WO2021032020A1 - 一种识别afp的高亲和力t细胞受体 - Google Patents

一种识别afp的高亲和力t细胞受体 Download PDF

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WO2021032020A1
WO2021032020A1 PCT/CN2020/109351 CN2020109351W WO2021032020A1 WO 2021032020 A1 WO2021032020 A1 WO 2021032020A1 CN 2020109351 W CN2020109351 W CN 2020109351W WO 2021032020 A1 WO2021032020 A1 WO 2021032020A1
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tcr
variable domain
chain variable
sghvs
fnyeaq
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PCT/CN2020/109351
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French (fr)
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张婷婷
唐先青
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广东香雪精准医疗技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of biotechnology, and more specifically to a T cell receptor (TCR) capable of recognizing a polypeptide derived from an AFP protein.
  • TCR T cell receptor
  • the invention also relates to the preparation and use of the receptor.
  • TCR T cell receptor
  • TCR is the only receptor for specific antigen peptides presented on the main histocompatibility complex (MHC). This exogenous or endogenous peptide may be the only sign of abnormal cells.
  • MHC main histocompatibility complex
  • APC antigen-presenting cells
  • the MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily but have specificity for antigen presentation. Different individuals have different MHCs, which can present different shortcomings in a protein antigen. Peptides to the surface of the respective APC cells. Human MHC is usually called HLA gene or HLA complex.
  • AFP ( ⁇ Fetoprotein), also known as ⁇ fetoprotein, is a protein expressed during embryonic development and the main component of embryonic serum. During development, AFP has a relatively high expression level in the yolk sac and liver, and is subsequently inhibited. In hepatocellular carcinoma, the expression of AFP is activated (Butterfield et al. J Immunol., 2001, Apr 15; 166(8): 5300-8). After AFP is produced in the cell, it is degraded into small molecule polypeptides, and combined with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface.
  • FMNKFIYEI is a short peptide derived from AFP antigen and is a target for the treatment of AFP-related diseases.
  • the FMNKFIYEI-HLA A0201 complex provides a marker for TCR to target tumor cells.
  • the TCR that can be combined with FMNKFIYEI-HLA A0201 complex has high application value for tumor treatment.
  • TCR that can target the tumor cell marker can be used to deliver cytotoxic agents or immunostimulants to target cells, or be transformed into T cells, so that T cells expressing the TCR can destroy tumor cells, so as to be called Adoptive immunotherapy is given to patients during the course of treatment.
  • the ideal TCR has a high affinity, so that the TCR can reside on the targeted cells for a long time.
  • it is preferable to use a medium affinity TCR it is preferable to use a medium affinity TCR. Therefore, those skilled in the art devote themselves to developing TCRs that can be used for different purposes to target tumor cell markers.
  • the purpose of the present invention is to provide a TCR with higher affinity to the FMNKFIYEI-HLA A0201 complex.
  • Another object of the present invention is to provide a method for preparing the above type of TCR and use of the above type of TCR.
  • the first aspect of the present invention provides a T cell receptor (TCR), which has the activity of binding to the FMNKFIYEI-HLA A0201 complex.
  • the T cell receptor has the activity of binding FMNKFIYEI-HLA A0201 complex, and the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain, and the TCR ⁇ chain variable The domain contains 3 CDR regions, and the reference sequence of the 3 CDR regions of the TCR ⁇ chain variable domain is as follows,
  • CDR3 ⁇ AVETSYDKVI, and contains at least one of the following mutations:
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the reference sequence of the 3 CDR regions of the TCR ⁇ chain variable domain is as follows,
  • CDR3 ⁇ ASSYGAGGPLDTQY, and contains at least one of the following mutations:
  • the mutation occurs in one or more CDR regions of the ⁇ chain and/or ⁇ chain variable domain.
  • the number of mutations in the 3 CDR regions of the TCR ⁇ chain variable domain is 1 to 9, and/or the number of mutations in the 3 CDR regions of the TCR ⁇ chain variable domain From 1 to 10.
  • the number of mutations in the CDR region of the TCR ⁇ chain may be 2, 3, 4, 5, 6, 7, 8, or 9.
  • the number of mutations in the CDR region of the TCR ⁇ chain can be 3, 4, 5, 6, 7, 8, 9, or 10.
  • the number of mutations in the CDR3 ⁇ of the ⁇ chain of the TCR is 4;
  • the affinity of the TCR and the FMNKFIYEI-HLA A0201 complex is at least twice that of the wild-type TCR.
  • the affinity of the TCR and the FMNKFIYEI-HLA A0201 complex is at least 2 times that of wild-type TCR; preferably, at least 5 times; more preferably, at least 10 times.
  • the affinity of the TCR and FMNKFIYEI-HLA A0201 complex is at least 50 times that of the wild-type TCR; preferably, at least 100 times; more preferably, at least 500 times.
  • the affinity of the TCR complex FMNKFIYEI-HLA A0201 is at least 103-fold of wild-type TCR; preferably, at least 5 * 10 3 times; more preferably, at least 104-fold; even more preferably Ground, at least 5*10 4 times.
  • the dissociation equilibrium constant KD of the TCR to the FMNKFIYEI-HLA A0201 complex is ⁇ 20 ⁇ M;
  • the dissociation equilibrium constant of the TCR to the FMNKFIYEI-HLA A0201 complex is 5 ⁇ M ⁇ KD ⁇ 10 ⁇ M; preferably, 0.1 ⁇ M ⁇ KD ⁇ 1 ⁇ M; more preferably, 1nM ⁇ KD ⁇ 100 nM.
  • amino acid sequence of the ⁇ chain variable domain of the wild-type TCR is SEQ ID NO:1
  • amino acid sequence of the ⁇ chain variable domain is SEQ ID NO: 2.
  • the alpha chain variable domain of the TCR comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:1; and/or the beta chain of the TCR The variable domain includes an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 2.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , characterized in that the amino acid sequence of CDR1 ⁇ is SGHVS.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • the amino acid sequence of the CDR3 ⁇ is: AS[ 3 ⁇ X1][3 ⁇ X2][3 ⁇ X3][3 ⁇ X4]GGPL[3 ⁇ X5][3 ⁇ X6]Q[3 ⁇ X7], where [3 ⁇ X1] is A or S; and/or [3 ⁇ X2] is Y, L, P, R, K, Q or F; and/or [3 ⁇ X3] is G, F, M, Y, H, S, W, or A; and/or [3 ⁇ X4] is A, S, P, or G; and/or [3 ⁇ X5] is D , S, G, R, M, or E; and/or [3 ⁇ X6] is T, G, E, S, M, or A; and/or [3 ⁇ X7] is
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , characterized in that the CDR3 ⁇ is selected from the following group:
  • ASALMSGGPLDTQY ASARYPGGPLDTQY, ASARHAGGPLDTQY, ASSYGAGGPLDTQY, ASALFSGGPLDTQY, ASAPFSGGPLDTQY, ASAKMSGGPLDTQY, ASSYGAGGPLGEQW, ASSYGAGGPLGAQA, ASSQSGGGPLDTQY, ASSYGAGGPLGEQV, ASSYGAGGPLMAQA, ASALYSGGPLDTQY, ASSPFSGGPLDTQY, ASSYGAGGPLGAQK, ASSYGAGGPLEGQV, ASSYGAGGPLSSQI, ASSLFGGGPLDTQY, ASSYGAGGPLSGQI, ASSYGAGGPLASQY, ASSYGAGGPLRTQM, ASSYGAGGPLGSQQ, ASSYGAGGPLGSQV, ASSYGAGGPLGSQA, ASSLFSGGPLDTQY, ASSLWSGGPLDTQY, ASSFAGGGPLDTQY and
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , characterized in that the amino acid sequence of CDR2 ⁇ is selected from FNYEAQ and FNYVSI.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , characterized in that the amino acid sequence of CDR1 ⁇ is selected from the following group: VGISA , AGLQA, VGLQA and PGLQA.
  • variable domain of the TCR ⁇ chain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • variable domain of the TCR ⁇ chain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , characterized in that the amino acid sequence of CDR2 ⁇ is selected from the following group: LSSGK , LPFGK and LPYQT.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , characterized in that the amino acid sequence of CDR3 ⁇ is selected from the following group: AVETSYDKVI , AVETTRDKVI and AVETFDKVI.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , characterized in that the amino acid sequence of CDR1 ⁇ is VGISA, CDR2 ⁇ amino acid The sequence is LSSGK, and the amino acid sequence of CDR3 ⁇ is AVETSYDKVI.
  • amino acid sequence of the alpha chain variable domain of the TCR is SEQ ID NO:1.
  • the mutation occurs in one or more CDR regions of the ⁇ chain and/or ⁇ chain variable domain.
  • the T cell receptor has the activity of binding to the FMNKFIYEI-HLA A2 complex and comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain.
  • the TCR A mutation occurs in the ⁇ chain variable domain shown in SEQ ID NO:1, and the mutated amino acid residue positions include one or more of 27V, 29I, 30S, 50S, 51S, 52G, 53K, 92S, and 93Y
  • the numbering of the amino acid residues adopts the ⁇ -chain variable domain numbering shown in SEQ ID NO: 1; and/or the TCR is mutated in the ⁇ -chain variable domain shown in SEQ ID NO: 2
  • the mutated amino acid residue positions include one or more of 52E, 53A, 54Q, 95S, 96Y, 97G, 98A, 103D, 104T, 106Y, wherein the amino acid residue numbering adopts SEQ ID NO: 2. The number shown;
  • the TCR ⁇ chain variable domain after mutation includes one or more amino acid residues selected from the group consisting of: 27A or 27P; 29L; 30Q; 50P; 51F or 51Y; 52Q; 53T; 92T or 92F and 93R Or 93N, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:1; and/or the TCR ⁇ chain variable domain after mutation includes one or more amino acid residues selected from the group consisting of: 52V, 53S , 54I, 95A, 96L or 96P or 96R or 96K or 96Q or 96F, 97F or 97M or 97Y or 97H or 97S or 97W or 97A, 98S or 98P or 98G, 103G or 103S or 103M or 103E or 103A or 103R, 104S Or 104A or 104E or 104G or 104M, 106A or 106V or
  • the TCR has a CDR selected from the following group:
  • the TCR is soluble.
  • the TCR is ⁇ heterodimeric TCR or single-stranded TCR.
  • the amino acid sequence of the ⁇ chain variable domain of the TCR is selected from: SEQ ID NO: 9-13 and 45-51; and/or the amino acid sequence of the ⁇ chain variable domain of the TCR is selected from: SEQ ID NO: 14-40 and 52-79.
  • the TCR of the present invention is an ⁇ heterodimeric TCR.
  • the TCR has an ⁇ chain constant region sequence TRAC*01 and a ⁇ chain constant region sequence TRBC1*01 or TRBC2*01.
  • the TCR is an ⁇ heterodimeric TCR
  • the ⁇ chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO:1; preferably, at least 92% ; More preferably, at least 94% (eg, can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology)
  • the amino acid sequence of sequence homology; and/or the ⁇ chain variable domain of the TCR contains at least 90%, preferably at least 92%, and more preferably at least 94% of the amino acid sequence shown in SEQ ID NO: 2 % (Eg, may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) of the amino acid sequence.
  • the TCR comprises (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) And (ii) both comprise the variable domain and at least a part of the constant domain of the TCR chain.
  • the TCR is an ⁇ heterodimeric TCR, and the ⁇ chain variable region and the ⁇ chain constant region of the TCR contain artificial interchain disulfide bonds.
  • cysteine residues forming artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region of the TCR are substituted with one or more groups selected from the following point:
  • the TCR containing an artificial inter-chain disulfide bond between the ⁇ -chain variable region and the ⁇ -chain constant region includes the ⁇ -chain variable domain, the ⁇ -chain variable domain, and all or all except the transmembrane domain. Part of the ⁇ chain constant domain, but it does not contain the ⁇ chain constant domain, the ⁇ chain variable domain of the TCR and the ⁇ chain form a heterodimer.
  • the TCR containing an artificial interchain disulfide bond between the variable region of the ⁇ chain and the constant region of the ⁇ chain comprises (i) all or part of the TCR ⁇ chain except for its transmembrane domain, and (ii) All or part of the TCR ⁇ chain except its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least a part of the constant domain of the TCR chain.
  • the TCR is an ⁇ heterodimeric TCR, which comprises (i) all or part of the TCR ⁇ chain except its transmembrane domain, and (ii) all except its transmembrane domain Or part of the TCR ⁇ chain, wherein (i) and (ii) both comprise the variable domain and at least a part of the constant domain of the TCR chain, and the constant region of the ⁇ chain and the constant region of the ⁇ chain contain artificial interchain disulfide bonds.
  • the ⁇ -chain constant region and the ⁇ -chain constant region of the TCR contain artificial interchain disulfide bonds.
  • cysteine residues forming artificial interchain disulfide bonds between the constant regions of the TCR ⁇ and ⁇ chains are substituted for one or more sets of sites selected from the following:
  • the TCR is selected from the following group:
  • the TCR is a single-chain TCR.
  • the TCR is a single-chain TCR composed of an ⁇ -chain variable domain and a ⁇ -chain variable domain, and the ⁇ -chain variable domain and ⁇ -chain variable domain are composed of a flexible short peptide sequence (linker )connection.
  • the hydrophobic core of the TCR ⁇ chain variable domain and/or ⁇ chain variable domain is mutated.
  • the TCR with a mutation in the hydrophobic core is a single-chain TCR composed of an ⁇ variable domain and a ⁇ variable domain, and the ⁇ variable domain and ⁇ variable domain are composed of a flexible short peptide sequence ( linker) connection.
  • the TCR of the present invention is a single-chain TCR
  • the ⁇ -chain variable domain of the TCR contains at least 85%, preferably at least 90%, of the amino acid sequence shown in SEQ ID NO: 3; More preferably, at least 92%; most preferably, at least 94% (eg, it can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% sequence homology); and/or the ⁇ chain variable domain of the TCR contains at least 85% of the amino acid sequence shown in SEQ ID NO: 4, Preferably, at least 90%; more preferably, at least 92%; most preferably, at least 94% (eg, it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% sequence homology) of the amino acid sequence.
  • the TCR is selected from the following group:
  • a conjugate is bound to the C- or N-terminus of the ⁇ chain and/or ⁇ chain of the TCR.
  • the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modified part or a combination of any of these substances.
  • the therapeutic agent that binds to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the ⁇ or ⁇ chain of the TCR.
  • the second aspect of the present invention provides a multivalent TCR complex comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present invention.
  • the third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or the multivalent TCR complex described in the second aspect of the present invention or its complement sequence;
  • the fourth aspect of the present invention provides a vector containing the nucleic acid molecule described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a host cell that contains the vector of the fourth aspect of the present invention or the nucleic acid molecule of the third aspect of the present invention integrated into the chromosome.
  • the sixth aspect of the present invention provides an isolated cell that expresses the TCR described in the first aspect of the present invention.
  • the seventh aspect of the present invention provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, Or the cell described in the sixth aspect of the present invention.
  • the eighth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the present invention to a subject in need of treatment.
  • the ninth aspect of the present invention provides the use of the TCR according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, or the use of the cell according to the sixth aspect of the present invention, for preparing and treating tumors
  • the tumor is hepatocellular carcinoma.
  • the T cell receptor, the TCR complex described in claim 29, or the cell described in claim 33 are used as drugs for treating tumors.
  • the tumor is an AFP positive tumor; more preferably, the tumor is hepatocellular carcinoma.
  • the eleventh aspect of the present invention provides a method for preparing the T cell receptor according to the first aspect of the present invention, including the steps:
  • Figure 1a and Figure 1b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chain variable domains that can specifically bind to the FMNKFIYEI-HLA A0201 complex.
  • Figure 2a and Figure 2b are the amino acid sequence of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain of the single-chain template TCR constructed in the present invention.
  • Figures 3a and 3b are respectively the DNA sequence of the ⁇ chain variable domain and the DNA sequence of the ⁇ chain variable domain of the single-stranded template TCR constructed in the present invention.
  • Figures 4a and 4b are respectively the amino acid sequence and nucleotide sequence of the linker of the single-stranded template TCR constructed in the present invention.
  • Figures 5(1)-(5) respectively show the amino acid sequence of the alpha chain variable domain of the single-chain TCR with high affinity for the FMNKFIYEI-HLA A0201 complex, and the mutated residues are underlined.
  • Figures 6(1)-(27) respectively show the amino acid sequence of the ⁇ -chain variable domain of the single-chain TCR with high affinity for the FMNKFIYEI-HLA A0201 complex, and the mutated residues are underlined.
  • Figure 7a and Figure 7b are the amino acid sequence and DNA sequence of the single-stranded template TCR constructed in the present invention, respectively.
  • Figure 8a and Figure 8b respectively show the amino acid sequences of the soluble reference TCR alpha and beta chains in the present invention.
  • Figures 9(1)-(7) respectively show the amino acid sequence of the ⁇ chain variable domain of a heterodimeric TCR with high affinity for the FMNKFIYEI-HLA A0201 complex, and the mutated residues are underlined.
  • Figures 10(1)-(28) respectively show the amino acid sequence of the ⁇ -chain variable domain of a heterodimeric TCR with high affinity to the FMNKFIYEI-HLA A0201 complex, and the mutated residues are underlined.
  • Figure 11a and Figure 11b respectively show the extracellular amino acid sequences of wild-type TCR ⁇ and ⁇ chains that can specifically bind to the FMNKFIYEI-HLA A0201 complex.
  • Figure 12a and Figure 12b respectively show the amino acid sequences of wild-type TCR ⁇ and ⁇ chains that can specifically bind to the FMNKFIYEI-HLA A0201 complex.
  • Figure 13 is the binding curve of the soluble reference TCR, that is, the wild-type TCR and the FMNKFIYEI-HLA A0201 complex.
  • Figures 14a and 14b are the experimental results of the activation function of the effector cells transfected with the high-affinity TCR of the present invention.
  • Figure 15 shows the results of the killing function experiment of the effector cell LDH transfected with the high affinity TCR of the present invention.
  • the present invention has obtained a high-affinity T cell receptor (TCR) that recognizes the FMNKFIYEI short peptide (derived from the AFP protein).
  • the FMNKFIYEI short peptide is in the form of a peptide-HLA A0201 complex. Submit.
  • the high-affinity TCR is in the 3 CDR regions of its ⁇ chain variable domain:
  • CDR3 ⁇ A mutation in AVETSYDKVI; and/or in the 3 CDR regions of the ⁇ chain variable domain:
  • CDR3 ⁇ Mutation occurs in ASSYGAGGPLDTQY; and after the mutation, the affinity and/or binding half-life of the TCR of the present invention for the above-mentioned FMNKFIYEI-HLA A0201 complex is at least twice that of the wild-type TCR.
  • TCR T cell receptor
  • the International Immunogenetics Information System can be used to describe TCR.
  • the natural ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain. Broadly speaking, each chain includes a variable region, a connecting region, and a constant region.
  • the beta chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often regarded as a part of the connecting region.
  • the unique IMGT TRAJ and TRBJ are used to determine the TCR connection region, and the IMGT TRAC and TRBC are used to determine the TCR constant region.
  • Each variable region contains 3 CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, chimeric in the framework sequence.
  • the different numbers of TRAV and TRBV refer to different types of V ⁇ and V ⁇ respectively.
  • the alpha chain constant domain has the following symbols: TRAC*01, where "TR” represents the T cell receptor gene; "A” represents the alpha chain gene; C represents the constant region; "*01” represents alleles Gene 1.
  • the ⁇ chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR" represents the T cell receptor gene; "B” represents the ⁇ chain gene; C represents the constant region; "*01” represents the allele 1.
  • the constant region of the ⁇ chain is uniquely determined.
  • TCR ⁇ chain variable domain refers to the connected TRAV and TRAJ regions
  • TCR ⁇ chain variable domain refers to the connected TRBV and TRBD/TRBJ regions.
  • the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ ; the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ , respectively.
  • the framework sequence of the TCR variable domain of the present invention can be murine or human, preferably human.
  • the constant domain of TCR contains an intracellular part, a transmembrane region and an extracellular part.
  • the TCR of the present invention preferably does not include a transmembrane region.
  • the amino acid sequence of the TCR of the present invention refers to the extracellular amino acid sequence of the TCR.
  • the TCR sequence used in the present invention is of human origin.
  • the alpha chain amino acid sequence and the beta chain amino acid sequence of the "wild-type TCR" in the present invention are SEQ ID NO: 82 and SEQ ID NO: 83, respectively, as shown in Figures 12a and 12b.
  • the alpha chain amino acid sequence and the beta chain amino acid sequence of the "reference TCR” in the present invention are SEQ ID NO: 43 and SEQ ID NO: 44, as shown in Figures 8a and 8b.
  • the extracellular amino acid sequences of the alpha chain and beta chain of the "wild-type TCR" in the present invention are SEQ ID NO: 80 and SEQ ID NO: 81, respectively, as shown in Figures 11a and 11b.
  • amino acid sequences of the alpha and beta chain variable domains of the wild-type TCR capable of binding to the FMNKFIYEI-HLA A0201 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Figures 1a and 1b.
  • polypeptide of the present invention TCR of the present invention
  • T cell receptor of the present invention are used interchangeably.
  • the position numbers of the amino acid sequence of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered from N-terminal to C-terminal.
  • N The 60th amino acid in the sequence from end to C end is P (proline), then it can be described as Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, or it can be expressed as TRBC1* 01 or TRBC2*01 exon 1’s 60th amino acid, as in TRBC1*01 or TRBC2*01, the 61st amino acid from N-terminal to C-terminal is Q (glutamine), then this In the present invention, it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1, or it can be expressed as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, and so on.
  • the position numbers of the amino acid sequences of TRAV and TRBV in the variable regions are numbered according to the position numbers listed in IMGT.
  • the position number listed in IMGT is 46, then it is described as the 46th amino acid of TRAV in the present invention, and so on.
  • sequence position numbers of other amino acids have special instructions, follow the special instructions.
  • tumor is meant to include all types of cancer cell growth or carcinogenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of the pathological type or the stage of infection.
  • tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include: malignant tumors of different organ systems, such as sarcoma, lung squamous cell carcinoma and cancer.
  • sarcoma for example: infected prostate, lung, breast, lymph, gastrointestinal (for example: colon), and genitourinary tract (for example: kidney, epithelial cells), pharynx.
  • Lung squamous cell carcinoma includes malignant tumors, for example, most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer and esophageal cancer.
  • malignant tumors for example, most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer and esophageal cancer.
  • the above-mentioned metastatic lesions of cancer can also be treated and prevented by the method and composition of the present invention.
  • the ⁇ chain variable domain and ⁇ chain variable domain of TCR each contain 3 CDRs, which are similar to the complementarity determining regions of antibodies.
  • CDR3 interacts with short antigen peptides
  • CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigen short peptide-HLA complex.
  • the amino acid sequence of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain of the wild-type TCR that can bind the antigen short peptide FMNKFIYEI and HLA A0201 complex are SEQ ID NO:1 and SEQ, respectively ID NO: 2, this sequence is the first discovery by the inventor. It has the following CDR regions:
  • the present invention obtains a high-affinity TCR whose affinity with the FMNKFIYEI-HLA A0201 complex is at least twice the affinity of the wild-type TCR and the FMNKFIYEI-HLA A0201 complex by screening the above-mentioned CDR regions for mutation.
  • the present invention provides a T cell receptor (TCR), which has the activity of binding FMNKFIYEI-HLA A0201 complex.
  • the T cell receptor comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain.
  • the TCR ⁇ chain variable domain comprises 3 CDR regions.
  • the reference sequences of the 3 CDR regions of the TCR ⁇ chain variable domain are as follows:
  • CDR3 ⁇ AVETSYDKVI, and contains at least one of the following mutations:
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the reference sequence of the 3 CDR regions of the TCR ⁇ chain variable domain is as follows,
  • CDR3 ⁇ ASSYGAGGPLDTQY, and contains at least one of the following mutations:
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO:1, namely CDR1, CDR2, and CDR3 are located at positions 27-31, 49-53, and 88-97 of SEQ ID NO:1, respectively .
  • amino acid residue numbering adopts the numbering shown in SEQ ID NO:1, 27V is the first V of CDR1 ⁇ , 29I is the third I of CDR1 ⁇ , 30S is the fourth S of CDR1 ⁇ , and 50S is It is the second S of CDR2 ⁇ , 51S is the third S of CDR2 ⁇ , 52G is the fourth G of CDR2 ⁇ , 53K is the fifth K of CDR2 ⁇ , and 92S is the fifth S and 93Y of CDR3 ⁇ . It is the 5th Y of CDR3 ⁇ .
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2 and CDR3 are located at positions 27-31, 49-54 and 93 of SEQ ID NO: 2, respectively. -106 people. Therefore, the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 2, 52E is the fourth position E of CDR2 ⁇ , 53A is the fifth position A of CDR2 ⁇ , and 54Q is the sixth position Q and 95S of CDR2 ⁇ .
  • the 3rd S and 96Y of CDR3 ⁇ are the 4th Y of CDR3 ⁇
  • 97G is the 5th G of CDR3 ⁇
  • 98A is the 6th A of CDR3 ⁇
  • 103D is the 3rd D and 104T of CDR11 ⁇ .
  • the 12th T and 106Y of CDR3 ⁇ are the 14th Y of CDR3 ⁇ .
  • the TCR ⁇ chain variable domain after mutation includes one or more amino acid residues selected from the group consisting of: 27A or 27P; 29L; 30Q; 50P; 51F or 51Y; 52Q; 53T; 92T or 92F and 93R Or 93N, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:1; and/or the TCR ⁇ chain variable domain after mutation includes one or more amino acid residues selected from the group consisting of: 52V, 53S , 54I, 95A, 96L or 96P or 96R or 96K or 96Q or 96F, 97F or 97M or 97Y or 97H or 97S or 97W or 97A, 98S or 98P or 98G, 103G or 103S or 103M or 103E or 103A or 103R, 104S Or 104A or 104E or 104G or 104M, 106A or 106V or
  • the specific form of the mutation in the ⁇ chain variable domain includes one or several groups of V27A/P, I29L, S30Q, S50P, S51F/Y, G52Q, K53T, S92T/F, Y93R/N;
  • Specific forms of the mutations in the ⁇ chain variable domain include E52V, A53S, Q54I, S95A, Y96L/P/R/K/Q/F, G97F/M/Y/H/S/W/A, A98S/P
  • the TCR according to the present invention includes a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain, and the TCR ⁇ chain variable domain includes CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ , and the TCR ⁇
  • the chain variable domain includes CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ .
  • the CDR1 ⁇ includes the sequence: [1 ⁇ X1]G[1 ⁇ X2][1 ⁇ X3]A, wherein [1 ⁇ X1], [1 ⁇ X2], and [1 ⁇ X3] are each independently selected from any natural amino acid residues.
  • the [1 ⁇ X1] is V or A or P.
  • the [1 ⁇ X2] is I or L.
  • the [1 ⁇ X3] is S or Q.
  • the [1 ⁇ X1] is V or A or P
  • [1 ⁇ X2] is I or L
  • [1 ⁇ X3] is S or Q.
  • the CDR1 ⁇ comprises a sequence selected from the group consisting of VGISA, AGLQA, VGLQA and PGLQA.
  • the CDR2 ⁇ contains the sequence: L[2 ⁇ X1][2 ⁇ X2][2 ⁇ X3][2 ⁇ X4], wherein [2 ⁇ X1], [2 ⁇ X2], [2 ⁇ X3], [2 ⁇ X4] are each independently selected from any Natural amino acid residues.
  • the [2 ⁇ X1] is S or P.
  • the [2 ⁇ X2] is S or F or Y.
  • the [2 ⁇ X3] is G or Q.
  • the [2 ⁇ X4] is K or T.
  • the [2 ⁇ X1] is S or P
  • [2 ⁇ X2] is S or F or Y
  • [2 ⁇ X3] is G or Q
  • [2 ⁇ X4] is K or T.
  • the CDR2 ⁇ comprises a sequence selected from the group consisting of LSSGK, LPFGK and LPYQT.
  • the CDR3 ⁇ includes the sequence: AVET[3 ⁇ X1][3 ⁇ X2]DKVI, wherein [3 ⁇ X1] and [3 ⁇ X2] are each independently selected from any natural amino acid residue.
  • the [3 ⁇ X1] is T or F.
  • the [3 ⁇ X2] is R or N.
  • the [3 ⁇ X1] is T or F
  • [3 ⁇ X2] is R or N.
  • the CDR3 ⁇ comprises a sequence selected from the group consisting of AVETSYDKVI, AVETTRDKVI and AVETFDKVI.
  • the TCR includes a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
  • the TCR ⁇ chain variable domain includes CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇
  • the TCR ⁇ chain variable domain The domain includes CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , wherein the CDR1 ⁇ includes the sequence: SGHVS.
  • the CDR2 ⁇ comprises the sequence: FNY[2 ⁇ X1][2 ⁇ X2][2 ⁇ X3], wherein [2 ⁇ X1], [2 ⁇ X2], and [2 ⁇ X3] are each independently selected from any natural amino acid residues.
  • the [2 ⁇ X1] is Q or H.
  • the [2 ⁇ X2] is N or G.
  • the [2 ⁇ X3] is E or D.
  • the CDR2 ⁇ comprises a sequence selected from the group consisting of FNYEAQ and FNYVSI.
  • the CDR3 ⁇ includes the sequence: AS[3 ⁇ X1][3 ⁇ X2][3 ⁇ X3][3 ⁇ X4]GGPL[3 ⁇ X5][3 ⁇ X6]Q[3 ⁇ X7].
  • [3 ⁇ X1], [3 ⁇ X2], [3 ⁇ X3], [3 ⁇ X4], [3 ⁇ X5], [3 ⁇ X6] and [3 ⁇ X7] are each independently selected from any natural amino acid residues.
  • the [3 ⁇ X1] is A or S.
  • the [3 ⁇ X2] is Y or L or P or R or K or Q or F.
  • the [3 ⁇ X3] is G or F or M or Y or H or S or W or A.
  • the [3 ⁇ X4] is S or A or P or G.
  • the [3 ⁇ X5] is D, G, S, M, E, A or R.
  • the [3 ⁇ X6] is T, S, A, E, G or M.
  • the [3 ⁇ X7] is Y, A, V, I, W, K, M, Q or R.
  • the TCR ⁇ chain variable domain comprises CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , characterized in that the amino acid sequence of CDR1 ⁇ is VGISA, the amino acid sequence of CDR2 ⁇ is LSSGK, and the amino acid sequence of CDR3 ⁇ is AVESTSYDKVI.
  • the number of mutations in the CDR region of the TCR ⁇ chain may be 3, 4, 5, 6, 7, 8, or 9; and/or the number of mutations in the CDR region of the TCR ⁇ chain It can be 4, 5, 6, 7, 8, 9, or 10.
  • the TCR of the present invention is an ⁇ heterodimeric TCR, and the ⁇ chain variable domain of the TCR contains at least 90% of the amino acid sequence shown in SEQ ID NO:1; preferably, at least 92%; more preferably Ground, at least 94% (eg, can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) sequence homology
  • the ⁇ -chain variable domain of the TCR contains at least 90%, preferably at least 92%; more preferably, at least 94% (e.g., SEQ ID NO: 2 , It can be an amino acid sequence with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology).
  • the TCR of the present invention is a single-chain TCR, and the alpha chain variable domain of the TCR contains at least 85%, preferably at least 90%, and more preferably at least the amino acid sequence shown in SEQ ID NO: 3; 92%; most preferably, at least 94% (eg, it can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % Sequence homology); and/or the ⁇ chain variable domain of the TCR contains at least 85% of the amino acid sequence shown in SEQ ID NO: 4, preferably at least 90% %; More preferably, at least 92%; Most preferably, at least 94% (eg, it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Sequence homology) sequence homology amino acid sequence.
  • the TCR comprises (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) and (ii) Both contain the variable domain and at least a part of the constant domain of the TCR chain.
  • the Thr48 of the wild-type TCR ⁇ chain constant region TRAC*01 exon 1 was mutated to cysteine, and the ⁇ chain constant region TRBC1*01 or TRBC2*01 exon 1 Ser57 is mutated to cysteine to obtain a reference TCR.
  • the amino acid sequence of the reference TCR is shown in Figures 8a and 8b.
  • the cysteine residues after mutation are shown in bold letters.
  • the above cysteine substitution can form artificial interchain disulfide bonds between the constant regions of the ⁇ and ⁇ chains of the reference TCR to form a more stable soluble TCR, which makes it easier to evaluate the complex of TCR and FMNKFIYEI-HLA A2
  • the binding affinity and/or binding half-life between substances It should be understood that the CDR region of the TCR variable region determines its affinity with the pMHC complex. Therefore, the above-mentioned cysteine substitution in the TCR constant region will not affect the binding affinity and/or binding half-life of the TCR.
  • the measured binding affinity between the reference TCR and the FMNKFIYEI-HLA A0201 complex is considered to be the binding affinity between the wild-type TCR and the FMNKFIYEI-HLA A0201 complex.
  • the binding affinity between the TCR of the present invention and the FMNKFIYEI-HLA A0201 complex is at least 10 times that of the reference TCR and the FMNKFIYEI-HLA A0201 complex, it is equivalent to the TCR of the present invention and FMNKFIYEI.
  • the binding affinity between the HLA A0201 complex is at least 10 times that between the wild-type TCR and the FMNKFIYEI-HLA A0201 complex.
  • the binding affinity (inversely proportional to the dissociation equilibrium constant K D ) and the binding half-life (denoted as T 1/2 ) can be determined by any suitable method. Such as the use of surface plasmon resonance technology for detection. It should be understood that doubling the affinity of TCR will cause K D to be halved. T 1/2 is calculated as In2 divided by the dissociation rate (K off ). Therefore, doubling T 1/2 will cause K off to be halved.
  • the same test protocol is used to detect the binding affinity or binding half-life of a given TCR several times, for example, 3 times or more, and the results are averaged.
  • the surface plasmon resonance (BIAcore) method in the examples herein is used to detect the affinity of soluble TCR, and the conditions are: a temperature of 25° C. and a pH of 7.1-7.5.
  • This method detects that the dissociation equilibrium constant K D of the reference TCR for the FMNKFIYEI-HLA A2 complex is 9.89E-06M, which is 9.89 ⁇ M.
  • the dissociation equilibrium of the FMNKFIYEI-HLA A2 complex by the wild-type TCR is considered The constant K D is also 9.89 ⁇ M.
  • the affinity of the TCR and the FMNKFIYEI-HLA A0201 complex is at least 2 times that of wild-type TCR; preferably, at least 5 times; more preferably, at least 10 times.
  • the affinity of the TCR and FMNKFIYEI-HLA A0201 complex is at least 50 times that of the wild-type TCR; preferably, at least 100 times; more preferably, at least 500 times.
  • the affinity of the TCR complex FMNKFIYEI-HLA A0201 is at least 103-fold of wild-type TCR; preferably, at least 5 * 10 3 times; more preferably, at least 104-fold; even more preferably Ground, at least 5*10 4 times.
  • PCR polymerase chain reaction
  • LIC ligation-independent cloning
  • the method for producing the TCR of the present invention can be, but is not limited to, screening a TCR with high affinity for the FMNKFIYEI-HLA-A2 complex from a diverse library of phage particles displaying such TCR, as shown in the literature (Li, et al. (2005) Nature Biotech 23(3):349-354).
  • genes expressing wild-type TCR alpha and beta chain variable domain amino acids or genes expressing slightly modified wild-type TCR alpha and beta chain variable domain amino acids can be used to prepare template TCRs.
  • the DNA encoding the variable domain of the template TCR then introduces the changes required to produce the high-affinity TCR of the present invention.
  • the high-affinity TCR of the present invention comprises one of the amino acid sequence of the ⁇ -chain variable domain SEQ ID NO: 45-51 and/or one of the amino acid sequence of the ⁇ -chain variable domain SEQ ID NO: 52-79. Therefore, the TCR ⁇ chain containing the wild-type TCR ⁇ chain variable domain amino acid sequence (SEQ ID NO:1) can be combined with the TCR ⁇ chain containing one of SEQ ID NOs: 52-79 to form a heterodimeric TCR or a single-chain TCR molecular.
  • the TCR ⁇ chain containing the ⁇ variable domain amino acid sequence of the wild-type TCR can be combined with the TCR ⁇ chain comprising one of SEQ ID NOs: 45-51 to form a heterodimeric TCR or single-stranded TCR molecule .
  • the TCR ⁇ chain comprising one of the TCR ⁇ chain variable domain amino acid sequence SEQ ID NO: 45-51 may be combined with the TCR ⁇ chain comprising one of the TCR ⁇ chain variable domain amino acid sequence SEQ ID NO: 52-79 to form a heterogeneous two Poly TCR or single chain TCR molecule.
  • the amino acid sequences of the ⁇ -chain variable domain and ⁇ -chain variable domain forming the heterodimeric TCR molecule are preferably from Table 1 below:
  • the TCR of the present invention is a part having at least one TCR ⁇ and/or TCR ⁇ chain variable domain. They usually contain both the TCR ⁇ chain variable domain and the TCR ⁇ chain variable domain. They can be ⁇ heterodimers or single-stranded forms or any other forms that can exist stably. In adoptive immunotherapy, the full-length chain of ⁇ heterodimeric TCR (including cytoplasmic and transmembrane domains) can be transfected.
  • the TCR of the present invention can be used as a targeting agent for delivering therapeutic agents to antigen-presenting cells or combined with other molecules to prepare bifunctional polypeptides to target effector cells. In this case, the TCR is preferably in a soluble form.
  • the prior art discloses that the introduction of artificial interchain disulfide bonds between the ⁇ and ⁇ chain constant domains of TCR can obtain soluble and stable TCR molecules, as described in patent document PCT/CN2015/093806 Narrated. Therefore, the TCR of the present invention may be a TCR in which an artificial interchain disulfide bond is introduced between the residues of the constant domain of its ⁇ and ⁇ chains. Cysteine residues form artificial interchain disulfide bonds between the alpha and beta chain constant domains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate positions in the natural TCR to form artificial interchain disulfide bonds.
  • Thr48 in TRAC*01 exon 1 and replacing Ser57 in TRBC1*01 or TRBC2*01 exon 1 to form a disulfide bond can also be: Thr45 of TRAC*01 exon 1 and TRBC1*01 or Ser77 of TRBC2*01 exon 1; TRAC*01 exon Tyr10 of 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and TRBC1*01 or TRBC2*01 exon 1 Glu15; TRAC*01 exon 1 Arg53 and TRBC1*01 or TRBC2*01 exon 1 Ser54; TRAC*01 exon 1 Pro89 and Ala19 of TRBC1*01 or TRBC2*01 exon 1; or Tyr10 of TRAC*01 exon 1 and Glu20
  • cysteine residues replace any set of positions in the constant domains of the ⁇ and ⁇ chains.
  • One or more C-terminals of the TCR constant domain of the present invention can be truncated up to 15, or up to 10, or up to 8 or less amino acids so that it does not include cysteine residues to achieve the deletion of natural
  • the purpose of interchain disulfide bonds can also be achieved by mutating the cysteine residues that form natural interchain disulfide bonds to another amino acid.
  • the TCR of the present invention may contain artificial interchain disulfide bonds introduced between the residues of the constant domains of its ⁇ and ⁇ chains. It should be noted that, with or without the introduced artificial disulfide bonds between the constant domains, the TCR of the present invention can contain the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence.
  • the TRAC constant domain sequence of TCR and the TRBC1 or TRBC2 constant domain sequence can be connected by natural interchain disulfide bonds present in the TCR.
  • patent document PCT/CN2016/077680 also discloses that the introduction of artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region of the TCR can significantly improve the stability of the TCR. Therefore, the high-affinity TCR of the present invention may also contain artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region.
  • cysteine residue that forms an artificial interchain disulfide bond between the ⁇ chain variable region and the ⁇ chain constant region of the TCR is substituted: the 46th amino acid of TRAV and TRBC1*01 or TRBC2* The 60th amino acid of 01 exon 1; the 47th amino acid of TRAV and the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1; the 46th amino acid of TRAV and the TRBC1*01 or TRBC2*01 exon The 61st amino acid of sub 1; or the 47th amino acid of TRAV and the 60th amino acid of TRBC1*01 or TRBC2*01 exon 1.
  • such a TCR may comprise (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) and (ii) ) Contains the variable domain and at least a part of the constant domain of the TCR chain, and the ⁇ chain and the ⁇ chain form a heterodimer. More preferably, such a TCR may include an ⁇ chain variable domain and a ⁇ chain variable domain and all or part of the ⁇ chain constant domain except the transmembrane domain, but it does not include the ⁇ chain constant domain. The chain variable domain and the ⁇ chain form a heterodimer.
  • the TCR of the present invention also includes TCRs with mutations in the hydrophobic core region.
  • the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the TCR of the present invention, as described in Publication No. It is described in the patent document of WO2014/206304.
  • the TCR with mutation in the hydrophobic core region of the present invention may be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the ⁇ and ⁇ chains of the TCR.
  • the CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex. Mutations in the hydrophobic core can make the TCR more stable, but it will not affect its affinity with the short peptide-HLA complex.
  • the flexible peptide chain in the present invention can be any peptide chain suitable for connecting the variable domains of the TCR ⁇ and ⁇ chains.
  • the template chain constructed in Example 1 of the present invention for screening high-affinity TCRs is the high-stability single-chain TCR containing the hydrophobic core mutation. Using TCR with higher stability can more conveniently evaluate the affinity between TCR and FMNKFIYEI-HLA-A0201 complex.
  • the CDR regions of the ⁇ -chain variable domain and ⁇ -chain variable domain of the single-chain template TCR are exactly the same as those of the wild-type TCR. That is, the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : VGISA, CDR2 ⁇ : LSSGK, CDR3 ⁇ : AVETSYDKVI, and the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : SGHVS, CDR2 ⁇ : FNYEAQ, and CDR3 ⁇ : ASSYGAGGPLDTQY.
  • the amino acid sequence (SEQ ID NO: 41) and nucleotide sequence (SEQ ID NO: 42) of the single-stranded template TCR are shown in Figures 7a and 7b, respectively. Based on this, a single-chain TCR composed of ⁇ -chain variable domain and ⁇ -chain variable domain with high affinity to FMNKFIYEI-HLA A0201 complex was screened out.
  • the three CDRs of the single-stranded template TCR ⁇ chain variable domain SEQ ID NO: 3, namely CDR1, CDR2 and CDR3 are located at positions 27-31, 49-55 and 90-102 of SEQ ID NO: 3, respectively Bit.
  • the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 3, 27V is the first V of CDR1 ⁇ , 29I is the third I of CDR1 ⁇ , 30S is the fourth S of CDR1 ⁇ , and 50S is the It is the second S of CDR2 ⁇ , 51S is the third S of CDR2 ⁇ , 52G is the fourth G of CDR2 ⁇ , 53K is the fifth K of CDR2 ⁇ , and 92S is the fifth S and 93Y of CDR3 ⁇ . It is the 5th Y of CDR3 ⁇ .
  • the three CDRs of the single-stranded template TCR ⁇ chain variable domain SEQ ID NO: 4, namely CDR1, CDR2, and CDR3, are located at positions 27-31, 49-54, and No. 2 of SEQ ID NO: 2. 93-102 digits. Therefore, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 4, 52E is the 4th E of CDR2 ⁇ , 53A is the 5th A of CDR2 ⁇ , 54Q is the 6th Q and 95S of CDR2 ⁇ .
  • the 3rd S and 96Y of CDR3 ⁇ are the 4th Y of CDR3 ⁇
  • 97G is the 5th G of CDR3 ⁇
  • 98A is the 6th A of CDR3 ⁇
  • 103D is the 3rd D and 104T of CDR11 ⁇ .
  • the 12th T and 106Y of CDR3 ⁇ are the 14th Y of CDR3 ⁇ .
  • the ⁇ heterodimer with high affinity to the FMNKFIYEI-HLA-A0201 complex of the present invention is obtained by transferring the CDR regions of the ⁇ and ⁇ chain variable domains of the selected high-affinity single-chain TCR To the corresponding positions of the wild-type TCR ⁇ chain variable domain (SEQ ID NO: 1) and ⁇ chain variable domain (SEQ ID NO: 2). Another part is obtained by artificial combination based on the mutation sites of the CDR regions obtained by screening.
  • the high-affinity TCR of the present invention further comprises one of the amino acid sequence of the alpha chain variable domain of SEQ ID NO: 9-13 and/or one of the amino acid sequence of the beta chain variable domain of SEQ ID NO: 14-40. Therefore, the above-mentioned high-stability single-chain TCR ⁇ chain variable domain (SEQ ID NO: 3) as a template chain can be combined with the TCR ⁇ chain variable domain of one of SEQ ID NO: 14-40 to form the single chain TCR molecule.
  • the above-mentioned high-stability single-chain TCR ⁇ chain variable domain (SEQ ID NO: 4) as a template chain can be combined with the TCR ⁇ chain variable domain of one of SEQ ID NOs: 9-13 to form the single chain TCR molecule.
  • one of the TCR alpha chain variable domain SEQ ID NO: 9-13 and one of the TCR beta chain variable domain SEQ ID NO: 14-40 are combined to form the single-chain TCR molecule.
  • the amino acid sequences of the ⁇ -chain variable domain and ⁇ -chain variable domain of the high-affinity single-chain TCR molecule are preferably from Table 2 below:
  • the TCR of the present invention can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention contains two, three, four or more TCRs of the present invention combined to form a polymer.
  • the tetramerization domain of p53 can be used to generate a tetramer, or more A complex formed by combining the TCR of the present invention with another molecule.
  • the TCR complex of the present invention can be used to track or target cells presenting a specific antigen in vitro or in vivo, and can also be used to produce intermediates of other multivalent TCR complexes with such applications.
  • the TCR of the present invention can be used alone or combined with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the FMNKFIYEI-HLA-A0201 complex), a therapeutic agent, a PK (protein kinase) modified portion or any of the above Combination of substances combined or coupled.
  • Detectable markers used for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or capable of producing detectable products Of enzymes.
  • Therapeutic agents that can be combined or coupled with the TCR of the present invention include but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , Nature 339,394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy 51,565); 3. Cytokines such as IL-2, etc.
  • Gold nanoparticles/nano Stick (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Virus particles (Peng et al., 2004, Gene Treatment (Genetherapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nano magnetic particles; 10. Prodrug activating enzymes (for example, DT-cardiac Diazyme (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. Chemotherapeutics (for example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-cardiac Diazyme
  • BPHL biphenyl hydrolase-like protein
  • the antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies.
  • the combination of the aforementioned antibodies or fragments with TCR can affect effector cells. Orientation to better target target cells.
  • a preferred embodiment is that the TCR of the present invention is combined with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody.
  • the fusion molecule of the TCR and the anti-CD3 single chain antibody of the present invention includes the amino acid sequence of the variable domain of the TCR ⁇ chain selected from the group of SEQ ID NO: 9-13, 45-51 and/or the TCR ⁇ chain selected from the group The variable domain amino acid sequence SEQ ID NO: 14-40, 52-79.
  • the invention also relates to a nucleic acid molecule encoding the TCR of the invention.
  • the nucleic acid molecule of the present invention may be in the form of DNA or RNA.
  • DNA can be a coding strand or a non-coding strand.
  • the nucleic acid sequence encoding the TCR of the present invention may be the same as the nucleic acid sequence shown in the drawings of the present invention or a degenerate variant.
  • degenerate variant refers to a protein sequence that encodes SEQ ID NO: 41, but has the same sequence as SEQ ID NO: 42 Different nucleic acid sequences.
  • the full-length sequence of the nucleic acid molecule of the present invention or its fragments can usually be obtained by but not limited to PCR amplification method, recombination method or artificial synthesis method.
  • the DNA sequence encoding the TCR (or a fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the present invention also relates to a vector containing the nucleic acid molecule of the present invention, and a host cell produced by genetic engineering using the vector or coding sequence of the present invention.
  • the invention also includes isolated cells expressing the TCR of the invention, particularly T cells.
  • T cells There are many methods suitable for T cell transfection with DNA or RNA encoding the high-affinity TCR of the present invention (eg, Robbins et al., (2008) J. Immunol. 180: 6116-6131).
  • T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy.
  • Those skilled in the art can know many suitable methods for adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).
  • the present invention also provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell presenting the TCR of the present invention.
  • the present invention also provides a method for treating diseases, which comprises administering an appropriate amount of the TCR of the present invention, or the TCR complex of the present invention, or cells presenting the TCR of the present invention, or the pharmaceutical composition of the present invention to a subject in need of treatment.
  • V27A/P representing that the V at position 27 is replaced by A or replaced by P.
  • I29L represents that I at position 29 is replaced by L. The other analogy is similar.
  • the TCR of the present invention also includes at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid (especially the amino acid located outside the CDR region) of the TCR of the present invention. Or similar amino acids are replaced, and still maintain its functional TCR.
  • the present invention also includes a TCR slightly modified from the TCR of the present invention.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of the TCR of the present invention such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those TCRs produced by glycosylation modification during the synthesis and processing of the TCR of the present invention or in further processing steps. This modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase).
  • Modified forms also include sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). It also includes TCR that has been modified to improve its resistance to proteolysis or optimize its solubility.
  • the TCR, TCR complex of the present invention or T cells transfected with the TCR of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCR, multivalent TCR complex or cell of the present invention is usually provided as part of a sterile pharmaceutical composition, which usually includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in any suitable form (depending on the desired method of administration to the patient). It can be provided in a unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such kits (but not required) include instructions for use. It may include a plurality of such unit dosage forms.
  • the TCR of the present invention can be used alone, or can be combined or coupled with other therapeutic agents (for example, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent.
  • medicament carriers they themselves do not induce the production of antibodies that are harmful to the individual receiving the composition and do not have excessive toxicity after administration.
  • Such vectors are well known to those of ordinary skill in the art.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol.
  • these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
  • the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • an injectable such as a liquid solution or suspension
  • it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • composition of the invention can be administered by conventional routes, including (but not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal
  • the outside includes subcutaneous, intramuscular or intravenous.
  • the objects to be prevented or treated can be animals; especially humans.
  • composition of the present invention When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the use situation. Preferably, injections and oral preparations can be exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a usual manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (Isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a usual manner according to the dosage form.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained-release formulation.
  • the TCR of the present invention can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like, preferably exemplified are biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the TCR or TCR complex of the present invention as the active ingredient or the cells presenting the TCR of the present invention can be based on the weight, age, sex, and degree of symptoms of each patient to be treated. The reasonable dosage is determined by the doctor.
  • the affinity and/or binding half-life of the TCR of the present invention for the FMNKFIYEI-HLA-A2 complex is at least 2 times, preferably at least 10 times, that of the wild-type TCR.
  • the affinity and/or binding half-life of the TCR of the present invention for the FMNKFIYEI-HLA-A2 complex is at least 100 times, preferably at least 500 times, and more preferably up to 10 3 -5 than the wild-type TCR *10 4 times.
  • the effector cells transduced with the high-affinity TCR of the present invention have a strong killing effect on target cells.
  • E. coli DH5 ⁇ was purchased from Tiangen
  • E. coli BL21 (DE3) was purchased from Tiangen
  • E. coli Tuner (DE3) was purchased.
  • plasmid pET28a was purchased from Novagen.
  • Example 1 Stability of hydrophobic core mutations. Generation of single-stranded TCR template chains
  • the present invention uses the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-stranded TCR molecule composed of a flexible short peptide (linker) connecting TCR ⁇ and ⁇ chain variable domains, its amino acids and DNA
  • the sequences are SEQ ID NO: 41 and SEQ ID NO: 42, as shown in Figure 7a and Figure 7b.
  • the amino acid sequences of the ⁇ variable domain (SEQ ID NO: 3) and ⁇ variable domain (SEQ ID NO: 4) of the template chain are shown in Figure 2a and 2b; the corresponding DNA sequences are respectively SEQ ID NO: 5 And 6, as shown in Figures 3a and 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are SEQ ID NO: 7 and 8, respectively, as shown in Figures 4a and 4b.
  • the target gene carrying the template chain was digested with NcoI and NotI, and then connected to the pET28a vector that was digested with NcoI and NotI.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, and incubated overnight at 37°C. Positive clones were selected for PCR screening, the positive recombinants were sequenced, and the recombinant plasmids were extracted after confirming the correct sequence.
  • E.coli BL21(DE3) for expression.
  • Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1
  • the inclusion bodies were dissolved in a buffer (20mM Tris-HCl pH8.0, 8M urea), centrifuged at a high speed to remove insoluble materials, the supernatant was quantified by BCA method, then aliquoted, and stored at -80°C for use.
  • a syringe to drop the single-stranded TCR treated above into 125mL of refolding buffer (100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine), Stir at 4°C for 10 minutes, then put the refolding solution into a cellulose membrane dialysis bag with a cutoff of 4kDa, place the dialysis bag in 1L of pre-cooled water, and stir slowly at 4°C overnight.
  • refolding buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
  • the collected elution fractions were analyzed by SDS-PAGE, and the fractions containing single-stranded TCR were concentrated and further purified with a gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
  • the eluted fractions used for BIAcore analysis were further tested for purity by gel filtration.
  • the conditions are: Column Agilent Bio SEC-3 (300A, ), the mobile phase is 150mM phosphate buffer, the flow rate is 0.5mL/min, the column temperature is 25°C, and the UV detection wavelength is 214nm.
  • the BIAcore T200 real-time analysis system was used to detect the binding activity of TCR molecules and the FMNKFIYEI-HLA-A0201 complex.
  • the coupling level is about 15,000 RU.
  • the conditions are: the temperature is 25°C and the PH value is 7.1-7.5.
  • the TCR was diluted with HEPES-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20, pH 7.4) into several different concentrations at a flow rate of 30 ⁇ L/min , Flow through the chip surface in sequence, the binding time of each injection is 120s, and let it dissociate for 600s after the last injection. After each round of measurement, the chip was regenerated with 10mM Gly-HCl at pH 1.75. Use BIAcore Evaluation software to calculate kinetic parameters.
  • the synthetic short peptide FMNKFIYEI (Beijing Saibaisheng Gene Technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml.
  • the inclusion bodies of the light chain and the heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA were added before renaturation to further denature.
  • FMNKFIYEI peptide at 25mg/L (final concentration) to refolding buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C), then add 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain is added in three times, 8h/time), and renaturate at 4°C for at least 3 days Upon completion, SDS-PAGE will test whether the refolding is successful.
  • refolding buffer 0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C
  • the protein-containing fractions were combined, concentrated with a Millipore ultrafiltration tube, the protein concentration was determined by BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules in aliquots at -80°C.
  • Phage display technology is a means of generating TCR high-affinity variant libraries to screen high-affinity variants.
  • the TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) was applied to the single-stranded TCR template in Example 1.
  • a high-affinity TCR library was established and panned. After several rounds of panning, the phage library has specific binding to the corresponding antigen, and a single clone is selected from it, and sequence analysis is performed.
  • the K D value of the interaction between the reference TCR and the FMNKFIYEI-HLA-A0201 complex is 9.39 ⁇ M, and the interaction curve is shown in Figure 12, that is, the wild-type TCR interacts with the FMNKFIYEI-HLA-A0201 complex
  • the KD value is also 9.39 ⁇ M, which is 9.39E-06M.
  • the alpha chain variable domains of these high-affinity TCR mutants are mutated at one or more of the following amino acids: 27V, 29I, 30S, 50S, 51S, 52G, 53K, 92S and 93Y and/or adopt the numbering shown in SEQ ID NO: 2.
  • the ⁇ chain variable domains of these high-affinity TCR mutants are located at one or more of the following positions 52E, 53A, 54Q, 95S, Mutations occurred in 96Y, 97G, 98A, 103D, 104T, and 106Y.
  • the alpha chain variable domains of these high-affinity TCRs contain one or more amino acid residues 27A or 27P; 29L; 30Q; 50P; 51F or 51Y; 52Q; 53T; 92T or 92F and 93R or 93N; and/or the numbering shown in SEQ ID NO: 2, the ⁇ -chain variable domains of these high-affinity TCRs contain one or more amino acid residues selected from the following group Base 52V, 53S, 54I, 95A, 96L or 96P or 96R or 96K or 96Q or 96F, 97F or 97M or 97Y or 97H or 97S or 97W or 97A, 98S or 98P or 98G, 103G or 103S or 103M or 103E or 103A Or 103R, 104S or 104A or 104E or 104G or 104M, 106A or 106V or
  • the CDR region mutation of the high-affinity single-chain TCR screened in Example 4 was introduced into the corresponding position of the variable domain of ⁇ heterodimeric TCR, and the complex with FMNKFIYEI-HLA-A0201 was detected by BIAcore Affinity.
  • the introduction of the above-mentioned high-affinity mutation points in the CDR region adopts a site-directed mutation method well known to those skilled in the art.
  • the amino acid sequences of the alpha chain and beta chain variable domains of the wild-type TCR are shown in Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.
  • the ⁇ heterodimeric TCR can be constant in the ⁇ and ⁇ chains.
  • a cysteine residue was introduced into the regions to form the TCR of the artificial inter-chain disulfide bond.
  • the amino acid sequences of the TCR ⁇ and ⁇ chains after the introduction of cysteine residues are shown in Figure 8a (SEQ ID NO : 43) and 8b (SEQ ID NO: 44), the introduced cysteine residues are indicated by bold letters.
  • the extracellular sequence genes of the TCR ⁇ and ⁇ chains to be expressed were synthesized and inserted into the expression vector by the standard method described in "Molecular Cloning a Laboratory Manual” (third edition, Sambrook and Russell) pET28a+ (Novagene), the upstream and downstream cloning sites are NcoI and NotI respectively. Mutations in the CDR region are introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The inserted fragment was confirmed by sequencing.
  • the ⁇ and ⁇ chains of TCR were expressed
  • the inclusion bodies formed later were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution.
  • the inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA) ), 20mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains are quickly mixed with 5M urea, 0.4M arginine, 20mM Tris (pH8.1), 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1, and the final concentration is It is 60mg/mL.
  • 5M urea 20mM Tris (pH8.1)
  • 20mM Tris 20mM Tris (pH8.1)
  • cystamine 3.7mM cystamine
  • 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1
  • the final concentration is It is 60mg/mL.
  • the solution is filtered by a 0.45 ⁇ M filter membrane and purified by an anion exchange column (HiTrap Q HP, 5ml, GE Healthcare).
  • the eluted peak contains the TCR of the successfully renatured ⁇ and ⁇ dimers and confirmed by SDS-PAGE gel.
  • TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Example 3 The method described in Example 3 was used to detect the affinity of the ⁇ heterodimeric TCR introduced into the high-affinity CDR region and the FMNKFIYEI-HLA-A0201 complex.
  • the CDR regions selected from the high-affinity single-chain TCR ⁇ and ⁇ chains are transferred to the corresponding positions of the wild-type TCR ⁇ chain variable domain SEQ ID NO:1 and ⁇ chain variable domain SEQ ID NO: 2 to form ⁇ Quality dimerization TCR.
  • the mutation sites in the CDR regions obtained by the screening were artificially combined to form ⁇ heterodimeric TCR, and the new TCR ⁇ and ⁇ chain variable domain amino acid sequences were obtained as shown in Figure 9(1)-(7). As shown in Figure 10(1)-(28).
  • the ⁇ heterodimeric TCR introduced into the mutation point of the CDR region maintains a high affinity for the FMNKFIYEI-HLA-A0201 complex.
  • the affinity of the heterodimeric TCR is at least 2 times that of the wild-type TCR for the FMNKFIYEI-HLA-A0201 complex.
  • Example 8 Expression, renaturation and purification of fusion of anti-CD3 antibody and high-affinity single-chain TCR
  • the high-affinity single-chain TCR molecule of the present invention is fused with a single-chain molecule (scFv) of an anti-CD3 antibody to construct a fusion molecule.
  • scFv single-chain molecule
  • design primers to connect the genes of anti-CD3 antibody and high-affinity single-chain TCR molecule design the middle linker peptide (linker) as GGGGS, and bring the gene fragment of the fusion molecule Restriction endonuclease sites NcoI and NotI.
  • the PCR amplified product was digested with NcoI and NotI, and then connected to the pET28a vector that was digested with NcoI and NotI.
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, spread on LB plates containing kanamycin, and incubated overnight at 37°C. Positive clones were selected for PCR screening, and the positive recombinants were sequenced to determine the correct sequence and then extracted The recombinant plasmid is transformed into E. coli BL21(DE3) competent cells for expression.
  • the expression plasmid containing the target gene was transformed into E. coli strain BL21 (DE3), and spread on an LB plate (kanamycin 50 ⁇ g/ml) and incubated at 37°C overnight. On the next day, pick the clones and inoculate them into 10ml LB liquid medium (kanamycin 50 ⁇ g/ml) for 2-3 hours, and inoculate them into 1L LB medium (kanamycin 50 ⁇ g/ml) at a volume ratio of 1:100, continue Cultivate to an OD 600 of 0.5-0.8, and then use IPTG at a final concentration of 0.5 mM to induce the expression of the target protein. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min.
  • the cells were washed once with PBS buffer and divided into cells.
  • the cells equivalent to 200ml of bacterial culture were lysed with 5ml BugBuster Master Mix (Novagen), and the inclusion bodies were collected by centrifugation at 6000g for 15min. Then 4 washings with detergent were performed to remove cell debris and membrane components. Then, the inclusion bodies are washed with a buffer such as PBS to remove detergent and salt. Finally, the inclusion bodies were dissolved in a Tris buffer solution containing 8M urea, and the concentration of the inclusion bodies was measured, and the inclusion bodies were divided and stored at -80°C for storage.
  • inclusion bodies Take out about 10 mg of inclusion bodies from the -80°C ultra-low temperature refrigerator to thaw, add dithiothreitol (DTT) to a final concentration of 10mM, and incubate at 37°C for 30min to 1 hour to ensure that the disulfide bonds are completely opened. Then the inclusion body sample solution was dropped into 200ml of 4°C pre-cooled refolding buffer (100mM Tris pH 8.1, 400mM L-arginine, 2mM EDTA, 5M urea, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine), 4°C slowly Stir for about 30 minutes.
  • 4°C pre-cooled refolding buffer 100mM Tris pH 8.1, 400mM L-arginine, 2mM EDTA, 5M urea, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
  • the refolding solution was dialyzed with 8 volumes of pre-cooled H 2 O for 16-20 hours. Then dialyzed twice with 8 times the volume of 10mM Tris pH 8.0, and continue the dialysis at 4°C for about 8 hours. After the dialysis, the sample is filtered and the following purification is performed.
  • the dialyzed refolded product (in 10mM Tris pH 8.0) uses POROS HQ/20 anion exchange chromatography prepacked column (Applied Biosystems), and gradient elution with 0-600mM NaCl in AKTA purifier (GE Healthcare). The individual components were analyzed by SDS-PAGE stained with Coomassie brilliant blue and then combined.
  • Example 9 Expression, renaturation and purification of the fusion of anti-CD3 antibody and high affinity ⁇ heterodimeric TCR
  • the anti-CD3 single chain antibody (scFv) was fused with ⁇ heterodimeric TCR to prepare a fusion molecule.
  • the anti-CD3 scFv is fused with the ⁇ chain of the TCR.
  • the TCR ⁇ chain may include any of the above-mentioned high-affinity ⁇ heterodimeric TCR ⁇ -chain variable domains
  • the TCR ⁇ chain of the fusion molecule may include any of the above-mentioned high-affinity The alpha chain variable domain of a sexual alpha beta heterodimeric TCR.
  • the target gene carrying the ⁇ chain of ⁇ heterodimeric TCR was digested with NcoI and NotI, and then connected to the pET28a vector that was digested with NcoI and NotI.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, and incubated overnight at 37°C. Positive clones were selected for PCR screening, positive recombinants were sequenced, and the recombinant plasmids were extracted after the sequence was correct. Transform to E.coli Tuner (DE3) for expression.
  • primers are designed to connect the anti-CD3 scFv and the high affinity heterodimeric TCR ⁇ chain gene, the linker in the middle is GGGGS, and the anti-CD3 scFv is linked to the high affinity
  • the gene fragment of the fusion protein of the affinity heterodimeric TCR ⁇ chain carries restriction endonuclease sites Nco I (CCATGG) and Not I (GCGGCCGC).
  • CCATGG restriction endonuclease sites Nco I
  • Not I GCGGCCGC
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, spread on LB plates containing kanamycin, and incubated overnight at 37°C. Positive clones were selected for PCR screening, and the positive recombinants were sequenced to determine the correct sequence and then extracted The recombinant plasmid is transformed into E. coli Tuner (DE3) competent cells for expression.
  • the expression plasmids were respectively transformed into E. coli Tuner (DE3) competent cells, spread on LB plates (kanamycin 50 ⁇ g/mL) and incubated overnight at 37°C. On the next day, pick clones and inoculate 10mL LB liquid medium (kanamycin 50 ⁇ g/mL) for 2-3 hours, inoculate into 1L LB medium at a volume ratio of 1:100, continue to cultivate until OD600 is 0.5-0.8, add The final concentration is 1mM IPTG induces the expression of the target protein. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min. Wash the cells once with PBS buffer, and divide them into cells.
  • the dissolved TCR ⁇ chain and anti-CD3(scFv)- ⁇ chain are quickly mixed with 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1, 3.7 at a mass ratio of 2:5 mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C), the final concentration of ⁇ chain and anti-CD3 (scFv)- ⁇ chain are 0.1mg/mL and 0.25mg/mL respectively.
  • the TCR fusion molecule is then further purified by size exclusion chromatography (S-100 16/60, GE healthcare), and again purified by anion exchange column (HiTrap Q HP 5ml, GE healthcare).
  • the purity of the purified TCR fusion molecule was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Example 10 Activation function experiment of effector cells transfected with high-affinity TCR of the present invention
  • the TCR of the present invention is randomly selected to transfect PBL isolated from the blood of healthy volunteers as effector cells.
  • TCR13 ⁇ chain variable domain SEQ ID NO: 45, ⁇ chain variable domain SEQ ID NO: 2
  • TCR21 ⁇ chain variable domain SEQ ID NO: 46, ⁇ chain variable domain SEQ ID NO: 2
  • TCR9 ⁇ chain variable domain SEQ ID NO: 1, ⁇ chain variable domain SEQ ID NO: 60
  • TCR4 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 55
  • TCR1 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 52
  • TCR2 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 53
  • TCR12 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 63
  • TCR5 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 56
  • TCR3 ⁇ chain variable domain SEQ ID NO: 1, ⁇ chain variable domain SEQ ID NO: 54
  • the target cell lines were HepG2, HCCC9810-AFP (ie AFP transfected with HCCC9810), SNU-398-AFP (ie AFP transfected with SNU-398), Huh-7, SNU-398 and HCCC9810 cells.
  • the target cell lines HepG2, HCCC9810-AFP and SNU-398-AFP are positive tumor cell lines; Huh-7, SNU-398 and HCCC9810 are negative tumor cell lines as controls.
  • TCR14 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 64
  • TCR26 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 75
  • TCR18 ⁇ chain variable domain SEQ ID NO: 1, ⁇ chain variable domain SEQ ID NO: 68
  • TCR19 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 69
  • TCR10 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 61
  • TCR22 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 71
  • TCR23 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 72
  • TCR24 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: 73
  • TCR11 ⁇ chain variable domain SEQ ID NO:1, ⁇ chain variable domain SEQ ID NO: ⁇ chain variable domain SEQ ID NO
  • the target cell lines were HepG2, HCCC9810-AFP (ie AFP transfected with HCCC9810), SK-HEP-1-AFP (ie AFP transfected with SK-HEP-1), Huh-7, HCCC9810 and SK-HEP-1 cells.
  • the target cell lines HepG2, HCCC9810-AFP and SK-HEP-1-AFP are positive tumor cell lines; Huh-7, HCCC9810 and SK-HEP-1 are negative tumor cell lines as controls.
  • ELISPOT plate was activated and coated with ethanol at 4°C overnight.
  • the plate was washed and subjected to secondary detection and color development, the plate was dried, and then the spots formed on the membrane were counted with an immunospot plate reader (ELISPOT READER system; AID20 company).
  • Example 11 LDH killing function experiment of effector cells transfected with high-affinity TCR of the present invention
  • a non-radioactive cytotoxicity experiment was used to measure the release of LDH to verify the killing function of the cells transduced with the TCR of the present invention.
  • This test is a colorimetric alternative to the 51 Cr release cytotoxicity test, which quantitatively measures the lactate dehydrogenase (LDH) released after cell lysis.
  • LDH lactate dehydrogenase
  • a 30-minute coupled enzyme reaction is used to detect the LDH released in the medium.
  • LDH can convert a tetrazolium salt (INT) into red formazan (formazan).
  • the amount of red product produced is proportional to the number of cells lysed.
  • CD3 + T cells isolated from the blood of healthy volunteers by transfecting the TCR of the present invention are randomly selected as effector cells.
  • the TCR and its number are known from Table 4. They are TCR4 ( ⁇ chain variable domain SEQ ID NO: 1 and ⁇ chain variable domain SEQ ID NO: 55), TCR1 ( ⁇ chain variable domain SEQ ID NO: 1 , ⁇ chain variable domain SEQ ID NO: 52), TCR2 ( ⁇ chain variable domain SEQ ID NO: 1, ⁇ chain variable domain SEQ ID NO: 53), TCR12 ( ⁇ chain variable domain SEQ ID NO: 1 , ⁇ -chain variable domain SEQ ID NO: 63) and TCR5 ( ⁇ -chain variable domain SEQ ID NO: 1, ⁇ -chain variable domain SEQ ID NO: 56), the control effector cells are labeled as wild-type TCR (transfection Wild-type TCR cells) and A6 (cells transfected with other TCR).
  • the target cell lines are HepG2, HC
  • the results of the experiment are shown in Figure 15.
  • the cells transduced with the TCR of the present invention have a strong killing effect on positive target cells, and the killing effect is much higher than the cells transduced with wild-type TCR; while the cells transduced with other TCRs are positive
  • the target cell basically has no killing effect.

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Abstract

提供一种T细胞受体(TCR)，其具有结合FMNKFIYEI-HLA A0201复合物的特性；并且所述TCR对所述FMNKFIYEI-HLA A0201复合物的结合亲和力是野生型TCR对FMNKFIYEI-HLA A0201复合物的结合亲和力的至少2倍。还提供了此类TCR与治疗剂的融合分子。此类TCR可以单独使用，也可与治疗剂联用，以靶向呈递FMNKFIYEI-HLA A0201复合物肿瘤细胞。

Description

一种识别AFP的高亲和力T细胞受体 技术领域
本发明涉及生物技术领域，更具体地涉及能够识别衍生自AFP蛋白多肽的T细胞受体(T cell receptor,TCR)。本发明还涉及所述受体的制备和用途。
背景技术
仅仅有两种类型的分子能够以特异性的方式识别抗原。其中一种是免疫球蛋白或抗体；另一种是T细胞受体(TCR)，它是由α链/β链或者γ链/δ链以异二聚体形式存在的细胞膜表面的糖蛋白。免疫系统的TCR总谱的组成是在胸腺中通过V(D)J重组，然后进行阳性和阴性选择而产生的。在外周环境中，TCR介导了T细胞对主组织相容性复合体-肽复合物(pMHC)的特异性识别，因此其对免疫系统的细胞免疫功能是至关重要的。
TCR是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的唯一受体，这种外源肽或内源肽可能会是细胞出现异常的唯一迹象。在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
与TCR相对应的MHC I类和II类分子配体也是免疫球蛋白超家族的蛋白质但对于抗原的呈递具有特异性，不同的个体有不同的MHC，从而能呈递一种蛋白抗原中不同的短肽到各自的APC细胞表面。人类的MHC通常称为HLA基因或HLA复合体。
AFP(αFetoprotein)也称α胎蛋白，是胚胎发育过程中表达的一种蛋白，是胚胎血清的主要成分。在发育过程中，AFP在卵黄囊及肝脏中有比较高的表达水平，随后被抑制。在肝细胞癌中，AFP的表达被激活(Butterfield et al.J Immunol.,2001,Apr 15；166(8):5300-8)。AFP在细胞内生成后被降解成小分子多肽，并与MHC(主组织相容性复合体)分子结合形成复合物，被呈递到细胞表面。FMNKFIYEI是衍生自AFP抗原的短肽，是AFP相关疾病治疗的一种靶标。
因此，FMNKFIYEI-HLA A0201复合物提供了一种TCR可靶向肿瘤细胞的标记。能够结合FMNKFIYEI-HLA A0201复合物的TCR对肿瘤的治疗具有很高的应用价值。例如，能够靶向该肿瘤细胞标记的TCR可用于将细胞毒性剂或免疫刺激剂递送到靶细胞，或被转化入T细胞，使表达该TCR的T细胞能够破坏肿瘤细胞，以便在被称为过继免疫治疗的治疗过程中给予患者。对于前一目的，理想的TCR是具有较高的亲和力的，从而使该TCR能够长期驻留在所靶向的细胞上面。对于后一目的，则优选使用中等亲和力的TCR。因此，本领域技术人员致力于开发可用于满足不同目的的靶向肿瘤细胞标记的TCR。
发明内容
本发明的目的在于提供一种对FMNKFIYEI-HLA A0201复合物具有较高亲和力的TCR。
本发明的再一目的是提供一种上述类型TCR的制备方法及上述类型TCR的用途。
本发明的第一方面，提供了一种T细胞受体(TCR)，其具有结合FMNKFIYEI-HLA A0201复合物的活性。
在另一优选例中，所述T细胞受体(TCR)具有结合FMNKFIYEI-HLA A0201复合物的活性，并且所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包含3个CDR区，所述TCRα链可变域的3个CDR区的基准序列如下，
CDR1α：VGISA
CDR2α：LSSGK
CDR3α：AVETSYDKVI，并且含有至少一个下列突变：
突变前的残基 突变后的残基
CDR1α的第1位V A或P
CDR1α的第3位I L
CDR1α的第4位S Q
CDR2α的第2位S P
CDR2α的第3位S F或Y
CDR2α的第4位G Q
CDR2α的第5位K T
CDR3α的第5位S T或F
CDR3α的第6位Y R或N
和/或，所述TCRβ链可变域包含3个CDR区，所述TCRβ链可变域的3个CDR区的基准序列如下，
CDR1β：SGHVS
CDR2β：FNYEAQ
CDR3β：ASSYGAGGPLDTQY，并且含有至少一个下列突变：
突变前的残基 突变后的残基
CDR2β的第4位E V
CDR2β的第5位A S
CDR2β的第6位Q I
CDR3β的第3位S A
CDR3β的第4位Y L或P或R或K或Q或F
CDR3β的第5位G F或M或Y或H或S或W或A
CDR3β的第6位A S或P或G
CDR3β的第11位D G或S或M或E或A或R
CDR3β的第12位T S或A或E或G或M
CDR3β的第14位Y A或V或I或W或K或M或Q或R。
在另一优选例中，所述突变发生在α链和/或β链可变域的一个或多个CDR区中。
在另一优选例中，所述TCRα链可变域的3个CDR区中的突变个数为1至9个，和/或所述TCRβ链可变域的3个CDR区中的突变个数为1至10个。
在另一优选例中，所述TCRα链CDR区的突变个数可以为2个、3个、4个、5个、6个、7个、8个或9个。
在另一优选例中，所述TCRβ链CDR区的突变个数可以为3个、4个、5个、6个、7个、8个、9个或10个。
在另一优选例中，所述TCR在β链的CDR3β中的突变个数为4个；
在另一优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少2倍。
在本发明的一个优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少2倍；优选地，至少5倍；更优选地，至少10倍。
在另一优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少50倍；优选地，至少100倍；更优选地，至少500倍。
在另一优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少10 3倍；优选地，至少5*10 3倍；更优选地，至少10 4倍；更优选地，至少5*10 4倍。
具体地，所述TCR对FMNKFIYEI-HLA A0201复合物的解离平衡常数KD≤20μM；
在另一优选例中，所述TCR对FMNKFIYEI-HLA A0201复合物的解离平衡常数5μM≤KD≤10μM；优选地，0.1μM≤KD≤1μM；更优选地，1nM≤KD≤100nM。
在另一优选例中，所述野生型TCR的α链可变域的氨基酸序列为SEQ ID NO:1，β链可变域的氨基酸序列为SEQ ID NO:2。
在另一优选例中，所述TCR的α链可变域包含与SEQ ID NO:1所示的氨基酸序列有至少90％的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO:2所示的氨基酸序列有至少90％的序列同源性的氨基酸序列。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其特征在于，CDR1β的氨基酸序列为SGHVS。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其特征在于，所述CDR3β的氨基酸序列为：AS[3βX1][3βX2][3βX3][3βX4]GGPL[3βX5][3βX6]Q[3βX7]，其中，[3βX1]为A或S；和/或[3βX2]为Y、L、P、R、K、Q或F；和/或[3βX3]为G、F、M、Y、H、S、W或A；和/或[3βX4]为A、S、P或G；和/或[3βX5]为D、S、G、R、M或E；和/或[3βX6]为T、G、E、S、M或A；和/或[3βX7]为Y、I、V、M、Q、R、A、W或K。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其特征在于，所述CDR3β选自下组：
ASALMSGGPLDTQY、ASARYPGGPLDTQY、ASARHAGGPLDTQY、ASSYGAGGPLDTQY、ASALFSGGPLDTQY、ASAPFSGGPLDTQY、ASAKMSGGPLDTQY、ASSYGAGGPLGEQW、ASSYGAGGPLGAQA、ASSQSGGGPLDTQY、ASSYGAGGPLGEQV、ASSYGAGGPLMAQA、ASALYSGGPLDTQY、ASSPFSGGPLDTQY、ASSYGAGGPLGAQK、ASSYGAGGPLEGQV、ASSYGAGGPLSSQI、ASSLFGGGPLDTQY、ASSYGAGGPLSGQI、ASSYGAGGPLASQY、ASSYGAGGPLRTQM、ASSYGAGGPLGSQQ、ASSYGAGGPLGSQV、ASSYGAGGPLGSQA、ASSLFSGGPLDTQY、ASSLWSGGPLDTQY、ASSFAGGGPLDTQY和ASSYGAGGPLGMQR。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其特征在于，CDR2β的氨基酸序列选自FNYEAQ和FNYVSI。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其特征在于，CDR1α的氨基酸序列选自下组：VGISA、AGLQA、VGLQA和PGLQA。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其特征在于，CDR2α的氨基酸序列选自下组：LSSGK、LPFGK和LPYQT。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其特征在于，CDR3α的氨基酸序列选自下组：AVETSYDKVI、AVETTRDKVI和AVETFNDKVI。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其特征在于，CDR1α的氨基酸序列为VGISA，CDR2α的氨基酸序列为LSSGK，并且CDR3α的氨基酸序列为AVETSYDKVI。
在另一优选例中，所述TCR的α链可变域氨基酸序列为SEQ ID NO:1。
在另一优选例中，所述突变发生在α链和/或β链可变域的一个或多个CDR区中。
在本发明的一个优选的实施方式中，所述T细胞受体(TCR)，其具有结合FMNKFIYEI-HLA A2复合物的活性，并包含TCRα链可变域和TCRβ链可变域，所述TCR在SEQ ID NO:1所示的α链可变域中发生突变，所述突变的氨基酸残基位点包括27V、29I、30S、50S、51S、52G、53K、92S和93Y中的一个或多个，其中，氨基酸残基编号采用SEQ ID NO:1所示的所示的β链可变域编号；和/或所述TCR在SEQ ID NO:2所示的β链可变域中发生突变，所述突变的氨基酸残基位点包括52E、53A、54Q、95S、96Y、97G、98A、103D、104T、106Y中的一个或多个，其中，氨基酸残基编号采用SEQ ID NO:2所示的编号；
优选地，突变后的所述TCRα链可变域包括选自下组的一个或多个氨基酸残基：27A或27P；29L；30Q；50P；51F或51Y；52Q；53T；92T或92F和93R或93N，其中，氨基酸残基编号采用SEQ ID NO:1所示的编号；和/或突变后的所述TCRβ链可变域包括选自下组的一个或多个氨基酸残基：52V、53S、54I、95A、96L或96P或96R或96K或96Q或96F、97F或97M或97Y或97H或97S或97W或97A、98S或98P或98G、103G或103S或103M或103E或103A或103R、104S或104A或104E或104G或104M、106A或106V或106I或106W或106K或106M或106Q或106R，其中，氨基酸残基编号采用SEQ ID NO:2所示的编号。
在另一优选例中，所述TCR具有选自下组的CDR：
CDR编号 A-CDR1 A-CDR2 A-CDR3 B-CDR1 B-CDR2 B-CDR3
1 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASALMSGGPLDTQY
2 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASARYPGGPLDTQY
3 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASARHAGGPLDTQY
4 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASAPFSGGPLDTQY
5 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASAKMSGGPLDTQY
6 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGEQW
7 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGAQA
8 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSQSGGGPLDTQY
9 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASALFSGGPLDTQY
10 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGEQV
11 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLMAQA
12 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASALYSGGPLDTQY
13 VGISA LSSGK AVETTRDKVI SGHVS FNYEAQ ASSYGAGGPLDTQY
14 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSPFSGGPLDTQY
15 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGAQK
16 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLEGQV
17 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLSSQI
18 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSLFGGGPLDTQY
19 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLSGQI
20 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLASQY
21 VGISA LSSGK AVETFNDKVI SGHVS FNYEAQ ASSYGAGGPLDTQY
22 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLRTQM
23 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGSQQ
24 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGSQV
25 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGSQA
26 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSLFSGGPLDTQY
27 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSLWSGGPLDTQY
28 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSFAGGGPLDTQY
29 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGMQR
30 VGLQA LPFGK AVETTRDKVI SGHVS FNYEAQ ASALFSGGPLDTQY
31 AGLQA LPFGK AVETSYDKVI SGHVS FNYEAQ ASALFSGGPLDTQY
32 PGLQA LPFGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLDTQY
33 AGLQA LPYQT AVETSYDKVI SGHVS FNYEAQ ASALFSGGPLDTQY
34 AGLQA LPYQT AVETTRDKVI SGHVS FNYEAQ ASALFSGGPLDTQY
35 VGLQA LPFGK AVETTRDKVI SGHVS FNYVSI ASALFSGGPLDTQY
36 AGLQA LPYQT AVETSYDKVI SGHVS FNYVSI ASALFSGGPLDTQY。
在另一优选例中，所述TCR是可溶的。
在另一优选例中，所述TCR为αβ异质二聚TCR或单链TCR。
在另一优选例中，所述TCR的α链可变域氨基酸序列选自：SEQ ID NO:9-13和45-51；和/或所述TCR的β链可变域氨基酸序列选自：SEQ ID NO:14-40和52-79。
在另一优选例中，本发明所述TCR是αβ异质二聚TCR，优选地，所述TCR具有α链恒定区序列TRAC*01和β链恒定区序列TRBC1*01或TRBC2*01。
在另一优选例中，所述TCR是αβ异质二聚TCR，所述TCR的α链可变域包含与SEQ ID NO:1所示的氨基酸序列有至少90％；优选地，至少92％；更优选地，至少94％(如，可以是至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO:2所示的氨基酸序列有至少90％，优选地，至少92％；更优选地，至少94％(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列。
在另一优选例中，所述TCR包含(i)除其跨膜结构域以外的全部或部分TCRα链，和(ii)除其跨膜结构域以外的全部或部分TCRβ链，其中(i)和(ii)均包含TCR链的可变域和至少一部分恒定域。
在另一优选例中，所述TCR是αβ异质二聚TCR，所述TCR的α链可变区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，在所述TCR的α链可变区与β链恒定区之间形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。
在另一优选例中，α链可变区与β链恒定区之间含有人工链间二硫键的TCR包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
在另一优选例中，α链可变区与β链恒定区之间含有人工链间二硫键的TCR包含(i)除其跨膜结构域以外的全部或部分TCRα链，和(ii)除其跨膜结构域以外的全部或部分TCRβ链，其中(i)和(ii)均包含TCR链的可变域和至少一部分恒定域。
在另一优选例中，所述TCR为αβ异质二聚TCR，其包含(i)除其跨膜结构域以外的全部或部分TCRα链，和(ii)除其跨膜结构域以外的全部或部分TCRβ链，其中(i)和(ii)均包含TCR链的可变域和至少一部分恒定域，α链恒定区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，在所述TCRα与β链的恒定区之间形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
和TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
在另一优选例中，所述TCR选自下组：
Figure PCTCN2020109351-appb-000001
Figure PCTCN2020109351-appb-000002
在另一优选例中，所述TCR为单链TCR。
在另一优选例中，所述TCR是由α链可变域和β链可变域组成的单链TCR，所述α链可变域和β链可变域由一柔性短肽序列(linker)连接。
在另一优选例中，所述TCRα链可变域和/或β链可变域的疏水芯发生突变。
在另一优选例中，所述疏水芯发生突变的TCR是由α可变域和β可变域组成的单链TCR，所述α可变域和β可变域由一柔性短肽序列(linker)连接。
在另一优选例中，本发明所述TCR是单链TCR，所述TCR的α链可变域包含与SEQ ID NO:3所示的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％(如，可以是至少88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO:4所示的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列。
在另一优选例中，所述TCR选自下组：
Figure PCTCN2020109351-appb-000003
Figure PCTCN2020109351-appb-000004
在另一优选例中，所述TCR的α链和/或β链的C-或N-末端结合有偶联物。
在另一优选例中，与所述TCR结合的偶联物为可检测标记物、治疗剂、PK修饰部分或任何这些物质的组合。
在另一优选例中，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
本发明的第二方面，提供了一种多价TCR复合物，包含至少两个TCR分子，并且其中的至少一个TCR分子为本发明第一方面所述的TCR。
本发明的第三方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述的TCR分子或者本发明第二方面所述的多价TCR复合物的核酸序列或其互补序列；
本发明的第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的所述的核酸分子。
本发明的第五方面，提供了一种宿主细胞，所述的宿主细胞中含有本发明第四方面所述的载体或染色体中整合有外源的本发明第三方面所述的核酸分子。
本发明的第六方面，提供了一种分离的细胞，所述细胞表达本发明第一方面所述的TCR。
本发明的第七方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的TCR、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞。
本发明的第八方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的TCR、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞、或本发明第七方面所述的药物组合物。
本发明的第九方面，提供了本发明第一方面所述的TCR、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞的用途，用于制备治疗肿瘤的药物，优选地，所述肿瘤为肝细胞癌。
本发明的第十方面，所述的T细胞受体、权利要求29中所述的TCR复合物或权利要求33中所述的细胞用作治疗肿瘤的药物。优选地，所述肿瘤为AFP阳性肿瘤；更优选地，所述肿瘤为肝细胞癌。
本发明的第十一方面，提供了一种制备本发明第一方面所述的T细胞受体的方法，包括步骤：
(i)培养本发明第五方面所述的宿主细胞，从而表达本发明第一方面所述的T细胞受体；
(ii)分离或纯化出所述的T细胞受体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1a和图1b分别显示了对FMNKFIYEI-HLA A0201复合物能够特异性结合的野生型TCRα与β链可变域氨基酸序列。
图2a和图2b分别为本发明构建的单链模板TCR的α链可变域的氨基酸序列和β链可变域的氨基酸序列。
图3a和图3b分别为本发明构建的单链模板TCR的α链可变域的DNA序列和β链可变域的DNA序列。
图4a和图4b分别为本发明构建的单链模板TCR的连接短肽(linker)的氨基酸序列和核苷酸序列。
图5(1)-(5)分别显示了对FMNKFIYEI-HLA A0201复合物具有高亲和力的单链TCR的α链可变域氨基酸序列，突变的残基以加下划线表示。
图6(1)-(27)分别显示了对FMNKFIYEI-HLA A0201复合物具有高亲和力的单链TCR的β链可变域氨基酸序列，突变的残基以加下划线表示。
图7a和图7b分别为本发明构建的单链模板TCR的氨基酸序列和DNA序列。
图8a和图8b分别显示了本发明中可溶性参比TCRα与β链的氨基酸序列。
图9(1)-(7)分别显示了对FMNKFIYEI-HLA A0201复合物具有高亲和力的异质二聚TCR的α链可变域氨基酸序列，突变的残基以加下划线表示。
图10(1)-(28)分别显示了对FMNKFIYEI-HLA A0201复合物具有高亲和力的异质二聚TCR的β链可变域氨基酸序列，突变的残基以加下划线表示。
图11a和图11b分别显示了对FMNKFIYEI-HLA A0201复合物能够特异性结合的野生型TCRα与β链胞外氨基酸序列。
图12a和图12b分别显示了对FMNKFIYEI-HLA A0201复合物能够特异性结合的野生型TCRα与β链的氨基酸序列。
图13为可溶性参比TCR即野生型TCR与FMNKFIYEI-HLA A0201复合物的结合曲线。
图14a和14b为转染本发明高亲和力TCR的效应细胞的激活功能实验结果。
图15为转染本发明高亲和力TCR的效应细胞LDH的杀伤功能实验结果。
具体实施方式
本发明通过广泛而深入的研究，获得一种识别FMNKFIYEI短肽(衍生自AFP蛋白)的高亲和性T细胞受体(TCR)，所述FMNKFIYEI短肽以肽-HLA A0201复合物的形式被呈递。所述高亲和性TCR在其α链可变域的3个CDR区：
CDR1α：VGISA
CDR2α：LSSGK
CDR3α：AVETSYDKVI中发生突变；和/或在其β链可变域的3个CDR区：
CDR1β：SGHVS
CDR2β：FNYEAQ
CDR3β：ASSYGAGGPLDTQY中发生突变；并且，突变后本发明TCR对上述FMNKFIYEI-HLA A0201复合物的亲和力和/或结合半衰期是野生型TCR的至少2倍。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且其意图不是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普 通技术人员通常理解的相同含义。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
术语
T细胞受体(T cell receptor,TCR)
可以采用国际免疫遗传学信息系统(IMGT)来描述TCR。天然αβ异质二聚TCR具有α链和β链。广义上讲，各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。通过独特的IMGT的TRAJ和TRBJ确定TCR的连接区，通过IMGT的TRAC和TRBC确定TCR的恒定区。
各可变区包含嵌合在框架序列中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。在IMGT命名法中，TRAV和TRBV的不同编号分别指代不同Vα类型和Vβ的类型。在IMGT系统中，α链恒定结构域具有以下的符号：TRAC*01，其中“TR”表示T细胞受体基因；“A”表示α链基因；C表示恒定区；“*01”表示等位基因1。β链恒定结构域具有以下的符号：TRBC1*01或TRBC2*01，其中“TR”表示T细胞受体基因；“B”表示β链基因；C表示恒定区；“*01”表示等位基因1。α链的恒定区是唯一确定的，在β链的形式中，存在两个可能的恒定区基因“C1”和“C2”。本领域技术人员通过公开的IMGT数据库可以获得TCRα与β链的恒定区基因序列。
TCR的α和β链一般看作各有两个“结构域”即可变域和恒定结构域。可变域由连接的可变区和连接区构成。因此，在本申请的说明书和权利要求书中，“TCRα链可变域”指连接的TRAV和TRAJ区，同样地，“TCRβ链可变域”指连接的TRBV和TRBD/TRBJ区。TCRα链可变域的3个CDR分别为CDR1α、CDR2α和CDR3α；TCRβ链可变域的3个CDR分别为CDR1β、CDR2β和CDR3β。本发明TCR可变域的框架序列可以为鼠源的或人源的，优选为人源的。TCR的恒定结构域包含胞内部分、跨膜区和胞外部分。为获得可溶性TCR，以便测定TCR与FMNKFIYEI-HLA A2复合物之间的亲和力，本发明TCR优选地不包含跨膜区。更优选地，本发明TCR的氨基酸序列是指TCR的胞外氨基酸序列。
本发明中所用的TCR序列为人源的。本发明中所述“野生型TCR”的α链氨基酸序列及β链氨基酸序列分别为SEQ ID NO:82和SEQ ID NO:83，如图12a和12b所示。本发明中所述“参比TCR”的α链氨基酸序列及β链氨基酸序列分别为SEQ ID NO:43和SEQ ID NO:44，如图8a和8b所示。本发明中所述“野生型TCR”的α链及β链的胞外氨基酸序列分别为SEQ ID NO:80和SEQ ID NO:81，如图11a和11b所示。本发明中，能够结合FMNKFIYEI-HLA A0201复合物的野生型TCR的α与β链可变域氨基酸序列分别为SEQ ID NO:1和SEQ ID NO:2，如图1a和1b所示。在本发明中，术语“本发明多肽”、“本发明的TCR”、“本发明的T细胞受体”可互换使用。
天然链间二硫键与人工链间二硫键
在天然TCR的近膜区Cα与Cβ链间存在一组二硫键，本发明中称为“天然链间二硫键”。在本发明中，将人工引入的，位置与天然链间二硫键的位置不同的链间共价二硫键称为“人工链间二硫键”。
为方便描述，本发明中TRAC*01与TRBC1*01或TRBC2*01氨基酸序列的位置编号按从N端到C端依次的顺序进行位置编号，如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第60个氨基酸为P(脯氨酸)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Pro60，也可将其表述为TRBC1*01或TRBC2*01外显子1的第60位氨基酸，又如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第61个氨基酸为Q(谷氨酰胺)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Gln61，也可将其表述为TRBC1*01或TRBC2*01外显子1的第61位氨基酸，其他以此类推。本发明中，可变区TRAV与TRBV的氨基酸序列的位置编号，按照IMGT中列出的位置编号。如TRAV中的某个氨基酸，IMGT中列出的位置编号为46，则本发明中将其描述为TRAV第46位氨基酸，其他以此类推。本发明中，其他氨基酸的序列位置编号有特殊说明的，则按特殊说明。
肿瘤
术语“肿瘤”指包括所有类型的癌细胞生长或致癌过程，转移性组织或恶性转化细胞、组织或器官，不管病理类型或侵染的阶段。肿瘤的实施例非限制性地包括：实体瘤，软组织瘤，和转移性病灶。实体瘤的实施例包括：不同器官系统的恶性肿瘤，例如肉瘤，肺鳞癌和癌症。例如：感染的前列腺，肺，乳房，淋巴，肠胃(例如：结肠)，和生殖泌尿道(例如：肾脏，上皮细胞)，咽头。肺鳞癌包括恶性肿瘤，例如，多数的结肠癌，直肠癌，肾细胞癌，肝癌，肺部的非小细胞癌，小肠癌和食道癌。上述癌症的转移性病变可同样用本发明的方法和组合物来治疗和预防。
发明详述
众所周知，TCR的α链可变域与β链可变域各含有3个CDR，类似于抗体的互补决定区。CDR3与抗原短肽相互作用，CDR1和CDR2与HLA相互作用。因此，TCR分子的CDR决定了其与抗原短肽-HLA复合物的相互作用。能够结合抗原短肽FMNKFIYEI与HLA A0201复合物(即，FMNKFIYEI-HLA A0201复合物)的野生型TCR的α链可变域氨基酸序列与β链可变域氨基酸序列分别为SEQ ID NO:1和SEQ ID NO:2,该序列为本发明人首次发现。其具有下列CDR区：
α链可变域CDR
CDR1α：VGISA
CDR2α：LSSGK
CDR3α：AVETSYDKVI
β链可变域CDR
CDR1β：SGHVS
CDR2β：FNYEAQ
CDR3β：ASSYGAGGPLDTQY
本发明通过对上述CDR区进行突变筛选，获得了与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR与FMNKFIYEI-HLA A0201复合物亲和力至少2倍的高亲和力TCR。
本发明提供了一种T细胞受体(TCR)，其具有结合FMNKFIYEI-HLA A0201复合物的活性。
所述T细胞受体包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包含3个CDR区，所述TCRα链可变域的3个CDR区的基准序列如下，
CDR1α：VGISA
CDR2α：LSSGK
CDR3α：AVETSYDKVI，并且含有至少一个下列突变：
突变前的残基 突变后的残基
CDR1α的第1位V A或P
CDR1α的第3位I L
CDR1α的第4位S Q
CDR2α的第2位S P
CDR2α的第3位S F或Y
CDR2α的第4位G Q
CDR2α的第5位K T
CDR3α的第5位S T或F
CDR3α的第6位Y R或N
和/或，所述TCRβ链可变域包含3个CDR区，所述TCRβ链可变域的3个CDR区的基准序列如下，
CDR1β：SGHVS
CDR2β：FNYEAQ
CDR3β：ASSYGAGGPLDTQY，并且含有至少一个下列突变：
突变前的残基 突变后的残基
CDR2β的第4位E V
CDR2β的第5位A S
CDR2β的第6位Q I
CDR3β的第3位S A
CDR3β的第4位Y L或P或R或K或Q或F
CDR3β的第5位G F或M或Y或H或S或W或A
CDR3β的第6位A S或P或G
CDR3β的第11位D G或S或M或E或A或R
CDR3β的第12位T S或A或E或G或M
CDR3β的第14位Y A或V或I或W或K或M或Q或R。
本发明中野生型TCRα链可变域SEQ ID NO:1的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO:1的第27-31位、第49-53位和第88-97位。据此，氨基酸残基编号采用SEQ ID NO:1所示的编号，27V即为CDR1α的第1位V，29I即为CDR1α的第3位I， 30S即为CDR1α的第4位S，50S即为CDR2α的第2位S、51S即为CDR2α的第3位S、52G即为CDR2α的第4位G、53K即为CDR2α的第5位K、92S即为CDR3α的第5位S和93Y即为CDR3α的第5位Y。
同理，本发明中野生型TCRβ链可变域SEQ ID NO:2的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO:2的第27-31位、第49-54位和第93-106位。因此，氨基酸残基编号采用SEQ ID NO:2所示的编号，52E即为CDR2β的第4位E、53A即为CDR2β的第5位A、54Q即为CDR2β的第6位Q、95S即为CDR3β的第3位S、96Y即为CDR3β的第4位Y、97G即为CDR3β的第5位G、98A即为CDR3β的第6位A、103D即为CDR11β的第3位D、104T即为CDR3β的第12位T、106Y即为CDR3β的第14位Y。
优选地，突变后的所述TCRα链可变域包括选自下组的一个或多个氨基酸残基：27A或27P；29L；30Q；50P；51F或51Y；52Q；53T；92T或92F和93R或93N，其中，氨基酸残基编号采用SEQ ID NO:1所示的编号；和/或突变后的所述TCRβ链可变域包括选自下组的一个或多个氨基酸残基：52V、53S、54I、95A、96L或96P或96R或96K或96Q或96F、97F或97M或97Y或97H或97S或97W或97A、98S或98P或98G、103G或103S或103M或103E或103A或103R、104S或104A或104E或104G或104M、106A或106V或106I或106W或106K或106M或106Q或106R，其中，氨基酸残基编号采用SEQ ID NO:2所示的编号。
更具体地，α链可变域中所述突变的具体形式包括V27A/P、I29L、S30Q、S50P、S51F/Y、G52Q、K53T、S92T/F、Y93R/N中的一组或几组；β链可变域中所述突变的具体形式包括E52V、A53S、Q54I、S95A、Y96L/P/R/K/Q/F、G97F/M/Y/H/S/W/A、A98S/P/G、D103G/S/M/E/A/R、T104S/A/E/G/M、Y106A/V/I/W/K/M/Q/R中的一组或几组。
在本发明地一个较佳地实施方式中，根据本发明的TCR，包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包括CDR1α、CDR2α、和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β。
在另一优选例中，所述CDR1α包含序列：[1αX1]G[1αX2][1αX3]A,其中，[1αX1]、[1αX2]、[1αX3]各自独立地选自任意的天然氨基酸残基。
在另一优选例中，所述[1αX1]为V或A或P。
在另一优选例中，所述[1αX2]为I或L。
在另一优选例中，所述[1αX3]为S或Q。
在另一优选例中，所述[1αX1]为V或A或P，[1αX2]为I或L，并且[1αX3]为S或Q。
在另一优选例中，所述CDR1α包含选自下组的序列：VGISA、AGLQA、VGLQA和PGLQA。
在另一优选例中，所述CDR2α包含序列：L[2αX1][2αX2][2αX3][2αX4]，其中[2αX1]、[2αX2]、[2αX3]、[2αX4]各自独立地选自任意的天然氨基酸残基。
在另一优选例中，所述[2αX1]为S或P。
在另一优选例中，所述[2αX2]为S或F或Y。
在另一优选例中，所述[2αX3]为G或Q。
在另一优选例中，所述[2αX4]为K或T。
在另一优选例中，所述[2αX1]为S或P，[2αX2]为S或F或Y，并且[2αX3]为G或Q，并且[2αX4]为K或T。
在另一优选例中，所述CDR2α包含选自下组的序列：LSSGK、LPFGK和LPYQT。
在另一优选例中，所述CDR3α包含序列：AVET[3αX1][3αX2]DKVI，其中，[3αX1]和[3αX2]各独立地选自任意的天然氨基酸残基。
在另一优选例中，所述[3αX1]为T或F。
在另一优选例中，所述[3αX2]为R或N。
在另一优选例中，所述[3αX1]为T或F，[3αX2]为R或N。
在另一优选例中，所述CDR3α包含选自下组的序列：AVETSYDKVI、AVETTRDKVI和AVETFNDKVI。
在本发明地一个较佳地实施方式中，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包括CDR1α、CDR2α、和CDR3α，和所述TCRβ链可变域包含CDR1β、CDR2β和CDR3β，其中，所述CDR1β包含序列：SGHVS。
在另一优选例中，所述CDR2β包含序列：FNY[2βX1][2βX2][2βX3]，其中[2βX1]、[2βX2]、[2βX3]各自独立地选自任意的天然氨基酸残基。
在另一优选例中，所述[2βX1]为Q或H。
在另一优选例中，所述[2βX2]为N或G。
在另一优选例中，所述[2βX3]为E或D。
在另一优选例中，所述CDR2β包含选自下组的序列：FNYEAQ和FNYVSI。
在另一优选例中，所述CDR3β包含序列：AS[3βX1][3βX2][3βX3][3βX4]GGPL[3βX5][3βX6]Q[3βX7]。其中，[3βX1]、[3βX2]、[3βX3]、[3βX4]、[3βX5]、[3βX6]和[3βX7]各自独立地选自任意的天然氨基酸残基。
在另一优选例中，所述[3βX1]为A或S。
在另一优选例中，所述[3βX2]为Y或L或P或R或K或Q或F。
在另一优选例中，所述[3βX3]为G或F或M或Y或H或S或W或A。
在另一优选例中，所述[3βX4]为S或A或P或G。
在另一优选例中，所述[3βX5]为D、G、S、M、E、A或R。
在另一优选例中，所述[3βX6]为T、S、A、E、G或M。
在另一优选例中，所述[3βX7]为Y、A、V、I、W、K、M、Q或R。
在另一优选例中，所述CDR3β包含选自下组的序列：ASALMSGGPLDTQY、ASARYPGGPLDTQY、ASARHAGGPLDTQY、ASSYGAGGPLDTQY、ASALFSGGPLDTQY、ASAPFSGGPLDTQY、ASAKMSGGPLDTQY、ASSYGAGGPLGEQW、ASSYGAGGPLGAQA、ASSQSGGGPLDTQY、ASSYGAGGPLGEQV、ASSYGAGGPLMAQA、ASALYSGGPLDTQY、ASSPFSGGPLDTQY、ASSYGAGGPLGAQK、ASSYGAGGPLEGQV、ASSYGAGGPLSSQI、 ASSLFGGGPLDTQY、ASSYGAGGPLSGQI、ASSYGAGGPLASQY、ASSYGAGGPLRTQM、ASSYGAGGPLGSQQ、ASSYGAGGPLGSQV、ASSYGAGGPLGSQA、ASSLFSGGPLDTQY、ASSLWSGGPLDTQY、ASSFAGGGPLDTQY和ASSYGAGGPLGMQR。
在另一优选例中，所述TCRα链可变域包含CDR1α、CDR2α和CDR3α，其特征在于，CDR1α的氨基酸序列为VGISA，CDR2α的氨基酸序列为LSSGK，并且CDR3α的氨基酸序列为AVETSYDKVI。
更详细地，所述TCRα链CDR区的突变个数可以为3个、4个、5个、6个、7个、8个或9个；和/或所述TCRβ链CDR区的突变个数可以为4个、5个、6个、7个、8个、9个或10个。
进一步，本发明所述TCR是αβ异质二聚TCR，所述TCR的α链可变域包含与SEQ ID NO:1所示的氨基酸序列有至少90％；优选地，至少92％；更优选地，至少94％(如，可以是至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO:2所示的氨基酸序列有至少90％，优选地，至少92％；更优选地，至少94％(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列。
进一步，本发明所述TCR是单链TCR，所述TCR的α链可变域包含与SEQ ID NO:3所示的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％(如，可以是至少88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO:4所示的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列。
优选地，所述TCR包含(i)除其跨膜结构域以外的全部或部分TCRα链，和(ii)除其跨膜结构域以外的全部或部分TCRβ链，其中(i)和(ii)均包含TCR链的可变域和至少一部分恒定域。
根据本领域技术人员熟知的定点突变的方法，将野生型TCRα链恒定区TRAC*01外显子1的Thr48突变为半胱氨酸，β链恒定区TRBC1*01或TRBC2*01外显子1的Ser57突变为半胱氨酸，即得到参比TCR，其氨基酸序列分别如图8a和8b所示，突变后的半胱氨酸残基以加粗字母表示。上述半胱氨酸取代能使参比TCR的α与β链的恒定区之间形成人工链间二硫键，以形成更加稳定的可溶性TCR，从而能够更加方便地评估TCR与FMNKFIYEI-HLA A2复合物之间的结合亲和力和/或结合半衰期。应理解，TCR可变区的CDR区决定了其与pMHC复合物之间的亲和力，因此，上述TCR恒定区的半胱氨酸取代并不会对TCR的结合亲和力和/或结合半衰期产生影响。所以，在本发明中，测得的参比TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力即认为是野生型TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力。同样地，如果测得本发明TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力是参比TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力的至少10倍，即等同于本 发明TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力是野生型TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力的至少10倍。
可通过任何合适的方法测定结合亲和力(与解离平衡常数K D成反比)和结合半衰期(表示为T 1/2)。如采用表面等离子共振技术进行检测。应了解，TCR的亲和力翻倍将导致K D减半。T 1/2计算为In2除以解离速率(K off)。因此，T 1/2翻倍会导致K off减半。优选采用相同的试验方案检测给定TCR的结合亲和力或结合半衰期数次，例如3次或更多，取结果的平均值。在优选的实施方式中，采用本文实施例中的表面等离振子共振(BIAcore)方法检测可溶性TCR的亲和力，条件为：温度25℃，PH值为7.1-7.5。该方法检测到参比TCR对FMNKFIYEI-HLA A2复合物的解离平衡常数K D为9.89E-06M，即9.89μM，本发明中即认为野生型TCR对FMNKFIYEI-HLA A2复合物的解离平衡常数K D也为9.89μM。由于TCR的亲和力翻倍将导致K D减半，所以若检测到高亲和力TCR对FMNKFIYEI-HLA A2复合物的解离平衡常数K D为9.89E-07M，即9.89E-01μM，则说明该高亲和力TCR对FMNKFIYEI-HLA A2复合物的亲和力是野生型TCR对FMNKFIYEI-HLA A2复合物的亲和力的10倍。本领域技术人员熟知K D值单位间的换算关系，即1M＝10 6μM,1μM＝1000nM，1nM＝1000pM。
在本发明的一个优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少2倍；优选地，至少5倍；更优选地，至少10倍。
在另一优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少50倍；优选地，至少100倍；更优选地，至少500倍。
在另一优选例中，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少10 3倍；优选地，至少5*10 3倍；更优选地，至少10 4倍；更优选地，至少5*10 4倍。
在另一优选例中，所述TCR对FMNKFIYEI-HLA A0201复合物的解离平衡常数5μM≤K D≤10μM；优选地，0.1μM≤K D≤1μM；更优选地，1nM≤K D≤100nM；
可采用任何合适的方法进行突变，包括但不限于依据聚合酶链式反应(PCR)的那些、依据限制性酶的克隆或不依赖连接的克隆(LIC)方法。许多标准分子生物学教材详述了这些方法。聚合酶链式反应(PCR)诱变和依据限制性酶的克隆的更多细节可参见Sambrook和Russell,(2001)分子克隆-实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)CSHL出版社。LIC方法的更多信息可见(Rashtchian,(1995)Curr Opin Biotechnol 6(1):30-6)。
产生本发明的TCR的方法可以是但不限于从展示此类TCR的噬菌体颗粒的多样性文库中筛选出对FMNKFIYEI-HLA-A2复合物具有高亲和性的TCR，如文献(Li,et al(2005)Nature Biotech 23(3):349-354)中所述。
应理解，表达野生型TCRα和β链可变域氨基酸的基因或者表达略作修饰的野生型TCR的α和β链可变域氨基酸的基因都可用来制备模板TCR。然后在编码该模板TCR的可变域的DNA中引入产生本发明的高亲和力TCR所需的改变。
本发明的高亲和性TCR包含α链可变域氨基酸序列SEQ ID NO:45-51之一和/或β链可变域氨基酸序列SEQ ID NO:52-79之一。因此，含有野生型TCR的α链可变域氨基酸序列(SEQ ID NO:1)的TCRα链可与包含SEQ ID NO:52-79之一的TCRβ链组合形成异质二聚TCR或单 链TCR分子。或者，含有野生型TCR的β可变域氨基酸序列(SEQ ID NO:2)的TCRβ链可与包含SEQ ID NO:45-51之一的TCRα链组合形成异质二聚TCR或单链TCR分子。又或者，包含TCRα链可变域氨基酸序列SEQ ID NO:45-51之一的TCRα链可与包含TCRβ链可变域氨基酸序列SEQ ID NO:52-79之一的TCRβ链组合形成异质二聚TCR或单链TCR分子。本发明中，形成异质二聚TCR分子的α链可变域与β链可变域的氨基酸序列优选自下表1：
表1
Figure PCTCN2020109351-appb-000005
基于本发明的目的，本发明TCR是具有至少一个TCRα和/或TCRβ链可变域的部分。 它们通常同时包含TCRα链可变域和TCRβ链可变域。它们可以是αβ异质二聚体或是单链形式或是其他任何能够稳定存在的形式。在过继性免疫治疗中，可将αβ异质二聚TCR的全长链(包含胞质和跨膜结构域)进行转染。本发明TCR可用作将治疗剂递送至抗原呈递细胞的靶向剂或与其他分子结合制备双功能多肽来定向效应细胞，此时TCR优选为可溶形式。
对于稳定性而言，现有技术中公开了在TCR的α与β链恒定域之间引入人工链间二硫键能够获得可溶且稳定的TCR分子，如专利文献PCT/CN2015/093806中所述。因此，本发明TCR可以是在其α和β链恒定域的残基之间引入人工链间二硫键的TCR。半胱氨酸残基在所述TCR的α和β链恒定域间形成人工链间二硫键。半胱氨酸残基可以取代在天然TCR中合适位点的其他氨基酸残基以形成人工链间二硫键。例如，取代TRAC*01外显子1的Thr48和取代TRBC1*01或TRBC2*01外显子1的Ser57来形成二硫键。引入半胱氨酸残基以形成二硫键的其他位点还可以是：TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；或TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。即半胱氨酸残基取代了上述α与β链恒定域中任一组位点。可在本发明TCR恒定域的一个或多个C末端截短最多15个、或最多10个、或最多8个或更少的氨基酸，以使其不包括半胱氨酸残基来达到缺失天然链间二硫键的目的，也可通过将形成天然链间二硫键的半胱氨酸残基突变为另一氨基酸来达到上述目的。
如上所述，本发明的TCR可以包含在其α和β链恒定域的残基间引入的人工链间二硫键。应注意，恒定域间含或不含上文所述的引入的人工二硫键，本发明的TCR均可含有TRAC恒定域序列和TRBC1或TRBC2恒定域序列。TCR的TRAC恒定域序列和TRBC1或TRBC2恒定域序列可通过存在于TCR中的天然链间二硫键连接。
另外，对于稳定性而言，专利文献PCT/CN2016/077680还公开了在TCR的α链可变区与β链恒定区之间引入人工链间二硫键能够使TCR的稳定性显著提高。因此，本发明的高亲和力TCR的α链可变区与β链恒定区之间还可以含有人工链间二硫键。具体地，在所述TCR的α链可变区与β链恒定区之间形成人工链间二硫键的半胱氨酸残基取代了：TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。优选地，这样的TCR可以包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的可变域和至少一部分恒定域，α链与β链形成异质二聚体。更优选地，这样的TCR可以包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
对于稳定性而言，另一方面，本发明TCR还包括在其疏水芯区域发生突变的TCR，这些疏水芯区域的突变优选为能够使本发明TCR的稳定性提高的突变，如在公开号为WO2014/206304的专利文献中所述。这样的TCR可在其下列可变域疏水芯位置发生突变：(α和/或β链)可变区氨基酸第11、13、19、21、53、76、89、91、94位，和/或α链J基因(TRAJ)短肽氨基酸位置倒数第3、5、7位和/或β链J基因(TRBJ)短肽氨基酸位置倒数第2、4、6位，其中氨基酸序列的位置编号按国际免疫遗传学信息系统(IMGT)中列出的位置编号。本领域技术人员知晓上述国际免疫遗传学信息系统，并可根据该数据库得到不同TCR的氨基酸残基在IMGT中的位置编号。
更具体地，本发明中疏水芯区域发生突变的TCR可以是由一柔性肽链连接TCR的α与β链的可变域而构成的高稳定性单链TCR。TCR可变区的CDR区决定了其与短肽-HLA复合物之间的亲和力，疏水芯的突变能够使TCR更加稳定，但并不会影响其与短肽-HLA复合物之间的亲和力。应注意，本发明中柔性肽链可以是任何适合连接TCRα与β链可变域的肽链。本发明实施例1中构建的用于筛选高亲和性TCR的模板链即为上述含有疏水芯突变的高稳定性单链TCR。采用稳定性较高的TCR，能够更方便的评估TCR与FMNKFIYEI-HLA-A0201复合物之间的亲和力。
该单链模板TCR的α链可变域及β链可变域的CDR区与野生型TCR的CDR区完全相同。即α链可变域的3个CDR分别为CDR1α：VGISA，CDR2α：LSSGK，CDR3α：AVETSYDKVI和β链可变域的3个CDR分别为CDR1β：SGHVS，CDR2β：FNYEAQ，CDR3β：ASSYGAGGPLDTQY。该单链模板TCR的氨基酸序列(SEQ ID NO:41)及核苷酸序列(SEQ ID NO:42)分别如图7a和7b所示。以此筛选出对FMNKFIYEI-HLA A0201复合物具有高亲和性的由α链可变域和β链可变域构成的单链TCR。
本发明中单链模板TCRα链可变域SEQ ID NO:3的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO:3的第27-31位、第49-55位和第90-102位。据此，氨基酸残基编号采用SEQ ID NO:3所示的编号，27V即为CDR1α的第1位V，29I即为CDR1α的第3位I，30S即为CDR1α的第4位S，50S即为CDR2α的第2位S、51S即为CDR2α的第3位S、52G即为CDR2α的第4位G、53K即为CDR2α的第5位K、92S即为CDR3α的第5位S和93Y即为CDR3α的第5位Y。
同理，本发明中单链模板TCRβ链可变域SEQ ID NO:4的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO:2的第27-31位、第49-54位和第93-102位。因此，氨基酸残基编号采用SEQ ID NO:4所示的编号，52E即为CDR2β的第4位E、53A即为CDR2β的第5位A、54Q即为CDR2β的第6位Q、95S即为CDR3β的第3位S、96Y即为CDR3β的第4位Y、97G即为CDR3β的第5位G、98A即为CDR3β的第6位A、103D即为CDR11β的第3位D、104T即为CDR3β的第12位T、106Y即为CDR3β的第14位Y。
本发明的对FMNKFIYEI-HLA-A0201复合物具有高亲和性的αβ异质二聚体的获得是通过将筛选出的高亲和性单链TCR的α与β链可变域的CDR区转移到野生型TCRα链可变域(SEQ ID NO:1)与β链可变域(SEQ ID NO:2)的相应位置而得到。还有一部分是根据筛选得到 的CDR区的突变位点进行人工组合而得到。
本发明的高亲和性TCR还包含α链可变域氨基酸序列SEQ ID NO:9-13之一和/或β链可变域氨基酸序列SEQ ID NO:14-40之一。因此，上述作为模板链的高稳定性单链TCRα链可变域(SEQ ID NO:3)可与氨基酸序列为SEQ ID NO:14-40之一的TCRβ链可变域组合形成所述单链TCR分子。或者，上述作为模板链的高稳定性单链TCRβ链可变域(SEQ ID NO:4)可与氨基酸序列为SEQ ID NO:9-13之一的TCRα链可变域组合形成所述单链TCR分子。又或者，TCRα链可变域SEQ ID NO:9-13之一与TCRβ链可变域SEQ ID NO:14-40之一组合形成所述单链TCR分子。本发明中，高亲和力单链TCR分子的α链可变域与β链可变域的氨基酸序列优选自下表2：
表2
Figure PCTCN2020109351-appb-000006
Figure PCTCN2020109351-appb-000007
本发明的TCR也可以多价复合体的形式提供。本发明的多价TCR复合体包含两个、三个、四个或更多个本发明TCR相结合而形成的多聚物，如可以用p53的四聚结构域来产生四聚体，或多个本发明TCR与另一分子结合而形成的复合物。本发明的TCR复合物可用于体外或体内追踪或靶向呈递特定抗原的细胞，也可用于产生具有此类应用的其他多价TCR复合物的中间体。
本发明的TCR可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物包括可检测标记物(为诊断目的，其中所述TCR用于检测呈递FMNKFIYEI-HLA-A0201复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明TCR结合或偶联的治疗剂包括但不限于：1.放射性核素(Koppe等，2005，癌转移评论(Cancer metastasis reviews)24，539)；2.生物毒(Chaudhary等，1989，自然(Nature)339，394；Epel等，2002，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51，565)；3.细胞因子如IL-2等(Gillies等，1992，美国国家科学院院刊(PNAS)89，1428；Card等，2004，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53，345；Halin等，2003，癌症研究(Cancer Research)63，3202)；4.抗体Fc片段(Mosquera等，2005，免疫学杂志(The Journal Of Immunology)174，4381)；5.抗体scFv片段(Zhu等，1995，癌症国际期刊(International Journal of Cancer)62,319)；6.金纳米颗粒/纳米棒(Lapotko等，2005，癌症通信(Cancer letters)239，36；Huang等，2006，美国化学学会杂志(Journal of the American Chemical Society)128，2115)；7.病毒颗粒(Peng等，2004，基因治疗(Gene therapy)11，1234)；8.脂质体(Mamot等，2005，癌症研究(Cancer research)65，11631)；9.纳米磁粒；10.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；11.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
与本发明TCR结合的抗体或其片段包括抗-T细胞或NK-细胞决定抗体，如抗-CD3或抗-CD28或抗-CD16抗体，上述抗体或其片段与TCR的结合能够对效应细胞进行定向来更好地靶向靶细胞。一个优选的实施方式是本发明TCR与抗-CD3抗体或所述抗-CD3抗体的功能片段或变体结合。具体地，本发明的TCR与抗CD3单链抗体的融合分子包括选自下组 的TCRα链可变域氨基酸序列SEQ ID NO:9-13、45-51和/或选自下组的TCRβ链可变域氨基酸序列SEQ ID NO:14-40、52-79。
本发明还涉及编码本发明TCR的核酸分子。本发明的核酸分子可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。例如，编码本发明TCR的核酸序列可以与本发明附图中所示的核酸序列相同或是简并的变异体。举例说明“简并的变异体”的含义，如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO:41的蛋白序列，但与SEQ ID NO:42的序列有差别的核酸序列。
本发明的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明TCR(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的核酸分子的载体，以及用本发明的载体或编码序列经基因工程产生的宿主细胞。
本发明还包括表达本发明TCR的分离细胞，特别是T细胞。有许多方法适合于用编码本发明的高亲和力TCR的DNA或RNA进行T细胞转染(如，Robbins等.，(2008)J.Immunol.180:6116-6131)。表达本发明高亲和性TCR的T细胞可以用于过继免疫治疗。本领域技术人员能够知晓进行过继性治疗的许多合适方法(如，Rosenberg等.，(2008)Nat Rev Cancer 8(4):299-308)。
本发明还提供一种药物组合物，所述药物组合物含有药学上可接受的载体以及本发明TCR、或本发明TCR复合物、或呈递本发明TCR的细胞。
本发明还提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明TCR、或本发明TCR复合物、或呈递本发明TCR的细胞、或本发明的药物组合物。
应理解，本文中氨基酸名称采用国际通用的单英文字母标识，与其相对应的氨基酸名称三英文字母简写分别是：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)；本发明中，Pro60或者60P均表示第60位脯氨酸。另外，本发明中所述突变的具体形式的表述方式如“V27A/P”代表第27位的V被A取代或被P取代，同理，“I29L”代表第29位的I被L取代。其他以此类推。
在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的结构和功能。因此，本发明TCR还包括本发明TCR的至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸(尤其是位于CDR区之外的氨基酸)，被性质相似或相近的氨基酸所替换，并仍能够保持其功能性的TCR。
本发明还包括对本发明TCR略作修饰后的TCR。修饰(通常不改变一级结构)形式包括：本发明TCR的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在本发明TCR的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的TCR。这种修饰可以通过将TCR暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括 具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的TCR。
本发明的TCR、TCR复合物或本发明TCR转染的T细胞可与药学上可接受的载体一起在药物组合物中提供。本发明的TCR、多价TCR复合物或细胞通常作为无菌药物组合物的一部分提供，所述组合物通常包括药学上可接受的载体。该药物组合物可以是任何合适的形式(取决于给予患者的所需方法)。其可采用单位剂型提供，通常在密封的容器中提供，可作为试剂盒的一部分提供。此类试剂盒(但非必需)包括使用说明书。其可包括多个所述单位剂型。
此外，本发明的TCR可以单用，也可与其他治疗剂结合或偶联在一起使用(如配制在同一药物组合物中)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在雷明顿药物科学(Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991))中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂、及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：眼内、肌内、静脉内、皮下、皮内、或局部给药，优选为胃肠外包括皮下、肌肉内或静脉内。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有针剂、口服剂等。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明TCR可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明TCR或TCR复合物或呈递本发明TCR的细胞，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定，最终由医师决定合理的用量。
本发明的主要优点在于：
(1)本发明的TCR对所述FMNKFIYEI-HLA-A2复合物的亲和力和/或结合半衰期是野生型TCR的至少2倍，优选地，至少10倍。
(2)本发明的TCR对所述FMNKFIYEI-HLA-A2复合物的亲和力和/或结合半衰期是野生型TCR的至少100倍，优选地，至少500倍，更优选地，可达10 3-5*10 4倍。
(3)转导本发明的高亲和力TCR的效应细胞对靶细胞有很强的杀伤作用。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
材料和方法
本发明实施例中所用的实验材料如无特殊说明均可从市售渠道获得，其中，E.coli DH5α购自Tiangen、E.coli BL21(DE3)购自Tiangen、E.coli Tuner(DE3)购自Novagen、质粒pET28a购自Novagen。
实施例1 疏水芯突变的稳定性单链TCR模板链的产生
本发明利用定点突变的方法，根据专利文献WO2014/206304中所述，构建了以一个柔性短肽(linker)连接TCRα与β链可变域而构成的稳定性单链TCR分子，其氨基酸及DNA序列分别为SEQ ID NO:41和SEQ ID NO:42，如图7a和图7b所示。并以该单链TCR分子为模板进行高亲和性TCR分子的筛选。该模板链的α可变域(SEQ ID NO:3)及β可变域(SEQ ID NO:4)的氨基酸序列如图2a和2b所示；其对应的DNA序列分别为SEQ ID NO:5和6，如图3a和3b所示；柔性短肽(linker)的氨基酸序列及DNA序列分别为SEQ ID NO:7和8，如图4a和4b所示。
将携带模板链的目的基因经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli BL21(DE3)，用于表达。
实施例2 实施例1中构建的稳定性单链TCR的表达、复性和纯化
将实施例1中制备的含有重组质粒pET28a-模板链的BL21(DE 3)菌落全部接种于含有卡那霉素的LB培养基中，37℃培养至OD 600为0.6-0.8，加入IPTG至终浓度为0.5mM，37℃继续培养4h。5000rpm离心15min收获细胞沉淀物，用Bugbuster Master Mix(Merck)裂解细胞 沉淀物，6000rpm离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000rpm离心15min，收集包涵体。将包涵体溶解在缓冲液(20mM Tris-HCl pH8.0,8M尿素)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。
向5mg溶解的单链TCR包涵体蛋白中，加入2.5mL缓冲液(6M Gua-HCl，50mM Tris-HCl pH 8.1，100mM NaCl，10mM EDTA)，再加入DTT至终浓度为10mM,37℃处理30min。用注射器向125mL复性缓冲液(100mM Tris-HCl pH 8.1,0.4M L-精氨酸,5M尿素,2mM EDTA,6.5mMβ-mercapthoethylamine,1.87mM Cystamine)中滴加上述处理后的单链TCR，4℃搅拌10min，然后将复性液装入截留量为4kDa的纤维素膜透析袋,透析袋置于1L预冷的水中，4℃缓慢搅拌过夜。17小时后，将透析液换成1L预冷的缓冲液(20mM Tris-HCl pH 8.0)，4℃继续透析8h，然后将透析液换成相同的新鲜缓冲液继续透析过夜。17小时后，样品经0.45μm滤膜过滤，真空脱气后通过阴离子交换柱(HiTrap Q HP,GE Healthcare)，用20mM Tris-HCl pH 8.0配制的0-1M NaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析，包含单链TCR的组分浓缩后进一步用凝胶过滤柱(Superdex 75 10/300,GE Healthcare)进行纯化，目标组分也进行SDS-PAGE分析。
用于BIAcore分析的洗脱组分进一步采用凝胶过滤法测试其纯度。条件为：色谱柱Agilent Bio SEC-3(300A，
Figure PCTCN2020109351-appb-000008
)，流动相为150mM磷酸盐缓冲液，流速0.5mL/min，柱温25℃，紫外检测波长214nm。
实施例3 结合表征
BIAcore分析
使用BIAcore T200实时分析系统检测TCR分子与FMNKFIYEI-HLA-A0201复合物的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，pH4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15，000RU。条件为：温度25℃，PH值为7.1-7.5。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将FMNKFIYEI-HLA-A0201复合物流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。采用单循环动力学分析方法测定其亲和力，将TCR用HEPES-EP缓冲液(10mM HEPES,150mM NaCl，3mM EDTA,0.005％P20，pH 7.4)稀释成几个不同的浓度，以30μL/min的流速，依次流过芯片表面，每次进样的结合时间为120s，最后一次进样结束后让其解离600s。每一轮测定结束后用pH 1.75的10mM Gly-HCl再生芯片。利用BIAcore Evaluation软件计算动力学参数。
上述FMNKFIYEI-HLA-A0201复合物的制备过程如下：
a.纯化
收集100ml诱导表达重链或轻链的E.coli菌液，于4℃8000g离心10min后用10ml PBS洗涤菌体一次，之后用5ml BugBuster Master Mix Extraction Reagents(Merck)剧烈震荡重悬菌 体，并于室温旋转孵育20min，之后于4℃，6000g离心15min，弃去上清，收集包涵体。
将上述包涵体重悬于5ml BugBuster Master Mix中，室温旋转孵育5min；加30ml稀释10倍的BugBuster，混匀，4℃6000g离心15min；弃去上清，加30ml稀释10倍的BugBuster重悬包涵体，混匀，4℃6000g离心15min，重复两次，加30ml 20mM Tris-HCl pH 8.0重悬包涵体，混匀，4℃6000g离心15min，最后用20mM Tris-HCl 8M尿素溶解包涵体，SDS-PAGE检测包涵体纯度，BCA试剂盒测浓度。
b.复性
将合成的短肽FMNKFIYEI(北京赛百盛基因技术有限公司)溶解于DMSO至20mg/ml的浓度。轻链和重链的包涵体用8M尿素、20mM Tris pH 8.0、10mM DTT来溶解，复性前加入3M盐酸胍、10mM醋酸钠、10mM EDTA进一步变性。将FMNKFIYEI肽以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mM Tris pH 8.3、2mM EDTA、0.5mM氧化性谷胱甘肽、5mM还原型谷胱甘肽、0.2mM PMSF，冷却至4℃)，然后依次加入20mg/L的轻链和90mg/L的重链(终浓度，重链分三次加入，8h/次)，复性在4℃进行至少3天至完成，SDS-PAGE检测能否复性成功。
c.复性后纯化
用10体积的20mM Tris pH 8.0作透析来更换复性缓冲液，至少更换缓冲液两次来充分降低溶液的离子强度。透析后用0.45μm醋酸纤维素滤膜过滤蛋白质溶液，然后加载到HiTrap Q HP(GE通用电气公司)阴离子交换柱上(5ml床体积)。利用Akta纯化仪(GE通用电气公司)，20mM Tris pH 8.0配制的0-400mM NaCl线性梯度液洗脱蛋白，pMHC约在250mM NaCl处洗脱，收集诸峰组分，SDS-PAGE检测纯度。
d.生物素化
用Millipore超滤管将纯化的pMHC分子浓缩，同时将缓冲液置换为20mM Tris pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mM MgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
e.纯化生物素化后的复合物
用Millipore超滤管将生物素化标记后的pMHC分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的pMHC，利用Akta纯化仪(GE通用电气公司)，用过滤过的PBS预平衡HiPrep TM16/60S200HR柱(GE通用电气公司)，加载1ml浓缩过的生物素化pMHC分子，然后用PBS以1ml/min流速洗脱。生物素化的pMHC分子在约55ml时作为单峰洗脱出现。合并含有蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，加入蛋白酶抑制剂cocktail(Roche)将生物素化的pMHC分子分装保存在-80℃。
实施例4 高亲和性单链TCR的产生
噬菌体展示技术是产生TCR高亲和力变体文库以筛选高亲和力变体的一种手段。将Li等((2005)Nature Biotech 23(3):349-354)描述的TCR噬菌体展示和筛选方法应用于实施例1中的单链TCR模板。通过突变该模板链的CDR区来建立高亲和性TCR的文库并进行淘选。经 过几轮淘选后的噬菌体文库均和相应抗原有特异性结合，从中挑取单克隆，并进行序列分析。
采用实施例3中BIAcore方法分析TCR分子与FMNKFIYEI-HLA-A0201复合物的相互作用，筛选出了亲和力和/或结合半衰期是野生型TCR的至少2倍的高亲和性TCR，即筛选出的高亲和性TCR结合FMNKFIYEI-HLA-A0201复合物的解离平衡常数K D小于等于野生型TCR结合FMNKFIYEI-HLA-A0201复合物的解离平衡常数K D的二分之一，结果如下表3所示。利用上述方法检测到参比TCR与FMNKFIYEI-HLA-A0201复合物相互作用的K D值为9.39μM，其相互作用曲线如图12所示，即野生型TCR与FMNKFIYEI-HLA-A0201复合物相互作用的KD值也为9.39μM，即9.39E-06M。
具体地，采用SEQ ID NO:1中所示的编号，这些高亲和力TCR突变体的α链可变域在下列一个或多个位点的氨基酸发生突变：27V、29I、30S、50S、51S、52G、53K、92S和93Y和/或采用SEQ ID NO:2中所示的编号，这些高亲和力TCR突变体的β链可变域在下列一个或多个位点52E、53A、54Q、95S、96Y、97G、98A、103D、104T和106Y中发生突变。
更具体地，采用SEQ ID NO:1所示的编号，这些高亲和力TCR的α链可变域包含选自下组的一个或多个氨基酸残基27A或27P；29L；30Q；50P；51F或51Y；52Q；53T；92T或92F和93R或93N；和/或采用SEQ ID NO:2所示的编号，这些高亲和力TCR的β链可变域包含选自下组的一个或多个氨基酸残基52V、53S、54I、95A、96L或96P或96R或96K或96Q或96F、97F或97M或97Y或97H或97S或97W或97A、98S或98P或98G、103G或103S或103M或103E或103A或103R、104S或104A或104E或104G或104M、106A或106V或106I或106W或106K或106M或106Q或106R。
高亲和性单链TCR的α链可变域(SEQ ID NO:9-13)和β链可变域(SEQ ID NO:14-40)的具体氨基酸序列分别如图5(1)-(5)和图6(1)-(27)所示。
表3
Figure PCTCN2020109351-appb-000009
Figure PCTCN2020109351-appb-000010
实施例5 高亲和性αβ异质二聚TCR的产生
将实施例4中筛选到的高亲和力的单链TCR的CDR区突变引入到αβ异质二聚TCR的可变域的相应位点中，并通过BIAcore来检测其与FMNKFIYEI-HLA-A0201复合物的亲和力。上述CDR区高亲和力突变点的引入采用本领域技术人员熟知的定点突变的方法。上述野生型TCR的α链与β链可变域氨基酸序列分别如图1a(SEQ ID NO:1)和1b(SEQ ID NO:2)所示。
应注意，为获得更加稳定的可溶性TCR，以便更方便地评估TCR与FMNKFIYEI-HLA A0201复合物之间的结合亲和力和/或结合半衰期，αβ异质二聚TCR可以是在α和β链的恒定区中分别引入了一个半胱氨酸残基以形成人工链间二硫键的TCR，本实施例中引入半胱氨酸残基后TCRα与β链的氨基酸序列分别如图8a(SEQ ID NO:43)和8b所示(SEQ ID NO:44)，引入的半胱氨酸残基以加粗字母表示。
通过《分子克隆实验室手册》(Molecular Cloning a Laboratory Manual)(第三版，Sambrook和Russell)中描述的标准方法将待表达的TCRα和β链的胞外序列基因经合成后分别插入到表达载体pET28a+(Novagene)，上下游的克隆位点分别是NcoI和NotI。CDR区的突变通过本领域技术人员熟知的重叠PCR(overlap PCR)引入。插入片段经过测序确认无误。
实施例6 αβ异质二聚TCR的表达、复性和纯化
将TCRα和β链的表达载体分别通过化学转化法转化进入表达细菌BL21(DE3),细菌用LB培养液生长，于OD 600＝0.6时用终浓度0.5mM IPTG诱导，TCR的α和β链表达后形成的包涵体通过BugBuster Mix(Novagene)进行提取，并且经BugBuster溶液反复多次洗涤，包涵体最后溶解于6M盐酸胍,10mM二硫苏糖醇(DTT),10mM乙二胺四乙酸(EDTA),20mM Tris(pH 8.1)中。
溶解后的TCRα和β链以1:1的质量比快速混合于5M尿素,0.4M精氨酸,20mM Tris(pH8.1),3.7mM cystamine,6.6mMβ-mercapoethylamine(4℃)中，终浓度为60mg/mL。混合后将溶液置于10倍体积的去离子水中透析(4℃)，12小时后将去离子水换成缓冲液(20mM Tris,pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP,5ml,GE Healthcare)纯化。洗脱峰含有复性成功的α和β二聚体的TCR通过SDS-PAGE胶确认。TCR随后通过凝胶过滤层析(HiPrep 16/60，Sephacryl S-100HR，GE Healthcare)进一步纯化。纯化后的TCR纯度经过SDS-PAGE测定大于90％，浓度由BCA法确定。
实施例7 BIAcore分析结果
采用实施例3中所述方法检测引入高亲和力CDR区的αβ异质二聚TCR与FMNKFIYEI-HLA-A0201复合物的亲和力。
将高亲和性单链TCRα与β链中筛选出的CDR区分别转移到野生型TCRα链可变域SEQ ID NO:1和β链可变域SEQ ID NO:2的相应位置，形成αβ异质二聚TCR。另外，还根据筛选得到的CDR区的突变位点进行人工组合来形成αβ异质二聚TCR，得到的新的TCRα和β链可变域氨基酸序列，分别如图9(1)-(7)和图10(1)-(28)所示。由于TCR分子的CDR区决定了其与相应的pMHC复合物的亲和力，所以本领域技术人员能够预料引入高亲和力突变点的αβ异质二聚TCR也具有对FMNKFIYEI-HLA-A0201复合物的高亲和力。利用实施例5中所述方法构建表达载体，利用实施例6中所述方法对上述引入高亲和力突变的αβ异质二聚TCR进行表达、复性和纯化，然后利用BIAcore T200测定其与FMNKFIYEI-HLA-A0201复合物的亲和力，如下表4所示。
表4
Figure PCTCN2020109351-appb-000011
Figure PCTCN2020109351-appb-000012
由上表4可知，引入CDR区突变点的αβ异质二聚TCR保持了对FMNKFIYEI-HLA-A0201复合物的高亲和力。所述异质二聚TCR的亲和力是野生型TCR对FMNKFIYEI-HLA-A0201复合物的亲和力的至少2倍。
实施例8.抗-CD3抗体与高亲和性单链TCR的融合体的表达、复性和纯化
将本发明的高亲和性单链TCR分子与抗CD3抗体的单链分子(scFv)进行融合，构建融合分子。通过重叠(overlap)PCR的方法，设计引物,连接抗-CD3抗体及高亲和性单链TCR分子的基因，设计中间的连接短肽(linker)为GGGGS，并且使融合分子的基因片段带上限制性内切酶位点NcoⅠ和NotⅠ。将PCR扩增产物经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α感受态细胞，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli BL21(DE3)感受态细胞，用于表达。
融合蛋白的表达
将含有目的基因的表达质粒转化入大肠杆菌菌株BL21(DE3)中，涂布LB平板(卡那霉素50μg/ml)置于37℃培养过夜。次日，挑克隆接种至10ml LB液体培养基(卡那霉素50μg/ml)培养2-3h，按体积比1:100接种至1L LB培养基(卡那霉素50μg/ml)中，继续培养至OD 600为0.5-0.8，然后使用终浓度为0.5mM的IPTG诱导目的蛋白的表达。诱导4小时以后，以6000rpm离心10min收获细胞。PBS缓冲液洗涤菌体一次，并且分装菌体，取相当于200ml的细菌培养物的菌体用5ml BugBuster Master Mix(Novagen)裂解细菌，以6000g离心15min收集包涵体。然后进行4次洗涤剂洗涤以去除细胞碎片和膜组分。然后，用缓冲液如PBS洗涤包涵体以除去洗涤剂和盐。最终，将包涵体用含8M尿素的Tris缓冲溶液溶解，并测定包涵体浓度，将其分装后置于-80℃冷冻保存。
融合蛋白的重折叠
从-80℃超低温冰箱中取出约10mg包涵体解冻，加二硫苏糖醇(DTT)至终浓度为10mM，在37℃中温育30min到1小时以确保二硫键完全打开。然后将包涵体样品溶液分别滴入200ml 4℃预冷重折叠缓冲液(100mM Tris pH 8.1，400mM L-精氨酸，2mM EDTA，5M尿素，6.5mMβ-mercapthoethylamine,1.87mM Cystamine)，4℃缓慢搅拌约30分钟。复性溶液用8倍体积预冷的H 2O透析16-20小时。再用8倍体积的10mM Tris pH 8.0透析两次，4℃继续透析约8小时，透析后样品过滤后进行以下纯化。
融合蛋白的第一步纯化
经过透析的重折叠物(10mM Tris pH 8.0中)使用POROS HQ/20阴离子交换层析预装柱(Applied Biosystems)，在AKTA纯化仪(GE Healthcare)用0-600mM NaCl进行梯度洗脱。通过考马斯亮蓝染色的SDS-PAGE分析各个组分，然后合并。
融合蛋白的第二步纯化
将第一步纯化合并的样品溶液浓缩以供此步纯化，利用在PBS缓冲液中预平衡的Superdex 75 10/300GL凝胶过滤层析预装柱(GE Healthcare)纯化融合蛋白，考马斯亮蓝染色的SDS-PAGE分析出峰的组分，然后合并。
实施例9.抗-CD3抗体与高亲和性αβ异质二聚TCR的融合体的表达、复性和纯化
将抗-CD3的单链抗体(scFv)与αβ异质二聚TCR融合，制备融合分子。抗-CD3的scFv与TCR的β链融合，该TCRβ链可以包含任一上述高亲和性αβ异质二聚TCR的β链可变 域，融合分子的TCRα链可以包含任一上述高亲和性αβ异质二聚TCR的α链可变域。
融合分子表达载体的构建
1.α链表达载体的构建
将携带αβ异质二聚TCR的α链的目的基因经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布于含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Tuner(DE3),用于表达。
2.抗-CD3(scFv)-β链表达载体的构建
通过重叠(overlap)PCR的方法，设计引物将抗-CD3scFv和高亲和性异质二聚TCRβ链基因连接起来，中间的连接短肽(linker)为GGGGS，并且使抗-CD3的scFv与高亲和性异质二聚TCRβ链的融合蛋白的基因片段带上限制性内切酶位点NcoⅠ(CCATGG)和NotⅠ(GCGGCCGC)。将PCR扩增产物经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α感受态细胞，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Tuner(DE3)感受态细胞，用于表达。
融合蛋白的表达、复性及纯化
将表达质粒分别转化进入E.coli Tuner(DE3)感受态细胞，涂布LB平板(卡那霉素50μg/mL)置于37℃培养过夜。次日，挑克隆接种至10mL LB液体培养基(卡那霉素50μg/mL)培养2-3h，按体积比1:100接种至1L LB培养基中，继续培养至OD600为0.5-0.8，加入终浓度为1mM IPTG诱导目的蛋白的表达。诱导4小时以后，以6000rpm离心10min收获细胞。PBS缓冲液洗涤菌体一次，并且分装菌体，取相当于200mL的细菌培养物的菌体用5mL BugBuster Master Mix(Merck)裂解细菌，以6000g离心15min收集包涵体。然后进行4次洗涤剂洗涤以去除细胞碎片和膜组分。然后，用缓冲液如PBS洗涤包涵体以除去洗涤剂和盐。最终，将包涵体用含6M盐酸胍,10mM二硫苏糖醇(DTT),10mM乙二胺四乙酸(EDTA),20mM Tris,pH 8.1缓冲溶液溶解，并测定包涵体浓度，将其分装后置于-80℃冷冻保存。
溶解后的TCRα链和抗-CD3(scFv)-β链以2：5的质量比快速混合于5M尿素(urea),0.4M L-精氨酸(L-arginine),20mM Tris pH 8.1,3.7mM cystamine,6.6mMβ-mercapoethylamine(4℃)，终浓度α链和抗-CD3(scFv)-β链分别为0.1mg/mL，0.25mg/mL。
混合后将溶液置于10倍体积的去离子水中透析(4℃)，12小时后将去离子水换成缓冲液(10mM Tris,pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP 5ml,GE healthcare)纯化。洗脱峰含有复性成功的TCRα链与抗-CD3(scFv)-β链二聚体的TCR通过SDS-PAGE胶确认。TCR融合分子随后通过尺寸排阻色谱法(S-100 16/60，GE healthcare)进一步纯化，以及阴离子交换柱(HiTrap Q HP 5ml,GE healthcare)再次纯化。纯化后的TCR融合分子纯度经过SDS-PAGE测定大于90％，浓度由BCA法测定。
实施例10.转染本发明高亲和力TCR的效应细胞的激活功能实验
本实施例验证了转染本发明高亲和力TCR的效应细胞对靶细胞有很好的特异性激活作用。通过ELISPOT实验检测本发明高亲和力TCR在细胞中的功能及特异性。
本领域技术人员熟知利用ELISPOT实验检测细胞功能的方法。随机选择本发明TCR转染从健康志愿者的血液中分离到的PBL，作为效应细胞。
以下两个批次(Ⅰ)、(Ⅱ)先后进行实验(其中，TCR、靶细胞系均有不同)：
(I)所述TCR以及其编号从表4获悉，分别为TCR13(α链可变域SEQ ID NO:45，β链可变域SEQ ID NO:2)、TCR21(α链可变域SEQ ID NO:46，β链可变域SEQ ID NO:2)、TCR9(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:60)、TCR4(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:55)、TCR1(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:52)、TCR2(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:53)、TCR12(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:63)、TCR5(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:56)、TCR3(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:54)和TCR8(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:59)，对照组效应细胞标号为野生型TCR(转染野生型TCR的细胞)和A6(转染其他TCR的细胞)。靶细胞系为HepG2、HCCC9810-AFP(即AFP转染HCCC9810)、SNU-398-AFP(即AFP转染SNU-398)、Huh-7、SNU-398和HCCC9810细胞。其中，靶细胞系HepG2、HCCC9810-AFP和SNU-398-AFP为阳性肿瘤细胞系；Huh-7、SNU-398和HCCC9810为阴性肿瘤细胞系，作为对照。
(II)所述TCR以及其编号从表4获悉，分别为TCR14(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:64)、TCR26(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:75)、TCR18(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:68)、TCR19(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:69)、TCR10(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:61)、TCR22(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:71)、TCR23(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:72)、TCR24(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:73)、TCR11(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:62)、TCR25(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:74)、TCR6(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:57)、TCR17(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:67)、TCR7(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:58)、TCR15(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:65)、TCR16(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:66)和TCR20(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:70)，对照组效应细胞标号为野生型TCR(转染野生型TCR的细胞)和A6(转染其他TCR的细胞)。靶细胞系为HepG2、HCCC9810-AFP(即AFP转染HCCC9810)、SK-HEP-1-AFP(即AFP转染SK-HEP-1)、Huh-7、HCCC9810和SK-HEP-1细胞。其中，靶细胞系HepG2、HCCC9810-AFP和SK-HEP-1-AFP为阳性肿瘤细胞系；Huh-7、HCCC9810和SK-HEP-1为阴性肿瘤细胞系，作为对照。
上述的两批次均进行以下实验步骤：首先准备ELISPOT平板。ELISPOT平板乙醇活化包被，4℃过夜。实验第1天，去掉包被液，洗涤封闭，室温下孵育两个小时，去除封闭液， 将试验的各个组分加入ELISPOT平板：靶细胞系为2*10 4个/孔，效应细胞为10 3个/孔(按转染的阳性率计算)，并设置二个复孔。温育过夜(37℃，5％CO 2)。实验第2天，洗涤平板并进行二级检测和显色，干燥平板，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。
两批次实验结果分别如图14a、14b所示，针对阳性靶细胞系，转染本发明高亲和力TCR的效应细胞产生了很好的特异性激活作用，功能要远远好于转染野生型TCR的效应细胞。而转导其他TCR的细胞基本没有产生激活作用。
实施例11.转染本发明高亲和力TCR的效应细胞的LDH杀伤功能实验
本实施例通过非放射性细胞毒性实验，测定LDH的释放，从而验证转导本发明TCR的细胞的杀伤功能。该试验是 51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的乳酸脱氢酶(LDH)。采用30分钟偶联的酶反应来检测释放在培养基中的LDH，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。可以用标准的96孔读板计收集490nm可见光吸光值数据。计算公式:％细胞毒性＝100×(实验-效应细胞自发-靶细胞自发)/(靶细胞最大-靶细胞自发)
本领域技术人员熟知利用LDH的释放实验检测细胞功能的方法。随机选择本发明TCR转染从健康志愿者的血液中分离到的CD3 +T细胞，作为效应细胞。所述TCR以及其编号从表4获悉，分别为TCR4(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:55)、TCR1(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:52)、TCR2(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:53)、TCR12(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:63)和TCR5(α链可变域SEQ ID NO:1，β链可变域SEQ ID NO:56)，对照组效应细胞标号为野生型TCR(转染野生型TCR的细胞)和A6(转染其他TCR的细胞)。靶细胞系为HepG2、HCCC9810和SNU-398细胞。其中，HepG2为阳性肿瘤细胞系；HCCC9810和SNU-398为阴性肿瘤细胞系，作为对照。
首先准备LDH平板。实验第1天，将试验的各个组分加入平板：靶细胞系3*10 4个细胞/孔、效应细胞3*10 4个细胞/孔，并设置三个复孔。同时设置效应细胞自发孔，靶细胞自发孔，靶细胞最大孔，体积校正对照孔及培养基背景对照孔。温育过夜(37℃，5％CO 2)。实验第2天，检测显色，终止反应后用酶标仪(Bioteck)在490nm记录吸光值。
实验结果如图15所示，转导本发明TCR的细胞对阳性靶细胞都具有很强的杀伤作用，杀伤效力远远高于转导野生型TCR的细胞；而转导其他TCR的细胞对阳性靶细胞基本没有杀伤作用。

Claims (34)

  1. 一种T细胞受体(TCR)，其特征在于，所述TCR具有结合FMNKFIYEI-HLA A0201复合物的活性，并且，所述TCR的α链可变域包含与SEQ ID NO:1所示的氨基酸序列有至少90％的序列同源性的氨基酸序列；和所述TCR的β链可变域包含与SEQ ID NO:2所示的氨基酸序列有至少90％的序列同源性的氨基酸序列。
  2. 如权利要求1所述的TCR，其特征在于，所述TCR与FMNKFIYEI-HLA A0201复合物的亲和力是野生型TCR的至少2倍。
  3. 如权利要求1所述的TCR，其特征在于，所述TCR是可溶的。
  4. 如权利要求1所述的TCR，其特征在于，所述TCR为αβ异质二聚TCR；优选地，所述TCR具有α链恒定区序列TRAC*01和β链恒定区序列TRBC1*01或TRBC2*01。
  5. 如权利要求1所述的TCR，其特征在于，所述TCRβ链可变域包含3个CDR区，CDR1β、CDR2β和CDR3β，其中CDR1β的氨基酸序列为SGHVS，和CDR2β的氨基酸序列为FNYEAQ。
  6. 如权利要求1所述的TCR，其特征在于，所述TCRα链可变域包含3个CDR区，CDR1α、CDR2α和CDR3α，其中CDR1α的氨基酸序列为VGISA，CDR2α的氨基酸序列为LSSGK，和CDR3α的氨基酸序列为AVETSYDKVI。
  7. 如权利要求1所述的TCR，其中，所述TCRβ链可变域包含3个CDR区，CDR1β、CDR2β和CDR3β，CDR3β的基准氨基酸序列为ASSYGAGGPLDTQY，其特征在于，所述TCR在CDR3β中发生至少一个突变，并且，所述突变包含：
    突变前的残基 突变后的残基 CDR3β的第3位S A。
  8. 如权利要求1所述TCR，其中，所述TCRβ链可变域包含3个CDR区，CDR1β、CDR2β和CDR3β，CDR3β的基准氨基酸序列为ASSYGAGGPLDTQY，其特征在于，所述TCR在CDR3β中发生至少一个突变，并且，所述突变包含：
    突变前的残基 突变后的残基 CDR3β的第6位A S。
  9. 如权利要求1所述TCR，其中，所述TCRβ链可变域包含3个CDR区，CDR1β、CDR2β和CDR3β，CDR3β的基准氨基酸序列为ASSYGAGGPLDTQY，其特征在于，所述TCR在CDR3β中发生至少一个突变，并且，所述突变包含：
    突变前的残基 突变后的残基 CDR3β的第3位S A；和 CDR3β的第6位A S。
  10. 如权利要求1所述TCR，所述TCRα链可变域包含3个CDR区，CDR1α、CDR2α和CDR3α，其特征在于，所述TCRα链可变域的3个CDR区的基准序列如下，
    CDR1α：VGISA
    CDR2α：LSSGK
    CDR3α：AVETSYDKVI，并且含有至少一个下列突变：
    突变前的残基 突变后的残基 CDR1α的第1位V A或P CDR1α的第3位I L CDR1α的第4位S Q CDR2α的第2位S P CDR2α的第3位S F或Y CDR2α的第4位G Q CDR2α的第5位K T CDR3α的第5位S T或F CDR3α的第6位Y R或N。
  11. 如权利要求1所述TCR，所述TCRβ链可变域包含3个CDR区，CDR1β、CDR2β和CDR3β，其特征在于，所述TCRβ链可变域的3个CDR区的基准序列如下，
    CDR1β：SGHVS
    CDR2β：FNYEAQ
    CDR3β：ASSYGAGGPLDTQY，并且含有至少一个下列突变：
    突变前的残基 突变后的残基 CDR2β的第4位E V CDR2β的第5位A S CDR2β的第6位Q I CDR3β的第3位S A CDR3β的第4位Y L或P或R或K或Q或F CDR3β的第5位G F或M或Y或H或S或W或A CDR3β的第6位A S或P或G CDR3β的第11位D G或S或M或E或A或R CDR3β的第12位T S或A或E或G或M CDR3β的第14位Y A或V或I或W或K或M或Q或R。
  12. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域包含与SEQ ID NO:1所示的氨基酸序列有至少95％的序列同源性的氨基酸序列；和所述TCR的β链可变域包含与SEQ ID NO:2所示的氨基酸序列有至少95％的序列同源性的氨基酸序列。
  13. 如权利要求1所述的TCR，其中，所述TCRα链可变域包含3个CDR，CDR1α、CDR2α和CDR3α，其特征在于，CDR1α的氨基酸序列选自下组：VGISA、AGLQA、VGLQA和PGLQA；和/或CDR2α的氨基酸序列选自下组：LSSGK、LPFGK和LPYQT；和/或CDR3α的氨基酸序列选自下组：AVETSYDKVI、AVETTRDKVI和AVETFNDKVI。
  14. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域氨基酸序列为SEQ ID NO:1。
  15. 如权利要求1所述的TCR，其特征在于，所述TCRβ链可变域包含3个CDR区，CDR1β、CDR2β和CDR3β，其中，所述TCR的CDR3β选自下组：ASALMSGGPLDTQY、ASARYPGGPLDTQY、ASARHAGGPLDTQY、ASAPFSGGPLDTQY和ASAKMSGGPLDTQY。
  16. 如权利要求1所述的TCR，其特征在于，所述TCRα链可变域包含3个CDR，CDR1α、CDR2α和CDR3α，和所述TCRβ链可变域包含3个CDR区，CDR1β、CDR2β和CDR3β，并且所述TCRα和β链可变域具有选自下组的CDR:
    CDR编号 A-CDR1 A-CDR2 A-CDR3 B-CDR1 B-CDR2 B-CDR3 1 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASALMSGGPLDTQY 2 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASARYPGGPLDTQY 3 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASARHAGGPLDTQY 4 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASAPFSGGPLDTQY 5 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASAKMSGGPLDTQY 6 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGEQW 7 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGAQA 8 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSQSGGGPLDTQY 9 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASALFSGGPLDTQY 10 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGEQV 11 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLMAQA 12 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASALYSGGPLDTQY 13 VGISA LSSGK AVETTRDKVI SGHVS FNYEAQ ASSYGAGGPLDTQY 14 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSPFSGGPLDTQY 15 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGAQK 16 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLEGQV 17 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLSSQI 18 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSLFGGGPLDTQY 19 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLSGQI 20 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLASQY 21 VGISA LSSGK AVETFNDKVI SGHVS FNYEAQ ASSYGAGGPLDTQY 22 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLRTQM 23 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGSQQ 24 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGSQV 25 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGSQA 26 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSLFSGGPLDTQY 27 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSLWSGGPLDTQY 28 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSFAGGGPLDTQY 29 VGISA LSSGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLGMQR 30 VGLQA LPFGK AVETTRDKVI SGHVS FNYEAQ ASALFSGGPLDTQY 31 AGLQA LPFGK AVETSYDKVI SGHVS FNYEAQ ASALFSGGPLDTQY 32 PGLQA LPFGK AVETSYDKVI SGHVS FNYEAQ ASSYGAGGPLDTQY 33 AGLQA LPYQT AVETSYDKVI SGHVS FNYEAQ ASALFSGGPLDTQY 34 AGLQA LPYQT AVETTRDKVI SGHVS FNYEAQ ASALFSGGPLDTQY 35 VGLQA LPFGK AVETTRDKVI SGHVS FNYVSI ASALFSGGPLDTQY 36 AGLQA LPYQT AVETSYDKVI SGHVS FNYVSI ASALFSGGPLDTQY。
  17. 如权利要求1所述的TCR，其特征在于，所述TCR包含(i)除其跨膜结构域以外的全部或部分TCRα链，和(ii)除其跨膜结构域以外的全部或部分TCRβ链，其中(i)和(ii)均包含TCR链的可变域和至少一部分恒定域。
  18. 如权利要求17所述的TCR，其特征在于，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
  19. 如权利要求18所述的TCR，其特征在于，在所述TCRα与β链的恒定区之间形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
    TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
    TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
    TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
    TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
    TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
    和TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
  20. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域氨基酸序列选自：SEQ ID NO:45-51；和/或所述TCR的β链可变域氨基酸序列选自：SEQ ID NO:52-79。
  21. 如权利要求1所述的TCR，其特征在于，所述TCR选自下组：
    Figure PCTCN2020109351-appb-100001
  22. 如权利要求1所述的TCR，其特征在于，所述TCR为单链TCR。
  23. 如权利要求1所述的TCR，其特征在于，所述TCR是由α链可变域和β链可变域组成的单链TCR，所述α链可变域和β链可变域由一柔性短肽序列(linker)连接。
  24. 如以上任一权利要求所述的TCR，其特征在于，所述TCR的α链和/或β链的C- 或N-末端结合有偶联物，优选地，所述偶联物为可检测标记物；更优选地，所述偶联物为抗-CD3抗体。
  25. 一种多价TCR复合物，其特征在于，所述TCR复合物包含至少两个TCR分子，并且其中的至少一个TCR分子为上述任一权利要求中所述的TCR。
  26. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1-24中任一项所述的TCR的核酸序列或其互补序列。
  27. 一种载体，其特征在于，所述的载体含有权利要求26中所述的核酸分子。
  28. 一种宿主细胞，其特征在于，所述的宿主细胞中含有权利要求27中所述的载体或染色体中整合有外源的权利要求26中所述的核酸分子。
  29. 一种分离的细胞，其特征在于，所述细胞表达权利要求1-24中任一项所述的TCR。
  30. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1-24中任一项所述的TCR、或权利要求25中所述的TCR复合物、或权利要求29中所述的细胞。
  31. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用权利要求1-24中任一项所述的TCR、或权利要求25中所述的TCR复合物、或权利要求29中所述的细胞、或权利要求30中所述的药物组合物。
  32. 权利要求1-24中任一项所述的T细胞受体、权利要求25中所述的TCR复合物或权利要求29中所述细胞的用途，其特征在于，用于制备治疗肿瘤的药物，优选地，所述肿瘤为肝细胞癌。
  33. 权利要求1-24中任一项所述的T细胞受体、权利要求25中所述的TCR复合物或权利要求29中所述的细胞用作治疗肿瘤的药物。优选地，所述肿瘤为AFP阳性肿瘤；更优选地，所述肿瘤为肝细胞癌。
  34. 一种制备权利要求1-24中任一项所述的T细胞受体的方法，其特征在于，包括步骤：
    (i)培养权利要求28中所述的宿主细胞，从而表达权利要求1-28中任一项所述的T细胞受体；
    (ii)分离或纯化出所述的T细胞受体。
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