WO2023179768A1 - 一种识别mage-a4抗原的高亲和力tcr及其序列和应用 - Google Patents

一种识别mage-a4抗原的高亲和力tcr及其序列和应用 Download PDF

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WO2023179768A1
WO2023179768A1 PCT/CN2023/083730 CN2023083730W WO2023179768A1 WO 2023179768 A1 WO2023179768 A1 WO 2023179768A1 CN 2023083730 W CN2023083730 W CN 2023083730W WO 2023179768 A1 WO2023179768 A1 WO 2023179768A1
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tcr
chain
variable domain
seq
amino acid
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French (fr)
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战凯
黄金花
郑文静
殷倩霞
唐先青
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香雪生命科学技术（广东）有限公司
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • 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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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • C—CHEMISTRY; METALLURGY
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to the field of biotechnology, and more specifically to a T cell receptor (TCR) capable of recognizing polypeptides derived from MAGE-A4 protein.
  • TCR T cell receptor
  • the invention also relates to the preparation and use of said receptors.
  • TCR T cell receptor
  • TCR is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). This exogenous or endogenous peptide may be the only sign of cellular abnormalities.
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • the MHC class I and class II molecular ligands corresponding to the TCR are also proteins of the immunoglobulin superfamily but are specific for antigen presentation. Different individuals have different MHC, which can present different short molecules in a protein antigen. peptides to the respective APC cell surface. Human MHC is often called HLA genes or HLA complexes.
  • MAGE protein family has regions of homology that closely match the sequences of other MAGE proteins and contains peptides that are displayed as HLA/peptide complexes in immune recognition.
  • Some MAGE gene family proteins (MAGE-A to MAGE-C families) are expressed only in germ cells and cancer; others (MAGE-D to MAGE-H) are widely expressed in normal tissues.
  • MAGE-A4 is degraded into small molecule peptides after being produced in cells, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface.
  • MHC major histocompatibility complex
  • MAGE-A4 protein is expressed in a variety of tumor types, including melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, etc.
  • GLYDGREHSV is a short peptide derived from MAGE-A8 and has high identity with GVYDGREHTV, a short peptide derived from MAGE-A4.
  • chemotherapy and radiotherapy can be used, but they will cause damage to their own normal cells.
  • the GVYDGREHTV-HLA A0201 complex provides a TCR-targetable marker for tumor cells.
  • TCR that can bind to the GVYDGREHTV-HLA A0201 complex has high application value in the treatment of tumors.
  • a TCR capable of targeting this tumor cell marker could be used to deliver a cytotoxic or immunostimulatory agent to the target cell, or be transformed into a T cell such that the T cell expressing the TCR is able to destroy the tumor cell in order to achieve the desired outcome in what is known as Adoptive immunotherapy is administered to patients during treatment.
  • the ideal TCR is one with high affinity, allowing the TCR to reside on the targeted cells for a long time.
  • the first aspect of the present application provides a TCR.
  • the technical solution is a TCR that includes an alpha chain variable domain and a beta chain variable domain, and the TCR has the ability to bind GVYDGREHTV-HLA A0201 activity of the complex;
  • amino acid sequence of the TCR ⁇ chain variable domain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1
  • amino acid sequence of the TCR ⁇ chain variable domain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.2
  • the amino acid sequences have at least 90% sequence identity.
  • the amino acid sequence of the TCR ⁇ chain variable domain and the amino acid sequence of the TCR ⁇ chain variable domain are not simultaneously the amino acid sequence of the wild-type TCR ⁇ chain variable domain and the amino acid sequence of the wild-type TCR ⁇ chain variable domain. sequence.
  • the amino acid sequence of the TCR ⁇ chain variable domain is not the amino acid sequence shown in SEQ ID NO.1.
  • the ⁇ chain variable domain of the TCR contains at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, An amino acid sequence that has 98% or 99% sequence identity, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids relative to the sequence set forth in SEQ ID NO.1 Residue insertions, deletions, substitutions or combinations thereof.
  • the ⁇ -chain variable domain of the TCR is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or more similar to the sequence shown in SEQ ID NO. 2.
  • An amino acid sequence that has 98%, 99% or 100% sequence identity, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more relative to the sequence shown in SEQ ID NO.2 11 amino acid residues are inserted, deleted, replaced or a combination thereof.
  • the amino acid sequence of the TCR ⁇ chain variable domain has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence of the TCR ⁇ chain variable domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO.
  • the amino acid sequence shown in ID NO.2 has at least 95% sequence identity.
  • the TCR also has the activity of binding GLYDGREHSV-HLA A0201 complex.
  • CDR1 ⁇ in the TCR ⁇ chain variable domain is: DSSSTY (SEQ ID NO. 37), and CDR2 ⁇ is IFSNMDM (SEQ ID NO. 38).
  • the CDR3 ⁇ of the TCR ⁇ chain variable domain is selected from AEHTKQNEKLT (SEQ ID NO. 39), AWSQFGNEKLT (SEQ ID NO. 40), AVSNFGNEKLT (SEQ ID NO. 41) and AASEFGNEKLT (SEQ ID NO. .42).
  • the three CDRs of the TCR ⁇ chain variable domain are:
  • amino acid sequence of the TCR ⁇ chain variable domain is SEQ ID NO. 2.
  • the number of amino acid mutations in the TCR ⁇ chain variable domain is 3-4, for example, it can be 3 or 4.
  • amino acid mutation sites of the TCR ⁇ chain variable domain are the 3rd, 4th, 5th and 6th positions of CDR3 ⁇ ; or the 2nd, 3rd and 6th positions of CDR3 ⁇ 4 bit.
  • the reference sequences of the three CDR regions (complementarity determining regions) of the TCR ⁇ chain variable domain are as follows:
  • CDR3 ⁇ contains at least one mutation in Table 1:
  • amino acid mutations in CDR3 ⁇ include the mutations in Table 2:
  • the affinity of the TCR to the GVYDGREHTV-HLA A0201 complex is at least 5 times that of the wild-type TCR.
  • the TCR is mutated in the alpha chain variable domain shown in SEQ ID NO.1, and the mutation is selected from E91W/V/A/T/S/H/Q/N/Y , Q92H/S, S93T/Q/N/E/D, F94K/R/H and G95Q, one or more groups, in which the amino acid residue numbering adopts the numbering shown in SEQ ID NO.1.
  • the TCR has a CDR selected from the group shown in Table 3:
  • ⁇ CDR3 The sequence of ⁇ CDR3 is as follows:
  • the TCR is soluble.
  • the TCR is an ⁇ heterodimeric TCR, and the TCR includes an ⁇ chain TRAC constant region sequence, and a ⁇ chain TRBC1 or TRBC2 constant region sequence.
  • the TCR includes:
  • the TCR includes an ⁇ chain constant region and a ⁇ chain constant region, and there is an artificial interchain disulfide bond between the ⁇ chain constant region and the ⁇ chain constant region of the TCR.
  • cysteine residues that form artificial interchain disulfide bonds between the constant regions of the TCR ⁇ and ⁇ chains are replaced with one or more groups of sites selected from the following:
  • the amino acid sequence of the ⁇ -chain variable domain of the TCR is one of SEQ ID NO. 13-32; and/or the amino acid sequence of the ⁇ -chain variable domain of the TCR is SEQ ID NO.2.
  • the TCR is selected from each group in Table 4:
  • the TCR is of human origin.
  • the TCR is isolated and purified.
  • the TCR is a single-chain TCR.
  • the TCR is a single-chain TCR composed of an ⁇ chain variable domain and a ⁇ chain variable domain, and the ⁇ chain variable domain and the ⁇ chain variable domain are composed of a flexible short peptide sequence (linker )connect.
  • the TCR includes an ⁇ chain constant region and a ⁇ chain constant region, the ⁇ chain constant region is a murine constant region and/or the ⁇ chain constant region is a murine constant region.
  • the TCR includes an ⁇ chain constant region and a ⁇ chain constant region, the ⁇ chain constant region is a murine constant region and/or the ⁇ chain constant region is a murine constant region.
  • a conjugate is bound to the C- or N-terminus of the ⁇ chain of the TCR, and/or a conjugate is bound to the C- or N-terminus of the ⁇ chain of the TCR.
  • the conjugate includes any one or a combination of at least two of detection markers, therapeutic agents or PK modifying moieties.
  • the therapeutic agent that binds to the TCR is an anti-CD3 antibody connected to the C- or N-terminus of the ⁇ chain of the TCR, or connected to the C- or N-terminus of the ⁇ chain of the TCR. N-terminal anti-CD3 antibody.
  • the second aspect of the present application provides a multivalent TCR complex
  • the multivalent TCR complex includes any two or at least three TCRs, and at least one of the TCRs is the third TCR.
  • the TCR of one aspect is the second aspect of the present application.
  • the third aspect of the present application provides a nucleic acid molecule, the nucleic acid molecule comprising a nucleotide sequence encoding the TCR described in the first aspect of the present application or the first aspect of the present application.
  • nucleic acid sequence of the multivalent TCR complex described in the second aspect of the application or the complementary sequence of the nucleic acid sequence of the multivalent TCR complex described in the second aspect of the application.
  • the fourth aspect of the present application provides a vector, the vector containing the nucleic acid molecule described in the third aspect of the present application.
  • the fifth aspect of the present application provides a host cell, the host cell contains the vector described in the fourth aspect of the present application, or the chromosome of the host cell has an external
  • the nucleic acid molecule according to the third aspect of the present application is derived from the source.
  • the sixth aspect of the present application provides an isolated cell expressing the TCR molecule described in the first aspect of the present application.
  • the isolated cells include any one or a combination of at least two of T cells, NK cells or NKT cells.
  • the isolated cells are T 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 second aspect of the present application The TCR complex described in the aspect or the isolated cell described in the sixth aspect of the present application.
  • the eighth aspect of the present application provides the TCR described in the first aspect, the TCR complex described in the second aspect, the isolated cell described in the sixth aspect or the seventh aspect.
  • the present application also provides a method for treating a disease, which includes administering an appropriate amount of the TCR described in the first aspect of the present application and the TCR complex described in the second aspect of the present application to a subject in need of treatment. , the isolated cells described in the sixth aspect of the present application or the pharmaceutical composition described in the seventh aspect of the present application.
  • the disease is a MAGE-A4 positive tumor.
  • the ninth aspect of the present application provides the TCR described in the first aspect of the present application, the TCR complex described in the second aspect of the present application, or the isolated cell described in the sixth aspect of the present application.
  • the disease is a MAGE-A4 positive tumor.
  • the tenth aspect of the present application provides a preparation method for the TCR described in the first aspect of the present application, the preparation method including the steps:
  • the affinity and/or binding half-life of the high-affinity TCR of the present invention for the GVYDGREHTV-HLA A0201 complex is at least 5 times that of the wild-type TCR.
  • the high-affinity TCR of the present invention can specifically bind to the GVYDGREHTV-HLA A0201, and at the same time, cells transfected with the high-affinity TCR of the present invention can be specifically activated.
  • Figure 1 shows the binding curve of soluble reference TCR, that is, wild-type TCR, and GVYDGREHTV-HLA A0201 complex;
  • Figures 2a and 2b are respectively the activation function experimental results of effector cells transfected with the high-affinity TCR of the present application on T2 cells loaded with the short peptide GVYDGREHTV and T2 cells loaded with the short peptide GLYDGREHSV;
  • Figure 3a and Figure 3b are the experimental results of the activation function of effector cells transfected with the high-affinity TCR of the present application on tumor cell lines;
  • Figure 4a and Figure 4b are the results of the killing function LDH experiment of effector cells transfected with the high-affinity TCR of the present application on tumor cell lines;
  • Figure 5 shows the results of ELISPOT detection of IFN- ⁇ release from effector cells transfected with high-affinity TCR on human normal tissue cells
  • Figure 6 is the experimental results of the in vivo efficacy of the high-affinity TCR of the present application in mice.
  • TCR high-affinity T cell receptor
  • CDR3 ⁇ AEQSFGNEKLT Mutation occurs in SEQ ID NO.62, and/or in the 3 CDR regions of the ⁇ -chain variable domain of the high-affinity TCR:
  • CDR3 ⁇ ASSLGRAYEQY SEQ ID NO.61 is mutated; and, after mutation, the affinity and/or binding half-life of the TCR to the GVYDGREHTV-HLA A0201 complex is at least 5 times that of the wild-type TCR.
  • TCR T cell receptor
  • the International Immunogenetics Information System can be used to describe TCRs.
  • Natural ⁇ heterodimeric TCRs have ⁇ and ⁇ chains. Broadly speaking, each chain contains 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 this variable region is often regarded as part of the connecting region.
  • the junction region of the TCR is determined by the unique TRAJ and TRBJ of IMGT, and the constant region of the TCR is determined by TRAC and TRBC of IMGT.
  • Each variable region contains three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, embedded in the framework sequence.
  • CDR1, CDR2 and CDR3 embedded in the framework sequence.
  • different numbers for TRAV and TRBV refer to different V ⁇ types and V ⁇ types, respectively.
  • the alpha chain constant domain has the following symbols: TRAC*01, where "TR” represents the T cell receptor gene; "A” represents the alpha chain gene; C represents the constant region; "*01” represents the allele Gene 1.
  • the beta chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR" represents the T cell receptor gene; "B” represents the beta chain gene; C represents the constant region; "*01” represents the allele 1.
  • the constant region of the alpha chain is uniquely determined, and in the form of the beta chain, there are two possible constant region genes, "C1" and “C2.” Those skilled in the art can obtain the constant region gene sequences of TCR ⁇ and ⁇ chains through the public IMGT database.
  • TCR alpha chain variable domain refers to the linked TRAV and TRAJ regions
  • TCR beta chain variable domain refers to the linked TRBV and TRBD/TRBJ regions.
  • the three CDRs in the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ ; the three CDRs in the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ , and CDR3 ⁇ .
  • the framework sequence of the TCR variable domain of the present application can be of murine or human origin, preferably of human origin.
  • the constant domain of a TCR contains an intracellular part, a transmembrane region, and an extracellular part.
  • amino acid sequences of the ⁇ and ⁇ chain variable domains of the wild-type TCR capable of binding to the GVYDGREHTV-HLA A0201 complex are the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.
  • the alpha chain amino acid sequence and beta chain amino acid sequence of the soluble "reference TCR" described in this application are the amino acid sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12 respectively.
  • the ⁇ -chain extracellular amino acid sequence and ⁇ -chain extracellular amino acid sequence of the “wild-type TCR” described in this application are the amino acid sequences shown in SEQ ID NO. 33 and SEQ ID NO. 34 respectively.
  • the TCR sequences used in this application are of human origin.
  • the ⁇ -chain amino acid sequence and ⁇ -chain amino acid sequence of the "wild-type TCR” described in this application are the amino acid sequences shown in SEQ ID NO. 35 and SEQ ID NO. 36 respectively.
  • the terms "polypeptide of the present application”, “TCR of the present application” and “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 order from the N end to the C end.
  • the position numbers are from N
  • the 60th amino acid in sequence from end to C end is P (proline)
  • Pro60 of TRBC1*01 or TRBC2*01 exon 1 or it can also be expressed as TRBC1* 01 or the 60th amino acid of exon 1 of TRBC2*01
  • the 61st amino acid in order from the N terminus to the C terminus is Q (glutamine), then this
  • it can be described as Gln61 in exon 1 of TRBC1*01 or TRBC2*01, or as the 61st amino acid in exon 1 of TRBC1*01 or TRBC2*01, and so on.
  • the position numbering of the amino acid sequences of variable regions TRAV and TRBV follows the position numbering listed in IMGT. For example, for a certain amino acid in TRAV, the position number listed in IMGT is 46, then this application will describe it as the 46th amino acid of TRAV, and so on. In this application, if there are special instructions for the sequence position numbers of other amino acids, the special instructions will apply.
  • tumor is meant to include all types of cancer cell growth or carcinogenic processes, metastatic tissue or malignantly transformed cells, tissues or organs, regardless of pathological type or stage of infection.
  • tumors include, without limitation: solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include malignancies of different organ systems, such as sarcomas, lung squamous cell carcinoma, and cancer. Examples include infected prostate, lungs, breasts, lymph, gastrointestinal (e.g., colon), genitourinary tract (e.g., kidneys and epithelial cells), and pharynx.
  • Lung squamous cell carcinoma includes malignant tumors such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell carcinomas of the lungs, small bowel cancers, and esophageal cancers. Metastatic lesions of the above-mentioned cancers can also be treated and prevented using the methods and compositions of the present application.
  • the ⁇ chain variable domain and ⁇ chain variable domain of TCR each contain three CDRs, similar to the complementarity determining regions of antibodies.
  • CDR3 interacts with short antigenic peptides
  • CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigen peptide-HLA complex.
  • the amino acid sequence of the ⁇ chain variable domain and the ⁇ chain variable domain amino acid sequence of the wild-type TCR capable of binding the antigen short peptide GVYDGREHTV-HLA A0201 complex are SEQ ID NO.1 and SEQ respectively ID NO.2, the sequence was discovered for the first time by the inventor. It has the following CDR regions:
  • CDR3 ⁇ ASSLGRAYEQY SEQ ID NO.61.
  • This application conducted mutation screening on the CDR region and obtained a high-affinity TCR whose affinity to the GVYDGREHTV-HLA A0201 complex is at least 5 times that of the wild-type TCR and the GVYDGREHTV-HLA A0201 complex.
  • the TCR described in this application is an ⁇ heterodimeric TCR, and the ⁇ chain variable domain of the TCR contains at least 85% of the amino acid sequence shown in SEQ ID NO. 1; preferably, at least 90%; more preferably Preferably, at least 92%; more preferably, at least 94% (for example, it can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% sequence identity) amino acid sequence with sequence identity;
  • the beta chain variable domain of the TCR contains at least 90%, preferably at least 92%, with the amino acid sequence shown in SEQ ID NO. 2; more preferably, at least 94% (for example, it can be at least 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) of the amino acid sequence.
  • the TCR described in the present application is a single-chain TCR, and the alpha chain variable domain of the TCR contains at least 85%, preferably at least 90%, of the amino acid sequence shown in SEQ ID NO. 3; more preferably, at least 92%; most preferably, at least 94% (for example, it can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Sequence identity) amino acid sequence of sequence identity;
  • the ⁇ -chain variable domain of the TCR contains at least 85%, preferably at least 90%, of the amino acid sequence shown in SEQ ID NO. 4; more preferably, at least 92%; most preferably, at least 94% %; (for example, it can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) of the amino acid sequence.
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO.1, namely CDR1, CDR2 and CDR3, are located at positions 26-31, 49-55 and 90-100 of SEQ ID NO.1 respectively.
  • the amino acid residue numbering adopts the numbering shown in SEQ ID NO.1, 91E is the second E of CDR3 ⁇ , 92Q is the third Q of CDR3 ⁇ , 93S is the fourth S of CDR3 ⁇ , and 94F is It is the 5th F of CDR3 ⁇ , and 95G is the 6th G of CDR3 ⁇ .
  • the specific forms of mutations in the alpha chain variable domain include E91W/V/A/T/S/H/Q/N/Y, Q92H/S, S93T/Q/N/E/D, F94K/ One or more groups of R/H and G95Q.
  • amino acid names in this article adopt the internationally accepted single English letter identifier, and the corresponding three English letter abbreviations of the amino acid names are: Ala(A), Arg(R), Asn(N), Asp(D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V);
  • Pro60 or 60P both represent proline at position 60.
  • the specific expression of the mutation described in this application is such as "Q92H/S", which means that Q at position 92 is replaced by H or replaced by S, and so on.
  • Thr48 of exon 1 of the wild-type TCR ⁇ chain constant region TRAC*01 is mutated to cysteine
  • exon 1 of the ⁇ chain constant region TRBC1*01 or TRBC2*01 is mutated to cysteine.
  • the Ser57 of Ser57 is mutated to cysteine to obtain a reference TCR.
  • the amino acid sequences of the reference TCR are the amino acid sequences shown in SEQ ID NO.11 and SEQ ID NO.12 respectively.
  • the cysteine residue after mutation Bases are represented by bold letters.
  • the above-mentioned cysteine substitution can form an artificial inter-chain disulfide bond between the constant regions of the ⁇ and ⁇ chains of the reference TCR to form a more stable soluble TCR, thereby making it easier to evaluate the complex between TCR and GVYDGREHTV-HLA A0201 Binding affinity and/or binding half-life between substances. It should be understood that the CDR region of the TCR variable region determines the affinity between it and the pMHC complex. Therefore, the above-mentioned cysteine substitution of the TCR constant region will not affect the binding affinity and/or binding half-life of the TCR.
  • the measured binding affinity between the reference TCR and the GVYDGREHTV-HLA A0201 complex is considered to be the binding affinity between the wild-type TCR and the GVYDGREHTV-HLA A0201 complex.
  • the measured binding affinity between the TCR of the present application and the GVYDGREHTV-HLA A0201 complex is at least 10 times that of the binding affinity between the reference TCR and the GVYDGREHTV-HLA A0201 complex, it is equivalent to the TCR of the present application and GVYDGREHTV -The binding affinity between the HLA A0201 complex is at least 10 times greater than the binding affinity between the wild-type TCR and the GVYDGREHTV-HLA A0201 complex.
  • Binding affinity (inversely proportional to the dissociation equilibrium constant KD) and binding half-life (expressed as T1/2) can be determined by any suitable method, such as detection using surface plasmon resonance technology. It should be understood that doubling the affinity of the TCR will result in the KD being halved. T1/2 is calculated as In2 divided by the off-rate (Koff). Therefore, doubling T1/2 will cause Koff to be halved.
  • the binding affinity or binding half-life of a given TCR is measured several times, for example three or more times, using the same experimental protocol, and the results are averaged.
  • the surface plasmon resonance (BIAcore) method in the embodiments of this article is used to detect the affinity of soluble TCR.
  • the conditions are: the temperature is 25°C and the pH value is 7.1-7.5.
  • This method detects that the dissociation equilibrium constant KD of the reference TCR for the GVYDGREHTV-HLA A0201 complex is 1.14E-04M, which is 114.00 ⁇ M.
  • the dissociation equilibrium constant of the wild-type TCR for the GVYDGREHTV-HLA A0201 complex is considered The KD is also 114.00 ⁇ M.
  • the affinity of the GVYDGREHTV-HLA A0201 complex is 10 times greater than the affinity of the wild-type TCR for the GVYDGREHTV-HLA A0201 complex.
  • the affinity of the TCR to the GVYDGREHTV-HLA A0201 complex is at least 5 times that of the wild-type TCR.
  • Mutations may be performed using any suitable method, including, but not limited to, those based on polymerase chain reaction (PCR), restriction enzyme based cloning, or ligation independent cloning (LIC) methods. These methods are detailed in many standard molecular biology texts. Polymerase chain reaction (PCR) More details on mutagenesis and cloning based on restriction enzymes can be found in Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (3rd ed.) CSHL Publishing. More information on the LIC method can be found in (Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6).
  • PCR polymerase chain reaction
  • LIC ligation independent cloning
  • the method of generating the TCR of the present application can be, but is not limited to, screening out TCRs with high affinity for the GVYDGREHTV-HLA A0201 complex from a diversity library of phage particles displaying such TCRs, as described in the literature (Li, et al ( 2005) Nature Biotech 23(3):349-354).
  • genes expressing the amino acids of the ⁇ and ⁇ chain variable domains of wild-type TCR or genes expressing the amino acids of the ⁇ and ⁇ chain variable domains of slightly modified wild-type TCR can be used to prepare template TCRs.
  • the changes required to produce the high affinity TCR of the present application are then introduced into the DNA encoding the variable domain of the template TCR.
  • the high-affinity TCR of the present application includes an ⁇ chain variable domain amino acid sequence that is one of SEQ ID NO. 13-32; and/or the ⁇ chain variable domain amino acid sequence of the TCR is SEQ ID NO. 2.
  • the amino acid sequences of the ⁇ chain variable domain and ⁇ chain variable domain of the heterodimeric TCR molecule described in this application are preferably from Table 4.
  • a TCR of the present application is a portion having at least one TCR ⁇ and/or TCR ⁇ chain variable domain. They usually contain both TCR ⁇ chain variable domains and TCR ⁇ chain variable domains. They can be ⁇ heterodimers or single-chain forms or any other form that can exist stably.
  • the full-length chain of ⁇ heterodimeric TCR (including cytoplasmic and transmembrane domains) can be transfected.
  • the TCR of the present application can be used as a targeting agent to deliver therapeutic agents to antigen-presenting cells or combined with other molecules to prepare bifunctional polypeptides to target effector cells.
  • the TCR is preferably in a soluble form.
  • the TCR of the present application may be a TCR in which artificial interchain disulfide bonds are introduced between residues in the constant domains of its ⁇ and ⁇ chains.
  • Cysteine residues form artificial interchain disulfide bonds between the ⁇ and ⁇ chain constant domains of the TCR. Cysteine residues can replace other amino acid residues at appropriate positions in the native TCR to form artificial interchain disulfide bonds.
  • cysteine residue replaces any set of positions in the constant domains of the above-mentioned ⁇ and ⁇ chains.
  • Deletion of native can be achieved by truncating up to 15, or up to 10, or up to 8 or less amino acids at one or more C termini of the TCR constant domain of the present application so that it does not include cysteine residues.
  • the purpose of interchain disulfide bonds can also be achieved by mutating the cysteine residue that forms the natural interchain disulfide bond to another amino acid.
  • the TCR of the present application may contain artificial interchain disulfide bonds introduced between residues in the constant domains of its ⁇ and ⁇ chains. It should be noted that the TCR of the present application may contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the introduced artificial disulfide bonds described above.
  • the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence can be linked by natural interchain disulfide bonds present in the TCR.
  • the patent document PCT/CN2016/077680 also discloses that the introduction of artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region of TCR can significantly improve the stability of TCR. Therefore, the high-affinity TCR of the present application may also contain artificial inter-chain disulfide bonds between the ⁇ -chain variable region and the ⁇ -chain constant region. Specifically, the cysteine residues that form an artificial interchain disulfide bond between the alpha chain variable region and the beta chain constant region of the TCR are replaced with:
  • the TCR may comprise: (i) all or part of the TCR ⁇ chain except its transmembrane domain, and (ii) all or part of the TCR ⁇ chain except its transmembrane domain, wherein (i) and (i) ii) Both contain the variable domain of the TCR chain and at least part of the constant domain, and the ⁇ chain and ⁇ chain form a heterodimer.
  • the 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 an ⁇ chain constant domain, and the ⁇ chain of the TCR
  • the chain variable domain forms a heterodimer with the ⁇ chain.
  • the TCR of the present application also includes TCRs with mutations in its hydrophobic core region.
  • the mutations in the hydrophobic core region are preferably mutations that can improve the stability of the TCR of the present application, as described in Publication No. It is described in the patent document WO2014/206304.
  • the TCR can be mutated at the following hydrophobic core positions of its variable domain:
  • IMGT International Immunogenetic Information System
  • the TCR with mutations in the hydrophobic core region in this application can be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the ⁇ chain and ⁇ chain of the TCR.
  • the CDR region of the TCR variable region determines its affinity with short peptide-HLA complexes. Mutation of the hydrophobic core can make the TCR more stable, but does not affect its affinity with short peptide-HLA complexes.
  • the flexible peptide chain in this application can be any peptide chain suitable for connecting the variable domains of TCR ⁇ and ⁇ chain.
  • the template chain constructed in Example 1 of the present application for screening high-affinity TCRs is the above-mentioned high-stability single-chain TCR containing hydrophobic core mutations. Using TCR with higher stability can more conveniently evaluate the affinity between TCR and GVYDGREHTV-HLA A0201 complex.
  • the CDR regions of the ⁇ chain variable domain and ⁇ chain variable domain of the single-chain template TCR are identical to the CDR regions of wild-type TCR. That is, the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : DSSSTY; CDR2 ⁇ : IFSNMDM; CDR3 ⁇ : AEQSFGNEKLT and the three CDRs of the ⁇ chain variable domain are CDR1 ⁇ : MNHEY; CDR2 ⁇ : SVGEGT; CDR3 ⁇ : ASSLGRAYEQY.
  • the amino acid sequence of the single-stranded template TCR is the amino acid sequence shown in SEQ ID NO. 9, and the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO. 10. In this way, single-chain TCRs composed of ⁇ -chain variable domains and ⁇ -chain variable domains with high affinity for the GVYDGREHTV-HLA A0201 complex were screened out.
  • the ⁇ heterodimer with high affinity for the GVYDGREHTV-HLA A0201 complex of the present application is obtained by transferring the CDR regions of the ⁇ and ⁇ chain variable domains of the screened high-affinity single-chain TCR to Obtained from the corresponding positions of the wild-type TCR ⁇ chain variable domain (SEQ ID NO.1) and ⁇ chain variable domain (SEQ ID NO.2).
  • the TCR of the present application can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present application includes any two or at least three (for example, it can be two, three, four or more) polymers formed by combining the TCR of the present application, such as p53. tetramerization domain to produce a tetramer, or a complex formed by combining multiple TCRs of the present application with another molecule.
  • the TCR complex 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, or can be combined with a conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate includes any one of a detection marker (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the GVYDGREHTV-HLA A0201 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or at least A combination of two (the combination includes combining or coupling).
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing a detectable product of enzymes.
  • Therapeutic agents that can be combined or coupled to the TCR of the present application include, but are not limited to:
  • Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539);
  • Cytokines such as IL-2 (Gillies et al., 1992, Proceedings of the National Academy of Sciences (PNAS) 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 53, 345; Halin et al. , 2003, Cancer Research (Cancer Research) 63, 3202);
  • Prodrug-activating enzymes eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)
  • Chemotherapeutic agents eg, cisplatin or any form of nanoparticles, etc.
  • Antibodies or fragments thereof that bind to the TCR of the present application include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies.
  • the combination of the above antibodies or their fragments with the TCR can affect effector cells. Orientation to better target target cells.
  • a preferred embodiment is that the TCR of the present application is combined with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody.
  • the fusion molecule of the TCR and the anti-CD3 single chain antibody of the present application includes one of the amino acid sequences of the variable domain of the TCR ⁇ chain selected from SEQ ID NO. 13-32; and/or the amino acids of the ⁇ chain variable domain of the TCR. The sequence is SEQ ID NO.2.
  • the present application also relates to nucleic acid molecules encoding the TCRs of the present application.
  • the nucleic acid molecules of the present application may be in the form of DNA or RNA.
  • DNA can be a coding strand or a non-coding strand.
  • the nucleic acid sequence encoding the TCR of the present application may be the same as or a degenerate variant of the nucleic acid sequence shown in the drawings of the present application.
  • degenerate variant refers to a protein sequence encoding SEQ ID NO.3, but different from the sequence of SEQ ID NO.5 Different nucleic acid sequences.
  • 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 described in this application can be obtained entirely through chemical synthesis.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • the present application also relates to vectors comprising the nucleic acid molecules described in the present application, as well as host cells genetically engineered using the vectors or coding sequences of the present application.
  • the present application also includes isolated cells, particularly T cells, expressing the TCRs described herein.
  • isolated cells particularly T cells, expressing the TCRs described herein.
  • T cell transfection with DNA or RNA encoding the high-affinity TCR of the present application eg, Robbins et al., (2008) J. Immunol. 180:6116-6131.
  • T cells expressing the high-affinity TCR described in this application can be used for adoptive immunotherapy.
  • Those skilled in the art will be aware of many suitable methods for conducting adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
  • the present application also provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR described in the present application, the TCR complex described in the present application, or a cell presenting the TCR described in the present application.
  • This application also provides a method for treating diseases, which includes administering an appropriate amount of the TCR described in this application, the TCR complex described in this application, the cells presenting the TCR described in this application, or the pharmaceutical composition of this application to a subject in need of treatment. .
  • the TCR of the present application also includes at most 5, preferably at most 3, more preferably at most 2, and optimally 1 amino acid (especially the amino acid located outside the CDR region) of the TCR of the present application, with similar properties. Or a TCR that is replaced with a similar amino acid and still maintains its functionality.
  • This application also includes a slightly modified TCR of the TCR of this application.
  • Modified forms (generally without changing the primary structure) include: chemically derivatized forms of the TCR of the present application such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation modifications during the synthesis and processing of the TCRs of the present application or during further processing steps. This modification can be accomplished by exposing the TCR to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences having phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to increase their resistance to proteolysis or to optimize their solubility properties.
  • the TCR, TCR complex described herein, or the TCR-transfected T cell described herein can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCR, the multivalent TCR complex or the cell described herein are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It is available in unit dosage form, usually in sealed containers, and may be provided as part of a kit. Such kits (but not required) include instructions for use. It may include a plurality of such unit dosage forms.
  • the TCR of the present application can be used alone, or can be used in combination or coupling with other therapeutic agents (such as formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent. body.
  • the carrier refers to pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the pharmaceutical composition and do not cause undue toxicity after administration.
  • These vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).
  • Such carriers include (but are not limited to): any one or a combination of at least two of saline, buffer, glucose, water, glycerol, ethanol or adjuvants.
  • Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerin, and ethanol.
  • the carrier may also contain auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc.
  • the therapeutic compositions may be prepared as injectables, such as liquid solutions or suspensions; they may also be prepared in solid forms suitable for solution or suspension in liquid vehicles prior to injection.
  • compositions of the present application may be administered by conventional routes, including but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal External including subcutaneous, intramuscular or intravenous.
  • the subject to be prevented or treated can be an animal; especially a human.
  • compositions in various dosage forms can be used according to the usage conditions.
  • injections, oral agents, etc. can be exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and cosolvents, and the preparation process can be carried out in a conventional manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and cosolvents, and the preparation process can be carried out in a conventional manner according to the dosage form.
  • the pharmaceutical composition of the present application can also be administered in the form of a sustained-release preparation.
  • the TCR described herein can be incorporated into a pellet or microcapsule using a sustained-release polymer as a carrier, and then the pellet or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers include ethylene-vinyl acetate copolymer, polyhydroxymethacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, Lactic acid-glycolic acid copolymers and the like, preferably biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers can be exemplified.
  • the TCR or TCR complex of the present application as the active ingredient or the cells presenting the TCR of the present application can be determined according to the weight, age, gender, and symptom severity of each patient to be treated. It is determined rationally, and ultimately it is up to the physician to determine the reasonable dosage.
  • the affinity and/or binding half-life of the high-affinity TCR of the present application for the GVYDGREHTV-HLA A0201 complex is at least 5 times that of the wild-type TCR.
  • the high-affinity TCR of the present application can specifically bind to the GVYDGREHTV-HLA A0201, and at the same time, cells transfected with the high-affinity TCR of the present application can be specifically activated.
  • E.coli DH5 ⁇ is purchased from Tiangen
  • E.coli BL21 (DE3) is purchased from Tiangen
  • E.coli Tuner (DE3) is purchased from Tiangen
  • Plasmid pET28a was purchased from Novagen.
  • This application uses the method of site-directed mutagenesis and constructs a stable single-chain TCR molecule composed of a flexible short peptide (linker) connecting TCR ⁇ and ⁇ chain variable domains, and its amino acids and DNA are constructed according to the patent document WO2014/206304.
  • the sequences are the sequences shown in SEQ ID NO.9 and SEQ ID NO.10 respectively.
  • the ⁇ chain variable domain of the template chain is the amino acid sequence shown in SEQ ID NO.3, and the ⁇ chain variable domain is the amino acid sequence shown in SEQ ID NO.4; their corresponding DNA sequences are SEQ ID NO.5 respectively.
  • the nucleotide sequence shown in SEQ ID NO.6; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 respectively.
  • the target gene carrying the template strand is double digested by Nco I and Not I, and ligated with the pET28a vector that has been double digested by Nco I and Not I.
  • the ligation product is transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, and incubated overnight at 37°C. Positive clones are selected for PCR screening, and the positive recombinants are sequenced. After confirming that the sequence is correct, the recombinant plasmid is extracted and transformed. to E. coli BL21(DE3) for expression.
  • the protein was purified using a 0-1M NaCl linear gradient eluent prepared with 20mM Tris-HCl pH8.0. , collected elution The fractions were analyzed by SDS-PAGE. The fractions containing single-chain TCR were concentrated and further purified using a gel filtration column (Superdex 75 10/300, GE Healthcare). The target fraction was also analyzed by SDS-PAGE.
  • the eluted fractions used for BIAcore analysis were further tested for purity using gel filtration.
  • the conditions are: chromatographic 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.
  • BIAcore T200 real-time analysis system to detect the binding activity of TCR molecules and GVYDGREHTV-HLA A0201 complex.
  • the coupling level is approximately 15,000RU.
  • the conditions are: temperature 25°C, pH value 7.1-7.5.
  • Flow low-concentration streptavidin through the antibody-coated chip surface then flow the GVYDGREHTV-HLA A0201 complex through the detection channel, and use the other channel as the reference channel, and then add 0.05mM biotin at 10 ⁇ L/min.
  • the flow rate flows through the chip for 2 minutes to block the remaining binding sites of streptavidin.
  • a single cycle kinetic analysis method was used to determine its affinity.
  • the TCR was diluted into several different concentrations with HEPES-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20, pH7.4), and the concentration was 30 ⁇ L/min.
  • Refolding Dissolve the synthesized short peptide GVYDGREHTV (Jiangsu GenScript Biotechnology Co., Ltd.) 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.
  • GVYDGREHTV peptide to the refolding buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 25mg/L (final concentration), 0.2mM PMSF, cooled to 4°C), then add 20mg/L light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain is added in three times, 8h/time), renaturation is carried out at 4°C for at least 3 days Upon completion, SDS-PAGE will test whether the renaturation can be successful.
  • the refolding buffer 0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 25mg/L (final concentration), 0.2mM PMSF, cooled to 4°C
  • 20mg/L light chain and 90mg/L heavy chain in sequence final concentration
  • Biotinylation Use Millipore ultrafiltration tube to concentrate the purified pMHC molecules, and replace the buffer with 20mM Tris pH 8.0, then add biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50 ⁇ M D- Biotin, 100 ⁇ g/mL BirA enzyme (GST-BirA), incubate the mixture at room temperature overnight, and check whether biotinylation is complete by SDS-PAGE.
  • the binding curve of the soluble reference TCR that is, wild-type TCR, and the GVYDGREHTV-HLA A0201 complex was measured.
  • the detection results are shown in Figure 1.
  • Phage display technology is a means to generate TCR high-affinity variant libraries for screening high-affinity variants.
  • the TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) was applied to the single-chain TCR template in Example 1.
  • a high-affinity TCR library is established and panned. After several rounds of panning, the phage library has specific binding to the corresponding antigen, and single clones are selected and analyzed.
  • the CDR region mutation of the screened high-affinity single-chain TCR was introduced into the corresponding site of the variable domain of the ⁇ heterodimeric TCR, and its affinity with the GVYDGREHTV-HLA A0201 complex was detected by BIAcore.
  • the above-mentioned high-affinity mutation points in the CDR region are introduced using site-directed mutagenesis methods that are well known to those skilled in the art.
  • the amino acid sequences of the ⁇ chain and ⁇ chain variable domains of the wild-type TCR are the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.
  • the ⁇ heterodimeric TCR can be a constant in the ⁇ and ⁇ chains.
  • a cysteine residue is introduced into the TCR region to form an artificial inter-chain disulfide bond.
  • the amino acid sequences of the TCR ⁇ and ⁇ chains after the cysteine residue is introduced are as follows: SEQ ID NO.11 and the amino acid sequence shown in SEQ ID NO. 12, the introduced cysteine residues are represented by bold and underlined letters.
  • the extracellular sequence genes of TCR ⁇ and ⁇ chains to be expressed are synthesized and inserted into the expression vector respectively through the standard method described in "Molecular Cloning a Laboratory Manual” (3rd edition, Sambrook and Russell) pET28a+ (Novagene), the upstream and downstream cloning sites are Nco I and Not I respectively. Mutations in the CDR regions are introduced by overlap PCR, which is well known to those skilled in the art. The insert was confirmed to be correct by sequencing.
  • the expression vectors of TCR ⁇ and ⁇ chains were transformed into expression bacteria BL21 (DE3) through chemical transformation.
  • the ⁇ and ⁇ chains of TCR were expressed.
  • the inclusion bodies formed were extracted with BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution.
  • the inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), and 10mM ethylenediaminetetraacetic acid (EDTA). ), in 20mM Tris (pH8.1).
  • the dissolved TCR ⁇ and ⁇ chains were quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH8.1), 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1. The concentration is 60mg/mL.
  • the solution was dialyzed in 10 times the volume of deionized water (4°C). After 12 hours, the deionized water was replaced with buffer (20mM Tris, pH 8.0) and continued to be dialyzed at 4°C for 12 hours. After dialysis, the solution was filtered through a 0.45 ⁇ M membrane and purified through an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare).
  • TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as measured by SDS-PAGE, and the concentration was determined by the BCA method.
  • Example 3 The method described in Example 3 was used to detect the affinity of the ⁇ heterodimeric TCR introduced into the high-affinity CDR region and the GVYDGREHTV-HLA A0201 complex.
  • the affinity of the heterodimeric TCR is at least 5 times the affinity of the wild-type TCR for the GVYDGREHTV-HLA A0201 complex.
  • the anti-CD3 single-chain antibody (scFv) is fused to ⁇ heterodimeric TCR to prepare a fusion molecule.
  • the anti-CD3 scFv is fused to the ⁇ chain of the TCR.
  • the TCR ⁇ chain can include any of the ⁇ chain variable domains of the above-mentioned high-affinity ⁇ heterodimeric TCR.
  • the TCR ⁇ chain of the fusion molecule can include any of the above-mentioned high-affinity ⁇ heterodimeric TCRs.
  • ⁇ chain expression vector The target gene carrying the ⁇ chain of ⁇ heterodimeric TCR is double digested by NcoI and NotI, and connected to the pET28a vector that has been double digested by NcoI and NotI.
  • the ligation product is transformed into E.coli DH5 ⁇ , spread on an LB plate containing kanamycin, and incubated overnight at 37°C. Positive clones are picked for PCR screening, and the positive recombinants are sequenced. After confirming that the sequence is correct, the recombinant plasmid is extracted. Transformed into E.coli Tuner(DE3) for expression.
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, spread on LB plates containing kanamycin, and incubated overnight at 37°C. Positive clones were picked for PCR screening, and the positive recombinants were sequenced to confirm that the sequence was correct and then extracted.
  • the recombinant plasmid was transformed into E.coli Tuner (DE3) competent cells for expression.
  • the expression plasmids were transformed into E.coli Tuner (DE3) competent cells, and the cells were coated on LB plates (kanamycin 50 ⁇ g/mL) and cultured at 37°C overnight. The next day, pick the clone and inoculate it into 10mL LB liquid medium (kanamycin 50 ⁇ g/mL) and culture it for 2-3 hours, inoculate it into 1L LB medium at a volume ratio of 1:100, and continue to culture until the OD 600 is 0.5-0.8. Add a final concentration of 1mM IPTG to induce the expression of the target protein. After 4 hours of induction, cells were harvested by centrifugation at 6000 rpm for 10 min. Wash the cells once with PBS buffer and aliquot the cells.
  • the dissolved TCR ⁇ chain and anti-CD3 (scFv)- ⁇ chain were quickly mixed with 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH8.1 at a mass ratio of 2:5. 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C), final concentrations of ⁇ chain and anti-CD3 (scFv)- ⁇ chain are 0.1mg/mL and 0.25mg/mL respectively.
  • the solution was dialyzed in 10 times the volume of deionized water (4°C). After 12 hours, the deionized water was replaced with buffer (10mM Tris, pH 8.0) and continued dialysis at 4°C for 12 hours. After dialysis, the solution was filtered through a 0.45 ⁇ M membrane and purified through an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The elution peak containing TCR of successfully renatured TCR ⁇ chain and anti-CD3(scFv)- ⁇ chain dimer was confirmed by SDS-PAGE gel.
  • TCR fusion molecules were then further purified by size exclusion chromatography (S-100 16/60, GE healthcare) and again by an anion exchange column (HiTrap Q HP 5ml, GE healthcare).
  • the purity of the purified TCR fusion molecule was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • IFN- ⁇ is a powerful immune regulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN- ⁇ is detected through the ELISPOT experiment well known to those skilled in the art to verify the activation of cells transfected with the high-affinity TCR of the present application. Function and antigen specificity.
  • the high-affinity TCR of this application (TCR number and its sequence number are obtained from Table 4) were transfected into CD3 + T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteers were used to transfect other TCRs (A6 ) or CD3 + T cells transfected with wild-type TCR (WT-TCR) were used as controls.
  • the target cells used were T2 loaded with MAGE-A4 antigen short peptide GVYDGREHTV, T2 loaded with other antigen short peptides, or empty T2.
  • ELISPOT plate First prepare the ELISPOT plate. First, add 1 ⁇ 10 4 cells/well for target cells and 2 ⁇ 10 3 cells/well for effector cells (calculated according to the positive transfection rate) into the corresponding wells, and then add MAGE-A4 antigen to the experimental group. Short peptide GVYDGREHTV solution, adding other antibodies to the control group The original short peptide solution was used, and the final concentration of the short peptide was 10-6M. An equal volume of culture medium was added to the blank group, and two duplicate wells were set up. Incubate overnight (37°C, 5% CO2 ). On the second day of the experiment, the plates were washed and secondary detection and color development were performed. The plates were dried and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 Company).
  • ELISPOT READER system AID20 Company
  • the effector cells transfected with the high-affinity TCR of the present application have a more obvious activation effect than the effector cells transfected with the wild-type TCR.
  • Effector cells transfected with other TCRs are inactive; at the same time, effector cells transfected with the TCR of the present application are basically inactive against target cells loaded with other antigen short peptides or empty.
  • GLYDGREHSV is a short peptide derived from MAGE-A8.
  • the target cells were T2 loaded with the MAGE-A8 antigen short peptide GLYDGREHSV, T2 loaded with other short antigen peptides, or empty T2, and the effector cells were CD3 + T cells transfected with the high-affinity TCR of the present application.
  • the effector cells transfected with the high-affinity TCR of the present application have a more obvious activation effect than the effector cells transfected with the wild-type TCR.
  • Effector cells transfected with other TCRs are basically inactive; at the same time, effector cells transfected with the TCR of the present application are basically inactive against target cells loaded with other antigen short peptides or empty.
  • This example uses tumor cell lines to once again verify the activation function and specificity of effector cells transfected with the high-affinity TCR of the present application.
  • the detection is also carried out through the ELISPOT experiment, which is well known to those skilled in the art.
  • the high-affinity TCR of the present application is transfected into CD3 + T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteers are transfected with other TCRs (A6) or wild-type TCRs (WT-TCRs). ) CD3 + T cells were used as controls.
  • the tumor cell lines used in the examples are NCI-H1299, A375, U-2OS, 293T, HT1080, and NUGC4 respectively.
  • NCI-H1299 was purchased from the Cell Bank of the Chinese Academy of Sciences
  • A375, 293T, and HT1080 were purchased from ATCC
  • U-2OS was purchased from Guangzhou Saiku Biotechnology Co., Ltd.
  • NUGC4 was purchased from Sciencell. The following experiments are conducted in two batches (I) and (II):
  • the high affinity TCRs can be learned from Table 4 and are respectively TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR9, TCR10, TCR11, TCR12, TCR13, TCR14, TCR15, TCR16, TCR19 and TCR20.
  • the MAGE-A4 positive tumor cell lines used in this batch are NCI-H1299-A0201 (HLA-A0201 overexpression), A375 (MAGE-A4 positive), U-2OS (MAGE-A4, A8 double positive) and 293T-MAGE -A4 (MAGE-A4 overexpression), negative cell lines are 293T and HT1080.
  • the high affinity TCRs can be learned from Table 4, which are TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR8, TCR9, TCR10, TCR11, TCR12, TCR13, TCR14, TCR15, TCR16, TCR17, TCR19 and TCR20.
  • the MAGE-A4 positive tumor cell lines used in this batch are U-2OS (MAGE-A4 and A8 double positive) and 293T-MAGE-A4 (MAGE-A4 overexpression), and the negative cell lines are 293T and NUGC4.
  • ELISPOT plates are ethanol-activated and coated at 4°C overnight.
  • the plates were washed and secondary detection and color development were performed. The plates were dried and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 Company).
  • Lactate dehydrogenase is abundant in the cytoplasm and cannot pass through the cell membrane normally. It can be released outside the cell when the cell is damaged or dead. At this time, the LDH activity in the cell culture medium is proportional to the number of cell death.
  • This example also measures the release of LDH through non-radioactive cytotoxicity experiments well known to those skilled in the art, thereby verifying the killing function of cells transfected with the TCR of the present application.
  • CD3 + T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of the application as effector cells, and the same volunteers were used to transfect CD3 + T cells with other TCR (A6) as controls. .
  • the tumor cell lines used in the examples are U2-OS, A375, 293T, NUGC4 and MS751 respectively.
  • U-2OS and MS751 were purchased from Guangzhou Saiku Biotechnology Co., Ltd.
  • A375 and 293T were purchased from ATCC
  • NUGC4 was purchased from Sciencell.
  • the following experiments are conducted in two batches (I) and (II):
  • the high-affinity TCRs can be learned from Table 4 and are TCR5, TCR17 and TCR20 respectively.
  • the MAGE-A4 positive tumor cell lines used in this batch are U-2OS (MAGE-A4 and A8 double positive) and A375 (MAGE-A4 positive), and the negative tumor cell line is 293T.
  • the high-affinity TCRs can be learned from Table 4 and are TCR3, TCR5 and TCR19 respectively.
  • the MAGE-A4 positive tumor cell lines used in this batch are U-2OS (MAGE-A4 and A8 double positive) and 293T-MAGE-A4 (293T cells transfected with MAGE-A4), and the negative tumor cell lines are NUGC4 and MS751 and 293T.
  • This example uses human normal tissue cells to verify the specificity of effector cells that transduce the high-affinity TCR of the present application.
  • the detection is also carried out through the ELISPOT experiment, which is well known to those skilled in the art.
  • the high-affinity TCR of this application (the TCR number and its sequence number are obtained from Table 4) are transduced by lentivirus into CD3 + T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteers are used to transduce other TCR (A6) or null transduced (NC) CD3+ T cells served as controls.
  • the positive tumor cell line used in the experiment was US-OS (MAGE-A4, A8 double-positive cells), and the human normal tissue cells used were: human lung fibroblasts (HPF-a), human aortic endothelial cells (HAEC-2A) , human cardiac fibroblasts (HCF-4A), human aortic smooth muscle cells (HASMC-6B), human renal mesangial cells (HRMC-11A), human gastric smooth muscle cells (HGSMC-13B), human renal epithelial cells (HREpiC- 14A), human bladder stromal fibroblasts (HBdSF-39), human choroidal longitudinal myofibroblasts (HCPF-20), human esophageal fibroblasts (HEF-27), human breast fibroblasts (HMF-40), Human spleen fibroblasts (HSF-17) and human brain vascular adventitial fibroblasts (HBVAF-22).
  • the human normal tissue cells used were purchased from Sciencell.
  • ELISPOT plates are ethanol-activated and coated at 4°C overnight.
  • the plates were washed and secondary detection and color development were performed. The plates were dried and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 Company).
  • the experimental results are shown in Figure 5.
  • the effector cells transduced with the high-affinity TCR of the present application are basically inactive against human normal tissue cells, further verifying the high specificity of the TCR of the present application.
  • the T cells transfected with the high-affinity TCR of the present application were injected into mice of the human melanoma xenograft model, and their in vivo tumor inhibitory effects were detected.
  • mice Female, experimental age 6-8 weeks
  • A375 tumor cell (ATCC) suspension 1* was collected and suspended after culture.
  • 10 ⁇ 7 mice/mouse were injected subcutaneously on one side of the abdomen to establish a human melanoma xenograft mouse model.
  • TCR-T cell injection After TCR-T cell injection, take the prepared IL-2 solution (50000IU) and inject 200 ⁇ l into each mouse intraperitoneally, and then do it every day for the next 4 days. Equal volumes of IL-2 solution were injected continuously. From the beginning of the experiment, the mouse tumor diameter and calculated volume were measured every 2 days according to the above method, and continued until the mice were affected by excessive tumors or the tumors receded. The above data were compiled and statistically analyzed and processed for the tumor volume data of each group of mice. .

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Abstract

提供一种识别MAGE-A4抗原的高亲和力TCR及其序列和应用，TCR具有结合GVYDGREHTV-HLA A0201复合物的特性；还提供了一种多价TCR复合物、编码TCR的核酸分子、包含核酸分子的载体、表达TCR的细胞以及包含前述物质的药物组合物及其制备方法。

Description

一种识别MAGE-A4抗原的高亲和力TCR及其序列和应用
相关申请
本申请要求2022年03月24日申请的，申请号为202210298377.4，名称为“一种识别MAGE-A4抗原的高亲和力TCR及其序列和应用”的中国专利申请的优先权，在此将其全文引入作为参考。
技术领域
本发明涉及生物技术领域，更具体地涉及能够识别衍生自MAGE-A4蛋白多肽的T细胞受体(T cell receptor，TCR)。本发明还涉及所述受体的制备和用途。
背景技术
仅仅有两种类型的分子能够以特异性的方式识别抗原。其中一种是免疫球蛋白或抗体；另一种是T细胞受体(TCR)，它是由α链/β链或者γ链/δ链以异二聚体形式存在的细胞膜表面的糖蛋白。免疫***的TCR总谱的组成是在胸腺中通过V(D)J重组，然后进行阳性和阴性选择而产生的。在外周环境中，TCR介导了T细胞对主组织相容性复合体-肽复合物(pMHC)的特异性识别，因此其对免疫***的细胞免疫功能是至关重要的。
TCR是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的唯一受体，这种外源肽或内源肽可能会是细胞出现异常的唯一迹象。在免疫***中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
与TCR相对应的MHC I类和II类分子配体也是免疫球蛋白超家族的蛋白质但对于抗原的呈递具有特异性，不同的个体有不同的MHC，从而能呈递一种蛋白抗原中不同的短肽到各自的APC细胞表面。人类的MHC通常称为HLA基因或HLA复合体。
MAGE蛋白家族具有与其他MAGE蛋白的序列紧密匹配的同源区，并且包含在免疫识别中展现为HLA/肽复合物的肽。一些MAGE基因家族蛋白(MAGE-A至MAGE-C家族)仅在生殖细胞和癌症中表达；其他(MAGE-D至MAGE-H)在正常组织中广泛表达。MAGE-A4作为一种内源性肿瘤抗原，在细胞内生成后被降解成小分子多肽，并与MHC(主组织相容性复合体)分子结合形成复合物，被呈递到细胞表面。研究显示，GVYDGREHTV是衍生自MAGE-A4的短肽。MAGE-A4蛋白在多种肿瘤类型中均有表达，包括黑色素瘤，以及其他固体肿瘤如胃癌、肺癌、食道癌、膀胱癌、头颈部鳞状细胞癌等。GLYDGREHSV是衍生于MAGE-A8的短肽，与衍生自MAGE-A4的短肽GVYDGREHTV具有高度同一性。对于上述疾病的治疗，可以采用化疗和放射性治疗等方法，但都会对自身的正常细胞造成损害。
因此，GVYDGREHTV-HLA A0201复合物提供了一种TCR可靶向肿瘤细胞的标记。能够结合GVYDGREHTV-HLA A0201复合物的TCR对肿瘤的治疗具有很高的应用价值。例如，能够靶向该肿瘤细胞标记的TCR可用于将细胞毒性剂或免疫刺激剂递送到靶细胞，或被转化入T细胞，使表达该TCR的T细胞能够破坏肿瘤细胞，以便在被称为过继免疫治疗的治疗过程中给予患者。对于前一目的，理想的TCR是具有较高的亲和力的，从而使该TCR能够长期驻留在所靶向的细胞上面。对于后一目的，则优选使用中等亲和力的TCR。因此，本领域技术人员致力于开发可用于满足不同目的的靶向肿瘤细胞标记的TCR。
发明内容
根据本申请的各实施例，本申请的第一方面，提供一种TCR，技术方案为一种包含了α链可变域和β链可变域的TCR，所述TCR具有结合GVYDGREHTV-HLA A0201复合物的活性；
并且所述TCRα链可变域的氨基酸序列与SEQ ID NO.1所示的氨基酸序列有至少90％的序列同一性，和所述TCRβ链可变域的氨基酸序列与SEQ ID NO.2所示的氨基酸序列有至少90％的序列同一性。
SEQ ID NO.1：

SEQ ID NO.2：
在一优选例中，所述TCRα链可变域的氨基酸序列和所述TCRβ链可变域的氨基酸序列不同时为野生型TCRα链可变域的氨基酸序列和野生型TCRβ链可变域的氨基酸序列。
在进一步的优选例中，所述TCRα链可变域的氨基酸序列不是SEQ ID NO.1所示的氨基酸序列。
在另一优选例中，所述TCR的α链可变域包含与SEQ ID NO.1所示的序列具有至少91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，或相对于SEQ ID NO.1所示的序列，有1、2、3、4、5、6、7、8、9、10或11个氨基酸残基***、缺失、替换或其组合。
在另一优选例中，所述TCR的β链可变域为与SEQ ID NO.2所示的序列有至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性的氨基酸序列，或相对于SEQ ID NO.2所示的序列，有1、2、3、4、5、6、7、8、9、10或11个氨基酸残基***、缺失、替换或其组合。
在另一优选例中，所述TCRα链可变域的氨基酸序列与SEQ ID NO.1所示的氨基酸序列有至少95％的序列同一性，和所述TCRβ链可变域的氨基酸序列与SEQ ID NO.2所示的氨基酸序列有至少95％的序列同一性。
在另一优选例中，所述TCR还具有结合GLYDGREHSV-HLA A0201复合物的活性。
在另一优选例中，所述TCRα链可变域中CDR1α为：DSSSTY(SEQ ID NO.37)，和CDR2α为IFSNMDM(SEQ ID NO.38)。
在另一优选例中，所述TCRα链可变域的CDR3α选自AEHTKQNEKLT(SEQ ID NO.39)、AWSQFGNEKLT(SEQ ID NO.40)、AVSNFGNEKLT(SEQ ID NO.41)和AASEFGNEKLT(SEQ ID NO.42)。
在另一优选例中，所述TCRβ链可变域的3个CDR为：
CDR1β：MNHEY                    SEQ ID NO.59；
CDR2β：SVGEGT                        SEQ ID NO.60；
和CDR3β：ASSLGRAYEQY           SEQ ID NO.61。
在另一优选例中，所述TCRβ链可变域的氨基酸序列为SEQ ID NO.2。
在另一优选例中，所述TCRα链可变域的氨基酸突变个数为3-4个，例如可以是3或4。
在另一优选例中，所述TCRα链可变域的氨基酸突变位点为CDR3α的第3位、第4位、第5位和第6位；或者CDR3α的第2位、第3位和第4位。
在另一优选例中，所述TCRα链可变域的3个CDR区(互补决定区)的基准序列如下，
CDR1α：DSSSTY                   SEQ ID NO.37
CDR2α：IFSNMDM                  SEQ ID NO.38
CDR3α：AEQSFGNEKLT              SEQ ID NO.62，
并且CDR3α含有至少一个表1中的突变：
表1
在另一优选例中，所述CDR3α中氨基酸突变包含表2中的突变：
表2
在另一优选例中，所述TCR与GVYDGREHTV-HLA A0201复合物的亲和力是野生型TCR的至少5倍。
在另一优选例中，所述TCR在SEQ ID NO.1所示的α链可变域中发生突变，所述突变选自E91W/V/A/T/S/H/Q/N/Y、Q92H/S、S93T/Q/N/E/D、F94K/R/H和G95Q中的一组或几组，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
在另一优选例中，所述TCR具有选自表3所示的CDR：
表3
αCDR3的序列如下：
SEQ ID NO.39：AEHTKQNEKLT
SEQ ID NO.40：AWSQFGNEKLT
SEQ ID NO.41：AVSNFGNEKLT
SEQ ID NO.42：AASEFGNEKLT
SEQ ID NO.43：AASTFGNEKLT
SEQ ID NO.44：AASQFGNEKLT
SEQ ID NO.45：AASQRGNEKLT
SEQ ID NO.46：AVSQFGNEKLT
SEQ ID NO.47：AVSDFGNEKLT
SEQ ID NO.48：AVSSFGNEKLT
SEQ ID NO.49：ATSTFGNEKLT
SEQ ID NO.50：ATSQFGNEKLT
SEQ ID NO.51：ATSDFGNEKLT
SEQ ID NO.52：ASSQFGNEKLT
SEQ ID NO.53：AWSSFGNEKLT
SEQ ID NO.54：AHSQFGNEKLT
SEQ ID NO.55：AQSQFGNEKLT
SEQ ID NO.56：ANSTFGNEKLT
SEQ ID NO.57：AYSQFGNEKLT
SEQ ID NO.58：AEHTHQNEKLT。
在另一优选例中，所述TCR是可溶的。
在另一优选例中，所述TCR为αβ异质二聚TCR，所述TCR包含α链TRAC恒定区序列，和β链TRBC1或TRBC2恒定区序列。
在另一优选例中，所述TCR包含：
(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的可变域和至少一部分恒定域。
在另一优选例中，所述TCR包含α链恒定区与β链恒定区，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，在所述TCRα与β链的恒定区之间形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
和TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
在另一优选例中，所述TCR的α链可变域氨基酸序列为SEQ ID NO.13-32之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO.2。
在另一优选例中，所述TCR选自表4中各组：
表4

在另一优选例中，所述TCR是人源的。
在另一优选例中，所述TCR是分离与纯化的。
在另一优选例中，所述TCR为单链TCR。
在另一优选例中，所述TCR是由α链可变域和β链可变域组成的单链TCR，所述α链可变域和β链可变域由一柔性短肽序列(linker)连接。
在另一优选例中，所述TCR包含α链恒定区与β链恒定区，所述α链恒定区为鼠的恒定区和/或所述β链恒定区为鼠的恒定区。
在另一优选例中，所述TCR包含α链恒定区与β链恒定区，所述α链恒定区为鼠源的恒定区和/或所述β链恒定区为鼠源的恒定区。
在另一优选例中，所述TCR的α链的C-或N-末端结合有偶联物，和/或所述TCR的β链的C-或N-末端结合有偶联物。
优选地，所述偶联物包括检测标记物、治疗剂或PK修饰部分中任意一种或至少两种的组合。
在另一优选例中，与所述TCR结合的治疗剂为连接于所述TCR的α链的C-或N-末端的抗-CD3抗体，或连接于所述TCR的β链的C-或N-末端的抗-CD3抗体。
根据本申请的各实施例，本申请的第二方面，提供了一种多价TCR复合物，所述多价TCR复合物包含任意两个或至少三个TCR，并且其中的至少一个TCR为第一方面所述的TCR。
根据本申请的各实施例，本申请的第三方面，提供了一种核酸分子，所述核酸分子包含编码本申请第一方面所述的TCR的核苷酸序列或本申请第一方面所述的TCR的核苷酸序列的互补序列；
或者本申请第二方面所述的多价TCR复合物的核酸序列或本申请第二方面所述的多价TCR复合物的核酸序列的互补序列。
根据本申请的各实施例，本申请的第四方面，提供了一种载体，所述的载体含有本申请第三方面所述的核酸分子。
根据本申请的各实施例，本申请的第五方面，提供了一种宿主细胞，所述的宿主细胞中含有本申请第四方面所述的载体，或所述宿主细胞的染色体中整合有外源的本申请第三方面所述的核酸分子。
根据本申请的各实施例，本申请的第六方面，提供了一种分离的细胞，所述细胞表达本申请第一方面所述的TCR分子。
优选地，所述分离的细胞包括T细胞、NK细胞或NKT细胞中任意一种或至少两种的组合。
最优选地，所述分离的细胞是T细胞。
根据本申请的各实施例，本申请的第七方面，提供了一种药物组合物，所述药物组合物含有药学上可接受的载体以及本申请第一方面所述的TCR、本申请第二方面所述的TCR复合物或本申请第六方面所述的分离的细胞。
根据本申请的各实施例，本申请的第八方面，提供了第一方面中所述的TCR、第二方面所述的TCR复合物、第六方面所述的分离的细胞或第七方面所述的药物组合物在MAGEA4阳性肿瘤治疗中的应用。
根据本申请的各实施例，本申请还提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本申请第一方面所述的TCR、本申请第二方面所述的TCR复合物、本申请第六方面所述的分离的细胞或本申请第七方面所述的药物组合物。
优选地，所述疾病为MAGE-A4阳性肿瘤。
根据本申请的各实施例，本申请的第九方面，提供了本申请第一方面所述的TCR、本申请第二方面所述的TCR复合物或本申请第六方面所述的分离的细胞的用途，用于制备***的药物，优选地，所述疾病为MAGE-A4阳性肿瘤。
根据本申请的各实施例，本申请的第十方面，提供了一种本申请第一方面所述的TCR的制备方法，所述制备方法包括步骤：
(i)培养本申请第五方面所述的宿主细胞，从而表达本申请第一方面所述的TCR；
(ii)分离或纯化出所述的TCR。
应理解，在本申请范围内中，本申请的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
本申请所述的数值范围不仅包括上述例举的点值，还包括没有例举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本申请不再穷尽列举所述范围包括的具体点值。
相对于现有技术，本申请具有以下有益效果：
(1)本发明的高亲和力TCR对所述GVYDGREHTV-HLA A0201复合物的亲和力和/或结合半衰期是野生型TCR的至少5倍。
(2)本发明的高亲和力TCR能够与所述GVYDGREHTV-HLA A0201特异性结合，同时转染了本发明高亲和力TCR的细胞能够被特异性激活。
(3)转染本发明的高亲和力TCR的效应细胞具有强的特异性杀伤作用。
本申请的一个或多个实施例细节在下面的描述中提出，本申请的其他特征、目的和优点将从说明书及其权利要求书变得明显。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他实施例的附图。
图1为可溶性参比TCR即野生型TCR与GVYDGREHTV-HLA A0201复合物的结合曲线；
图2a和图2b分别为针对负载短肽GVYDGREHTV的T2细胞和负载短肽GLYDGREHSV的T2细胞，转染本申请高亲和力TCR的效应细胞的激活功能实验结果；
图3a和图3b为针对肿瘤细胞系，转染本申请高亲和力TCR的效应细胞的激活功能实验结果；
图4a和图4b为针对肿瘤细胞系，转染本申请高亲和力TCR的效应细胞的杀伤功能LDH实验结果；
图5为ELISPOT检测转染高亲和力TCR的效应细胞在人类正常组织细胞上的IFN-γ释放结果；
图6为本申请高亲和力TCR小鼠体内效力的实验结果。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
本申请通过广泛而深入的研究，获得一种识别GVYDGREHTV短肽(衍生自MAGE-A4蛋白)的高亲和性T细胞受体(TCR)，所述GVYDGREHTV短肽以肽-HLA A0201复合物的形式被呈递。所述高亲和性TCR的α链可变域的3个CDR区：
CDR1α：DSSSTY               SEQ ID NO.37
CDR2α：IFSNMDM              SEQ ID NO.38
CDR3α：AEQSFGNEKLT          SEQ ID NO.62中发生突变，和/或在所述高亲和性TCR的β链可变域的3个CDR区：
CDR1β：MNHEY                SEQ ID NO.59
CDR2β：SVGEGT                   SEQ ID NO.60
CDR3β：ASSLGRAYEQY         SEQ ID NO.61中发生突变；并且，突变后所述TCR对所述GVYDGREHTV-HLA A0201复合物的亲和力和/或结合半衰期是野生型TCR的至少5倍。
在描述本申请之前，应当理解本申请不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且其意图不是限制性的，本申请的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本申请所属领域的普通技术人员通常理解的相同含义。
虽然在本申请的实施或测试中可以使用与本申请中所述相似或等价的任何方法和材料，本文在此处例 举优选的方法和材料。
术语
T细胞受体(T cell receptor，TCR)
可以采用国际免疫遗传学信息***(IMGT)来描述TCR。天然αβ异源二聚TCR具有α链和β链。广义上讲，各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。通过独特的IMGT的TRAJ和TRBJ确定TCR的连接区，通过IMGT的TRAC和TRBC确定TCR的恒定区。
各可变区包含嵌合在框架序列中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。在IMGT命名法中，TRAV和TRBV的不同编号分别指代不同Vα类型和Vβ的类型。在IMGT***中，α链恒定结构域具有以下的符号：TRAC*01，其中“TR”表示T细胞受体基因；“A”表示α链基因；C表示恒定区；“*01”表示等位基因1。β链恒定结构域具有以下的符号：TRBC1*01或TRBC2*01，其中“TR”表示T细胞受体基因；“B”表示β链基因；C表示恒定区；“*01”表示等位基因1。α链的恒定区是唯一确定的，在β链的形式中，存在两个可能的恒定区基因“C1”和“C2”。本领域技术人员通过公开的IMGT数据库可以获得TCRα与β链的恒定区基因序列。
TCR的α和β链一般看作各有两个“结构域”即可变域和恒定结构域。可变域由连接的可变区和连接区构成。因此，在本申请的说明书和权利要求书中，“TCRα链可变域”指连接的TRAV和TRAJ区，同样地，“TCRβ链可变域”指连接的TRBV和TRBD/TRBJ区。TCRα链可变域的3个CDR分别为CDR1α、CDR2α和CDR3α；TCRβ链可变域的3个CDR分别为CDR1β、CDR2β和CDR3β。本申请TCR可变域的框架序列可以为鼠源的或人源的，优选为人源的。TCR的恒定结构域包含胞内部分、跨膜区和胞外部分。
本申请中，能够结合GVYDGREHTV-HLA A0201复合物的野生型TCR的α与β链可变域氨基酸序列分别为SEQ ID NO.1和SEQ ID NO.2所示的氨基酸序列。本申请中所述可溶性“参比TCR”的α链氨基酸序列及β链氨基酸序列分别为SEQ ID NO.11和SEQ ID NO.12所示的氨基酸序列。本申请中所述“野生型TCR”的α链胞外氨基酸序列及β链胞外氨基酸序列分别为SEQ ID NO.33和SEQ ID NO.34所示的氨基酸序列。本申请中所用的TCR序列为人源的。本申请中所述“野生型TCR”的α链氨基酸序列及β链氨基酸序列分别为SEQ ID NO.35和SEQ ID NO.36所示的氨基酸序列。在本申请中，术语“本申请多肽”、“本申请的TCR”、“本申请的T细胞受体”可互换使用。
SEQ ID NO.33：
SEQ ID NO.34：
SEQ ID NO.35：
SEQ ID NO.36：
天然链间二硫键与人工链间二硫键
在天然TCR的近膜区Cα与Cβ链间存在一组二硫键，本申请中称为“天然链间二硫键”。在本申请中，将人工引入的，位置与天然链间二硫键的位置不同的链间共价二硫键称为“人工链间二硫键”。
为方便描述，本申请中TRAC*01与TRBC1*01或TRBC2*01氨基酸序列的位置编号按从N端到C端依次的顺序进行位置编号，如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第60个氨基酸为P(脯氨酸)，则本申请中可将其描述为TRBC1*01或TRBC2*01外显子1的Pro60，也可将其表述为TRBC1*01或TRBC2*01外显子1的第60位氨基酸，又如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第61个氨基酸为Q(谷氨酰胺)，则本申请中可将其描述为TRBC1*01或TRBC2*01外显子1的Gln61，也可将其表述为TRBC1*01或TRBC2*01外显子1的第61位氨基酸，其他以此类推。本申请中，可变区TRAV与TRBV的氨基酸序列的位置编号，按照IMGT中列出的位置编号。如TRAV中的某个氨基酸，IMGT中列出的位置编号为46，则本申请中将其描述为TRAV第46位氨基酸，其他以此类推。本申请中，其他氨基酸的序列位置编号有特殊说明的，则按特殊说明。
肿瘤
术语“肿瘤”指包括所有类型的癌细胞生长或致癌过程，转移性组织或恶性转化细胞、组织或器官，不管病理类型或侵染的阶段。肿瘤的实施例非限制性地包括：实体瘤、软组织瘤和转移性病灶。实体瘤的实施例包括：不同器官***的恶性肿瘤，例如肉瘤、肺鳞癌和癌症。例如：感染的***、肺、***、淋巴、肠胃(例如：结肠)、生殖泌尿道(例如：肾脏和上皮细胞)和咽头。肺鳞癌包括恶性肿瘤，例如，多数的结肠癌、直肠癌、肾细胞癌、肝癌、肺部的非小细胞癌、小肠癌和食道癌。上述癌症的转移性病变可同样用本申请的方法和组合物来治疗和预防。
申请详述
众所周知，TCR的α链可变域与β链可变域各含有3个CDR，类似于抗体的互补决定区。CDR3与抗原短肽相互作用，CDR1和CDR2与HLA相互作用。因此，TCR分子的CDR决定了其与抗原短肽-HLA复合物的相互作用。能够结合抗原短肽GVYDGREHTV-HLA A0201复合物(即，GVYDGREHTV-HLA A0201复合物)的野生型TCR的α链可变域氨基酸序列与β链可变域氨基酸序列分别为SEQ ID NO.1和SEQ ID NO.2，所述序列为本发明人首次发现。其具有下列CDR区：
α链可变域CDR：
CDR1α：DSSSTY               SEQ ID NO.37
CDR2α：IFSNMDM              SEQ ID NO.38
CDR3α：AEQSFGNEKLT          SEQ ID NO.62，
和β链可变域CDR：
CDR1β：MNHEY                SEQ ID NO.59
CDR2β：SVGEGT                    SEQ ID NO.60
CDR3β：ASSLGRAYEQY          SEQ ID NO.61。
本申请通过对CDR区进行突变筛选，获得了与GVYDGREHTV-HLA A0201复合物的亲和力是野生型TCR与GVYDGREHTV-HLA A0201复合物亲和力至少5倍的高亲和力TCR。
进一步，本申请所述TCR是αβ异质二聚TCR，所述TCR的α链可变域包含与SEQ ID NO.1所示的氨基酸序列有至少85％；优选地，至少90％；更优选地，至少92％；更优选地，至少94％(例如可以是至少88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性)的序列同一性的氨基酸序列；
和/或所述TCR的β链可变域包含与SEQ ID NO.2所示的氨基酸序列有至少90％，优选地，至少92％；更优选地，至少94％(例如可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性)的序列同一性的氨基酸序列。
进一步，本申请所述TCR是单链TCR，所述TCR的α链可变域包含与SEQ ID NO.3所示的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％(例如可以是至少88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性)的序列同一性的氨基酸序列；
和/或所述TCR的β链可变域包含与SEQ ID NO.4所示的氨基酸序列有至少85％，优选地，至少90％；更优选地，至少92％；最优选地，至少94％；(例如可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性)的序列同一性的氨基酸序列。
SEQ ID NO.3：
SEQ ID NO.4：
本申请中野生型TCRα链可变域SEQ ID NO.1的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO.1的第26-31位、第49-55位和第90-100位。据此，氨基酸残基编号采用SEQ ID NO.1所示的编号，91E即为CDR3α的第2位E、92Q即为CDR3α的第3位Q、93S即为CDR3α的第4位S、94F即为CDR3α的第5位F、95G即为CDR3α的第6位G。
具体地，α链可变域中所述突变的具体形式包括E91W/V/A/T/S/H/Q/N/Y、Q92H/S、S93T/Q/N/E/D、F94K/R/H、G95Q中的一组或几组。
应理解，本文中氨基酸名称采用国际通用的单英文字母标识，与其相对应的氨基酸名称三英文字母简写分别是：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)；
本申请中，Pro60或者60P均表示第60位脯氨酸。另外，本申请中所述突变的具体形式的表述方式如“Q92H/S”代表第92位的Q被H取代或被S取代，其他以此类推。
根据本领域技术人员熟知的定点突变的方法，将野生型TCRα链恒定区TRAC*01外显子1的Thr48突变为半胱氨酸，β链恒定区TRBC1*01或TRBC2*01外显子1的Ser57突变为半胱氨酸，即得到参比TCR，所述参比TCR的氨基酸序列分别为SEQ ID NO.11和SEQ ID NO.12所示的氨基酸序列，突变后的半胱氨酸残基以加粗字母表示。上述半胱氨酸取代能使参比TCR的α与β链的恒定区之间形成人工链间二硫键，以形成更加稳定的可溶性TCR，从而能够更加方便地评估TCR与GVYDGREHTV-HLA A0201复合物之间的结合亲和力和/或结合半衰期。应理解，TCR可变区的CDR区决定了其与pMHC复合物之间的亲和力，因此，上述TCR恒定区的半胱氨酸取代并不会对TCR的结合亲和力和/或结合半衰期产生影响。所以，在本申请中，测得的参比TCR与GVYDGREHTV-HLA A0201复合物之间的结合亲和力即认为是野生型TCR与GVYDGREHTV-HLA A0201复合物之间的结合亲和力。同样地，如果测得本申请TCR与GVYDGREHTV-HLA A0201复合物之间的结合亲和力是参比TCR与GVYDGREHTV-HLA A0201复合物之间的结合亲和力的至少10倍，即等同于本申请TCR与GVYDGREHTV-HLA A0201复合物之间的结合亲和力是野生型TCR与GVYDGREHTV-HLA A0201复合物之间的结合亲和力的至少10倍。
可通过任何合适的方法测定结合亲和力(与解离平衡常数KD成反比)和结合半衰期(表示为T1/2)，如采用表面等离子共振技术进行检测。应了解，TCR的亲和力翻倍将导致KD减半。T1/2计算为In2除以解离速率(Koff)。因此，T1/2翻倍会导致Koff减半。优选采用相同的试验方案检测给定TCR的结合亲和力或结合半衰期数次，例如3次或更多，取结果的平均值。在优选的实施方式中，采用本文实施例中的表面等离振子共振(BIAcore)方法检测可溶性TCR的亲和力，条件为：温度25℃，PH值为7.1-7.5。该方法检测到参比TCR对GVYDGREHTV-HLA A0201复合物的解离平衡常数KD为1.14E-04M，即114.00μM，本申请中即认为野生型TCR对GVYDGREHTV-HLA A0201复合物的解离平衡常数KD也为114.00μM。由于TCR的亲和力翻倍将导致KD减半，所以若检测到高亲和力TCR对GVYDGREHTV-HLA A0201复合物的解离平衡常数KD为1.14E-05，即11.40μM，则说明所述高亲和力TCR对GVYDGREHTV-HLA A0201复合物的亲和力是野生型TCR对GVYDGREHTV-HLA A0201复合物的亲和力的10倍。本领域技术人员熟知KD值单位间的换算关系，即1M＝106μM，1μM＝1000nM，1nM＝1000pM。在本申请中，所述TCR与GVYDGREHTV-HLA A0201复合物的亲和力是野生型TCR的至少5倍。
可采用任何合适的方法进行突变，包括但不限于依据聚合酶链式反应(PCR)的那些、依据限制性酶的克隆或不依赖连接的克隆(LIC)方法。许多标准分子生物学教材详述了这些方法。聚合酶链式反应(PCR) 诱变和依据限制性酶的克隆的更多细节可参见Sambrook和Russell，(2001)分子克隆-实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)CSHL出版社。LIC方法的更多信息可见(Rashtchian,(1995)Curr Opin Biotechnol 6(1):30-6)。
产生本申请的TCR的方法可以是但不限于从展示此类TCR的噬菌体颗粒的多样性文库中筛选出对GVYDGREHTV-HLA A0201复合物具有高亲和性的TCR，如文献(Li,et al(2005)Nature Biotech 23(3):349-354)中所述。
应理解，表达野生型TCRα和β链可变域氨基酸的基因或者表达略作修饰的野生型TCR的α和β链可变域氨基酸的基因都可用来制备模板TCR。然后在编码该模板TCR的可变域的DNA中引入产生本申请的高亲和力TCR所需的改变。
本申请的高亲和性TCR包含α链可变域氨基酸序列为SEQ ID NO.13-32之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO.2。本申请所述异质二聚TCR分子的α链可变域与β链可变域的氨基酸序列优选自表4。
基于本申请的目的，本申请TCR是具有至少一个TCRα和/或TCRβ链可变域的部分。它们通常同时包含TCRα链可变域和TCRβ链可变域。它们可以是αβ异源二聚体或是单链形式或是其他任何能够稳定存在的形式。在过继性免疫治疗中，可将αβ异源二聚TCR的全长链(包含胞质和跨膜结构域)进行转染。本申请TCR可用作将治疗剂递送至抗原呈递细胞的靶向剂或与其他分子结合制备双功能多肽来定向效应细胞，此时TCR优选为可溶形式。
对于稳定性而言，现有技术中公开了在TCR的α与β链恒定域之间引入人工链间二硫键能够获得可溶且稳定的TCR分子，如专利文献PCT/CN2015/093806中所述。因此，本申请TCR可以是在其α和β链恒定域的残基之间引入人工链间二硫键的TCR。半胱氨酸残基在所述TCR的α和β链恒定域间形成人工链间二硫键。半胱氨酸残基可以取代在天然TCR中合适位点的其他氨基酸残基以形成人工链间二硫键。
例如，取代TRAC*01外显子1的Thr48和取代TRBC1*01或TRBC2*01外显子1的Ser57来形成二硫键。引入半胱氨酸残基以形成二硫键的其他位点还可以是：
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
或TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
即半胱氨酸残基取代了上述α与β链恒定域中任一组位点。可在本申请TCR恒定域的一个或多个C末端截短最多15个、或最多10个、或最多8个或更少的氨基酸，以使其不包括半胱氨酸残基来达到缺失天然链间二硫键的目的，也可通过将形成天然链间二硫键的半胱氨酸残基突变为另一氨基酸来达到上述目的。
如上所述，本申请的TCR可以包含在其α和β链恒定域的残基间引入的人工链间二硫键。应注意，恒定域间含或不含上文所述的引入的人工二硫键，本申请的TCR均可含有TRAC恒定域序列和TRBC1或TRBC2恒定域序列。TCR的TRAC恒定域序列和TRBC1或TRBC2恒定域序列可通过存在于TCR中的天然链间二硫键连接。
另外，对于稳定性而言，专利文献PCT/CN2016/077680还公开了在TCR的α链可变区与β链恒定区之间引入人工链间二硫键能够使TCR的稳定性显著提高。因此，本申请的高亲和力TCR的α链可变区与β链恒定区之间还可以含有人工链间二硫键。具体地，在所述TCR的α链可变区与β链恒定区之间形成人工链间二硫键的半胱氨酸残基取代了：
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；
或TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。
优选地，所述TCR可以包含：(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的可变域和至少一部分恒定域，α链与β链形成异质二聚体。
更优选地，所述TCR可以包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
对于稳定性而言，另一方面，本申请TCR还包括在其疏水芯区域发生突变的TCR，所述疏水芯区域的突变优选为能够使本申请TCR的稳定性提高的突变，如在公开号为WO2014/206304的专利文献中所述。所述TCR可在其下列可变域疏水芯位置发生突变：
(α和/或β链)可变区氨基酸第11、13、19、21、53、76、89、91和94位，和/或α链J基因(TRAJ)短肽氨基酸位置倒数第3、5和7位，和/或β链J基因(TRBJ)短肽氨基酸位置倒数第2、4和6位，其中氨基酸序列的位置编号按国际免疫遗传学信息***(IMGT)中列出的位置编号。本领域技术人员知晓上述国际免疫遗传学信息***，并可根据该数据库得到不同TCR的氨基酸残基在IMGT中的位置编号。
更具体地，本申请中疏水芯区域发生突变的TCR可以是由一柔性肽链连接TCR的α链与β链的可变域而构成的高稳定性单链TCR。TCR可变区的CDR区决定了其与短肽-HLA复合物之间的亲和力，疏水芯的突变能够使TCR更加稳定，但并不会影响其与短肽-HLA复合物之间的亲和力。应注意，本申请中柔性肽链可以是任何适合连接TCRα与β链可变域的肽链。本申请实施例1中构建的用于筛选高亲和性TCR的模板链即为上述含有疏水芯突变的高稳定性单链TCR。采用稳定性较高的TCR，能够更方便的评估TCR与GVYDGREHTV-HLA A0201复合物之间的亲和力。
所述单链模板TCR的α链可变域及β链可变域的CDR区与野生型TCR的CDR区完全相同。即α链可变域的3个CDR分别为CDR1α：DSSSTY；CDR2α：IFSNMDM；CDR3α：AEQSFGNEKLT和β链可变域的3个CDR分别为CDR1β：MNHEY；CDR2β：SVGEGT；CDR3β：ASSLGRAYEQY。所述单链模板TCR的氨基酸序列为SEQ ID NO.9所示的氨基酸序列，及核苷酸序列为SEQ ID NO.10所示的核苷酸序列。以此筛选出对GVYDGREHTV-HLA A0201复合物具有高亲和性的由α链可变域和β链可变域构成的单链TCR。
本申请的对GVYDGREHTV-HLA A0201复合物具有高亲和性的αβ异质二聚体的获得是通过将筛选出的高亲和性单链TCR的α与β链可变域的CDR区转移到野生型TCRα链可变域(SEQ ID NO.1)与β链可变域(SEQ ID NO.2)的相应位置而得到。
本申请的TCR也可以多价复合体的形式提供。本申请的多价TCR复合体包含任意两个或至少三个(例如可以是两个、三个、四个或更多个)本申请TCR相结合而形成的多聚物，如可以用p53的四聚结构域来产生四聚体，或多个本申请TCR与另一分子结合而形成的复合物。本申请的TCR复合物可用于体外或体内追踪或靶向呈递特定抗原的细胞，也可用于产生具有此类应用的其他多价TCR复合物的中间体。
本申请的TCR可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物包括检测标记物(为诊断目的，其中所述TCR用于检测呈递GVYDGREHTV-HLA A0201复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分中任意一种或至少两种的组合(所述组合包括结合或偶联)。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本申请TCR结合或偶联的治疗剂包括但不限于：
1.放射性核素(Koppe等，2005，癌转移评论(Cancer metastasis reviews)24，539)；
2.生物毒(Chaudhary等，1989，自然(Nature)339，394；Epel等，2002，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51，565)；
3.细胞因子如IL-2等(Gillies等，1992，美国国家科学院院刊(PNAS)89，1428；Card等，2004，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53，345；Halin等，2003，癌症研究(Cancer Research)63，3202)；
4.抗体Fc片段(Mosquera等，2005，免疫学杂志(The Journal Of Immunology)174，4381)；
5.抗体scFv片段(Zhu等，1995，癌症国际期刊(International Journal of Cancer)62,319)；
6.金纳米颗粒/纳米棒(Lapotko等，2005，癌症通信(Cancer letters)239，36；Huang等，2006，美国化学学会杂志(Journal of the American Chemical Society)128，2115)；
7.病毒颗粒(Peng等，2004，基因治疗(Gene therapy)11，1234)；
8.脂质体(Mamot等，2005，癌症研究(Cancer research)65，11631)；
9.纳米磁粒；
10.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；11.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
与本申请TCR结合的抗体或其片段包括抗-T细胞或NK-细胞决定抗体，如抗-CD3或抗-CD28或抗-CD16抗体，上述抗体或其片段与TCR的结合能够对效应细胞进行定向来更好地靶向靶细胞。一个优选的实施方式是本申请TCR与抗-CD3抗体或所述抗-CD3抗体的功能片段或变体结合。具体地，本申请的TCR与抗CD3单链抗体的融合分子包括选自TCRα链可变域氨基酸序列为SEQ ID NO.13-32之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO.2。
本申请还涉及编码本申请TCR的核酸分子。本申请的核酸分子可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。例如，编码本申请TCR的核酸序列可以与本申请附图中所示的核酸序列相同或是简并的变异体。举例说明“简并的变异体”的含义，如本文所用，“简并的变异体”在本申请中是指编码具有SEQ ID NO.3的蛋白序列，但与SEQ ID NO.5的序列有差别的核酸序列。
本申请的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本申请所述TCR(或其片段，或其衍生物)的DNA序列。然后可将所述DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本申请也涉及包含本申请所述的核酸分子的载体，以及用本申请的载体或编码序列经基因工程产生的宿主细胞。
本申请还包括表达本申请所述TCR的分离细胞，特别是T细胞。有许多方法适合于用编码本申请的高亲和力TCR的DNA或RNA进行T细胞转染(如，Robbins等.，(2008)J.Immunol.180:6116-6131)。表达本申请所述高亲和性TCR的T细胞可以用于过继免疫治疗。本领域技术人员能够知晓进行过继性治疗的许多合适方法(如，Rosenberg等.，(2008)Nat Rev Cancer8(4)：299-308)。
本申请还提供一种药物组合物，所述药物组合物含有药学上可接受的载体以及本申请所述TCR、本申请所述TCR复合物或呈递本申请所述TCR的细胞。
本申请还提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本申请所述TCR、本申请所述TCR复合物、呈递本申请所述TCR的细胞或本申请的药物组合物。
在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的结构和功能。因此，本申请TCR还包括本申请TCR的至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸(尤其是位于CDR区之外的氨基酸)，被性质相似或相近的氨基酸所替换，并仍能够保持其功能性的TCR。
本申请还包括对本申请TCR略作修饰后的TCR。修饰(通常不改变一级结构)形式包括：本申请TCR的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在本申请TCR的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的TCR。这种修饰可以通过将TCR暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的TCR。
本申请所述的TCR、TCR复合物或本申请所述的TCR转染的T细胞可与药学上可接受的载体一起在药物组合物中提供。本申请所述的TCR、所述的多价TCR复合物或所述的细胞通常作为无菌药物组合物的一部分提供，所述组合物通常包括药学上可接受的载体。所述药物组合物可以是任何合适的形式(取决于给予患者的所需方法)。其可采用单位剂型提供，通常在密封的容器中提供，可作为试剂盒的一部分提供。此类试剂盒(但非必需)包括使用说明书。其可包括多个所述单位剂型。
此外，本申请的TCR可以单用，也可与其他治疗剂结合或偶联在一起使用(如配制在同一药物组合物中)。
所述药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载 体。所述载体指这样一些药剂载体：它们本身不诱导产生对接受所述药物组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在雷明顿药物科学(Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991))中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇或佐剂中任意一种或至少两种的组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，所述载体中还可能存在辅助性的物质，如润湿剂或乳化剂，pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本申请的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：眼内、肌内、静脉内、皮下、皮内、或局部给药，优选为胃肠外包括皮下、肌肉内或静脉内。待预防或治疗的对象可以是动物；尤其是人。
当本申请的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有针剂、口服剂等。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本申请的药物组合物还可以缓释剂形式给药。例如，本申请所述TCR可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本申请的药物组合物被用于实际治疗时，作为活性成分的本申请TCR或TCR复合物或呈递本申请TCR的细胞，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定，最终由医师决定合理的用量。
本申请的主要优点在于：
(1)本申请的高亲和力TCR对所述GVYDGREHTV-HLA A0201复合物的亲和力和/或结合半衰期是野生型TCR的至少5倍。
(2)本申请的高亲和力TCR能够与所述GVYDGREHTV-HLA A0201特异性结合，同时转染了本申请高亲和力TCR的细胞能够被特异性激活。
(3)转染本申请的高亲和力TCR的效应细胞具有强的特异性杀伤作用。
下面的具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
材料和方法
本申请实施例中所用的实验材料如无特殊说明均可从市售渠道获得，其中，E.coli DH5α购自Tiangen、E.coli BL21(DE3)购自Tiangen、E.coli Tuner(DE3)购自Novagen、质粒pET28a购自Novagen。
实施例1
疏水芯突变的稳定性单链TCR模板链的产生
本申请利用定点突变的方法，根据专利文献WO2014/206304中所述，构建了以一个柔性短肽(linker)连接TCRα与β链可变域而构成的稳定性单链TCR分子，其氨基酸及DNA序列分别为SEQ ID NO.9和SEQ ID NO.10所示的序列。并以所述单链TCR分子为模板进行高亲和性TCR分子的筛选。模板链的α链可变域为SEQ ID NO.3所示的氨基酸序列，及β链可变域为SEQ ID NO.4所示的氨基酸序列；其对应的DNA序列分别为SEQ ID NO.5和SEQ ID NO.6所示的核苷酸序列；柔性短肽(linker)的氨基酸序列及DNA序列分别为SEQ ID NO.7和SEQ ID NO.8所示的核苷酸序列。
SEQ ID NO.5：
SEQ ID NO.6：
SEQ ID NO.7：
SEQ ID NO.8：
SEQ ID NO.9：
SEQ ID NO.10：
将携带模板链的目的基因经Nco Ⅰ和Not Ⅰ双酶切，与经过Nco Ⅰ和Not Ⅰ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli BL21(DE3)，用于表达。
实施例2
实施例1中构建的稳定性单链TCR的表达、复性和纯化
将实施例1中制备的含有重组质粒pET28a-模板链的BL21(DE 3)菌落全部接种于含有卡那霉素的LB培养基中，37℃培养至OD600为0.6-0.8，加入IPTG至终浓度为0.5mM，37℃继续培养4h。5000rpm离心15min收获细胞沉淀物，用Bugbuster Master Mix(Merck)裂解细胞沉淀物，6000rpm离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000rpm离心15min,收集包涵体。将包涵体溶解在缓冲液(20mM Tris-HCl pH8.0，8M尿素)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。
向5mg溶解的单链TCR包涵体蛋白中，加入2.5mL缓冲液(6M Gua-HCl，50mM Tris-HCl pH 8.1，100mM NaCl，10mM EDTA)，再加入DTT至终浓度为10mM，37℃处理30min。用注射器向125mL复性缓冲液(100mM Tris-HCl pH 8.1，0.4M L-精氨酸，5M尿素，2mM EDTA，6.5mM β-mercapthoethylamine，1.87mM Cystamine)中滴加上述处理后的单链TCR，4℃搅拌10min，然后将复性液装入截留量为4kDa的纤维素膜透析袋，透析袋置于1L预冷的水中，4℃缓慢搅拌过夜。17小时后，将透析液换成1L预冷的缓冲液(20mM Tris-HCl pH8.0)，4℃继续透析8h，然后将透析液换成相同的新鲜缓冲液继续透析过夜。17小时后，样品经0.45μm滤膜过滤，真空脱气后通过阴离子交换柱(HiTrap Q HP，GE Healthcare)，用20mM Tris-HCl pH8.0配制的0-1M NaCl线性梯度洗脱液纯化蛋白，收集的洗脱 组分进行SDS-PAGE分析，包含单链TCR的组分浓缩后进一步用凝胶过滤柱(Superdex 75 10/300,GE Healthcare)进行纯化，目标组分也进行SDS-PAGE分析。
用于BIAcore分析的洗脱组分进一步采用凝胶过滤法测试其纯度。条件为：色谱柱Agilent Bio SEC-3(300A，)，流动相为150mM磷酸盐缓冲液，流速0.5mL/min，柱温25℃，紫外检测波长214nm。
实施例3
结合表征
BIAcore分析
使用BIAcore T200实时分析***检测TCR分子与GVYDGREHTV-HLA A0201复合物的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，pH4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15000RU。条件为：温度25℃，PH值为7.1-7.5。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将GVYDGREHTV-HLA A0201复合物流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。采用单循环动力学分析方法测定其亲和力，将TCR用HEPES-EP缓冲液(10mM HEPES，150mM NaCl，3mM EDTA，0.005％P20，pH7.4)稀释成几个不同的浓度，以30μL/min的流速，依次流过芯片表面，每次进样的结合时间为120s，最后一次进样结束后让其解离600s。每一轮测定结束后用pH1.75的10mM Gly-HCl再生芯片。利用BIAcore Evaluation软件计算动力学参数。
上述GVYDGREHTV-HLA A0201复合物的制备过程如下：
a.纯化：收集100mL诱导表达重链或轻链的E.coli菌液，于4℃，8000g离心10min后用10mL PBS洗涤菌体一次，之后用5ml BugBuster Master Mix Extraction Reagents(Merck)剧烈震荡重悬菌体，并于室温旋转孵育20min，之后于4℃，6000g离心15min，弃去上清，收集包涵体。
将上述包涵体重悬于5mL BugBuster Master Mix中，室温旋转孵育5min；加30mL稀释10倍的BugBuster，混匀，4℃，6000g离心15min；弃去上清，加30mL稀释10倍的BugBuster重悬包涵体，混匀，4℃，6000g离心15min，重复两次，加30mL 20mM Tris-HCl pH 8.0重悬包涵体，混匀，4℃，6000g离心15min，最后用20mM Tris-HCl 8M尿素溶解包涵体，SDS-PAGE检测包涵体纯度，BCA试剂盒测浓度。
b.复性：将合成的短肽GVYDGREHTV(江苏金斯瑞生物科技有限公司)溶解于DMSO至20mg/mL的浓度。轻链和重链的包涵体用8M尿素、20mM Tris pH 8.0、10mM DTT来溶解，复性前加入3M盐酸胍、10mM醋酸钠、10mM EDTA进一步变性。将GVYDGREHTV肽以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mM Tris pH 8.3、2mM EDTA、0.5mM氧化性谷胱甘肽、5mM还原型谷胱甘肽、0.2mM PMSF，冷却至4℃)，然后依次加入20mg/L的轻链和90mg/L的重链(终浓度，重链分三次加入，8h/次)，复性在4℃进行至少3天至完成，SDS-PAGE检测能否复性成功。
c.复性后纯化：用10体积的20mM Tris pH8.0作透析来更换复性缓冲液，至少更换缓冲液两次来充分降低溶液的离子强度。透析后用0.45μm醋酸纤维素滤膜过滤蛋白质溶液，然后加载到HiTrap Q HP(GE通用电气公司)阴离子交换柱上(5mL床体积)。利用Akta纯化仪(GE通用电气公司)，20mM Tris pH8.0配制的0-400mM NaCl线性梯度液洗脱蛋白，pMHC约在250mM NaCl处洗脱，收集诸峰组分，SDS-PAGE检测纯度。
d.生物素化：用Millipore超滤管将纯化的pMHC分子浓缩，同时将缓冲液置换为20mM Tris pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mM MgOAc、50μM D-Biotin、100μg/mL BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
e.纯化生物素化后的复合物：用Millipore超滤管将生物素化标记后的pMHC分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的pMHC，利用Akta纯化仪(GE通用电气公司)，用过滤过的PBS预平衡HiPrepTM 16/60 S200 HR柱(GE通用电气公司)，加载1mL浓缩过的生物素化pMHC分子，然后用PBS以1mL/min流速洗脱。生物素化的pMHC分子在约55mL时作为单峰洗脱出现。合并含有蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，加入蛋白酶抑制剂cocktail(Roche)将生 物素化的pMHC分子分装保存在-80℃。
测量可溶性参比TCR即野生型TCR与GVYDGREHTV-HLA A0201复合物的结合曲线，检测结果如图1所示。
实施例4
高亲和力TCR的产生
噬菌体展示技术是产生TCR高亲和力变体文库以筛选高亲和力变体的一种手段。将Li等((2005)Nature Biotech 23(3):349-354)描述的TCR噬菌体展示和筛选方法应用于实施例1中的单链TCR模板。通过突变该模板链的CDR区来建立高亲和性TCR的文库并进行淘选。经过几轮淘选后的噬菌体文库均和相应抗原有特异性结合，从中挑取单克隆，并进行分析。
将筛选到的高亲和力的单链TCR的CDR区突变引入到αβ异质二聚TCR的可变域的相应位点中，并通过BIAcore来检测其与GVYDGREHTV-HLA A0201复合物的亲和力。上述CDR区高亲和力突变点的引入采用本领域技术人员熟知的定点突变的方法。所述野生型TCR的α链与β链可变域氨基酸序列分别为如SEQ ID NO.1和SEQ ID NO.2所示的氨基酸序列。
应注意，为获得更加稳定的可溶性TCR，以便更方便地评估TCR与GVYDGREHTV-HLA A0201复合物之间的结合亲和力和/或结合半衰期，αβ异质二聚TCR可以是在α和β链的恒定区中分别引入了一个半胱氨酸残基以形成人工链间二硫键的TCR，本实施例中引入半胱氨酸残基后TCRα与β链的氨基酸序列分别为如SEQ ID NO.11和SEQ ID NO.12所示的氨基酸序列，引入的半胱氨酸残基以加粗并划线字母表示。
SEQ ID NO.11：
SEQ ID NO.12：
通过《分子克隆实验室手册》(Molecular Cloning a Laboratory Manual)(第三版，Sambrook和Russell)中描述的标准方法将待表达的TCRα和β链的胞外序列基因经合成后分别***到表达载体pET28a+(Novagene)，上下游的克隆位点分别是Nco I和Not I。CDR区的突变通过本领域技术人员熟知的重叠PCR(overlap PCR)引入。***片段经过测序确认无误。
实施例5
高亲和力TCR的表达、复性和纯化
将TCRα和β链的表达载体分别通过化学转化法转化进入表达细菌BL21(DE3)，细菌用LB培养液生长，于OD600＝0.6时用终浓度0.5mM IPTG诱导，TCR的α和β链表达后形成的包涵体通过BugBuster Mix(Novagene)进行提取，并且经BugBuster溶液反复多次洗涤，包涵体最后溶解于6M盐酸胍，10mM二硫苏糖醇(DTT)，10mM乙二胺四乙酸(EDTA)，20mM Tris(pH8.1)中。
溶解后的TCRα和β链以1:1的质量比快速混合于5M尿素，0.4M精氨酸，20mM Tris(pH8.1)，3.7mM cystamine，6.6mM β-mercapoethylamine(4℃)中，终浓度为60mg/mL。混合后将溶液置于10倍体积的去离子水中透析(4℃)，12小时后将去离子水换成缓冲液(20mM Tris，pH8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP,5ml,GE Healthcare)纯化。洗脱峰含有复性成功的α和β二聚体的TCR通过SDS-PAGE胶确认。TCR随后通过凝胶过滤层析(HiPrep 16/60，Sephacryl S-100HR，GE Healthcare)进一步纯化。纯化后的TCR纯度经过SDS-PAGE测定大于90％，浓度由BCA法确定。
实施例6
BIAcore分析结果
采用实施例3中所述方法检测引入高亲和力CDR区的αβ异质二聚TCR与GVYDGREHTV-HLA A0201复合物的亲和力。
本申请得到新的TCRα链可变域氨基酸序列，为如SEQ ID NO.13-32所示的氨基酸序列。由于TCR分子的CDR区决定了其与相应的pMHC复合物的亲和力，所以本领域技术人员能够预料引入高亲和力突变点的αβ异质二聚TCR也具有对GVYDGREHTV-HLA A0201复合物的高亲和力。利用实施例4中所述方法构建表达载体，利用实施例5中所述方法对上述引入高亲和力突变的αβ异质二聚TCR进行表达、复性和纯化，然后利用BIAcore T200测定其与GVYDGREHTV-HLA A0201复合物的亲和力，如表5所示。
表5
由表5可知，所述异质二聚TCR的亲和力是野生型TCR对GVYDGREHTV-HLA A0201复合物的亲和力的至少5倍。
SEQ ID NO.13：
SEQ ID NO.14
SEQ ID NO.15
SEQ ID NO.16
SEQ ID NO.17
SEQ ID NO.18
SEQ ID NO.19
SEQ ID NO.20
SEQ ID NO.21
SEQ ID NO.22
SEQ ID NO.23
SEQ ID NO.24
SEQ ID NO.25
SEQ ID NO.26
SEQ ID NO.27
SEQ ID NO.28
SEQ ID NO.29
SEQ ID NO.30
SEQ ID NO.31
SEQ ID NO.32

实施例7
抗-CD3抗体与高亲和性αβ异质二聚TCR的融合体的表达、复性和纯化
将抗-CD3的单链抗体(scFv)与αβ异质二聚TCR融合，制备融合分子。抗-CD3的scFv与TCR的β链融合，该TCRβ链可以包含任一上述高亲和性αβ异质二聚TCR的β链可变域，融合分子的TCRα链可以包含任一上述高亲和性αβ异质二聚TCR的α链可变域。
融合分子表达载体的构建
(1)α链表达载体的构建：将携带αβ异质二聚TCR的α链的目的基因经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布于含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Tuner(DE3)，用于表达。
(2)抗-CD3(scFv)-β链表达载体的构建：通过重叠(overlap)PCR的方法，设计引物将抗-CD3scFv和高亲和性异质二聚TCRβ链基因连接起来，中间的连接短肽(linker)为GGGGS，并且使抗-CD3的scFv与高亲和性异质二聚TCRβ链的融合蛋白的基因片段带上限制性内切酶位点NcoⅠ(CCATGG)和NotⅠ(GCGGCCGC)。将PCR扩增产物经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α感受态细胞，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Tuner(DE3)感受态细胞，用于表达。
融合蛋白的表达、复性及纯化
将表达质粒分别转化进入E.coli Tuner(DE3)感受态细胞，涂布LB平板(卡那霉素50μg/mL)置于37℃培养过夜。次日，挑克隆接种至10mL LB液体培养基(卡那霉素50μg/mL)培养2-3h，按体积比1:100接种至1L LB培养基中，继续培养至OD600为0.5-0.8，加入终浓度为1mM IPTG诱导目的蛋白的表达。诱导4小时以后，以6000rpm离心10min收获细胞。PBS缓冲液洗涤菌体一次，并且分装菌体，取相当于200mL的细菌培养物的菌体用5mL BugBuster Master Mix(Merck)裂解细菌，以6000g离心15min收集包涵体。然后进行4次洗涤剂洗涤以去除细胞碎片和膜组分。然后，用缓冲液如PBS洗涤包涵体以除去洗涤剂和盐。最终，将包涵体用含6M盐酸胍，10mM二硫苏糖醇(DTT)，10mM乙二胺四乙酸(EDTA)，20mM Tris，pH8.1缓冲溶液溶解，并测定包涵体浓度，将其分装后置于-80℃冷冻保存。
溶解后的TCRα链和抗-CD3(scFv)-β链以2:5的质量比快速混合于5M尿素(urea)，0.4M L-精氨酸(L-arginine)，20mM Tris pH8.1，3.7mM cystamine，6.6mM β-mercapoethylamine(4℃)，终浓度α链和抗-CD3(scFv)-β链分别为0.1mg/mL、0.25mg/mL。
混合后将溶液置于10倍体积的去离子水中透析(4℃)，12小时后将去离子水换成缓冲液(10mM Tris，pH8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP 5ml,GE healthcare)纯化。洗脱峰含有复性成功的TCRα链与抗-CD3(scFv)-β链二聚体的TCR通过SDS-PAGE胶确认。TCR融合分子随后通过尺寸排阻色谱法(S-100 16/60，GE healthcare)进一步纯化，以及阴离子交换柱(HiTrap Q HP 5ml，GE healthcare)再次纯化。纯化后的TCR融合分子纯度经过SDS-PAGE测定大于90％，浓度由BCA法测定。
实施例8
针对负载短肽的T2细胞，转染本申请高亲和力TCR的效应细胞的激活功能实验
IFN-γ是活化T淋巴细胞产生的一种强有力的免疫调节因子，因此本实施例通过本领域技术人员熟知的ELISPOT实验检测IFN-γ数以验证转染本申请高亲和力TCR的细胞的激活功能及抗原特异性。将本申请高亲和力TCR(TCR编号及其序列号从表4获悉)转染至从健康志愿者的血液中分离到的CD3+T细胞作为效应细胞，并以同一志愿者转染其他TCR(A6)的或转染野生型TCR(WT-TCR)的CD3+T细胞作为对照。所用的靶细胞为负载MAGE-A4抗原短肽GVYDGREHTV的T2、负载了其他抗原短肽的T2、或空载的T2。
首先准备ELISPOT平板，先按靶细胞1×104个细胞/孔、效应细胞2×103个细胞/孔(按转染阳性率计算)加入对应孔中，然后在实验组加入MAGE-A4抗原短肽GVYDGREHTV溶液，在对照组加入其他抗 原短肽溶液，且使短肽终浓度为10-6M，空白组加入等体积培养基，并设置二个复孔。温育过夜(37℃，5％CO2)。实验第2天，洗涤平板并进行二级检测和显色，干燥平板，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。
实验结果如图2a所示，针对负载了MAGE-A4抗原短肽GVYDGREHTV的靶细胞，转染本申请高亲和力TCR的效应细胞相比于转染野生型TCR的效应细胞起更明显的激活效应，而转染其他TCR的效应细胞无活性；同时，转染本申请TCR的效应细胞对负载其他抗原短肽的或空载的靶细胞基本无活性。
同时，利用上述ELISOPT实验测得转染本申请高亲和力TCR的效应细胞也具有结合GLYDGREHSV-HLA A0201复合物的特性，GLYDGREHSV是衍生于MAGE-A8的短肽。实验中靶细胞为负载MAGE-A8抗原短肽GLYDGREHSV的T2、负载了其他抗原短肽的T2、或空载的T2，效应细胞为转染本申请高亲和力TCR的CD3+T细胞。
实验结果如图2b所示，针对负载了MAGE-A8抗原短肽GLYDGREHSV的靶细胞，转染本申请高亲和力TCR的效应细胞相比于转染野生型TCR的效应细胞起更明显的激活效应，而转染其他TCR的效应细胞基本无活性；同时，转染本申请TCR的效应细胞对负载其他抗原短肽的或空载的靶细胞基本无活性。
实施例9
针对肿瘤细胞系，转染本申请高亲和力TCR的效应细胞的激活功能实验
本实施例利用肿瘤细胞系再次验证转染本申请高亲和力TCR的效应细胞的激活功能及特异性。同样是通过本领域技术人员熟知的ELISPOT实验进行检测。将本申请高亲和力TCR转染至从健康志愿者的血液中分离到的CD3+T细胞作为效应细胞，并以同一志愿者转染其他TCR(A6)的或转染野生型TCR(WT-TCR)的CD3+T细胞作为对照。实施例中所用肿瘤细胞系分别为NCI-H1299、A375、U-2OS、293T、HT1080、NUGC4。其中，NCI-H1299购自中国科学院细胞库，A375、293T、HT1080均购自ATCC，U-2OS购自广州赛库生物技术有限公司，NUGC4购自Sciencell。以下分两批次(Ⅰ)、(Ⅱ)先后进行实验：
(Ⅰ)所述高亲和力TCR可从表4获悉，分别为TCR1、TCR2、TCR3、TCR4、TCR5、TCR6、TCR8、TCR9、TCR10、TCR11、TCR12、TCR13、TCR14、TCR15、TCR16、TCR19和TCR20。该批次使用的MAGE-A4阳性肿瘤细胞系为NCI-H1299-A0201(HLA-A0201过表达)、A375(MAGE-A4阳性)、U-2OS(MAGE-A4、A8双阳性)和293T-MAGE-A4(MAGE-A4过表达)，阴性细胞系为293T和HT1080。
(Ⅱ)所述高亲和力TCR可从表4获悉，分别为TCR1、TCR2、TCR3、TCR4、TCR5、TCR6、TCR8、TCR9、TCR10、TCR11、TCR12、TCR13、TCR14、TCR15、TCR16、TCR17、TCR19和TCR20。该批次使用的MAGE-A4阳性肿瘤细胞系为U-2OS(MAGE-A4、A8双阳性)和293T-MAGE-A4(MAGE-A4过表达)，阴性细胞系为293T和NUGC4。
进行以下步骤：首先准备ELISPOT平板。ELISPOT平板乙醇活化包被，4℃过夜。实验第1天，去掉包被液，洗涤封闭，室温下孵育两个小时，去除封闭液，将试验的各个组分加入ELISPOT平板：靶细胞为2×104个/孔，效应细胞为2×103个/孔(按转染的阳性率计算)，并设置二个复孔。温育过夜(37℃，5％CO2)。实验第2天，洗涤平板并进行二级检测和显色，干燥平板，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。
实验结果如图3a和图3b所示，针对MAGE-A4阳性肿瘤细胞系，仅靶细胞无活性，转染本申请高亲和力TCR的效应细胞相比于转染野生型的效应细胞起更明显的激活效应，而转染其他TCR的效应细胞无活性；同时，转染本申请高亲和力TCR的效应细胞对MAGE-A4阴性细胞系无活性。
实施例10
针对肿瘤细胞系，转染本申请高亲和力TCR的效应细胞的杀伤功能实验
乳酸脱氢酶(LDH)在胞浆内含量丰富，正常时不能通过细胞膜，当细胞受损伤或死亡时可释放到细胞外，此时细胞培养液中LDH活性与细胞死亡数目成正比。本实施例同样通过本领域技术人员熟知的非放射性细胞毒性实验，测定LDH的释放，从而验证转染本申请TCR的细胞的杀伤功能。本实施例LDH实验用从健康志愿者的血液中分离到的CD3+T细胞转染本申请高亲和力TCR作为效应细胞，并以同一志愿者转染其他TCR(A6)的CD3+T细胞作为对照。实施例中所用肿瘤细胞系分别为U2-OS、A375、293T、NUGC4和MS751。其中，U-2OS和MS751购自广州赛库生物技术有限公司，A375和293T均购自ATCC，NUGC4购自Sciencell。以下分两批次(Ⅰ)、(Ⅱ)先后进行实验：
(Ⅰ)所述高亲和力TCR可从表4获悉，分别为TCR5、TCR17和TCR20。该批次使用的MAGE-A4阳性肿瘤细胞系为U-2OS(MAGE-A4、A8双阳性)和A375(MAGE-A4阳性)，阴性肿瘤细胞系为293T。
(Ⅱ)所述高亲和力TCR可从表4获悉，分别为TCR3、TCR5和TCR19。该批次使用的MAGE-A4阳性肿瘤细胞系为U-2OS(MAGE-A4、A8双阳性)和293T-MAGE-A4(转染MAGE-A4的293T细胞)，阴性肿瘤细胞系为NUGC4、MS751和293T。
实验步骤：首先准备LDH平板，按以下顺序将试验的各个组分加入平板：第(Ⅰ)批次靶细胞3×104个细胞/孔、效应细胞3×104个细胞/孔(按转染阳性率计算)；第(Ⅱ)批次靶细胞2.5×104个细胞/孔、效应细胞7.5×104个细胞/孔(按转染阳性率计算)，加入对应孔中，并设置三个复孔。同时设置效应细胞自发孔，靶细胞自发孔，靶细胞最大孔，体积校正对照孔及培养基背景对照孔。温育过夜(37℃，5％CO2)。实验第2天，检测显色，终止反应后用酶标仪(Bioteck)在490nm记录吸光值。
实验结果如图4a和图4b所示，针对MAGE-A4阳性肿瘤细胞系，转染本申请高亲和力TCR的效应细胞仍表现出强杀伤效力，而转染其他TCR的T细胞、未转染任何TCR的T细胞不起反应，同时，转染本申请高亲和力TCR的T细胞对阴性肿瘤细胞系几乎无杀伤。
实施例11
转导本申请高亲和力TCR的效应细胞在人类正常组织细胞上的特异性的验证
本实施例利用人类正常组织细胞验证转导本申请高亲和力TCR的效应细胞的特异性。同样是通过本领域技术人员熟知的ELISPOT实验进行检测。将本申请高亲和力TCR(TCR编号及其序列号从表4获悉)经慢病毒转导至从健康志愿者的血液中分离到的CD3+T细胞作为效应细胞，并以同一志愿者转导其他TCR(A6)的或空转导(NC)的CD3+T细胞作为对照。
实验使用的阳性肿瘤细胞系为US-OS(MAGE-A4、A8双阳性细胞)，使用的人类正常组织细胞为：人肺成纤维细胞(HPF-a)、人类大动脉内皮细胞(HAEC-2A)、人类心肌成纤维细胞(HCF-4A)、人类大动脉平滑肌细胞(HASMC-6B)、人类肾脏系膜细胞(HRMC-11A)、人胃平滑肌细胞(HGSMC-13B)、人肾上皮细胞(HREpiC-14A)、人膀胱基质成纤维细胞(HBdSF-39)、人脉络纵肌成纤维细胞(HCPF-20)、人食管成纤维细胞(HEF-27)、人乳腺成纤维细胞(HMF-40)、人脾脏成纤维细胞(HSF-17)和人脑血管外膜成纤维细胞(HBVAF-22)。使用的人类正常组织细胞购自Sciencell公司。
首先准备ELISPOT平板。ELISPOT平板乙醇活化包被，4℃过夜。实验第1天，去掉包被液，洗涤封闭，室温下孵育两个小时，去除封闭液，将试验的各个组分加入ELISPOT平板：靶细胞为2×104个/孔，效应细胞为2×103个/孔(按转导的阳性率计算)，并设置二个复孔。温育过夜(37℃，5％CO2)。实验第2天，洗涤平板并进行二级检测和显色，干燥平板，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。
实验结果如图5所示，转导本申请高亲和力TCR的效应细胞对人类正常组织细胞基本无活性，进一步验证了本申请TCR的高特异性。
实施例12
本申请高亲和TCR分子的体内效力
将本申请高亲和力TCR转染的T细胞注射进人黑色素瘤异种移植模型的小鼠体内，检测其在体内对肿瘤的抑制效果。
实验采用NSG小鼠(百奥塞图)(雌性，实验周龄6-8周)作为实验对象，实验开始前10天以培养后收集并混悬好的A375肿瘤细胞(ATCC)悬液1*10^7个/只对小鼠进行腹部单侧皮下注射，建立人黑色素瘤异种移植小鼠模型。
实验开始当天用游标卡尺对每只小鼠已成型肿瘤的长径(a)、短径(b)分别进行测量，并按如下公式计算肿瘤面积：V＝(a*b^2)/2；随后根据分组设置：对照组(转染无关TCR的T细胞)5只、转染TCR1(α链可变域SEQ ID NO：13，β链可变域SEQ ID NO：2)的T细胞组5只、转染TCR2(α链可变域SEQ ID NO:14，β链可变域SEQ ID NO:2)的T细胞组5只和转染TCR4(α链可变域SEQ ID NO:16，β链可变域SEQ ID NO:2)的T细胞组5只，按肿瘤体积对小鼠进行随机分组。分组完毕后取已制备好的T细胞按1*10^7个/只分别对上述小鼠进行尾部静脉注射。
TCR-T细胞注射后，再取制备好的IL-2溶液(50000IU)对每只小鼠腹腔注射200μl，随后4天每天 连续注射等量IL-2溶液。自实验开始，按上述方法每2天测量一次小鼠肿瘤瘤径和计算体积，持续至小鼠因肿瘤过大影响行动或者肿瘤消退为止，整理以上数据对每组小鼠肿瘤体积数据统计分析处理。
所得实验结果如图6所示，注射了转染本申请高亲和力TCR的T细胞的小鼠组，肿瘤的生长明显受到抑制并且呈现缩小的趋势，而注射了转染无关TCR的T细胞组的小鼠组的肿瘤体积增长依然很快。
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。
在本申请提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。

Claims (26)

  1. 一种T细胞受体(TCR)，所述T细胞受体包含TCRα链可变域和TCRβ链可变域，其特征在于，其具有结合GVYDGREHTV-HLA A0201复合物的活性，并且所述TCRα链可变域的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少90％的序列同源性和所述TCRβ链可变域的氨基酸序列与SEQ ID NO:2所示的氨基酸序列有至少90％的序列同源性。
  2. 如权利要求1所述的TCR，其特征在于，所述TCRβ链可变域的3个CDR为：CDR1β：MNHEY；CDR2β：SVGEGT；和CDR3β：ASSLGRAYEQY。
  3. 如权利要求1所述的TCR，其特征在于，所述TCRβ链可变域的氨基酸序列为SEQ ID NO:2。
  4. 如权利要求1所述的TCR，其特征在于，所述TCR还具有结合GLYDGREHSV-HLA A0201复合物的活性。
  5. 如权利要求1所述的TCR，其特征在于，所述TCRα链可变域的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少95％的序列同源性和所述TCRβ链可变域的氨基酸序列与SEQ ID NO:2所示的氨基酸序列有至少95％的序列同源性。
  6. 如权利要求1所述的TCR，其特征在于，所述TCR与GVYDGREHTV-HLA A0201复合物的亲和力是野生型TCR的至少5倍。
  7. 如权利要求1所述的TCR，其特征在于，所述TCRα链可变域的3个CDR区(互补决定区)的基准序列如下，
    CDR1α：DSSSTY
    CDR2α：IFSNMDM
    CDR3α：AEQSFGNEKLT，并且CDR3α含有至少一个下列突变：
  8. 如权利要求9所述的TCR，其特征在于，所述CDR3α中氨基酸突变包含：
  9. 如权利要求1所述的TCR，其特征在于，所述TCR具有选自下组的CDR:

  10. 如权利要求1所述的TCR，其特征在于，所述TCR是可溶的。
  11. 如权利要求1所述的TCR，其特征在于，所述TCR为αβ异质二聚TCR，包含α链TRAC恒定区序列和β链TRBC1或TRBC2恒定区序列。
  12. 如权利要求1所述的TCR，其特征在于，所述TCR包含(ⅰ)TCRα链可变域和除跨膜结构域以外的全部或部分TCRα链恒定区；和(ⅱ)TCRβ链可变域和除跨膜结构域以外的全部或部分TCRβ链恒定区。
  13. 如权利要求1所述的TCR，其特征在于，所述TCR包含α链恒定区与β链恒定区，并且所述α链恒定区与β链恒定区之间含有人工链间二硫键；优选地，在所述TCRα与β链的恒定区之间形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
    TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
    TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
    TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
    TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
    TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
    和TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
  14. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域氨基酸序列为SEQ ID NO:16、14、13、15或17-32之一；和所述TCR的β链可变域氨基酸序列为SEQ ID NO:2。
  15. 如权利要求1所述的TCR，其特征在于，所述TCR为单链TCR。
  16. 如权利要求1所述的TCR，其特征在于，所述TCR是由α链可变域和β链可变域组成的单链TCR，所述α链可变域和β链可变域由一柔性短肽序列(linker)连接。
  17. 如权利要求1所述的TCR，其特征在于，所述TCR的α链和/或β链的C-或N-末端结合有偶联物；
    优选地，所述偶联物为可检测标记物或治疗剂；
    更优选地，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
  18. 一种多价TCR复合物，其特征在于，包含至少两个TCR分子，并且其中的至少一个TCR分子为上述权利要求中任一项所述的TCR。
  19. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1-17中任一项所述的TCR的核酸序列或其互补序列。
  20. 一种载体，其特征在于，所述的载体含有权利要求19中所述的核酸分子。
  21. 一种宿主细胞，其特征在于，所述的宿主细胞中含有权利要求20中所述的载体或染色体中整合有外源的权利要求19中所述的核酸分子。
  22. 一种分离的细胞，其特征在于，所述细胞表达权利要求1-17中任一项所述的TCR，优选地所述分离的细胞为T细胞。
  23. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1-17中任一项所述的TCR、 或权利要求18中所述的TCR复合物、或权利要求22中所述的细胞。
  24. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用权利要求1-17中任一项所述的TCR、或权利要求18中所述的TCR复合物、或权利要求22中所述的细胞、或权利要求23中所述的药物组合物，优选地，所述疾病为MAGE-A4阳性肿瘤。
  25. 权利要求1-17中任一项所述的T细胞受体、权利要求18中所述的TCR复合物或权利要求22中所述细胞的用途，其特征在于，用于制备***的药物，优选地，所述肿瘤为MAGE-A4阳性肿瘤。
  26. 一种制备权利要求1-17中任一项所述的T细胞受体的方法，其特征在于，包括步骤：
    (i)培养权利要求21中所述的细胞，从而表达权利要求1-17中任一项所述的T细胞受体；
    (ii)分离或纯化出所述的T细胞受体。
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