WO2016095783A1 - T cell receptor for identifying eb virus short peptide - Google Patents

T cell receptor for identifying eb virus short peptide Download PDF

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WO2016095783A1
WO2016095783A1 PCT/CN2015/097312 CN2015097312W WO2016095783A1 WO 2016095783 A1 WO2016095783 A1 WO 2016095783A1 CN 2015097312 W CN2015097312 W CN 2015097312W WO 2016095783 A1 WO2016095783 A1 WO 2016095783A1
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tcr
seq
cells
exon
cell
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PCT/CN2015/097312
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French (fr)
Chinese (zh)
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李懿
李友佳
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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 CN201580062782.4A priority Critical patent/CN107001444B/en
Publication of WO2016095783A1 publication Critical patent/WO2016095783A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a TCR capable of recognizing an Epstein Barr virus (EBV) antigen, and to EBV-specific T cells obtained by transducing the above TCR, and their use in the prevention and treatment of EBV-related diseases .
  • EBV Epstein Barr virus
  • EBV is a human herpesvirus that is ubiquitous worldwide. Studies have shown that more than 95% of adults have antibodies to the virus, which means they have been infected with the virus at some stage. Most infected people have EBV in their lives, and there are few problems. However, in some cases, EBV is associated with the development of some cancers, including Burkitt's lymphoma, Hodgkin lymphoma, EBV-positive lymphoproliferative disease (PTLD) or nasopharyngeal Cancer, etc. For example, LMP1 and LMP2 are latent membrane proteins belonging to EBV and are expressed by most nasopharyngeal carcinoma cells (Raab-Traub N. Epstein-Barr virus in the pathogenesis of NPC [J]. Semin Cancer Biol, 2002, 12(6) :431-441.). For the treatment of the above diseases, chemotherapy and radiotherapy can be used, but all of them will cause damage to their normal cells.
  • chemotherapy and radiotherapy can be used, but all of them will
  • 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 the corresponding target cell antigen.
  • APCs antigen presenting cells
  • pMHC complex short peptide-major histocompatibility complex
  • T cells and APC The other cell membrane surface molecules interact to cause 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.
  • APCs antigen presenting cells
  • pMHC complex short peptide-major histocompatibility complex
  • those skilled in the art are directed to isolating TCRs specific for EBV antigens, and transducing TCRs to T cells to obtain T cells specific for EBV antigens, thereby enabling them to play a role in cellular immunotherapy.
  • the invention also provides cells that transduce the TCR of the invention.
  • a TCR comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, and said TCR alpha chain variable domain comprises three complementarity determining regions (CDRs):
  • ⁇ CDR1 TTSDR (SEQ ID NO: 10)
  • ⁇ CDR2 LLSNGAV (SEQ ID NO: 11)
  • ⁇ CDR3 AISTGFQKLV (SEQ ID NO: 12) and/or the TCR ⁇ chain variable domain comprises three complementarity determining regions:
  • ⁇ CDR2 FYNNEI (SEQ ID NO: 14)
  • ⁇ CDR3 ASSEGPSGSSYEQY (SEQ ID NO: 15).
  • the TCR is capable of specifically binding to a short peptide derived from Epstein Barr Virus latent membrane protein (LMP-2).
  • LMP-2 Epstein Barr Virus latent membrane protein
  • the short peptide is: SSCSSCPLSK.
  • the TCR is capable of specifically binding to the SSCSSCPLSK-HLA A1101 complex.
  • the TCR alpha chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% sequence identity to SEQ ID NO:1;
  • the TCR ⁇ chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% sequence identity to SEQ ID NO:5.
  • the TCR comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1.
  • the TCR comprises the ⁇ chain variable domain amino acid sequence of SEQ ID NO: 5.
  • the TCR is an alpha beta heterodimer.
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 3.
  • the ⁇ chain amino acid sequence of the TCR is SEQ ID NO: 7.
  • the TCR is soluble.
  • cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.
  • cysteine residue forming an artificial disulfide bond in the TCR replaces one or more sets of sites selected from the group consisting of:
  • the 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
  • a conjugate is incorporated at the C- or N-terminus of the alpha chain and/or beta chain of the TCR.
  • the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modified moiety, or a combination thereof.
  • the detectable label comprises: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
  • MRI magnetic resonance imaging
  • CT electron computed tomography
  • the therapeutic agent comprises: a radionuclide, a biotoxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a virus particle, a liposome, Nanomagnetic particles, prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin) or any form of nanoparticles, and the like.
  • a radionuclide e.g, a biotoxin, a cytokine (such as IL-2, etc.)
  • an antibody an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a virus particle, a liposome, Nanomagnetic particles, prodrug activating enzymes (eg, DT-diaphorase
  • the therapeutic agent that binds to the T cell receptor is an anti-CD3 antibody linked to the C- or N-terminus of the alpha or beta chain of the TCR or any protein that specifically binds to CD3 , small molecule compounds or organic macromolecular compounds.
  • nucleic acid molecule comprising a T cell receptor according to any one of the first aspects 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 any of the second aspects of the invention is provided.
  • the vector is a viral vector.
  • the vector is a lentiviral vector.
  • an isolated host cell comprising the vector of any of the third aspects of the invention or the chromosome of the second aspect of the invention integrated with exogenous Any of the nucleic acid molecules described.
  • a cell which is transduced with the nucleic acid molecule of any one of the second aspects of the invention or the vector of any of the third aspect of the invention.
  • the cell is a T cell.
  • the cell is a stem cell.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of the first aspects of the invention, or a second of the invention A nucleic acid molecule according to any one of the aspects of the invention, or a cell according to any of the fifth aspect of the invention.
  • a method of treating a disease comprising administering to a subject in need of treatment an appropriate amount of a TCR according to any one of the first aspects of the invention, or a fifth aspect of the invention Said cell or a pharmaceutical composition as described in the sixth aspect of the invention.
  • the disease is EBV-positive Hodgkin's lymphoma, EBV-positive nasopharyngeal carcinoma, EBV-positive post-transplant lymphoproliferative disease, or Burkitt's lymphoma.
  • 1a, 1b, 1c and 1d 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 and the TCR alpha chain nucleotide sequence, respectively.
  • 2a, 2b, 2c and 2d are the TCR ⁇ chain variable domain amino acid sequence, the TCR ⁇ chain variable domain nucleotide sequence, the TCR ⁇ chain amino acid sequence and the TCR ⁇ chain nucleotide sequence, respectively.
  • Figure 3 shows the results of double positive staining of CD8 + and EBV-tetramer-PE in monoclonal cells.
  • Figure 4 is a graph showing the results of experiments with tetrameric stained TCR-transduced primary T cells.
  • Figure 5 is a graph showing the results of an ELISPOT experiment in which TCR-transduced T cells specifically activate a target cell.
  • Figure 6 is a graph showing the results of an experimental study on the killing effect of TCR-transduced T cells of the present invention on EBV LMp2A 340-349 SSCSSCPLSK short peptide-specific target cells and non-specific target cells by a non-radioactive cytotoxicity assay.
  • Figure 7 is a graph showing experimental results of detecting effector cell-specific killing ability by loading short peptides on different target cells.
  • the inventors have extensively and intensively studied to find a TCR capable of specifically binding to the EBV antigen LMP-2A 340-349 (SSCSSCPLSK) (SEQ ID NO: 9) in the form of the SSCSSCPLSK-HLA A1101 complex. Being presented.
  • 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 T cell receptor is a glycoprotein on the surface of a cell membrane in the form of a heterodimer formed by an ⁇ chain/ ⁇ chain or a ⁇ chain/ ⁇ chain.
  • the TCR heterodimer consists of alpha and beta chains in 95% of T cells, while 5% of T cells have a TCR consisting of gamma and delta chains.
  • the native ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain, and the ⁇ chain and the ⁇ chain constitute a subunit of the ⁇ heterodimeric TCR.
  • each of the alpha and beta chains comprises a variable region, a junction region, and a constant region
  • the beta chain typically also contains a short polymorphic region between the variable region and the junction region, but the polymorphic region is often considered as a junction region. a part of.
  • Each variable region comprises three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in framework regions.
  • the CDR regions determine the binding of the TCR to the pMHC complex, wherein the CDR3 is recombined from the variable region and the junction region and is referred to as the hypervariable region.
  • the alpha and beta chains of TCR are generally considered to have two "domains", namely a variable domain and a constant domain, and the variable domain consists of linked variable and linking regions.
  • the alpha and beta chains of TCR also contain a transmembrane and cytoplasmic regions with a short cytoplasmic region.
  • LMP-2 refers to two EBV-associated viral proteins, LMP-2A and LMP-2B, which are transmembrane proteins that block tyrosine kinase signaling.
  • 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.
  • the constant domain of the TCR molecule of the invention is a human constant domain.
  • IMGT International Immunogenetics Information System
  • 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.
  • polypeptide of the present invention TCR of the present invention
  • T cell receptor of the present invention T cell receptor of the present invention
  • a first aspect of the invention provides a TCR molecule capable of specifically binding to the SSCSSCPLSK-HLA A1101 complex.
  • the TCR molecule is isolated or purified.
  • the alpha and beta strands of the TCR each have three complementarity determining regions (CDRs).
  • the alpha chain comprises a CDR having the following amino acid sequence:
  • the beta strand comprises a CDR having the following amino acid sequence:
  • the chimeric TCR can be prepared by embedding the above-described CDR region amino acid sequences of the present invention into any suitable framework structure.
  • the framework structure is compatible with the CDR regions of the TCRs of the present invention, one skilled in the art can design or synthesize TCR molecules having corresponding functions in accordance with the CDR regions disclosed herein.
  • a TCR molecule of the invention refers to a TCR molecule comprising the above-described alpha and/or beta chain CDR region sequences and any suitable framework structure.
  • the TCR molecule of the invention is a heterodimer composed of alpha and beta chains.
  • the alpha chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the alpha chain variable domain amino acid sequence comprising the CDR1 (SEQ ID NO: 10), CDR2 (SEQ) ID NO: 11) and CDR3 (SEQ ID NO: 12).
  • the TCR molecule comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1. More preferably, the alpha chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO: 1.
  • the beta strand of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the beta strand variable domain amino acid sequence comprising the CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID) NO: 14) and CDR3 (SEQ ID NO: 15).
  • the TCR molecule comprises a beta chain variable domain amino acid sequence of SEQ ID NO:5. More preferably, the beta strand variable domain amino acid sequence of the TCR molecule is SEQ ID NO:5.
  • the TCR molecule of the invention is a single-chain TCR molecule consisting of part or all of the alpha chain and/or part or all of the beta chain.
  • a description of single-chain TCR molecules can be found in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658.
  • One skilled in the art can readily construct single-chain TCR molecules comprising the CDRs regions of the invention, as described in the literature.
  • the single-chain TCR molecule comprises V ⁇ , V ⁇ and C ⁇ , preferably linked in order from N-terminus to C-terminus.
  • the single-chain TCR molecule consists of V ⁇ , V ⁇ and a linker as described in patent document PCT/CN2014/080773.
  • the alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above alpha chain.
  • the single-chain TCR molecule comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1. More preferably, the alpha chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO: 1.
  • the ⁇ chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above-described ⁇ chain.
  • the single-chain TCR molecule comprises the ⁇ -chain variable domain amino acid sequence of SEQ ID NO: 5. More preferably, the ⁇ chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO:5.
  • the constant domain of the TCR molecule of the invention is a human constant domain.
  • a human constant domain amino acid sequence by consulting a related book or a public database of IMGT (International Immunogenetics Information System).
  • IMGT International Immunogenetics Information System
  • the amino acid sequence of the ⁇ chain of the TCR molecule of the present invention is SEQ ID NO: 3, and/or the amino acid sequence of the ⁇ chain is SEQ ID NO: 7.
  • TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane domain.
  • TCR can also be developed for diagnosis and treatment, when soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCR has a wide range of uses, not only for studying the interaction of TCR with pMHC, but also as a diagnostic tool for detecting infection or as a marker for autoimmune diseases.
  • soluble TCR can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells that present specific antigens.
  • soluble TCRs can also bind to other molecules (eg, anti-CD3 antibodies). To redirect T cells so that they target cells that present a particular antigen. Those skilled in the art are aware of methods for obtaining 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.
  • the position of the disulfide bond and the method for preparing the soluble TCR can be referred to the literature (Jonathan M. Boulter et al., 2003, Protein Engineering 16(9): 707-711) and the patent document PCT/CN2015/093806.
  • 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: TRAC*01 Thr45 of exon 1 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01; Ter17 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC2*01; TRAC*01 Sp45 of subunit 1 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; Ser15 of TRACL*01 exon 1 and Glu15 of exon 1 of TRBC2*01; TRAC*01 exon 1 Arg53 and TRBC1*01 or TRBC2*01 exon 1 of Ser54; TRAC*01 exon 1 of Pro89 and TRBC1*01 or TRBC2*01 exon 1 of Ala19
  • 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 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 identified by the international common single letter, and the corresponding amino acid names are abbreviated as: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V);
  • 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:
  • 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 ⁇ chain of the invention
  • the nucleotide sequence includes SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
  • 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.
  • nucleotide sequence of a nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention.
  • 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: 2.
  • the nucleotide sequence can be codon optimized. Different cells are different in the utilization of specific codons, and the number of expressions can be increased by changing the codons in the sequence depending on the type of the cell. Codon selection tables for mammalian cells as well as a variety of other organisms are well known to those skilled in the art.
  • the full length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification, recombinant methods or synthetic methods. At present, it has been possible to obtain a DNA sequence encoding the TCR (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. The DNA can be a coding strand or a non-coding strand.
  • the invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, ie, constructs that are capable of expression in vivo or in vitro.
  • expression vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.
  • Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.
  • AAV adeno-associated virus
  • the vector can transfer a nucleotide of the invention into a cell, such as a T cell, such that the cell expresses an EBV-specific TCR.
  • a cell such as a T cell
  • the vector should be capable of sustained high levels of expression in T cells.
  • the invention also relates to host cells genetically engineered using the vectors or coding sequences of the invention.
  • 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 an EBV-associated disease in a subject comprising the step of adoptively transferring EBV-specific T cells to the subject.
  • the EBV-specific T cells recognize the SSCSSCPLSK-HLA A1101 complex.
  • EBV-specific T cells can be used to treat any EBV-associated disease presenting the antigen LMP-2A 340-349 (SSCSSCPLSK). These include, but are not limited to, Burkitt's lymphoma, EBV-positive Hodgkin's lymphoma, EBV-positive nasopharyngeal carcinoma, or EBV-positive post-transplant lymphoproliferative disease (PTLD).
  • SSCSSCPLSK antigen LMP-2A 340-349
  • PTLD EBV-positive post-transplant lymphoproliferative disease
  • Burkitt's lymphoma is the most common childhood malignancy in the equatorial region of Africa. Genetic studies have shown that the vast majority of Burkitt's lymphoma in the equatorial region of Africa is derived from EBV-infected lymphocytes. EBV genetic material is found in up to 50% of Burkitt's lymphoma cases in certain geographic regions and patient populations.
  • Nasopharyngeal carcinoma refers to a malignant tumor that occurs on the top and side walls of the nasopharyngeal cavity. It is one of the high-grade malignant tumors in China, and the incidence rate is the first of the malignant tumors of the ear, nose and throat. Immunological and biochemical studies have confirmed that Epstein-Barr virus is closely related to nasopharyngeal carcinoma.
  • Post-transplant lymphoproliferative disease refers to a disease that may form in humans after organ transplantation.
  • the EBV virus is implicated in most PTLD cases.
  • the manifestations can vary, from an increase in the number of lymphocytes in the blood to a malignant growth of blood cells, such as B-cell lymphoma.
  • the T cells of a patient or volunteer having an EBV-associated disease can be isolated and introduced into the above T cells by the TCR of the present invention, and then these genetically engineered cells are returned to the patient for treatment.
  • the present invention provides a method of treating an EBV-associated disease comprising administering an isolated T cell expressing a TCR of the invention, preferably, the T cell is derived from the patient itself and is administered to the 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 is capable of specifically binding to the EBV antigen LMP-2A 340-349 (SSCSSCPLSK) (SEQ ID NO: 9), while the cells transduced with the TCR of the present invention are specifically activated and have a target cell Very strong killing.
  • SSCSSCPLSK EBV antigen LMP-2A 340-349
  • Peripheral blood lymphocytes from healthy volunteers with genotype HLA-A1101 were stimulated with synthetic short peptide EBV LMp2A 340-349 SSCSSCPLSK (Beijing Cypress Biotech Co., Ltd.).
  • the EBV LMp2A 340-349 SSCSSCPLSK short peptide was renatured with biotinylated HLA-A*1101 to prepare a pHLA haploid.
  • These haploids were combined with PE-labeled streptavidin (BD) into PE-labeled tetramers, and the tetramer and anti-CD8-APC double positive cells were sorted.
  • the sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonalization by limiting dilution. Monoclonal cells were stained with tetramers and the double positive clones screened are shown in Figure 3.
  • Example 2 Extracted with Quick-RNA TM MiniPrep (ZYMO research ) in Example 1 to EBV LMp2A 340-349 screened specific, HLA-A1101 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 ⁇ chain and ⁇ chain of the TCR expressed by the double positive clone are shown in Fig. 1 and Fig. 2, respectively.
  • 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:
  • the full-length genes of the TCR alpha chain and the beta chain were cloned into the lentiviral expression vector pLenti (addgene) by overlap PCR, 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.
  • Lentiviral expression vector and TCR ⁇ -2A-TCR ⁇ was digested to obtain the pLenti-EBVTRA-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.
  • Lentiviruses containing the gene encoding the desired TCR were packaged using a third generation lentiviral packaging system.
  • a third generation lentiviral packaging system Using the Express-In-mediated transient transfection (Open Biosystems), four plasmids (containing the pLenti-EBVTRA-2A-TRB-IRES-NGFR described in Example 2) A lentiviral vector, and three plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles, were transfected into 293T/17 cells.
  • 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 concentrating tube Merck Millipore, catalog number UFC905096 was concentrated to remove most of the supernatant, finally concentrated to 1 ml, and aliquoted at -80 ° C for cryopreservation.
  • 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).
  • EBV LMp2A 340-349 SSCSSCPLSK short peptide was renatured with biotinylated HLA-A*1101 to prepare pHLA haploid. These haploids were combined with PE-labeled streptavidin (BD) into a PE-labeled tetramer called EBV-tetramer-PE. This tetramer can label T cells expressing an EBV-specific T cell receptor gene as positive cells.
  • the transfected T cell samples in (b) were incubated with EBV-tetramer-PE for 30 minutes on ice, then anti-CD8-APC (biolegend) antibody was added and incubation was continued for 15 minutes on ice.
  • the following assays were performed to demonstrate the activation response of TCR-transduced T cells 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 cell of this example is an Epstein-Barr virus (EBV) transformed immortalized lymphoblastoid cell line (LCL).
  • 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.
  • TPA tetradecanoyl phorbol ester
  • the ELISPOT assay targets HLA-A11 as a target cell.
  • T cells The effector cells (T cells) of the present assay were CD8+ T cells expressing EBV-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 (life technologies), transduced with lentivirus carrying the EBV-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.
  • EBV CD8+ T cells (EBV TCR transduced T cells, effector cells), CD8+ T cells (negative control effector cells) and LCL cells (target cells) were prepared as described in Example 3, which includes specific target cell LCL -A11 (LCL cells with genotype HLA-A11) and non-specific target cells LCL-A24 (LCL cells with genotype HLA-A24) and add corresponding short peptides in the corresponding experimental group, wherein P EBV is LMp2A 340-349 SSCSSCPLSK short peptide, P A24 is a non-EBV TCR specific binding short peptide.
  • effector cells 1000 EBV TCR 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.
  • IFN-[gamma] release by EBV TCR transduced T cells against EBV LMp2A 340-349 SSCSSCPLSK short peptide-specific target cells and non-specific target cells was examined by ELISPOT assay (described above). The number of ELSPOT spots observed in each well was plotted using graphpad prism6.
  • the T cells transducing the TCR of the present invention have an activation reaction only to target cells of a specific genotype carrying a specific short peptide, and substantially no activation reaction to other non-specific target cells.
  • the activation reaction of T cells which are not transduced with the TCR of the present invention is poor.
  • This test is a colorimetric substitution test for the 51 Cr 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, Gibco), phenol red containing 90% RPMI 1640 (Gibco, catalog number C11875500bt), 1% penicillin/streptococcus Prime (Jibuco, catalog number 15070-063).
  • Microporous round bottom 96-well tissue culture plate (Nunc, catalog number 163320)
  • the target cell LCL used in this assay is the same as the target cell preparation method in the ELISPOT protocol of Example 4.
  • the target cells used in this protocol include LCL-A11, LCL-A02 (LCL cells with genotype HLA-A02) and LCL-A24.
  • Target cells were prepared in the test medium: the target cell concentration was adjusted to 3 ⁇ 10 5 /ml, and 50 ⁇ l per well was taken to obtain 1.5 ⁇ 10 4 cells/well.
  • the effector cells (T cells) of this assay were CD8+ T cells expressing EBV-specific TCR by flow cytometry in Example 3.
  • the ratio of effector cells to target cells was 1:1 (3 ⁇ 10 5 /ml, 50 ⁇ l per well to obtain 1.5 ⁇ 10 4 cells/well).
  • LMp2A 340-349 SSCSSCPLSK short peptide was diluted with 10% FBS in RPMI 1640 medium to a 10 -4 M-10 -12 M working solution, and added to the experimental group with a final concentration gradient of 10 - 5 M-10 -13 M.
  • 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:
  • Unloaded short peptide experimental group (untreated LCL-A11): containing 50 ul of effector cells and 50 ul of target cells.
  • 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)
  • Three target cells LCL-A11, LCL-A02 and LCL-A24, were added to 10 -5 M LMp2A 340-349 SSCSSCPLSK (PEBV) short peptide and incubated for 2 hours at 37 °C.
  • the medium containing the short peptide was removed, and the cells were resuspended to 3 ⁇ 10 5 /ml with a new test medium, and 50 ⁇ l per well to obtain 1.5 ⁇ 10 4 cells/well.
  • Another three target cells were not loaded with short peptides as a blank control group.
  • the effector cells (T cells) of this assay were CD8+ T cells expressing EBV-specific TCR by flow cytometry in Example 3.
  • the ratio of effector cells to target cells was 1:1 (3 ⁇ 10 5 /ml, 50 ⁇ l per well to obtain 1.5 ⁇ 10 4 cells/well).
  • 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:
  • Unloaded short peptide experimental group containing 50 ul of effector cells and 50 ul of target cells.
  • Figure 7 shows the specific killing effect of EBV CD8+ T cells on LCL-A11 target cells loaded with the short peptide LMp2A 340-349 SSCSSCPLSK (P EBV ). There is no obvious killing effect on other target cells that do not load short peptides or load short peptides.

Abstract

Provided is a T cell receptor (TCR) capable of combining peptide derived from Epstein barr virus (EB virus) latent membrane protein (LMP-2). The peptide is presented in the form of a SSCSSCPLSK-HLA A1101 compound. Also provided are a nucleic acid molecule for encoding the TCR, carrier comprising the nucleic acid molecule, and cell for transferring the TCR.

Description

识别EB病毒短肽的T细胞受体T cell receptor recognizing Epstein-Barr virus short peptide 技术领域Technical field
本发明涉及能够识别源自EB病毒(Epstein Barr virus,EBV)抗原的TCR，本发明还涉及转导上述TCR来获得的EBV特异性的T细胞，及他们在预防和治疗EBV相关疾病中的用途。The present invention relates to a TCR capable of recognizing an Epstein Barr virus (EBV) antigen, and to EBV-specific T cells obtained by transducing the above TCR, and their use in the prevention and treatment of EBV-related diseases .
背景技术Background technique
EBV是全球范围普遍存在的一种人类疱疹病毒。研究显示，超过95％的成年人体内含有针对这种病毒的抗体，这也就意味着他们在某一阶段被这种病毒感染过。大多数被感染过的人的体内一生都会存在EBV，一般很少出现问题。然而，在某些情况下，EBV与一些癌症的发生相关，包括伯基特淋巴瘤(Burkitt’s lymphoma)、霍奇金淋巴瘤(Hodgkin lymphoma)、EBV阳性移植后淋巴增生病(PTLD)或鼻咽癌等。比如，LMP1和LMP2是属于EBV的潜伏期膜蛋白，会被多数鼻咽癌细胞表达(Raab-Traub N.Epstein-Barr virus in the pathogenesis of NPC[J].Semin Cancer Biol,2002,12(6):431-441.)。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。EBV is a human herpesvirus that is ubiquitous worldwide. Studies have shown that more than 95% of adults have antibodies to the virus, which means they have been infected with the virus at some stage. Most infected people have EBV in their lives, and there are few problems. However, in some cases, EBV is associated with the development of some cancers, including Burkitt's lymphoma, Hodgkin lymphoma, EBV-positive lymphoproliferative disease (PTLD) or nasopharyngeal Cancer, etc. For example, LMP1 and LMP2 are latent membrane proteins belonging to EBV and are expressed by most nasopharyngeal carcinoma cells (Raab-Traub N. Epstein-Barr virus in the pathogenesis of NPC [J]. Semin Cancer Biol, 2002, 12(6) :431-441.). 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抗原。在免疫***中，通过抗原特异性的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EBV抗原具有特异性的TCR，以及将该TCR转导T细胞来获得对EBV抗原具有特异性的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 the corresponding target cell antigen. In the immune system, direct binding of T cells to antigen presenting cells (APCs) by antigen-specific TCR binding to a short peptide-major histocompatibility complex (pMHC complex), followed by T cells and APC The other cell membrane surface molecules interact to cause 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. Thus, those skilled in the art are directed to isolating TCRs specific for EBV antigens, and transducing TCRs to T cells to obtain T cells specific for EBV antigens, thereby enabling them to play a role in cellular immunotherapy.
发明内容Summary of the invention
本发明的目的是提供一种能够结合SSCSSCPLSK-HLA A1101复合物的TCR，以及编码所述TCR的核酸分子和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。It is an object of the present invention to provide a TCR capable of binding to the SSCSSCPLSK-HLA A1101 complex, and a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule. In addition, the invention also provides cells that transduce the TCR of the invention.
在本发明的第一方面，提供了一种TCR，其包含TCRα链可变域和TCRβ链可变域，并且所述TCRα链可变域包含3个互补决定区(CDR)：In a first aspect of the invention, there is provided a TCR comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, and said TCR alpha chain variable domain comprises three complementarity determining regions (CDRs):
αCDR1：TTSDR    (SEQ ID NO:10)αCDR1: TTSDR (SEQ ID NO: 10)
αCDR2：LLSNGAV    (SEQ ID NO:11)和αCDR2: LLSNGAV (SEQ ID NO: 11) and
αCDR3：AISTGFQKLV  (SEQ ID NO:12)和/或所述TCRβ链可变域包含3个互补决定区： αCDR3: AISTGFQKLV (SEQ ID NO: 12) and/or the TCR β chain variable domain comprises three complementarity determining regions:
βCDR1：SNHLY  (SEQ ID NO:13)βCDR1:SNHLY (SEQ ID NO:13)
βCDR2：FYNNEI  (SEQ ID NO:14)和βCDR2: FYNNEI (SEQ ID NO: 14) and
βCDR3：ASSEGPSGSSYEQY  (SEQ ID NO:15)。βCDR3: ASSEGPSGSSYEQY (SEQ ID NO: 15).
在另一优选例中，所述TCR能够特异性结合源自于EB病毒(Epstein Barr Virus)潜伏膜蛋白(LMP-2)的短肽。In another preferred embodiment, the TCR is capable of specifically binding to a short peptide derived from Epstein Barr Virus latent membrane protein (LMP-2).
在另一优选例中，所述短肽为：SSCSSCPLSK。In another preferred embodiment, the short peptide is: SSCSSCPLSK.
在另一优选例中，所述TCR能够特异性结合SSCSSCPLSK-HLA A1101复合物。In another preferred embodiment, the TCR is capable of specifically binding to the SSCSSCPLSK-HLA A1101 complex.
在另一优选例中，所述TCRα链可变域包含与SEQ ID NO:1具有至少90％，优选地至少95％，更优选地至少98％序列相同性的氨基酸序列；In another preferred embodiment, the TCR alpha chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% sequence identity to SEQ ID NO:1;
在另一优选例中，所述TCRβ链可变域包含与SEQ ID NO:5具有至少90％，优选地至少95％，更优选地至少98％序列相同性的氨基酸序列。In another preferred embodiment, the TCR β chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% 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: 1.
在另一优选例中，所述TCR包含β链可变域氨基酸序列SEQ ID NO:5。In another preferred embodiment, the TCR comprises the β chain variable domain amino acid sequence of SEQ ID NO: 5.
在另一优选例中，所述TCR为αβ异质二聚体。In another preferred embodiment, the TCR is an alpha beta heterodimer.
在另一优选例中，所述TCR的α链氨基酸序列为SEQ ID NO:3。In another preferred embodiment, the amino acid sequence of the α chain of the TCR is SEQ ID NO: 3.
在另一优选例中，所述TCR的β链氨基酸序列为SEQ ID NO:7。In another preferred embodiment, the β chain amino acid sequence of the TCR is SEQ ID NO: 7.
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，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，在所述TCR的α链和/或β链的C-或N-末端结合有偶联物。 In another preferred embodiment, a conjugate is incorporated at the C- or N-terminus of the alpha chain and/or beta chain of the TCR.
在另一优选例中，与所述TCR结合的偶联物为可检测标记物、治疗剂、PK修饰部分或其组合。In another preferred embodiment, the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modified moiety, or a combination thereof.
优选地，所述可检测标记物包括：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。Preferably, the detectable label comprises: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
优选地，所述治疗剂包括：放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。Preferably, the therapeutic agent comprises: a radionuclide, a biotoxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a virus particle, a liposome, Nanomagnetic particles, prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin) or any form of nanoparticles, and the like.
在另一优选例中，与所述T细胞受体结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-CD3抗体或任何与CD3特异性结合的蛋白质、小分子化合物或有机大分子化合物。In another preferred embodiment, the therapeutic agent that binds to the T cell receptor is an anti-CD3 antibody linked to the C- or N-terminus of the alpha or beta chain of the TCR or any protein that specifically binds to CD3 , small molecule compounds or organic macromolecular compounds.
在本发明的第二方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面中任一所述的T细胞受体或其互补序列。In a second aspect of the invention, a nucleic acid molecule comprising a T cell receptor according to any one of the first aspects 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.
在本发明的第三方面，提供了一种载体，所述的载体含有本发明第二方面中任一所述的核酸分子。In a third aspect of the invention, a vector comprising the nucleic acid molecule of any of the second aspects of the invention is provided.
在另一优选例中，所述的载体为病毒载体。In another preferred embodiment, the vector is a viral vector.
在另一优选例中，所述的载体为慢病毒载体。In another preferred embodiment, the vector is a lentiviral vector.
在本发明的第四方面，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三方面中任一所述的载体或染色体中整合有外源的本发明第二方面中任一所述的核酸分子。In a fourth aspect of the invention, there is provided an isolated host cell comprising the vector of any of the third aspects of the invention or the chromosome of the second aspect of the invention integrated with exogenous Any of the nucleic acid molecules described.
在本发明的第五方面，提供了一种细胞，所述细胞转导本发明第二方面中任一所述的核酸分子或本发明第三方面中任一所述载体。In a fifth aspect of the invention, there is provided a cell, which is transduced with the nucleic acid molecule of any one of the second aspects of the invention or the vector of any of the third aspect of the invention.
在另一优选例中，所述细胞为T细胞。In another preferred embodiment, the cell is a T cell.
在另一优选例中，所述细胞为干细胞。In another preferred embodiment, the cell is a stem cell.
在本发明的第六方面，提供了一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及本发明第一方面中任一所述的TCR、或本发明第二方面任一所述的核酸分子、或本发明第五方面中任一所述的细胞。In a sixth aspect of the invention, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of the first aspects of the invention, or a second of the invention A nucleic acid molecule according to any one of the aspects of the invention, or a cell according to any of the fifth aspect of the invention.
在本发明的第七方面，提供了本发明第一方面中任一项所述的T细胞受体或本发明第五方面中任一所述的细胞的用途，其特征在于，用于制备*** 或HCV病毒感染的药物。In a seventh aspect of the invention, the use of the T cell receptor of any one of the first aspect of the invention, or the cell of any of the fifth aspect of the invention, for use in the preparation of a treatment Tumor Or a drug infected with HCV virus.
在本发明的第八方面，提供了一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用适量的本发明第一方面中任一所述的TCR、或本发明第五方面中所述的细胞或本发明第六方面中所述的药物组合物。In an eighth aspect of the invention, a method of treating a disease, comprising administering to a subject in need of treatment an appropriate amount of a TCR according to any one of the first aspects of the invention, or a fifth aspect of the invention Said cell or a pharmaceutical composition as described in the sixth aspect of the invention.
在另一优选例中，所述的疾病为EBV阳性霍奇金淋巴瘤、EBV阳性鼻咽癌、EBV阳性移植后淋巴增生病或伯基特淋巴瘤。In another preferred embodiment, the disease is EBV-positive Hodgkin's lymphoma, EBV-positive nasopharyngeal carcinoma, EBV-positive post-transplant lymphoproliferative disease, or Burkitt's lymphoma.
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。1a, 1b, 1c and 1d 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 and the TCR alpha chain nucleotide sequence, respectively.
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。2a, 2b, 2c and 2d are the TCR β chain variable domain amino acid sequence, the TCR β chain variable domain nucleotide sequence, the TCR β chain amino acid sequence and the TCR β chain nucleotide sequence, respectively.
图3为单克隆细胞的CD8+及EBV-四聚体-PE双阳性染色结果。Figure 3 shows the results of double positive staining of CD8 + and EBV-tetramer-PE in monoclonal cells.
图4为四聚体染色TCR转导的原代T细胞的实验结果图。Figure 4 is a graph showing the results of experiments with tetrameric stained TCR-transduced primary T cells.
图5为TCR-转导的T细胞对靶细胞特异性地激活反应的ELISPOT实验结果图。Figure 5 is a graph showing the results of an ELISPOT experiment in which TCR-transduced T cells specifically activate a target cell.
图6为通过非放射性细胞毒性实验检测本发明TCR转导的T细胞对负载EBV LMp2A 340-349 SSCSSCPLSK短肽特异性靶细胞和非特异性靶细胞的杀伤作用的实验结果图。Figure 6 is a graph showing the results of an experimental study on the killing effect of TCR-transduced T cells of the present invention on EBV LMp2A 340-349 SSCSSCPLSK short peptide-specific target cells and non-specific target cells by a non-radioactive cytotoxicity assay.
图7为通过不同靶细胞负载短肽检测效应细胞特异性杀伤能力的实验结果图。Figure 7 is a graph showing experimental results of detecting effector cell-specific killing ability by loading short peptides on different 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EBV抗原LMP-2A 340-349(SSCSSCPLSK)(SEQ ID NO:9)能够特异性结合的TCR，所述抗原以SSCSSCPLSK-HLA A1101复合物的形式被呈递。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 extensively and intensively studied to find a TCR capable of specifically binding to the EBV antigen LMP-2A 340-349 (SSCSSCPLSK) (SEQ ID NO: 9) in the form of the SSCSSCPLSK-HLA A1101 complex. Being presented. 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
T细胞受体(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。此外，TCR的α和β链还包含跨膜区和胞质区，胞质区很短。The T cell receptor (TCR) is a glycoprotein on the surface of a cell membrane in the form of a heterodimer formed by an α chain/β chain or a γ chain/δ chain. The TCR heterodimer consists of alpha and beta chains in 95% of T cells, while 5% of T cells have a TCR consisting of gamma and delta chains. The native αβ heterodimeric TCR has an α chain and a β chain, and the α chain and the β chain constitute a subunit of the αβ 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. In addition, the alpha and beta chains of TCR also contain a transmembrane and cytoplasmic regions with a short cytoplasmic region.
LMP-2是指两种与EB病毒相关的病毒蛋白，LMP-2A和LMP-2B，它们是发挥阻断酪氨酸激酶信号传导作用的跨膜蛋白。LMP-2 refers to two EBV-associated viral proteins, LMP-2A and LMP-2B, which are transmembrane proteins that block tyrosine kinase signaling.
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.
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。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.
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.
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特异性结合SSCSSCPLSK-HLA A1101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 specifically binding to the SSCSSCPLSK-HLA A1101 complex. Preferably, the TCR molecule is isolated or purified. The alpha and beta strands of the TCR each have three complementarity determining regions (CDRs).
α链包含具有以下氨基酸序列的CDR：The alpha chain comprises a CDR having the following amino acid sequence:
α  CDR1-TTSDR      (SEQ ID NO:10)α CDR1-TTSDR (SEQ ID NO: 10)
α  CDR2-LLSNGAV      (SEQ ID NO:11)α CDR2-LLSNGAV (SEQ ID NO: 11)
α  CDR3-AISTGFQKLV      (SEQ ID NO:12)α CDR3-AISTGFQKLV (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-SNHLY      (SEQ ID NO:13)β CDR1-SNHLY (SEQ ID NO: 13)
β  CDR2-FYNNEI      (SEQ ID NO:14)β CDR2-FYNNEI (SEQ ID NO: 14)
β  CDR3-ASSEGPSGSSYEQY    (SEQ ID NO:15)β CDR3-ASSEGPSGSSYEQY (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。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.
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: 1. More preferably, the alpha chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO: 1. 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 the 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:5. More preferably, the beta strand variable domain amino acid sequence of the TCR molecule is SEQ ID NO:5.
在本发明的一个优选例中，本发明的TCR分子是由α链的部分或全部和/或β链的部分或全部组成的单链TCR分子。有关单链TCR分子的描述可以参考文献Chung et al(1994)Proc.Natl.Acad.Sci.USA 91,12654-12658。根据文献中所述，本领域技术人员能够容易地构建包含本发明CDRs区的单链TCR分子。具体地，所述单链TCR分子包含Vα、Vβ和Cβ，优选地按照从N端到C端的顺序连接。或者，所述单链TCR分子由Vα、Vβ和连接序列(linker)构成，如专利文献PCT/CN2014/080773中所述。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。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. Alternatively, the single-chain TCR molecule consists of Vα, Vβ and a linker as described in patent document PCT/CN2014/080773. 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: 1. More preferably, the alpha chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO: 1. 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: 5. More preferably, the β chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO:5.
在本发明的一个优选例中，本发明的TCR分子的恒定域是人的恒定域。本领域技术人员知晓或可以通过查阅相关书籍或IMGT(国际免疫遗传学信息***)的公开数据库来获得人的恒定域氨基酸序列。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). 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: 7.
天然存在的TCR是一种膜蛋白，通过其跨膜区得以稳定。如同免疫球蛋白(抗体)作为抗原识别分子一样，TCR也可以被开发应用于诊断和治疗，这时需要获得可溶性的TCR分子。可溶性的TCR分子不包括其跨膜区。可溶性TCR有很广泛的用途，它不仅可用于研究TCR与pMHC的相互作用，也可用作检测感染的诊断工具或作为自身免疫病的标志物。类似地，可溶性TCR可以被用来将治疗剂(如细胞毒素化合物或免疫刺激性化合物)输送到呈递特异性抗原的细胞，另外，可溶性TCR还可与其他分子(如，抗-CD3抗体)结合来重新定向T细胞，从而使其靶向呈递特定抗原的细胞。本领域技术人员知晓获得可溶性TCR的方法。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. Those skilled in the art are aware of methods for obtaining 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。二硫键的位置以及制备可溶性TCR的方法可以参考文献(Jonathan M.Boulter etal.,2003,Protein Engineering 16(9):707-711)以及专利文献PCT/CN2015/093806。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. The position of the disulfide bond and the method for preparing the soluble TCR can be referred to the literature (Jonathan M. Boulter et al., 2003, Protein Engineering 16(9): 707-711) and the patent document PCT/CN2015/093806. 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: TRAC*01 Thr45 of exon 1 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01; Ter17 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC2*01; TRAC*01 Sp45 of subunit 1 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; Ser15 of TRACL*01 exon 1 and Glu15 of exon 1 of TRBC2*01; TRAC*01 exon 1 Arg53 and TRBC1*01 or TRBC2*01 exon 1 of Ser54; TRAC*01 exon 1 of Pro89 and TRBC1*01 or TRBC2*01 exon 1 of Ala19; or TRAC*01 exon 1 Glu20 of exon 1 of Tyr10 and TRBC1*01 or 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.
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标识，与其相对应的氨基酸名称三英文字母简写分别是：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)；It should be understood that the amino acid names in this article are identified by the international common single letter, and the corresponding amino acid names are abbreviated as: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V);
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-accacttcagacaga    (SEQ ID NO:16)α CDR1-accacttcagacaga (SEQ ID NO: 16)
α  CDR2-ttgctatcaaatggagcagtg  (SEQ ID NO:17) α CDR2-ttgctatcaaatggagcagtg (SEQ ID NO: 17)
α  CDR3-gccatctcaacaggctttcagaaacttgta    (SEQ ID NO:18)α CDR3-gccatctcaacaggctttcagaaacttgta (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-tctaatcacttatac    (SEQ ID NO:19)β CDR1-tctaatcacttatac (SEQ ID NO: 19)
β  CDR2-ttttataataatgaaatc    (SEQ ID NO:20)β CDR2-ttttataataatgaaatc (SEQ ID NO: 20)
β  CDR3-gccagcagtgaaggccctagcgggagctcctacgagcagtac   (SEQ ID NO:21)β CDR3-gccagcagtgaaggcccctagcgggagctcctacgagcagtac (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。优选地，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:2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:6。更优选地，本发明核酸分子的核苷酸序列为SEQ ID NO:4和/或SEQ ID NO:8。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: 21. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR alpha chain of the invention comprises SEQ ID NO: 2 and/or the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR beta chain of the invention comprises SEQ ID NO: 6. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention is SEQ ID NO: 4 and/or SEQ ID NO: 8.
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。一般情况下，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。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. In general, the nucleotide sequence of a nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention. 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: 2.
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EBV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 an EBV-specific TCR. 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).
EBV相关疾病EBV related diseases
本发明还涉及在受试者中治疗和/或预防与EBV相关疾病的方法，其包括过继性转移EBV特异性T细胞至该受试者的步骤。该EBV特异性T细胞可识别SSCSSCPLSK-HLA A1101复合物。The invention also relates to a method of treating and/or preventing an EBV-associated disease in a subject comprising the step of adoptively transferring EBV-specific T cells to the subject. The EBV-specific T cells recognize the SSCSSCPLSK-HLA A1101 complex.
EBV特异性的T细胞可用于治疗任何呈递抗原LMP-2A 340-349(SSCSSCPLSK)的EBV相关疾病。包括但不限于伯基特淋巴瘤(Burkitt’s lymphoma)、EBV阳性霍奇金淋巴瘤、EBV阳性鼻咽癌或EBV阳性移植后淋巴增生病(PTLD)。EBV-specific T cells can be used to treat any EBV-associated disease presenting the antigen LMP-2A 340-349 (SSCSSCPLSK). These include, but are not limited to, Burkitt's lymphoma, EBV-positive Hodgkin's lymphoma, EBV-positive nasopharyngeal carcinoma, or EBV-positive post-transplant lymphoproliferative disease (PTLD).
伯基特淋巴瘤是非洲赤道区域最常见的儿童恶性肿瘤。遗传研究已显示在非洲赤道区域的绝大多数伯基特淋巴瘤源自EBV感染的淋巴细胞。某些地理区域和患者人群中，在高至50％的伯基特淋巴瘤病例中发现了EBV遗传材料。Burkitt's lymphoma is the most common childhood malignancy in the equatorial region of Africa. Genetic studies have shown that the vast majority of Burkitt's lymphoma in the equatorial region of Africa is derived from EBV-infected lymphocytes. EBV genetic material is found in up to 50% of Burkitt's lymphoma cases in certain geographic regions and patient populations.
鼻咽癌是指发生于鼻咽腔顶部和侧壁的恶性肿瘤。是中国高发恶性肿瘤之一，发病率为耳鼻咽喉恶性肿瘤之首。免疫学和生物化学研究证实EB病毒与鼻咽癌关系密切。Nasopharyngeal carcinoma refers to a malignant tumor that occurs on the top and side walls of the nasopharyngeal cavity. It is one of the high-grade malignant tumors in China, and the incidence rate is the first of the malignant tumors of the ear, nose and throat. Immunological and biochemical studies have confirmed that Epstein-Barr virus is closely related to nasopharyngeal carcinoma.
移植后淋巴增生病(PTLD)是指可能在器官移植后的人中形成的一种疾病。EBV病毒与大多数PTLD病例有牵连。表现形式可以是不同的，从血液中淋巴细胞数量的增加到血细胞恶性生长，如B细胞淋巴瘤。Post-transplant lymphoproliferative disease (PTLD) refers to a disease that may form in humans after organ transplantation. The EBV virus is implicated in most PTLD cases. The manifestations can vary, from an increase in the number of lymphocytes in the blood to a malignant growth of blood cells, such as B-cell lymphoma.
治疗方法treatment method
可以通过分离患有EBV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EBV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 volunteer having an EBV-associated disease can be isolated and introduced into the above T cells by the TCR of the present invention, and then these genetically engineered cells are returned to the patient for treatment. Accordingly, the present invention provides a method of treating an EBV-associated disease comprising administering an isolated T cell expressing a TCR of the invention, preferably, the T cell is derived from the patient itself and is administered to the 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EBV抗原LMP-2A 340-349(SSCSSCPLSK)(SEQ ID NO:9)特异性结合，同时转导了本发明TCR的细胞能够被特异性激活并且对靶细胞具有很强的杀伤性。(1) The TCR of the present invention is capable of specifically binding to the EBV antigen LMP-2A 340-349 (SSCSSCPLSK) (SEQ ID NO: 9), while the cells transduced with the TCR of the present invention are specifically activated and have a target cell Very strong killing.
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EBV特异性T细胞Example 1 Cloning of EBV-specific T cells
利用合成短肽EBV LMp2A 340-349 SSCSSCPLSK(北京赛百盛基因技术有限公司)刺激来自于基因型为HLA-A1101的健康志愿者的外周血淋巴细胞(PBL)。将EBV LMp2A 340-349 SSCSSCPLSK短肽与带有生物素标记的HLA-A*1101复性,制备pHLA单倍体。这些单倍体与用PE标记的链霉亲和素(BD公司)组合成PE标记的四聚体，分选该四聚体及抗-CD8-APC双阳性细胞。扩增分选的细胞，并按上述方法进行二次分选，随后用有限稀释法进行单克隆。单克隆细胞用四聚体染色，筛选到的双阳性克隆如图3所示。Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A1101 were stimulated with synthetic short peptide EBV LMp2A 340-349 SSCSSCPLSK (Beijing Cypress Biotech Co., Ltd.). The EBV LMp2A 340-349 SSCSSCPLSK short peptide was renatured with biotinylated HLA-A*1101 to prepare a pHLA haploid. These haploids were combined with PE-labeled streptavidin (BD) into PE-labeled tetramers, and the tetramer and anti-CD8-APC double positive cells were sorted. The sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonalization by limiting dilution. Monoclonal cells were stained with tetramers and the double positive clones screened are shown in Figure 3.
实施例2 获取EBV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 EBV-Specific T Cell Clones
用Quick-RNATM MiniPrep(ZYMO research)抽提实施例1中筛选到的EBV LMp2A 340-349特异性、HLA-A1101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 ) in Example 1 to EBV LMp2A 340-349 screened specific, HLA-A1101 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-TTSDR      (SEQ ID NO:10)α CDR1-TTSDR (SEQ ID NO: 10)
α  CDR2-LLSNGAV      (SEQ ID NO:11)α CDR2-LLSNGAV (SEQ ID NO: 11)
α  CDR3-AISTGFQKLV    (SEQ ID NO:12)α CDR3-AISTGFQKLV (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-SNHLY      (SEQ ID NO:13)β CDR1-SNHLY (SEQ ID NO: 13)
β  CDR2-FYNNEI      (SEQ ID NO:14)β CDR2-FYNNEI (SEQ ID NO: 14)
β  CDR3-ASSEGPSGSSYEQY    (SEQ ID NO:15)。β CDR3-ASSEGPSGSSYEQY (SEQ ID NO: 15).
通过重叠(overlap)PCR分别将TCRα链和β链的全长基因克隆至慢病毒表达载体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-EBVTRA-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 full-length genes of the TCR alpha chain and the beta chain were cloned into the lentiviral expression vector pLenti (addgene) by overlap PCR, 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. Lentiviral expression vector and TCRα -2A-TCRβ was digested to obtain the pLenti-EBVTRA-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 EBV特异性T细胞受体慢病毒包装与原代T细胞转染EBV TCRExample 3 EBV-specific T cell receptor lentiviral packaging and primary T cell transfection EBV 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-EBVTRA-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. Using the Express-In-mediated transient transfection (Open Biosystems), four plasmids (containing the pLenti-EBVTRA-2A-TRB-IRES-NGFR described in Example 2) A lentiviral vector, and 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-EBVTRA-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，等分分装后-80℃冻存。取假病毒样品进行病毒滴度测定，步骤参照p24 ELISA(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-EBVTRA-2A-TRB-IRES-NGFR and pLenti-eGFP pseudoviruses were packaged. 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 concentrating tube (Merck Millipore, catalog number UFC905096) was concentrated to remove most of the supernatant, finally concentrated to 1 ml, and aliquoted at -80 ° C for cryopreservation. 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EBV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 an EBV-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，life technologies，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 coated with 1 x 10 6 cells/ml (0.5 ml/well) and pre-washed anti-CD3/CD28 antibody-coated beads (T cells, life technologies, Cat. No. 11452D) was incubated overnight for stimulation, cell: beads = 3:1.
刺激过夜后，根据p24 ELISA试剂盒所测到的病毒滴度，按MOI＝10的比例加入已浓缩的EBV特异性T细胞受体基因的慢病毒，32℃，900g离心感染1小时。感染完毕后去除慢病毒感染液，用含有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 concentrated EBV-specific T cell receptor gene lentivirus was added at a MOI=10 ratio 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 containing 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
EBV LMp2A 340-349 SSCSSCPLSK短肽与带有生物素标记的HLA-A*1101复性，制备pHLA单倍体。这些单倍体用PE标记的链霉亲和素(BD)组合成PE标记的四聚体,称为EBV-tetramer-PE。此四聚体能把表达了EBV特异性T细胞受体基因的T细胞标记为阳性细胞。把(b)中经转染的T细胞样品与EBV-tetramer-PE在冰上孵育30分钟，然后加入anti-CD8-APC(biolegend)抗体，继续冰上孵育15分钟。样品用含有2％FBS的PBS清洗2次后用BD Calibur或BD Arial检测或分选表达了EBV特异性T细胞受体基因的EBV-tetramer-PE和CD8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。EBV LMp2A 340-349 SSCSSCPLSK short peptide was renatured with biotinylated HLA-A*1101 to prepare pHLA haploid. These haploids were combined with PE-labeled streptavidin (BD) into a PE-labeled tetramer called EBV-tetramer-PE. This tetramer can label T cells expressing an EBV-specific T cell receptor gene as positive cells. The transfected T cell samples in (b) were incubated with EBV-tetramer-PE for 30 minutes on ice, then anti-CD8-APC (biolegend) antibody was added and incubation was continued for 15 minutes on ice. Samples were washed twice with PBS containing 2% FBS and EBV-tetramer-PE and CD8 double positive T cells expressing EBV-specific T cell receptor gene were detected or sorted by BD Calibur or BD Arial. Data analysis was performed using CellQuest. Software (BD) or FlowJo software (Tree Star Inc, Ashland, OR) analysis.
经检测分析，结果如图4所示，用EBV-tetramer-PE和抗CD8抗体染色后，未经TCR慢病毒感染的NC组T细胞无EBV-tetramer-PE阳性细胞，而经TCR慢病毒感染的T细胞出现较多表达TCR的EBV-tetramer-PE阳性细胞(EBV tetramer组)，当用非EBV-tetramer-PE的其他tetramer-PE(Nonspecific tetramer组)染色时只有少量非特异性的阳性细胞。After analysis and analysis, the results are shown in Figure 4. After staining with EBV-tetramer-PE and anti-CD8 antibody, NC group T cells without TCR lentivirus infection were free of EBV-tetramer-PE positive cells, but infected with TCR lentivirus. T cells with more TCR-expressing EBV-tetramer-PE positive cells (EBV tetramer group) showed only a small amount of non-specific positive cells when stained with other tetramer-PE (Nonspecific tetramer group) other than EBV-tetramer-PE.
实施例4 转导本发明TCR的T细胞的特异性激活实验Example 4 Specific Activation Experiment of T Cells Transducing 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的激活反应。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 the activation response of TCR-transduced T cells 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)，RPMI 1640(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-γELISPOT 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(LCL)。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(LCL)。本ELISPOT试验以HLA-A11为靶细胞。The target cell of this example is an Epstein-Barr virus (EBV) transformed immortalized lymphoblastoid cell line (LCL). 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 the peripheral blood lymphocytes (PBL) of healthy volunteers with genotype HLA-A11/A02/A24, take 10 ml of PBL suspension with a concentration of 2×10 7 /ml in a 25 cm square culture flask and add the ring. After incubating for 1 hour in a 37 ° C / CO 2 incubator, one EBV was quickly thawed, added to the above cells in 1/10 dilution, gently shaken and placed upright at 37 ° C / CO 2 Culture in an incubator. After 12 days of culture, the culture was continued by adding 10 ml of the medium, and after about 30 days, the culture was further expanded and subjected to flow detection, wherein CD19+CD23hiCD58+ was an immortalized lymphoblastoid cell line (LCL). The ELISPOT assay targets HLA-A11 as a 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EBV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，life technologies)刺激T细胞，用携带EBV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 EBV-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 (life technologies), transduced with lentivirus carrying the EBV-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.
EBV CD8+T细胞(EBV TCR转导的T细胞，效应细胞)、CD8+T细胞(阴性对照效应细胞)和LCL细胞(靶细胞)依据实施例3所述制备，LCL包括特异性靶细胞LCL-A11(基因型为HLA-A11的LCL细胞)和非特异性靶细胞LCL-A24(基因型为HLA-A24的LCL细胞)并在相应实验组加入对应短肽，其中PEBV为LMp2A 340-349 SSCSSCPLSK短肽，PA24为非EBV TCR特异结合短肽。EBV CD8+ T cells (EBV TCR transduced T cells, effector cells), CD8+ T cells (negative control effector cells) and LCL cells (target cells) were prepared as described in Example 3, which includes specific target cell LCL -A11 (LCL cells with genotype HLA-A11) and non-specific target cells LCL-A24 (LCL cells with genotype HLA-A24) and add corresponding short peptides in the corresponding experimental group, wherein P EBV is LMp2A 340-349 SSCSSCPLSK short peptide, P A24 is a non-EBV TCR specific binding short peptide.
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EBV TCR阳性T细胞)。50 microliters of effector cells (1000 EBV TCR positive T cells).
20微升10-4摩尔/升的EBV LMp2A 340-349 SSCSSCPLSK/PA24短肽溶液(终浓度为10-5摩尔/升)。20 μl of 10-45 mol/L EBV LMp2A 340-349 SSCSSCPLSK/PA24 short peptide solution (final concentration 10-5 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1：10000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。100 μl of diluted streptavidin-alkaline phosphatase was added to each well with 1:10000 dilution of streptavidin-alkaline phosphatase in PBS containing 10% FBS and the well plate was incubated 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 the BCIP/NBT solution and rinse the plate with double distilled water to stop the development reaction, dry, then remove the bottom of the plate and dry the plate at room temperature. Until each well was completely dry, spots formed by the basement membrane of the well plate were counted using an immunospot plate counter (CTL, Cellular Technology Limited).
结果result
通过ELISPOT试验(如上所述)检验EBV TCR转导的T细胞对负载EBV LMp2A 340-349 SSCSSCPLSK短肽特异性靶细胞和非特异性靶细胞起反应的IFN-γ释放。利用graphpad prism6绘制各孔中观察到的ELSPOT斑点数量。IFN-[gamma] release by EBV TCR transduced T cells against EBV LMp2A 340-349 SSCSSCPLSK short peptide-specific target cells and non-specific target cells was examined by ELISPOT assay (described above). The number of ELSPOT spots observed in each well was plotted using graphpad prism6.
实验结果如图5所示，转导本发明TCR的T细胞仅对特定基因型的负载其特异的短肽的靶细胞有激活反应，而对其他非特异靶细胞基本没有激活反应。同时，也可以看出未转导本发明TCR的T细胞的激活反应很差。As shown in Fig. 5, the T cells transducing the TCR of the present invention have an activation reaction only to target cells of a specific genotype carrying a specific short peptide, and substantially no activation reaction to other non-specific target cells. At the same time, it can also be seen that the activation reaction of T cells which are not transduced with the TCR of the present invention is poor.
实施例5 转导本发明TCR的T细胞的非放射性细胞毒性试验Example 5 Non-radioactive cytotoxicity test for transduction of TCR T cells of the present invention
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 51 Cr 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 PCTCN2015097312-appb-000001
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 PCTCN2015097312-appb-000001
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(Gibco))，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, Gibco), phenol red containing 90% RPMI 1640 (Gibco, catalog number C11875500bt), 1% penicillin/streptococcus Prime (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
该试验所用的靶细胞LCL与实施例4的ELISPOT方案中的靶细胞制备方法一样，本方案中用到的靶细胞包括LCL-A11、LCL-A02(基因型为HLA-A02的LCL细胞)和LCL-A24。在试验培养基中制备靶细胞：靶细胞浓度调至3×105个/毫升，每孔取50微升从而得1.5×104个细胞/孔。The target cell LCL used in this assay is the same as the target cell preparation method in the ELISPOT protocol of Example 4. The target cells used in this protocol include LCL-A11, LCL-A02 (LCL cells with genotype HLA-A02) and LCL-A24. Target cells were prepared in the test medium: the target cell concentration was adjusted to 3 × 10 5 /ml, and 50 μl per well was taken to obtain 1.5 × 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EBV特异性TCR的CD8+T细胞。效应细胞与靶细胞之比采用1：1(3×105个/毫升，每孔取50微升从而得1.5×104个细胞/孔)。The effector cells (T cells) of this assay were CD8+ T cells expressing EBV-specific TCR by flow cytometry in Example 3. The ratio of effector cells to target cells was 1:1 (3 × 10 5 /ml, 50 μl per well to obtain 1.5 × 10 4 cells/well).
短肽溶液制备Preparation of short peptide solution
LMp2A 340-349 SSCSSCPLSK短肽用含10％FBS的RPMI 1640培养基通过10倍稀释依次稀释成10-4M-10-12M的工作液，使其加入到实验组后最终浓度梯度为10-5M-10-13M。LMp2A 340-349 SSCSSCPLSK short peptide was diluted with 10% FBS in RPMI 1640 medium to a 10 -4 M-10 -12 M working solution, and added to the experimental group with a final concentration gradient of 10 - 5 M-10 -13 M.
(a)通过靶细胞负载不同浓度的短肽检测效应细胞杀伤能力(a) Detection of effector cell killing ability by loading different concentrations of short peptides on target cells
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:
-50ul靶细胞(如上所述制备)加入各孔 -50 ul of target cells (prepared as described above) were added to each well
-50ul效应细胞(如上所述制备)加入各孔-50 ul of effector cells (prepared as described above) were added to each well
-12ul短肽溶液加入各孔-12ul short peptide solution added to each well
-8ul培养补入孔(不负载短肽实验组直接补20ul培养基)。-8 ul culture supplemental wells (not loaded with short peptide experimental group directly supplemented with 20 ul of medium).
如下所述制备对照组：A control group was prepared as follows:
-不负载短肽实验组(未处理的LCL-A11)：含有50ul效应细胞和50ul靶细胞。- Unloaded short peptide experimental group (untreated LCL-A11): containing 50 ul of effector cells and 50 ul of target cells.
-效应细胞自发释放：仅有50ul效应细胞。- Spontaneous release of effector cells: only 50 ul of effector cells.
-靶细胞自发释放：仅有50ul靶细胞。- Spontaneous release of target cells: only 50 ul of target cells.
-靶细胞最大释放：仅有50ul靶细胞。- Maximum release of target cells: only 50 ul of target cells.
-试剂培养基对照：仅有120ul培养基。- Reagent medium control: only 120 ul of medium.
所有孔一式三份制备，终体积为120ul(不够的用培养基补足)。All wells were prepared in triplicate with a final volume of 120 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. Prior to collection of all well supernatants, the target cells were maximally released. Control cells were placed at -70 ° C for approximately 30 minutes and then thawed at 37 ° C for 15 minutes to allow total lysis of 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EBV TCR转导的T细胞对负载EBV LMp2A 340-349 SSCSSCPLSK短肽特异性靶细胞和非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。LDH release by EBV TCR transduced T cells against EBV LMp2A 340-349 SSCSSCPLSK short peptide-specific target cells and non-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6所示，在10-5M-10-13M的短肽浓度范围内EBV CD8+T细胞随着短肽浓度的升高，其对靶细胞LCL-A11的杀伤作用增强；对不负载短肽的靶细胞LCL-A11无杀伤作用。The statistical results of the experimental data are shown in Figure 6. In the short peptide concentration range of 10 -5 M -10 -13 M, the killing effect of EBV CD8+ T cells on the target cell LCL-A11 with the increase of the concentration of short peptides. Enhanced; no killing effect on target cell LCL-A11 without loading short peptide.
(b)通过不同靶细胞负载短肽检测效应细胞特异性杀伤能力(b) Detection of effector cell-specific killing ability by loading short peptides on different target cells
靶细胞制备Target cell preparation
LCL-A11、LCL-A02和LCL-A24三种靶细胞加入10-5M LMp2A 340-349 SSCSSCPLSK(PEBV)短肽，37℃温育2小时。去除含短肽的培养基，用新的试验培养基重悬细胞至3×105个/毫升，每孔取50微升从而得1.5×104个细胞/孔。另设三种靶细胞不负载短肽作为空白对照组。Three target cells, LCL-A11, LCL-A02 and LCL-A24, were added to 10 -5 M LMp2A 340-349 SSCSSCPLSK (PEBV) short peptide and incubated for 2 hours at 37 °C. The medium containing the short peptide was removed, and the cells were resuspended to 3 × 10 5 /ml with a new test medium, and 50 μl per well to obtain 1.5 × 10 4 cells/well. Another three target cells were not loaded with short peptides as a blank control group.
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EBV特异性TCR的CD8+T细胞。效应细胞与靶细胞之比采用1：1(3×105个/毫升，每孔取50微升从而得1.5×104个细胞/孔)。 The effector cells (T cells) of this assay were CD8+ T cells expressing EBV-specific TCR by flow cytometry in Example 3. The ratio of effector cells to target cells was 1:1 (3 × 10 5 /ml, 50 μl per well to obtain 1.5 × 10 4 cells/well).
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:
-50ul靶细胞(如上所述制备)加入各孔-50 ul of target cells (prepared as described above) were added to each well
-50ul效应细胞(如上所述制备)加入各孔-50 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:
-不负载短肽实验组：含有50ul效应细胞和50ul靶细胞。- Unloaded short peptide experimental group: containing 50 ul of effector cells and 50 ul of target cells.
-效应细胞自发释放：仅有50ul效应细胞。- Spontaneous release of effector cells: only 50 ul of effector cells.
-靶细胞自发释放：仅有50ul靶细胞。- Spontaneous release of target cells: only 50 ul of target cells.
-靶细胞最大释放：仅有50ul靶细胞。- Maximum release of target cells: only 50 ul of target cells.
-试剂培养基对照：仅有100ul培养基。- Reagent medium control: only 100 ul of medium.
所有孔一式三份制备，终体积为120ul(不够的用培养基补足)。All wells were prepared in triplicate with a final volume of 120 ul (not enough to make up with medium).
细胞培养、样品处理及检测和计算结果方法与本实施例(a)试验一样。The method of cell culture, sample processing, and detection and calculation results was the same as that of the experiment of Example (a).
结果result
图7显示EBV CD8+T细胞对负载了短肽LMp2A 340-349 SSCSSCPLSK(PEBV)的LCL-A11靶细胞的特异性杀伤作用。而对不负载短肽或负载短肽的其他靶细胞无明显杀伤作用。Figure 7 shows the specific killing effect of EBV CD8+ T cells on LCL-A11 target cells loaded with the short peptide LMp2A 340-349 SSCSSCPLSK (P EBV ). There is no obvious killing effect on other target cells that do not load short peptides or load short peptides.
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 (24)

  1. 一种T细胞受体(TCR)，其特征在于，其包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域包含3个互补决定区(CDR)：A T cell receptor (TCR) comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain comprising three complementarity determining regions (CDRs):
    αCDR1：TTSDR(SEQ ID NO:10)αCDR1: TTSDR (SEQ ID NO: 10)
    αCDR2：LLSNGAV(SEQ ID NO:11)和αCDR2: LLSNGAV (SEQ ID NO: 11) and
    αCDR3：AISTGFQKLV(SEQ ID NO:12)和/或所述TCRβ链可变域包含3个互补决定区：αCDR3: AISTGFQKLV (SEQ ID NO: 12) and/or the TCR β chain variable domain comprises three complementarity determining regions:
    βCDR1：SNHLY(SEQ ID NO:13)βCDR1: SNHLY (SEQ ID NO: 13)
    βCDR2：FYNNEI(SEQ ID NO:14)和βCDR2: FYNNEI (SEQ ID NO: 14) and
    βCDR3：ASSEGPSGSSYEQY(SEQ ID NO:15)。βCDR3: ASSEGPSGSSYEQY (SEQ ID NO: 15).
  2. 如权利要求1所述的TCR，其特征在于，所述TCRα链可变域包含与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链可变域包含与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, wherein said TCR alpha chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; and/or said TCR beta chain variable domain comprises SEQ ID NO: 5 has an amino acid sequence of at least 90% sequence identity.
  3. 如权利要求1或2所述的TCR，其特征在于，所述TCR包含α链可变域氨基酸序列SEQ ID NO:1。The TCR according to claim 1 or 2, wherein the TCR comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1.
  4. 如权利要求1-3中任一所述的TCR，其特征在于，所述TCR包含β链可变域氨基酸序列SEQ ID NO:5。The TCR according to any one of claims 1 to 3, wherein the TCR comprises the β chain variable domain amino acid sequence of SEQ ID NO: 5.
  5. 如以上任一权利要求所述TCR，其特征在于，所述TCR为αβ异质二聚体。A TCR according to any of the preceding claims, wherein the TCR is an alpha beta heterodimer.
  6. 如权利要求5所述的TCR，其特征在于，其包含TCRα链恒定区TRAC*01和TCRβ链恒定区TRBC1*01或TRBC2*01。The TCR according to claim 5, which comprises a TCR alpha chain constant region TRAC*01 and a TCR beta chain constant region TRBC1*01 or TRBC2*01.
  7. 如权利要求6中所述的TCR，其特征在于，所述TCR的α链氨基酸序列为SEQ ID NO：3。The TCR according to claim 6, wherein the amino acid sequence of the α chain of the TCR is SEQ ID NO: 3.
  8. 如权利要求6或7中所述TCR，其特征在于，所述TCR的β链氨基酸序列为SEQ ID NO:7。The TCR according to claim 6 or 7, wherein the β chain amino acid sequence of the TCR is SEQ ID NO: 7.
  9. 如权利要求1-6中任一所述TCR，其特征在于，所述TCR是可溶的。A TCR according to any one of claims 1 to 6, wherein the TCR is soluble.
  10. 如权利要求9所述的TCR，其特征在于，半胱氨酸残基在所述TCR的α和β链恒定域之间形成人工二硫键。The TCR according to claim 9, wherein the cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of said TCR.
  11. 如权利要求10所述的TCR，其特征在于，在所述TCR中形成人工二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：The TCR according to claim 10, wherein the cysteine residue forming an artificial disulfide bond in said TCR replaces one or more groups 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.
  12. 如权利要求9所述的TCR，其特征在于，所述TCR为单链。 The TCR of claim 9 wherein said TCR is single stranded.
  13. 如权利要求12所述的TCR，其特征在于，所述TCR是由α链可变域与β链可变域通过肽连接序列连接而成。The TCR according to claim 12, wherein the TCR is formed by linking an α chain variable domain and a β chain variable domain through a peptide linking sequence.
  14. 如权利要求13所述的TCR，其特征在于，所述TCR在α链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或α链J基因短肽氨基酸倒数第3位、倒数第5位或倒数第7位中具有一个或多个突变；和/或所述TCR在β链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或β链J基因短肽氨基酸倒数第2位、倒数第4位或倒数第6位中具有一个或多个突变，其中氨基酸位置编号按IMGT(国际免疫遗传学信息***)中列出的位置编号。The TCR according to claim 13, wherein the TCR is at the 11th, 13, 19, 21, 53, 76, 89, 91, or 94th, and/or alpha chain of the alpha chain variable region amino acid. J gene short peptide amino acid has the one or more mutations in the third, the fifth or the third of the penultimate amino acid; and/or the TCR is in the beta chain variable region amino acids 11, 13, 19, 21, 53 , 76, 89, 91, or 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 The location number listed in the International Immunogenetics Information System.
  15. 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.
  16. 如权利要求15所述的核酸分子，其特征在于，其包含编码TCRα链可变域的核苷酸序列SEQ ID NO：2。The nucleic acid molecule according to claim 15, which comprises a nucleotide sequence of SEQ ID NO: 2 encoding a TCR alpha chain variable domain.
  17. 如权利要求15或16所述的核酸分子，其特征在于，其包含编码TCRβ链可变域的核苷酸序列SEQ ID NO：6。A nucleic acid molecule according to claim 15 or 16, which comprises a nucleotide sequence of SEQ ID NO: 6 encoding a TCR β chain variable domain.
  18. 如权利要求15-17中任一所述的核酸分子，其特征在于，其包含编码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。A nucleic acid molecule according to any one of claims 15-17, which comprises the nucleotide sequence SEQ ID NO: 4 encoding the TCR alpha chain and/or comprises the nucleotide sequence encoding the TCR beta chain SEQ ID NO: 8.
  19. 一种载体，其特征在于，所述的载体含有权利要求15-18中任一所述的核酸分子；优选地，所述的载体为病毒载体；更优选地，所述的载体为慢病毒载体。A vector, characterized in that the vector comprises the nucleic acid molecule of any one of claims 15-18; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector .
  20. 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19中所述的载体或染色体中整合有外源的权利要求15-18中任一所述的核酸分子。An isolated host cell, characterized in that the host cell comprises the vector of claim 19 or the nucleic acid molecule of any one of claims 15-18 integrated into the chromosome.
  21. 一种细胞，其特征在于，所述细胞转导权利要求15-18中任一所述的核酸分子或权利要求19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 any one of claims 15 to 18 or the vector of claim 19; preferably, the cell is a T cell or a stem cell.
  22. 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1-14中任一项所述的TCR、或权利要求15-18中任一所述的核酸分子、或权利要求21中所述的细胞。A pharmaceutical composition, comprising a pharmaceutically acceptable carrier, and the TCR according to any one of claims 1 to 14, or the nucleic acid molecule of any one of claims 15 to 18. Or the cell of claim 21.
  23. 权利要求1-14中任一项所述的T细胞受体或权利要求21中所述的细胞的用途，其特征在于，用于制备***或EB病毒感染的药物。Use of a T cell receptor according to any one of claims 1 to 14 or a cell according to claim 21 for the preparation of a medicament for the treatment of a tumor or an EB virus infection.
  24. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用适量的权利要求1-14中任一所述的TCR、或权利要求21中所述的细胞或权利要求22中所述的药物组合物；A method for treating a disease, which comprises administering to a subject in need of treatment an appropriate amount of the TCR according to any one of claims 1 to 14, or the cell of claim 21 or the method of claim 22. Pharmaceutical composition;
    优选地，所述的疾病为EBV阳性霍奇金淋巴瘤、EBV阳性鼻咽癌、EBV阳性移植后淋巴增生病或伯基特淋巴瘤。 Preferably, the disease is EBV-positive Hodgkin's lymphoma, EBV-positive nasopharyngeal carcinoma, EBV-positive post-transplant lymphoproliferative disease or Burkitt's lymphoma.
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