WO2021170116A1 - Récepteur de lymphocytes t reconnaissant l'afp - Google Patents

Récepteur de lymphocytes t reconnaissant l'afp Download PDF

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WO2021170116A1
WO2021170116A1 PCT/CN2021/078276 CN2021078276W WO2021170116A1 WO 2021170116 A1 WO2021170116 A1 WO 2021170116A1 CN 2021078276 W CN2021078276 W CN 2021078276W WO 2021170116 A1 WO2021170116 A1 WO 2021170116A1
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tcr
chain
amino acid
seq
exon
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Chinese (zh)
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李懿
杨东雪
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香雪生命科学技术（广东）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
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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]
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/00041—Use of virus, viral particle or viral elements as a vector
    • C12N2730/00043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to TCRs capable of recognizing short peptides derived from AFP antigens and their coding sequences.
  • the present invention also relates to AFP-specific T cells obtained by transducing the above-mentioned TCRs, and their use in the prevention and treatment of AFP-related diseases.
  • 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 level of expression 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.
  • KWVESIFLIF (SEQ ID NO: 9) is a short peptide derived from AFP antigen and a target for the treatment of AFP-related diseases.
  • T cell adoptive immunotherapy is the transfer of reactive T cells that are specific to target cell antigens into the patient's body to make them work against the target cells.
  • T cell receptor TCR
  • T cell receptor is a membrane protein on the surface of T cells that can recognize short antigenic peptides on the surface of corresponding target cells.
  • pMHC complex antigen-presenting cells
  • those skilled in the art are dedicated to isolating the TCR specific to the AFP antigen short peptide, and transducing the TCR to T cells to obtain T cells specific to the AFP antigen short peptide, so that they can be used in cellular immunotherapy. Play a role.
  • the purpose of the present invention is to provide a T cell receptor that recognizes short peptides of AFP antigen.
  • the first aspect of the present invention provides a T cell receptor (TCR), which can bind to the KWVESIFLIF-HLA A2402 complex.
  • TCR T cell receptor
  • the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
  • the amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is AVRRRNYGQNFV (SEQ ID NO: 12); and/or the The amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is ASSLIAQNHNEQF (SEQ ID NO: 15).
  • the three complementarity determining regions (CDRs) of the TCR ⁇ chain variable domain are:
  • the three complementarity determining regions of the variable domain of the TCR ⁇ chain are:
  • the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
  • the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1; and/ Or the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5.
  • the TCR comprises an alpha chain variable domain amino acid sequence SEQ ID NO:1.
  • the TCR comprises the ⁇ -chain variable domain amino acid sequence SEQ ID NO: 5.
  • the TCR is an ⁇ heterodimer, which comprises a TCR ⁇ chain constant region TRAC*01 and a TCR ⁇ chain constant region TRBC1*01 or TRBC2*01.
  • the alpha chain amino acid sequence of the TCR is SEQ ID NO: 3 and/or the beta chain amino acid sequence of the TCR is SEQ ID NO: 7.
  • the TCR is soluble.
  • the TCR is single-stranded.
  • the TCR is formed by linking the ⁇ chain variable domain and the ⁇ chain variable domain through a peptide linking sequence.
  • the TCR is at the 11th, 13th, 19th, 21st, 53, 76, 89, 91, or 94th amino acid positions of the ⁇ chain variable region, and/or the reciprocal of the ⁇ chain J gene short peptide amino acid There are one or more mutations in position 3, position 5, or position 7; and/or the TCR has amino acids 11, 13, 19, 21, 53, 76, 89, 91 in the ⁇ chain variable region , Or position 94, and/or ⁇ -chain J gene short peptide amino acid in the penultimate position 2, the last 4 position or the last 6 position has one or more mutations, wherein the amino acid position number is according to IMGT (International Immunogenetics Information) The position number listed in the system).
  • IMGT International Immunogenetics Information
  • the amino acid sequence of the ⁇ chain variable domain of the TCR comprises SEQ ID NO: 32 and/or the amino acid sequence of the ⁇ chain variable domain of the TCR comprises SEQ ID NO: 34.
  • amino acid sequence of the TCR is SEQ ID NO: 30.
  • the TCR includes (a) all or part of the TCR ⁇ chain except the transmembrane domain; and (b) all or part of the TCR ⁇ chain except the transmembrane domain;
  • (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a part of the constant domain of the TCR chain.
  • cysteine residues form an artificial disulfide bond between the ⁇ and ⁇ chain constant domains of the TCR.
  • cysteine residues forming artificial disulfide bonds in the TCR are substituted for one or more sets of sites selected from the following:
  • the alpha chain amino acid sequence of the TCR is SEQ ID NO: 26 and/or the beta chain amino acid sequence of the TCR is SEQ ID NO: 28.
  • the alpha chain variable region and the beta chain constant region of the TCR contain artificial interchain disulfide bonds.
  • cysteine residues forming the artificial interchain disulfide bond in the TCR are substituted for one or more sets of sites selected from the following:
  • the TCR comprises 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 comprise the ⁇ chain constant domain.
  • the TCR The ⁇ chain variable domain and ⁇ chain form a heterodimer.
  • 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 T cell receptor is a detectable marker, a therapeutic agent, a PK modified portion, or a combination of any of these substances.
  • the therapeutic agent is an anti-CD3 antibody.
  • 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, the nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule of the first aspect of the present invention or its complementary sequence.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2 or SEQ ID NO: 33 encoding the variable domain of the TCR ⁇ chain.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 6 or SEQ ID NO: 35 encoding the variable domain of the TCR ⁇ chain.
  • the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 4 encoding a TCR ⁇ chain and/or a nucleotide sequence of SEQ ID NO: 8 encoding a TCR ⁇ chain.
  • the fourth aspect of the present invention provides a vector containing the nucleic acid molecule described in the third aspect of the present invention; preferably, the vector is a viral vector; more preferably, the vector is a slow Viral vector.
  • the fifth aspect of the present invention provides an isolated host cell containing the vector according to the fourth aspect of the present invention or the nucleic acid molecule according to the third aspect of the present invention integrated into the genome .
  • the sixth aspect of the present invention provides a cell that transduces the nucleic acid molecule of the third aspect of the present invention or the vector of the fourth aspect of the present invention; preferably, the cell is a T cell or a stem cell .
  • the seventh aspect of the present invention provides a pharmaceutical composition containing a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present invention, the TCR complex described in the second aspect of the present invention, and the The nucleic acid molecule according to the third aspect of the invention, the vector according to the fourth aspect of the invention, or the cell according to the sixth aspect of the invention.
  • the eighth aspect of the present invention provides the T cell receptor according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, and the fourth aspect of the present invention.
  • the use of the vector described in this aspect or the cell described in the sixth aspect of the present invention is used to prepare a drug for treating tumors or autoimmune diseases.
  • the tumor is hepatocellular carcinoma.
  • the ninth aspect of the present invention provides the T cell receptor according to the first aspect of the present invention, or the TCR complex according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, and the fourth aspect of the present invention.
  • the vector described in this aspect or the cell described in the sixth aspect of the present invention is used as a medicine for treating tumors or autoimmune diseases; preferably, the tumor is hepatocellular carcinoma.
  • the tenth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the T cell receptor according to the first aspect of the present invention or the TCR complex according to the second aspect of the present invention to a subject in need of treatment ,
  • the disease is a tumor, preferably the tumor is hepatocellular carcinoma.
  • Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e and Figure 1f are the TCR ⁇ chain variable domain amino acid sequence, TCR ⁇ chain variable domain nucleotide sequence, TCR ⁇ chain amino acid sequence, TCR ⁇ chain nucleotide sequence, The amino acid sequence of the TCR ⁇ chain of the leader sequence and the nucleotide sequence of the TCR ⁇ chain with the leader sequence.
  • Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the TCR ⁇ chain variable domain amino acid sequence, TCR ⁇ chain variable domain nucleotide sequence, TCR ⁇ chain amino acid sequence, TCR ⁇ chain nucleotide sequence, The amino acid sequence of the TCR ⁇ chain of the leader sequence and the nucleotide sequence of the TCR ⁇ chain with the leader sequence.
  • Figure 3 shows the CD8 + and tetramer-PE double positive staining results of monoclonal cells.
  • Figures 4a and 4b show the amino acid sequence and nucleotide sequence of the soluble TCR ⁇ chain, respectively.
  • Figures 5a and 5b show the amino acid sequence and nucleotide sequence of the soluble TCR ⁇ chain, respectively.
  • Figures 6a and 6b are gel images of soluble TCR obtained after purification. Among them, the right lanes of Fig. 6a and Fig. 6b are non-reducing glue and reducing glue, respectively, and the left lanes are all molecular weight markers.
  • Figures 7a and 7b are the amino acid sequence and nucleotide sequence of the single-stranded TCR, respectively.
  • Figures 8a and 8b are the amino acid sequence and nucleotide sequence of the variable domain of the single-chain TCR ⁇ chain, respectively.
  • Figures 9a and 9b are the amino acid sequence and nucleotide sequence of the variable domain of the single-stranded TCR ⁇ chain, respectively.
  • Figures 10a and 10b are the amino acid sequence and nucleotide sequence of the single-stranded TCR linker, respectively.
  • Figure 11 is a gel image of a soluble single-chain TCR obtained after purification.
  • the leftmost lane is reducing glue
  • the middle lane is molecular weight markers
  • the rightmost lane is non-reducing glue.
  • Figure 12 is a BIAcore kinetic map of the combination of soluble TCR and KWVESIFLIF-HLA A2402 complex of the present invention.
  • Figure 13 is a BIAcore kinetic map of the combination of the soluble single-chain TCR of the present invention and the KWVESIFLIF-HLA A2402 complex.
  • Figure 14 shows the result of the ELISPOT activation function verification of the obtained T cell clone.
  • Figure 15 shows the results of the ELISPOT activation function verification of the effector cells transduced with the TCR of the present invention.
  • Figure 16 shows the results of the LDH killing function verification of the effector cells transduced with the TCR of the present invention.
  • the present invention also provides a nucleic acid molecule encoding the TCR and a vector containing the nucleic acid molecule. In addition, the present invention also provides cells transducing the TCR of the present invention.
  • MHC molecules are proteins of the immunoglobulin superfamily, and can be class I or class II MHC molecules. Therefore, it is specific for the presentation of antigens. Different individuals have different MHCs and can present different short peptides in a protein antigen to the surface of their respective APC cells. Human MHC is usually called HLA gene or HLA complex.
  • T cell receptor is the only receptor for specific antigen peptides presented on the main histocompatibility complex (MHC).
  • MHC main histocompatibility complex
  • APC antigen presenting cells
  • T cells with different antigen specificities exerted immune effects on their target cells.
  • TCR is a glycoprotein on the surface of the cell membrane that exists as a heterodimer of ⁇ chain/ ⁇ chain or ⁇ chain/ ⁇ chain.
  • TCR heterodimers are composed of ⁇ and ⁇ chains, while 5% of T cells have TCRs composed of ⁇ and ⁇ chains.
  • the natural ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain, and the ⁇ chain and the ⁇ chain constitute subunits of the ⁇ heterodimeric TCR.
  • each chain of ⁇ and ⁇ includes a variable region, a connecting region and a constant region.
  • the ⁇ chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often regarded as the connecting region.
  • Each variable region contains 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are embedded in framework regions.
  • the CDR region determines the combination of TCR and pMHC complex, where CDR3 is recombined from the variable region and the connecting region, which is called the hypervariable region.
  • the ⁇ and ⁇ chains of TCR are generally regarded as having two "domains" each, that is, a variable domain and a constant domain.
  • the variable domain is composed of a connected variable region and a connecting region.
  • the sequence of the constant domain of TCR can be found in the public database of the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the sequence of the constant domain of the ⁇ chain of the TCR molecule is "TRAC*01”
  • the sequence of the constant domain of the ⁇ chain of the TCR molecule is "TRBC1*" 01" or "TRBC2*01”.
  • the ⁇ and ⁇ chains of TCR also contain transmembrane and cytoplasmic regions, which are very short.
  • polypeptide of the present invention TCR of the present invention
  • T cell receptor of the present invention T cell receptor of the present invention
  • the position numbers of the amino acid sequence of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered in sequence from the N-terminus to the C-terminus, such as TRBC1*01 or TRBC2*01
  • the 60th amino acid is P (proline)
  • Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention or It is expressed as the 60th amino acid of TRBC1*01 or TRBC2*01 exon 1.
  • the 61st amino acid is Q(glutamine) in sequence from N-terminal to C-terminal.
  • Amide 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 other And so on.
  • the position numbers of the amino acid sequences of TRAV and TRBV in the variable regions follow the position numbers listed in IMGT.
  • the position number listed in IMGT is 46, it is described as the 46th amino acid of TRAV in the present invention, and the rest can be deduced by analogy.
  • sequence position numbers of other amino acids have special instructions, follow the special instructions.
  • the antigen In the process of antigen processing, the antigen is degraded inside the cell and then carried to the cell surface by MHC molecules. T cell receptors can recognize peptide-MHC complexes on the surface of antigen-presenting cells. Therefore, the first aspect of the present invention provides a TCR molecule capable of binding to the KWVESIFLIF-HLA A2402 complex. Preferably, the TCR molecule is isolated or purified. The ⁇ and ⁇ chains of the TCR each have three complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • the ⁇ chain of the TCR includes a CDR having the following amino acid sequence:
  • the three complementarity determining regions of the variable domain of the TCR ⁇ chain are:
  • the TCR molecule of the present invention refers to a TCR molecule comprising the aforementioned ⁇ and/or ⁇ chain CDR region sequence and any suitable framework structure.
  • the TCR ⁇ chain variable domain of the present invention is an amino acid sequence that has at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO:1; and/or the TCR ⁇ chain variable domain of the present invention is the same as SEQ ID NO:5 has an amino acid sequence with at least 90%, preferably 95%, more preferably 98% sequence identity.
  • the TCR molecule of the present invention is a heterodimer composed of ⁇ and ⁇ chains.
  • the ⁇ chain of the heterodimeric TCR molecule includes a variable domain and a constant domain, and the amino acid sequence of the ⁇ chain variable domain includes the CDR1 (SEQ ID NO: 10) and CDR2 (SEQ ID NO: 10) of the above ⁇ chain. ID NO: 11) and CDR3 (SEQ ID NO: 12).
  • the TCR molecule comprises an alpha chain variable domain amino acid sequence SEQ ID NO:1. More preferably, the alpha chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO:1.
  • the ⁇ chain of the heterodimeric TCR molecule includes a variable domain and a constant domain
  • the amino acid sequence of the ⁇ chain variable domain includes CDR1 (SEQ ID NO: 13) and CDR2 (SEQ ID NO: 13) of the above ⁇ chain. NO: 14) and CDR3 (SEQ ID NO: 15).
  • the TCR molecule comprises the ⁇ -chain variable domain amino acid sequence SEQ ID NO: 5. More preferably, the amino acid sequence of the ⁇ chain variable domain of the TCR molecule is SEQ ID NO: 5.
  • the TCR molecule of the present invention is a single-stranded TCR molecule composed of part or all of the alpha chain and/or part or all of the beta chain.
  • single-chain TCR molecules please refer to Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. According to the literature, those skilled in the art can easily construct single-chain TCR molecules containing the CDRs of the present invention.
  • the single-stranded TCR molecule includes V ⁇ , V ⁇ , and C ⁇ , and is preferably connected in order from N-terminal to C-terminal.
  • the alpha chain variable domain amino acid sequence of the single-chain TCR molecule includes CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the aforementioned ⁇ chain.
  • the single-chain TCR molecule comprises an ⁇ -chain variable domain amino acid sequence SEQ ID NO:1. More preferably, the alpha chain variable domain amino acid sequence of the single-chain TCR molecule is SEQ ID NO:1.
  • the ⁇ -chain variable domain amino acid sequence of the single-chain TCR molecule includes CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the ⁇ -chain.
  • the single-chain TCR molecule comprises the ⁇ -chain variable domain amino acid sequence SEQ ID NO: 5. More preferably, the ⁇ -chain variable domain amino acid sequence of the single-chain TCR molecule is SEQ ID NO: 5.
  • the constant domain of the TCR molecule of the present invention is a human constant domain.
  • the constant domain sequence of the alpha chain of the TCR molecule of the present invention can be "TRAC*01”
  • the constant domain sequence of the beta chain of the TCR molecule can be "TRBC1*01” or "TRBC2*01”.
  • the 53rd position of the amino acid sequence given in IMGT's TRAC*01 is Arg, which is represented here as: Arg53 of TRAC*01 exon 1, and so on.
  • the amino acid sequence of the alpha chain of the TCR molecule of the present invention is SEQ ID NO: 3, and/or the amino acid sequence of the beta chain is SEQ ID NO: 7.
  • TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane domain.
  • TCRs can also be developed for diagnosis and treatment, and it is necessary to obtain soluble TCR molecules.
  • Soluble TCR molecules do not include their transmembrane regions. Soluble TCR has a wide range of uses. It can be used not only to study the interaction between TCR and pMHC, but also as a diagnostic tool for detecting infections or as a marker for autoimmune diseases.
  • soluble TCR can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells presenting specific antigens.
  • soluble TCR can also be combined with other molecules (such as anti-CD3 antibodies). To redirect T cells to target cells that present specific antigens.
  • the present invention also obtains the soluble TCR specific to the AFP antigen short peptide.
  • the TCR of the present invention may be a TCR with artificial disulfide bonds introduced between the residues of the constant domains 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. For example, replacing Thr48 of TRAC*01 exon 1 and replacing the cysteine residue of Ser57 of TRBC1*01 or TRBC2*01 exon 1 to form a disulfide bond.
  • Other sites for introducing cysteine residues to form disulfide bonds 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 Glu15 of TRBC1*01 or TRBC2*01 exon 1; Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1; Pro89 and Pro89 of TRAC*01 exon 1 Ala19 of TRBC1*01 or TRBC2*01 exon 1; or Tyr10 of TRAC*01 exon 1 and Glu20 of TRBC1*01 or TRBC2*01 exon 1.
  • cysteine residues replace any set of positions in the constant domains of the ⁇ and ⁇ chains.
  • a maximum of 50, or a maximum of 30, or a maximum of 15, or a maximum of 10, or a maximum of 8 or less amino acids can be truncated at one or more C-termini of the TCR constant domain of the present invention, so that it does not include Cysteine residues can be used to delete natural disulfide bonds, and the cysteine residues that form natural disulfide bonds can also be mutated to another amino acid to achieve the above purpose.
  • the TCR of the present invention may contain artificial 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 disulfide bonds present in the TCR.
  • the TCR of the present invention also includes a TCR that has mutations in its hydrophobic core region.
  • These mutations in the hydrophobic core region are preferably mutations that can improve the stability of the soluble TCR of the present invention, as described in Publication No. It is described in the patent document of WO2014/206304.
  • Such a TCR can be mutated at the following variable domain hydrophobic core positions: ( ⁇ and/or ⁇ chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/ Or alpha chain J gene (TRAJ) short peptide amino acid position is the 3rd, 5th and 7th position from the bottom, and/or ⁇ chain J gene (TRBJ) short peptide amino acid position is the 2nd, 4th and 6th position from the bottom, the position number of the amino acid sequence According to the position number listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR in which the hydrophobic core region is mutated may be a stable soluble single-chain TCR composed of a flexible peptide chain connecting the variable domains of the ⁇ and ⁇ chains of the TCR.
  • 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 ⁇ -chain variable domain amino acid sequence is SEQ ID NO: 32
  • the encoded nucleotide sequence is SEQ ID NO: 33
  • ⁇ -chain variable domain amino acid sequence It is SEQ ID NO: 34
  • the encoded nucleotide sequence is SEQ ID NO: 35.
  • Patent Document 201680003540.2 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 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 can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention.
  • 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 marker (for diagnostic purposes, the TCR is used to detect the presence of cells presenting the KWVESIFLIF-HLA A2402 complex), a therapeutic agent, a PK (protein kinase) modified part or any of the above substances The combination of binding or coupling.
  • 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 computed tomography technology) 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 TCR of the present invention may also be a hybrid TCR containing sequences derived from more than one species.
  • the TCR of the present invention may include a human variable domain and a murine constant domain. The disadvantage of this method is that it may trigger an immune response. Therefore, when it is used in adoptive T cell therapy, there should be a regulatory scheme for immunosuppression to allow the implantation of T cells expressing murine.
  • the second aspect of the present invention provides a nucleic acid molecule encoding the TCR molecule of the first aspect of the present invention or a part thereof, which may be one or more CDRs, variable domains of ⁇ and/or ⁇ chains, and ⁇ chains and/ Or beta chain.
  • nucleotide sequence encoding the ⁇ chain CDR region of the TCR molecule of the first aspect of the present invention is as follows:
  • nucleotide sequence encoding the ⁇ chain CDR region of the TCR molecule of the first aspect of the present invention is as follows:
  • the nucleotide sequence of the nucleic acid molecule of the present invention encoding the TCR ⁇ chain of the present invention includes SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the TCR ⁇ chain of the present invention.
  • the nucleotide sequence includes SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
  • the nucleotide sequence of the nucleic acid molecule of the present invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns.
  • the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but can encode the polypeptide of the present invention.
  • the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR ⁇ chain of the present invention includes SEQ ID NO: 2 and / Or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR ⁇ chain of the present invention includes SEQ ID NO: 6.
  • the nucleotide sequence of the nucleic acid molecule of the present invention encoding the TCR ⁇ chain variable domain of the present invention includes SEQ ID NO: 33 and/or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the TCR ⁇ chain variable domain of the present invention includes SEQ ID NO: 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the present invention comprises SEQ ID NO: 4 and/or SEQ ID NO: 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention is SEQ ID NO: 31.
  • 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. Taking one of the examples in the present invention to illustrate, "degenerate variant” refers to a nucleic acid sequence that encodes a protein sequence of SEQ ID NO: 1, but differs from the sequence of SEQ ID NO: 2.
  • the nucleotide sequence can be codon optimized. Different cells use specific codons differently. According to the cell type, the codons in the sequence can be changed to increase the amount of expression. Codon usage tables for mammalian cells and many other organisms are well known to those skilled in the art.
  • 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. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. DNA can be a coding strand or a non-coding strand.
  • the present invention also relates to vectors containing the nucleic acid molecules of the present invention, including expression vectors, that is, constructs that can be expressed in vivo or in vitro.
  • expression vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.
  • Virus delivery systems include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors.
  • AAV adeno-associated virus
  • the vector can transfer the nucleotide of the present invention into a cell, such as a T cell, so that the cell expresses a TCR specific for the AFP antigen.
  • a cell such as a T cell
  • the vector should be able to continuously express at a high level in T cells.
  • the present invention also relates to a host cell produced by genetic engineering using the vector or coding sequence of the present invention.
  • the host cell contains the vector of the present invention or the nucleic acid molecule of the present invention is integrated into the chromosome.
  • the host cell is selected from: prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast cells, CHO cells and the like.
  • the present invention also includes isolated cells expressing the TCR of the present invention, especially T cells.
  • the T cells may be derived from T cells isolated from the subject, or may be a mixed cell population isolated from the subject, such as part of a peripheral blood lymphocyte (PBL) population.
  • PBL peripheral blood lymphocyte
  • the cells can be isolated from peripheral blood mononuclear cells (PBMC), and can be CD4 + helper T cells or CD8 + cytotoxic T cells.
  • PBMC peripheral blood mononuclear cells
  • the cells can be in a mixed population of CD4 + helper T cells/CD8 + cytotoxic T cells.
  • the cells can be activated with antibodies (eg, anti-CD3 or anti-CD28 antibodies) so that they can be more easily transfected, for example, with a vector containing a nucleotide sequence encoding the TCR molecule of the present invention. dye.
  • antibodies eg, anti-CD3 or anti-CD28 antibodies
  • the cells of the present invention can also be or derived from stem cells, such as hematopoietic stem cells (HSC).
  • stem cells such as hematopoietic stem cells (HSC).
  • HSC hematopoietic stem cells
  • T cell transfection with DNA or RNA encoding the TCR of the present invention There are many methods suitable for T cell transfection with DNA or RNA encoding the TCR of the present invention (eg, Robbins et al., (2008) J. Immunol. 180: 6116-6131). T cells expressing the 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 relates to a method for treating and/or preventing AFP-related diseases in a subject, which includes the step of adoptively transferring AFP-specific T cells to the subject.
  • the AFP-specific T cells can recognize the KWVESIFLIF-HLA A2402 complex.
  • the AFP-specific T cells of the present invention can be used to treat any AFP-related diseases that present the AFP antigen short peptide KWVESIFLIF-HLA A2402 complex, including but not limited to tumors, such as hepatocellular carcinoma.
  • Treatment can be carried out by isolating T cells from patients or volunteers suffering from AFP antigen-related diseases, introducing the TCR of the present invention into the above-mentioned T cells, and then infusing these genetically engineered cells back into the patient's body for treatment. Therefore, the present invention provides a method for the treatment of AFP-related diseases, including the isolated T cell expressing the TCR of the present invention, preferably, the T cell is derived from the patient itself and transferred into the patient's body.
  • the present invention includes (1) isolation of patient's T cells, (2) in vitro transduction of T cells with nucleic acid molecules of the present invention or nucleic acid molecules capable of encoding TCR molecules of the present invention, and (3) infusion of genetically engineered T cells into patients in vivo.
  • the number of isolated, transfected, and reinfused cells can be determined by the physician.
  • the TCR of the present invention can specifically bind to the AFP antigen short peptide complex KWVESIFLIF-HLA A2402, and the effector cells transduced with the TCR of the present invention can be specifically activated;
  • the effector cells transduced with the TCR of the present invention have a strong killing function on positive tumor cells.
  • the synthetic short peptide KWVESIFLIF (SEQ ID NO: 9; Beijing Saibaisheng Gene Technology Co., Ltd.) was used to stimulate peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A2402.
  • the KWVESIFLIF short peptide was renatured with biotin-labeled HLA-A2402 to prepare pHLA haploids. These haploids were combined with PE-labeled streptavidin (BD Company) to form a PE-labeled tetramer, and the tetramer and anti-CD8-APC double positive cells were sorted.
  • T2 T2 cells transfected with HLA-A2402 loaded with AFP antigen short peptide, SNU398-AFP (AFP) Overexpression
  • HepG2 cell lines are positive target cells
  • T2 T2 cells transfected with HLA-A2402
  • SNU398 loaded with other short peptides are used as negative target cells.
  • the ELISPOT plate First prepare the ELISPOT plate.
  • the test components to the ELISPOT plate in the following order: 20,000 T2 cells/well, 2000 effector cells/well, and then add 20 ⁇ l of the TCR of the present invention to the experimental group
  • 20 ⁇ l of other short peptides were added to the control group
  • 20 ⁇ l of medium (test medium) was added to the blank group
  • 2 replicate wells were set.
  • the test components to the ELISPOT plate in the following order: 20,000 target cells/well, 2,000 effector cells/well, and set 2 multiple wells. Then incubate overnight (37°C, 5% CO 2 ). Subsequently, the plate was washed and subjected to secondary detection and color development. The plate was dried for 1 hour, and then the spots formed on the membrane were counted with an immunospot plate reader (ELISPOT READER system; AID company).
  • ELISPOT READER system an immunospot plate reader
  • the results of the experiment are shown in Figure 14.
  • the T cell clones obtained were loaded with AFP short peptides, T2, AFP overexpressed SNU398-AFP, and AFP overexpressed HEPG2 had a significant activation response, while for T2 loaded with other short peptides.
  • the cells and SNU398 cells were not activated.
  • the cDNA synthesis uses clontech’s SMART RACE cDNA amplification kit, and the primers used are designed in the C-terminal conserved region of the human TCR gene.
  • the sequence was cloned into the T vector (TAKARA) for sequencing. It should be noted that this sequence is a complementary sequence and does not contain introns. After sequencing, the structure of the ⁇ chain and ⁇ chain sequence of the TCR expressed by the double-positive clone is shown in Figure 1 and Figure 2, respectively.
  • Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e and Figure 1f are the TCR ⁇ chain Variable domain amino acid sequence, TCR ⁇ chain variable domain nucleotide sequence, TCR ⁇ chain amino acid sequence, TCR ⁇ chain nucleotide sequence, TCR ⁇ chain amino acid sequence with leader sequence, and TCR ⁇ chain nucleotide sequence with leader sequence;
  • Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the TCR ⁇ chain variable domain amino acid sequence, the TCR ⁇ chain variable domain nucleotide sequence, the TCR ⁇ chain amino acid sequence, the TCR ⁇ chain nucleotide sequence, and the leader sequence. TCR ⁇ chain amino acid sequence and TCR ⁇ chain nucleotide sequence with leader sequence.
  • the ⁇ chain contains CDRs with the following amino acid sequence:
  • the full-length genes of TCR alpha chain and beta chain were cloned into the lentiviral expression vector pLenti (addgene) by overlap PCR. Specifically: using overlap PCR to connect the full-length gene of the TCR ⁇ chain and the TCR ⁇ chain to obtain the TCR ⁇ -2A-TCR ⁇ fragment.
  • the lentiviral expression vector and TCR ⁇ -2A-TCR ⁇ were digested and ligated to obtain the pLenti-TRA-2A-TRB-IRES-NGFR plasmid.
  • a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. Then use 293T/17 to package the fake virus.
  • the ⁇ and ⁇ chains of the TCR molecule of the present invention may only contain the variable domain and part of the constant domain, respectively, and a cysteine residue is introduced into the constant domains of the ⁇ and ⁇ chains.
  • the positions of introducing cysteine residues are Thr48 of TRAC*01 exon 1 and Ser57 of TRBC2*01 exon 1; the amino acid sequence and nucleotides of its ⁇ chain The sequence is shown in Figure 4a and Figure 4b, respectively, and the amino acid sequence and nucleotide sequence of the ⁇ chain are shown in Figure 5a and Figure 5b, respectively.
  • the target gene sequences of the above-mentioned TCR ⁇ and ⁇ chains were synthesized and inserted into the expression vector pET28a+ (Novagene ), the upstream and downstream cloning sites are NcoI and NotI respectively. The inserted fragment was confirmed by sequencing.
  • TCR ⁇ and ⁇ chains were transformed into expressing bacteria BL21(DE3) by chemical transformation method respectively.
  • 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 in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1, and the final concentration is 60mg/mL.
  • 5M urea 20mM Tris (pH 8.1)
  • 20mM Tris 20mM Tris (pH 8.1)
  • cystamine 3.7mM cystamine
  • 6.6mM ⁇ -mercapoethylamine 4°C
  • the solution is filtered through a 0.45 ⁇ M filter membrane and purified by an anion exchange column (HiTrap Q HP, 5ml, GE Healthcare).
  • the elution peak contains the successfully renatured ⁇ and ⁇ dimer TCRs, which are confirmed by SDS-PAGE gel.
  • the TCR is 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.
  • the SDS-PAGE gel image of the soluble TCR obtained by the present invention is shown in FIG. 6.
  • variable domains of the TCR alpha and beta chains in Example 2 were constructed into a stable soluble single-chain TCR molecule connected by a flexible short peptide (linker) by using the method of site-directed mutagenesis.
  • the amino acid sequence and nucleotide sequence of the single-stranded TCR molecule are shown in Figure 7a and Figure 7b, respectively.
  • the amino acid sequence and nucleotide sequence of the ⁇ chain variable domain are shown in Figure 8a and Figure 8b, respectively; the amino acid sequence and nucleotide sequence of the ⁇ chain variable domain are shown in Figure 9a and Figure 9b, respectively; its linker The amino acid sequence and nucleotide sequence of the sequence are shown in Figure 10a and Figure 10b, respectively.
  • the target gene 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, incubated overnight at 37°C, and selected positive clones for PCR screening, and sequenced the positive recombinants to confirm the correct sequence and extract the recombinant plasmid for transformation To E.coli BL21(DE3), for expression.
  • the inclusion bodies were dissolved in a buffer (20mM Tris-HCl pH 8.0, 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 later use.
  • a syringe to drop the single-stranded TCR after the above treatment 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 slowly stir overnight at 4°C.
  • 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 SDS-PAGE gel image of the soluble single-chain TCR obtained in the present invention is shown in FIG. 11.
  • the BIAcore T200 real-time analysis system was used to detect the binding activity of the TCR molecules obtained in Example 3 and Example 5 with the KWVESIFLIF-HLA A2402 complex.
  • the unreacted activated surface was sealed with ethanolamine hydrochloric acid solution to complete the coupling process, and the coupling level was about 15,000 RU.
  • the synthetic short peptide KWVESIFLIF (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 are dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and 3M guanidine hydrochloride, 10mM sodium acetate, and 10mM EDTA are added before renaturation to further denature.
  • the protein-containing fractions were combined, concentrated with a Millipore ultrafiltration tube, the protein concentration was determined by the BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules in aliquots at -80°C.
  • the soluble TCR molecules of the present invention and the kinetic profiles of the combination of the soluble single-chain TCR molecules constructed by the present invention and the KWVESIFLIF-HLA A2402 complex are shown in Figure 12 and Figure 13, respectively.
  • the map shows that both the soluble TCR molecules and the soluble single-chain TCR molecules obtained in the present invention can bind to the KWVESIFLIF-HLA A2402 complex.
  • the above method was used to detect the binding activity of the soluble TCR molecule of the present invention with several other unrelated antigen short peptides and HLA complexes, and the results showed that the TCR molecule of the present invention did not bind to other unrelated antigens.
  • ELISPOT assay to detect cell function. Construct a lentiviral vector containing the target gene of the TCR of the present invention, transduce the T cell, and perform an ELISPOT test to prove the specific activation response of the T cell transduced by the TCR of the present invention to the target cell.
  • the IFN- ⁇ production detected by the ELISPOT test was used as the readout value of T cell activation.
  • the target cells used in this experiment were T2 cells (T2 cells transfected with HLA-A2402) loaded with the AFP antigen short peptide KWVESIFLIF, and the effector cells used were CD3 + T cells of the TCR of the present invention, and were transfected by the same volunteer CD3 + T cells stained with other TCR (A6) were used as a control group.
  • the cells are then resuspended in the test medium at 2 ⁇ the desired final concentration.
  • the negative control effector cells were treated in the same way.
  • the corresponding short peptides were added to the corresponding target cell (T2) experimental group, so that the final concentration of the short peptides in the ELISPOT well plate was 1 ⁇ 10 -11 M to 1 ⁇ 10 -6 M, a total of 6 gradients.
  • test components to the ELISPOT well plate: target cells 2 ⁇ 10 4 /well, effector cells 2 ⁇ 10 3 /well (calculated according to the antibody positive rate), and set up two duplicate wells. The plate was then incubated overnight (37°C/5% CO 2 ).
  • the detection antibody was diluted 1:200 with PBS containing 10% FBS, and 100 ⁇ l/well was added to each well. Incubate the well plate for 2 hours at room temperature, and then wash it 3 times with washing buffer. Tap the well on a paper towel to remove excess washing buffer. Dilute streptavidin-alkaline phosphatase 1:100 with PBS containing 10% FBS, add 100 microliters of diluted streptavidin-alkaline phosphatase to each well and incubate the plate at room temperature 1 hour.
  • the ELISPOT experiment (as described above) was used to test the IFN- ⁇ release of the T cells transduced with the TCR of the present invention in response to the target cells loaded with the AFP antigen short peptide KWVESIFLIF.
  • Use graphpad prism6 to plot the number of ELSPOT spots observed in each well.
  • the results of the experiment are shown in Figure 15.
  • the T cells transduced with the TCR of the present invention have an obvious activation response when the concentration of the antigen short peptide is low, while the T cells transduced with other TCRs Has not been activated since the beginning.
  • Example 8 LDH killing function experiment of effector cells transduced with TCR of the present invention
  • a non-radioactive cytotoxicity experiment was used to measure the release of LDH, so as to verify the killing function of the cells transduced with the TCR of the present invention.
  • This test is a colorimetric alternative to the 51Cr 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 culture 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.
  • a standard 96-well plate reader can be used to collect 490nm visible light absorbance data.
  • CD3+ T cells transduced with TCR of the present invention were used as effector cells, and CD3+ T cells transduced with other TCR (A6) from the same volunteer were used as control group.
  • CD3+ T cells transduced with other TCR (A6) from the same volunteer were used as control group.
  • HepG2 was used as the positive tumor cell line
  • NCI-H226 and SK-HEP-1 were used as the negative tumor cell line.
  • the LDH plate First prepare the LDH plate. On the first day of the experiment, add the test components to the plate in the following order: target cell line 3 ⁇ 104 cells/well, effector cell 3 ⁇ 104 cells/well (calculated according to the positive rate of transfection), and set three Multiple holes. At the same time, set up the spontaneous hole for effector cells, the spontaneous hole for target cells, the largest hole for target cells, the volume correction control hole and the medium background control hole. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the color development was detected. After the reaction was terminated, the absorbance value was recorded at 490nm with a microplate reader (Bioteck).
  • the effector cells that transduce the TCR of the present invention have a strong killing function on the positive target cells, while the cells that transduce other TCRs have no killing effect; at the same time, the TCR of the present invention is transduced
  • the effector cells did not kill the negative tumor cells.

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Abstract

La présente invention concerne un récepteur de lymphocytes T (TCR) pouvant se lier spécifiquement à un peptide court KWVESIFLIF dérivé d'un antigène AFP, le peptide court antigénique KWVESIFLIF pouvant former un complexe avec HLA A2402 et être présenté ensemble à une surface cellulaire. La présente invention concerne en outre une molécule d'acide nucléique codant pour le TCR et un vecteur comprenant la molécule d'acide nucléique. De plus, la présente invention concerne également une cellule pour la transduction du TCR.
PCT/CN2021/078276 2020-02-28 2021-02-26 Récepteur de lymphocytes t reconnaissant l'afp WO2021170116A1 (fr)

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CN104087592A (zh) * 2014-05-13 2014-10-08 天津医科大学总医院 Afp158-166特异性tcr基因及其转基因t细胞及体外增殖方法及用途
WO2017089787A1 (fr) * 2015-11-23 2017-06-01 Immunocore Limited Peptides dérivés de l'alpha-fœtoprotéine (afp), complexes comprenant ces peptides liés à des molécules du cmh
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CN114057864B (zh) * 2020-07-30 2023-12-15 香雪生命科学技术（广东）有限公司 一种识别afp的高亲和力tcr

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