WO2022262842A1 - 一种针对afp抗原的高亲和力t细胞受体 - Google Patents

一种针对afp抗原的高亲和力t细胞受体 Download PDF

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WO2022262842A1
WO2022262842A1 PCT/CN2022/099383 CN2022099383W WO2022262842A1 WO 2022262842 A1 WO2022262842 A1 WO 2022262842A1 CN 2022099383 W CN2022099383 W CN 2022099383W WO 2022262842 A1 WO2022262842 A1 WO 2022262842A1
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tcr
seq
chain
variable domain
amino acid
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PCT/CN2022/099383
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French (fr)
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郭姗姗
温桥生
黄姣
翁志明
陈建君
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香雪生命科学技术（广东）有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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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
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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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2319/00—Fusion polypeptide

Definitions

  • the present application belongs to the field of biotechnology, and more specifically relates to a T cell receptor (T cell receptor, TCR) capable of recognizing a polypeptide derived from an AFP protein.
  • TCR T cell receptor
  • the present application also relates to the preparation and use of said receptors.
  • TCR T cell receptor
  • TCR is the sole receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC), and such exogenous or endogenous peptides may be the only sign of abnormalities in cells.
  • MHC major histocompatibility complex
  • the combination of antigen-specific TCR and pMHC complexes triggers direct physical contact between T cells and antigen-presenting cells (APCs), and then other cell membrane surface molecules of T cells and APCs interact. It causes a series of subsequent cell signal transmission and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells.
  • APCs antigen-presenting cells
  • MHC class I and class II molecules corresponding to TCR are also proteins of the immunoglobulin superfamily, but they are specific for the presentation of antigens. Different individuals have different MHCs, so they can present different short sequences of a protein antigen. Peptides to the surface of the respective APC cells. Human MHC is often referred to as HLA genes or HLA complexes.
  • AFP ( ⁇ Fetoprotein), also known as ⁇ -fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP has relatively high expression levels in the yolk sac and liver, and is subsequently repressed. In liver cancer, the expression of AFP is activated. AFP is processed into antigenic peptides in cells, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented on the cell surface.
  • TSSELMAITR (SEQ ID NO: 32) is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.
  • the TSSELMAITR (SEQ ID NO:32)-HLA A1101 complex provides a TCR-targetable marker for tumor cells.
  • the TCR capable of binding TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex has high application value for the treatment of tumors.
  • a TCR capable of targeting this tumor cell marker can be used to deliver a cytotoxic or immunostimulatory agent to the target cell, or be transformed into a T cell, enabling the T cell expressing the TCR to destroy the tumor cell, so that in what is known as Administered to patients during the course of treatment with adoptive immunotherapy.
  • the ideal TCR is one with high affinity, allowing the TCR to reside on the targeted cell for a long period of time.
  • the use of intermediate affinity TCRs is preferred. Therefore, those skilled in the art are devoting themselves to developing TCRs targeting tumor cell markers that can be used for different purposes.
  • the application provides a TCR with high affinity for TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex.
  • the present application further provides a preparation method of the above-mentioned TCR and an application of the above-mentioned TCR.
  • the first aspect of the present application provides a T cell receptor (TCR) comprising an ⁇ -chain variable domain and a ⁇ -chain variable domain, which has the ability to bind TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex Activity, and the amino acid sequence of the TCR ⁇ chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence of the TCR ⁇ chain variable domain and SEQ ID NO: 2 The amino acid sequences shown have at least 90% sequence identity.
  • TCR T cell receptor
  • the amino acid sequence of the variable domain of the TCR ⁇ chain and the amino acid sequence of the variable domain of the TCR ⁇ chain are different from the amino acid sequence of the variable domain of the wild-type TCR ⁇ chain and the amino acid sequence of the variable domain of the wild-type TCR ⁇ chain sequence.
  • the amino acid sequence of the TCR ⁇ chain variable domain is not the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence of the TCR ⁇ chain variable domain is not the amino acid sequence shown in SEQ ID NO: 2 sequence.
  • the ⁇ -chain variable domain of the TCR comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% sequence homology.
  • the amino acid sequence of the variable domain of the TCR ⁇ chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO:1.
  • the ⁇ chain variable domain of the TCR is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98%, 99% or 100% sequence homology.
  • the amino acid sequence of the TCR ⁇ chain variable domain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO:2.
  • the CDR1 ⁇ in the variable domain of the TCR ⁇ chain is: DSVNN (SEQ ID NO: 33), and the CDR2 ⁇ is IPSGT (SEQ ID NO: 34).
  • the CDR3 ⁇ of the TCR ⁇ chain variable domain is selected from SGGSGFRLT (SEQ ID NO: 35), SGGQGYKLT (SEQ ID NO: 36) and SGGSNYKLT (SEQ ID NO: 37).
  • CDR1 ⁇ in the variable domain of the TCR ⁇ chain is DSVNN (SEQ ID NO: 33)
  • CDR2 ⁇ is IPSGT (SEQ ID NO: 34)
  • CDR3 ⁇ is selected from SGGSGFRLT (SEQ ID NO: 35), SGGQGYKLT (SEQ ID NO: 36) and SGGSNYKLT (SEQ ID NO: 37).
  • the three CDRs of the TCR ⁇ chain variable domain are: CDR1 ⁇ : SEHNR (SEQ ID NO: 38); CDR2 ⁇ : FQNEAQ (SEQ ID NO: 39); and CDR3 ⁇ : ASSPGTGVGYT (SEQ ID NO :40).
  • amino acid sequence of the TCR ⁇ chain variable domain is SEQ ID NO:2.
  • the number of amino acid mutations in the variable domain of the TCR ⁇ chain is 1-3; preferably 3.
  • amino acid mutation sites of the TCR ⁇ chain variable domain are the 5th, 6th, and 7th positions of CDR3 ⁇ .
  • amino acid mutation sites of the TCR ⁇ chain variable domain are the 4th and 5th positions of CDR3 ⁇ .
  • the reference sequences of the three CDR regions (complementarity determining regions) of the variable domain of the TCR ⁇ chain are as follows:
  • CDR1 ⁇ DSVNN (SEQ ID NO: 33);
  • CDR3 ⁇ SGGSNYKLT (SEQ ID NO: 37), and CDR3 ⁇ contains at least one of the following mutations:
  • amino acid mutation in CDR3 ⁇ comprises:
  • the reference sequences of the three CDR regions (complementarity determining regions) of the TCR ⁇ chain variable domain are as follows,
  • CDR1 ⁇ SEHNR (SEQ ID NO: 38);
  • CDR3 ⁇ ASSPGTGVGYT (SEQ ID NO: 40), and CDR3 ⁇ contains at least one of the following mutations:
  • amino acid mutation in the CDR3 ⁇ comprises:
  • the affinity between the TCR and the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex is at least 2 times that of the wild-type TCR.
  • the TCR is mutated in the ⁇ -chain variable domain shown in SEQ ID NO: 1, and the mutation is selected from one or more groups of S91Q, N92G, Y93F, and K94R, wherein the amino acid Residue numbering adopts the numbering shown in SEQ ID NO:1.
  • the TCR is mutated in the ⁇ chain variable domain shown in SEQ ID NO: 1, and the mutation is selected from P96H, G97P/Q, T98Q/H, V100I, G101Q/R/S One or several groups in /I/L/V, Y102Q/F/M, T103E/Q/H/K/L/R, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:2.
  • the TCR has a CDR selected from the group consisting of:
  • the TCR is soluble.
  • the TCR is an ⁇ heterodimeric TCR, comprising a TRAC constant region sequence of the ⁇ chain and a TRBC1 or TRBC2 constant region sequence of the ⁇ chain.
  • the TCR comprises (i) TCR ⁇ chain variable domain and all or part of the TCR ⁇ chain constant region except the transmembrane domain; and (ii) TCR ⁇ chain variable domain and except the transmembrane domain All or part of the TCR ⁇ chain constant region.
  • an artificial interchain disulfide bond is contained between the constant region of the ⁇ chain and the constant region of the ⁇ chain of the TCR.
  • cysteine residues forming an artificial interchain disulfide bond between the constant regions of the TCR ⁇ and ⁇ chains are substituted for 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: 1, 5-7; and/or the amino acid sequence of the ⁇ -chain variable domain of the TCR is SEQ ID NO: 2.
  • the TCR is selected from the following group:
  • the TCR is of human origin.
  • 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 comprises an ⁇ -chain constant region and a ⁇ -chain constant region
  • the ⁇ -chain constant region is a mouse constant region and/or the ⁇ -chain constant region is a mouse constant region.
  • the C- or N-terminus of the ⁇ chain and/or ⁇ chain of the TCR is bound with a conjugate, preferably, the conjugate is a detectable marker, a therapeutic agent, a PK modification part or combination of any of these substances.
  • the therapeutic agent that binds to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the ⁇ or ⁇ chain of the TCR.
  • the second aspect of the present application provides a multivalent TCR complex, which comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present application.
  • the third aspect of the present application provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present application or the multivalent TCR complex described in the second aspect of the present application or its complement sequence.
  • the fourth aspect of the present application provides a 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 containing the vector of the fourth aspect of the present application or the exogenous nucleic acid molecule of the third aspect of the present application integrated in the chromosome.
  • the sixth aspect of the present application provides an isolated cell expressing the TCR described in the first aspect of the application, preferably, the isolated cell is a T cell, NK cell or NKT cell, most preferably , 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, or the TCR complex described in the second aspect of the present application, Or the cell described in the sixth aspect of the present application.
  • the eighth aspect of the present application provides a method for treating diseases, comprising administering an appropriate amount of the TCR described in the first aspect of the present application, or the TCR complex described in the second aspect of the present application, or the For the cell described in the sixth aspect of the application, or the pharmaceutical composition described in the seventh aspect of the application, preferably, the disease is an AFP-positive tumor, more preferably, the tumor is liver cancer.
  • the ninth aspect of the present application provides the use of the TCR described in the first aspect of the present application, or the TCR complex described in the second aspect of the present application, or the use of the cells described in the sixth aspect of the present application, for preparing and treating tumors
  • the disease is an AFP-positive tumor, more preferably, the tumor is liver cancer.
  • the tenth aspect of the present application provides a method for preparing the T cell receptor described in the first aspect of the present application, comprising the steps of:
  • Figure 1a and Figure 1b respectively show the amino acid sequences of the variable domains of the wild-type TCR ⁇ chain and ⁇ chain that can specifically bind to the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex.
  • Figure 2a and Figure 2b are the amino acid sequences of the soluble reference TCR ⁇ chain and ⁇ chain in this application, respectively.
  • Figure 3 (1)-(3) shows the ⁇ chain variable domain amino acid sequence of the heterogeneous dimerization TCR with high affinity to TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex respectively, the residue of mutation is added underlined.
  • Figure 4 (1)-(20) shows the amino acid sequence of the ⁇ chain variable domain of the heterogeneous dimerization TCR with high affinity to TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex respectively, the residue of mutation is added underlined.
  • Figure 5a and Figure 5b show the extracellular amino acid sequences of the wild-type TCR ⁇ chain and ⁇ chain that can specifically bind to the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex.
  • Figure 6a and Figure 6b respectively show the amino acid sequences of the wild-type TCR ⁇ chain and ⁇ chain that can specifically bind to the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex.
  • Fig. 7 is the binding curve of soluble reference TCR, ie, wild-type TCR, and TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex.
  • Fig. 8 is the experimental result of the activation function of T2 cells loaded with short peptides and effector cells transfected with the high-affinity TCR of the present application.
  • Figure 9a and Figure 9b are the results of the activation function experiment of the effector cells transfected with the high-affinity TCR of the present application for tumor cell lines.
  • Figures 10a and 10b are the results of killing function experiments of effector cells transfected with the high-affinity TCR of the present application for tumor cell lines.
  • TCR T cell receptor
  • CDR1 ⁇ DSVNN (SEQ ID NO: 33);
  • CDR3 ⁇ Mutations in SGGSNYKLT (SEQ ID NO: 37); and/or in the 3 CDR regions of its ⁇ chain variable domain:
  • CDR1 ⁇ SEHNR (SEQ ID NO: 38);
  • CDR3 ⁇ Mutation in ASSPGTGVGYT (SEQ ID NO: 40).
  • the affinity and/or binding half-life of the TCR of the present application to the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex after mutation is at least 2 times that of the wild-type TCR.
  • T cell receptor T cell receptor, TCR
  • TCRs can be described using the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • Natural ⁇ heterodimeric TCRs have an ⁇ chain and a ⁇ chain.
  • each chain comprises a variable region, a connecting region, and a constant region
  • the beta strands usually also contain a short variable region between the variable and connecting regions, but this variable region is often considered 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 the TRAC and TRBC of IMGT.
  • Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimeric in the framework sequence.
  • TRAV and TRBV refer to different V ⁇ types and V ⁇ types, respectively.
  • the ⁇ -chain constant domain has the following symbols: TRAC*01, where "TR” indicates the T cell receptor gene; "A” indicates the ⁇ -chain gene; C indicates the constant region; "*01” indicates allele Gene 1.
  • the beta chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR” indicates the T cell receptor gene; "B” indicates the beta chain gene; C indicates the constant region; "*01” indicates the allele 1.
  • the constant region of the alpha chain is uniquely defined, 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 published IMGT database.
  • TCR ⁇ chain variable domain refers to the connected TRAV and TRAJ regions
  • TCR ⁇ chain variable domain refers to the connected TRBV and TRBD/TRBJ regions.
  • the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ ; the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ .
  • 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 comprises an intracellular portion, a transmembrane region and an extracellular portion.
  • the ⁇ -chain extracellular amino acid sequence and ⁇ -chain extracellular amino acid sequence of the "wild-type TCR” described in this application are SEQ ID NO: 28 and SEQ ID NO: 29, respectively, as shown in Figure 5a and Figure 5b.
  • 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 SEQ ID NO: 30 and SEQ ID NO: 31, respectively, as shown in Figure 6a and Figure 6b.
  • the terms "polypeptide of the present application", “TCR of the present application”, “T cell receptor of the present application” are used interchangeably.
  • the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in this application are numbered in sequence from the N-terminal to the C-terminal, such as in TRBC1*01 or TRBC2*01, according to the sequence from N
  • the 60th amino acid in the sequence from terminal to C-terminal is P (proline)
  • it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01 in this application and it can also be expressed as TRBC1* 01 or the 60th amino acid of exon 1 of TRBC2*01, and for example in TRBC1*01 or TRBC2*01
  • the 61st amino acid is Q (glutamine) in the order from N-terminal to C-terminal, then this
  • it can be described as Gln61 of exon 1 of TRBC1*01 or TRBC2*01, or it can be expressed as amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01,
  • the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are according to the position numbers listed in IMGT. For example, if a certain amino acid in TRAV is numbered 46 in IMGT, it will be described as the 46th amino acid in TRAV in this application, and so on. In this application, if there are special instructions for the sequence position numbers of other amino acids, the special instructions shall be followed.
  • tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include: malignant tumors of different organ systems, such as sarcomas, squamous cell carcinomas of the lung, and carcinomas. Examples: Infected prostate, lung, breast, lymphatic, gastrointestinal (eg colon), and genitourinary (eg kidney, epithelial), pharynx.
  • Squamous cell carcinoma of the lung includes malignancies such as most colon, rectal, renal cell, liver, non-small cell carcinomas of the lung, small intestine, and esophagus. Metastatic lesions of the above cancers can also be treated and prevented by the methods and compositions of the present application.
  • ⁇ -chain variable domain and ⁇ -chain variable domain of TCR each contain 3 CDRs, which are similar to the complementarity determining regions of antibodies.
  • CDR3 interacts with antigenic short peptides
  • CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigen short peptide-HLA complex.
  • TSSELMAITR antigen short peptide
  • HLA A1101 complex ie, TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex
  • the amino acid sequences of the variable domains are respectively SEQ ID NO: 1 and SEQ ID NO: 2, which were discovered by the inventors for the first time. It has the following CDR regions:
  • CDR1 ⁇ DSVNN (SEQ ID NO: 33);
  • CDR1 ⁇ SEHNR (SEQ ID NO: 38);
  • CDR3 ⁇ ASSPGTGVGYT (SEQ ID NO: 40).
  • the present application obtains a TCR with improved affinity with TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex and improved cell function by performing mutation screening on the above CDR region.
  • the TCR described in the present application is an ⁇ heterodimeric TCR or a single-chain TCR comprising an ⁇ chain variable domain and a ⁇ chain variable domain, and the ⁇ chain variable domain of the TCR comprises the same sequence as SEQ ID NO: 1.
  • At least 90%; more preferably, at least 92%; more preferably, at least 94% (e.g., may be at least 88%, 89%, 90%, 91%, 92% of the amino acid sequence shown) %, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) amino acid sequence of sequence homology); and/or the ⁇ chain variable domain of the TCR comprises At least 90%, preferably at least 92%; more preferably at least 94% (e.g., at least 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in SEQ ID NO: 2 , 96%, 97%, 98%, 99% or 100% sequence homology) amino acid sequences with sequence homology).
  • the three CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO: 1, namely CDR1, CDR2 and CDR3 are respectively located at positions 27-31, 49-53 and 88-96 of SEQ ID NO: 1 . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1, 91S is the 4th S of CDR3 ⁇ , 92N is the 5th N of CDR3 ⁇ , 93Y is the 6th Y of CDR3 ⁇ , and 94K is It is the 7th K of CDR3 ⁇ .
  • the specific form of the mutation in the ⁇ -chain variable domain includes one or several groups of S91Q, N92G, Y93F, and K94R.
  • the 3 CDRs of the wild-type TCR ⁇ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2 and CDR3 are respectively located at positions 27-31, 49-54 and 93-103 of SEQ ID NO: 2 .
  • the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1
  • 96P is the 4th P of CDR3 ⁇
  • 97G is the 5th G of CDR3 ⁇
  • 98T is the 6th T of CDR3 ⁇
  • 100V is The 8th V of CDR3 ⁇
  • 101G is the 9th G of CDR3 ⁇
  • 102Y is the 10th Y of CDR3 ⁇
  • 103T is the 11th T of CDR3 ⁇ .
  • variable domain of the ⁇ chain examples include P96H, G97P/Q, T98Q/H, V100I, G101Q/R/S/I/L/V, Y102Q/F/M, T103E/Q/ One or several groups of H/K/L/R.
  • amino acids in this article are identified by international single English letters, and the corresponding three-letter abbreviations of 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 the 60th proline.
  • expression of the specific form of the mutation mentioned in this application is such as "N92G” means that the N at the 92nd position is replaced by G, “G97P/Q” means that the G at the 97th position is replaced by P or Q, and others are as follows analogy.
  • the Pro89 of the exon 1 of the TRAC*01 exon of the wild-type TCR ⁇ chain constant region was mutated to cysteine, and the ⁇ chain constant region TRBC1*01 or TRBC2*01 exon 1
  • the Ala19 of Ala19 is mutated to cysteine, that is, the reference TCR is obtained, and its amino acid sequences are SEQ ID NO: 3 and SEQ ID NO: 4, respectively, as shown in Figure 2a and Figure 2b, the mutated cysteine residue Indicated in bold letters.
  • the cysteine substitutions described above enable the formation of an artificial interchain disulfide bond between the constant regions of the ⁇ and ⁇ chains of the reference TCR to form a more stable soluble TCR, thereby allowing for a more convenient evaluation of TCR and TSSELMAITR (SEQ ID NO :32)-Binding affinity and/or binding half-life between HLA A1101 complexes. It should be understood that the CDR region of the TCR variable region determines its affinity with the pMHC complex, therefore, the above-mentioned cysteine substitution in the TCR constant region will not affect the binding affinity and/or binding half-life of the TCR.
  • the measured binding affinity between the reference TCR and TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex is considered to be the binding affinity between wild-type TCR and TSSELMAITR (SEQ ID NO: 32)-HLA A1101 Binding affinity between complexes.
  • the binding affinity between the TCR of the present application and the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex is determined to be the binding affinity between the reference TCR and the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex At least 2 times of the affinity, which is equivalent to the binding affinity between the TCR of the present application and the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex is higher than that of the wild-type TCR and the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex At least 2 times the binding affinity between.
  • Binding affinity (inversely proportional to the dissociation equilibrium constant KD) and binding half-life (expressed as T 1/2 ) can be determined by any suitable method, such as by surface plasmon resonance techniques. It will be appreciated that doubling the affinity of a TCR will result in a halving of the KD. T 1/2 was calculated as In2 divided by the off-rate (K off ). Therefore, doubling T 1/2 causes K off to be halved.
  • the same assay protocol is used to detect the binding affinity or binding half-life of a given TCR several times, eg 3 times or more, and the results are averaged.
  • the surface plasmon resonance (BIAcore) method in the examples herein is used to detect the affinity of soluble TCR, and the conditions are: temperature 25° C., pH value 7.1-7.5.
  • This method detects that the dissociation equilibrium constant KD of the reference TCR to the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex is 4.16E-05M, that is, 41.6 ⁇ M.
  • the wild-type TCR has a TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex.
  • the dissociation equilibrium constant KD of the NO:32)-HLA A1101 complex was also 41.6 ⁇ M.
  • 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 textbooks. More details on polymerase chain reaction (PCR) mutagenesis and restriction enzyme based cloning can be found in Sambrook and Russell, (2001) Molecular Cloning - A Laboratory Manual (Third Edition) CSHL publishing house. 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 for producing the TCR of the present application may be, but not limited to, screening a TCR with high affinity for TSSELMAITR (SEQ ID NO: 32)-HLA-A1101 complex from a diversity library of phage particles displaying such TCR, As described in the literature (Li, et al (2005) Nature Biotech 23(3):349-354).
  • genes expressing the variable domain amino acids of the alpha and beta chains of wild-type TCRs or genes expressing the amino acids of the variable domains of the alpha and beta chains of wild-type TCRs with slight modifications can be used to make template TCRs.
  • the changes required to generate the high affinity TCRs of the present application are then introduced into the DNA encoding the variable domains of this template TCR.
  • the high-affinity TCR of the present application comprises the amino acid sequence of the ⁇ -chain variable domain as one of SEQ ID NO: 1, 5-7; and/or the amino acid sequence of the ⁇ -chain variable domain of the TCR as SEQ ID NO: 2, One of 8-27.
  • the amino acid sequences of the ⁇ -chain variable domain and the ⁇ -chain variable domain of the heterodimeric TCR molecule of the present application are preferably selected from the following table 1:
  • a TCR of the present application is a portion having at least one variable domain of a TCR alpha and/or TCR beta chain. They usually contain both TCR ⁇ chain variable domains and TCR ⁇ chain variable domains. They may be ⁇ heterodimers or single-chain forms or any other forms that can exist stably.
  • the full-length chain of the ⁇ heterodimeric TCR (including the 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 be combined with other molecules to prepare bifunctional polypeptides to target effector cells. At this time, TCR is preferably in a soluble form.
  • the TCR of the present application may be a TCR that introduces an artificial interchain disulfide bond between residues of its alpha and beta chain constant domains.
  • Cysteine residues form artificial interchain disulfide bonds between the alpha and beta chain constant domains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate sites in native TCRs to form artificial interchain disulfide bonds.
  • cysteine residues replace any one group of positions in the above-mentioned ⁇ and ⁇ chain constant domains.
  • a maximum of 15, or a maximum of 10, or a maximum of 8 or fewer amino acids may be truncated at one or more C-terminal ends of the TCR constant domains of the present application, so that it does not include a cysteine residue to achieve the deletion of the native
  • 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 TCRs of the present application may contain artificial interchain disulfide bonds introduced between residues in the constant domains of their alpha and beta chains. It should be noted that the TCR of the present application can contain both the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence, with or without the introduced artificial disulfide bond between the constant domains as described above.
  • the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by native interchain disulfide bonds present in the TCR.
  • the patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond 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 an artificial interchain disulfide bond between the variable region of the ⁇ chain and the constant region of the ⁇ chain.
  • cysteine residue that forms an artificial interchain disulfide bond between the ⁇ -chain variable region and the ⁇ -chain constant region of the TCR is substituted for: amino acid 46 of TRAV and TRBC1*01 or TRBC2* Amino acid 60 of exon 1 of 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01; amino acid 46 of TRAV and exon of TRBC1*01 or TRBC2*01 Amino acid 61 of exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.
  • such a TCR may comprise (i) all or part of a TCR alpha chain except for its transmembrane domain, and (ii) all or part of a TCR beta chain except for its transmembrane domain, wherein (i) and (ii ) all contain the variable domain of the TCR chain and at least a part of the constant domain, and the ⁇ chain and the ⁇ chain form a heterodimer.
  • such a TCR may comprise an ⁇ -chain variable domain and a ⁇ -chain variable domain and all or part of a ⁇ -chain constant domain except the transmembrane domain, but it does not contain an ⁇ -chain constant domain, and the ⁇ -chain of said TCR Chain variable domains form heterodimers with beta chains.
  • the TCRs of the present application also include TCRs that undergo mutations in their hydrophobic core regions, and these mutations in the hydrophobic core regions are preferably mutations that can improve the stability of the TCRs of the present application, as disclosed in Publication No. described in the patent literature of WO2014/206304.
  • TCRs may have mutations at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the 3rd, 5th, and 7th positions from the bottom of the amino acid position of the ⁇ -chain J gene (TRAJ) short peptide, and/or the 2nd, 4th, and 6th position from the bottom of the amino acid position of the ⁇ -chain J gene (TRBJ) short peptide, wherein the position number of the amino acid sequence By position number as listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR with mutations in the hydrophobic core region of the present application may be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the ⁇ chain and the ⁇ chain of the TCR.
  • the CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex.
  • the mutation of the hydrophobic core can make the TCR more stable, but it will not affect its affinity with the short peptide-HLA complex.
  • the flexible peptide chain in this application can be any peptide chain suitable for linking the variable domains of TCR ⁇ and ⁇ chains.
  • the TCRs of the present application may also be provided in the form of multivalent complexes.
  • the multivalent TCR complex of the present application comprises two, three, four or more multimers formed by combining the TCRs of the present application, such as the tetramerization domain of p53 can be used to produce tetramers, or multimers A complex formed by the combination of a TCR of the present application and another molecule.
  • the TCR complexes of the present application can be used to track or target cells presenting specific antigens in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes with such applications.
  • the TCR of the present application can be used alone, and can also be combined with a conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugates include detectable markers (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the TSSELMAITR (SEQ ID NO: 32)-HLA-A1101 complex), therapeutic agents, PK (protein kinase ) modifying moieties or combinations or couplings of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or products capable of producing detectable enzymes.
  • Therapeutic agents that can be combined or coupled with the TCR of the present application include but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2. Biological toxicity (Chaudhary et al., 1989 , Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2 etc.
  • Gold nanoparticles /Nanorods (Lapotko et al., 2005, Cancer letters (Cancer letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society (Journal of the American Chemical Society) 128, 2115); 7. Virus particles (Peng et al., 2004 , Gene therapy (Gene therapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nanomagnetic particles; 10. Prodrug activating enzymes (for example, DT - diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); and 11. A chemotherapeutic agent (eg cisplatin) or any form of nanoparticle or the like.
  • DTD DT - diaphorase
  • BPHL biphenylhydrolase-like protein
  • Antibodies or fragments thereof that bind to the TCR of the present application include anti-T cells or NK-cell determination antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies, and the combination of the above-mentioned antibodies or fragments thereof and TCR can carry out effects on 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 of the present application and the anti-CD3 single-chain antibody comprises an amino acid sequence selected from the variable domain of the TCR ⁇ chain as one of SEQ ID NO: 1, 5-7; and/or the variable ⁇ chain of the TCR
  • the domain amino acid sequence is one of SEQ ID NO: 2, 8-27.
  • 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 either the coding strand or the 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 that encodes a protein having SEQ ID NO: 3, but is identical to 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 of the present application (or its fragments, or its derivatives) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the present application also relates to vectors comprising the nucleic acid molecules of the present application, and host cells produced by genetic engineering using the vectors or coding sequences of the present application.
  • the present application also includes isolated cells expressing the TCRs of the present application, especially T cells.
  • T cells There are many methods suitable for T cell transfection with DNA or RNA encoding the high-affinity TCR of the present application (eg, Robbins et al., (2008) J. Immunol. 180:6116-6131).
  • T cells expressing the high-affinity TCR of this application can be used for adoptive immunotherapy.
  • Many suitable methods of performing adoptive therapy are known to those skilled in the art (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
  • the present application also provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR of the present application, or the TCR complex of the present application, or a cell presenting the TCR of the present application.
  • the present application also provides a method for treating diseases, comprising administering an appropriate amount of the TCR of the present application, or the TCR complex of the present application, or the cells presenting the TCR of the present application, or the pharmaceutical composition of the present application to the 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 most preferably 1 amino acid (especially the amino acid located outside the CDR region) of the TCR of the present application, which are similar in nature or similar amino acid replacement, and still be able to maintain its functional TCR.
  • the present application also includes the slightly modified TCR of the present application.
  • Modified (generally without altering the primary structure) forms include: chemically derivatized forms of the TCRs of the present application such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those TCRs produced by 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 an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme.
  • Modified forms also include sequences with 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 solubility.
  • the TCR of the present application, the TCR complex or the T cells transfected by the TCR of the present application can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCRs, multivalent TCR complexes or cells of the present application 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 can be presented in unit dosage form, usually in a hermetically sealed container, which can be provided as part of a kit. Such kits, but not necessarily, include instructions for use. It may comprise a plurality of such unit dosage forms.
  • the TCR of the present application can be used alone, or combined or coupled 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 for the administration of a therapeutic agent.
  • pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon 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., N.J. 1991).
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids, such as water, saline, glycerol and ethanol.
  • liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances in these carriers such as wetting agents or emulsifying agents, pH buffering substances and the like.
  • therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension, in liquid carriers prior to injection can also be prepared.
  • compositions of the present application may be administered by conventional routes, including but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical, preferably gastrointestinal External includes subcutaneous, intramuscular or intravenous.
  • the subject to be prevented or treated can be an animal; especially a human.
  • compositions of the present application When the pharmaceutical composition of the present application is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the usage conditions. Preferably, injections, oral preparations and the like 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 agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.
  • the pharmaceutical composition of the present application can also be administered in the form of sustained release formulations.
  • the TCR of the present application can be incorporated into a pill or microcapsule with a slow-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers include ethylene-vinyl acetate copolymers, polyhydroxymethacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the TCR or TCR complex of the present application or the cells presenting the TCR of the present application as the active ingredient can be selected according to the body weight, age, sex, and degree of symptoms of each patient to be treated. However, it should be reasonably determined, and finally the doctor will determine the reasonable dosage.
  • the TCR of the present application can specifically bind to the TSSELMAITR (SEQ ID NO: 32)-HLA A1101, and 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 From Novagen
  • plasmid pET28a was purchased from Novagen.
  • BIAcore T200 real-time analysis system was used to detect the binding activity of TCR molecules and TSSELMAITR (SEQ ID NO: 32)-HLA-A1101 complex.
  • the conditions are: temperature 25°C, pH value 7.1-7.5.
  • a low concentration of streptavidin was flowed over the surface of the antibody-coated chip, and then the TSSELMAITR (SEQ ID NO: 32)-HLA-A1101 complex was flowed through the detection channel, and the other channel was used as a reference channel, and then 0.05 mM biotin flowed through the chip at a flow rate of 10 ⁇ L/min for 2 min to block the remaining binding sites of streptavidin.
  • the affinity was determined by single-cycle kinetic analysis, and the TCR was diluted into several different concentrations with HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) at a flow rate of 30 ⁇ L/min , flowing over the surface of the chip in turn, the binding time of each injection is 120s, and it is allowed to dissociate for 600s after the last injection.
  • the chip was regenerated with 10 mM Gly-HCl pH 1.75 after each run. Kinetic parameters were calculated using BIAcore Evaluation software.
  • the binding curve of the soluble reference TCR that is, the wild-type TCR, and the TSSELMAITR (SEQ ID NO: 32)-HLA A1101 complex is shown in Figure 7.
  • TSSELMAITR The synthetic short peptide TSSELMAITR (SEQ ID NO: 32, Jiangsu GenScript Biotechnology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. The inclusion bodies of the light chain and heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, and 10mM DTT. Before refolding, 3M guanidine hydrochloride, 10mM sodium acetate, and 10mM EDTA were added for further denaturation.
  • TSSELMAITR peptide (SEQ ID NO: 32) was added to refolding buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM Reduced glutathione, 0.2mM PMSF, cooled to 4°C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain is added in three times, 8h/time), renaturation At 4°C for at least 3 days to complete, SDS-PAGE test whether the renaturation is successful.
  • Biotinylation Concentrate the purified pMHC molecules with Millipore ultrafiltration tubes, and replace the buffer with 20mM Tris pH 8.0, then add biotinylation reagents 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50 ⁇ M D- Biotin, 100 ⁇ g/ml BirA enzyme (GST-BirA), incubate the mixture overnight at room temperature, and check whether the biotinylation is complete by SDS-PAGE.
  • Biotinylated complex use Millipore ultrafiltration tube to concentrate the biotinylated pMHC molecules to 1ml, use gel filtration chromatography to purify the biotinylated pMHC, and use Akta purifier (GE General Electric) Company), pre-equilibrated HiPrep TM 16/60 S200 HR column (GE General Electric Company) with filtered PBS, loaded 1 ml of concentrated biotinylated pMHC molecules, and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules elute as a single peak at about 55 ml.
  • the protein-containing fractions were pooled, concentrated with Millipore ultrafiltration tubes, and the protein concentration was determined by the BCA method (Thermo).
  • the biotinylated pMHC molecules were subpackaged and stored at -80°C by adding protease inhibitor cocktail (Roche).
  • Phage display technology is a means to generate a library of TCR high-affinity variants to screen for 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 wild-type TCR templates.
  • a site-directed mutagenesis method well known to those skilled in the art is used to mutate the CDR region of the template chain to establish a high-affinity TCR library and perform panning. After several rounds of panning, the phage library was specifically combined with the corresponding antigen, and single clones were picked and analyzed.
  • the extracellular sequence genes of the TCR ⁇ and ⁇ chains to be expressed were synthesized and inserted into the expression vectors, respectively, by standard methods described in Molecular Cloning a Laboratory Manual (Third Edition, Sambrook and Russell) For pET28a+ (Novagene), the upstream and downstream cloning sites are NcoI and NotI, respectively. Mutations in the CDR regions were introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The insert was confirmed by sequencing.
  • the dissolved TCR ⁇ and ⁇ chains were quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1. 60mg/mL. After mixing, the solution was dialyzed (4°C) in 10 times the volume of deionized water, and after 12 hours, the deionized water was replaced with buffer solution (20mM Tris, pH 8.0) and continued to be dialyzed at 4°C for 12 hours.
  • the solution was filtered through a 0.45 ⁇ M filter membrane, and then purified by an anion exchange column (HiTrap Q HP, 5ml, GE Healthcare).
  • the elution peaks containing TCRs of successfully refolded ⁇ and ⁇ dimers were confirmed by SDS-PAGE.
  • TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100 HR, GE Healthcare). The purity of the purified TCR was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • Embodiment 4 BIAcore analysis result
  • Example 1 The method described in Example 1 was used to detect the affinity between the ⁇ heterodimeric TCR of the high-affinity CDR of the present application and the TSSELMAITR (SEQ ID NO: 32)-HLA-A1101 complex.
  • the amino acid sequences of the new TCR ⁇ chain and ⁇ chain variable domain obtained in the present application are shown in Fig. 3(1)-(3) and Fig. 4(1)-(20), respectively.
  • Using the method described in Example 2 to construct an expression vector using the method described in Example 3 to express, anneal and purify the ⁇ heterodimeric TCR with high affinity mutation, and then use BIAcore T200 to determine its relationship with TSSELMAITR (SEQ The affinity of ID NO:32)-HLA-A1101 complex is shown in Table 2 below.
  • the affinity of the heterodimeric TCR is at least 2 times that of the wild-type TCR to the TSSELMAITR (SEQ ID NO: 32)-HLA-A1101 complex.
  • Example 5 Expression, refolding and purification of the fusion of anti-CD3 antibody and high-affinity ⁇ heterodimeric TCR
  • a fusion molecule was prepared by fusing an anti-CD3 single-chain antibody (scFv) to an ⁇ heterodimeric TCR.
  • the scFv of anti-CD3 is fused with the ⁇ chain of TCR.
  • the TCR ⁇ chain can contain any of the ⁇ chain variable domains of the above-mentioned high-affinity ⁇ heterodimeric TCR, and the TCR ⁇ chain of the fusion molecule can contain any of the above-mentioned high-affinity The ⁇ -chain variable domain of a sex ⁇ heterodimeric TCR.
  • the target gene carrying the ⁇ chain of the ⁇ heterodimeric TCR was digested with Nco I and Not I, and connected to the pET28a vector that was digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ , spread on LB plates containing kanamycin, cultured upside down at 37°C overnight, picked positive clones for PCR screening, sequenced positive recombinants, and extracted recombinant plasmids after confirming the correct sequence Transformed into E.coli Tuner (DE3) for expression.
  • anti-CD3 (scFv)-beta chain expression vector by overlapping (overlap) PCR, design primers to connect anti-CD3 scFv and high-affinity heterogeneous dimeric TCR beta chain gene, the middle connection is short
  • the peptide (linker) is GGGGS (SEQ ID NO: 61)
  • the gene fragment of the fusion protein of the scFv of anti-CD3 and the fusion protein of the high-affinity heterogeneous dimerization TCR ⁇ chain is brought on the restriction endonuclease site Nco I( CCATGG (SEQ ID NO: 62)) and Not I (GCGGCCGC (SEQ ID NO: 63)).
  • the PCR amplified product was digested with Nco I and Not I, and then ligated with the pET28a vector that had been digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ competent cells, coated with LB plates containing kanamycin, cultured upside down at 37°C overnight, picked positive clones for PCR screening, sequenced the positive recombinants, and extracted after confirming the correct sequence
  • the recombinant plasmid was transformed into E.coli Tuner (DE3) competent cells for expression.
  • the expression plasmids were respectively transformed into E.coli Tuner (DE3) competent cells, spread on LB plates (kanamycin 50 ⁇ g/mL) and cultured at 37°C overnight. The next day, pick clones and inoculate them into 10 mL LB liquid medium (kanamycin 50 ⁇ g/mL) for 2-3 hours, then inoculate them into 1L LB medium at a volume ratio of 1:100, and continue culturing until the OD600 is 0.5-0.8, adding The final concentration was 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. The bacteria were washed once with PBS buffer, and the bacteria were subpackaged.
  • the bacteria equivalent to 200 mL of bacterial culture were lysed with 5 mL of BugBuster Master Mix (Merck), and the inclusion bodies were collected by centrifugation at 6000 g for 15 min. Four detergent washes were then performed to remove cell debris and membrane components. Then, the inclusion bodies are washed with a buffer such as PBS to remove detergents and salts. Finally, the inclusion bodies were dissolved with a buffer solution containing 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris, pH 8.1, and the concentration of the inclusion bodies was measured, and then packed Store in -80°C freezer.
  • DTT dithiothreitol
  • EDTA ethylenediaminetetraacetic acid
  • the dissolved TCR ⁇ chain and anti-CD3 (scFv)- ⁇ chain are quickly mixed in 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1, 3.7 at a mass ratio of 2:5 mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C), final concentrations of ⁇ chain and anti-CD3(scFv)- ⁇ chain were 0.1mg/mL, 0.25mg/mL, respectively.
  • the solution was dialyzed (4°C) in 10 times the volume of deionized water, and after 12 hours, the deionized water was replaced with buffer solution (10mM Tris, pH 8.0) and continued to be dialyzed at 4°C for 12 hours.
  • buffer solution 10mM Tris, pH 8.0
  • the solution was filtered through a 0.45 ⁇ M filter membrane, and then purified by an anion exchange column (HiTrap Q HP 5ml, GE healthcare).
  • the elution peak contained the successfully refolded TCR ⁇ chain and anti-CD3(scFv)- ⁇ chain dimer TCR confirmed by SDS-PAGE gel.
  • the TCR fusion molecule was 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 is determined by SDS-PAGE to be greater than 90%, and the concentration is determined by the BCA method.
  • Example 6 Activation function experiment of effector cells transfected with high-affinity TCR of the application for T2 cells loaded with short peptides
  • IFN- ⁇ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN- ⁇ is detected by the ELISPOT experiment well known to those skilled in the art to verify the activation of cells transfected with the high-affinity TCR of this application Function and antigen specificity.
  • Transfect the high-affinity TCR of this application (TCR numbering and its sequence number are learned from Table 2) to CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and transfect other TCRs (A6 ) CD3 + T cells as a control.
  • the target cells used are T2-A11 loaded with AFP antigen short peptide TSSELMAITR (SEQ ID NO: 32), loaded with other antigen short peptides, or empty (referring to T2 cells transfected with HLA-A1101).
  • ELISPOT plate First prepare the ELISPOT plate, add 1 ⁇ 104 cells/well of target cells and 2 ⁇ 103 cells/well of effector cells (calculated according to the positive rate of transfection) into the corresponding wells, and then add AFP antigen short peptide to the experimental group
  • TSSELMAITR SEQ ID NO: 32
  • other antigenic short peptide solutions were added to the control group, and the final concentration of the short peptides was 10 -6 M.
  • An equal volume of medium was added to the blank group, and two replicate wells were set. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted by an immunospot plate reader (ELISPOT READER system; AID20 company).
  • Example 7 For the cell line, the activation function experiment of the effector cells transfected with the high-affinity TCR of the application
  • tumor cell lines were used to verify again the activation function and specificity of the effector cells transfected with the high-affinity TCR of the present application. It is also detected by ELISPOT experiment well known to those skilled in the art. Transfect the high-affinity TCR of this application (the TCR number and its sequence number are obtained from Table 2) to CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and empty transfection (NC) with the same volunteer CD3 + T cells transfected with other TCR (A6) or transfected with wild-type TCR (WT-TCR) served as controls.
  • TCR number and its sequence number are obtained from Table 2
  • the positive tumor cell lines used are HepG2-A11-B2M (HLA-A1101 and ⁇ 2M overexpression), SK-MEL-28-AFP (AFP overexpression), and the negative cell lines are HepG2, SK-MEL-28, SNU423, HUCCT1 .
  • ELISPOT plates were prepared. ELISPOT plates were activated with ethanol and coated at 4°C overnight. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add each component of the test to the ELISPOT plate: 2 *10 cells/well for target cells, 2* for effector cells 10 3 cells/well (calculated according to the positive rate of transfection), and set up two duplicate holes. Incubate overnight (37°C, 5% CO 2 ). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company).
  • ELISPOT READER system an immunospot plate reader
  • the effector cells transfected with the high-affinity TCR of the present application had a more obvious activation effect than the effector cells transfected with the wild type, while the effector cells transfected with other TCRs,
  • the effector cells transfected with empty transfection are basically inactive; at the same time, the effector cells transfected with the high-affinity TCR of the present application are basically inactive against AFP-negative cell lines.
  • non-radioactive cytotoxicity experiments well known to those skilled in the art were used to measure the release of LDH, thereby verifying the killing function of the cells transfected with the high-affinity TCR of the present application.
  • This assay is a colorimetric alternative to the 51Cr release cytotoxicity assay and quantifies LDH released after cell lysis.
  • LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to the red formazan.
  • INT tetrazolium salt
  • CD3 + T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of this application (the TCR number and its sequence number are obtained from Table 2) as effector cells, and the same volunteers were transfected Other TCR (A6), empty transfected (NC) CD3 + T cells were used as controls.
  • the following two batches (I), (II) were successively tested:
  • the positive tumor cell lines used are HepG2-A11-B2M, SK-MEL-28-AFP, and the negative cell lines are HepG2, SK-MEL-28, HUCCT1, SNU423.
  • the positive tumor cell line used is HepG2-A11-B2M, and the negative tumor cell line is HepG2.

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Abstract

本申请提供了一种T细胞受体(TCR)，其具有结合TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的特性。本申请还提供了多价TCR复合物、编码此类TCR的核酸分子、包含这些核酸的载体、表达此类TCR的细胞以及包含前述物质的药物组合物，它们可用于诊断、治疗和预防AFP阳性疾病。本申请还提供了此类TCR的制备方法。

Description

一种针对AFP抗原的高亲和力T细胞受体 技术领域
本申请属于生物技术领域，更具体地涉及能够识别衍生自AFP蛋白多肽的T细胞受体(T cell receptor，TCR)。本申请还涉及所述受体的制备和用途。
背景技术
仅仅有两种类型的分子能够以特异性的方式识别抗原。其中一种是免疫球蛋白或抗体；另一种是T细胞受体(TCR)，它是由α链/β链或者γ链/δ链以异二聚体形式存在的细胞膜表面的糖蛋白。免疫系统的TCR总谱的组成是在胸腺中通过V(D)J重组，然后进行阳性和阴性选择而产生的。在外周环境中，TCR介导了T细胞对主组织相容性复合体-肽复合物(pMHC)的特异性识别，因此其对免疫系统的细胞免疫功能是至关重要的。
TCR是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的唯一受体，这种外源肽或内源肽可能会是细胞出现异常的唯一迹象。在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
与TCR相对应的MHC I类和II类分子配体也是免疫球蛋白超家族的蛋白质但对于抗原的呈递具有特异性，不同的个体有不同的MHC，从而能呈递一种蛋白抗原中不同的短肽到各自的APC细胞表面。人类的MHC通常称为HLA基因或HLA复合体。
AFP(αFetoprotein)也称α胎蛋白，是胚胎发育过程中表达的一种蛋白，是胚胎血清的主要成分。在发育过程中，AFP在卵黄囊及肝脏中有比较高的表达水平，随后被抑制。在肝癌中，AFP的表达被激活。AFP在细胞内加工处理成抗原肽，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。TSSELMAITR(SEQ ID NO：32)是衍生自AFP抗原的短肽，是AFP相关疾病治疗的一种靶标。
因此，TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物提供了一种TCR可靶向肿瘤细胞的标记。能够结合TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的TCR对肿瘤的治疗具有很高的应用价值。例如，能够靶向该肿瘤细胞标记的TCR可用于将细胞毒性剂或免疫刺激剂递送到靶细胞，或被转化入T细胞，使表达该TCR的T细胞能够破坏肿瘤细胞，以便在被称为过继免疫治疗的治疗过程中给予患者。对于前一目的，理想的TCR是具有较高的亲和力的，从而使该TCR能够长期驻留在所靶向的细胞上面。对于后一目的，则优选使用中等亲和力的TCR。因此，本领域技术人员致力于开发可用于满足不同目的的靶向肿瘤细胞标记的TCR。
发明内容
本申请提供了一种对TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物高亲和力提高的TCR。
本申请进一步提供了一种上述类型TCR的制备方法及上述类型TCR的用途。
本申请的第一方面，提供了一种包含了α链可变域和β链可变域的T细胞受体(TCR)，其具有结合TSSELMAITR(SEQ ID NO：32)-HLA A1101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。
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，或所述TCRβ链可变域的氨基酸序列不是SEQ ID NO：2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所示的序列有至少91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列。优选地，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。
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％的序列同源性的氨基酸序列。优选地，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。
在另一优选例中，所述TCRα链可变域中CDR1α为：DSVNN(SEQ ID NO：33)，和CDR2α为IPSGT(SEQ ID NO：34)。
在另一优选例中，所述TCRα链可变域的CDR3α选自SGGSGFRLT(SEQ ID NO：35)、SGGQGYKLT(SEQ ID NO：36)和SGGSNYKLT(SEQ ID NO：37)。
在另一优选例中，所述TCRα链可变域中CDR1α为DSVNN(SEQ ID NO：33)，CDR2α为IPSGT(SEQ ID NO：34)，和CDR3α选自SGGSGFRLT(SEQ ID NO：35)、SGGQGYKLT(SEQ ID NO：36)和SGGSNYKLT(SEQ ID NO：37)。
在另一优选例中，所述TCRβ链可变域的3个CDR为：CDR1β：SEHNR(SEQ ID NO：38)；CDR2β：FQNEAQ(SEQ ID NO：39)；和CDR3β：ASSPGTGVGYT(SEQ ID NO：40)。
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1-3个；优选地为3个。
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5位、第6位、第7位。
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4位、第5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：DSVNN(SEQ ID NO：33)；
CDR2α：IPSGT(SEQ ID NO：34)；和
CDR3α：SGGSNYKLT(SEQ ID NO：37)，并且CDR3α含有至少一个下列突变：
突变前的残基 突变后的残基
CDR3α的第5位N G
CDR3α的第6位Y F
CDR3α的第7位K R
CDR3α的第4位S Q
在另一优选例中，所述CDR3α中的氨基酸突变包含：
突变前的残基 突变后的残基
CDR3α的第5位N G
在另一优选例中，所述TCRβ链可变域的3个CDR区(互补决定区)的基准序列如下，
CDR1β：SEHNR(SEQ ID NO：38)；
CDR2β：FQNEAQ(SEQ ID NO：39)；和
CDR3β：ASSPGTGVGYT(SEQ ID NO：40)，并且CDR3β含有至少一个下列突变：
突变前的残基 突变后的残基
CDR3β的第4位P H
CDR3β的第5位G P或Q
CDR3β的第6位T Q或H
CDR3β的第8位V I
CDR3β的第9位G Q或R或S或I或L或V
CDR3β的第10位Y Q或F或M
CDR3β的第11位T E或Q或H或K或R或L
优选地，所述CDR3β中的氨基酸突变包含：
突变前的残基 突变后的残基
CDR3β的第8位V I
在另一优选例中，所述TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的亲和力是野生型TCR的至少2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S91Q、N92G、Y93F、K94R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在SEQ ID NO：1所示的β链可变域中发生突变，所述突变选自P96H、G97P/Q、T98Q/H、V100I、G101Q/R/S/I/L/V、Y102Q/F/M、T103E/Q/H/K/L/R中的一组或几组，其中氨基酸残基编号采用SEQ ID NO：2所示的编号。
在另一优选例中，所述TCR具有选自下组的CDR：
Figure PCTCN2022099383-appb-000001
Figure PCTCN2022099383-appb-000002
Figure PCTCN2022099383-appb-000003
Figure PCTCN2022099383-appb-000004
Figure PCTCN2022099383-appb-000005
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，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)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。
在另一优选例中，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，在所述TCRα与β链的恒定区之间形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；
和TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
在另一优选例中，所述TCR的α链可变域氨基酸序列为SEQ ID NO：1、5-7之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO：2、8-27之一。
在另一优选例中，所述TCR选自下组：
Figure PCTCN2022099383-appb-000006
在另一优选例中，所述TCR是人源的。
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、治疗剂、PK修饰部分或任何这些物质的组合。
在另一优选例中，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
本申请的第二方面，提供了一种多价TCR复合物，其包含至少两个TCR分子，并 且其中的至少一个TCR分子为本申请第一方面所述的TCR。
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分子或者本申请第二方面所述的多价TCR复合物的核酸序列或其互补序列。
本申请的第四方面，提供了一种载体，所述的载体含有本申请第三方面所述的所述的核酸分子。
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染色体中整合有外源的本申请第三方面所述的核酸分子。
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，优选地，所述分离的细胞为T细胞、NK细胞或NKT细胞，最优选地，所述分离的细胞是T细胞。
本申请的第七方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本申请第一方面所述的TCR、或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细胞。
本申请的第八方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本申请第一方面所述的TCR、或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AFP阳性肿瘤，更优选地，所述肿瘤为肝癌。
本申请的第九方面，提供了本申请第一方面所述的TCR、或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细胞的用途，用于制备治疗肿瘤的药物，优选地，所述疾病为AFP阳性肿瘤，更优选地，所述肿瘤为肝癌。
本申请的第十方面，提供了一种制备本申请第一方面所述的T细胞受体的方法，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T细胞受体；
(ii)分离或纯化出所述的T细胞受体。
应理解，在本申请范围内中，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。
附图说明
图1a和图1b分别显示了对TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物能够特异性结合的野生型TCRα链、β链的可变域氨基酸序列。
图2a、图2b分别为本申请中可溶性参比TCRα链与β链的氨基酸序列。
图3(1)-(3)分别显示了对TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物具有高亲和力的异质二聚TCR的α链可变域氨基酸序列，突变的残基以加下划线表示。
图4(1)-(20)分别显示了对TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物具有高亲和力的异质二聚TCR的β链可变域氨基酸序列，突变的残基以加下划线表示。
图5a和图5b分别显示了对TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物能够 特异性结合的野生型TCRα链与β链的胞外氨基酸序列。
图6a和图6b分别显示了对TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物能够特异性结合的野生型TCRα链与β链的氨基酸序列。
图7为可溶性参比TCR即野生型TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的结合曲线。
图8为针对负载短肽的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结果。
图9a和图9b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10a和10b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实验结果。
具体实施方式
本申请通过广泛而深入的研究，获得一种识别TSSELMAITR(SEQ ID NO：32)短肽(衍生自AFP蛋白)的T细胞受体(TCR)，所述TSSELMAITR(SEQ ID NO：32)短肽以肽-HLA A1101复合物的形式被呈递。所述TCR在其α链可变域的3个CDR区：
CDR1α：DSVNN(SEQ ID NO：33)；
CDR2α：IPSGT(SEQ ID NO：34)；和
CDR3α：SGGSNYKLT(SEQ ID NO：37)中发生突变；和/或在其β链可变域的3个CDR区：
CDR1β：SEHNR(SEQ ID NO：38)；
CDR2β：FQNEAQ(SEQ ID NO：39)；和
CDR3β：ASSPGTGVGYT(SEQ ID NO：40)中发生突变。
在另一优选例中，突变后本申请TCR对上述TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的亲和力和/或结合半衰期是野生型TCR的至少2倍。
在描述本申请之前，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系统(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系统中，α链恒定结构域具有以下的符号：TRAC*01，其中“TR”表示T细胞受体基因；“A”表示α链基因；C表示恒定区；“*01”表示等位基因1。β链恒定结构域具有以下的符号：TRBC1*01或TRBC2*01，其中“TR”表示T细胞受体基因；“B”表示β链基因；C表示恒定区；“*01”表示等位基因1。α链的恒定区是唯一确定的，在β链的形式中，存在两个可能的恒定区基因“C1”和“C2”。本领域技术人员通过公开的IMGT数据库可以获得TCRα与β链的恒定区基因序列。
TCR的α和β链一般看作各有两个“结构域”即可变域和恒定结构域。可变域由连接的可变区和连接区构成。因此，在本申请的说明书和权利要求书中，“TCRα链可变域”指连接的TRAV和TRAJ区，同样地，“TCRβ链可变域”指连接的TRBV和TRBD/TRBJ区。TCRα链可变域的3个CDR分别为CDR1α、CDR2α和CDR3α；TCRβ链可变域的3个CDR分别为CDR1β、CDR2β和CDR3β。本申请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：28和SEQ ID NO：29，如图5a和图5b所示。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：30和SEQ ID NO：31，如图6a和图6b所示。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。
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：不同器官系统的恶性肿瘤，例如肉瘤， 肺鳞癌和癌症。例如：感染的前列腺，肺，乳房，淋巴，肠胃(例如：结肠)，和生殖泌尿道(例如：肾脏，上皮细胞)，咽头。肺鳞癌包括恶性肿瘤，例如，多数的结肠癌，直肠癌，肾细胞癌，肝癌，肺部的非小细胞癌，小肠癌和食道癌。上述癌症的转移性病变可同样用本申请的方法和组合物来治疗和预防。
发明详述
众所周知，TCR的α链可变域与β链可变域各含有3个CDR，类似于抗体的互补决定区。CDR3与抗原短肽相互作用，CDR1和CDR2与HLA相互作用。因此，TCR分子的CDR决定了其与抗原短肽-HLA复合物的相互作用。能够结合抗原短肽TSSELMAITR(SEQ ID NO：32)与HLA A1101复合物(即，TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物)的野生型TCR的α链可变域氨基酸序列与β链可变域氨基酸序列分别为SEQ ID NO：1和SEQ ID NO：2，该序列为本发明人首次发现。其具有下列CDR区：
α链可变域CDR：
CDR1α：DSVNN(SEQ ID NO：33)；
CDR2α：IPSGT(SEQ ID NO：34)；和
CDR3α：SGGSNYKLT(SEQ ID NO：37)；
和β链可变域CDR：
CDR1β：SEHNR(SEQ ID NO：38)；
CDR2β：FQNEAQ(SEQ ID NO：39)；和
CDR3β：ASSPGTGVGYT(SEQ ID NO：40)。
本申请通过对上述CDR区进行突变筛选，获得了与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的亲和力提高以及使细胞功能改善的TCR。
进一步，本申请所述TCR是包含了α链可变域和β链可变域的αβ异质二聚TCR或单链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％(如，可以是至少88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同源性)的序列同源性的氨基酸序列；和/或所述TCR的β链可变域包含与SEQ ID NO：2所示的氨基酸序列有至少90％，优选地，至少92％；更优选地，至少94％(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性)的序列同源性的氨基酸序列。
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27-31位、第49-53位和第88-96位。据此，氨基酸残基编号采用SEQ ID NO：1所示的编号，91S即为CDR3α的第4位S、92N即为CDR3α的第5位N、93Y即为CDR3α的第6位Y、94K即为CDR3α的第7位K。
具体地，α链可变域中所述突变的具体形式包括S91Q、N92G、Y93F、K94R中的一组或几组。
本申请中野生型TCRβ链可变域SEQ ID NO：2的3个CDR即CDR1、CDR2和CDR3分别位于SEQ ID NO：2的第27-31位、第49-54位和第93-103位。据此，氨基酸残基编号采用SEQ ID NO：1所示的编号，96P即为CDR3β的第4位P、97G即为CDR3β的 第5位G、98T即为CDR3β的第6位T、100V即为CDR3β的第8位V、101G即为CDR3β的第9位G、102Y即为CDR3β的第10位Y、103T即为CDR3β的第11位T。
具体地，β链可变域中所述突变的具体形式包括P96H、G97P/Q、T98Q/H、V100I、G101Q/R/S/I/L/V、Y102Q/F/M、T103E/Q/H/K/L/R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“N92G”代表第92位的N被G取代，“G97P/Q”代表第97位的G被P取代或被Q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Pro89突变为半胱氨酸，β链恒定区TRBC1*01或TRBC2*01外显子1的Ala19突变为半胱氨酸，即得到参比TCR，其氨基酸序列分别为SEQ ID NO：3和SEQ ID NO：4，如图2a和图2b所示，突变后的半胱氨酸残基以加粗字母表示。上述半胱氨酸取代能使参比TCR的α与β链的恒定区之间形成人工链间二硫键，以形成更加稳定的可溶性TCR，从而能够更加方便地评估TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101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TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物之间的结合亲和力即认为是野生型TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物之间的结合亲和力。同样地，如果测得本申请TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物之间的结合亲和力是参比TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物之间的结合亲和力的至少2倍，即等同于本申请TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物之间的结合亲和力是野生型TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物之间的结合亲和力的至少2倍。
可通过任何合适的方法测定结合亲和力(与解离平衡常数K D成反比)和结合半衰期(表示为T 1/2)，如采用表面等离子共振技术进行检测。应了解，TCR的亲和力翻倍将导致K D减半。T 1/2计算为In2除以解离速率(K off)。因此，T 1/2翻倍会导致K off减半。优选采用相同的试验方案检测给定TCR的结合亲和力或结合半衰期数次，例如3次或更多，取结果的平均值。在优选的实施方式中，采用本文实施例中的表面等离振子共振(BIAcore)方法检测可溶性TCR的亲和力，条件为：温度25℃，PH值为7.1-7.5。该方法检测到参比TCR对TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的解离平衡常数KD为4.16E-05M，即41.6μM，本申请中即认为野生型TCR对TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的解离平衡常数KD也为41.6μM。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TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的解离平衡常数KD为4.16E-06M，即4.16μM，则说明该高亲和力TCR对TSSELMAITR (SEQ ID NO：32)-HLA A1101复合物的亲和力是野生型TCR对TSSELMAITR(SEQ ID NO：32)-HLA A1101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＝10 6μM，1μM＝1000nM。
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TSSELMAITR(SEQ ID NO：32)-HLA-A1101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：1、5-7之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO：2、8-27之一。本申请异质二聚TCR分子的α链可变域与β链可变域的氨基酸序列优选自下表1：
表1
Figure PCTCN2022099383-appb-000007
Figure PCTCN2022099383-appb-000008
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Pro89和取代TRBC1*01或TRBC2*01外显子1的Ala19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G1u15；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Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；或TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的G1u20。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，这样的TCR可以包含(i)除其跨膜结构域以外的全部或部分TCRα链，和(ii)除其跨膜结构域以外的全部或部分TCRβ链，其中(i)和(ii)均包含TCR链的可变域和至少一部分恒定域，α链与β链形成异质二聚体。更优选地，这样的TCR可以包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
对于稳定性而言，另一方面，本申请TCR还包括在其疏水芯区域发生突变的TCR，这些疏水芯区域的突变优选为能够使本申请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。这样的TCR可在其下列可变域疏水芯位置发生突变：(α和/或β链)可变区氨基酸第11，13，19，21，53，76，89，91，94位，和/或α链J基因(TRAJ)短肽氨基酸位置倒数第3，5，7位，和/或β链J基因(TRBJ)短肽氨基酸位置倒数第2，4，6位，其中氨基酸序列的位置编号按国际免疫遗传学信息系统(IMGT)中列出的位置编号。本领域技术人员知晓上述国际免疫遗传学信息系统，并可根据该数据库得到不同TCR的氨基酸残基在IMGT中的位置编号。
更具体地，本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。所述偶联物包括可检测标记物(为诊断目的，其中所述TCR用于检测呈递TSSELMAITR(SEQ ID NO：32)-HLA-A1101复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本申请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))；和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：1、5-7之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO：2、8-27之一。
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TCR(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本申请也涉及包含本申请的核酸分子的载体，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TCR、或本申请TCR复合物、或呈递本申请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TCR、或本申请TCR复合物、或呈递本申请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。
本申请的TCR、TCR复合物或本申请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。本申请的TCR、多价TCR复合物或细胞通常作为无菌药物组合物的一部分提供，所述组合物通常包括药学上可接受的载体。该药物组合物可以是任何合适的形式(取决于给予患者的所需方法)。其可采用单位剂型提供，通常在密封的容器中提供，可作为试剂盒的一部分提供。此类试剂盒(但非必需)包括使用说明书。其可包括多个所述单位剂型。
此外，本申请的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)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在雷明顿药物科学(Remington′s Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991))中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂、及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本申请的组合物，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TCR能够与所述TSSELMAITR(SEQ ID NO：32)-HLA A1101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。
(2)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结合表征BIAcore分析
使用BIAcore T200实时分析系统检测TCR分子与TSSELMAITR(SEQ ID NO：32)-HLA-A1101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，pH 4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15，000RU。条件为：温度25℃，PH值为7.1-7.5。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将TSSELMAITR(SEQ ID NO：32)-HLA-A1101复合物流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。采用单循环动力学分析方法测定其亲和力，将TCR用HEPES-EP缓冲液(10mM HEPES，150mM NaCl，3mM EDTA，0.005％P20，pH 7.4)稀释成几个不同的浓度，以30μL/min的流速，依次流过芯片表面，每次进样的结合时间为120s，最后一次进样结束后让其解离600s。每一轮测定结束后用pH 1.75的10mM Gly-HCl再生芯片。利用BIAcore Evaluation软件计算动力学参数。可溶性参比TCR即野生型TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的结合曲线如图7所示。
上述TSSELMAITR(SEQ ID NO：32)-HLA-A1101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TSSELMAITR(SEQ ID NO：32，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TSSELMAITR肽(SEQ ID NO：32)以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mM Tris pH 8.3、2mM EDTA、0.5mM氧化性谷胱甘肽、5mM还原型谷胱甘肽、0.2mM PMSF，冷却至4℃)，然后依次加入20mg/L的轻链和90mg/L的重链(终浓度，重链分三次加入，8h/次)，复性在4℃进行至少3天至完成，SDS-PAGE检测能否复性成功。
c.复性后纯化：用10体积的20mM Tris pH 8.0作透析来更换复性缓冲液，至少更换缓冲液两次来充分降低溶液的离子强度。透析后用0.45μm醋酸纤维素滤膜过滤蛋白质溶液，然后加载到HiTrap Q HP(GE通用电气公司)阴离子交换柱上(5ml床体积)。利用Akta纯化仪(GE通用电气公司)，20mM Tris pH 8.0配制的0-400mM NaCl线性梯度液洗脱蛋白，pMHC约在250mM NaCl处洗脱，收集诸峰组分，SDS-PAGE检测纯度。
d.生物素化：用Millipore超滤管将纯化的pMHC分子浓缩，同时将缓冲液置换为20mM Tris pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mM MgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
e.纯化生物素化后的复合物：用Millipore超滤管将生物素化标记后的pMHC分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的pMHC，利用Akta纯化仪(GE通用电气公司)，用过滤过的PBS预平衡HiPrep TM 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℃。
实施例2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野生型TCR模板。采用本领域技术人员熟知的定点突变的方法，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。
通过BIAcore来检测其与TSSELMAITR(SEQ ID NO：32)-HLA-A1101复合物的亲和力。上述野生型TCR的α链与β链可变域氨基酸序列分别如图1a(SEQ ID NO：1)和1b(SEQ ID NO：2)所示。
通过《分子克隆实验室手册》(Molecular Cloning a Laboratory Manual)(第三版，Sambrook和Russell)中描述的标准方法将待表达的TCRα和β链的胞外序列基因经合成后分别插入到表达载体pET28a+(Novagene)，上下游的克隆位点分别是NcoI和NotI。CDR区的突变通过本领域技术人员熟知的重叠PCR(overlap PCR)引入。插入片段经过测序确认无误。
实施例3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OD 600＝0.6时用终浓度0.5mM IPTG诱导，TCR的α和β链表达后形成的包涵体通过BugBuster Mix(Novagene)进行提取，并且经BugBuster溶液反复多次洗涤，包涵体最后溶解于6M盐酸胍，10mM二硫苏糖醇(DTT)，10mM乙二胺四乙酸(EDTA)，20mM Tris(pH 8.1)中。
溶解后的TCRα和β链以1∶1的质量比快速混合于5M尿素，0.4M精氨酸，20mM Tris(pH 8.1)，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，pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP，5ml，GE Healthcare)纯化。洗脱峰含有复性成功的α和β二聚体的TCR通过SDS-PAGE胶确认。TCR随后通过凝胶过滤层析(HiPrep 16/60，Sephacryl S-100 HR，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4 BIAcore分析结果
采用实施例1中所述方法检测本申请高亲和力CDR的αβ异质二聚TCR与TSSELMAITR(SEQ ID NO：32)-HLA-A1101复合物的亲和力。
本申请得到新的TCRα链及β链可变域氨基酸序列分别如图3(1)-(3)、图4(1)-(20)所示。利用实施例2中所述方法构建表达载体，利用实施例3中所述方法对上述高亲和力突变的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TSSELMAITR(SEQ ID NO：32)-HLA-A1101复合物的亲和力，如下表2所示。
表2
Figure PCTCN2022099383-appb-000009
Figure PCTCN2022099383-appb-000010
由上表2可知，所述异质二聚TCR的亲和力是野生型TCR对TSSELMAITR(SEQ ID NO：32)-HLA-A1101复合物的亲和力的至少2倍。
实施例5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(1inker)为GGGGS(SEQ ID NO：61)，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Nco I(CCATGG(SEQ ID NO：62))和Not I(GCGGCCGC(SEQ ID NO：63))。将PCR扩增产物经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感受态细胞，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Tuner(DE3)感受态细胞，用于表达。
融合蛋白的表达、复性及纯化
将表达质粒分别转化进入E.coli Tuner(DE3)感受态细胞，涂布LB平板(卡那霉素50μg/mL)置于37℃培养过夜。次日，挑克隆接种至10mL LB液体培养基(卡那霉素50μg/mL)培养2-3h，按体积比1∶100接种至1L LB培养基中，继续培养至OD600为0.5-0.8，加入终浓度为1mM IPTG诱导目的蛋白的表达。诱导4小时以后，以6000rpm离心10min收获细胞。PBS缓冲液洗涤菌体一次，并且分装菌体，取相当于200mL的细菌培养物的菌体用5mL BugBuster Master Mix(Merck)裂解细菌，以6000g离心15min收集包涵体。然后进行4次洗涤剂洗涤以去除细胞碎片和膜组分。然后，用缓冲液如PBS洗涤包涵体以除去洗涤剂和盐。最终，将包涵体用含6M盐酸胍，10mM二硫苏糖醇(DTT)，10mM乙二胺四乙酸(EDTA)，20mM Tris，pH 8.1缓冲溶液溶解，并测定包涵体浓度，将其分装后置于-80℃冷冻保存。
溶解后的TCRα链和抗-CD3(scFv)-β链以2：5的质量比快速混合于5M尿素(urea)，0.4M L-精氨酸(L-arginine)，20mM Tris pH 8.1，3.7mM cystamine，6.6mM 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，pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP 5ml，GE healthcare)纯化。洗脱峰含有复性成功的TCRα链与抗-CD3(scFv)-β链二聚体的TCR通过SDS-PAGE胶确认。TCR融合分子随后通过尺寸排阻色谱法(S-100 16/60，GE healthcare)进一步纯化，以及阴离子交换柱(HiTrap Q HP 5ml，GE healthcare)再次纯化。纯化后的TCR融合分子纯度经过SDS-PAGE测定大于90％，浓度由BCA法测定。
实施例6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2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CD3 +T细胞作为对照。所用的靶细胞为负载AFP抗原短肽TSSELMAITR(SEQ ID NO：32)的、负载了其他抗原短肽的、或空载的T2-A11(指转染了HLA-A1101的T2细胞)。
首先准备ELISPOT平板，先按靶细胞1×10 4个细胞/孔、效应细胞2×10 3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AFP抗原短肽TSSELMAITR(SEQ ID NO：32)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8所示，针对负载了AFP抗原短肽TSSELMAITR(SEQ ID NO：32)的靶细胞，转染本申请高亲和力TCR的效应细胞起明显的激活效应，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无活性。
实施例7针对细胞系，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(TCR编号及其序列号从表2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，并以同一志愿者空转染(NC)的、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。使用的阳性肿瘤细胞系为HepG2-A11-B2M(HLA-A1101和β2M过表达)、SK-MEL-28-AFP(AFP过表达)，阴性细胞系为HepG2、SK-MEL-28、SNU423、HUCCT1。
分两个批次先后进行实验，皆进行以下步骤：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*10 4个/孔，效应细胞为2*10 3个/孔(按转染的阳性率计算)，并设置二个复孔。温育过夜(37℃，5％CO 2)。实验第2天，洗涤平板并进行二级检测和显色，干燥平板，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。
实验结果如图9a和9b所示，针对阳性肿瘤细胞系，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、空转染的效应细胞基本无活性；同时，转染本申请高亲和力TCR的效应细胞对AFP阴性细胞系基本无活性。
实施例8转染本申请高亲和力TCR的效应细胞的杀伤功能实验
本实施例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高亲和力TCR的细胞的杀伤功能。该试验是51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的LDH。采用30分钟偶联的酶反应来检测释放在培养基中的LDH，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臌(formazan)。生成的红色产物的量与裂解的细胞数成正比。可以用标准的96孔读板计收集490nm可见光吸光值数据。计算公式：％细胞毒性＝100％×(实验-效应细胞自发-靶细胞自发)/(靶细胞最大-靶细胞自发)。
本实施例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(TCR编号及其序列号从表2获悉)作为效应细胞，并以同一志愿者转染其他TCR(A6)的、空转染(NC)的CD3 +T细胞作为对照。以下分两个批次(I)、(II)先后进行实验：
(I)使用的阳性肿瘤细胞系为HepG2-A11-B2M、SK-MEL-28-AFP，阴性细胞系为HepG2、SK-MEL-28、HUCCT1、SNU423。
(II)使用的阳性肿瘤细胞系为HepG2-A11-B2M，阴性肿瘤细胞系为HepG2。
以上两批次都进行如下实验步骤：首先准备LDH平板，按以下顺序将试验的各个组分加入平板：靶细胞3×10 4个细胞/孔、效应细胞3×10 4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％CO 2)。实验第2天，检测显色，终止反应后用酶标仪(Bioteck)在490nm记录吸光值。
实验结果如图10a和10b所示，针对阳性肿瘤细胞系，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、空转染的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基本无杀伤。
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。

Claims (26)

  1. 一种T细胞受体(TCR)，其包含TCRα链可变域和TCRβ链可变域，其中，所述T细胞受体具有结合TSSELMAITR(SEQ ID NO：32)-HLA A1101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，其中，所述TCR与TSSELMAITR(SEQ ID NO：32)-HLA A1101复合物的亲和力是野生型TCR的至少2倍。
  3. 如权利要求1所述的TCR，其中，所述TCR在TCRα链可变域的CDR3α中发生突变，并且突变个数为1-3个，优选地为3个。
  4. 如权利要求1所述的TCR，其中，所述TCRα链可变域中CDR1α为DSVNN(SEQ ID NO：33)，CDR2α为IPSGT(SEQ ID NO：34)，和CDR3α选自SGGSGFRLT(SEQ ID NO：35)、SGGQGYKLT(SEQ ID NO：36)和SGGSNYKLT(SEQ ID NO：37)。
  5. 如权利要求1所述的TCR，其中，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％的序列同源性和/或所述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，其中，所述TCRβ链可变域的3个CDR为：CDR1β：SEHNR(SEQ ID NO：38)；CDR2β：FQNEAQ(SEQ ID NO：39)；和CDR3β：ASSPGTGVGYT(SEQ ID NO：40)；优选地，所述TCRβ链可变域的氨基酸序列为SEQ ID NO：2。
  7. 如权利要求1所述的TCR，其中，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：DSVNN(SEQ ID NO：33)；
    CDR2α：IPSGT(SEQ ID NO：34)；和
    CDR3α：SGGSNYKLT(SEQ ID NO：37)，并且CDR3α含有至少一个下列突变：
    突变前的残基 突变后的残基 CDR3α的第5位N G CDR3α的第6位Y F CDR3α的第7位K R CDR3α的第4位S Q
    优选地，所述CDR3α中的氨基酸突变包含：
    突变前的残基 突变后的残基 CDR3α的第5位N G
  8. 如权利要求1所述的TCR，其中，所述TCRβ链可变域的3个CDR区(互补决定区)的基准序列如下，
    CDR1β：SEHNR(SEQ ID NO：38)；
    CDR2β：FQNEAQ(SEQ ID NO：39)；和
    CDR3β：ASSPGTGVGYT(SEQ ID NO：40)，并且CDR3β含有至少一个下列突变：
    突变前的残基 突变后的残基
    CDR3β的第4位P H CDR3β的第5位G P或Q CDR3β的第6位T Q或H CDR3β的第8位V I CDR3β的第9位G Q或R或S或I或L或V CDR3β的第10位Y Q或F或M CDR3β的第11位T E或Q或H或K或R或L
    优选地，所述CDR3β中的氨基酸突变包含：
    突变前的残基 突变后的残基 CDR3β的第8位V I
  9. 如权利要求1所述的TCR，其中，所述TCR具有选自下组的CDR：
    Figure PCTCN2022099383-appb-100001
    Figure PCTCN2022099383-appb-100002
    Figure PCTCN2022099383-appb-100003
  10. 如权利要求1所述的TCR，其中，所述TCR是可溶的。
  11. 如权利要求1所述的TCR，其中，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，其中，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，其中，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，其中，所述TCR的α链可变域氨基酸序列为SEQ ID NO：1、5-7之一；和/或所述TCR的β链可变域氨基酸序列为SEQ ID NO：2、8-27之一。
  15. 如权利要求1所述的TCR，其中，所述TCR选自下组：
    Figure PCTCN2022099383-appb-100004
  16. 如权利要求1所述的TCR，其中，所述TCR为单链TCR。优选地，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. 如以上任一权利要求所述的TCR，其中，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，所述治疗剂为抗-CD3抗体。
  18. 一种多价TCR复合物，其包含至少两个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，其含有权利要求19中所述的核酸分子。
  21. 一种宿主细胞，其含有权利要求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，其含有药学上可接受的载体以及权利要求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AFP阳性肿瘤，更优选地，所述肿瘤为肝癌。
  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，其用于制备治疗肿瘤的药物；优选地，所述肿瘤为AFP阳性肿瘤，更优选地，所述肿瘤为肝癌。
  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；以及
    (ii)分离或纯化出所述的T细胞受体。
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