WO2016184258A1 - 一种可溶且稳定的异质二聚tcr - Google Patents

一种可溶且稳定的异质二聚tcr Download PDF

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WO2016184258A1
WO2016184258A1 PCT/CN2016/077680 CN2016077680W WO2016184258A1 WO 2016184258 A1 WO2016184258 A1 WO 2016184258A1 CN 2016077680 W CN2016077680 W CN 2016077680W WO 2016184258 A1 WO2016184258 A1 WO 2016184258A1
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tcr
chain
exon
disulfide bond
trbc2
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PCT/CN2016/077680
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English (en)
French (fr)
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李懿
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广州市香雪制药股份有限公司
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Priority to CN201680003540.2A priority Critical patent/CN107108717B/zh
Priority to EP16795745.5A priority patent/EP3299389A4/en
Priority to US15/573,692 priority patent/US10316087B2/en
Priority to CA2986273A priority patent/CA2986273A1/en
Priority to JP2018512466A priority patent/JP6640994B2/ja
Publication of WO2016184258A1 publication Critical patent/WO2016184258A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention belongs to the field of biomedicine, and in particular, to a soluble T cell receptor, a process for its preparation, and an application.
  • TCR T cell receptor
  • the TCR heterodimer consists of alpha and beta chains in 95% of T cells, while 5% of T cells have a TCR consisting of gamma and delta chains.
  • the native ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain, and the ⁇ chain and the ⁇ chain constitute a subunit of the ⁇ heterodimeric TCR.
  • the alpha and beta chains of TCR are generally considered to have two “domains” each, namely the TCR alpha chain variable domain (V alpha) and the TCR alpha chain constant domain (C alpha), the TCR beta chain variable domain (V beta) and the TCR beta chain constant domain ( C ⁇ ).
  • 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 is critical for the cellular immune function of the immune system.
  • 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).
  • therapeutic agents such as cytotoxic compounds or immunostimulatory compounds
  • T cells such as those that react with autoimmune peptide antigens.
  • soluble TCRs can also bind to other molecules (eg, anti-CD3 antibodies) to redirect T cells, thereby targeting cells that present specific antigens, killing.
  • TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region, and for obtaining a soluble TCR protein, when the TCR is separated from the membrane, it is soluble and maintains its ability to bind to its original ligand (ie, pMHC).
  • Stable TCR is a very difficult task (Shin, et al., (1993) science 259:1901). Its instability and low protein yield are major obstacles to the development of therapeutic or diagnostic agents with TCR or fragments thereof.
  • Some literature describes a truncated form of TCR that contains only the extracellular domain or only the extracellular and cytoplasmic regions, although such TCRs can be recognized by TCR-specific antibodies, but the yield is low and cannot be observed at low concentrations. Identification of the main histocompatibility complex-peptide complex indicates that it is easily denatured and not stable enough. Those skilled in the art are directed to the development of soluble, stable T cell receptors.
  • an ⁇ heterodimeric TCR wherein an artificial chain disulfide bond is contained between the ⁇ chain variable region of the TCR and the ⁇ chain constant region.
  • the artificial interchain disulfide bond of the TCR is located between the FR2 of the alpha chain variable region and the constant region of the beta chain.
  • cysteine residue forming the artificial interchain disulfide bond of the TCR is substituted for The 46th or 47th amino acid residue of TRAV.
  • cysteine residue forming the artificial interchain disulfide bond of the TCR replaces the 60th or 61st amino acid residue of exon 1 of TRBC1*01 or TRBC2*01.
  • cysteine residue forming the artificial interchain disulfide bond of the TCR is substituted:
  • the TCR is soluble.
  • the TCR comprises an alpha chain variable domain and a beta chain variable domain and all or part of a beta chain constant domain other than a transmembrane domain, but which does not comprise an alpha chain constant domain, said TCR
  • the alpha chain variable domain forms a heterodimer with the beta chain.
  • cysteine residue forming the natural interchain disulfide bond in the ⁇ -chain constant domain is replaced with another amino acid, preferably, alanine or serine.
  • the C-terminus of the constant domain of the TCR ⁇ chain is truncated to remove a cysteine residue that forms a natural interchain disulfide bond.
  • 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 variable domain of the TCR chain and at least a portion of the constant domain.
  • the C-terminus of the TCR alpha chain and/or the beta chain constant region is truncated to remove a cysteine residue that forms a natural interchain disulfide bond.
  • cysteine residue forming the natural interchain disulfide bond in the TCR alpha chain and/or the beta chain constant region is replaced with another residue.
  • an artificial interchain disulfide bond is contained between the alpha chain constant region of the TCR and the beta chain constant region.
  • cysteine residue forming an artificial interchain disulfide bond between the constant region of the TCR alpha chain and the constant region of the beta chain is substituted:
  • the C- or N-terminus of the alpha chain and/or beta strand of the TCR incorporates a conjugate.
  • the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modified moiety, or a combination thereof.
  • the therapeutic agent that binds to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the alpha or beta chain of the TCR.
  • the TCR has a Tm value ⁇ 45 ° C, preferably ⁇ 50 ° C, more preferably ⁇ 52 ° C, and most preferably ⁇ 55 ° C.
  • 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.
  • 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 T cell receptor complex comprising one or more TCRs according to the first aspect of the invention.
  • 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 fifth aspect of the invention or the invention
  • the T cell receptor complex of the seventh aspect is provided.
  • a tenth aspect of the invention provides a method for treating a disease, comprising administering a TCR according to the first aspect of the invention, a cell according to the fifth aspect of the invention, to a subject in need of treatment, the seventh aspect of the invention a T cell receptor complex or the pharmaceutical composition of the ninth aspect of the invention;
  • the diseases include: tumors, autoimmune diseases, and viral infectious diseases.
  • Figure 1a and Figure 1b are the ⁇ -chain variable domain amino acid sequences of the three-domain 1G4 TCR molecule forming an artificial interchain disulfide bond at position 46 of TRAV and position 60 of TRBC1*01 or TRBC2*01 exon 1, respectively. And beta chain amino acid sequence.
  • 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 TCR alpha chain variable domain and a beta chain refolded by a gel filtration chromatography column shown in Figures 1a and 1b.
  • Figure 4 is a SEC map of the TCR alpha chain variable domain and beta chain refolded and protein purified as shown in Figures 1a and 1b.
  • Figure 5 is a graph showing the TCR alpha chain variable domain and the beta chain shown in Figure 1a and Figure 1b after refolding and protein purification. DSC thermogram.
  • Figure 6 is a graph showing the binding curves of different concentrations of 1G4 TCR molecules and their corresponding antigens obtained after TCR alpha chain variable domain and beta chain refolding and protein purification shown in Figures 1a and 1b.
  • Figure 7a and Figure 7b are the ⁇ -chain variable domain amino acid sequences of the three-domain JM22 TCR molecule forming the artificial interchain disulfide bond at position 46 of TRAV and position 60 of TRBC1*01 or TRBC2*01 exon 1, respectively. And beta chain amino acid sequence.
  • 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 TCR alpha chain variable domain and a beta chain refolded by a gel filtration chromatography column shown in Figures 7a and 7b.
  • Figure 10 is a SEC map of the TCR alpha chain variable domain and beta chain refolded and protein purified as shown in Figures 7a and 7b.
  • Figure 11 is a DSC thermogram measured after TCR alpha chain variable domain and beta chain refolding and protein purification shown in Figures 7a and 7b.
  • Figure 12 is a graph showing the binding curves of different concentrations of the JM22 TCR molecule to the corresponding antigen obtained after refolding and protein purification of the TCR alpha chain variable domain and the beta chain shown in Figures 7a and 7b.
  • Figure 13a and Figure 13b are the alpha-chain variable domain amino acid sequences of the three-domain LC13TCR molecule that forms an artificial interchain disulfide bond at position 46 of TRAV and position 60 of TRBC1*01 or TRBC2*01 exon 1, respectively. And beta chain amino acid sequence.
  • 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 the TCR alpha chain variable domain and the beta chain refolded after gel filtration column shown in Figures 13a and 13b.
  • Figure 16 is a SEC map of the TCR alpha chain variable domain and beta chain refolded and protein purified as shown in Figures 13a and 13b.
  • Figure 17 is a DSC thermogram measured after TCR alpha chain variable domain and beta chain refolding and protein purification shown in Figures 13a and 13b.
  • Figure 18 is a graph showing the binding curves of different concentrations of LC13 TCR molecules and their corresponding antigens obtained after TCR alpha chain variable domain and beta chain refolding and protein purification shown in Figures 13a and 13b.
  • Figure 19 is an alpha chain amino acid sequence of a four domain 1G4 molecule that forms an artificial interchain disulfide bond at position 46 of TRAV and at position 60 of TRBC1*01 or TRBC2*01 exon 1.
  • Figure 20 is a nucleotide sequence corresponding to the amino acid of Figure 19.
  • Figure 21 shows the four-domain 1G4 TCR alpha chain and the ⁇ -strand refolding at the 46th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 by gel filtration chromatography. The elution curve of the column.
  • Figure 22 is a SEC map of the four-domain 1G4 TCR ⁇ chain and ⁇ -chain refolding and protein purification of the inter-chain disulfide bond at position 46 of TRAV and TRBC1*01 or TRBC2*01 exon 1. .
  • Figure 23 shows the four-domain 1G4 TCR alpha chain and the ⁇ -chain refolding and protein purification after the 46th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond. DSC thermogram.
  • Figure 24 shows the different concentrations of the four-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 46th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond.
  • Figure 25 is an ⁇ -chain amino acid sequence of a four-domain JM22 molecule which forms an artificial interchain disulfide bond at position 46 of TRAV and at position 60 of TRBC1*01 or TRBC2*01 exon 1.
  • Figure 26 is a nucleotide sequence corresponding to the amino acid of Figure 25.
  • Figure 27 is a four-domain JM22TCR ⁇ chain and ⁇ -strand refolding at the 46th position of TRAV and TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond.
  • Figure 28 shows the formation of the 46th position of TRAV and the 60th position of exon 1 of TRBC1*01 or TRBC2*01
  • Figure 29 is a four-domain JM22 TCR alpha chain and beta chain refolding and protein purification after the 46th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond. DSC thermogram.
  • Figure 30 shows the different concentrations of the four-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 46th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond.
  • Figure 31 is an alpha-chain amino acid sequence of a four-domain LC13 molecule forming an artificial interchain disulfide bond at position 46 of TRAV and at position 60 of TRBC1*01 or TRBC2*01 exon 1.
  • Figure 32 is a nucleotide sequence corresponding to the amino acid of Figure 31.
  • Figure 33 is a four-domain LC13 TCR alpha chain and ⁇ -chain refolding at the 46th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 by gel filtration chromatography The elution curve of the column.
  • Figure 34 is a SEC map of the four-domain LC13TCR ⁇ chain and ⁇ -chain refolding and protein purification of the inter-ligand disulfide bond at position 46 of TRAV and TRBC1*01 or TRBC2*01 exon 1. .
  • Figures 35a and 35b are the amino acid sequences of TRBC1*01 and TRBC2*01 listed in IMGT, respectively.
  • Figure 36 shows the different concentrations of the four-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 46th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond.
  • Figure 37a and Figure 37b are the ⁇ -chain variable domain amino acid sequences of the three-domain 1G4 TCR molecule forming an artificial interchain disulfide bond at position 47 of TRAV and position 61 of TRBC1*01 or TRBC2*01 exon 1, respectively. And beta chain amino acid sequence.
  • 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 the TCR alpha chain variable domain and the beta chain refolded after gel filtration column shown in Figures 37a and 37b.
  • Figure 40 is a SEC map of the TCR alpha chain variable domain and beta chain refolded and protein purified as shown in Figures 37a and 37b.
  • Figure 41 is a DSC thermogram measured after TCR alpha chain variable domain and beta chain refolding and protein purification shown in Figures 37a and 37b.
  • Figure 42 is a graph showing the binding curves of different concentrations of TCR molecules and their corresponding antigens obtained after TCR alpha chain variable domain and beta chain refolding and protein purification shown in Figures 37a and 37b.
  • Figure 43 is an ⁇ -chain amino acid sequence of a four-domain 1G4 TCR molecule which forms an artificial interchain disulfide bond at position 47 of TRAV and position 61 of TRBC1*01 or TRBC2*01.
  • Figure 44 is a nucleotide sequence corresponding to the amino acid in Figure 43.
  • Figure 45 is a gel filtration chromatography of a four-domain TCR ⁇ chain and a ⁇ -chain refolded at the 47th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond.
  • the elution curve of the column is a gel filtration chromatography of a four-domain TCR ⁇ chain and a ⁇ -chain refolded at the 47th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond.
  • Figure 46 is a SEC map of the four-domain TCR ⁇ chain and ⁇ -chain refolding and protein purification of the 47th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 forming an artificial interchain disulfide bond. .
  • Figure 47 shows the four-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 47th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond. DSC thermogram.
  • Figure 48 shows the different concentrations of the four-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 47th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond.
  • Figure 49 is a gel filtration chromatography of a three-domain TCR ⁇ chain and a ⁇ -strand refolded at the 46th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond.
  • the elution curve of the column is a gel filtration chromatography of a three-domain TCR ⁇ chain and a ⁇ -strand refolded at the 46th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond.
  • Figure 50 shows the formation of the 46th position of TRAV and the 61st position of exon 1 of TRBC1*01 or TRBC2*01
  • Figure 51 shows the three-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 46th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond. DSC thermogram.
  • Figure 52 shows the different concentrations of the three-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 46th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond.
  • Figure 53 is a gel filtration chromatography of a four-domain TCR ⁇ chain and a ⁇ -chain refolded at the 46th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond.
  • the elution curve of the column is a gel filtration chromatography of a four-domain TCR ⁇ chain and a ⁇ -chain refolded at the 46th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond.
  • Figure 54 is a SEC map of the four-domain TCR ⁇ chain and ⁇ -chain refolding and protein purification of the inter-ligand disulfide bond at position 46 of TRAV and TR561*01 or TRBC2*01 exon 1. .
  • Figure 55 shows the four-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 46th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond. DSC thermogram.
  • Figure 56 shows the different concentrations of the four-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 46th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond.
  • Figure 57 is a three-domain TCR alpha chain and a ⁇ -strand refolded at position 47 of TRAV and TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond.
  • Figure 58 is a SEC map of the three-domain TCR ⁇ chain and ⁇ -chain refolding and protein purification of the inter-chain disulfide bond forming the artificial interchain disulfide bond at position 47 of TRAV and TRBC1*01 or TRBC2*01 exon 1. .
  • Figure 59 shows the three-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 47th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond. DSC thermogram.
  • Figure 60 shows the different concentrations of the three-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 47th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond.
  • Figure 61 is a gel filtration chromatography of a four-domain TCR ⁇ chain and a ⁇ -chain refolded at the 47th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond.
  • the elution curve of the column is a gel filtration chromatography of a four-domain TCR ⁇ chain and a ⁇ -chain refolded at the 47th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 to form an artificial interchain disulfide bond. The elution curve of the column.
  • Figure 62 is a SEC map of the four-domain TCR ⁇ chain and ⁇ -chain refolding and protein purification of the 47th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 forming an artificial interchain disulfide bond. .
  • Figure 63 shows the four-domain TCR ⁇ chain and ⁇ -chain refolding and protein purification after the 47th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond. DSC thermogram.
  • Figure 64 shows the different concentrations of the four-domain TCR ⁇ chain and the ⁇ -chain refolding and protein purification after the 47th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 form an artificial interchain disulfide bond.
  • Figure 65 is a gel diagram of a three domain soluble protein containing an artificial interchain disulfide bond at different positions between the alpha chain variable region of the 1G4 TCR molecule and the beta chain constant region.
  • Figure 66 is a gel diagram of a three domain soluble protein containing an artificial interchain disulfide bond between the alpha chain variable region and the beta chain constant region of different TCR molecules.
  • Figure 67 is a gel diagram of a four domain soluble protein containing an artificial interchain disulfide bond at different positions between the alpha chain variable region of the 1G4 TCR molecule and the beta chain constant region.
  • Figure 68 is a gel diagram of a four domain soluble protein containing an artificial interchain disulfide bond between the alpha chain variable region and the beta chain constant region of different TCR molecules.
  • the present inventors have unexpectedly obtained a soluble and stable T cell receptor by extensive and intensive research.
  • the TCR of the present invention is an ⁇ heterodimer, and a covalent inter-chain disulfide bond is contained between the ⁇ chain variable region of the TCR of the present invention and the ⁇ chain constant region. More specifically, the artificial interchain disulfide bond of the TCR of the present invention is located in the alpha chain Between the constant regions of the FR2 and beta chains.
  • the invention also provides for the use of the TCR, and a process for its preparation.
  • the native ⁇ heterodimeric TCR has an alpha chain and a beta chain, and the alpha chain and the beta chain constitute two subunits of the ⁇ heterodimeric TCR.
  • the alpha and beta chains of TCR are generally considered to have two "domains" each, namely the TCR alpha chain variable domain (V alpha) and the TCR alpha chain constant domain (C alpha), the TCR beta chain variable domain (V beta) and the TCR beta chain constant domain ( C ⁇ ).
  • V alpha TCR alpha chain variable domain
  • C alpha TCR alpha chain constant domain
  • V beta TCR beta chain variable domain
  • C ⁇ TCR beta chain constant domain
  • 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 both refer to the present invention between the variable region of the alpha chain and the constant region of the beta chain.
  • a heterodimeric TCR that invents an artificial interchain disulfide bond.
  • the TCR ⁇ and ⁇ chains each comprise a variable region, a junction region and a constant region
  • the ⁇ chain usually also contains a short polymorphic region between the variable region and the junction region, but the polymorphic region is often regarded as a junction region. a part of.
  • the alpha and beta chains of TCR are generally considered to have two "domains", ie, a variable domain and a constant domain, and the variable domain is composed of a linked variable region and a junction region, and the constant domain further comprises a transmembrane region and a cytoplasmic region.
  • the cytoplasmic area is very short.
  • 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 region of TCR, wherein “TR” represents a T cell receptor gene, "A” represents an ⁇ chain gene, C represents a constant region, and "01” represents an allele. 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.
  • IMGT International Immunogenetics Information System
  • TCR denotes an alpha chain variable region of TCR
  • TR denotes a T cell receptor gene
  • A denotes an alpha chain gene
  • V denotes a variable region
  • TRBV denotes the beta chain variable region of the TCR.
  • Each variable region comprises three framework regions (FR) and three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3 which are chimeric in the framework structure.
  • the CDR regions, particularly CDR3, determine the diversity of TCR and the binding of TCR to the pMHC complex.
  • the three skeleton structures are respectively FR1, their position numbers in the IMGT are 1-26; FR2, whose position number in the IMGT is 39-55; FR3, whose position number in the IMGT is 66-104.
  • the skeletal structures of the different TCR molecules are quite similar (K. Christopher Garcia, et al., Annu. Rev. Immunol. 1999. 17: 369-397), which is widely known to those skilled in the art and can be obtained from the TCR given in IMGT.
  • the variable region framework structure and its position number in the IMGT can be found, for example, in the IMGT public database (http://www.imgt.org/).
  • the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 are numbered in order from N-terminal to C-terminal, such as TRBC1*01 or TRBC2*01, according to N.
  • the 60th amino acid in the order from the end to the C-end is P (valine), which may be described as 60P of TRBC1*01 or TRBC2*01 exon 1 in the present invention, and may also be expressed as TRBC1* 01 or TRBC2*01, the 60th amino acid of exon 1, and TRBC1*01 or TRBC2*01, the 61st amino acid is Q (glutamine) in order from N to C.
  • TRBC1*01 or TRBC2*01 exon 1 can also be expressed as amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01, and so on.
  • Push. The amino acid sequences of TRBC1*01 and TRBC2*01 from the N-terminus to the C-terminus are shown in Figures 35a and 35b, respectively.
  • the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are numbered according to the positions listed in the IMGT.
  • the position number listed in IMGT is 46, which is described in the present invention as amino acid 46 of TRAV, and so on.
  • the positions of the amino acids in the TRAV mentioned in the present invention are numbered according to the position of the amino acid sequence listed in the IMGT, and the positions of the amino acids in the TRBC1*01 or TRBC2*01 are in the order from the N-terminus to the C-terminus.
  • the position number of the sequence It should be noted that the position number of the amino acid sequence listed in the IMGT is not exactly the same as the position number of the amino acid sequence in the order from the N-terminus to the C-terminus.
  • the alpha chain of TCR has a unique constant region, TRAC*01, and the two constant regions of the beta chain differ only slightly.
  • TRBC1*01 has 4N, 5K and 37F in its exon 1, and TRBC2*01 is prominent in it.
  • Sub-1 has 4K, 5N and 37Y. Therefore, the constant region of the ⁇ chain of the TCR molecule is TRBC1*01 or TRBC2*01 is substantially indistinguishable.
  • the constant region of the ⁇ chain selected in the examples of the present invention is TRBC2*01.
  • stability refers to any aspect of protein stability. Including renaturation ability, expression ability, protein refolding yield, thermal stability, anti-aggregation and anti-unfolding; more preferably, protein reproducibility yield and thermal stability.
  • three domain TCR means that the TCR comprises an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain except for the transmembrane domain, but which does not comprise an alpha chain constant domain, alpha chain
  • the variable domain forms a heterodimer with the beta strand, and the inter-chain disulfide bond connects the alpha chain variable region of the TCR to the beta chain constant region.
  • TCR tetra-domain TCR
  • 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 variable domain of the TCR chain and at least a portion of the constant domain, the alpha chain and the beta chain form a heterodimer, and the interchain disulfide bond connects the alpha chain variable region and the beta chain of the TCR Constant zone.
  • the present invention obtains a soluble and stable heterodimeric T cell receptor by introducing a covalent artificial interchain disulfide bond between the alpha chain variable region of the TCR and the beta chain constant region.
  • the artificial interchain disulfide bond of the TCR of the present invention is located between the FR2 of the ⁇ chain variable region (TRAV) and the constant region of the ⁇ chain.
  • the site for forming an artificial interchain disulfide bond may be between a suitable amino acid residue at position 46 or 47 of the TRAV and a constant region of the beta chain.
  • the site for the formation of an artificial interchain disulfide bond may also be between the 60th or 61st amino acid residue of exon 1 of TRBC1*01 or TRBC2*01 and the appropriate site of the alpha chain variable region. .
  • cysteine residue forming the interchain disulfide bond of the TCR of the invention is substituted:
  • the amino acid at position 46 of TRAV may be D, A, P, T, S, C, L, H, Y or K; the amino acid at position 47 of TRAV may be G, N, S, R, W, A Or K.
  • the TCR of the present invention is a three domain TCR, that is, the TCR comprises an alpha chain variable domain and a beta chain variable domain, and all or part of the beta chain except the transmembrane domain.
  • the constant domain but which does not comprise an alpha chain constant domain, forms a heterodimer with the beta chain, and the interchain disulfide bond connects the alpha chain variable region of the TCR to the beta chain constant region.
  • the beta strand of the three domain TCR of the invention comprises all constant structures except the transmembrane domain Domain (ie containing extracellular and cytoplasmic domains).
  • the cysteine residue forming a natural interchain disulfide bond in the ⁇ chain is preferably mutated to other amino acid residues which do not participate in the formation of a disulfide bond, preferably alanine or serine.
  • the beta strand of the three domain TCR of the invention comprises a partial constant domain other than the transmembrane domain, in which case a cysteine residue forming a natural interchain disulfide bond in the beta strand It is preferred to mutate to other amino acid residues which are not involved in the formation of disulfide bonds, preferably alanine or serine.
  • the C-terminus of the TCR ⁇ chain constant domain may be truncated to remove a cysteine residue forming a natural interchain disulfide bond.
  • it may be truncated at a distance of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from a cysteine residue forming a disulfide bond between the natural chains, The cysteine forming the natural interchain disulfide bond is removed.
  • the TCR of the invention is a four domain TCR, ie the TCR comprises (i) all or part of the TCR alpha chain except for its transmembrane domain, and (ii) All or part of the TCR ⁇ chain outside the transmembrane domain, wherein (i) and (ii) both comprise a variable domain of the TCR chain and at least a portion of the constant domain, the alpha chain and the beta chain form a heterodimer, and the artificial strand A sulfur bond connects the alpha chain variable region of the TCR to the beta chain constant region.
  • the natural interchain disulfide bond is not included in the four domain TCR of the invention.
  • the alpha and/or beta strands of the four domain TCRs of the invention can comprise all of the constant domains except the transmembrane domain (ie, comprising extracellular and cytoplasmic domains).
  • the cysteine residue forming a natural interchain disulfide bond in each chain is preferably mutated to another amino acid residue which does not participate in the formation of a disulfide bond, preferably alanine or serine.
  • the alpha and/or beta strands of the four domain TCRs of the invention may comprise a partial constant domain other than a transmembrane domain, in which case a natural TCR interchain disulfide bond is formed in each strand.
  • the cysteine residue is preferably mutated to other amino acid residues that are not involved in the formation of disulfide bonds, preferably alanine or serine. More preferably, the C-terminus of the TCR alpha and/or beta chain constant domains is truncated to remove cysteine residues that form a natural interchain disulfide bond.
  • the natural interchain disulfide bond may also be included in the TCR of the present invention.
  • the ⁇ domain and the ⁇ chain constant domain of the four domain TCR of the present invention may also contain an artificial interchain disulfide bond, and the cysteine residue forming the above interchain disulfide bond is substituted:
  • 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.
  • the constant domain of TCR is not directly involved in the binding of TCR to pMHC, and truncation of a certain number of amino acid residues at the C-terminus of the constant domain does not substantially affect the function of TCR, and thus the various chains of the TCR of the present invention It can also be shorter.
  • 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). In a preferred embodiment of the invention, the binding of the TCR to 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/or Or substitution, add one or several at the C-terminus and / or N-terminus (usually within 5, compared A good area of 3 or less, more preferably 2 or less) amino acids.
  • 1-6 usually 1-5, preferably 1-3, more preferably 1-2, optimally 1 amino acid deletions, insertions and/or Or substitution
  • add one or several at the C-terminus and / or N-terminus usually within 5, compared A good area of 3 or less, more preferably 2 or less
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus generally does not alter the structure and function of the protein.
  • the introduction of an artificial interchain disulfide bond between the alpha chain variable region of the TCR and the beta chain constant region provides a soluble and stable T cell receptor of the invention. Further, the present invention also identifies suitable sites in the alpha chain variable region and the beta chain constant region which are capable of mutating to cysteine to form an artificial interchain disulfide bond.
  • the TCR of the present invention not only contains human TCR, but one skilled in the art can obtain soluble and stable TCR of other species according to the information provided by the present invention.
  • alpha chain variable region and/or beta chain constant region of the TCR of other species may not be 100% identical to the corresponding portion of the human TCR chain, one skilled in the art will be able to identify the equivalent portion of the corresponding TCR to be mutated Cysteine residue.
  • ClustalW obtained from the European Institute of Bioinformatics can be used to compare TCR chains of other species with corresponding parts of human TCR chains to obtain corresponding sites.
  • the present invention comprises an artificially interchain disulfide-linked human soluble and stable ⁇ heterodimeric TCR, and other mammalian inter-chain disulfide-linked ⁇ TCRs, including but not limited to goats, Sheep, pigs, mice and rats.
  • 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.
  • a polypeptide fragment, derivative or analog of the invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more a polypeptide having a substituent group in one amino acid residue, or (iii) a polypeptide formed by fusing a TCR of the present invention with another compound (such as a compound which prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid.
  • polypeptide formed by the fusion of the polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His). These fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.
  • 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, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, Malay. Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include 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 may be identical or degenerate to the coding region sequence set forth in SEQ ID NO: 3, 4, 7, 8, 11, 12, 14, 16, 18, 21, 22, 24.
  • Variant As used herein, "degenerate variant" in the present invention refers to an amino acid sequence having the sequence of SEQ ID NO: 1, 2, 5, 6, 9, 10, 13, 15, 17, 19, 20, 23. a protein, but a nucleic acid sequence that differs 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 a vector comprising a polynucleotide of the invention, and to a vector or coding sequence of the invention Host cells produced by genetic engineering.
  • a cysteine residue forming an artificial interchain disulfide bond may be by any suitable method including, but not limited to, those based on polymerase chain reaction (PCR), cloning by restriction enzymes, or linkage-independent Cloning (LIC) method.
  • PCR polymerase chain reaction
  • LIC linkage-independent Cloning
  • the polypeptide of the invention may be a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptides of the invention may be chemically synthesized or recombinant. Accordingly, the polypeptide of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.
  • the polynucleotide of the present invention can be utilized to express or produce a recombinant polypeptide of the present invention by conventional recombinant DNA techniques. Generally there are the following steps:
  • TCR polypeptide of the present invention is isolated and purified from a culture medium or a cell.
  • the soluble and 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 with a conjugate, preferably in a covalent manner.
  • 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-myocardium) Yellow enzyme (DTD) or biphenyl hydrolase-like protein (BPHL); 11. chemotherapeutic agent (eg, cisplatin) and the like.
  • prodrug activating enzymes eg, DT-myocardium
  • DTD dephen
  • An antibody or fragment thereof that binds (preferably, covalently binds) to a TCR of the invention comprises an anti-T cell or an NK-cell determining antibody, such as an anti-CD3 or anti-CD28 or anti-CD16 antibody, preferably an anti-CD3 antibody.
  • an anti-CD3 or anti-CD28 or anti-CD16 antibody preferably an anti-CD3 antibody
  • 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.
  • a pharmaceutical composition of various dosage forms may be employed depending on the use.
  • an injection, an oral preparation, or the like can be exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity 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 with existing drugs such as HIV, SIV, EBV, CMV, HCV, HBV
  • the drug is near the infected cell area It is also beneficial to release or activate the activation function.
  • 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 soluble TCR of the invention may also bind to other molecules, preferably anti-CD3 antibodies, to redirect T cells such that they target target cells that present a particular antigen and kill.
  • the present invention is a soluble and stable T cell receptor which can be used for the purpose of studying the interaction between TCR and pMHC (peptide-primary histocompatibility complex) and for the diagnosis and treatment of diseases.
  • the present invention obtains a soluble and stable T cell receptor, and the TCR of the present invention can be well renatured, refolded, purified, and capable of specifically binding to its original ligand.
  • the T cell receptor of the present invention has a high Tm value.
  • 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.
  • TCR molecule 1G4 for antigen short peptide HLA-A2/SLLMWITQC (SEQ ID NO: 25), NY-ESO-1 tumor specific antigen
  • TRAV to cysteine
  • TRBC1* The amino acid at position 60 of 01 or TRBC2*01 exon 1 is mutated to cysteine to form an artificial interchain disulfide bond.
  • the designed primers are as follows:
  • the expression plasmid pET28a+ (Novagene) containing the 1G4 TCR ⁇ variable domain and the ⁇ chain gene was mutated as follows using the above ⁇ chain variable domain and ⁇ chain primer, respectively.
  • the reaction was carried out in a Bio-Rad PCR machine. After initial denaturation at 94 ° C for 2 min, 18 cycles of amplification (94 ° C 15 sec denaturation, 55 ° C 30 sec annealing and 68 ° C 6 min extension) were performed. It was then digested with 10 units of DpnI restriction enzyme (New England Biolabs) for 1 hour at 37 °C. 10 ⁇ L of the digested product was transformed into competent E. coli DH5 ⁇ bacteria and grown at 37 ° C for 16 hours. Monoclones were picked and cultured overnight in 5 mL LB + carnamycin. Plasmid DNA was purified using the Zyppy Plasmid Kit (ZYMO RESEARCH) according to the manufacturer's instructions and sent to Invitrogen for sequencing verification for correct mutation for downstream expression.
  • ZYMO RESEARCH Zyppy Plasmid Kit
  • the ⁇ -chain variable domain and the ⁇ -strand extracellular amino acid sequence of the three-domain TCR molecule 1G4 containing the artificial interchain disulfide bond of the present invention are shown in Figures 1a and 1b, respectively, and the corresponding nucleotide sequences are as shown in Figure 2a and As shown in 2b, the introduced cysteine residue is represented by a bold and underlined letter.
  • TCR ⁇ and ⁇ chain target gene sequences were synthesized and inserted into the expression vector pET28a+ (Novagene) by standard methods described in the Molecular Cloning a Laboratory Manual (3rd edition, Sambrook and Russell). ), the upstream and downstream cloning sites are NcoI and NotI, respectively. The insert was sequenced to confirm that it was correct.
  • the expression plasmid containing the TCR ⁇ chain variable domain and the ⁇ 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. Then, the inclusion body sample solution (9.2 mg ⁇ chain and 10 mg ⁇ chain) was sequentially dropped into 200 ml of 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 hydrochloric acid cysteine).
  • DTT dithiothreitol
  • the amine and 1.87 mM cystamine dihydrochloride were slowly stirred 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 using a Superdex 100 160/300 GL gel filtration chromatography prepacked column (GE Healthcare) pre-equilibrated in PBS buffer, at 46 of TRAV. Position and the 60th position of exon 1 of TRBC1*01 or TRBC2*01 are introduced into the artificial chain of the present invention.
  • the elution curve of the three-domain TCR molecule obtained after the disulfide bond is 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 1 and 6 of Figure 65.
  • the eluted single peak is a soluble TCR molecule linked by an artificial interchain disulfide bond.
  • the molecule forms a single band in the SDS gel and is stably present, and a separate ⁇ chain can be formed after reduction.
  • the variable domain and the beta chain are soluble TCR molecule linked by an artificial interchain disulfide bond.
  • 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 (Size Exclusion Chromatography) spectrum of the TCR molecule is shown in Figure 4.
  • the HPLC elution peak of the three-domain TCR molecule containing the artificial interchain disulfide bond of the present invention is single and symmetrical, indicating that the protein structure is stable, and there is no occurrence of aggregation or unfolding, and the purity is very high.
  • 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 the 1G4 TCR molecule which forms an artificial interchain disulfide bond between the 46th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1 is 49%.
  • the high yield indicates that the artificial chain interchain disulfide triple domain TCR molecule of the present invention is soluble and stable in the alpha chain variable region of the TCR and the beta chain constant region.
  • Example 2 1 ml of the 1G4 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 1G4 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
  • thermogram of the TCR of the ⁇ chain variable region and the ⁇ chain constant region of the present invention containing an artificial interchain disulfide bond is shown in Fig. 5, and its Tm value can reach 53 °C.
  • the thermogram can reflect that at room temperature, even at a temperature of 43-44 ° 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 several different concentrations (typically 64, 32, 16, 8, 4, 0uM) using PBST buffer (PBS + 0.005% Tween 20, pH 7.4). ), the affinity of the corresponding pMHC is 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 1G4 TCR molecules containing the artificial interchain disulfide bond of the present invention to their corresponding antigens are shown in FIG. 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, and the low concentration of the TCR molecule exhibits the same binding time as the high concentration TCR molecule, and can also be described from the side.
  • the soluble tridomain TCR molecule of the disulfide bond between artificial strands 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.
  • the specific detection method of the TCR protein containing the artificial interchain disulfide bond of the present invention is as follows: the corresponding antigen pMHC complex of TCR (biotinylated) and selected other unrelated antigen pMHC complexes (biotinylated) ) are loaded separately onto the surface of the SA sensor and then interact with each TCR protein to be tested; finally, the signals generated by their interaction are analyzed.
  • the 1G4 TCR protein containing the artificial interchain disulfide bond of the present invention binds only to its corresponding antigen and has no interaction with other unrelated antigens.
  • Example 5 Three-domain TCR molecule forming an artificial interchain disulfide bond at position 46 of TRAV and position 60 of TRBC1*01 or TRBC2*01 exon 1
  • This example further demonstrates that a soluble and stable three-domain TCR can be obtained after forming an artificial interchain disulfide bond at position 46 of the TCR molecule TRAV and at position 60 of TRBC1*01 or TRBC2*01 exon 1. molecule.
  • the TCR molecule JM22 (for antigen short peptide HLA-A2/GILGFVFTL (SEQ ID NO: 30), derived from influenza virus matrix protein) and LC13 (for antigen short peptide HLA-B4405: EEYLKAWTF (SEQ ID NO: 31)) TRAV
  • the amino acid at position 46 is mutated to cysteine, and the amino acid at position 60 of exon 1 of TRBC1*01 or TRBC2*01 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 ⁇ -chain variable domain and the ⁇ -strand extracellular amino acid sequence of the three-domain TCR molecule JM22 of the present invention containing an artificial interchain disulfide bond are shown in Figures 7a and 7b, respectively, and the corresponding nucleotide sequences are shown in Figure 8a and As shown in 8b, the introduced cysteine residues are indicated by bolded and underlined letters.
  • the elution profile and gel map are shown in lanes 2 (reducing gel) and lane 5 (non-reducing gel) of Figures 9 and 66, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 25%. Its Tm value is 54 ° C, the corresponding DSC spectrum is shown in Figure 11.
  • the binding curve of the JM22 molecule to its corresponding antigen is shown in FIG.
  • the ⁇ -chain variable domain and the ⁇ -chain extracellular amino acid sequence of the three-domain TCR molecule LC13 of the present invention containing an artificial interchain disulfide bond are shown in Figures 13a and 13b, respectively, and their corresponding nucleotide sequences are as shown in Figure 14a and As shown at 14b, the introduced cysteine residue is indicated by bolded and underlined letters.
  • the elution profile and gel map are shown in lanes 1 (reducing gel) and lane 4 (non-reducing gel) of Figures 15 and 66, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield is also quite high, reaching 21%. Its Tm value can reach 60 ° C, the corresponding DSC spectrum is shown in Figure 17.
  • the binding curve of the LC13 molecule to its corresponding antigen is shown in FIG.
  • the eluted peak component is a disulfide-bonded soluble TCR molecule of the artificial chain of the invention, forms a single band in the SDS gel and is stably present, and is reduced. A separate alpha chain variable domain and beta chain are then formed. The protein refolding yield is also high.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bonds of the present invention are also high, indicating that they maintain proper folding at a higher temperature and maintain the desired activity, indicating 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.
  • these TCR molecules of the present invention which introduce an artificial interchain disulfide bond bind only to their respective antigens and do not interact with other unrelated antigens, demonstrating good specificity. Therefore, the above experimental data demonstrate that the artificial interchain disulfide bond is introduced at position 46 of TRAV and at position 60 of exon 1 of TRBC1*01 or TRBC2*01, and a soluble and stable three-domain TCR of the present invention can be obtained. protein.
  • Example 6 forms a person at position 46 of TRAV and position 60 of exon 1 of TRBC1*01 or TRBC2*01 Four-domain TCR molecule with disulfide bond between chains
  • This example demonstrates that a soluble and stable four-domain TCR molecule can be obtained after forming an artificial interchain disulfide bond at position 46 of the TCR molecule TRAV and at position 60 of TRBC1*01 or TRBC2*01 exon 1. .
  • TCR molecule 1G4 for antigen short peptide HLA-A2/SLLMWITQC, NY-ESO-1 tumor-specific antigen
  • JM22 for antigen short peptide HLA-A2/GILGFVFTL, derived from influenza virus matrix protein
  • LC13 for Antigen short peptide HLA-B4405: EEYLKAWTF
  • the amino acid at position 46 of TRAV is mutated to cysteine
  • the amino acid at position 60 of exon 1 of TRBC1*01 or TRBC2*01 is mutated to cysteine to form Artificial interchain disulfide bond.
  • the primers and procedures used for the mutation are referred to the above examples.
  • the TCR was subjected to PCR, renaturation and performance testing in the manner described in Examples 1 to 4. The difference is that in the TCR refolding step of Example 2, the amounts of the inclusion bodies of the TCR ⁇ chain and the ⁇ chain used were 15 mg and 10 mg, respectively.
  • the ⁇ -chain and ⁇ -strand extracellular amino acid sequences of the four-domain TCR molecule 1G4 of the present invention containing an artificial interchain disulfide bond are shown in Figures 19 and 1b, respectively, and the corresponding nucleotide sequences are shown in Figures 20 and 2b, respectively.
  • the introduced cysteine residue is indicated by bolded and underlined letters.
  • the elution profile and gel map are shown in lanes 1 (reducing gel) and lane 6 (non-reducing gel) of Figures 21 and 67, respectively.
  • the HPLC elution peak is single and symmetric as shown in FIG.
  • the protein refolding yield reached 35%. Its Tm value is 56 ° C, and the corresponding DSC spectrum is shown in FIG. 23 .
  • the binding curve of the 1G4 molecule to its corresponding antigen is shown in FIG.
  • the ⁇ -chain and ⁇ -chain extracellular amino acid sequences of the four-domain TCR molecule JM22 of the present invention containing an artificial interchain disulfide bond are shown in Figures 25 and 7b, respectively, and the corresponding nucleotide sequences are shown in Figures 26 and 8b, respectively.
  • the introduced cysteine residue is indicated by bolded and underlined letters.
  • the elution profile and gel map are shown in lanes 2 (reducing gel) and lane 5 (non-reducing gel) of Figures 27 and 68, respectively.
  • the HPLC elution peak is single and symmetric as shown in FIG.
  • the protein refolding yield reached 20%. Its Tm value is 53 ° C, and the corresponding DSC spectrum is shown in FIG.
  • the binding curve of the JM22 molecule to its corresponding antigen is shown in FIG.
  • the alpha-chain variable domain and the beta-strand extracellular amino acid sequence of the four-domain TCR molecule LC13 of the present invention containing an artificial interchain disulfide bond are shown in Figures 31 and 13b, respectively, and the corresponding nucleotide sequences are shown in Figure 32 and As shown at 14b, the introduced cysteine residue is indicated by bolded and underlined letters.
  • the elution profile and gel map are shown in lanes 1 (reducing gel) and lane 4 (non-reducing gel) of Figures 33 and 68, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield was also quite high, reaching 22%. Its Tm value can reach 60 °C.
  • the binding curve of the LC13 molecule to its corresponding antigen is shown in FIG.
  • the eluted peak component is a soluble tetradomain TCR molecule which is disulfide-bonded between the artificial strands of the invention, forms a single band in the SDS gel and stably exists. After reduction, separate alpha and beta chains are formed. The protein refolding yield is also high.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bonds of the present invention are also high, indicating that they maintain proper folding at a higher temperature and maintain the desired activity, indicating 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.
  • these TCR molecules which introduce the artificial interchain disulfide bond of the present invention bind only to their corresponding antigens and do not interact with other unrelated antigens, exhibiting good specificity. Therefore, the above experimental data demonstrate that the artificial interchain disulfide bond is introduced at position 46 of TRAV and at position 60 of exon 1 of TRBC1*01 or TRBC2*01, and a soluble and stable four-domain TCR of the present invention can be obtained. protein.
  • Example 7 Three-domain TCR molecule forming an artificial interchain disulfide bond at position 47 of TRAV and position 61 of TRBC1*01 or TRBC2*01 exon 1
  • This example demonstrates that a soluble and stable three-domain domain of the invention can be obtained after the formation of an artificial interchain disulfide bond at position 47 of the TCR molecule TRAV and at position 61 of TRBC1*01 or TRBC2*01 exon 1 TCR molecule.
  • Mutant amino acid 47 of the 1G4 TCR molecule TRAV was mutated to cysteine, and the amino acid at position 61 of TRBC1*01 or TRBC2*01 exon 1 was mutated to cysteine to form inter-chain disulfide. key.
  • 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 ⁇ -chain variable domain and the ⁇ -chain extracellular amino acid sequence of the three-domain TCR molecule of the present invention containing an artificial interchain disulfide bond are shown in Figures 37a and 37b, respectively, and the corresponding nucleotide sequences are shown in Figures 38a and 38b, respectively.
  • the introduced cysteine residues are indicated by bolded and underlined letters.
  • the elution profile and gel map are shown in lanes 4 (reducing gel) and lane 9 (non-reducing gel) of Figures 39 and 65, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 36%. Its Tm value is 52 ° C, and the corresponding DSC spectrum is shown in Figure 41.
  • the binding curve of the TCR molecule to its corresponding antigen is shown in FIG.
  • the eluted peak component is a soluble tri-domain TCR molecule which is disulfide-bonded between the artificial strands of the invention, forms a single band in the SDS gel and is stably present, and is reduced. A separate alpha chain variable domain and beta chain are then formed. Protein refolding yield is also higher.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bonds of the present invention are high, indicating that they maintain proper folding at a higher temperature and maintain the desired activity, indicating 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.
  • these TCR molecules which introduce the artificial interchain disulfide bond of the present invention bind only to their corresponding antigens and do not interact with other unrelated antigens, exhibiting good specificity. Therefore, the above experimental data demonstrate that the artificial interchain disulfide bond is introduced at the 47th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1, and a soluble and stable three-domain TCR of the present invention can be obtained. protein.
  • Example 8 Four-domain TCR molecule forming an artificial interchain disulfide bond at position 47 of TRAV and position 61 of TRBC1*01 or TRBC2*01 exon 1
  • Mutant amino acid of amino acid 47 of TCR molecule TRAV was mutated to cysteine, and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01 was mutated to cysteine to form inter-chain disulfide bond. .
  • the primers and procedures used for the mutation are referred to the above examples.
  • the TCR was subjected to PCR, renaturation and performance testing in the manner described in Examples 1 to 4. The difference is that in the TCR refolding step of Example 2, the amounts of the inclusion bodies of the TCR ⁇ chain and the ⁇ chain used were 15 mg and 10 mg, respectively.
  • the four-domain TCR molecule ⁇ -chain and ⁇ -strand extracellular amino acid sequence of the present invention containing an artificial interchain disulfide bond are shown in Figures 43 and 37b, respectively, and the corresponding nucleotide sequences are shown in Figures 44 and 38b, respectively. Cysteine residues are indicated by bolded and underlined letters.
  • the elution profile and gel map are shown in lanes 4 (reducing gel) and lane 9 (non-reducing gel) of Figures 45 and 67, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 43%. Its Tm value is 56 ° C, and the corresponding DSC spectrum is shown in FIG. 47 .
  • the binding curve of the TCR molecule to its corresponding antigen is shown in FIG.
  • the eluted peak component is a soluble tetradomain TCR molecule which is disulfide-bonded between the artificial strands of the invention, forms a single band in the SDS gel and is stably present, and is reduced.
  • the separated alpha and beta chains are then formed. Protein refolding yield is also higher.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bonds of the present invention are high, indicating that they maintain proper folding at a higher temperature and maintain the desired activity, indicating high stability.
  • the binding curve of the TCR molecule to its original ligand shows that the concentration of TCR The reduction 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.
  • these TCR molecules which introduce the artificial interchain disulfide bond of the present invention bind only to their corresponding antigens and do not interact with other unrelated antigens, exhibiting good specificity. Therefore, the above experimental data demonstrate that the artificial interchain disulfide bond is introduced at the 47th position of TRAV and the 61st position of TRBC1*01 or TRBC2*01 exon 1 to obtain a soluble and stable four-domain TCR of the present invention. protein.
  • Example 9 A three-domain TCR molecule forming an artificial interchain disulfide bond at position 46 of TRAV and position 61 of TRBC1*01 or TRBC2*01 exon 1
  • Mutant amino acid of amino acid 46 of 1G4 TCR molecule TRAV was mutated to cysteine, and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01 was mutated to cysteine to form inter-chain disulfide. key.
  • the primers and procedures used for the mutation are referred to the above examples.
  • the TCR was subjected to PCR, renaturation and performance testing in the manner described in Examples 1 to 4.
  • the elution profile and gel map of the three domain TCR molecule of the present invention containing an artificial interchain disulfide bond are shown in lanes 2 (reducing gel) and lane 7 (non-reducing gel) of Figs. 49 and 65, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 37%.
  • Its Tm value is 48 ° C, and the corresponding DSC spectrum is shown in Figure 51.
  • the binding curve of the TCR molecule to its corresponding antigen is shown in FIG.
  • the eluted peak component is a soluble tri-domain TCR molecule which is disulfide-bonded between the artificial strands of the invention, forms a single band in the SDS gel and is stably present, and is reduced. A separate alpha chain variable domain and beta chain are then formed. Protein refolding yield is also higher.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bonds of the present invention are high, indicating that they maintain proper folding at a higher temperature and maintain the desired activity, indicating 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.
  • these TCR molecules which introduce the artificial interchain disulfide bond of the present invention bind only to their corresponding antigens and do not interact with other unrelated antigens, exhibiting good specificity. Therefore, the above experimental data demonstrate that the artificial interchain disulfide bond is introduced at position 46 of TRAV and at position 61 of exon 1 of TRBC1*01 or TRBC2*01, and a soluble and stable three-domain TCR of the present invention can be obtained. protein.
  • Example 10 Four-domain TCR molecule forming an artificial interchain disulfide bond at position 46 of TRAV and position 61 of TRBC1*01 or TRBC2*01 exon 1
  • Mutant amino acid of amino acid 46 of 1G4 TCR molecule TRAV was mutated to cysteine, and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01 was mutated to cysteine to form inter-chain disulfide. key.
  • the primers and procedures used for the mutation are referred to the above examples.
  • the TCR was subjected to PCR, renaturation and performance testing in the manner described in Examples 1 to 4. The difference is that in the TCR refolding step of Example 2, the amounts of the inclusion bodies of the TCR ⁇ chain and the ⁇ chain used were 15 mg and 10 mg, respectively.
  • the elution profile and gel map of the four domain TCR molecules of the present invention containing an artificial interchain disulfide bond are shown in lanes 2 (reducing gel) and lane 7 (non-reducing gel) of Figures 53 and 67, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 38%.
  • Its Tm value is 50 ° C, and the corresponding DSC spectrum is shown in FIG. 55 .
  • the binding curve of the TCR molecule to its corresponding antigen is shown in Figure 56.
  • the eluted peak component is a soluble tetradomain TCR molecule which is disulfide-bonded between the artificial strands of the invention, forms a single band in the SDS gel and is stably present, and is reduced.
  • the separated alpha and beta chains are then formed. Protein refolding yield is also higher.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bonds of the present invention are high, indicating that they maintain proper folding at a higher temperature and maintain the desired activity, indicating high stability.
  • the binding curve of the TCR molecule to its original ligand shows that the concentration of TCR The reduction 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.
  • these TCR molecules which introduce the artificial interchain disulfide bond of the present invention bind only to their corresponding antigens and do not interact with other unrelated antigens, exhibiting good specificity. Therefore, the above experimental data demonstrate that the introduction of an artificial interchain disulfide bond at position 46 of TRAV and position 61 of TRBC1*01 or TRBC2*01 exon 1 can obtain a soluble and stable four-domain TCR of the present invention. protein.
  • Example 11 Three-domain TCR molecule forming an artificial interchain disulfide bond at position 47 of TRAV and position 60 of TRBC1*01 or TRBC2*01 exon 1
  • This example demonstrates that a soluble and stable three-domain domain of the invention can be obtained after the formation of an artificial interchain disulfide bond at position 47 of the TCR molecule TRAV and at position 60 of TRBC1*01 or TRBC2*01 exon 1 TCR molecule.
  • Mutant amino acid 47 of 1G4 TCR molecule TRAV was mutated to cysteine, and amino acid 60 of TRBC1*01 or TRBC2*01 exon 1 was mutated to cysteine to form artificial interchain disulfide. key.
  • the primers and procedures used for the mutation are referred to the above examples.
  • the TCR was subjected to PCR, renaturation and performance testing in the manner described in Examples 1 to 4.
  • the elution profile and gel map of the three domain TCR molecule of the present invention containing an artificial interchain disulfide bond are shown in lanes 3 (reducing gel) and lane 8 (non-reducing gel) of Figs. 57 and 65, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 22%.
  • Its Tm value is 48 ° C, and the corresponding DSC spectrum is shown in FIG. 59 .
  • the binding curve of the TCR molecule to its corresponding antigen is shown in FIG.
  • the eluted peak component is a soluble tri-domain TCR molecule which is disulfide-bonded between the artificial strands of the invention, forms a single band in the SDS gel and is stably present, and is reduced. A separate alpha chain variable domain and beta chain are then formed. Protein refolding yield is also higher.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bonds of the present invention are high, indicating that they maintain proper folding at a higher temperature and maintain the desired activity, indicating 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.
  • these TCR molecules which introduce the artificial interchain disulfide bond of the present invention bind only to their corresponding antigens and do not interact with other unrelated antigens, exhibiting good specificity. Therefore, the above experimental data demonstrate that the artificial interchain disulfide bond is introduced at the 47th position of TRAV and the 60th position of TRBC1*01 or TRBC2*01 exon 1, and a soluble and stable three-domain TCR of the present invention can be obtained. protein.
  • Example 12 Four-domain TCR molecule forming an artificial interchain disulfide bond at position 47 of TRAV and position 60 of TRBC1*01 or TRBC2*01 exon 1
  • Mutant amino acid of amino acid 46 of 1G4 TCR molecule TRAV was mutated to cysteine, and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01 was mutated to cysteine to form inter-chain disulfide. key.
  • the primers and procedures used for the mutation are referred to the above examples.
  • the TCR was subjected to PCR, renaturation and performance testing in the manner described in Examples 1 to 4. The difference is that in the TCR refolding step of Example 2, the amounts of the inclusion bodies of the TCR ⁇ chain and the ⁇ chain used were 15 mg and 10 mg, respectively.
  • the elution profile and gel map of the tetradomain TCR molecule of the present invention containing an artificial interchain disulfide bond are shown in lanes 3 (reducing gel) and lane 8 (non-reducing gel) of Fig. 61 and Fig. 67, respectively.
  • the HPLC elution peaks are single and symmetric as shown in FIG.
  • the protein refolding yield reached 31%. Its Tm value is 52 ° C, and the corresponding DSC spectrum is shown in Fig. 63.
  • the binding curve of the TCR molecule to its corresponding antigen is shown in FIG.
  • the eluted peak component is a soluble tetradomain TCR molecule which is disulfide-bonded between the artificial strands of the invention, forms a single band in the SDS gel and is stably present, and is reduced.
  • the separated alpha and beta chains are then formed. Protein refolding yield is also higher.
  • the Tm values of the TCR molecules linked by the artificial interchain disulfide bonds of the present invention are high, indicating that they maintain proper folding at a higher temperature and maintain the desired activity, indicating high stability.
  • the binding curve of the TCR molecule to its original ligand shows that the concentration of TCR The reduction 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.
  • these TCR molecules which introduce the artificial interchain disulfide bond of the present invention bind only to their corresponding antigens and do not interact with other unrelated antigens, exhibiting good specificity. Therefore, the above experimental data demonstrate that the artificial interchain disulfide bond is introduced at position 47 of TRAV and at position 60 of exon 1 of TRBC1*01 or TRBC2*01, and a soluble and stable four-domain TCR of the present invention can be obtained. protein.

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Abstract

本发明公开了一种在其α链可变区与β链恒定区之间含有人工链间二硫键的异质二聚TCR及其制备方法，及所述TCR的用途。

Description

一种可溶且稳定的异质二聚TCR 技术领域
本发明属于生物医药领域，具体地说，本发明涉及一种可溶性T细胞受体及其制法、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。在95％的T细胞中TCR异质二聚体由α和β链组成，而5％的T细胞具有由γ和δ链组成的TCR。天然αβ异质二聚TCR具有α链和β链，α链和β链构成αβ异源二聚TCR的亚单位。TCR的α和β链一般看作各有两个“结构域”，即TCRα链可变域(Vα)和TCRα链恒定域(Cα)，TCRβ链可变域(Vβ)和TCRβ链恒定域(Cβ)。
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。因此，TCR对免疫系统的细胞免疫功能是至关重要的。
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,起到杀伤作用。
天然存在的TCR是一种膜蛋白，通过其跨膜区得以稳定，对于获得可溶性TCR蛋白而言，当TCR与膜分离开时，获得保持与其原配体(即pMHC)结合能力的可溶且稳定的TCR是一件非常困难的事情(Shin,et al.,(1993)science259:1901)。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。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。本领域技术人员致力于开发出可溶的、稳定性T细胞受体。
发明内容
本发明的目的在于提供一种可溶且稳定的异质二聚TCR及其应用。
本发明的第一方面，提供了一种αβ异质二聚TCR，所述TCR的α链可变区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，所述TCR的人工链间二硫键位于α链可变区的FR2及β链的恒定区之间。
在另一优选例中，形成所述TCR的人工链间二硫键的半胱氨酸残基取代了位于 TRAV的第46位或第47位氨基酸残基。
在另一优选例中，形成所述TCR的人工链间二硫键的半胱氨酸残基取代了TRBC1*01或TRBC2*01外显子1的第60位或第61位氨基酸残基。
在另一优选例中，形成所述TCR的人工链间二硫键的半胱氨酸残基取代了：
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。
在另一优选例中，所述TCR是可溶的。
在另一优选例中，所述TCR包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
在另一优选例中，β链恒定域中形成天然链间二硫键的半胱氨酸残基被替换为另一氨基酸，优选地，被替换为丙氨酸或丝氨酸。
在另一优选例中，在所述TCRβ链恒定域的C末端截短以去除形成天然链间二硫键的半胱氨酸残基。
在另一优选例中，所述TCR包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的可变域和至少一部分恒定域。
在另一优选例中，所述TCRα与β链恒定域之间不存在天然链间二硫键。
在另一优选例中，在所述TCRα链和/或β链恒定区的C末端截短以去除形成天然链间二硫键的半胱氨酸残基。
在另一优选例中，所述TCRα链和/或β链恒定区中形成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，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，在TCRα链恒定区与β链恒定区之间形成人工链间二硫键的半胱氨酸残基取代了：
TRAC*01外显子1的48T和TRBC1*01或TRBC2*01外显子1的57S；
TRAC*01外显子1的45T和TRBC1*01或TRBC2*01外显子1的77S；
TRAC*01外显子1的10Y和TRBC1*01或TRBC2*01外显子1的17S；
TRAC*01外显子1的45T和TRBC1*01或TRBC2*01外显子1的59D；
TRAC*01外显子1的15S和TRBC1*01或TRBC2*01外显子1的15E；
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。
在另一优选例中，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
在另一优选例中，所述TCR的Tm值≥45℃，优选地≥50℃，更优选地≥52℃，最优选的≥55℃。
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。
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。
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、本发明第五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，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TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域1G4TCR分子的α链可变域氨基酸序列及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链可变域与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链可变域与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链可变域与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链可变域与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7a和图7b分别是在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域JM22TCR分子的α链可变域氨基酸序列及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链可变域与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链可变域与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链可变域与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链可变域与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13a和图13b分别是在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域LC13TCR分子的α链可变域氨基酸序列及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链可变域与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链可变域与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链可变域与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链可变域与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19是在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域1G4分子的α链氨基酸序列。
图20是图19中氨基酸所对应的核苷酸序列。
图21为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域1G4TCRα链与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图22为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域1G4TCRα链与β链重折叠及蛋白纯化后的SEC图谱。
图23为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域1G4TCRα链与β链重折叠及蛋白纯化后测得的DSC热谱图。
图24为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域TCRα链与β链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25是在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域JM22分子的α链氨基酸序列。
图26是图25中氨基酸所对应的核苷酸序列。
图27为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域JM22TCRα链与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图28为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人 工链间二硫键的四结构域JM22TCRα链与β链重折叠及蛋白纯化后的SEC图谱。
图29为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域JM22TCRα链与β链重折叠及蛋白纯化后测得的DSC热谱图。
图30为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域TCRα链与β链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31是在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域LC13分子的α链氨基酸序列。
图32是图31中氨基酸所对应的核苷酸序列。
图33为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域LC13TCRα链与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图34为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域LC13TCRα链与β链重折叠及蛋白纯化后的SEC图谱。
图35a和图35b分别为IMGT中列出的TRBC1*01和TRBC2*01的氨基酸序列。
图36为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域TCRα链与β链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37a和图37b分别是在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的三结构域1G4TCR分子的α链可变域氨基酸序列及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链可变域与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链可变域与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链可变域与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链可变域与β链经重折叠及蛋白纯化后所得不同浓度的TCR分子与其相应抗原的结合曲线。
图43是在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域1G4TCR分子的α链氨基酸序列。
图44是图43中氨基酸所对应的核苷酸序列。
图45为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCRα链与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图46为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCRα链与β链重折叠及蛋白纯化后的SEC图谱。
图47为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCRα链与β链重折叠及蛋白纯化后测得的DSC热谱图。
图48为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCRα链与β链重折叠及蛋白纯化后所得不同浓度的TCR分子与其相应抗原的结合曲线。
图49为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的三结构域TCRα链与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图50为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人 工链间二硫键的三结构域TCRα链与β链重折叠及蛋白纯化后的SEC图谱。
图51为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的三结构域TCRα链与β链重折叠及蛋白纯化后测得的DSC热谱图。
图52为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的三结构域TCRα链与β链重折叠及蛋白纯化后所得不同浓度的TCR分子与其相应抗原的结合曲线。
图53为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCRα链与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图54为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCRα链与β链重折叠及蛋白纯化后的SEC图谱。
图55为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCRα链与β链重折叠及蛋白纯化后测得的DSC热谱图。
图56为在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCRα链与β链重折叠及蛋白纯化后所得不同浓度的TCR分子与其相应抗原的结合曲线。
图57为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域TCRα链与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图58为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域TCRα链与β链重折叠及蛋白纯化后的SEC图谱。
图59为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域TCRα链与β链重折叠及蛋白纯化后测得的DSC热谱图。
图60为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域TCRα链与β链重折叠及蛋白纯化后所得不同浓度的TCR分子与其相应抗原的结合曲线。
图61为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域TCRα链与β链重折叠后经凝胶过滤层析柱的洗脱曲线。
图62为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域TCRα链与β链重折叠及蛋白纯化后的SEC图谱。
图63为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域TCRα链与β链重折叠及蛋白纯化后测得的DSC热谱图。
图64为在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域TCRα链与β链重折叠及蛋白纯化后所得不同浓度的TCR分子与其相应抗原的结合曲线。
图65为在1G4 TCR分子α链可变区与β链恒定区之间不同位置含有人工链间二硫键的三结构域可溶蛋白的胶图。
图66为在不同TCR分子α链可变区与β链恒定区之间含有人工链间二硫键的三结构域可溶蛋白的胶图。
图67为在1G4 TCR分子α链可变区与β链恒定区之间不同位置含有人工链间二硫键的四结构域可溶蛋白的胶图。
图68为在不同TCR分子α链可变区与β链恒定区之间含有人工链间二硫键的四结构域可溶蛋白的胶图。
具体实施方式
本发明通过广泛而深入的研究，意外地获得了一种可溶且稳定的T细胞受体。具体地，本发明TCR为αβ异质二聚体，并且本发明TCR的α链可变区与β链恒定区之间含有共价的人工链间二硫键。更具体地，本发明TCR的人工链间二硫键位于α链 的FR2及β链的恒定区之间。本发明还提供了所述TCR的用途，及其制备方法。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且其意图不是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
术语
T细胞受体
天然αβ异质二聚TCR具有α链和β链，α链和β链构成αβ异质二聚TCR的两个亚单位。TCR的α和β链一般看作各有两个“结构域”，即TCRα链可变域(Vα)和TCRα链恒定域(Cα)，TCRβ链可变域(Vβ)和TCRβ链恒定域(Cβ)。在天然TCR的近膜区Cα与Cβ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，都指在α链可变区与β链恒定区之间含有本发明人工链间二硫键的异质二聚TCR。
广义上讲，TCRα和β链各包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。TCR的α和β链一般看作各有两个“结构域”即可变域和恒定域，可变域由连接的可变区和连接区构成，恒定域还包含跨膜区和胞质区，胞质区很短。
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链恒定区，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”。
本领域的技术人员广为知晓并可得到IMGT中给出的TRAC*01与TRBC1*01或TRBC2*01的序列，例如可在IMGT的公开数据库中找到(http://www.imgt.org/)。
“TRAV”表示TCR的α链可变区，其中“TR”表示T细胞受体基因，“A”表示α链基因，V表示可变区。同样地，“TRBV”表示TCR的β链可变区。各可变区包含3个骨架结构(framework regions，FR)以及嵌合在骨架结构中的3个CDR(complementary determining region，互补决定区)，CDR1、CDR2和CDR3。CDR区，尤其是CDR3决定了TCR的多样性以及TCR与pMHC复合物的结合。3个骨架结构分别为FR1，其在IMGT中的位置编号为1-26；FR2，其在IMGT中的位置编号为39-55；FR3，其在IMGT中的位置编号为66-104。不同TCR分子的骨架结构是十分相似的(K.Christopher Garcia,et al.,Annu.Rev.Immunol.1999.17:369-397)，本领域的技术人员广为知晓并可得到IMGT中给出的TCR可变区骨架结构及其在IMGT中的位置编号，例如可在IMGT的公开数据库中找到(http://www.imgt.org/)。
为方便描述，本发明中TRAC*01与TRBC1*01或TRBC2*01氨基酸序列的位置编号按从N端到C端依次的顺序进行位置编号，如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第60个氨基酸为P(脯氨酸)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的60P，也可将其表述为TRBC1*01或TRBC2*01外显子1的第60位氨基酸，又如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第61个氨基酸为Q(谷氨酰胺)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的61Q，也可将其表述为TRBC1*01或TRBC2*01外显子1的第61位氨基酸，其他以此类 推。TRBC1*01和TRBC2*01从N端到C端的氨基酸序列分别如图35a和35b所示。本发明中，可变区TRAV与TRBV的氨基酸序列的位置编号，按照IMGT中列出的位置编号。如TRAV中的某个氨基酸，IMGT中列出的位置编号为46，则本发明中将其描述为TRAV第46位氨基酸，其他以此类推。综上，本发明中提及的TRAV中氨基酸的位置是按照IMGT中列出的氨基酸序列的位置编号，TRBC1*01或TRBC2*01中氨基酸的位置则是按其从N端到C端依次的顺序进行的位置编号。应注意，IMGT中列出的氨基酸序列的位置编号与将氨基酸的序列按从N端到C端依次的顺序进行的位置编号并不完全相同。
TCR的α链具有唯一的恒定区TRAC*01，β链的两种恒定区仅有微小差别，TRBC1*01在其外显子1中具有4N、5K和37F，而TRBC2*01在其外显子1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。本发明实施例中选用的β链的恒定区为TRBC2*01。
稳定性
术语“稳定性”指蛋白质稳定性的任何方面。包括复性能力、表达能力、蛋白复性收率、热稳定性、抗聚集及抗解折叠等方面；更佳地，是指蛋白复性收率及热稳定性方面。
三结构域TCR
术语“三结构域TCR”指所述TCR包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，α链可变域与β链形成异质二聚体，人工链间二硫键连接所述TCR的α链可变区与β链恒定区。
四结构域TCR
术语“四结构域TCR”指所述TCR包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的可变域和至少一部分恒定域，α链与β链形成异质二聚体，人工链间二硫键连接所述TCR的α链可变区与β链恒定区。
发明详述
本发明通过在TCR的α链可变区与β链恒定区之间引入共价人工链间二硫键，获得了一种可溶且稳定的异质二聚T细胞受体。具体地，本发明TCR的人工链间二硫键位于α链可变区(TRAV)的FR2及β链的恒定区之间。更具体地，形成人工链间二硫键的位点可在TRAV的第46位或第47位氨基酸残基与β链恒定区适宜的位点之间。同样地，形成人工链间二硫键的位点也可在TRBC1*01或TRBC2*01外显子1的第60位或第61位氨基酸残基与α链可变区适宜的位点之间。
在本发明的优选例中，形成本发明TCR的人工链间二硫键的半胱氨酸残基取代了：
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。
优选地，TRAV的第46位氨基酸可以是D、A、P、T、S、C、L、H、Y或K；TRAV的第47位氨基酸可以是G、N、S、R、W、A或K。
在本发明的一个较佳实施方式中，本发明的TCR为三结构域TCR，即所述TCR包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，α链可变域与β链形成异质二聚体，人工链间二硫键连接所述TCR的α链可变区与β链恒定区。
优选地，本发明的三结构域TCR的β链包含除跨膜结构域以外的全部恒定结构 域(即包含胞外和胞质结构域)。在这种情况下，β链中形成天然链间二硫键的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，优选地，丙氨酸或丝氨酸。
更优选地，本发明的三结构域TCR的β链包含除跨膜结构域以外的部分恒定结构域，在这种情况下，β链中形成天然链间二硫键的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，优选地，丙氨酸或丝氨酸。或者，也可以在所述TCRβ链恒定域的C末端截短以去除形成天然链间二硫键的半胱氨酸残基。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。
在本发明的另一较佳实施方式中，本发明的TCR为四结构域TCR，即所述TCR包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的可变域和至少一部分恒定域，α链与β链形成异质二聚体，人工链间二硫键连接所述TCR的α链可变区与β链恒定区。
优选地，本发明的四结构域TCR中不包含天然链间二硫键。一方面，本发明的四结构域TCR的α和/或β链可以包含除跨膜结构域以外的全部恒定结构域(即包含胞外和胞质结构域)。在这种情况下，各链中形成天然链间二硫键的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，优选地，丙氨酸或丝氨酸。另一方面，本发明的四结构域TCR的α和/或β链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，各链中形成天然TCR链间二硫键的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，优选地，丙氨酸或丝氨酸。更优选地，在所述TCRα和/或β链恒定域的C末端截短以去除形成天然链间二硫键的半胱氨酸残基。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。但应当指出，本发明的TCR中也可包含天然的链间二硫键。
本发明的四结构域TCR的α和β链恒定域间也可以包含人工链间二硫键，形成上述人工链间二硫键的半胱氨酸残基取代了：
TRAC*01外显子1的48T和TRBC1*01或TRBC2*01外显子1的57S；
TRAC*01外显子1的45T和TRBC1*01或TRBC2*01外显子1的77S；
TRAC*01外显子1的10Y和TRBC1*01或TRBC2*01外显子1的17S；
TRAC*01外显子1的45T和TRBC1*01或TRBC2*01外显子1的59D；
TRAC*01外显子1的15S和TRBC1*01或TRBC2*01外显子1的15E；
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。
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。
应理解，TCR的恒定结构域并不直接参与TCR与pMHC的结合，在恒定域的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)。在本发明的优选例中，TCR与其相应pMHC的结合通过forteBIO Oke进行测定，如在本发明实施例4中所述。
可在本发明TCR链中引入适量的突变而不影响其功能性。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。
在TCR的α链可变区与β链恒定区之间引入人工链间二硫键可以获得可溶且稳定的本发明T细胞受体。进一步，本发明还鉴定出了α链可变区与β链恒定区中能够突变为半胱氨酸以形成人工链间二硫键的合适位点。本发明的TCR不仅包含人类的TCR，本领域技术人员可根据本发明提供的信息，获得其他物种的可溶且稳定的TCR。
虽然其他物种的TCR的α链可变区和/或β链恒定区可能与人类TCR链的相应部分不具有100％相同性，但本领域技术人员能够鉴定出相应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链与人类TCR链的相应部分进行比对，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可溶且稳定的αβ异质二聚TCR，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。
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。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 PCTCN2016077680-appb-000001
Figure PCTCN2016077680-appb-000002
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:3、4、7、8、11、12、14、16、18、21、22、24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:1、2、5、6、9、10、13、15、17、19、20、23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。
制备方法
形成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且稳定的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，优选地以共价方式结合。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(优选地,共价结合)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，优选地抗-CD3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。
另外，可溶性的本发明TCR还可与其他分子，优选地抗-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靶细胞并进行杀伤。
工业应用性
本发明可溶且稳定的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)本发明获得了可溶且稳定的T细胞受体，本发明的TCR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。
(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，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TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域1G4分子的引物设计和PCR突变
将TCR分子1G4(针对抗原短肽HLA-A2/SLLMWITQC(SEQ ID NO:25)，NY-ESO-1肿瘤特异性抗原)TRAV的第46位氨基酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第60位氨基酸突变为半胱氨酸，以形成人工链间二硫键。
上述TCR的TRAV的第46位氨基酸突变为半胱氨酸时，所设计的引物如下：
Figure PCTCN2016077680-appb-000003
上述TCR的TRBC1*01或TRBC2*01外显子1的第60位氨基酸突变为半胱氨酸时，所设计的引物如下：
Figure PCTCN2016077680-appb-000004
进行PCR的步骤如下：
含1G4 TCRα可变域和β链基因的表达质粒pET28a+(Novagene)分别用上述α链可变域和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。
含有本发明人工链间二硫键的三结构域TCR分子1G4的α链可变域与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的字母表示。
通过《分子克隆实验室手册》(Molecular Cloning a Laboratory Manual)(第三版，Sambrook和Russell)中描述的标准方法将上述TCRα和β链的目的基因序列经合成后分别插入到表达载体pET28a+(Novagene)，上下游的克隆位点分别是NcoI和NotI。插入片段经过测序确认无误。
实施例2 TCR的表达、重折叠和纯化及其结果测定
TCR蛋白的表达
将含有TCRα链可变域与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(9.2mgα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 100 160/300GL凝胶过滤层析预装柱(GE Healthcare)纯化蛋白，在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位引入本发明的人工链间 二硫键后得到的三结构域TCR分子的洗脱曲线如图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65的泳道1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凝胶中形成单一条带并稳定存在，经还原后形成分开的α链可变域和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三结构域TCR分子的HPLC洗脱峰单一且对称，说明该蛋白结构稳定，并没有聚集或解折叠等现象的发生，纯度非常高。
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TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位之间形成人工链间二硫键的1G4 TCR分子的蛋白复性收率为49％。收率很高，说明在TCR的α链可变区与β链恒定区具有本发明人工链间二硫键三结构域TCR分子可溶且稳定。
实施例3 TCR的α链可变区与β链恒定区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1G4 TCR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。本发明的α链可变区与β链恒定区含有人工链间二硫键的TCR的DSC热谱图如图5所示，其Tm值可以达到53℃。该热谱图能够反映在室温下，甚至温度达到43-44℃，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，pH7.4)将TCR蛋白稀释成几个不同的浓度(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 16/60 S200HR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℃。
不同浓度的含有本发明人工链间二硫键的1G4 TCR分子与其相应抗原的结合曲线如图6所示。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分子相同的结合时间，也能够从侧面说明具有本发明人工链间二硫键的可溶性三结构域TCR分子是较稳定的。
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。含有本发明人工链间二硫键的TCR蛋白的特异性检测方式如下：将TCR的相应抗原pMHC复合物(已生物素化)及选定的几种其他无关抗原pMHC复合物(已生物素化)分别加载到SA传感器的表面，然后，与各个待测的TCR蛋白相互作用；最后，分析其互作所产生的信号。
按上述测定方法，含本发明人工链间二硫键的1G4 TCR蛋白只与其相应抗原有结合，与其他无关抗原均无相互作用。
实施例5在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域TCR分子
本实施例进一步验证了在TCR分子TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键后，能够获得可溶且稳定的三结构域TCR分子。
将TCR分子JM22(针对抗原短肽HLA-A2/GILGFVFTL(SEQ ID NO:30)，源自流感病毒基质蛋白)和LC13(针对抗原短肽HLA-B4405：EEYLKAWTF(SEQ ID NO:31))TRAV的第46位氨基酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第60位氨基酸突变为半胱氨酸，以形成人工链间二硫键。
上述JM22 TCR的TRAV的第46位氨基酸突变为半胱氨酸时，所设计的引物如下：
Figure PCTCN2016077680-appb-000005
上述JM22 TCR的TRBC1*01或TRBC2*01外显子1的第60位氨基酸突变为半胱氨酸时，所设计的引物如下：
Figure PCTCN2016077680-appb-000006
上述LC13 TCR的TRAV的第46位氨基酸突变为半胱氨酸时，所设计的引物如下：
Figure PCTCN2016077680-appb-000007
上述LC13 TCR的TRBC1*01或TRBC2*01外显子1的第60位氨基酸突变为半胱氨酸时，所设计的引物如下：
Figure PCTCN2016077680-appb-000008
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。
含有人工链间二硫键的本发明三结构域TCR分子JM22的α链可变域与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。其洗脱曲线和胶图分别如图9和图66的泳道2(还原胶)和泳道5(非还原胶)所示。HPLC洗脱峰单一且对称如图10所示。蛋白复性收率达到了25％。其Tm值为54℃，对应的DSC谱图如图11所示。JM22分子与其相应抗原的结合曲线如图12所示。
含有人工链间二硫键的本发明三结构域TCR分子LC13的α链可变域与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。其洗脱曲线和胶图分别如图15和图66的泳道1(还原胶)和泳道4(非还原胶)所示。HPLC洗脱峰单一且对称如图16所示。蛋白复性收率也相当高，达到了21％。其Tm值可以达到60℃，对应的DSC谱图如图17所示。LC13分子与其相应抗原的结合曲线如图18所示。
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凝胶中形成单一条带并稳定存在，经还原后形成分开的α链可变域和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，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本发明TCR分子只与其相应抗原结合，与其他几种无关抗原并无相互作用，展示了很好的特异性。因此，上述实验数据证明了在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位引入人工链间二硫键，可以获得可溶且稳定的本发明三结构域TCR蛋白。
实施例6在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人 工链间二硫键的四结构域TCR分子
本实施例验证了在TCR分子TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键后，能够获得可溶且稳定的四结构域TCR分子。
分别将TCR分子1G4(针对抗原短肽HLA-A2/SLLMWITQC，NY-ESO-1肿瘤特异性抗原)、JM22(针对抗原短肽HLA-A2/GILGFVFTL，源自流感病毒基质蛋白)和LC13(针对抗原短肽HLA-B4405：EEYLKAWTF)TRAV的第46位氨基酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第60位氨基酸突变为半胱氨酸，以形成人工链间二硫键。突变所用引物及步骤参照上述实施例。
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。稍有不同之处在于，实施例2中TCR重折叠步骤中，所用TCRα链与β链的包涵体的量分别为15mg及10mg。
含有人工链间二硫键的本发明四结构域TCR分子1G4的α链与β链胞外氨基酸序列分别如图19和1b所示，其对应的核苷酸序列分别如图20和2b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21和图67的泳道1(还原胶)和泳道6(非还原胶)所示。HPLC洗脱峰单一且对称如图22所示。蛋白复性收率达到了35％。其Tm值为56℃，对应的DSC谱图如图23所示。1G4分子与其相应抗原的结合曲线如图24所示。
含有人工链间二硫键的本发明四结构域TCR分子JM22的α链与β链胞外氨基酸序列分别如图25和7b所示，其对应的核苷酸序列分别如图26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。其洗脱曲线和胶图分别如图27和图68的泳道2(还原胶)和泳道5(非还原胶)所示。HPLC洗脱峰单一且对称如图28所示。蛋白复性收率达到了20％。其Tm值为53℃，对应的DSC谱图如图29所示。JM22分子与其相应抗原的结合曲线如图30所示。
含有人工链间二硫键的本发明四结构域TCR分子LC13的α链可变域与β链胞外氨基酸序列分别如图31和13b所示，其对应的核苷酸序列分别如图32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。其洗脱曲线和胶图分别如图33和图68的泳道1(还原胶)和泳道4(非还原胶)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22％。其Tm值可以达到60℃。LC13分子与其相应抗原的结合曲线如图36所示。
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四结构域TCR分子，在SDS凝胶中形成单一条带并稳定存在，经还原后形成分开的α链和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，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，这些引入本发明人工链间二硫键的TCR分子只与其相应抗原结合，与其他几种无关抗原并无相互作用，展示了很好的特异性。因此，上述实验数据证明了在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第60位引入人工链间二硫键，可以获得可溶且稳定的本发明四结构域TCR蛋白。
实施例7在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的三结构域TCR分子
本实施例验证了在TCR分子TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键后，能够获得可溶且稳定的本发明三结构域TCR分子。
将1G4 TCR分子TRAV的第47位氨基酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第61位氨基酸突变为半胱氨酸，以形成人工链间二硫键。
上述TCR的TRAV的第47位氨基酸突变为半胱氨酸时，所设计的引物如下：
Figure PCTCN2016077680-appb-000009
上述TCR的TRBC1*01或TRBC2*01外显子1的第61位氨基酸突变为半胱氨酸时，所设计的引物如下：
Figure PCTCN2016077680-appb-000010
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。
含有人工链间二硫键的本发明三结构域TCR分子的α链可变域与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65的泳道4(还原胶)和泳道9(非还原胶)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36％。其Tm值为52℃，对应的DSC谱图如图41所示。TCR分子与其相应抗原的结合曲线如图42所示。
由以上的洗脱曲线和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三结构域TCR分子，在SDS凝胶中形成单一条带并稳定存在，经还原后形成分开的α链可变域和β链。蛋白复性收率也较高。另外，由本发明人工链间二硫键连接的TCR分子的Tm值很高，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，这些引入本发明人工链间二硫键的TCR分子只与其相应抗原结合，与其他几种无关抗原并无相互作用，展示了很好的特异性。因此，上述实验数据证明了在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位引入人工链间二硫键，可以获得可溶且稳定的本发明三结构域TCR蛋白。
实施例8在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCR分子
本实施例验证了在TCR分子TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键后，能够获得可溶且稳定的本发明四结构域TCR分子。
将TCR分子TRAV的第47位氨基酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第61位氨基酸突变为半胱氨酸，以形成人工链间二硫键。突变所用引物及步骤参照上述实施例。
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。稍有不同之处在于，实施例2中TCR重折叠步骤中，所用TCRα链与β链的包涵体的量分别为15mg及10mg。
含有人工链间二硫键的本发明四结构域TCR分子α链与β链胞外氨基酸序列分别如图43和37b所示，其对应的核苷酸序列分别如图44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45和图67的泳道4(还原胶)和泳道9(非还原胶)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43％。其Tm值为56℃，对应的DSC谱图如图47所示。TCR分子与其相应抗原的结合曲线如图48所示。
由以上的洗脱曲线和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四结构域TCR分子，在SDS凝胶中形成单一条带并稳定存在，经还原后形成分开的α链和β链。蛋白复性收率也较高。另外，由本发明人工链间二硫键连接的TCR分子的Tm值很高，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，这些引入本发明人工链间二硫键的TCR分子只与其相应抗原结合，与其他几种无关抗原并无相互作用，展示了很好的特异性。因此，上述实验数据证明了在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第61位引入人工链间二硫键，可以获得可溶且稳定的本发明四结构域TCR蛋白。
实施例9在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的三结构域TCR分子
本实施例验证了在TCR分子TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键后，能够获得可溶且稳定的本发明三结构域TCR分子。
将1G4 TCR分子TRAV的第46位氨基酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第61位氨基酸突变为半胱氨酸，以形成人工链间二硫键。突变所用引物及步骤参照上述实施例。
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。
含有人工链间二硫键的本发明三结构域TCR分子的其洗脱曲线和胶图分别如图49和图65的泳道2(还原胶)和泳道7(非还原胶)所示。HPLC洗脱峰单一且对称如图50所示。蛋白复性收率达到了37％。其Tm值为48℃，对应的DSC谱图如图51所示。TCR分子与其相应抗原的结合曲线如图52所示。
由以上的洗脱曲线和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三结构域TCR分子，在SDS凝胶中形成单一条带并稳定存在，经还原后形成分开的α链可变域和β链。蛋白复性收率也较高。另外，由本发明人工链间二硫键连接的TCR分子的Tm值很高，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，这些引入本发明人工链间二硫键的TCR分子只与其相应抗原结合，与其他几种无关抗原并无相互作用，展示了很好的特异性。因此，上述实验数据证明了在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位引入人工链间二硫键，可以获得可溶且稳定的本发明三结构域TCR蛋白。
实施例10在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键的四结构域TCR分子
本实施例验证了在TCR分子TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位形成人工链间二硫键后，能够获得可溶且稳定的本发明四结构域TCR分子。
将1G4 TCR分子TRAV的第46位氨基酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第61位氨基酸突变为半胱氨酸，以形成人工链间二硫键。突变所用引物及步骤参照上述实施例。
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。稍有不同之处在于，实施例2中TCR重折叠步骤中，所用TCRα链与β链的包涵体的量分别为15mg及10mg。
含有人工链间二硫键的本发明四结构域TCR分子的其洗脱曲线和胶图分别如图53和图67的泳道2(还原胶)和泳道7(非还原胶)所示。HPLC洗脱峰单一且对称如图54所示。蛋白复性收率达到了38％。其Tm值为50℃，对应的DSC谱图如图55所示。TCR分子与其相应抗原的结合曲线如图56所示。
由以上的洗脱曲线和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四结构域TCR分子，在SDS凝胶中形成单一条带并稳定存在，经还原后形成分开的α链和β链。蛋白复性收率也较高。另外，由本发明人工链间二硫键连接的TCR分子的Tm值很高，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，这些引入本发明人工链间二硫键的TCR分子只与其相应抗原结合，与其他几种无关抗原并无相互作用，展示了很好的特异性。因此，上述实验数据证明了在TRAV的第46位和TRBC1*01或TRBC2*01外显子1的第61位引入人工链间二硫键，可以获得可溶且稳定的本发明四结构域TCR蛋白。
实施例11在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的三结构域TCR分子
本实施例验证了在TCR分子TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键后，能够获得可溶且稳定的本发明三结构域TCR分子。
将1G4 TCR分子TRAV的第47位氨基酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第60位氨基酸突变为半胱氨酸，以形成人工链间二硫键。突变所用引物及步骤参照上述实施例。
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。
含有人工链间二硫键的本发明三结构域TCR分子的其洗脱曲线和胶图分别如图57和图65的泳道3(还原胶)和泳道8(非还原胶)所示。HPLC洗脱峰单一且对称如图58所示。蛋白复性收率达到了22％。其Tm值为48℃，对应的DSC谱图如图59所示。TCR分子与其相应抗原的结合曲线如图60所示。
由以上的洗脱曲线和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三结构域TCR分子，在SDS凝胶中形成单一条带并稳定存在，经还原后形成分开的α链可变域和β链。蛋白复性收率也较高。另外，由本发明人工链间二硫键连接的TCR分子的Tm值很高，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，这些引入本发明人工链间二硫键的TCR分子只与其相应抗原结合，与其他几种无关抗原并无相互作用，展示了很好的特异性。因此，上述实验数据证明了在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位引入人工链间二硫键，可以获得可溶且稳定的本发明三结构域TCR蛋白。
实施例12在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键的四结构域TCR分子
本实施例验证了在TCR分子TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位形成人工链间二硫键后，能够获得可溶且稳定的本发明四结构域TCR分子。
将1G4 TCR分子TRAV的第46位氨基酸突变为半胱氨酸，并将其TRBC1*01或TRBC2*01外显子1的第61位氨基酸突变为半胱氨酸，以形成人工链间二硫键。突变所用引物及步骤参照上述实施例。
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。稍有不同之处在于，实施例2中TCR重折叠步骤中，所用TCRα链与β链的包涵体的量分别为15mg及10mg。
含有人工链间二硫键的本发明四结构域TCR分子的其洗脱曲线和胶图分别如图61和图67的泳道3(还原胶)和泳道8(非还原胶)所示。HPLC洗脱峰单一且对称如图62所示。蛋白复性收率达到了31％。其Tm值为52℃，对应的DSC谱图如图63所示。TCR分子与其相应抗原的结合曲线如图64所示。
由以上的洗脱曲线和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四结构域TCR分子，在SDS凝胶中形成单一条带并稳定存在，经还原后形成分开的α链和β链。蛋白复性收率也较高。另外，由本发明人工链间二硫键连接的TCR分子的Tm值很高，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，这些引入本发明人工链间二硫键的TCR分子只与其相应抗原结合，与其他几种无关抗原并无相互作用，展示了很好的特异性。因此，上述实验数据证明了在TRAV的第47位和TRBC1*01或TRBC2*01外显子1的第60位引入人工链间二硫键，可以获得可溶且稳定的本发明四结构域TCR蛋白。
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 (27)

  1. 一种α β异质二聚TCR，其特征在于，所述TCR的α链可变区与β链恒定区之间含有人工链间二硫键。
  2. 如权利要求1所述的TCR，其特征在于，所述TCR的人工链间二硫键位于α链可变区的FR2及β链的恒定区之间。
  3. 如权利要求2所述的TCR，其特征在于，形成所述TCR的人工链间二硫键的半胱氨酸残基取代了位于TRAV的第46位或第47位氨基酸残基。
  4. 如权利要求2或3所述的TCR，其特征在于，形成所述TCR的人工链间二硫键的半胱氨酸残基取代了TRBC1*01或TRBC2*01外显子1的第60位或第61位氨基酸残基。
  5. 如以上任一权利要求所述的TCR，其特征在于，形成所述TCR的人工链间二硫键的半胱氨酸残基取代了：
    TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；
    TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；
    TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或
    TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。
  6. 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。
  7. 如以上任一权利要求所述的TCR，其特征在于，所述TCR包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
  8. 如权利要求7所述的TCR，其特征在于，β链恒定域中形成天然链间二硫键的半胱氨酸残基被替换为另一氨基酸，优选地，被替换为丙氨酸或丝氨酸。
  9. 如权利要求7所述的TCR，其特征在于，在所述TCRβ链恒定域的C末端截短以去除形成天然链间二硫键的半胱氨酸残基。
  10. 如权利要求1-6中任一所述的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链的可变域和至少一部分恒定域。
  11. 如权利要求10所述的TCR，其特征在于，所述TCRα与β链恒定域之 间不存在天然链间二硫键。
  12. 如权利要求11所述的TCR，其特征在于，在所述TCRα链和/或β链恒定区的C末端截短以去除形成天然链间二硫键的半胱氨酸残基。
  13. 如权利要求11所述的TCR，其特征在于，所述TCRα链和/或β链恒定区中形成天然链间二硫键的半胱氨酸残基被替换为另一残基。
  14. 如权利要求10-13中任一所述的TCR，其特征在于，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
  15. 如权利要求14所述的TCR，其特征在于，在TCRα链恒定区与β链恒定区之间形成人工链间二硫键的半胱氨酸残基取代了：
    TRAC*01外显子1的48T和TRBC1*01或TRBC2*01外显子1的57S；
    TRAC*01外显子1的45T和TRBC1*01或TRBC2*01外显子1的77S；
    TRAC*01外显子1的10Y和TRBC1*01或TRBC2*01外显子1的17S；
    TRAC*01外显子1的45T和TRBC1*01或TRBC2*01外显子1的59D；
    TRAC*01外显子1的15S和TRBC1*01或TRBC2*01外显子1的15E；
    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。
  16. 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。
  17. 如权利要求16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。
  18. 如权利要求17所述的TCR，其特征在于，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体。
  19. 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。
  20. 一种载体，所述的载体含有权利要求19所述的核酸分子。
  21. 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20所述的载体或染色体中整合有外源的权利要求19所述的核酸分子。
  22. 一种分离的细胞，其特征在于，其表达权利要求1-18中任一所述的TCR。
  23. 一种制备权利要求1-18中任一所述的T细胞受体的方法，包括步骤：
    (i)培养权利要求21所述的宿主细胞，从而表达权利要求1-18中任一所述的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。
  24. 一种T细胞受体复合物，其特征在于，所述的复合物含有一个或多个权利要求1-18中任一所述的TCR。
  25. 一种权利要求1-18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。
  26. 一种药物组合物，其特征在于，含有药学上可接受的载体以及安全有效量的权利要求1-18中任一所述的TCR、权利要求22所述的细胞或权利要求24所述的T细胞受体复合物。
  27. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用权利要求1-18中任一所述的TCR、权利要求22所述的细胞、权利要求24所述的T细胞受体复合物或权利要求26所述的药物组合物；
    较佳地，所述的疾病包括：肿瘤、自身免疫疾病和病毒感染性疾病。
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