WO2022262835A1 - Tcr pour identifier un antigène afp et séquence codant pour celui-ci - Google Patents

Tcr pour identifier un antigène afp et séquence codant pour celui-ci Download PDF

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WO2022262835A1
WO2022262835A1 PCT/CN2022/099352 CN2022099352W WO2022262835A1 WO 2022262835 A1 WO2022262835 A1 WO 2022262835A1 CN 2022099352 W CN2022099352 W CN 2022099352W WO 2022262835 A1 WO2022262835 A1 WO 2022262835A1
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tcr
chain
seq
amino acid
exon
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Chinese (zh)
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杨东雪
钟时
廖有平
郑文静
温桥生
马瑞娟
孙含丽
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香雪生命科学技术（广东）有限公司
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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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]
    • 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
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    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
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    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
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    • A61K39/464481—Alpha-feto protein
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Definitions

  • This application belongs to the field of biotechnology, and specifically relates to the TCR and its coding sequence that can recognize short peptides derived from the AFP antigen.
  • the application also relates to the AFP-specific T cells obtained by transducing the above-mentioned TCR, and their role in the prevention and treatment of AFP. use in disease.
  • AFP ( ⁇ Fetoprotein), also known as ⁇ -fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP has relatively high expression levels in the yolk sac and liver, and is subsequently repressed. 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 cells, it is degraded into small molecular polypeptides, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface.
  • TSSELMAITR (SEQ ID NO: 9) is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.
  • T cell adoptive immunotherapy is the transfer of reactive T cells specific to target cell antigens into the patient's body so that they can act against the target cells.
  • T cell receptor TCR
  • T cell receptor is a membrane protein on the surface of T cells, which can recognize short antigenic peptides on the surface of corresponding target cells.
  • APCs antigen-presenting cells
  • pMHC complex short peptide-major histocompatibility complex
  • the present application provides a T cell receptor that recognizes short peptides of AFP antigens.
  • a T cell receptor (TCR) is provided, and the TCR can bind to the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex.
  • 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 SGGSNYKLT (SEQ ID NO: 12); and/or the The amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is ASSPGTGVGYT (SEQ ID NO: 15).
  • the three complementarity determining regions (CDRs) of the variable domain of the TCR ⁇ chain are:
  • the three complementarity-determining regions of the TCR ⁇ chain variable domain 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 to SEQ ID NO: 1
  • the TCR beta chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5.
  • the TCR comprises the amino acid sequence of an alpha chain variable domain of SEQ ID NO: 1.
  • the TCR comprises the amino acid sequence of the ⁇ -chain variable domain of 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.
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 3 and/or the amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 7.
  • the TCR described in this application is of human origin.
  • the TCR is soluble.
  • the TCR is a single chain.
  • the TCR is formed by linking the ⁇ -chain variable domain and the ⁇ -chain variable domain through a peptide linker sequence.
  • the constant regions of the ⁇ and ⁇ chains of the TCR are the constant regions of the murine ⁇ and ⁇ chains, respectively.
  • the TCR is at the 11th, 13th, 19th, 21st, 53rd, 76, 89, 91st, or 94th amino acid position in the ⁇ -chain variable region, and/or at the reciprocal amino acid position of the J gene short peptide of the ⁇ -chain
  • IMGT International Immunogenetics Information system
  • 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 comprises (i) TCR ⁇ chain variable domain and all or part of the TCR ⁇ chain constant region except the transmembrane domain; and (ii) TCR ⁇ chain variable domain and except the transmembrane domain All or part of the TCR ⁇ chain constant region.
  • cysteine residue forms 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 groups of sites selected from the following:
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 26 and/or the amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 28.
  • the TCR contains an artificial interchain disulfide bond between the ⁇ -chain variable region and the ⁇ -chain constant region.
  • cysteine residues forming artificial interchain disulfide bonds in the TCR are substituted for one or more groups 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 contain an ⁇ -chain constant domain, and the TCR The ⁇ -chain variable domain forms a heterodimer with the ⁇ -chain.
  • 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 modification moiety or any combination of these substances;
  • the therapeutic agent is an anti-CD3 antibody.
  • the second aspect of the present application provides a multivalent TCR complex, which comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present application.
  • the third aspect of the present application provides a nucleic acid molecule comprising the nucleic acid sequence encoding the TCR molecule described in the first aspect of the present application 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 encoding a TCR ⁇ chain of SEQ ID NO: 4 and/or comprises a nucleotide sequence encoding a TCR ⁇ chain of SEQ ID NO: 8.
  • the fourth aspect of the present application provides a vector containing the nucleic acid molecule described in the third aspect of the present application; preferably, the vector is a viral vector; more preferably, the vector is a lentivirus Viral vector.
  • the fifth aspect of the present application provides an isolated host cell containing the vector described in the fourth aspect of the present application or the exogenous nucleic acid molecule described in the third aspect of the application integrated in the genome .
  • the sixth aspect of the present application provides a cell transduced with the nucleic acid molecule described in the third aspect of the present application or the vector described in the fourth aspect of the present application; preferably, the cell is a T cell, NK cells, NKT cells or stem cells.
  • the seventh aspect of the present application provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present application, the TCR complex described in the second aspect of the present application, the TCR complex described in the second aspect of the present application, The nucleic acid molecule described in the third aspect of the application, the vector described in the fourth aspect of the application, or the cell described in the sixth aspect of the application.
  • the eighth aspect of the present application provides the use of the T cell receptor described in the first aspect of the present application, or the TCR complex described in the second aspect of the present application, or the cells described in the sixth aspect of the present application, for Medicines for treating tumors or autoimmune diseases are prepared, preferably, the tumors are liver cancers.
  • the ninth aspect of the present application provides the T cell receptor described in the first aspect of the present application, or the TCR complex described in the second aspect of the present application, or the cells described in the sixth aspect of the present application for treating tumors or Drugs for autoimmune diseases; preferably, the tumor is liver cancer.
  • the tenth aspect of the present application provides a method for treating diseases, comprising administering an appropriate amount of the T cell receptor described in the first aspect of the present application or the TCR complex described in the second aspect of the present application to a subject in need of treatment , or the cell described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application; preferably, the disease is a tumor, preferably, the tumor is liver cancer.
  • Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e and Figure 1f are the amino acid sequence of the TCR ⁇ chain variable domain, the nucleotide sequence of the TCR ⁇ chain variable domain, the amino acid sequence of the TCR ⁇ chain, the nucleotide sequence of the TCR ⁇ chain, and The amino acid sequence of the TCR ⁇ chain of the leader sequence and the nucleotide sequence of the TCR ⁇ chain having the leader sequence.
  • Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of the TCR ⁇ chain variable domain, the TCR ⁇ chain variable domain nucleotide sequence, the TCR ⁇ chain amino acid sequence, the TCR ⁇ chain nucleotide sequence, and 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 double positive staining results of CD8 + -APC and tetramer-PE of monoclonal cells.
  • Figure 4a and Figure 4b are the amino acid sequence and nucleotide sequence of the soluble TCR ⁇ chain, respectively.
  • Figure 5a and Figure 5b are the amino acid sequence and nucleotide sequence of the soluble TCR ⁇ chain, respectively.
  • Figures 6a and 6b are gel images of purified soluble TCRs. Wherein, the right swimming lanes of Figures 6a and 6b are reducing gels and non-reducing gels respectively, and the left swimming lanes are molecular weight markers.
  • Figure 7a and Figure 7b are the amino acid sequence and nucleotide sequence of the single-chain TCR, respectively, and the amino acid sequence and nucleotide sequence of the linker are underlined.
  • Figure 8a and Figure 8b are the amino acid sequence and nucleotide sequence of the variable domain of the single-chain TCR ⁇ chain, respectively.
  • Figure 9a and Figure 9b are the amino acid sequence and nucleotide sequence of the single-chain TCR ⁇ chain variable domain, respectively.
  • Figures 10a and 10b are gel images of purified soluble single-chain TCRs.
  • the right swimming lanes in Figures 10a and 10b are reducing gels and non-reducing gels respectively, and the left swimming lanes are molecular weight markers.
  • Figure 11 is a BIAcore kinetic profile of the combination of the soluble TCR of the present application and the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex.
  • Figure 12 is the BIAcore kinetic profile of the combination of the soluble single-chain TCR of the present application and the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex.
  • Figure 13 shows the results of the ELISPOT activation function verification of the obtained T cell clones.
  • Figure 14 is the result of ELISPOT activation function verification of T2 cells transfected with TCR effector cells of the present application.
  • Fig. 15 is the result of ELISPOT activation function verification of effector cells transfected with the TCR of the present application for tumor cell lines.
  • Figure 16 is the verification result of the killing function of the effector cells transfected with the TCR of the present application.
  • the present application also provides a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule.
  • the present application also provides cells transduced with the TCR of the present application.
  • 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, and different individuals have different MHCs, which can present different short peptides in a protein antigen to the surface of their respective APC cells.
  • Human MHC is often referred to as HLA genes or HLA complexes.
  • T cell receptor is the sole receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • T cells with different antigen specificities exert immune effects on their target cells.
  • TCR is a glycoprotein on the cell membrane surface that exists in the form of a heterodimer of ⁇ chain/ ⁇ chain or ⁇ chain/ ⁇ chain.
  • TCR heterodimers consist of alpha and beta chains in 95% of T cells, whereas 5% of T cells have TCRs consisting of gamma and delta chains.
  • the native ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain, which constitute the subunits of the ⁇ heterodimeric TCR.
  • each chain of ⁇ and ⁇ contains a variable region, a connecting region, and a constant region, and the ⁇ chain usually also contains a short variable region between the variable region and the connecting region, but this variable region is often regarded as the connecting region a part of.
  • Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are embedded in framework regions.
  • the CDR region determines the combination of the TCR and the pMHC complex, in which CDR3 is recombined from the variable region and the linker region, known as the hypervariable region.
  • the ⁇ and ⁇ chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domains are composed of linked variable and linker regions.
  • the sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the constant domain sequence of the ⁇ chain of the TCR molecule is "TRAC*01”
  • the constant domain sequence of the ⁇ chain of the TCR molecule is "TRBC1* 01" or "TRBC2*01”.
  • the ⁇ and ⁇ chains of TCR also contain a transmembrane region and a cytoplasmic region, which is very short.
  • polypeptide of the present application In the present application, the terms “polypeptide of the present application”, “TCR of the present application”, “T cell receptor of the present application” are used interchangeably.
  • the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in this application are numbered in sequence from the N-terminal to the C-terminal, such as TRBC1*01 or TRBC2*01
  • the 60th amino acid in the sequence from N-terminal to C-terminal is P (proline)
  • Pro60 of exon 1 of TRBC1*01 or TRBC2*01 in this application can also be described as It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01
  • the 61st amino acid in the order from N-terminal to C-terminal is Q (glutamine amide), then it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1 in this application, and it can also be expressed as the 61st amino acid of TRBC
  • the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are according to the position numbers listed in IMGT. For example, if a certain amino acid in TRAV is numbered 46 in IMGT, it will be described as the 46th amino acid in TRAV in this application, and so on. In this application, if there are special instructions for the sequence position numbers of other amino acids, the special instructions shall be followed.
  • the first aspect of the application provides a TCR molecule capable of binding TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex.
  • said TCR molecule is isolated or purified.
  • the ⁇ and ⁇ chains of this TCR each have 3 complementarity determining regions (CDRs).
  • the ⁇ chain of the TCR comprises a CDR having the following amino acid sequence:
  • the three complementarity-determining regions of the TCR ⁇ chain variable domain are:
  • a chimeric TCR can be prepared by embedding the amino acid sequences of the above CDR regions of the present application into any suitable framework structure.
  • the framework structure is compatible with the CDR region of the TCR of the present application, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR region disclosed in the present application. Therefore, the TCR molecule in the present application refers to a TCR molecule comprising the above-mentioned ⁇ and/or ⁇ chain CDR region sequence and any suitable framework structure.
  • the TCR ⁇ chain variable domain of the present application is an amino acid sequence having 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 application is an amino acid sequence identical to SEQ ID NO: 5 amino acid sequences having at least 90%, preferably 95%, more preferably 98% sequence identity.
  • the TCR molecule of the present application is a heterodimer composed of ⁇ and ⁇ chains.
  • the ⁇ chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain
  • the amino acid sequence of the variable domain of the ⁇ chain comprises CDR1 (SEQ ID NO: 10), 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 amino acid sequence of the ⁇ -chain variable domain of the TCR molecule is SEQ ID NO:1.
  • the ⁇ chain of the heterogeneous dimeric TCR molecule comprises a variable domain and a constant domain
  • the amino acid sequence of the variable domain of the ⁇ chain comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 13) of the above ⁇ chain. NO: 14) and CDR3 (SEQ ID NO: 15).
  • the TCR molecule comprises the amino acid sequence of the ⁇ chain variable domain of 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 application is a single-chain TCR molecule composed of part or all of the ⁇ chain and/or part or all of the ⁇ chain.
  • single-chain TCR molecules For the description of single-chain TCR molecules, reference can be made 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 comprising the CDRs region of the present application.
  • the single-chain TCR molecule comprises V ⁇ , V ⁇ and C ⁇ , preferably connected in order from N-terminus to C-terminus.
  • the ⁇ -chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above-mentioned ⁇ -chain.
  • the single-chain TCR molecule comprises the amino acid sequence of an ⁇ -chain variable domain of SEQ ID NO: 1. More preferably, the amino acid sequence of the ⁇ -chain variable domain of the single-chain TCR molecule is SEQ ID NO:1.
  • the ⁇ chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above ⁇ chain.
  • the single-chain TCR molecule comprises the amino acid sequence of the ⁇ -chain variable domain of SEQ ID NO: 5. More preferably, the amino acid sequence of the ⁇ -chain variable domain of the single-chain TCR molecule is SEQ ID NO: 5.
  • the constant domain of the TCR molecule of the present application is a human constant domain.
  • the constant domain sequence of the ⁇ chain of the TCR molecule in the present application may be "TRAC*01”
  • the constant domain sequence of the ⁇ chain of the TCR molecule may be "TRBC1*01” or "TRBC2*01”.
  • the 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is expressed here as: Arg53 of exon 1 of TRAC*01, and so on.
  • the amino acid sequence of the ⁇ chain of the TCR molecule of the present application is SEQ ID NO: 3, and/or the amino acid sequence of the ⁇ chain is SEQ ID NO: 7.
  • TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region. Like immunoglobulin (antibody) as an antigen recognition molecule, TCR can also be developed for diagnosis and treatment, and soluble TCR molecules need to be obtained at this time. Soluble TCR molecules do not include their transmembrane domains. Soluble TCR has a wide range of applications. It can not only be used to study the interaction between TCR and pMHC, but also can be used as a diagnostic tool for detecting infection or as a marker for autoimmune diseases.
  • soluble TCRs can be used to deliver therapeutic agents, such as cytotoxic or immunostimulatory compounds, to cells presenting specific antigens, and additionally, soluble TCRs can be conjugated to other molecules, such as anti-CD3 antibodies To redirect T cells so that they target cells presenting specific antigens.
  • therapeutic agents such as cytotoxic or immunostimulatory compounds
  • soluble TCRs can be conjugated to other molecules, such as anti-CD3 antibodies To redirect T cells so that they target cells presenting specific antigens.
  • the present application also obtained a soluble TCR specific for the short peptide of the AFP antigen.
  • the TCR of the present application can be a TCR that introduces an artificial disulfide bond 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 sites in native TCRs to form artificial interchain disulfide bonds. For example, substitution of Thr48 of TRAC*01 exon 1 and substitution of cysteine residues of Ser57 of TRBC1*01 or TRBC2*01 exon 1 to form disulfide bonds.
  • Other sites for introducing cysteine residues to form disulfide bonds can also be: Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon Tyr10 of 1 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01; Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; of exon 1 of TRAC*01 Ser15 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01; Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01; Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.
  • cysteine residues replace any one group of positions in the above-mentioned ⁇ and ⁇ chain constant domains.
  • 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 may be truncated at the C-terminus of one or more TCR constant domains of the present application, so that it does not include Cysteine residues can be used to delete natural disulfide bonds, or by mutating a cysteine residue that forms a natural disulfide bond to another amino acid.
  • the TCRs of the present application may contain artificial disulfide bonds introduced between residues in the constant domains of their alpha and beta chains. It should be noted that the TCR of the present application can contain both the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence, with or without the introduced artificial disulfide bond between the constant domains as described above.
  • the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by native disulfide bonds present in the TCR.
  • the TCR of the present application also includes TCRs with mutations in its hydrophobic core region, and these mutations in the hydrophobic core region are preferably mutations that can improve the stability of the soluble TCR of the present application. described in the patent literature of WO2014/206304.
  • TCRs may have mutations at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the penultimate 3rd, 5th, and 7th amino acid positions of the ⁇ -chain J gene (TRAJ) short peptide, and/or the penultimate 2nd, 4th, and 6th amino acid positions of the ⁇ -chain J gene (TRBJ) short peptide amino acid position, where the position number of the amino acid sequence By position number as listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR with mutations in the hydrophobic core region 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 this application can be any peptide chain suitable for linking the variable domains of TCR ⁇ and ⁇ chains.
  • the amino acid sequence of the ⁇ -chain variable domain is SEQ ID NO: 32
  • the encoded nucleotide sequence is SEQ ID NO: 33
  • the amino acid sequence of the ⁇ -chain variable domain 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 TCR can significantly improve the stability of TCR. Therefore, the TCR of the present application may also contain an artificial interchain disulfide bond between the variable region of the ⁇ chain and the constant region of the ⁇ chain.
  • cysteine residue that forms an artificial interchain disulfide bond between the ⁇ -chain variable region and the ⁇ -chain constant region of the TCR is substituted for: amino acid 46 of TRAV and TRBC1*01 or TRBC2* Amino acid 60 of exon 1 of 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01; amino acid 46 of TRAV and exon of TRBC1*01 or TRBC2*01 Amino acid 61 of exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.
  • such a TCR may comprise (i) all or part of a TCR alpha chain except for its transmembrane domain, and (ii) all or part of a TCR beta chain except for its transmembrane domain, wherein (i) and (ii ) all contain the variable domain of the TCR chain and at least a part of the constant domain, and the ⁇ chain and the ⁇ chain form a heterodimer.
  • such a TCR may comprise an ⁇ -chain variable domain and a ⁇ -chain variable domain and all or part of a ⁇ -chain constant domain except the transmembrane domain, but it does not contain an ⁇ -chain constant domain, and the ⁇ -chain of said TCR Chain variable domains form heterodimers with beta chains.
  • the TCRs of the present application may also be provided in the form of multivalent complexes.
  • the multivalent TCR complex of the present application comprises two, three, four or more multimers formed by combining the TCRs of the present application, such as the tetramerization domain of p53 can be used to produce tetramers, or multimers A complex formed by the combination of a TCR of the present application and another molecule.
  • the TCR complexes of the present application can be used to track or target cells presenting specific antigens in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes with such applications.
  • the TCR of the present application can be used alone, and can also be combined with a conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugates include detectable markers (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the TSSELMAITR (SEQ ID NO:9)-HLA A1101 complex), therapeutic agents, PK (protein kinase) A modifying moiety or any combination of the above is combined or coupled.
  • Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or products capable of producing detectable enzymes.
  • Therapeutic agents that can be combined or coupled with the TCR of the present application include but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2. Biological toxicity (Chaudhary et al., 1989 , Nature (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 /Nanorods (Lapotko et al., 2005, Cancer letters (Cancer letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society (Journal of the American Chemical Society) 128, 2115); 7. Virus particles (Peng et al., 2004 , Gene therapy (Gene therapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nanomagnetic particles; 10. Prodrug activating enzymes (for example, DT - diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); and 11. A chemotherapeutic agent (eg cisplatin) or any form of nanoparticle or the like.
  • DTD DT - diaphorase
  • BPHL biphenylhydrolase-like protein
  • the TCRs of the present application may also be hybrid TCRs comprising sequences derived from more than one species.
  • murine TCRs are more efficiently expressed in human T cells than human TCRs.
  • the TCRs of the present application may comprise human variable domains and murine constant domains.
  • a drawback of this approach is the potential for eliciting an immune response. Therefore, its use in adoptive T cell therapy should have regulatory protocols for immunosuppression to allow engraftment of murine-expressing T cells.
  • the second aspect of the present application provides a nucleic acid molecule encoding the TCR molecule of the first aspect of the present application or a part thereof, the part may be one or more CDRs, variable domains of ⁇ and/or ⁇ chains, and ⁇ chains and/or or beta strand.
  • the nucleotide sequence encoding the ⁇ -chain CDR region of the TCR molecule in the first aspect of the application is as follows:
  • the nucleotide sequence encoding the CDR region of the ⁇ chain of the TCR molecule in the first aspect of the application is as follows:
  • nucleotide sequence of the nucleic acid molecule of the present application encoding the TCR alpha chain of the present application comprises SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or the nucleic acid molecule of the present application encoding the TCR beta chain of the present application
  • Nucleotide sequences include SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
  • the nucleotide sequence of the nucleic acid molecule of the present application 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 application does not contain introns but can encode the polypeptide of the present application, for example, the nucleotide sequence of the nucleic acid molecule of the present application encoding the TCR ⁇ chain variable domain of the present application includes SEQ ID NO: 2 and /Or the nucleotide sequence of the nucleic acid molecule of the present application encoding the TCR ⁇ chain variable domain of the present application comprises SEQ ID NO:6.
  • the nucleotide sequence of the nucleic acid molecule encoding the variable domain of the TCR ⁇ chain of the application comprises SEQ ID NO: 33 and/or the nucleotide sequence of the nucleic acid molecule of the application encoding the variable domain of the TCR ⁇ chain of the application comprises SEQ ID NO: 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the present application comprises SEQ ID NO: 4 and/or SEQ ID NO: 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present application is SEQ ID NO: 31.
  • nucleic acid sequence encoding the TCR of the present application may be the same as the nucleic acid sequence shown in the drawings of the present application or a degenerate variant.
  • degenerate variant refers to a nucleic acid sequence that encodes a protein sequence with SEQ ID NO: 1, but differs from the sequence of SEQ ID NO: 2.
  • Nucleotide sequences may be codon optimized. Different cells use different codons, and the codons in the sequence can be changed to increase the expression according to the cell type. Codon usage tables for mammalian cells, as well as for a variety of other organisms, are well known to those skilled in the art.
  • the full-length sequence of the nucleic acid molecule of the present application or its fragments can usually be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis.
  • the DNA sequence encoding the TCR of the present application (or its fragments, or its derivatives) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. DNA can be either the coding strand or the non-coding strand.
  • the present application also relates to vectors comprising the nucleic acid molecules of the present application, including expression vectors, ie constructs capable of expression in vivo or in vitro.
  • vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.
  • Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpesviral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.
  • AAV adeno-associated viral
  • the vector can transfer the nucleotide of the present application into cells, such as T cells, so that the cells express AFP antigen-specific TCR.
  • the vector should be consistently expressed at high levels in T cells.
  • the present application also relates to host cells produced by genetic engineering using the vectors or coding sequences of the present application.
  • the host cell contains the vector of the present application or the nucleic acid molecule of the present application is integrated in 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 application also includes isolated cells expressing the TCR of the present application, which can be but not limited to T cells, NK cells, NKT cells, stem cells, especially T cells.
  • the T cells may be derived from T cells isolated from the subject, or may be part of a mixed cell population isolated from the subject, such as 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 may be in a mixed population of CD4 + helper T cells/CD8 + cytotoxic T cells.
  • the cells can be activated with antibodies (such as anti-CD3 or anti-CD28 antibodies) so that they can be more easily transfected, for example, transfected with a vector comprising a nucleotide sequence encoding a TCR molecule of the present application dye.
  • antibodies such as anti-CD3 or anti-CD28 antibodies
  • the cells of the present application may also be or be derived from stem cells, such as hematopoietic stem cells (HSC).
  • stem cells such as hematopoietic stem cells (HSC).
  • HSCs hematopoietic stem cells
  • T cell transfection with DNA or RNA encoding the TCR of the present application There are many methods suitable for T cell transfection with DNA or RNA encoding the TCR of the present application (eg, Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the TCR of the present application can be used for adoptive immunotherapy. Many suitable methods of performing adoptive therapy are known to those skilled in the art (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
  • the present application 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 recognize the TSSELMAITR (SEQ ID NO:9)-HLA A1101 complex.
  • the AFP-specific T cells of the present application can be used to treat any AFP-related diseases that present the AFP antigen short peptide TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex, including but not limited to tumors such as liver cancer.
  • Treatment can be carried out by isolating T cells from patients or volunteers suffering from diseases related to AFP antigens, introducing the TCR of the present application into the above T cells, and then returning these genetically modified cells to the patients. Therefore, the present application provides a method for treating AFP-related diseases, comprising injecting isolated T cells expressing the TCR of the present application, preferably, the T cells are derived from the patient itself, into the patient. Generally, it includes (1) isolating T cells from patients; (2) transducing T cells in vitro with nucleic acid molecules of the present application or nucleic acid molecules capable of encoding TCR molecules of the present application; and (3) importing T cells modified by genetic engineering into inside the patient. Wherein, the number of isolated, transfected and reinfused cells can be determined by the physician.
  • the TCR of the present application can specifically bind to the AFP antigen short peptide complex TSSELMAITR (SEQ ID NO: 9)-HLA A1101, and at the same time, the effector cells transduced with the TCR of the present application can be specifically activated.
  • the effector cells transduced with the TCR of the present application can specifically kill AFP-positive target cells.
  • Peripheral blood lymphocytes from healthy volunteers with genotype HLA-A1101 were stimulated with the synthetic short peptide TSSELMAITR (SEQ ID NO: 9; Jiangsu GenScript Biotechnology Co., Ltd.). Refold TSSELMAITR (SEQ ID NO: 9) short peptide with biotin-labeled HLA-A1101 to prepare pMHC haploids. These haploids were combined with PE-labeled streptavidin (BD Company) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double-positive cells were sorted.
  • TSSELMAITR synthetic short peptide TSSELMAITR
  • Sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonalization by limiting dilution. Monoclonal cells were stained with tetramers, and the screened double-positive clones are shown in Figure 3. The double-positive clones obtained through layers of screening still need to meet further functional tests.
  • IFN- ⁇ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN- ⁇ is detected by the ELISPOT experiment well known to those skilled in the art to verify the activation function of cells transfected with the TCR of this application and Antigen specificity. The function and specificity of the T cell clone were further detected by ELISPOT experiment.
  • the effector cells used in the IFN- ⁇ ELISPOT experiment of this embodiment are the T cell clones obtained in this application, and the target cells are T2-A11 (referring to T2 cells transfected with HLA-A1101) loaded with TSSELMAITR (SEQ ID NO: 9) short peptide cells), SK-MEL-28-AFP (AFP overexpression), and the control group were T2 cells loaded with other antigen short peptides and SK-MEL-28.
  • ELISPOT plate First prepare the ELISPOT plate, and the ELISPOT experiment steps are as follows: Add the components of the test to the ELISPOT plate in the following order: 20,000 target cells/well and 2,000 effector cells/well, then add 20 ⁇ L of the corresponding short peptide to the experimental group and the control group , so that the final concentration of the short peptide was 10 -5 M, add 20 ⁇ L medium (test medium) to the blank group, and set up 2 duplicate wells. It was then incubated overnight (37°C, 5% CO 2 ). Then the plate was washed for secondary detection and color development, and the plate was dried for 1 hour, and the spots formed on the membrane were counted by an immunospot plate reader (ELISPOT READER system; AID company).
  • ELISPOT READER system AID company
  • T cell clones released high IFN- ⁇ to T2-A11 and SK-MEL-28-AFP loaded with TSSELMAITR (SEQ ID NO: 9) short peptide, but to T2-A11 and SK-MEL-28-AFP loaded with other antigens
  • TSSELMAITR SEQ ID NO: 9
  • RNA of the HLA-A1101-restricted T cell clone specific to the short antigen peptide TSSELMAITR (SEQ ID NO: 9) screened in Example 1 was extracted with Quick-RNA TM MiniPrep (ZYMO research).
  • the cDNA was synthesized using clontech's SMART RACE cDNA amplification kit, and the primers used were designed at the C-terminal conserved region of the human TCR gene.
  • the sequence was cloned into T vector (TAKARA) for sequencing. It should be noted that this sequence is complementary and does not contain introns. After sequencing, the sequence structures of the TCR ⁇ chain and ⁇ chain expressed by the double-positive clone are shown in Figure 1 and Figure 2, respectively.
  • Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of the TCR ⁇ chain variable domain, the nucleotide sequence of the TCR ⁇ chain variable domain, the amino acid sequence of the TCR ⁇ chain, the nucleotide sequence of the TCR ⁇ chain, and the TCR ⁇ chain amino acid sequence and TCR ⁇ chain nucleotide sequence with leader sequence.
  • the alpha chain was identified to contain CDRs with the following amino acid sequence:
  • the beta strand contains CDRs with the following amino acid sequence:
  • the full-length genes of TCR ⁇ chain and ⁇ chain were respectively cloned into the lentiviral expression vector pLenti(addgene) by overlapping PCR. Specifically: use overlap PCR to connect the full-length genes 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 application may only contain their variable domains and part of the constant domains, respectively, and a cysteine residue is introduced into the constant domains of the ⁇ and ⁇ chains respectively
  • the amino acid sequence and nucleotide sequence of its ⁇ chain are shown in Figure 4a and Figure 4b, respectively, and the amino acid sequence and nucleotide sequence of its ⁇ chain are shown in Figure 5a and Figure 5b, respectively .
  • the target gene sequences of the above 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 insert was confirmed by sequencing.
  • the dissolved TCR ⁇ and ⁇ chains were 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 was 60mg/mL.
  • the solution was dialyzed (4°C) in 10 times the volume of deionized water, and after 12 hours, the deionized water was replaced with buffer solution (20mM Tris, pH 8.0) and continued to be dialyzed at 4°C for 12 hours.
  • the solution was filtered through a 0.45 ⁇ M filter membrane, and then purified by an anion exchange column (HiTrap Q HP, 5ml, GE Healthcare).
  • the elution peaks containing TCRs of successfully refolded ⁇ and ⁇ dimers were confirmed by SDS-PAGE.
  • 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.
  • the SDS-PAGE gel images of the soluble TCR obtained in this application are shown in Figures 6a and 6b.
  • variable domains of the TCR ⁇ and ⁇ chains in Example 2 were constructed by site-directed mutagenesis into a stable soluble single-chain TCR molecule linked by a flexible short peptide (linker).
  • the amino acid sequence and nucleotide sequence of the single-chain TCR molecule are shown in Figure 7a and Figure 7b respectively, and the amino acid sequence and nucleotide sequence of the linker are underlined.
  • the amino acid sequence and nucleotide sequence of its alpha chain variable domain are shown in Figure 8a and Figure 8b, respectively; the amino acid sequence and nucleotide sequence of its beta chain variable domain are shown in Figure 9a and Figure 9b, respectively.
  • the target gene was digested with Nco I and Not I, and connected to the pET28a vector that was digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ , coated with kanamycin-containing LB plates, cultured upside down at 37°C overnight, and positive clones were picked for PCR screening, and the positive recombinants were sequenced, and the recombinant plasmids were extracted and transformed after confirming the sequence was correct to E.coli BL21(DE3) for expression.
  • Example 5 Expression, refolding and purification of soluble single-chain TCR specific for short peptides of AFP antigen
  • the inclusion bodies were dissolved in the buffer solution (20mM Tris-HCl pH 8.0, 8M urea), and the insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by the BCA method, then aliquoted, and stored at -80°C for later use.
  • dialysate was replaced with 1 L of pre-cooled buffer solution (20mM Tris-HCl pH 8.0), and dialysis was continued at 4°C for 8 hours, and then the dialysate was replaced with the same fresh buffer solution to continue dialysis overnight.
  • the sample was filtered through a 0.45 ⁇ m filter membrane, vacuum degassed and then passed through an anion exchange column (HiTrap Q HP, GE Healthcare), and the protein was purified with a 0-1M NaCl linear gradient eluent prepared with 20mM Tris-HCl pH 8.0, The collected eluted fractions were analyzed by SDS-PAGE, and the fractions containing single-chain TCR were concentrated and further purified by gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
  • the eluted fractions for BIAcore analysis were further tested for purity by gel filtration.
  • the conditions are: chromatographic column Agilent Bio SEC-3 (300A, ), the mobile phase was 150mM phosphate buffer, the flow rate was 0.5mL/min, the column temperature was 25°C, and the ultraviolet detection wavelength was 214nm.
  • BIAcore T200 real-time analysis system was used to detect the binding activity of the TCR molecule obtained in Example 3 and Example 5 to the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex.
  • TSSELMAITR SEQ ID NO: 9
  • HLA A1101 complex Let a low concentration of streptavidin flow over the surface of the antibody-coated chip, then flow the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex through the detection channel, and the other channel as a reference channel, and then 0.05mM Biotin flowed through the chip at a flow rate of 10 ⁇ L/min for 2 min to block the remaining binding sites of streptavidin.
  • the synthetic short peptide TSSELMAITR (SEQ ID NO: 9) was dissolved in DMSO to a concentration of 20 mg/mL.
  • the inclusion bodies of the light chain and heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, and 10mM DTT.
  • 3M guanidine hydrochloride, 10mM sodium acetate, and 10mM EDTA were added for further denaturation.
  • TSSELMAITR (SEQ ID NO: 9) peptide was added 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 sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain is added in three times, 8h/time), renaturation At 4°C for at least 3 days to complete, SDS-PAGE test whether the renaturation is successful.
  • biotinylated pMHC molecules were concentrated to 1 mL with Millipore ultrafiltration tubes, and the biotinylated pMHC was purified by gel filtration chromatography, and the HiPrep was pre-equilibrated with filtered PBS using an Akta purification instrument (GE General Electric Company). TM 16/60 S200 HR column (GE General Electric Company), loaded with 1 mL of concentrated biotinylated pMHC molecules, and then eluted with PBS at a flow rate of 1 mL/min. Biotinylated pMHC molecules elute as a single peak at approximately 55 mL.
  • the protein-containing fractions were pooled, concentrated with Millipore ultrafiltration tubes, and the protein concentration was determined by the BCA method (Thermo).
  • the biotinylated pMHC molecules were subpackaged and stored at -80°C by adding protease inhibitor cocktail (Roche).
  • Kinetic parameters were calculated using BIAcore Evaluation software to obtain the kinetic profiles of the soluble TCR molecule of the application and the combination of the soluble single-chain TCR molecule constructed by the application and the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex, respectively as shown in Figure 11 and Figure 12 shows.
  • the map shows that both the soluble TCR molecule and the soluble single-chain TCR molecule obtained in the present application can bind to the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex.
  • the above method was also used to detect the binding activity of the soluble TCR molecules of the present application to the complexes of several other irrelevant antigen short peptides and HLA, and the results showed that the TCR molecules of the present application did not bind to other irrelevant antigens.
  • the effector cells used in this experiment were CD3 + T cells expressing the TCR of the present application, and CD3 + T cells transfected with other TCR (A6) from the same volunteer were used as the control group.
  • the target cells used were T2-A11 loaded with AFP antigen short peptide TSSELMAITR (SEQ ID NO: 9), and empty T2-A11 loaded with other antigen short peptides was used as a control.
  • TSSELMAITR (SEQ ID NO: 9) short peptide was added to the corresponding wells, so that the final concentration of the short peptide in the ELISPOT well plate was 10 ⁇ 6 M.
  • Dilute streptavidin-alkaline phosphatase 1:100 with PBS containing 10% FBS add 100 ⁇ l of diluted streptavidin-alkaline phosphatase to each well and incubate the plate at room temperature 1 hour. Then wash 4 times with wash buffer and 2 times with PBS, tapping the well plate on paper towels to remove excess wash buffer and PBS. After washing, 100 microliters/well of BCIP/NBT solution provided in the kit was added for development. Cover the well plate with tin foil to avoid light during the development period, and let it stand for 5-15 minutes. During this period, the spots on the developed well plate were routinely detected to determine the optimal time to terminate the reaction.
  • Example 8 For tumor cell lines, the activation function experiment of the effector cells transfected with the TCR of the present application
  • the function and specificity of the TCR of the present application in cells are also detected by ELISPOT experiment.
  • the effector cells used were CD3 + T cells expressing the specific TCR of the AFP antigen short peptide of the application, and the same volunteers transfected with other TCR (A6) and empty transfected (NC) CD3 + T cells were used as the control group.
  • the target cells are tumor cell lines, and the positive tumor cell lines used are HepG2-A11-B2M (overexpression of HLA A1101 and ⁇ 2M), SK-MEL-28-AFP; the negative tumor cell lines used are HepG2, SK-MEL-28, SNU423 and HUCCT1, as a control group.
  • ELISPOT plates were activated with ethanol and coated at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add each component of the test to the ELISPOT plate: 2 ⁇ 10 cells/well for target cells, 2 ⁇ for effector cells 10 3 cells/well (calculated according to the positive rate of transfection), and set up two duplicate holes. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted by an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system AID20 company
  • the release of LDH was measured by non-radioactive cytotoxicity experiments well known to those skilled in the art, so as to verify the killing function of the cells transfected with the TCR of the present application.
  • CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the TCR of this application as effector cells, and the same volunteers were used to transfect other TCR (A6) or empty transfection (NC) CD3 + T cells served as negative controls.
  • the target cells are tumor cell lines, and the positive tumor cell lines used are HepG2-A11-B2M (overexpression of HLA A1101 and ⁇ 2M), SK-MEL-28-AFP; the negative tumor cell lines used are HepG2, SK-MEL-28, SNU423 and HUCCT1, as a control group.
  • the positive tumor cell lines used are HepG2-A11-B2M (overexpression of HLA A1101 and ⁇ 2M), SK-MEL-28-AFP; the negative tumor cell lines used are HepG2, SK-MEL-28, SNU423 and HUCCT1, as a control group.

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Abstract

La présente demande concerne un récepteur de lymphocytes T (TCR) capable de se lier de manière spécifique à un peptide court TSSELMAITR (SEQ ID NO : 9) dérivé d'un antigène AFP, le peptide court d'antigène TSSELMAITR (SEQ ID NO : 9) peut former un complexe avec HLA A1101 et peut être présenté sur la surface d'une cellule conjointement avec HLA A1101. La présente demande concerne en outre une molécule d'acide nucléique codant pour le TCR et un vecteur contenant la molécule d'acide nucléique. De plus, la présente demande concerne également une cellule pour la transduction du TCR selon la présente invention.
PCT/CN2022/099352 2021-06-18 2022-06-17 Tcr pour identifier un antigène afp et séquence codant pour celui-ci WO2022262835A1 (fr)

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WO2021022447A1 (fr) * 2019-08-05 2021-02-11 广东香雪精准医疗技术有限公司 Récepteur de slymphocytes t capable de reconnaître un peptide court dérivé d'un antigène afp

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