WO2016070814A1 - 一种可溶的异质二聚t细胞受体及其制法和应用 - Google Patents

一种可溶的异质二聚t细胞受体及其制法和应用 Download PDF

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WO2016070814A1
WO2016070814A1 PCT/CN2015/093806 CN2015093806W WO2016070814A1 WO 2016070814 A1 WO2016070814 A1 WO 2016070814A1 CN 2015093806 W CN2015093806 W CN 2015093806W WO 2016070814 A1 WO2016070814 A1 WO 2016070814A1
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tcr
chain
exon
disulfide bond
cell receptor
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PCT/CN2015/093806
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English (en)
French (fr)
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李懿
樊辉
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广州市香雪制药股份有限公司
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Priority to CN201580060677.7A priority Critical patent/CN107223133B/zh
Priority to US15/524,370 priority patent/US11851469B2/en
Priority to EP15857032.5A priority patent/EP3216801B1/en
Priority to JP2017524406A priority patent/JP6415716B2/ja
Priority to CA2967073A priority patent/CA2967073A1/en
Publication of WO2016070814A1 publication Critical patent/WO2016070814A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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 [IGs], 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the invention belongs to the field of biomedicine, and in particular to a method and a method for preparing a highly stable soluble T cell receptor.
  • 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 abnormalities in the cell.
  • MHC major histocompatibility complex
  • APC antigen presenting cells
  • TCR On the T cell membrane, the TCR binds to the constant protein CD3 involved in signaling to form a complex.
  • TCR exists in many forms and is structurally similar, however 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 antibodies. 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 various Different short peptide fragments are added to the surface of APC cells.
  • TCR can also be developed for diagnosis and treatment.
  • 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 T cells (such as those that react with autoimmune peptide antigens). T cells).
  • soluble TCRs can also bind to other molecules (eg, anti-CD3 antibodies) to redirect T cells, thereby targeting cells that present a particular antigen.
  • soluble TCR in E. coli, when TCR is separated from the membrane, its instability and low protein yield are major obstacles to the development of therapeutic or diagnostic agents with TCR or fragments thereof.
  • TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region, so that for the expression of soluble TCR in bacteria, a high stability TCR that retains its ability to specifically bind to its original ligand (ie, pMHC) is one.
  • pMHC original ligand
  • the stability of the TCR can be improved by mutating the non-cysteine residue of the constant domain of the TCR to a cysteine to increase the stability of the TCR, but between the antibody constant domain and the TCR constant domain. Without such homology, this technique cannot be used to identify suitable sites for new interchain disulfide bonds between TCR constant domains.
  • a T cell receptor TCR
  • a cysteine residue is introduced in a constant region of the alpha chain and the beta chain of the TCR to form an artificial interchain disulfide bond
  • the T cell value of the T cell receptor containing an artificial interchain disulfide bond is ⁇ 45 °C.
  • substitution site for the cysteine residue introduced in the beta chain constant region of the TCR is selected from the group consisting of: TRBC1*01 or TRBC2*01 exon 1 of 54S, 19A and 20E.
  • the substitution site for the cysteine residue introduced in the alpha chain constant region of the TCR is selected from the group consisting of: 53R, 89P and 10Y of exon 1 of TRAC*01.
  • the TCR comprises (i) all or part of a TCR alpha chain other than its transmembrane domain, and (ii) all or part of a TCR beta chain other than its transmembrane domain, wherein (i) And (ii) both comprise a functional variable domain of the TCR chain and at least a portion of the constant domain.
  • the TCR is soluble.
  • the natural interchain disulfide bond is absent from the TCR.
  • the C-terminus of the native TCR is truncated in the TCR to remove a cysteine residue that forms the native interchain disulfide bond.
  • cysteine residue forming the natural interchain disulfide bond in the TCR is replaced with another residue.
  • an unpaired cysteine residue is absent in the beta chain constant region of the TCR.
  • the unpaired cysteine residue in the constant region of the TCR ⁇ chain is replaced with alanine or serine.
  • cysteine residue forming an artificial interchain disulfide bond is replaced by:
  • the T cell receptor comprises an extracellular alpha chain amino acid sequence selected from the group consisting of an extracellular beta chain amino acid sequence:
  • a conjugate is incorporated at the C- or N-terminus of the alpha chain and/or beta chain of the TCR.
  • the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modified moiety, or a combination thereof.
  • the detectable label comprises: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
  • MRI magnetic resonance imaging
  • CT electron computed tomography
  • 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 (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin) or any form of nanoparticles, and the like.
  • a radionuclide e.g, 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 (eg, DT-diaphorase
  • the therapeutic agent that binds to the T cell receptor is an anti-CD3 antibody linked to the C- or N-terminus of the alpha or beta chain of the TCR or any protein that specifically binds to CD3 , small molecule compounds or organic macromolecular compounds.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an alpha chain and/or a beta chain of a TCR of the first aspect 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 or genetically engineered engineered cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell, such as E. coli, yeast cells, CHO cells, and the like.
  • an isolated cell expressing the TCR of the first aspect of the invention in a fifth aspect of the invention, there is provided an isolated cell expressing the TCR of the first aspect of the invention.
  • a method for the preparation of the T cell receptor of the first aspect of the invention comprising the steps of:
  • a T cell receptor complex comprising one or more TCRs according to the first aspect of the invention.
  • the complex comprises a complex formed by binding of a T 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 TCR according to the first aspect of the invention for the preparation of a medicament for the treatment of a tumor, a viral infection or an autoimmune disease or for the preparation of a reagent for detecting an MHC-peptide complex.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a safe and effective amount of the TCR according to the first aspect of the invention, the cell of the fourth aspect of the invention or the invention
  • the T cell receptor complex of the seventh aspect is provided.
  • a method for treating a disease comprising administering a TCR according to the first aspect of the present invention, a cell of the fifth aspect of the present invention, and a seventh aspect of the present invention to a subject in need of treatment
  • the T cell receptor complex or the pharmaceutical composition of the ninth aspect of the invention comprising administering a TCR according to the first aspect of the present invention, a cell of the fifth aspect of the present invention, and a seventh aspect of the present invention.
  • the diseases include: tumors, autoimmune diseases, and viral infectious diseases.
  • Figure 1a and Figure 1b are the extracellular alpha-chain amino acid sequence of LC13TCR introduced into the cysteine at position 53 of exon 1 of TRAC*01 and 54th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • the extracellular beta chain amino acid sequence of the LC13 TCR of cysteine was introduced.
  • Figures 2a and 2b are the nucleotide sequences corresponding to the amino acids in Figures 1a and 1b, respectively.
  • Figure 3 is an elution curve of a gel filtration chromatography column after refolding of the TCR ⁇ and ⁇ chains shown in Figures 1a and 1b.
  • Figure 4 is a SEC map of the TCR alpha and beta chains shown in Figures 1a and 1b after refolding and protein purification.
  • Figure 5 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 1a and 1b.
  • Figure 6 is a graph showing the binding curves of different concentrations of LC13 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 1a and 1b.
  • Figure 7a and Figure 7b are the extracellular alpha-chain amino acid sequence of the 1G4 TCR introducing cysteine at position 53 of exon 1 of TRAC*01 and 54th of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • the extracellular beta chain amino acid sequence of the 1G4 TCR of cysteine was introduced.
  • Figures 8a and 8b are the nucleotide sequences corresponding to the amino acids in Figures 7a and 7b, respectively.
  • Figure 9 is an elution curve of a gel filtration chromatography column after refolding of the TCR ⁇ and ⁇ chains shown in Figures 7a and 7b.
  • Figure 10 is a SEC map of the TCR alpha and beta chains shown in Figures 7a and 7b after refolding and protein purification.
  • Figure 11 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 7a and 7b.
  • Figure 12 is a graph showing the binding curves of different concentrations of 1G4 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 7a and 7b.
  • Figure 13a and Figure 13b are the extracellular alpha chain amino acid sequence of JM22 TCR introducing cysteine at position 53 of exon 1 of TRAC*01 and 54th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • the extracellular beta chain amino acid sequence of the cysteine-derived JM22 TCR was introduced.
  • Figures 14a and 14b are the nucleotide sequences corresponding to the amino acids in Figures 13a and 13b, respectively.
  • Figure 15 is an elution curve of a gel filtration chromatography column after TCR ⁇ and ⁇ chain refolding shown in Figures 13a and 13b.
  • Figure 16 is a SEC map of the TCR alpha and beta chains shown in Figures 13a and 13b after refolding and protein purification.
  • Figure 17 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 13a and 13b.
  • Figure 18 is a graph showing the binding curves of different concentrations of JM22 TCR molecules to their corresponding antigens after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 13a and 13b.
  • Figure 19a and Figure 19b are the extracellular alpha chain amino acid sequence of the MGA3 TCR introduced into the cysteine at position 53 of exon 1 of TRAC*01 and 54th of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • Figure 20a and Figure 20b are the nucleotide sequences corresponding to the amino acids in Figures 19a and 19b, respectively.
  • Figure 21 is an elution curve of a gel filtration chromatography column after refolding of the TCR? and ? chains shown in Figures 19a and 19b.
  • Figure 22 is a SEC map of the TCR alpha and beta chains shown in Figures 19a and 19b after refolding and protein purification.
  • Figure 23 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 19a and 19b.
  • Figure 24 is a graph showing the binding curves of different concentrations of MGA3 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 19a and 19b.
  • Figure 25a and Figure 25b are the extracellular alpha chain amino acid sequence of the LC13 TCR introduced into the cysteine at position 89 of exon 1 of TRAC*01 and the 19th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • the extracellular beta chain amino acid sequence of the LC13 TCR of cysteine was introduced.
  • Figure 26a and Figure 26b are the nucleotide sequences corresponding to the amino acids in Figures 25a and 25b, respectively.
  • Figure 27 is an elution curve of a gel filtration chromatography column after refolding of the TCR ⁇ and ⁇ chains shown in Figures 25a and 25b.
  • Figure 28 is a SEC map of the TCR alpha and beta chains shown in Figures 25a and 25b after refolding and protein purification.
  • Figure 29 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 25a and 25b.
  • Figure 30 is a graph showing the binding curves of different concentrations of LC13 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 25a and 25b.
  • Figure 31a and Figure 31b show the extracellular alpha chain amino acid sequence of the 1G4 TCR introducing cysteine at position 89 of exon 1 of TRAC*01 and the 19th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • the extracellular beta chain amino acid sequence of the 1G4 TCR of cysteine was introduced.
  • Figures 32a and 32b are the nucleotide sequences corresponding to the amino acids in Figures 31a and 31b, respectively.
  • Figure 33 is an elution curve of a gel filtration chromatography column after refolding of the TCR ⁇ and ⁇ chains shown in Figures 31a and 31b.
  • Figure 34 is a SEC map of the TCR alpha and beta chains shown in Figures 31a and 31b after refolding and protein purification.
  • Figure 35 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 31a and 31b.
  • Figure 36 is a graph showing the binding curves of different concentrations of 1G4 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 31a and 31b.
  • Figure 37a and Figure 37b show the extracellular alpha-chain amino acid sequence of the JM22 TCR introduced into the cysteine at position 89 of exon 1 of TRAC*01 and the 19th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • the extracellular beta chain amino acid sequence of the cysteine-derived JM22 TCR was introduced.
  • Figure 38a and Figure 38b are the nucleotide sequences corresponding to the amino acids in Figures 37a and 37b, respectively.
  • Figure 39 is an elution curve of a gel filtration chromatography column after refolding of the TCR? and ? chains shown in Figs. 37a and 37b.
  • Figure 40 is a SEC map of the TCR alpha and beta chains shown in Figures 37a and 37b after refolding and protein purification.
  • Figure 41 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 37a and 37b.
  • Figure 42 is a graph showing the binding curves of different concentrations of JM22 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 37a and 37b.
  • Figure 43a and Figure 43b are the extracellular alpha chain amino acid sequence of the MGA3 TCR introduced into the cysteine at position 89 of exon 1 of TRAC*01 and the 19th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • Figure 44a and Figure 44b are the nucleotide sequences corresponding to the amino acids in Figures 43a and 43b, respectively.
  • Figure 45 is an elution curve of a gel filtration chromatography column after refolding of the TCR? and ? chains shown in Figures 43a and 43b.
  • Figure 46 is a SEC map of the TCR alpha and beta chains shown in Figures 43a and 43b after refolding and protein purification.
  • Figure 47 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 43a and 43b.
  • Figure 48 is a graph showing the binding curves of different concentrations of MGA3 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 43a and 43b.
  • Figure 49a and Figure 49b are the extracellular alpha-chain amino acid sequence of LC13TCR which introduces cysteine at position 10 of exon 1 of TRAC*01 and the 20th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • the extracellular beta chain amino acid sequence of the LC13 TCR of cysteine was introduced.
  • Figures 50a and 50b are the nucleotide sequences corresponding to the amino acids in Figures 49a and 49b, respectively.
  • Figure 51 is an elution curve of a gel filtration chromatography column after refolding of the TCR? and ? chains shown in Figures 49a and 49b.
  • Figure 52 is a SEC map of the TCR alpha and beta chains shown in Figures 49a and 49b after refolding and protein purification.
  • Figure 53 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 49a and 49b.
  • Figure 54 is a graph showing the binding curves of different concentrations of LC13 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 49a and 49b.
  • Figure 55a and Figure 55b are the extracellular alpha chain amino acid sequence of the 1G4 TCR introducing cysteine at position 10 of exon 1 of TRAC*01 and the 20th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • the extracellular beta chain amino acid sequence of the 1G4 TCR of cysteine was introduced.
  • Figure 56a and Figure 56b are the nucleotide sequences corresponding to the amino acids in Figures 49a and 49b, respectively.
  • Figure 57 is an elution curve of a gel filtration chromatography column after refolding of the TCR ⁇ and ⁇ chains shown in Figures 55a and 55b.
  • Figure 58 is a SEC map of the TCR alpha and beta chains shown in Figures 55a and 55b after refolding and protein purification.
  • Figure 59 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 55a and 55b.
  • Figure 60 is a graph showing the binding curves of different concentrations of 1G4 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 55a and 55b.
  • Figure 61a and Figure 61b are the extracellular alpha-chain amino acid sequence of JM22 TCR introduced into the cysteine at position 10 of exon 1 of TRAC*01 and the 20th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • the extracellular beta chain amino acid sequence of the cysteine-derived JM22 TCR was introduced.
  • Figures 62a and 62b are the nucleotide sequences corresponding to the amino acids in Figures 61a and 61b, respectively.
  • Figure 63 is an elution curve of a gel filtration chromatography column after refolding of the TCR? and ? chains shown in Fig. 61a and Fig. 61b.
  • Figure 64 is a SEC map of the TCR alpha and beta chains shown in Figure 61a and Figure 61b after refolding and protein purification.
  • Figure 65 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 61a and 61b.
  • Figure 66 is a graph showing the binding curves of different concentrations of JM22 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Fig. 61a and Fig. 61b.
  • Figure 67a and Figure 67b are the extracellular alpha chain amino acid sequence of the MGA3 TCR introduced into the cysteine at position 10 of exon 1 of TRAC*01 and the 20th position of exon 1 of TRBC1*01 or TRBC2*01, respectively.
  • Figures 68a and 68b are the nucleotide sequences corresponding to the amino acids in Figures 67a and 67b, respectively.
  • Figure 69 is an elution curve of a gel filtration chromatography column after refolding of the TCR? and ? chains shown in Figures 67a and 67b.
  • Figure 70 is a SEC map of the TCR alpha and beta chains shown in Figures 67a and 67b after refolding and protein purification.
  • Figure 71 is a DSC thermogram measured after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 67a and 67b.
  • Figure 72 is a graph showing the binding curves of different concentrations of MGA3 TCR molecules and their corresponding antigens obtained after TCR ⁇ and ⁇ chain refolding and protein purification shown in Figures 67a and 67b.
  • Figure 73 is a reduced and non-reduced gel of LC13 TCR molecule incorporating an artificial interchain disulfide bond, wherein lane 4 is a molecular weight marker.
  • Figure 74 is a reduced and non-reduced gel of a 1G4 TCR molecule incorporating an inter-chain disulfide bond, wherein lane 4 is a molecular weight marker.
  • Figure 75 is a reduced and non-reduced gel of JM22 TCR molecule incorporating an artificial interchain disulfide bond, wherein lane 4 is a molecular weight marker.
  • Figure 76 is a reduced and non-reduced gel map of an MGA3 TCR molecule incorporating an inter-chain disulfide bond, wherein lane 4 is a molecular weight marker.
  • the inventors have unexpectedly obtained a highly stable soluble T cell receptor having a Tm value greater than 45 ° C through extensive and intensive research. Specifically, the present inventors mutated many different sites in the ⁇ and ⁇ chains of TCR to cysteine to introduce an inter-chain disulfide bond, and obtained a large class of highly stable soluble T cells after extensive screening. body.
  • a specific site in the ⁇ and ⁇ chain constant domains of the TCR of the present invention is mutated to a cysteine to form a new interchain disulfide bond, and a TCR containing a new interchain disulfide bond has high stability and a Tm value thereof.
  • the invention also provides for the use of the TCR, and a process for its preparation.
  • TCR T cell receptor
  • the native TCR consists of two polypeptide chains, each in the alpha beta form or the gamma delta form. Each polypeptide has a near membrane constant domain and a distal membrane variable domain. Each constant domain and variable domain comprises an intrachain disulfide bond.
  • the extracellular constant domain of TCR has a membrane proximal region and an immunoglobulin region.
  • In natural TCR There is a set of disulfide bonds between the two strands of the membrane proximal region, which is referred to herein as "natural interchain disulfide bonds".
  • an inter-chain covalent disulfide bond which is artificially introduced and whose position is different from the position of a disulfide bond between natural chains is referred to as "artificial interchain disulfide bond".
  • the terms "polypeptide of the present invention”, “TCR of the present invention”, and “T cell receptor of the present invention” are used interchangeably and refer to a TCR containing an artificial interchain disulfide bond of the present invention.
  • the naming method of the TCR of the present invention adopts the naming manner of TCR in the International Immunogenetics Information System (IMGT). That is, in this system, "TRAC*01” represents the ⁇ chain constant domain of TCR, wherein “TR” represents a T cell receptor gene, “A” represents an ⁇ chain gene, C represents a constant region, and “01” represents Gene 1. Similarly, “TRBC1*01” or “TRBC2*01” represents a ⁇ -chain constant domain. There are two possible constant region genes “C1” and "C2" in the beta chain. The domain translated by each allele can be composed of genetic codes from several exons.
  • IMGT International Immunogenetics Information System
  • TCR constant domains given in IMGT for example, found in the public database of IMGT.
  • the 53rd position of the amino acid sequence given in TRAC*01 of IMGT is R, which is represented here as: 53R of exon 1 of TRAC*01, and so on.
  • the ⁇ chain of TCR has a unique constant domain, TRAC*01, and the two constant domains of the ⁇ chain differ only slightly.
  • TRBC1*01 has 4N, 5K and 37F in its exon, while TRBC2*01 is outside. There are 4K, 5N and 37Y in the exon.
  • the constant region of the ⁇ chain of the TCR molecule is TRBC1*01 or TRBC2*01 is substantially indistinguishable.
  • the spatial structure of the constant regions of different TCRs is also considered to be the same.
  • 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 refolding yield is higher, thermal stability is increased; more preferably, protein refolding yield is higher and/or thermal stability is increased.
  • the non-cysteine residue on each strand of the TCR can be mutated to a cysteine to form an artificial interchain disulfide bond.
  • the disulfide bond is preferably a constant region located in each of the TCR chains.
  • the site in which cysteine is introduced to form an artificial interchain disulfide bond is:
  • the TCR of the invention may comprise an entire constant domain (ie comprising extracellular and cytoplasmic domains) in addition to the transmembrane domain.
  • one or more cysteine residues forming a natural TCR interchain disulfide bond are preferably mutated to other amino acid residues that are not involved in disulfide bond formation.
  • the TCR of the invention may comprise a partial constant domain other than a transmembrane domain, in which case one or more half of the native TCR interchain disulfide bond is formed.
  • the cystine residue is mutated to other amino acid residues that are not involved in disulfide bond formation, or one or more of these residues are deleted.
  • the TCR is free of natural interchain disulfide bonds.
  • Deletion of the natural chain can be achieved by mutating a cysteine that forms a natural interchain disulfide bond to another amino acid or by truncating the corresponding strand so that it does not include a cysteine residue that forms a natural interchain disulfide bond. The purpose of the inter-disulfide bond.
  • the high stability TCR of the invention comprises a constant region of a C-terminally truncated native TCR alpha and beta strand, preferably a cysteine residue at a distance from the disulfide bond forming the natural chain Truncated at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acids to remove cysteine residues forming a natural interchain disulfide bond, such a TCR Contains no natural interchain disulfide bonds. It should be noted, however, that the natural interchain disulfide bond may also be included in the TCR of the present invention.
  • TCR chain has a cysteine forming a natural interchain disulfide bond for linking a TCR molecule having an artificial interchain disulfide bond of the present invention with another molecule.
  • a free unpaired cysteine residue is contained, and in the present invention, the cysteine is preferably mutated to another amino acid, such as a mutation to serine or alanine.
  • Each strand of the TCR of the invention also contains an intrachain disulfide bond.
  • the constant domain of TCR is not directly involved in the binding of TCR to pMHC.
  • the truncation of a certain number of amino acid residues at the C-terminus does not substantially affect the function of TCR, so the chains of the TCR of the present invention can be further short.
  • the binding affinity of the TCR of the invention to its corresponding antigen can be determined by any suitable method (in inverse proportion to the dissociation equilibrium constant KD). It should be understood that doubling the affinity of the TCR will result in a halving of KD.
  • the dissociation equilibrium constant KD of the TCR and its corresponding pMHC is determined by forteBIO Oke, as described in Example 4 of the present invention.
  • mutated forms 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 And/or substitution, 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 And/or substitution, 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 usually 1-5, preferably 1-3, more preferably 1-2, optimally 1 amino acid deletions, insertions, and And/or substitution, 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.
  • the function of the protein is generally
  • the present invention identifies suitable sites in the TCR chain that are capable of mutating to cysteine to form an inter-chain disulfide bond to stabilize the TCR.
  • the TCR of the present invention not only comprises the human TCR, but is also obtainable by a suitable site provided by those skilled in the art according to the present invention for the soluble high stability TCR of other species. For example, one skilled in the art can determine the residue to be mutated by looking for the following motif in the corresponding TCR strand (the bolded and underlined residue is the residue used to mutate to cysteine):
  • ⁇ chain constant region 19A EVAVFEPSE EISHTQKATL.
  • TCR chain of other species may not have 100% identical regions to the above motif, one skilled in the art will be able to identify the equivalent portion of the corresponding TCR based on the above motif to obtain the cysteine residue to be mutated.
  • ClustalW obtained from the European Institute of Bioinformatics can be used to compare TCR chains of other species with the above motifs to obtain corresponding sites.
  • the present invention encompasses artificially stable disulfide-linked human stable alpha beta TCRs, as well as other mammalian inter-chain disulfide-linked alpha beta TCRs including, but not limited to, goats, sheep, pigs, mice, and large mouse.
  • a site in which a cysteine residue is introduced into a mouse to form an artificial interchain disulfide bond can be identified as follows:
  • 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), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V).
  • the invention also includes 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 an amino acid residue, Or (iii) a polypeptide formed by fusing a TCR of the invention with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by the additional amino acid sequence fused to the polypeptide sequence (and A fusion protein formed by fusion of 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 conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • the invention also provides analogs of the TCRs of the invention.
  • the difference between these analogs and the original TCR polypeptide of the present invention may be a difference in amino acid sequence, a difference in a modified form which does not affect the sequence, or a combination thereof.
  • Analogs also include analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, gamma-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 derived 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, phosphorus Acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, carbamates or other conventional "prodrugs".
  • the polypeptides of the invention may be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the invention comprises two, three, four or more T cell receptor molecules linked to another molecule.
  • the invention also relates to polynucleotides encoding TCRs of the invention.
  • the full-length nucleotide sequence of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification, recombinant methods, or synthetic methods.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vectors or coding sequences of the invention.
  • the invention also relates to polynucleotides encoding TCRs of the invention, including polynucleotides encoding alpha and/or beta chains of the T cell receptors of the invention.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide can be SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35
  • the coding region sequences shown in 37, 39, 41, 43, 45, and 47 are the same or degenerate variants.
  • degenerate variant in the present invention means having the coding with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 a protein of the amino acid sequence shown by 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, but a nucleic acid sequence different from the sequence of the corresponding coding region described above.
  • the full-length nucleotide sequence of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification, recombinant methods, or synthetic methods. At present, it has been possible to obtain a DNA sequence encoding a polypeptide of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as 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.
  • a cysteine residue forming a new interchain disulfide bond may be by any suitable method including, but not limited to, those based on polymerase chain reaction (PCR), cloning or non-dependency based on restriction enzymes. Connected clone (LIC) method. Many standard molecular biology textbooks detail these methods. For more details on polymerase chain reaction (PCR) mutagenesis and cloning based on restriction enzymes, see Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (Third Edition) CSHL Publishing house. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6).
  • 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 soluble, highly stable TCR of the invention can be obtained by expression in the form of inclusion bodies in bacteria such as E. coli and in vitro refolding.
  • 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 TCR, multivalent TCR complexes and cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in any suitable form (depending on the method desired for administration to a 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.
  • the TCR of the present invention may be used alone or in combination or coupled to a conjugate.
  • the conjugate includes a detectable label, a therapeutic agent, a PK (protein kinase) modified moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agents, or capable of producing detectable products Enzyme.
  • 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 metastasis reviews 24, 539); 2. Biotoxins (Chaudhary et al, 1989) , Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines (Gillies et al., 1992, Proceedings of the National Academy of Sciences (PNAS) 89 , 1428; Card et al, 2004, Cancer Immunology and Immunotherapy 53, 345, Halin et al, 2003, Cancer Research 63, 3202); 4.
  • Antibody Fc fragment (Mosquera et al, 2005) , The Journal Of Immunology 174, 4381); 5. Antibody scFv fragment (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 (Gene the Rap) 11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nanomagnetic particles; 10. prodrug activating enzymes (eg, DT-diaphorase ( DTD) or biphenyl hydrolase-like protein (BPHL); 11. chemotherapeutic agent (eg, cisplatin) and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the antibody or fragment thereof to be combined with the TCR of the present invention includes an anti-T cell or an NK-cell determining antibody, such as an anti-CD3 or an anti-CD28 or an anti-CD16 antibody, and the binding of the above antibody or a fragment thereof to the TCR can effect the effector cell. Targeting better targets target cells.
  • 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 can be formulated as 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 subject to be prevented or treated may be an animal; especially a human.
  • composition of the present invention When used for actual treatment, various different agents may be used depending on the use.
  • Type of pharmaceutical composition Preferably, 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 Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and solubilizers, 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 Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and solubilizers, 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 include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, and lactic acid polymer.
  • a lactic acid-glycolic acid copolymer or the like is preferably exemplified by a biodegradable polymer such as a lactic acid polymer and a lactic acid-glycolic acid copolymer.
  • 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 drug or a 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.
  • autoimmune disease For cancer, localization to 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 is already present 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 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.
  • the highly stable T 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 for the diagnosis and treatment of diseases.
  • the T cell receptor of the present invention has high stability, can be renatured, refolded, purified, and is capable of specifically binding to its original ligand.
  • the T cell receptor of the present invention has a high Tm value and a Tm value of more than 45 °C.
  • the T cell receptor of the present invention has high protein refolding yield, is easy to prepare on a large scale, and is advantageous in reducing production cost.
  • the present invention will be further described in detail below with reference to specific embodiments. It should be understood that due to the identity of the amino acid sequence and the spatial structure of the constant region of different TCRs, the introduction of the artificial interchain disulfide bond of the present invention into the constant region of a TCR to obtain a highly stable TCR molecule is sufficient to illustrate the artificial chain of the present invention. The role of the inter-disulfide bond.
  • the following examples further illustrate the introduction of the artificial interchain disulfide bond of the present invention into a TCR molecule in combination with several different molecules, and can obtain a soluble TCR with good refolding effect, high refolding yield, and high stability. It is to be understood that the examples are not intended to limit the scope of the invention.
  • Example 1 Primer design and PCR mutation of LC13 molecule introducing an artificial interchain disulfide bond at position 53 of exon 1 of TRACT*01 and position 54 of exon 1 of TRBC1*01 or TRBC2*01
  • TRACE molecule LC13 for the antigen short peptide HLA-B4405: EEYLKAWTF (SEQ ID NO.: 49) was mutated to cysteine, and its TRBC1* The serine at position 54 of 01 or TRBC2*01 exon 1 is mutated to cysteine to form an artificial interchain disulfide bond.
  • the expression plasmid containing the LC13TCR ⁇ and ⁇ chain genes was subjected to the following mutations using the above ⁇ and ⁇ chain primers, respectively.
  • the gene of interest carrying the template strand was digested with NcoI and NotI and ligated with the pET28a (Novagen) vector digested with NcoI and NotI.
  • the ligation product was transformed into E. coli DH5 ⁇ (Tiangen), and the kanamycin-containing LB plate was coated, cultured at 37 ° C overnight, and clones were picked for PCR screening, and the positive recombinants were sequenced.
  • the expression plasmid containing TCR ⁇ and ⁇ chain was separately transformed into Escherichia coli strain BL21 (DE3), and LB plate (Kanamycin 50 ⁇ g/ml) was applied and cultured at 37 ° C overnight. On the next day, the clones were inoculated into 10 ml of LB liquid medium (kanamycin 50 ⁇ g/ml) for 2-3 h, and inoculated into 1 L of LB medium (kanamycin 50 ⁇ g/ml) at a volume ratio of 1:100. The culture was carried out until the OD 600 was 0.5-0.8, and then the expression of the protein of interest was induced using IPTG at a final concentration of 1 mM.
  • the cells were harvested by centrifugation at 6000 rpm for 10 min.
  • the cells were washed once in PBS buffer, and the cells were dispensed, and the cells corresponding to 200 ml of the bacterial culture were lysed with 5 ml of BugBuster Master Mix (Novagen), and the inclusion bodies were collected by centrifugation at 6000 g for 15 minutes.
  • a detergent wash was then performed 4 times to remove cell debris and membrane components.
  • the inclusion bodies are then washed with a buffer such as PBS to remove detergent and salt.
  • the inclusion bodies were dissolved in a buffer solution containing 6 M guanidine hydrochloride, and the inclusion body concentration was measured, and the package was divided and stored at -80 ° C for cryopreservation.
  • the inclusion bodies were taken out from the -80 ° C ultra-low temperature freezer and thawed, and dithiothreitol (DTT) was added to a final concentration of 10 mM, and incubated at 37 ° C for 30 min to 1 hour to ensure complete opening of the disulfide bond.
  • the inclusion body sample solution (15 mg ⁇ chain and 10 mg ⁇ chain) was then dropped into 200 ml 4 ° C pre-cooled refolding buffer (100 mM Tris pH 8.1, 400 mM L-arginine, 2 mM EDTA, 5 M urea, 6.5 mM cysteamine hydrochloride). And 1.87 mM cystamine dihydrochloride), stir slowly at 4 ° C for about 30 minutes.
  • the renaturation solution was dialyzed against 8 volumes of pre-cooled H 2 O for 16-20 hours. It was further dialyzed twice with 8 volumes of 20 mM Tris pH 8.0, and dialysis was continued at 4 ° C for about 8 hours. After dialysis, the sample was filtered and subjected to the following purification.
  • the dialyzed refolded material (in 20 mM Tris pH 8.0) was eluted with a gradient gradient of 0-600 mM NaCl using an GE Hitrap Q anion exchange chromatography prepacked column (GE Healthcare) on an AKTA Purifier (GE Healthcare). Each component was analyzed by Coomassie brilliant blue stained SDS-PAGE and then combined.
  • the first step of the purified sample solution was concentrated for purification in this step, and the protein was purified by Superdex 100160/300GL gel filtration chromatography prepacked column (GE Healthcare) pre-equilibrated in PBS buffer, eluting the TCR molecule LC13.
  • the curves are shown in Figure 3, respectively.
  • the peak fractions were analyzed by Coomassie blue stained SDS-PAGE, and the reduced and non-reduced gel maps are shown in lanes 2 and 6 of Figure 73. According to the elution peak and the gel map, the eluted single peak is an artificial TCS-bonded soluble TCR molecule, which is stably present in the SDS gel and is reduced to form separate ⁇ and ⁇ chains.
  • the eluted fraction was tested for purity by HPLC.
  • the conditions were: Agilent 1260, column Bio SEC-3 (300A, ⁇ 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.
  • the SEC (space exclusion chromatography) spectrum of the TCR molecule LC13 is shown in FIG.
  • the HPLC elution peak of the TCR molecule containing the artificial interchain disulfide bond of the present invention is single and symmetrical.
  • the method for calculating the refolding yield of TCR protein in the present invention is as follows:
  • Protein refolding yield (%) 100 * The amount of protein (mg) obtained after purification was completed / the amount (mg) of inclusion bodies used for renaturation.
  • the protein refolding yield of LC13TCR which forms an artificial interchain disulfide bond between the 53rd position of exon 1 of TRACT*01 and the 54th position of exon 1 of TRBC1*01 or TRBC2*01 It is 43.30%. Very good yield High, indicating that the soluble TCR molecule having the artificial interchain disulfide bond of the present invention is very stable.
  • Example 2 1 ml of the LC13 TCR protein (concentration: 0.5 mg/ml) obtained in Example 2 was dialyzed into PBS, and the TCR protein was subjected to thermal stability measurement using a differential scanning calorimeter (Nano DSC) of TA (waters), USA. The temperature range of detection was 10-90 ° C and the heating rate was 1 ° C/min. The dialysis solution PBS was used as a control, and the baseline was measured 3 times. After the baseline was stabilized, the protein sample was further examined. After collecting the data, the Tm value of the TCR was measured using the analysis software TA_DSC_NanoAnalyze, and the DSC thermogram was obtained.
  • TA_DSC_NanoAnalyze 1 ml of the LC13 TCR protein obtained in Example 2 was dialyzed into PBS, and the TCR protein was subjected to thermal stability measurement using a differential scanning calorimeter (Nano DSC) of TA (waters
  • the DSC thermogram of the LC13TCR of the present invention containing an artificial interchain disulfide bond obtained by in vitro soluble expression is shown in Fig. 5, and its Tm value can reach 55.82 °C.
  • the thermogram can reflect that at room temperature, even at a temperature of 41-43 ° C, the TCR molecule containing the artificial interchain disulfide bond of the present invention can maintain proper folding and maintain the desired activity, indicating that the stability is high.
  • the fortBIO Oke real-time analysis system was used to detect the binding activity of the TCR protein to its corresponding antigen pMHC complex.
  • a biotinylated pMHC complex of about 2 nm was immobilized on the surface of the SA sensor, and 0.05 mM of biotin was flowed through the chip at a flow rate of 10 ⁇ L/min for 120 s to block the remaining binding sites of streptavidin.
  • the affinity was determined by kinetic analysis and the TCR protein was diluted to 5 different concentrations (typically 64, 32, 16, 8, 4, 0 uM) using PBST buffer (PBS + 0.005% Tween 20, pH 7.4). , the affinity of the corresponding pMHC was determined.
  • the kinetic parameters were calculated using the Evaluation software in a 1:1 combined model using the Evaluation software.
  • E. coli bacterial solution inducing expression of heavy or light chain 100 ml of E. coli bacterial solution inducing expression of heavy or light chain was collected, and the cells were washed once with 8000 g of PBS at 10 ° C for 10 min, and then resuspended by vigorous shaking with 5 ml of BugBuster Master Mix Extraction Reagents (Merck). Incubate for 20 min at room temperature, then centrifuge at 6000 g for 15 min at 4 ° C, discard the supernatant, and collect inclusion bodies.
  • the above-mentioned inclusion weight was suspended in 5 ml BugBuster Master Mix, and incubated at room temperature for 5 min; 30 ml of BugBuster diluted 10 times, mixed, centrifuged at 6000 g for 15 min at 4 ° C; the supernatant was discarded, and 30 ml of BugBuster resuspended inclusion body was diluted 10 times.
  • the short peptide required for the synthesis was dissolved in DMSO to a concentration of 20 mg/ml.
  • the inclusion bodies of the light and heavy chains were dissolved with 8 M urea, 20 mM Tris pH 8.0, 10 mM DTT, and further denatured by adding 3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA before renaturation.
  • the short peptide was added to the refolding buffer (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, at 25 mg/L (final concentration), 0.2 mM PMSF, cooled to 4 ° C), then add 20 mg / L light chain and 90 mg / L heavy chain (final concentration, heavy chain added three times, 8h / time), renaturation at 4 ° C for at least 3 days By the time of completion, SDS-PAGE can be used to detect renaturation.
  • the refolding buffer 0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, at 25 mg/L (final concentration), 0.2 mM PMSF, cooled to 4 ° C
  • 20 mg / L light chain and 90 mg / L heavy chain
  • the renaturation buffer was replaced with 10 volumes of 20 mM Tris pH 8.0 for dialysis, and at least two buffers were exchanged to substantially reduce the ionic strength of the solution.
  • the protein solution was filtered through a 0.45 ⁇ m cellulose acetate filter and then loaded onto a HiTrap Q HP (GE General Electric Company) anion exchange column (5 ml bed volume).
  • the protein was eluted using a linear gradient of 0-400 mM NaCl prepared by an Akta Purifier (GE General Electric Company), 20 mM Tris pH 8.0, pMHC was eluted at approximately 250 mM NaCl, peak fractions were collected, and purity was determined by SDS-PAGE.
  • the purified pMHC molecule was concentrated using a Millipore ultrafiltration tube while the buffer was replaced with 20 mM Tris pH 8.0, followed by biotinylation reagent 0.05M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml BirA
  • the enzyme (GST-BirA) was incubated overnight at room temperature and SDS-PAGE was used to determine if biotinylation was complete.
  • Biotinylated labeled pMHC molecules were concentrated to 1 ml using a Millipore ultrafiltration tube, biotinylated pMHC was purified by gel filtration chromatography, and HiPrepTM was pre-equilibrated with filtered PBS using an Akta Purifier (GE General Electric). A 16/60 S200 HR column (GE General Electric Company) was loaded with 1 ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. The biotinylated pMHC molecule appeared as a single peak elution at about 55 ml.
  • the protein-containing fractions were pooled, concentrated using a Millipore ultrafiltration tube, protein concentration was determined by BCA method (Thermo), and biotinylated pMHC molecules were dispensed at -80 °C by adding protease inhibitor cocktail (Roche).
  • the binding curves of different concentrations of LC13 molecules to their corresponding antigens are shown in Figure 6.
  • the KD value is 10.5 ⁇ M. It can be seen from the binding curve that the decrease in concentration does not affect the binding of the TCR molecule of the present invention to its corresponding antigen, low concentration.
  • the TCR molecule exhibits the same binding activity as the high concentration TCR molecule, and it can also be seen from the side that the TCR having the artificial interchain disulfide bond of the present invention is relatively stable.
  • the forteBIO Oke real-time analysis system was used to detect the specificity of the TCR protein for its corresponding antigen pMHC complex.
  • Six different biotinylated antigens (concentration 0.5 ⁇ M) were loaded onto the surface of six SA sensors; then, interacted with each TCR protein to be tested (concentration 2-20 ⁇ M); finally, analyzed The signal produced by the interaction.
  • Example 5 forms an artificial interchain disulfide bond 1G4 molecule at position 53 of exon 1 of TRACT*01 and at position 54 of exon 1 of TRBC1*01 or TRBC2*01
  • the primers and the PCR step described in Example 1 were used for mutation.
  • the ⁇ -chain and ⁇ -chain extracellular amino acid sequences of the TCR molecule 1G4 were shown in Figures 7a and 7b, respectively, and the corresponding nucleotide sequences are shown in Figure 8a, respectively.
  • the introduced cysteine residues are indicated by bolded and underlined letters.
  • the 1G4 TCR was expressed, refolded and purified by the method described in Example 2.
  • the elution curve after the second purification was as shown in FIG.
  • the peak fractions were analyzed by Coomassie blue stained SDS-PAGE, and the reduced and non-reduced gel maps are shown in lanes 2 and 6 of Figure 74.
  • the eluted single peak is an artificial TCS-bonded soluble TCR molecule, which is stably present in the SDS gel and is reduced to form separate ⁇ and ⁇ chains.
  • the purity of the 1G4 TCR protein was determined according to the method described in Example 2 and the yield was calculated.
  • the SEC spectrum was obtained as shown in Fig. 10.
  • the HPLC elution peak of the 1G4 TCR molecule containing the artificial interchain disulfide bond of the present invention was single and symmetrical. Its yield reached 40%.
  • the stability of 1G4 TCR containing an artificial interchain disulfide bond was measured by the method described in Example 3.
  • the DSC thermogram is shown in Fig. 11, and the Tm value was 55.21 °C.
  • the thermogram can be reflected at room temperature or even temperature At 47-48 ° C, the TCR molecules containing the artificial interchain disulfide bond of the present invention can maintain proper folding and maintain the desired activity, indicating that the stability is high.
  • the binding activity and specificity of the 1G4 TCR protein and its corresponding antigen pMHC complex were detected by the method described in Example 4.
  • the binding curve was obtained as shown in Fig. 12, and the KD value was 6.96 ⁇ M; from the binding curve, the concentration was The decrease does not affect the binding of the stable TCR molecule of the present invention to its corresponding antigen, the low concentration of the TCR molecule exhibits the same binding activity as the high concentration TCR molecule, and the TCR having the interchain disulfide bond of the present invention can also be described from the side. More stable.
  • the TCR molecule of the present invention is also highly specific and binds only to its corresponding pMHC complex, while other unrelated antigens include B4405: EEYLKAWTF (SEQ ID NO.: 49), A0201: GILGFVFTL (SEQ ID NO. :56), A0101: EVDPIGHLY (SEQ ID NO.: 57), A1101: SSCSSCPLSK (SEQ ID NO.: 58) and A2402: KYKDYFPVI (SEQ ID NO.: 59) did not bind.
  • Example 6 JM22 molecule which forms an artificial interchain disulfide bond at position 53 of exon 1 of TRACT*01 and position 54 of exon 1 of TRBC1*01 or TRBC2*01
  • the primers and the PCR step described in Example 1 were used for mutation.
  • the ⁇ -chain and ⁇ -chain extracellular amino acid sequences of the TCR molecule JM22 were shown in Figures 13a and 13b, respectively, and the corresponding nucleotide sequences are shown in Figure 14a, respectively.
  • the introduced cysteine residues are indicated by bolded and underlined letters.
  • the JM22 TCR was expressed, refolded and purified by the method described in Example 2.
  • the elution curve after the second purification was as shown in FIG.
  • the peak fraction was analyzed by Coomassie-stained SDS-PAGE, and the reduced and non-reduced gel images are shown in lanes 2 and 6 of Figure 75.
  • the eluted single peak is an artificial TCS-bonded soluble TCR molecule, which is stably present in the SDS gel and is reduced to form separate ⁇ and ⁇ chains.
  • the purity of the JM22 TCR protein was determined according to the method described in Example 2 and the yield was calculated.
  • the SEC spectrum was obtained as shown in Fig. 16.
  • the HPLC elution peak of the JM22TCR molecule containing the artificial interchain disulfide bond of the present invention was single and symmetrical. The yield reached 31.65%.
  • the stability of JM22TCR containing an artificial interchain disulfide bond was measured by the method described in Example 3.
  • the DSC thermogram is shown in Fig. 17, and the Tm value was 49.06 °C.
  • the thermogram can reflect that at room temperature, even at a temperature of 40 ° C, the TCR molecule containing the artificial interchain disulfide bond of the present invention can maintain proper folding and maintain the desired activity, indicating that the stability is high.
  • the binding activity and specificity of the JM22 TCR protein and its corresponding antigen pMHC complex were detected by the method described in Example 4.
  • the binding curve was obtained as shown in Fig. 18, and the KD value was 7.14 ⁇ M. From the binding curve, the concentration was observed. The decrease does not affect the binding of the stable TCR molecule of the present invention to its corresponding antigen, the low concentration of the TCR molecule exhibits the same binding activity as the high concentration TCR molecule, and the TCR having the interchain disulfide bond of the present invention can also be described from the side. More stable.
  • the TCR molecule of the present invention is also highly specific and binds only to its corresponding pMHC complex, while other unrelated antigens include B4405: EEYLKAWTF (SEQ ID NO.: 49), A0201: SLLMWITQC (SEQ ID NO. :55), A0101: EVDPIGHLY (SEQ ID NO.: 57), A1101: SSCSSCPLSK (SEQ ID NO.: 58) and A2402: KYKDYFPVI (SEQ ID NO.: 59) did not bind.
  • Example 7 MGA3 molecule forming an artificial interchain disulfide bond at position 53 of exon 1 of TRACT*01 and position 54 of exon 1 of TRBC1*01 or TRBC2*01
  • the TCR molecule MGA3 (for the antigen short peptide HLA-A1: EVDPIGHLY (SEQ ID NO.: 57), MageA3
  • the tumor-specific antigen is mutated to cysteine at position 53 of exon 1 of TRAC*01, and the serine at position 54 of exon 1 of TRBC1*01 or TRBC2*01 is mutated to cysteine. Amino acid to form an artificial interchain disulfide bond.
  • the primers and the PCR step described in Example 1 were used for mutation.
  • the ⁇ -chain and ⁇ -chain extracellular amino acid sequences of the TCR molecule MGA3 were shown in Figures 19a and 19b, respectively, and the corresponding nucleotide sequences are shown in Figure 20a, respectively.
  • the introduced cysteine residues are indicated by bolded and underlined letters.
  • the MGA3 TCR was expressed, refolded and purified by the method described in Example 2.
  • the elution curve after the second purification was as shown in FIG.
  • the peak fractions were analyzed by Coomassie blue stained SDS-PAGE, and the reduced and non-reduced gel maps are shown in lanes 2 and 6 of Figure 76.
  • the eluted single peak is an artificial TCS-bonded soluble TCR molecule, which is stably present in the SDS gel and is reduced to form separate ⁇ and ⁇ chains.
  • the purity of the MGA3 TCR protein was determined according to the method described in Example 2 and the yield was calculated.
  • the SEC spectrum was obtained as shown in Fig. 22.
  • the HPLC elution peak of the MGA3 TCR molecule containing the artificial interchain disulfide bond of the present invention was single and symmetrical. The yield reached 30.14%.
  • the stability of MGA3TCR containing an artificial interchain disulfide bond was measured by the method described in Example 3.
  • the DSC thermogram is shown in Fig. 23, and its Tm value is 53.86 °C.
  • the thermogram can reflect that at room temperature, even at a temperature of 45-46 ° C, the TCR molecule containing the artificial interchain disulfide bond of the present invention can maintain proper folding and maintain the desired activity, indicating that the stability is high.
  • the binding activity and specificity of the MGA3 TCR protein and its corresponding antigen pMHC complex were detected by the method described in Example 4.
  • the binding curve was obtained as shown in Fig. 24, and the KD value was 1.42 ⁇ M. From the binding curve, the concentration was observed. The decrease does not affect the binding of the stable TCR molecule of the present invention to its corresponding antigen, the low concentration of the TCR molecule exhibits the same binding activity as the high concentration TCR molecule, and the TCR having the interchain disulfide bond of the present invention can also be described from the side. More stable.
  • the TCR molecule of the present invention is also highly specific and binds only to its corresponding pMHC complex, while other unrelated antigens include B4405: EEYLKAWTF (SEQ ID NO.: 49), A0201: SLLMWITQC (SEQ ID NO.: 55). ), A0201: GILGFVFTL (SEQ ID NO.: 56), A1101: SSCSSCPLSK (SEQ ID NO.: 58), and A2402: KYKDYFPVI (SEQ ID NO.: 59) did not bind.
  • Example 8 Performance test of a molecule forming an artificial interchain disulfide bond at position 89 of exon 1 of TRACT*01 and position 19 of exon 1 of TRBC1*01 or TRBC2*01
  • the 89th proline of the TRAC molecule LC13, 1G4, JM22 and MGA3 of the TRAC*01 exon 1 was mutated to cysteine, and the TRBC1*01 or TRBC2*01 exon 1 was 19th.
  • the alanine is mutated to cysteine to form an artificial interchain disulfide bond.
  • the designed primers are as follows:
  • the TCR was subjected to PCR, renaturation and performance testing in the manner described in Examples 1 to 4.
  • the amino acid sequences of the ⁇ chain and ⁇ chain of the TCR molecule LC13 are shown in Figures 25a and 25b, respectively, and the corresponding nucleotide sequences are shown in Figures 26a and 26b, respectively, and the cysteine residues introduced are added. Thick and underlined Line letters are indicated.
  • the elution curve and the gel chart are shown in lanes 3 and 7 of Figures 27 and 73, respectively.
  • the HPLC elution peak is single and symmetric as shown in FIG.
  • the protein refolding yield was also quite high, reaching 42.82%.
  • Its Tm value is 55.65 ° C, and the corresponding DSC spectrum is shown in FIG. 29 .
  • the binding curve of the LC13 molecule to its corresponding antigen is shown in Figure 30, and the KD value was 10.3 ⁇ M.
  • the ⁇ -chain and ⁇ -chain extracellular amino acid sequences of the TCR molecule 1G4 are shown in Figures 31a and 31b, respectively, and the corresponding nucleotide sequences are shown in Figures 32a and 32b, respectively, and the introduced cysteine residues are added. Thick and underlined letters.
  • the elution curve and the gel chart are shown in lanes 3 and 7 of Figures 33 and 74, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield was also quite high, reaching 48%. Its Tm value is 55.82 ° C, and the corresponding DSC spectrum is shown in FIG.
  • the binding curve of the 1G4 molecule to its corresponding antigen is shown in Figure 36, and the KD value was 6.63 ⁇ M.
  • the ⁇ -chain and ⁇ -chain extracellular amino acid sequences of the TCR molecule JM22 are shown in Figures 37a and 37b, respectively, and the corresponding nucleotide sequences are shown in Figures 38a and 38b, respectively, and the introduced cysteine residues are added. Thick and underlined letters.
  • the elution profile and the gel chart are shown in lanes 3 and 7 of Figures 39 and 75, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 14.93%. Its Tm value is 51.08 ° C, and the corresponding DSC spectrum is shown in Figure 41.
  • the binding curve of the JM22 molecule to its corresponding antigen is shown in Figure 42 with a KD value of 7.61 ⁇ M.
  • the ⁇ -chain and ⁇ -chain extracellular amino acid sequences of the TCR molecule MGA3 are shown in Figures 43a and 43b, respectively, and the corresponding nucleotide sequences are shown in Figures 44a and 44b, respectively, and the introduced cysteine residues are added. Thick and underlined letters.
  • the elution curve and the gel chart are shown in lanes 3 and 7 of Fig. 45 and Fig. 76, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 13.76%. Its Tm value is 54.49 ° C, and the corresponding DSC spectrum is shown in Figure 47.
  • the binding curve of the MGA3 molecule to its corresponding antigen is shown in Figure 48, and the KD value was 2.04 ⁇ M.
  • the eluted peak component is a disulfide-bonded soluble TCR molecule of the artificial chain of the present invention, which is stably present in the SDS gel and forms a separate ⁇ after reduction. And beta chain.
  • the protein refolding yield is also high.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bond of the present invention are also high, both being greater than 45 ° C, indicating that they can maintain proper folding at a higher temperature and maintain the desired activity, indicating that High stability.
  • the binding curve of the TCR molecule to its original ligand shows that the decrease in the concentration of TCR does not affect the binding to its ligand, and it can also be described from the side that the TCR molecule having the interchain disulfide bond of the present invention is stable. In the specificity test, these TCR molecules that introduce inter-chain disulfide bonds also showed good specificity.
  • Example 9 Performance test of a molecule forming an artificial interchain disulfide bond at position 10 of exon 1 of TRACT*01 and at position 20 of exon 1 of TRBC1*01 or TRBC2*01
  • the tyrosine at position 10 of the TRC molecule LC13, 1G4, JM22 and MGA3 of the TRAC*01 exon 1 was mutated to cysteine, respectively, and the 20th of its TRBC1*01 or TRBC2*01 exon 1 was The glutamic acid is mutated to cysteine to form an artificial interchain disulfide bond.
  • the designed primers are as follows:
  • the TCR was subjected to PCR, renaturation and performance testing in the manner described in Examples 1 to 4.
  • the amino acid sequences of the ⁇ chain and ⁇ chain of the TCR molecule LC13 are shown in Figures 49a and 49b, respectively, and the corresponding nucleotide sequences are shown in Figures 50a and 50b, respectively, and the cysteine residues introduced are added. Thick and underlined letters.
  • the elution curve and the gel chart are shown in lanes 1 and 5 of Fig. 51 and Fig. 73, respectively.
  • the HPLC elution peak is single and symmetric as shown in FIG.
  • the protein refolding yield reached 16.19%.
  • Its Tm value is 50.42 ° C, and the corresponding DSC spectrum is shown in FIG. 53 .
  • the binding curve of the LC13 molecule to its corresponding antigen is shown in Figure 54 with a KD value of 10 ⁇ M.
  • the amino acid sequences of the ⁇ chain and ⁇ chain of the TCR molecule 1G4 are shown in Figures 55a and 55b, respectively, and the corresponding nucleotide sequences are shown in Figures 56a and 56b, respectively, and the introduced cysteine residues are added. Thick and underlined letters.
  • the elution curve and the gel chart are shown in lanes 1 and 5 of Figs. 57 and 74, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 29%. Its Tm value is 54.68 ° C, and the corresponding DSC spectrum is shown in Figure 59.
  • the binding curve of the 1G4 molecule to its corresponding antigen is shown in Figure 60, and the KD value is 6.68 ⁇ M.
  • the amino acid sequences of the ⁇ chain and ⁇ chain of the TCR molecule JM22 are shown in Fig. 61a and Fig. 61b, respectively, and the corresponding nucleotide sequences are shown in Fig. 62a and 62b, respectively, and the introduced cysteine residue is added. Thick and underlined letters.
  • the elution curve and the gel chart are shown in lanes 1 and 5 of Figs. 63 and 75, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 10.50%. Its Tm value is 49.95 ° C, and the corresponding DSC spectrum is shown in FIG. 65 .
  • the binding curve of the JM22 molecule to its corresponding antigen is shown in Figure 66, and the KD value is 5.54 ⁇ M.
  • the ⁇ -chain and ⁇ -chain extracellular amino acid sequences of the TCR molecule MGA3 are shown in Figures 67a and 67b, respectively, and the corresponding nucleotide sequences are shown in Figures 68a and 68b, respectively, and the introduced cysteine residues are added. Thick and underlined letters.
  • the elution curve and the gel chart are shown in lanes 1 and 5 of Fig. 69 and Fig. 76, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG. Its protein refolding yield reached 4.53%. Its Tm value is 53.38 ° C, and the corresponding DSC spectrum is shown in Fig. 71.
  • the binding curve of the MGA3 molecule to its corresponding antigen is shown in Figure 72, and the KD value is 3.45 ⁇ M.
  • the eluted main peak component is a disulfide-bonded soluble TCR molecule of the artificial chain of the present invention, which is stably present in the SDS gel and forms a separate ⁇ after reduction. And beta chain.
  • the protein refolding yield is also high.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bond of the present invention are also high, both being greater than 45 ° C, indicating that they can maintain proper folding at a higher temperature and maintain the desired activity, indicating that High stability.
  • the binding curve of the TCR molecule to its original ligand shows that the decrease in the concentration of TCR does not affect the binding to its ligand, and it can also be described from the side that the TCR molecule having the interchain disulfide bond of the present invention is stable. In the specificity test, these TCR molecules that introduce inter-chain disulfide bonds also showed good specificity.
  • the TCR molecule of the present invention obtained by introducing the artificial interchain disulfide bond of the present invention into the TCR constant region has high stability and a Tm value of more than 45 °C. And it can be well renatured, refolded and purified, and the refolding yield is high while being able to specifically bind to its original ligand.

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Abstract

本发明提供了一种高稳定性的T细胞受体（TCR），所述TCR包含（ⅰ）除其跨膜结构域以外的全部或部分TCRα链，和（ⅱ）除其跨膜结构域以外的全部或部分TCRβ链，其中（ⅰ）和（ⅱ）均包含TCR链的功能性可变结构域和至少一部分恒定结构域。人工链间二硫键连接所述TCRα与β链的恒定区，并且所述T细胞受体的Tm值大于或等于45℃。

Description

一种可溶的异质二聚T细胞受体及其制法和应用 技术领域
本发明属于生物医药领域，具体地说，本发明涉及一种高稳定的可溶性T细胞受体其制法、和应用。
背景技术
仅仅有两种类型的分子能够以特异性的方式识别抗原。其中一种是免疫球蛋白或抗体；另一种是T细胞受体(TCR)，它是由α链/β链或者γ链/δ链以异二聚体形式存在的细胞膜表面的糖蛋白。免疫系统的TCR总谱的组成是在胸腺中通过V(D)J重组，然后进行阳性和阴性选择而产生的。在外周环境中，TCR介导了T细胞对主组织相容性复合体-肽复合物(pMHC)的特异性识别，因此其对免疫系统的细胞免疫功能是至关重要的。
TCR是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的唯一受体，这种外源肽或内源肽可能会是细胞出现异常的唯一迹象。在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
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II类分子配体也是免疫球蛋白超家族的蛋白质但对于抗原的呈递具有特异性，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。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刺激性化合物)输送到呈递特异性抗原的细胞，或者用来抑制T细胞(如那些与自身免疫性肽抗原进行反应的T细胞)。另外，可溶性TCR还可与其他分子(如，抗-CD3抗体)结合来重新定向T细胞，从而使其靶向呈递特定抗原的细胞。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。
天然存在的TCR是一种膜蛋白，通过其跨膜区得以稳定，因此对于在细菌中表达可溶性TCR而言，获得保持与其原配体(即pMHC)特异性结合能力的高稳定性TCR是一件非常困难的事情，如专利文献WO99/18129中所述。有些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，尽管这样的TCR可以被TCR特异性的抗体识别，但是产率很低，并且低浓度时不能识别主组织相容性复合体-肽复合物，说明其很容易变性，不够稳定。
Reiter等，在免疫学(Immunity)，1995,2:281-287中描述了二硫键稳定的TCR α和β可变结构域的可溶性分子的构建，其中的一个可变结构域与截短形式的假单胞菌外毒素(PE38)连接。TCR可变结构域内新二硫键的位置通过与抗体可变结构域的同源性得以鉴定(参见Brinkmann等(1993)，美国国家科学院院刊(Proc.Natl.Acad.Sci.USA)90:7538-7542,和Reiter等(1994)生物化学(Biochemistry)33:5451-5459)。可以通过将TCR各条链恒定域的非半胱氨酸残基突变为半胱氨酸来形成人工链间二硫键而提高TCR的稳定性，但在抗体恒定结构域和TCR恒定结构域间不存在此类同源性，该技术不能用于鉴定TCR恒定结构域间新链间二硫键的合适位点。
理论上在TCR中能够形成人工链间二硫键的位点非常多，但找到在TCR中形成人工链间二硫键的合适位点使得含有人工链间二硫键的TCR都能够被成功复性、重折叠来得到高产量、高稳定的，并且具有与其原配体特异性结合活性的TCR是非常困难的。本领域技术人员致力于开发含有人工链间二硫键的，能够被很好地复性、重折叠、纯化且具有高稳定性，复性收率高同时能够与其原配体特异性结合的TCR。
发明内容
本发明的目的在于提供一种高稳定的可溶性T细胞受体其制法、和应用。
本发明的第一方面，提供了一种T细胞受体(TCR)，在所述TCR的α链和β链的恒定区中引入半胱氨酸残基以形成人工链间二硫键，并且所述含人工链间二硫键的T细胞受体的Tm值≥45℃。
在另一优选例中,在所述TCR的β链恒定区中引入的半胱氨酸残基的替换位点选自：TRBC1*01或TRBC2*01外显子1的54S、19A和20E。
在另一优选例中,在所述TCR的α链恒定区中引入的半胱氨酸残基的替换位点选自：TRAC*01外显子1的53R、89P和10Y。
在另一优选例中,所述TCR包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的功能性可变结构域和至少一部分恒定结构域。
在另一优选例中,所述TCR是可溶的。
在另一优选例中,所述TCR中不存在天然链间二硫键。
在另一优选例中,在所述TCR中将天然TCR的C末端截短以去除形成所述天然链间二硫键的半胱氨酸残基。
在另一优选例中,在所述TCR中形成所述天然链间二硫键的半胱氨酸残基被替换为另一残基。
在另一优选例中,在所述TCR的β链恒定区中不存在未配对的半胱氨酸残基。
在另一优选例中,所述TCRβ链恒定区中未配对的半胱氨酸残基被替换为丙氨酸或丝氨酸。
在另一优选例中，形成人工链间二硫键的半胱氨酸残基替换了：
TRAC*01外显子1的53R和TRBC1*01或TRBC2*01外显子1的54S；
TRAC*01外显子1的89P和TRBC1*01或TRBC2*01外显子1的19A；或
TRAC*01外显子1的10Y和TRBC1*01或TRBC2*01外显子1的20E。
在另一优选例中，所述T细胞受体包含选自下组的胞外α链氨基酸序列和胞外β链氨基酸序列：
SEQ ID NO.:2所示的胞外α链氨基酸序列和SEQ ID NO.:4所示的胞外β链氨基酸序列；
SEQ ID NO.:6所示的胞外α链氨基酸序列和SEQ ID NO.:8所示的胞外β链氨基酸序列；
SEQ ID NO.:10所示的胞外α链氨基酸序列和SEQ ID NO.:12所示的胞外β链氨基酸序列；
SEQ ID NO.:14所示的胞外α链氨基酸序列和SEQ ID NO.:16所示的胞外β链氨基酸序列；
SEQ ID NO.:18所示的胞外α链氨基酸序列和SEQ ID NO.:20所示的胞外β链氨基酸序列；
SEQ ID NO.:22所示的胞外α链氨基酸序列和SEQ ID NO.:24所示的胞外β链氨基酸序列；
SEQ ID NO.:26所示的胞外α链氨基酸序列和SEQ ID NO.:28所示的胞外β链氨基酸序列；
SEQ ID NO.:30所示的胞外α链氨基酸序列和SEQ ID NO.:32所示的胞外β链氨基酸序列；
SEQ ID NO.:34所示的胞外α链氨基酸序列和SEQ ID NO.:36所示的胞外β链氨基酸序列；
SEQ ID NO.:38所示的胞外α链氨基酸序列和SEQ ID NO.:40所示的胞外β链氨基酸序列；
SEQ ID NO.:42所示的胞外α链氨基酸序列和SEQ ID NO.:44所示的胞外β链氨基酸序列；
SEQ ID NO.:46所示的胞外α链氨基酸序列和SEQ ID NO.:48所示的胞外β链氨基酸序列。
在另一优选例中，在所述TCR的α链和/或β链的C-或N-末端结合有偶联物。
在另一优选例中，与所述TCR结合的偶联物为可检测标记物、治疗剂、PK修饰部分或其组合。
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，与所述T细胞受体结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体或任何与CD3特异性结合的蛋白质、小分子化合物或有机大分子化合物。
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所述的TCR的α链和/或β链的核酸序列或其互补序列。
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。
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，所述的宿主细胞或遗传改造的工程细胞选自：原核细胞和真核细胞，例如大肠杆菌、酵母细胞、CHO细胞等。
本发明的第五方面，提供了一种分离的细胞，其表达本发明第一方面所述的TCR。
本发明的第六方面，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，从而表达本发明第一方面所述的T细胞受体的α链和/或β链；
(ii)分离或纯化出所述的α链和/或β链；
(iii)重折叠所述的α链和/或β链，获得所述T细胞受体。
本发明的第七方面，提供了一种T细胞受体复合物，所述的复合物含有一个或多个本发明第一方面所述的TCR。
在另一优选例中，所述的复合物包括本发明的T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。
本发明的第八方面，提供了本发明第一方面所述的TCR的用途，用于制备治疗肿瘤、病毒感染或自身免疫疾病的药物或用于制备检测MHC-肽复合体的试剂。
本发明的第九方面，提供了一种药物组合物，其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所述的TCR、本发明第四方面所述的细胞或本发明第七方面所述的T细胞受体复合物。
本发明的第十方面，提供了一种治疗疾病的方法，其中包括给需要治疗的对象施用本发明第一方面所述的TCR、本发明第五方面所述的细胞、本发明第七方面所述的T细胞受体复合物或本发明第九方面所述的药物组合物；
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。
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1a和图1b分别是在TRAC*01外显子1的第53位引入半胱氨酸的LC13TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第54位引入半胱氨酸的LC13TCR的胞外β链氨基酸序列。
图2a和图2b分别是图1a和图1b中氨基酸所对应的核苷酸序列。
图3为图1a和图1b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图4为图1a和图1b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图5为图1a和图1b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图6为图1a和图1b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的LC13TCR分子与其相应抗原的结合曲线。
图7a和图7b分别是在TRAC*01外显子1的第53位引入半胱氨酸的1G4TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第54位引入半胱氨酸的1G4TCR的胞外β链氨基酸序列。
图8a和图8b分别是图7a和图7b中氨基酸所对应的核苷酸序列。
图9为图7a和图7b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图10为图7a和图7b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图11为图7a和图7b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图12为图7a和图7b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的1G4TCR分子与其相应抗原的结合曲线。
图13a和图13b分别是在TRAC*01外显子1的第53位引入半胱氨酸的JM22TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第54位引入半胱氨酸的JM22TCR的胞外β链氨基酸序列。
图14a和图14b分别是图13a和图13b中氨基酸所对应的核苷酸序列。
图15为图13a和图13b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图16为图13a和图13b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图17为图13a和图13b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图18为图13a和图13b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的JM22TCR分子与其相应抗原的结合曲线。
图19a和图19b分别是在TRAC*01外显子1的第53位引入半胱氨酸的MGA3TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第54位引入半胱氨酸的MGA3TCR的胞外β链氨基酸序列。
图20a和图20b分别是图19a和图19b中氨基酸所对应的核苷酸序列。
图21为图19a和图19b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图22为图19a和图19b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图23为图19a和图19b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图24为图19a和图19b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的MGA3TCR分子与其相应抗原的结合曲线。
图25a和图25b分别是在TRAC*01外显子1的第89位引入半胱氨酸的LC13TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第19位引入半胱氨酸的LC13TCR的胞外β链氨基酸序列。
图26a和图26b分别是图25a和图25b中氨基酸所对应的核苷酸序列。
图27为图25a和图25b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图28为图25a和图25b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图29为图25a和图25b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图30为图25a和图25b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的LC13TCR分子与其相应抗原的结合曲线。
图31a和图31b分别是在TRAC*01外显子1的第89位引入半胱氨酸的1G4TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第19位引入半胱氨酸的1G4TCR的胞外β链氨基酸序列。
图32a和图32b分别是图31a和图31b中氨基酸所对应的核苷酸序列。
图33为图31a和图31b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图34为图31a和图31b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图35为图31a和图31b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图36为图31a和图31b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的1G4TCR分子与其相应抗原的结合曲线。
图37a和图37b分别是在TRAC*01外显子1的第89位引入半胱氨酸的JM22TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第19位引入半胱氨酸的JM22TCR的胞外β链氨基酸序列。
图38a和图38b分别是图37a和图37b中氨基酸所对应的核苷酸序列。
图39为图37a和图37b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图40为图37a和图37b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图41为图37a和图37b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图42为图37a和图37b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的JM22TCR分子与其相应抗原的结合曲线。
图43a和图43b分别是在TRAC*01外显子1的第89位引入半胱氨酸的MGA3TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第19位引入半胱氨酸的MGA3TCR的胞外β链氨基酸序列。
图44a和图44b分别是图43a和图43b中氨基酸所对应的核苷酸序列。
图45为图43a和图43b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图46为图43a和图43b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图47为图43a和图43b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图48为图43a和图43b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的MGA3TCR分子与其相应抗原的结合曲线。
图49a和图49b分别是在TRAC*01外显子1的第10位引入半胱氨酸的LC13TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第20位引入半胱氨酸的LC13TCR的胞外β链氨基酸序列。
图50a和图50b分别是图49a和图49b中氨基酸所对应的核苷酸序列。
图51为图49a和图49b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图52为图49a和图49b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图53为图49a和图49b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图54为图49a和图49b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的LC13TCR分子与其相应抗原的结合曲线。
图55a和图55b分别是在TRAC*01外显子1的第10位引入半胱氨酸的1G4TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第20位引入半胱氨酸的1G4TCR的胞外β链氨基酸序列。
图56a和图56b分别是图49a和图49b中氨基酸所对应的核苷酸序列。
图57为图55a和图55b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图58为图55a和图55b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图59为图55a和图55b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图60为图55a和图55b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的1G4TCR分子与其相应抗原的结合曲线。
图61a和图61b分别是在TRAC*01外显子1的第10位引入半胱氨酸的JM22TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第20位引入半胱氨酸的JM22TCR的胞外β链氨基酸序列。
图62a和图62b分别是图61a和图61b中氨基酸所对应的核苷酸序列。
图63为图61a和图61b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图64为图61a和图61b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图65为图61a和图61b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图66为图61a和图61b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的JM22TCR分子与其相应抗原的结合曲线。
图67a和图67b分别是在TRAC*01外显子1的第10位引入半胱氨酸的MGA3TCR的胞外α链氨基酸序列和在TRBC1*01或TRBC2*01外显子1的第20位引入半胱氨酸的MGA3TCR的胞外β链氨基酸序列。
图68a和图68b分别是图67a和图67b中氨基酸所对应的核苷酸序列。
图69为图67a和图67b中所示TCRα与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图70为图67a和图67b中所示TCRα与β链经重折叠及蛋白纯化后的SEC图谱。
图71为图67a和图67b中所示TCRα与β链经重折叠及蛋白纯化后测得的DSC热谱图。
图72为图67a和图67b中所示TCRα与β链经重折叠及蛋白纯化后所得不同浓度的MGA3TCR分子与其相应抗原的结合曲线。
图73为引入人工链间二硫键的LC13TCR分子的还原与非还原胶图，其中泳道4为分子量标记。
图74为引入人工链间二硫键的1G4TCR分子的还原与非还原胶图，其中泳道4为分子量标记。
图75为引入人工链间二硫键的JM22TCR分子的还原与非还原胶图，其中泳道4为分子量标记。
图76为引入人工链间二硫键的MGA3TCR分子的还原与非还原胶图，其中泳道4为分子量标记。
具体实施方式
本发明人通过广泛而深入的研究，意外地获得一种Tm值大于45℃的高稳定性可溶T细胞受体。具体地，本发明人将TCR的α和β链中的很多个不同位点突变为半胱氨酸以引入人工链间二硫键，经过大量筛选获得了一类高稳定性可溶T细胞受体。将本发明TCR的α和β链恒定域中特定位点突变为半胱氨酸以形成新的链间二硫键，含有新的人工链间二硫键的TCR具有高稳定性，其Tm值大于45℃，并且能够被很好地复性、重折叠并被纯化，复性收率高同时能够与其原配体特异性结合。本发明还提供了所述TCR的用途，及其制备方法。
T细胞受体(TCR)
天然的TCR由两条多肽链组成，分别为αβ形式或γδ形式。每一条多肽均具有一近膜恒定结构域和一远膜的可变结构域。每一恒定结构域和可变结构域中均包含一链内二硫键。TCR的胞外恒定结构域具有一近膜区和一免疫球蛋白区。在天然TCR 的近膜区的两条链间存在一组二硫键，本发明中称为“天然链间二硫键”。在本发明中，将人工引入的，位置与天然链间二硫键的位置不同的链间共价二硫键称为“人工链间二硫键”。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，都指含有本发明人工链间二硫键的TCR。
本发明TCR的命名方式采用国际免疫遗传学信息系统(IMGT)中对TCR的命名方式。即，在该系统中，“TRAC*01”表示TCR的α链恒定结构域，其中“TR”表示T细胞受体基因，“A”表示α链基因，C表示恒定区，“01”表示等位基因1。同样地，“TRBC1*01”或“TRBC2*01”表示β链恒定结构域。在β链中存在两个可能的恒定区基因“C1”和“C2”。由每一等位基因编码而翻译的结构域可由来自几个外显子的遗传密码组成。因此，本领域的技术人员广为知晓并可得到IMGT中给出的TCR恒定结构域的序列，例如可在IMGT的公开数据库中找到。IMGT的TRAC*01中给出的氨基酸序列的第53位为R，在此表示为：TRAC*01外显子1的53R，其他以此类推。TCR的α链具有唯一的恒定结构域TRAC*01，β链的两种恒定结构域仅有微小差别，TRBC1*01在其外显子中具有4N、5K和37F，而TRBC2*01在其外显子中具有4K、5N和37Y。因此，TCR分子β链的恒定区为TRBC1*01或为TRBC2*01基本没有区别。综上，由于不同TCR的恒定区氨基酸序列的固定性，不同的TCR其恒定区的空间结构也认为是相同的。
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；更佳地，是指蛋白复性收率更高和/或热稳定性增加。
可以将TCR各条链上的非半胱氨酸残基突变为半胱氨酸来形成人工链间二硫键。该二硫键优选为位于TCR各条链的恒定区。
在本发明较佳地实施方式中，引入半胱氨酸以形成人工链间二硫键的位点为：
TRAC*01外显子1的53R和TRBC1*01或TRBC2*01外显子1的54S；
TRAC*01外显子1的89P和TRBC1*01或TRBC2*01外显子1的19A；或
TRAC*01外显子1的10Y和TRBC1*01或TRBC2*01外显子1的20E。
在本发明的一个较佳地实施方式中，本发明的TCR可以包含除跨膜结构域外的完整的恒定结构域(即包含胞外和胞质结构域)。在这种情况下，形成天然TCR链间二硫键的一个或多个半胱氨酸残基优选突变为不参与二硫键形成的其他氨基酸残基。
在本发明的另一个较佳地实施方式中，本发明TCR可以包含除跨膜结构域以外的部分恒定结构域，在这种情况下，形成天然TCR链间二硫键的一个或多个半胱氨酸残基突变为不参与二硫键形成的其他氨基酸残基，或将这些残基中的一个或多个进行缺失。
在本发明的一个较佳地实施方式中，所述TCR不存在天然链间二硫键。可以通过将形成天然链间二硫键的半胱氨酸突变为其他氨基酸或者将相应链截短以使其不包括形成天然链间二硫键的半胱氨酸残基从而来达到缺失天然链间二硫键的目的。
在本发明的一个优选例中，本发明的高稳定性TCR包含C末端截短的天然TCRα和β链的恒定区，优选地，可在距形成天然链间二硫键的半胱氨酸残基1、2、3、4、5、6、7、8、9或10个或更多个氨基酸处截短，以去掉形成天然链间二硫键的半胱氨酸残基，这样的TCR不含天然链间二硫键。但应当指出，本发明的TCR中也可包含天然的链间二硫键。应注意，在某些情况下，仅一条TCR链具有形成天然链间二硫键的半胱氨酸，该半胱氨酸用于连接具有本发明人工链间二硫键的TCR分子与另外的分子。在TCR的β链中含有一个游离的未配对半胱氨酸残基，在本发明中优选将该半胱氨酸突变为另一氨基酸，如突变为丝氨酸或丙氨酸。本发明TCR的各条链也包含其链内二硫键。
应理解，TCR的恒定结构域并不直接参与TCR与pMHC的结合，在C末端截短一定数量的氨基酸残基基本不会对TCR的功能产生影响，因此本发明TCR的各条链还可以更短。可通过任何合适的方法测定本发明TCR与其相应抗原的结合亲和力(与解离平衡常数KD成反比)。应了解，TCR的亲和力翻倍将导致KD减半。在本发明的优选例中，TCR与其相应pMHC的解离平衡常数KD通过forteBIO Oke进行测定，如在本发明实施例4中所述。
并非TCR链中每个氨基酸残基对其抗原特异性和功能性都至关重要，因此，可在本发明TCR链中引入适量的突变而不影响其抗原特异性和功能性。其他突变形式包括(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。
本发明鉴定出了TCR链中能够突变为半胱氨酸来形成人工链间二硫键以使TCR稳定的合适位点。本发明的TCR不仅包含人类的TCR，对于其他物种的可溶高稳定性TCR，本领域技术人员可根据本发明提供的合适位点，也同样能够获得。例如，本领域技术人员可通过在相应的TCR链中寻找下列基序来确定待突变的残基(加粗并带下划线的残基为用以突变成半胱氨酸的残基)：
α链恒定区10Y:    IQNPDPAV
Figure PCTCN2015093806-appb-000001
QLRDSKSSDKS
α链恒定区53R：    ITDKTVLDM
Figure PCTCN2015093806-appb-000002
SMDFKSNSAV
α链恒定区89P：    SIIPEDTFFCS
Figure PCTCN2015093806-appb-000003
ESSSAAAL
β链恒定区20E:    EVAVFEPSEA
Figure PCTCN2015093806-appb-000004
ISHTQKATL
β链恒定区54S:    WWVNGKEVH
Figure PCTCN2015093806-appb-000005
GVSTDPQPLK和
β链恒定区19A:    EVAVFEPSE
Figure PCTCN2015093806-appb-000006
EISHTQKATL。
虽然其他物种的TCR链可能与以上基序并不具有100％相同的区域，但本领域技术人员能够根据上述基序鉴定出相应TCR的等同部分而得到待突变的半胱氨酸残基。比如，可利用欧洲生物信息学学院网站获得的ClustalW来将其他物种的TCR链与以上基序进行比较，来得到相应的位点。
本发明包含人工链间二硫键连接的人类的稳定性αβTCR，以及其他哺乳动物的人工链间二硫键连接的αβTCR，所述哺乳动物包括但不限于山羊、绵羊、猪、小鼠和大鼠。例如，根据本发明，可以鉴定出在小鼠中引入半胱氨酸残基以形成人工链间二硫键的位点(以加粗并带下划线的字母表示)如下：
人的α链的10Y的鼠的等同物：IQNPEPAV
Figure PCTCN2015093806-appb-000007
QLKDPRSQDSTLCLF
人的α链的53R的鼠的等同物:GTFITDKTVLDM
Figure PCTCN2015093806-appb-000008
AMDSKSNGA
人的α链的89P的鼠的等同物:QDIFKETNATY
Figure PCTCN2015093806-appb-000009
SS
人的β链的20E的鼠的等同物：FPPEVAVFEPSEA
Figure PCTCN2015093806-appb-000010
ISHTQKATLVCLAT
人的β链的54S鼠的等同物:LSWWVNGKEVH
Figure PCTCN2015093806-appb-000011
GVSTDPQAYKESN
人的β链的19A的鼠的等同物:FPPEVAVFEPSE
Figure PCTCN2015093806-appb-000012
EISHTQKATLVCLAT。
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)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
本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(ii)在一个或多个氨基酸残基中具有取代基团的多肽， 或(iii)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6His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 PCTCN2015093806-appb-000013
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、γ-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。
本发明还涉及编码本发明TCR的多核苷酸。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DNA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。
编码序列
本发明还涉及编码本发明TCR的多核苷酸，包括编码本发明所述的T细胞受体的α链和/或β链的多核苷酸。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。例如，编码成熟多肽的编码区序列可以与SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47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:2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48所示氨基酸序列的蛋白质，但与上述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DNA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
形成新的人工链间二硫键的半胱氨酸残基的引入可采用任何合适的方法，包括但不限于依据聚合酶链式反应(PCR)的那些、依据限制性酶的克隆或不依赖连接的克隆(LIC)方法。许多标准分子生物学教材详述了这些方法。聚合酶链式反应(PCR)诱变和依据限制性酶的克隆的更多细节可参见Sambrook和Russell,(2001)分子克隆-实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)CSHL出版社。LIC方法的更多信息可见(Rashtchian,(1995)Curr Opin Biotechnol 6(1):30-6)。
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。
优选地，可通过在细菌如大肠杆菌中以包涵体形式表达并进行体外重折叠来获得本发明的可溶性、高稳定TCR。
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。
本发明的TCR可以单独使用，也可与偶联物结合或偶联。所述偶联物包括可检测标记物、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明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 metastasis reviews)24，539)；2.生物毒素(Chaudhary等，1989，自然(Nature)339，394；Epel等，2002，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51，565)；3.细胞因子(Gillies等，1992，美国国家科学院院刊(PNAS)89，1428；Card等，2004，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53，345；Halin等，2003，癌症研究(Cancer Research)63，3202)；4.抗体Fc片段(Mosquera等，2005，免疫学杂志(The Journal Of Immunology)174，4381)；5.抗体scFv片段(Zhu等，1995，癌症国际期刊(International Journal of Cancer)62,319)；6.金纳米颗粒/纳米棒(Lapotko等，2005，癌症通信(Cancer letters)239，36；Huang等，2006，美国化学学会杂志(Journal of the American Chemical Society)128，2115)；7.病毒颗粒(Peng等，2004，基因治疗(Gene therapy)11，1234)；8.脂质体(Mamot等，2005，癌症研究(Cancer research)65，11631)；9.纳米磁粒；10.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；11.化疗剂(例如，顺铂)等。
与本发明TCR结合的抗体或其片段包括抗-T细胞或NK-细胞决定抗体，如抗-CD3或抗-CD28或抗-CD16抗体，上述抗体或其片段与TCR的结合能够对效应细胞进行定向更好地靶向靶细胞。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在雷明顿药物科学(Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991))中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括但并不限于：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂、及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，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(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本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刺激物的效果。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。
工业应用性
本发明的高稳定性T细胞受体，可用于研究TCR与pMHC(肽-主组织相容性复合体)之间的相互作用及用于疾病的诊断和治疗等目的。
本发明的主要优点在于：
(1)本发明的T细胞受体具有高稳定性，能够被很好地复性、重折叠、纯化同时能够与其原配体特异性结合。
(2)本发明的T细胞受体具有较高的Tm值，Tm值大于45℃。
(3)本发明的T细胞受体的蛋白复性收率高，易于大规模制备，并有利于降低生产成本。
下面结合具体实施例，进一步详陈本发明。应理解，由于不同TCR的恒定区氨基酸序列和空间结构的相同性，将本发明的人工链间二硫键引入到一种TCR的恒定区中得到高稳定的TCR分子就足以说明本发明人工链间二硫键的作用。下面的实施例结合几种不同的分子进一步阐述将本发明的人工链间二硫键引入到TCR分子中，能够得到重折叠效果好、复性收率高、稳定性高的可溶TCR。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，否则百分比和份数按重量计算。本发明实施例中所用的实验材料如无特殊说明均可从市售渠道获得。
实施例1在TRAC*01外显子1的第53位和TRBC1*01或TRBC2*01外显子1的第54位引入人工链间二硫键的LC13分子的引物设计和PCR突变
将TCR分子LC13(针对抗原短肽HLA-B4405：EEYLKAWTF(SEQ ID NO.:49))的TRAC*01外显子1的第53位精氨酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第54位丝氨酸突变为半胱氨酸，以形成人工链间二硫键。
上述TCR的TRAC*01外显子1的第53位精氨酸突变为半胱氨酸时，所设计的引物如下：
5’-3’
Figure PCTCN2015093806-appb-000014
上述TCR的TRBC1*01或TRBC2*01外显子1的第54位丝氨酸突变为半胱氨酸时，所设计的引物如下：
5’-3’
Figure PCTCN2015093806-appb-000015
进行PCR的步骤如下：
含LC13TCRα和β链基因的表达质粒分别用上述α和β链引物进行如下突变。每个PCR点突变反应中，将10-30ng的质粒DNA与5μL 10×KOD plus缓冲液、5μL 2.5mM dNTP Mix、3μL 2mM MgSO4、1单位的KOD plus聚合酶(东洋纺上海生物科技有限公司)，10μM的上、下游引物各1μL，最终以H2O补充至50μL。混匀后，至于Bio-Rad PCR仪中反应。94℃2min初始变性之后，进行18个循环的扩增(94℃15sec变性、55℃30sec退火和68℃6min延伸)。然后用10单位的DpnⅠ限制酶(New England Biolabs)37℃消化1小时。将10μL消化产物转化到感受态E.coli DH5α细菌中并在37℃下生长16小时。挑取单克隆在5mL LB+卡纳青霉素中过夜培养。根据生产商的使用说明书应用Zyppy质粒小提试剂盒(ZYMO RESEARCH)纯化质粒DNA，并送至Invitrogen公司测序验证，正确突变后用于下游表达。
经突变后的TCR分子LC13的α链与β链胞外氨基酸序列分别如图1a和1b所示，其对应的核苷酸序列分别如图2a和2b所示，引入的半胱氨酸残基以加粗并带下划线并带下划线字母表示。
实施例2TCR的表达、重折叠和纯化及其结果测定
TCR蛋白的表达
将携带模板链的目的基因经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a(Novagen)载体连接。连接产物转化至E.coli DH5α(Tiangen)，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取克隆进行PCR筛选，对阳性重组子进行测序。
将含有TCRα与β链的表达质粒分别转化入大肠杆菌菌株BL21(DE3)中，涂布LB平板(卡那霉素50μg/ml)置于37℃培养过夜。次日，挑克隆接种至10ml LB液体培养基(卡那霉素50μg/ml)培养2-3h，按体积比1:100接种至1L LB培养基(卡那霉素50μg/ml)中，继续培养至OD600为0.5-0.8，然后使用终浓度为1mM的IPTG诱导目的蛋白的表达。诱导4小时以后，以6000rpm离心10min收获细胞。PBS缓冲液洗涤菌体一次，并且分装菌体，取相当于200ml的细菌培养物的菌体用5ml BugBuster Master Mix(Novagen)裂解细菌，以6000g离心15min收集包涵体。然后进行4次洗涤剂洗涤以去除细胞碎片和膜组分。然后，用缓冲液如PBS洗涤包涵体以除去洗涤剂和盐。最终，将包涵体用含6M盐酸胍缓冲溶液溶解，并测定包涵体浓度，将其分装后置于-80℃冷冻保存。
TCR蛋白的重折叠
从-80℃超低温冰箱中取出包涵体解冻，加二硫苏糖醇(DTT)至终浓度为10mM，在37℃中温育30min到1小时以确保二硫键完全打开。然后将包涵体样品溶液(15mgα链和10mgβ链)先后滴入200ml 4℃预冷重折叠缓冲液(100mM Tris pH 8.1，400mM L-精氨酸，2mM EDTA，5M尿素，6.5mM盐酸半胱胺和1.87mM二盐酸胱胺)，4℃缓慢搅拌约30分钟。复性溶液用8倍体积预冷的H2O透析16-20小时。再用8倍体积的20mM Tris pH 8.0透析两次，4℃继续透析约8小时，透析后样品过滤后进行以下纯化。
TCR蛋白的第一步纯化
经过透析的重折叠物(20mM Tris pH 8.0中)使用GE Hitrap Q阴离子交换层析预装柱(GE Healthcare)，在AKTA纯化仪(GE Healthcare)用0-600mM NaCl进行梯度洗脱。通过考马斯亮蓝染色的SDS-PAGE分析各个组分，然后合并。
TCR蛋白的第二步纯化
将第一步纯化合并的样品溶液浓缩以供此步纯化，利用在PBS缓冲液中预平衡的Superdex 100160/300GL凝胶过滤层析预装柱(GE Healthcare)纯化蛋白，TCR分子LC13的洗脱曲线分别如图3所示。考马斯亮蓝染色的SDS-PAGE分析出峰的组分，其还原和非还原胶图如图73的泳道2和泳道6所示。根据洗脱峰及胶图可知，洗脱单峰为人工链间二硫键连接的可溶性TCR分子，该分子在SDS凝胶中稳定存在，经还原后形成分开的α和β链。
HPLC法测定TCR蛋白的纯度
TCR蛋白在经过两步纯化合并后，将洗脱组分用HPLC测试其纯度。条件为：Agilent 1260,色谱柱Bio SEC-3(300A，φ7.8×300mm)，流动相为150mM磷酸盐缓冲液，流速0.5mL/min，柱温25℃，紫外检测波长214nm。TCR分子LC13的SEC(空间排阻色谱)谱图如图4所示。含有本发明人工链间二硫键的TCR分子的HPLC洗脱峰单一且对称。
TCR蛋白复性收率的计算
本发明中TCR蛋白复性收率的计算方式如下：
蛋白复性收率(％)＝100*纯化完成后所得蛋白量(mg)/复性所用包涵体的量(mg)。根据上述计算方式，在TRAC*01外显子1的第53位和TRBC1*01或TRBC2*01外显子1的第54位之间形成人工链间二硫键的LC13TCR的蛋白复性收率为43.30％。收率很 高，说明具有本发明人工链间二硫键的可溶性TCR分子很稳定。
实施例3含有人工链间二硫键的TCR的稳定性测试
将实施例2中获得的LC13TCR蛋白(浓度0.5mg/ml)1ml透析至PBS中，利用美国TA(waters)公司的差示扫描量热仪(Nano DSC)对TCR蛋白进行热稳定性测定。检测的温度范围为10-90℃，升温速率为1℃/min。用透析外液PBS作为对照，测定基线3次，待基线稳定之后，再检测蛋白样品。采集数据之后，用分析软件TA_DSC_NanoAnalyze测定TCR的Tm值，并得到其DSC热谱图。经过体外可溶表达得到的含有人工链间二硫键的本发明LC13TCR的DSC热谱图如图5所示，其Tm值可以达到55.82℃。该热谱图能够反映在室温下，甚至温度达到41-43℃，含有本发明人工链间二硫键的TCR分子都能维持正确折叠，并保持应有的活性，说明其稳定性很高。
实施例4结合表征及特异性检测
使用forteBIO Oke实时分析系统检测TCR蛋白与其对应抗原pMHC复合物的结合活性。
在SA传感器表面固定了约2nm的生物素化的pMHC复合物，再将0.05mM的生物素以10μL/min的流速流过芯片120s，封闭链霉亲和素剩余的结合位点。采用动力学分析方法测定其亲和力，使用PBST缓冲液(PBS+0.005％吐温20，pH 7.4)将TCR蛋白稀释成5个不同的浓度(一般为64、32、16、8、4、0uM)，测定与其相对应的pMHC的亲和力。使用Evaluation软件以1:1结合的模型拟合计算动力学参数。
上述pMHC复合物的制备过程如下：
a.纯化
收集100ml诱导表达重链或轻链的E.coli菌液，于4℃8000g离心10min后用10ml PBS洗涤菌体一次，之后用5ml BugBuster Master Mix Extraction Reagents(Merck)剧烈震荡重悬菌体，并于室温旋转孵育20min，之后于4℃，6000g离心15min，弃去上清，收集包涵体。
将上述包涵体重悬于5ml BugBuster Master Mix中，室温旋转孵育5min；加30ml稀释10倍的BugBuster，混匀，4℃6000g离心15min；弃去上清，加30ml稀释10倍的BugBuster重悬包涵体，混匀，4℃6000g离心15min，重复两次，加30ml 20mM Tris-HCl pH 8.0重悬包涵体，混匀，4℃6000g离心15min，最后用20mM Tris-HCl 8M尿素溶解包涵体，SDS-PAGE检测包涵体纯度，BCA试剂盒测浓度。
b.复性
合成所需的短肽(北京赛百盛基因技术有限公司)溶解于DMSO至20mg/ml的浓度。轻链和重链的包涵体用8M尿素、20mM Tris pH 8.0、10mM DTT来溶解，复性前加入3M盐酸胍、10mM醋酸钠、10mM EDTA进一步变性。将短肽以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mM Tris pH 8.3、2mM EDTA、0.5mM氧化性谷胱甘肽、5mM还原型谷胱甘肽、0.2mM PMSF，冷却至4℃)，然后依次加入20mg/L的轻链和90mg/L的重链(终浓度，重链分三次加入，8h/次)，复性在4℃进行至少3天至完成，SDS-PAGE检测能否复性成功。
c.复性后纯化
用10体积的20mM Tris pH 8.0作透析来更换复性缓冲液，至少更换缓冲液两次来充分降低溶液的离子强度。透析后用0.45μm醋酸纤维素滤膜过滤蛋白质溶液，然后加载到HiTrap Q HP(GE通用电气公司)阴离子交换柱上(5ml床体积)。利用Akta纯化仪(GE通用电气公司)，20mM Tris pH 8.0配制的0-400mM NaCl线性梯度液洗脱蛋白，pMHC约在250mM NaCl处洗脱，收集诸峰组分，SDS-PAGE检测纯度。
d.生物素化
用Millipore超滤管将纯化的pMHC分子浓缩，同时将缓冲液置换为20mM Tris pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mM MgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
e.纯化生物素化后的复合物
用Millipore超滤管将生物素化标记后的pMHC分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的pMHC，利用Akta纯化仪(GE通用电气公司)，用过滤过的PBS预平衡HiPrepTM 16/60S200HR柱(GE通用电气公司)，加载1ml浓缩过的生物素化pMHC分子，然后用PBS以1ml/min流速洗脱。生物素化的pMHC分子在约55ml时作为单峰洗脱出现。合并含有蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，加入蛋白酶抑制剂cocktail(Roche)将生物素化的pMHC分子分装保存在-80℃。
不同浓度的LC13分子与其相应抗原的结合曲线如图6所示，KD值为10.5μM；从该结合曲线中可以看出，浓度的降低并没有影响本发明TCR分子与其相应抗原的结合，低浓度的TCR分子表现出与高浓度TCR分子相同的结合活性，也能够从侧面说明具有本发明人工链间二硫键的TCR是较稳定的。
TCR蛋白的特异性检测
应用forteBIO Oke实时分析系统检测TCR蛋白对其相应抗原pMHC复合物的特异性。将6种不同的已生物素化的抗原(浓度为0.5μM)分别加载到6根SA传感器的表面；然后，与各个待测的TCR蛋白(浓度为2-20μM)相互作用；最后，分析其互作所产生的信号。结果显示，引入人工链间二硫键的LC13TCR只与其相应抗原pMHC复合物结合，与其他无关抗原包括A0201：KLVALGINAV(SEQ ID NO.:54)、A0201:SLLMWITQC(SEQ ID NO.:55)、A0201:GILGFVFTL(SEQ ID NO.:56)、A0101:EVDPIGHLY(SEQ ID NO.:57)、A1101:SSCSSCPLSK(SEQ ID NO.:58)和A2402:KYKDYFPVI(SEQ ID NO.:59)没有结合。
实施例5在TRAC*01外显子1的第53位和TRBC1*01或TRBC2*01外显子1的第54位形成人工链间二硫键的1G4分子
将TCR分子1G4(针对抗原短肽HLA-A2/SLLMWITQC(SEQ ID NO.:55)，NY-ESO-1肿瘤特异性抗原)的TRAC*01外显子1的第53位精氨酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第54位丝氨酸突变为半胱氨酸，以形成人工链间二硫键。
采用实施例1中所述的引物及PCR步骤进行突变，突变后TCR分子1G4的α链与β链胞外氨基酸序列分别如图7a和7b所示，其对应的核苷酸序列分别如图8a和8b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。
采用实施例2中所述方法对1G4TCR进行表达、重折叠及纯化，经过第二步纯化的洗脱曲线如图9所示。考马斯亮蓝染色的SDS-PAGE分析出峰的组分，其还原和非还原胶图如图74的泳道2和泳道6所示。根据洗脱峰及胶图可知，洗脱单峰为人工链间二硫键连接的可溶性TCR分子，该分子在SDS凝胶中稳定存在，经还原后形成分开的α和β链。
根据实施例2中所述方法测定1G4TCR蛋白的纯度及计算其收率。得到其SEC谱图如图10所示，含有本发明人工链间二硫键的1G4TCR分子的HPLC洗脱峰单一且对称。其收率达到了40％。
采用实施例3中所述方法测定含有人工链间二硫键的1G4TCR的稳定性，其DSC热谱图如图11所示，得到其Tm值为55.21℃。该热谱图能够反映在室温下，甚至温度 达到47-48℃，含有本发明人工链间二硫键的TCR分子都能维持正确折叠，并保持应有的活性，说明其稳定性很高。
采用实施例4中所述方式检测1G4TCR蛋白与其对应抗原pMHC复合物的结合活性及特异性，得到结合曲线如图12所示，KD值为6.96μM；从该结合曲线中可以看出，浓度的降低并没有影响本发明的稳定性TCR分子与其相应抗原的结合，低浓度的TCR分子表现出与高浓度TCR分子相同的结合活性，也能够从侧面说明具有本发明链间二硫键的TCR是较稳定的。
同时，本发明的TCR分子也具有很强的特异性，只与其对应的pMHC复合物结合，而与其他无关抗原包括B4405:EEYLKAWTF(SEQ ID NO.:49)、A0201:GILGFVFTL(SEQ ID NO.:56)、A0101:EVDPIGHLY(SEQ ID NO.:57)、A1101:SSCSSCPLSK(SEQ ID NO.:58)和A2402:KYKDYFPVI(SEQ ID NO.:59)没有结合。
实施例6在TRAC*01外显子1的第53位和TRBC1*01或TRBC2*01外显子1的第54位形成人工链间二硫键的JM22分子
将TCR分子JM22(针对抗原短肽HLA-A2/GILGFVFTL(SEQ ID NO.:56)，源自流感病毒基质蛋白)的TRAC*01外显子1的第53位精氨酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第54位丝氨酸突变为半胱氨酸，以形成人工链间二硫键。
采用实施例1中所述的引物及PCR步骤进行突变，突变后TCR分子JM22的α链与β链胞外氨基酸序列分别如图13a和13b所示，其对应的核苷酸序列分别如图14a和14b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。
采用实施例2中所述方法对JM22TCR进行表达、重折叠及纯化，经过第二步纯化的洗脱曲线如图15所示。考马斯亮蓝染色的SDS-PAGE分析出峰的组分，其还原和非还原胶图如图75的泳道2和泳道6所示。根据洗脱峰及胶图可知，洗脱单峰为人工链间二硫键连接的可溶性TCR分子，该分子在SDS凝胶中稳定存在，经还原后形成分开的α和β链。
根据实施例2中所述方法测定JM22TCR蛋白的纯度及计算其收率。得到其SEC谱图如图16所示，含有本发明人工链间二硫键的JM22TCR分子的HPLC洗脱峰单一且对称。其收率达到了31.65％。
采用实施例3中所述方法测定含有人工链间二硫键的JM22TCR的稳定性，其DSC热谱图如图17所示，得到其Tm值为49.06℃。该热谱图能够反映在室温下，甚至温度达到40℃，含有本发明人工链间二硫键的TCR分子都能维持正确折叠，并保持应有的活性，说明其稳定性很高。
采用实施例4中所述方式检测JM22TCR蛋白与其对应抗原pMHC复合物的结合活性及特异性，得到结合曲线如图18所示，KD值为7.14μM；从该结合曲线中可以看出，浓度的降低并没有影响本发明的稳定性TCR分子与其相应抗原的结合，低浓度的TCR分子表现出与高浓度TCR分子相同的结合活性，也能够从侧面说明具有本发明链间二硫键的TCR是较稳定的。
同时，本发明的TCR分子也具有很强的特异性，只与其对应的pMHC复合物结合，而与其他无关抗原包括B4405:EEYLKAWTF(SEQ ID NO.:49)、A0201:SLLMWITQC(SEQ ID NO.:55)、A0101:EVDPIGHLY(SEQ ID NO.:57)、A1101:SSCSSCPLSK(SEQ ID NO.:58)和A2402:KYKDYFPVI(SEQ ID NO.:59)没有结合。
实施例7在TRAC*01外显子1的第53位和TRBC1*01或TRBC2*01外显子1的第54位形成人工链间二硫键的MGA3分子
将TCR分子MGA3(针对抗原短肽HLA-A1：EVDPIGHLY(SEQ ID NO.:57)，MageA3 肿瘤特异性抗原)的TRAC*01外显子1的第53位精氨酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第54位丝氨酸突变为半胱氨酸，以形成人工链间二硫键。
采用实施例1中所述的引物及PCR步骤进行突变，突变后TCR分子MGA3的α链与β链胞外氨基酸序列分别如图19a和19b所示，其对应的核苷酸序列分别如图20a和20b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。
采用实施例2中所述方法对MGA3TCR进行表达、重折叠及纯化，经过第二步纯化的洗脱曲线如图21所示。考马斯亮蓝染色的SDS-PAGE分析出峰的组分，其还原和非还原胶图如图76的泳道2和泳道6所示。根据洗脱峰及胶图可知，洗脱单峰为人工链间二硫键连接的可溶性TCR分子，该分子在SDS凝胶中稳定存在，经还原后形成分开的α和β链。
根据实施例2中所述方法测定MGA3TCR蛋白的纯度及计算其收率。得到其SEC谱图如图22所示，含有本发明人工链间二硫键的MGA3TCR分子的HPLC洗脱峰单一且对称。其收率达到了30.14％。
采用实施例3中所述方法测定含有人工链间二硫键的MGA3TCR的稳定性，其DSC热谱图如图23所示，得到其Tm值为53.86℃。该热谱图能够反映在室温下，甚至温度达到45-46℃，含有本发明人工链间二硫键的TCR分子都能维持正确折叠，并保持应有的活性，说明其稳定性很高。
采用实施例4中所述方式检测MGA3TCR蛋白与其对应抗原pMHC复合物的结合活性及特异性，得到结合曲线如图24所示，KD值为1.42μM；从该结合曲线中可以看出，浓度的降低并没有影响本发明的稳定性TCR分子与其相应抗原的结合，低浓度的TCR分子表现出与高浓度TCR分子相同的结合活性，也能够从侧面说明具有本发明链间二硫键的TCR是较稳定的。
本发明的TCR分子也具有很强的特异性，只与其对应的pMHC复合物结合，而与其他无关抗原包括B4405:EEYLKAWTF(SEQ ID NO.:49)、A0201:SLLMWITQC(SEQ ID NO.:55)、A0201:GILGFVFTL(SEQ ID NO.:56)、A1101:SSCSSCPLSK(SEQ ID NO.:58)和A2402:KYKDYFPVI(SEQ ID NO.:59)没有结合。
实施例8在TRAC*01外显子1的第89位和TRBC1*01或TRBC2*01外显子1的第19位形成人工链间二硫键的分子的性能测试
分别将TCR分子LC13、1G4、JM22和MGA3的TRAC*01外显子1的第89位脯氨酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第19位丙氨酸突变为半胱氨酸，以形成人工链间二硫键。
上述几种TCR的TRAC*01外显子1的第89位脯氨酸突变为半胱氨酸时，所设计的引物如下：
5’-3’
Figure PCTCN2015093806-appb-000016
上述几种TCR的TRBC1*01或TRBC2*01外显子1的第19位丙氨酸突变为半胱氨酸时，所设计的引物如下：
5’-3’
Figure PCTCN2015093806-appb-000017
采用实施例1至实施例4中所述方式对TCR进行PCR、复性及性能测试。
突变后TCR分子LC13的α链与β链胞外氨基酸序列分别如图25a和25b所示，其对应的核苷酸序列分别如图26a和26b所示，引入的半胱氨酸残基以加粗并带下划 线字母表示。其洗脱曲线和胶图分别如图27和图73的泳道3和泳道7所示。HPLC洗脱峰单一且对称如图28所示。蛋白复性收率也相当高，达到了42.82％。其Tm值为55.65℃，对应的DSC谱图如图29所示。LC13分子与其相应抗原的结合曲线如图30所示，KD值为10.3μM。
突变后TCR分子1G4的α链与β链胞外氨基酸序列分别如图31a和31b所示，其对应的核苷酸序列分别如图32a和32b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。其洗脱曲线和胶图分别如图33和图74的泳道3和泳道7所示。HPLC洗脱峰单一且对称如图34所示。蛋白复性收率也相当高，达到了48％。其Tm值为55.82℃，对应的DSC谱图如图35所示。1G4分子与其相应抗原的结合曲线如图36所示，KD值为6.63μM。
突变后TCR分子JM22的α链与β链胞外氨基酸序列分别如图37a和37b所示，其对应的核苷酸序列分别如图38a和38b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。其洗脱曲线和胶图分别如图39和图75的泳道3和泳道7所示。HPLC洗脱峰单一且对称如图40所示。蛋白复性收率达到了14.93％。其Tm值为51.08℃，对应的DSC谱图如图41所示。JM22分子与其相应抗原的结合曲线如图42所示，KD值为7.61μM。
突变后TCR分子MGA3的α链与β链胞外氨基酸序列分别如图43a和43b所示，其对应的核苷酸序列分别如图44a和44b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。其洗脱曲线和胶图分别如图45和图76的泳道3和泳道7所示。HPLC洗脱峰单一且对称如图46所示。蛋白复性收率达到了13.76％。其Tm值为54.49℃，对应的DSC谱图如图47所示。MGA3分子与其相应抗原的结合曲线如图48所示，KD值为2.04μM。
由以上几种分子的洗脱曲线和SDS胶图可知，洗脱峰组份为本发明人工链间二硫键连接的可溶性TCR分子，在SDS凝胶中稳定存在，经还原后形成分开的α和β链。蛋白复性收率也都较高。另外，由本发明人工链间二硫键连接的TCR分子的Tm值也都很高，都大于45℃，说明在较高的温度下它们都能维持正确折叠，并保持应有的活性，显示其稳定性很高。同时，TCR分子与其原配体的结合曲线显示，TCR浓度的降低并没有影响与其配体的结合，也能够从侧面说明具有本发明链间二硫键的TCR分子是稳定的。在特异性测试中，这些引入人工链间二硫键的TCR分子也都展示了很好的特异性。
实施例9在TRAC*01外显子1的第10位和TRBC1*01或TRBC2*01外显子1的第20位形成人工链间二硫键的分子的性能测试
分别将TCR分子LC13、1G4、JM22和MGA3的TRAC*01外显子1的第10位酪氨酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第20位谷氨酸突变为半胱氨酸，以形成人工链间二硫键。
上述几种TCR的TRAC*01外显子1的第10位酪氨酸突变为半胱氨酸时，所设计的引物如下：
5’-3’
Figure PCTCN2015093806-appb-000018
上述几种TCR的TRBC1*01或TRBC2*01外显子1的第20位谷氨酸突变为半胱氨酸时，所设计的引物如下：
5’-3’
Figure PCTCN2015093806-appb-000019
采用实施例1至实施例4中所述方式对TCR进行PCR、复性及性能测试。
突变后TCR分子LC13的α链与β链胞外氨基酸序列分别如图49a和49b所示，其对应的核苷酸序列分别如图50a和50b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。其洗脱曲线和胶图分别如图51和图73的泳道1和泳道5所示。HPLC洗脱峰单一且对称如图52所示。蛋白复性收率达到了16.19％。其Tm值为50.42℃，对应的DSC谱图如图53所示。LC13分子与其相应抗原的结合曲线如图54所示，KD值为10μM。
突变后TCR分子1G4的α链与β链胞外氨基酸序列分别如图55a和55b所示，其对应的核苷酸序列分别如图56a和56b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。其洗脱曲线和胶图分别如图57和图74的泳道1和泳道5所示。HPLC洗脱峰单一且对称如图58所示。蛋白复性收率达到29％。其Tm值为54.68℃，对应的DSC谱图如图59所示。1G4分子与其相应抗原的结合曲线如图60所示，KD值为6.68μM。
突变后TCR分子JM22的α链与β链胞外氨基酸序列分别如图61a和61b所示，其对应的核苷酸序列分别如图62a和62b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。其洗脱曲线和胶图分别如图63和图75的泳道1和泳道5所示。HPLC洗脱峰单一且对称如图64所示。蛋白复性收率达到了10.50％。其Tm值为49.95℃，对应的DSC谱图如图65所示。JM22分子与其相应抗原的结合曲线如图66所示，KD值为5.54μM。
突变后TCR分子MGA3的α链与β链胞外氨基酸序列分别如图67a和67b所示，其对应的核苷酸序列分别如图68a和68b所示，引入的半胱氨酸残基以加粗并带下划线字母表示。其洗脱曲线和胶图分别如图69和图76的泳道1和泳道5所示。HPLC洗脱峰单一且对称如图70所示。其蛋白复性收率达到了4.53％。其Tm值为53.38℃，对应的DSC谱图如图71所示。MGA3分子与其相应抗原的结合曲线如图72所示，KD值为3.45μM。
由以上几种分子的洗脱曲线和SDS胶图可知，洗脱主峰组份为本发明人工链间二硫键连接的可溶性TCR分子，在SDS凝胶中稳定存在，经还原后形成分开的α和β链。蛋白复性收率也都较高。另外，由本发明人工链间二硫键连接的TCR分子的Tm值也都很高，都大于45℃，说明在较高的温度下它们都能维持正确折叠，并保持应有的活性，显示其稳定性很高。同时，TCR分子与其原配体的结合曲线显示，TCR浓度的降低并没有影响与其配体的结合，也能够从侧面说明具有本发明链间二硫键的TCR分子是稳定的。在特异性测试中，这些引入人工链间二硫键的TCR分子也都展示了很好的特异性。
以上的实施例，证明了将本发明的人工链间二硫键引入到TCR恒定区中得到的本发明TCR分子具有高稳定性，其Tm值大于45℃。并且能够被很好地复性、重折叠并被纯化，复性收率高同时能够与其原配体特异性结合。
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 (23)

  1. 一种T细胞受体(TCR)，其特征在于，在所述TCR的α链和β链的恒定区中引入半胱氨酸残基以形成人工链间二硫键，并且所述T细胞受体的Tm值≥45℃。
  2. 如权利要求1所述的TCR，其特征在于，在所述TCR的β链恒定区中引入的半胱氨酸残基的替换位点选自：TRBC1*01或TRBC2*01外显子1的54S、19A和20E。
  3. 如权利要求1或2所述的TCR，其特征在于，在所述TCR的α链恒定区中引入的半胱氨酸残基的替换位点选自：TRAC*01外显子1的53R、89P和10Y。
  4. 如以上任一权利要求所述的TCR，其特征在于，所述TCR包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的功能性可变结构域和至少一部分恒定结构域。
  5. 如以上任一权利要求所述的TCR，其特征在于，所述TCR是可溶的。
  6. 如以上任一权利要求所述的TCR，其特征在于，所述TCR中不存在天然链间二硫键。
  7. 如权利要求6所述的TCR，其特征在于，所述TCR中将天然TCR的C末端截短以去除形成所述天然链间二硫键的半胱氨酸残基。
  8. 如权利要求6所述的TCR，其特征在于，所述TCR中形成所述天然链间二硫键的半胱氨酸残基被替换为另一残基。
  9. 如以上任一权利要求所述的TCR，其特征在于，所述TCR的β链恒定区中不存在未配对的半胱氨酸残基。
  10. 如权利要求9所述的TCR，其特征在于，所述TCRβ链恒定区中未配对的半胱氨酸残基被替换为丙氨酸或丝氨酸。
  11. 如以上任一权利要求所述的TCR，其特征在于，形成人工链间二硫键的半胱氨酸残基替换了：
    TRAC*01外显子1的53R和TRBC1*01或TRBC2*01外显子1的54S；
    TRAC*01外显子1的89P和TRBC1*01或TRBC2*01外显子1的19A；或
    TRAC*01外显子1的10Y和TRBC1*01或TRBC2*01外显子1的20E。
  12. 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链的C-或N-末端结合有偶联物。
  13. 如权利要求12所述的TCR，其特征在于，与所述TCR结合的偶联物为可检测标记物、治疗剂、PK修饰部分或其组合。
  14. 如权利要求13所述的T细胞受体，其特征在于，与所述T细胞受体结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
  15. 一种核酸分子，其特征在于，所述核酸分子包含编码以上权利要求中任一所述的TCR的α链和/或β链的核酸序列或其互补序列。
  16. 一种载体，所述的载体含有权利要求15所述的核酸分子。
  17. 一种宿主细胞或遗传改造的工程细胞，所述的细胞含有权利要求15所述的载体或染色体中整合有外源的权利要求15所述的核酸分子。
  18. 一种分离的细胞，其特征在于，其呈递权利要求1所述的TCR。
  19. 一种制备权利要求1所述的T细胞受体的方法，包括步骤：
    (i)培养权利要求17所述的宿主细胞，从而表达权利要求1所述的T细胞受体的α链和/或β链；
    (ii)分离或纯化出所述的α链和/或β链；
    (iii)重折叠所述的α链和/或β链，获得所述T细胞受体。
  20. 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1-14中任一所述的TCR。
  21. 一种权利要求1-14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或用于制备检测MHC-肽复合体的试剂。
  22. 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1-14中任一所述的TCR、权利要求18所述的细胞或权利要求20所述的T细胞受体复合物。
  23. 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1-14中任一所述的TCR、权利要求18所述的细胞、权利要求20所述的T细胞受体复合物或权利要求22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。
PCT/CN2015/093806 2014-11-07 2015-11-04 一种可溶的异质二聚t细胞受体及其制法和应用 WO2016070814A1 (zh)

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