1β, CDR2β and CDR3β complementarity-determining regions: wherein, The amino acid sequence of CDR1α is as shown in SEQ ID NO:1: NSASDY; The amino acid sequence of CDR2α is as shown in SEQ ID NO:2: LYSTMDK; The amino acid sequence of CDR3α is as shown in SEQ ID NO:3: AENSDGTSYGKLT; The amino acid sequence of CDR1β is as shown in SEQ ID NO:4: SGHAT; The amino acid sequence of CDR2β is as shown in SEQ ID NO:5: FQNNGV; The amino acid sequence of CDR3β is as shown in SEQ ID NO:6: ASSLVGSPDYEQY.

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

8.

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

10.

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

5. The affinity-enhanced T cell receptor according to any one of claims 1-4, wherein, The amino acid sequence of the KRAS G12V epitope is as shown in SEQ ID NO:11: VVVGAVGVGK.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains the nucleic acid sequence encoding the affinity-enhanced T cell receptor according to any one of claims 1-5.

7. The nucleic acid molecule according to claim 6, wherein, The nucleic acid molecule contains the nucleotide sequence SEQ ID NO:13 encoding the variable region of the TCR α chain, or the nucleic acid molecule contains the nucleotide sequence SEQ ID NO:14 encoding the TCR α chain; and / or The nucleic acid molecule contains the nucleotide sequence SEQ ID NO:15 encoding the variable region of the TCR β chain, or the nucleic acid molecule contains 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 affinity-enhanced T cell receptor according to any one of claims 1-5, or carries the nucleic acid molecule according to claim 6 or 7, or contains the vector according to claim 8.

10. A drug for treating KRAS G12V-positive tumors, the drug contains the affinity-enhanced T cell receptor according to any one of claims 1-5, 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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