WO2014206304A1 - 高稳定性的t细胞受体及其制法和应用 - Google Patents

高稳定性的t细胞受体及其制法和应用 Download PDF

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Publication number
WO2014206304A1
WO2014206304A1 PCT/CN2014/080773 CN2014080773W WO2014206304A1 WO 2014206304 A1 WO2014206304 A1 WO 2014206304A1 CN 2014080773 W CN2014080773 W CN 2014080773W WO 2014206304 A1 WO2014206304 A1 WO 2014206304A1
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Prior art keywords
amino acid
tcr
chain variable
variable domain
acid sequence
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English (en)
French (fr)
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李懿
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GUANGZHOU XIANGXUE PHARMACEUTICAL Co Ltd
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GUANGZHOU XIANGXUE PHARMACEUTICAL Co Ltd
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Priority to CA2916960A priority Critical patent/CA2916960C/en
Priority to KR1020167002293A priority patent/KR101904385B1/ko
Priority to EP14817065.7A priority patent/EP3015477B1/en
Priority to AU2014301777A priority patent/AU2014301777B2/en
Priority to US14/901,344 priority patent/US10654906B2/en
Priority to RU2016101711A priority patent/RU2645256C2/ru
Priority to CN201480036288.6A priority patent/CN105683215B/zh
Priority to MX2016000231A priority patent/MX375056B/es
Priority to NZ715815A priority patent/NZ715815A/en
Application filed by GUANGZHOU XIANGXUE PHARMACEUTICAL Co Ltd filed Critical GUANGZHOU XIANGXUE PHARMACEUTICAL Co Ltd
Priority to JP2016522222A priority patent/JP6549564B2/ja
Publication of WO2014206304A1 publication Critical patent/WO2014206304A1/zh
Anticipated expiration legal-status Critical
Priority to IL243365A priority patent/IL243365B/en
Priority to AU2017204047A priority patent/AU2017204047B2/en
Ceased legal-status Critical Current

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    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a highly stable soluble sputum receptor (TCR) receptor (TCR) in a hydrophobic core region.
  • TCR soluble sputum receptor
  • the invention also relates to the preparation and use of such receptors. Background technique
  • TCR T cell receptor
  • TCR is the only receptor for a specific antigenic peptide presented on the major histocompatibility complex (MHC), which may be the only sign of abnormal cell appearance.
  • MHC major histocompatibility complex
  • APCs antigen presenting cells
  • TCR On the T cell membrane, TCR binds to the constant protein CD3 involved in signal transduction to form a complex. TCR exists in many forms and is structurally similar, whereas T cells expressing these TCRs may exist in different anatomical locations and may have different functions.
  • the extracellular portion of the TCR consists of two near-membrane constant domains and two distal membrane variable domains with polymorphic loops similar to the complementarity determining regions (CDRs) of the antibody. It is these loops that form the binding site for T cell receptor molecules and determine peptide specificity.
  • MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily but are specific for antigen presentation, and they have polymorphic peptide binding sites that enable them to present a variety of Different short peptide fragments are added to the surface of APC cells.
  • TCR Like immunoglobulins (antibodies) as antigen recognition molecules, TCR can also be developed and applied. Diagnosis and treatment. However, it is difficult to prepare such a protein consisting of more than one polypeptide subunit and having a transmembrane region in a (water) soluble form, since, in many cases, such a protein is stabilized by its transmembrane region. . This is the case with TCR, which has been reflected in the scientific literature. The literature describes a truncated form of TCR that contains only extracellular regions or only extracellular and cytoplasmic regions. Such TCRs can be specifically TCR-specific. Antibody recognition (this indicates that the recombinant TCR portion recognized by the antibody has been correctly folded), but the yield is not high, is not stable enough at low concentrations and/or does not recognize the primary histocompatibility complex-peptide complex.
  • Soluble TCR has a wide range of uses, not only for studying TCR-pMHC interactions, but also as a diagnostic tool for detecting infections or as a marker for autoimmune diseases.
  • soluble TCR can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells that present specific antigens, or to inhibit tau cells (such as those that react with autoimmune peptide antigens). T cells).
  • therapeutic agents such as cytotoxic compounds or immunostimulatory compounds
  • tau cells such as those that react with autoimmune peptide antigens.
  • modification of the TCR protein is critical.
  • heterologous expression of TCR in prokaryotic or eukaryotic systems is important.
  • TCRs can be recognized by TCR-specific antibodies, they only show recognition of natural ligands at relatively high concentrations, suggesting that the recognition is unstable.
  • Another object of the present invention is to provide a process and use of the high stability T cell receptor.
  • TCR T-cell receptor
  • the stability of the TCR is higher than the TCR of its corresponding hydrophobic core being wild type.
  • the "mutation" means that the hydrophobic core region of the TCR of the present invention is mutated relative to the corresponding wild-type TCR hydrophobic core region.
  • the "stability higher than" indicates that the TCR of the present invention has a stability improvement of at least 5%, preferably at least 30%, more preferably than the corresponding hydrophobic core of the wild type. At least 80%.
  • wild-type TCR hydrophobic core refers to a hydrophobic core which is the same as the hydrophobic core amino acid residue (sequence) in the naturally occurring TCR without mutation.
  • the phrase "the corresponding hydrophobic core is a wild type TCR” means that the hydrophobic core is wild type, and the other regions are different from the TCR which is mutated in the hydrophobic core region of the present invention. Invented the same TCR for TCR. Additionally or preferably, said "the corresponding hydrophobic core is a wild-type TCR” refers to a naturally occurring wild-type TCR that does not contain any mutation sites, particularly its a-chain variable domain and beta-chain. The variable domain is a wild-type sTv molecule, and representative examples include LC13-WT.
  • the CDR region of the TCR is identical to the wild type, or contains an affinity A sudden increase in mutation.
  • the affinity refers to the binding affinity between the TCR molecule and its corresponding antigen.
  • the T cell receptor variable region framework and the hydrophobic region of the side chain in the constant region position are mutated toward the surface.
  • the variable domain backbone of the TCR and the amino acid residues exposed to the surface in the constant domain are mutated.
  • the mutated amino acid residue is an amino acid residue exposed to the surface of the a chain and/or the beta chain variable domain of the TCR.
  • the amino acid sites exposed to the surface in the variable domain include the 4th, 12th, 16th, 93rd, 97th, and 100th positions of the TCR a chain variable domain amino acid,
  • the amino acid position number is numbered according to the position listed in IMGT (International Immunogenetics Information System).
  • the T cell receptor variable region framework side chain is directed to the surface (the variable domain is exposed to the surface) and the mutant form of the hydrophobic residue includes, but is not limited to, a chain : I7S, A9S, A10S, V20S, ⁇ 92 ⁇ , A93S, J gene
  • the amino acid position of the short peptide from the penultimate position changes from I to ⁇ , ⁇ chain: I 12S, or any combination of the above mutations, where the amino acid position number is listed in IMGT The location number.
  • the T cell receptor is soluble.
  • the T cell receptor is a membrane protein.
  • the T cell receptor comprises (a) all or part of a TCR a chain other than a transmembrane domain; and (b) all or part of a TCR ⁇ chain other than a transmembrane domain;
  • the TCR is a single-chain TCR composed of a flexible peptide chain linking the variable domains of the a and ⁇ chains of the TCR.
  • the mutation comprises a mutation in at least one hydrophobic core position.
  • the T cell receptor has one or more mutations at the position, a and/or ⁇ chain amino acid sequence variable region hydrophobic core position: i.e., variable region amino acid number 11, 13, 19 , 21, 53, 76, 89, 91, 94, and/or ⁇ chain J gene short peptide amino acid position reciprocal number 3, 5, 7 and / or ⁇ chain J gene short peptide amino acid position reciprocal 2, 4, 6 digits, where the amino acid position number is based on top GT (country The location number listed in the Immune Genetics Information System.
  • top GT country The location number listed in the Immune Genetics Information System.
  • the a-chain variable domain of the TCR is mutated in one or more of the following positions: alpha chain variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91 Or, at position 94, and/or the alpha chain J gene short peptide amino acid reciprocal number 3, the penultimate number 5 or the penultimate number 7, wherein the amino acid position number is in the position listed in IMGT (International Immunogenetics Information System) Numbering.
  • IMGT International Immunogenetics Information System
  • the TCR is at one or more of the following positions in the alpha chain variable domain set forth in SEQ ID NO: 9 or SEQ ID NO: 29 or SEQ ID NO: 31 or SEQ ID NO: Mutations occur: a chain variable region amino acid 11th, 13th, 19th, 21st, 53th, 76th, 89th, 91th, or 94th, and/or a chain J gene short peptide amino acid reciprocal 3rd, the penultimate 5th Bit 7 or the last 7th digit, where the amino acid position number is numbered according to the position listed in IMGT.
  • the ⁇ chain variable domain of the TCR is mutated in one or more of the following positions: ⁇ chain variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91 , or the 94th, and / or ⁇ chain J gene short peptide amino acid reciprocal number 2, the last 4th or the last 6th, wherein the amino acid position number is numbered according to the position listed in IMGT.
  • the TCR is at SEQ ID NO: 11 or SEQ ID NO: 30 or SEQ ID NO:
  • a mutation occurs in one or more of the following positions of the ⁇ chain variable domain represented by NO: 32 or SEQ ID NO: 34: ⁇ chain variable region amino acids 11, 11, 19, 21, 53, 76, 89, 91, or the 94th, and / or ⁇ chain J gene short peptide amino acid reciprocal number 2, the last 4th or the last 6th, wherein the amino acid position number is numbered according to the position listed in IMGT.
  • the a-chain variable domain of the TCR comprises one or more amino acid residues selected from the group consisting of: 11L, 11M or 11E; 13V, 13R or 13K; 19V; 211; 91L or 911; 94V or 941; and/or the beta chain variable domain of the TCR comprises one or more amino acid residues selected from the group consisting of: 11L or 11V; 13V; 19V; 89L; 91F or 911; 94V or 94L;
  • the sixth reciprocal of the J gene is the fourth position of the T and ⁇ chain J genes, and the fourth position is M; wherein the amino acid position numbers are numbered according to the positions listed in the IMGT.
  • the amino acid residue exposed to the surface of the a chain and/or the ⁇ chain variable domain of the TCR is mutated.
  • the TCR comprises one or more of the a chain variable domain amino acid residues 4L selected from the group consisting of: 12N; 16S; 93N or 93R; 97N; 100G; 105S; and a chain J gene reciprocal
  • the first position is D
  • the TCR comprises one or more ⁇ chain variable domain amino acid residues 41 selected from the group consisting of: 101L; the first position of the ⁇ chain J gene is the reciprocal of the D and ⁇ chain J genes.
  • the third digit is E.
  • the TCR comprises one of the following alpha chain variable domain amino acid sequences SEQ ID NO: 15, 17, 35, 37, 39, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85,
  • the TCR comprises one of the following ⁇ chain variable domain amino acid sequences SEQ ID NO: 16, 18, 36, 38, 40, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96,
  • the combination of the a-chain variable domain and the beta-chain variable domain of the TCR is selected from one of the following combinations:
  • the T cell receptor a chain variable region hydrophobic core has at least one of the following mutations: the 19th amino acid mutation is V, the 21st amino acid mutation is I, and the 91st amino acid mutation is L; And/or the beta chain variable region hydrophobic core has at least one of the following mutations: the amino acid mutation at position 91 is F or the mutation is I; and/or the amino acid at position 4 of the amino acid sequence of the short peptide of the ⁇ chain J gene is mutated to M.
  • the mutation is selected from the group consisting of:
  • the 19th amino acid mutation of the a chain variable region is V, the 21st amino acid mutation is I, the 91st amino acid mutation is ⁇ chain variable region 91th amino acid mutation is F, ⁇ chain J gene short peptide
  • the fourth position of the amino acid sequence is mutated to M; or (ii) the 19th amino acid mutation of the ⁇ chain variable region is V, the amino acid mutation at position 21 is I, and the amino acid mutation at position 91 of the ⁇ chain variable region is I;
  • the amino acid mutation at position 19 of the ⁇ chain variable region is V
  • the amino acid mutation at position 21 is I
  • the amino acid mutation at position 91 is changed to the amino acid at position 91 of the ⁇ chain variable region.
  • the ⁇ cell receptor a chain variable region hydrophobic core has at least one of the following mutations: L19V, L21I, I91L; and/or the ⁇ chain variable region hydrophobic core has at least one of the following mutations: V91F or The fourth position of the amino acid sequence of the V91I; and/or ⁇ chain J gene short peptide is mutated from L to M.
  • the mutation is selected from the group consisting of:
  • the amino acid position number is numbered according to the position listed in the top GT.
  • the sputum cell receptor further has a disulfide bond connecting the TCR ⁇ chain constant region and the ⁇ chain constant region.
  • the disulfide bond is present in the natural TCR or introduced artificially.
  • the artificially introduced disulfide bond is located between the constant domains of TCRcc and the beta strand.
  • the artificially introduced cysteine residue forming an interchain disulfide bond replaces an amino acid residue including, but not limited to, at least one of the a and ⁇ chains in the following positions:
  • the T cell receptor is screened by phage display technology.
  • the tau cell receptor binds (covalently or otherwise) to a conjugate.
  • the conjugate is selected from one or more of the group consisting of:
  • the detectable label comprises: a fluorescent or luminescent label, a radioactive label, an MRI
  • Magnetic resonance imaging or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products.
  • the therapeutic agent comprises: a radionuclide, a biotoxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a virus particle, a liposome , nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (; BPHL), chemotherapeutic agents (eg, cisplatin;) or any form of nano Particles, etc.
  • a radionuclide for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (; BPHL), chemotherapeutic agents (eg, cisplatin;) or any form of nano Particles, etc.
  • the conjugate is an anti-CD3 antibody linked to the C- or ⁇ -terminus of the a and/or ⁇ chain of the TCR.
  • nucleic acid molecule comprising a sputum cell receptor according to any one of the first aspects of the invention, or a complement thereof, is provided.
  • a vector comprising the nucleic acid molecule of the second aspect of the invention is provided.
  • a host cell or genetically engineered engineered cell comprising the vector of the third aspect of the invention or the chromosome of the second aspect of the invention integrated with exogenous Nucleic acid molecule.
  • the host cell is selected from the group consisting of: a prokaryotic cell and a eukaryotic cell, such as Escherichia coli, yeast cells, CH0 cells, and the like.
  • a method of preparing a T cell receptor according to the first aspect of the invention comprising the steps of:
  • the T cell receptor is isolated or purified.
  • the invention provides a T cell receptor complex, characterized in that the complex comprises the T cell receptor according to any one of the first aspects of the invention.
  • the complex comprises a complex formed by binding of a tau cell receptor of the present invention to a therapeutic agent, or a complex formed by binding to a detectable label.
  • the complex comprises two or more T cell receptor molecules.
  • a seventh aspect of the invention there is provided the use of the T cell receptor of the invention described above for use in the manufacture of a medicament for the treatment of a tumor, a viral infection or an autoimmune disease.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a safe and effective amount of the T cell receptor of any one of the first aspects of the invention.
  • a method of treating a disease comprising administering a T cell receptor according to any one of the first aspects of the invention, or a T cell according to the sixth aspect, to a subject in need of treatment A receptor complex, or a pharmaceutical composition as described in the eighth aspect.
  • the disease comprises: a tumor, an autoimmune disease, and a viral infectious disease.
  • a method for the preparation of the T cell receptor of the first aspect of the invention comprising the steps of:
  • the screening methods include, but are not limited to, phage display technology.
  • step (i i) a T cell receptor which is mutated in the hydrophobic core region is displayed by phage display technology and screened.
  • the method further comprises the step of determining the sequence, activity and/or other characteristics of the selected T cell receptor.
  • Figure 1 shows a schematic diagram of the variable domain structure of a typical TCR with a cancer antigen MAGE
  • Figures 2a and 2b show the TCR a chain variable domain amino acid sequence and nucleotide sequence (SEQ ID NOS: 9 and 10), respectively, after site-directed mutagenesis.
  • the amino acid sequence is further optimized for the a-chain variable domain amino acid sequence of the TCR disclosed in the patent document (WO2012/013913), and more specifically, the hydrophobic residue exposed to the surface in the variable domain is further mutated to hydrophilic. Or a polar residue in which the bolded and underlined letter is the mutated amino acid residue.
  • Figures 3a and 3b show the TCR i chain variable domain amino acid sequence and nucleotide sequence (SEQ ID NOS: 11 and 12), respectively, after site-directed mutagenesis.
  • the amino acid sequence is further optimized for the TCR i chain variable domain amino acid sequence disclosed in the patent document (WO2012/013913), and more specifically, the hydrophobic residue exposed to the surface in the variable domain is further mutated to hydrophilic or A polar residue in which the bolded and underlined letters are the mutated amino acid residues.
  • Figure 4 shows the connection of each primer when constructing MAGE-sTv-WT.
  • Figures 5a and 5b are the amino acid sequence and nucleotide sequence (SEQ ID NOS: 13 and 14) of the a and ⁇ chain conjugates when constructing the sTv mutant library, respectively.
  • Figures 6a and 6b are the a-chain variable domain amino acid sequence and the ⁇ -chain variable domain amino acid sequence (SEQ ID NOS: 15 and 16), respectively, of the sTv mutant MG29, and the mutated residues are in bold relative to MAGE-sTv-WT. Words and underscores are displayed.
  • Figures 7a and 7b are the a-chain variable domain amino acid sequence and the ⁇ -chain variable domain amino acid sequence (SEQ ID ⁇ 0: 17 and 18) of the sTv mutant P8F1, respectively, and the mutated residues are in bold relative to MAGE-sTv-WT. Words and underscores are displayed.
  • Figures 8a and 8b are the a-chain variable domain amino acid sequence and the ⁇ -chain variable domain amino acid sequence (SEQ ID ⁇ 0: 15 and 18) of the sTv mutant P8F2, respectively, and the mutated residues are in bold relative to MAGE-sTv-WT. Words and underscores are displayed.
  • Figure 9 is a ELISA experimental 0D value of different mutant strains and MAGE-sTv-WT against antigen MAGEA3, EBV, Flu, NY-ES0.
  • Figure 10a and Figure 10b are the a-chain variable domains (SEQ ID NO: 29) and ⁇ -chain variable domains of LC13-WT, respectively. (SEQ ID NO: 30) amino acid sequence.
  • Figure 11a and Figure lib are the amino acid sequences of the ⁇ chain variable domain (SEQ ID NO: 31) and the ⁇ chain variable domain (SEQ ID NO: 32), respectively, of JM22-WT.
  • Figure 12a and Figure 12b are the amino acid sequences of the ⁇ G chain variable domain (SEQ ID NO: 33) and the ⁇ chain variable domain (SEQ ID NO: 34), respectively, of 1G4-WT.
  • Figure 13a and Figure 13b are the amino acid sequences of the LC13-sTva chain variable domain (SEQ ID NO: 35) and the LC13-sTv beta chain variable domain (SEQ ID NO: 36), respectively.
  • Figure 14a and Figure 14b show the amino acid sequences of the JM22-sTva chain variable domain (SEQ ID NO: 37) and the JM22-sTv beta chain variable domain (SEQ ID NO: 38), respectively.
  • Figure 15a and Figure 15b are the amino acid sequences of the 1G4-sTv alpha chain variable domain (SEQ ID NO: 39) and the 1G4-sTvP chain variable domain (SEQ ID NO: 40), respectively.
  • Figure 16 is the amino acid sequence (SEQ ID NO: 41) of the ligated linker used to construct the sTv single-stranded molecule.
  • Figure 17 is an SDS-PAGE gel of the purified protein LC13-WT and LC13-sTv. Lane 1: molecular marker, lane 2: LC13-WT, lane 3: LC13-sTv.
  • Figure 18a and Figure 18b show the SEC profiles of the purified proteins LC13-WT and LC13-sTv, respectively.
  • Figure 19 is an SDS-PAGE gel of the purified proteins JM22-WT and JM22-sTv.
  • Lane 1 molecular weight marker
  • lane 2 JM22-WT
  • lane 3 JM22-sTv.
  • Figure 20a and Figure 20b show the SEC profiles of the purified proteins JM22-WT and JM22-sTv, respectively.
  • Figure 21 is an SDS-PAGE gel of the purified protein 1G4-WT and 1G4-STV.
  • Lane 1 molecular weight marker
  • lane 2 1G4-WT
  • Figure 22a and Figure 22b show the SEC profiles of the purified proteins 1G4-WT and 1G4-STV, respectively.
  • Figure 23 is the amino acid sequence of 1G4-STV (SEQ ID NO: 42).
  • Figure 24 shows the 0D values of the 1G4-STV mutant for different antigens.
  • Figure 25 shows the ⁇ chain variable domain amino acid sequence of the 1G4-STV high stability mutant screen (SEQ ID NO: 1G4-STV high stability mutant screen (SEQ ID NO: 1G4-STV high stability mutant screen
  • Figure 26 is a ⁇ chain variable domain amino acid sequence (SEQ ID NO: 86-96) of the screened 1G4-STV high stability mutant.
  • Figure 27 is a DSC graph of a 1G4-STV high stability mutant.
  • Figure 28 is a DSC graph of 1G4-WT.
  • Figure 29a and Figure 29b show the amino acid sequences of the highly stable G15a chain variable domain (SEQ ID NO: 97) and the ⁇ chain variable domain (SEQ ID NO: 98), respectively.
  • Figure 30 is an SDS-PAGE gel of the purified proteins 1G4-WT, 1G4-sTv, G13, G15, G9.
  • Lane 1 molecular weight marker
  • lane 2 1G4-WT
  • lane 4 lane G13
  • lane 5 G15
  • lane 6 molecular weight marker
  • lane 7 G9.
  • Figures 31a, 31b and 31c are SEC profiles of purified proteins G9, G13 and G15, respectively.
  • Figure 32a and Figure 32b are the amino acid sequences of the LC13-G9 chain variable domain (SEQ ID NO: 99) and the ⁇ chain variable domain (SEQ ID NO: 100), respectively.
  • Figure 33a and Figure 33b are the amino acid sequences of the LC13-G15 alpha chain variable domain (SEQ ID NO: 101) and the beta chain variable domain (SEQ ID NO: 102), respectively.
  • Figure 34a and Figure 34b show the amino acid sequences of the JM22-G9a chain variable domain (SEQ ID NO: 103) and the ⁇ chain variable domain (SEQ ID NO: 104), respectively.
  • Figure 35a and Figure 35b are the amino acid sequences of the JM22-G15 chain variable domain (SEQ ID NO: 105) and the beta chain variable domain (SEQ ID NO: 106), respectively.
  • Figure 36 is a SDS-PAGE gel of the purified proteins LC13-WT, LC13-sTv, LC13-G15, LC13-G9.
  • Lane 1 molecular weight marker
  • lane 2 LC13-WT
  • lane 3 LC13-sTv
  • lane 4 LC13-G15
  • lane 5 molecular weight marker
  • lane 6 LC13-G9o
  • Figure 37 is a SEC map of the purified protein LC13-G9.
  • Figure 38 is a SEC map of the purified protein LC13-G15.
  • Figure 39 is a SDS-PAGE gel of the purified proteins JM22-WT, JM22-sTv, JM22-G15, JM22-G9.
  • Lane 1 molecular weight marker
  • lane 2 JM22-WT
  • lane 3 JM22-sTv
  • lane 4 JM22-G15
  • lane 5 JM22-G9o
  • Figure 40 is a SEC map of the purified protein JM22-G9.
  • Figure 41 is a SEC map of the purified protein JM22-G15.
  • Figure 42a and Figure 42b show the amino acid sequences of the MAGE-G15 chain variable domain (SEQ ID NO: 107) and the ⁇ chain variable domain (SEQ ID NO: 108), respectively.
  • Figure 43 is an SDS-PAGE gel of the purified protein MAGE-G15.
  • Lane 1 molecular weight marker
  • lane 2 MAGE - G15.
  • Figure 44 is a SEC map of the purified protein MAGE-G15.
  • Figure 45 is a DSC graph of the purified protein MAGE-G15.
  • Figure 46 is a DSC graph of the purified protein G15.
  • Figure 47 is a DSC graph of the purified protein LC13-sTv.
  • Figures 48a and 48b are DSC plots of purified proteins JM22-WT and JM22-sTv, respectively.
  • Figures 49a and 49b are DSC plots of purified proteins LC13-G9 and LC13-G15, respectively.
  • the inventors have extensively and intensively studied and found for the first time that a highly stable mutant TCR, particularly soluble TCR, can be unexpectedly obtained by targeted mutation of the hydrophobic core region of a T cell receptor.
  • the present invention has been completed on this basis.
  • the inventors used an optimized TCR protein structure to construct a highly stable TCR molecule by changing the hydrophobic core of the TCR.
  • the present invention constructs a novel single-stranded TCR variable domain, and the optimal hydrophobic core is isolated by directed molecular evolution.
  • a TCR fragment having a novel hydrophobic core can be further improved by replacing the hydrophobic residue exposed to the surface in the TCR variable domain with a hydrophilic or polar residue.
  • each strand comprises a variable region, a junction region, and a constant region
  • the beta strand typically also contains a short polymorphic region between the variable region and the junction region, but the variable region is often considered part of the junction region.
  • the three CDRs (complementarity determining regions) of each variable region are chimeric in the framework of the variable region, and the hydrophobic core is also located in the framework of the variable region.
  • the alpha chain variable region (V a ) can be divided into several classes, and the ⁇ chain variable region ( ⁇ ⁇ ) can also be classified into several classes.
  • the Va type and the ⁇ type are referred to by the unique TRAV number and the TRBV number, respectively, and TRAJ and TRBJ refer to the TCR junction area.
  • the ⁇ chain J gene used in the present invention means TRAJ, and the ⁇ chain J gene means TRBJ.
  • the a and ⁇ chains of TCR are often treated as having two "domains", namely "variable domains" and “constant domains".
  • the variable domain consists of variable and connected regions that are connected together.
  • TCR a variable domain refers to TRAV and TRAJ linked together
  • TCR i variable domain refers to TRBV and TRBJ joined together.
  • the TCR field is widely known and available to obtain the amino acid sequence of the TCR given in the top GT and its variable domain framework including the specific position number of the hydrophobic core position in the IMGT. For example, it can be found in the IMGT public database.
  • the TCR amino acid position numbers are numbered according to the positions listed in the IMGT unless otherwise stated. If there is a change in the position number listed in the IMGT in the future, the amino acid sequence position number of the TCR listed in the IMGT version of the January 1, 2013 version shall prevail.
  • hydrophobic core which may also be referred to as “hydrophobic core” means that when any protein is dissolved in water, the protein domain typically comprises a core region comprised of a hydrophobic amino acid, typically within its molecular structure.
  • the hydrophobic core of the TCR ci chain variable domain is the amino acid positions of the variable region amino acids 1, 1, 13, 19, 21, 53, 76, 89, 91, 94 and the ⁇ chain J gene (TRAJ) short peptide.
  • TRAJ ⁇ chain J gene
  • the third, fifth, and seventh digits of the reciprocal; the hydrophobic core of the TCR i chain variable domain is the variable region amino acid 1, 1, 13, 19, 21, 53, 76, 89, 91, 94 and the ⁇ chain J gene (TRAJ)
  • the amino acid position of the short peptide is in the second, fourth, and sixth positions.
  • the above location number uses the location number listed in the top GT.
  • Figure 1 is a schematic diagram showing the structure of the variable region of the wild-type TCR having the specificity of the cancer antigen MAGE A3 HLA A1.
  • the bold amino acid residues in the lower left and lower right sides of the schematic are respectively a and A hydrophobic core at the position of the ⁇ chain variable region framework.
  • the antigen binding site of TCR is in the CDR region, and the CDR region determines the binding affinity between TCR and its corresponding antigen.
  • the hydrophobic core is not in the CDR region, and its mutation should not affect the binding and binding affinity of the TCR to its corresponding antigen, but according to the inventors' study, the change of the hydrophobic core will result in the stability of the TCR molecule. influences.
  • Tv refers to a single-chain TCR consisting of a variable domain of the a and beta chains of a TCR linked by a flexible peptide chain
  • the flexible peptide chain can be any peptide suitable for linking the TCR a to the beta variable domain.
  • the chain, the number of amino acid residues of the peptide chain may be 1 to 50, but is not limited to 1 to 50.
  • the term "stability" refers to any aspect of protein stability. Compared to the original wild-type protein, the highly stable protein obtained by screening has one or more of the following characteristics: more resistant to unfolding, more resistant to inappropriate or undesired folding, more renaturation, and more expressive ability Strong, protein reproducible yields, increased thermal stability, increased stability in a variety of environments (eg, ra value, salt concentration, presence of detergent, presence of denaturant, etc.).
  • the final receptor When a phage display system is used to isolate a receptor, the final receptor is usually screened by two important properties, the first being the binding strength or affinity of the receptor to the ligand, and the second being the receptor on the surface of the phage. Show density.
  • the first property is the basis for the evolution of protein affinity, which guides the development of all methodologies that produce high affinity receptors. A brief description is as follows: When a receptor display library is loaded onto a ligand, receptors with higher binding strength bind to the ligand at a faster rate and/or longer retention time and are able to withstand higher Intensive washing. Therefore, these receptors and their coding genes will be Capture and zoom in on subsequent processes.
  • the affinity factor does not contribute to the screening, and the display density dominates the evolutionary result.
  • the receptor and the coding gene have more opportunities and matches. In vivo binding, more of this receptor should be retained under defined wash conditions, thereby being captured and expanded during subsequent screening.
  • more stable proteins can be isolated by using phage display technology or other directed molecular display techniques.
  • the inventors have devised a directed evolutionary library of TCR protein hydrophobic cores to isolate more stable proteins or TCRs. Such a hydrophobic core has been shown to have substantially no effect on the binding strength of TCR to its ligand pMHC or pHLA, since TCR binds to pMHC through its complementarity determining region (CDR).
  • phage display technology can be used to isolate more stable protein constructs.
  • this hypothesis is tested with a TCR (hereinafter referred to as MAGE A3 TCR) extracellular domain specific for the cancer antigen MAGE A3 HLA A1.
  • MAGE A3 TCR TCR
  • the extracellular domain is synthesized according to the sequence in the patent literature.
  • ELI SA enzyme-linked immunosorbent assay
  • the A and ⁇ chains of the TCR displayed by the phage are detected by ELI SA.
  • No binding function was found in the single-chain TCR form (sTv) composed of the variable domains.
  • the library of restriction-random mutations of the single-chain TCR (sTv) variable domain hydrophobic core was cloned into the phage display vector and after several rounds of screening, some highly stable clones were unexpectedly obtained, and then detected by ELISA. These clones can be found to bind to the corresponding pMHC.
  • TCR T cell receptor
  • polypeptides of the invention may also include other mutations outside of the hydrophobic core region, particularly those that increase affinity and mutations in amino acid residues that are exposed to the surface in the TCR variable domain.
  • hydrophobic core regions include, but are not limited to: 1-6 (usually 1-5, preferably 1-3, more preferably 1-2, optimally 1) Amino acid deletions, insertions and/or substitutions, one or several (usually 5 or less, preferably 3 or less, more preferably 2 or less) amino acids are added to the C-terminus and/or the N-terminus.
  • 1-6 usually 1-5, preferably 1-3, more preferably 1-2, optimally 1
  • Amino acid deletions, insertions and/or substitutions one or several (usually 5 or less, preferably 3 or less, more preferably 2 or less) amino acids are added to the C-terminus and/or the N-terminus.
  • amino acids when substituted with amino acids of similar or similar properties, the function of the protein is generally not altered. Add one or both at the C-terminus and / or N-terminus Several amino acids usually do not alter the structure and function of the protein.
  • the term also encompasses polypeptides of the invention in both monomeric and multimeric forms
  • amino acid names in this article are identified by the internationally accepted single letter, and the corresponding amino acid names are abbreviated as: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gin (Q), Glu (E), Gly (G), Hi s (H), l ie (1), Leu (L), Lys (K), Met (M), Phe (F) , Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).
  • amino acid substitution is written in such a way that L19V represents the substitution of V (valine) at the 19th position of the position number given in the top GT, and the meaning of other identical amino acid substitutions is referred to this example. .
  • the invention also encompasses active fragments, derivatives and analogs of the polypeptides of the invention.
  • fragment refers to a polypeptide that binds to a ligand molecule.
  • a polypeptide fragment, derivative or analog of the invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more a polypeptide having a substituent group in one amino acid residue, or (iii) a polypeptide formed by fusing a TCR of the present invention with another compound (such as a compound which prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid.
  • a polypeptide formed by the fusion of the polypeptide sequence a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His).
  • a preferred class of reactive derivatives means having up to 5, preferably up to 3, more preferably up to 2, and optimally 1 amino acid is replaced by an amino acid of similar or similar nature to form a polypeptide.
  • These conservation variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the invention also provides analogs of the TCR of the invention.
  • the difference between these analogs and the pro-TCR polypeptide of the present invention may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, ⁇ -amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the polypeptides of the invention may also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include, but are not limited to, salts formed with: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, malay Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include: salts with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, carbamates or other conventional The form of "prodrugs".
  • the polypeptides of the invention may be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention comprises two, three, four or more T cell receptor molecules linked to another molecule. Coding sequence
  • the invention also relates to polynucleotides encoding the TCRs of the invention.
  • the polynucleotide of the present invention may be in the form of DNA or RNA. ⁇ can be a coded chain or a non-coded chain.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 10 or a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 9, but differs from the corresponding coding region sequence in SEQ ID NO: 10.
  • the full-length nucleotide sequence of the present invention or a fragment thereof can be usually obtained by, but not limited to, PCR amplification, recombinant methods or synthetic methods.
  • DNA sequences encoding the polypeptides of the present invention (or fragments thereof, or derivatives thereof) have been obtained by chemical synthesis.
  • the DNA sequence can then be introduced into various existing Sa molecules (e.g., vectors) and cells known in the art.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and to host cells genetically engineered using the vectors or coding sequences of the invention.
  • the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific for a TCR polypeptide of the invention.
  • One method of producing the TCR of the present invention is to select a highly stable TCR from a diverse library of phage particles displaying such TCR.
  • Mutations can be carried out by any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme-based cloning or linkage-independent cloning (LIC) methods. Many standard molecular biology textbooks detail these methods. Further details of polymerase chain reaction (PCR) mutagenesis and cloning based on restriction enzymes can be found in Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (Third Edition). CSHL Press. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Bi otechnol 6 (1): 30-6).
  • PCR polymerase chain reaction
  • LIC linkage-independent cloning
  • the polypeptide of the invention may be a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptide of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.
  • the polynucleotide of the present invention can be utilized to express or produce a recombinant polypeptide of the present invention by conventional recombinant DNA techniques. Generally there are the following steps:
  • TCR polypeptide of the present invention is isolated and purified from a culture medium or a cell.
  • the recombinant polypeptide can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Pharmaceutical composition and method of administration include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration
  • the TCR of the invention and the TCR transfected T cells of the invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCRs, multivalent TCR complexes and cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in any suitable form (depending on the method desired for administration to the patient). It can be provided in unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such kits (but not required) include instructions for use. It can include a plurality of said unit dosage forms.
  • polypeptides of the invention may be used alone or in combination or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
  • Therapeutic agents that can be combined or coupled to the TCRs of the invention include, but are not limited to: 1. Radionuclides (Koppe et al, 2005, Cancer metastas is reviews 24, 539); 2. Biotoxins (Chaudhary et al, 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines (Gi llies et al., 1992, Proceedings of the National Academy of Sciences) (PNAS) 89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy 53 , 345; Hal in et al, 2003, Cancer Research 63, 3202); 4.
  • Antibody Fc Fragments (Mosquera et al, 2005, The Journal Of Immunology 174, 4381); 5. Antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62, 319); 6. Gold nanoparticles/nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Viral particles (Peng et al, 2004, Gene therapy 1 1, 1234); 8. Liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. A prodrug activating enzyme (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BraD); 1 1. a chemotherapeutic agent (for example, cisplatin) or the like.
  • DTD DT-diaphorase
  • BrainD biphenyl hydrolase-like protein
  • 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 themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. These vectors are well known to those of ordinary skill in the art. A full 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.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the therapeutic compositions may be in the form of injectables, such as liquid solutions or suspensions; solid forms such as liquid carriers, which may be formulated in solution or suspension prior to injection.
  • composition of the invention can be administered by conventional routes including, but not limited to, intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration.
  • the object to be prevented or treated may be an animal; especially a human.
  • composition of the present invention When used for actual treatment, a pharmaceutical composition of various different dosage forms may be employed depending on the use.
  • a pharmaceutical composition of various different dosage forms may be employed depending on the use.
  • an injection, an oral preparation, or 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, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity (i sotonic i ties preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be carried out in a customary manner depending on the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity (i sotonic i ties preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be carried out in a customary manner depending on the dosage form.
  • compositions of the invention may also be administered in the form of sustained release agents.
  • the polypeptide of the present invention can be incorporated into a pill or microcapsule in which the sustained release polymer is used as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • the sustained-release polymer an ethylene-vinyl acetate copolymer, polyhydroxymethacrylate (polyhydrometaacrylate polyacrylamide, polyvinylpyrrolidone, or the like) may be mentioned.
  • Methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the dose of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may be based on the weight, age, sex, and degree of symptoms of each patient to be treated. Determine it reasonably.
  • the TCR of the present invention can be used as a pharmaceutical or diagnostic reagent. Modifications or other modifications may be made to obtain features that are more suitable for use as a drug or diagnostic agent.
  • the medicament or diagnostic reagent can be used to treat or diagnose a variety of different diseases including, but not limited to: cancer (eg, kidney cancer, ovarian cancer, head and neck cancer, testicular cancer, lung cancer, stomach cancer, cervical cancer, bladder) Cancer, prostate cancer or melanoma, etc., autoimmune diseases, viral infectious diseases, transplant rejection and graft versus host disease.
  • Drug localization or targeted administration can be achieved by the specificity of the TCR of the present invention, thereby improving the therapeutic or diagnostic effects of various diseases.
  • targeting tumors or metastatic cancer can increase the effects of toxins or immune stimuli.
  • an autoimmune disease it is possible to specifically inhibit an immune response to normal cells or tissues, or to slowly release an immunosuppressive drug, so that it produces more local effects over a longer period of time, thereby The impact of immunity is minimized.
  • the role of immunosuppression can be optimized in the same way in preventing transplant rejection.
  • viral diseases in which a drug already exists such as HIV, SIV, EBV, CMV, HCV, HBV, it is also beneficial that the drug releases or exerts an activating function in the vicinity of the infected cell region.
  • the TCR of the present invention can be used to modulate T cell activation, and the TCR of the present invention inhibits T cell activation by binding to specific pMHC.
  • Autoimmune diseases involving T cell-mediated inflammation and/or tissue damage may be suitable for this method, such as type I diabetes.
  • the TCR of the present invention can also be used for the purpose of delivering cytotoxic agents to cancer cells, or can be used to transfect T cells, thereby enabling them to destroy tumor cells presenting HLA complexes, in a process known as adoptive immunotherapy. Give the patient.
  • the TCR of the present invention can also be used as a diagnostic reagent.
  • the TCR of the present invention is labeled with a detectable label, such as a label for diagnostic purposes, to detect binding between the MHC-peptide and the MHC-peptide specific TCR of the invention.
  • a detectable label such as a label for diagnostic purposes
  • Fluorescently labeled TCR multimers are suitable for FACS analysis and can be used to detect antigen presenting cells carrying TCR-specific peptides.
  • TCR of the invention that binds to a conjugate, including but not limited to an anti-CD3 antibody
  • the T cells are redirected to target cells that present a particular antigen, such as tumor cells.
  • the highly stable sputum cell receptor of the present invention can be used for the purpose of studying the interaction between TCR and pMHC (peptide-major histocompatibility complex) and the diagnosis and treatment of diseases.
  • TCR polypeptide of the invention has high stability
  • TCR polypeptides with high stability and high affinity can be further screened.
  • the primers designed as shown in Table 1 were subjected to directed point mutation of the chemically synthesized TCR ci chain and the ⁇ chain variable domain amino acid sequence (refer to Patent Document W02012/013913, respectively). These mutations cause the hydrophobic residues exposed to the surface in the TCR alpha and beta chain variable domains to become hydrophilic or polar residues for use in making templates for hydrophobic core mutant libraries.
  • the process of mutating the surface hydrophobic residue V at position 20 of the a chain to the hydrophilic residue S is accomplished by site-directed mutagenesis when constructing a hydrophobic core mutant library.
  • YW800, YW80U YW802, YW803, YW804 are primers designed for site-directed mutagenesis ⁇ chain variable domain
  • YW806 and YW807 are primers designed to direct the mutant ⁇ chain variable domain
  • YW805 is a ligation primer for introducing flexible peptide chains.
  • the sTv was constructed by PCR according to the primer connection method shown in Fig. 4.
  • the sTv was named MAGE-sTv-WT.
  • the first step PCR uses the following primer pairs for YW801/YW803 (a chain), YW802/YW804 (a chain), YW806/YW807 ( ⁇ chain) for PCR.
  • the reaction procedure was: denaturation at 98 °C for 30 seconds, repeated cycles of 25 cycles of 94 ° C for 5 seconds, 55 ° C for 10 seconds, and 72 ° C for 20 seconds.
  • the second step PCR uses the first step PCR purified product and the chemically synthesized flexible peptide single-stranded DNA as a template, and YW800/YW807 as a primer for the second step PCR.
  • the reaction procedure was: denaturation at 98 ° C for 30 seconds, repeated cycles of 30 cycles of 94 ° C for 5 seconds, 55 ° C for 10 seconds, 72 ° C for 30 seconds, and once for 72 ° C for 5 minutes.
  • the purified PCR product of the second step is digested and ligated into the phage display vector.
  • Ncol and Notll-cleaved DNA sequence encoding MAGE-sTv-WT was ligated into the pET-28a vector (Novagen) cleaved with Ncol and Notll.
  • the ligated plasmid was transformed into a conventional competent E. coli ( ⁇ cAer ⁇ ac ⁇ ) strain BL21
  • Figures 2a and 3a show the alpha-chain variable domain and the beta-chain variable domain amino acid sequence of MAGE-sTv-WT, respectively.
  • FIGS. 9 and 11 the optimized amino acid positions are shown in bold and underlined.
  • Figures 2b and 3b show the alpha-chain variable domain and beta-chain variable domain nucleotide sequences of MAGE-sTv-WT, respectively (SEQ ID NOS: 10 and 12).
  • Example 3 Expression, renaturation and purification of MAGE-sTv-WT
  • the expression plasmid containing MAGE-sTv-WT obtained in Example 2 was transformed into a medium plate of Escherichia coli strain Rosetta (DE3) (Merck), cultured at 37 ° C overnight, and the single colony was contained at 37 ° C.
  • the natamycin medium was cultured to 0D 6 . .
  • protein expression was then induced with 0.5 mM IPTG for 4 hours, and cells were harvested by centrifugation at 5,000 rpm for 15 minutes using a Fisher Thermo Sovall R6+ centrifuge. Cell pellets were lysed with Bugbuster MasterMix (Merck).
  • the inclusion body pellet was recovered by centrifugation at 6,000 g for 15 minutes using a FisherThermo Sovall X1R centrifuge. The inclusion bodies were then washed three times with a 10-fold diluted Bugbuster solution to remove cell debris and membrane components. The inclusion bodies were then dissolved using the following buffer: 20 mM Tris, pH 9.0, 8 M urea. After quantification by BCA method, the fraction was 10 mg per tube and frozen at -80 °C.
  • the dialyzed renaturation MAGE-sTv-WT was centrifuged and the anion exchange column Q HP 5 ml (GE) was washed with a linear gradient of 0-1 M NaCl prepared with 20 mM Tris pH 9.0 using an AKTA Purifier (GE).
  • the debound protein was 10 column volumes, and the elution peak (relative molecular mass of approximately 28 kD) was collected and analyzed by SDS-PAGE (Bio-Rad).
  • the fraction containing MAGE-sTv-WT was concentrated and further purified by a gel filtration column (Superdex 75 10/300, GE Healthcare).
  • the target component is run on SDS-PAGE gel and analyzed, and then the target component is stored at 4 °C.
  • the target peak fractions were combined and concentrated and replaced with 10 mM HEPES buffer pH 7.4.
  • the eluted fraction was further tested for purity by gel filtration.
  • the conditions are: Column Agilent Bio SEC-3 (300 ⁇ , ⁇ 7.8 ⁇ 300 mm), mobile phase 150 mM phosphate buffer, flow rate 0.5 mL/min, column temperature 25 ° C, UV detection wavelength 214 nm.
  • a library of MAGE-sTv-WT hydrophobic core variants was generated using phage display technology to screen and identify highly stable mutants.
  • the hydrophobic core mutant library was constructed by mutagenesis of the hydrophobic core site of MAGE-sTv-WT, and the library was panned and screened.
  • TCR phage library differs in the design of hydrophobic core mutant library.
  • Primers are designed based on the hydrophobic core sites of the template strand, while the high affinity TCR library is constructed by designing primers based on the CDR regions of the template strand.
  • the designed primers are shown in Table 2 below.
  • the hydrophobic core of the highly stable sTv mutant strain screened by the above method was mutated.
  • the highly stable mutant strains screened were named MG29, P8F1 and P8F2.
  • the amino acid of one or more of the following hydrophobic core positions of the ⁇ chain variable domain is mutated: 19th, 21st, 91st; and/or its ⁇ chain variable domain
  • the amino acid of one or more of the following hydrophobic core positions is mutated: position 91, the fourth strand of the amino acid sequence of the ⁇ chain J gene short peptide. More specifically, according to the position number in the top GT, it has one or more of the following alpha chain variable domain amino acid residues 19V, 211, 91L and/or one or more of the following beta chain variable domain amino acid residues 91F or
  • the specific a-chain variable domain amino acid sequences are SEQ ID NOs: 15 and 17; the ⁇ -chain variable domain amino acid sequences are SEQ ID NO: 16 and 18.
  • the amino acid sequences of the ⁇ and ⁇ chain variable domains constituting the mutant strain MG29 are SEQ ID NOs: 15 and 16, respectively, as shown in Figures 6a and 6b.
  • amino acid sequences of the ⁇ and ⁇ chain variable domains of the mutant strain P8F1 are SEQ ID NOs: 17 and 18, respectively, as shown in Figures 7a and 7b; the amino acid sequences of the a and ⁇ chain variable domains of the mutant P8F2 SEQ ID NO: 15 and 18, respectively, as shown in Figures 8a and 8b;
  • the high-stability mutants MG29, P8F1, P8F2 with high 0D values and the MAGE-sTv-WT with no hydrophobic mutations were compared for their 0D values, and the specificity of the mutants was verified.
  • the supernatant of the overnight culture was collected by centrifugation, and the phage in the supernatant was precipitated with 1/4 volume ratio of PEG/NaCl, placed on ice for 1 h, and the pellet was collected by centrifugation and resuspended in 3 mL of PBS.
  • Hydrophobic core-optimized clones can display sTv to varying degrees and specifically bind to their original ligand MAGE A3 pHLA-Al antigen, but have no junction with other unrelated antigens such as EBV, influenza and NY-ES0-1 antigen. Hehe.
  • the binding of the sTv of the hydrophobic core mutation detected by phage display to MG29, P8F1 and P8F2 and the specific antigen should not be due to the fact that its affinity is stronger than that of the wild TCR, which was proved in Example 15.
  • the partially hydrophobic core of the high stability variant screened in Example 4 was introduced into several other TCR molecules according to a site-directed mutagenesis method well known to those skilled in the art to construct a highly stable sTv molecule.
  • variable domains of the alpha and beta chains of the wild-type TCR molecule of the specific antigen were constructed into single-stranded forms of the above several molecules, respectively, and designated as LC13-WT, JM22-WT and 1G4-WT, respectively.
  • amino acid sequences of the ⁇ 13 and ⁇ chain variable domains of LC13-WT are SEQ ID NO: 29 and SEQ ID NO: 30, respectively, as shown in Figures 10a and 10b; the ⁇ -chain and ⁇ -chain variable domains of JM22-WT
  • the amino acid sequences are SEQ ID NO: 31 and SEQ ID NO: 32, respectively, as shown in Figures 11a and lib; the amino acid sequences of the ⁇ G and ⁇ chain variable domains of 1G4-WT are SEQ ID NO: 33 and SEQ ID NO, respectively. : 34, as shown in Figures 12a and 12b.
  • the partial hydrophobic core of the high stability variant screened in Example 4 was separately introduced into the LC13-WT, JM22-WT and 1G4-WT molecules by site-directed mutagenesis well known to those skilled in the art, and the mutation was introduced.
  • the molecules are designated LC13-sTv, JM22-sTv, and 1G4-STV, respectively, and the introduced hydrophobic core is indicated by an underlined bold letter.
  • amino acid sequences of the a chain and the ⁇ chain variable domain of LC13-sTv are SEQ ID ⁇ 0:35 and SEQ ID ⁇ 0:36, respectively, as shown in Figures 13a and 13b, wherein the hydrophobic core introduced in the ⁇ chain variable domain is 11L, 13V, 211 and 911, the hydrophobic core introduced in the ⁇ chain variable domain is 94L; the amino acid sequences of the ⁇ chain and ⁇ chain variable domains of JM22-sTv are SEQ ID NO: 37 and SEQ ID NO: 38, respectively.
  • the hydrophobic core introduced in the ⁇ chain variable domain is 19V and 211, and the hydrophobic core introduced in the ⁇ chain variable domain is 911 and 94L;
  • the ⁇ chain and ⁇ chain of 1G4-STV can be
  • the variable domain amino acid sequences are SEQ ID NO: 39 and SEQ ID NO: 40, respectively, as shown in Figures 15a and 15b, and the hydrophobic core introduced in the ⁇ chain variable domain is 19V and 211, and the ⁇ chain variable domain thereof
  • the hydrophobic core introduced in the 19V, 911, 94L and the sixth of the J gene are the T.
  • the above location numbers are based on the location numbers listed in IMGT.
  • the linker for constructing the above single-stranded molecule may be any suitable sequence, and the preferred amino acid sequence of the present invention is SEQ ID NO: 41, as shown in FIG.
  • Expression, renaturation, and protein LC13-WT and LC13-sTv were expressed by the method described in Example 3. After purification, the gel filtration column was purified and run SDS-PAGE gel, and the SEC map of the two proteins was made by gel filtration, and the expression amount, the amount of protein obtained after purification and the protein refolding yield were calculated. Among them, the expression amount was 1 L of E. coli induced expression and purification of the inclusion body. The amount of protein obtained after purification was 1 L of E. coli induced expression and purification, and the obtained inclusion body was renatured and purified to obtain the amount of protein.
  • Protein refolding yield (%) 100 * The amount of protein obtained after purification (mg) / the amount of inclusion body used for renaturation (mg).
  • the expression amount and protein refolding yield referred to in the present invention are calculated according to the above calculation methods, unless otherwise specified.
  • the Tm values of the purified proteins LC13-WT and LC13-sTv were determined by a differential scanning calorimeter (Nano DSC) of TA (waters), USA.
  • the scanning range is 10-9CTC
  • the heating rate is 1 °C /min
  • the loading is 90 ( ⁇ L.
  • the Tm value is obtained by fitting the two-dimensional scaled fitting model of the analysis software Nanoanalyze.
  • Table 3 below lists the expression levels of LC13-WT and LC13-sTv, the amount of protein obtained after purification, and the refolding yield of the protein.
  • Figure 17 is a SDS-PAGE gel of the protein LC13-WT and LC13-sTv obtained after purification through a gel filtration column (Superdex 75 10/300, GE Healthcare) as described in Example 3.
  • the gel map shows that the bands formed by the LC13-WT protein after purification are not uniform, and the LC13-sTv can form a single band with high purity.
  • the renaturation of LC13-sTv is much better than that of LC13-WT.
  • Figures 18a and 18b show the SEC profiles of the proteins LC13-WT and LC13-sTv, respectively. From the map, the purified protein LC13-WT has no peak, and LC13-sTv can form a single and symmetric elution peak. The renaturation of LC13-sTv was significantly better than LC13-WT.
  • LC13-WT After refolding, LC13-WT showed minimal protein content in the correct conformation, no obvious protein unfolding endothermic peak, and the Tm value of LC13-sTv after hydrophobic core mutation was not obtained by the analysis software Nanoanalyze.
  • the temperature is 43. 6 ° C, and its DSC curve is shown in Figure 47. This indicates that LC13-sTv is more versatile, more resistant to unfolding, more resistant to inappropriate or undesirable folding, and has a significantly improved thermal stability compared to LC13-WT.
  • LC13-sTv modified by hydrophobic core
  • the unmodified LC13-WT is more renaturation, more resistant to unfolding, more resistant to inappropriate or undesirable folding, higher yield of protein refolding, and a significant increase in thermal stability. Therefore, LC13-sTv has a significant improvement over LC 13-WT stability.
  • the amount of stability improvement was calculated from the data of protein refolding yield, and the stability of LC13-sTv was 35 times higher than that of LC13-WT in the present invention.
  • the proteins JM22-WT and JM22-sTv were expressed, renatured and purified by the method described in Example 3.
  • the gel filtration column was purified and run SDS-PAGE gel, and the SEC pattern of the two proteins was determined by gel filtration.
  • the expression amount, the amount of protein obtained after purification, and the protein refolding yield were calculated, and the Tm value was determined by the method described in Example 6.
  • Table 4 lists the expression levels of JM22-WT and JM22-sTv, the amount of protein obtained after purification, and the protein refolding yield.
  • Figure 19 is an SDS-PAGE gel of proteins JM22-WT and JM22-sTv obtained after purification through a gel filtration column (Superdex 75 10/300, GE Healthcare) as described in Example 3.
  • the glue map shows that the monomer bands formed by the renaturation of JM22-WT are not uniform, and there are three bands, and JM22-sTv can form a single band of monomers with high purity.
  • the renaturation of JM22-sTv is much better than that of JM22-WT.
  • Figures 20a and 20b show the SEC profiles of the proteins JM22-WT and JM22-sTv, respectively. From the map, the purified protein JM22-WT forms a single elution peak with a low signal, and the purified JM22-sTv can A single and symmetric elution peak is formed, indicating that the renaturation of JM22-sTv is significantly better than JM22-WT.
  • Figures 48a and 48b are DSC plots of proteins JM22-WT and JM22-sTv, respectively. Due to the minimal protein content of the correct conformation of JM22- WT, there is no obvious protein unfolding endothermic peak. The Tm value is not obtained by the analysis software Nanoanalyze, and the Tm of JM22-sTv after the hydrophobic core mutation. The value is 43. 7 °C. From the above DSC curve, JM22-sTv is more versatile than JM22-WT. Stronger, more resistant to unfolding, more resistant to improper or undesirable folding and a significant increase in thermal stability.
  • the hydrophobic core modified JM22-sTv Compared with the hydrophobic core, the unmodified JM22-WT has stronger renaturation ability, more anti-folding, more resistance to inappropriate or undesired folding and thermal stability, and a significant increase in protein refolding yield. Therefore, the JM22-sTv of the present invention has a significantly improved stability compared to JM22-WT.
  • the amount of stability improvement was calculated from the data of the refolding yield of the protein. In the present invention, the stability of JM22-sTv was improved by 4200% with respect to JM22-WT.
  • the proteins 1G4-WT and 1G4-STV were expressed, renatured and purified by the method described in Example 3.
  • the gel filtration column was purified and run SDS-PAGE gel, and the SEC pattern of the two proteins was determined by gel filtration. At the same time, the expression amount, the amount of protein obtained after purification, and the yield were calculated.
  • Table 5 below lists the expression levels of 1G4-WT and 1G4-STV, the amount of protein obtained after purification, and the protein reproducibility yield.
  • the 1G4-STV protein introduced into the hydrophobic core was increased by 2.6 times compared with the 1G4-WT protein in which the hydrophobic core was not mutated.
  • Figure 21 is an SDS-PAGE gel of Protein 1G4-WT and lG4-sTv obtained after purification through a gel filtration column (Superdex 75 10/300, GE Healthcare) as described in Example 3.
  • the gel image shows that the bands formed by the 1G4-WT protein after purification are not uniform, forming two bands, and 1G4-STV can form a single band of monomers with high purity.
  • the renaturation of 1G4-STV is much better than that of 1G4-WT.
  • Figures 22a and 22b show the SEC profiles of the proteins 1G4-WT and 1G4-STV, respectively. From the map, the purified protein 1G4-WT has a single elution peak and a lower signal, while the purified 1G4-STV can A single and symmetric elution peak is formed, indicating that the renaturation of 1G4-STV is significantly better than that of 1G4-WT.
  • 1G4-STV modified by hydrophobic core is more hydrophobic than 1G4-STV.
  • the core unmodified 1G4-WT has stronger renaturation ability, higher expression level, and higher protein refolding yield. Therefore, the 1G4-STV of the present invention has a significantly improved stability compared to 1G4-WT. Take The data of protein refolding yield was calculated for the amount of stability improvement. In the present invention, the stability of 1G4-STV was increased by 260% relative to 1G4-WT.
  • the surface amino acid residues of the hydrophobic core and the variable domain were mutated by using 1G4-STV as a template to construct a library and screen for highly stable molecules.
  • the hydrophobic core site to be mutated has been indicated in bold underlined in SEQ ID NO: 42 and the surface amino acid residues that need to be mutated are shown in bold, as shown in Figure 23.
  • Example 4 The basic method employed in library construction has been described in Example 4.
  • three libraries were constructed for sites that required mutation, and the hydrophobic core sites required for mutation were all in library 1, and libraries 2 and 3 were constructed for surface amino acid residues.
  • the 1G4-STV plasmid was used as a template, and the mutated DNA fragment was obtained by overlapping PCR with the designed mutant primer, and then the fragment was cloned into the pUC19-based phage plasmid vector pLitmus28 by Ncol/Notl digestion. (NEB).
  • the ligated DNA was electroporated into TGI competent cells (lucigen), and a total of three phage plasmid vector libraries were obtained, and the storage capacity was about 1 X 10 9 -3 X 10 9 in terms of colony number.
  • the lawns grown from the three libraries were scraped off and added to a final concentration of 20% glycerol and stored at -80 °C.
  • Table 8, Table 9, and Table 10 below are primers designed for Library 1, Library 2, and Library 3, respectively.
  • Primer name Primer sequence (5' -3') SEQ ID NO :
  • the phage grown in the library is concentrated after precipitation.
  • Example 10 Stability verification of clones screened in Example 9
  • Example 9 The 11 clones screened in Example 9 were examined by the ELISA test procedure described in Example 4. The 0D value was measured and the antigen specificity was verified, and the results are shown in Fig. 24. This result shows that 11 clones have high 0D values and can specifically bind to their original ligand antigen HLA-A2/SLL ⁇ ITQC (NY-ES0-1 tumor-specific antigen), and have no binding to other unrelated antigens. The binding of these sTvs detected by phage display to the antigen HLA-A2/SLLMWITQC should not be due to their affinity being stronger than wild TCR, as demonstrated in Example 11.
  • the above 11 mutants are mutated at one or more of the following hydrophobic core positions: the 11th, 13th or 94th and/or ⁇ chain of the alpha chain variable region
  • the 11th, 13th or 94th position of the variable zone comprises one or more of the following alpha chain variable region hydrophobic core amino acid residues 11M, 11E, 13R, 13K, 94V or 941 and/or beta chain variable region hydrophobic core amino acid residues 11L, 11V, 13V or 94V.
  • the clones we screened also contain one or more of the following alpha chain variable region amino acid residues 4L, 12N, 16S, 93N, 93R, 97N, 100G, 105S or ⁇ chain J gene reciprocal first D and/or ⁇ chain variable region amino acid residues 41, 101 ⁇ chain J gene reciprocal first position is D or the third last digit is E.
  • the selected 11 clones were ligated, expressed, renatured and purified by the methods described in Example 2 and Example 3.
  • the Tm values of the above 11 clones were determined using a differential scanning calorimeter (Nano DSC) of TA (waters), USA. The scanning range is 10-90 ° C, the heating rate is rC /min, and the loading is 90 ( ⁇ L.
  • the Tm value is obtained by fitting the two-statescaled fitting model of the analysis software Nanoanalyze. The results are shown in Fig. 27 and As shown in Table 9, the Tm value is not less than 37. 9 ° C, and has a distinct protein unfolding endothermic peak.
  • the 1G4-WT is identical to the expression, renaturation, purification process and DSC experimental conditions of the above clones.
  • the DSC results are shown in Figure 28. As can be seen from the figure, there is no obvious endothermic peak of protein unfolding, indicating that the protein content of the correct conformation is extremely small. Compare the DSC map of the above 11 clones with the 1G4-WT The DSC map results showed that the clones screened were more resistant to unfolding than 1G4-WT, more resistant to inappropriate or undesirable folding, more renaturation and a significant increase in thermal stability. Therefore, we screened clones. The stability is much higher than that of the hydrophobic core without mutation 1G4 - WT.
  • the mutant strains G9 G13 and G15 described in Example 10 were expressed, renatured and purified by the method described in Example 3. After purification by gel filtration column, SDS-PAGE gel was run, and gel filtration was used. The SEC profile of the protein was simultaneously calculated for its expression, the amount of protein obtained after purification, and the refolding yield of the protein, and compared with 1G4-WT.
  • Table 10 lists the expression levels of 1G4-WT G9 G13 and G15, the amount of protein obtained after purification, and the reproducibility yield of the protein.
  • Figure 30 is an SDS-PAGE gel of Protein 1G4-WT, G9, G13 and G15 obtained after purification through a gel filtration column (Superdex 75 10/300, GE Healthcare) as described in Example 3.
  • the gel map shows that the bands formed by the 1G4-WT protein after purification are not uniform, and the modified G9, G13 and G15 are capable of forming a single band of monomers with high purity. Note that the renaturation of G9, G13 and G15 is better than 1G4-WT.
  • Figures 31a, 31b, and 31c show the SEC profiles of proteins G9, G13, and G15, respectively. From the SEC profile of 1G4-WT in Figure 22a, the purified protein 1G4O L
  • the elution peak formed by OO LWT is not single and the signal is low, and the purified G9, G13 and G15 can form a single OG-OL and symmetric elution peak, indicating that the renaturation of G9, G13 and G15 is significant. Better than 1G4-WT.
  • the Tm value of G15 was determined by the method described in Example 10 to be 46. 6 ° C, and the DSC chart is shown in Fig. 46. According to the results of the measurement in Example 10, the Tm values of the mutant strains G9 and G13 were also relatively high, being 49.55 ° C and 49. 63 ° C, respectively.
  • the BIAcore T200 real-time analysis system was used to detect the binding of proteins G9, G13 and G15 to their ligands.
  • the results showed that the affinity of the three sTv proteins to the antigen HLA-A2/SLLMWITQC was not superior to that of the wild-type 1G4 TCR.
  • the dissociation equilibrium constant of the 1G4TCR binding antigen HLA-A2/SLLMWITQC is 32 ⁇ (Refer to Li et al.
  • a highly stable sTv molecule was constructed from the hydrophobic core of the high stability variant screened in Example 9 and the surface amino acid residue of the variable region backbone.
  • amino acid sequences of the ⁇ chain and the ⁇ chain variable domain of LC13-G9 are SEQ ID NO: 99 and
  • the hydrophobic core introduced in the alpha chain variable domain is 13V, 211, 911 and 941.
  • the hydrophobic core introduced in the ⁇ chain variable domain is 11V, 13V and 94V.
  • the amino acid sequences of the ⁇ chain and ⁇ chain variable domains of LC13-G15 are SEQ ID NO: 101 and SEQ ID NO: 102, respectively, as shown in Figures 33a and 33b, and the hydrophobic core introduced in the ⁇ chain variable domain is 11L.
  • the hydrophobic core introduced in the ⁇ chain variable domain is 11L, 13V and 94V;
  • the amino acid sequences of the ⁇ chain and ⁇ chain variable domain of JM22-G9 are SEQ ID NO: 103 and SEQ, respectively ID NO: 104, as shown in Figures 34a and 34b, the hydrophobic cores introduced in the ⁇ chain variable domain are 11M, 13V, 19V, 211 and 941, and the hydrophobic core introduced in the ⁇ chain variable domain is 11V, 13V.
  • the amino acid sequences of the alpha and beta chain variable domains of JM22-G15 are SEQ ID NO: 105 and SEQ ID NO: 106, respectively, as shown in Figures 35a and 35b, introduced in the alpha chain variable domain
  • the hydrophobic cores are 13V, 19V, 211 and 941, and the hydrophobic cores introduced in the ⁇ -chain variable domains are 13V, 911 and 94V;
  • the ⁇ -chain and ⁇ -chain of MAGE-G15 can be
  • the amino acid sequence of the domain is SEQ ID ⁇ 0: 107 and SEQ ID NO: 108, respectively, as shown in Figures 42a and 42b, the hydrophobic core introduced in the ⁇ chain variable domain is 19V, 211 and 941, and the ⁇ chain variable domain thereof
  • the hydrophobic cores introduced are 13V, 89L, 911 and 94V.
  • the above location number uses the location number listed in the top GT.
  • the ligated linker used to construct the above single-stranded molecule may be any suitable sequence, and the preferred amino acid sequence of the present invention is SEQ ID NO: 41, as shown in FIG.
  • the proteins LC13-G9 and LC13-G15 were expressed, renatured and purified by the method described in Example 3.
  • the gel filtration column was purified and run SDS-PAGE gel, and the SEC map of the two proteins was made by gel filtration.
  • the expression amount, the amount of protein obtained after purification, and the protein refolding yield were calculated.
  • Table 11 lists the expression levels of LC13-G9 and LC13-G15, the amount of protein obtained after purification, and the refolding yield of the protein. The data of LC13-WT are also listed for analysis.
  • Protein name Expression amount (mg/L) Protein amount after purification (mg/L) Yield (%)
  • the revival yield of the 1G4-WT protein was 5.4% higher than that of the 1G4-WT protein, which was not mutated by the hydrophobic core, respectively. And 57. 9 times.
  • Figure 36 is an SDS-PAGE gel of proteins LC 13-G9 and LC13-G15 obtained after purification through a gel filtration column (Superdex 75 10/300, GE Healthcare) as described in Example 3.
  • the gel map shows that the bands formed by LC 13-WT are not uniform, and the modified LC13-G9 and LC13-G15 are capable of forming a single band of monomers with high purity. Note The renaturation of LC13-G9 and LC13-G15 is much better than that of LX13-WT.
  • Figure 37 and ⁇ 38 are the SEC spectra of the proteins LC13-G9 and LC13-G15, respectively.
  • the SEC map of LC 13-WT shows no peak, while the proteins LC 13-G9 and LC13-G15 can form a single and symmetrical
  • the elution peak indicated that the renaturation of LC13-G9 and LC13-G15 was significantly better than that of LC13-WT.
  • Tm values of the proteins LC13-G O9 and LC13-G15 were determined by the method described in Example 10.
  • Figures 49a and 49b are DSC curves of the proteins LC13-G9 and LC13-G15, respectively, with Tm values of 43 °C. And 50. 5 °C.
  • LC13-WT has a low protein content in the correct conformation after renaturation, and there is no obvious protein unfolding endothermic peak, and its Tm value cannot be obtained.
  • the thermal stability of LC13-G9 and LC13-G15 of the present invention is at least double that of LC13-WT.
  • the LC13-G9 of the present invention is more resistant to unfolding, more resistant to inappropriate or undesirable folding and renaturation than LC 13-WT.
  • the LC 13-G9 of the present invention has a markedly improved stability compared to LC 13-WT compared to LC13-G15.
  • the stability of the LC13-G9 and the LC13-G15 were increased by 5.4% and 57.9 times, respectively, with respect to the LC13-WT, respectively.
  • the proteins JM22-G9 and JM22-G15 were expressed, renatured and purified by the method described in Example 3.
  • the gel filtration column was purified and run SDS-PAGE gel, and the SEC pattern of the two proteins was determined by gel filtration. At the same time, the expression amount, the amount of protein obtained after purification, and the protein refolding yield were calculated.
  • Table 12 lists the expression levels of JM22-G9 and JM22-G15, the amount of protein obtained after purification, and protein. The data of the renaturation yield, together with the relevant data of JM22-WT, for analysis.
  • Figure 39 is a gel filtration column as described in Example 3 (Superdex 75 10/300, GE
  • Heal thcare was purified to obtain SDS-PAGE gel images of proteins JM22-G9 and JM22-G15.
  • the glue map shows that the monomer bands formed by the renaturation of JM22-WT are not uniform, and there are three bands, and the modified JM22-G9 and JM22-G15 can form a single band of monomers with high purity. Note The renaturation of JM22-G9 and JM22-G15 is much better than JM22-WT.
  • Figure 40 and ⁇ 41 are the SEC spectra of proteins JM22-G9 and JM22-G15, respectively.
  • the SEC map of JM22-WT shows that the elution peaks formed are not single and the signal is very low, while the proteins JM22-G9 and JM22-G15 can basically A single and symmetric elution peak was formed, further indicating that the renaturation of JM22-G9 and JM22-G15 was significantly better than JM22-WT.
  • JM22-G9 and JM22-G15 have stronger renaturation ability, higher expression amount and protein refolding yield than the hydrophobic core unmodified JM22-WT. Therefore, the JM22-G9 of the present invention has a significant improvement in stability compared to JM22-G15 compared to JM22-WT.
  • the stability of the protein was calculated by the data of the protein refolding yield. In the present invention, the stability of JM22-G9 and JM22-G15 relative to JM22-WT was increased by 28. 5 times and 127.2 times, respectively.
  • the protein MAGE-G15 was expressed, renatured and purified by the method described in Example 3. After purification by gel filtration column, SDS-PAGE gel was run, and the SEC spectrum of the protein was determined by gel filtration, and the expression amount was calculated. , the amount of protein obtained after purification and protein refolding yield.
  • Table 13 lists the expression levels of MAGE-sTv-WT and MAGE-G15, the amount of protein obtained after purification, and the refolding yield of protein.
  • the SDS-PAGE gel of the protein obtained after purification of the hydrophobic core-modified MAGE-G15 via a gel filtration column is shown in Fig. 43.
  • the gel image shows that MAGE-G15 is able to form a single band with high purity, indicating that the refolding ability of protein MAGE-G15 is much stronger than MAGE-sTv-WT.
  • the SEC map of MAGE-G15 shown in Figure 44 has a single and symmetric elution peak, which also indicates that the refolding ability of protein MAGE-G15 is much stronger than that of MAGE-sTv-WT.
  • Figure 45 is a DSC curve of the protein MAGE-G15, which was obtained by fitting the two-dimensional scaled model of the analysis software Nanoanalyze to obtain a Tm value of 46. 7 °C.
  • the BIAcore T200 real-time analysis system was used to detect the binding of the protein MAGE-G15 to its ligand.
  • the results showed that the affinity of the protein MAGE-G15 to its ligand was not superior to its corresponding wild-type TCR, and its K D value was 30. 4 ⁇ . Hey.
  • the amount of stability improvement was calculated from the data of protein refolding yield. As can be seen from Table 13, the MAGE-G15 of the present invention has an infinite-fold improvement (at least 10,000 times) compared to the MAGE-sTv-WT stability. ).
  • the protein we constructed was purified by a gel filtration column (Superdex 75 10/300, GE Heal thcare) and its molecular weight was determined by mass spectrometry. The molecular weight of the mass spectrometry was determined to be consistent with its theoretical molecular weight. Whether the protein sequence is identical to the original design sequence.
  • the full protein molecular weight of the sample was determined using a mass spectrometer (Eks igent nano LC (nanflex) - Triple T0F 5600 LC/MS system) from AB SCIEX, USA.
  • the sample was diluted with 10% acetonitrile (Fi sherA955-4), 1% formic acid (Fi sher A11750) and water (Si gma39253-1 L-R) and analyzed by mass spectrometry.
  • the system analysis conditions are as follows:
  • the Triple T0F 5600 with Nanospray source analyzes the eluent in the column
  • the collected mass spectrometry data was deconvoluted by Bioanalyst software to obtain the complete protein molecular weight information of the sample.
  • the molecular weight of the whole protein determined by mass spectrometry is consistent with its theoretical molecular weight, indicating that the purified protein sequence is identical to the original design protein sequence.
  • the hydrophobic core screened by the present invention can significantly improve the stability of TCR molecules.
  • the above examples demonstrate that the introduction of the hydrophobic core screened by the present invention into other TCR molecules also serves to enhance stability.

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Abstract

本发明提供了一种高稳定性的T细胞受体，所述T细胞受体在其疏水芯区域发生了突变，引起了其稳定性的提高。本发明还提供了所述T细胞受体的制备方法和应用。

Description

高稳定性的 τ细胞受体及其制法和应用 技术领域
本发明涉及生物技术领域， 更具体地涉及疏水芯区域突变的、 高稳定性的 可溶性 Τ 细胞受体（T ce l l receptor, TCR)。 本发明还涉及所述受体的制备和 用途。 背景技术
仅仅有两种类型的分子能够以特异性的方式识别抗原。 其中一种是免疫球 蛋白或抗体； 另一种是 T 细胞受体（TCR)， 它是由 a链 / β链或者 Y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 τ细胞对其靶细胞发挥免疫效应。
在 T细胞膜上， TCR与参与信号传导的恒定蛋白 CD3结合而形成复合物。 TCR以多种形式存在并在结构上相似， 然而表达这些 TCR的 T 细胞可存在于不 同的解剖学位置并可能具有不同的功能。 TCR 的胞外部分由两个近膜的恒定结 构域和两个远膜的可变结构域组成， 所述可变结构域具有与抗体的互补决定区 (CDRs)相似的多态环。 正是这些环形成了 T细胞受体分子的结合位点以及决定 了肽特异性。 与 TCR相对应的 MHC I类和 I I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， 它们具有多态的肽结合位点， 这些 位点使它们能够呈递各种不同的短肽片段到 APC细胞表面。
如同免疫球蛋白（抗体）作为抗原识别分子一样， TCR 也可以被开发应用于 诊断和治疗。 然而， 很难以（水）可溶形式制备这种由超过一种多肽亚基所组成 的且同时具有一个跨膜区的蛋白质， 因为， 在很多情况下， 这样的蛋白质通过 其跨膜区得以稳定。 TCR 的情况就是这样， 这在科学文献中已经有所反映， 有 文献描述了截短形式的 TCR， 它仅仅包含胞外区或者仅仅包含胞外和胞质区， 这样的 TCR可以被 TCR特异性的抗体识别（这表明， 被抗体所识别的重组 TCR 部分已经正确折叠）， 但是产量并不高， 在低浓度时不够稳定和 /或不能识别主 组织相容性复合体 -肽复合物。
可溶性 TCR有很广泛的用途， 它不仅可用于研究 TCR-pMHC 的相互作用， 也可用作检测感染的诊断工具或作为自身免疫病的标志物。类似地，可溶性 TCR 可以被用来将治疗剂（如细胞毒素化合物或免疫剌激性化合物）输送到呈递特 异性抗原的细胞， 或者用来抑制 τ 细胞（如那些与自身免疫性肽抗原进行反应 的 T细胞）。 为了这些目的， TCR蛋白的修饰是至关重要的。 尤其， 在原核或真 核生物系统中异源表达 TCR是很重要的。
一方面， 对于在大肠杆菌中表达可溶性 TCR而言， 当 TCR与膜分离开时， 其不稳定性和蛋白产量低成为用 TCR或其片段来开发治疗剂或诊断试剂的主要 障碍。 为了克服单链 TCR固有的不稳定性， 一些文献描述了 TCR异二聚体的制 备， 所述异二聚体具有连接各亚基的天然二硫键（Garboczi，等，（1996)， 自然 (Nature) 384 (6605)： 134-41； Garboczi, 等 , (1996) , 免 疫 学 杂 志 (J Immunol) 157 (12)： 5403-10 ; Chang 等， (1994) , PNAS USA 91 : 11408-11412 ; Davodeau 等， (1993)，生物化学杂志（J. Biol. Chem. ) 268 (21)： 15455-15460 ; Golden 等， （1997)， 免疫方法学杂志（J. Imm. Meth. ) 206： 163-169 ; US专利号 6080840) o 尽管这样的 TCR可以被 TCR-特异性的抗体识别， 但其仅能在相对高 的浓度下显示出对天然配体的识别， 提示该识别是不稳定的。
另一方面，对于具有原始抗原特异性的 TCR的制备，在怎样提高水溶性 TCR 片段稳定性方面有很多研究， 包括： 单链 TCR可变域（Novotny，等（1991) PNAS USA 88 : 8646-8650) , 异质二聚体 TCR 胞外区域（Garcial 等（1996)科学 (Science) 274 : 209-219)，或这些分子的改良形式（Shusta 等（2000)自然生物技 术（Nature Biotechnology) 18 : 754-759) , Boulter 等（2003) 蛋白质工程 (Protein Engineering) 16： 707- 711)。 在这些研究中， Novotny 等用柔性肽连 接单链可变域来构建 TCR， 然而在用含水溶性侧链的亲水性残基取代暴露于表 面疏水性残基后， 才能得到稳定的分子。 Shusta等通过在整个分子中引入随机 突变然后展示在酵母菌的表面并用流式细胞仪 FACS 进行筛选的方法， 来进一 步修饰 TCR单链可变域结构。 Garcia等构建 a / i TCR2C胞外结构域， 其中天 然的链间二硫键被保留在结构中。 而 Boulter等通过引入包埋在两个恒定区之 间的一个人工二硫键， 来改善 α / β异二聚体的构建物。
利用恒定区之间二硫键的方法， 已经被用于噬菌体展示 TCR载体， 这种载 体已用于产生很多高亲和性 TCR (Li 等（2005) 自然生物技术（Nature Biotechnology) 34 : 349-354 ; Liddy 等 （2012) 自 然 医 学 （Nature Medicine) 18 : 980-987) 然而， 本发明人研究已经发现， 使用这种构建物成功 地产生高亲和 TCR的机率仍很低， 而且很难获得高亲和性且高稳定性的 TCR。 因此， 有必要开发新的方法来制备水溶性的 TCR、 高亲和性且高稳定性的 TCR、 及它们的活性片段。 发明内容
本发明的目的是提供一种高稳定性的 τ细胞受体。
本发明的另一目的是提供所述高稳定性 T细胞受体的制法和用途。
在本发明的第一方面， 提供了一种 T 细胞受体（T-cell receptor, TCR) , 所述 T细胞受体具有以下特征：
( i )所述 TCR的疏水芯区域发生突变； 并且
( ii )所述 TCR的稳定性高于其对应的疏水芯为野生型的 TCR。
在另一优选例中， 所述 "突变" 是指本发明所述的 TCR的疏水芯区域相对 于与其对应的野生型的 TCR疏水芯区域发生突变。
在另一优选例中， 所述的 "稳定性高于" 指出本发明 TCR与其对应的疏水 芯为野生型的 TCR相比，稳定性提高至少 5%，较佳地至少 30%，更佳地至少 80%。
在另一优选例中， 所述的 "野生型的 TCR疏水芯" 是指与天然产生的 TCR 中疏水芯氨基酸残基（序列）相同， 未发生突变的疏水芯。
在另一优选例中， 所述的 "其对应的疏水芯为野生型的 TCR" 是指与本发 明的疏水芯区域发生突变的 TCR 相比， 除了疏水芯为野生型外，其他区域与本 发明 TCR相同的 TCR。 另外地或较佳地， 所述的 "其对应的疏水芯为野生型的 TCR"指天然产生的， 不含任何突变位点的野生型 TCR， 尤其是其 a链可变域和 β链可变域为野生型的 sTv分子， 代表性的例子包括 LC13-WT。
在另一优选例中， 所述 TCR的 CDR区与野生型相同， 或含有导致亲和力上 升的突变。
在另一优选例中， 所述的亲和力指该 TCR分子与其相应抗原之间的结合亲 和力。
在另一优选例中， 所述的 T 细胞受体可变区骨架（Framework)和恒定区位 置中的侧链指向表面的疏水性残基发生突变。 gP， 所述 TCR的可变域骨架和恒 定域中暴露于表面的氨基酸残基发生突变。 较佳地， 所述发生突变的氨基酸残 基为 TCR的 a链和 /或 β链可变域中暴露于表面的氨基酸残基。 更具体地， 所 述的可变域中暴露于表面的氨基酸位点包括 TCR a链可变域氨基酸第 4位、第 12位、 第 16位、 第 93位、 第 97位、 第 100位、 第 105位和 α链 J基因倒数 第 1位和 TCR i 链可变域氨基酸第 4位、 第 101位、 β链 J基因倒数第 1位和 β链 J基因倒数第 3位。 其中， 氨基酸位置编号按 IMGT (国际免疫遗传学信 息系统） 中列出的位置编号。 在另一优选例中， 所述的 T 细胞受体可变区骨架（Framework)侧链指向表 面（可变域中暴露于表面）的疏水性残基的突变形式包括（但不限于） a链： I7S, A9S, A10S, V20S, Α92Ε, A93S, J gene 短肽氨基酸位置倒数第二位由 I变为 Τ， β链： I 12S， 或上述突变的任意组合， 其中氨基酸位置编号按 IMGT 中列 出的位置编号。
在另一优选例中， 所述的 T细胞受体是可溶性的。
在另一优选例中， 所述 T细胞受体是膜蛋白。
在另一优选例中， 所述 T 细胞受体包括（a)除跨膜结构域以外的全部或部 分 TCR a链； 以及（b)除跨膜结构域以外的全部或部分 TCR β链；
并且 (a)和（b)各自包含功能性可变结构域， 或包含功能性可变结构域和所 述 TCR链恒定结构域的至少一部分。
在另一优选例中， 所述 TCR是由一柔性肽链连接 TCR的 a与 β链的可变域 而构成的单链 TCR。
在另一优选例中， 所述的突变中包括至少一个疏水芯位置的突变。
在另一优选例中， 所述的 T细胞受体在以下位置具有一个或多个突变， a 和 /或 β链氨基酸序列可变区疏水芯位置： 即可变区氨基酸第 11， 13， 19， 21， 53， 76， 89， 91， 94位， 和 /或 α链 J基因短肽氨基酸位置倒数第 3， 5， 7位和 / 或 β链 J基因短肽氨基酸位置倒数第 2， 4， 6位，其中氨基酸位置编号按頂 GT (国 际免疫遗传学信息系统）中列出的位置编号。
在另一优选例中， 所述 TCR的 a链可变域在一个或多个下列位点中发生突 变： α链可变区氨基酸第 11、 13、 19、 21、 53、 76、 89、 91、 或第 94位， 和 / 或 α链 J基因短肽氨基酸倒数第 3位、 倒数第 5位或倒数第 7位， 其中氨基酸 位置编号按 IMGT (国际免疫遗传学信息系统） 中列出的位置编号。
在另一优选例中， 所述 TCR在 SEQ ID NO:9或 SEQ ID NO:29或 SEQ ID NO:31或 SEQ ID NO:33所示的 α链可变域的一个或多个下列位点中发生突变： a链可变区氨基酸第 11、 13、 19、 21、 53、 76、 89、 91、 或第 94位， 和 /或 a 链 J基因短肽氨基酸倒数第 3位、 倒数第 5位或倒数第 7位， 其中氨基酸位置 编号按 IMGT中列出的位置编号。
在另一优选例中， 所述 TCR的 β链可变域在一个或多个下列位点中发生突 变： β链可变区氨基酸第 11、 13、 19、 21、 53、 76、 89、 91、 或第 94位， 和 / 或 β链 J基因短肽氨基酸倒数第 2位、 倒数第 4位或倒数第 6位， 其中氨基酸 位置编号按 IMGT中列出的位置编号。
在另一优选例中， 所述 TCR在 SEQ IDNO:ll或 SEQ IDNO:30或 SEQ ID
NO:32或 SEQ ID NO:34所示的 β链可变域的一个或多个下列位点中发生突变： β链可变区氨基酸第 11、 13、 19、 21、 53、 76、 89、 91、 或第 94位， 和 /或 β 链 J基因短肽氨基酸倒数第 2位、 倒数第 4位或倒数第 6位， 其中氨基酸位置 编号按 IMGT中列出的位置编号。
在另一优选例中，所述 TCR的 a链可变域包含选自下组的一个或多个氨基 酸残基： 11L、 11M或 11E; 13V、 13R或 13K; 19V; 211； 91L或 911; 94V 或 941; 和 /或所述 TCR的 β链可变域包含选自下组的一个或多个氨基酸残基： 11L或 11V; 13V; 19V; 89L; 91F或 911; 94V或 94L; β链 J基因倒数第 6 位为 T和 β链 J基因倒数第 4位为 M; 其中氨基酸位置编号按 IMGT中列出的 位置编号。
在另一优选例中， 所述 TCR的 a链和 /或 β链可变域中暴露于表面的氨基 酸残基发生突变。
在另一优选例中，所述 TCR包含选自下组的一个或多个 a链可变域氨基酸 残基 4L; 12N; 16S; 93N或 93R; 97N; 100G; 105S; 和 a链 J基因倒数第 1 位为 D，和 /或所述 TCR包含选自下组的一个或多个 β链可变域氨基酸残基 41; 101L; β链 J基因倒数第 1位为 D和 β链 J基因倒数第 3位为 E。 在另一优选例中， 所述 TCR包含下列 α链可变域氨基酸序列之一 SEQ ID NO: 15、 17、 35、 37、 39、 75、 76、 77、 78、 79、 80、 81、 82、 83、 84、 85、
97、 99、 101、 103、 105和 107。
在另一优选例中， 所述 TCR包含下列 β链可变域氨基酸序列之一 SEQ ID NO: 16、 18、 36、 38、 40、 86、 87、 88、 89、 90、 91、 92、 93、 94、 95、 96、
98、 100、 102、 104、 106和 108。
在另一优选例中，所述 TCR的 a链可变域和 β链可变域组合选自下列组合 之一：
( a) 链可变域氨基酸序列 SEQ ID NO: 15和 β链可变域氨基酸序列 SEQ ID NO: 16
(b ) 链可变域氨基酸序列 SEQ ID NO: 17和 β链可变域氨基酸序列 SEQ ID NO: 18
( c) 链可变域氨基酸序列 SEQ ID NO: 15和 β链可变域氨基酸序列 SEQ
ID NO: 18
( d) 链可变域氨基酸序列 SEQ ID NO:35和 β链可变域氨基酸序列 SEQ ID NO:36
( e ) 链可变域氨基酸序列 SEQ ID NO:37和 β链可变域氨基酸序列 SEQ ID NO:38
(f) 链可变域氨基酸序列 SEQ ID NO:39和 β链可变域氨基酸序列 SEQ
ID NO:40
(g ) a链可变域氨基酸序列 SEQ ID NO:75和 β链可变域氨基酸序列 SEQ ID NO:86
(h) a链可变域氨基酸序列 SEQ ID NO:76和 β链可变域氨基酸序列 SEQ
ID NO:87
(i) 链可变域氨基酸序列 SEQ ID NO:77和 β链可变域氨基酸序列 SEQ
ID NO:88
链可变域氨基酸序列 SEQ ID NO:78和 β链可变域氨基酸序列 SEQ
ID NO:89;
(k) 链可变域氨基酸序列 SEQ ID NO:79和 β链可变域氨基酸序列 SEQ
ID NO:90;
(1) 链可变域氨基酸序列 SEQ ID NO:80和 β链可变域氨基酸序列 SEQ ID NO:91;
(m) α链可变域氨基酸序列 SEQ ID NO:81和 β链可变域氨基酸序列 SEQ ID NO:92;
(n) a链可变域氨基酸序列 SEQ ID NO:82和 β链可变域氨基酸序列 SEQ ID NO:93;
(o) a链可变域氨基酸序列 SEQIDNO:83和 β链可变域氨基酸序列 SEQ ID NO:94;
(p) a链可变域氨基酸序列 SEQ ID NO:84和 β链可变域氨基酸序列 SEQ ID NO:95;
(q) a链可变域氨基酸序列 SEQ ID NO:85和 β链可变域氨基酸序列 SEQ
ID NO:96;
(r) a链可变域氨基酸序列 SEQ ID NO:97和 β链可变域氨基酸序列 SEQ ID NO:98;
(s) 链可变域氨基酸序列 SEQ ID NO:99和 β链可变域氨基酸序列 SEQ ID NO:100;
(t) 链可变域氨基酸序列 SEQ ID NO: 101和 β链可变域氨基酸序列 SEQ ID NO:102;
(u) 链可变域氨基酸序列 SEQ ID NO: 103和 β链可变域氨基酸序列 SEQ ID NO:104;
(v) 链可变域氨基酸序列 SEQ ID NO: 105和 β链可变域氨基酸序列 SEQ
ID NO: 106; 和
(w) a链可变域氨基酸序列 SEQ ID NO: 107禾卩 β链可变域氨基酸序列 SEQ
Figure imgf000008_0001
在另一优选例中， 所述 T细胞受体 a链可变区疏水芯具有至少一个下列突 变： 第 19位氨基酸突变为 V、 第 21位氨基酸突变为 I、 第 91位氨基酸突变为 L; 和 /或 β链可变区疏水芯具有至少一个下列突变： 第 91 位氨基酸突变为 F 或突变为 I； 和 /或 β链 J基因短肽氨基酸序列倒数第 4位氨基酸突变为 M。
在另一优选例中， 所述的突变选自下组：
( i ) a链可变区第 19位氨基酸突变为 V、 第 21位氨基酸突变为 I、 第 91 位氨基酸突变为 ^ β链可变区第 91位氨基酸突变为 F， β链 J基因短肽氨基 酸序列倒数第 4位突变为 M; 或 (ii) α链可变区第 19位氨基酸突变为 V、 第 21位氨基酸突变为 I， β链 可变区第 91位氨基酸突变为 I； 或
(iii) α链可变区第 19位氨基酸突变为 V、 第 21位氨基酸突变为 I、 第 91 位氨基酸突变为 ^ β链可变区第 91位氨基酸突变为 I。
在另一优选例中， 所述 Τ细胞受体 a链可变区疏水芯具有至少一个下列突 变： L19V、 L21I、 I91L; 和 /或 β链可变区疏水芯具有至少一个下列突变： V91F 或 V91I; 和 /或 β链 J基因短肽氨基酸序列倒数第 4位由 L突变为 M。
在另一优选例中， 所述的突变选自下组：
( i ) 链可变区的 L19V、 L21I、 I91L, β链可变区的 V91F， β链 J基因短 肽氨基酸序列倒数第 4位由 L变为 M; 或
(ii) α链可变区的 L19V、 L21I， β链可变区的 V91I; 或
(iii) α链可变区的 L19V、 L21I、 I91L， β链可变区的 V91I;
其中氨基酸位置编号按頂 GT中列出的位置编号。
在另一优选例中， 所述的 Τ细胞受体还具有连接 TCR α链恒定区和 β链恒 定区的二硫键。
在另一优选例中， 所述的二硫键是天然 TCR中存在的或人工引入的。
在另一优选例中， 所述的人工引入的二硫键位于 TCRcc与 β链的恒定结构 域之间。
在另一优选例中,所述的人工引入的形成链间二硫键的半胱氨酸残基替换 了包括但不限于下列位置中至少一组 a和 β链的氨基酸残基：
(a) TCR α链恒定区的第 48位 Τ和 TCR β链恒定区的第 57位 S；或
(b) TCR α链恒定区的第 45位 Τ和 TCR β链恒定区的第 77位 S；或
(c) TCRa链恒定区的第 10位 T和 TCRP链恒定区的第 17位 S;或
(d) TCR α链恒定区的第 45位 Τ和 TCRP链恒定区的第 59位 D;或
(e) TCR α链恒定区的第 15位 S和 TCR β链恒定区的第 15位 E;或
(f) TCRa链恒定区的第 61位 S和 TCRP链恒定区的第 57位 S;或
(g) TCR α链恒定区的第 50位 L和 TCR β链恒定区的第 57位 S；或
(h) TCRa链恒定区的第 15位 S和 TCRP链恒定区的第 13位 V；或
(i) TCR α链恒定区的第 12位 L和 TCR β链恒定区的第 17位 S;或
(j)TCRa链恒定区的第 61位 S和 TCRP链恒定区的第 79位 R;或
(k) TCR α链恒定区的第 12位 L和 TCRP链恒定区的第 14位 F;或 (1) TCR α链恒定区的第 22位 V和 TCR β链恒定区的第 14位 F；或
(m) TCR a链恒定区的第 43位 Y和 TCRP链恒定区的第 63位 L;或
(n)TCRa链恒定区的第 10位 Y和 TCRi 链恒定区的第 17位 S。
该处 TCRci链与 β链恒定区被取代的氨基酸的位置编号按文献 "在结晶化 及治疗应用中稳定的可溶 Τ 细胞受体" （Stable, souble T-cell receptor molecules for crystallization and therapeutics) (Jonathan M. Boulter等， 2003， 蛋白质工程（Protein Engineering) 16 (9)： 707-711)里面给出的位置编 号。
在另一优选例中， 所述的 T细胞受体是通过噬菌体展示技术筛选出的。 在另一优选例中， 所述的 τ细胞受体结合（以共价或其他方式）有偶联物。 在另一优选例中， 所述偶联物选自下组的一个或多个：
(1) 可检测标记物；
(2) 治疗剂； 和 /或
(3) PK修饰部分。
优选地， 所述可检测标记物包括： 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。
优选地， 所述治疗剂包括： 放射性核素、 生物毒素、 细胞因子 (如 IL-2等；)、 抗 体、 抗体 Fc片段、 抗体 scFv片段、 金纳米颗粒 /纳米棒、 病毒颗粒、 脂质体、 纳 米磁粒、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》、 化疗剂 (例如， 顺铂;)或任何形式的纳米颗粒等。
在另一优选例中， 所述偶联物为连接于所述 TCR的 a和 /或 β链的 C-或 Ν-末端 的抗 -CD3的抗体。
在本发明的第二方面， 提供了一种核酸分子， 所述核酸分子包含编码本发 明第一方面中任一所述的 Τ细胞受体或其互补序列。
在本发明的第三方面， 提供了一种载体， 所述的载体含有本发明第二方面 所述的核酸分子。
在本发明的第四方面， 提供了一种宿主细胞或遗传改造的工程细胞， 所述 的细胞含有本发明第三方面所述的载体或染色体中整合有外源的本发明第二 方面所述的核酸分子。 在另一优选例中， 所述的宿主细胞选自： 原核细胞和真核细胞， 例如大肠 杆菌、 酵母细胞、 CH0细胞等。
在本发明的第五方面， 提供了一种制备本发明第一方面所述的 T细胞受体 的方法， 包括步骤：
(i) 培养本发明第四方面所述的宿主细胞， 从而表达本发明所述的 T细胞 受体；
(ϋ) 分离或纯化出所述的 T细胞受体。
在本发明的第六方面， 提供了一种 T细胞受体复合物， 其特征在于， 所述 的复合物含有本发明第一方面中任一所述的 T细胞受体。
在另一优选例中， 所述的复合物包括本发明的 τ细胞受体与治疗剂的结合 所形成的复合物、 或与可检测标记物的结合所形成的复合物。
在另一优选例中， 所述的复合物包含 2个或多个 T细胞受体分子。
在本发明的第七方面， 提供了本发明上述所述的 T细胞受体的用途， 它被 用于制备治疗肿瘤、 病毒感染或自身免疫疾病的药物。
在本发明的第八方面， 提供了一种药物组合物， 它含有药学上可接受的载 体以及安全有效量的本发明第一方面中任一所述的 T细胞受体。
在本发明的第九方面， 提供了一种治疗疾病的方法， 包括给需要治疗的对 象施用本发明第一方面中任一所述的 T细胞受体、 或第六方面中所述的 T细胞 受体复合物、 或第八方面中所述的药物组合物。
在另一优选例中， 所述的疾病包括： 肿瘤、 自身免疫疾病和病毒感染性疾 病。
在本发明的第十方面， 提供了一种制备本发明第一方面所述的 T细胞受体 的方法， 包括步骤：
(i)对 T细胞受体的疏水芯区域引入氨基酸残基突变； 和
(i i)筛选稳定性显著提高的 T细胞受体， 从而获得本发明第一方面中所述 的 T细胞受体。
在另一优选例中， 所述筛选方法包括但不限于噬菌体展示技术。
在另一优选例中， 在步骤（i i)中， 利用噬菌体展示技术展示疏水芯区域发 生突变的 T细胞受体， 并进行筛选。
在另一优选例中，所述方法还包括步骤：测定所筛选出 T细胞受体的序列、 活性和 /或其他特性。 应理解， 在本发明范围内中， 本发明的上述各技术特征和在下文（如实施 例）中具体描述的各技术特征之间都可以互相组合， 从而构成新的或优选的技 术方案。 限于篇幅， 在此不再一一累述。
附图说明
图 1显示了一种典型 TCR的可变域结构示意图，该 TCR为具有癌症抗原 MAGE
A3 HLA A1特异性的野生型 TCR。
图 2a和 2b分别显示定点突变后的 TCR a链可变域氨基酸序列和核苷酸序 列（SEQ ID NO : 9和 10)。 所述氨基酸序列为对专利文献（W02012/013913)中公 开的 TCR的 a链可变域氨基酸序列进一步优化， 更具体地， 即将可变域中暴露 于表面的疏水性残基进一步突变为亲水或极性残基， 其中加粗的并带下划线的 字母为突变后的氨基酸残基。
图 3a和 3b分别显示定点突变后的 TCR i 链可变域氨基酸序列和核苷酸序 列（SEQ ID NO : 11 和 12)。 所述氨基酸序列为对专利文献（W02012/013913)中 公开的 TCR i 链可变域氨基酸序列进一步优化， 更具体地， 即将可变域中暴露 于表面的疏水性残基进一步突变为亲水或极性残基， 其中加粗的并带下划线的 字母为突变后的氨基酸残基。
图 4为构建 MAGE-sTv-WT时各引物的连接方式。
图 5a和 5b分别为构建 sTv突变株文库时 a与 β链连接物的氨基酸序列和 核苷酸序列（SEQ ID NO : 13和 14)。
图 6a和 6b分别为 sTv突变株 MG29的 a链可变域氨基酸序列与 β链可变 域氨基酸序列（SEQ ID NO : 15和 16)， 相对于 MAGE-sTv-WT , 突变的残基以黑体 字和下划线显示。
图 7a和 7b分别为 sTv突变株 P8F1 的 a链可变域氨基酸序列与 β链可变 域氨基酸序列（SEQ ID Ν0 : 17和 18)， 相对于 MAGE-sTv-WT , 突变的残基以黑体 字和下划线显示。
图 8a和 8b分别为 sTv突变株 P8F2的 a链可变域氨基酸序列与 β链可变 域氨基酸序列（SEQ ID Ν0 : 15和 18)， 相对于 MAGE-sTv-WT , 突变的残基以黑体 字和下划线显示。
图 9为筛选出的不同突变株及 MAGE-sTv-WT对抗原 MAGEA3、 EBV、 Flu, NY - ES0的 ELISA实验 0D值。 图 10a与图 10b分别为 LC13-WT的 a链可变域（SEQ ID NO : 29)与 β链可变域 (SEQ ID NO: 30)的氨基酸序列。
图 11a与图 lib分别为 JM22-WT的 α链可变域（SEQ ID NO: 31)与 β链可 变域（SEQ ID NO: 32)的氨基酸序列。
图 12a与图 12b分别为 1G4-WT的 α链可变域（SEQ ID NO: 33)与 β链可变 域（SEQ ID NO: 34)氨基酸序列。 图 13a与图 13b分别为 LC13- sTva链可变域（SEQ ID NO: 35)与 LC13- sTv β链可变域（SEQ ID NO: 36)氨基酸序列。 图 14a与图 14b分别为 JM22- sTva链可变域（SEQ ID NO: 37)与 JM22- sTv β链可变域（SEQ ID NO: 38)氨基酸序列。
图 15a与图 15b分别为 1G4- sTv α链可变域（SEQ ID NO: 39)与 1G4- sTvP 链可变域（SEQ ID NO: 40)氨基酸序列。 图 16为构建 sTv单链分子所用的连接短肽（ linker)的氨基酸序列（SEQ ID NO: 41)。 图 17为纯化后的蛋白 LC13-WT与 LC13-sTv的 SDS-PAGE胶图。 泳道 1: 分子 量标记， 泳道 2: LC13-WT, 泳道 3: LC13-sTv。
图 18a与图 18b分别为纯化后的蛋白 LC13-WT与 LC13-sTv的 SEC图谱。
图 19为纯化后的蛋白 JM22-WT与 JM22-sTv的 SDS-PAGE胶图。 泳道 1: 分子量标记， 泳道 2: JM22-WT, 泳道 3: JM22-sTv。
图 20a与图 20b分别为纯化后的蛋白 JM22-WT与 JM22-sTv的 SEC图谱。
图 21为纯化后的蛋白 1G4-WT与 1G4-STV的 SDS-PAGE胶图。 泳道 1: 分子量标 记， 泳道 2: 1G4-WT, 泳道 3.1G4-sTv。
图 22a与图 22b分别为纯化后的蛋白 1G4-WT与 1G4-STV的 SEC图谱。
图 23为 1G4-STV的氨基酸序列（SEQ ID NO: 42)。
图 24 为 1G4-STV突变株针对不同抗原的 0D值。
图 25 为筛选到的 1G4-STV高稳定性突变株的 α链可变域氨基酸序列（SEQ
ID NO: 75-85)。
图 26为筛选到的 1G4-STV高稳定性突变株的 β链可变域氨基酸序列（SEQ ID NO: 86-96)。
图 27为 1G4-STV 高稳定性突变株的 DSC曲线图。 图 28为 1G4-WT的 DSC曲线图。
图 29a与图 29b分别为高稳定性 G15a链可变域 （SEQ ID NO: 97) 与 β链可变域 （SEQ ID NO: 98) 氨基酸序列。
图 30为纯化后蛋白 1G4- WT、 1G4- sTv、 G13、 G15、 G9的 SDS- PAGE胶图。 泳 道 1: 分子量标记， 泳道 2: 1G4-WT, 泳道 31G4-sTv， 泳道 4: G13 泳道 5: G15 ， 泳道 6: 分子量标记， 泳道 7: G9。
图 31a、 31b和 31c分别为纯化后的蛋白 G9、 G13和 G15的 SEC图谱。 图 32a与图 32b分别为 LC13-G9 链可变域 （SEQ ID NO: 99) 与 β链可变 域 （SEQ ID NO: 100) 氨基酸序列。
图 33a与图 33b分别为 LC13- G15 α链可变域 （SEQ ID N0: 101) 与 β链可 变域 （SEQ ID NO: 102) 氨基酸序列。
图 34a与图 34b分别为 JM22-G9a链可变域 （SEQ ID NO: 103) 与 β链可 变域 （SEQ ID NO: 104) 氨基酸序列。
图 35a与图 35b分别为 JM22- G15 链可变域 （SEQ ID NO: 105) 与 β链可 变域 （SEQ ID NO: 106) 氨基酸序列。
图 36为纯化后蛋白 LC13- WT、 LC13- sTv、 LC13- G15、 LC13- G9的 SDS- PAGE胶 图。 泳道 1:分子量标记，泳道 2:LC13-WT，泳道 3:LC13-sTv 泳道 4: LC13-G15， 泳道 5: 分子量标记， 泳道 6: LC13-G9o
图 37为纯化后蛋白 LC13-G9的 SEC图谱。
图 38为纯化后蛋白 LC13-G15的 SEC图谱。
图 39为纯化后蛋白 JM22- WT、 JM22- sTv、 JM22- G15、 JM22- G9的 SDS- PAGE胶 图。 泳道 1: 分子量标记， 泳道 2: JM22-WT , 泳道 3: JM22-sTv 泳道 4: JM22-G15, 泳道 5: JM22-G9o
图 40为纯化后蛋白 JM22-G9的 SEC图谱。
图 41为纯化后蛋白 JM22-G15的 SEC图谱。
图 42a与图 42b分别为 MAGE-G15 链可变域 （SEQ ID NO: 107) 与 β链可 变域 （SEQ ID NO: 108) 氨基酸序列。
图 43为纯化后蛋白 MAGE-G15的 SDS-PAGE胶图。 泳道 1: 分子量标记， 泳 道 2： MAGE - G15。
图 44为纯化后蛋白 MAGE-G15的 SEC图谱。
图 45为纯化后蛋白 MAGE-G15的 DSC曲线图。
图 46为纯化后蛋白 G15的 DSC曲线图。
图 47为纯化后蛋白 LC13-sTv的 DSC曲线图。 图 48a和 48b分别为纯化后蛋白 JM22-WT和 JM22-sTv的 DSC曲线图。 图 49a和 49b分别为纯化后蛋白 LC13-G9与 LC13-G15的 DSC曲线图。 具体实施方式
本发明人经过广泛而深入的研究， 首次发现， 通过有针对性地对 T细胞受 体的疏水芯区域进行突变， 可意外地获得高稳定性的突变型 TCR， 尤其是可溶 性 TCR。 在此基础上完成了本发明。
本发明人使用了优化的 TCR蛋白结构， 通过改变 TCR的疏水芯， 构建出一 种高稳定性的 TCR分子。 本发明构建了新型的单链 TCR可变域， 通过定向的分 子进化法， 分离出了最佳的疏水芯。 具有新型疏水芯的 TCR片段， 可通过用亲 水或极性残基取代 TCR可变域中暴露于表面的疏水残基， 来进一步改进。 术语
TCR
天然 α - β异源二聚 TCR 具有 a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。 各可变区的 3个 CDR (互补决定区） 嵌合在可变 区的框架 （framework ) 中， 疏水芯也位于可变区的框架中。 α链可变区 （V a ) 可分成几类， β链可变区 （ν β ) 也可分成几类。 在国际免疫遗传学信息系统 ( IMGT ) 中， 以唯一的 TRAV编号和 TRBV编号分别指代 V a类型和 ν β类型， TRAJ和 TRBJ指代 TCR的连接区。 本发明中所用的 α链 J基因即指 TRAJ， β链 J基因指 TRBJ。 TCR的 a和 β链常视作各自具有两个 "结构域" ， 即 "可变域" 和 "恒定域" 。 可变域由连接在一起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，术语" TCR a可变域 "指连接在一起的 TRAV和 TRAJ， 术语 " TCR i 可变域" 指连接在一起的 TRBV和 TRBJ。
TCR领域的工作人员广为知晓并可得到頂 GT中给出的 TCR的氨基酸序列及 其可变域框架（framework )包括疏水芯位置在 IMGT中的具体位置编号。例如， 可以在 IMGT公开数据库中找到。 本发明中， 所述 TCR氨基酸位置编号均按照 IMGT中列出的位置编号， 除非另有说明。 如果今后 IMGT中列出的位置编号有 变动， 则以 2013年 1月 1 日版本的 IMGT中列出的 TCR的氨基酸序列位置号为 准。 如本文所用， 术语 "疏水芯" 又可称为 "疏水核心" 指在任何蛋白质溶于 水时， 蛋白结构域中一般包含在其分子结构内部， 大部分由疏水性氨基酸构成 的核心区域。 在 TCR中， TCR ci链可变域的疏水芯为可变区氨基酸第 1 1， 13， 19， 21， 53， 76， 89， 91， 94位和 α链 J基因 （TRAJ ) 短肽氨基酸位置倒数第 3 , 5, 7 位； TCR i 链可变域的疏水芯为可变区氨基酸第 1 1， 13， 19， 21， 53， 76， 89， 91， 94位和 β链 J基因 （TRAJ ) 短肽氨基酸位置倒数第 2， 4， 6位。 上 述位置编号采用頂 GT中列出的位置编号。
如附图 1所示为具有癌症抗原 MAGE A3 HLA A1特异性的野生型 TCR的可 变区结构示意图， 示意图的左下方与右下方的两个椭圆中加粗的氨基酸残基即 分别为 a与 β链可变区骨架（framework)位置的疏水芯。 TCR的抗原结合位点在 CDR区， CDR区决定了 TCR与其相应抗原间的结合亲和力。 从图中可以看出， 疏水芯并不在 CDR区， 它的突变并不应该影响 TCR与其相应抗原的结合以及结 合亲和力， 但根据本发明人的研究疏水芯的改变会对 TCR分子的稳定性产生影 响。
应理解， 该 TCR的结构示意图仅用于说明本发明， 而不能以任何方式限制 本发明的范围。
术语 " sTv " 指功能结构域由一柔性肽链连接的 TCR 的 a与 β链的可变域 而构成的单链 TCR， 其柔性肽链可以是任何适合连接 TCR a与 β可变域的肽链， 肽链的氨基酸残基数量可以是 1到 50个， 但不限于 1到 50个。
术语"稳定性 "指蛋白质稳定性的任何方面。 与初始野生型的蛋白质相比， 经过筛选得到的高稳定性蛋白质具有一个或一个以上的下列特征： 更抗解折 叠、 更抗不适当或不希望的折叠、 复性能力更强、 表达能力更强、 蛋白复性收 率更高、 热稳定性增加、 在多种环境 （例如， ra值、 盐浓度、 存在洗涤剂、 存 在变性剂等） 下稳定性增加。
噬菌体展示系统和筛选髙稳定性的 TCR
当使用噬菌体展示系统来分离受体的时候， 通常是通过两种重要的性质筛 选得到最终的受体， 第一是受体与配体的结合强度或亲和力， 第二是受体在噬 菌体表面的展示密度。 第一个性质是蛋白质亲和力进化的基础， 它指导产生高 亲和性受体的所有方法论的发展。 一个简单的描述如下： 当受体展示文库上样 于配体时， 具有更高结合强度的受体会以更快的速度和 /或更长的保留时间结 合于配体， 并能够抵抗更高强度的洗涤。 因此， 这些受体及其编码基因就会被 捕获并在后续过程中放大。 另一方面， 当受体-配体相互作用的亲和力没被改 变或改变程度不大， 甚至变低， 亲和力因素就对于筛选不起作用， 展示密度就 会主导进化结果。 这就意味着当更多正确折叠的受体分子展示在一个噬菌体粒 子上面或者更多的噬菌体粒子展示一个或多个这样的受体时， 该受体和编码基 因就有更多的机会与配体结合， 在规定洗涤条件下应滞留更多的该受体， 从而 被捕获并在后续的筛选过程中扩大。 基于第二个性质， 通过使用噬菌体展示技 术或其他定向的分子展示技术可以分离出更稳定的蛋白质。 本发明人已经设计 出了 TCR蛋白质疏水芯的定向进化文库， 来分离更稳定的蛋白质或 TCR。 已证 实， 这样的疏水芯对 TCR与其配体 pMHC或 pHLA的结合强度基本没有影响， 因 为 TCR是通过它的互补决定区（CDR)与 pMHC结合。
在本发明中， 可使用噬菌体展示技术来分离更稳定的蛋白构建物。 在一个 优选例中，用具有癌症抗原 MAGE A3 HLA A1特异性的 TCR (以下简称 MAGE A3 TCR) 胞外结构域来测试这个设想。 根据专利文献里面的序列来合成胞外结构域， 当 它表达在丝状噬菌体的表面时， TCR与 pMHC 的结合就可以通过 ELI SA (酶联免 疫吸附测定法）进行检测并得到其相互作用强度。 然而， 按已发表的改进方法 如将可变域中暴露于表面的疏水残基突变为亲水或极性的残基后， 用 ELI SA检 测通过噬菌体展示的该 TCR的 a与 β链的可变域组成的单链 TCR 形式（sTv)时 没有发现任何结合功能。可是将该单链 TCR (sTv)可变域疏水芯的限制性随机突 变的文库克隆入噬菌体展示载体并通过几轮的筛选后， 却意外地得到了一些高 稳定性的克隆， 再使用 ELISA检测这些克隆就可以査到与相应 pMHC的结合。
活性多肽
在本发明中， 术语 "本发明多肽" 、 "本发明的 TCR " 、 "本发明的 T细 胞受体" 可互换使用， 都指这样的 T细胞受体（TCR)， 所述 TCR的疏水芯区域发 生突变， 并且该 TCR的稳定性显著高于其对应的疏水芯为野生型的 TCR。
此外， 本发明的多肽还可包括在疏水芯区域之外的其他突变， 尤其是那些 可以提高亲和力的突变以及 TCR可变域中暴露于表面的氨基酸残基的突变。
这些疏水芯区域之外的其他变异形式包括（但不限于）： 1-6个（通常为 1-5 个， 较佳地 1-3个， 更佳地 1-2个， 最佳地 1个）氨基酸的缺失、 插入和 /或取 代， 在 C末端和 /或 N末端添加一个或数个（通常为 5个以内， 较佳地为 3个以 内， 更佳地为 2个以内）氨基酸。 例如， 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。 此外， 所述术语还包括单体 和多聚体形式的本发明多肽。
应理解， 本文中氨基酸名称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)、 Gin (Q)、 Glu (E)、 Gly (G)、 Hi s (H)、 l ie (1)、 Leu (L)、 Lys (K)、 Met (M)、 Phe (F)、 Pro (P)、 Ser (S)、 Thr (T)、 Trp (W)、 Tyr (Y)、 Val (V)。 本文中氨基酸取代的书写方式如 L19V代表按照頂 GT中给出的位置编号第 19位 的 L (亮氨酸）被 V (缬氨酸)取代，其他相同的氨基酸取代书写方式的含义参照本 例。
本发明还包括本发明多肽的活性片段、 衍生物和类似物。 如本文所用， 术 语 "片段" 、 "衍生物" 和 "类似物" 是指能与配体分子结合的多肽。 本发明 的多肽片段、衍生物或类似物可以是（i)有一个或多个保守或非保守性氨基酸残 基 (优选保守性氨基酸残基)被取代的多肽， 或（i i)在一个或多个氨基酸残基中 具有取代基团的多肽， 或（i i i)本发明 TCR与另一个化合物（比如延长多肽半衰 期的化合物， 例如聚乙二醇）融合所形成的多肽， 或（iv)附加的氨基酸序列融合 于此多肽序列而形成的多肽（与前导序列、 分泌序列或 6Hi s等标签序列融合而 形成的融合蛋白）。 根据本文的教导， 这些片段、 衍生物和类似物属于本领域熟 练技术人员公知的范围。
一类优选的活性衍生物指具有至多 5个， 较佳地至多 3个， 更佳地至多 2 个， 最佳地 1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。 这些保 守性变异多肽最好根据表 A进行氨基酸替换而产生。
表 A
Figure imgf000018_0001
Gly (G) Pro; Ala Ala
His (H) Asn; Gin; Lys ; Arg Arg
He (I) Leu; Val ; Met; Ala; Phe Leu
Leu (L) He ; Val ; Met; Ala; Phe lie
Lys (K) Arg; Gin; Asn Arg
Met (M) Leu; Phe ; lie Leu
Phe (F) Leu; Val； lie ; Ala; Tyr Leu
Pro (P) Ala Ala
Ser (S) Thr Thr
Thr (T) Ser Ser
Trp (W) Tyr; Phe Tyr
Tyr (Y) Trp; Phe ; Thr; Ser Phe
Val (V) l ie ; Leu; Met; Phe ; Ala Leu 发明还提供本发明 TCR的类似物。 这些类似物与本发明原 TCR多肽的差别 可以是氨基酸序列上的差异， 也可以是不影响序列的修饰形式上的差异， 或者 兼而有之。类似物还包括具有不同于天然 L-氨基酸的残基（如 D-氨基酸）的类似 物， 以及具有非天然存在的或合成的氨基酸（如 β、 Υ -氨基酸）的类似物。 应 理解， 本发明的多肽并不限于上述例举的代表性的多肽。
修饰（通常不改变一级结构）形式包括： 体内或体外的多肽的化学衍生形式 如乙酰化或羧基化。 修饰还包括糖基化， 如那些在多肽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多肽。 这种修饰可以通过将多肽暴露于 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多肽。
本发明多肽还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使 用。 这些盐包括（但不限于）与如下酸形成的盐： 氢氯酸、 氢溴酸、 硫酸、 柠檬 酸、 酒石酸、 磷酸、 乳酸、 丙酮酸、 乙酸、 琥珀酸、 草酸、 富马酸、 马来酸、 草酰乙酸、 甲磺酸、 乙磺酸、 苯磺酸、 或羟乙磺酸。 其他盐包括： 与碱金属或 碱土金属（如钠、 钾、 钙或镁）形成的盐， 以及以酯、 氨基甲酸酯或其他常规的 "前体药物" 的形式。
本发明的多肽可以多价复合体的形式提供。 本发明的多价 TCR复合体包含 两个、 三个、 四个或更多个与另一分子相连的 T细胞受体分子。 编码序列
本发明还涉及编码本发明 TCR的多核苷酸。
本发明的多核苷酸可以是 DNA形式或 RNA形式。 匪可以是编码链或非编 码链。 例如， 编码成熟多肽的编码区序列可以与 SEQ ID NO : 10所示的编码区序 列相同或者是简并的变异体。 如本文所用， "简并的变异体" 在本发明中是指 编码具有 SEQ ID NO : 9蛋白质， 但与 SEQ ID NO : 10中相应编码区序列有差别 的核酸序列。
本发明的核苷酸全长序列或其片段通常可以用但不限于 PCR扩增法、 重组 法或人工合成的方法获得。 目前， 已经可以完全通过化学合成来得到编码本发 明多肽（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薩分子（如载体）和细胞中。
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或编码 序列经基因工程产生的宿主细胞。
另一方面， 本发明还包括对本发明 TCR多肽具有特异性的多克隆抗体和单 克隆抗体， 尤其是单克隆抗体。 制备方法
产生本发明的 TCR的一种方法是从展示此类 TCR的噬菌体颗粒的多样性文 库中选择出高稳定性的 TCR。
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 Russel l, (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 Bi otechnol 6 (1) : 30-6)。
本发明多肽可以是重组多肽或合成多肽。本发明的多肽可以是化学合成的， 或重组的。 相应地， 本发明多肽可用常规方法人工合成， 也可用重组方法生产。 通过常规的重组 DNA技术， 可利用本发明的多核苷酸来表达或生产重组的 本发明多肽。 一般来说有以下步骤：
(1) 用编码本发明 TCR多肽的多核苷酸 (或变异体）， 或用含有该多核苷酸 的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、 纯化出本发明 TCR多肽。
重组多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如果需要， 可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规 的复性处理、 用蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超处理、 超离 心、 分子筛层析（凝胶过滤）、 吸附层析、 离子交换层析、 高效液相层析 (HPLC) 和其它各种液相层析技术及这些方法的结合。 药物组合物和施用方法
本发明的 TCR和本发明 TCR转染的 T细胞可与药学上可接受的载体一起在 药物组合物中提供。 本发明的 TCR、 多价 TCR复合物和细胞通常作为无菌药物 组合物的一部分提供， 所述组合物通常包括药学上可接受的载体。 该药物组合 物可以是任何合适的形式（取决于给予患者的所需方法）。 其可采用单位剂型提 供， 通常在密封的容器中提供， 可作为试剂盒的一部分提供。 此类试剂盒 (但非 必需）包括使用说明书。 其可包括多个所述单位剂型。
此外， 本发明的多肽可以单用， 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）。
可与本发明 TCR 结合或偶联的治疗剂包括但不限于： 1. 放射性核素 (Koppe等， 2005， 癌转移评论（Cancer metastas i s reviews) 24， 539)； 2. 生 物毒素（Chaudhary等， 1989， 自然（Nature) 339， 394； Epel等， 2002 , 癌症免 疫学禾口免疫治疗 (Cancer Immunology and Immunotherapy) 51 , 565)； 3. 细胞 因子（Gi l l ies等， 1992， 美国国家科学院院刊（PNAS) 89， 1428； Card等， 2004， 癌症免疫学禾口免疫治疗 (Cancer Immunology and Immunotherapy) 53 , 345； Hal in 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 Soc iety) 128 , 2115)； 7. 病毒颗粒（Peng等， 2004， 基因治疗（Gene therapy) 1 1， 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 (BraD ) ; 1 1.化疗剂（例如， 顺铂）等。
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 Sc i ences (Mack Pub. Co.， N. J. 1991) )中可找到关于药学上可接受的赋形剂的 充分讨论。 这类载体包括但并不限于： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙 醇、 佐剂、 及其组合。
治疗性组合物中药学上可接受的载体可含有液体， 如水、 盐水、 甘油和乙 醇。 另外， 这些载体中还可能存在辅助性的物质， 如润湿剂或乳化剂、 pH缓冲 物质等。
通常， 可将治疗性组合物制成可注射剂， 例如液体溶液或悬液； 还可制成 在注射前适合配入溶液或悬液中、 液体载体的固体形式。
一旦配成本发明的组合物， 可将其通过常规途径进行给药， 其中包括（但并 不限于）： 眼内、 肌内、 静脉内、 皮下、 皮内、 或局部给药。 待预防或治疗的对 象可以是动物； 尤其是人。
当本发明的药物组合物被用于实际治疗时， 可根据使用情况而采用各种不 同剂型的药物组合物。 较佳地， 可以例举的有针剂、 口服剂等。
这些药物组合物可根据常规方法通过混合、 稀释或溶解而进行配制， 并且 偶尔添加合适的药物添加剂， 如赋形剂、 崩解剂、 粘合剂、 润滑剂、 稀释剂、 缓冲剂、 等渗剂（i sotonic i ties 防腐剂、 润湿剂、 乳化剂、 分散剂、 稳定剂 和助溶剂， 而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。 例如， 本发明多肽可被掺入 以缓释聚合物为载体的药丸或微囊中， 然后将该药丸或微囊通过手术植入待治 疗的组织。 作为缓释聚合物的例子， 可例举的有乙烯-乙烯基乙酸酯共聚物、 聚 羟基甲基丙烯酸酯（polyhydrometaacrylate 聚丙烯酰胺、 聚乙烯吡咯烷酮、 甲基纤维素、 乳酸聚合物、 乳酸 -乙醇酸共聚物等， 较佳地可例举的是可生物降 解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时， 作为活性成分的本发明多肽或 其药学上可接受的盐的剂量， 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。 本发明 TCR的用途
本发明的 TCR可用作药物或诊断试剂。 可通过修饰或其他改进以使其获得 更适于作为药物或诊断试剂使用的特征。 该药物或诊断试剂可用于治疗或诊断 多种不同的疾病， 所述疾病包括但不限于： 癌症（例如肾癌、 卵巢癌、头和颈癌、 睾丸癌、 肺癌、 胃癌、 子宫颈癌、 膀胱癌、 前列腺癌或黑素瘤等）、 自身免疫病、 病毒感染性疾病、 移植排斥和移植物抗宿主病。
通过本发明 TCR的特异性可实现药物定位或靶向给药， 从而提高多种疾病 的治疗或诊断效果。
对于癌症， 定位于肿瘤或转移癌的附近可提高毒素或免疫剌激物的效果。 在自身免疫病中，可特异性地抑制对正常细胞或组织的免疫反应，或缓慢释放免 疫抑制药，使其在更长的时间范围内产生更多的局部效果，从而对受试者的整体 免疫能力的影响减至最小。 在防止移植排斥中， 可以同样的方式优化免疫抑制 的作用。 对于已存在药物的病毒性疾病， 例如 HIV、 SIV、 EBV、 CMV、 HCV、 HBV， 药物在感染细胞区域附近释放或发挥激活功能也是有益的。
本发明的 TCR可用于调节 T细胞激活， 本发明的 TCR通过结合特异的 pMHC 并由此抑制 T细胞活化。涉及 T细胞介导的炎症和 /或组织损伤的自身免疫病可 适于此方法， 例如 I型糖尿病。
本发明的 TCR也可用于将细胞毒性剂递送至癌细胞的目的， 或可用于转染 T细胞， 从而使得它们能够破坏呈递 HLA复合物的肿瘤细胞， 以便在称为过继 免疫治疗的治疗过程中给予患者。
本发明的 TCR也可用作诊断试剂。 用可检测标记物对本发明的 TCR进行标 记， 如用适用于诊断目的的标记物标记， 来检测 MHC-肽与 MHC-肽特异性的本 发明 TCR之间的结合。 荧光标记的 TCR多聚体适用于 FACS分析， 可用来检测 携带 TCR特异性的肽的抗原呈递细胞。
与偶联物结合的本发明 TCR， 所述偶联物包括但不限于抗 -CD3抗体， 可以 重新定向 T细胞， 从而使其靶向呈递特定抗原的细胞， 如肿瘤细胞。
工业应用性
本发明的高稳定性 Τ细胞受体，可用于研究 TCR与 pMHC (肽-主组织相容性 复合体）之间的相互作用及疾病的诊断和治疗等目的。
本发明的主要优点包括：
(a) 本发明 TCR多肽的稳定性高；
(b) 可以高效、 简便地筛选获得高稳定性的 TCR多肽。
(c) 可进一步筛选获得高稳定性和高亲和性的 TCR多肽。
下面的具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方 法， 通常按照常规条件， 例如 Sambrook和 Russell等人， 分子克隆： 实验室 手册 (Molecular Cloning-A Laboratory Manual) (第三版）（2001)CSHL出版社 中 所述的条件， 或按照制造厂商所建议的条件。 除非另外说明， 否则百分比和份 数是重量百分比和重量份数。 实施例 1 初级单链 TCR可变域（sTv)的构建和序列的优化
设计如表 1所示引物分别将化学合成的 TCR ci链和 β链可变域氨基酸序列 (引用于专利文献 W02012/013913)进行定向点突变。 这些突变使该 TCR α链和 β链可变域中暴露于表面的疏水残基变为亲水或极性残基， 以用来制作疏水芯 突变文库的模板。 对于 a链第 20位的表面疏水残基 V突变为亲水残基 S的过 程是在构建疏水芯突变文库时， 利用定点突变技术完成的。
表 1 用来定点突变氨基酸序列的引物及连接物
Figure imgf000024_0001
YW806 gcgaaggaggctccggaggcaaggctggagtcactcaaac 7
YW807 ctagatgcggccgcctctgtgaccgtgagcctg 8
其中 YW800、 YW80U YW802、 YW803、 YW804为定点突变 α链可变域所设计 的引物， YW806、 YW807为定向突变 β链可变域所设计的引物， YW805为连接引 物用于引入柔性肽链。 按照附图 4所示引物连接方式进行 PCR构建 sTv， 将此 sTv命名为 MAGE- sTv- WT。
具体的 PCR诱变实验步骤如下：
第一步 PCR，分别用合成的 a或 β链为模板，使用下列引物对 YW801/YW803 (a链）， YW802/YW804 ( a链）， YW806/YW807 ( β链）进行 PCR。 反应程序是： 98 °C变性 30秒一次， 25次的重复循环 94°C5秒， 55°C10秒， 72°C20秒。
第二步 PCR， 以重叠 PCR (overlap PCR) 方法， 用第一步 PCR纯化后的产 物和化学合成的柔性肽单链 DNA为模板， YW800/YW807为引物进行第二步 PCR。 其反应程序是： 98°C变性 30秒一次， 30次的重复循环 94°C5秒， 55°C10秒， 72°C30秒， 1次 72°C5分钟。 纯化后的第二步 PCR产物经酶切后连接入噬菌体 展示载体。
实施例 2 将 MAGE-sTv-WT序列克隆入基于 pET_28a的表达质粒
通过《分子克隆实验室手册》(Molecular Cloning a Laboratory
Manual) (第三版， Sambrook禾卩 Russell)中描述的标准方法将 MAGE- sTv- WT克 隆入 pET-28a(Novagen)表达质粒。 利用 ABI公司的 3730 DNA分析仪测序质粒。
将 Ncol禾卩 Notll切割的编码 MAGE-sTv-WT的 DNA序列连接入用 Ncol和 Notll切割的 pET- 28a载体（Novagen)。
将连接的质粒转化入常规的感受态大肠杆菌（^cAer ^ ac^ )菌株 BL21
(DE3) (购自 merck公司） 细胞，接种在含有 50 g/mL卡那霉素的 LB/琼脂板 上。 37°C温育过夜后，挑取单个菌落， 37°C，在含有 50 g/mL卡那霉素的 5mlLB 中震荡生长过夜。 利用 Zymo公司的质粒提取试剂盒（Zyppy Plasmid Midiprep Kit, Zymo)纯化克隆的质粒， 由 ABI公司的 3730 DNA测序仪测序插入物。
图 2a和 3a分别显示 MAGE-sTv-WT的 α链可变域与 β链可变域氨基酸序列
(SEQ ID NO: 9和 11)， 优化后的氨基酸位点以加粗和下划线显示。 图 2b和 3b 分别显示 MAGE-sTv-WT的 α链可变域与 β链可变域核苷酸序列（SEQ ID NO: 10 和 12)。
实施例 3 MAGE-sTv-WT的表达、 复性和纯化 将实施例 2 中得到的含有 MAGE-sTv-WT 的表达质粒转化到大肠杆菌菌株 Rosetta (DE3) ( Merck)的培养基平板上， 在 37°C培养过夜， 挑单菌落在 37°C 含有卡那霉素培养基中培养至 0D6。。为 0.6-0.8， 然后用 0.5 mM IPTG诱导蛋白 质表达 4小时， 用 FisherThermo Sovall R6+离心机以 5, 000 rpm 离心 15分 钟收获细胞。 用 Bugbuster MasterMix (Merck)裂解细胞沉淀物。 用 FisherThermo Sovall X1R离心机以 6, 000 g 离心 15分钟以回收包涵体沉淀 物。 之后用 10倍稀释的 Bugbuster溶液三次洗涤包涵体以除去细胞碎片和膜 组分。 然后利用以下缓冲液溶解包涵体： 20 mM Tris, pH 9.0， 8 M 尿素。 用 BCA法定量后分装为 10 mg每管在 -80°C冷冻。
解冻 10 mg溶解的 MAGE-sTv-WT包涵体蛋白， 滴到以下的 200 ml 复性缓 冲液中： 100 mM Tris, pH 9.0,400 mM L-精氨酸， 2 mM EDTA。 加入氧化型和 还原型谷胱甘肽氧化还原对至终浓度分别为 1 mM和 10 mM， 溶液在 10 V搅 拌 10分钟后以 100 rpm摇 1-2天。 4-8°C下， 用分子截留量为 4 kD的纤维素 膜透析袋和 5 L 20 mM Tris pH 9.0透析复性 MAGE-sTv-WT 8小时， 再更换相 同的新鲜缓冲液透析两次。
将透析后的复性 MAGE-sTv-WT离心后上阴离子交换柱 Q HP 5ml(GE公司）， 利用 AKTA纯化仪（GE公司）， 用 20 mM Tris pH 9.0配制的 0-1 M NaCl线性 梯度洗脱结合的蛋白质 10 个柱体积， 收集洗脱峰（相对分子质量约为 28 kD) 并跑 SDS-PAGE (Bio-Rad)进行分析。 包含 MAGE-sTv-WT的组分浓缩后进一步用 凝胶过滤柱（Superdex 75 10/300, GE Healthcare)进行纯化。 纯化后如果能 够得到目标组分则将目标组分跑 SDS-PAGE 胶并分析并， 之后将目标组分保存 于 4°C。 合并目标峰组分并浓缩， 置换到 10mM pH7.4的 HEPES缓冲液。
洗脱组分进一步采用凝胶过滤法测试其纯度。 条件为： 色谱柱 Agilent Bio SEC-3(300 Α， φ7.8Χ300 mm), 流动相为 150 mM 磷酸盐缓冲液， 流速 0.5 mL/min, 柱温 25°C， 紫外检测波长 214 nm。
实施例 4 产生 MAGE sTv WT的高稳定性变体
利用噬菌体展示技术产生 MAGE-sTv-WT疏水芯变体文库， 用以筛选并鉴定 高稳定性的突变体。 通过诱变 MAGE-sTv-WT的疏水芯位点来构建疏水芯突变文 库， 并对文库进行淘选和筛选。 上述疏水芯文库构建及筛选方法可以参照 Li 等在（（2005) Nature Biotech 23 (3)： 349-354)中描述的噬菌体展示和高亲和性
TCR 噬菌体文库的构建及筛选方法， 设计上的不同点在于构建疏水芯突变文库 时， 需要根据模板链的疏水芯位点来设计引物， 而高亲和性 TCR文库的构建是 根据模板链的 CDR区来设计引物。 构建疏水芯突变文库时， 设计的引物如下表 2所示。
表 2 构建 MAGE-sTv-WT疏水芯变体文库时所设计的引物
Figure imgf000027_0001
本发明中所用的简并碱基，如本领域技术人员熟知的那样，可以分别代表的碱 基类型如下： B=C或 G或 T; D=A或 G或 T; H=A或 C或 T; K=G或 T; M=A或 C; N=A 或 C或 G或 T; R=A或 G; S=C或 G; V=A或 C或 G; W=A或 T; Y=C或 T。
为了让稳定性差的突变株在噬菌体展示过程中被淘汰，更稳定的突变株被筛选 到，采用以下三种处理方法： 1.在 37°C展示 sTv； 2.加入诱导剂（如 IPTG)诱导 sTv 在噬菌体表面展示； 3.筛选前将展示 sTv的噬菌体在 55°C孵育 60分钟。
经测序鉴定， 利用上述方法筛选到的高稳定性 sTv突变株的疏水芯均发生 了突变。 筛选到的高稳定性的突变株命名为 MG29、 P8F1和 P8F2。 根据頂 GT中 的位置编号， 其 α链可变域的一个或多个下列疏水芯位置的氨基酸发生突变： 第 19位、 第 21位、 第 91位； 和 /或其 β链可变域的一个或多个下列疏水芯位 置的氨基酸发生突变： 第 91位、 β链 J gene短肽氨基酸序列倒数第 4位。 更 具体地， 根据 頂 GT 中的位置编号， 其具有一个或多个下列 α链可变域氨基酸 残基 19V、 211、 91L和 /或具有一个或多个下列 β链可变域氨基酸残基 91F或
911 ; β链 J基因倒数第 4位为 M。具体的 a链可变域氨基酸序列为 SEQ ID NO : 15 和 17 ; β链可变域氨基酸序列为 SEQ ID NO : 16和 18。 其中， 组成突变株 MG29 的 α与 β链可变域的氨基酸序列分别为 SEQ ID NO : 15和 16， 如图 6a和 6b所 示； 组成突变株 P8F1 的 α与 β链可变域的氨基酸序列分别为 SEQ ID NO : 17 和 18， 如图 7a和 7b所示； 组成突变株 P8F2的 a与 β链可变域的氨基酸序列 分别为 SEQ ID NO : 15禾卩 18， 如图 8a禾卩 8b所示；
将上述筛选到的 0D值较高的高稳定性突变株 MG29、 P8F1、 P8F2及疏水芯 未发生突变的 MAGE-sTv-WT进行 ELISA实验比较其 0D值， 并验证突变株的特 异性。
ELISA实验步骤：
1 ·分别接种 MAGE-sTv-WT、 MG29、 P8F1、 P8F2甘油菌至 5mL 2xTY (100
Figure imgf000028_0001
氨苄青霉素， 2% 葡萄糖）， 250 rpm/min, 37 °C培养过夜。
2. 分别接种培养过夜的菌液 50 L至新鲜的 5mL 2xTY (100 g/mL氨苄青霉 素， 2% 葡萄糖）， 250 rpm/min, 37°C培养至 0D600=0. 4; 用 5 L (6. 5 x 1010)。 KM13辅助噬菌体 （Source Biosc ience ) 感染， 37 °C静止 30min后， 200 rpm/min, 37 °C振荡 30 min， 离心将沉淀重悬于 30mL 2xTY (100 g/mL氨苄青霉素， 50 g/mL 卡那霉素， 0. 1% 葡萄糖）， 250 rpm/min, 30 °C培养过夜。
3. 用 10 g/mL链霉亲和素（ PBS， pH=7. 4) 包被免疫吸附板（NUNC)， 100 μΐ 每孔， 4°C过夜。
4. 离心收集过夜培养的菌液上清，用 1/4体积比的 PEG/NaCl沉淀上清液中的 噬菌体， 冰上放置 1 h， 离心收集沉淀， 重悬于 3 mL PBS。
5. 用 0. 1% PBST洗板 3次后， 每孔加 400 μΐ, 3%Marvel-PBS (Cadbury Schweppes ) , 37 °C封闭 2h; PBST洗板 3次， 每孔加 100 μΐ 10 ug/mL的 pMHC， 室 温放置 lh; 洗板 3次， 每孔加 100 噬菌体样品（10 μΐ PEG沉淀的样品与 3%的 Marvel -PBS室温孵育 1 h)，室温放置 lh;洗板 3次后，每孔加 100 uL， anti-M13-HRP conjugate (GE Healthcare ) (1 : 5000稀释于 3%的 Marvel- PBS中）， 室温放置 lh; 洗板 6次， 每孔加 100 μΐ TMD， 放置 5 min后， 每孔加 10( L， 1M硫酸终止。
6. 读取 450 nm、 650 nm吸光值。
上述突变株的 ELISA实验 0D值如附图 9所示， 此结果显示了疏水芯优化后的 sTv特异性保持与疏水芯为野生型的 MAGE-sTv-WT相同。在本实验中， MAGE-sTv-WT 由于展示太差， 其 0D值极低， 说明即使将 a链和 β链可变域中暴露于表面的疏水 残基变为亲水或极性残基，其蛋白稳定性仍相对较差， 因此仍需要对疏水芯进行优 化。 疏水芯被优化的克隆都能不同程度地展示 sTv，并与其原配体 MAGE A3 pHLA-Al 抗原发生特异性结合， 但与其它无关抗原， 如 EBV， 流感和 NY-ES0-1抗原没有结 合。 MG29、 P8F1和 P8F2等这些通过噬菌体展示检测到的疏水芯突变的 sTv与特异 性抗原的结合不应是由于其亲和力较野生的 TCR强的原因， 这一点在实施例 15得 到了证明
实施例 5 构建疏水芯突变的高稳定性 sTv分子
根据本领域技术人员熟知的定点突变方法将实施例 4中筛选到的高稳定性 变体的部分疏水芯引入到几种其他的 TCR分子中， 构建高稳定性 sTv分子。
根据几种分别针对抗原短肽 HLA-B8/FLRGRAYGL (源自 EB 病毒抗原 EBNA3A) 、 HLA-A2/GILGFVFTL( 源 自 流 感 病 毒 基 质 蛋 白 ） 和 HLA-A2/SLL丽 ITQC (NY-ES0-1肿瘤特异性抗原）的野生型 TCR分子的 α和 β链的 可变域来分别构建上述几种分子的单链形式， 并分别命名为 LC13-WT、 JM22-WT 和 1G4-WT。其中， LC13-WT的 α链和 β链可变域氨基酸序列分别为 SEQ ID N0:29 和 SEQ ID NO :30,如图 10a和 10b所示； JM22-WT的 α链和 β链可变域氨基酸 序列分别为 SEQ ID NO: 31禾卩 SEQ ID NO :32， 如图 11a和 lib所示； 1G4- WT的 α链和 β链可变域氨基酸序列分别为 SEQ ID NO: 33 和 SEQ ID NO: 34， 如图 12a和 12b所示。
将实施例 4中筛选到的高稳定性变体的部分疏水芯通过本领域技术人员熟 知的定点突变的方法分别引入到 LC13-WT、 JM22-WT和 1G4-WT分子中， 将引入 突变后得到的分子分别命名为 LC13-sTv、 JM22-sTv和 1G4-STV， 被引入的疏水 芯以带下划线的加粗字母表示。 其中， LC13-sTv的 a链和 β链可变域氨基酸序 列分别为 SEQ ID Ν0:35和 SEQ ID Ν0: 36， 如图 13a和 13b所示， 其 α链可变 域中引入的疏水芯为 11L、 13V、 211和 911， 其 β链可变域中引入的疏水芯为 94L; JM22-sTv的 α链和 β链可变域氨基酸序列分别为 SEQ ID NO :37和 SEQ ID NO: 38， 如图 14a和 14b所示， 其 α链可变域中引入的疏水芯为 19V和 211， 其 β链可变域中引入的疏水芯为 911和 94L; 1G4-STV 的 α链和 β链可变域氨 基酸序列分别为 SEQ ID NO :39禾卩 SEQ ID NO: 40， 如图 15a禾卩 15b所示， 其 α 链可变域中引入的疏水芯为 19V和 211， 其 β链可变域中引入的疏水芯为 19V、 911、 94L和 J基因倒数第 6位为 T。 上述位置编号采用 IMGT中列出的位置编 号。 构建上述单链分子所用的连接短肽 （linker) 可以为任何合适的序列， 本 发明优选的氨基酸序列为 SEQ ID N0:41， 如图 16所示。
实施例 6 蛋白 LC13-WT与 LC13-sTv的稳定性测试
采用实施例 3中所述的方法对蛋白 LC13-WT和 LC13-sTv进行表达、 复性、 纯化， 凝胶过滤柱纯化后跑 SDS-PAGE胶， 并用凝胶过滤法作出 2种蛋白的 SEC 图谱， 同时计算其表达量、 纯化后所得蛋白量和蛋白复性收率。 其中， 表达量 为 1L大肠杆菌诱导表达并提纯后的包涵体的产量。 纯化后所得蛋白量为 1L大 肠杆菌诱导表达并提纯后所得包涵体经复性、 纯化后得到的蛋白的量。 蛋白复 性收率的计算式如下： 蛋白复性收率 （％) =100*纯化后所得蛋白量 （mg ) /复 性所用包涵体的量 （mg )。 在本发明中提及的表达量和蛋白复性收率均按上述 计算方法进行计算， 有特别说明的除外。
利用美国 TA (waters ) 公司的差示扫描量热仪 （Nano DSC ) 测定上述纯化 后蛋白 LC13-WT与 LC13-sTv的 Tm值。 其扫描范围为 10-9CTC， 升温速率为 1 °C /min，上样量为 90(^L。 其中， Tm值是通过分析软件 Nanoanalyze 的拟合模 型 TwostateScaled拟合而得到。
下表 3列出了 LC13-WT与 LC13-sTv的表达量、 纯化后所得蛋白量及蛋白 复性收率的数据。
表 3
Figure imgf000030_0001
由上表的数据可知， 经纯化后， 引入疏水芯突变的 LC13-sTv 蛋白相较于 疏水芯未发生突变的 LC13-WT蛋白复性收率提高了 35倍。
图 17 为按实施例 3 中所述经过凝胶过滤柱（Superdex 75 10/300, GE Heal thcare)纯化后得到蛋白 LC13- WT与 LC13-sTv的 SDS- PAGE胶图。 胶图显 示， 纯化后得到 LC13-WT蛋白形成的条带不均一， 而 LC13-sTv能够形成单一 的条带且纯度很高。 说明 LC13-sTv的复性情况要远好于 LC13-WT。
图 18a和 18b分别为蛋白 LC13-WT与 LC13-sTv的 SEC图谱， 从图谱上来 看， 纯化后的蛋白 LC13-WT并未出峰， 而 LC13-sTv能够形成单一且对称的洗 脱峰， 说明 LC13-sTv的复性要显著优于 LC13-WT。
LC13-WT 复性纯化后得到正确构象的蛋白含量极少， 没有明显的蛋白去折 叠吸热峰， 利用分析软件 Nanoanalyze得不到其 Tm值， 而经疏水芯突变后的 LC13-sTv的 Tm值为 43. 6°C， 其 DSC曲线如图 47所示。 说明 LC13-sTv相较于 LC13-WT 复性能力更强、 更抗解折叠、 更抗不适当或不希望的折叠以及热稳定 性有显著提高。 通过蛋白 LC13-WT与 LC 13-sTv 的表达量、 纯化后所得蛋白量、 蛋白复性 收率、 SDS-PAGE胶图、 SEC 图谱及 Tm值的对比分析可知， 经过疏水芯改造的 LC13-sTv相较于疏水芯未经改造的 LC13-WT复性能力更强、 更抗解折叠、 更抗 不适当或不希望的折叠、 蛋白复性收率更高以及热稳定性有显著提高。 因此， LC13-sTv相较于 LC 13-WT稳定性有显著提高。以蛋白复性收率的数据来对稳定 性提高的量进行计算，本发明中 LC13-sTv相对于 LC13-WT稳定性提高了 35倍。
实施例 7 蛋白 JM22-WT与 JM22-STV的稳定性测试
采用实施例 3中所述的方法对蛋白 JM22-WT和 JM22-sTv进行表达、 复性、 纯化， 凝胶过滤柱纯化后跑 SDS-PAGE胶， 并用凝胶过滤法作出 2种蛋白的 SEC 图谱， 同时计算其表达量、 纯化后所得蛋白量和蛋白复性收率， 并利用实施例 6中所述方法测定其 Tm值。
下表 4列出了 JM22-WT与 JM22-sTv的表达量、 纯化后所得蛋白量及蛋白 复性收率的数据。
表 4
Figure imgf000031_0001
由上表的数据可知， 经纯化后， 引入疏水芯突变的 JM22-sTv 蛋白相较于 疏水芯未发生突变的 JM22-WT蛋白复性收率提高了 42倍。
图 19 为按实施例 3 中所述经过凝胶过滤柱（Superdex 75 10/300, GE Heal thcare)纯化后得到蛋白 JM22-WT和 JM22-sTv的 SDS-PAGE胶图。 胶图显 示， JM22-WT复性形成的单体条带不均一， 有 3个条带， 而 JM22-sTv能够形成 单一条带的单体且纯度很高。 说明 JM22-sTv的复性情况要远好于 JM22-WT。
图 20a和 20b分别为蛋白 JM22-WT与 JM22-sTv的 SEC图谱， 从图谱上来 看， 纯化后的蛋白 JM22-WT 形成的洗脱峰不单一而且信号很低， 而纯化后的 JM22-sTv能够形成单一且对称的洗脱峰， 说明 JM22-sTv的复性情况要显著优 于 JM22- WT。
图 48a和 48b分别为蛋白 JM22- WT与 JM22- sTv的 DSC曲线图。由于 JM22- WT 复性纯化后得到正确构象的蛋白含量极少， 没有明显的蛋白去折叠吸热峰， 利 用分析软件 Nanoanalyze得不到其 Tm值，而经疏水芯突变后的 JM22-sTv的 Tm 值为 43. 7 °C。 由上述 DSC 曲线图可知， JM22-sTv相较于 JM22-WT复性能力更 强、 更抗解折叠、 更抗不适当或不希望的折叠以及热稳定性有显著提高。
通过蛋白 JM22-WT与 JM22-sTv 的表达量、 纯化后所得蛋白量、 蛋白复性 收率、 SDS-PAGE胶图、 DSC曲线图及 SEC图谱的对比分析可知， 经过疏水芯改 造的 JM22-sTv相较于疏水芯未经改造的 JM22-WT复性能力更强、 更抗解折叠、 更抗不适当或不希望的折叠以及热稳定性有显著提高同时蛋白复性收率也有 明显提高。 因此， 本发明 JM22-sTv相较于 JM22-WT稳定性有显著提高。 以蛋 白复性收率的数据来对稳定性提高的量进行计算， 本发明中 JM22-sTv 相对于 JM22-WT稳定性提高了 4200%。
实施例 8 蛋白 1G4 WT与 1G4 STV的稳定性测试
采用实施例 3 中所述的方法对蛋白 1G4-WT和 1G4-STV进行表达、 复性、 纯化， 凝胶过滤柱纯化后跑 SDS-PAGE胶， 并用凝胶过滤法作出 2种蛋白的 SEC 图谱， 同时计算其表达量、 纯化后所得蛋白量和收率。
下表 5列出了 1G4-WT与 1G4-STV的表达量、 纯化后所得蛋白量及蛋白复 性收率的数据。
表 5
Figure imgf000032_0001
由上表的数据可知， 经纯化后， 引入疏水芯突变的 1G4-STV蛋白相较于疏 水芯未发生突变的 1G4-WT蛋白复性收率提高了 2.6倍。
图 21 为按实施例 3 中所述经过凝胶过滤柱（Superdex 75 10/300, GE Healthcare)纯化后得到蛋白 1G4-WT和 lG4-sTv的 SDS-PAGE胶图。 胶图显示， 纯化后得到 1G4-WT蛋白形成的条带不均一， 形成了 2个条带， 而 1G4-STV能 够形成单一条带的单体且纯度很高。说明 1G4-STV的复性情况要远好于 1G4-WT。
图 22a和 22b分别为蛋白 1G4-WT与 1G4-STV的 SEC图谱， 从图谱上来看， 纯化后的蛋白 1G4-WT形成的洗脱峰不单一而且信号较低，而纯化后的 1G4-STV 能够形成单一且对称的洗脱峰， 说明 1G4-STV的复性情况要显著优于 1G4-WT。
通过蛋白 1G4-WT与 1G4-STV 的表达量、 纯化后所得蛋白量、 蛋白复性收 率、 SDS-PAGE胶图及 SEC图谱的对比分析可知， 经过疏水芯改造的 1G4-STV相 较于疏水芯未经改造的 1G4-WT 具有更强的复性能力、 更高的表达量、 更高的 蛋白复性收率。 因此， 本发明 1G4-STV相较于 1G4-WT稳定性有显著提高。 以 蛋白复性收率的数据来对稳定性提高的量进行计算， 本发明中 1G4-STV相对于 1G4-WT稳定性提高了 260%。
实施例 9 以 1G4-STV为模板对分子的稳定性进一步优化
以 1G4-STV为模板对其疏水芯及可变域的表面氨基酸残基进行突变， 构建 文库并筛选高稳定性的分子。 需要进行突变的疏水芯位点已在序列 SEQ IDN0: 42 中以带下划线的黑体字标出， 需要进行突变的表面氨基酸残基以黑体字标 出， 如图 23所示。
文库构建所采用的基本方法已经在实施例 4中有所描述。 在本实施例中， 针对需要进行突变的位点构建 3个文库， 需要突变的疏水芯位点全部在文库 1 中， 文库 2和 3是针对表面氨基酸残基而构建的。 更具体地， 以 1G4-STV质粒 作为模板， 以设计好的突变引物进行重叠 PCR (Overlap PCR) 得到突变 DNA片 段后， 通过 Ncol/Notl酶切， 将片段克隆至基于 pUC19骨架的噬菌体质粒载体 pLitmus28 (NEB) 中。 连接后的 DNA电转 TGI感受态细胞（lucigen)， 得到共 3个噬菌体质粒载体文库， 其库容按菌落数计约为 1 X 109-3 X 109。 分别将这 3 个文库生长出的菌苔刮下，加入至终浓度为 20%的甘油于 -80°C保存。 以下表 8、 表 9和表 10分别为针对文库 1、 文库 2和文库 3所设计的引物。
表 6 构建文库 1所设计的引物
Figure imgf000033_0001
L2-02 GATTGAGACATTTTCMYYTTCCGGGACAG 53
L2-03 GTCACTGTCTGTCCCGGAARRKGAAAATGTCTCAATC 54
L2-04 CACAAAAGTACGTCGCMYYGTCMYYCGGCTGCGATGAT 55
L2-05 CGACGTACTTTTGTGCGGTTCGTCC 56
L2-06 CAGCTTCGTACCCTTGCCGAAGGTC 57
L2-07 TTCGGCAAGGGTACGAAGCTGRRKGTCACGCC 58
L2-08 CATTGCAGGGTCACMYYCTGMYYCGTMYYCAGGCTCTGGT 59
L2-09 GTGACCCTGCAATGCGCCCAGGATATG 60
L2-10 CACAGATAAACTGCGGAGTCMYYCGGGGTCAG 61
L2-1 1 GACTCCGCAGTTTATCTGTGTGCTTCGTCC 62
L2-12 GAGTGCGGCCGCCGTCACMYYCAGGCGCGTG 63
表 8 构建文库 3所设计的引物
引物名称 引物序列 （5' -3' ) SEQ ID NO :
L3-01 CCGGCCATGGCCAAGCAGGAAGTCACGCAATCCCCGTCGTC 64
L3-02 ACGCAATCCCCGTCGTCACTGRRKGTCCCGGAAGG 65
L3-03 GGAGTCACCCGGCTGMANMYYTTCAATATACAGGGTAC 66
L3-04 CAGCCGGGTGACTCCRRKACGTACTTTTGTGCG 67
L3-05 TTCGCTGCCGCCCCCMYYCGTGACGCTCAGCTT 68
L3-06 AAGCTGAGCGTCACGRRKGGGGGCGGCAGC 69
L3-07 GCATTGCAGGGTCACAGACTGACCMYYCTTCAGGCTC 70
L3-08 GTCAGTCTGTGACCCTGCAATGCGCCCAGGATATG 71
L3-09 CTGCGGAGTCACTCGGMYYCAGMYYTTCAATGCG 72
L3-10 CCGAGTGACTCCGCAGTTTATCTGTGTGCTTCGTCC 73
L3-1 1 AGTGCGGCCGCMYYCACCGTCAG 74
为得到高稳定性的 sTv 克隆， 库中长出的噬菌体在沉淀浓缩后， 进行 65
°C热激处理， 同时为进一步加强筛选力度还加入了 0. 02%的 SDS共孵育， 而后 将此处理过的噬菌体做后续筛选。 将于 3个库中筛选到的 0D值较高的克隆进 行组合最终得到 11个克隆。
实施例 10 对实施例 9中筛选到的克隆的稳定性验证
将实施例 9中筛选到的 11个克隆按实施例 4中所述的 ELISA实验步骤检 测其 0D值并验证抗原特异性， 结果如图 24所示。 此结果显示了 11个克隆的 0D值均较高， 能够与其原配体抗原 HLA-A2/SLL丽 ITQC (NY-ES0-1肿瘤特异性抗 原）特异性结合， 与其他无关抗原基本没有结合。 这些通过噬菌体展示检测到 的 sTv与抗原 HLA-A2/SLLMWITQC的结合不应是由于其亲和力较野生的 TCR强 的原因， 这一点在实施例 11得到了证明。
根据頂 GT中列出的位置编号， 上述 11个突变株在下列一个或多个疏水芯 位置发生了突变： α链可变区第 11位、 第 13位或第 94位和 /或 β链可变区第 11位、 第 13位或第 94位。 具体地， 其包含一个或多个下列 α链可变区疏水芯 氨基酸残基 11M、 11E、 13R、 13K、 94V或 941和 /或 β链可变区疏水芯氨基酸 残基 11L、 11V、 13V或 94V。 除了疏水芯外， 我们筛选到的克隆还包含一个或 多个下列 α链可变区氨基酸残基 4L、 12N、 16S、 93N、 93R、 97N、 100G、 105S 或 α链 J基因倒数第 1位是 D和 /或包含 β链可变区氨基酸残基 41、 101 β 链 J基因倒数第 1位是 D或倒数第 3位是 E。
筛选到的高稳定性的上述克隆的 a链可变域的氨基酸序列 （SEQ ID NO : 75-85 ) 和 β链可变域氨基酸序列 （SEQ ID NO : 86-96 ) 分别如图 25 和图 26 所示。
采用实施例 2和实施例 3 中所述的方法对筛选到的 1 1个克隆进行连接、 表达、 复性和纯化。 利用美国 TA ( waters ) 公司的差示扫描量热仪（Nano DSC ) 测定上述 11种克隆的 Tm值。 其扫描范围为 10-90°C， 升温速率为 rC /min，上 样量为 90(^L。 其中， Tm 值是通过分析软件 Nanoanalyze 的拟合模型 TwostateScaled拟合而得到。结果如图 27及表 9所示，其 Tm值均不小于 37. 9 °C， 并具有明显的蛋白去折叠吸热峰。 而与上述克隆的表达、 复性、 纯化过程 及 DSC实验条件完全相同的 1G4-WT的 DSC结果如图 28所示，从图中可以看出， 其没有明显的蛋白去折叠的吸热峰， 说明正确构象的蛋白含量极少。 对比上述 11 个克隆的 DSC 图谱与 1G4-WT 的 DSC 图谱结果可知， 筛选到的克隆相较于 1G4-WT更抗解折叠、 更抗不适当或不希望的折叠、 复性能力更强以及热稳定性 显著增加。 因此， 我们筛选到的克隆的稳定性要远远高于疏水芯未经突变的 1G4 - WT。
由于上述 1G4-WT 复性纯化后得到正确构象的蛋白含量极少， 没有明显的 蛋白去折叠吸热峰， 利用软件得不到其 Tm值， 而上述疏水芯突变 TCR具有约 38°C或更高的 Tm值，这提示，本发明的上述 G3-G7以及 G9-G14等 1 1种突变 TCR 的稳定性都有极其显著的提高（至少提高 1倍）。
表 9
Figure imgf000036_0001
本领域技术人员可以对上述筛选到的高稳定性突变点进行重新组合来构 建新的稳定性突变株。 我们对上述突变点进行重新组合， 构建出了新的 α链可 变域 （SEQ ID N0 : 97)和 β链可变域 （SEQ ID NO : 98) , 其氨基酸序列分别如图 29a和图 29b所示。 以此 α链可变域和 β链可变域构建 sTv分子， 命名为 G15 实施例 11 1G4 WT突变株稳定性的进一步测试
采用实施例 3 中所述的方法对实施例 10中所述的突变株 G9 G13和 G15 进行表达、 复性、 纯化， 凝胶过滤柱纯化后跑 SDS-PAGE 胶， 并用凝胶过滤法 作出 3种蛋白的 SEC图谱， 同时计算其表达量、 纯化后所得蛋白量和蛋白复性 收率， 并与 1G4-WT进行比较。
表 10列出了 1G4-WT G9 G13和 G15 的表达量、 纯化后所得蛋白量及蛋 白复性收率的数据。
表 10
Figure imgf000036_0002
G9 356 15. 5%
G13 223 45. 4%
G15 279 129. 7 46. 5% 由上表的数据可知， 经纯化后， 突变株 G9、 G13和 G15相较于 1G4-WT蛋 白复性收率均有显著提高， 分别提高了 4. 5倍、 15. 2倍和 15. 6倍。
图 30 为按实施例 3 中所述经过凝胶过滤柱（Superdex 75 10/300, GE Heal thcare)纯化后得到蛋白 1G4-WT、 G9、 G13和 G15的 SDS- PAGE胶图。 胶图 显示， 纯化后得到 1G4-WT蛋白形成的条带不均一， 而经过改造的 G9、 G13和 G15均能够形成单一条带的单体且纯度很高。 说明 G9、 G13和 G15的复性情况 均要好于 1G4-WT。
图 31a、 31b和 31 c分别为蛋白 G9、 G13和 G15的 SEC图谱， 从图 22a的 1G4-WT的 SEC图谱可知， 纯化后的蛋白 1G4O L
O-O LWT形成的洗脱峰不单一而且信号 较低， 而纯化后的 G9、 G13和 G15均能够形成单O G一O L 且对称的洗脱峰， 说明 G9、 G13和 G15的复性情况均要显著优于 1G4-WT。
采用实施例 10中所述方法测定 G15的 Tm值为 46. 6°C，其 DSC曲线图如图 46所示。 而根据实施例 10中的测定结果， 突变株 G9和 G13的 Tm值也相对较 高， 分别为 49. 55 °C和 49. 63 °C。
使用 BIAcore T200实时分析系统检测蛋白 G9、 G13和 G15与其配体的结 合， 结果显示 3种 sTv蛋白与抗原 HLA-A2/SLLMWITQC的亲合力并不优于野生 型 1G4TCR与该抗原的结合， 野生型 1G4TCR结合抗原 HLA-A2/SLLMWITQC的解 离平衡常数是 32 μ Μ (参照 Li等（（2005) Nature Bi otech 23 (3) : 349-354) ) 0 通过蛋白 1G4-WT与 G9、 G13和 G15 的表达量、 纯化后所得蛋白量、 蛋白 复性收率、 SDS-PAGE胶图及 SEC图谱的对比分析可知， 经过疏水芯改造的突变 株的复性能力、 热稳定性和蛋白复性收率均要远远高于疏水芯未经改造的 1G4-WT 。 因此， 经疏水芯改造的突变株的稳定性相较于 1G4-WT 有显著提高。 以蛋白复性收率的数据来对稳定性提高的量进行计算，本发明中 G9、G13和 G15 相对于 1G4-WT稳定性分别提高了 450%、 1520%和 1560%。
实施例 12 构建疏水芯突变的高稳定性 sTv分子
根据实施例 9中筛选到的高稳定性变体的疏水芯及可变区骨架的表面氨基 酸残基构建高稳定性 sTv分子。
将实施例 9中筛选到的高稳定性变体的部分疏水芯及可变区骨架的表面氨 基酸残基通过本领域技术人员熟知的定点突变的方法分别引入到 LC13-WT、 JM22-WT 和 MAGE-sTv-WT 分子中， 将引入突变后得到的分子分别命名为 LC13- G9、 LC13- G15、 JM22- G9、 JM22-G15 和 MAGE- G15， 被引入的疏水芯以带 下划线的加粗字母表示。
其中， LC13-G9的 α链和 β链可变域氨基酸序列分别为 SEQ ID NO: 99和
SEQ ID NO: 100，如图 32a和 32b所示， 其 α链可变域中引入的疏水芯为 13V、 211、 911和 941其 β链可变域中引入的疏水芯为 11V、 13V和 94V; LC13-G15 的 α链和 β链可变域氨基酸序列分别为 SEQ ID NO: 101和 SEQ ID NO: 102， 如 图 33a和 33b所示， 其 α链可变域中引入的疏水芯为 11L、 13V、 211、 911和 941， 其 β链可变域中引入的疏水芯为 11L、 13V和 94V; JM22-G9 的 α链和 β 链可变域氨基酸序列分别为 SEQ ID NO: 103和 SEQ ID NO: 104， 如图 34a和 34b所示， 其 α链可变域中引入的疏水芯为 11M、 13V、 19V、 211和 941， 其 β 链可变域中引入的疏水芯为 11V、 13V、 911和 94V; JM22-G15的 α链和 β链可 变域氨基酸序列分别为 SEQ ID NO: 105和 SEQ ID NO: 106， 如图 35a和 35b 所示， 其 α链可变域中引入的疏水芯为 13V、 19V、 211和 941， 其 β链可变域 中引入的疏水芯为 13V、 911和 94V; MAGE-G15 的 α链和 β链可变域氨基酸序 列分别为 SEQ ID Ν0: 107禾卩 SEQ ID NO: 108， 如图 42a和 42b所示， 其 α链可 变域中引入的疏水芯为 19V、 211和 941，其 β链可变域中引入的疏水芯为 13V、 89L、 911禾口 94V。
上述位置编号采用 頂 GT 中列出的位置编号。 构建上述单链分子所用的连 接短肽 （linker) 可以为任何合适的序列， 本发明优选的氨基酸序列为 SEQ ID N0:41， 如图 16所示。
实施例 13 蛋白 LC13-G9与 LC13-G15的稳定性测试
采用实施例 3中所述的方法对蛋白 LC13-G9和 LC13-G15进行表达、 复性、 纯化， 凝胶过滤柱纯化后跑 SDS-PAGE胶， 并用凝胶过滤法作出 2种蛋白的 SEC 图谱， 同时计算其表达量、 纯化后所得蛋白量和蛋白复性收率。
下表 11列出了 LC13-G9与 LC13-G15的表达量、 纯化后所得蛋白量及蛋白 复性收率方面的数据， 同时列出了 LC13-WT的相关数据， 以便分析。
表 11
蛋白名称 表达量 (mg/L) 纯化后所得蛋白量（mg/L) 收率 （％)
LC13-WT 231 1.3 0.56% LC13-G9 233 1. 37 0. 59%
LC13-G15 185 33% 由上表的数据可知， 经纯化后， LC13-G9蛋白和 LC13-G15蛋白相较于疏水 芯未发生突变的 1G4-WT蛋白复性收率分别提高了 5. 4%和 57. 9倍。
图 36 为按实施例 3 中所述经过凝胶过滤柱（Superdex 75 10/300, GE Heal thcare)纯化后得到蛋白 LC 13-G9和 LC13-G15 的 SDS-PAGE胶图。 胶图显 示， LC 13-WT形成的条带不均一， 而经过改造的 LC13-G9和 LC13-G15均能形成 单一条带的单体且纯度很高。 说明 LC13-G9和 LC13-G15 的复性情况要远好于 LX13 - WT。
图 37禾卩 38分别为蛋白 LC13- G9与 LC13- G15的 SEC图谱， LC 13- WT的 SEC 图谱显示其并未出峰， 而蛋白 LC 13-G9与 LC13-G15均能够形成单一且对称的 洗脱峰， 说明 LC13-G9与 LC13-G15的复性情况均要显著优于 LC13-WT。
采用实施例 10中所述方法测定蛋白 LC13-G O9与 LC13-G15的 Tm值， 图 49a 和图 49b分别为蛋白 LC13-G9与 LC13-G15的 DSC 曲线图， 其 Tm值分别为 43 °C和 50. 5 °C。 而 LC13-WT由于复性后得到正确构象的蛋白含量很低， 没有明显 的蛋白去折叠吸热峰， 无法得到其 Tm值。这说明，本发明 LC13-G9与 LC13-G15 的热稳定性相对于 LC13-WT 提高了至少一倍。 同时说明， 本发明 LC13-G9 与 LC13-G15相较于 LC 13-WT更抗解折叠、 更抗不适当或不希望的折叠、 复性能力 更强。
通过蛋白 LC13-G9与 LC 13-G15 的表达量、 纯化后所得蛋白量、 蛋白复性 收率、 SDS-PAGE胶图、 DSC曲线图及 SEC图谱与 LC13-WT的相关数据对比分析 可知， 经过疏水芯改造的 LC13-G9 与 LC13-G15 相较于疏水芯未经改造的 LC13-WT 更抗解折叠、 更抗不适当或不希望的折叠、 复性能力更强、 热稳定性 更高、 蛋白复性收率更高。 因此， 本发明 LC 13-G9与 LC13-G15相较于 LC 13-WT 稳定性有显著提高。 以蛋白复性收率的数据来对稳定性提高的量进行计算， 本 发明中 LC13-G9与 LC 13-G15相对于 LC13-WT稳定性分别提高了 5. 4%和 57. 9倍。
实施例 14 蛋白 JM22-G9与 JM22-G15的稳定性测试
采用实施例 3中所述的方法对蛋白 JM22-G9和 JM22-G15进行表达、 复性、 纯化， 凝胶过滤柱纯化后跑 SDS-PAGE胶， 并用凝胶过滤法作出 2种蛋白的 SEC 图谱， 同时计算其表达量、 纯化后所得蛋白量和蛋白复性收率。
下表 12列出了 JM22-G9与 JM22-G15的表达量、 纯化后所得蛋白量及蛋白 复性收率的数据， 同时列出了 JM22-WT的相关数据， 以便分析。
表 12
Figure imgf000040_0001
由上表的数据可知， 经纯化后， JM22-G9蛋白和 JM22-G15蛋白相较于疏水 芯未发生突变的 JM22-WT蛋白复性收率分别提高了 28. 5倍和 127. 25倍。
图 39 为按实施例 3 中所述经过凝胶过滤柱（Superdex 75 10/300, GE
Heal thcare)纯化后得到蛋白 JM22-G9和 JM22-G15 的 SDS-PAGE胶图。 胶图显 示， JM22-WT复性形成的单体条带不均一， 有 3个条带， 而经过改造的 JM22-G9 和 JM22-G15均能形成单一条带的单体且纯度很高。 说明 JM22-G9和 JM22-G15 的复性情况要远好于 JM22-WT。
图 40禾卩 41分别为蛋白 JM22- G9与 JM22- G15的 SEC图谱， JM22- WT的 SEC 图谱显示其形成的洗脱峰不单一而且信号很低， 而蛋白 JM22-G9 与 JM22-G15 基本能够形成单一且对称的洗脱峰， 进一步说明 JM22-G9与 JM22-G15 的复性 情况均要显著优于 JM22-WT。
通过蛋白 JM22-G9与 JM22-G15 的表达量、 纯化后所得蛋白量、 蛋白复性 收率、 SDS-PAGE胶图及 SEC图谱与 JM22-WT的相关数据对比分析可知， 经过疏 水芯改造的 JM22-G9与 JM22-G15相较于疏水芯未经改造的 JM22-WT具有更强 的复性能力、更高的表达量和蛋白复性收率。因此，本发明 JM22-G9与 JM22-G15 相较于 JM22-WT稳定性有显著提高。 以蛋白复性收率的数据来对稳定性提高的 量进行计算， 本发明中 JM22-G9与 JM22-G15相对于 JM22-WT稳定性分别提高 了 28. 5倍和 127. 25倍。
实施例 15 蛋白 MAGE-sTv-WT与 MAGE- G15的稳定性测试
采用实施例 3 中所述的方法对蛋白 MAGE-G15进行表达、 复性、 纯化， 凝 胶过滤柱纯化后跑 SDS-PAGE胶， 并用凝胶过滤法作出蛋白的 SEC 图谱， 同时 计算其表达量、 纯化后所得蛋白量和蛋白复性收率。
下表 13列出了 MAGE-sTv-WT与 MAGE-G15的表达量、 纯化后所得蛋白量及 蛋白复性收率的数据。
表 13 蛋白名称 表达量 ( mg/L ) 纯化后所得蛋白量（mg/L ) 收率 （％)
MAGE-sTv-WT 270 0 0
MAGE - G 15 220 19. 8 9% 蛋白 MAGE-sTv-WT经实施例 3中所述的凝胶过滤柱后，没有得到目标组分， 因此无法得到其 SDS-PAGE胶图、 SEC图谱和 DSC曲线图 （Tm值）。
而经过疏水芯改造后的 MAGE-G15经凝胶过滤柱（Superdex 75 10/300, GE Heal thcare)纯化后得到蛋白的 SDS-PAGE 胶图如图 43 所示。 该胶图显示， MAGE-G15能够形成单一条带且纯度很高， 说明蛋白 MAGE-G15 的复性能力要远 远强于 MAGE-sTv-WT。 同时， 图 44所示的 MAGE-G15的 SEC图谱， 具有单一且 对称的洗脱峰，也能够说明蛋白 MAGE-G15的复性能力要远远强于 MAGE-sTv-WT。
图 45为蛋白 MAGE-G15的 DSC曲线图， 利用分析软件 Nanoanalyze的拟合 模型 TwostateScaled拟合得到其 Tm值为 46. 7 °C。
使用 BIAcore T200实时分析系统检测蛋白 MAGE-G15与其配体的结合， 结 果显示， 蛋白 MAGE-G15与其配体的亲和力并不优于其相对应的野生型 TCR， 其 KD值为 30. 4 μ Μ。
以蛋白复性收率的数据来对稳定性提高的量进行计算， 从表 13 中可以看 出，本发明的 MAGE-G15相较于 MAGE-sTv-WT稳定性提高了无穷大倍（至少 10000 倍）。
由以上数据可知， 本发明的 MAGE-G15 的复性能力、 蛋白复性收率及热稳 定性相较于 MAGE-sTv-WT都有极其显著的提高， 因此本发明 MAGE-G15 的稳定 性相较于 MAGE-sTv-WT有了显著的提高。 实施例 16质谱分析
我们构建的蛋白经过凝胶过滤柱（Superdex 75 10/300, GE Heal thcare) 纯化后通过质谱仪测定其全蛋白分子量， 并分析质谱测定的分子量与其理论分 子量是否相符， 以此检验纯化后得到的蛋白序列与原设计序列是否相同。
利用美国 AB SCIEX公司的质谱仪（Eks igent nano LC ( nanoflex ) - Triple T0F 5600 液质联用系统） 测定样品的全蛋白分子量。 用 10%乙腈 ( Fi sherA955- 4 )、 1 %甲酸 （Fi sher A11750 ) 和水 （ Si gma39253- 1L- R ) 对 样品稀释后上质谱分析。 系统分析条件如下：
L C部分 AB SCIEX的 Eksigent nano LC ( nanof lex)
保护柱： C4-3 μ m 30θΑ 200μ mXO.5mm; Lot 804-00019
分析柱： C4; 3um, 300A; 75um*15cm， Lot 804-00018
流动相 A: 2 %乙腈， 0.1%甲酸
流动相 B : 98%乙腈， 0.1%甲酸
流速： 300nl I min
梯度： 1 0分钟内 B液的比例从 5 %升至 9 0 %, 总运行时间为 3 0分钟。 质谱部分
带有 Nanospray源的 Triple T0F 5600分析柱子中的洗脱液，
数据采集方法： 正离子 M S，
数据采集范围： 400— 200m/z。
采集到的质谱数据经 Bioanalyst 软件去卷积处理后得到样品的完整蛋白 分子量信息。
经分析， 本发明中构建的分子经表达、 复性和纯化后， 利用质谱仪测定的 全蛋白分子量与其理论分子量均相符， 说明纯化后得到的蛋白序列与原设计蛋 白序列相同。
本发明筛选到的疏水芯能够显著提高 TCR分子的稳定性。同时，上述实施例证 明将本发明筛选到的疏水芯引入到其他的 TCR 分子中也能够起到增强稳定性的作 用。
在本发明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， 在阅读了本发明的上述讲授内容之后， 本领域 技术人员可以对本发明作各种改动或修改，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

权 利 要 求 书
1. 一种 T细胞受体（TCR)， 其特征在于：
( i )所述 TCR的疏水芯发生突变； 并且
( ii )所述 TCR的稳定性高于其对应的疏水芯为野生型的 TCR。
2. 如权利要求 1所述的 T细胞受体， 其特征在于， 所述 TCR的 CDR区与 野生型 TCR的 CDR区相同， 或含有导致亲和力上升的突变。
3. 如权利要求 1所述的 T细胞受体， 其特征在于， 所述的 T细胞受体是 可溶性的。
4. 如权利要求 1所述的 T细胞受体， 其特征在于， 所述的 T细胞受体包 括（a)除跨膜结构域以外的全部或部分 TCR a链； 以及（b)除跨膜结构域以外的 全部或部分 TCR i 链；
并且（a)和（b)各自包含功能性可变结构域，或包含功能性可变结构域和所 述 TCR链恒定结构域的至少一部分。
5. 如以上任一权利要求所述的 T细胞受体， 其特征在于， 所述 TCR是由 一柔性肽链 （l inker ) 连接 TCR的 α与 β链的可变域而构成的单链 TCR。
6.如以上任一权利要求所述的 TCR， 其特征在于， 所述 TCR的 a链可变域 在一个或多个下列位点中发生突变： α链可变区氨基酸第 11、 13、 19、 21、 53、 76、 89、 91、 或第 94位， 和 /或 α链 J基因短肽氨基酸倒数第 3位、 倒数第 5 位或倒数第 7位，其中氨基酸位置编号按 IMGT (；国际免疫遗传学信息系统；)中列 出的位置编号。
7.如以上任一权利要求所述的 TCR，其特征在于，所述 TCR在 SEQ ID NO:9 或 SEQ ID NO:29或 SEQ ID NO:3 1或 SEQ ID NO:33所示的 α链可变域的一个 或多个下列位点中发生突变： a链可变区氨基酸第 11、 13、 19、 21、 53、 76、 89、 91、 或第 94位， 和 /或 a链 J基因短肽氨基酸倒数第 3位、 倒数第 5位或 倒数第 7位， 其中氨基酸位置编号按 IMGT中列出的位置编号。
8.如以上任一权利要求所述的 TCR， 其特征在于， 所述 TCR的 β链可变域 在一个或多个下列位点中发生突变： β链可变区氨基酸第 11、 13、 19、 21、 53、 76、 89、 91、 或第 94位， 和 /或 β链 J基因短肽氨基酸倒数第 2位、 倒数第 4 位或倒数第 6位， 其中氨基酸位置编号按 IMGT中列出的位置编号。
9.如以上任一权利要求所述的 TCR，其特征在于，所述 TCR在 SEQ ID NO: 1 1 或 SEQ ID NO:30或 SEQ ID NO:32或 SEQ ID NO:34所示的 β链可变域的一个 或多个下列位点中发生突变： β链可变区氨基酸第 11、 13、 19、 21、 53、 76、 89、 91、 或第 94位， 和 /或 β链 J基因短肽氨基酸倒数第 2位、 倒数第 4位或 倒数第 6位， 其中氨基酸位置编号按 IMGT中列出的位置编号。
10.如以上任一权利要求所述的 TCR， 其特征在于， 所述 TCR的 a链可变 域包含选自下组的一个或多个氨基酸残基： 11L、 11M 或 11E;13V、 13R 或 13K;19V;21I;91L或 911;禾 Π 94V或 941;和 /或所述 TCR的 β链可变域包含选自下 组的一个或多个氨基酸残基： 11L或 11V;13V;19V;89L;91F或 91I;94V或 94L; β链 J基因倒数第 6位为 T;和 β链 J基因倒数第 4位为 M;其中氨基酸序列位置 编号按 IMGT中列出的位置编号。
11.如以上任一权利要求所述的 TCR， 其特征在于， 所述 TCR 的 α链和 / 或 β链可变域中暴露于表面的氨基酸残基发生突变。
12.如权利要求 11所述的 TCR， 其特征在于， 所述 TCR包含选自下组的一 个或多个 α链可变域氨基酸残基 4L;12N;16S;93N或 93R;97N;100G;105S;和 α 链 J基因倒数第 1位为 D;和 /或所述 TCR包含选自下组的一个或多个 β链可变 域氨基酸残基 4I;101L; β链 J基因倒数第 1位为 D;和 β链 J基因倒数第 3位为 E;其中氨基酸位置编号按 IMGT中列出的位置编号。
13.如以上任一权利要求所述的 TCR， 其特征在于， 所述 TCR包含下列 a 链可变域氨基酸序列之一 SEQ IDNO:15、 17、 35、 37、 39、 75、 76、 77、 78、 79、 80、 81、 82、 83、 84、 85、 97、 99、 101、 103、 105和 107; 和 /或
所述 TCR包含下列 β链可变域氨基酸序列之一 SEQ ID NO: 16、 18、 36、 38、 40、 86、 87、 88、 89、 90、 91、 92、 93、 94、 95、 96、 98、 100、 102、 104、 106禾口 108。
14. 如以上任一权利要求所述的 TCR， 其特征在于， 所述 TCR的 a链可变 域和 β链可变域组合选自下列组合之一：
(a) α链可变域氨基酸序列 SEQIDNO:15和 β链可变域氨基酸序列 SEQ ID NO:16;
(b) α链可变域氨基酸序列 85010^):17和3链可变域氨基酸序列 SEQ ID NO:18;
(c) α链可变域氨基酸序列 SEQ ID NO: 15和 β链可变域氨基酸序列 SEQ
ID NO:18; ( d) α链可变域氨基酸序列 850 10 ^):35和3链可变域氨基酸序列 SEQ ID NO:36;
( e) a链可变域氨基酸序列 SEQ ID NO:37和β链可变域氨基酸序列 SEQ ID NO:38;
(f) 链可变域氨基酸序列 SEQ ID NO:39和 β链可变域氨基酸序列 SEQ
ID NO:40;
(g ) a链可变域氨基酸序列 SEQ ID NO:75和 β链可变域氨基酸序列 SEQ ID NO:86;
(h) a链可变域氨基酸序列 SEQ ID NO:76和 β链可变域氨基酸序列 SEQ ID NO:87;
(i) a链可变域氨基酸序列 SEQ ID NO:77和 β链可变域氨基酸序列 SEQ ID NO:88;
(j ) a链可变域氨基酸序列 SEQ ID NO:78和 β链可变域氨基酸序列 SEQ ID NO:89;
(k) a链可变域氨基酸序列 SEQ ID NO:79和 β链可变域氨基酸序列 SEQ
ID NO:90;
(1) a链可变域氨基酸序列 SEQ ID NO:80和 β链可变域氨基酸序列 SEQ ID NO:91 ;
(m) a链可变域氨基酸序列 SEQ ID NO:81和 β链可变域氨基酸序列 SEQ ID NO:92;
(n) a链可变域氨基酸序列 SEQ ID NO:82和 β链可变域氨基酸序列 SEQ ID NO:93 ;
( o ) a链可变域氨基酸序列 SEQ ID NO:83和 β链可变域氨基酸序列 SEQ ID NO:94;
(p) 链可变域氨基酸序列 SEQ ID NO:84和 β链可变域氨基酸序列 SEQ
ID NO:95 ;
( q) 链可变域氨基酸序列 SEQ ID NO:85和 β链可变域氨基酸序列 SEQ ID NO:96;
(r) a链可变域氨基酸序列 SEQ ID NO:97和 β链可变域氨基酸序列 SEQ ID NO:98;
( s ) 链可变域氨基酸序列 SEQ ID NO:99和 β链可变域氨基酸序列 SEQ ID NO: 100 ;
( t) 链可变域氨基酸序列 SEQ ID NO: 101和 β链可变域氨基酸序列 SEQ ID NO: 102 ;
( u) 链可变域氨基酸序列 SEQ ID NO: 103和 β链可变域氨基酸序列 SEQ ID NO: 104 ;
( v) 链可变域氨基酸序列 SEQ ID NO: 105和 β链可变域氨基酸序列 SEQ ID NO: 106 ; 和
( w) a链可变域氨基酸序列 SEQ ID NO: 107 和 β链可变域氨基酸序列 SEQ ID NO: 108。
15.如权利要求 4所述的 TCR，其特征在于，所述 TCR是 α β异质二聚 TCR， 其具有 a和 β链恒定域序列，其半胱氨酸残基在所述 TCR的 a和 β链恒定域之 间形成二硫键。
16.如权利要求 15所述的 TCR， 其特征在于， 半胱氨酸残基在所述 TCR的 a和 β链恒定域之间形成人工二硫键。
17.如以上任一权利要求所述的 TCR， 其特征在于， 所述 TCR的 a链和 /或 β链的 C-或 Ν-末端结合有偶联物。
18. 如权利要求 17所述的 Τ细胞受体， 其特征在于， 与所述 Τ细胞受体 结合的偶联物为可检测标记物、 治疗剂、 ΡΚ修饰部分或任何这些物质的组合； 优选地， 与所述 Τ细胞受体结合的治疗剂为连接于所述 TCR 的 a或 β链的 C- 或 Ν-末端的抗 -CD3抗体。
19. 如权利要求 1所述的 TCR， 其特征在于， 所述的 T细胞受体中的突变 是通过噬菌体展示技术筛选出的。
20. 一种核酸分子， 其特征在于， 所述核酸分子包含编码权利要求 1-19中 任一所述的 TCR或其互补序列。
21. 一种载体，其特征在于，所述的载体含有权利要求 20所述的核酸分子。
22. 一种宿主细胞， 其特征在于， 所述的宿主细胞含有权利要求 21所述的 载体或染色体中整合有外源的权利要求 20所述的核酸分子。
23. 一种制备权利要求 1-19任一所述的 TCR的方法， 其特征在于， 包括 步骤：
(i) 培养权利要求 22所述的宿主细胞，从而表达权利要求 1-19所述的 TCR;
(ii) 分离或纯化出所述的 TCR。
24. 一种 T细胞受体复合物， 其特征在于， 所述的复合物含有一个或多个 权利要求 1-19中任一所述的 TCR。
25. 一种权利要求 1-19中任一所述的 TCR的用途， 其特征在于， 用于制 备治疗肿瘤、 病毒感染或自身免疫疾病的药物。
26. 一种药物组合物， 其特征在于， 含有药学上可接受的载体以及安全有 效量的权利要求 1-19中任一所述的 TCR。
27. 一种治疗疾病的方法， 其特征在于， 包括给需要治疗的对象施用权利 要求 1-19中任一所述的 TCR、 权利要求 24所述的 T细胞受体复合物或权利要 求 26所述的药物组合物；
较佳地， 所述的疾病包括： 肿瘤、 自身免疫疾病和病毒感染性疾病。
28. 一种分离的细胞， 其特征在于， 其呈递权利要求 1所述的 TCR。
29. 一种制备权利要求 1-19任一所述的 TCR的方法， 其特征在于， 包括 步骤：
(i)对 TCR的疏水芯区域引入氨基酸残基突变； 和
(ii)筛选稳定性显著提高的 TCR， 从而获得权利要求 1-19 中任一所述的
TCR。
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