WO2018077242A1 - Récepteur de lymphocytes t pour la reconnaissance de polypeptide à chaîne courte de l'antigène sage1 - Google Patents

Récepteur de lymphocytes t pour la reconnaissance de polypeptide à chaîne courte de l'antigène sage1 Download PDF

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

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a TCR capable of recognizing a short peptide derived from a SAGE1 antigen, and to a SAGE1-specific T cell obtained by transducing the above TCR, and their use in the prevention and treatment of a disease associated with SAGE1.
  • SAGE1 As an endogenous tumor antigen, SAGE1 is degraded into small molecule polypeptides after intracellular production, and combined with MHC (main histocompatibility complex) molecules to form a complex, which is presented to the cell surface.
  • MHC main histocompatibility complex
  • VFSTVPPAFI is a short peptide derived from SAGE1.
  • SAGE1 antigen is expressed in tumor tissues such as melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, and squamous cell carcinoma, but not in most normal tissues except testis (Martelange V1, De Smet C, De Plaen E, Lurquin C, Boon T. Cancer Res.
  • T cell adoptive immunotherapy is the transfer of reactive T cells specific for the target cell antigen into the patient to act on the target cells.
  • the T cell receptor (TCR) is a membrane protein on the surface of T cells that recognizes antigenic short peptides on the surface of the corresponding target cells.
  • APCs antigen presenting cells
  • pMHC complex short peptide-primary histocompatibility complex
  • TCR T cell receptor
  • the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain
  • the amino acid sequence of the CDR3 of the TCR alpha chain variable domain is CAVLYTGANSKLTF (SEQ ID NO. 12); and/or The amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is CASSLVGKQPQHF (SEQ ID NO. 15).
  • the three complementarity determining regions (CDRs) of the TCR alpha chain variable domain are:
  • the three complementarity determining regions of the TCR ⁇ chain variable domain are:
  • the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID NO.
  • the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5.
  • the TCR comprises an alpha chain variable domain amino acid sequence of SEQ ID NO.
  • the TCR comprises the ⁇ chain variable domain amino acid sequence of SEQ ID NO.
  • the TCR is an alpha beta heterodimer comprising a TCR alpha chain constant region TRAC*01 and a TCR beta chain constant region TRBC1*01 or TRBC2*01.
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 3 and/or the ⁇ chain amino acid sequence of the TCR is SEQ ID NO.
  • the TCR is soluble.
  • the TCR is single stranded.
  • the TCR is formed by linking an alpha chain variable domain to a beta chain variable domain via a peptide linker sequence.
  • the TCR is in the alpha chain variable region amino acid at the 11th, 13th, 19th, 21st, 53th, 76th, 89th, 91th or 94th position, and/or the alpha chain J gene short peptide amino acid reciprocal One or more mutations in the third position, the fifth last position or the seventh in the last number; and/or the TCRs in the ⁇ chain variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91 Or the 94th, and/or ⁇ chain J gene short peptide amino acid reciprocal number 2, the last 4th or the last 6th position has one or more mutations, wherein the amino acid position number according to IMGT (International Immunogenetics Information The location number listed in the system).
  • IMGT International Immunogenetics Information
  • the TCR comprises (a) all or part of a TCR alpha chain other than a transmembrane domain; and (b) all or part of a TCR beta chain other than a transmembrane domain;
  • cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.
  • cysteine residue forming an artificial disulfide bond in the TCR replaces one or more sets of sites selected from the group consisting of:
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO. 26 and/or the ⁇ chain amino acid sequence of the TCR is SEQ ID NO.
  • an artificial interchain disulfide bond is contained between the alpha chain variable region of the TCR and the beta chain constant region.
  • cysteine residue forming an artificial interchain disulfide bond in the TCR replaces one or more sets of sites selected from the group consisting of:
  • the TCR comprises an alpha chain variable domain and a beta chain variable domain and all or part of a beta chain constant domain other than a transmembrane domain, but which does not comprise an alpha chain constant domain, said TCR
  • the alpha chain variable domain forms a heterodimer with the beta chain.
  • the C- or N-terminus of the alpha chain and/or beta strand of the TCR incorporates a conjugate.
  • the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modified moiety, or a combination of any of these.
  • the therapeutic agent is an anti-CD3 antibody.
  • a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one TCR molecule is the TCR of the first aspect of the invention.
  • a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule of the first aspect of the invention or a complement thereof is provided.
  • the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 2 encoding a TCR alpha chain variable domain.
  • the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 6 encoding a TCR ⁇ chain variable domain.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 4 encoding the TCR alpha chain and/or comprises the nucleotide sequence SEQ ID NO: 8 encoding the TCR beta chain.
  • a vector comprising the nucleic acid molecule of the third aspect of the invention is provided; preferably, the vector is a viral vector; more preferably, the vector is slow Viral vector.
  • an isolated host cell comprising the vector of the fourth aspect of the invention or the nucleic acid molecule of the third aspect of the invention integrated with exogenous in the genome .
  • the invention provides a cell which is transduced with the nucleic acid molecule of the third aspect of the invention or the vector of the fourth aspect of the invention; preferably, the cell is a T cell or a stem cell .
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a TCR according to the first aspect of the present invention, a TCR complex according to the second aspect of the present invention, and a present invention are provided.
  • the T cell receptor of the first aspect of the invention, or the TCR complex of the second aspect of the invention, the nucleic acid molecule of the third aspect of the invention, the fourth aspect of the invention The use of the vector of the aspect, or the cell of the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumor or an autoimmune disease.
  • a method of treating a disease comprising administering an appropriate amount of the T cell receptor of the first aspect of the invention, or the TCR complex of the second aspect of the invention to a subject in need of treatment
  • the nucleic acid molecule of the third aspect of the invention, the vector of the fourth aspect of the invention, or the cell of the sixth aspect of the invention, or the pharmaceutical composition of the seventh aspect of the invention comprising administering an appropriate amount of the T cell receptor of the first aspect of the invention, or the TCR complex of the second aspect of the invention to a subject in need of treatment.
  • the disease is a tumor, preferably the tumor comprises melanoma, and other tumors Tumors such as gastric cancer, lung cancer (eg, lung squamous cell carcinoma), esophageal cancer, bladder cancer, head and neck cancer (eg, head and neck squamous cell carcinoma), prostate cancer, breast cancer, colon cancer, ovarian cancer, kidney Cell carcinoma, Hodgkin's lymphoma, sarcoma, medulloblastoma, leukemia, etc.
  • Tumors such as gastric cancer, lung cancer (eg, lung squamous cell carcinoma), esophageal cancer, bladder cancer, head and neck cancer (eg, head and neck squamous cell carcinoma), prostate cancer, breast cancer, colon cancer, ovarian cancer, kidney Cell carcinoma, Hodgkin's lymphoma, sarcoma, medulloblastoma, leukemia, etc.
  • the tumor comprises melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, and squamous cell carcinoma.
  • Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e and Figure 1f are the TCR alpha chain variable domain amino acid sequence, the TCR alpha chain variable domain nucleotide sequence, the TCR alpha chain amino acid sequence, the TCR alpha chain nucleotide sequence, respectively.
  • 2a, 2b, 2c, 2d, 2e, and 2f are a TCR ⁇ chain variable domain amino acid sequence, a TCR ⁇ chain variable domain nucleotide sequence, a TCR ⁇ chain amino acid sequence, a TCR ⁇ chain nucleotide sequence, respectively.
  • Figure 3 shows the results of double positive staining of CD8 + and tetramer-PE in monoclonal cells.
  • Figures 4a and 4b are the amino acid sequence and nucleotide sequence of the soluble TCR alpha chain, respectively.
  • Figures 5a and 5b are the amino acid sequence and nucleotide sequence of the soluble TCR ⁇ chain, respectively.
  • Figure 6 is a gel diagram of the soluble TCR obtained after purification.
  • the leftmost lane is the reducing gel
  • the middle lane is the molecular weight marker
  • the rightmost lane is the non-reducing gel.
  • Figure 7 is a ProteOn combining the soluble TCR with the VFSTVPPAFI-HLA A2402 complex of the present invention.
  • Figure 8 shows the results of primary T-fine detection of tetrameric stained TCR transduction.
  • Figure 9 shows the results of the ELISPOT test.
  • Figure 10 is a graph showing the killing effect of TCR transduced T cells of the present invention on specific target cells.
  • the inventors have found extensively and intensively, and found a TCR capable of specifically binding to the SAGE1 antigen short peptide VFSTVPPAFI (SEQ ID NO. 9), which can form a complex with HLA A2402 and be presented together.
  • Cell surface The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules.
  • the invention also provides cells that transduce the TCR of the invention.
  • the MHC molecule is a protein of the immunoglobulin superfamily and may be a class I or class II MHC molecule. Therefore, it is specific for the presentation of antigens, and different individuals have different MHCs that can present different short peptides of a protein antigen to the surface of the respective APC cells.
  • Human MHC is commonly referred to as the HLA gene or the HLA complex.
  • T cell receptor is the only receptor that presents a specific antigenic peptide on the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • APC antigen presenting cells
  • TCR is a glycoprotein on the surface of a cell membrane in the form of a heterodimer formed by an alpha chain/beta chain or a gamma chain/delta chain.
  • the TCR heterodimer consists of alpha and beta chains in 95% of T cells, while 5% of T cells have a TCR consisting of gamma and delta chains.
  • Natural ⁇ heterodimeric TCR has ⁇ chain and ⁇ chain, ⁇ chain and ⁇ The strand constitutes a subunit of the alpha beta heterodimeric TCR.
  • each of the alpha and beta chains comprises a variable region, a junction region, and a constant region
  • the beta chain typically also contains a short polymorphic region between the variable region and the junction region, but the polymorphic region is often considered as a junction region. a part of.
  • Each variable region comprises three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in framework regions.
  • the CDR regions determine the binding of the TCR to the pMHC complex, wherein the CDR3 is recombined from the variable region and the junction region and is referred to as the hypervariable region.
  • the alpha and beta chains of TCR are generally considered to have two "domains", namely a variable domain and a constant domain, and the variable domain consists of linked variable and linking regions.
  • the sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the constant domain sequence of the TCR molecule ⁇ chain is “TRAC*01”
  • the constant domain sequence of the TCR molecule ⁇ chain is “TRBC1*”. 01" or "TRBC2*01”.
  • the alpha and beta chains of TCR also contain a transmembrane and cytoplasmic regions with a short cytoplasmic region.
  • polypeptide of the present invention TCR of the present invention
  • T cell receptor of the present invention T cell receptor of the present invention
  • the position numbers of the amino acid sequences of TROC*01 and TRBC1*01 or TRBC2*01 are numbered in order from N-terminus to C-terminus, such as TRBC1*01 or TRBC2*01.
  • the 60th amino acid is P (valine), which may be described as Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, or may be It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and in the case of TRBC1*01 or TRBC2*01, the 61st amino acid is Q in the order from N to C.
  • Amide which may be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, or may be expressed as amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01, other And so on.
  • the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are numbered according to the positions listed in the IMGT.
  • the position number listed in IMGT is 46, which is described in the present invention as amino acid 46 of TRAV, and so on.
  • special instructions will be given.
  • a first aspect of the invention provides a TCR molecule capable of binding to a VFSTVPPAFI-HLA A2402 complex.
  • the TCR molecule is isolated or purified.
  • the alpha and beta strands of the TCR each have three complementarity determining regions (CDRs).
  • the alpha chain of the TCR comprises a CDR having the following amino acid sequence:
  • the three complementarity determining regions of the TCR ⁇ chain variable domain are:
  • the chimeric TCR can be prepared by embedding the above-described CDR region amino acid sequences of the present invention into any suitable framework structure.
  • a TCR molecule of the invention refers to a TCR molecule comprising the above-described alpha and/or beta chain CDR region sequences and any suitable framework structure.
  • the TCR alpha chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID NO. 1; and/or the TCR ⁇ chain variable domain of the invention is SEQ ID NO: 5 has an amino acid sequence of at least 90%, preferably 95%, more preferably 98% sequence identity.
  • the TCR molecule of the invention is a heterodimer composed of alpha and beta chains.
  • the alpha chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the alpha chain variable domain amino acid sequence comprising the CDR1 (SEQ ID NO: 10), CDR2 (SEQ) ID NO: 11) and CDR3 (SEQ ID NO. 12).
  • the TCR molecule comprises an alpha chain variable domain amino acid sequence of SEQ ID NO. More preferably, the alpha chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO.
  • the beta strand of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the beta strand variable domain amino acid sequence comprising CDR1 (SEQ ID NO. 13), CDR2 (SEQ ID) NO: 14) and CDR3 (SEQ ID NO. 15).
  • the TCR molecule comprises a beta chain variable domain amino acid sequence of SEQ ID NO. More preferably, the ⁇ chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO.
  • the TCR molecule of the invention is a single-chain TCR molecule consisting of part or all of the alpha chain and/or part or all of the beta chain.
  • a description of single-chain TCR molecules can be found in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658.
  • One skilled in the art can readily construct single-chain TCR molecules comprising the CDRs regions of the invention, as described in the literature.
  • the single-chain TCR molecule comprises V ⁇ , V ⁇ and C ⁇ , preferably linked in order from N-terminus to C-terminus.
  • the alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above alpha chain.
  • the single-chain TCR molecule comprises an alpha chain variable domain amino acid sequence of SEQ ID NO. More preferably, the alpha chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO.
  • the ⁇ chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above-described ⁇ chain.
  • the single-chain TCR molecule comprises the ⁇ -chain variable domain amino acid sequence of SEQ ID NO. More preferably, the ⁇ chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO.
  • the constant domain of the TCR molecule of the invention is a human constant domain.
  • the constant domain sequence of the ⁇ chain of the TCR molecule of the present invention may be "TRAC*01”
  • the constant domain sequence of the ⁇ chain of the TCR molecule may be "TRBC1*01” or "TRBC2*01”.
  • the 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is represented here as: Arg53 of exon 1 of TRAC*01, and so on.
  • the amino acid sequence of the ⁇ chain of the TCR molecule of the present invention is SEQ ID NO. 3, and/or the amino acid sequence of the ⁇ chain is SEQ ID NO.
  • TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane domain.
  • TCR can also be developed for diagnosis and treatment, when soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCR has a wide range of uses, not only for studying the interaction of TCR with pMHC, but also as a diagnostic tool for detecting infection or as a marker for autoimmune diseases.
  • soluble TCR can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells that present specific antigens.
  • soluble TCRs can also bind to other molecules (eg, anti-CD3 antibodies). To redirect T cells so that they target cells that present a particular antigen.
  • the SAGE1 antigen short peptide is also obtained by the present invention. Has a specific soluble TCR.
  • the TCR of the invention can be a TCR that introduces an artificial disulfide bond between the residues of its alpha and beta chain constant domains.
  • the cysteine residue forms an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR.
  • a cysteine residue can replace other amino acid residues at a suitable position in the native TCR to form an artificial interchain disulfide bond. For example, a Thr248 residue of the exon 1 of TRAC*01 and a cysteine residue of Ser57 of the exon 1 of TRBC1*01 or TRBC2*01 are substituted to form a disulfide bond.
  • Other sites for introducing a cysteine residue to form a disulfide bond may also be: Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 of Tyr10 and TRBC1*01 or TRBC2*01 exon 1 of Ser17; TRAC*01 exon 1 of Thr45 and TRBC1*01 or TRBC2*01 exon 1 of Asp59; TRAC*01 exon 1 Ser15 and TRBC1*01 or TRBC2*01 exon 1 of Glu15; TRAC*01 exon 1 of Arg53 and TRBC1*01 or TRBC2*01 exon 1 of Ser54; TRAC*01 exon 1 of Pro89 and ABC19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 and TRBC1*01 of exon 1 of TRAC*01 or Glu20 of exon 1 of TRBC2*01.
  • a cysteine residue replaces any of the above-mentioned sites in the ⁇ and ⁇ chain constant domains.
  • a maximum of 50, or a maximum of 30, or a maximum of 15, or a maximum of 10, or a maximum of 8 or fewer amino acids may be truncated at one or more C-termini of the TCR constant domains of the invention such that they are not included
  • the cysteine residue is used for the purpose of deleting the natural disulfide bond, and the above object can also be achieved by mutating the cysteine residue forming the natural disulfide bond to another amino acid.
  • the TCR of the present invention may comprise an artificial disulfide bond introduced between residues of its ⁇ and ⁇ chain constant domains.
  • the constant domains may or may not contain the introduced artificial disulfide bonds as described above, and the TCRs of the present invention may each contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence.
  • the TRAC constant domain sequence of TCR and the TRBC1 or TRBC2 constant domain sequence can be joined by a native disulfide bond present in the TCR.
  • the TCR of the present invention further comprises a TCR having a mutation in its hydrophobic core region, and the mutation of these hydrophobic core regions is preferably a mutation capable of improving the stability of the soluble TCR of the present invention, as in the publication number It is described in the patent document of WO2014/206304.
  • Such a TCR can be mutated at its position in the following variable domain hydrophobic core: (alpha and/or beta chain) variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and / Or the ⁇ -chain J gene (TRAJ) short peptide amino acid position reciprocal position 3, 5, 7 and/or ⁇ chain J gene (TRBJ) short peptide amino acid position reciprocal position 2, 4, 6 where the amino acid sequence position number The location number listed in the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the TCR in which the hydrophobic core region is mutated in the present invention may be a stable soluble single-chain TCR composed of a flexible peptide chain linking the variable domains of the ⁇ and ⁇ chains of the TCR.
  • the flexible peptide chain of the present invention may be any peptide chain suitable for linking the TCR alpha and beta chain variable domains.
  • the patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the alpha chain variable region of the TCR and the beta chain constant region can significantly improve the stability of the TCR. Therefore, the ⁇ chain variable region of the high affinity TCR of the present invention and the ⁇ chain constant region may further contain an artificial interchain disulfide bond.
  • cysteine residue forming an artificial interchain disulfide bond between the ⁇ chain variable region of the TCR and the ⁇ chain constant region is substituted with: amino acid 46 of TRAV and TRBC1*01 or TRBC2* 01 amino acid at position 60 of exon 1; amino acid at position 47 of TRAV and amino acid at position 61 of exon 1 of TRBC1*01 or TRBC2*01; amino acid at position 46 of TRAV and TRBC1*01 or TRBC2*01 The amino acid at position 61 of the 1st; or the amino acid at position 47 of TRAV and the amino acid at position 60 of exon 1 of TRBC1*01 or TRBC2*01.
  • such a TCR may comprise (i) all or part of a TCR alpha chain other than its transmembrane domain, and (ii) all or part of a TCR beta chain other than its transmembrane domain, wherein (i) and (ii) ) both contain a variable domain of the TCR chain and at least a portion In the constant domain, the alpha chain forms a heterodimer with the beta chain. More preferably, such a TCR may comprise an alpha chain variable domain and a beta chain variable domain and all or part of a beta chain constant domain other than a transmembrane domain, but which does not comprise an alpha chain constant domain, said TCR alpha The chain variable domain forms a heterodimer with the beta chain.
  • the TCR of the present invention can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention, such as a tetrameric domain of p53 to produce a tetramer, or more A complex formed by combining a TCR of the invention with another molecule.
  • the TCR complexes of the invention can be used to track or target cells that present a particular antigen in vitro or in vivo, as well as intermediates that produce other multivalent TCR complexes for such applications.
  • the TCR of the present invention may be used singly or in combination with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate comprises a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of a cell presenting a VFSTVPPAFI-HLA A2402 complex), a therapeutic agent, a PK (protein kinase) modified moiety, or any of these The combination is combined or coupled.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agents, or capable of producing detectable products Enzyme.
  • Therapeutic agents that can be combined or coupled to the TCRs of the invention include, but are not limited to: 1. Radionuclides (Koppe et al, 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al, 1989) , Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc.
  • liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nanomagnetic particles; 10. prodrug activating enzymes (eg, DT-diaphorase) (DTD) or biphenyl hydrolase-like protein (BPHL); 11. chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.
  • prodrug activating enzymes eg, DT-diaphorase) (DTD) or biphenyl hydrolase-like protein (BPHL)
  • chemotherapeutic agent eg, cisplatin or any form of nanoparticles, and the like.
  • the TCR of the invention may also be a hybrid TCR comprising sequences derived from more than one species.
  • the TCR of the invention may comprise a human variable domain and a murine constant domain.
  • a drawback of this approach is that it may trigger an immune response. Therefore, there should be a regulatory regimen for immunosuppression when used in adoptive T cell therapy to allow for the implantation of murine T cells.
  • amino acid names in this article are represented by the international single letter or three English letters.
  • the correspondence between the single English letters of the amino acid name and the three English letters is as follows: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).
  • a second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention, or a portion thereof, which may be one or more CDRs, a variable domain of an alpha and/or beta chain, and an alpha chain and/or Or beta chain.
  • nucleotide sequence encoding the CDR region of the alpha chain of the TCR molecule of the first aspect of the invention is as follows:
  • nucleotide sequence encoding the CDR region of the ⁇ chain of the TCR molecule of the first aspect of the invention is as follows:
  • nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR alpha chain of the invention comprises SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18, and/or a nucleic acid molecule of the invention encoding a TCR ⁇ chain of the invention.
  • the nucleotide sequence includes SEQ ID NO. 19, SEQ ID NO. 20, and SEQ ID NO.
  • the nucleotide sequence of the nucleic acid molecule of the present invention may be single-stranded or double-stranded, and the nucleic acid molecule may be RNA or DNA, and may or may not contain an intron.
  • the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, for example, the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID NO. 2 and / or the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR beta chain variable domain of the invention comprises SEQ ID NO.
  • the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. It will be appreciated that due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide.
  • a nucleic acid sequence encoding a TCR of the invention may be the same or a degenerate variant of the nucleic acid sequence set forth in the Figures of the invention.
  • a "degenerate variant" refers to a nucleic acid sequence which encodes a protein sequence having SEQ ID NO. 1, but differs from the sequence of SEQ ID NO.
  • the nucleotide sequence can be codon optimized. Different cells are different in the utilization of specific codons, and the number of expressions can be increased by changing the codons in the sequence depending on the type of the cell. Codon selection tables for mammalian cells as well as a variety of other organisms are well known to those skilled in the art.
  • the full length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification, recombinant methods or synthetic methods. At present, it has been possible to obtain a DNA sequence encoding the TCR (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. The DNA can be a coding strand or a non-coding strand.
  • the invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, ie, constructs that are capable of expression in vivo or in vitro.
  • expression vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.
  • Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.
  • AAV adeno-associated virus
  • the vector can transfer a nucleotide of the invention into a cell, such as a T cell, such that the cell expresses a TAGE specific for the SAGE1 antigen.
  • the vector should be capable of sustained high levels of expression in T cells.
  • the invention also relates to host cells genetically engineered using the vectors or coding sequences of the invention.
  • the host cell contains the vector of the present invention or a nucleic acid molecule of the present invention in which the chromosome is integrated.
  • the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, such as E. coli, yeast cells, CHO cells, and the like.
  • the invention also encompasses isolated cells, particularly T cells, which express the TCR of the invention.
  • the T cell can be derived from a T cell isolated from the subject, or can be a mixed cell population isolated from the subject, such as a portion of a peripheral blood lymphocyte (PBL) population.
  • PBL peripheral blood lymphocyte
  • the cells can be isolated from peripheral blood mononuclear cells (PBMC), which can be CD4 + helper T cells or CD8 + cytotoxic T cells.
  • PBMC peripheral blood mononuclear cells
  • the cells can be in a mixed population of CD4 + helper T cells/CD8 + cytotoxic T cells.
  • the cells can be activated with antibodies (e.g., anti-CD3 or anti-CD28 antibodies) to enable them to be more readily transfected, e.g., with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention. dye.
  • antibodies e.g., anti-CD3 or anti-CD28 antibodies
  • the cells of the invention may also be or be derived from stem cells, such as hematopoietic stem cells (HSCs). Transfer of the gene to HSC does not result in the expression of TCR on the cell surface because the stem cell surface does not express CD3 molecules. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of thymocytes.
  • stem cells differentiate into lymphoid precursors that migrate to the thymus
  • CD3 molecule will initiate expression of the introduced TCR molecule on the surface of thymocytes.
  • T cell transfection with DNA or RNA encoding the TCR of the invention e.g., Robbins et al., (2008) J. Immunol. 180: 6116-6131.
  • T cells expressing the TCR of the present invention can be used in adoptive immunotherapy.
  • Those skilled in the art will be aware of many suitable methods for performing adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).
  • the invention also relates to a method of treating and/or preventing a disease associated with SAGE1 in a subject, comprising the step of adoptively transferring SAGE1-specific T cells to the subject.
  • the SAGE1-specific T cells recognize the VFSTVPPAFI-HLA A2402 complex.
  • the SAGE1-specific T cells of the invention can be used to treat any SAGE1-related disease presenting the SAGE1 antigen short peptide VFSTVPPAFI-HLA A2402 complex.
  • SAGE1-related disease presenting the SAGE1 antigen short peptide VFSTVPPAFI-HLA A2402 complex.
  • tumors such as melanoma, and other solid tumors such as gastric cancer, lung cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, ovarian cancer, and the like.
  • the T cells of a patient or a volunteer having a disease associated with the SAGE1 antigen can be isolated, and the TCR of the present invention can be introduced into the above T cells, and then these genetically engineered cells can be returned to the patient for treatment.
  • the present invention provides a method of treating a SAGE1-related disease comprising administering an isolated TCR expressing a TCR of the present invention, preferably, the T cell is derived from a patient itself and is administered to a patient.
  • it comprises (1) isolating a patient's T cells, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding the TCR molecule of the invention, and (3) inputting genetically engineered T cells into the patient in vivo.
  • the number of cells that are isolated, transfected, and returned can be determined by the physician.
  • the TCR of the present invention can bind to the SAGE1 antigen short peptide complex VFSTVPPAFI-HLA A2402, and the cells transduced with the TCR of the present invention can be specifically activated and have a strong killing effect on target cells.
  • Peripheral blood lymphocytes from healthy volunteers with genotype HLA-A2402 were stimulated with the synthetic short peptide SAGE1PX149 597-606 VFSTVPPAFI (Beijing Cypress Biotech Co., Ltd.).
  • SAGE1 PX149 597-606 VFSTVPPAFI short peptide was renatured with biotinylated HLA-A*2402 to prepare a pHLA monomer.
  • moners were combined with PE-labeled streptavidin (BD) into PE-labeled tetramers, which were sorted and anti-CD8-APC double positive cells.
  • the sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonal culture by limiting dilution. Monoclonal cells were stained with tetramers and the double positive clones screened are shown in Figure 3.
  • TCR ⁇ chain variable domain amino acid sequence and TCR ⁇ are the TCR ⁇ chain variable domain amino acid sequence and TCR ⁇ , respectively.
  • Fig. 2a, Fig. 2b, Fig. 2c and Fig. 2d are TCR ⁇ chain variable domain amino acid sequence, TCR ⁇ chain variable domain nucleotide, respectively Sequence, TCR ⁇ chain amino acid sequence and TCR ⁇ chain nucleotide sequence.
  • the alpha chain has been identified to comprise a CDR having the following amino acid sequence:
  • the beta strand comprises a CDR having the following amino acid sequence:
  • variable domains of the TCR alpha chain and the beta chain are each spliced into the full length gene and linked to the lentiviral expression vector pLenti (addgene) by overlap PCR with the conserved domains of the mouse TCR alpha chain and the beta chain, respectively.
  • the TCR ⁇ -2A-TCR ⁇ fragment was obtained by ligating the full-length genes of the TCR ⁇ chain and the TCR ⁇ chain by overlap PCR.
  • the lentiviral expression vector and TCR ⁇ -2A-TCR ⁇ were digested to obtain the pLenti-SAGE1TRA-2A-TRB-IRES-NGFR plasmid.
  • a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. The 1981T/17 is then used to package the pseudovirus.
  • the ⁇ and ⁇ chains of the TCR molecule of the present invention may comprise only their variable domains and partial constant domains, respectively, and a cysteine residue is introduced in the constant domains of the ⁇ and ⁇ chains, respectively.
  • the positions at which cysteine residues are introduced are Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC2*01, respectively; amino acid sequence and nucleotide of ⁇ chain thereof
  • the sequences are shown in Figures 4a and 4b, respectively, and the amino acid sequence and nucleotide sequence of the ⁇ chain are shown in Figures 5a and 5b, respectively, and the introduced cysteine residues are indicated by bold and underlined letters.
  • the above TCR ⁇ and ⁇ chain target gene sequences were synthesized and inserted into the expression vector pET28a+ (Novagene) by standard methods described in the Molecular Cloning a Laboratory Manual (3rd edition, Sambrook and Russell). ), the upstream and downstream cloning sites are NcoI and NotI, respectively. The insert was sequenced to confirm that it was correct.
  • the TCR ⁇ and ⁇ chain expression vectors were transformed into the expression bacterium BL21 (DE3) by chemical transformation, respectively, and the bacteria were grown in LB medium.
  • the formed inclusion bodies were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution. The inclusion bodies were finally dissolved in 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA). , 20 mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains were rapidly mixed in a mass ratio of 1:1 in 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, 6.6 mM ⁇ -mercapoethylamine (4 ° C), and the final concentration was 60 mg. /mL. After mixing, the solution was placed in 10 volumes of deionized water. After analysis (4 ° C), 12 hours later, the deionized water was replaced with a buffer (20 mM Tris, pH 8.0) and dialysis was continued at 4 ° C for 12 hours.
  • the solution after completion of dialysis was filtered through a 0.45 ⁇ M filter, and then purified by an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare).
  • the TCR containing the refolding successful alpha and beta dimers was confirmed by SDS-PAGE gel.
  • the TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare).
  • the purified TCR purity was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.
  • the SDS-PAGE gel of the soluble TCR obtained by the present invention is shown in Fig. 6.
  • the binding activity of the TCR molecule obtained in Example 3 to the VFSTVPPAFI-HLA A2402 complex was examined using a BIAcore T200 real-time analysis system.
  • the anti-streptavidin antibody (GenScript) was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the surface of the chip. Finally, the unreacted activated surface was blocked with a solution of ethanolamine in hydrochloric acid to complete the coupling process at a coupling level of about 15,000 RU.
  • a low concentration of streptavidin is passed over the surface of the coated antibody chip, then the VFSTVPPAFI-HLA A2402 complex is flowed through the detection channel, and the other channel is used as a reference channel, and then 0.05 mM biotin is 10 ⁇ L/min. The flow rate was passed through the chip for 2 min, blocking the remaining binding sites of streptavidin.
  • E. coli bacterial solution inducing expression of heavy or light chain 100 ml of E. coli bacterial solution inducing expression of heavy or light chain was collected, and the cells were washed once with 8000 g of PBS at 10 ° C for 10 min, and then resuspended by vigorous shaking with 5 ml of BugBuster Master Mix Extraction Reagents (Merck). Incubate for 20 min at room temperature, then centrifuge at 6000 g for 15 min at 4 ° C, discard the supernatant, and collect inclusion bodies.
  • the above-mentioned inclusion weight was suspended in 5 ml BugBuster Master Mix, and incubated at room temperature for 5 min; 30 ml of BugBuster diluted 10 times, mixed, centrifuged at 6000 g for 15 min at 4 ° C; the supernatant was discarded, and 30 ml of BugBuster resuspended inclusion body was diluted 10 times.
  • the synthesized short peptide VFSTVPPAFI (Beijing Saibaisheng Gene Technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml.
  • the inclusion bodies of the light and heavy chains were dissolved with 8 M urea, 20 mM Tris pH 8.0, 10 mM DTT, and further denatured by the addition of 3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA before renaturation.
  • the VFSTVPPAFI peptide was added to the refolding buffer at 25 mg/L (final concentration) (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 0.2 mM PMSF, cooled to 4 ° C), then add 20 mg / L light chain and 90 mg / L heavy chain (final concentration, heavy chain added three times, 8h / time), renaturation at 4 ° C for at least 3 days to Upon completion, SDS-PAGE can detect whether the renaturation is successful.
  • the renaturation buffer was replaced with 10 volumes of 20 mM Tris pH 8.0 for dialysis, and at least two buffers were exchanged to substantially reduce the ionic strength of the solution.
  • the protein solution was filtered through a 0.45 ⁇ m cellulose acetate filter and then loaded onto a HiTrap Q HP (GE General Electric Company) anion exchange column (5 ml bed volume).
  • HiTrap Q HP GE General Electric Company
  • Akta Purifier GE General Electric
  • 20 Akta Purifier 20 mM Tris pH 8.0
  • the protein was eluted with a linear gradient of 0-400 mM NaCl.
  • the pMHC was eluted at approximately 250 mM NaCl.
  • the peak fractions were collected and the purity was determined by SDS-PAGE.
  • the purified pMHC molecule was concentrated using a Millipore ultrafiltration tube while the buffer was replaced with 20 mM Tris pH 8.0, followed by biotinylation reagent 0.05M Bicine pH 8.3, 10 mM ATP, 10 mM MgAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml BirA enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and biotinylation was detected by SDS-PAGE.
  • Biotinylated labeled pMHC molecules were concentrated to 1 ml using a Millipore ultrafiltration tube, biotinylated pMHC was purified by gel filtration chromatography, and HiPrepTM was pre-equilibrated with filtered PBS using an Akta Purifier (GE General Electric). A 16/60 S200 HR column (GE General Electric Company) was loaded with 1 ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. The biotinylated pMHC molecule appeared as a single peak elution at about 55 ml.
  • the protein-containing fractions were pooled, concentrated using a Millipore ultrafiltration tube, protein concentration was determined by BCA method (Thermo), and biotinylated pMHC molecules were dispensed at -80 °C by adding protease inhibitor cocktail (Roche).
  • the kinetic parameters of the soluble TCR molecules of the present invention were determined by using the BIAcore Evaluation software to calculate the kinetic parameters as shown in FIG.
  • Lentiviruses containing the gene encoding the desired TCR were packaged using a third generation lentiviral packaging system.
  • Four plasmids (containing one of pLenti-SAGE1 TRA-2A-TRB-IRES-NGFR described in Example 2) using Express-In-mediated transient transfection (Open Biosystems)
  • the ratio of transfection reagent PEI-MAX to plasmid was 2:1, and the usage per plate was 114.75 micrograms.
  • the specific operation is as follows: the expression plasmid and the packaging plasmid are added to a medium of 1800 ⁇ l of OPTI-MEM (Gibco, catalog number 31985-070), and uniformly mixed, and allowed to stand at room temperature for 5 minutes to become a DNA mixture; The corresponding amount of PEI was mixed well with 1800 ⁇ l of OPTI-MEM medium, and allowed to stand at room temperature for 5 minutes to become a PEI mixture. The DNA mixture and the PEI mixture were mixed together and allowed to stand at room temperature for 30 minutes, and then 3150 ⁇ l of OPTI was added.
  • -MEM medium mix well, add to 293T/17 cells that have been converted to 11.25 ml of OPTI-MEM, gently shake the dish, mix the medium evenly, and incubate at 37 ° C / 5% CO 2 . After -7 hours, the transfection medium was removed and replaced with DMEM (Gibco, catalog number C11995500bt) containing 10% fetal bovine serum, and cultured at 37 ° C / 5% CO 2 . The medium supernatant containing the packaged lentivirus was collected on day 3 and day 4.
  • DMEM Gibco, catalog number C11995500bt
  • the collected culture supernatant was centrifuged for 15 minutes for 15 minutes to remove cell debris and then passed through a 0.22 micron filter (Merckmi Merck Millipore, catalog number SLGP033RB), finally intercepted with 50KD
  • the concentrated tube Merck Millipore, catalog number UFC905096 was concentrated to remove most of the supernatant, finally concentrated to 1 ml, and aliquoted and stored at -80 ° C.
  • the pseudovirus sample was taken for virus.
  • the titer was measured by the p24 ELISA (Clontech, Cat. No. 632200) kit instructions.
  • the pseudovirus of pLenti-eGFP was also included.
  • the cells were counted every two days, and fresh medium containing 50 IU/ml IL-2 and 10 ng/ml IL-7 was replaced or added to maintain the cells at 0.5 x 10 6 - 1 x 10 6 cells/ml.
  • Cells were analyzed by flow cytometry starting on day 3 and were used for functional assays from day 5 (eg, ELISPOT and non-radioactive cytotoxicity assays for IFN- ⁇ release).
  • SAGE1PX149 597-606 VFSTVPPAFI short peptide was renatured with biotinylated HLA-A*2402 to prepare pHLA monomer. These monomers are combined with PE-labeled streptavidin (BD) into a PE-labeled tetramer called PX149-tetramer-PE. This tetramer can label T cells expressing a SAGE1-specific T cell receptor gene as positive cells.
  • the transduced T cell samples in (b) were incubated with PX149-tetramer-PE for 30 minutes on ice, then anti-mouse ⁇ -chain-APC antibody was added and incubation was continued for 15 minutes on ice.
  • BD Calibur or BD Arial was used to detect or sort PX149-tetramer-PE expressing the SAGE1-specific T cell receptor gene and anti-mouse ⁇ -chain-APC double positive T Cells were analyzed by CellQuest software (BD) or FlowJo software (Tree Star Inc, Ashland, OR).
  • the following assays were performed to demonstrate activation of TCR-transduced T cells specifically responding to target cells.
  • the IFN- ⁇ production detected by the ELISPOT assay was used as a readout value for T cell activation.
  • Test medium 10% FBS (Gibco, catalog number 16000-044), RPMI 1640 (Gibco, catalog number C11875500bt)
  • PVDF ELISPOT 96-well plate (Merck Millipore, catalog number MSIPS4510)
  • the human IFN- ⁇ ELISPOT PVDF-Enzyme Kit contains all the other reagents required (capture and detection antibodies, streptavidin-alkaline phosphatase and BCIP/NBT solutions)
  • the target cells of this example are Epstein-Barr virus (EBV) transformed immortalized lymphoblastoid cell lines (LCLs).
  • B95-8 cells were induced to produce EBV-containing medium supernatant by tetradecanoyl phorbol ester (TPA), centrifuged at 4 ° C / 600 g for 10 minutes to remove impurities, and then passed through a 0.22 micron filter, aliquoted -70 ° C save.
  • EBV Epstein-Barr virus
  • TPA tetradecanoyl phorbol ester
  • PBL peripheral blood lymphocytes
  • CD19 + CD23 hi CD58 + was an immortalized lymphoblastic cell line (LCLs).
  • This ELISPOT assay uses HLA-A24 as a specific target cell.
  • T cells The effector cells (T cells) of the present assay were CD8 + T cells expressing SAGE1-specific TCR by flow cytometry in Example 3, and CD8 + T of the same volunteer was used as a negative control effector cell.
  • T cells were stimulated with anti-CD3/CD28 coated beads (T cell amplicon, LifeTechnologies), transduced with lentivirus carrying the SAGE1-specific TCR gene (according to Example 3), containing 50 IU/ml IL-2 and 10 ng /ml IL-7 in 1040 medium containing 10% FBS was amplified until 9-12 days after transduction, and then these cells were placed in a test medium, and washed by centrifugation at 300 g for 10 minutes at room temperature. The cells were then resuspended in test medium at 2 x the desired final concentration. Negative control effector cells were also treated.
  • SAGE1CD8 + T cells SAGE1 TCR transduced T cells, effector cells VF3CD8+ T cells), CD8+ T cells (negative control effector cells) and LCL-A24/A02 (target cells) were prepared as described in Example 3, and The corresponding experimental group was added with the corresponding short peptide, wherein PX149 was SAGE1PX149597-606VFSTVPPAFI short peptide, and PA11, PA02, PA24-1, PA24-2 and PA24-3 were non-SAGE1 TCR specific binding short peptides.
  • effector cells 1000 SAGE1 TCR double positive T cells.
  • the plates were then incubated overnight (37 ° C / 5% CO 2 ) for the next day, the medium was discarded, the plates were washed twice with double distilled water, washed 3 times with wash buffer, and tapped on a paper towel to remove residuals. Wash buffer.
  • the primary antibody was then diluted with PBS containing 10% FBS and added to each well at 100 ⁇ L/well. The well plates were incubated for 2 hours at room temperature and washed 3 times with wash buffer and the well plates were tapped on paper towels to remove excess wash buffer.
  • the TCR-transduced T cells of the present invention were tested for IFN- ⁇ release in response to target cells bearing the SAGE1 PX149 597-606 VFSTVPPAFI short peptide and target cells of the non-specific short peptide by the ELISPOT assay (described above).
  • the number of ELSPOT spots observed in each well was plotted using graphpad prism6.
  • the experimental results are shown in Fig. 9.
  • the CD8 + T cells transducing the TCR of the present invention have a strong activation effect on the LCLs of the load-related short peptides, releasing more IFN- ⁇ , and the LCLs supporting the non-specific short peptides are basically There was no response; at the same time, CD8 + T cells that did not transduce the TCR of the present invention responded very little to LCLs loaded with short peptides, with only a small amount of IFN- ⁇ released.
  • This test is a colorimetric substitution test for the 51Cr release cytotoxicity assay to quantify the lactate dehydrogenase (LDH) released after cell lysis.
  • LDH lactate dehydrogenase
  • the LDH released in the medium was detected using a 30 minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) to red formazan.
  • INT tetrazolium salt
  • the amount of red product produced is directly proportional to the number of cells lysed.
  • the 490 nm visible light absorbance data can be collected using a standard 96-well plate reader.
  • CytoTox Non-radioactive cytotoxicity assays (Promega, G1780) contain a substrate mixture, assay buffer, lysis solution, and stop buffer.
  • Test medium 10% FBS (heat inactivated, Gibbco, catalog number 16000-044), phenol red containing 90% RPMI 1640 (Gibco, catalog number C11875500bt), 1% penicillin / Streptomycin (Jibuco, catalog number 15070-063).
  • Microporous round bottom 96-well tissue culture plate (Nunc, catalog number 163320)
  • Target cells were prepared in the test medium: the target cell concentration was adjusted to 334 cells/ml, and 45 microliters per well was taken to obtain 1.5 x 10 4 cells/well.
  • T cells The effector cells (T cells) of this assay were analyzed by flow cytometry in Example 3 to express CD8 + T cells expressing SAGE1-specific TCR.
  • the ratio of effector cells to target cells was 10:1, 5:1, 2.5:1, 1.25:1, and 0.625:1.
  • a homologous CD8 + T cell plus target cell control group (10:1) was set.
  • the components of the assay were added to a microwell round bottom 96-well tissue culture plate in the following sequence:
  • a control group was prepared as follows:
  • the plate was centrifuged at 250 g for 4 minutes. 50 ul of the supernatant from each well of the assay plate was transferred to the corresponding well of a 96-well immunoplate Maxisorb plate. The substrate mixture was reconstituted using assay buffer (12 ml) and then 50 ul was added to each well of the plate. The plate was capped and incubated for 30 minutes at room temperature in the dark. 50 ul of the stop solution was added to each well of the plate to terminate the reaction. The absorbance at 490 nm was counted and recorded within 1 hour after the addition of the stop solution.
  • Absorbance values of the medium background were subtracted from all absorbance values of the experimental group, the target cell spontaneous release group, and the effector cell self-release group.
  • % cytotoxicity 100 ⁇ (experimental - effector cell spontaneous - target cell spontaneous) / (target cell max - target cell spontaneous)
  • the statistical results of the experimental data are shown in Fig. 10. As the ratio of the effective target increases, the killing effect of the TCR-transduced T cells of the present invention on the specific target cells K562-24 and NCI H1299-A24 is enhanced; for the non-specific target cells IM 9 The killing effect is very weak. The homologous CD8 + T cells that did not transduce the TCR of the present invention showed no significant killing of the target cell K562-24.

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Abstract

L'invention concerne un récepteur de lymphocytes T (TCR) capable de se lier spécifiquement à un polypeptide VFSTVPPAFI à chaîne courte dérivé d'un antigène SAGE1, une molécule d'acide nucléique codant pour le TCR, un vecteur comprenant la molécule d'acide nucléique, et une cellule de transduction du TCR. Le polypeptide à chaîne courte d'antigène VFSTVPPAFI peut former un complexe avec HLA A2402 et être transduit conjointement avec la surface cellulaire.
PCT/CN2017/108081 2016-10-27 2017-10-27 Récepteur de lymphocytes t pour la reconnaissance de polypeptide à chaîne courte de l'antigène sage1 WO2018077242A1 (fr)

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CN107936109A (zh) * 2016-10-12 2018-04-20 广东香雪精准医疗技术有限公司 衍生自sage1的肿瘤抗原短肽

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