CN115160432A - T cell receptor recognizing AFP - Google Patents

T cell receptor recognizing AFP Download PDF

Info

Publication number
CN115160432A
CN115160432A CN202110363579.8A CN202110363579A CN115160432A CN 115160432 A CN115160432 A CN 115160432A CN 202110363579 A CN202110363579 A CN 202110363579A CN 115160432 A CN115160432 A CN 115160432A
Authority
CN
China
Prior art keywords
tcr
ser
seq
gly
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110363579.8A
Other languages
Chinese (zh)
Inventor
李懿
李振娟
何均仪
钟时
吴万里
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xiangxue Life Science Technology Guangdong Co ltd
Original Assignee
Xiangxue Life Science Technology Guangdong Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xiangxue Life Science Technology Guangdong Co ltd filed Critical Xiangxue Life Science Technology Guangdong Co ltd
Priority to CN202110363579.8A priority Critical patent/CN115160432A/en
Priority to PCT/CN2022/084190 priority patent/WO2022206861A1/en
Publication of CN115160432A publication Critical patent/CN115160432A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46448—Cancer antigens from embryonic or fetal origin
    • A61K39/464481—Alpha-feto protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Pregnancy & Childbirth (AREA)
  • Reproductive Health (AREA)
  • Oncology (AREA)

Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding a short peptide KWVESIFLIF derived from an AFP antigen, which can form a complex with HLA a2402 and be presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.

Description

T cell receptor recognizing AFP
Technical Field
The present invention relates to a TCR capable of recognising short peptides derived from the AFP antigen and its coding sequence, to AFP-specific T cells obtained by transduction of the above TCR, and to their use in the prevention and treatment of AFP-related diseases.
Background
AFP (alpha Fetoprotein), also called alpha Fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver, and is subsequently inhibited. In hepatocellular carcinoma, AFP expression is activated (Butterfield et al.J. Immunol.,2001, apr15 166 (8): 5300-8). AFP is degraded into small polypeptides after intracellular production and binds to MHC (major histocompatibility complex) molecules to form complexes, which are presented on the cell surface. KWVESIFILIF (SEQ ID NO: 9) is a short peptide derived from the AFP antigen, and is a target for the treatment of AFP-related diseases.
T cell adoptive immunotherapy is the transfer of reactive T cells specific for a target cell antigen into a patient to act on the target cell. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that recognizes a corresponding short peptide antigen on the surface of a target cell. In the immune system, the direct physical contact between T cells and Antigen Presenting Cells (APC) is initiated by the combination of antigen short peptide specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then other cell membrane surface molecules of the T cells and APC interact to cause a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities can exert immune effects on target cells. Accordingly, those skilled in the art have focused on isolating TCRs specific for short AFP antigen peptides and transducing the TCRs into T cells to obtain T cells specific for short AFP antigen peptides, thereby allowing them to function in cellular immunotherapy.
Disclosure of Invention
The invention aims to provide a T cell receptor for recognizing AFP antigen short peptide.
In a first aspect of the invention, there is provided a T Cell Receptor (TCR) capable of binding to the KWVESIFLIF-HLA a2402 complex.
In another preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the amino acid sequence of CDR3 of the TCR alpha chain variable domain is AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSYPGSYGYT (SEQ ID NO: 15).
In another preferred embodiment, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:
αCDR1-VGISA(SEQ ID NO:10)
αCDR2-LSSGK(SEQ ID NO:11)
α CDR3-AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-MNHEY(SEQ ID NO:13)
βCDR2-SVGAGI(SEQ ID NO:14)
βCDR3-ASSYPGSYGYT(SEQ ID NO:15)。
in another preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 5.
In another preferred embodiment, the TCR comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1.
In another preferred embodiment, the TCR comprises the beta chain variable domain amino acid sequence SEQ ID NO 5.
In another preferred embodiment, the TCR is an α β heterodimer comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC1 01 or TRBC2 01.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO 3 and/or the β chain amino acid sequence of the TCR is SEQ ID NO 7.
In another preferred embodiment, the TCR is of human origin.
In another preferred embodiment, the TCR is isolated and purified.
In another preferred embodiment, the TCR is soluble.
In another preferred embodiment, the TCR is single-chain.
In another preferred embodiment, the TCR is formed by linking an α chain variable domain to a β chain variable domain via a peptide linker.
In another preferred embodiment, the TCR comprises an alpha chain constant region and a beta chain constant region, the alpha chain constant region being murine constant region and/or the beta chain constant region being murine constant region.
In another preferred embodiment, the TCR has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the α chain variable region, and/or in the penultimate 3-, 5-, or 7-position of the short peptide amino acid of the α chain J gene; and/or the TCR has one or more mutations in beta chain variable region amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 th, and/or beta chain J gene short peptide amino acid penultimate 2,4 or 6 th, wherein the amino acid position numbering is according to the position numbering listed in IMGT (international immunogenetic information system).
In another preferred embodiment, the α chain variable domain amino acid sequence of the TCR comprises SEQ ID NO 32 and/or the β chain variable domain amino acid sequence of the TCR comprises SEQ ID NO 34.
In another preferred embodiment, the amino acid sequence of the TCR is SEQ ID NO 30.
In another preferred embodiment, the TCR comprises (a) all or part of a TCR α chain, excluding the transmembrane domain; and (b) all or part of the TCR β chain, excluding the transmembrane domain;
and (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a portion of the TCR chain constant domain.
In another preferred embodiment, cysteine residues form an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.
In another preferred embodiment, the cysteine residues forming the artificial disulfide bond in the TCR are substituted at one or more groups of sites selected from the group consisting of:
thr48 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser57 of TRBC2 × 01 exon 1;
thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser77 of TRBC2 × 01 exon 1;
tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01;
thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1;
ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15;
arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1;
pro89 of exon 1 TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 of Ala19 of exon 1; and Tyr10 of exon 1 TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 of Glu20 of exon 1.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO 26 and/or the β chain amino acid sequence of the TCR is SEQ ID NO 28.
In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond between the α chain variable region and the β chain constant region.
In another preferred embodiment, the cysteine residues that form the artificial interchain disulfide bond in the TCR replace one or more groups of sites selected from the group consisting of:
amino acid 46 of TRAV and amino acid 60 of exon 1 TRBC1 x 01 or TRBC2 x 01;
amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01;
amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; or
Amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01.
In another preferred embodiment, the TCR comprises an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain, excluding the transmembrane domain, but which does not comprise an alpha chain constant domain, the alpha chain variable domain of the TCR forming a heterodimer with the beta chain.
In another preferred embodiment, the TCR has a conjugate attached to the C-or N-terminus of the alpha and/or beta chain.
In another preferred embodiment, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these. Preferably, the therapeutic agent is an anti-CD 3 antibody.
In a second aspect of the invention, there is provided a multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR according to the first aspect of the invention.
In a third aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to the first aspect of the invention, or the complement thereof.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 2 or SEQ ID NO 33 encoding the variable domain of the TCR alpha chain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 6 or SEQ ID NO 35 encoding the variable domain of the TCR β chain.
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.
In a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the third aspect of the invention; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
In a fifth aspect of the invention, there is provided an isolated host cell comprising a vector according to the fourth aspect of the invention or a genome into which has been integrated an exogenous nucleic acid molecule according to the third aspect of the invention.
In a sixth aspect of the invention, there is provided a cell transduced with a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention; preferably, the cell is a T cell, NK cell, NKT cell or stem cell.
In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention.
In an eighth aspect of the invention there is provided the use of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, in the manufacture of a medicament for the treatment of a tumour or an autoimmune disease, preferably wherein the tumour is liver cancer.
According to a ninth aspect of the invention there is provided a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, for use as a medicament for the treatment of a tumour or an autoimmune disease; preferably, the tumor is liver cancer.
In a tenth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention; preferably, the disease is a tumor, more preferably, the tumor is liver cancer.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.
Drawings
FIG. 1a, FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e and FIG. 1f are the amino acid sequence of the TCR α chain variable domain, the nucleotide sequence of the TCR α chain variable domain, the amino acid sequence of the TCR α chain, the nucleotide sequence of the TCR α chain, the amino acid sequence of the TCR α chain with leader sequence and the nucleotide sequence of the TCR α chain with leader sequence, respectively.
Fig. 2a, fig. 2b, fig. 2c, fig. 2d, fig. 2e and fig. 2f are a TCR β chain variable domain amino acid sequence, a TCR β chain variable domain nucleotide sequence, a TCR β chain amino acid sequence, a TCR β chain nucleotide sequence with a leader sequence, and a TCR β chain nucleotide sequence with a leader sequence, respectively.
FIG. 3 shows the double positive staining results of monoclonal cells for CD8+ and tetramer-PE.
Figure 4a and figure 4b are the amino acid and nucleotide sequences, respectively, of a soluble TCR alpha chain.
Figure 5a and figure 5b are the amino acid and nucleotide sequences, respectively, of a soluble TCR β chain.
Fig. 6a and 6b are gel maps of the soluble TCRs obtained after purification. In FIGS. 6a and 6b, the lanes on the right side are reducing gel and non-reducing gel, respectively, and the lanes on the left side are molecular weight markers (marker).
FIGS. 7a and 7b show the amino acid and nucleotide sequences, respectively, of a single-chain TCR, with the amino and nucleotide sequences of the linker sequence underlined.
FIGS. 8a and 8b are the amino acid and nucleotide sequences, respectively, of the variable domain of the single-chain TCR α chain.
FIGS. 9a and 9b are the amino acid and nucleotide sequences, respectively, of the variable domain of the single-chain TCR β chain.
Figure 10 is a gel diagram of the soluble single chain TCR obtained after purification. The leftmost lane is a non-reducing gel, the middle lane is a molecular weight marker (marker), and the rightmost lane is a reducing gel.
FIG. 11 is a BIAcore kinetic profile of binding of soluble TCRs of the invention to the KWVESIFILIF-HLA A2402 complex.
FIG. 12 is a BIAcore kinetic profile of binding of soluble single chain TCRs of the invention to the KWVESIFILIF-HLA A2402 complex.
FIG. 13 shows the results of functional verification of the ELISPOT activation of the resulting T cell clones.
FIG. 14 is a graphical representation of the results of functional confirmation of ELISPOT activation of effector cells transduced with the TCRs of the invention.
Fig. 15 is a validation result of LDH killing function of effector cells transduced with the TCR of the present invention.
Detailed Description
The present inventors have extensively and intensively studied to find a TCR capable of specifically binding to AFP antigen short peptide KWVESIFLIF (SEQ ID NO: 9) which can form a complex with HLA A2402 and be presented together to the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.
Term(s)
MHC molecules are proteins of the immunoglobulin superfamily and may be MHC class I or II molecules. Therefore, it is specific for antigen presentation, different individuals have different MHC, and different short peptides in one protein antigen can be presented on the cell surface of respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.
The T Cell Receptor (TCR), is the only receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which leads to a series of subsequent cell signaling and other physiological responses, thereby allowing T cells of different antigen specificities to exert an immune effect on their target cells.
TCRs are cell membrane surface glycoproteins that exist as heterodimers from either the α chain/β chain or the γ chain/δ chain. In 95% of T cells the TCR heterodimer consists of α and β chains, while 5% of T cells have TCRs consisting of γ and δ chains. Native α β heterodimeric TCRs have an α chain and a β chain, which constitute subunits of an α β heterodimeric TCR. Broadly, each of the α and β chains comprises a variable region, a linker region and a constant region, and the β chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered to be part of the linker region. Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in framework structures (framework regions). The CDR regions determine the binding of the TCR to the pMHC complex, with CDR3 being recombined from variable and connecting regions, referred to as hypervariable regions. The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domain being made up of linked variable and linking regions. The sequences of TCR constant domains can be found in public databases of the international immunogenetic information system (IMGT), e.g. the constant domain sequence of the α chain of the TCR molecule is "TRAC 01", the constant domain sequence of the β chain of the TCR molecule is "TRBC1 01" or "TRBC2 01". In addition, the α and β chains of the TCR also comprise a transmembrane region and a cytoplasmic region, the cytoplasmic region being very short.
In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.
Natural interchain disulfide bond and artificial interchain disulfide bond
A set of disulfide bonds, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as an "artificial interchain disulfide bond".
For convenience of description of the positions of disulfide bonds, the positions of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 amino acid sequences are numbered in the sequential order from the N-terminus to the C-terminus, and in TRBC1 × 01 or TRBC2 × 01, the 60 th amino acid in the sequential order from the N-terminus to the C-terminus is P (proline), and thus in the present invention, it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Pro60, and also as TRBC1 × 01 or TRBC2 × 01 exon 1 60, in TRBC1 × 01 or TRBC2 × 01, and in TRBC1 × 01 or TRBC2 × 01, the 61 th amino acid in the sequential order from the N-terminus to the C-terminus is Q (glutamine), and in the present invention, it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Gln61, and also as TRBC1 × 01 or TRBC2 × 01, and so on, the other TRBC1 × 01 or TRBC2 × 01 amino acid. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.
Detailed Description
TCR molecules
During antigen processing, antigens are degraded within cells and then carried to the cell surface by MHC molecules. T cell receptors are capable of recognizing peptide-MHC complexes on the surface of antigen presenting cells. Accordingly, in a first aspect the invention provides a TCR molecule capable of binding to the KWVESIFLIF-HLA a2402 complex. Preferably, the TCR molecule is isolated or purified. The α and β chains of the TCR each have 3 Complementarity Determining Regions (CDRs).
In a preferred embodiment of the invention, the α chain of the TCR comprises CDRs having the amino acid sequence:
αCDR1-VGISA(SEQ ID NO:10)
αCDR2-LSSGK(SEQ ID NO:11)
α CDR3-AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-MNHEY(SEQ ID NO:13)
βCDR2-SVGAGI(SEQ ID NO:14)
βCDR3-ASSYPGSYGYT(SEQ ID NO:15)。
chimeric TCRs can be prepared by embedding the above-described amino acid sequences of the CDR regions of the invention into any suitable framework. One skilled in the art can design or synthesize a TCR molecule with the corresponding function based on the CDR regions disclosed herein, so long as the framework structure is compatible with the CDR regions of the TCR of the invention. Thus, the TCR molecules of the invention are those which comprise the above-described α and/or β chain CDR region sequences and any suitable framework structure. The TCR α chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 5.
In a preferred embodiment of the invention, the TCR molecules of the invention are heterodimers consisting of α and β chains. In particular, in one aspect the α chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the α chain variable domain amino acid sequence comprising CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above-described α chain. Preferably, the TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the amino acid sequence of the α chain variable domain of the TCR molecule is SEQ ID NO 1. In another aspect, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence 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 TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO 5.
In a preferred embodiment of the invention, the TCR molecules of the invention are single chain TCR molecules consisting of part or all of the α chain and/or part or all of the β chain. Single chain TCR molecules are described in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91,12654-12658. From the literature, those skilled in the art are readily able to construct single chain TCR molecules comprising the CDRs regions of the invention. In particular, the single chain TCR molecule comprises V α, V β and C β, preferably linked in order from N-terminus to C-terminus.
The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the alpha chain described above. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the variable domain of the beta chain 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 beta chain described above. Preferably, the single chain TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the single chain TCR molecule is SEQ ID NO 5.
In a preferred embodiment of the invention, the constant domain of the TCR molecules of the invention is a human constant domain. The person skilled in the art knows or can obtain the human constant domain amino acid sequence by consulting public databases of relevant books or IMGT (international immunogenetic information system). For example, the constant domain sequence of the α chain of the TCR molecules of the invention can be "TRAC 01", and the constant domain sequence of the β chain of the TCR molecules can be "TRBC1 01" or "TRBC2 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, here denoted: TRAC × 01 Arg53 of exon 1, and so on. Preferably, the amino acid sequence of the α chain of the TCR molecule of the invention is SEQ ID NO 3 and/or the amino acid sequence of the β chain is SEQ ID NO 7.
Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane region. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The present invention also provides soluble TCRs with specificity for AFP antigen short peptides.
To obtain a soluble TCR, in one aspect, the inventive TCR may be one in which an artificial disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of exon 1 of TRAC × 01 and a cysteine residue of Ser57 of exon 1 of TRBC1 × 01 or TRBC2 × 01. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser77 of TRBC2 × 01 exon 1; tyr10 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Ser17; thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1; ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15; arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1; pro89 of TRAC × 01 exon 1 and TRBC1 × 01 or Ala19 of TRBC2 × 01 exon 1; or Tyr10 of exon 1 TRAC 01 and TRBC1 01 or TRBC 201 of Glu20 of exon 1. I.e. a cysteine residue has substituted any of the above-mentioned groups of positions in the constant domains of the alpha and beta chains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include a cysteine residue for the purpose of deleting the native disulphide bond, or by mutating the cysteine residue forming the native disulphide bond to another amino acid.
As mentioned above, the inventive TCR may comprise an artificial disulfide bond introduced between residues of its alpha and beta chain constant domains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by the native disulfide bond present in the TCR.
To obtain a soluble TCR, the inventive TCR may, on the other hand, also comprise a TCR having a mutation in its hydrophobic core region, preferably a mutation that results in an improved stability of the inventive soluble TCR, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or positions 3,5,7 from the last amino acid position of the short peptide of the alpha chain J gene (TRAJ) and/or positions 2,4,6 from the last amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbers of the amino acid sequences are according to the position numbers listed in the International Immunogenetic information System (IMGT). The skilled person is aware of the above international immunogenetic information system and can derive from this database the position numbering of the amino acid residues of different TCRs in IMGT.
The TCR with the mutated hydrophobic core region of the present invention may be a stable soluble single chain TCR consisting of a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains. The single-chain soluble TCR constructed as in example 4 of the invention has an alpha chain variable domain amino acid sequence of SEQ ID NO. 32 and an encoded nucleotide sequence of SEQ ID NO. 33; the amino acid sequence of the beta chain variable domain is SEQ ID NO. 34, and the coded nucleotide sequence is SEQ ID NO. 35.
In addition, for stability, patent document 201680003540.2 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of a TCR can significantly improve the stability of the TCR. Thus, the TCR of the invention may also comprise an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of exon 1 TRBC1 x 01 or TRBC2 x 01; amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; or amino acid 47 of TRAV and amino acid 60 of exon 1 TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.
The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be formed by tetramer formation with the tetrameric domain of p53, or complexes formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the invention may be used alone or may be covalently or otherwise associated, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the KWVESIFLIF-HLA a2402 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2. biotoxics (Chaudhary et al, 1989, nature 339, 394, epel et al, 2002, cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 etc (Gillies et al, 1992, national institute of sciences (PNAS) 89, 1428, card et al, 2004, cancer Immunology and Immunotherapy) 53, 345, haiin et al, 2003, cancer Research (Cancer Research) 63, 3202; 4. antibody Fc fragment (Mosquera et al, 2005, journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, international Journal of Cancer 62, 319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, cancer letters (Cancer letters) 239, 36, huang et al, 2006, journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, gene therapy 11, 1234); 8. liposomes (Mamot et al, 2005, cancer research 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.
In addition, the TCRs of the invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the inventive TCR may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regimen to immunosuppresse when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.
It should be understood that the amino acid names herein are expressed in terms of international single-letter or three-letter english letters, and the single-letter english letter and three-letter english letters of the amino acid names correspond to the following relationships: 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).
Nucleic acid molecules
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, variable domains of the alpha and/or beta chains, and the alpha and/or beta chains.
The nucleotide sequence encoding the α chain CDR regions of the TCR molecules of the first aspect of the invention is as follows:
CDR1α-gtaggaataagtgcc(SEQ ID NO:16)
CDR2α-ctgagctcagggaag(SEQ ID NO:17)
CDR3α-gctgtcagggatgctggtggtactagctatggaaagctgaca(SEQ ID NO:18)
the nucleotide sequence encoding the CDR regions of the β chain of the TCR molecules of the first aspect of the invention is as follows:
CDR1β-atgaaccatgaatac(SEQ ID NO:19)
CDR2β-tcagttggtgctggtatc(SEQ ID NO:20)
CDR3β-gccagcagttacccaggttcttatggctacacc(SEQ ID NO:21)
thus, the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR alpha chain of the invention comprises SEQ ID NO 16, 17 and 18 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR beta chain of the invention comprises SEQ ID NO 19, 20 and 21.
The nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not comprise an intron. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID NO 2 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR beta chain variable domain of the invention comprises SEQ ID NO 6. Alternatively, the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR α chain variable domain of the invention comprises SEQ ID NO 33 and/or the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR β chain variable domain of the invention comprises SEQ ID NO 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention is SEQ ID NO. 31.
It is understood that due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Thus, the nucleic acid sequence encoding the TCR of the present invention may be identical to or a degenerate variant of the nucleic acid sequences shown in the figures of the present invention. As illustrated by one of the examples herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO. 1, but differs from the sequence of SEQ ID NO. 2.
The nucleotide sequence may be codon optimized. Different cells differ in the utilization of specific codons, and the expression level can be increased by changing the codons in the sequence according to the type of the cell. Codon usage tables for mammalian cells as well as for 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 be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be the coding strand or the non-coding strand.
Carrier
The invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, i.e. constructs capable of expression in vivo or in vitro. Commonly used 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, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.
Preferably, the vector can transfer the nucleotide of the invention into a cell, e.g., a T cell, such that the cell expresses a TCR specific for the AFP antigen. Ideally, the vector should be capable of sustained high level expression in T cells.
Cells
The invention also relates to genetically engineered host cells using the vectors or coding sequences of the invention. The host cell comprises the vector of the invention or has the nucleic acid molecule of the invention integrated into the chromosome. The host cell is selected from: prokaryotic and eukaryotic cells, such as E.coli, yeast cells, CHO cells, and the like.
In addition, the invention also includes isolated cells expressing the TCRs of the invention, which may be, but are not limited to, T cells, NK cells, NKT cells, and particularly T cells. The T cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMC), which may be CD4 + Helper T cell or CD8 + Cytotoxic T cells. The cells can be in CD4 + Helper T cell/CD 8 + A mixed population of cytotoxic T cells. Generally, the cells can be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to make them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.
Alternatively, the cell of the invention may also be or be derived from a stem cell, such as a Hematopoietic Stem Cell (HSC). Gene transfer to HSCs does not result in TCR expression on the cell surface, since the CD3 molecule is not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors (lymphoid precursors) that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.
There are many methods suitable for T cell transfection using DNA or RNA encoding the TCR of the invention (e.g., robbins et al, (2008) J.Immunol.180: 6116-6131). T cells expressing the TCRs of the invention may be used for adoptive immunotherapy. One skilled in the art will be aware of many suitable methods for adoptive therapy (e.g., rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).
AFP antigen associated diseases
The present invention also relates to a method for the treatment and/or prevention of a disease associated with AFP in a subject, comprising the step of adoptive transfer of AFP-specific T cells to the subject. The AFP-specific T cells recognize the KWVESIFLIF-HLA A2402 complex.
The AFP-specific T cells of the invention can be used for treating any AFP-related disease presenting AFP antigen short peptide KWVESIFLIF-HLA A2402 complex, including but not limited to tumors, such as liver cancer and the like.
Method of treatment
Treatment may be effected by isolating T cells from patients or volunteers suffering from a disease associated with the AFP antigen and introducing the TCR of the invention into such T cells, followed by reinfusion of these genetically engineered cells into the patient. Accordingly, the present invention provides a method of treating an AFP-related disease comprising infusing into a patient an isolated T cell expressing a TCR of the invention, preferably, the T cell is derived from the patient itself. Generally, this involves (1) isolating T cells from the patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing the genetically modified T cells into the patient. The number of cells isolated, transfected and transfused can be determined by a physician.
The main advantages of the invention are:
(1) The TCR of the invention can be specifically combined with AFP antigen short peptide complex KWVESIFLIF-HLA A2402, and the effector cells transduced with the TCR of the invention can be specifically activated.
(2) Effector cells transduced with the inventive TCR are capable of specifically killing the target cell.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1 cloned AFP antigen short peptide specific T cells
Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA A2402 were stimulated using the synthetic short peptide KWVESIFLIF (SEQ ID NO:9; kingsu King Biotech, inc.). The KWVESIFLIF short peptide and HLA A2402 with biotin labels are renatured to prepare the pHLA haploid. These haploids were combined with streptavidin labeled with PE (BD Co.) to form PE-labeled tetramers, which were sorted from anti-CD 8-APC double positive cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by single cloning by limiting dilution. Monoclonal cells were stained with tetramer and double positive clones were selected as shown in FIG. 3. The double positive clones obtained by layer-by-layer screening also need to meet the requirement of further functional test.
The function and specificity of the T cell clone were further tested by ELISPOT assay. Methods for detecting cell function using the ELISPOT assay are well known to those skilled in the art. The effector cells used in the IFN-gamma ELISPOT experiment in the present example were T cell clones obtained in the present invention, the target cells were T2-A24 (T2 cells transfected with HLA-A2402) loaded with short peptides of KWVESSIFLIF, SNU398-AFP (AFP overexpression), and HepG2, and the control group was T2-A24 and SNU398 loaded with other short peptides.
First, ELISPOT plates were prepared and the components of the assay were added to the ELISPOT plates in the following order: after 20,000 target cells/well and 2000 effector cells/well, 20. Mu.l of the corresponding short peptide was added to the experimental group and the control group, and 20. Mu.l of the medium (test medium) was added to the blank group, and 2 wells were set. Then incubated overnight (37 ℃,5% CO) 2 ). The plate was then washed and subjected to secondary detection and color development, the plate was dried for 1 hour, and spots formed on the membrane were counted using an immuno spot plate READER (ELISPOT READER system; AID Co.). The experimental results are shown in fig. 13, and the obtained T cell clone has obvious activation reaction on target cells loaded with KWVESIFNLIF short peptide and SNU398-AFP and HepG2 over-expressing AFP antigen, but has no reaction on cells loaded with other short peptide and SNU398.
Example 2 construction of TCR Gene and vector for obtaining AFP antigen short peptide specific T cell clone
Using Quick-RNA TM The total RNA of the T cell clone specific to the antigen short peptide KWVESIFLIF and restricted by HLA A2402 selected in example 1 was extracted by MiniPrep (ZYMO research). The cDNA was synthesized using a SMART RACE cDNA amplification kit from clontech, using primers designed in the C-terminal conserved region of the human TCR gene. The sequences were cloned into the T vector (TAKARA) and sequenced. It should be noted that the sequence is a complementary sequence, not including introns. The alpha chain and beta chain sequence structures of the TCR expressed by the double positive clone were sequenced as shown in fig. 1 and fig. 2, respectively.
The alpha chain was identified to comprise CDRs with the following amino acid sequences:
αCDR1-VGISA(SEQ ID NO:10)
αCDR2-LSSGK(SEQ ID NO:11)
αCDR3-AVRDAGGTSYGKLT(SEQ ID NO:12)
the beta chain comprises CDRs having the amino acid sequences:
βCDR1-MNHEY(SEQ ID NO:13)
βCDR2-SVGAGI(SEQ ID NO:14)
βCDR3-ASSYPGSYGYT(SEQ ID NO:15)。
the full-length genes of the TCR α and β chains were cloned into the lentiviral expression vector pllenti (addendum) by overlap (overlap) PCR, respectively. The method specifically comprises the following steps: the TCR alpha chain and the TCR beta chain are connected by overlap PCR to obtain the TCR alpha-2A-TCR beta segment. The lentivirus expression vector and the TCR alpha-2A-TCR beta are connected by enzyme digestion to obtain a pLenti-TRA-2A-TRB-IRES-NGFR plasmid. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. The pseudovirus was then packaged again at 293T/17.
Example 3 expression, refolding and purification of soluble TCR specific for short peptide AFP antigen
To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the invention may comprise only the variable and part of the constant domains thereof, respectively, and one cysteine residue has been introduced into the constant domains of the α and β chains, respectively, to form an artificial interchain disulfide bond, the amino acid and nucleotide sequences of the α chain being as shown in figures 4a and 4b, respectively, and the amino acid and nucleotide sequences of the β chain being as shown in figures 5a and 5b, respectively. The above-mentioned desired gene sequences for the TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning A Laboratory Manual (third edition, sambrook and Russell), and the upstream and downstream Cloning sites were NcoI and NotI, respectively. The insert was confirmed by sequencing without error.
The expression vectors of TCR alpha and beta chains are respectively transformed into expression bacteria BL21 (DE 3) by a chemical transformation method, and the bacteria grow by LB culture solution and OD 600 At 0.6 induction with final concentration of 0.5mM IPTG, inclusion bodies formed after expression of the α and β chains of the TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris (pH 8.1).
The TCR α and β chains after lysis are separated by a 1:1 was rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine,6.6mM beta-merimepoethylamine (4 ℃ C.) to a final concentration of 60mg/mL. After mixing, the solution was dialyzed against 10 times the volume of deionized water (4 ℃ C.), and after 12 hours, the deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. Mu.M filter and then purified by an anion exchange column (HiTrap Q HP,5ml, GE Healthcare). The TCR eluted with peaks containing successfully renatured α and β dimers was confirmed by SDS-PAGE gel. The TCR was subsequently further purified by gel filtration chromatography (HiPrep 16/60, sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA. The SDS-PAGE gel of the soluble TCR of the invention is shown in FIGS. 6a and 6 b.
Example 4 Generation of soluble Single chain TCR specific for short peptides of AFP antigen
The variable domains of TCR α and β chains in example 2 were constructed as a stable soluble single-chain TCR molecule linked by a flexible short peptide (linker) using site-directed mutagenesis as described in WO 2014/206304. The amino acid sequence and the nucleotide sequence of the single-chain TCR molecule are respectively shown in FIG. 7a and FIG. 7b, wherein the amino acid sequence and the nucleotide sequence of the linker sequence are underlined; the amino acid sequence and the nucleotide sequence of the alpha chain variable domain are shown in FIG. 8a and FIG. 8b respectively; the amino acid sequence and nucleotide sequence of the beta-chain variable domain are shown in FIGS. 9a and 9b, respectively.
The target gene was digested simultaneously with Nco I and Not I, and ligated to pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into e.coli DH5 α, spread on LB plates containing kanamycin, cultured at 37 ℃ for overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, recombinant plasmids were extracted and transformed into e.coli BL21 (DE 3) for expression.
Example 5 expression, renaturation and purification of soluble Single chain TCR specific for short peptides of the AFP antigen
The BL21 (DE 3) colonies containing the recombinant plasmid pET28 a-template strand prepared in example 4 were all inoculated in LB medium containing kanamycin, cultured at 37 ℃ to OD600 of 0.6 to 0.8, IPTG was added to a final concentration of 0.5mM, and the culture was continued at 37 ℃ for 4 hours. The cell pellet was harvested by centrifugation at 5000rpm for 15min, the cell pellet was lysed by Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000rpm for 15min, washed with Bugbuster (Merck) to remove cell debris and membrane components, centrifuged at 6000rpm for 15min, and the inclusion bodies were collected. The inclusion bodies were dissolved in buffer (20 mM Tris-HCl pH 8.0,8M urea), the insoluble material was removed by high speed centrifugation, the supernatant was quantified by BCA method and split charged, and stored at-80 ℃ for further use.
To 5mg of solubilized single-chain TCR inclusion body protein, 2.5mL of buffer (6M Gua-HCl,50mM Tris-HCl pH 8.1, 100mM NaCl,10mM EDTA) was added, DTT was added to a final concentration of 10mM, and treatment was carried out at 37 ℃ for 30min. The treated single-chain TCR was added dropwise to 125mL of renaturation buffer (100 mM Tris-HCl pH 8.1,0.4M L-arginine, 5M urea, 2mM EDTA,6.5 mM. Beta. -captoethylamine, 1.87mM Cystamine) with a syringe, stirred at 4 ℃ for 10min, and then the renaturation solution was filled into a cellulose membrane dialysis bag with an interception amount of 4kDa, and the dialysis bag was placed in 1L of precooled water and slowly stirred at 4 ℃ overnight. After 17 hours, the dialysate was changed to 1L of pre-chilled buffer (20 mM Tris-HCl pH 8.0), dialysis was continued at 4 ℃ for 8h, and then dialysis was continued overnight with the same fresh buffer. After 17 hours, the sample was filtered through a 0.45 μ M filter, vacuum degassed and then passed through an anion exchange column (HiTrap Q HP, GE Healthcare), the protein was purified by a 0-1M NaCl linear gradient eluent formulated in 20mM Tris-HCl pH 8.0, the fractions collected were subjected to SDS-PAGE analysis, the fractions containing single-chain TCR were concentrated and then further purified by a gel filtration column (Superdex 75/300, GE Healthcare), and the target fraction was also subjected to SDS-PAGE analysis.
The eluted fractions for BIAcore analysis were further tested for purity using gel filtration. The conditions are as follows: the chromatography column Agilent Bio SEC-3 (300A,
Figure BDA0003006507670000181
) The mobile phase is 150mM phosphate buffer solution, the flow rate is 0.5mL/min, the column temperature is 25 ℃, and the ultraviolet detection wavelength is 214nm.
The SDS-PAGE gel of the soluble single-chain TCR obtained by the invention is shown in FIG. 10.
Example 6 binding characterization
This example demonstrates that soluble TCR molecules of the invention are capable of specifically binding to the KWVESIFLIF-HLA a2402 complex.
Binding activity of the TCR molecules obtained in examples 3 and 5 to the KWVESIFLIF-HLA a2402 complex was examined using a BIAcore T200 real-time assay system. Anti-streptavidin antibody (GenScript) was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine in hydrochloric acid to complete the coupling process at a coupling level of about 15,000RU. The low concentration of streptavidin through the coated chip surface, then the KWVESIFLIF-HLA A2402 complex through the detection channel, another channel as a reference channel, and 0.05mM biotin at 10 u L/min flow rate through the chip for 2min, closed streptavidin the remaining binding sites.
The preparation process of the KWVESIFLIF-HLA A2402 complex is as follows:
a. purification of
Collecting 100ml of E.coli bacterial liquid for inducing expression of heavy chain or light chain, centrifuging at 8000g at 4 ℃ for 10min, washing the bacterial cells once with 10ml of PBS, then resuspending the bacterial cells by vigorous shaking with 5ml of BugBuster Master Mix Extraction Reagents (Merck), carrying out rotary incubation at room temperature for 20min, centrifuging at 6000g at 4 ℃ for 15min, discarding supernatant, and collecting inclusion bodies.
Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15min; discarding supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl with pH of 8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.
b. Renaturation
The synthesized short peptide KWVESIFLIF (King-Murray Biotech Co., ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion bodies of light and heavy chains were solubilized with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. KWVESIFILIF peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in sequence (final concentration, addition of heavy chain in three portions, 8 h/time), and renaturation was carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE was checked for success of the renaturation.
c. Purification after renaturation
The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5 ml bed volume). The protein was eluted using a linear gradient of 0-400mM NaCl prepared using an Akta purifier (GE general electric) at 20mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, and the peak fractions were collected and subjected to purity detection using SDS-PAGE.
d. Biotinylation of the compound
The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while replacing the buffer with 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu.M D-Biotin, 100. Mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine whether biotinylation was complete.
e. Purification of the biotinylated Complex
The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric Co., ltd.) TM A16/60S200 HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a single peak elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using Millipore ultrafiltration tubes, protein concentration was measured by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots by adding the protease inhibitor cocktail (Roche)-80℃。
Kinetic parameters are calculated by using BIAcore Evaluation software, and kinetic maps of the soluble TCR molecule and the soluble single-chain TCR molecule combined with the KWVESIFILIF-HLA A2402 complex are respectively shown in fig. 11 and fig. 12. The map shows that the soluble TCR molecule and the soluble single-chain TCR molecule obtained by the invention can be combined with the KWVESIFLIF-HLA A2402 compound. Meanwhile, the method is used for detecting the binding activity of the soluble TCR molecule and the short peptides of other unrelated antigens and the HLA complex, and the result shows that the TCR molecule is not bound with other unrelated antigens.
Example 7 ELISPOT activation Effect assay of Effect cells transfected with a TCR of the invention
IFN-. Gamma.is a potent immunomodulatory factor produced by activated T lymphocytes, and thus the present example examined the IFN-. Gamma.counts by ELISPOT assay well known to those skilled in the art to verify the activation function and antigen specificity of cells transfected with the TCR of the invention.
The effector cells used in this experiment were CD3 expressing the TCR of the invention + T cells and CD3 transfected with other TCR (A6) or free-running (NC) in the same volunteer + T cells served as a control group. The target cells used were T2-A24 loaded with the AFP antigen short peptide, KWVESIFLIF, and T2-A24 loaded with other irrelevant peptides or unloaded as control. The components of the assay were added to ELISPOT well plates: target cell 1X 10 4 2X 10 cells/well, effector cells 3 One/well (calculated as transfection positivity) and two duplicate wells were set. Then adding KWVESIFILIF short peptide solution, other short peptide solutions and the culture medium with the same volume into corresponding holes respectively to ensure that the final concentration of the KWVESIFILIF short peptide in the ELISPOT hole plate is 1 multiplied by 10 in sequence -6 M to 1X 10 -12 M, 7 gradients in total; the final concentration of the irrelevant peptide in the ELISPOT well plate is 1X 10 -6 M to 1X 10 -8 M,3 gradients in total. Then incubated overnight (37 ℃,5% 2 ). On day 2 of the experiment, the plate was washed, subjected to secondary detection and color development, dried, and counted using an immuno-spot plate READER (AID 20 Co., ltd.) to determine spots formed on the membrane。
The experimental results are shown in fig. 14, for the target cells loaded with the KWVESIFLIF short peptide, the T cells transfected with the TCR of the invention have obvious activation effect, while the T cells transfected with other TCRs or free-transfected cells have no response from the beginning; at the same time, T cells transfected with the TCR of the invention are not activated by other short peptide-loaded or unloaded cells.
Example 8 experiment of LDH killing function of Effector cells transfected with TCR of the invention
This example demonstrates the killing function of cells transfected with a TCR of the invention by measuring LDH release by non-radioactive cytotoxicity assays well known to those skilled in the art. This test is a colorimetric substitution test for the 51 Cr-release cytotoxicity test, and quantitatively determines the Lactate Dehydrogenase (LDH) released after cell lysis. LDH released in the medium was detected using a 30min coupled enzymatic reaction in which LDH converted a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. 490nm visible absorbance data can be collected using a standard 96-well plate reader. The formula is calculated as% cytotoxicity =100 × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximal-target cell spontaneous)
The LDH assay of this example uses isolated CD3+ T cells to transfect the TCR of the invention as effector cells and the same volunteer to transfect other TCRs (A6) or CD3+ T cells free for staining (NC) as a control. The target cells used were T2-A24 loaded with the AFP antigen short peptide, KWVESIFLIF, and T2-A24 loaded with other irrelevant peptides or unloaded as control.
LDH plates were prepared first, and target cells 3X 10 were first 4 One/well, effector cells 3 x 10 4 Adding each well into the corresponding well, and then adding a KWVESIFLIF short peptide solution, other short peptide solutions and an equal volume of culture medium into the corresponding wells respectively to ensure that the final concentration of the KWVESIFLIF short peptide in the LDH well plate is 1 multiplied by 10 in sequence -6 M to 1X 10 -13 M, 8 gradients in total; the final concentration of the unrelated peptide in LDH well plates was 1X 10 -6 M to 1X 10 -8 M,3 gradients total. And three multiple holes are arranged. Simultaneously setting effector cell spontaneous pores, target cell spontaneous pores and target cells to be maximumWells, volume corrected control wells and media background control wells. Incubation overnight (37 ℃,5% CO) 2 ). On day 2 of the experiment, color development was detected, and after termination of the reaction, the absorbance was recorded at 490nm using a microplate reader (Bioteck).
The experimental results are shown in fig. 15, the effector cells transfected with the TCR of the present invention have strong killing effect against the target cells loaded with AFP antigen short peptide KWVESIFLIF, and react at a lower concentration of the short peptide, while the effector cells transfected with other TCRs or empty transfection have no killing effect from the beginning; at the same time, effector cells transfected with the inventive TCR did not kill other short peptide-loaded or unloaded target cells.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.
Sequence listing
<110> Scent and snow Life sciences technology (Guangdong) Co., ltd
<120> T cell receptor recognizing AFP
<130> P2021-0620
<160> 35
<170> PatentIn version 3.5
<210> 1
<211> 112
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 1
Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Ala Gln Glu Gly
1 5 10 15
Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu
20 25 30
His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met
35 40 45
Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn
50 55 60
Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg
65 70 75 80
Asp Ser Ala Val Tyr Ile Cys Ala Val Arg Asp Ala Gly Gly Thr Ser
85 90 95
Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr Ile Leu Thr Val His Pro
100 105 110
<210> 2
<211> 336
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 2
aaaaatgaag tggagcagag tcctcagaac ctgactgccc aggaaggaga atttatcaca 60
atcaactgca gttactcggt aggaataagt gccttacact ggctgcaaca gcatccagga 120
ggaggcattg tttccttgtt tatgctgagc tcagggaaga agaagcatgg aagattaatt 180
gccacaataa acatacagga aaagcacagc tccctgcaca tcacagcctc ccatcccaga 240
gactctgccg tctacatctg tgctgtcagg gatgctggtg gtactagcta tggaaagctg 300
acatttggac aagggaccat cttgactgtc catcca 336
<210> 3
<211> 253
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 3
Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Ala Gln Glu Gly
1 5 10 15
Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu
20 25 30
His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met
35 40 45
Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn
50 55 60
Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg
65 70 75 80
Asp Ser Ala Val Tyr Ile Cys Ala Val Arg Asp Ala Gly Gly Thr Ser
85 90 95
Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr Ile Leu Thr Val His Pro
100 105 110
Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys
115 120 125
Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr
130 135 140
Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr
145 150 155 160
Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala
165 170 175
Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser
180 185 190
Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp
195 200 205
Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe
210 215 220
Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala
225 230 235 240
Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250
<210> 4
<211> 759
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 4
aaaaatgaag tggagcagag tcctcagaac ctgactgccc aggaaggaga atttatcaca 60
atcaactgca gttactcggt aggaataagt gccttacact ggctgcaaca gcatccagga 120
ggaggcattg tttccttgtt tatgctgagc tcagggaaga agaagcatgg aagattaatt 180
gccacaataa acatacagga aaagcacagc tccctgcaca tcacagcctc ccatcccaga 240
gactctgccg tctacatctg tgctgtcagg gatgctggtg gtactagcta tggaaagctg 300
acatttggac aagggaccat cttgactgtc catccaaata tccagaaccc tgaccctgcc 360
gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420
gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480
gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540
tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600
cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 660
aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 720
gggtttaatc tgctcatgac gctgcggctg tggtccagc 759
<210> 5
<211> 112
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 5
Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly
1 5 10 15
Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met
20 25 30
Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr
35 40 45
Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr
50 55 60
Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser
65 70 75 80
Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Pro
85 90 95
Gly Ser Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val
100 105 110
<210> 6
<211> 336
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 6
aatgctggtg tcactcagac cccaaaattc caggtcctga agacaggaca gagcatgaca 60
ctgcagtgtg cccaggatat gaaccatgaa tacatgtcct ggtatcgaca agacccaggc 120
atggggctga ggctgattca ttactcagtt ggtgctggta tcactgacca aggagaagtc 180
cccaatggct acaatgtctc cagatcaacc acagaggatt tcccgctcag gctgctgtcg 240
gctgctccct cccagacatc tgtgtacttc tgtgccagca gttacccagg ttcttatggc 300
tacaccttcg gttcggggac caggttaacc gttgta 336
<210> 7
<211> 289
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 7
Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly
1 5 10 15
Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met
20 25 30
Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr
35 40 45
Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr
50 55 60
Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser
65 70 75 80
Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Pro
85 90 95
Gly Ser Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val
100 105 110
Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro
115 120 125
Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu
130 135 140
Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn
145 150 155 160
Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys
165 170 175
Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu
180 185 190
Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys
195 200 205
Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp
210 215 220
Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg
225 230 235 240
Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser
245 250 255
Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala
260 265 270
Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp
275 280 285
Phe
<210> 8
<211> 867
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 8
aatgctggtg tcactcagac cccaaaattc caggtcctga agacaggaca gagcatgaca 60
ctgcagtgtg cccaggatat gaaccatgaa tacatgtcct ggtatcgaca agacccaggc 120
atggggctga ggctgattca ttactcagtt ggtgctggta tcactgacca aggagaagtc 180
cccaatggct acaatgtctc cagatcaacc acagaggatt tcccgctcag gctgctgtcg 240
gctgctccct cccagacatc tgtgtacttc tgtgccagca gttacccagg ttcttatggc 300
tacaccttcg gttcggggac caggttaacc gttgtagagg acctgaacaa ggtgttccca 360
cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420
ctggtgtgcc tggccacagg cttcttcccc gaccacgtgg agctgagctg gtgggtgaat 480
gggaaggagg tgcacagtgg ggtcagcacg gacccgcagc ccctcaagga gcagcccgcc 540
ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 600
aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660
tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 720
gcagactgtg gctttacctc ggtgtcctac cagcaagggg tcctgtctgc caccatcctc 780
tatgagatcc tgctagggaa ggccaccctg tatgctgtgc tggtcagcgc ccttgtgttg 840
atggccatgg tcaagagaaa ggatttc 867
<210> 9
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 9
Lys Trp Val Glu Ser Ile Phe Leu Ile Phe
1 5 10
<210> 10
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 10
Val Gly Ile Ser Ala
1 5
<210> 11
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 11
Leu Ser Ser Gly Lys
1 5
<210> 12
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 12
Ala Val Arg Asp Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr
1 5 10
<210> 13
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 13
Met Asn His Glu Tyr
1 5
<210> 14
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 14
Ser Val Gly Ala Gly Ile
1 5
<210> 15
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 15
Ala Ser Ser Tyr Pro Gly Ser Tyr Gly Tyr Thr
1 5 10
<210> 16
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 16
gtaggaataa gtgcc 15
<210> 17
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 17
ctgagctcag ggaag 15
<210> 18
<211> 42
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 18
gctgtcaggg atgctggtgg tactagctat ggaaagctga ca 42
<210> 19
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 19
atgaaccatg aatac 15
<210> 20
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 20
tcagttggtg ctggtatc 18
<210> 21
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 21
gccagcagtt acccaggttc ttatggctac acc 33
<210> 22
<211> 275
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 22
Met Val Lys Ile Arg Gln Phe Leu Leu Ala Ile Leu Trp Leu Gln Leu
1 5 10 15
Ser Cys Val Ser Ala Ala Lys Asn Glu Val Glu Gln Ser Pro Gln Asn
20 25 30
Leu Thr Ala Gln Glu Gly Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser
35 40 45
Val Gly Ile Ser Ala Leu His Trp Leu Gln Gln His Pro Gly Gly Gly
50 55 60
Ile Val Ser Leu Phe Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg
65 70 75 80
Leu Ile Ala Thr Ile Asn Ile Gln Glu Lys His Ser Ser Leu His Ile
85 90 95
Thr Ala Ser His Pro Arg Asp Ser Ala Val Tyr Ile Cys Ala Val Arg
100 105 110
Asp Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr
115 120 125
Ile Leu Thr Val His Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr
130 135 140
Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr
145 150 155 160
Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val
165 170 175
Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys
180 185 190
Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala
195 200 205
Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser
210 215 220
Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr
225 230 235 240
Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile
245 250 255
Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu
260 265 270
Trp Ser Ser
275
<210> 23
<211> 825
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 23
atggtgaaga tccggcaatt tttgttggct attttgtggc ttcagctaag ctgtgtaagt 60
gccgccaaaa atgaagtgga gcagagtcct cagaacctga ctgcccagga aggagaattt 120
atcacaatca actgcagtta ctcggtagga ataagtgcct tacactggct gcaacagcat 180
ccaggaggag gcattgtttc cttgtttatg ctgagctcag ggaagaagaa gcatggaaga 240
ttaattgcca caataaacat acaggaaaag cacagctccc tgcacatcac agcctcccat 300
cccagagact ctgccgtcta catctgtgct gtcagggatg ctggtggtac tagctatgga 360
aagctgacat ttggacaagg gaccatcttg actgtccatc caaatatcca gaaccctgac 420
cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480
gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540
aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600
aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660
ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720
gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 780
gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 825
<210> 24
<211> 308
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 24
Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala
1 5 10 15
Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu
20 25 30
Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His
35 40 45
Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu
50 55 60
Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro
65 70 75 80
Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg
85 90 95
Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser
100 105 110
Ser Tyr Pro Gly Ser Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu
115 120 125
Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val
130 135 140
Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
145 150 155 160
Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp
165 170 175
Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln
180 185 190
Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser
195 200 205
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His
210 215 220
Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp
225 230 235 240
Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala
245 250 255
Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly
260 265 270
Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr
275 280 285
Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys
290 295 300
Arg Lys Asp Phe
305
<210> 25
<211> 924
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 25
atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 60
gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120
cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 180
gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 240
aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 300
gctccctccc agacatctgt gtacttctgt gccagcagtt acccaggttc ttatggctac 360
accttcggtt cggggaccag gttaaccgtt gtagaggacc tgaacaaggt gttcccaccc 420
gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480
gtgtgcctgg ccacaggctt cttccccgac cacgtggagc tgagctggtg ggtgaatggg 540
aaggaggtgc acagtggggt cagcacggac ccgcagcccc tcaaggagca gcccgccctc 600
aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660
ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720
acccaggata gggccaaacc cgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780
gactgtggct ttacctcggt gtcctaccag caaggggtcc tgtctgccac catcctctat 840
gagatcctgc tagggaaggc caccctgtat gctgtgctgg tcagcgccct tgtgttgatg 900
gccatggtca agagaaagga tttc 924
<210> 26
<211> 207
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 26
Met Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Ala Gln Glu
1 5 10 15
Gly Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala
20 25 30
Leu His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe
35 40 45
Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile
50 55 60
Asn Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro
65 70 75 80
Arg Asp Ser Ala Val Tyr Ile Cys Ala Val Arg Asp Ala Gly Gly Thr
85 90 95
Ser Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr Ile Leu Thr Val His
100 105 110
Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser
115 120 125
Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln
130 135 140
Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys
145 150 155 160
Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val
165 170 175
Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn
180 185 190
Ser Ile Ile Pro Glu Asp Thr Phe Phe Cys Ser Pro Glu Ser Ser
195 200 205
<210> 27
<211> 621
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 27
atgaaaaacg aagtggaaca gagtcctcag aacctgactg cccaggaagg agaatttatc 60
acaatcaact gcagttactc ggtaggaata agtgccttac actggctgca acagcatcca 120
ggaggaggca ttgtttcctt gtttatgctg agctcaggga agaagaagca tggaagatta 180
attgccacaa taaacataca ggaaaagcac agctccctgc acatcacagc ctcccatccc 240
agagactctg ccgtctacat ctgtgctgtc agggatgctg gtggtactag ctatggaaag 300
ctgacatttg gacaagggac catcttgact gtccatccaa atatccagaa ccctgaccct 360
gccgtttatc agctgcgtga tagcaaaagc agcgataaaa gcgtgtgcct gttcaccgat 420
tttgatagcc agaccaacgt gagccagagc aaagatagcg atgtgtacat caccgataaa 480
accgtgctgg atatgcgcag catggatttc aaaagcaata gcgcggttgc gtggagcaac 540
aaaagcgatt ttgcgtgcgc gaacgcgttt aacaacagca tcatcccgga agatacgttc 600
ttctgcagcc cagaaagttc c 621
<210> 28
<211> 243
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 28
Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr
1 5 10 15
Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr
20 25 30
Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His
35 40 45
Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly
50 55 60
Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu
65 70 75 80
Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr
85 90 95
Pro Gly Ser Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Cys Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp
<210> 29
<211> 729
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 29
atgaacgcgg gcgtgaccca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60
acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120
ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180
gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240
tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcagttaccc aggttcttat 300
ggctacacct tcggttcggg gaccaggtta accgttgtag aggacctgaa aaacgtgttc 360
ccacccgagg tcgctgtgtt tgagccatca gaatgcgaaa ttagccatac ccagaaagcg 420
accctggttt gtctggcgac cggtttttat ccggatcatg tggaactgtc ttggtgggtg 480
aacggcaaag aagtgcatag cggtgtttct accgatccgc agccgctgaa agaacagccg 540
gcgctgaatg atagccgtta tgcgctgtct agccgtctgc gtgttagcgc gaccttttgg 600
caaaatccgc gtaaccattt tcgttgccag gtgcagtttt atggcctgag cgaaaacgat 660
gaatggaccc aggatcgtgc gaagccggtt acccagattg ttagcgcgga agcctggggc 720
cgcgcagat 729
<210> 30
<211> 250
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 30
Met Ala Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Val Gln
1 5 10 15
Glu Gly Glu Asn Val Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser
20 25 30
Ala Leu His Trp Leu Arg Gln Asp Pro Gly Gly Gly Pro Val Ser Leu
35 40 45
Phe Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr
50 55 60
Ile Asn Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Val His
65 70 75 80
Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Val Arg Asp Ala Gly Gly
85 90 95
Thr Ser Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr Lys Leu Thr Val
100 105 110
His Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
115 120 125
Glu Gly Gly Gly Ser Glu Gly Gly Thr Gly Asn Ala Gly Val Thr Gln
130 135 140
Thr Pro Lys Tyr Leu Ser Val Lys Thr Gly Gln Ser Val Thr Leu Gln
145 150 155 160
Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser Trp Tyr Arg Gln Asp
165 170 175
Pro Gly Gln Gly Leu Arg Leu Ile His Tyr Ser Val Gly Ala Gly Ile
180 185 190
Thr Asp Gln Gly Glu Val Pro Asn Arg Tyr Asn Val Ser Arg Ser Thr
195 200 205
Thr Glu Asp Phe Pro Leu Arg Ile Glu Ser Val Thr Pro Ser Asp Thr
210 215 220
Ser Val Tyr Phe Cys Ala Ser Ser Tyr Pro Gly Ser Tyr Gly Tyr Thr
225 230 235 240
Phe Gly Ser Gly Thr Arg Leu Thr Val Asp
245 250
<210> 31
<211> 750
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 31
atggctaaaa atgaagttga acagagcccg cagaatctga ccgttcaaga aggcgaaaac 60
gtcaccatca actgcagcta cagcgttggc attagcgcac tgcattggct gcgtcaagat 120
ccgggcggcg gtccggtttc tctgtttatg ctgagtagcg gcaaaaagaa acacggtcgt 180
ctgaacgcga ccattaacat ccaggaaaag cacagcagtc tgcatattac cgcagttcat 240
ccgcgcgata gcgcggttta cttctgcgca gttcgcgatg caggcggtac cagttacggt 300
aaactgacct tcggccaagg taccaaactg accgttcatc cgggtggggg ttctgaaggc 360
ggcggtagtg aaggcggcgg tagcgaaggc ggcggttctg aaggcggtac cggtaacgca 420
ggcgtaaccc aaaccccgaa atacctgagc gtcaaaaccg gtcagagcgt gaccctgcag 480
tgcgctcagg atatgaacca cgagtacatg agctggtatc gtcaagatcc gggtcaaggt 540
ctgcgtctga ttcattacag cgttggcgcg ggtattaccg atcaaggcga agttccgaac 600
cgttataacg tcagccgtag caccaccgaa gattttccgc tgcgtattga aagcgttacc 660
ccgtctgata ccagcgtgta cttctgcgcg agcagttatc caggtagcta cggttacacc 720
ttcggttctg gtacccgtct gaccgttgat 750
<210> 32
<211> 112
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 32
Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Val Gln Glu Gly
1 5 10 15
Glu Asn Val Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu
20 25 30
His Trp Leu Arg Gln Asp Pro Gly Gly Gly Pro Val Ser Leu Phe Met
35 40 45
Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile Asn
50 55 60
Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Val His Pro Arg
65 70 75 80
Asp Ser Ala Val Tyr Phe Cys Ala Val Arg Asp Ala Gly Gly Thr Ser
85 90 95
Tyr Gly Lys Leu Thr Phe Gly Gln Gly Thr Lys Leu Thr Val His Pro
100 105 110
<210> 33
<211> 336
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 33
aaaaatgaag ttgaacagag cccgcagaat ctgaccgttc aagaaggcga aaacgtcacc 60
atcaactgca gctacagcgt tggcattagc gcactgcatt ggctgcgtca agatccgggc 120
ggcggtccgg tttctctgtt tatgctgagt agcggcaaaa agaaacacgg tcgtctgaac 180
gcgaccatta acatccagga aaagcacagc agtctgcata ttaccgcagt tcatccgcgc 240
gatagcgcgg tttacttctg cgcagttcgc gatgcaggcg gtaccagtta cggtaaactg 300
accttcggcc aaggtaccaa actgaccgtt catccg 336
<210> 34
<211> 112
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 34
Asn Ala Gly Val Thr Gln Thr Pro Lys Tyr Leu Ser Val Lys Thr Gly
1 5 10 15
Gln Ser Val Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met
20 25 30
Ser Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile His Tyr
35 40 45
Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Arg Tyr
50 55 60
Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Ile Glu Ser
65 70 75 80
Val Thr Pro Ser Asp Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Pro
85 90 95
Gly Ser Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Asp
100 105 110
<210> 35
<211> 336
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 35
aacgcaggcg taacccaaac cccgaaatac ctgagcgtca aaaccggtca gagcgtgacc 60
ctgcagtgcg ctcaggatat gaaccacgag tacatgagct ggtatcgtca agatccgggt 120
caaggtctgc gtctgattca ttacagcgtt ggcgcgggta ttaccgatca aggcgaagtt 180
ccgaaccgtt ataacgtcag ccgtagcacc accgaagatt ttccgctgcg tattgaaagc 240
gttaccccgt ctgataccag cgtgtacttc tgcgcgagca gttatccagg tagctacggt 300
tacaccttcg gttctggtac ccgtctgacc gttgat 336

Claims (10)

1. A T Cell Receptor (TCR), wherein the TCR is capable of binding to the KWVESIFLIF-HLA a2402 complex; preferably, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the amino acid sequence of CDR3 of the TCR alpha chain variable domain is AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSYPGSYGYT (SEQ ID NO: 15);
more preferably, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:
αCDR1-VGISA(SEQ ID NO:10)
αCDR2-LSSGK(SEQ ID NO:11)
α CDR3-AVRDAGGTSYGKLT (SEQ ID NO: 12); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-MNHEY(SEQ ID NO:13)
βCDR2-SVGAGI(SEQ ID NO:14)
βCDR3-ASSYPGSYGYT(SEQ ID NO:15)。
2. a TCR as claimed in claim 1 which comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 5.
3. A TCR as claimed in claim 1 wherein a conjugate is attached to the C-or N-terminus of the α and/or β chain of the TCR; preferably, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these; preferably, the therapeutic agent is an anti-CD 3 antibody.
4. A multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR as claimed in any one of the preceding claims.
5. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to any preceding claim, or the complement thereof;
preferably, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 2 or SEQ ID NO 33 encoding the variable domain of the TCR alpha chain; and/or
The nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 6 or SEQ ID NO 35 encoding the variable domain of the TCR beta chain.
6. A vector comprising the nucleic acid molecule of claim 5; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
7. An isolated host cell comprising the vector of claim 6 or a nucleic acid molecule of claim 5 integrated into the chromosome.
8. A cell which transduces the nucleic acid molecule of claim 5 or the vector of claim 6; preferably, the cell is a T cell or a stem cell.
9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1-3, a TCR complex according to claim 4, a nucleic acid molecule according to claim 5, or a cell according to claim 8.
10. Use of a T cell receptor according to any one of claims 1 to 3, or a TCR complex according to claim 4 or a cell according to claim 8, for the preparation of a medicament for the treatment of a tumour or an autoimmune disease.
CN202110363579.8A 2021-04-02 2021-04-02 T cell receptor recognizing AFP Pending CN115160432A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202110363579.8A CN115160432A (en) 2021-04-02 2021-04-02 T cell receptor recognizing AFP
PCT/CN2022/084190 WO2022206861A1 (en) 2021-04-02 2022-03-30 T cell receptor for identifying afp

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110363579.8A CN115160432A (en) 2021-04-02 2021-04-02 T cell receptor recognizing AFP

Publications (1)

Publication Number Publication Date
CN115160432A true CN115160432A (en) 2022-10-11

Family

ID=83458010

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110363579.8A Pending CN115160432A (en) 2021-04-02 2021-04-02 T cell receptor recognizing AFP

Country Status (2)

Country Link
CN (1) CN115160432A (en)
WO (1) WO2022206861A1 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201313377D0 (en) * 2013-07-26 2013-09-11 Adaptimmune Ltd T cell receptors
CN111171137A (en) * 2018-11-13 2020-05-19 中国科学院广州生物医药与健康研究院 T cell receptor for identifying AFP antigen short peptide and its coding sequence
CN112390875B (en) * 2019-08-16 2023-01-24 香雪生命科学技术（广东）有限公司 High-affinity T cell receptor for identifying AFP

Also Published As

Publication number Publication date
WO2022206861A1 (en) 2022-10-06

Similar Documents

Publication Publication Date Title
CN106632660B (en) TCR for recognizing NY-ESO-1 antigen short peptide
CN110950949B (en) T cell receptor for recognizing SSX2 antigen
CN107197625B (en) T cell receptor for recognizing NY-ESO-1 antigen short peptide
AU2020363138B2 (en) T cell receptor recognising KRAS mutation and encoding sequence thereof
CN106749620B (en) T cell receptor for recognizing MAGE-A1 antigen short peptide
CN106831978B (en) T cell receptor recognizing PRAME antigen
CN110343166B (en) T cell receptor recognizing AFP antigen short peptides
CN110343167B (en) T cell receptor recognizing SSX2 antigen short peptide
CN110776562B (en) T cell receptor for identifying AFP antigen
CN110577591B (en) T cell receptor for identifying AFP antigen short peptide and its coding sequence
CN113321726B (en) T cell receptor for recognizing HPV
CN106632658B (en) TCR for recognizing NY-ESO-1 antigen short peptide
CN109575121B (en) T cell receptor recognizing AFP antigen short peptides
WO2021170116A1 (en) T cell receptor recognizing afp
CN113321727B (en) T cell receptor for identifying AFP antigen short peptide and coding sequence thereof
CN109251243B (en) T cell receptor for recognizing SAGE1 antigen and nucleic acid for encoding receptor
CN108948184B (en) T cell receptor for recognizing PRAME antigen-derived short peptide
WO2021139699A1 (en) T cell receptor for recognizing afp and coding sequence thereof
CN110627894B (en) T cell receptor for recognizing NY-ESO-1 antigen short peptide and coding sequence thereof
CN110577590B (en) TCR capable of recognizing AFP antigen and encoding nucleic acid thereof
CN110407927B (en) TCR (T cell receptor) capable of recognizing AFP (alpha fetoprotein) antigen
CN109575120B (en) TCR for identifying PRAME antigen short peptide and coding sequence thereof
CN109400696B (en) TCR for identifying PRAME antigen short peptide
CN108659114B (en) TCR for recognizing PASD1 antigen short peptide
CN115160432A (en) T cell receptor recognizing AFP

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination