WO2016177339A1 - 识别ny-eso-1抗原短肽的t细胞受体 - Google Patents

识别ny-eso-1抗原短肽的t细胞受体 Download PDF

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WO2016177339A1
WO2016177339A1 PCT/CN2016/081180 CN2016081180W WO2016177339A1 WO 2016177339 A1 WO2016177339 A1 WO 2016177339A1 CN 2016081180 W CN2016081180 W CN 2016081180W WO 2016177339 A1 WO2016177339 A1 WO 2016177339A1
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tcr
seq
chain
amino acid
variable domain
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PCT/CN2016/081180
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French (fr)
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李懿
区裕升
吴万里
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广州市香雪制药股份有限公司
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Priority to CN201680001318.9A priority Critical patent/CN107197625B/zh
Publication of WO2016177339A1 publication Critical patent/WO2016177339A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464488—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a TCR capable of recognizing a short peptide derived from the NY-ESO-1 antigen, and to a NY-ESO-1 specific T cell obtained by transducing the above TCR, and their prevention and treatment of NY-ESO- 1 use in related diseases.
  • NY-ESO-1 belongs to the family of Cancer-Testis Antigen (CTA). It can be expressed in testis, ovarian tissue and many different types of tumor tissues, but not in other normal tissues. It is a kind of specificity. Strong tumor antigens.
  • NY-ESO-1 is an endogenous antigen that is degraded into small molecule polypeptides after intracellular production and binds to MHC (main histocompatibility complex) molecules to form a complex that is presented to the cell surface.
  • SLLMWITQC (157-165) is a short peptide derived from the NY-ESO-1 antigen and is a target for the treatment of NY-ESO-1 related diseases.
  • T cell adoptive immunotherapy is the transfer of reactive T cells specific for the target cell antigen into the patient to act on the target cells.
  • the T cell receptor (TCR) is a membrane protein on the surface of T cells that recognizes antigenic short peptides on the surface of the corresponding target cells.
  • APCs antigen presenting cells
  • pMHC complex short peptide-primary histocompatibility complex
  • a T cell receptor capable of specifically binding to a SLLMWITQC-HLA complex comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, and
  • the TCR alpha chain variable domain is an amino acid sequence having at least 90% (preferably at least 95%, more preferably at least 98%, optimally at least 99%) sequence identity to SEQ ID NO: 1.
  • the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5.
  • the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain
  • the amino acid sequence of the CDR3 of the TCR alpha chain variable domain is ALTLNNAGNMLT (SEQ ID NO: 12); and/or The amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is ASLDPRAGTDTQY (SEQ ID NO: 15).
  • the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:
  • the three complementarity determining regions of the TCR ⁇ chain variable domain are:
  • the TCR comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1.
  • the TCR comprises the ⁇ chain variable domain amino acid sequence of SEQ ID NO: 5.
  • the TCR is an alpha beta heterodimer comprising a TCR alpha chain constant region TRAC*01 and a TCR beta chain constant region TRBC1*01 or TRBC2*01.
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 3 and/or the ⁇ chain amino acid sequence of the TCR is SEQ ID NO: 7.
  • the TCR is soluble.
  • the TCR is single stranded.
  • the TCR is formed by linking an alpha chain variable domain to a beta chain variable domain via a peptide linker sequence.
  • the TCR is in the alpha chain variable region amino acid at the 11th, 13th, 19th, 21st, 53th, 76th, 89th, 91th or 94th position, and/or the alpha chain J gene short peptide amino acid reciprocal One or more mutations in the third position, the fifth last position or the seventh in the last number; and/or the TCRs in the ⁇ chain variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91 Or the 94th, and/or ⁇ chain J gene short peptide amino acid reciprocal number 2, the last 4th or the last 6th position has one or more mutations, wherein the amino acid position number according to IMGT (International Immunogenetics Information The location number listed in the system).
  • IMGT International Immunogenetics Information
  • the alpha chain variable domain amino acid sequence of the TCR comprises SEQ ID NO: 32 and/or the beta chain variable domain amino acid sequence of the TCR comprises SEQ ID NO:34.
  • amino acid sequence of the TCR is SEQ ID NO:30.
  • the TCR comprises (a) all or part of a TCR alpha chain other than a transmembrane domain; and (b) all or part of a TCR beta chain other than a transmembrane domain;
  • cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.
  • cysteine residue forming an artificial disulfide bond in the TCR replaces one or more sets of sites selected from the group consisting of:
  • the amino acid sequence of the O chain of the TCR is SEQ ID NO: 26 and/or the ⁇ chain amino acid sequence of the TCR is SEQ ID NO: 28.
  • 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 T cell receptor is a peptide, a detectable label, a therapeutic agent, a PK modified moiety, or a combination of any of these.
  • the therapeutic agent is an anti-CD3 antibody.
  • a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one TCR molecule is the TCR of the first aspect of the invention.
  • a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule of the first aspect of the invention or a complement thereof is provided.
  • the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 33 encoding a TCR alpha chain variable domain.
  • the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 35 encoding a TCR ⁇ chain variable domain.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 4 encoding the TCR alpha chain and/or comprises the nucleotide sequence SEQ ID NO: 8 encoding the TCR beta chain.
  • a vector comprising the nucleic acid molecule of the third aspect of the invention is provided; preferably, the vector is a viral vector; more preferably, the vector is slow Viral vector.
  • an isolated host cell comprising the vector of the fourth aspect of the invention or the nucleic acid molecule of the third aspect of the invention integrated with exogenous in the genome .
  • the invention provides a cell which is transduced with the nucleic acid molecule of the third aspect of the invention or the vector of the fourth aspect of the invention; preferably, the cell is a T cell or a stem cell .
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, the TCR according to the first aspect of the invention, the TCR complex of the second aspect of the invention The nucleic acid molecule of the third aspect of the invention, or the cell of the sixth aspect of the invention.
  • the T cell receptor of the first aspect of the invention, or the TCR complex of the second aspect of the invention, the nucleic acid molecule of the third aspect of the invention, the fourth aspect of the invention Use of the vector of the aspect or the cell of the sixth aspect of the invention for the preparation of a medicament for the treatment of a tumor or an autoimmune disease.
  • a method for treating a disease comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, the TCR complex of the second aspect of the present invention, and the present invention to a subject in need of treatment
  • the cell of the sixth aspect or the pharmaceutical composition of the seventh aspect of the invention comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, the TCR complex of the second aspect of the present invention, and the present invention to a subject in need of treatment.
  • the disease is neuroblastoma, sarcoma, malignant melanoma, prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous cell carcinoma, and esophageal cancer.
  • Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e and Figure 1f are the TCR alpha chain variable domain amino acid sequence, the TCR alpha chain variable domain nucleotide sequence, the TCR alpha chain amino acid sequence, the TCR alpha chain nucleotide sequence, respectively.
  • 2a, 2b, 2c, 2d, 2e, and 2f are a TCR ⁇ chain variable domain amino acid sequence, a TCR ⁇ chain variable domain nucleotide sequence, a TCR ⁇ chain amino acid sequence, a TCR ⁇ chain nucleotide sequence, respectively.
  • Figure 3 shows the results of double positive staining of CD8 + and tetramer-PE in monoclonal cells.
  • Figures 4a and 4b are the amino acid sequence and nucleotide sequence of the soluble TCR alpha chain, respectively.
  • Figures 5a and 5b are the amino acid sequence and nucleotide sequence of the soluble TCR ⁇ chain, respectively.
  • Figure 6 is a gel diagram of the soluble TCR obtained after purification.
  • Figures 7a and 7b are the amino acid sequence and nucleotide sequence of a single-chain TCR, respectively.
  • Figures 8a and 8b are the amino acid sequence and nucleotide sequence, respectively, of a single-chain TCR alpha chain.
  • Figures 9a and 9b are the amino acid sequence and nucleotide sequence of a single-chain TCR ⁇ chain, respectively.
  • Figures 10a and 10b are the amino acid sequence and nucleotide sequence, respectively, of a single-chain TCR linker.
  • Figure 11 is a BIAcore kinetic map of the soluble TCR of the present invention in combination with the SLLMWITQC-HLA A0201 complex.
  • Figure 12a and Figure 12b show the results of the ELISPOT experiment for the function and specificity detection of TCR of the present invention.
  • the inventors have found extensive and intensive research to find a TCR capable of specifically binding to the NY-ESO-1 antigen short peptide SLLMWITQC (157-165) (SEQ ID NO: 9), which can be associated with HLA.
  • A0201 forms a complex and is presented together to the cell surface.
  • the invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules.
  • the invention also provides cells that transduce the TCR of the invention.
  • the MHC molecule is a protein of the immunoglobulin superfamily and may be a class I or class II MHC molecule. Therefore, it is specific for the presentation of antigens, and different individuals have different MHCs that can present different short peptides of a protein antigen to the surface of the respective APC cells.
  • Human MHC is commonly referred to as the HLA gene or the HLA complex.
  • T cell receptor is the only receptor that presents a specific antigenic peptide on the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • APC antigen presenting cells
  • TCR is a glycoprotein on the surface of a cell membrane in the form of a heterodimer formed by an alpha chain/beta chain or a gamma chain/delta chain.
  • 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.
  • each of the alpha and beta chains comprises a variable region, a junction region, and a constant region
  • the beta chain typically also contains a short polymorphic region between the variable region and the junction region, but the polymorphic region is often considered as a junction region. a part of.
  • Each variable region comprises three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in framework regions.
  • the CDR regions determine the binding of the TCR to the pMHC complex, wherein the CDR3 is recombined from the variable region and the junction region and is referred to as the hypervariable region.
  • the alpha and beta chains of TCR are generally considered to have two "domains", namely a variable domain and a constant domain, and the variable domain consists of linked variable and linking regions.
  • the sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the constant domain sequence of the TCR molecule ⁇ chain is “TRAC*01”
  • the constant domain sequence of the TCR molecule ⁇ chain is “TRBC1*”. 01" or "TRBC2*01”.
  • the alpha and beta chains of TCR also contain a transmembrane and cytoplasmic regions with a short cytoplasmic region.
  • polypeptide of the present invention TCR of the present invention
  • T cell receptor of the present invention T cell receptor of the present invention
  • a first aspect of the invention provides a TCR molecule capable of specifically binding to the SLLMWITQC-HLA A0201 complex.
  • the TCR molecule is isolated or purified.
  • the alpha and beta strands of the TCR each have three complementarity determining regions (CDRs).
  • the alpha chain comprises a CDR having the following amino acid sequence:
  • the beta strand comprises a CDR having the following amino acid sequence:
  • the chimeric TCR can be prepared by embedding the above-described CDR region amino acid sequences of the present invention into any suitable framework structure.
  • the framework structure is compatible with the CDR regions of the TCRs of the present invention, one skilled in the art can design or synthesize TCR molecules having corresponding functions in accordance with the CDR regions disclosed herein.
  • a TCR molecule of the invention refers to a TCR molecule comprising the above-described alpha and/or beta chain CDR region sequences and any suitable framework structure.
  • the TCR alpha chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID NO: 1; and/or the TCR ⁇ chain variable domain of the invention is An amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID NO:5.
  • the TCR molecule of the invention is a heterodimer composed of alpha and beta chains.
  • the alpha chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the alpha chain variable domain amino acid sequence comprising the CDR1 (SEQ ID NO: 10), CDR2 (SEQ) ID NO: 11) and CDR3 (SEQ ID NO: 12).
  • the TCR molecule comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1. More preferably, the alpha chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO: 1.
  • the beta strand of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the beta strand variable domain amino acid sequence comprising the CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID) NO: 14) and CDR3 (SEQ ID NO: 15).
  • the TCR molecule comprises a beta chain variable domain amino acid sequence of SEQ ID NO:5. More preferably, the beta strand variable domain amino acid sequence of the TCR molecule is SEQ ID NO:5.
  • the TCR molecule of the invention is a single-chain TCR molecule consisting of part or all of the alpha chain and/or part or all of the beta chain.
  • a description of single-chain TCR molecules can be found in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658.
  • One skilled in the art can readily construct single-chain TCR molecules comprising the CDRs regions of the invention, as described in the literature.
  • the single-chain TCR molecule comprises V ⁇ , V ⁇ and C ⁇ , preferably linked in order from N-terminus to C-terminus.
  • the alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above alpha chain.
  • the single-chain TCR molecule comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1. More preferably, the alpha chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO: 1.
  • the ⁇ chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above-described ⁇ chain.
  • the single-chain TCR molecule comprises the ⁇ -chain variable domain amino acid sequence of SEQ ID NO: 5. More preferably, the ⁇ chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO:5.
  • the constant domain of the TCR molecule of the invention is a human constant domain.
  • the constant domain sequence of the ⁇ chain of the TCR molecule of the present invention may be "TRAC*01”
  • the constant domain sequence of the ⁇ chain of the TCR molecule may be "TRBC1*01” or "TRBC2*01”.
  • the 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is represented here as: Arg53 of exon 1 of TRAC*01, and so on.
  • the amino acid sequence of the ⁇ chain of the TCR molecule of the present invention is SEQ ID NO: 3, and/or the amino acid sequence of the ⁇ chain is SEQ ID NO: 7.
  • TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane domain.
  • TCR can also be developed for diagnosis and treatment, when soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCR has a wide range of uses, not only for studying the interaction of TCR with pMHC, but also as a diagnostic tool for detecting infection 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.
  • soluble TCRs can also bind to other molecules (eg, anti-CD3 antibodies). To redirect T cells so that they target cells that present a particular antigen.
  • the present invention also obtains the NY-ESO-1 antigen Short peptides have specific soluble TCRs.
  • the TCR of the invention can be a TCR that introduces an artificial disulfide bond between the residues of its alpha and beta chain constant domains.
  • the cysteine residue forms an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR.
  • a cysteine residue can replace other amino acid residues at a suitable position in the native TCR to form an artificial interchain disulfide bond. For example, a Thr248 residue of the exon 1 of TRAC*01 and a cysteine residue of Ser57 of the exon 1 of TRBC1*01 or TRBC2*01 are substituted to form a disulfide bond.
  • Other sites for introducing a cysteine residue to form a disulfide bond may also be: Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 of Tyr10 and TRBC1*01 or TRBC2*01 exon 1 of Ser17; TRAC*01 exon 1 of Thr45 and TRBC1*01 or TRBC2*01 exon 1 of Asp59; TRAC*01 exon 1 Ser15 and TRBC1*01 or TRBC2*01 exon 1 of Glu15; TRAC*01 exon 1 of Arg53 and TRBC1*01 or TRBC2*01 exon 1 of Ser54; TRAC*01 exon 1 of Pro89 and ABC19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 and TRBC1*01 of exon 1 of TRAC*01 or Glu20 of exon 1 of TRBC2*01.
  • a cysteine residue replaces any of the above-mentioned sites in the ⁇ and ⁇ chain constant domains.
  • a maximum of 50, or a maximum of 30, or a maximum of 15, or a maximum of 10, or a maximum of 8 or fewer amino acids may be truncated at one or more C-termini of the TCR constant domains of the invention such that they are not included
  • the cysteine residue is used for the purpose of deleting the natural disulfide bond, and the above object can also be achieved by mutating the cysteine residue forming the natural disulfide bond to another amino acid.
  • the TCR of the present invention may comprise an artificial disulfide bond introduced between residues of its ⁇ and ⁇ chain constant domains.
  • the constant domains may or may not contain the introduced artificial disulfide bonds as described above, and the TCRs of the present invention may each contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence.
  • the TRAC constant domain sequence of TCR and the TRBC1 or TRBC2 constant domain sequence can be joined by a native disulfide bond present in the TCR.
  • the TCR of the present invention further comprises a TCR having a mutation in its hydrophobic core region, and the mutation of these hydrophobic core regions is preferably a mutation capable of improving the stability of the soluble TCR of the present invention, as in the publication number It is described in the patent document of WO2014/206304.
  • Such a TCR can be mutated at its position in the following variable domain hydrophobic core: (alpha and/or beta chain) variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and / Or the ⁇ -chain J gene (TRAJ) short peptide amino acid position reciprocal position 3, 5, 7 and/or ⁇ chain J gene (TRBJ) short peptide amino acid position reciprocal position 2, 4, 6 where the amino acid sequence position number The location number listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR in which the hydrophobic core region is mutated in the present invention may be a stable soluble single-chain TCR composed of a flexible peptide chain linking the variable domains of the ⁇ and ⁇ chains of the TCR.
  • the flexible peptide chain of the present invention may be any peptide chain suitable for linking the TCR alpha and beta chain variable domains.
  • the single-chain soluble TCR constructed in Example 4 of the present invention has the ⁇ chain variable domain amino acid sequence of SEQ ID NO: 32, the encoded nucleotide sequence of SEQ ID NO: 33, and the ⁇ chain variable domain amino acid sequence.
  • SEQ ID NO: 34 the nucleotide sequence encoded is SEQ ID NO:35.
  • the TCR of the present invention can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention, such as a tetrameric domain of p53 to produce a tetramer, or more A complex formed by combining a TCR of the invention with another molecule.
  • the TCR complex of the present invention can be used to track or target cells presenting a specific antigen in vitro or in vivo. It can also be used to produce intermediates for other multivalent TCR complexes of such applications.
  • the TCR of the present invention may be used singly or in combination with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate comprises a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of a cell presenting the SLLMWITQC-HLA A0201 complex), a therapeutic agent, a PK (protein kinase) modified moiety or any of these substances The combination is combined or coupled.
  • 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. Biotoxicity (Chaudhary et al, 1989) , Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc.
  • liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nanomagnetic particles; 10. prodrug activating enzymes (eg, DT-diaphorase) (DTD) or biphenyl hydrolase-like protein (BPHL); 11. chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.
  • prodrug activating enzymes eg, DT-diaphorase) (DTD) or biphenyl hydrolase-like protein (BPHL)
  • chemotherapeutic agent eg, cisplatin or any form of nanoparticles, and the like.
  • the TCR of the invention may also be a hybrid TCR comprising sequences derived from more than one species.
  • the TCR of the invention may comprise a human variable domain and a murine constant domain.
  • a drawback of this approach is that it may trigger an immune response. Therefore, there should be a regulatory regimen for immunosuppression when used in adoptive T cell therapy to allow for the implantation of murine T cells.
  • amino acid names in this article are represented by the international single letter or three English letters.
  • the correspondence between the single English letters of the amino acid name and the three English letters is as follows: 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).
  • a second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention, or a portion thereof, which may be one or more CDRs, a variable domain of an alpha and/or beta chain, and an alpha chain and/or Or beta chain.
  • nucleotide sequence encoding the CDR region of the alpha chain of the TCR molecule of the first aspect of the invention is as follows:
  • nucleotide sequence encoding the CDR region of the ⁇ chain of the TCR molecule of the first aspect of the invention is as follows:
  • nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR alpha chain of the invention comprises SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or a nucleic acid molecule of the invention encoding a TCR ⁇ chain of the invention
  • the nucleotide sequence includes SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
  • the nucleotide sequence of the nucleic acid molecule of the present invention may be single-stranded or double-stranded, and the nucleic acid molecule may be RNA or DNA, and may or may not contain an intron.
  • the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, for example, the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID NO: 2 and / or the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR beta chain variable domain of the invention comprises SEQ ID NO: 6.
  • the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID NO: 33 and/or the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR beta chain variable domain of the invention comprising the SEQ ID NO: 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO: 4 and/or SEQ ID NO: 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention is SEQ ID NO:31.
  • nucleic acid sequence encoding a TCR of the invention may be the same or a degenerate variant of the nucleic acid sequence set forth in the Figures of the invention.
  • a "degenerate variant” refers to a nucleic acid sequence which encodes a protein sequence having SEQ ID NO: 1, but differs from the sequence of SEQ ID NO: 2.
  • the nucleotide sequence can be codon optimized. Different cells are different in the utilization of specific codons, and the number of expressions can be increased by changing the codons in the sequence depending on the type of the cell. Codon selection tables for mammalian cells as well as a variety of other organisms are well known to those skilled in the art.
  • the full length sequence of the nucleic acid molecule 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 the TCR (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. The DNA can be a coding strand or a non-coding strand.
  • the invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, ie, constructs that are capable of expression in vivo or in vitro.
  • expression vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.
  • Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.
  • AAV adeno-associated virus
  • the vector can transfer a nucleotide of the invention into a cell, such as a T cell, such that the cell expresses a TCR specific for the NY-ESO-1 antigen.
  • a cell such as a T cell
  • the vector should be capable of sustained high levels of expression in T cells.
  • the invention also relates to host cells genetically engineered using the vectors or coding sequences of the invention.
  • Place The host cell contains the vector of the present invention or a nucleic acid molecule of the present invention integrated into the chromosome.
  • the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, such as E. coli, yeast cells, CHO cells, and the like.
  • the invention also encompasses isolated cells, particularly T cells, which express the TCR of the invention.
  • the T cell can be derived from a T cell isolated from the subject, or can be a mixed cell population isolated from the subject, such as a portion of a peripheral blood lymphocyte (PBL) population.
  • PBL peripheral blood lymphocyte
  • the cells can be isolated from peripheral blood mononuclear cells (PBMC), which can be CD4 + helper T cells or CD8 + cytotoxic T cells.
  • PBMC peripheral blood mononuclear cells
  • the cells can be in a mixed population of CD4 + helper T cells/CD8 + cytotoxic T cells.
  • the cells can be activated with antibodies (e.g., anti-CD3 or anti-CD28 antibodies) to enable them to be more readily transfected, e.g., with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention. dye.
  • antibodies e.g., anti-CD3 or anti-CD28 antibodies
  • the cells of the invention may also be or be derived from stem cells, such as hematopoietic stem cells (HSCs). Transfer of the gene to HSC does not result in the expression of TCR on the cell surface because the stem cell surface does not express CD3 molecules. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of thymocytes.
  • stem cells differentiate into lymphoid precursors that migrate to the thymus
  • CD3 molecule will initiate expression of the introduced TCR molecule on the surface of thymocytes.
  • T cell transfection with DNA or RNA encoding the TCR of the invention e.g., Robbins et al., (2008) J. Immunol. 180: 6116-6131.
  • T cells expressing the TCR of the present invention can be used in adoptive immunotherapy.
  • Those skilled in the art will be aware of many suitable methods for performing adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).
  • the invention also relates to a method of treating and/or preventing a NY-ESO-1 related disease in a subject comprising the step of adoptively transferring NY-ESO-1 specific T cells to the subject.
  • the NY-ESO-1 specific T cell recognizes the SLLMWITQC-HLA A0201 complex.
  • the NY-ESO-1 specific T cells of the present invention are useful for treating any NY-ESO-1 related disease presenting the NY-ESO-1 antigen short peptide SLLMWITQC-HLA A0201 complex.
  • NY-ESO-1 related disease presenting the NY-ESO-1 antigen short peptide SLLMWITQC-HLA A0201 complex.
  • These include, but are not limited to, neuroblastoma, sarcoma, malignant melanoma, prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous cell carcinoma, and esophageal cancer.
  • the T cells of a patient or a volunteer having a disease associated with the NY-ESO-1 antigen can be isolated, and the TCR of the present invention can be introduced into the above T cells, and then these genetically engineered cells can be returned to the patient. treatment.
  • the present invention provides a method of treating a NY-ESO-1 related disease comprising administering an isolated T cell expressing a TCR of the present invention, preferably, the T cell is derived from a patient itself and is administered to a patient.
  • it comprises (1) isolating a patient's T cells, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding the TCR molecule of the invention, and (3) inputting genetically engineered T cells into the patient in vivo.
  • the number of cells that are isolated, transfected, and returned can be determined by the physician.
  • the TCR of the present invention can specifically bind to the NY-ESO-1 antigen short peptide complex SLLMWITQC-HLA A0201, and the cells transducing the TCR of the present invention can be specifically activated and have a strong killing effect on target cells. effect.
  • Peripheral blood lymphocytes from healthy volunteers with genotype HLA-A0201 were stimulated with the synthetic short peptide SLLMWITQC (Beijing Cypress Biotech Co., Ltd.).
  • SLLMWITQC short peptide was renatured with biotinylated HLA-A*0201 to prepare a pHLA haploid.
  • haploids were combined with PE-labeled streptavidin (BD) into PE-labeled tetramers, and the tetramer and anti-CD8-APC double positive cells were sorted.
  • the sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonalization by limiting dilution. Monoclonal cells were stained with tetramers and the double positive clones screened are shown in Figure 3.
  • Example 2 Extracted with Quick-RNA TM MiniPrep (ZYMO research ) in Example 1 were screened SLLMWITQC peptide antigen specific, HLA-A0201 restricted T cell clones Total RNA.
  • the cDNA was synthesized using clontech's SMART RACE cDNA Amplification Kit, and the primers were designed to be conserved in the C-terminal region of the human TCR gene.
  • the sequence was cloned into a T vector (TAKARA) for sequencing. It should be noted that this sequence is a complementary sequence and does not contain introns. After sequencing, the ⁇ chain and ⁇ chain sequence structures of the TCR expressed by the double positive clone are shown in FIG. 1 and FIG. 2 respectively, and FIG. 1a, FIG.
  • FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e and FIG. 1f are respectively TCR ⁇ chains.
  • Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the TCR ⁇ chain variable domain amino acid sequence, the TCR ⁇ chain variable domain nucleotide sequence, the TCR ⁇ chain amino acid sequence, the TCR ⁇ chain nucleotide sequence, and the leader sequence, respectively.
  • the alpha chain has been identified to comprise a CDR having the following amino acid sequence:
  • the beta strand comprises a CDR having the following amino acid sequence:
  • the full-length genes of the TCR alpha chain and the beta chain were cloned into a lentiviral expression vector by overlap PCR, respectively.
  • a lentiviral vector expressing eGFP was also constructed.
  • the 1981T/17 is then used to package the pseudovirus.
  • the ⁇ and ⁇ chains of the TCR molecule of the present invention may contain only a variable domain and a partial constant domain, and a cysteine residue is introduced in the constant domains of the ⁇ and ⁇ chains, respectively, to form an artificial interchain disulfide bond, and the position of introducing a cysteine residue is TRAC*01, respectively.
  • TCR ⁇ and ⁇ chain were transformed into expression plasmid BL21(DE3) by chemical transformation, respectively, and the bacteria were grown in LB medium.
  • the resulting inclusion bodies were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution.
  • the inclusion bodies were finally dissolved in 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA). ), in 20 mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains were rapidly mixed in 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, 6.6 mM ⁇ -mercapoethylamine (4 ° C) at a final concentration of 1:1. 60 mg/mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ° C), and after 12 hours, deionized water was exchanged for buffer (20 mM Tri s, pH 8.0) and dialysis was continued at 4 ° C for 12 hours.
  • the solution was filtered through a 0.45 ⁇ M filter and purified through an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare).
  • the TCR containing the refolding successful alpha and beta dimers was confirmed by SDS-PAGE gel.
  • the TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare).
  • the purified TCR purity was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • the SDS-PAGE gel of the soluble TCR obtained by the present invention is shown in Fig. 6.
  • variable domains of TCR ⁇ and ⁇ -chain in Example 2 were constructed as a stable soluble single-chain TCR molecule linked by a flexible short linker using the method of site-directed mutagenesis as described in the patent document WO2014/206304.
  • the amino acid sequence and nucleotide sequence of the single-chain TCR molecule are shown in Figures 7a and 7b, respectively.
  • the amino acid sequence and nucleotide sequence of the ⁇ chain variable domain are shown in Figure 8a and Figure 8b, respectively; the amino acid sequence and nucleotide sequence of the ⁇ chain variable domain are shown in Figure 9a and Figure 9b, respectively;
  • the amino acid sequence and nucleotide sequence of the sequence are shown in Figures 10a and 10b, respectively.
  • the gene of interest was digested with NcoI and NotI and ligated with the pET28a vector digested with NcoI and NotI.
  • the ligation product was transformed into E. coli DH5 ⁇ , coated with kanamycin-containing LB plate, inverted culture at 37 ° C overnight, and the positive clones were picked for PCR screening.
  • the positive recombinants were sequenced to determine the correct sequence and the recombinant plasmid was extracted.
  • E. coli BL21 (DE3) for expression.
  • the BL21(DE 3) colonies containing the recombinant plasmid pET28a-template strand prepared in Example 4 were all inoculated into LB medium containing kanamycin, cultured at 37 ° C until the OD600 was 0.6-0.8, and IPTG was added to the final concentration. The culture was continued at 37 ° C for 4 h at 0.5 mM.
  • Harvesting cell pellet by centrifugation at 5000 rpm for 15 min The cell pellet was lysed with Bugbuster Master Mix (Merck), and the inclusion bodies were recovered by centrifugation at 6000 rpm for 15 min, followed by washing with Bugbuster (Merck) to remove cell debris and membrane fraction, and centrifuged at 6000 rpm for 15 min to collect inclusion bodies.
  • the inclusion body was dissolved in a buffer (20 mM Tris-HCl pH 8.0, 8 M urea), and the insoluble matter was removed by high-speed centrifugation. The supernatant was fractionated by the BCA method, and then stored at -80 ° C until use.
  • the reconstituted solution was placed in a cellulose membrane dialysis bag with a cut-off amount of 4 kDa, and the dialysis bag was placed in 1 L of pre-cooled water and slowly stirred at 4 ° C overnight. After 17 hours, the dialysate was changed to 1 L of pre-cooled buffer (20 mM Tris-HCl pH 8.0), dialysis was continued for 8 h at 4 ° C, and the dialysate was replaced with the same fresh buffer to continue dialysis overnight.
  • pre-cooled buffer 20 mM Tris-HCl pH 8.0
  • the sample was filtered through a 0.45 ⁇ m filter, and the protein was purified by vacuum degassing through an anion exchange column (HiTrap Q HP, GE Healthcare) in a linear gradient of 0-mM NaCl prepared with 20 mM Tris-HCl pH 8.0.
  • the collected fractions were subjected to SDS-PAGE analysis, and the fractions containing the single-chain TCR were concentrated and further purified by a gel filtration column (Superdex 7510/300, GE Healthcare), and the target components were also subjected to SDS-PAGE analysis.
  • the eluted fraction for BIAcore analysis was further tested for purity using gel filtration.
  • the conditions were as follows: column Agilent 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 binding activity of the TCR molecule obtained in Example 3 to the SLLMWITQC-HLA A0201 complex was examined using a BIAcore T200 real-time analysis system.
  • the anti-streptavidin antibody (GenScript) was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the surface of the chip. Finally, the unreacted activated surface was blocked with a solution of ethanolamine in hydrochloric acid to complete the coupling process at a coupling level of about 15,000 RU.
  • a low concentration of streptavidin is passed over the surface of the coated antibody chip, then the SLLMWITQC-HLA A0201 complex is flowed through the detection channel, the other channel is used as a reference channel, and 0.05 mM biotin is then added at 10 ⁇ L/min. The flow rate was passed through the chip for 2 min, blocking the remaining binding sites of streptavidin.
  • 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 inclusion weight was suspended in 5 ml BugBuster Master Mix, and incubated at room temperature for 5 min; Add 30ml of BugBuster diluted 10 times, mix well, centrifuge at 6000g for 15min at 4°C; discard the supernatant, add 30ml of BugBuster diluted 10 times and resuspend the inclusion body, mix, centrifuge at 6000g for 15min at 4°C, repeat twice, add 30ml 20mM
  • the inclusion bodies were resuspended in Tris-HCl pH 8.0, mixed, centrifuged at 6000g for 15min at 4°C, and finally the inclusion bodies were dissolved in 20mM Tris-HCl 8M urea.
  • the purity of inclusion bodies was determined by SDS-PAGE and the concentration was measured by BCA kit.
  • the synthesized short peptide SLLMWITQC (Beijing Saibaisheng Gene Technology Co., Ltd.) 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 SLLMWITQC 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
  • 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 molecules were 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/
  • the ml BirA enzyme (GST-BirA) was incubated overnight at room temperature and SDS-PAGE was used to determine if biotinylation was complete.
  • the biotinylated labeled pMHC molecule was concentrated to 1 ml using a Millipore ultrafiltration tube, biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using an Akta Purifier (GE General Electric Company).
  • Akta Purifier GE General Electric Company
  • a TM 16/60S200 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 kinetic parameters of the soluble TCR molecule of the present invention in combination with the SLLMWITQC-HLA A0201 complex were calculated using BIAcore Evaluation software to calculate the kinetic parameters as shown in FIG.
  • the binding activity of the soluble TCR molecule of the present invention to other antigenic short peptides including the KLVALGINAV-HLA A0201 complex and the like was also examined by the above method, and the results showed that the TCR molecule of the present invention did not bind to other unrelated antigenic short peptides.
  • TCR transduced PBLs obtained by the present invention are capable of specifically recognizing An illustration of a NY-ESO-1 antigen positive cancer cell.
  • Lentiviruses containing the gene encoding the desired TCR were packaged using a third generation lentiviral packaging system.
  • Four plasmids were transiently transfected with 293T (containing pGZ178-HI WT TRA-2A-TRB lentiviral vector and three plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles) using PEI cell.
  • the ratio of transfection reagent PEI-MAX to plasmid was 2:1, and the usage per plate was 114.75 micrograms.
  • the specific operation is as follows: the expression plasmid and the packaging plasmid are added to a medium of 1800 ⁇ l of OPTI-MEM (Gibco, catalog number 31985-070), and uniformly mixed, and allowed to stand at room temperature for 5 minutes to become a DNA mixture; The corresponding amount of PEI was mixed well with 1800 ⁇ l of OPTI-MEM medium, and allowed to stand at room temperature for 5 minutes to become a PEI mixture. The DNA mixture and the PEI mixture were mixed together and allowed to stand at room temperature for 30 minutes, and then 3150 ⁇ l of OPTI was added.
  • -MEM medium mix well and add to 293T cells which have been converted into 11.25 ml of OPTI-MEM, gently shake the culture dish, mix the medium evenly, and incubate at 37 ° C / 5% CO2. Transfection for 5-7 hours The transfection medium was removed and replaced with DMEM (Gibco, Cat. No. C11995500bt) containing 10% fetal bovine serum, and cultured at 37 ° C / 5% CO 2 . The medium supernatant containing the packaged lentivirus was collected for 4 days.
  • DMEM Gibco, Cat. No. C11995500bt
  • the collected culture supernatant was centrifuged for 15 minutes for 15 minutes to remove cell debris and then passed through a 0.22 micron filter (Merck) Millipore), catalog number SLGP033RB) filtered, and finally with 50KD cut-off Concentrate tube (Merck Millipore, catalog number UFC905096) was concentrated to remove most of the supernatant, finally concentrated to 1 ml, aliquoted and stored at -80 ° C.
  • the pseudovirus sample was taken for virus drop. For the determination, the procedure was as follows: p24 ELISA (Clontech, catalog number 632200) kit instructions. As a control, pseudoviruses of pLenti-eGFP were also included.
  • the cells were counted every two days, and fresh medium containing 50 IU/ml IL-2 was replaced or added to maintain the cells at 0.5 x 10 6 - 1 x 10 6 cells/ml.
  • lentivirus-transduced PBL was quantified by flow cytometry by staining with a tetramer containing SLLMWITQC short peptide and HLA-A*0201. When the tetramer detection positive rate is greater than 10%, it is used for functional testing (eg, ELISPOT and non-radioactive cytotoxicity assay for IFN- ⁇ release) on day 5.
  • NY-ESO-1 157-165 SLLMWITQC short peptide (p1A) was renatured with biotinylated HLA-A*0201 to prepare pHLA haploid. These haploids were combined with PE-labeled streptavidin (BD) into a PE-labeled tetramer called p1A-tetramer-PE. This tetramer can label T cells expressing the NY-ESO-1 157-165 SLLMWITQC-specific T cell receptor gene as positive cells.
  • BD PE-labeled streptavidin
  • the transfected T cell samples in (b) were incubated with p1A-tetramer-PE for 30 minutes on ice, then anti-CD8-APC (BioLegend) antibody was added and incubation was continued for 15 minutes on ice.
  • the samples were washed twice with PBS containing 2% FBS, and then the p1A-tetramer-PE and CD8 double positive T cells expressing the NY-ESO-1 157-165 SLLMWITQC-specific T cell receptor gene were detected by Guava 16HT. FlowJo software (Tree Star Inc, Ashland, OR) analysis.
  • the function and specificity of transducing the PBL of the present invention were further examined by ELISPOT assay.
  • Methods for detecting cellular function using ELISPOT assays are well known to those skilled in the art.
  • the effector cells used in the IFN- ⁇ ELISPOT assay of the present example are the PBL of the virus-transduced HI-WT gene obtained in the present invention, and the target cell line is the antigen (NY-ESO-1)-positive IM9 (ATCC) and U266B1 (ATCC).
  • the cells and the control group were 293T cells negative for antigen (NY-ESO-1).
  • the ELISPOT experiment was carried out as follows: 15 components of the test were added to the ELISPOT plate in the following order: each group was added to the ELISPOT plate: the T2 cells were adjusted to 2 X 10 5 cells/ml with the medium, according to Final volume and final peptide concentration The specific short peptide and non-specific short peptide were separately added to the corresponding T2 cell dilution solution, and the corresponding amount of medium was added to the blank group. After mixing, 100 ⁇ L of T2 cells and target cell line were taken.
  • the virus obtained by the present invention transduced the PBL of the HI-WT gene to the T2 cell and antigen carrying the specific polypeptide (NY-ESO-1).
  • the positive IM9 and U266B1 cell lines have a specific response.

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Abstract

本发明提供了一种能够特异性结合衍生自NY-ESO-1抗原短肽SLLMWITQC的T细胞受体(TCR)，所述抗原短肽SLLMWITQC可与HLA A0201形成复合物并一起被呈递到细胞表面。本发明还提供了编码所述TCR的核酸分子以及包含所述核酸分子的载体。另外，本发明还提供了转导本发明TCR的细胞。

Description

识别NY-ESO-1抗原短肽的T细胞受体 技术领域
本发明涉及能够识别源自NY-ESO-1抗原短肽的TCR，本发明还涉及转导上述TCR来获得的NY-ESO-1特异性的T细胞，及他们在预防和治疗NY-ESO-1相关疾病中的用途。
背景技术
NY-ESO-1属于肿瘤-睾丸抗原(Cancer-Testis Antigen,CTA)家族，能在睾丸、卵巢组织以及多种不同类型的肿瘤组织中表达，而在其他正常组织中不表达，是一种特异性较强的肿瘤抗原。NY-ESO-1是一种内源性抗原，在细胞内生成后被降解成小分子多肽，并与MHC(主组织相容性复合体)分子结合形成复合物，被呈递到细胞表面。SLLMWITQC(157-165)是衍生自NY-ESO-1抗原的短肽，是NY-ESO-1相关疾病治疗的一种靶标。研究显示，NY-ESO-1在多种肿瘤组织中均有表达，在神经母细胞瘤(Rodolfo M,et al.,Cancer Res,2003,63(20):6948-6955)、肉瘤(Jungbhth A A et al.,Int J Cancer,200l,94(2):252-256)、恶性黑色素瘤(Barrow C,et al.,Cl in Cancer Res,2006 ,12(3Pt 1):764-771)中有非常高的表达，同时在前列腺癌、膀胱癌、乳腺癌、多发性骨髓瘤、肝细胞癌、口腔鳞癌(Ries J,et al.,Anticancer RES,2009,29(12):5125-5130)、以及食管癌(Fuj ita S,Clin Cancer Res,2004,10(19):6551-6558)中也有较高的表达。对于上述疾病的治疗，可以采用化疗和放射性治疗等方法，但都会对自身的正常细胞造成损害。
T细胞过继免疫治疗是将对靶细胞抗原具有特异性的反应性T细胞转入病人体内，使其针对靶细胞发挥作用。T细胞受体(TCR)是T细胞表面的一种膜蛋白，其能够识别相应的靶细胞表面的抗原短肽。在免疫系统中，通过抗原短肽特异性的TCR与短肽-主组织相容性复合体(pMHC复合物)的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，引起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，本领域技术人员致力于分离出对NY-ESO-1抗原短肽具有特异性的TCR，以及将该TCR转导T细胞来获得对NY-ESO-1抗原短肽具有特异性的T细胞，从而使他们在细胞免疫治疗中发挥作用。
发明内容
本发明的目的在于提供一种识别NY-ESO-1抗原短肽的T细胞受体及其应用。
本发明的第一方面，提供了一种T细胞受体(TCR)，所述TCR能够与SLLMWITQC-HLA复合物特异性地结合,其包含TCRα链可变域和TCRβ链可变域，并且所述TCRα链可变域为与SEQ ID NO:1具有至少90％(较佳地至少95％，更佳地至少98％，最佳的至少99％)序列相同性的氨基酸序列。
在另一优选例中，所述TCRβ链可变域为与SEQ ID NO：5具有至少90％序列相同性的氨基酸序列。
在另一优选例中，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域的CDR3的氨基酸序列为ALTLNNAGNMLT(SEQ ID NO:12)；和/或所述TCRβ链可变域的CDR3的氨基酸序列为ASLDPRAGTDTQY(SEQ ID NO:15)。
在另一优选例中，所述TCRα链可变域的3个互补决定区(CDR)为：
α CDR1-ATGYPS     (SEQ ID NO:10)；
α CDR2-ATKADDK     (SEQ ID NO:11)；和
α CDR3-ALTLNNAGNMLT  (SEQ ID NO:12)；和/或
所述TCRβ链可变域的3个互补决定区为：
β CDR1-SNHLY     (SEQ ID NO:13)；
β CDR2-FYNNEI     (SEQ ID NO:14)；和
β CDR3-ASLDPRAGTDTQY  (SEQ ID NO:15)。
在另一优选例中，所述TCR包含α链可变域氨基酸序列SEQ ID NO:1。
在另一优选例中，所述TCR包含β链可变域氨基酸序列SEQ ID NO:5。
在另一优选例中，所述TCR为αβ异质二聚体，其包含TCRα链恒定区TRAC*01和TCRβ链恒定区TRBC1*01或TRBC2*01。
在另一优选例中，所述TCR的α链氨基酸序列为SEQ ID NO：3和/或所述TCR的β链氨基酸序列为SEQ ID NO:7。
在另一优选例中，所述TCR是可溶的。
在另一优选例中，所述TCR为单链。
在另一优选例中，所述TCR是由α链可变域与β链可变域通过肽连接序列连接而成。
在另一优选例中，所述TCR在α链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或α链J基因短肽氨基酸倒数第3位、倒数第5位或倒数第7位中具有一个或多个突变；和/或所述TCR在β链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或β链J基因短肽氨基酸倒数第2位、倒数第4位或倒数第6位中具有一个或多个突变，其中氨基酸位置编号按IMGT(国际免疫遗传学信息系统)中列出的位置编号。
在另一优选例中，所述TCR的α链可变域氨基酸序列包含SEQ ID NO:32和/或所述TCR的β链可变域氨基酸序列包含SEQ ID NO:34。
在另一优选例中，所述TCR的氨基酸序列为SEQ ID NO:30。
在另一优选例中，所述TCR包括(a)除跨膜结构域以外的全部或部分TCRα链；以及(b)除跨膜结构域以外的全部或部分TCRβ链；
并且(a)和(b)各自包含功能性可变结构域,或包含功能性可变结构域和所述TCR链恒定结构域的至少一部分。
在另一优选例中，半胱氨酸残基在所述TCR的α和β链恒定域之间形成人工二硫键。
在另一优选例中，在所述TCR中形成人工二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；和
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
在另一优选例中，所述TCR的α链氨基酸序列为SEQ ID NO:26和/或所述TCR的β链氨基酸序列为SEQ ID NO:28。
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T细胞受体结合的偶联物为肽、可检测标记物、治疗剂、PK修饰部分或任何这些物质的组合。优选地，所述治疗剂为抗-CD3抗体。
本发明的第二方面，提供了一种多价TCR复合物，其包含至少两个TCR分子，并且其中的至少一个TCR分子为本发明第一方面所述的TCR。
本发明的第三方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述的TCR分子的核酸序列或其互补序列。
在另一优选例中，所述核酸分子包含编码TCRα链可变域的核苷酸序列SEQ ID NO：2或SEQ ID NO:33。
在另一优选例中，所述核酸分子包含编码TCRβ链可变域的核苷酸序列SEQ ID NO：6或SEQ ID NO:35。
在另一优选例中，所述核酸分子包含编码TCRα链的核苷酸序列SEQ ID NO：4和/或包含编码TCRβ链的核苷酸序列SEQ ID NO：8。
本发明的第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的核酸分子；优选地，所述的载体为病毒载体；更优选地，所述的载体为慢病毒载体。
本发明的第五方面，提供了一种分离的宿主细胞，所述的宿主细胞中含有本发明第四方面所述的载体或基因组中整合有外源的本发明第三方面所述的核酸分子。
本发明的第六方面，提供了一种细胞，所述细胞转导本发明第三方面所述的核酸分子或本发明第四方面所述的载体；优选地，所述细胞为T细胞或干细胞。
本发明的第七方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的所述的TCR、本发明第二方面所述的TCR复合物、本发明第三方面所述的核酸分子、或本发明第六方面所述的细胞。
本发明的第八方面，提供了本发明第一方面所述的T细胞受体、或本发明第二方面所述的TCR复合物、本发明第三方面所述的核酸分子、本发明第四方面所述的载体或本发明第六方面所述的细胞的用途，其特征在于，用于制备治疗肿瘤或自身免疫疾病的药物。
本发明的第九方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的TCR、本发明第二方面所述的TCR复合物、本发明第六方面所述的细胞或本发明第七方面所述的药物组合物；
优选地，所述的疾病为神经母细胞瘤、肉瘤、恶性黑色素瘤、前列腺癌、膀胱癌、乳腺癌、多发性骨髓瘤、肝细胞癌、口腔鳞癌以及食管癌。
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、图1b、图1c、图1d、图1e和图1f分别为TCRα链可变域氨基酸序列、TCRα链可变域核苷酸序列、TCRα链氨基酸序列、TCRα链核苷酸序列、具有前导序列的TCRα链氨基酸序列以及具有前导序列的TCRα链核苷酸序列。
图2a、图2b、图2c、图2d、图2e和图2f分别为TCRβ链可变域氨基酸序列、TCRβ链可变域核苷酸序列、TCRβ链氨基酸序列、TCRβ链核苷酸序列、具有前导序列的TCRβ链氨基酸序列以及具有前导序列的TCRβ链核苷酸序列。
图3为单克隆细胞的CD8+及四聚体-PE双阳性染色结果。
图4a和图4b分别为可溶性TCRα链的氨基酸序列和核苷酸序列。
图5a和图5b分别为可溶性TCRβ链的氨基酸序列和核苷酸序列。
图6为纯化后得到的可溶性TCR的胶图。
图7a和图7b分别为单链TCR的氨基酸序列和核苷酸序列。
图8a和图8b分别为单链TCRα链的氨基酸序列和核苷酸序列。
图9a和图9b分别为单链TCRβ链的氨基酸序列和核苷酸序列。
图10a和图10b分别为单链TCR连接序列(linker)的氨基酸序列和核苷酸序列。
图11为本发明可溶性TCR与SLLMWITQC-HLA A0201复合物结合的BIAcore动力学图谱。
图12a和图12b为本发明TCR的功能及特异性检测的ELISPOT实验结果。
具体实施方式
本发明人经过广泛而深入的研究，找到了与NY-ESO-1抗原短肽SLLMWITQC(157-165)(SEQ ID NO:9)能够特异性结合的TCR，所述抗原短肽SLLMWITQC可与HLA A0201形成复合物并一起被呈递到细胞表面。本发明还提供了编码所述TCR的核酸分子以及包含所述核酸分子的载体。另外，本发明还提供了转导本发明TCR的细胞。
术语
MHC分子是免疫球蛋白超家族的蛋白质，可以是Ⅰ类或Ⅱ类MHC分子。因此，其对于抗原的呈递具有特异性，不同的个体有不同的MHC，能呈递一种蛋白抗原中不同的短肽到各自的APC细胞表面。人类的MHC通常称为HLA基因或HLA复合体。
T细胞受体(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。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。
TCR是由α链/β链或者γ链/δ链以异质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。广义上讲，α和β各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。各可变区包含嵌合在框架结构(framework regions)中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。CDR区决定了TCR与pMHC复合物的结合，其中CDR3由可变区和连接区重组而成，被称为超变区。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。TCR恒定域的序列可以在国际免疫遗传学信息系统(IMGT)的公开数据库中找到，如TCR分子α链的恒定域序列为“TRAC*01”，TCR分子β链的恒定域序列为“TRBC1*01”或“TRBC2*01”。此外，TCR的α和β链还包含跨膜区和胞质区，胞质区很短。
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。
发明详述
TCR分子
在抗原加工过程中，抗原在细胞内被降解，然后通过MHC分子携带至细胞表面。T细胞受体能够识别抗原呈递细胞表面的肽-MHC复合物。因此，本发明的第一方面提供了一种能够特异性结合SLLMWITQC-HLA A0201复合物的TCR分子。优选地，所述TCR分子是分离的或纯化的。该TCR的α和β链各具有3个互补决定区(CDR)。
α链包含具有以下氨基酸序列的CDR：
α CDR1-ATGYPS   (SEQ ID NO:10)
α CDR2-ATKADDK   (SEQ ID NO:11)
α CDR3-ALTLNNAGNMLT   (SEQ ID NO:12)
β链包含具有以下氨基酸序列的CDR：
β CDR1-SNHLY   (SEQ ID NO:13)
β CDR2-FYNNEI   (SEQ ID NO:14)
β CDR3-ASLDPRAGTDTQY   (SEQ ID NO:15)
可以将上述本发明的CDR区氨基酸序列嵌入到任何适合的框架结构中来制备嵌合TCR。只要框架结构与本发明的TCR的CDR区兼容，本领域技术人员根据本发明公开的CDR区就能够设计或合成出具有相应功能的TCR分子。因此，本发明TCR分子是指包含上述α和/或β链CDR区序列及任何适合的框架结构的TCR分子。本发明TCRα链可变域为与SEQ ID NO:1具有至少90％，优选地95％，更优选地98％序列相同性的氨基酸序列；和/或本发明TCRβ链可变域为 与SEQ ID NO：5具有至少90％，优选地95％，更优选地98％序列相同性的氨基酸序列。
在本发明的一个优选例中，本发明的TCR分子是由α与β链构成的异质二聚体。具体地，一方面所述异质二聚TCR分子的α链包含可变域和恒定域，所述α链可变域氨基酸序列包含上述α链的CDR1(SEQ ID NO：10)、CDR2(SEQ ID NO：11)和CDR3(SEQ ID NO:12)。优选地，所述TCR分子包含α链可变域氨基酸序列SEQ ID NO:1。更优选地，所述TCR分子的α链可变域氨基酸序列为SEQ ID NO:1。另一方面，所述异质二聚TCR分子的β链包含可变域和恒定域，所述β链可变域氨基酸序列包含上述β链的CDR1(SEQ ID NO:13)、CDR2(SEQ ID NO：14)和CDR3(SEQ ID NO:15)。优选地，所述TCR分子包含β链可变域氨基酸序列SEQ ID NO:5。更优选地，所述TCR分子的β链可变域氨基酸序列为SEQ ID NO:5。
在本发明的一个优选例中，本发明的TCR分子是由α链的部分或全部和/或β链的部分或全部组成的单链TCR分子。有关单链TCR分子的描述可以参考文献Chung et al(1994)Proc.Natl.Acad.Sci.USA 91,12654-12658。根据文献中所述，本领域技术人员能够容易地构建包含本发明CDRs区的单链TCR分子。具体地，所述单链TCR分子包含Vα、Vβ和Cβ，优选地按照从N端到C端的顺序连接。
所述单链TCR分子的α链可变域氨基酸序列包含上述α链的CDR1(SEQ ID NO：10)、CDR2(SEQ ID NO：11)和CDR3(SEQ ID NO:12)。优选地，所述单链TCR分子包含α链可变域氨基酸序列SEQ ID NO:1。更优选地，所述单链TCR分子的α链可变域氨基酸序列为SEQ ID NO:1。所述单链TCR分子的β链可变域氨基酸序列包含上述β链的CDR1(SEQ ID NO：13)、CDR2(SEQ ID NO：14)和CDR3(SEQ ID NO：15)。优选地，所述单链TCR分子包含β链可变域氨基酸序列SEQ ID NO:5。更优选地，所述单链TCR分子的β链可变域氨基酸序列为SEQ ID NO:5。
在本发明的一个优选例中，本发明的TCR分子的恒定域是人的恒定域。本领域技术人员知晓或可以通过查阅相关书籍或IMGT(国际免疫遗传学信息系统)的公开数据库来获得人的恒定域氨基酸序列。例如，本发明TCR分子α链的恒定域序列可以为“TRAC*01”，TCR分子β链的恒定域序列可以为“TRBC1*01”或“TRBC2*01”。IMGT的TRAC*01中给出的氨基酸序列的第53位为Arg，在此表示为：TRAC*01外显子1的Arg53，其他以此类推。优选地，本发明TCR分子α链的氨基酸序列为SEQ ID NO:3，和/或β链的氨基酸序列为SEQ ID NO:7。
天然存在的TCR是一种膜蛋白，通过其跨膜区得以稳定。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，这时需要获得可溶性的TCR分子。可溶性的TCR分子不包括其跨膜区。可溶性TCR有很广泛的用途，它不仅可用于研究TCR与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，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。本发明也获得了对NY-ESO-1抗原 短肽具有特异性的可溶性TCR。
为获得可溶性TCR，一方面，本发明TCR可以是在其α和β链恒定域的残基之间引入人工二硫键的TCR。半胱氨酸残基在所述TCR的α和β链恒定域间形成人工链间二硫键。半胱氨酸残基可以取代在天然TCR中合适位点的其他氨基酸残基以形成人工链间二硫键。例如，取代TRAC*01外显子1的Thr48和取代TRBC1*01或TRBC2*01外显子1的Ser57的半胱氨酸残基来形成二硫键。引入半胱氨酸残基以形成二硫键的其他位点还可以是：TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；或TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。即半胱氨酸残基取代了上述α与β链恒定域中任一组位点。可在本发明TCR恒定域的一个或多个C末端截短最多50个、或最多30个、或最多15个、或最多10个、或最多8个或更少的氨基酸，以使其不包括半胱氨酸残基来达到缺失天然二硫键的目的，也可通过将形成天然二硫键的半胱氨酸残基突变为另一氨基酸来达到上述目的。
如上所述，本发明的TCR可以包含在其α和β链恒定域的残基间引入的人工二硫键。应注意，恒定域间含或不含上文所述的引入的人工二硫键，本发明的TCR均可含有TRAC恒定域序列和TRBC1或TRBC2恒定域序列。TCR的TRAC恒定域序列和TRBC1或TRBC2恒定域序列可通过存在于TCR中的天然二硫键连接。
为获得可溶性TCR，另一方面，本发明TCR还包括在其疏水芯区域发生突变的TCR，这些疏水芯区域的突变优选为能够使本发明可溶性TCR的稳定性提高的突变，如在公开号为WO2014/206304的专利文献中所述。这样的TCR可在其下列可变域疏水芯位置发生突变：(α和/或β链)可变区氨基酸第11，13，19，21，53，76，89，91，94位，和/或α链J基因(TRAJ)短肽氨基酸位置倒数第3,5,7位,和/或β链J基因(TRBJ)短肽氨基酸位置倒数第2,4,6位，其中氨基酸序列的位置编号按国际免疫遗传学信息系统(IMGT)中列出的位置编号。本领域技术人员知晓上述国际免疫遗传学信息系统，并可根据该数据库得到不同TCR的氨基酸残基在IMGT中的位置编号。
本发明中疏水芯区域发生突变的TCR可以是由一柔性肽链连接TCR的α与β链的可变域而构成的稳定性可溶单链TCR。应注意，本发明中柔性肽链可以是任何适合连接TCRα与β链可变域的肽链。如在本发明实施例4中构建的单链可溶性TCR，其α链可变域氨基酸序列为SEQ ID NO:32，编码的核苷酸序列为SEQ ID NO:33；β链可变域氨基酸序列为SEQ ID NO:34，编码的核苷酸序列为SEQ ID NO:35。
本发明的TCR也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、四个或更多个本发明TCR相结合而形成的多聚物，如可以用p53的四聚结构域来产生四聚体，或多个本发明TCR与另一分子结合而形成的复合物。本发明的TCR复合物可用于体外或体内追踪或靶向呈递特定抗原的细胞， 也可用于产生具有此类应用的其他多价TCR复合物的中间体。
本发明的TCR可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物包括可检测标记物(为诊断目的，其中所述TCR用于检测呈递SLLMWITQC-HLA A0201复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明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(Chaudhary等，1989，自然(Nature)339，394；Epel等，2002，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51，565)；3.细胞因子如IL-2等(Gillies等，1992，美国国家科学院院刊(PNAS)89，1428；Card等，2004，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53，345；Halin等，2003，癌症研究(Cancer Research)63，3202)；4.抗体Fc片段(Mosquera等，2005，免疫学杂志(The Journal Of Immunology)174，4381)；5.抗体scFv片段(Zhu等，1995，癌症国际期刊(International Journal of Cancer)62,319)；6.金纳米颗粒/纳米棒(Lapotko等，2005，癌症通信(Cancer letters)239，36；Huang等，2006，美国化学学会杂志(Journal of the American Chemical Society)128，2115)；7.病毒颗粒(Peng等，2004，基因治疗(Gene therapy)11，1234)；8.脂质体(Mamot等，2005，癌症研究(Cancer research)65，11631)；9.纳米磁粒；10.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；11.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
另外，本发明的TCR还可以是包含衍生自超过一种物种序列的杂合TCR。例如，有研究显示鼠科TCR在人T细胞中比人TCR能够更有效地表达。因此，本发明TCR可包含人可变域和鼠的恒定域。这一方法的缺陷是可能引发免疫应答。因此，在其用于过继性T细胞治疗时应当有调节方案来进行免疫抑制，以允许表达鼠科的T细胞的植入。
应理解，本文中氨基酸名称采用国际通用的单英文字母或三英文字母表示，氨基酸名称的单英文字母与三英文字母的对应关系如下：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)。
核酸分子
本发明的第二方面提供了编码本发明第一方面TCR分子或其部分的核酸分子，所述部分可以是一个或多个CDR，α和/或β链的可变域，以及α链和/或β链。
编码本发明第一方面TCR分子α链CDR区的核苷酸序列如下：
α CDR1-gccacaggatacccttcc       (SEQ ID NO:16)
α CDR2-gccacgaaggctgatgacaag    (SEQ ID NO:17)
α CDR3-gctctgacccttaataatgcaggcaacatgctcacc  (SEQ ID NO:18)
编码本发明第一方面TCR分子β链CDR区的核苷酸序列如下：
β CDR1-tctaatcacttatac      (SEQ ID NO:19)
β CDR2-ttttataataatgaaatc     (SEQ ID NO:20)
β CDR3-gccagcctggacccacgagcgggcacagatacgcagtat  (SEQ ID NO:21)
因此，编码本发明TCRα链的本发明核酸分子的核苷酸序列包括SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18，和/或编码本发明TCRβ链的本发明核酸分子的核苷酸序列包括SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21。
本发明核酸分子的核苷酸序列可以是单链或双链的，该核酸分子可以是RNA或DNA，并且可以包含或不包含内含子。优选地，本发明核酸分子的核苷酸序列不包含内含子但能够编码本发明多肽，例如编码本发明TCRα链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:2和/或编码本发明TCRβ链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:6。或者，编码本发明TCRα链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:33和/或编码本发明TCRβ链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:35。更优选地，本发明核酸分子的核苷酸序列包含SEQ ID NO:4和/或SEQ ID NO:8。或者，本发明核酸分子的核苷酸序列为SEQ ID NO:31。
应理解，由于遗传密码的简并，不同的核苷酸序列可以编码相同的多肽。因此，编码本发明TCR的核酸序列可以与本发明附图中所示的核酸序列相同或是简并的变异体。以本发明中的其中一个例子来说明，“简并的变异体”是指编码具有SEQ ID NO:1的蛋白序列，但与SEQ ID NO:2的序列有差别的核酸序列。
核苷酸序列可以是经密码子优化的。不同的细胞在具体密码子的利用上是不同的，可以根据细胞的类型，改变序列中的密码子来增加表达量。哺乳动物细胞以及多种其他生物的密码子选择表是本领域技术人员公知的。
本发明的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明TCR(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。DNA可以是编码链或非编码链。
载体
本发明还涉及包含本发明的核酸分子的载体，包括表达载体，即能够在体内或体外表达的构建体。常用的载体包括细菌质粒、噬菌体和动植物病毒。
病毒递送系统包括但不限于腺病毒载体、腺相关病毒(AAV)载体、疱疹病毒载体、逆转录病毒载体、慢病毒载体、杆状病毒载体。
优选地，载体可以将本发明的核苷酸转移至细胞中，例如T细胞中，使得该细胞表达NY-ESO-1抗原特异性的TCR。理想的情况下，该载体应当能够在T细胞中持续高水平地表达。
细胞
本发明还涉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。所 述宿主细胞中含有本发明的载体或染色体中整合有本发明的核酸分子。宿主细胞选自：原核细胞和真核细胞，例如大肠杆菌、酵母细胞、CHO细胞等。
另外，本发明还包括表达本发明的TCR的分离的细胞，特别是T细胞。该T细胞可衍生自从受试者分离的T细胞，或者可以是从受试者中分离的混合细胞群，诸如外周血淋巴细胞(PBL)群的一部分。如，该细胞可以分离自外周血单核细胞(PBMC)，可以是CD4+辅助T细胞或CD8+细胞毒性T细胞。该细胞可在CD4+辅助T细胞/CD8+细胞毒性T细胞的混合群中。一般地，该细胞可以用抗体(如，抗-CD3或抗-CD28的抗体)活化，以便使它们能够更容易接受转染，例如用包含编码本发明TCR分子的核苷酸序列的载体进行转染。
备选地，本发明的细胞还可以是或衍生自干细胞，如造血干细胞(HSC)。将基因转移至HSC不会导致在细胞表面表达TCR，因为干细胞表面不表达CD3分子。然而，当干细胞分化为迁移至胸腺的淋巴前体(lymphoid precursor)时，CD3分子的表达将启动在胸腺细胞的表面表达该引入的TCR分子。
有许多方法适合于用编码本发明TCR的DNA或RNA进行T细胞转染(如，Robbins等.，(2008)J.Immunol.180:6116-6131)。表达本发明TCR的T细胞可以用于过继免疫治疗。本领域技术人员能够知晓进行过继性治疗的许多合适方法(如，Rosenberg等.，(2008)Nat Rev Cancer8(4)：299-308)。
NY-ESO-1抗原相关疾病
本发明还涉及在受试者中治疗和/或预防与NY-ESO-1相关疾病的方法，其包括过继性转移NY-ESO-1特异性T细胞至该受试者的步骤。该NY-ESO-1特异性T细胞可识别SLLMWITQC-HLA A0201复合物。
本发明的NY-ESO-1特异性的T细胞可用于治疗任何呈递NY-ESO-1抗原短肽SLLMWITQC-HLA A0201复合物的NY-ESO-1相关疾病。包括但不限于神经母细胞瘤、肉瘤、恶性黑色素瘤、前列腺癌、膀胱癌、乳腺癌、多发性骨髓瘤、肝细胞癌、口腔鳞癌以及食管癌。
治疗方法
可以通过分离患有与NY-ESO-1抗原相关疾病的病人或志愿者的T细胞，并将本发明的TCR导入上述T细胞中，随后将这些基因工程修饰的细胞回输到病人体内来进行治疗。因此，本发明提供了一种治疗NY-ESO-1相关疾病的方法，包括将分离的表达本发明TCR的T细胞，优选地，该T细胞来源于病人本身，输入到病人体内。一般地，包括(1)分离病人的T细胞，(2)用本发明核酸分子或能够编码本发明TCR分子的核酸分子体外转导T细胞，(3)将基因工程修饰的T细胞输入到病人体内。分离、转染及回输的细胞的数量可以由医师决定。
本发明的主要优点在于：
(1)本发明的TCR能够与NY-ESO-1抗原短肽复合物SLLMWITQC-HLA A0201特异性结合，同时转导了本发明TCR的细胞能够被特异性激活并且对靶细胞具有很强的杀伤作用。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方 法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1克隆NY-ESO-1抗原短肽特异性T细胞
利用合成短肽SLLMWITQC(北京赛百盛基因技术有限公司)刺激来自于基因型为HLA-A0201的健康志愿者的外周血淋巴细胞(PBL)。将SLLMWITQC短肽与带有生物素标记的HLA-A*0201复性,制备pHLA单倍体。这些单倍体与用PE标记的链霉亲和素(BD公司)组合成PE标记的四聚体，分选该四聚体及抗-CD8-APC双阳性细胞。扩增分选的细胞，并按上述方法进行二次分选，随后用有限稀释法进行单克隆。单克隆细胞用四聚体染色，筛选到的双阳性克隆如图3所示。
实施例2获取NY-ESO-1抗原短肽特异性T细胞克隆的TCR基因与载体的构建
用Quick-RNATMMiniPrep(ZYMO research)抽提实施例1中筛选到的抗原短肽SLLMWITQC特异性、HLA-A0201限制性的T细胞克隆的总RNA。cDNA的合成采用clontech的SMART RACE cDNA扩增试剂盒，采用的引物是设计在人类TCR基因的C端保守区。将序列克隆至T载体(TAKARA)上进行测序。应注意，该序列为互补序列，不包含内含子。经测序，该双阳性克隆表达的TCR的α链和β链序列结构分别如图1和图2所示，图1a、图1b、图1c、图1d、图1e和图1f分别为TCRα链可变域氨基酸序列、TCRα链可变域核苷酸序列、TCRα链氨基酸序列、TCRα链核苷酸序列、具有前导序列的TCRα链氨基酸序列以及具有前导序列的TCRα链核苷酸序列；图2a、图2b、图2c、图2d、图2e和图2f分别为TCRβ链可变域氨基酸序列、TCRβ链可变域核苷酸序列、TCRβ链氨基酸序列、TCRβ链核苷酸序列、具有前导序列的TCRβ链氨基酸序列以及具有前导序列的TCRβ链核苷酸序列。
经鉴定，α链包含具有以下氨基酸序列的CDR：
α CDR1-ATGYPS      (SEQ ID NO:10)
α CDR2-ATKADDK    (SEQ ID NO:11)
α CDR3-ALTLNNAGNMLT   (SEQ ID NO:12)
β链包含具有以下氨基酸序列的CDR：
β CDR1-SNHLY      (SEQ ID NO:13)
β CDR2-FYNNEI     (SEQ ID NO:14)
β CDR3-ASLDPRAGTDTQY  (SEQ ID NO:15)
通过重叠(overlap)PCR分别将TCRα链和β链的全长基因克隆至慢病毒表达载体。作为对照用,同时也构建表达eGFP的慢病毒载体。之后再用293T/17包装假病毒。
实施例3 NY-ESO-1抗原短肽特异性可溶TCR的表达、重折叠和纯化
为获得可溶的TCR分子，本发明的TCR分子的α和β链可以分别只包含其 可变域及部分恒定域，并且α和β链的恒定域中分别引入了一个半胱氨酸残基以形成人工链间二硫键，引入半胱氨酸残基的位置分别为TRAC*01外显子1的Thr48和TRBC2*01外显子1的Ser57；其α链的氨基酸序列与核苷酸序列分别如图4a和图4b所示，其β链的氨基酸序列与核苷酸序列分别如图5a和图5b所示，引入的半胱氨酸残基以加粗字母表示。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。
将TCRα和β链的表达载体分别通过化学转化法转化进入表达细菌BL21(DE3),细菌用LB培养液生长，于OD600＝0.6时用终浓度0.5mM IPTG诱导，TCR的α和β链表达后形成的包涵体通过BugBuster Mix(Novagene)进行提取，并且经BugBuster溶液反复多次洗涤，包涵体最后溶解于6M盐酸胍,10mM二硫苏糖醇(DTT),10mM乙二胺四乙酸(EDTA),20mM Tris(pH 8.1)中。
溶解后的TCRα和β链以1：1的质量比快速混合于5M尿素,0.4M精氨酸,20mM Tris(pH 8.1),3.7mM cystamine,6.6mMβ-mercapoethylamine(4℃)中，终浓度为60mg/mL。混合后将溶液置于10倍体积的去离子水中透析(4℃)，12小时后将去离子水换成缓冲液(20mM Tri s,pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45 μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP,5ml,GE Healthcare)纯化。洗脱峰含有复性成功的α和β二聚体的TCR通过SDS-PAGE胶确认。TCR随后通过凝胶过滤层析(HiPrep 16/60，Sephacryl S-100HR，GE Healthcare)进一步纯化。纯化后的TCR纯度经过SDS-PAGE测定大于90％，浓度由BCA法确定。本发明得到的可溶性TCR的SDS-PAGE胶图如图6所示。
实施例4 NY-ESO-1抗原短肽特异性的可溶性单链TCR的产生
根据专利文献WO2014/206304中所述，利用定点突变的方法将实施例2中TCRα与β链的可变域构建成了一个以柔性短肽(linker)连接的稳定的可溶性单链TCR分子。该单链TCR分子的氨基酸序列及核苷酸序列分别如图7a和图7b所示。其α链可变域的氨基酸序列及核苷酸序列分别如图8a和图8b所示；其β链可变域的氨基酸序列及核苷酸序列分别如图9a和图9b所示；其linker序列的氨基酸序列及核苷酸序列分别如图10a和图10b所示。
将目的基因经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli BL21(DE3),用于表达。
实施例5 NY-ESO-1抗原短肽特异性的可溶性单链TCR的表达、复性和纯化
将实施例4中制备的含有重组质粒pET28a-模板链的BL21(DE 3)菌落全部接种于含有卡那霉素的LB培养基中,37℃培养至OD600为0.6-0.8，加入IPTG至终浓度为0.5mM，37℃继续培养4h。5000rpm离心15min收获细胞沉淀 物，用Bugbuster Master Mix(Merck)裂解细胞沉淀物，6000rpm离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000rpm离心15min,收集包涵体。将包涵体溶解在缓冲液(20mM Tris-HCl pH 8.0,8M尿素)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。
向5mg溶解的单链TCR包涵体蛋白中，加入2.5mL缓冲液(6M Gua-HCl，50mM Tris-HCl pH 8.1，100mM NaCl，10mM EDTA)，再加入DTT至终浓度为10mM,37℃处理30min。用注射器向125mL复性缓冲液(100mM Tris-HCl pH 8.1,0.4M L-精氨酸,5M尿素,2mM EDTA,6.5mMβ-mercapthoethylamine,1.87mM Cystamine)中滴加上述处理后的单链TCR，4℃搅拌10min，然后将复性液装入截留量为4kDa的纤维素膜透析袋,透析袋置于1L预冷的水中，4℃缓慢搅拌过夜。17小时后，将透析液换成1L预冷的缓冲液(20mM Tris-HCl pH 8.0)，4℃继续透析8h，然后将透析液换成相同的新鲜缓冲液继续透析过夜。17小时后，样品经0.45μm滤膜过滤，真空脱气后通过阴离子交换柱(HiTrap Q HP,GE Healthcare)，用20mM Tris-HCl pH 8.0配制的0-1M NaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析，包含单链TCR的组分浓缩后进一步用凝胶过滤柱(Superdex 7510/300,GE Healthcare)进行纯化，目标组分也进行SDS-PAGE分析。
用于BIAcore分析的洗脱组分进一步采用凝胶过滤法测试其纯度。条件为：色谱柱Agilent Bio SEC-3(300A，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。
实施例6结合表征
BIAcore分析
本实施例证明了可溶性的本发明TCR分子能够与SLLMWITQC-HLA A0201复合物特异性结合。
使用BIAcore T200实时分析系统检测实施例3中得到的TCR分子与SLLMWITQC-HLA A0201复合物的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，pH 4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15，000RU。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将SLLMWITQC-HLA A0201复合物流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。
上述SLLMWITQC-HLA A0201复合物的制备过程如下：
a.纯化
收集100ml诱导表达重链或轻链的E.coli菌液，于4℃8000g离心10min后用10ml PBS洗涤菌体一次，之后用5ml BugBuster Master Mix Extraction Reagents(Merck)剧烈震荡重悬菌体，并于室温旋转孵育20min，之后于4℃，6000g离心15min，弃去上清，收集包涵体。
将上述包涵体重悬于5ml BugBuster Master Mix中，室温旋转孵育5min； 加30ml稀释10倍的BugBuster，混匀，4℃6000g离心15min；弃去上清，加30ml稀释10倍的BugBuster重悬包涵体，混匀，4℃6000g离心15min，重复两次，加30ml 20mM Tris-HCl pH 8.0重悬包涵体，混匀，4℃6000g离心15min，最后用20mM Tris-HCl 8M尿素溶解包涵体，SDS-PAGE检测包涵体纯度，BCA试剂盒测浓度。
b.复性
将合成的短肽SLLMWITQC(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SLLMWITQC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、50 μM D-Biotin、100 μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
e.纯化生物素化后的复合物
用Millipore超滤管将生物素化标记后的pMHC分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的pMHC，利用Akta纯化仪(GE通用电气公司)，用过滤过的PBS预平衡HiPrepTM 16/60S200HR柱(GE通用电气公司)，加载1ml浓缩过的生物素化pMHC分子，然后用PBS以1ml/min流速洗脱。生物素化的pMHC分子在约55ml时作为单峰洗脱出现。合并含有蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，加入蛋白酶抑制剂cocktail(Roche)将生物素化的pMHC分子分装保存在-80℃。
利用BIAcore Evaluation软件计算动力学参数，得到本发明可溶性的TCR分子与SLLMWITQC-HLA A0201复合物结合的动力学图谱如图11所示。同时，还利用上述方法检测了本发明可溶性的TCR分子与其他抗原短肽包括KLVALGINAV-HLA A0201复合物等的结合活性，结果显示本发明TCR分子与其他无关抗原短肽均无结合。
实施例7 TCR的功能及特异性
本实施例证明了经本发明所获得的TCR转导的PBL能够特异性识别 NY-ESO-1抗原阳性癌细胞的例证。
(a)通过293T细胞的快速介导瞬时转染(Express-In-mediated transient transfection)制备慢病毒
利用第三代慢病毒包装系统包装含有编码所需TCR的基因的慢病毒。利用PEI将4种质粒，用(含有pGZ178-HI WT TRA-2A-TRB慢病毒载体，以及含有构建传染性但非复制型慢病毒颗粒所必需的其他组分的3种质粒)瞬时转染293T细胞。
为进行转染，第0天种细胞，在15厘米培养皿，种上1.7×107个293T细胞，使细胞均匀分布在培养皿上，汇合度略高于50％。第1天转染质粒，包装pGZ178-HIWTTRA-2A-TRB和pLenti-eGFP假病毒，将以上表达质粒与包装质粒pMDLg/pRRE,pRSV-REV和pMD.2G混匀，一个15厘米直径平皿的用量如下：22.5微克：15微克:15微克：7.5微克。转染试剂PEI-MAX与质粒的比例是2:1，每个平皿的使用量为114.75微克。具体操作为：把表达质粒与包装质粒加入1800微升OPTI-MEM((吉布可公司(Gibco)，目录号31985-070)培养基中混合均匀，室温静置5分钟成为DNA混合液；取相应量PEI与1800微升OPTI-MEM培养基混合均匀，室温静置5分钟成为PEI混合液。把DNA混合液和PEI混合液混合在一起并在室温静置30分钟，再添加3150微升OPTI-MEM培养基，混合均匀后加入到已经转换成11.25毫升OPTI-MEM的293T细胞中，轻轻晃动培养皿，使培养基混合均匀，37℃/5％CO2下培养。转染5-7小时，去除转染培养基，换成含有10％胎牛血清的DMEM((吉布可公司(Gibco)，目录号C11995500bt))完全培养基，37℃/5％CO2下培养。第3和第4天收集含有包装的慢病毒的培养基上清。为收获包装的慢病毒，把所收集到的培养上清3000g离心15分钟去除细胞碎片，再经0.22微米过滤器(默克密理博(Merck Millipore)，目录号SLGP033RB)过滤，最后用50KD截留量的浓缩管(默克密理博(Merck Millipore)，目录号UFC905096)进行浓缩，除去大部分上清液，最后浓缩到1毫升，等分分装后-80℃冻存。取假病毒样品进行病毒滴度测定，步骤参照p24ELISA(Clontech，目录号632200)试剂盒说明书。作为对照用，同时也包转pLenti-eGFP的假病毒。
(b)用含有HI-WT特异性T细胞受体基因的慢病毒转导PBL
从健康志愿者的血液中分离到PBL细胞，再用包装的慢病毒转导。计数这些细胞，在48孔板中，在含有100IU/ml IL-2的含10％FBS(吉布可公司(Gibco)，目录号C10010500BT)的1640(吉布可公司(Gibco)，目录号C11875500bt)培养基中以1×106个细胞/毫升(0.5毫升/孔)与预洗涤的抗CD3/CD28抗体-包被小珠(T细胞扩增物，life technologies，目录号11452D)共孵育过夜刺激，细胞：珠＝3：1。
刺激过夜后，根据p24ELISA试剂盒所测到的病毒滴度，按MOI＝10的比例加入已浓缩的HI-WT特异性T细胞受体基因的慢病毒，32℃，900g离心感染1小时。感染完毕后去除慢病毒感染液，用含有100IU/ml IL-2的含10％FBS的1640培养基重悬细胞，37℃/5％CO2下培养3天。转导3天后计数细胞，稀释细胞至0.5×106个细胞/毫升。每两天计数一次细胞，替换或加入含有50IU/ml IL-2的新鲜培养基，维持细胞在0.5×106-1×106个细胞/毫升。从第3天开始,经含有SLLMWITQC短肽与HLA-A*0201的四聚体染色，慢病毒转导的PBL通过 流式细胞术的定量分析。当四聚体检测阳性率大于10％，则于第5天开始用于功能试验(例如，IFN-γ释放的ELISPOT和非放射性细胞毒性检测)。
(c)四聚体染色TCR转导的PBL
NY-ESO-1 157-165 SLLMWITQC短肽(p1A)与带有生物素标记的HLA-A*0201复性，制备pHLA单倍体。这些单倍体用PE标记的链霉亲和素(BD)组合成PE标记的四聚体,称为p1A-tetramer-PE。此四聚体能把表达了NY-ESO-1 157-165 SLLMWITQC特异性T细胞受体基因的T细胞标记为阳性细胞。把(b)中经转染的T细胞样品与p1A-tetramer-PE在冰上孵育30分钟，然后加入anti-CD8-APC(BioLegend)抗体，继续冰上孵育15分钟。样品用含有2％FBS的PBS清洗2次后用Guava 16HT检测表达了NY-ESO-1 157-165SLLMWITQC特异性T细胞受体基因的p1A-tetramer-PE和CD8双阳性的T细胞，数据分析采用FlowJo软件(Tree Star Inc,Ashland,OR)分析。
通过ELISPOT实验进一步检测转导本发明的PBL的功能及特异性。本领域技术人员熟知利用ELISPOT实验检测细胞功能的方法。本实施例IFN-γELISPOT实验中所用的效应细胞为本发明中获得的病毒转导HI-WT基因的PBL，靶细胞系为抗原(NY-ESO-1)阳性的IM9(ATCC)和U266B1(ATCC)细胞，对照组为抗原(NY-ESO-1)阴性的293T细胞。
首先准备ELISPOT平板，ELISPOT实验步骤如下：按以下顺序将试验的各15个组分加入ELISPOT平板：个组分别加入ELISPOT平板：将T2细胞用培养基调整到2 X 105个细胞/毫升，根据终体积与短肽终浓度将特异性短肽及非特异性短肽分别加到对应的T2细胞稀释液中，空白组加入相应量的培养基，混合均匀后取100 μL T2细胞与靶细胞系2 X 105个细胞/毫升(即20，000个细胞/孔)、100 μL效应细胞2 X 104个细胞/毫升(即20，00个病毒转导细胞/孔)，并设置三个复孔。然后温育过夜(37℃，5％CO2)。随后洗涤平板并进行二级检测和显色，干燥平板1小时，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。实验结果如图12(a和b)所示，相较于对照组293T细胞，本发明得到的病毒转导HI-WT基因的PBL对负载特异性多肽的T2细胞及抗原(NY-ESO-1)阳性的IM9和U266B1细胞系有特异性反应。
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 (31)

  1. 一种T细胞受体(TCR)，其特征在于，所述TCR能够与SLLMWITQC-HLA复合物特异性地结合,其包含TCR α链可变域和TCR β链可变域，并且所述TCR α链可变域为与SEQ ID NO:1具有至少90％序列相同性的氨基酸序列。
  2. 如权利要求1所述的TCR，其特征在于所述TCR β链可变域为与SEQ ID NO：5具有至少90％序列相同性的氨基酸序列。
  3. 如权利要求1或2所述的TCR，其包含TCR α链可变域和TCR β链可变域，其特征在于，所述TCR α链可变域的CDR3的氨基酸序列为ALTLNNAGNMLT(SEQ ID NO:12)；和/或所述TCR β链可变域的CDR3的氨基酸序列为ASLDPRAGTDTQY(SEQ ID NO:15)。
  4. 如权利要求3所述的TCR，其特征在于，所述TCR α链可变域的3个互补决定区(CDR)为：
    α CDR1-ATGYPS         (SEQ ID NO:10)
    α CDR2-ATKADDK        (SEQ ID NO:11)
    α CDR3-ALTLNNAGNMLT   (SEQ ID NO:12)和/或所述TCR β链可变域的3个互补决定区为：
    β CDR1-SNHLY          (SEQ ID NO:13)
    β CDR2-FYNNEI         (SEQ ID NO:14)
    β CDR3-ASLDPRAGTDTQY  (SEQ ID NO:15)
  5. 如以上任一权利要求所述的TCR，其特征在于，所述TCR包含α链可变域氨基酸序列SEQ ID NO:1。
  6. 如以上任一权利要求所述的TCR，其特征在于，所述TCR包含β链可变域氨基酸序列SEQ ID NO:5。
  7. 如以上任一权利要求所述的TCR，其特征在于，所述TCR为α β异质二聚体，其包含TCR α链恒定区TRAC*01和TCR β链恒定区TRBC1*01或TRBC2*01。
  8. 如权利要求7中所述的TCR，其特征在于，所述TCR的α链氨基酸序列为SEQ ID NO：3和/或所述TCR的β链氨基酸序列为SEQ ID NO:7。
  9. 如权利要求1-6中任一所述的TCR，其特征在于，所述TCR是可溶的。
  10. 如权利要求9所述的TCR，其特征在于，所述TCR为单链。
  11. 如权利要求10所述的TCR，其特征在于，所述TCR是由α链可变域与β链可变域通过肽连接序列连接而成。
  12. 如权利要求11所述的TCR，其特征在于，所述TCR在α链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或α链J基因短肽氨基酸倒数第3位、倒数第5位或倒数第7位中具有一个或多个突变；和/或所述TCR在β链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或β链J基因短肽氨基酸倒数第2位、倒数第4位或倒数第6位中具有一个或多个突变，其中氨基酸位置编号按IMGT(国际免疫遗传学信息系统)中列出的位置编号。
  13. 如权利要求12所述的TCR，其特征在于，所述TCR的α链可变域氨基酸序列包含SEQ ID NO:32和/或所述TCR的β链可变域氨基酸序列包含SEQ ID  NO:34。
  14. 如权利要求13所述的TCR，其特征在于，所述TCR的氨基酸序列为SEQ ID NO:30。
  15. 如权利要求9所述的TCR，其特征在于，所述TCR包括(a)除跨膜结构域以外的全部或部分TCR α链；以及(b)除跨膜结构域以外的全部或部分TCR β链；
    并且(a)和(b)各自包含功能性可变结构域,或包含功能性可变结构域和所述TCR链恒定结构域的至少一部分。
  16. 如权利要求15所述的TCR，其特征在于，半胱氨酸残基在所述TCR的α和β链恒定域之间形成人工二硫键。
  17. 如权利要求16所述的TCR，其特征在于，在所述TCR中形成人工二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
    TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
    TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
    TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
    TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
    TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；和
    TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
  18. 如权利要求17所述的TCR，其特征在于，所述TCR的α链氨基酸序列为SEQ ID NO:26和/或所述TCR的β链氨基酸序列为SEQ ID NO:28。
  19. 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。
  20. 如权利要求19所述的T细胞受体，其特征在于，与所述T细胞受体结合的偶联物为可检测标记物、治疗剂、PK修饰部分或任何这些物质的组合。优选地，所述治疗剂为抗-CD3抗体。
  21. 一种多价TCR复合物，其特征在于，包含至少两个TCR分子，并且其中的至少一个TCR分子为上述权利要求中任一项所述的TCR。
  22. 一种核酸分子，其特征在于，所述核酸分子包含编码上述任一权利要求所述的TCR分子的核酸序列或其互补序列。
  23. 如权利要求22所述的核酸分子，其特征在于，其包含编码TCR α链可变域的核苷酸序列SEQ ID NO：2或SEQ ID NO:33。
  24. 如权利要求22或23所述的核酸分子，其特征在于，其包含编码TCR β链可变域的核苷酸序列SEQ ID NO：6或SEQ ID NO:35。
  25. 如权利要求22所述的核酸分子，其特征在于，其包含编码TCR α链的核苷酸序列SEQ ID NO：4和/或包含编码TCR β链的核苷酸序列SEQ ID NO：8。
  26. 一种载体，其特征在于，所述的载体含有权利要求22-25中任一所述的核酸分子；优选地，所述的载体为病毒载体；更优选地，所述的载体为慢病毒载体。
  27. 一种分离的宿主细胞，其特征在于，所述的宿主细胞中含有权利要求 26中所述的载体或染色体中整合有外源的权利要求22-25中任一所述的核酸分子。
  28. 一种细胞，其特征在于，所述细胞转导权利要求22-25中任一所述的核酸分子或权利要求26中所述载体；优选地，所述细胞为T细胞或干细胞。
  29. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1-20中任一项所述的TCR、权利要求21中所述的TCR复合物、权利要求22-25中任一所述的核酸分子、或权利要求28中所述的细胞。
  30. 权利要求1-20中任一项所述的T细胞受体、或权利要求21中所述的TCR复合物或权利要求28中所述的细胞的用途，其特征在于，用于制备治疗肿瘤或自身免疫疾病的药物。
  31. 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适量的权利要求1-20中任一所述的TCR、权利要求21中所述TCR复合物、权利要求28中所述的细胞或权利要求29中所述的药物组合物；
    优选地，所述的疾病为神经母细胞瘤、肉瘤、恶性黑色素瘤、前列腺癌、膀胱癌、乳腺癌、多发性骨髓瘤、肝细胞癌、口腔鳞癌以及食管癌。
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