WO2018077242A1 - T cell receptor for recognizing sage1 antigen short-chain polypeptide - Google Patents

T cell receptor for recognizing sage1 antigen short-chain polypeptide Download PDF

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WO2018077242A1
WO2018077242A1 PCT/CN2017/108081 CN2017108081W WO2018077242A1 WO 2018077242 A1 WO2018077242 A1 WO 2018077242A1 CN 2017108081 W CN2017108081 W CN 2017108081W WO 2018077242 A1 WO2018077242 A1 WO 2018077242A1
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tcr
seq
cell
cells
variable domain
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PCT/CN2017/108081
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French (fr)
Chinese (zh)
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李懿
陈安安
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中国科学院广州生物医药与健康研究院
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Priority to CN201780066440.9A priority Critical patent/CN109890839B/en
Publication of WO2018077242A1 publication Critical patent/WO2018077242A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a TCR capable of recognizing a short peptide derived from a SAGE1 antigen, and to a SAGE1-specific T cell obtained by transducing the above TCR, and their use in the prevention and treatment of a disease associated with SAGE1.
  • SAGE1 As an endogenous tumor antigen, SAGE1 is degraded into small molecule polypeptides after intracellular production, and combined with MHC (main histocompatibility complex) molecules to form a complex, which is presented to the cell surface.
  • MHC main histocompatibility complex
  • VFSTVPPAFI is a short peptide derived from SAGE1.
  • SAGE1 antigen is expressed in tumor tissues such as melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, and squamous cell carcinoma, but not in most normal tissues except testis (Martelange V1, De Smet C, De Plaen E, Lurquin C, Boon T. Cancer Res.
  • 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
  • TCR T cell receptor
  • 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 CAVLYTGANSKLTF (SEQ ID NO. 12); and/or The amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is CASSLVGKQPQHF (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 a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID NO.
  • the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5.
  • the TCR comprises an alpha chain variable domain amino acid sequence of SEQ ID NO.
  • the TCR comprises the ⁇ chain variable domain amino acid sequence of SEQ ID NO.
  • 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.
  • 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 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:
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO. 26 and/or the ⁇ chain amino acid sequence of the TCR is SEQ ID NO.
  • an artificial interchain disulfide bond is contained between the alpha chain variable region of the TCR and the beta chain constant region.
  • cysteine residue forming an artificial interchain disulfide bond in the TCR replaces one or more sets of sites selected from the group consisting of:
  • 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.
  • 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 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 encoding a TCR alpha chain variable domain.
  • the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 6 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, a TCR according to the first aspect of the present invention, a TCR complex according to the second aspect of the present invention, and a present invention are provided.
  • 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 The use of the vector of the aspect, or the cell of the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumor or an autoimmune disease.
  • a method of treating a disease comprising administering an appropriate amount of the T cell receptor of the first aspect of the invention, or the TCR complex of the second aspect of the invention to a subject in need of treatment
  • the nucleic acid molecule of the third aspect of the invention, the vector of the fourth aspect of the invention, or the cell of the sixth aspect of the invention, or the pharmaceutical composition of the seventh aspect of the invention comprising administering an appropriate amount of the T cell receptor of the first aspect of the invention, or the TCR complex of the second aspect of the invention to a subject in need of treatment.
  • the disease is a tumor, preferably the tumor comprises melanoma, and other tumors Tumors such as gastric cancer, lung cancer (eg, lung squamous cell carcinoma), esophageal cancer, bladder cancer, head and neck cancer (eg, head and neck squamous cell carcinoma), prostate cancer, breast cancer, colon cancer, ovarian cancer, kidney Cell carcinoma, Hodgkin's lymphoma, sarcoma, medulloblastoma, leukemia, etc.
  • Tumors such as gastric cancer, lung cancer (eg, lung squamous cell carcinoma), esophageal cancer, bladder cancer, head and neck cancer (eg, head and neck squamous cell carcinoma), prostate cancer, breast cancer, colon cancer, ovarian cancer, kidney Cell carcinoma, Hodgkin's lymphoma, sarcoma, medulloblastoma, leukemia, etc.
  • the tumor comprises melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, and squamous cell carcinoma.
  • 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.
  • the leftmost lane is the reducing gel
  • the middle lane is the molecular weight marker
  • the rightmost lane is the non-reducing gel.
  • Figure 7 is a ProteOn combining the soluble TCR with the VFSTVPPAFI-HLA A2402 complex of the present invention.
  • Figure 8 shows the results of primary T-fine detection of tetrameric stained TCR transduction.
  • Figure 9 shows the results of the ELISPOT test.
  • Figure 10 is a graph showing the killing effect of TCR transduced T cells of the present invention on specific target cells.
  • the inventors have found extensively and intensively, and found a TCR capable of specifically binding to the SAGE1 antigen short peptide VFSTVPPAFI (SEQ ID NO. 9), which can form a complex with HLA A2402 and be presented together.
  • 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.
  • Natural ⁇ heterodimeric TCR has ⁇ chain and ⁇ chain, ⁇ chain and ⁇ The strand constitutes a subunit of the alpha beta 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
  • the position numbers of the amino acid sequences of TROC*01 and TRBC1*01 or TRBC2*01 are numbered in order from N-terminus to C-terminus, such as TRBC1*01 or TRBC2*01.
  • the 60th amino acid is P (valine), which may be described as Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, or may be It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and in the case of TRBC1*01 or TRBC2*01, the 61st amino acid is Q in the order from N to C.
  • Amide which may be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, or may be expressed as amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01, other And so on.
  • 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.
  • special instructions will be given.
  • a first aspect of the invention provides a TCR molecule capable of binding to a VFSTVPPAFI-HLA A2402 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 of the TCR comprises a CDR having the following amino acid sequence:
  • the three complementarity determining regions of the TCR ⁇ chain variable domain are:
  • 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.
  • 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 SEQ ID NO: 5 has an amino acid sequence of at least 90%, preferably 95%, more preferably 98% sequence identity.
  • 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. More preferably, the alpha chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO.
  • 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 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. More preferably, the ⁇ chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO.
  • 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. More preferably, the alpha chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO.
  • 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. More preferably, the ⁇ chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO.
  • 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.
  • 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 SAGE1 antigen short peptide is also obtained by the present invention. Has a specific soluble TCR.
  • 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 patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the alpha chain variable region of the TCR and the beta chain constant region can significantly improve the stability of the TCR. Therefore, the ⁇ chain variable region of the high affinity TCR of the present invention and the ⁇ chain constant region may further contain an artificial interchain disulfide bond.
  • cysteine residue forming an artificial interchain disulfide bond between the ⁇ chain variable region of the TCR and the ⁇ chain constant region is substituted with: amino acid 46 of TRAV and TRBC1*01 or TRBC2* 01 amino acid at position 60 of exon 1; amino acid at position 47 of TRAV and amino acid at position 61 of exon 1 of TRBC1*01 or TRBC2*01; amino acid at position 46 of TRAV and TRBC1*01 or TRBC2*01 The amino acid at position 61 of the 1st; or the amino acid at position 47 of TRAV and the amino acid at position 60 of exon 1 of TRBC1*01 or TRBC2*01.
  • such a TCR may comprise (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 contain a variable domain of the TCR chain and at least a portion In the constant domain, the alpha chain forms a heterodimer with the beta chain. More preferably, such a TCR may comprise 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 alpha The chain variable domain forms a heterodimer with the beta chain.
  • 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 complexes of the invention can be used to track or target cells that present a particular antigen in vitro or in vivo, as well as intermediates that produce other multivalent TCR complexes for 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 a VFSTVPPAFI-HLA A2402 complex), a therapeutic agent, a PK (protein kinase) modified moiety, or any of these 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.
  • 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 the TCR beta chain variable domain of the invention comprises SEQ ID NO.
  • the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. It will be appreciated that due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide.
  • a 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.
  • 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 TAGE specific for the SAGE1 antigen.
  • 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.
  • the host cell contains the vector of the present invention or a nucleic acid molecule of the present invention in which the chromosome is integrated.
  • 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 disease associated with SAGE1 in a subject, comprising the step of adoptively transferring SAGE1-specific T cells to the subject.
  • the SAGE1-specific T cells recognize the VFSTVPPAFI-HLA A2402 complex.
  • the SAGE1-specific T cells of the invention can be used to treat any SAGE1-related disease presenting the SAGE1 antigen short peptide VFSTVPPAFI-HLA A2402 complex.
  • SAGE1-related disease presenting the SAGE1 antigen short peptide VFSTVPPAFI-HLA A2402 complex.
  • tumors such as melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, ovarian cancer, and the like.
  • the T cells of a patient or a volunteer having a disease associated with the SAGE1 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 for treatment.
  • the present invention provides a method of treating a SAGE1-related disease comprising administering an isolated TCR 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 bind to the SAGE1 antigen short peptide complex VFSTVPPAFI-HLA A2402, and the cells transduced with the TCR of the present invention can be specifically activated and have a strong killing effect on target cells.
  • Peripheral blood lymphocytes from healthy volunteers with genotype HLA-A2402 were stimulated with the synthetic short peptide SAGE1PX149 597-606 VFSTVPPAFI (Beijing Cypress Biotech Co., Ltd.).
  • SAGE1 PX149 597-606 VFSTVPPAFI short peptide was renatured with biotinylated HLA-A*2402 to prepare a pHLA monomer.
  • moners were combined with PE-labeled streptavidin (BD) into PE-labeled tetramers, which were sorted and anti-CD8-APC double positive cells.
  • the sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonal culture by limiting dilution. Monoclonal cells were stained with tetramers and the double positive clones screened are shown in Figure 3.
  • TCR ⁇ chain variable domain amino acid sequence and TCR ⁇ are the TCR ⁇ chain variable domain amino acid sequence and TCR ⁇ , respectively.
  • Fig. 2a, Fig. 2b, Fig. 2c and Fig. 2d are TCR ⁇ chain variable domain amino acid sequence, TCR ⁇ chain variable domain nucleotide, respectively Sequence, TCR ⁇ chain amino acid sequence and TCR ⁇ chain nucleotide sequence.
  • 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:
  • variable domains of the TCR alpha chain and the beta chain are each spliced into the full length gene and linked to the lentiviral expression vector pLenti (addgene) by overlap PCR with the conserved domains of the mouse TCR alpha chain and the beta chain, respectively.
  • the TCR ⁇ -2A-TCR ⁇ fragment was obtained by ligating the full-length genes of the TCR ⁇ chain and the TCR ⁇ chain by overlap PCR.
  • the lentiviral expression vector and TCR ⁇ -2A-TCR ⁇ were digested to obtain the pLenti-SAGE1TRA-2A-TRB-IRES-NGFR plasmid.
  • a lentiviral vector pLenti-eGFP 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 comprise only their variable domains and partial constant domains, respectively, and a cysteine residue is introduced in the constant domains of the ⁇ and ⁇ chains, respectively.
  • the positions at which cysteine residues are introduced are Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC2*01, respectively; amino acid sequence and nucleotide of ⁇ chain thereof
  • the sequences are shown in Figures 4a and 4b, respectively, and the amino acid sequence and nucleotide sequence of the ⁇ chain are shown in Figures 5a and 5b, respectively, and the introduced cysteine residues are indicated by bold and underlined letters.
  • the above 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 TCR ⁇ and ⁇ chain expression vectors were transformed into the expression bacterium BL21 (DE3) by chemical transformation, respectively, and the bacteria were grown in LB medium.
  • the formed 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). , 20 mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains were rapidly mixed in a mass ratio of 1:1 in 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, 6.6 mM ⁇ -mercapoethylamine (4 ° C), and the final concentration was 60 mg. /mL. After mixing, the solution was placed in 10 volumes of deionized water. After analysis (4 ° C), 12 hours later, the deionized water was replaced with a buffer (20 mM Tris, pH 8.0) and dialysis was continued at 4 ° C for 12 hours.
  • the solution after completion of dialysis was filtered through a 0.45 ⁇ M filter, and then purified by 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.
  • the binding activity of the TCR molecule obtained in Example 3 to the VFSTVPPAFI-HLA A2402 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 VFSTVPPAFI-HLA A2402 complex is flowed through the detection channel, and the other channel is used as a reference channel, and then 0.05 mM biotin is 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-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 synthesized short peptide VFSTVPPAFI (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 the addition of 3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA before renaturation.
  • the VFSTVPPAFI peptide was added to the refolding buffer at 25 mg/L (final concentration) (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 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 to Upon completion, SDS-PAGE can detect whether the renaturation is successful.
  • 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).
  • HiTrap Q HP GE General Electric Company
  • Akta Purifier GE General Electric
  • 20 Akta Purifier 20 mM Tris pH 8.0
  • the protein was eluted with a linear gradient of 0-400 mM NaCl.
  • the pMHC was eluted at approximately 250 mM NaCl.
  • the peak fractions were collected and the 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 MgAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml BirA enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and biotinylation was detected by SDS-PAGE.
  • 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 kinetic parameters of the soluble TCR molecules of the present invention were determined by using the BIAcore Evaluation software to calculate the kinetic parameters as shown in FIG.
  • Lentiviruses containing the gene encoding the desired TCR were packaged using a third generation lentiviral packaging system.
  • Four plasmids (containing one of pLenti-SAGE1 TRA-2A-TRB-IRES-NGFR described in Example 2) using Express-In-mediated transient transfection (Open Biosystems)
  • 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, add to 293T/17 cells that have been converted to 11.25 ml of OPTI-MEM, gently shake the dish, mix the medium evenly, and incubate at 37 ° C / 5% CO 2 . After -7 hours, the transfection medium was removed and replaced with DMEM (Gibco, catalog number C11995500bt) containing 10% fetal bovine serum, and cultured at 37 ° C / 5% CO 2 . The medium supernatant containing the packaged lentivirus was collected on day 3 and day 4.
  • DMEM Gibco, catalog number 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 (Merckmi Merck Millipore, catalog number SLGP033RB), finally intercepted with 50KD
  • the concentrated tube Merck Millipore, catalog number UFC905096 was concentrated to remove most of the supernatant, finally concentrated to 1 ml, and aliquoted and stored at -80 ° C.
  • the pseudovirus sample was taken for virus.
  • the titer was measured by the p24 ELISA (Clontech, Cat. No. 632200) kit instructions.
  • the pseudovirus of pLenti-eGFP was also included.
  • the cells were counted every two days, and fresh medium containing 50 IU/ml IL-2 and 10 ng/ml IL-7 was replaced or added to maintain the cells at 0.5 x 10 6 - 1 x 10 6 cells/ml.
  • Cells were analyzed by flow cytometry starting on day 3 and were used for functional assays from day 5 (eg, ELISPOT and non-radioactive cytotoxicity assays for IFN- ⁇ release).
  • SAGE1PX149 597-606 VFSTVPPAFI short peptide was renatured with biotinylated HLA-A*2402 to prepare pHLA monomer. These monomers are combined with PE-labeled streptavidin (BD) into a PE-labeled tetramer called PX149-tetramer-PE. This tetramer can label T cells expressing a SAGE1-specific T cell receptor gene as positive cells.
  • the transduced T cell samples in (b) were incubated with PX149-tetramer-PE for 30 minutes on ice, then anti-mouse ⁇ -chain-APC antibody was added and incubation was continued for 15 minutes on ice.
  • BD Calibur or BD Arial was used to detect or sort PX149-tetramer-PE expressing the SAGE1-specific T cell receptor gene and anti-mouse ⁇ -chain-APC double positive T Cells were analyzed by CellQuest software (BD) or FlowJo software (Tree Star Inc, Ashland, OR).
  • the following assays were performed to demonstrate activation of TCR-transduced T cells specifically responding to target cells.
  • the IFN- ⁇ production detected by the ELISPOT assay was used as a readout value for T cell activation.
  • Test medium 10% FBS (Gibco, catalog number 16000-044), RPMI 1640 (Gibco, catalog number C11875500bt)
  • PVDF ELISPOT 96-well plate (Merck Millipore, catalog number MSIPS4510)
  • the human IFN- ⁇ ELISPOT PVDF-Enzyme Kit contains all the other reagents required (capture and detection antibodies, streptavidin-alkaline phosphatase and BCIP/NBT solutions)
  • the target cells of this example are Epstein-Barr virus (EBV) transformed immortalized lymphoblastoid cell lines (LCLs).
  • B95-8 cells were induced to produce EBV-containing medium supernatant by tetradecanoyl phorbol ester (TPA), centrifuged at 4 ° C / 600 g for 10 minutes to remove impurities, and then passed through a 0.22 micron filter, aliquoted -70 ° C save.
  • EBV Epstein-Barr virus
  • TPA tetradecanoyl phorbol ester
  • PBL peripheral blood lymphocytes
  • CD19 + CD23 hi CD58 + was an immortalized lymphoblastic cell line (LCLs).
  • This ELISPOT assay uses HLA-A24 as a specific target cell.
  • T cells The effector cells (T cells) of the present assay were CD8 + T cells expressing SAGE1-specific TCR by flow cytometry in Example 3, and CD8 + T of the same volunteer was used as a negative control effector cell.
  • T cells were stimulated with anti-CD3/CD28 coated beads (T cell amplicon, LifeTechnologies), transduced with lentivirus carrying the SAGE1-specific TCR gene (according to Example 3), containing 50 IU/ml IL-2 and 10 ng /ml IL-7 in 1040 medium containing 10% FBS was amplified until 9-12 days after transduction, and then these cells were placed in a test medium, and washed by centrifugation at 300 g for 10 minutes at room temperature. The cells were then resuspended in test medium at 2 x the desired final concentration. Negative control effector cells were also treated.
  • SAGE1CD8 + T cells SAGE1 TCR transduced T cells, effector cells VF3CD8+ T cells), CD8+ T cells (negative control effector cells) and LCL-A24/A02 (target cells) were prepared as described in Example 3, and The corresponding experimental group was added with the corresponding short peptide, wherein PX149 was SAGE1PX149597-606VFSTVPPAFI short peptide, and PA11, PA02, PA24-1, PA24-2 and PA24-3 were non-SAGE1 TCR specific binding short peptides.
  • effector cells 1000 SAGE1 TCR double positive T cells.
  • the plates were then incubated overnight (37 ° C / 5% CO 2 ) for the next day, the medium was discarded, the plates were washed twice with double distilled water, washed 3 times with wash buffer, and tapped on a paper towel to remove residuals. Wash buffer.
  • the primary antibody was then diluted with PBS containing 10% FBS and added to each well at 100 ⁇ L/well. The well plates were incubated for 2 hours at room temperature and washed 3 times with wash buffer and the well plates were tapped on paper towels to remove excess wash buffer.
  • the TCR-transduced T cells of the present invention were tested for IFN- ⁇ release in response to target cells bearing the SAGE1 PX149 597-606 VFSTVPPAFI short peptide and target cells of the non-specific short peptide by the ELISPOT assay (described above).
  • the number of ELSPOT spots observed in each well was plotted using graphpad prism6.
  • the experimental results are shown in Fig. 9.
  • the CD8 + T cells transducing the TCR of the present invention have a strong activation effect on the LCLs of the load-related short peptides, releasing more IFN- ⁇ , and the LCLs supporting the non-specific short peptides are basically There was no response; at the same time, CD8 + T cells that did not transduce the TCR of the present invention responded very little to LCLs loaded with short peptides, with only a small amount of IFN- ⁇ released.
  • This test is a colorimetric substitution test for the 51Cr release cytotoxicity assay to quantify the lactate dehydrogenase (LDH) released after cell lysis.
  • LDH lactate dehydrogenase
  • the LDH released in the medium was detected using a 30 minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) to red formazan.
  • INT tetrazolium salt
  • the amount of red product produced is directly proportional to the number of cells lysed.
  • the 490 nm visible light absorbance data can be collected using a standard 96-well plate reader.
  • CytoTox Non-radioactive cytotoxicity assays (Promega, G1780) contain a substrate mixture, assay buffer, lysis solution, and stop buffer.
  • Test medium 10% FBS (heat inactivated, Gibbco, catalog number 16000-044), phenol red containing 90% RPMI 1640 (Gibco, catalog number C11875500bt), 1% penicillin / Streptomycin (Jibuco, catalog number 15070-063).
  • Microporous round bottom 96-well tissue culture plate (Nunc, catalog number 163320)
  • Target cells were prepared in the test medium: the target cell concentration was adjusted to 334 cells/ml, and 45 microliters per well was taken to obtain 1.5 x 10 4 cells/well.
  • T cells The effector cells (T cells) of this assay were analyzed by flow cytometry in Example 3 to express CD8 + T cells expressing SAGE1-specific TCR.
  • the ratio of effector cells to target cells was 10:1, 5:1, 2.5:1, 1.25:1, and 0.625:1.
  • a homologous CD8 + T cell plus target cell control group (10:1) was set.
  • the components of the assay were added to a microwell round bottom 96-well tissue culture plate in the following sequence:
  • a control group was prepared as follows:
  • the plate was centrifuged at 250 g for 4 minutes. 50 ul of the supernatant from each well of the assay plate was transferred to the corresponding well of a 96-well immunoplate Maxisorb plate. The substrate mixture was reconstituted using assay buffer (12 ml) and then 50 ul was added to each well of the plate. The plate was capped and incubated for 30 minutes at room temperature in the dark. 50 ul of the stop solution was added to each well of the plate to terminate the reaction. The absorbance at 490 nm was counted and recorded within 1 hour after the addition of the stop solution.
  • Absorbance values of the medium background were subtracted from all absorbance values of the experimental group, the target cell spontaneous release group, and the effector cell self-release group.
  • % cytotoxicity 100 ⁇ (experimental - effector cell spontaneous - target cell spontaneous) / (target cell max - target cell spontaneous)
  • the statistical results of the experimental data are shown in Fig. 10. As the ratio of the effective target increases, the killing effect of the TCR-transduced T cells of the present invention on the specific target cells K562-24 and NCI H1299-A24 is enhanced; for the non-specific target cells IM 9 The killing effect is very weak. The homologous CD8 + T cells that did not transduce the TCR of the present invention showed no significant killing of the target cell K562-24.

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Abstract

Provided is are a T cell receptor (TCR) capable of specific binding of short-chain polypeptide VFSTVPPAFI derived from a SAGE1 antigen, a nucleic acid molecule coding the TCR, a vector comprising the nucleic acid molecule, and a cell transducing the TCR. The antigen short-chain polypeptide VFSTVPPAFI can form a complex with HLA A2402 and be transduced together to cell surface.

Description

识别SAGE1抗原短肽的T细胞受体T cell receptor recognizing SAGE1 antigen short peptide 技术领域Technical field
本发明涉及能够识别源自SAGE1抗原短肽的TCR，本发明还涉及转导上述TCR来获得的SAGE1特异性的T细胞，及他们在预防和治疗SAGE1相关疾病中的用途。The present invention relates to a TCR capable of recognizing a short peptide derived from a SAGE1 antigen, and to a SAGE1-specific T cell obtained by transducing the above TCR, and their use in the prevention and treatment of a disease associated with SAGE1.
背景技术Background technique
SAGE1作为一种内源性肿瘤抗原，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。研究显示，VFSTVPPAFI是衍生自SAGE1的短肽。SAGE1抗原在黑色素瘤、膀胱癌、肝癌、表皮样癌、非小细胞肺癌和鳞状细胞癌等肿瘤组织中均有表达，而在除睾丸外的多数正常组织中不表达(Martelange V1,De Smet C,De Plaen E,Lurquin C,Boon T.Cancer Res.2000；60(14):3848-55；Atanackovic D,etal.,Cancer BiolTher.2006；5(9):1218-25)。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。As an endogenous tumor antigen, SAGE1 is degraded into small molecule polypeptides after intracellular production, and combined with MHC (main histocompatibility complex) molecules to form a complex, which is presented to the cell surface. Studies have shown that VFSTVPPAFI is a short peptide derived from SAGE1. SAGE1 antigen is expressed in tumor tissues such as melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, and squamous cell carcinoma, but not in most normal tissues except testis (Martelange V1, De Smet C, De Plaen E, Lurquin C, Boon T. Cancer Res. 2000; 60(14): 3848-55; Atanackovic D, et al., Cancer Biol Ther. 2006; 5(9): 1218-25). For the treatment of the above diseases, chemotherapy and radiotherapy can be used, but all of them will cause damage to their normal cells.
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***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SAGE1抗原短肽具有特异性的TCR，使其发挥作用，或者将该TCR转导T细胞来获得对SAGE1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。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. In the immune system, direct binding of T cells to antigen presenting cells (APCs) by TCR binding to the short peptide-primary histocompatibility complex (pMHC complex), and then T cells and The other cell membrane surface molecules of APC interact, causing a series of subsequent cell signaling and other physiological responses, allowing different antigen-specific T cells to exert an immune effect on their target cells. Therefore, those skilled in the art are working to isolate a TCR specific for a SAGE1 antigen short peptide to function, or to transduce the TCR to obtain a T cell specific for the SAGE1 antigen short peptide, thereby They play a role in cellular immunotherapy.
发明内容Summary of the invention
本发明的目的在于提供一种识别SAGE1抗原短肽的T细胞受体。It is an object of the present invention to provide a T cell receptor that recognizes a SAGE1 antigen short peptide.
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VFSTVPPAFI-HLA A2402复合物结合。In a first aspect of the invention, there is provided a T cell receptor (TCR) capable of binding to a VFSTVPPAFI-HLA A2402 complex.
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CAVLYTGANSKLTF(SEQ ID NO.12)；和/或所述TCRβ链可变域的CDR3的氨基酸序列为CASSLVGKQPQHF(SEQ ID NO.15)。In another preferred embodiment, 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 CAVLYTGANSKLTF (SEQ ID NO. 12); and/or The amino acid sequence of CDR3 of the TCR β chain variable domain is CASSLVGKQPQHF (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：In another preferred embodiment, the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:
α CDR1-DSAIYN(SEQ ID NO.10)α CDR1-DSAIYN (SEQ ID NO. 10)
α CDR2-IQSSQRE(SEQ ID NO.11)α CDR2-IQSSQRE (SEQ ID NO. 11)
α CDR3-CAVLYTGANSKLTF(SEQ ID NO.12)；和/或α CDR3-CAVLYTGANSKLTF (SEQ ID NO. 12); and/or
所述TCRβ链可变域的3个互补决定区为：The three complementarity determining regions of the TCR β chain variable domain are:
β CDR1-SGHDT(SEQ ID NO.13)β CDR1-SGHDT (SEQ ID NO. 13)
β CDR2-YYEEEE(SEQ ID NO.14)β CDR2-YYEEEE (SEQ ID NO. 14)
β CDR3-CASSLVGKQPQHF(SEQ ID NO.15)。β CDR3-CASSLVGKQPQHF (SEQ ID NO. 15).
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链可变域为与SEQ ID NO.1具有至少90％序列相同性的氨基酸序列；和/或所述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。In another preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID NO. Or the TCR β chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5.
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。 In another preferred embodiment, the TCR comprises an alpha chain variable domain amino acid sequence of SEQ ID NO.
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。In another preferred embodiment, the TCR comprises the β chain variable domain amino acid sequence of SEQ ID NO.
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。In another preferred embodiment, 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.
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。In another preferred embodiment, the amino acid sequence of the α chain of the TCR is SEQ ID NO: 3 and/or the β chain amino acid sequence of the TCR is SEQ ID NO.
在另一优选例中，所述TCR是可溶的。In another preferred embodiment, the TCR is soluble.
在另一优选例中，所述TCR为单链。In another preferred embodiment, the TCR is single stranded.
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。In another preferred embodiment, the TCR is formed by linking an alpha chain variable domain to a beta chain variable domain via a peptide linker sequence.
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***)中列出的位置编号。In another preferred embodiment, 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 β chain variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91 Or the 94th, and/or β 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).
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；In another preferred embodiment, 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;
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。And (a) and (b) each comprise a functional variable domain or comprise at least a portion of a functional variable domain and said TCR chain constant domain.
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。In another preferred embodiment, the cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.
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：In another preferred embodiment, the cysteine residue forming an artificial disulfide bond in the TCR replaces one or more sets of sites selected from the group consisting of:
TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；Thr48 of TRAC*01 exon 1 and Ser57 of TRBC1*01 or TRBC2*01 exon 1;
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；Thr45 of TRAC*01 exon 1 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01;
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；Tyr10 and TRBC1*01 of exon 1 of TRAC*01 or Ser17 of exon 1 of TRBC2*01;
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1;
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；Ser15 and TRBC1*01 of exon 1 of TRAC*01 or Glu15 of exon 1 of TRBC2*01;
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1;
TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；和Pro89 and TRBC1*01 of exon 1 of TRAC*01 or Ala19 of exon 1 of TRBC2*01;
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。Tyr10 and TRBC1*01 of exon 1 of TRAC*01 or Glu20 of exon 1 of TRBC2*01.
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。In another preferred embodiment, the amino acid sequence of the α chain of the TCR is SEQ ID NO. 26 and/or the β chain amino acid sequence of the TCR is SEQ ID NO.
在另一优选例中，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。In another preferred embodiment, an artificial interchain disulfide bond is contained between the alpha chain variable region of the TCR and the beta chain constant region.
在另一优选例中，其特征在于，在所述TCR中形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：In another preferred embodiment, the cysteine residue forming an artificial interchain disulfide bond in the TCR replaces one or more sets of sites selected from the group consisting of:
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；The amino acid at position 46 of TRAV and the amino acid at position 60 of exon 1 of TRBC1*01 or TRBC2*01;
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；The amino acid at position 47 of TRAV and the amino acid at position 61 of exon 1 of TRBC1*01 or TRBC2*01;
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The amino acid at position 46 of TRAV and the amino acid at position 61 of exon 1 of TRBC1*01 or TRBC2*01;
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。 The 47th amino acid of TRAV and the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01.
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。In another preferred embodiment, 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.
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。In another preferred embodiment, the C- or N-terminus of the alpha chain and/or beta strand of the TCR incorporates a conjugate.
在另一优选例中，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。In another preferred embodiment, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modified moiety, or a combination of any of these. Preferably, the therapeutic agent is an anti-CD3 antibody.
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。In a second aspect of the invention, there is provided 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.
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。In a third 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.
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。In another preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 2 encoding a TCR alpha chain variable domain.
在另一优选例中，所述的核酸分子包含编码TCRβ链可变域的核苷酸序列SEQ ID NO：6。In another preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 6 encoding a TCR β chain variable domain.
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。In another preferred embodiment, 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.
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。According to a fourth aspect of the invention, 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.
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。According to a fifth aspect of the invention, there is provided 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 .
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。According to a sixth aspect of the invention, 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 .
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、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。According to a seventh aspect of the present invention, a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a TCR according to the first aspect of the present invention, a TCR complex according to the second aspect of the present invention, and a present invention are provided. The nucleic acid molecule according to the third aspect of the invention, the vector according to the fourth aspect of the invention, or the cell of the sixth aspect of the invention.
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，用于制备***或自身免疫疾病的药物。According to an eighth 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 The use of the vector of the aspect, or the cell of the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumor or an autoimmune disease.
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、或本发明第七方面所述的药物组合物；According to a ninth aspect of the invention, a method of treating a disease comprising administering an appropriate amount of the T cell receptor of the first aspect of the invention, or the TCR complex of the second aspect of the invention to a subject in need of treatment The nucleic acid molecule of the third aspect of the invention, the vector of the fourth aspect of the invention, or the cell of the sixth aspect of the invention, or the pharmaceutical composition of the seventh aspect of the invention;
优选地，所述的疾病为肿瘤，优选地所述肿瘤包括黑色素瘤，以及其他肿 瘤如胃癌、肺癌(如，肺鳞状细胞癌)、食道癌、膀胱癌、头颈部肿瘤(如，头颈部鳞状细胞癌)、***癌、乳腺癌、结肠癌、卵巢癌、肾细胞癌、霍杰金氏淋巴瘤、肉瘤、成神经管细胞瘤、白血病等。Preferably, the disease is a tumor, preferably the tumor comprises melanoma, and other tumors Tumors such as gastric cancer, lung cancer (eg, lung squamous cell carcinoma), esophageal cancer, bladder cancer, head and neck cancer (eg, head and neck squamous cell carcinoma), prostate cancer, breast cancer, colon cancer, ovarian cancer, kidney Cell carcinoma, Hodgkin's lymphoma, sarcoma, medulloblastoma, leukemia, etc.
在另一优选例中，所述肿瘤包括黑色素瘤、膀胱癌、肝癌、表皮样癌、非小细胞肺癌和鳞状细胞癌。In another preferred embodiment, the tumor comprises melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, and squamous cell carcinoma.
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。It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.
附图说明DRAWINGS
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。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. The TCR alpha chain amino acid sequence of the leader sequence and the TCR alpha chain nucleotide sequence having the leader sequence.
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。2a, 2b, 2c, 2d, 2e, and 2f are a TCR β chain variable domain amino acid sequence, a TCR β chain variable domain nucleotide sequence, a TCR β chain amino acid sequence, a TCR β chain nucleotide sequence, respectively. The TCR β chain amino acid sequence of the leader sequence and the TCR β chain nucleotide sequence having the leader sequence.
图3为单克隆细胞的CD8+及四聚体-PE双阳性染色结果。Figure 3 shows the results of double positive staining of CD8 + and tetramer-PE in monoclonal cells.
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。Figures 4a and 4b are the amino acid sequence and nucleotide sequence of the soluble TCR alpha chain, respectively.
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。Figures 5a and 5b are the amino acid sequence and nucleotide sequence of the soluble TCR β chain, respectively.
图6为纯化后得到的可溶性TCR的胶图。最左侧泳道为还原胶，中间泳道为分子量标记(marker)，最右侧泳道为非还原胶。Figure 6 is a gel diagram of the soluble TCR obtained after purification. The leftmost lane is the reducing gel, the middle lane is the molecular weight marker, and the rightmost lane is the non-reducing gel.
图7为本发明可溶性TCR与VFSTVPPAFI-HLA A2402复合物结合的ProteOnFigure 7 is a ProteOn combining the soluble TCR with the VFSTVPPAFI-HLA A2402 complex of the present invention.
动力学图谱。Kinetic map.
图8为四聚体染色TCR转导的原代T细检测结果。Figure 8 shows the results of primary T-fine detection of tetrameric stained TCR transduction.
图9为ELISPOT试验检测结果。Figure 9 shows the results of the ELISPOT test.
图10本发明TCR转导的T细胞对特异性靶细胞的杀伤作用结果图。Figure 10 is a graph showing the killing effect of TCR transduced T cells of the present invention on specific target cells.
具体实施方式detailed description
本发明人经过广泛而深入的研究，找到了与SAGE1抗原短肽VFSTVPPAFI(SEQ ID NO.9)能够特异性结合的TCR，所述抗原短肽VFSTVPPAFI可与HLA A2402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。The inventors have found extensively and intensively, and found a TCR capable of specifically binding to the SAGE1 antigen short peptide VFSTVPPAFI (SEQ ID NO. 9), which can form a complex with HLA A2402 and be presented together. Cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention also provides cells that transduce the TCR of the invention.
术语the term
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。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***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。The T cell receptor (TCR) is the only receptor that presents a specific antigenic peptide on the major histocompatibility complex (MHC). In the immune system, the binding of antigen-specific TCR to the pMHC complex triggers direct physical contact between T cells and antigen presenting cells (APC), and then the other cell membrane surface molecules of both T cells and APC interact. This leads to a series of subsequent cell signaling and other physiological responses, allowing different antigen-specific T cells to exert an immune effect on their target cells.
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***(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。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. Natural αβ heterodimeric TCR has α chain and β chain, α chain and β The strand constitutes a subunit of the alpha beta heterodimeric TCR. Broadly speaking, each of the alpha and beta chains comprises a variable region, a junction region, and a constant region, and 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). For example, the constant domain sequence of the TCR molecule α chain is “TRAC*01”, and the constant domain sequence of the TCR molecule β chain is “TRBC1*”. 01" or "TRBC2*01". In addition, the alpha and beta chains of TCR also contain a transmembrane and cytoplasmic regions with a short cytoplasmic region.
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。In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention", and "T cell receptor of the present invention" are used interchangeably.
天然链间二硫键与人工链间二硫键Natural interchain disulfide bond and artificial interchain disulfide bond
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”。There is a set of disulfide bonds between the Cα and Cβ chains in the membrane proximal region of the native TCR, which is referred to herein as the "natural interchain disulfide bond". In the present invention, 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".
为方便描述二硫键的位置，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Pro60，也可将其表述为TRBC1*01或TRBC2*01外显子1的第60位氨基酸，又如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第61个氨基酸为Q(谷氨酰胺)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Gln61，也可将其表述为TRBC1*01或TRBC2*01外显子1的第61位氨基酸，其他以此类推。本发明中，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。本发明中，其他氨基酸的序列位置编号有特殊说明的，则按特殊说明。In order to facilitate the description of the position of the disulfide bond, in the present invention, the position numbers of the amino acid sequences of TROC*01 and TRBC1*01 or TRBC2*01 are numbered in order from N-terminus to C-terminus, such as TRBC1*01 or TRBC2*01. In the order from the N-terminus to the C-terminus, the 60th amino acid is P (valine), which may be described as Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, or may be It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and in the case of TRBC1*01 or TRBC2*01, the 61st amino acid is Q in the order from N to C. Amide), which may be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, or may be expressed as amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01, other And so on. In the present invention, 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. As for an amino acid in TRAV, the position number listed in IMGT is 46, which is described in the present invention as amino acid 46 of TRAV, and so on. In the present invention, if the sequence position numbers of other amino acids are specifically described, special instructions will be given.
发明详述Detailed description of the invention
TCR分子TCR molecule
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结合VFSTVPPAFI-HLA A2402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)。During antigen processing, the antigen is degraded within the cell and then carried to the cell surface by MHC molecules. The T cell receptor is capable of recognizing the peptide-MHC complex on the surface of the antigen presenting cell. Accordingly, a first aspect of the invention provides a TCR molecule capable of binding to a VFSTVPPAFI-HLA A2402 complex. Preferably, the TCR molecule is isolated or purified. The alpha and beta strands of the TCR each have three complementarity determining regions (CDRs).
在本发明的一个优选地实施方式中，所述TCR的α链包含具有以下氨基酸序列的CDR：In a preferred embodiment of the invention, the alpha chain of the TCR comprises a CDR having the following amino acid sequence:
α CDR1-DSAIYN(SEQ ID NO.10)α CDR1-DSAIYN (SEQ ID NO. 10)
α CDR2-IQSSQRE(SEQ ID NO.11)α CDR2-IQSSQRE (SEQ ID NO. 11)
α CDR3-CAVLYTGANSKLTF(SEQ ID NO.12)；和/或α CDR3-CAVLYTGANSKLTF (SEQ ID NO. 12); and/or
所述TCRβ链可变域的3个互补决定区为：The three complementarity determining regions of the TCR β chain variable domain are:
β CDR1-SGHDT(SEQ ID NO.13)β CDR1-SGHDT (SEQ ID NO. 13)
β CDR2-YYEEEE(SEQ ID NO.14)β CDR2-YYEEEE (SEQ ID NO. 14)
β CDR3-CASSLVGKQPQHF(SEQ ID NO.15)。 β CDR3-CASSLVGKQPQHF (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。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. As long as 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. Thus, 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 β chain variable domain of the invention is SEQ ID NO: 5 has an amino acid sequence of at least 90%, preferably 95%, more preferably 98% sequence identity.
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。In a preferred embodiment of the invention, the TCR molecule of the invention is a heterodimer composed of alpha and beta chains. Specifically, in one aspect, 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). Preferably, the TCR molecule comprises an alpha chain variable domain amino acid sequence of SEQ ID NO. More preferably, the alpha chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO. In another aspect, 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 CDR1 (SEQ ID NO. 13), CDR2 (SEQ ID) NO: 14) and CDR3 (SEQ ID NO. 15). Preferably, the TCR molecule comprises a beta chain variable domain amino acid sequence of SEQ ID NO. More preferably, the β chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO.
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。In a preferred embodiment of the invention, 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. Specifically, the single-chain TCR molecule comprises Vα, Vβ and Cβ, preferably linked in order from N-terminus to C-terminus.
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。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. Preferably, the single-chain TCR molecule comprises an alpha chain variable domain amino acid sequence of SEQ ID NO. More preferably, the alpha chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO. The β 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 β chain. Preferably, the single-chain TCR molecule comprises the β-chain variable domain amino acid sequence of SEQ ID NO. More preferably, the β chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO.
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***)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。In a preferred embodiment of the invention, the constant domain of the TCR molecule of the invention is a human constant domain. Those skilled in the art are aware of or can obtain a human constant domain amino acid sequence by consulting a related book or a public database of IMGT (International Immunogenetics Information System). For example, the constant domain sequence of the α chain of the TCR molecule of the present invention may be "TRAC*01", and the constant domain sequence of the β 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. Preferably, the amino acid sequence of the α chain of the TCR molecule of the present invention is SEQ ID NO. 3, and/or the amino acid sequence of the β chain is SEQ ID NO.
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SAGE1抗原短肽 具有特异性的可溶性TCR。The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane domain. Like immunoglobulins (antibodies) as antigen-recognizing molecules, 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. Similarly, soluble TCR can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells that present specific antigens. In addition, 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 SAGE1 antigen short peptide is also obtained by the present invention. Has a specific soluble 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。To obtain a soluble TCR, in one aspect, 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. That is, a cysteine residue replaces any of the above-mentioned sites in the α and β 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.
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。As described above, the TCR of the present invention may comprise an artificial disulfide bond introduced between residues of its α and β chain constant domains. It should be noted that 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.
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***(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***，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。In order to obtain a soluble TCR, in another aspect, 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 α-chain J gene (TRAJ) short peptide amino acid position reciprocal position 3, 5, 7 and/or β 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). Those skilled in the art are aware of the above-described international immunogenetic information system and can obtain the position number of the amino acid residues of different TCRs in the IMGT according to the database.
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。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 α and β chains of the TCR. It should be noted that the flexible peptide chain of the present invention may be any peptide chain suitable for linking the TCR alpha and beta chain variable domains.
另外，对于稳定性而言，专利文献PCT/CN2016/077680还公开了在TCR的α链可变区与β链恒定区之间引入人工链间二硫键能够使TCR的稳定性显著提高。因此，本发明的高亲和力TCR的α链可变区与β链恒定区之间还可以含有人工链间二硫键。具体地，在所述TCR的α链可变区与β链恒定区之间形成人工链间二硫键的半胱氨酸残基取代了：TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。优选地，这样的TCR可以包含(ⅰ)除其跨膜结构域以外的全部或部分TCRα链，和(ⅱ)除其跨膜结构域以外的全部或部分TCRβ链，其中(ⅰ)和(ⅱ)均包含TCR链的可变域和至少一部分 恒定域，α链与β链形成异质二聚体。更优选地，这样的TCR可以包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。In addition, for stability, the patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the alpha chain variable region of the TCR and the beta chain constant region can significantly improve the stability of the TCR. Therefore, the α chain variable region of the high affinity TCR of the present invention and the β chain constant region may further contain an artificial interchain disulfide bond. Specifically, a cysteine residue forming an artificial interchain disulfide bond between the α chain variable region of the TCR and the β chain constant region is substituted with: amino acid 46 of TRAV and TRBC1*01 or TRBC2* 01 amino acid at position 60 of exon 1; amino acid at position 47 of TRAV and amino acid at position 61 of exon 1 of TRBC1*01 or TRBC2*01; amino acid at position 46 of TRAV and TRBC1*01 or TRBC2*01 The amino acid at position 61 of the 1st; or the amino acid at position 47 of TRAV and the amino acid at position 60 of exon 1 of TRBC1*01 or TRBC2*01. Preferably, such a TCR may comprise (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 contain a variable domain of the TCR chain and at least a portion In the constant domain, the alpha chain forms a heterodimer with the beta chain. More preferably, such a TCR may comprise 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 alpha The chain variable domain forms a heterodimer with the beta chain.
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。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 complexes of the invention can be used to track or target cells that present a particular antigen in vitro or in vivo, as well as intermediates that produce other multivalent TCR complexes for such applications.
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VFSTVPPAFI-HLA A2402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。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 a VFSTVPPAFI-HLA A2402 complex), a therapeutic agent, a PK (protein kinase) modified moiety, or any of these The combination is combined or coupled.
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。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.
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。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. (Gillies et al., 1992, 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); (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 / nanometer Rod (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 Treatment (Ge Ne therapy) 11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nanomagnetic particles; 10. prodrug activating enzymes (eg, DT-diaphorase) (DTD) or biphenyl hydrolase-like protein (BPHL); 11. chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.
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。Additionally, the TCR of the invention may also be a hybrid TCR comprising sequences derived from more than one species. For example, studies have shown that murine TCR can be expressed more efficiently in human T cells than human TCR. Thus, 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.
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)。It should be understood that the 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).
核酸分子Nucleic acid molecule
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。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.
编码本发明第一方面TCR分子α链CDR区的核苷酸序列如下：The nucleotide sequence encoding the CDR region of the alpha chain of the TCR molecule of the first aspect of the invention is as follows:
α CDR1-
Figure PCTCN2017108081-appb-000001
(SEQ ID NO.16) α CDR1-
Figure PCTCN2017108081-appb-000001
(SEQ ID NO. 16)
α CDR2-
Figure PCTCN2017108081-appb-000002
(SEQ ID NO.17)α CDR2-
Figure PCTCN2017108081-appb-000002
(SEQ ID NO. 17)
α CDR3-
Figure PCTCN2017108081-appb-000003
(SEQ ID NO.18)α CDR3-
Figure PCTCN2017108081-appb-000003
(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：The nucleotide sequence encoding the CDR region of the β chain of the TCR molecule of the first aspect of the invention is as follows:
β CDR1-
Figure PCTCN2017108081-appb-000004
(SEQ ID NO.19)β CDR1-
Figure PCTCN2017108081-appb-000004
(SEQ ID NO. 19)
β CDR2-
Figure PCTCN2017108081-appb-000005
(SEQ ID NO.20)β CDR2-
Figure PCTCN2017108081-appb-000005
(SEQ ID NO. 20)
β CDR3-
Figure PCTCN2017108081-appb-000006
(SEQ ID NO.21)β CDR3-
Figure PCTCN2017108081-appb-000006
(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。Thus, the 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 β chain of the invention. The nucleotide sequence includes SEQ ID NO. 19, SEQ ID NO. 20, and SEQ ID NO.
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。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。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。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. Preferably, 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 the TCR beta chain variable domain of the invention comprises SEQ ID NO. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. It will be appreciated that due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Thus, a 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. As an example of the present 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.
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。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.
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。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.
载体Carrier
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。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. Commonly used vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.
病毒递送***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。Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.
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SAGE1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。Preferably, the vector can transfer a nucleotide of the invention into a cell, such as a T cell, such that the cell expresses a TAGE specific for the SAGE1 antigen. Ideally, the vector should be capable of sustained high levels of expression in T cells.
细胞cell
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。The invention also relates to host cells genetically engineered using the vectors or coding sequences of the invention. The host cell contains the vector of the present invention or a nucleic acid molecule of the present invention in which the chromosome is integrated. 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.
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。In addition, 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. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMC), which can be CD4 + helper T cells or CD8 + cytotoxic T cells. The cells can be in a mixed population of CD4 + helper T cells/CD8 + cytotoxic T cells. Generally, 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.
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。Alternatively, 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.
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)。There are a number of methods suitable for 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).
SAGE1抗原相关疾病SAGE1 antigen related diseases
本发明还涉及在受试者中治疗和/或预防与SAGE1相关疾病的方法，其包括过继性转移SAGE1特异性T细胞至该受试者的步骤。该SAGE1特异性T细胞可识别VFSTVPPAFI-HLA A2402复合物。The invention also relates to a method of treating and/or preventing a disease associated with SAGE1 in a subject, comprising the step of adoptively transferring SAGE1-specific T cells to the subject. The SAGE1-specific T cells recognize the VFSTVPPAFI-HLA A2402 complex.
本发明的SAGE1特异性的T细胞可用于治疗任何呈递SAGE1抗原短肽VFSTVPPAFI-HLA A2402复合物的SAGE1相关疾病。包括但不限于肿瘤，如黑色素瘤，以及其他实体肿瘤如胃癌、肺癌、食道癌、膀胱癌、头颈部鳞状细胞癌、***癌、乳腺癌、结肠癌、卵巢癌等。The SAGE1-specific T cells of the invention can be used to treat any SAGE1-related disease presenting the SAGE1 antigen short peptide VFSTVPPAFI-HLA A2402 complex. These include, but are not limited to, tumors such as melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, ovarian cancer, and the like.
治疗方法treatment method
可以通过分离患有与SAGE1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SAGE1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。The T cells of a patient or a volunteer having a disease associated with the SAGE1 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 for treatment. Accordingly, the present invention provides a method of treating a SAGE1-related disease comprising administering an isolated TCR expressing a TCR of the present invention, preferably, the T cell is derived from a patient itself and is administered to a patient. Generally, 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.
本发明的主要优点在于：The main advantages of the invention are:
(1)本发明的TCR能够与SAGE1抗原短肽复合物VFSTVPPAFI-HLA A2402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。(1) The TCR of the present invention can bind to the SAGE1 antigen short peptide complex VFSTVPPAFI-HLA A2402, and the cells transduced with the TCR of the present invention can be specifically activated and have a strong killing effect on target cells.
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。The invention is further illustrated by the following specific examples. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually published under conventional conditions, for example, (Sambrook and Russell et al., Molecular Cloning-A Laboratory Manual (Third Edition) (2001) CSHL Publishing The conditions stated in the company, or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Percentages and parts are by weight unless otherwise stated. The experimental materials and reagents used in the following examples are available from commercially available sources unless otherwise specified.
实施例1 克隆抗原短肽特异性T细胞Example 1 Cloning of antigen short peptide-specific T cells
利用合成短肽SAGE1PX149 597-606 VFSTVPPAFI(北京赛百盛基因技术有限公司)刺激来自于基因型为HLA-A2402的健康志愿者的外周血淋巴细胞(PBL)。将SAGE1 PX149 597-606 VFSTVPPAFI短肽与带有生物素标记的HLA-A*2402复性,制备pHLA单聚体。这些单聚体与用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培养。 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。Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A2402 were stimulated with the synthetic short peptide SAGE1PX149 597-606 VFSTVPPAFI (Beijing Cypress Biotech Co., Ltd.). The SAGE1 PX149 597-606 VFSTVPPAFI short peptide was renatured with biotinylated HLA-A*2402 to prepare a pHLA monomer. These moners were combined with PE-labeled streptavidin (BD) into PE-labeled tetramers, which were sorted and anti-CD8-APC double positive cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonal culture by limiting dilution. Monoclonal cells were stained with tetramers and the double positive clones screened are shown in Figure 3.
实施例2 获取本发明抗原短肽特异性T细胞克隆的TCR基因与载体的构建Example 2 Construction of TCR Gene and Vector for Obtaining Antigen Short Peptide-Specific T Cell Clone of the Present Invention
用Quick-RNATM MiniPrep(ZYMO research)抽提实施例1中筛选到的SAGE1 PX149 597-606VFSTVPPAFI特异性、HLA-A2402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。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和图1d分别为TCRα链可变域氨基酸序列、TCRα链可变域核苷酸序列、TCRα链氨基酸序列和TCRα链核苷酸序列；图2a、图2b、图2c和图2d分别为TCRβ链可变域氨基酸序列、TCRβ链可变域核苷酸序列、TCRβ链氨基酸序列和TCRβ链核苷酸序列。Extracted with Quick-RNA TM MiniPrep (ZYMO research ) screened in Example 1 to SAGE1 PX149 597-606VFSTVPPAFI specific, HLA-A2402 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. After sequencing, the sequence of the α chain and β chain of the TCR expressed by the double positive clone are shown in Fig. 1 and Fig. 2, respectively. Fig. 1a, Fig. 1b, Fig. 1c and Fig. 1d are the TCR α chain variable domain amino acid sequence and TCRα, respectively. Chain variable domain nucleotide sequence, TCR alpha chain amino acid sequence and TCR alpha chain nucleotide sequence; Fig. 2a, Fig. 2b, Fig. 2c and Fig. 2d are TCR β chain variable domain amino acid sequence, TCR β chain variable domain nucleotide, respectively Sequence, TCR β chain amino acid sequence and TCR β chain nucleotide sequence.
经鉴定，α链包含具有以下氨基酸序列的CDR：The alpha chain has been identified to comprise a CDR having the following amino acid sequence:
α CDR1-DSAIYN(SEQ ID NO.10)α CDR1-DSAIYN (SEQ ID NO. 10)
α CDR2-IQSSQRE(SEQ ID NO.11)α CDR2-IQSSQRE (SEQ ID NO. 11)
α CDR3-CAVLYTGANSKLTF(SEQ ID NO.12)α CDR3-CAVLYTGANSKLTF (SEQ ID NO. 12)
β链包含具有以下氨基酸序列的CDR：The beta strand comprises a CDR having the following amino acid sequence:
β CDR1-SGHDT(SEQ ID NO.13)β CDR1-SGHDT (SEQ ID NO. 13)
β CDR2-YYEEEE(SEQ ID NO.14)β CDR2-YYEEEE (SEQ ID NO. 14)
β CDR3-CASSLVGKQPQHF(SEQ ID NO.15)。β CDR3-CASSLVGKQPQHF (SEQ ID NO. 15).
通过重叠(overlap)PCR分别将TCRα链和β链的可变域各自与小鼠TCRα链和β链的保守域拼接成全长基因并连接至慢病毒表达载体pLenti(addgene)。具体为：用overlap PCR将TCRα链和TCRβ链的全长基因进行连接得到TCRα-2A-TCRβ片段。将慢病毒表达载体及TCRα-2A-TCRβ酶切连接得到pLenti-SAGE1TRA-2A-TRB-IRES-NGFR质粒。作为对照用,同时也构建表达eGFP的慢病毒载体pLenti-eGFP。之后再用293T/17包装假病毒。The variable domains of the TCR alpha chain and the beta chain are each spliced into the full length gene and linked to the lentiviral expression vector pLenti (addgene) by overlap PCR with the conserved domains of the mouse TCR alpha chain and the beta chain, respectively. Specifically, the TCRα-2A-TCRβ fragment was obtained by ligating the full-length genes of the TCR α chain and the TCR β chain by overlap PCR. The lentiviral expression vector and TCRα-2A-TCRβ were digested to obtain the pLenti-SAGE1TRA-2A-TRB-IRES-NGFR plasmid. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. The 1981T/17 is then used to package the pseudovirus.
实施例3 本发明抗原短肽特异性可溶TCR的表达、重折叠和纯化Example 3 Expression, Refolding and Purification of Specific Soluble TCRs of Antigen Short Peptides of the Invention
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和加下划线字母表示。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***到表达载体pET28a+(Novagene)，上下游的克隆位点分别是NcoI和NotI。***片段经过测序确认无误。In order to obtain a soluble TCR molecule, the α and β chains of the TCR molecule of the present invention may comprise only their variable domains and partial constant domains, respectively, and a cysteine residue is introduced in the constant domains of the α and β chains, respectively. To form an artificial interchain disulfide bond, the positions at which cysteine residues are introduced are Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC2*01, respectively; amino acid sequence and nucleotide of α chain thereof The sequences are shown in Figures 4a and 4b, respectively, and the amino acid sequence and nucleotide sequence of the β chain are shown in Figures 5a and 5b, respectively, and the introduced cysteine residues are indicated by bold and underlined letters. The above TCRα and β 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.
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),20mMTris(pH 8.1)中。The TCRα and β chain expression vectors were transformed into the expression bacterium BL21 (DE3) by chemical transformation, respectively, and the bacteria were grown in LB medium. The OD600=0.6 was induced with a final concentration of 0.5 mM IPTG, and the TCR α and β chains were expressed. The formed 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). , 20 mM Tris (pH 8.1).
溶解后的TCR α和β链以1：1的质量比快速混合于5M尿素,0.4M精氨酸,20mMTris(pH 8.1),3.7mMcystamine,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(20mMTris,pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP,5ml,GE Healthcare)纯化。洗脱峰含有复性成功的α和β二聚体的TCR通过SDS-PAGE胶确认。TCR随后通过凝胶过滤层析(HiPrep16/60，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。The dissolved TCR α and β chains were rapidly mixed in a mass ratio of 1:1 in 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, 6.6 mM β-mercapoethylamine (4 ° C), and the final concentration was 60 mg. /mL. After mixing, the solution was placed in 10 volumes of deionized water. After analysis (4 ° C), 12 hours later, the deionized water was replaced with a buffer (20 mM Tris, pH 8.0) and dialysis was continued at 4 ° C for 12 hours. The solution after completion of dialysis was filtered through a 0.45 μM filter, and then purified by 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.
实施例4 结合表征Example 4 Binding Characterization
BIAcore分析BIAcore analysis
本实施例证明了可溶性的本发明TCR分子能够与VFSTVPPAFI-HLAA2402复合物特异性结合。This example demonstrates that soluble TCR molecules of the invention are capable of specifically binding to the VFSTVPPAFI-HLAA2402 complex.
使用BIAcore T200实时分析***检测实施例3中得到的TCR分子与VFSTVPPAFI-HLA A2402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。The binding activity of the TCR molecule obtained in Example 3 to the VFSTVPPAFI-HLA A2402 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.
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VFSTVPPAFI-HLA A2402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。A low concentration of streptavidin is passed over the surface of the coated antibody chip, then the VFSTVPPAFI-HLA A2402 complex is flowed through the detection channel, and the other channel is used as a reference channel, and then 0.05 mM biotin is 10 μL/min. The flow rate was passed through the chip for 2 min, blocking the remaining binding sites of streptavidin.
上述VFSTVPPAFI-HLA A2402复合物的制备过程如下：The preparation process of the above VFSTVPPAFI-HLA A2402 composite is as follows:
a.纯化Purification
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。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.
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 20mMTris-HCl pH 8.0重悬包涵体，混匀，4℃6000g离心15min，最后用20mMTris-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。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. , mix, centrifuge at 6000g for 15min at 4°C for 15min, repeat twice, add 30ml 20mM Tris-HCl pH 8.0, resuspend the inclusion body, mix, centrifuge at 6000g for 15min at 4°C, finally dissolve the inclusion body with 20mM Tris-HCl 8M urea, detect by SDS-PAGE Inclusion body purity, BCA kit measured concentration.
b.复性b. renaturation
将合成的短肽VFSTVPPAFI(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、20mMTris 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VFSTVPPAFI肽以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mMTris 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。The synthesized short peptide VFSTVPPAFI (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 the addition of 3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA before renaturation. The VFSTVPPAFI peptide was added to the refolding buffer at 25 mg/L (final concentration) (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 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 to Upon completion, SDS-PAGE can detect whether the renaturation is successful.
c.复性后纯化c. renaturation and purification
用10体积的20mMTris 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)，20mMTris pH 8.0配制 的0-400mMNaCl线性梯度液洗脱蛋白，pMHC约在250mMNaCl处洗脱，收集诸峰组分，SDS-PAGE检测纯度。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. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE General Electric Company) anion exchange column (5 ml bed volume). Formulated with Akta Purifier (GE General Electric), 20 mM Tris pH 8.0 The protein was eluted with a linear gradient of 0-400 mM NaCl. The pMHC was eluted at approximately 250 mM NaCl. The peak fractions were collected and the purity was determined by SDS-PAGE.
d.生物素化d. Biotinylation
用Mill ipore超滤管将纯化的pMHC分子浓缩，同时将缓冲液置换为20mMTris pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mMMgOAc、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。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 MgAc, 50 μM D-Biotin, 100 μg/ml BirA enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and biotinylation was detected by SDS-PAGE.
e.纯化生物素化后的复合物e. Purification of the biotinylated complex
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℃。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).
利用BIAcore Evaluation软件计算动力学参数，得到本发明可溶性的TCR分子与复合物结合的动力学图谱如图7所示。The kinetic parameters of the soluble TCR molecules of the present invention were determined by using the BIAcore Evaluation software to calculate the kinetic parameters as shown in FIG.
实施例5 T细胞受体慢病毒包装与原代T细胞转染SAGE1TCRExample 5 T cell receptor lentiviral packaging and primary T cell transfection of SAGE1 TCR
(a)通过293T/17细胞的快速介导瞬时转染(Express-In-mediated transient transfection)制备慢病毒(a) Preparation of lentivirus by fast-mediated transient transfection of 293T/17 cells
利用第三代慢病毒包装***包装含有编码所需TCR的基因的慢病毒。利用快速介导瞬时转染(Express-In-mediated transient transfection)(开放生物***公司(Open Biosystems))用4种质粒(含有实施例2所述pLenti-SAGE1TRA-2A-TRB-IRES-NGFR的一种慢病毒载体，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)转染293T/17细胞。Lentiviruses containing the gene encoding the desired TCR were packaged using a third generation lentiviral packaging system. Four plasmids (containing one of pLenti-SAGE1 TRA-2A-TRB-IRES-NGFR described in Example 2) using Express-In-mediated transient transfection (Open Biosystems) A lentiviral vector, as well as three plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles, were transfected into 293T/17 cells.
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/17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pLenti-SAGE1TRA-2A-TRB-IRES-NGFR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/17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，等份分装后-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。 For transfection, cells were seeded on day 0, and 1.7×10 7 293T/17 cells were seeded in a 15 cm culture dish to evenly distribute the cells on the culture dish at a confluency of slightly more than 50%. The plasmid was transfected on day 1, and the pLenti-SAGE1TRA-2A-TRB-IRES-NGFR and pLenti-eGFP pseudoviruses were packaged, and the above expression plasmid was mixed with the packaging plasmids pMDLg/pRRE, pRSV-REV and pMD.2G, one 15 cm. The amount of the diameter plate is as follows: 22.5 micrograms: 15 micrograms: 15 micrograms: 7.5 micrograms. 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 μl 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 μl 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 μl of OPTI was added. -MEM medium, mix well, add to 293T/17 cells that have been converted to 11.25 ml of OPTI-MEM, gently shake the dish, mix the medium evenly, and incubate at 37 ° C / 5% CO 2 . After -7 hours, the transfection medium was removed and replaced with DMEM (Gibco, catalog number C11995500bt) containing 10% fetal bovine serum, and cultured at 37 ° C / 5% CO 2 . The medium supernatant containing the packaged lentivirus was collected on day 3 and day 4. To harvest the packaged lentivirus, 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 (Merckmi Merck Millipore, catalog number SLGP033RB), finally intercepted with 50KD The concentrated tube (Merck Millipore, catalog number UFC905096) was concentrated to remove most of the supernatant, finally concentrated to 1 ml, and aliquoted and stored at -80 ° C. The pseudovirus sample was taken for virus. The titer was measured by the p24 ELISA (Clontech, Cat. No. 632200) kit instructions. As a control, the pseudovirus of pLenti-eGFP was also included.
(b)用含有SAGE1特异性T细胞受体基因的慢病毒转导原代T细胞(b) Transduction of primary T cells with a lentivirus containing a SAGE1-specific T cell receptor gene
从健康志愿者的血液中分离到CD8+T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50IU/ml IL-2和10ng/ml IL-7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，lifetechnologies，目录号11452D)共孵育过夜刺激，细胞：珠＝3：1。CD8 + T cells were isolated from the blood of healthy volunteers and transduced with packaged lentivirus. These cells were counted in a 40-well plate at 1640 (Gibco) containing 10% FBS (Gibco, Cat. No. C10010500BT) containing 50 IU/ml IL-2 and 10 ng/ml IL-7. (Gibco), catalog number C11875500bt) medium at 1 x 10 6 cells/ml (0.5 ml/well) with pre-washed anti-CD3/CD28 antibody-coated beads (T cell amplicon, lifetechnologies, catalog No. 11452D) was incubated overnight for stimulation, cells: beads = 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SAGE1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，用加入50IU/ml IL-2和10ng/ml IL-7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和10ng/ml IL-7的新鲜培养基，维持细胞在0.5×106-1×106个细胞/毫升。从第3天开始通过流式细胞术分析细胞，从第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)。从第10天开始或在细胞减缓***和尺寸变小之时，冷冻储存等分细胞，至少4×106个细胞/管(1×107个细胞/毫升，90％FBS/10％DMSO)。After overnight stimulation, the lentivirus of the concentrated SAGE1-specific T cell receptor gene was added at a ratio of MOI=10 according to the virus titer measured by the p24 ELISA kit, and the infection was centrifuged at 900 g for 1 hour at 32 ° C. After the infection was completed, the lentiviral infection solution was removed, and the cells were resuspended in 1640 medium containing 10% FBS supplemented with 50 IU/ml IL-2 and 10 ng/ml IL-7, and cultured at 37 ° C / 5% CO 2 for 3 days. After 3 days of transduction, the cells were counted and the cells were diluted to 0.5 x 10 6 cells/ml. The cells were counted every two days, and fresh medium containing 50 IU/ml IL-2 and 10 ng/ml IL-7 was replaced or added to maintain the cells at 0.5 x 10 6 - 1 x 10 6 cells/ml. Cells were analyzed by flow cytometry starting on day 3 and were used for functional assays from day 5 (eg, ELISPOT and non-radioactive cytotoxicity assays for IFN-γ release). Store frozen aliquots from day 10 or at least 4 x 10 6 cells/tube (1 x 10 7 cells/ml, 90% FBS/10% DMSO) at the time the cells are slowed down and the size is reduced. .
(c)四聚体染色TCR转导的原代T细胞(c) tetramer-stained TCR-transduced primary T cells
SAGE1PX149 597-606 VFSTVPPAFI短肽与带有生物素标记的HLA-A*2402复性，制备pHLA单聚体。这些单聚体用PE标记的链霉亲和素(BD)组合成PE标记的四聚体,称为PX149-tetramer-PE。此四聚体能把表达了SAGE1特异性T细胞受体基因的T细胞标记为阳性细胞。把(b)中经转导的T细胞样品与PX149-tetramer-PE混合在冰上孵育30分钟，然后加入抗小鼠β链-APC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BD Calibur或BD Arial检测或分选表达了SAGE1特异性T细胞受体基因的PX149-tetramer-PE和抗小鼠β链-APC双阳性的T细胞，数据分析采用CellQuest软件(BD)或者FlowJo软件(Tree Star Inc,Ashland,OR)分析。SAGE1PX149 597-606 VFSTVPPAFI short peptide was renatured with biotinylated HLA-A*2402 to prepare pHLA monomer. These monomers are combined with PE-labeled streptavidin (BD) into a PE-labeled tetramer called PX149-tetramer-PE. This tetramer can label T cells expressing a SAGE1-specific T cell receptor gene as positive cells. The transduced T cell samples in (b) were incubated with PX149-tetramer-PE for 30 minutes on ice, then anti-mouse β-chain-APC antibody was added and incubation was continued for 15 minutes on ice. The sample was washed twice with PBS containing 2% FBS, and then BD Calibur or BD Arial was used to detect or sort PX149-tetramer-PE expressing the SAGE1-specific T cell receptor gene and anti-mouse β-chain-APC double positive T Cells were analyzed by CellQuest software (BD) or FlowJo software (Tree Star Inc, Ashland, OR).
经检测分析，结果如图8所示，用PX149-tetramer-PE和抗小鼠β链-APC抗体染色后，未经TCR慢病毒感染的空白对照组T细胞无PX149-tetramer-PE和抗小鼠β链-APC双阳性细胞，而经TCR慢病毒感染的T细胞出现表达TCR的PX149-tetramer-PE和抗小鼠β链-APC双阳性细胞，当用非PX149-tetramer-PE的其他tetramer-PE染色时只有极少量非特异性的双阳性细胞。After analysis and analysis, the results are shown in Figure 8. After staining with PX149-tetramer-PE and anti-mouse β-chain-APC antibody, T control cells without TCR lentivirus infection had no PX149-tetramer-PE and anti-small. Rat β-chain-APC double positive cells, while TCR-infected T cells showed TCR-expressing PX149-tetramer-PE and anti-mouse β-chain-APC double positive cells when using other tetramers other than PX149-tetramer-PE - There are only a very small number of non-specific double positive cells in PE staining.
实施例6 转导本发明TCR的效应细胞的激活实验Example 6 Activation experiment of transducing effector cells of the TCR of the present invention
ELISPOT方案ELISPOT solution
进行以下试验以证明TCR-转导的T细胞对靶细胞特异性地起反应的激活。利用ELISPOT试验检测的IFN-γ产量作为T细胞激活的读出值。The following assays were performed to demonstrate activation of TCR-transduced T cells specifically responding to target cells. The IFN-γ production detected by the ELISPOT assay was used as a readout value for T cell activation.
试剂Reagent
试验培养基：10％FBS(吉布可公司(Gibco)，目录号16000-044)，RPMI1640(吉布可公司(Gibco)，目录号C11875500bt)Test medium: 10% FBS (Gibco, catalog number 16000-044), RPMI 1640 (Gibco, catalog number C11875500bt)
洗涤缓冲液：0.01M PBS/0.05％吐温20Wash buffer: 0.01M PBS / 0.05% Tween 20
PBS(吉布可公司(Gibco)，目录号C10010500BT)PBS (Gibco, catalog number C10010500BT)
PVDF ELISPOT 96孔板(默克密理博(Merck Millipore)，目录号MSIPS4510)PVDF ELISPOT 96-well plate (Merck Millipore, catalog number MSIPS4510)
人IFN-γ ELISPOT PVDF-酶试剂盒(BD)装有所需的所有其他试剂(捕捉和检测抗体，链霉亲和素-碱性磷酸酶和BCIP/NBT溶液)The human IFN-γ ELISPOT PVDF-Enzyme Kit (BD) contains all the other reagents required (capture and detection antibodies, streptavidin-alkaline phosphatase and BCIP/NBT solutions)
方法method
靶细胞制备 Target cell preparation
本实施例的靶细胞为Epstein-Barr病毒(EBV)转化的永生化淋巴母细胞系(LCLs)。B95-8细胞经十四酰乙酸佛波醇酯(TPA)诱导生产含有EBV的培养基上清，4℃/600g离心10分钟去除杂质，然后过0.22微米过滤器，等分分装-70℃保存。从基因型为HLA-A11/A02/A24(包括纯合子和杂合子)的健康志愿者的外周血淋巴细胞(PBL),取10毫升浓度为2×107/毫升的PBL悬浮液于25平方厘米的培养瓶中，加入环孢霉素后在37℃/CO2培养箱中孵育1小时，快速解冻一份EBV，按1/10稀释加入到上述细胞中，轻轻摇匀并把培养瓶直立置于37℃/CO2培养箱中培养。培养12天后添加10毫升培养基继续培养，约30天后进一步扩大培养并进行流式检测，其中CD19+CD23hiCD58+为永生化淋巴母细胞系(LCLs)。本ELISPOT试验以HLA-A24为特异性靶细胞。The target cells of this example are Epstein-Barr virus (EBV) transformed immortalized lymphoblastoid cell lines (LCLs). B95-8 cells were induced to produce EBV-containing medium supernatant by tetradecanoyl phorbol ester (TPA), centrifuged at 4 ° C / 600 g for 10 minutes to remove impurities, and then passed through a 0.22 micron filter, aliquoted -70 ° C save. From peripheral blood lymphocytes (PBL) of healthy volunteers with genotype HLA-A11/A02/A24 (including homozygotes and heterozygotes), take 10 ml of PBL suspension at a concentration of 2 × 10 7 /ml at 25 square In a centimeter flask, add cyclosporine and incubate in a 37 ° C / CO 2 incubator for 1 hour, quickly thaw a portion of EBV, add 1/10 dilution to the above cells, gently shake and shake the flask The cells were erected in a 37 ° C / CO 2 incubator. After 12 days of culture, 10 ml of the medium was added to continue the culture, and after about 30 days, the culture was further expanded and subjected to flow detection, wherein CD19 + CD23 hi CD58 + was an immortalized lymphoblastic cell line (LCLs). This ELISPOT assay uses HLA-A24 as a specific target cell.
效应细胞制备Effector cell preparation
本试验的效应细胞(T细胞)是实施例3中经流式细胞术分析表达SAGE1特异性TCR的CD8+T细胞，并以同一志愿者的CD8+T作为阴性对照效应细胞。用抗CD3/CD28包被珠(T细胞扩增物，LifeTechnologies)刺激T细胞，用携带SAGE1特异性TCR基因的慢病毒转导(依据实施例3)，在含有50IU/ml IL-2和10ng/ml IL-7的含10％FBS的1640培养基扩增直至转导后9-12天，然后将这些细胞置于试验培养基中，300g常温离心10分钟进行洗涤。然后将细胞以2×所需终浓度重悬在试验培养基中。同样处理阴性对照效应细胞。The effector cells (T cells) of the present assay were CD8 + T cells expressing SAGE1-specific TCR by flow cytometry in Example 3, and CD8 + T of the same volunteer was used as a negative control effector cell. T cells were stimulated with anti-CD3/CD28 coated beads (T cell amplicon, LifeTechnologies), transduced with lentivirus carrying the SAGE1-specific TCR gene (according to Example 3), containing 50 IU/ml IL-2 and 10 ng /ml IL-7 in 1040 medium containing 10% FBS was amplified until 9-12 days after transduction, and then these cells were placed in a test medium, and washed by centrifugation at 300 g for 10 minutes at room temperature. The cells were then resuspended in test medium at 2 x the desired final concentration. Negative control effector cells were also treated.
ELISPOTELISPOT
按照生产商提供的说明书，如下所述准备孔板：以每块板10毫升无菌PBS按1：200稀释抗人IFN-γ捕捉抗体，然后将100微升的稀释捕捉抗体等分加入各孔。4℃下孵育孔板过夜。孵育后，洗涤孔板以除去多余的捕捉抗体。加入100微升/孔含有10％FBS的RPMI 1640培养基并在室温下温育孔板2小时以封闭孔板。然后从孔板中洗去培养基，通过在纸上轻弹和轻拍ELISPOT孔板以除去任何残余的洗涤缓冲液。Prepare wells as follows, following the manufacturer's instructions: Dilute anti-human IFN-γ capture antibody 1:200 in 10 ml sterile PBS per plate, then add 100 μl of diluted capture antibody aliquot to each well. . The plates were incubated overnight at 4 °C. After incubation, the well plates were washed to remove excess capture antibody. 100 microliters/well of RPMI 1640 medium containing 10% FBS was added and the well plates were incubated for 2 hours at room temperature to close the well plates. The medium is then washed from the well plate and any residual wash buffer is removed by flicking and tapping the ELISPOT plate on the paper.
SAGE1CD8+T细胞(SAGE1TCR转导的T细胞，效应细胞VF3CD8+T细胞)、CD8+T细胞(阴性对照效应细胞)和LCL-A24/A02(靶细胞)依据实施例3所述制备，并在相应实验组加入对应短肽，其中PX149为SAGE1PX149597-606VFSTVPPAFI短肽，PA11、PA02、PA24-1、PA24-2和PA24-3为非SAGE1TCR特异结合短肽。SAGE1CD8 + T cells (SAGE1 TCR transduced T cells, effector cells VF3CD8+ T cells), CD8+ T cells (negative control effector cells) and LCL-A24/A02 (target cells) were prepared as described in Example 3, and The corresponding experimental group was added with the corresponding short peptide, wherein PX149 was SAGE1PX149597-606VFSTVPPAFI short peptide, and PA11, PA02, PA24-1, PA24-2 and PA24-3 were non-SAGE1 TCR specific binding short peptides.
然后采用以下顺序将试验的诸组分加入ELISPOT孔板：The components of the test were then added to the ELISPOT well plate in the following order:
130微升靶细胞77000个细胞/毫升(得到总共约10000个靶细胞/孔)。130 microliters of target cells were 77,000 cells/ml (a total of approximately 10,000 target cells/well were obtained).
50微升效应细胞(1000个SAGE1TCR双阳性T细胞)。50 microliters of effector cells (1000 SAGE1 TCR double positive T cells).
20微升10-5摩尔/升的SAGE1PX149 597-606VFSTVPPAFI/非特异性短肽溶液(终浓度为10-6摩尔/升)。20 μl of 10 -5 mol/L SAGE1 PX149 597-606 VFSTVPPAFI/non-specific short peptide solution (final concentration 10-6 mol/L).
所有孔一式三份制备添加。All wells were prepared in triplicate.
然后温育孔板过夜(37℃/5％CO2)第二天，弃培养基，用双蒸水洗涤孔板2次，再用洗涤缓冲液洗涤3次，在纸巾上轻拍以除去残余的洗涤缓冲液。然后用含有10％FBS的PBS稀释检测一抗，按100微升/孔加入各孔。室温下温育孔板2小时，再用洗涤缓冲液洗涤3次，在纸巾上轻拍孔板以除去过量的洗涤缓冲液。The plates were then incubated overnight (37 ° C / 5% CO 2 ) for the next day, the medium was discarded, the plates were washed twice with double distilled water, washed 3 times with wash buffer, and tapped on a paper towel to remove residuals. Wash buffer. The primary antibody was then diluted with PBS containing 10% FBS and added to each well at 100 μL/well. The well plates were incubated for 2 hours at room temperature and washed 3 times with wash buffer and the well plates were tapped on paper towels to remove excess wash buffer.
用含有10％FBS的PBS按1：100稀释链霉亲和素-碱性磷酸酶，将100微升稀释的链霉亲和素-碱性磷酸酶加入各孔并在室温下温育孔板1小时。然后用洗涤缓冲液洗涤3次PBS洗涤2次，在纸巾上轻拍孔板以除去过量的洗涤缓冲液和PBS。洗涤完毕后加入试剂盒提供的BCIP/NBT溶液100微升/孔进行显影。在显影期间用锡箔纸覆盖孔板避光，静置5-15分钟。在此期间常规检测显影孔板的斑点，确定终止反应的最佳时间。去除BCIP/NBT溶液并用双蒸水 冲洗孔板以中止显影反应，甩干，然后将孔板底部去除，在室温下干燥孔板直至每个孔完全干燥，再利用免疫斑点平板计数计(CTL，细胞技术有限公司(Cellular Technology Limited))计数孔板内底膜形成的斑点。Add 100 μl of diluted streptavidin-alkaline phosphatase to each well with 1:100 dilution of streptavidin-alkaline phosphatase in PBS containing 10% FBS and incubate the wells at room temperature 1 hour. The PBS was then washed twice with wash buffer 3 times and the well plate was tapped on a paper towel to remove excess wash buffer and PBS. After the washing, 100 μl/well of the BCIP/NBT solution supplied from the kit was added for development. The plate was covered with tin foil during development to protect it from light and allowed to stand for 5-15 minutes. During this time, the spots of the developing well plates were routinely examined to determine the optimal time to terminate the reaction. Remove BCIP/NBT solution and use double distilled water Rinse the plate to stop the development reaction, spin dry, then remove the bottom of the plate, dry the plate at room temperature until each well is completely dry, and then use the immunospot plate counter (CTL, Cellular Technology Limited) ) Counting the spots formed by the base film in the orifice plate.
结果result
通过ELISPOT试验(如上所述)检验本发明TCR转导的T细胞对负载SAGE1PX149 597-606VFSTVPPAFI短肽的靶细胞和非特异性短肽的靶细胞起反应的IFN-γ释放。利用graphpad prism6绘制各孔中观察到的ELSPOT斑点数量。The TCR-transduced T cells of the present invention were tested for IFN-γ release in response to target cells bearing the SAGE1 PX149 597-606 VFSTVPPAFI short peptide and target cells of the non-specific short peptide by the ELISPOT assay (described above). The number of ELSPOT spots observed in each well was plotted using graphpad prism6.
实验结果如图9所示，转导本发明TCR的CD8+T细胞对负载相关短肽的LCLs有很强的激活作用，释放出较多IFN-γ，而对负载非特异短肽的LCLs基本没有反应；同时，未转导本发明TCR的CD8+T细胞对负载相关短肽的LCLs反应很小，仅有少量IFN-γ释放。The experimental results are shown in Fig. 9. The CD8 + T cells transducing the TCR of the present invention have a strong activation effect on the LCLs of the load-related short peptides, releasing more IFN-γ, and the LCLs supporting the non-specific short peptides are basically There was no response; at the same time, CD8 + T cells that did not transduce the TCR of the present invention responded very little to LCLs loaded with short peptides, with only a small amount of IFN-γ released.
实施例7 非放射性细胞毒性试验方案Example 7 Non-radioactive cytotoxicity test protocol
该试验是51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的乳酸脱氢酶(LDH)。采用30分钟偶联的酶反应来检测释放在培养基中的LDH，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。可以用标准的96孔读板计收集490nm可见光吸光值数据。This test is a colorimetric substitution test for the 51Cr release cytotoxicity assay to quantify the lactate dehydrogenase (LDH) released after cell lysis. The LDH released in the medium was detected using a 30 minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is directly proportional to the number of cells lysed. The 490 nm visible light absorbance data can be collected using a standard 96-well plate reader.
材料material
CytoTox
Figure PCTCN2017108081-appb-000007
非放射性细胞毒性检测(普罗迈格公司，G1780)含有底物混合物、试验缓冲液、裂解溶液和终止缓冲液。CytoTox
Figure PCTCN2017108081-appb-000007
Non-radioactive cytotoxicity assays (Promega, G1780) contain a substrate mixture, assay buffer, lysis solution, and stop buffer.
试验培养基：10％FBS(热灭活的，吉布可公司，目录号16000-044)，含酚红的90％RPMI 1640(吉布可公司(Gibco)，目录号C11875500bt)，1％青霉素/链霉素(吉布可公司，目录号15070-063)。Test medium: 10% FBS (heat inactivated, Gibbco, catalog number 16000-044), phenol red containing 90% RPMI 1640 (Gibco, catalog number C11875500bt), 1% penicillin / Streptomycin (Jibuco, catalog number 15070-063).
微孔圆底96孔组织培养板(纽克公司(Nunc)，目录号163320)Microporous round bottom 96-well tissue culture plate (Nunc, catalog number 163320)
96孔免疫平板Maxisorb(纽克公司(Nunc)，目录号442404)96-well immunization plate Maxisorb (Nunc, catalog number 442404)
方法method
靶细胞制备Target cell preparation
本试验采用K562-A24，NCI-H1299-A24和IM 9三株肿瘤细胞系为靶细胞。在试验培养基中制备靶细胞：靶细胞浓度调至334个/毫升，每孔取45微升从而得1.5×104个细胞/孔。In this experiment, three tumor cell lines, K562-A24, NCI-H1299-A24 and IM9, were used as target cells. Target cells were prepared in the test medium: the target cell concentration was adjusted to 334 cells/ml, and 45 microliters per well was taken to obtain 1.5 x 10 4 cells/well.
效应细胞制备Effector cell preparation
本试验的效应细胞(T细胞)是实施例3中经流式细胞术分析表达SAGE1特异性TCR的CD8+T细胞。效应细胞与靶细胞之比采用10：1.、5:1、2.5:1、1.25:1和0.625:1。设同源CD8+T细胞加靶细胞对照组(10:1)。The effector cells (T cells) of this assay were analyzed by flow cytometry in Example 3 to express CD8 + T cells expressing SAGE1-specific TCR. The ratio of effector cells to target cells was 10:1, 5:1, 2.5:1, 1.25:1, and 0.625:1. A homologous CD8 + T cell plus target cell control group (10:1) was set.
SAGE1特异性TCR转导的CD8+T细胞特异性杀伤肿瘤细胞试验SAGE1-specific TCR-transduced CD8 + T cell-specific killing tumor cell assay
试验准备Test preparation
采用以下顺序将试验的诸组分加入微孔圆底96孔组织培养板：The components of the assay were added to a microwell round bottom 96-well tissue culture plate in the following sequence:
-45ul靶细胞(如上所述制备)加入各孔-45 ul of target cells (prepared as described above) were added to each well
-45ul效应细胞(如上所述制备)加入各孔-45 ul of effector cells (prepared as described above) were added to each well
如下所述制备对照组：A control group was prepared as follows:
-效应细胞自发释放：仅有45ul效应细胞。- Spontaneous release of effector cells: only 45 ul of effector cells.
-靶细胞自发释放：仅有45ul靶细胞。- Spontaneous release of target cells: only 45 ul of target cells.
-靶细胞最大释放：仅有45ul靶细胞。- Maximum release of target cells: only 45 ul of target cells.
-培养基对照：仅有90ul培养基。- Medium control: only 90 ul of medium.
所有孔一式三份制备，终体积为90ul(不够的用培养基补足)。All wells were prepared in triplicate with a final volume of 90 ul (not enough to make up with medium).
37℃温育24小时。收集所有孔上清前，将靶细胞最大释放对照孔在-70℃ 放置细胞大约30分钟，再在37℃融化15分钟，以使靶细胞全部裂解。Incubate at 37 ° C for 24 hours. Collect all wells before clearing the supernatant and release the target cells to the maximum at -70 °C The cells were allowed to stand for about 30 minutes and then thawed at 37 ° C for 15 minutes to completely lyse the target cells.
在250g离心平板4分钟。将试验平板各孔的50ul上清液转移至96孔免疫平板Maxisorb板的相应孔。利用试验缓冲液(12ml)重建底物混合物，然后加50ul至平板各孔。平板盖上盖子后在阴暗处室温温育30分钟。将50ul终止溶液加入平板各孔以终止反应。加入终止溶液后1小时内计数记录490nm的吸光度。The plate was centrifuged at 250 g for 4 minutes. 50 ul of the supernatant from each well of the assay plate was transferred to the corresponding well of a 96-well immunoplate Maxisorb plate. The substrate mixture was reconstituted using assay buffer (12 ml) and then 50 ul was added to each well of the plate. The plate was capped and incubated for 30 minutes at room temperature in the dark. 50 ul of the stop solution was added to each well of the plate to terminate the reaction. The absorbance at 490 nm was counted and recorded within 1 hour after the addition of the stop solution.
计算结果Calculation results
从实验组、靶细胞自发释放组和效应细胞自释放组的所有吸光度值中扣除培养基背景的吸光度值。Absorbance values of the medium background were subtracted from all absorbance values of the experimental group, the target cell spontaneous release group, and the effector cell self-release group.
将上述中获得的经过校正的值带入下面公式，计算每个效靶比所产生的细胞毒性百分比。The corrected values obtained above were taken into the following formula to calculate the percentage of cytotoxicity produced by each of the target ratios.
％细胞毒性＝100×(实验-效应细胞自发-靶细胞自发)/(靶细胞最大-靶细胞自发)% cytotoxicity = 100 × (experimental - effector cell spontaneous - target cell spontaneous) / (target cell max - target cell spontaneous)
结果result
通过非放射性细胞毒性检测(如上所述)检验SAGE1TCR转导的T细胞对特异性靶细胞起反应的LDH释放。利用graphpad prism6绘制各孔中490nm可见光吸光值。The release of LDH by SAGE1 TCR-transduced T cells against specific target cells was examined by non-radioactive cytotoxicity assay (described above). The 490 nm visible light absorption value in each well was plotted using graphpad prism6.
实验数据统计结果如图10所示，随着效靶比例增加，本发明TCR转导的T细胞对特异性靶细胞K562-24和NCI H1299-A24的杀伤作用增强；对非特异性靶细胞IM 9杀伤作用很弱。未转导本发明TCR的同源CD8+T细胞对靶细胞K562-24无明显杀伤。The statistical results of the experimental data are shown in Fig. 10. As the ratio of the effective target increases, the killing effect of the TCR-transduced T cells of the present invention on the specific target cells K562-24 and NCI H1299-A24 is enhanced; for the non-specific target cells IM 9 The killing effect is very weak. The homologous CD8 + T cells that did not transduce the TCR of the present invention showed no significant killing of the target cell K562-24.
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。 All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (10)

  1. 一种T细胞受体(TCR)，其特征在于，所述TCR能够与VFSTVPPAFI-HLAA2402复合物结合；优选地，所述的TCR包含TCRα链可变域和TCRβ链可变域，其特征在于，所述TCRα链可变域的CDR3的氨基酸序列为CAVLYTGANSKLTF(SEQ ID NO.12)；和/或所述TCRβ链可变域的CDR3的氨基酸序列为CASSLVGKQPQHF(SEQ ID NO.15)；A T cell receptor (TCR), characterized in that the TCR is capable of binding to a VFSTVPPAFI-HLAA2402 complex; preferably, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, characterized in that The amino acid sequence of the CDR3 of the TCR alpha chain variable domain is CAVLYTGANSKLTF (SEQ ID NO. 12); and/or the amino acid sequence of the CDR3 of the TCR β chain variable domain is CASSLVGKQPQHF (SEQ ID NO. 15);
    更优选地，所述TCRα链可变域的3个互补决定区(CDR)为：More preferably, the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:
    αCDR1-DSAIYN      (SEQ ID NO.10)αCDR1-DSAIYN (SEQ ID NO.10)
    αCDR2-IQSSQRE     (SEQ ID NO.11)αCDR2-IQSSQRE (SEQ ID NO.11)
    αCDR3-CAVLYTGANSKLTF   (SEQ ID NO.12)；和/或αCDR3-CAVLYTGANSKLTF (SEQ ID NO. 12); and/or
    所述TCRβ链可变域的3个互补决定区为：The three complementarity determining regions of the TCR β chain variable domain are:
    βCDR1-SGHDT          (SEQ ID NO.13)βCDR1-SGHDT (SEQ ID NO. 13)
    βCDR2-YYEEEE           (SEQ ID NO.14)βCDR2-YYEEEE (SEQ ID NO. 14)
    βCDR3-CASSLVGKQPQHF     (SEQ ID NO.15)。βCDR3-CASSLVGKQPQHF (SEQ ID NO. 15).
  2. 如权利要求1所述的TCR，其特征在于，其包含TCRα链可变域和TCRβ链可变域，所述TCRα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；和/或所述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。The TCR according to claim 1, which comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, said TCR alpha chain variable domain being an amino acid having at least 90% sequence identity to SEQ ID NO. The sequence; and/or the TCR β chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5.
  3. 如权利要求1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；优选地，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。The TCR according to claim 1, wherein the C- or N-terminus of the α chain and/or the β chain of the TCR is bound to a conjugate; preferably, the T cell receptor is bound to The conjugate is a detectable label, a therapeutic, a PK modified moiety or a combination of any of these; preferably, the therapeutic agent is an anti-CD3 antibody.
  4. 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。A multivalent TCR complex characterized by comprising at least two TCR molecules, and wherein at least one TCR molecule is the TCR of any of the preceding claims.
  5. 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；A nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule of any of the preceding claims or a complement thereof;
    优选地，所述的核酸分子包含编码TCRα链可变域的核苷酸序列SEQ ID NO：2；和/或Preferably, said nucleic acid molecule comprises a nucleotide sequence encoding a TCR alpha chain variable domain of SEQ ID NO: 2; and/or
    所述的核酸分子包含编码TCRβ链可变域的核苷酸序列SEQ ID NO：6。The nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 6 encoding a TCR β chain variable domain.
  6. 一种载体，其特征在于，所述的载体含有权利要求5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。A vector comprising the nucleic acid molecule of claim 5; Preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
  7. 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6中所述的载体或染色体中整合有外源的权利要求5所述的核酸分子。An isolated host cell comprising the vector of claim 6 or the nucleic acid molecule of claim 5 integrated with an exogenous source in the host cell.
  8. 一种细胞，其特征在于，所述细胞转导权利要求5所述的核酸分子或权利要求6中所述载体；优选地，所述细胞为T细胞或干细胞。A cell characterized by transducing the nucleic acid molecule of claim 5 or the vector of claim 6; preferably, the cell is a T cell or a stem cell.
  9. 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1-3中任一项所述的TCR、权利要求4中所述的TCR复合物、权利要求5所述的核酸分子、或权利要求8中所述的细胞。A pharmaceutical composition comprising a pharmaceutically acceptable carrier, a TCR according to any one of claims 1 to 3, a TCR complex according to claim 4, claim 5 The nucleic acid molecule, or the cell of claim 8.
  10. 权利要求1-3中任一项所述的T细胞受体、或权利要求4中所述的TCR复合物或权利要求8中所述的细胞的用途，其特征在于，用于制备***或自身免疫疾病的药物。 Use of a T cell receptor according to any one of claims 1 to 3, or a TCR complex as claimed in claim 4 or a cell according to claim 8, for use in the preparation of a tumor or A drug for autoimmune diseases.
PCT/CN2017/108081 2016-10-27 2017-10-27 T cell receptor for recognizing sage1 antigen short-chain polypeptide WO2018077242A1 (en)

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