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WO2016095783A1 - Récepteur de lymphocytes t pour identifier le peptide court du virus d'epstein-barr (eb) - Google Patents

Récepteur de lymphocytes t pour identifier le peptide court du virus d'epstein-barr (eb) Download PDF

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Publication number
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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English (en)
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/zh
Publication of WO2016095783A1 publication Critical patent/WO2016095783A1/fr

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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.

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Abstract

L'invention concerne un récepteur de lymphocytes T (TCR) pouvant se combiner avec un peptide dérivé d'une protéine de membrane latente (LMP-2) du virus d'Epstein barr (virus EB). Le peptide se présente sous la forme d'un composé SSCSSCPLSK-HLA A1101. L'invention concerne également une molécule d'acide nucléique permettant de coder pour le TCR, un support comprenant la molécule d'acide nucléique et une cellule permettant de transférer le TCR.
PCT/CN2015/097312 2014-12-17 2015-12-14 Récepteur de lymphocytes t pour identifier le peptide court du virus d'epstein-barr (eb) WO2016095783A1 (fr)

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WO2020082130A1 (fr) * 2018-10-25 2020-04-30 The Council Of The Queensland Institute Of Medical Research Récepteurs de lymphocytes t et utilisations associées
EP3786178A1 (fr) * 2019-08-30 2021-03-03 Max-Delbrück-Centrum für Molekulare Medizin in der Helmholtz-Gemeinschaft Constructions tcr spécifiques pour antigènes dérivés du virus d'epstein-barr (ebv)
CN112940108A (zh) * 2021-03-19 2021-06-11 河南省肿瘤医院 识别ebv抗原的t细胞受体以及该t细胞受体的应用
CN112940109A (zh) * 2021-03-19 2021-06-11 河南省肿瘤医院 识别ebv抗原的t细胞受体及其应用
CN113423724A (zh) * 2018-12-27 2021-09-21 深圳华大生命科学研究院 Ebv表位高亲和力t细胞受体
WO2021243695A1 (fr) * 2020-06-05 2021-12-09 Guangdong Tcrcure Biopharma Technology Co., Ltd. Thérapie cellulaire tcr-t ciblant le virus d'epstein-barr
US20230079750A1 (en) * 2021-04-05 2023-03-16 Janssen Biotech, Inc. Calr and jak2 t-cell receptors
US12139523B2 (en) 2018-11-27 2024-11-12 Duke University Anti-LMP2 TCR-T cell therapy for the treatment of EBV-associated cancers

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CN108997481B (zh) * 2017-06-07 2022-06-03 中国科学院广州生物医药与健康研究院 源自于lmp1的抗原短肽
CN110407926B (zh) * 2018-04-26 2022-09-09 香雪生命科学技术（广东）有限公司 一种识别lmp1抗原短肽的tcr及其编码序列
CN113308465A (zh) * 2021-06-19 2021-08-27 广东天科雅生物医药科技有限公司 一种针对表位点为sscsscplsk的tcr所设计的引物及其应用

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Cited By (11)

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Publication number Priority date Publication date Assignee Title
WO2020082130A1 (fr) * 2018-10-25 2020-04-30 The Council Of The Queensland Institute Of Medical Research Récepteurs de lymphocytes t et utilisations associées
US12139523B2 (en) 2018-11-27 2024-11-12 Duke University Anti-LMP2 TCR-T cell therapy for the treatment of EBV-associated cancers
CN113423724A (zh) * 2018-12-27 2021-09-21 深圳华大生命科学研究院 Ebv表位高亲和力t细胞受体
CN113423724B (zh) * 2018-12-27 2023-11-24 深圳华大生命科学研究院 Ebv表位高亲和力t细胞受体
EP3786178A1 (fr) * 2019-08-30 2021-03-03 Max-Delbrück-Centrum für Molekulare Medizin in der Helmholtz-Gemeinschaft Constructions tcr spécifiques pour antigènes dérivés du virus d'epstein-barr (ebv)
WO2021038031A1 (fr) * 2019-08-30 2021-03-04 Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft Constructions de tcr spécifiques des antigènes dérivés du veb
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WO2021243695A1 (fr) * 2020-06-05 2021-12-09 Guangdong Tcrcure Biopharma Technology Co., Ltd. Thérapie cellulaire tcr-t ciblant le virus d'epstein-barr
CN112940108A (zh) * 2021-03-19 2021-06-11 河南省肿瘤医院 识别ebv抗原的t细胞受体以及该t细胞受体的应用
CN112940109A (zh) * 2021-03-19 2021-06-11 河南省肿瘤医院 识别ebv抗原的t细胞受体及其应用
US20230079750A1 (en) * 2021-04-05 2023-03-16 Janssen Biotech, Inc. Calr and jak2 t-cell receptors

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