[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2021135178A1 - 一种增强型t细胞受体star及其应用 - Google Patents

一种增强型t细胞受体star及其应用 Download PDF

Info

Publication number
WO2021135178A1
WO2021135178A1 PCT/CN2020/103687 CN2020103687W WO2021135178A1 WO 2021135178 A1 WO2021135178 A1 WO 2021135178A1 CN 2020103687 W CN2020103687 W CN 2020103687W WO 2021135178 A1 WO2021135178 A1 WO 2021135178A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
constant region
amino acid
antigen binding
cell receptor
Prior art date
Application number
PCT/CN2020/103687
Other languages
English (en)
French (fr)
Inventor
芮魏
伍春燕
刘芳
Original Assignee
华夏英泰（北京）生物技术有限公司
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 华夏英泰（北京）生物技术有限公司 filed Critical 华夏英泰（北京）生物技术有限公司
Priority to KR1020227026190A priority Critical patent/KR20220131253A/ko
Priority to JP2022564686A priority patent/JP2023508608A/ja
Priority to EP20910062.7A priority patent/EP4086281A4/en
Priority to CN202080091762.0A priority patent/CN116034113A/zh
Publication of WO2021135178A1 publication Critical patent/WO2021135178A1/zh

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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A61K39/0005—Vertebrate antigens
    • 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
    • A61K39/463—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
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464404—Epidermal growth factor receptors [EGFR]
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464413—CD22, BL-CAM, siglec-2 or sialic acid binding Ig-related lectin 2
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464424—CD20
    • 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
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • 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
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • 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
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the invention relates to the field of biomedicine, in particular to an enhanced T cell receptor STAR and its application.
  • the present invention discloses an improved synthetic T-Cell Receptor and Antigen Receptor (STAR), T cells containing the synthetic T-Cell Receptor and Antigen Receptor, and their use.
  • STAR synthetic T-Cell Receptor and Antigen Receptor
  • Immune cells in the human body eliminate tumor cells through the following ways: dendritic cells recognize the mutated gene of tumor cells and display this mutation information to T cells. The T cells will then look for tumor cells that carry the mutation and kill them. Sometimes, tumor cells will down-regulate MHC molecules that can display mutant peptides, so that they can escape the killing of T cells and gradually form uncontrollable cancers.
  • Chimeric antigen receptor T cell (CAR-T) therapy is an anti-cancer immunotherapy that has achieved good results in recent years. Different from the way that natural T cells recognize tumor cells, CAR-T cells do not rely on MHC molecules to recognize tumor cells.
  • the CAR molecule contains three major parts: the extracellular domain is the antigen recognition domain derived from the antibody and is responsible for recognizing the target antigen; the transmembrane domain; the intracellular domain is the signal molecule and costimulatory signal molecule derived from the T cell receptor and is responsible for Transmit T cell activation signal after receiving stimulation. Its working principle is as follows: when CAR molecules bind to their corresponding antigens, CAR molecules will aggregate, thereby increasing the level of local phosphorylation, activating downstream signals, and finally starting the effector function of T cells and killing target tumor cells.
  • CD19-CAR-T chimeric antigen receptor T cell
  • CD19-CAR-T Two clinical applications of chimeric antigen receptor T cell (CD19-CAR-T) therapies targeting CD19 protein have been approved for marketing in the United States for the treatment of relapsed and refractory B-cell lymphoma.
  • CAR-T therapy encounters difficulties when used in the treatment of solid tumors.
  • T cell therapy There are multiple factors for the failure of T cell therapy in the treatment of solid tumors.
  • CAR-T cells are inhibited in the tumor microenvironment and T cells are prone to exhaustion and apoptosis. Recent studies have shown that the disability of this T cell may be related to the nature of the chimeric antigen receptor signaling pathway.
  • the T cell receptor (TCR) complex molecule contains multiple chains.
  • the TCR ⁇ chain and the TCR ⁇ chain are responsible for recognizing MHC-polypeptide molecules.
  • the other 6 CD3 subunits are combined with the TCR ⁇ / ⁇ chain to function as signal transduction.
  • the natural TCR complex contains a total of 10 ITAM signal sequences, which can theoretically transmit stronger signals than CAR. Previous studies have shown that although the TCR signal is slower than the CAR signal, the TCR signal is more durable. Therefore, using the signal transduction function of natural TCR, it will be possible to construct a new type of receptor to alleviate the disability of T cells, so that it can better exert its anti-solid tumor effect.
  • TCR variable region sequence can be replaced with antibody variable region sequence to obtain synthetic T cell receptor antigen receptor (Snythetic TCR and Antigen Receptor, STAR), It not only has the specificity of antibodies, but also has the superior signal transduction function of natural TCR, which can mediate complete T cell activation.
  • STAR derived from natural TCR still has disadvantages such as poor membrane stability, low ⁇ / ⁇ chain pairing ability, mismatch with endogenous TCR, and difficulty in introducing T cells. Therefore, there is still a need for improved STAR in this field.
  • Figure 1 shows the structure of the STAR.
  • A Schematic diagram of the structure of the prototype of STAR;
  • B Schematic diagram of the structure of the improved STAR;
  • C Schematic diagram of the dual-STAR targeting different target antigens.
  • Figure 2 shows the sequence alignment results of the human and murine T cell receptor ⁇ chain constant regions and ⁇ chain constant regions.
  • Figure 3 shows the results of the sequence alignment of the N-terminal 18 amino acids of the human and mouse TCR ⁇ chain constant regions.
  • UserSeq1 is a human sequence
  • UserSeq2 is a murine sequence.
  • Figure 4 shows the results of the sequence alignment of the 25 amino acid N-terminus of the human and mouse TCR ⁇ chain constant regions.
  • UserSeq1 is a human sequence
  • UserSeq2 is a murine sequence.
  • Figure 5 shows constant region mutants combining different modifications.
  • Figure 6 The function of each mutant of Cetux STAR targeting EGFR.
  • Figure 7 The function of each mutant of GC33 STAR targeting GPC3.
  • Figure 8 The function of the 334 STAR comprehensive mutant targeting CD19.
  • Figure 9 FMC63-2C6 STAR comprehensive mutant function targeting CD19/CD20.
  • Figure 10 FMC63-M791 STAR comprehensive mutant function targeting CD19/CD22.
  • the term “and/or” encompasses all combinations of items connected by the term, and should be treated as if each combination has been individually listed herein.
  • “A and/or B” encompasses “A”, “A and B”, and “B”.
  • “A, B, and/or C” encompasses "A”, “B”, “C”, “A and B”, “A and C”, “B and C”, and "A and B and C”.
  • the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nuclei at one or both ends of the protein or nucleic acid. Glycolic acid, but still has the activity described in the present invention.
  • methionine encoded by the start codon at the N-terminus of the polypeptide will be retained under certain practical conditions (for example, when expressed in a specific expression system), but does not substantially affect the function of the polypeptide.
  • the present invention provides a synthetic T cell receptor antigen receptor (STAR) comprising an alpha chain and a beta chain, the alpha chain comprising a first antigen binding region and a first constant region, and the beta chain comprising The second antigen binding region and the second constant region,
  • STAR synthetic T cell receptor antigen receptor
  • the first constant region is a variant of the natural T cell receptor alpha chain constant region, which relative to the natural T cell receptor alpha chain constant region comprises i) N-terminal modification; ii) cysteine substitution; and/or iii ) Hydrophobic amino acid substitutions, and/or
  • the second constant region is a variant of the natural T cell receptor ⁇ chain constant region, which contains i) N-terminal modification; and/or ii) cysteine substitution relative to the natural T cell receptor ⁇ chain constant region.
  • the antigen binding region is fused to the N-terminus of the constant region.
  • the ⁇ chain and ⁇ chain can form a functional TCR complex after being expressed in T cells.
  • the ⁇ chain and ⁇ chain can combine with the cell’s endogenous CD3 molecules (CD3 ⁇ , CD3 ⁇ , CD3 ⁇ ) to form a TCR complex of 8 subunits, and the TCR complex is displayed in the cell.
  • CD3 ⁇ , CD3 ⁇ , CD3 ⁇ endogenous CD3 molecules
  • Functional TCR complex refers to its ability to activate T cells after specifically binding to the target antigen.
  • N-terminal modification means that the N-terminal of the mentioned amino acid sequence (polypeptide or protein) contains one or more amino acid substitutions, deletions and/or additions.
  • the term may also include the mentioned amino acid sequence (polypeptide or protein).
  • the amino acid sequence (polypeptide or protein) is deleted from the N-terminus of one or more consecutive amino acids.
  • cyste substitution or “hydrophobic amino acid substitution” refers to the replacement of the original amino acid in the mentioned amino acid sequence (polypeptide or protein) with cysteine or hydrophobic amino acid.
  • the hydrophobic amino acid substitution may be the substitution of a hydrophilic amino acid by a hydrophobic amino acid, or the substitution of an amino acid with low hydrophobicity by an amino acid with high hydrophobicity.
  • the first constant region is derived from a human T cell receptor alpha chain constant region, a non-human primate T cell receptor alpha chain constant region, a rodent such as a mouse T cell receptor alpha chain Constant region.
  • An exemplary human T cell receptor alpha chain constant region includes the amino acid sequence shown in SEQ ID NO:1.
  • An exemplary mouse T cell receptor alpha chain constant region includes the amino acid sequence shown in SEQ ID NO: 2.
  • the first constant region is derived from a mouse T cell receptor alpha chain constant region.
  • the first constant region comprises an N-terminal 18 amino acid modification relative to the natural T cell receptor alpha chain constant region.
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and is missing SEQ ID NO: 3 (DIQNPEPAVYQLKDPRSQ) relative to the natural mouse T cell receptor ⁇ chain constant region Amino acid sequence.
  • the first constant region including the N-terminal modification includes the amino acid sequence shown in SEQ ID NO:4. (Corresponding to TCRac-N.DLT)
  • the first constant region comprises an N-terminal modification relative to the natural T cell receptor ⁇ chain constant region, wherein one or more or all of the 15-18th amino acids of the N-terminal are deleted, so
  • the amino acid numbers refer to SEQ ID NO: 2.
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 15-18 is deleted from the natural mouse T cell receptor ⁇ chain constant region.
  • the amino acid numbers refer to SEQ ID NO: 2.
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 15-18 is deleted from the natural mouse T cell receptor ⁇ chain constant region.
  • N-terminal amino acids 1-14 are replaced by the corresponding amino acids of the natural human T cell receptor alpha chain constant region.
  • the amino acid numbers refer to SEQ ID NO: 2.
  • the first constant region including the N-terminal modification includes the amino acid sequence shown in SEQ ID NO: 5. (Corresponding to TCRaC-N.Rec-4)
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and relative to the natural mouse T cell receptor ⁇ chain constant region, X consecutive amino acids from the N-terminus are deleted, And/or the amino acids from positions X+1 to 18 at the N-terminus are substituted with the corresponding amino acids in the constant region of the alpha chain of the natural human T cell receptor, where X is 0, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and relative to the natural mouse T cell receptor ⁇ chain constant region, the N-terminal 1-4 are deleted.
  • the amino acid at position 5-18 at the N-terminus is substituted with the corresponding amino acid in the constant region of the alpha chain of the natural human T cell receptor.
  • the amino acid number refers to SEQ ID NO: 2.
  • the first constant region including the N-terminal modification includes the amino acid sequence shown in SEQ ID NO: 34. (Corresponding to TCRaC-N.Rec-1)
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-8 is deleted from the natural mouse T cell receptor ⁇ chain constant region, And the 9-18th amino acid at the N-terminus is replaced by the corresponding amino acid of the natural human T cell receptor alpha chain constant region, and the amino acid number refers to SEQ ID NO: 2.
  • the first constant region including the N-terminal modification includes the amino acid sequence shown in SEQ ID NO:35. (Corresponding to TCRaC-N.Rec-2)
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-12 is deleted from the natural mouse T cell receptor ⁇ chain constant region, And wherein the 13th-18th amino acid at the N-terminus is replaced by the corresponding amino acid of the natural human T cell receptor ⁇ chain constant region, and the amino acid number refers to SEQ ID NO: 2.
  • the first constant region including the N-terminal modification includes the amino acid sequence shown in SEQ ID NO: 36. (Corresponding to TCRaC-N.Rec-3)
  • the first constant region relative to the natural T cell receptor alpha chain constant region, includes a cysteine substitution at position 48, and the amino acid number refers to SEQ ID NO: 2.
  • the threonine at position 48 of the first constant region is mutated to cysteine, and the amino acid number refers to SEQ ID NO: 2 .
  • the first constant region containing a cysteine substitution includes the amino acid sequence shown in SEQ ID NO: 6 (derived from the human T cell receptor alpha chain constant region).
  • the first constant region comprising a cysteine substitution comprises the amino acid sequence shown in SEQ ID NO: 7 (derived from the mouse T cell receptor alpha chain constant region). (Corresponding to TCRaC-Cys)
  • amino acid numbering refers to SEQ ID NO: x
  • SEQ ID NO: x is a specific sequence listed herein
  • amino acid correspondence can be determined according to sequence alignment methods known in the art. For example, the amino acid correspondence can be determined by the online comparison tool of EMBL-EBI (https://www.ebi.ac.uk/Tools/psa/), where two sequences can use the Needleman-Wunsch algorithm, using the default parameters to Aligned.
  • amino acid at position 46 from the N-terminus of a polypeptide is aligned with the amino acid at position 48 of SEQ ID NO: x in a sequence alignment
  • amino acid in the polypeptide can also be described herein as " For the alanine at position 48 of the polypeptide, the amino acid position refers to SEQ ID NO: x".
  • the first constant region includes a hydrophobic amino acid substitution in the transmembrane region, for example, the transmembrane region includes the amino acid sequence of positions 111-119 , The amino acid number refers to SEQ ID NO: 2.
  • the first constant region comprises hydrophobic amino acid substitutions at positions 112, 114, and/or 115, and the amino acid number refers to SEQ ID NO :2.
  • the first constant region includes hydrophobic amino acid substitutions at positions 112, 114, and 115, and the amino acid number refers to SEQ ID NO: 2 .
  • the serine at position 112 of the first constant region is replaced by leucine, and the methionine at position 114 is replaced by isoleucine , And/or the glycine at position 115 is replaced by valine, and the amino acid number refers to SEQ ID NO: 2.
  • the serine at position 112 of the first constant region is replaced by leucine, and the methionine at position 114 is replaced by isoleucine , And the glycine at position 115 is replaced by valine, and the amino acid number refers to SEQ ID NO: 2.
  • the first constant region containing hydrophobic amino acid substitutions includes the transmembrane region shown in SEQ ID NO: 8 (derived from the mouse T cell receptor alpha chain constant region).
  • the first constant region comprising hydrophobic amino acid substitutions comprises the amino acid sequence shown in SEQ ID NO: 9 (derived from the mouse T cell receptor alpha chain constant region).
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-4 is deleted from the natural mouse T cell receptor ⁇ chain constant region, And wherein the 5-18th amino acid at the N-terminus is replaced by the corresponding amino acid of the natural human T cell receptor alpha chain constant region, the threonine at the 48th position is mutated to cysteine, and the amino acid number refers to SEQ ID NO: 2.
  • the first constant region comprises the amino acid sequence of SEQ ID NO: 11. (Corresponding to TCRaC-Cys-N.Rec-1)
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-4 is deleted from the natural mouse T cell receptor ⁇ chain constant region, And where the 5-18th amino acid at the N-terminus is replaced by the corresponding amino acid in the constant region of the natural human T cell receptor ⁇ chain, the serine at position 112 is replaced by leucine, and the methionine at position 114 is replaced by isoleucine. The amino acid is substituted, and the glycine at position 115 is substituted by valine.
  • the amino acid number refers to SEQ ID NO: 2.
  • the first constant region comprises the amino acid sequence of SEQ ID NO: 12. (Corresponding to TCRaC-TM9-N.Rec-1)
  • the first constant region is derived from the mouse T cell receptor alpha chain constant region, and relative to the natural mouse T cell receptor alpha chain constant region, the threonine at position 48 is mutated Is cysteine, the serine at position 112 is replaced by leucine, the methionine at position 114 is replaced by isoleucine, and the glycine at position 115 is replaced by valine, the amino acid numbering reference SEQ ID NO: 2.
  • the first constant region comprises the amino acid sequence of SEQ ID NO: 10. (Corresponding to TCRaC-Cys-TM9)
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-4 is deleted from the natural mouse T cell receptor ⁇ chain constant region, And the amino acids 5-18 at the N-terminus were replaced by the corresponding amino acids in the constant region of the natural human T cell receptor ⁇ chain, the threonine at position 48 was mutated to cysteine, and the serine at position 112 was replaced by Leucine substitution, methionine at position 114 is replaced by isoleucine, and glycine at position 115 is replaced by valine.
  • the amino acid number refers to SEQ ID NO: 2.
  • the first constant region comprises the amino acid sequence of SEQ ID NO: 13. (Corresponding to TCRaC-Cys-TM9-N.Rec-1)
  • the second constant region is derived from a human T cell receptor ⁇ chain constant region, a non-human primate T cell receptor ⁇ chain constant region, a rodent such as a mouse T cell receptor ⁇ chain Constant region.
  • An exemplary human T cell receptor ⁇ chain constant region includes the amino acid sequence shown in SEQ ID NO: 14.
  • An exemplary mouse T cell receptor alpha chain constant region comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first constant region is derived from a mouse T cell receptor ⁇ chain constant region.
  • the second constant region comprises a modification of 25 amino acids at the N-terminus relative to the natural T cell receptor ⁇ chain constant region.
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and is missing SEQ ID NO: 16 (DLRNVTPPKVSLFEPSKAEIANKQK) relative to the natural mouse T cell receptor ⁇ chain constant region. Amino acid sequence.
  • the second constant region including N-terminal modification includes the amino acid sequence shown in SEQ ID NO:17. (Corresponding to TCRbC-N.DLT)
  • the second constant region comprises an N-terminal modification relative to the natural T cell receptor ⁇ chain constant region, wherein one or more or all of the 17th and 21-25th amino acids of the N-terminal are deleted ,
  • the amino acid number refers to SEQ ID NO: 15.
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal positions 17 and 21-25 are deleted from the natural mouse T cell receptor ⁇ chain constant region
  • One or more or all of the amino acids, and the amino acid number refers to SEQ ID NO: 15.
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal positions 17 and 21-25 are deleted from the natural mouse T cell receptor ⁇ chain constant region One or more or all of the amino acids, and the amino acids at positions 1-16 at the N-terminus are replaced by the corresponding amino acids in the constant region of the beta chain of the natural human T cell receptor.
  • the amino acid numbers refer to SEQ ID NO: 15.
  • the second constant region including N-terminal modification includes the amino acid sequence shown in SEQ ID NO: 18. (Corresponding to TCRbC-N.Rec-4)
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and relative to the natural mouse T cell receptor ⁇ chain constant region, X consecutive amino acids from the N-terminus are deleted, And/or the amino acids from positions X+1 to 25 at the N-terminus are substituted with the corresponding amino acids in the constant region of the ⁇ -chain of the natural human T cell receptor, where X is 0, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-6 is deleted relative to the natural mouse T cell receptor ⁇ chain constant region, And wherein the 7-25th amino acid at the N-terminus is replaced by the corresponding amino acid of the natural human T cell receptor ⁇ chain constant region, and the amino acid number refers to SEQ ID NO: 15.
  • the second constant region including N-terminal modification includes the amino acid sequence shown in SEQ ID NO: 37. (Corresponding to TCRbC-N.Rec-1)
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-12 is deleted from the natural mouse T cell receptor ⁇ chain constant region, And wherein the 13-25th amino acid at the N-terminus is replaced by the corresponding amino acid of the natural human T cell receptor ⁇ chain constant region, and the amino acid number refers to SEQ ID NO: 15.
  • the second constant region including N-terminal modification includes the amino acid sequence shown in SEQ ID NO: 38. (Corresponding to TCRbC-N.Rec-2)
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-18 is deleted from the natural mouse T cell receptor ⁇ chain constant region, And the amino acids 19-25 at the N-terminus are replaced by the corresponding amino acids in the constant region of the beta chain of the natural human T cell receptor, and the amino acid numbers refer to SEQ ID NO: 15.
  • the second constant region including N-terminal modification includes the amino acid sequence shown in SEQ ID NO: 39. (Corresponding to TCRbC-N.Rec-3)
  • the second constant region includes a cysteine substitution at position 56, and the amino acid number refers to SEQ ID NO: 15.
  • the serine at position 56 of the second constant region is mutated to cysteine, and the amino acid number refers to SEQ ID NO: 15.
  • the second constant region comprising a cysteine substitution comprises the amino acid sequence shown in SEQ ID NO: 19 (derived from the human T cell receptor ⁇ chain constant region).
  • the second constant region containing a cysteine substitution includes the amino acid sequence shown in SEQ ID NO: 20 (derived from the mouse T cell receptor ⁇ chain constant region). (Corresponding to TCRbC-Cys)
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-6 is deleted relative to the natural mouse T cell receptor ⁇ chain constant region, And wherein the 7-25th amino acid at the N-terminus is replaced by the corresponding amino acid of the constant region of the natural human T cell receptor ⁇ chain, the serine at position 56 is mutated to cysteine, and the amino acid number refers to SEQ ID NO: 15.
  • the second constant region comprises the amino acid sequence shown in SEQ ID NO:21. (Corresponding to TCRbC-Cys-N.Rec-1)
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-4 is deleted relative to the natural mouse T cell receptor ⁇ chain constant region , And wherein the 5-18th amino acid at the N-terminus is replaced by the corresponding amino acid of the natural human T cell receptor ⁇ chain constant region, the amino acid number refers to SEQ ID NO: 2; the second constant region is derived from mouse T The cell receptor ⁇ chain constant region, and relative to the natural mouse T cell receptor ⁇ chain constant region, the N-terminal amino acids 1-6 are deleted, and the N-terminal amino acids 7-25 are accepted by natural human T cells.
  • the amino acid number refers to SEQ ID NO: 15.
  • the first constant region includes the amino acid sequence shown in SEQ ID NO: 34
  • the second constant region includes the amino acid sequence shown in SEQ ID NO: 37.
  • the first constant region is derived from the mouse T cell receptor alpha chain constant region, and relative to the natural mouse T cell receptor alpha chain constant region, the threonine at position 48 is Mutation to cysteine, the amino acid number refers to SEQ ID NO: 2; the second constant region is derived from the mouse T cell receptor ⁇ chain constant region and is constant relative to the natural mouse T cell receptor ⁇ chain In the region, the serine at position 56 was mutated to cysteine, and the amino acid number refers to SEQ ID NO: 15.
  • the first constant region includes the amino acid sequence shown in SEQ ID NO: 7, and the second constant region includes the amino acid sequence shown in SEQ ID NO: 20.
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and relative to the natural mouse T cell receptor ⁇ chain constant region, the serine at position 112 is leucine. Acid substitution, the methionine at position 114 is replaced by isoleucine, and the glycine at position 115 is replaced by valine, the amino acid number refers to SEQ ID NO: 2; the second constant region is a natural small Mouse T cell receptor ⁇ chain constant region.
  • the first constant region includes the amino acid sequence shown in SEQ ID NO: 9, and the second constant region includes the amino acid sequence shown in SEQ ID NO: 15.
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-4 is deleted relative to the natural mouse T cell receptor ⁇ chain constant region And wherein the 5-18th amino acid at the N-terminus is replaced by the corresponding amino acid in the constant region of the natural human T cell receptor ⁇ chain, the threonine at the 48th position is mutated to cysteine, and the amino acid number refers to SEQ ID NO: 2;
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-6 is deleted from the natural mouse T cell receptor ⁇ chain constant region, and The 7-25th amino acids at the N-terminus are replaced by the corresponding amino acids in the constant region of the ⁇ -chain of the natural human T cell receptor, and the serine at position 56 is mutated to cysteine.
  • the amino acid number refers to SEQ ID NO: 15 .
  • the first constant region includes the amino acid sequence of SEQ ID NO:
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-4 is deleted relative to the natural mouse T cell receptor ⁇ chain constant region And the amino acids 5-18 at the N-terminus were replaced by the corresponding amino acids in the constant region of the natural human T cell receptor ⁇ chain, the serine at position 112 was replaced by leucine, and the methionine at position 114 was replaced by iso Leucine is substituted, and the glycine at position 115 is substituted by valine.
  • the amino acid number refers to SEQ ID NO: 2; the second constant region is derived from the mouse T cell receptor ⁇ chain constant region and is relative to The natural mouse T cell receptor ⁇ chain constant region lacks the N-terminal amino acids 1-6, and the N-terminal amino acids 7-25 are replaced by the corresponding amino acids in the natural human T cell receptor ⁇ chain constant region.
  • the amino acid numbers refer to SEQ ID NO: 15.
  • the first constant region includes the amino acid sequence of SEQ ID NO: 12
  • the second constant region includes the amino acid sequence of SEQ ID NO: 37.
  • the first constant region is derived from the mouse T cell receptor alpha chain constant region, and relative to the natural mouse T cell receptor alpha chain constant region, the threonine at position 48 is Mutation to cysteine, serine at position 112 was replaced by leucine, methionine at position 114 was replaced by isoleucine, and glycine at position 115 was replaced by valine, the amino acid numbering Refer to SEQ ID NO: 2; the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and compared to the natural mouse T cell receptor ⁇ chain constant region, the serine at position 56 is mutated to Cysteine, the amino acid number refers to SEQ ID NO: 15.
  • the first constant region includes the amino acid sequence of SEQ ID NO: 10
  • the second constant region includes the amino acid sequence of SEQ ID NO: 20.
  • the first constant region is derived from the mouse T cell receptor ⁇ chain constant region, and the N-terminal amino acid 1-4 is deleted relative to the natural mouse T cell receptor ⁇ chain constant region , And the 5-18th amino acids at the N-terminus were replaced by the corresponding amino acids in the constant region of the natural human T cell receptor ⁇ chain, the threonine at the 48th position was mutated to cysteine, and the serine at the 112th position Is replaced by leucine, the methionine at position 114 is replaced by isoleucine, and the glycine at position 115 is replaced by valine, the amino acid number refers to SEQ ID NO: 2; and,
  • the second constant region is derived from the mouse T cell receptor ⁇ chain constant region, and relative to the natural mouse T cell receptor ⁇ chain constant region, the N-terminal amino acids 1-6 are deleted, and the N-terminal amino acid is deleted.
  • the amino acids at positions 7-25 were replaced by the corresponding amino acids in the constant region of the beta chain of the natural human T cell receptor, and the serine at position 56 was mutated to cysteine.
  • the amino acid number refers to SEQ ID NO: 15.
  • the first constant region includes the amino acid sequence of SEQ ID NO: 13
  • the second constant region includes the amino acid sequence of SEQ ID NO: 21.
  • antigen binding region means that it alone or in combination with another antigen binding region can specifically bind to a target antigen.
  • the "antigen” or “target antigen” described herein can be any antigen that can be recognized and bound by an antibody, preferably a disease-related antigen, and more preferably a cancer-related antigen.
  • cancer-associated antigens include, but are not limited to, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cells Adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialylganglioside GD2, ductal epithelial mucin, gp36, TAG- 72.
  • Glycosphingolipids glioma-related antigens, ⁇ -human chorionic gonadotropin, ⁇ -fetoglobulin (AFP), lectin-responsive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostatase specific antigen (PSA), PAP, NY- ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22 , Insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, major histocompatibility complex (MHC) molecules that present tumor-specific peptide epitopes, 5
  • the antigen binding region can be derived from one or more known antibodies, including any commercially available antibodies, such as FMC63, rituximab, alemtuzumab, epalizumab (epratuzumab), trastuzumab, bivatuzumab, cetuximab, labetuzumab, palivizumab, Si Sevirumab, tuvirumab, basiliximab, daclizumab, infliximab, omalizumab, law Efalizumab, Keliximab, Siplizumab, Natalizumab, Clenoliximab, Pemtumomab, Pemtumomab Drolozumab (Edrecolomab), cantuzumab (Cantuzumab) and so on.
  • FMC63 rituximab
  • alemtuzumab alemtuzumab
  • the antigen binding region derived free deposit number is CGMCC No. 17095 (deposited on January 21, 2019 at the address of No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China, Chinese Academy of Sciences Microbiology Monoclonal antibody 334 produced by hybridoma cell 334 produced by the General Microbiology Center of the China Microbial Culture Collection and Management Committee of the Institute.
  • the first antigen binding region comprises a heavy chain variable region of an antibody that specifically binds to a target antigen
  • the second antigen binding region comprises a light chain variable region of the antibody.
  • the first antigen binding region includes the light chain variable region of an antibody that specifically binds to a target antigen
  • the second antigen binding region includes the heavy chain variable region of the antibody.
  • the first antigen binding region includes the amino acid sequence shown in SEQ ID NO: 22, and the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 23; or, the first antigen The binding region includes the amino acid sequence shown in SEQ ID NO: 23, and the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 22, so that the STAR specifically binds to EGFR.
  • the first antigen binding region includes the amino acid sequence shown in SEQ ID NO: 44
  • the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 45
  • the first antigen The binding region includes the amino acid sequence shown in SEQ ID NO: 45
  • the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 44, so that the STAR specifically binds to CD19.
  • the first antigen binding region includes the amino acid sequence shown in SEQ ID NO: 46
  • the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 47; or, the first antigen The binding region includes the amino acid sequence shown in SEQ ID NO: 47, and the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 46, so that the STAR specifically binds to CD20.
  • the first antigen binding region includes the amino acid sequence shown in SEQ ID NO: 48
  • the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 49
  • the first antigen The binding region includes the amino acid sequence shown in SEQ ID NO: 49
  • the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 48, so that the STAR specifically binds to CD22.
  • the first antigen binding region includes the amino acid sequence shown in SEQ ID NO: 24, and the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 25; or, the first antigen The binding region includes the amino acid sequence shown in SEQ ID NO: 25, and the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 24, so that the STAR specifically binds to GPC3.
  • the first antigen binding region includes a heavy chain variable region of an antibody that specifically binds to CD19
  • the heavy chain variable region includes heavy chain CDR1 shown in SEQ ID NO: 28, and SEQ ID NO:
  • the second antigen binding region includes the light chain variable region of an antibody that specifically binds to CD19
  • the light chain variable region includes SEQ ID
  • the light chain variable region includes the light chain CDR1 shown in SEQ ID NO: 31, the light chain CDR2 shown in SEQ ID NO: 32, and the light chain CDR3 shown in SEQ ID NO: 33
  • the second The antigen binding region specifically binds to the heavy chain variable region of an
  • the first antigen binding region includes the amino acid sequence shown in SEQ ID NO: 26, and the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 27; or, the first antigen The binding region includes the amino acid sequence shown in SEQ ID NO: 27, and the second antigen binding region includes the amino acid sequence shown in SEQ ID NO: 26, so that the STAR specifically binds to CD19.
  • the first antigen binding region comprises a single chain antibody (such as scFv) or a single domain antibody (such as camel antibody) that specifically binds to the target antigen.
  • the second antigen binding region comprises a single chain antibody (such as scFv) or a single domain antibody (such as camel antibody) that specifically binds to the target antigen.
  • the first antigen binding region and the second antigen binding region bind the same target antigen.
  • the first antigen binding region and the second antigen binding region bind to different regions (such as different epitopes) of the same target antigen.
  • the first antigen binding region and the second antigen binding region bind different target antigens.
  • the two antigen binding regions may bind CD19 and CD20, or CD19 and CD22, or CD38 and BCMA, or PDL1 and EGFR, respectively.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 22 and the amino acid sequence shown in SEQ ID NO: 23.
  • the first antigen binding region and/or the second antigen binding region specifically bind to EGFR.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 44 and the amino acid sequence shown in SEQ ID NO: 45.
  • the first antigen binding region and/or the second antigen binding region specifically bind to CD19.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 46 and the amino acid sequence shown in SEQ ID NO: 47.
  • the first antigen binding region and/or the second antigen binding region specifically bind to CD20.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 48 and the amino acid sequence shown in SEQ ID NO: 49.
  • the first antigen binding region and/or the second antigen binding region specifically bind to CD22.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 24 and the amino acid sequence shown in SEQ ID NO: 25.
  • the first antigen binding region and/or the second antigen binding region specifically bind to GPC3.
  • the single chain antibody contained in the first antigen binding region and/or the second antigen binding region comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises SEQ The heavy chain CDR1 shown in ID NO: 28, the heavy chain CDR2 shown in SEQ ID NO: 29, and the heavy chain CDR3 shown in SEQ ID NO: 30, and the light chain variable region includes the light chain CDR1 shown in SEQ ID NO: 31 , The light chain CDR2 shown in SEQ ID NO: 32 and the light chain CDR3 shown in SEQ ID NO: 33, whereby the first antigen binding region and/or the second antigen binding region specifically bind to CD19.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 26 and the amino acid sequence shown in SEQ ID NO: 27.
  • the first antigen binding region and/or the second antigen binding region specifically bind to CD19.
  • the present invention provides a synthetic T cell receptor antigen receptor (STAR) comprising an ⁇ chain and a ⁇ chain, the ⁇ chain comprising a first antigen binding region and a first constant region, and the ⁇ chain Contains a second antigen binding region and a second constant region,
  • STAR synthetic T cell receptor antigen receptor
  • first constant region is a natural T cell receptor ⁇ chain constant region or a variant of the natural T cell receptor ⁇ chain constant region as disclosed herein
  • second constant region is a natural T cell receptor ⁇ chain constant region or such as The natural T cell receptor beta chain constant region variants disclosed herein,
  • the first antigen binding region and the second antigen binding region specifically bind to different target antigens or different regions of the same target antigen.
  • the first antigen binding region and/or the second antigen binding region comprise single chain antibodies (such as scFv) or single domain antibodies (such as camel antibodies) that specifically bind to the target antigen.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 22 and the amino acid sequence shown in SEQ ID NO: 23.
  • the first antigen binding region and/or the second antigen binding region specifically bind to EGFR.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 44 and the amino acid sequence shown in SEQ ID NO: 45.
  • the first antigen binding region and/or the second antigen binding region specifically bind to CD19.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 46 and the amino acid sequence shown in SEQ ID NO: 47.
  • the first antigen binding region and/or the second antigen binding region specifically bind to CD20.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 48 and the amino acid sequence shown in SEQ ID NO: 49.
  • the first antigen binding region and/or the second antigen binding region specifically bind to CD22.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 24 and the amino acid sequence shown in SEQ ID NO: 25.
  • the first antigen binding region and/or the second antigen binding region specifically bind to GPC3.
  • the single chain antibody contained in the first antigen binding region and/or the second antigen binding region comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises SEQ The heavy chain CDR1 shown in ID NO: 28, the heavy chain CDR2 shown in SEQ ID NO: 29, and the heavy chain CDR3 shown in SEQ ID NO: 30, and the light chain variable region includes the light chain CDR1 shown in SEQ ID NO: 31 , The light chain CDR2 shown in SEQ ID NO: 32 and the light chain CDR3 shown in SEQ ID NO: 33, whereby the first antigen binding region and/or the second antigen binding region specifically bind to CD19.
  • the single-chain antibody contained in the first antigen-binding region and/or the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 26 and the amino acid sequence shown in SEQ ID NO: 27.
  • the first antigen binding region and/or the second antigen binding region specifically bind to CD19.
  • the two antigen binding regions may bind to CD19 and CD20, or CD19 and CD22, or CD38 and BCMA, or PDL1 and EGFR, respectively.
  • the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the alpha chain and/or beta chain of the synthetic T cell receptor antigen receptor (STAR) of the present invention.
  • STAR synthetic T cell receptor antigen receptor
  • the polynucleotide comprises i) a nucleotide sequence encoding the ⁇ chain, ii) a nucleotide sequence encoding the ⁇ chain, and iii) in the same reading frame. And ii) the nucleotide sequence encoding the self-cleavable peptide.
  • the nucleotide sequence encoding the ⁇ chain may be located at the 5'end or the 3'end of the nucleotide sequence encoding the ⁇ chain.
  • self-cleaving peptide means a peptide that can achieve self-cleavage within a cell.
  • the self-cleaving peptide may include a protease recognition site, so that it can be recognized and specifically cleaved by the protease in the cell.
  • the self-cleaving peptide may be a 2A polypeptide.
  • 2A polypeptide is a type of short peptide derived from viruses, and its self-cleavage occurs during translation. When 2A polypeptide is used to connect two different target proteins and expressed in the same reading frame, the two target proteins are almost produced at a ratio of 1:1.
  • Commonly used 2A polypeptides can be P2A from porcine techovirus-1, T2A from Thosea asignis virus, and E2A from equine rhinitis A virus. And F2A from foot-and-mouth disease virus. Among them, P2A has the highest cutting efficiency and is therefore preferred.
  • a variety of functional variants of these 2A polypeptides are also known in the art, and these variants can also be used in the present invention.
  • the invention provides an expression vector comprising the polynucleotide of the invention operably linked to a regulatory sequence.
  • the "expression vector" of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or may be RNA (such as mRNA) that can be translated.
  • the expression vector is a viral vector, such as a lentiviral vector.
  • regulatory sequence and “regulatory element” are used interchangeably and refer to the upstream (5' non-coding sequence), middle or downstream (3' non-coding sequence) of the coding sequence, and affect the transcription, RNA processing, or processing of the related coding sequence. Stability or translated nucleotide sequence.
  • An expression control element refers to a nucleotide sequence capable of controlling the transcription, RNA processing or stability, or translation of a nucleotide sequence of interest. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, enhancers, and polyadenylation recognition sequences.
  • operably linked refers to the connection of regulatory elements (for example, but not limited to, promoter sequences, transcription termination sequences, etc.) to nucleic acid sequences (for example, coding sequences or open reading frames) such that the nucleotides The transcription of the sequence is controlled and regulated by the transcription control element.
  • regulatory elements for example, but not limited to, promoter sequences, transcription termination sequences, etc.
  • nucleic acid sequences for example, coding sequences or open reading frames
  • the present invention provides a method for preparing therapeutic T cells, which includes expressing the synthetic T cell receptor antigen receptor (STAR) of the present invention in the T cells.
  • the method includes introducing a polynucleotide of the invention or an expression vector of the invention into a T cell.
  • the present invention provides a therapeutic T cell, which comprises the synthetic T cell receptor antigen receptor (STAR) of the present invention, or is obtained by the method of the present invention.
  • STAR synthetic T cell receptor antigen receptor
  • the T cells of the present invention can be obtained from many non-limiting sources by various non-limiting methods, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors.
  • the cells can be derived from a healthy donor or from a patient diagnosed with cancer.
  • the cell may be part of a mixed population of cells exhibiting different phenotypic characteristics.
  • T cells can be obtained by isolating peripheral blood mononuclear cells (PBMC), then using specific antibodies to activate and expand them.
  • PBMC peripheral blood mononuclear cells
  • the T cell is derived from an autologous cell of the subject.
  • autologous refers to cells, cell lines, or cell populations used to treat a subject derived from the subject.
  • the T cells are derived from allogeneic cells, such as from a donor that is compatible with the subject's human leukocyte antigen (HLA). Standard protocols can be used to transform cells from a donor into non-alloreactive cells and replicate as needed to produce cells that can be administered to one or more patients.
  • HLA human leukocyte antigen
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the therapeutic T cell of the present invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion).
  • the present invention provides the use of the therapeutic T cell of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating diseases such as cancer in a subject.
  • Subject refers to an organism suffering from or susceptible to diseases (such as cancer) that can be treated by the cells, methods, or pharmaceutical compositions of the present invention.
  • diseases such as cancer
  • Non-limiting examples include humans, cows, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals.
  • the subject is a human.
  • the present invention provides a method of treating a disease such as cancer in a subject, comprising administering to the subject a therapeutically effective amount of the therapeutic T cell of the present invention or the pharmaceutical composition of the present invention.
  • terapéuticaally effective amount or “therapeutically effective dose” or “effective amount” refers to the amount of a substance, compound, material, or cell that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is the amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of the disease or disorder.
  • an "effective amount" of the cells or pharmaceutical composition of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of injury or disability due to painful disease.
  • an "effective amount" of the cells or pharmaceutical composition of the present invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, and more It is preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.
  • the ability to inhibit tumor growth can be evaluated in an animal model system that predicts the efficacy of human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit the growth of tumor cells, and this inhibition can be determined in vitro by a test well known to those skilled in the art.
  • the dosage level of the cells in the pharmaceutical composition of the present invention may be changed to obtain an amount of the active ingredient that can effectively achieve the desired therapeutic response to a specific patient, composition, and mode of administration without being toxic to the patient.
  • the dosage level selected depends on a variety of pharmacokinetic factors, including the activity of the specific composition of the invention used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, and the specific application.
  • Other drugs, compounds and/or materials used in combination with the composition the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors known in the medical field.
  • T cells or pharmaceutical compositions or drugs according to the present invention can be carried out in any convenient way, including by injection, infusion, implantation or transplantation.
  • Administration of the cells or compositions described herein can be by intravenous, intralymphatic, intradermal, intratumoral, intramedullary, intramuscular, or intraperitoneal administration.
  • the cells or compositions of the invention are preferably administered by intravenous injection.
  • the cancer is selected from lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, Glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, intrauterine cancer Membrane cancer, vaginal cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , Chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid
  • the secreted antibody (Ab) or B cell receptor (BCR) produced by B cells is very similar to the T cell receptor (TCR) in gene structure, protein structure and spatial conformation.
  • Both antibodies and TCRs are composed of variable regions and constant regions, where the variable region plays the role of antigen recognition and binding, and the constant region domain plays the role of structural interaction and signal transduction.
  • TCR ⁇ and ⁇ chains or TCR ⁇ and ⁇ chains
  • VH heavy chain variable region
  • VL light chain variable region
  • STAR Called Synthetic T-Cell Receptor and Antibody Receptor
  • the STAR molecule has two chains.
  • the first chain is the antigen recognition sequence (such as the heavy chain variable region VH of an antibody) and the constant region (C ⁇ , human TCR ⁇ -WT, SEQ ID NO) of the T cell receptor ⁇ chain (TCR ⁇ ).
  • the second chain is the antigen recognition sequence (such as the light chain variable region VL of an antibody) and the constant region (C ⁇ , human TCRbC-WT, SEQ ID) of the T cell receptor ⁇ chain (TCR ⁇ ) NO:14) is obtained by fusion.
  • antigen recognition domains such as VH, VL or scFv, etc.
  • constant region domains TCR ⁇ , ⁇ , ⁇ and ⁇ constant regions
  • Immunoreceptor tyrosine activation motif (Immunoreceptor Tyrosine-based Activation Motif, ITAM) is a motif that plays a role in signal transduction in TCR molecules, and its conservative sequence is YxxL/V.
  • the intracellular region of CD3 ⁇ , ⁇ , ⁇ , and ⁇ chains contains 1 ITAM sequence, and the intracellular region of CD3 ⁇ chain contains 3 ITAM sequences, so a complete STAR complex contains a total of 10 ITAM sequences.
  • the intracellular ITAM sequence is phosphorylated successively, which activates downstream signaling pathways, activates transcription factors such as NF- ⁇ B, NFAT, and AP-1, and triggers the activation of T cells , Produce effect function.
  • the STAR prototype design uses human-derived TCR ⁇ / ⁇ chains (or TCR ⁇ and ⁇ chains) constant region sequences. Due to the constant region sequence of human, primate and murine TCR ⁇ / ⁇ chains (mouse TCRaC-WT, SEQ ID NO: 2; mouse TCRbC-WT, SEQ ID NO: 15) (or TCR ⁇ and ⁇ chains) (or TCR ⁇ and ⁇ chains)
  • TCRaC-WT human TCRaC-WT, SEQ ID NO: 2
  • mouse TCRbC-WT mouse TCRbC-WT, SEQ ID NO: 15
  • TCR ⁇ and ⁇ chains The functional conservation of the products is relatively high, and the key amino acid sequences are the same, so they can be replaced with each other.
  • the present invention replaces the constant region of the STAR molecule with a murine sequence to enhance the function of the STAR molecule after it is transferred into human T cells.
  • the alignment of the human sequence and the murine sequence is shown in Figure 2.
  • the present invention modifies the specific sequence in the STAR molecule, and one of the modification schemes is to delete the N-terminus of the constant region of the STAR molecule.
  • the N-terminal 18 amino acids of the TCR ⁇ chain constant region are deleted (the mouse sequence is DIQNPEPAVYQLKDPRSQ, SEQ ID NO: 3) (the resulting constant region sequence is mouse TCRaC-N.DLT, SEQ ID NO: 4), and the TCR ⁇ chain constant region is deleted N-terminal 25 amino acids (mouse sequence is DLRRVTPPKVSLFEPSKAEIANKQK, SEQ ID NO: 16) (The generated constant region sequence is mouse TCRbC-N.DLT, SEQ ID NO: 17).
  • the present invention rearranges the specific sequence of the constant region to obtain a better effect. Rearrangement means to delete part of the sequence while making humanized mutations to part of the sequence.
  • This regional modification has the following advantages: First, the functional optimization of the hinge region can reduce the mismatch between STAR and endogenous TCR. Second, the molecular size of the optimized STAR is more similar to that of the natural TCR, which is more conducive to the combination of costimulatory molecules, the formation and stabilization of immune synapses, and the subsequent effector functions of T cells.
  • the significance of humanized mutations is to reduce the non-human sequences in the STAR molecules as much as possible while ensuring the function of the STAR molecule, so as to avoid the possibility of STAR-T cells being rejected by the receptor in clinical applications.
  • the specific rearrangement scheme is described as follows.
  • Figure 3 shows the alignment of the 18 amino acids at the N-terminus of the murine TCR ⁇ chain constant region with the human sequence.
  • UserSeq1 is a human sequence
  • UserSeq2 is a murine sequence.
  • the mouse and human sequences are homogenous amino acid substitutions at E6D, K13R, R16K, and Q18S, while the non-polar amino acid at the P15S site is replaced by a polar amino acid. Therefore, it can be considered that the The properties of the protein near the site are not conserved and can be modified without affecting the function.
  • the present invention retains and humanizes the amino acid sequence at positions 1-14 at the N-terminus of the mouse TCR ⁇ chain constant region, and deletes amino acids at positions 15-18 to produce mouse TCRaC-N.Rec-4, SEQ ID NO: 5.
  • mouse TCRaC-N.Rec-2 the resulting constant region sequence is SEQ ID NO: 35; the amino acids 1-12 at the N-terminus of the mouse TCR ⁇ chain constant region are deleted, and amino acids 13-18 are humanized, resulting in the constant region sequence mouse TCRaC-N.Rec-3, SEQ ID NO: 36.
  • the amino acids 1-18 of the N-terminus of the mouse TCR ⁇ chain constant region were also randomly rearranged, resulting in the constant region sequence mouse TCRaC-N.rRec, SEQ ID NO: 40.
  • the 25 amino acid N-terminus of the TCR ⁇ chain constant region (mouse sequence is DLRNVTPPKVSLFEPSKAEIANKQK) rearrangement scheme:
  • Figure 4 shows the alignment of the 25 amino acids at the N-terminus of the murine TCR ⁇ chain constant region with the human sequence.
  • UserSeq1 is a human sequence
  • UserSeq2 is a murine sequence.
  • the present invention retains and humanizes the amino acid sequence at positions 1-16 of the N-terminus of the mouse TCR ⁇ chain constant region, and deletes the amino acids at positions 17 and 21-25, resulting in a constant region sequence of mouse TCRbC-N.Rec-4, SEQ ID NO: 18.
  • the sequence is mouse TCRbC-N.Rec-1, SEQ ID NO: 37; the amino acids 1-12 at the N-terminus of the mouse TCR ⁇ chain constant region were deleted, and the amino acids 13-25 were humanized, resulting in the constant region sequence mouse TCRbC -N.Rec-2, SEQ ID NO: 38; The amino acids 1-18 of the N-terminus of the mouse TCR ⁇ chain constant region were deleted, and amino acids 19-25 were humanized, resulting in the constant region sequence mouse TCRbC-N.Rec -3, SEQ ID NO: 39.
  • the amino acids 1-25 of the N-terminus of the mouse TCR ⁇ chain constant region were randomly rearranged, resulting in the constant region sequence mouse TCRbC-N.rRec, SEQ ID NO: 41.
  • the present invention also deletes 15 amino acids at the C-terminus of the mouse TCR ⁇ chain constant region, and the resulting constant region sequence is mouse TCRaC-C.DLT (SEQ ID NO: 42).
  • the present invention also deletes 15 amino acids at the C-terminus of the constant region of the mouse TCR ⁇ chain, and the resulting constant region sequence is mouse TCRbC-C.DLT (SEQ ID NO: 43).
  • the present invention modifies the specific sequence in the STAR molecule.
  • One of the modification schemes is to perform a cysteine point mutation on the STAR molecule to introduce intermolecular disulfide bonds and strengthen the two chains of the STAR molecule. Mutual pairing with each other to reduce mismatches with endogenous TCR.
  • the specific scheme is described as follows.
  • the present invention modifies the specific sequence of the STAR molecule.
  • One of the modifications is to replace the hydrophobic amino acid in the transmembrane region of the STAR molecule to increase the stability of the STAR molecule and help it to perform more durable.
  • the specific scheme is described as follows.
  • mice TCRaC-Cys-N.Rec-1 (SEQ ID NO: 11), mouse TCRaC-TM9-N. Rec-1 (SEQ ID NO: 12), mouse TCRaC-Cys-TM9 (SEQ ID NO: 10), mouse TCRaC-Cys-TM9-N.Rec-1 (SEQ ID NO: 13), and mouse TCRbC-Cys -N.Rec-1 (SEQ ID NO: 21).
  • variable region domain is composed of amino acid sequences with antigen recognition ability, and generally consists of the following types of molecules: antibody light chain variable region, antibody heavy chain variable region, single chain antibody (scFv), ligand-receptor binding District, Nanobody, etc.
  • the constant region domain is composed of TCR constant regions or related sequences that have the ability to assemble with CD3 subunits, and generally include the constant regions of the TCR ⁇ , ⁇ , ⁇ , and ⁇ chains.
  • the antigen recognition domain and constant region domain in the STAR construct can be permuted and combined to form a variety of constructs with different configurations but similar functions. Some possible permutations and combinations are shown in the following table:
  • STAR targets are mainly achieved through gene expression databases.
  • the expression levels of suitable STAR targets are generally higher or specifically expressed on disease or related cells than normal cells.
  • the antigen recognition domain that recognizes the target is generally an antibody, SCFV, VHH, or antigen-corresponding receptor.
  • the heavy chain variable region (VH), light chain variable region (VL), alpaca single-chain antibody (VHH), as well as receptors and ligands of antibodies can be obtained by immunizing mice, alpaca, sequencing, or through published The sequence is obtained.
  • the obtained mouse or alpaca sequence generally needs to be humanized and codon optimized to make it more conducive to expression and function in human cells.
  • lentiviral or retroviral vector After obtaining the appropriate sequence, connect the obtained sequence to a lentiviral or retroviral vector, check whether the vector is constructed correctly by sequencing method, and then express it in somatic cells through virus packaging and infection, using tag proteins RFP, GFP, MYC, HIS, etc. are used to detect STAR protein expression, membrane loading, and antigen binding.
  • Applicable targets include but are not limited to GPC3, EGFR VIII, CD22, CD20, MSLN, GD2, LIRB4, ROR1, Cllaudin 18.2, CD7, CD47, CD24.
  • a variety of target cell lines are used to illustrate the function of STAR, and the target cells can obtain the luciferase gene by means of genetic engineering, thereby facilitating in vitro function detection.
  • Luciferase is a common substance used for cell function research. The enzyme activity is judged by adding a luciferase substrate to the system, and the luciferase activity is closely related to the expression of the target gene, the strength of the binding, and the number of cells.
  • a target cell line stably expressing luciferase is established, and the amount of luciferase is used to refer to the number of target cells, and then to indicate the killing function of functional cells.
  • the specific cells are as follows:
  • Antigen recognition domains can be derived from antibodies that recognize antigens, single-chain variable regions, variable regions of antibodies, nanobodies, receptors that recognize antigens, and so on.
  • viruses are the main way of T cell transformation.
  • the packaging of lentivirus or retrovirus is related to the size of the packaging. Therefore, smaller antibody recognition units will be favored by larger ones, such as variable single-chain antibodies. Regions and Nanobodies are better choices in terms of size.
  • the existing antibodies as the existing technology are one of the ways to screen antibody recognition domains extensively.
  • the phage library is used to construct a fully human antibody library, affinity optimization, mouse hybridoma screening, alpaca monoclonal antibody screening, Humanization and other technologies obtain new antibodies that recognize antigens.
  • the antigen receptor as a specific molecule that can recognize the antigen, is also one of the main choices for the antigen recognition domain.
  • the vectors used in the present invention including lentiviral vectors, retroviral vectors, protein expression vectors, phagemids, lentiviral packaging plasmids, retroviral packaging plasmids, etc., are purchased and synthesized by commercial companies or commercial companies, and these vectors are obtained The full-length sequence of, a clear restriction site is known.
  • the gene fragments used in the present invention including signal peptides, antibody binding regions, hinge regions, TCR constant regions, tag proteins, etc., are all synthesized by commercial companies. By means of synthetic primer PCR, connect one or more target fragments to obtain the corresponding functional sequence.
  • the lentiviral vector used in the present invention is pHAGE-EF1 ⁇ -GFP, pHAGR-EF1A-WPRE-AMP vector is obtained by restriction endonuclease Not I/Cla I, and the fragment gene is obtained by synthesis and PCR method, and by homologous recombination method , The complete vector is obtained under the action of Gibsson/NEBuilder and other recombinases.
  • the retroviral vector RVKM-CMV-GFP obtains vector fragments by restriction endonuclease Xho I/BamH I, while the insert gene is obtained by synthesis and PCR methods, and the vector and fragments are connected by homologous recombination.
  • the prokaryotic expression vector is pET-28a vector, the restriction endonuclease sites used are XhoI and NcoI, the eukaryotic expression vector is PVRC8400, and the restriction endonuclease sites used are XhoI and XbaI, designed according to the human antigen gene Primer, add restriction enzyme cleavage sites to both ends of the primer, and amplify the truncated extracellular segment of the antigen and the his-tag of the C segment from the entire gene of the target cell for subsequent purification.
  • sticky-end PCR design primers according to the sticky end sense strand and antisense strand of the digested product.
  • the digested vector is recombined and ligated in one step. After ligation, 5 ⁇ L of ligation product is added to 50 ⁇ L of DH5 ⁇ competent cells. For transformation, place it upside down in an incubator at 37°C and incubate for 12-16hr.
  • the plasmid was extracted by alkaline lysis method, and the plasmid concentration was detected by light absorption at 260nm after the plasmid was extracted.
  • the plasmid with the correct result of the initial sequencing needs to be transformed to pick out a single clone, perform a small extraction, and identify whether the plasmid is correct by enzyme digestion.
  • the plasmid pHAGE-EF1A-IRES-RFP was obtained by the above method; the constant regions were TCRa/bC-WT (including TCRac-WT and TCRbC-WT), TCRa/bC-N.DLT (including TCRac-N.DLT and TCRbC- N.DLT), TCRa/bC-C.DLT (including TCRaC-C.DLT and TCRbC-CDLT), TCRa/bC-N.Rec-1 (including TCRaC-N.Rec-1 and TCRbC-N.Rec- 1), TCRa/bC-N.Rec-2 (including TCRaC-N.Rec-2 and TCRbC-N.Rec-2), TCRa/bC-N.Rec-3 (including TCRaC-N.Rec-3 and TCRbC-N.Rec-3), TCRa/bC-N.Rec-4 (including TCRaC-N.Rec-4 and T
  • TCRa/bC-Cys including TCRaC-Cys and TCRbC-Cys
  • TCRa/bC-TM9 including TCRaC-TM9 and TCRbC-WT
  • TCRa/bC-Cys-N.Rec -1 including TCRaC-Cys-N.Rec-1 and TCRbC-Cys-N.Rec-1
  • TCRa/bC-TM9-N.Rec-1 including TCRaC-TM9-N.Rec-1 and TCRbC- N.Rec-1
  • TCRa/bC-Cys-TM9 including TCRaC-Cys-TM9 and TCRbC-Cys
  • TCRa/bC-Cys-TM9-N.Rec-1 including TCRaC-Cys-TM9-N.Rec-1 (including TCRaC-Cys-TM9-N.Rec-1.
  • Rec-1 and TCRbC-Cys-N.Rec-1) a variety of pHAGE-EF1A-Cetux-VL-TCRaC-P2A-VH-TCRbC-IRES-RFP and pHAGE-EF1A-GC33-VL-TCRaC-P2A-VH -TCRbC-IRES-RFP plasmid; and for different targets, its TCR constant region is pHAGE-EF1A-334-VL-TCRbC-P2A-VH-TCRaC- TCRa/bC-Cys-TM9-N.Rec-1 IRES-RFP, pHAGE-EF1A-2C6-VL-(G4S)3-VH-TCRbC-P2A-FMC63-VL-(G4S)3-VH-TCRaC-IRES-RFP, pHAGE-EF1A-M971-VL-(G4S )3-VH-TCRb
  • pHAGE can stably insert the target gene into the target cell genome, which is an important way to construct a stable cell line.
  • Luciferase is an enzyme with catalytic activity, which can catalyze the chemiluminescence of the substrate.
  • the number of target cells can be indicated after the substrate is added, thereby reflecting the function of the cells. The influence of the target cell.
  • PHAGE-EF1A vector carries restriction endonuclease Not I/Cla I restriction sites
  • the vector is cut with these two enzymes, the luciferase and GFP sequences are obtained by NCBI, and the fragments are synthesized by the commercial company Ruibo Xingke Firstly, the luciferase gene and the GFP gene were combined by the overlap PCR method, and then the luciferase-GFP fragment was connected into the pHAGE vector by the homologous recombination method.
  • the forward and reverse primers were designed in EF1A and WPRE respectively, and the vector construction was obtained by sequencing. whether succeed.
  • the lentivirus is packaged by Lentix-293T, the lentiviral liquid is concentrated by the PEG8000 concentration method, and the virus titer is determined by the gradient dilution method, and then the target cells are infected including but not limited to the following table Medium cell line:
  • the luciferase substrate is used to incubate with the target cells to detect the expression and detection level of luciferin to determine the expression level.
  • TCR ⁇ - ⁇ -Jurkat cell line Based on the structure and sequence characteristics of TCR, guide sequences were designed in the constant regions of the ⁇ and ⁇ chains to construct the TCR ⁇ - ⁇ -Jurkat cell line. Obtain the exon sequences of the constant regions of the TCR ⁇ and ⁇ chains at NCBI, and submit the exon 1 sequences of the constant regions of the ⁇ and ⁇ chains to the tools.genome-engineering.org website to design the guide sequence, and synthesize the oligo sequence according to the results.
  • the sgRNA-LentiCRISPR lentiviral vector cut the CRISPR lentiviral vector with BsmBI restriction enzyme, remove the filter sequence, expose the connection end, and treat the plasmid with alkaline phosphatase at 37°C for 1 hour to reduce self-ligation. .
  • the digested system is run on agarose gel electrophoresis for gel recovery.
  • the plasmid backbone of the target fragment is about 11500 bp, and there is a cut filter band at ⁇ 2000 bp; the oligo single chain of the synthesized guide sequence is connected in vitro with T4 Enzyme and T4 PNK treatment, the anneal is double-stranded and a phosphate group is added at the end for ligation; the double-stranded guide sequence of the anneal is diluted 200 times with water, and then ligated with LentiCRISPR digested with BsmBI, transformed into Stbl3 E. coli, and extracted The plasmid is used to encapsulate virus infection.
  • the guide sequence of the ⁇ chain is connected with LentiCRISPR-puro, and the guide sequence of the ⁇ chain is connected with LentiCRISPR-BSD.
  • sgRNA-LentiCRISPR lentivirus packaging and concentration spread HEK-293T to 10cm dish in advance, when the cells grow to 80%-90%, prepare the transfection system, add the transfection system to HEK-293T, and put the cells back 37 degree incubator, cultivate. Count as 0 hours; 12 hours after transfection, change into fresh 10% FBS-DMEM.
  • the virus can be harvested within 48 hours and 72 hours of transfection; centrifuge and filter the virus-containing medium, add PEG8000 to mix, and let stand at 4 degrees for more than 12 hours, then centrifuge at 3500 rpm for 30 minutes; discard the supernatant and use a suitable volume of medium Resuspend the pellet. -80 Store frozen or use directly.
  • Centrifuge infection 1000 rpm, 32 degrees centrifugation for 90 minutes. After taking it out, put it in a 37°C incubator and count as 0 hours; change the medium after 10-12 hours; after 48 hours, add Puromycin and blasticidin to the appropriate final concentration, kill for 48 hours, it can be seen that all the uninfected control cells die .
  • the surviving cells were aspirated, centrifuged and cultured in complete medium to form the TCR ⁇ - ⁇ -Jurkat cell bank; the TCR ⁇ - ⁇ -Jurkat cell bank was sorted into 96-well plates by flow Aria, and after two weeks of culture, Aspirate the grown-up monoclonal cells and expand the culture; the monoclonal cell lines are identified with TCR ⁇ chain and ⁇ chain antibodies, and cell lines with defects in both chains are expanded to obtain Jurkat with endogenous TCR knockout. T cell line.
  • Lentix-293T cells were inoculated into a 10cm culture dish at 5 ⁇ 10 5 cells/mL, cultured in a 37°C, 5% CO2 incubator, and transfected when the cell density reached about 80% (observed under a microscope) .
  • Jurkat-C5 cells were seeded in a flat-bottom 96-well plate at 1.5 ⁇ 10 ⁇ 5 cells/mL, and 100uL 1640 medium containing 10% FBS and 0.2uL 1000 ⁇ polybrene was added to each well.
  • 1640 complete medium for 10-fold dilution. If it is a virus stock solution, the amount of virus in the first hole is 100uL, and if it is a concentrated solution, the amount of virus in the first hole is 1uL.
  • the diluted cells were added to the virus well, 100uL/well, mixed, 32°C, 1500rpm, centrifuged for 90min, 37°C, 5% CO2 incubator for 72h.
  • the cells on the 96-well flat bottom plate were sucked into the round-bottom 96-well plate, centrifuged at 4° C., 1800 rpm for 5 min, and the supernatant was discarded. After adding 200uL 1 ⁇ PBS, centrifuge at 4°C, 1800rpm for 5min, and discard the supernatant. Add 200uL 4% tissue fixative, store in the dark, and use the flow cytometer. Use a flow cytometer to measure the infection efficiency.
  • the viruses with the following plasmids are packaged by the above method: pHAGE-EF1A-IRES-RFP; the constant regions are respectively TCRa/bC-WT (including TCRac-WT and TCRbC-WT), TCRa/bC-N.DLT (including TCRac-N) .DLT and TCRbC-N.DLT), TCRa/bC-C.DLT (including TCRaC-C.DLT and TCRbC-CDLT), TCRa/bC-N.Rec-1 (including TCRaC-N.Rec-1 and TCRbC -N.Rec-1), TCRa/bC-N.Rec-2 (including TCRaC-N.Rec-2 and TCRbC-N.Rec-2), TCRa/bC-N.Rec-3 (including TCRaC-N .Rec-3 and TCRbC-N.Rec-3), TCRa/bC-N.Rec-4 (including TCR
  • the Jurkat T cell line was cultured in RPMI 1640 medium containing 10% FBS.
  • the culture density is 3*10 5 /ml, and the highest is not more than 3*10 6 /ml. Passage every 1-2 days. After counting the cells, take the required amount of cells, supplement the medium to adjust to the above density, and place it in CO 2 Cultivation in an incubator.
  • Count the cells take 1*10 6 /ml cell centrifugation and change the medium, resuspend it with 1ml RPMI 1640 medium containing 10% FBS, add it to a 24-well plate, add appropriate amount of virus solution, centrifuge at 1500rpm for 90min, and place it in a CO2 incubator bring up. After 12 hours of infection, the medium was completely changed to fresh RPMI 1640 medium containing 10% FBS, and the positive rate was detected 72 hours.
  • T cells After obtaining primary T cells by the Ficoil separation method, they were cultured in X-VIVO medium containing 10% FBS and 100IU/ml IL-2 at an initial culture density of 1*10 6 /ml.
  • CD3 and RetroNectin r-Fibronectin The final concentration is 5ug/ml) in a pre-coated orifice plate.
  • the later culture density is 5*10 5 /ml, and the highest is not more than 3*10 6 /ml. Passage is carried out every 1-2 days.
  • X-VIVO medium containing 10% FBS and 100IU/ml IL-2 was supplemented and transferred to the wells. At 72 hours, the infection efficiency was detected by tag protein or antibody.
  • the cells were blown and counted, centrifuged at 5*10 5 /ml, the supernatant was discarded, the staining solution was PBS + 2% FBS + 2 mM EDTA, the corresponding antibody was added for incubation, incubated for 30 minutes, and then washed twice with PBS. On-board testing.
  • Example 7 Method for detecting the expression of STAR receptor and its mutants in T cells
  • the expression of the target gene, the upper membrane, and its distribution on the cell surface affect the function of T cells. Therefore, the efficiency of virus infection is detected by fluorescent protein, and the myc antibody is connected to the N-terminal of STAR.
  • the constructed pHAGE-EF1A-myc-STAR-IRES-RFP plasmid was packaged with Lentix-293T for virus packaging, virus concentration and titer detection were carried out, and then Jurkat C5 and primary T cells were infected. After 72 hours of infection, the infection was collected The cells were stained with FITC-anti-myc antibody and APC-anti-mTCR- ⁇ antibody, and flow cytometry (BD Fortessa) was used to detect the flow cytometry RFP/FITC/APC fluorescence channel. If FITC or APC has obvious positive cell population, it means that STAR can be expressed on the membrane, if neither is expressed, it means that STAR is not on the membrane.
  • the constructed pHAGE-EF1A-myc-STAR-IRES-RFP plasmid was packaged with Lentix-293T for virus packaging, virus concentration and titer detection were carried out, and then Jurkat and primary T cells were infected. After 72 hours of infection, the infected were collected Cells were stained with FITC-anti-myc antibody and APC-anti-mTCR- ⁇ antibody, and flow cytometry (BD Fortessa f4) was used to detect flow cytometry RFP/FITC/APC fluorescence channels. The difference between the positive rate of RFP and the positive rate of FITC or APC.
  • Example 8 In vitro functional detection method of STAR receptor and its mutants in T cells
  • T cells include the T cell line Jurkat C5. Both Jurkat and primary cell lines are suspension cells, and target cells can be divided into suspension cells and adherent cells according to different antigens and cell lines, such as suspension target cells and T cells such as RAJI For co-incubation, at the same time, take the corresponding number of cells and mix with the target cell culture medium for centrifugal culture. If it is an adherent target cell, it is necessary to inoculate the target cell one day in advance, and then add a certain amount of T cells. The specific steps are as follows: The packaged and purified STAR virus infects T cell lines or primary T cells. Use flow cytometry the day before co-cultivation to determine the ratio of functional cells to target cells. Generally use ratios of 10:1, 5:1, and 1:1 Calculate the total number of T cells based on the infection efficiency, and the general use amount of target cells is 4E5/well (24-well plate).
  • the target of this declaration is generally a cell surface protein.
  • the antigen can be directly used for T cell activation to detect the function of T cells. Usually 300 microliters of 5 ⁇ g/ml antigen (1xcoating buffer dilution, 24-well plate as an example) is used. After being plated overnight, the antigen was discarded, 1E6/well positive T cells were added, and T cells were collected for 6, 12, and 24 hours after activation by centrifugation to test T cell function.
  • TNF- ⁇ , IFN- ⁇ , and IL-2 are released to help T cells kill target cells or promote the expansion of T cells themselves.
  • the common ones are TNF- ⁇ , IFN- ⁇ , and IL-2.
  • TNF- ⁇ , IFN- ⁇ , IL-2 ELISA kits use Human IL-2 Uncoated ELISA, Human TNF- ⁇ Uncoated ELISA, Human IFN- ⁇ Uncoated ELISA (the article numbers are 88-7025, 88-7346, 88- 7316).
  • the specific steps are: Dilute 10X Coating Buffer to 1X with ddH2O, add coating antibody (250X), mix well and add to 96-well plate (for ELISA), 100 ⁇ l/well. Seal the cling film overnight at 4°C, wash 3 times with 1X PBST (also known as Wash Buffer, 0.05% Tween 20 in 1X PBS), 260 ⁇ l/well each time, dilute 5X ELISA/ELISPOT Diluent to 1X with ddH2O, add 96 Orifice plate, 200 ⁇ l/well, stand at room temperature for 1h.
  • 1X PBST also known as Wash Buffer, 0.05% Tween 20 in 1X PBS
  • wash once with PBST dilute the standard song (range: 2 ⁇ 250, 4 ⁇ 500, 4 ⁇ 500), and use 1xDiluent to dilute the sample 20-50 times.
  • Add sample and standard song 100 microliters per well, two duplicate wells, after 2h incubation at room temperature, wash three times with PBST, add 1xDiluent diluted Detection antibody, after 1h incubation, wash three times with PBST, then add 1xDiluent diluted HRP, After incubating for 30 minutes, wash 6 times, add TMB for color development, the color development time does not exceed 15 minutes, add 2N H2SO4 to stop, 450nm light absorption to detect light absorption.
  • Example 9 Method for functional detection of STAR receptor and its mutants in T cells at the level of animal tumor models
  • mice used as a model.
  • the genotype mice NOD-Prkdc em26 Il2rg em26 / Nju the lack of T cells, B cells, NK cells and macrophages and which dendritic cells are also defective.
  • NSG mice are currently the most immunodeficient mouse strain. Because they will not reject transplanted tumors and T cells, they are widely used in preclinical studies of T cell therapy.
  • female NSG mice aged 6-8 weeks will be used, and the weight difference of the mice in each batch of experiments will be controlled within 2g.
  • Mice are raised in independent ventilated cages in a clean-level barrier free of specific pathogens (SPF), and they are provided with normal diet and drinking water with acidic pH to prevent pathogen contamination.
  • SPPF specific pathogens
  • the present invention uses a variety of tumor models to verify the function of STAR-T cells to verify the universality of their effects.
  • this experiment uses human Burkitt's lymphoma cell line Raji cells for xenotransplantation.
  • Raji cells are cell lines expressing the luciferase gene through lentiviral vectors.
  • the development and changes of Raji tumors are monitored in real time in mice through fluorescein chemiluminescence and in vivo imaging.
  • Raji-luciferase cells of different doses (generally around 1 to 3 ⁇ 10 6 cells) were inoculated into female NSG mice aged 6 to 8 weeks through tail vein reinfusion.
  • mice were intraperitoneally injected with fluorescein potassium salt solution, and the fluorescent signal of tumor cells in the body was detected by in vivo imaging.
  • Raji cells grow fast in mice and distribute throughout the body through the blood. They have a certain chance of infecting vital organs and forming solid tumors, causing symptoms such as weight loss in mice; without treatment, the burden of Raji tumors is 30-40 The mouse died in about days.
  • this experiment selected a variety of tumor types, including human epidermal cell carcinoma A431 cell line, human non-small cell lung cancer A549 cell line, human liver cancer Huh-7 and HepG2, human glioma U87 And U251 cell line, and human breast cancer MDA-MB-231 cell line. These cells express the luciferase gene through lentiviral vectors. In mice, the development and changes of tumors can be monitored in real time through fluorescein chemiluminescence and in vivo imaging.
  • cells of different doses are mixed with Matrigel and then inoculated into mice subcutaneously by subcutaneous injection.
  • intraperitoneal injection of fluorescein potassium salt solution can be used to detect the fluorescent signal of tumor cells in vivo by in vivo imaging.
  • Solid tumors inoculated subcutaneously are generally not invasive and difficult to metastasize, and often form dense tumor masses under the skin in situ. By measuring the length, width, and height of the tumor mass with a vernier caliper, the tumor progress can also be monitored by the size of the tumor.
  • the tumor masses formed by these solid tumor cells meet the requirements of animal welfare ethics standards in about 20-40 days, that is, when the maximum tumor mass exceeds 15mm, or the tumor mass exceeds 1500mm 3 , or subcutaneously
  • animal welfare ethics standards in about 20-40 days, that is, when the maximum tumor mass exceeds 15mm, or the tumor mass exceeds 1500mm 3 , or subcutaneously
  • the animal should be euthanized.
  • the operation methods of experimental animal mice include: capture and fixation, numbering, anesthesia, hair removal, drug administration, blood collection, sacrifice and dissection.
  • the numbering methods used in the experiment include: finger cut, ear mark, and coat color marking.
  • the anesthesia methods used in the experiment included isoflurane inhalation anesthesia and Avertin or pentobarbital injection anesthesia.
  • Hair removal methods include: using scissors or razors to remove hair on local parts of the mouse.
  • Administration methods include: intraperitoneal injection, tail vein injection, subcutaneous injection, fundus venous plexus injection, intracranial injection, etc.
  • Blood collection methods include: orbital blood collection, eyeball collection blood collection, tail vein blood collection, etc.
  • the methods of execution include neck removal, carbon dioxide execution and so on. All operations are performed after the approval of the Experimental Animal Research and Use Plan (Animal Protocol).
  • This experiment mainly uses two methods to detect tumor growth.
  • the first is the in vivo fluorescence imaging method: tumor cells with luciferase gene are injected into animals to colonize. Inject the fluorescein potassium salt solution into the abdominal cavity of the mouse. The substrate emits light of a specific wavelength under the action of the enzyme, and the fluorescent signal of the tumor cells in the body is detected by the sensitive CCD device of the in vivo imaging instrument. Furthermore, professional software can be used to quantitatively analyze the fluorescence signal and draw a heat map, which can intuitively and quantitatively reflect the tumor growth.
  • T cells in the body are directly related to their ultimate anti-tumor effect.
  • this experiment regularly collects blood from mice, and analyzes the proportion of STAR-T cells in the peripheral blood, cell status and cell grouping. The specific operation is: every about a week, the mice are anesthetized with isoflurane, and about 100ul of blood is collected from the orbit of the mice. After the blood sample undergoes operations such as anticoagulation, plasma collection, and red blood cell splitting, the remaining cells are subjected to flow staining to detect the ratio of CD4 and CD8, and CCR7, CD45RA, PD-1, LAG-3, TIM-3 And other molecules to perform T cell subpopulation analysis and cell state analysis.
  • the absolute number of STAR-T cells in the peripheral blood of the mouse was obtained by the method of flow cytometry or digital PCR.
  • the mice can be dissected and the proportion of T cells in the other immune organs of the mice can be detected.
  • STAR-T cells In order to evaluate the toxicity and safety of STAR-T cells, it is possible to detect whether the cells cause side effects on experimental animals. By observing the behavior of the mouse, performing pathological analysis of the mouse, and analyzing the slices of the important organs of the mouse, it is possible to evaluate whether the infused T cells have significant toxicity. At the same time, through the analysis of T cell infiltration in the non-tumor tissues of mice, it is also possible to determine whether T cells have off-target killing effects on them. In addition, by detecting the levels of cytokines in the blood of mice, such as IL-2, IFN- ⁇ , TNF ⁇ or IL-6, it is possible to determine whether T cells will cause a systemic cytokine storm.
  • cytokines in the blood of mice such as IL-2, IFN- ⁇ , TNF ⁇ or IL-6
  • T cells to infiltrate tumors is its core ability to challenge solid tumors.
  • the tumor tissue can be separated, digested and ground to obtain single cells, and the proportion of T cells in the tumor tissue can be detected by flow staining.
  • density gradient centrifugation such as Percoll gradient, Ficoll gradient, etc.
  • the antibody heavy chain variable region (anti-EGFR Cetux-VH, SEQ ID NO: 22) and the antibody light chain variable region (anti-EGFR Cetux-VL, SEQ ID NO: 23) select Cetuximab (Centuximab, Referred to as Cetux).
  • the sequence is derived from gene synthesis after codon optimization.
  • STAR contains two polypeptide chains.
  • the anti-EGFR Cetux-VL and mouse TCRbC-WT chains are fused into the first polypeptide segment, and the anti-EGFR Cetux-VH and mouse TCRaC-WT chains are fused into the second polypeptide segment. Both chains use GM-CSF signal peptide signal peptide.
  • the gene sequence of STAR is connected by the polypeptide segment of the Furin-SGSG-p2A protease cleavage site. The two polypeptide chains will be transcribed and translated together into a protein, and then cleaved by the protease corresponding to furin and p2A, and finally become two independent proteins. chain.
  • the two protein chains form a complex with the endogenous CD3 subunits ( ⁇ , ⁇ , ⁇ , ⁇ ) of T cells, and are transported and presented on the cell membrane under the guidance of the GM-CSF signal peptide (as shown in Figure 1 ).
  • the four fragments obtained through gene synthesis and cloning are "anti-EGFR Cetux-VL”, “mouse TCRbC-WT”, “anti-EGFR Cetux-VH”, and “mouse TCRac-WT”.
  • Each pair of primers has 25 bp bases that are homologous to the front and back, and the four fragments are recombined into the lentiviral vector pHAGE by Gibson Assembly method in one step. To get STAR.
  • the bacteria with the correct sequencing results were inoculated into LB liquid medium and cultured overnight. Use a kit with endotoxin removal function to extract plasmids. Plasmid concentration is measured by Nanodrop, the final concentration of plasmid is about 1000ng/ul, and the value of A260/A280 is greater than 1.8.
  • the EGFR-targeting STAR plasmid pHAGE-EF1A-Cetux-VL-TCRaC-P2A-VH-TCRbC-IRES-RFP with a constant region of mouse TCRa/bC-WT was obtained.
  • the constant regions were constructed as TCRa/bC-N.DLT (including TCRac-N.DLT and TCRbC-N.DLT), TCRa/bC-C.DLT (including TCRac-C.DLT and TCRbC-CDLT) , TCRa/bC-N.Rec-1 (including TCRaC-N.Rec-1 and TCRbC-N.Rec-1), TCRa/bC-N.Rec-2 (including TCRaC-N.Rec-2 and TCRbC- N.Rec-2), TCRa/bC-N.Rec-3 (including TCRaC-N.Rec-3 and TCRbC-N.Rec-3), TCRa/bC-N.Rec-4 (including TCRaC-N.
  • TCRa/bC-N.DLT including TCRac-N.DLT and TCRbC-N.DLT
  • TCRa/bC-C.DLT including TCRac-C
  • TCRa/bC-N.rRec including TCRaC-N.rRec and TCRbC-N.rRec
  • TCRa/bC-Cys including TCRaC-Cys and TCRbC-Cys
  • TCRa/bC-TM9 including TCRaC-TM9 and TCRbC-WT
  • TCRa/bC-Cys-N.Rec-1 including TCRaC-Cys-N.Rec-1 and TCRbC-Cys-N.Rec-1)
  • TCRa/bC-TM9-N.Rec-1 including TCRaC-TM9-N.Rec-1 and TCRbC-N.Rec-1
  • TCRa/bC-Cys-TM9 including TCRaC-Cys-TM9 and TCRbC- Cys
  • the packaging plasmids are pSPAX, PMD2.G ,
  • the virus solution was harvested for 48h and 72h, centrifuged at 20,000 rpm and 4° for 2h, resuspended in serum-free medium, concentrated the virus 100 times, and tested the virus titer.
  • the results showed that Mouse TCRa/bC-N.DLT STAR virus packaging The efficiency is better than Mouse TCRa/bC-WT STAR.
  • the same MOI was infected with Jurkat C5 and primary T cells, and there was no significant difference in infection efficiency.
  • the RFP and EGFR-his-APC staining in primary T cells showed that Mouse TCR/bC-N.
  • Mouse TCRa/bC-N.DLT had no killing ability. After stimulation with EGFR antigen, it did not secrete the cytokines IL-2, TNF ⁇ and IFN ⁇ .
  • the cysteine point mutation of the STAR receptor can add additional disulfide bonds, which can enhance the pairing and stabilize the complex.
  • the infection efficiency of Jurkat C5 and primary T cells was found to be no significant difference.
  • the RFP and EGFR-his-APC staining in primary T cells showed that Mouse TCRa/bC-Cys is more endogenous than Mouse TCRa/bC-WT.
  • the mismatch rate is significantly reduced.
  • After stimulation with EGFR antigen the secretion levels of cytokines IL-2, TNF ⁇ and IFN ⁇ are higher.
  • the substitution of hydrophobic amino acids at specific sites in the transmembrane region of the STAR receptor plays a key role.
  • the cysteine point mutation of the STAR receptor can add additional disulfide bonds to enhance the pairing and stabilize the complex.
  • the 72-hour virus solution was centrifuged at 20,000 rpm and 4° for 2 hours, and then resuspended in serum-free medium. The virus was concentrated 100 times and the virus titer was detected. The results showed that there was no significant difference in the virus titer between the two.
  • the infection efficiency of Jurkat C5 and primary T cells was found to be no significant difference.
  • the RFP and EGFR-his-APC staining in primary T cells showed that Mouse TCRa/bC-TM9 is more endogenous than Mouse TCRa/bC-WT. In terms of TCR mismatch, the mismatch rate is significantly reduced.
  • Mouse TCRa/bC-TM9 has a stronger ability to kill target cells than Mouse TCRa/bC-WT.
  • the secretion levels of cytokines IL-2, TNF ⁇ and IFN ⁇ are higher.
  • Example 11 Function of N-terminal modification and rearrangement mutants of the constant region of anti-GPC3 STAR receptor
  • the present invention carries out optimization schemes such as truncation, rearrangement, and rearrangement of the N-terminal sequence for the constant region.
  • the antibody sequence selects the monoclonal antibody GC33 against GPC3, and the antibody heavy chain
  • the variable region (anti-GPC3 GC33-VH, SEQ ID NO: 25) and the antibody light chain variable region (anti-GPC3 GC33-VL, SEQ ID NO: 24) use the phage-EF1A-IRES-RFP vector through Gibsson Assembly reorganized to construct Mouse GC33-VH TCRa-GC33-VL TCRbC-N.DLT STAR, Mouse GC33-VH TCRa-GC33-VL TCRbC-C.DLT STAR, Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec- 1 STAR, Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec- 1 STAR
  • the results are shown in Figure 7.
  • MouseGC33-VHTCRa-GC33-VLTCRbC-N.Rec-1 is better than MouseGC33-VHTCRa-GC33-VLTCRbC -WT STAR has stronger ability to kill target cells.
  • the cysteine point mutation of the STAR receptor can add additional disulfide bonds, which can enhance the pairing and stabilize the complex.
  • the phage-EF1A-IRES-RFP vector is used to construct Mouse GC33-VH TCRa- by Gibsson Assembly recombination.
  • GC33-VL TCRbC-Cys STAR and Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR vector using Lentix-293T cells to package the virus, the packaging plasmids are pSPAX, PMD2.G, and the packaging ratio is Transfer plasma: pSPAX: PMD2.
  • G 3:2:1 a total of 18 ⁇ g plasmid per 10cm culture dish, and the ratio of PEI-MAX is 1:3, that is, 54 ⁇ l PEI is used, the solution is changed 12h after transfection, the virus solution is harvested for 48h and 72h, and passed through 20000rpm, 4 ° After centrifugation for 2 hours, resuspend in serum-free medium, concentrate the virus 100 times, and detect the virus titer. The results show that there is no significant difference in the virus titer between the two. There is no significant difference in infection efficiency between Jurkat C5 and primary T cells.
  • the RFP and GPC3-FITC staining in primary T cells shows that Mouse GC33-VH TCRa-GC33-VL TCRbC-Cys is better than Mouse GC33-VH TCRa-GC33 -VL TCRbC-WT has a significantly lower mismatch rate with endogenous TCR.
  • Mouse GC33-VH TCRa-GC33-VL TCRbC-Cys is better than Mouse GC33- VH TCRa-GC33-VL TCRbC-WT has stronger ability to kill target cells.
  • Example 13 The function of hydrophobic amino acid substitution at specific sites in the transmembrane region of anti-GPC3 STAR receptor
  • the substitution of hydrophobic amino acids at specific sites in the transmembrane region of the STAR receptor plays a key role.
  • the cysteine point mutation of the STAR receptor can increase additional disulfide bonds, which can enhance the pairing and stabilize the complex.
  • Use phage- EF1A-IRES-RFP vector constructed by Gibsson Assembly recombination Mouse GC33-VH TCRa-GC33-VL TCRbC-TM9 STAR and Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR vector, using Lentix-293T cell packaging virus, packaging
  • Mouse GC33-VH TCRa-GC33-VL TCRbC-TM9 is better than Mouse GC33 -VH TCRa-GC33-VL TCRbC-WT has stronger ability to kill target cells.
  • Example 15 Function of anti-CD19 STAR comprehensive mutant based on 334-scFv
  • the inventors obtained a new CD19-targeting monoclonal antibody 334, which was deposited with the deposit number CGMCC No. 17095 (deposited on January 21, 2019 at the address of No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China.
  • Hybridoma cells 334 produced by the General Microbiology Center of the Chinese Microbial Culture Collection and Management Committee of the Institute of Microbiology, Chinese Academy of Sciences.
  • the antibody includes anti-CD19 334 VL (SEQ ID NO: 27) and anti-CD19 334 VH (SEQ ID NO: 26), wherein the anti-CD19 334 VH includes SEQ ID NO: 28 heavy chain CDR1 and SEQ The heavy chain CDR2 shown in ID NO: 29 and the heavy chain CDR3 shown in SEQ ID NO: 30, and the anti-CD19 334 VL includes the light chain CDR1 shown in SEQ ID NO: 31 and the light chain CDR2 shown in SEQ ID NO: 32 And the light chain CDR3 shown in SEQ ID NO: 33.
  • the ELISA method was used to identify the ability of 334 antibody to recognize the target antigen CD19 and non-target antigen CD20.
  • the plasmid expressing 334 antibody was transfected into 293T cells with the transfection reagent PEI. After 72 hours, the cell supernatant was collected and the antigen antibody was detected by ELISA method. The specific binding capacity. The experimental results showed that the 334 antibody showed significant specific binding to the CD19 target antigen, and no binding ability to the non-target antigen CD20.
  • the WB test was used to detect the recognition ability of the CD19-334 antibody and the human CD19 antigen.
  • Transfer membrane Use a semi-dry transfer system to transfer the target protein onto a 0.45 ⁇ m PVDF membrane.
  • the transfer membrane condition is 15V, 1h.
  • the test results showed that the 334 antibody can recognize the CD19 protein in Raji cells of the human lymphoma cell line, and the target band size is 95kD.
  • the test results showed that the binding curve fitted by the fluorescence intensity of Raji cells under different antibody concentrations, and the Kd value of CD19-334 antibody was calculated by GraphPad software to be 1.528 ⁇ 0.25nM.
  • the myc-Cys-N.Rec-1-TM9-STAR-334-anti-CD19 STAR structure was constructed on the basis of the pHAGE-IRES-RFP vector.
  • the antigen binding sequences used are anti-CD19 334 VL (SEQ ID NO: 27) and anti-CD19 334 VH (SEQ ID NO: 26), wherein the anti-CD19 334 VH includes the heavy chain shown in SEQ ID NO: 28 CDR1, heavy chain CDR2 shown in SEQ ID NO: 29 and heavy chain CDR3 shown in SEQ ID NO: 30, and the anti-CD19 334 VL includes light chain CDR1 shown in SEQ ID NO: 31 and the light chain CDR1 shown in SEQ ID NO: 32 The light chain CDR2 and the light chain CDR3 shown in SEQ ID NO: 33.
  • C ⁇ and C ⁇ can be connected to the VH and VL of scFv respectively, or different scFv can be connected, which makes it possible to dual-target or single-target dual recognition sites.
  • the myc-Cys-N.Rec-1-TM9-STAR-anti-CD19/CD20 STAR structure was constructed on the basis of the pHAGE-IRES-RFP vector, and the FMC63 monoclonal antibody selected for the target of CD19 (FMC63 VH seq No: 44 , FMC63 VL seq No: 45), select 2C6 monoclonal antibody for CD20, (2C6VH seq No: 46, 2C6VL seq No: 47), construct myc-Cys-N.Rec-1-TM9-STAR-FMC63-2C6 structure, After the virus was packaged with the second-generation packaging plasmid, it was infected with Jurkat C5 cells.
  • C ⁇ and C ⁇ can be connected to the VH and VL of scFv respectively, or different scFv can be connected, which makes it possible to dual-target or single-target dual recognition sites.
  • the myc-Cys-N.Rec-1-TM9-STAR-anti-CD19/CD22 STAR structure was constructed on the basis of the pHAGE-IRES-RFP vector, in which 334 monoclonal antibodies (334 VH seq No: 26) were selected for the target of CD19 , 334 VL seq No: 27), select M971 monoclonal antibody for CD22, (M971 VH seq No: 48, M971 VL seq No: 49), construct myc-Cys-N.Rec-1-TM9-STAR-334-M971 Structure.
  • the virus After the virus was packaged with the second-generation packaging plasmid, it was infected with Jurkat C5 cells. The flow cytometry result showed that STAR can cover the membrane normally. After the virus infects the primary T cells, the T cells and the lymphoma cell lines Raji, Raji-CD19KO, Raji- After CD22KO co-culture, it can obviously stimulate T cells to secrete IL-2, TNF- ⁇ and IFN ⁇ , and induce the killing of target cells, indicating that 334-M971 STAR has a good killing effect on both targets, which can be avoided in clinical practice. Tumor recurrence caused by the loss of one of the targets.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Dermatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Hospice & Palliative Care (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

涉及生物医药领域，尤其涉及一种增强型T细胞受体STAR及其应用。具体而言，公开了一种改进的合成T细胞受体抗原受体(Snythetic T-Cell Receptor and Antigen Receptor,STAR)、包含所述合成T细胞受体抗原受体的T细胞以及它们的用途。

Description

一种增强型T细胞受体STAR及其应用 技术领域
本发明涉及生物医药领域，尤其涉及一种增强型T细胞受体STAR及其应用。具体而言，本发明公开了一种改进的合成T细胞受体抗原受体(Snythetic T-Cell Receptor and Antigen Receptor,STAR)、包含所述合成T细胞受体抗原受体的T细胞以及它们的用途。
发明背景
人体中免疫细胞清除肿瘤细胞是通过以下途径：树突状细胞识别到肿瘤细胞的突变基因，并将这种突变信息展示给T细胞。T细胞随后会寻找携带该突变的肿瘤细胞并对其进行杀伤。有时，肿瘤细胞会下调能展示突变肽段的MHC分子，这样就能逃脱T细胞的杀伤，逐渐形成不可控制的癌症。
嵌合抗原受体T细胞(CAR-T)治疗是近年来收获良好疗效的一种抗癌免疫疗法。不同于天然的T细胞识别肿瘤细胞的方式，CAR-T细胞对肿瘤细胞的识别不依赖于MHC分子。CAR分子包含三大部分：胞外区是来源于抗体的抗原识别结构域，负责识别靶标抗原；跨膜区；胞内区是来源于T细胞受体的信号分子和共刺激信号分子，负责在接受刺激后传导T细胞激活信号。其工作原理如下：当CAR分子与其对应抗原结合的时候，CAR分子会发生聚集，从而局部磷酸化水平升高、激活下游信号，最终启动T细胞的效应功能、杀伤靶标肿瘤细胞。
去靶向CD19蛋白的嵌合抗原受体T细胞(CD19-CAR-T)疗法已有两项临床应用在美国获批上市，用于复发难治性B细胞淋巴瘤的治疗。但是CAR-T疗法用于实体肿瘤治疗时遇到困难。T细胞疗法未能在实体瘤治疗中取得较好疗效有多重因素，其中一个重要原因是CAR-T细胞在肿瘤微环境中功能受到抑制、T细胞容易发生耗竭而凋亡。近来研究表明，这种T细胞的失能可能与嵌合抗原受体的信号通路性质有关。
T细胞受体(TCR)复合物分子含有多条链，TCRα链和TCRβ链负责识别MHC-多肽分子，其它6个CD3亚基与TCRα/β链结合，起信号转导的功能。天然TCR复合体中共含有10个ITAM信号序列，理论上能传导比CAR更强的信号。之前研究表明，TCR的信号虽然比CAR的信号传导得缓慢一些，但是TCR的信号更为持久。因此，利用天然TCR的信号传导功能，将可能构建出一种新型受体来缓解T细胞失能，使其能够更好地发挥抗实体肿瘤作用。
TCR的胞外区和抗体的Fab结构域非常相似，因此可以把TCR可变区序列替换成抗体可变区序列，从而得到合成T细胞受体抗原受体(Snythetic TCR and Antigen Receptor,STAR)，其既具有抗体的特异性，也具有天然TCR的优越的信号传导功能，可以介导完全的T细胞激活。
然而，衍生自天然TCR的STAR仍然存在膜稳定性差、较低的α/β链配对能力、与内源性TCR存在错配、难以导入T细胞等缺点。因此本领域仍然需要改进的STAR。
附图简述
图1示出STAR的结构。A、STAR的原型的结构示意图；B、改进的STAR的结构示意图；C、靶向不同靶抗原的dual-STAR的结构示意图。
图2示出人和鼠T细胞受体α链恒定区以及β链恒定区的序列比对结果。
图3示出人和鼠的TCRα链恒定区N端的18个氨基酸的序列比对结果。UserSeq1为人源序列，UserSeq2为鼠源序列。
图4示出人和鼠的TCRβ链恒定区N端的25个氨基酸的序列比对结果。UserSeq1为人源序列，UserSeq2为鼠源序列。
图5示出组合不同的修饰的恒定区突变体。
图6靶向EGFR的Cetux STAR各突变体功能。
图7靶向GPC3的GC33 STAR各突变体功能。
图8靶向CD19的334 STAR综合突变体功能。
图9靶向CD19/CD20的FMC63-2C6 STAR综合突变体功能。
图10靶向CD19/CD22的FMC63-M791 STAR综合突变体功能。
发明内容
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook et al.,“Molecular Cloning:A Laboratory Manual”；Lewin,“Genes VIII”；及Roitt et al.,“Immunology”(第8版)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
“包含”一词在本文中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本发明所述的活性。此外，本领域技术人员清楚多肽N端由起始密码子编码的甲硫氨酸在某些实际情况下(例如在特定表达系统表达时)会被保留，但不实质影响多肽的功能。因此，本申请说明书和权利要求书中在描述具体的多肽氨基酸序列时，尽管其可能不包含N端由起始密码子编码的甲硫氨酸，然而此时也涵盖包含该甲硫氨酸的序列，相应地，其编码核苷酸序列也可以包含起始密码子；反之亦然。
在一方面，本发明提供一种合成T细胞受体抗原受体(STAR)，其包含α链和β链，所述α链包含第一抗原结合区和第一恒定区，所述β链包含第二抗原结合区和第二恒定区，
其中第一恒定区是天然T细胞受体α链恒定区的变体，其相对于天然T细胞受体α链恒定区包含i)N末端修饰；ii)半胱氨酸取代；和/或iii)疏水氨基酸取代，和/或
其中第二恒定区是天然T细胞受体β链恒定区的变体，其相对于天然T细胞受体β链恒定区包含i)N末端修饰；和/或ii)半胱氨酸取代。
在一些实施方案中，所述抗原结合区融合至所述恒定区的N末端。
在一些实施方案中，所述α链和β链在T细胞内表达后，能够形成功能性TCR复合物。例如，所述α链和β链在T细胞表达后，能够与细胞内源的CD3分子(CD3εδ、CD3γε、CD3ζζ)相结合形成8个亚基的TCR复合物，所述TCR复合物展示在细胞表面，并且在与靶抗原结合后激活T细胞。功能性TCR复合物指的是其能够在与靶抗原特异性结合后，激活T细胞。
如本文所述，“N末端修饰”指的是所提及的氨基酸序列(多肽或蛋白)的N末端包含一个或更多个氨基酸的取代、缺失和/或添加，该术语还可以包括所提及的氨基酸序列(多肽或蛋白)从N末端起一个或更多个连续氨基酸的缺失。
如本文所述，“半胱氨酸取代”或“疏水氨基酸取代”指的是所提及氨基酸序列(多肽或蛋白)中原始氨基酸被半胱氨酸或疏水氨基酸取代。其中疏水氨基酸取代可以是亲水氨基酸被疏水氨基酸取代，也可以是疏水性低的氨基酸被疏水性高的氨基酸取代。
在一些实施方案中，所述第一恒定区衍生自人T细胞受体α链恒定区、非人灵长类动物T细胞受体α链恒定区、啮齿动物例如小鼠T细胞受体α链恒定区。示例性的人T细胞受体α链恒定区包含SEQ ID NO:1所示氨基酸序列。示例性的小鼠T细胞受体α链恒定区包含SEQ ID NO:2所示氨基酸序列。在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区。
在一些实施方案中，所述第一恒定区相对于天然T细胞受体α链恒定区包含N末端18个氨基酸的修饰。在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失SEQ ID NO:3(DIQNPEPAVYQLKDPRSQ)所示氨基酸序列。在一些具体实施方案中，所述包含N末端修饰的第一恒定区包含SEQ ID NO:4所示氨基酸序列。(对应于TCRaC-N.DLT)
在一些实施方案中，所述第一恒定区相对于天然T细胞受体α链恒定区包含N末端修饰，其中N末端的第15-18位氨基酸中的一个或多个或全部被缺失，所述氨基酸编号参考SEQ ID NO:2。在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第15-18位氨基酸中的一个或多个或全部，所述氨基酸编号参考SEQ ID NO:2。在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第15-18位氨基酸中的一个或多个或全部，且其中N末端的第1-14位氨基 酸被天然人T细胞受体α链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些具体实施方案中，所述包含N末端修饰的第一恒定区包含SEQ ID NO:5所示氨基酸序列。(对应于TCRaC-N.Rec-4)
在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区，N末端起X个连续氨基酸被缺失，和/或N末端的第X+1位至第18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，其中X为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17或18。
例如，在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-4位氨基酸，且其中N末端的第5-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些具体实施方案中，所述包含N末端修饰的第一恒定区包含SEQ ID NO:34所示氨基酸序列。(对应于TCRaC-N.Rec-1)
在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-8位氨基酸，且其中N末端的第9-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些具体实施方案中，所述包含N末端修饰的第一恒定区包含SEQ ID NO:35所示氨基酸序列。(对应于TCRaC-N.Rec-2)
在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-12位氨基酸，且其中N末端的第13-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些具体实施方案中，所述包含N末端修饰的第一恒定区包含SEQ ID NO:36所示氨基酸序列。(对应于TCRaC-N.Rec-3)
在一些实施方案中，相对于天然T细胞受体α链恒定区，所述第一恒定区在第48位包含半胱氨酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些实施方案中，相对于天然T细胞受体α链恒定区，所述第一恒定区在第48位的苏氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:2。在一些具体实施方案中，所述包含半胱氨酸取代的第一恒定区包含SEQ ID NO:6所示氨基酸序列(衍生自人T细胞受体α链恒定区)。在一些具体实施方案中，所述包含半胱氨酸取代的第一恒定区包含SEQ ID NO:7所示氨基酸序列(衍生自小鼠T细胞受体α链恒定区)。(对应于TCRaC-Cys)
如本文所用，“氨基酸编号参考SEQ ID NO:x”(SEQ ID NO:x为本文所列的某一具体序列)指的是所描述的具体氨基酸的位置编号是该氨基酸在SEQ ID NO:x上对应的氨基酸的位置编号。不同序列中的氨基酸的对应性可以根据本领域公知的序列比对方法确定。例如氨基酸对应性可以通过EMBL-EBI的在线比对工具来确定(https://www.ebi.ac.uk/Tools/psa/)，其中两个序列可以使用Needleman-Wunsch算法，使用默认参数来对齐。例如，一多肽从其N末端起第46位的氨基酸与SEQ ID NO:x的第48位的氨基酸在序列比对中对齐，则该多肽中的该氨基酸在本文中也可以被描述为“在 该多肽的第48位的丙氨酸，所述氨基酸位置参考SEQ ID NO:x”。
在一些实施方案中，相对于天然T细胞受体α链恒定区，所述第一恒定区包含在跨膜区内的疏水氨基酸取代，所述跨膜区例如包含第111-119位的氨基酸序列，所述氨基酸编号参考SEQ ID NO:2。在一些实施方案中，相对于天然T细胞受体α链恒定区，所述第一恒定区包含在第112位、114位和/或115位的疏水氨基酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些实施方案中，相对于天然T细胞受体α链恒定区，所述第一恒定区包含在第112位、114位和115位的疏水氨基酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些实施方案中，相对于天然T细胞受体α链恒定区，所述第一恒定区在第112位的丝氨酸被亮氨酸取代，在114位的甲硫氨酸被异亮氨酸取代，和/或在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些实施方案中，相对于天然T细胞受体α链恒定区，所述第一恒定区在第112位的丝氨酸被亮氨酸取代，在114位的甲硫氨酸被异亮氨酸取代，且在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些实施方案中，所述包含疏水氨基酸取代的第一恒定区包含SEQ ID NO:8所示跨膜区(衍生自小鼠T细胞受体α链恒定区)。在一些具体实施方案中，所述包含疏水氨基酸取代的第一恒定区包含SEQ ID NO:9所示氨基酸序列(衍生自小鼠T细胞受体α链恒定区)。(对应于TCRaC-TM9)
在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-4位氨基酸，且其中N末端的第5-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，在第48位的苏氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:2。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:11的氨基酸序列。(对应于TCRaC-Cys-N.Rec-1)
在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-4位氨基酸，且其中N末端的第5-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，在第112位的丝氨酸被亮氨酸取代，在114位的甲硫氨酸被异亮氨酸取代，且在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:12的氨基酸序列。(对应于TCRaC-TM9-N.Rec-1)
在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区，在第48位的苏氨酸被突变为半胱氨酸，在第112位的丝氨酸被亮氨酸取代，在114位的甲硫氨酸被异亮氨酸取代，且在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:10的氨基酸序列。(对应于TCRaC-Cys-TM9)
在一些实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-4位氨基酸，且其中N末端的第5-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，在第48位的苏氨酸被突变为半胱氨酸，在第112位的丝氨酸被亮氨酸取代，在114位的甲硫氨酸被异亮氨酸取 代，且在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:13的氨基酸序列。(对应于TCRaC-Cys-TM9-N.Rec-1)
在一些实施方案中，所述第二恒定区衍生自人T细胞受体β链恒定区、非人灵长类动物T细胞受体β链恒定区、啮齿动物例如小鼠T细胞受体β链恒定区。示例性的人T细胞受体β链恒定区包含SEQ ID NO:14所示氨基酸序列。示例性的小鼠T细胞受体α链恒定区包含SEQ ID NO:15所示氨基酸序列。在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体β链恒定区。
在一些实施方案中，所述第二恒定区相对于天然T细胞受体β链恒定区包含N末端25个氨基酸的修饰。在一些实施方案中，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失SEQ ID NO:16(DLRNVTPPKVSLFEPSKAEIANKQK)所示氨基酸序列。在一些具体实施方案中，所述包含N末端修饰的第二恒定区包含SEQ ID NO:17所示氨基酸序列。(对应于TCRbC-N.DLT)
在一些实施方案中，所述第二恒定区相对于天然T细胞受体β链恒定区包含N末端修饰，其中N末端的第17和21-25位氨基酸中的一个或多个或全部被缺失，所述氨基酸编号参考SEQ ID NO:15。在一些实施方案中，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第17和21-25位氨基酸中的一个或多个或全部，所述氨基酸编号参考SEQ ID NO:15。在一些实施方案中，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第17和21-25位氨基酸中的一个或多个或全部，且其中N末端的第1-16位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述包含N末端修饰的第二恒定区包含SEQ ID NO:18所示氨基酸序列。(对应于TCRbC-N.Rec-4)
在一些实施方案中，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区，N末端起X个连续氨基酸被缺失，和/或N末端的第X+1位至第25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，其中X为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或25。
在一些实施方案中，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第1-6位氨基酸，且其中N末端的第7-25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述包含N末端修饰的第二恒定区包含SEQ ID NO:37所示氨基酸序列。(对应于TCRbC-N.Rec-1)
在一些实施方案中，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第1-12位氨基酸，且其中N末端的第13-25 位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述包含N末端修饰的第二恒定区包含SEQ ID NO:38所示氨基酸序列。(对应于TCRbC-N.Rec-2)
在一些实施方案中，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第1-18位氨基酸，且其中N末端的第19-25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述包含N末端修饰的第二恒定区包含SEQ ID NO:39所示氨基酸序列。(对应于TCRbC-N.Rec-3)
在一些实施方案中，相对于天然T细胞受体β链恒定区，所述第二恒定区在第56位包含半胱氨酸取代，所述氨基酸编号参考SEQ ID NO:15。在一些实施方案中，相对于天然T细胞受体β链恒定区，所述第二恒定区在第56位的丝氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述包含半胱氨酸取代的第二恒定区包含SEQ ID NO:19所示氨基酸序列(衍生自人T细胞受体β链恒定区)。在一些具体实施方案中，所述包含半胱氨酸取代的第二恒定区包含SEQ ID NO:20所示氨基酸序列(衍生自小鼠T细胞受体β链恒定区)。(对应于TCRbC-Cys)
在一些实施方案中，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第1-6位氨基酸，且其中N末端的第7-25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，在第56位的丝氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述第二恒定区包含SEQ ID NO:21所示的氨基酸序列。(对应于TCRbC-Cys-N.Rec-1)
在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-4位氨基酸，且其中N末端的第5-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:2；所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第1-6位氨基酸，且其中N末端的第7-25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:34所示氨基酸序列，第二恒定区包含SEQ ID NO:37所示氨基酸序列。
在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区，在第48位的苏氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:2；所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区，在第56位的丝氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:7所示氨基酸序列，所述第二恒定区包含SEQ ID NO:20所示氨基酸序列。
在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区，在第112位的丝氨酸被亮氨酸取代，在114位的 甲硫氨酸被异亮氨酸取代，且在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2；所述第二恒定区是天然小鼠T细胞受体β链恒定区。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:9所示氨基酸序列，所述所述第二恒定区包含SEQ ID NO:15所示氨基酸序列。
在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-4位氨基酸，且其中N末端的第5-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，在第48位的苏氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:2；所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第1-6位氨基酸，且其中N末端的第7-25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，在第56位的丝氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:11的氨基酸序列，所述第二恒定区包含SEQ ID NO:21所示的氨基酸序列。
在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-4位氨基酸，且其中N末端的第5-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，在第112位的丝氨酸被亮氨酸取代，在114位的甲硫氨酸被异亮氨酸取代，且在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2；所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第1-6位氨基酸，且其中N末端的第7-25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:12的氨基酸序列，所述第二恒定区包含SEQ ID NO:37所示氨基酸序列。
在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区，在第48位的苏氨酸被突变为半胱氨酸，在第112位的丝氨酸被亮氨酸取代，在114位的甲硫氨酸被异亮氨酸取代，且在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2；所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区，在第56位的丝氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:15。在一些具体实施方案中，所述第一恒定区包含SEQ ID NO:10的氨基酸序列，第二恒定区包含SEQ ID NO:20所示氨基酸序列。
在一些优选实施方案中，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-4位氨基酸，且其中N末端的第5-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，在第48位的苏氨酸被突变为半胱氨酸，在第112位的丝氨酸被亮氨酸取代，在114位的甲硫氨酸被异亮氨酸取代，且在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2；且，
所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体 β链恒定区缺失N末端的第1-6位氨基酸，且其中N末端的第7-25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，在第56位的丝氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:15。
在一些优选实施方案中，所述第一恒定区包含SEQ ID NO:13的氨基酸序列，且所述第二恒定区包含SEQ ID NO:21所示的氨基酸序列。
如本文所用，“抗原结合区”意指其独自或者和另一抗原结合区组合可以特异性结合靶抗原。
本文所述“抗原”或“靶抗原”可以是任何能被抗体识别并结合的抗原，优选是疾病相关抗原，更优选是癌症相关抗原。示例性的癌症相关抗原包括但不限于CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。
所述抗原结合区可以衍生自一或多种已知抗体，包括任何商业可获得的抗体，如FMC63、利妥昔单抗(rituximab)、阿仑珠单抗(alemtuzumab)、依帕珠单抗(epratuzumab)、曲妥珠单抗(trastuzumab)、比伐珠单抗(bivatuzumab)、西妥昔单抗(cetuximab)、拉贝珠单抗(labetuzumab)、帕利珠单抗(palivizumab)、司韦单抗(sevirumab)、妥韦单抗(tuvirumab)、巴利昔单抗(basiliximab)、达克珠单抗(daclizumab)、英利昔单抗(infliximab)、奥马珠单抗(omalizumab)、依法珠单抗(efalizumab)、凯利昔单抗(Keliximab)、西利珠单抗(siplizumab)、那他珠单抗(natalizumab)、克立昔单抗(clenoliximab)、培马单抗(pemtumomab)、依屈洛单抗(Edrecolomab)、坎妥珠单抗(Cantuzumab)等。在一些优选实施方式中，所述抗原结合区衍生自由保藏号编号为CGMCC No.17095(于2019年1月21日保藏于地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所的中国 微生物菌种保藏管理委员会普通微生物中心)的杂交瘤细胞334产生的单克隆抗体334。
在一些实施方案中，所述第一抗原结合区包含特异性结合靶抗原的抗体的重链可变区，且所述第二抗原结合区包含所述抗体的轻链可变区。在一些实施方案中，所述第一抗原结合区包含特异性结合靶抗原的抗体的轻链可变区，且所述第二抗原结合区包含所述抗体的重链可变区。
在一些实施方案中，所述第一抗原结合区包含SEQ ID NO:22所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:23所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:23所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:22所示氨基酸序列，由此所述STAR特异性结合EGFR。
在一些实施方案中，所述第一抗原结合区包含SEQ ID NO:44所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:45所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:45所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:44所示氨基酸序列，由此所述STAR特异性结合CD19。
在一些实施方案中，所述第一抗原结合区包含SEQ ID NO:46所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:47所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:47所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:46所示氨基酸序列，由此所述STAR特异性结合CD20。
在一些实施方案中，所述第一抗原结合区包含SEQ ID NO:48所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:49所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:49所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:48所示氨基酸序列，由此所述STAR特异性结合CD22。
在一些实施方案中，所述第一抗原结合区包含SEQ ID NO:24所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:25所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:25所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:24所示氨基酸序列，由此所述STAR特异性结合GPC3。
在一些实施方案中，所述第一抗原结合区包含特异性结合CD19的抗体的重链可变区，所述重链可变区包含SEQ ID NO:28所示重链CDR1、SEQ ID NO:29所示重链CDR2和SEQ ID NO:30所示重链CDR3，且所述第二抗原结合区包含特异性结合CD19的抗体的轻链可变区，所述轻链可变区包含SEQ ID NO:31所示轻链CDR1、SEQ ID NO:32所示轻链CDR2和SEQ ID NO:33所示轻链CDR3；或者，所述第一抗原结合区包含特异性结合CD19的抗体的轻链可变区，所述轻链可变区包含SEQ ID NO:31所示轻链CDR1、SEQ ID NO:32所示轻链CDR2和SEQ ID NO:33所示轻链CDR3，且所述第二抗原结合区特异性结合CD19的抗体的重链可变区，所述重链可变区包含SEQ ID NO:28所示重链CDR1、SEQ ID NO:29所示重链CDR2和SEQ ID NO:30所示重链CDR3，由此所述STAR特异性结合CD19。
在一些实施方案中，所述第一抗原结合区包含SEQ ID NO:26所示氨基酸序列，且 所述第二抗原结合区包含SEQ ID NO:27所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:27所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:26所示氨基酸序列，由此所述STAR特异性结合CD19。
在一些实施方案中，所述第一抗原结合区包含特异性结合靶抗原的单链抗体(如scFv)或单域抗体(如骆驼抗体)。在一些实施方案中，所述第二抗原结合区包含特异性结合靶抗原的单链抗体(如scFv)或单域抗体(如骆驼抗体)。在一些实施方案中，所述第一抗原结合区和所述第二抗原结合区结合相同的靶抗原。在一些实施方案中，所述第一抗原结合区和所述第二抗原结合区结合相同靶抗原的不同区域(如不同的表位)。在一些实施方案中，所述第一抗原结合区和所述第二抗原结合区结合不同的靶抗原。例如，在一些示例性实施方案中，所述两个抗原结合区可以分别结合CD19和CD20，或分别结合CD19和CD22，或分别结合CD38和BCMA，或分别结合PDL1和EGFR。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:22所示氨基酸序列以及SEQ ID NO:23所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合EGFR。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:44所示氨基酸序列以及SEQ ID NO:45所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD19。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:46所示氨基酸序列以及SEQ ID NO:47所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD20。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:48所示氨基酸序列以及SEQ ID NO:49所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD22。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:24所示氨基酸序列以及SEQ ID NO:25所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合GPC3。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:28所示重链CDR1、SEQ ID NO:29所示重链CDR2和SEQ ID NO:30所示重链CDR3，所述轻链可变区包含SEQ ID NO:31所示轻链CDR1、SEQ ID NO:32所示轻链CDR2和SEQ ID NO:33所示轻链CDR3，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD19。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:26所示氨基酸序列以及SEQ ID NO:27所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD19。
在另一方面，本发明提供一种合成T细胞受体抗原受体(STAR)，其包含α链和β链，所述α链包含第一抗原结合区和第一恒定区，所述β链包含第二抗原结合区和第二 恒定区，
其中第一恒定区是天然T细胞受体α链恒定区或如本文所公开的天然T细胞受体α链恒定区变体，其中第二恒定区是天然T细胞受体β链恒定区或如本文所公开的天然T细胞受体β链恒定区变体，
其中第一抗原结合区和第二抗原结合区特异性地结合不同的靶抗原或相同靶抗原的不同区域。
在一些实施方案中，所述第一抗原结合区和/或第二抗原结合区包含特异性结合靶抗原的单链抗体(如scFv)或单域抗体(如骆驼抗体)。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:22所示氨基酸序列以及SEQ ID NO:23所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合EGFR。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:44所示氨基酸序列以及SEQ ID NO:45所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD19。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:46所示氨基酸序列以及SEQ ID NO:47所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD20。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:48所示氨基酸序列以及SEQ ID NO:49所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD22。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:24所示氨基酸序列以及SEQ ID NO:25所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合GPC3。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:28所示重链CDR1、SEQ ID NO:29所示重链CDR2和SEQ ID NO:30所示重链CDR3，所述轻链可变区包含SEQ ID NO:31所示轻链CDR1、SEQ ID NO:32所示轻链CDR2和SEQ ID NO:33所示轻链CDR3，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD19。
在一些实施方案中，所述第一抗原结合区和/或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:26所示氨基酸序列以及SEQ ID NO:27所示氨基酸序列，由此所述第一抗原结合区和/或所述第二抗原结合区特异性结合CD19。
在一些示例性实施方案中，所述两个抗原结合区可以分别结合CD19和CD20，或分别结合CD19和CD22，或分别结合CD38和BCMA，或分别结合PDL1和EGFR。
在另一方面，本发明提供一种分离的多核苷酸，其包含编码本发明所述合成T细胞受体抗原受体(STAR)的α链和/或β链的核苷酸序列。
在一些实施方案中，所述多核苷酸包含在同一读码框内的i)编码所述α链的核苷酸 序列、ii)编码所述β链的核苷酸序列和iii)位于i)和ii)之间的编码自裂解肽的核苷酸序列。编码所述α链的核苷酸序列可以位于编码所述β链的核苷酸序列的5’端或3’端。
如本文所用“自裂解肽”意指可以在细胞内实现自剪切的肽。例如，所述自裂解肽可以包含蛋白酶识别位点，从而被细胞内的蛋白酶识别并特异性切割。
或者，所述自裂解肽可以是2A多肽。2A多肽是一类来自病毒的短肽，其自切割发生在翻译期间。当用2A多肽连接两种不同目的蛋白在同一读码框表达时，几乎以1:1的比例生成两种目的蛋白。常用的2A多肽可以是来自猪捷申病毒(porcine techovirus-1)的P2A、来自明脉扁刺蛾β四体病毒(Thosea asigna virus)的T2A、马甲型鼻病毒(equine rhinitis A virus)的E2A和来自口蹄疫病毒(foot-and-mouth disease virus)的F2A。其中P2A的切割效率最高，因此是优选的。本领域也已知多种这些2A多肽的功能性变体，这些变体也可以用于本发明。
在另一方面，本发明提供一种表达载体，其包含与调控序列可操作连接的本发明的多核苷酸。
本发明的“表达载体”可以是线性的核酸片段、环状质粒、病毒载体，或者可以是能够翻译的RNA(如mRNA)。在一些优选实施方案中，所述表达载体是病毒载体，例如慢病毒载体。
“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5'非编码序列)、中间或下游(3'非编码序列)，并且影响相关编码序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。表达调控元件指的是能够控制感兴趣的核苷酸序列转录、RNA加工或稳定性或者翻译的核苷酸序列。调控序列可包括但不限于启动子、翻译前导序列、内含子、增强子和多腺苷酸化识别序列。
如本文中所用，术语“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知的。
在另一方面，本发明提供一种制备治疗性T细胞的方法，包括在T细胞中表达本发明的合成T细胞受体抗原受体(STAR)。在一些实施方案中，所述方法包括将本发明的多核苷酸或本发明的表达载体导入T细胞。
在另一方面，本发明提供一种治疗性T细胞，其包含本发明的合成T细胞受体抗原受体(STAR)，或其通过本发明的方法获得。
本发明的T细胞可以通过各种非限制性方法从许多非限制性来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、腹水、胸腔积液、脾组织和肿瘤。在一些实施方案中，细胞可以衍生自健康供体或来自诊断为癌症的患者。在一些实施方案中，细胞可以是呈现不同表型特征的细胞的混合群体的一部分。例如，T细胞可以通过分离外周血单个核细胞(PBMC)，然后用特异性抗体活化、扩增获得。
在本发明各方面的一些实施方案中，所述T细胞衍生自对象的自体细胞。如本文所 用，“自体”是指用于治疗对象的细胞、细胞系或细胞群源自所述对象。在一些实施方案中，所述T细胞衍生自异体细胞，例如源自与所述对象人类白细胞抗原(HLA)相容的供体。可以使用标准方案将来自供体的细胞转化为非同种异体反应性细胞，并根据需要进行复制，从而产生可以施用至一个或多个患者的细胞。
在另一方面，本发明提供一种药物组合物，其包含本发明的治疗性T细胞，和药物可接受的载体。
本文使用的“药学上可接受的载体”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。
在另一方面，本发明提供本发明的治疗性T细胞或本发明的药物组合物在制备用于在对象中治疗疾病例如癌症的药物中的用途。
如本文所用“对象”是指患有或者易于患有可以通过本发明的细胞、方法、或药物组合物治疗的疾病(如癌症)的生物体。非限制性例子包括人、牛、大鼠、小鼠、狗、猴、山羊、绵羊、母牛、鹿，及其它非哺乳动物。在优选实施方案中，对象是人。
在另一方面，本发明提供一种在对象中治疗疾病例如癌症的方法，包括给所述对象施用治疗有效量的本发明的治疗性T细胞或本发明的药物组合物。
如本文所用，“治疗有效量”或“治疗有效剂量”或“有效量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或细胞的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。例如，“有效量”的本发明的细胞或药物组合物优选地导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。例如，对于肿瘤的治疗，相对于未接受治疗的对象，“有效量”的本发明的细胞或药物组合物优选地将肿瘤细胞生长或肿瘤生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。抑制肿瘤生长的能力可以在预测对人类肿瘤的疗效的动物模型系统中评价。或者，也可以通过检查抑制肿瘤细胞生长的能力来评价，这种抑制可以通过本领域技术人员公知的试验在体外测定。
实际应用中，本发明药物组合物中细胞的剂量水平可能改变，以获得可有效实现对特定患者、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择的剂量水平取决于多种药物代谢动力学因素，包括应用的本发明特定组合物的活性，给药途径，给药时间，应用的特定化合物的排泄速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的患者的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素。
根据本发明的治疗性T细胞或药物组合物或药物的施用可以以任何方便的方式进行，包括通过注射、输注、植入或移植。本文所述的细胞或组合物施用可以通过静脉内、淋巴内、皮内、肿瘤内、髓内、肌内或腹膜内施用。在一个实施方案中，本发明的细胞 或组合物优选通过静脉内注射施用。
在本发明各个方面的实施方案中，所述癌症选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤、骨肉瘤、骨癌、胰腺癌、皮肤癌、前列腺癌、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、霍奇金病、非霍奇金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。
实施例
实施例1.STAR受体及其突变体的恒定区结构域的设计
1.1 STAR的原型设计
B细胞产生的分泌抗体(Antibody，Ab)或B细胞受体(BCR)与T细胞受体(TCR)在基因结构、蛋白结构和空间构象上有很大的相似性。抗体和TCR都是由可变区和恒定区组成，其中可变区起到抗原识别和结合的作用，而恒定区结构域起到结构互作和信号转导的作用。通过把TCRα和β链(或TCRγ和δ链)的可变区替换为抗体的重链可变区(VH)和轻链可变区(VL)，可以构建出一种人工合成的嵌合分子，称为合成T细胞受体抗体受体(Synthetic T-Cell Receptor and Antibody Receptor,STAR)，其结构见图1。
STAR分子有两条链，第一条链是抗原识别序列(如抗体的重链可变区VH)与T细胞受体α链(TCRα)的恒定区(Cα，human TCRaC-WT，SEQ ID NO:1)相融合而得，第二条链是抗原识别序列(如抗体的轻链可变区VL)与T细胞受体β链(TCRβ)的恒定区(Cβ,human TCRbC-WT，SEQ ID NO:14)相融合而得。该构建体中抗原识别结构域(如VH、VL或scFv等)与恒定区结构域(TCRα、β、γ和δ的恒定区)可以进行排列组合，组成多种构型不同但功能类似的构建体。
STAR分子的第一、二条链在T细胞内表达后，会在内质网中与细胞内源的CD3εδ、CD3γε、CD3ζζ链相结合形成8个亚基的复合物，并以复合物的形式展示在细胞膜表面。免疫受体酪氨酸活化基序(Immunoreceptor Tyrosine-based Activation Motif，ITAM)是TCR分子中起到信号转导作用的基序，其保守序列为YxxL/V。CD3ε、δ、γ、ε链的胞内区含有1个ITAM序列，CD3ζ链的胞内区含有3个ITAM序列，所以一个完整STAR复合体共含有10个ITAM序列。当STAR受体的抗原识别序列与其特异性抗原结合后，胞内的ITAM序列会逐次被磷酸化，进而激活下游信号通路，激活NF-κΒ、NFAT和AP-1等转录因子，引发激活T细胞，产生效应功能。
1.2鼠源化STAR的设计
STAR原型设计使用的是人类来源的TCRα/β链(或TCRγ和δ链)的恒定区序列。由于人源、灵长类动物来源和鼠源TCRα/β链(mouse TCRaC-WT，SEQ ID NO:2；mouse TCRbC-WT，SEQ ID NO:15)(或TCRγ和δ链)的恒定区序列的功能保守性较高，且关键氨基酸序列相同，所以可以相互替换。
STAR分子被转导入T细胞中后，会通过恒定区与T细胞内源的TCR发生错配。这一错配问题一方面减少了STAR分子正确配对的效率、削弱其功能，另一方面增加了错配产生未知特异性的可能、增加了安全性风险。为解决这一问题，本发明将STAR分子的恒定区替换为鼠源序列，以增强STAR分子转入人类T细胞后的功能。人源序列和鼠源序列的比对见图2。
1.3 STAR的恒定区N端的修饰设计
为进一步优化STAR分子的设计，本发明对STAR分子中特定序列进行修改，其中一种修改方案为对STAR分子恒定区的N端进行删除。TCRα链恒定区删掉N端的18个氨基酸(鼠源序列为DIQNPEPAVYQLKDPRSQ，SEQ ID NO:3)(产生恒定区序列为mouse TCRaC-N.DLT，SEQ ID NO:4)，TCRβ链恒定区删掉N端的25个氨基酸(鼠源序列为DLRNVTPPKVSLFEPSKAEIANKQK，SEQ ID NO:16)(产生恒定区序列为mouse TCRbC-N.DLT，SEQ ID NO:17)。这两个区域位于恒定区的N端，不属于类免疫球蛋白C1型结构域(Ig-like C1 type domain)，也不参与分子内二硫键的形成，但主要起到铰链的功能，而这一功能对STAR的功能是否有影响，为此设计不同删除方案。本发明对恒定区特定序列进行重排，以获得更优效果。重排意为进行部分序列删除，同时对部分序列进行人源化突变。该区域性修饰具有以下优势：第一，铰链区功能优化能够减少STAR与内源TCR错配。第二，优化后的STAR的分子大小与天然TCR的大小更相似，更利于共刺激分子的结合、免疫突触的形成和稳定、以及后续T细胞的效应功能。
人源化突变的意义在于，在保证STAR分子功能的同时，尽可能减少STAR分子中的非人源序列，以最大程度规避STAR-T细胞在临床应用中被受体排斥的可能。具体重排方案阐述如下。
TCRα链恒定区N端的18个氨基酸(鼠源序列为DIQNPEPAVYQLKDPRSQ)重排方案：
图3示出鼠源TCRα链恒定区N端的18个氨基酸与人源序列的比对，UserSeq1为人源序列，UserSeq2为鼠源序列。通过氨基酸性质分析，发现鼠源和人源序列在E6D、K13R、R16K、Q18S这些位点均属同性质氨基酸替换，而在P15S位点属于非极性氨基酸替换为极性氨基酸，因此可认为该位点附近的蛋白质性质不保守、进而可以进行修改而不影响功能。综上，本发明将鼠源TCRα链恒定区N端的1-14位氨基酸序列保留并人源化、并删除15-18位氨基酸，产生mouse TCRaC-N.Rec-4，SEQ ID NO:5。
此外，为考虑此区域的铰链作用，全部截短后可能会对效果产生影响，还将鼠源TCRα链恒定区N端的1-4位氨基酸删除，5-18位氨基酸人源化，该修饰称作mouse TCRaC-N.Rec-1，产生恒定区序列为SEQ ID NO:34；将鼠源TCRα链恒定区N端的1-8位氨基酸删除，9-18位氨基酸人源化，该修饰称作mouse TCRaC-N.Rec-2，产生恒定区序列为SEQ ID NO:35；将鼠源TCRα链恒定区N端的1-12位氨基酸删除，13-18位氨基酸人源化，产生恒定区序列为mouse TCRaC-N.Rec-3，SEQ ID NO:36。作为对照，还对鼠源TCRα链恒定区N端的1-18位氨基酸进行了随机重排，产生恒定区序列为mouse TCRaC-N.rRec，SEQ ID NO:40。
TCRβ链恒定区N端的25个氨基酸(鼠源序列为DLRNVTPPKVSLFEPSKAEIANKQK)重排方案：
图4示出鼠源TCRβ链恒定区N端的25个氨基酸与人源序列的比对，UserSeq1为人源序列，UserSeq2为鼠源序列。通过氨基酸性质分析，发现鼠源和人源序列仅在R3K和L12V位点属同性质氨基酸替换，而在T6F、K8E、D11A、K17E、A21S、N22H和K23T位点均属不同氨基酸性质的替换，因此可认为该位点附近的蛋白质性质不保守、进而可以进行修改而不影响功能。综上，本发明将鼠源TCRβ链恒定区N端的1-16位氨基酸序列保留并人源化、并删除17、21-25位氨基酸，产生恒定区序列为mouse TCRbC-N.Rec-4，SEQ ID NO:18。
此外，为考虑此区域的铰链作用，全部截短后可能会对效果产生影响，还将鼠源TCRβ链恒定区N端的1-6位氨基酸删除，13-25位氨基酸人源化，产生恒定区序列为mouse TCRbC-N.Rec-1，SEQ ID NO:37；还将鼠源TCRβ链恒定区N端的1-12位氨基酸删除，13-25位氨基酸人源化，产生恒定区序列为mouse TCRbC-N.Rec-2，SEQ ID NO:38；还将鼠源TCRβ链恒定区N端的1-18位氨基酸删除，19-25位氨基酸人源化，产生恒定区序列为mouse TCRbC-N.Rec-3，SEQ ID NO:39。作为对照，还对鼠源TCRβ链恒定区N端的1-25位氨基酸进行了随机重排，产生恒定区序列为mouse TCRbC-N.rRec，SEQ ID NO:41。
1.4 STAR的恒定区C端的修饰设计
本发明还删除了鼠源TCRα链恒定区C端的15个氨基酸，产生恒定区序列为mouse TCRaC-C.DLT(SEQ ID NO:42)。
本发明还删除了鼠源TCRβ链恒定区C端的15个氨基酸，产生恒定区序列为mouse TCRbC-C.DLT(SEQ ID NO:43)。
1.5 STAR半胱氨酸点突变引入分子间二硫键的设计
为进一步优化STAR分子的设计，本发明对STAR分子中特定序列进行修改，其中一种修改方案为对STAR分子进行半胱氨酸点突变，以引入分子间二硫键，增强STAR分子两条链间的相互配对，减少与内源TCR的错配。具体方案阐述如下。
于TCRα链恒定区将48位苏氨酸T突变为半胱氨酸C(产生恒定区序列为human TCRaC-Cys,SEQ ID NO:6；mouse TCRaC-Cys,SEQ ID NO:7)，于TCRβ链恒定区将56位丝氨酸S突变为半胱氨酸C(产生恒定区序列为human TCRbC-Cys,SEQ ID NO:19；mouse TCRbC-Cys,SEQ ID NO:20)。这两个新增的半胱氨酸会在STAR的两条链间形成二硫键，减少STAR两条链与内源性TCR链的错配，帮助STAR分子形成更加稳定的复合体，进而获得更好的功能。
1.6 STAR跨膜区疏水氨基酸替换的设计
为进一步优化STAR分子的设计，本发明对STAR分子中特定序列进行修改，其中一种修改方案为对STAR分子的跨膜区进行疏水氨基酸替换，以增加STAR分子的稳定性，帮助其发挥更持久功能。具体方案阐述如下。
在TCRα链恒定区跨膜区的111位至119位氨基酸区域内进行3个氨基酸位点的突变，将112位丝氨酸S变成亮氨酸L、114位甲硫氨酸M变成异亮氨酸I、115位甘氨酸G变成颉氨酸V。该区域整体氨基酸序列由LSVMGLRIL变为LLVIVLRIL，该修饰称作mouse TCRaC-TM9，产生恒定区序列为SEQ ID NO:9。这一设计增加了跨膜区的疏水性，抵消了TCR跨膜区携带正电荷所导致的不稳定性，使得STAR分子能在细胞膜上更为稳定地存在，进而获得更好的功能。
1.7 N端修饰、半胱氨酸修饰和跨膜区修饰的组合
发明人对上述的N端修饰、半胱氨酸修饰和跨膜区修饰进行了组合，设计了mouse TCRaC-Cys-N.Rec-1(SEQ ID NO:11)、mouse TCRaC-TM9-N.Rec-1(SEQ ID NO:12)、mouse TCRaC-Cys-TM9(SEQ ID NO:10)、mouse TCRaC-Cys-TM9-N.Rec-1(SEQ ID NO:13)，以及mouse TCRbC-Cys-N.Rec-1(SEQ ID NO:21)。
1.8 STAR可变区和恒定区的交换排列组合
STAR分子可分为可变区结构域和恒定区结构域两部分。可变区结构域由具有抗原识别能力的氨基酸序列组成，一般由以下类别的分子构成：抗体轻链可变区、抗体重链可变区、单链抗体(scFv)、配体-受体结合区、Nanobody等等。恒定区结构域由具有和CD3亚基集合能力的TCR恒定区或相关序列组成，一般包括TCRα、β、γ和δ链的恒定区。
STAR构建体中抗原识别结构域与恒定区结构域可以进行排列组合，组成多种构型不同但功能类似的构建体。一些可能的排列组合如下表所示：
Figure PCTCN2020103687-appb-000001
Figure PCTCN2020103687-appb-000002
实施例2.STAR受体的抗原识别结构域的设计
1)靶点选择
STAR靶点选择主要通过基因表达的数据库实现，总的来说，合适的STAR靶点的表达水平相较于正常细胞一般在疾病或者相关细胞上高表达或者特异性表达。确定合适靶点后，识别靶点的抗原识别结构域一般为抗体、SCFV、VHH或抗原对应受体等。抗体的重链可变区(VH)、轻链可变区(VL)，羊驼单链抗体(VHH)，以及受体和配体可以通过免疫小鼠、羊驼、测序或者通过已经发表的序列获得。而获得的鼠源或者羊驼序列一般需要经过人源化和密码子优化使其更有利于在人体细胞中表达和行使功能。获得合适的序列后，将所得序列连接到慢病毒或者逆转录病毒载体中，通过测序方法检测载体构建是否正确，再通过病毒包装、感染使其表达与体细胞中，使用标签蛋白RFP、GFP、MYC、HIS等用于检测STAR蛋白表达、上膜、与抗原结合等。所适用的靶点包括但不限于GPC3、EGFR VIII、CD22、CD20、MSLN、GD2、LIRB4、ROR1、Cllaudin18.2、CD7、CD47、CD24。
2)靶细胞系及工具细胞系列表
在本发明中，使用了多种靶标细胞系用于说明STAR的功能，并通过基因工程手段使靶标细胞获得luciferase基因，从而利于体外功能检测。荧光素酶是常见的用于细胞功能研究的物质，通过在体系中添加荧光素酶底物从而判断酶活性，而荧光素酶活性与目的基因的表达、结合强弱、细胞数量等密切相关。本发明中，建立稳定表达荧光素酶的靶标细胞系，以荧光素酶的多少指代靶标细胞的数量，进而指示功能细胞的杀伤功能。具体细胞如下:
细胞系名称 靶点 对应癌症/疾病或功能
A431 EGFR 皮肤癌
A549 EGFR 肺癌
B16/htEGFR EGFR 黑色素瘤
HepG2 GPC3 肝癌
HUH7 GPC3 肝癌
RAJI CD19 淋巴瘤/白血病
RAJI-CD19KO CD19- 淋巴瘤/白血病
RAJI-CD20KO CD20- 淋巴瘤
RAJI-CD22KO CD22- 淋巴瘤
Jurkat   功能检测
Jurkat Clone 5(JC5)   功能检测/病毒滴度检测
3)抗原识别结构域的选择(抗体、scFv、Fab、VHH等)
抗原识别结构域可来源于识别抗原的抗体，抗体的单链可变区、可变区、纳米抗体，于抗原识别的受体等等。在STAR的设计中，病毒是主要的T细胞改造的途径，慢病毒或者逆转录病毒的包装与包装的尺寸大小相关，因此较小的抗体识别单位会受到较大青睐，如抗体单链可变区和纳米抗体从尺寸上是更好的选择。针对同一抗原，目前已有的抗体作为现有技术是广泛筛选抗体识别结构域的途径之一，同时通过噬菌体文库构建全人源抗体库、亲和力优化、鼠杂交瘤筛选、羊驼单抗筛选、人源化等技术获得全新的识别抗原的抗体。此外抗原的受体作为可识别抗原的一种特异性分子，也是抗原识别结构域的主要选择之一。
实施例3.STAR受体及其突变体的质粒构建
1)质粒来源
本发明中所用的载体，包括慢病毒载体、逆转录病毒载体、蛋白表达载体、噬菌粒、慢病毒包装质粒、逆转录病毒包装质粒等均购买与商业公司或商业公司合成，并获得这些载体的全长序列，已知明确的酶切位点。
2)片段来源
本发明中采用的基因片段包括信号肽、抗体结合区、铰链区、TCR恒定区、标签蛋白等均来源于商业化公司合成。通过合成引物PCR方式，连接一段或多段目的片段获得相应功能序列。
3)质粒构建与测序方法
本发明使用中的慢病毒载体为pHAGE-EF1α-GFP，通过限制性内切酶Not I/Cla I获得pHAGR-EF1A-WPRE-AMP载体，片段基因通过合成和PCR方法获得，通过同源重组方法，在Gibsson/NEBuilder等重组酶作用下获得完整载体。逆转录病毒载体RVKM-CMV-GFP通过限制性内切酶Xho I/BamH I获得载体片段，而插入片段基因通过合成和PCR方法获得，通过同源重组方法将载体和片段相连。原核表达载体为pET-28a载体，使用的限制性内切酶位点为XhoI和NcoI，真核表达载体为PVRC8400，使用的限制性内切酶位点为XhoI和XbaI，根据人源抗原基因设计引物，向引物两端加入限制性内切酶酶切位点，从靶细胞全基因中扩增出截断的抗原胞外段以及C段的his-tag，用于后续纯化。利用粘性末端PCR，根据酶切产物粘性末端正义链和反义链分别设计引物， 分别进行PCR后，与酶切后的载体进行一步重组连接，连接后向50μL DH5α感受态细胞中加入5μL连接产物进行转化，倒置于37℃培养箱中培养12-16hr。
4)质粒提取方法
质粒提取采用碱裂解法，质粒提取后通过260nm光吸收检测质粒浓度。初次测序结果正确的质粒，需要转化挑取单克隆，进行小提，并且酶切鉴定质粒是否正确。
通过以上方法获得质粒pHAGE-EF1A-IRES-RFP；恒定区分别为TCRa/bC-WT(包含TCRaC-WT和TCRbC-WT)、TCRa/bC-N.DLT(包含TCRaC-N.DLT和TCRbC-N.DLT)、TCRa/bC-C.DLT(包含TCRaC-C.DLT和TCRbC-CDLT)、TCRa/bC-N.Rec-1(包含TCRaC-N.Rec-1和TCRbC-N.Rec-1)、TCRa/bC-N.Rec-2(包含TCRaC-N.Rec-2和TCRbC-N.Rec-2)、TCRa/bC-N.Rec-3(包含TCRaC-N.Rec-3和TCRbC-N.Rec-3)、TCRa/bC-N.Rec-4(包含TCRaC-N.Rec-4和TCRbC-N.Rec-4)、TCRa/bC-N.rRec(包含TCRaC-N.rRec和TCRbC-N.rRec)、TCRa/bC-Cys(包含TCRaC-Cys和TCRbC-Cys)、TCRa/bC-TM9(包含TCRaC-TM9和TCRbC-WT)、TCRa/bC-Cys-N.Rec-1(包含TCRaC-Cys-N.Rec-1和TCRbC-Cys-N.Rec-1)、TCRa/bC-TM9-N.Rec-1(包含TCRaC-TM9-N.Rec-1和TCRbC-N.Rec-1)、TCRa/bC-Cys-TM9(包含TCRaC-Cys-TM9和TCRbC-Cys)、TCRa/bC-Cys-TM9-N.Rec-1(包含TCRaC-Cys-TM9-N.Rec-1和TCRbC-Cys-N.Rec-1)的多种pHAGE-EF1A-Cetux-VL-TCRaC-P2A-VH-TCRbC-IRES-RFP和pHAGE-EF1A-GC33-VL-TCRaC-P2A-VH-TCRbC-IRES-RFP质粒；以及针对不同靶点，其TCR恒定区均为TCRa/bC-Cys-TM9-N.Rec-1的pHAGE-EF1A-334-VL-TCRbC-P2A-VH-TCRaC-IRES-RFP、pHAGE-EF1A-2C6-VL-(G4S)3-VH-TCRbC-P2A-FMC63-VL-(G4S)3-VH-TCRaC-IRES-RFP、pHAGE-EF1A-M971-VL-(G4S)3-VH-TCRbC-P2A-334-VL-(G4S)3-VH-TCRaC-IRES-RFP等质粒。
实施例4.工具质粒和细胞系的构建
1)质粒pHAGE-luciferase-GFP构建
pHAGE作为一种慢病毒载体可将目的基因稳定的插入到目的细胞基因组中，是构建稳定细胞系的一种重要途径。荧光素酶(Luciferase)是一种具有催化活性的酶，可以催化底物发生化学自发光，通过将目的细胞稳定表达荧光素酶，加入底物后可指示目的细胞的数量，从而反应功能细胞对目的细胞的影响。PHAGE-EF1A载体携带限制性内切酶Not I/Cla I酶切位点，利用这两个酶将载体切开，利用NCBI获得荧光素酶和GFP序列，通过商业化公司睿博兴科合成片段，通过Overlap PCR方法首先将荧光素酶基因和GFP基因联合，再通过同源重组方法将荧光素酶-GFP片段连入pHAGE载体中，在EF1A和WPRE分别设计正反向引物，测序获得载体构建是否成功。
2)携带luciferase靶细胞系构建(含细胞系列表)
带有荧光素酶和GFP的慢病毒载体构建成功后，通过Lentix-293T包装慢病毒，PEG8000浓缩方法浓缩慢病毒液，使用梯度稀释法测定病毒滴度，然后感染目的细胞包括但不限于下表中细胞系：
名称 靶点 对应癌症/疾病或功能
A431 EGFR 皮肤癌
A549 EGFR 肺癌
B16/htEGFR EGFR 黑色素瘤
HepG2 GPC3 肝癌
HUH7 GPC3 肝癌
RAJI CD19 淋巴瘤/白血病
RAJI-CD19KO CD19- 淋巴瘤/白血病
RAJI-CD20KO CD20- 淋巴瘤
RAJI-CD22KO CD22- 淋巴瘤
感染后72小时通过荧光显微镜观察是否有GFP阳性细胞，然后通过流式分选仪分选出GFP阳性细胞，挑选单克隆，并建库保存。同时，使用荧光素酶底物与目的细胞共孵育，检测荧光酶素表达及检测水平，确定表达水平。
3)TCRa/b敲除的Jurkat细胞系构建
基于TCR的结构和序列特征，在α链和β链的恒定区设计guide序列，构建TCRα-β-Jurkat细胞系。在NCBI分别获取TCRα链和β链的恒定区exon序列，将α链和β链恒定区的exon 1的序列提交到tools.genome-engineering.org网站设计guide序列，根据结果合成oligo序列。然后构建sgRNA-LentiCRISPR慢病毒载体，将CRISPR慢病毒载体用BsmBI限制性酶进行酶切，去除filter序列，暴露连接末端，同时将质粒用碱性磷酸酶37℃处理1小时，以减少自连现象。酶切后的体系跑琼脂糖凝胶电泳进行胶回收，目的片段的质粒骨架约11500bp,同时在～2000bp处有切下来的filter条带；将合成的guide序列的oligo单链在体外用T4连接酶和T4 PNK处理，anneal成双链和并在末端添加磷酸基团用于连接；将anneal的双链guide序列用水稀释200倍，和BsmBI酶切的LentiCRISPR进行连接，转化到Stbl3大肠杆菌，提取质粒用于包毒感染。α链的guide序列与LentiCRISPR-puro进行连接，β链的guide序列与LentiCRISPR-BSD进行连接。sgRNA-LentiCRISPR慢病毒的包装，浓缩：将HEK-293T提前铺到10cm dish,待细胞长至80％-90％时，配制转染体系，将转染体系加入HEK-293T后，把细胞放回37度培养箱，培养。计为0小时；转染后12小时后，换入新鲜10％FBS-DMEM。转染48小时和72小时可收病毒；将含有病毒的培养基离心并过滤，加入PEG8000混匀，在4度静置12小时以上，然后3500rpm离心30min；弃上清，用合适体积的培养基重悬沉淀。 -80冻存，或直接使用。Jurkat T细胞的感染，药杀筛选和单克隆细胞系的鉴定：将Jurkat T细胞接种在12或24孔板里；细胞密度不宜太大；同时加入合适体积的α链和β链的sgRNA-LentiCRISPR病毒，以及polybrene(按总体积1:1000加入)，混匀。离心感染，1000rpm，32度离心90min。取出后放入37度培养箱，计为0小时；10-12小时后换液；48小时后，加入Puromycin和Blasticidin至合适的终浓度，药杀48小时，可见未感染的对照组细胞全部死亡。将存活的细胞吸出，离心后用完全培养基培养，即为TCRα-β-Jurkat细胞库；将TCRα-β-Jurkat细胞库用流式Aria分选单细胞到96孔板中，培养两周后，将长起来的单克隆吸出，扩大培养；将单克隆细胞系分别用TCRα链和β链的抗体进行鉴定,两条链都缺陷的细胞系进行扩增，即获得内源性TCR敲除的Jurkat T细胞系。
实施例5.转导T细胞的病毒体系的建立
1)慢病毒体系与包装方法(不同代次)
将Lentix-293T细胞按照5×10 5个/mL接种至10cm培养皿中，置于37℃、5％CO2培养箱中培养，待细胞密度达到80％(在显微镜下观察)左右时进行转染。将三质粒按照PMD2.G:PSPAX:transfer plamid＝1:2:3比例，与500uL无血清的DMEM混合均匀。将54uL PEI-max与500uL无血清的DMEM混合均匀，室温静置5min(PEI-Max和质粒的体积质量比为3:1)。将PEI-max混合液缓慢加至质粒混合液中，轻轻吹打，混匀，室温静置15min。将最终混合液缓慢加入培养基中，充分混匀后，放回培养箱中继续培养12h-16h，换到6％FBS DMEM培养基中继续培养，收取48h、72h病毒液。
2)病毒滴度测量方法
将Jurkat-C5细胞以1.5×10^5个/mL接种在平底96孔板中，每孔中加入100uL含10％FBS、0.2uL 1000×polybrene的1640培养基。病毒稀释时用1640完全培养基进行10倍倍比稀释，若为病毒原液测定时第一个孔病毒量为100uL，若为浓缩液第一个孔病毒用量为1uL。将稀释好的细胞加入病毒孔中，100uL/well，混合，32℃，1500rpm，离心90min，37℃、5％CO2培养箱中培养72h。将96孔平底板上的细胞吸入到圆底96孔板上，4℃，1800rpm离心5min，弃上清。加入200uL 1×PBS后，4℃，1800rpm离心5min，弃上清。加入200uL 4％组织固定液，避光保存，上流式细胞仪。用流式细胞仪测感染效率，计算滴度时，选择感染率在2-30％的孔，计算公式为：滴度(TU/mL)＝1.5×10^4×阳性率÷病毒体积(uL)×1000。
通过以上方法包装了下列质粒的病毒：pHAGE-EF1A-IRES-RFP；恒定区分别为TCRa/bC-WT(包含TCRaC-WT和TCRbC-WT)、TCRa/bC-N.DLT(包含TCRaC-N.DLT和TCRbC-N.DLT)、TCRa/bC-C.DLT(包含TCRaC-C.DLT和TCRbC-CDLT)、TCRa/bC-N.Rec-1(包含TCRaC-N.Rec-1和TCRbC-N.Rec-1)、TCRa/bC-N.Rec-2(包含TCRaC-N.Rec-2和TCRbC-N.Rec-2)、TCRa/bC-N.Rec-3(包含TCRaC-N.Rec-3和TCRbC-N.Rec-3)、TCRa/bC-N.Rec-4(包含TCRaC-N.Rec-4和TCRbC-N.Rec-4)、TCRa/bC-N.rRec(包含TCRaC- N.rRec和TCRbC-N.rRec)、TCRa/bC-Cys(包含TCRaC-Cys和TCRbC-Cys)、TCRa/bC-TM9(包含TCRaC-TM9和TCRbC-WT)、TCRa/bC-Cys-N.Rec-1(包含TCRaC-Cys-N.Rec-1和TCRbC-Cys-N.Rec-1)、TCRa/bC-TM9-N.Rec-1(包含TCRaC-TM9-N.Rec-1和TCRbC-N.Rec-1)、TCRa/bC-Cys-TM9(包含TCRaC-Cys-TM9和TCRbC-Cys)、TCRa/bC-Cys-TM9-N.Rec-1(包含TCRaC-Cys-TM9-N.Rec-1和TCRbC-Cys-N.Rec-1)的多种pHAGE-EF1A-Cetux-VL-TCRaC-P2A-VH-TCRbC-IRES-RFP和pHAGE-EF1A-GC33-VL-TCRaC-P2A-VH-TCRbC-IRES-RFP质粒；以及针对不同靶点，其TCR恒定区均为TCRa/bC-Cys-TM9-N.Rec-1的pHAGE-EF1A-334-VL-TCRbC-P2A-VH-TCRaC-IRES-RFP、pHAGE-EF1A-2C6-VL-(G4S)3-VH-TCRbC-P2A-FMC63-VL-(G4S)3-VH-TCRaC-IRES-RFP、pHAGE-EF1A-M971-VL-(G4S)3-VH-TCRbC-P2A-334-VL-(G4S)3-VH-TCRaC-IRES-RFP等质粒。
实施例6.T细胞的培养与感染方法的建立
1)Jurkat T细胞系的培养方法
Jurkat T细胞系用含10％FBS的RPMI 1640培养基培养。培养密度为3*10 5/ml，最高不超过3*10 6/ml，隔1-2天进行传代，细胞计数后取所需量细胞，并补充培养基调整至上述密度，置于CO 2培养箱培养。
2)Jurkat T细胞系的感染方法
细胞计数，取1*10 6/ml细胞离心换液，用1ml含10％FBS的RPMI 1640培养基重悬，加入24孔板中，加入适量病毒液，1500rpm，离心90min，置于C02培养箱培养。感染12h后彻底换液为新鲜含10％FBS的RPMI 1640培养基，72h检测阳性率。
3)人原代T细胞的培养方法
用Ficoil分离法获得原代T细胞后，用含10％FBS及100IU/ml IL-2的X-VIVO培养基培养，初始培养密度为1*10 6/ml，加入CD3及RetroNectin r-Fibronectin(终浓度均为5ug/ml)预包被的孔板中。后期培养密度为5*10 5/ml，最高不超过3*10 6/ml，隔1-2天进行传代。
4)人原代T细胞的感染方法
原代T细胞培养48h后用加入病毒液，MOI＝20，1500rpm，离心90min，置于C0 2培养箱培养。感染24h后补充含10％FBS及100IU/ml IL-2的X-VIVO培养基并转孔，72h通过标签蛋白或者抗体检测感染效率。
5)感染效率检测方法
感染72h后，细胞吹匀计数，取5*10 5/ml离心，弃上清，染色液为PBS+2％FBS+2mM EDTA，加入对应抗体进行孵育，孵育30min，然后加入PBS清洗两次，上机检测。
实施例7.STAR受体及其突变体在T细胞中的表达检测方法
通过基因改造的手段改造T细胞过程中，目的基因的表达、上膜、及其在细胞表面 的分布影响了T细胞功能，因此通过荧光蛋白检测病毒感染效率，通过在STAR N端连接myc抗体，使用流式抗体检测STAR上膜效率。因为STAR的结构起源于TCR，因此STAR是否和内源TCR产生错配会影响完整STAR的表达丰度，通过RFP阳性比率与STAR myc阳性比率比较，评价错配效率。
1)STAR受体上膜效率检测
将构建好的pHAGE-EF1A-myc-STAR-IRES-RFP质粒，通过Lentix-293T进行病毒包装，并进行病毒浓缩和滴度检测，然后感染Jurkat C5和原代T细胞，感染72h后，收集感染的细胞，使用FITC-anti-myc抗体、APC-anti-mTCR-β抗体进行染色，并使用流式细胞仪(BD Fortessa)检测流式RFP/FITC/APC荧光通道。若FITC或APC有明显的阳性细胞群，则说明STAR可以上膜表达，若二者都不表达则说明STAR没有上膜。
2)STAR受体与内源性TCR错配几率评估
将构建好的pHAGE-EF1A-myc-STAR-IRES-RFP质粒，通过Lentix-293T进行病毒包装，并进行病毒浓缩和滴度检测，然后感染Jurkat和原代T细胞，感染72h后，收集感染的细胞，使用FITC-anti-myc抗体、APC-anti-mTCR-β抗体进行染色，并使用流式细胞仪(BD Fortessa f4)检测流式RFP/FITC/APC荧光通道。检测RFP阳性率与FITC或者APC阳性率的区别。
实施例8.STAR受体及其突变体在T细胞中体外水平的功能检测方法
1)T细胞与靶细胞体外共培养方法
T细胞包括T细胞系Jurkat C5，Jurkat和原代细胞系均为悬浮细胞，而靶细胞根据不同的抗原和细胞系则可分为悬浮细胞和贴壁细胞，如RAJI等悬浮靶细胞和T细胞共孵育，则同时取相应细胞数量使用靶细胞培养基混匀离心培养即可，若为贴壁靶细胞，则需提前一天接种靶细胞，然后再加入一定量的T细胞，具体步骤为：利用包装并纯化好的STAR病毒感染T细胞系或原代T细胞，共培养前一天使用流式检测感染效率，确定功能细胞与靶细胞比值，一般使用10:1，5:1，1:1比例，根据感染效率计算总T细胞数量，靶细胞一般使用量为4E5/孔(24孔板)。
2)T细胞被靶标抗原刺激方法
本申明的靶点一般为细胞表面蛋白，抗原可直接用于T细胞的激活，以检测T细胞的功能，通常使用300微升5μg/ml抗原(1xcoating buffer稀释，以24孔板为例)包被孔板过夜，然后弃去抗原，加入1E6/孔的阳性T细胞，离心激活6、12、24h后收集T细胞检测T细胞功能。
3)T细胞分泌细胞因子分析：ELISA
T细胞激活过程中会释放大量细胞因子，以帮助T细胞杀伤靶细胞或者促进T细胞自身的扩增，常见的有TNF-α、IFN-γ、IL-2。T细胞经过与靶细胞或者抗原刺激后，收集T细胞，离心，取上清。TNF-α、IFN-γ、IL-2ELISA试剂盒使用的为Human IL-2 Uncoated ELISA、Human TNF-α Uncoated ELISA、Human IFN-γ Uncoated ELISA(货号分别为 88-7025、88-7346、88-7316)。具体步骤为：用ddH2O将10X Coating Buffer稀释为1X，加入包被抗体(250X)，混匀后加入96孔板(ELISA专用)，100μl/孔。保鲜膜密封后于4℃过夜，1X PBST(又名Wash Buffer，1X PBS中加0.05％Tween 20)洗3次，每次260μl/孔，用ddH2O将5X ELISA/ELISPOT Diluent稀释为1X，加入96孔板，200μl/孔，室温静置1h。PBST清洗1次，标曲稀释(范围分别为：2～250、4～500、4～500)，样品使用1xDiluent稀释20-50倍。加入样品和标曲，每孔100微升，两个复孔，常温孵育2h后，PBST清洗三次，加入1xDiluent稀释好的Detection antibody，孵育1h后，PBST清洗3次，然后加入1xDiluent稀释的HRP，孵育30分钟后，清洗6次，加入TMB显色，显色时间不超过15min，加入2N H2SO4终止，450nm光吸收检测光吸收。
4)T细胞杀伤功能验证：Luciferase检测
共培养24h后，轻轻吹匀细胞悬液，每孔取150μL细胞悬液加入到白色96孔板中，每孔取2个复孔，加入luciferase底物(promega)。振荡(低速)，共孵育10min后，使用多功能酶标仪检测化学发光值，增益值固定100。细胞杀伤计算：杀伤效率＝100％-(效应细胞-靶细胞孔值/对照细胞-靶细胞孔值)。
实施例9.STAR受体及其突变体在T细胞中于动物肿瘤模型水平的功能检测方法
1)实验动物模型
本实验采用NSG免疫缺陷型小鼠为模型。该小鼠基因型为NOD-Prkdc em26Il2rg em26/Nju，缺乏T细胞、B细胞、NK细胞，并且其巨噬细胞和树突状细胞也存在缺陷。NSG小鼠是目前免疫缺陷最完全的小鼠品系，因其不会对移植瘤、T细胞产生排斥反应，从而广泛应用于T细胞治疗的临床前研究。本实验将采用6～8周龄的雌性NSG小鼠，每批实验中小鼠体重差异控制在2g以内。小鼠饲养于无特定病原体(SPF)的清洁级屏障内的独立通风笼中，提供正常饮食和pH偏酸的饮用水，以防止病原体污染。
2)肿瘤模型建立
本发明采用多种肿瘤模型来验证STAR-T细胞的功能，以验证其作用的普适性。在构建血液肿瘤模型中，本实验采用人类Burkitt’s淋巴瘤细胞系Raji细胞进行异种移植。Raji细胞为通过慢病毒载体表达了荧光素酶(luciferase)基因的细胞株，在小鼠体内通过荧光素化学发光、活体成像的方式实时监测Raji肿瘤的发展变化。该模型中，不同剂量(一般在1～3x10 6细胞左右)的Raji-luciferase细胞通过尾静脉回输方式接种于6～8周的雌性NSG小鼠中。3天后给小鼠腹腔注射荧光素钾盐溶液，通过活体成像的方式检测到体内肿瘤细胞的荧光信号。Raji细胞在小鼠体内生长速度快，通过血液分布遍布全身，有一定几率侵染重要器官并形成实体肿瘤，引起小鼠体重降低等症状；在无治疗处理情况下，Raji肿瘤负担在30～40天左右导致小鼠死亡。
在构建异种移植实体肿瘤模型中，本实验选取了多种肿瘤类型，包括人类表皮细胞癌A431细胞系、人类非小细胞肺癌A549细胞系、人类肝癌Huh-7和HepG2、人类脑 胶质肿瘤U87和U251细胞系、以及人类乳腺癌MDA-MB-231细胞系。这些细胞通过慢病毒载体表达了荧光素酶(luciferase)基因，在小鼠体内可通过荧光素化学发光、活体成像的方式实时监测肿瘤的发展变化。该模型中，不同剂量(根据肿瘤恶性程度和特性不同，从2x10 5至3x10 6范围内)的细胞与基质胶混合后，通过皮下注射的模式接种于小鼠皮下。3天后给小鼠腹腔注射荧光素钾盐溶液，可通过活体成像的方式检测到体内肿瘤细胞的荧光信号。皮下接种的实体肿瘤一般不具有侵袭性、不易转移，常在原位皮下形成致密肿瘤包块。通过用游标卡尺测量肿瘤包块的长、宽、高，亦可通过肿瘤体积大小来监测肿瘤进程。在无治疗处理情况下，这些实体肿瘤细胞形成的肿瘤包块在20～40天左右达到动物福利伦理标准规定，即当肿瘤包块最大体积超过15mm、或肿瘤包块体积超过1500mm 3、或皮下肿瘤区域形成溃烂、或肿瘤显著影响到小鼠的活动和进食的时候，应对动物进行安乐死处理。
3)动物实验操作方法
实验动物小鼠的操作方法包括：捉拿和固定、编号、麻醉、去毛、给药、取血、处死和解剖。其中，实验中使用了的编号方法包括：剪指号、打耳号以及毛色标记法。实验中使用了的麻醉方法包括：异氟烷吸入式麻醉和Avertin或戊巴比妥注射式麻醉。去毛方法包括：使用剪刀或剃刀对小鼠局部部位脱毛。给药方式包括：腹腔注射、尾静脉注射、皮下注射、眼底静脉丛注射、颅内注射等。取血方法包括：眼眶采血、摘眼球取血、尾静脉采血等。处死方式包括脱颈处死、二氧化碳处死等。所有操作均在实验动物研究和使用计划(Animal protocol)的审批通过后进行。
4)肿瘤生长监测手段
本实验主要通过两种手段来检测肿瘤生长。第一种是活体荧光成像方法：带有荧光素酶基因的肿瘤细胞被打入动物体内定植。给小鼠腹腔注射荧光素钾盐溶液，该底物在酶的作用下发出特定波长的光，通过活体成像仪器敏感的CCD设备检测到体内肿瘤细胞的荧光信号。进一步，可以通过专业软件，对荧光信号进行定量分析和绘制热图，可以既直观又定量的反映肿瘤生长情况。第二种方法是测量皮下肿瘤的体积：用游标卡尺测量肿瘤的长、宽、高，并通过特定的计算公式得到体积值。常用的计算公司包括：体积＝长x宽x高；体积＝长x宽x宽；体积＝3x长x宽x高/π等。
5)T细胞在动物体内活性和扩增检测方法
T细胞在体内的存活和扩增情况与其最终抗肿瘤效果有直接联系。为检测T细胞在动物体内的活性和增殖情况，本实验定期对小鼠采血，并分析外周血中STAR-T细胞的比例、细胞状态和细胞分群。具体操作为：每隔约一周时间，用异氟烷麻醉小鼠，对小鼠进行眼眶采血约100ul。该血样经历抗凝、收集血浆、裂除红细胞等操作后，对剩下的细胞进行流式染色，以检测CD4和CD8的比例、及CCR7、CD45RA、PD-1、LAG-3、TIM-3等分子来进行T细胞亚群分析和细胞状态分析。同时，通过流式细胞仪计数或数字PCR的方法，得到STAR-T细胞在小鼠外周血中的绝对数量值。另外，在到达实验终点是，可解剖小鼠、检测T细胞在小鼠其它免疫器官里的比例。
6)T细胞在动物体内的安全性评估方法
为评估STAR-T细胞的毒性和安全性，可检测细胞对实验动物是否造成副作用。通过观察小鼠行为状态、进行小鼠病理分析、对小鼠重要器官切片分析等方式，可评估回输的T细胞是否有显著毒性。同时，通过对小鼠非肿瘤的组织进行T细胞浸润分析，也可判断T细胞是否对其有脱靶杀伤效应。此外，通过检测小鼠血液中的细胞因子水平，如IL-2、IFN-γ、TNFα或IL-6等，可以判断T细胞是否会造成系统性的细胞因子风暴。
7)T细胞肿瘤浸润能力评估方法
T细胞浸润肿瘤的能力是其挑战实体肿瘤的核心能力。为检测T细胞浸润能力，首先可以分离肿瘤组织、消化研磨得到单细胞，通过流式染色可以检测T细胞在肿瘤组织中的占比。同时，可以进一步通过密度梯度离心(如Percoll梯度、Ficoll梯度等)的方法将肿瘤悬液中的肿瘤细胞、肿瘤基质细胞与免疫细胞分离，以便获得纯化的肿瘤浸润T细胞，利用测序等方法细致分析其特性，包括趋化因子受体表达、T细胞耗竭程度等等。
实施例10.anti-EGFR STAR受体的构建以及恒定区修饰的鉴定
10.1.anti-EGFR STAR受体的构建
(1)靶向EGFR的抗体序列确定
抗体重链可变区(anti-EGFR Cetux-VH，SEQ ID NO:22)和抗体轻链可变区(anti-EGFR Cetux-VL，SEQ ID NO:23)选择西妥昔单抗(Centuximab，简称Cetux)。序列经过密码子优化后基因合成而得。
(2)靶向EGFR的STAR设计
STAR含有两条多肽链，将anti-EGFR Cetux-VL与mouse TCRbC-WT链融合为第一多肽段，将anti-EGFR Cetux-VH与mouse TCRaC-WT链融合为第二多肽段。两条链均使用GM-CSF signal peptide信号肽。STAR的基因序列通过Furin-SGSG-p2A蛋白酶切位点多肽段相连，两条多肽链将一同被转录并翻译表达成蛋白质，之后再被furin和p2A对应的蛋白酶切割，最终成为两个独立的蛋白链。两条蛋白链与T细胞内源的CD3亚基(ε,δ,γ,ζ)形成复合体，并且在GM-CSF信号肽的引导下，被转运并呈递在细胞膜上(如图1所示)。
(3)、基因片段的克隆与组装
通过基因合成和克隆的方式获得的四个片段“anti-EGFR Cetux-VL”，“mouse TCRbC-WT”，“anti-EGFR Cetux-VH”，“mouse TCRaC-WT”。每对引物上都带有与前后同源的25bp碱基，四个片段通过Gibson Assembly的方法一步重组连接到慢病毒载体pHAGE中。从而获得STAR。
(4).载体转化和测序
Gibson Assembly的产物被转化入了DH5α菌株，使其在含氨苄的LB平板上过夜生长。挑单克隆隆菌进行测序，测序引物选用pHAGE载体上的引物seq-pHAGE-F和seq-pHAGE-R。
(5).质粒提取
将测序结果正确的菌接种在LB液体培养基中，过夜培养。使用具有去内毒素功能的试剂盒提取质粒。质粒浓度用Nanodrop测量，质粒终浓度在1000ng/ul左右，A260/A280值大于1.8。由此获得恒定区为mouse TCRa/bC-WT的靶向EGFR的STAR质粒pHAGE-EF1A-Cetux-VL-TCRaC-P2A-VH-TCRbC-IRES-RFP。
类似地，构建了恒定区分别为TCRa/bC-N.DLT(包含TCRaC-N.DLT和TCRbC-N.DLT)、TCRa/bC-C.DLT(包含TCRaC-C.DLT和TCRbC-CDLT)、TCRa/bC-N.Rec-1(包含TCRaC-N.Rec-1和TCRbC-N.Rec-1)、TCRa/bC-N.Rec-2(包含TCRaC-N.Rec-2和TCRbC-N.Rec-2)、TCRa/bC-N.Rec-3(包含TCRaC-N.Rec-3和TCRbC-N.Rec-3)、TCRa/bC-N.Rec-4(包含TCRaC-N.Rec-4和TCRbC-N.Rec-4)、TCRa/bC-N.rRec(包含TCRaC-N.rRec和TCRbC-N.rRec)、TCRa/bC-Cys(包含TCRaC-Cys和TCRbC-Cys)、TCRa/bC-TM9(包含TCRaC-TM9和TCRbC-WT)、TCRa/bC-Cys-N.Rec-1(包含TCRaC-Cys-N.Rec-1和TCRbC-Cys-N.Rec-1)、TCRa/bC-TM9-N.Rec-1(包含TCRaC-TM9-N.Rec-1和TCRbC-N.Rec-1)、TCRa/bC-Cys-TM9(包含TCRaC-Cys-TM9和TCRbC-Cys)、TCRa/bC-Cys-TM9-N.Rec-1(包含TCRaC-Cys-TM9-N.Rec-1和TCRbC-Cys-N.Rec-1)的多种pHAGE-EF1A-Cetux-VL-TCRaC-P2A-VH-TCRbC-IRES-RFP质粒。
上述质粒包装成病毒，感染细胞并检测了STAR的上膜效率。结果见图6。
10.2.anti-EGFR STAR受体的恒定区N端修饰和重排突变体的功能
1)STAR恒定区N端特定序列删除使STAR失去功能
使用phge-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建Mouse TCRa/bC-N.DLT STAR和Mouse TCRa/bC-WT STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3：2：1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1：3，即使用54微升PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度，结果显示Mouse TCRa/bC-N.DLT STAR病毒包装效率要优于Mouse TCRa/bC-WT STAR。相同MOI感染Jurkat C5和原代T细胞发现其感染效率并无显著差异，通过原代T细胞中RFP与EGFR-his-APC染色发现Mouse TCRa/bC-N.DLT无法上膜，通过与靶细胞A431、B16-tEGFR、A549共培养后发现Mouse TCRa/bC-N.DLT没有杀伤能力，与EGFR抗原刺激后，不分泌细胞因子IL-2、TNFα和IFNγ。
2)STAR恒定区C端重排使STAR失去功能
使用phge-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建Mouse TCRa/bC-C.DLT STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3：2：1，10cm培养皿每皿 质粒共18μg，与PEI-MAX比例为1：3，即使用54微升PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度。感染Jurkat C5和原代T细胞，通过原代T细胞中蛋白质印记、RFP与EGFR-his-APC染色发现Mouse TCRa/bC-C.DLT有蛋白表达却无法上膜，说明Mouse TCRa/bC-C.DLT所删除的序列对STAR的功能很关键，说明STAR的重排并非是随意的。
3)STAR恒定区N端的重排起关键作用
使用phge-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建Mouse TCRa/bC-N.Rec-1 STAR、Mouse TCRa/bC-N.Rec-2 STAR、Mouse TCRa/bC-N.Rec-3 STAR、Mouse TCRa/bC-N.Rec-4 STAR和Mouse TCRa/bC-WT STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3：2：1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1：3，即使用54微升PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度，结果显示二者之间的病毒滴度无明显差异。感染Jurkat C5和原代T细胞发现其感染效率并无显著差异。通过原代T细胞中RFP与EGFR-his-APC染色发现Mouse TCRa/bC-N.Rec-1比Mouse TCRa/bC-WT STAR在与内源性TCR错配方面错配率更低，通过与靶细胞A431、B16-tEGFR、A549共培养后发现Mouse TCRa/bC-N.Rec-1比Mouse TCRa/bC-WT STAR具有更强的杀伤靶细胞能力。序列分析表明Mouse TCRa/bC-N.Rec-1比Mouse TCRa/bC-WT STAR的序列人源性更高，免疫原性预测分析表明Mouse TCRa/bC-N.Rec比Mouse TCRa/bC-WT STAR的免疫原性更低。同时Mouse TCRa/bC-N.Rec-2 STAR、Mouse TCRa/bC-N.Rec-3 STAR、Mouse TCRa/bC-N.Rec-4 STAR却使STAR丧失功能。
4)STAR恒定区N端的重排具有序列特异性，它种重排方式无效
使用phge-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建Mouse TCRa/bC-N.Rec-1 STAR和Mouse TCRa/bC-N.rRec STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3：2：1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1：3，即使用54μl PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度，结果显示二者之间的病毒滴度无明显差异。感染Jurkat C5和原代T细胞发现其感染效率并无显著差异。通过原代T细胞中RFP与EGFR-his-APC染色发现Mouse TCRa/bC-N.rRec与Mouse TCRa/bC-N.Rec-1相比，其错配率显著升高，通过与靶细胞A431、B16-tEGFR、A549共培养后发现Mouse TCRa/bC-N.rRec的杀伤功能几乎丧失，比Mouse TCRa/bC-N.Rec START功能差距较大。
10.3.anti-EGFR STAR受体的半胱氨酸点突变突变体的功能
STAR受体的半胱氨酸点突变能增加额外的二硫键，起到增强配对、稳定复合体的 作用，使用phge-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建Mouse TCRa/bC-Cys STAR和Mouse TCRa/bC-WT STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3：2：1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1：3，即使用54μl PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度，结果显示二者之间的病毒滴度无明显差异。感染Jurkat C5和原代T细胞发现其感染效率并无显著差异，通过原代T细胞中RFP与EGFR-his-APC染色发现Mouse TCRa/bC-Cys比Mouse TCRa/bC-WT在与内源性TCR错配方面错配率显著降低，通过与靶细胞A431、B16-tEGFR、A549共培养后发现Mouse TCRa/bC-Cys比Mouse TCRa/bC-WT具有更强的杀伤靶细胞能力。与EGFR抗原刺激后，其细胞因子IL-2、TNFα和IFNγ分泌水平更高。
10.4.anti-EGFR STAR受体跨膜区特定位点进行疏水氨基酸替换的功能
STAR受体跨膜区特定位点进行疏水氨基酸替换起到关键作用，STAR受体的半胱氨酸点突变能增加额外的二硫键，起到增强配对、稳定复合体的作用，使用phge-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建Mouse TCRa/bC-TM9 STAR和Mouse TCRa/bC-WT STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3：2：1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1：3，即使用54微升PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度，结果显示二者之间的病毒滴度无明显差异。感染Jurkat C5和原代T细胞发现其感染效率并无显著差异，通过原代T细胞中RFP与EGFR-his-APC染色发现Mouse TCRa/bC-TM9比Mouse TCRa/bC-WT在与内源性TCR错配方面错配率显著降低，通过与靶细胞A431、B16-tEGFR、A549共培养后发现Mouse TCRa/bC-TM9比Mouse TCRa/bC-WT具有更强的杀伤靶细胞能力。与EGFR抗原刺激后，其细胞因子IL-2、TNFα和IFNγ分泌水平更高。
10.5.anti-EGFR STAR综合突变体的功能
为比较恒定区修饰策略(N末端修饰、Cys取代和跨膜区疏水性修饰)三个组合、两两组合与单一修饰的效果，使用phge-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建下表中所有STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3：2：1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1：3，即使用54微升PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度，感染Jurkat C5和原代T细胞发现，相同MOI感染T细胞后感染效率即RFP阳性率无显著差异，通过RFP与EGFR-his-APC抗体染色发现，两两突 变组合的错配率要显著低于单一突变策略。相同RFP阳性细胞情况下，两两突变的STAR-T细胞对靶细胞A431、B16-htEGFR、A549的杀伤显著高于单个突变策略。细胞因子IL-2、TNFα和IFNγ分泌水平也更高。而将三种突变策略同时组合在一起时，则显示出最低的错配率、同时对靶细胞的杀伤也更强、细胞因子的分泌水平也更高。整体结果见图6。
Figure PCTCN2020103687-appb-000003
实施例11.anti-GPC3 STAR受体的恒定区N端修饰和重排突变体的功能
基于实施例1中所述突变策略，本发明针对恒定区进行N端序列截短、重排、C端重排等优化方案，针对GPC3，抗体序列选择针对GPC3的单克隆抗体GC33，抗体重链可变区(anti-GPC3 GC33-VH，SEQ ID NO:25)和抗体轻链可变区(anti-GPC3 GC33-VL，SEQ ID NO:24)使用phage-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建Mouse GC33-VH TCRa-GC33-VL TCRbC-N.DLT STAR、Mouse GC33-VH TCRa-GC33-VL TCRbC-C.DLT STAR、Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-1 STAR、Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-2 STAR、Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-3 STAR、Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-4 STAR和Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR等载体。利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3：2：1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1:3，即使用54微升PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度。结果见图7。
1)STAR恒定区N端特定序列截短使STAR失去功能
结果显示Mouse GC33-VH TCRa-GC33-VL TCRbC-N.DLT STAR和Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR。相同MOI感染Jurkat C5和原代T细胞发现其感染效率并无显著差异，通过原代T细胞中RFP与GPC3-FITC染色发现Mouse GC33-VH TCRa-GC33-VL TCRbC-N.DLT无法上膜，通过与靶细胞Huh-7和HepG2细胞共培养后发现Mouse GC33-VH TCRa-GC33-VL TCRbC-N.DLT没有杀伤能力。
2)STAR恒定区C端重排使STAR失去功能
感染Jurkat C5和原代T细胞，通过原代T细胞中蛋白质印记、RFP与GPC3-FITC染色发现Mouse GC33-VH TCRa-GC33-VL TCRbC-C.DLT无法上膜，说明Mouse GC33-VH TCRa-GC33-VL TCRbC-C.DLT所删除的序列对STAR的功能很关键，说明 STAR的重排并非是随意的，通过与靶细胞Huh-7和HepG2细胞共培养后发现其没有杀伤功能。
3)STAR恒定区N端的重排起关键作用
感染Jurkat C5和原代T细胞发现其感染效率并无显著差异。通过原代T细胞中RFP与EGFR-his-APC染色发现Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-1比Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR在与内源性TCR错配方面错配率更低，通过与靶细胞Huh-7和HepG2细胞共培养后发现MouseGC33-VH TCRa-GC33-VL TCRbC-N.Rec-1比Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR具有更强的杀伤靶细胞能力。序列分析表明Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-1比Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR的序列人源性更高，免疫原性预测分析表明Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec比Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR的免疫原性更低。同时Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-2 STAR、Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-3 STAR、Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-4 STAR却使STAR丧失功能。
4)STAR恒定区N端的重排具有序列特异性，它种重排方式无效
通过原代T细胞中RFP与GPC3-FITC染色发现Mouse GC33-VH TCRa-GC33-VL TCRbC-N.rRec与Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec-1相比，GC33 scFv无法上膜，通过与靶细胞Huh-7和HepG2细胞共培养后发现Mouse GC33-VH TCRa-GC33-VL TCRbC-N.rRec的杀伤功能丧失，与Mouse GC33-VH TCRa-GC33-VL TCRbC-N.Rec START功能差距较大。
实施例12.anti-GPC3 STAR受体的半胱氨酸点突变突变体的功能
STAR受体的半胱氨酸点突变能增加额外的二硫键，起到增强配对、稳定复合体的作用，使用phage-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建Mouse GC33-VH TCRa-GC33-VL TCRbC-Cys STAR和Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3:2:1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1:3，即使用54μl PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度，结果显示二者之间的病毒滴度无明显差异。感染Jurkat C5和原代T细胞发现其感染效率并无显著差异，通过原代T细胞中RFP与GPC3-FITC染色发现Mouse GC33-VH TCRa-GC33-VL TCRbC-Cys比Mouse GC33-VH TCRa-GC33-VL TCRbC-WT在与内源性TCR错配方面错配率显著降低，通过与靶细胞Huh-7和HepG2细胞共培养后发现Mouse GC33-VH TCRa-GC33-VL TCRbC-Cys比Mouse GC33-VH TCRa-GC33-VL TCRbC-WT具有更强的杀伤靶细胞能力。
实施例13.anti-GPC3 STAR受体跨膜区特定位点进行疏水氨基酸替换的功能
STAR受体跨膜区特定位点进行疏水氨基酸替换起到关键作用，STAR受体的半胱氨酸点突变能增加额外的二硫键，起到增强配对、稳定复合体的作用，使用phage-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建Mouse GC33-VH TCRa-GC33-VL TCRbC-TM9 STAR和Mouse GC33-VH TCRa-GC33-VL TCRbC-WT STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3：2：1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1：3，即使用54微升PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度，结果显示二者之间的病毒滴度无明显差异。感染Jurkat C5和原代T细胞发现其感染效率并无显著差异，通过原代T细胞中RFP与GPC3-FITC蛋白染色发现Mouse GC33-VH TCRa-GC33-VL TCRbC-TM9比Mouse GC33-VH TCRa-GC33-VL TCRbC-WT在与内源性TCR错配方面错配率显著降低，通过与靶细胞Huh-7和HepG2细胞共培养后发现Mouse GC33-VH TCRa-GC33-VL TCRbC-TM9比Mouse GC33-VH TCRa-GC33-VL TCRbC-WT具有更强的杀伤靶细胞能力。
实施例14.anti-GPC3 STAR综合突变体的功能
为比较突变策略三个组合、两两组合与单一突变的效果，使用使用phage-EF1A-IRES-RFP载体，通过Gibsson Assembly重组构建下表中所有STAR载体，利用Lentix-293T细胞包装病毒，包装质粒为pSPAX、PMD2.G，包装比例为Transfer plasmid：pSPAX：PMD2.G＝3:2:1，10cm培养皿每皿质粒共18μg，与PEI-MAX比例为1：3，即使用54微升PEI，转染后12h换液，收获48h及72h病毒液，通过20000rpm，4°离心2h后，使用无血清培养基重悬，100倍浓缩病毒，并检测病毒滴度，感染Jurkat C5和原代T细胞发现，相同MOI感染T细胞后感染效率即RFP阳性率无显著差异，通过RFP与GPC3-FITC蛋白染色发现，两两突变组合的上膜效率显著高于单一突变策略。相同RFP阳性细胞情况下，两两突变的STAR-T细胞对靶细胞Huh-7和HepG2细胞的杀伤显著高于单个突变策略。而将三种突变策略同时组合在一起时，则显示出最低的错配率、同时对靶细胞的杀伤也更强、细胞因子的分泌水平也更高。具体结果见图7。
针对GPC3 GC33 STAR突变体比较策略：
Figure PCTCN2020103687-appb-000004
实施例15.基于334-scFv的anti-CD19 STAR综合突变体的功能
本发明人获得了新的靶向CD19的单克隆抗体334，由保藏号编号为CGMCC No.17095(于2019年1月21日保藏于地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所的中国微生物菌种保藏管理委员会普通微生物中心)的杂交瘤细胞334产生。该抗体包含anti-CD19 334 VL(SEQ ID NO:27)和anti-CD19 334 VH(SEQ ID NO:26)，其中所述anti-CD19 334 VH包含SEQ ID NO:28所示重链CDR1、SEQ ID NO:29所示重链CDR2和SEQ ID NO:30所示重链CDR3，所述anti-CD19 334 VL包含SEQ ID NO:31所示轻链CDR1、SEQ ID NO:32所示轻链CDR2和SEQ ID NO:33所示轻链CDR3。
1)抗体334特异性识别CD19
采用ELISA方法鉴定了334抗体与靶抗原CD19和非靶抗原CD20的识别能力，将表达334抗体质粒利用转染试剂PEI转染293T细胞，72小时后，收取细胞上清，采用ELISA方法检测抗原抗体的特异性结合能力。实验结果显示，334抗体呈现显著的CD19靶抗原特异性结合，对非靶抗原CD20无结合能力。
为了验证CD19-334抗体可以特异性识别人源细胞表达的CD19抗原，采用WB试验，检测CD19-334抗体与人源CD19抗原的识别能力。
2)CD19阳性蛋白样本的制备：
常规培养人淋巴瘤细胞系Raji细胞(CD19抗原阳性高表达)，待细胞融合至90％，取6cm dish的悬浮细胞，离心后，收集细胞沉淀于1.5mL离心管内。加入200uL总蛋白提取裂解试剂(RIPA)，充分吹打混匀后，冰上裂解15分钟。将离心管置于4℃预冷的低温冷冻离心机内，4℃，12000rpm，15min，收集上清。加入5×loading buffer，100℃，5min，蛋白变性。将样本离心后，20冰上预冷待进行WB电泳(采用人肾癌细胞系M62作为CD19阴性对照组细胞)。
(2)WB电泳：制备浓缩胶为4％，分离胶为10％的SDS-PAGE电泳胶。每孔加入提取的CD19阳性蛋白样本和CD19阴性的蛋白样本40uL。80V，20min电泳，后调整电压为120V，40-60min。
(3)转膜：采用半干转系统，将目的蛋白转印至0.45μm的PVDF膜上。转膜条件为15V，1h。
(4)封闭：将转印完成的PVDF膜，置于5％的脱脂奶粉中封闭，室温，摇床1h。
(5)一抗孵育：将PVDF采用PBST将残留的奶粉洗净，并采用滤纸将残留buffer吸干，置于含有CD19抗原杂交瘤上清(5-8mL)的一抗孵育盒中。4℃，孵育过夜。
(6)PBST冲洗：5min x 3。
(7)二抗孵育：将PVDF置于HRP标记的抗鼠(1：20000)单抗中。室温，孵育1h。
(8)PBST冲洗：5min x 3。
(9)凝胶成像仪显色，拍照。
试验结果显示，334抗体可识别人淋巴瘤细胞系Raji细胞中的CD19蛋白，目标条带大小为95kD。
3)抗体334的Kd值检测
常规培养人淋巴瘤细胞系Raji细胞(CD19抗原阳性高表达)，待细胞融合至90％，取6cm dish的悬浮细胞，离心后，收集细胞沉淀于1.5mL离心管内，并用PBS重悬细胞沉淀并调整细胞密度为5×10 6个细胞/mL。
取5×10 6个Raji细胞每孔加入至96孔板中，并稀释纯化的334抗体为375nM、187.5nM、30nM、3nM、0.3nM、0.03nM、0.003nM和0nM，并加入至96孔板中的Raji细胞中，4℃孵育30min后，2000rpm，离心5min，弃上清，加入PBS清洗细胞一次后，加入goat-anti mouse AlexaFluor 555荧光二抗孵育Raji细胞30min。4℃孵育30min，2000rpm，离心5min，弃上清，加入PBS清洗细胞一次后，加入200μL PBS重悬细胞，并采用流式细胞术检测不同CD19-334抗体浓度下Raji细胞的荧光强度，并通过GraphPad软件计算得到CD19-334抗体的Kd值。
检测结果显示，不同抗体浓度下Raji细胞的荧光强度所拟合出的结合曲线，并通过GraphPad软件计算得到CD19-334抗体的Kd值为1.528±0.25nM。
4)基于334-scFv的anti-CD19 STAR综合突变体
在pHAGE-IRES-RFP载体基础上构建myc-Cys-N.Rec-1-TM9-STAR-334-anti-CD19 STAR结构。使用的抗原结合序列为anti-CD19 334 VL(SEQ ID NO:27)和anti-CD19 334 VH(SEQ ID NO:26)，其中所述anti-CD19 334 VH包含SEQ ID NO:28所示重链CDR1、SEQ ID NO:29所示重链CDR2和SEQ ID NO:30所示重链CDR3，所述anti-CD19 334 VL包含SEQ ID NO:31所示轻链CDR1、SEQ ID NO:32所示轻链CDR2和SEQ ID NO:33所示轻链CDR3。使用二代包装质粒包装病毒后，感染Jurkat C5细胞，流式检测结果发现STAR可正常上膜，与靶细胞Raji和抗原CD19蛋白结合可促进CD69、CD25等激活分子表达；病毒感染原代T细胞后，T细胞与靶细胞Raji、Jeko-1等共培养，可明显刺激T细胞分泌IL-2、TNF-α和IFNγ，并诱导杀伤靶细胞，同时T细胞也表达PD1、TIM3、LAG3等免疫抑制分子，进一步说明T细胞被激活，并行使杀伤功能。以Raji细胞在NSG小鼠上建立淋巴瘤模型，使用anti-CD19 STAR T细胞作用于荷瘤小鼠后，与对照组相比，其可明显对肿瘤产生杀伤作用。结果见图8。
实施例16.针对CD19-CD20的FMC63-2C6 STAR综合突变体功能
STAR的结构中，Cα和Cβ可分别连接scFv的VH和VL，也可以连接不同的scFv，这样就使得在双靶点或者单靶点双识别位点上成为可能。在pHAGE-IRES-RFP载体基础上构建myc-Cys-N.Rec-1-TM9-STAR-anti-CD19/CD20 STAR结构，其中针对CD19的靶点选择的FMC63单抗(FMC63 VH seq No:44，FMC63 VL seq No:45)，针对CD20选择2C6单抗，(2C6VH seq No:46，2C6VL seq No:47)，构建myc-Cys-N.Rec-1-TM9-STAR-FMC63-2C6结构，使用二代包装质粒包装病毒后，感染Jurkat C5细胞，流式检测结果发现STAR可正常上膜；病毒感染原代T细胞后，T细胞 与淋巴瘤细胞系Raji、Raji-CD19KO、Raji-CD20KO共培养后，可明显刺激T细胞分泌IL-2、TNF-α和IFNγ，并诱导杀伤靶细胞，说明FMC63-2C6 STAR对两个靶点均具有良好的杀伤，在临床上可避免这两个靶点其中一个丢失造的肿瘤复发。以90％Raji+5％Raji-CD19KO+5％Raji-CD20KO细胞在NSG小鼠上建立荷瘤模型，使用FMC63-2C6STAR细胞作用于荷瘤小鼠后，与对照组相比，其可明显可完全清除肿瘤，进一步证明其对这两个靶点都有杀伤作用。结果见图9。
实施例17.针对CD19-CD22 334-M971 STAR综合突变体功能
STAR的结构中，Cα和Cβ可分别连接scFv的VH和VL，也可以连接不同的scFv，这样就使得在双靶点或者单靶点双识别位点上成为可能。在pHAGE-IRES-RFP载体基础上构建myc-Cys-N.Rec-1-TM9-STAR-anti-CD19/CD22 STAR结构，其中针对CD19的靶点选择的334单抗(334 VH seq No:26，334 VL seq No:27)，针对CD22选择M971单抗，(M971 VH seq No:48，M971 VL seq No:49)，构建myc-Cys-N.Rec-1-TM9-STAR-334-M971结构，使用二代包装质粒包装病毒后，感染Jurkat C5细胞，流式检测结果发现STAR可正常上膜；病毒感染原代T细胞后，T细胞与淋巴瘤细胞系Raji、Raji-CD19KO、Raji-CD22KO共培养后，可明显刺激T细胞分泌IL-2、TNF-α和IFNγ，并诱导杀伤靶细胞，说明334-M971 STAR对两个靶点均具有良好的杀伤，在临床上可避免这两个靶点其中一个丢失造的肿瘤复发。以90％Raji+5％Raji-CD19KO+5％Raji-CD22KO细胞在NSG小鼠上建立荷瘤模型，使用334-M971STAR细胞作用于荷瘤小鼠后，与对照组相比，其可明显可完全清除肿瘤，进一步证明其对这两个靶点都有杀伤作用。结果见图10。
Figure PCTCN2020103687-appb-000005
Figure PCTCN2020103687-appb-000006
Figure PCTCN2020103687-appb-000007
Figure PCTCN2020103687-appb-000008
Figure PCTCN2020103687-appb-000009

Claims (42)

  1. 一种合成T细胞受体抗原受体(STAR)，其包含α链和β链，所述α链包含第一抗原结合区和第一恒定区，所述β链包含第二抗原结合区和第二恒定区，
    其中第一恒定区是天然T细胞受体α链恒定区的变体，其相对于天然T细胞受体α链恒定区包含i)N末端修饰；ii)半胱氨酸取代；和/或iii)疏水氨基酸取代，和/或
    其中第二恒定区是天然T细胞受体β链恒定区的变体，其相对于天然T细胞受体β链恒定区包含i)N末端修饰；和/或ii)半胱氨酸取代。
  2. 权利要求1的合成T细胞受体抗原受体，其中所述第一恒定区衍生自人T细胞受体α链恒定区、非人灵长类动物T细胞受体α链恒定区、啮齿动物例如小鼠T细胞受体α链恒定区，优选地，所述第一恒定区衍生自小鼠T细胞受体α链恒定区。
  3. 权利要求1或2的合成T细胞受体抗原受体，所述第一恒定区相对于天然T细胞受体α链恒定区包含N末端18个氨基酸的修饰。
  4. 权利要求1-3中任一项的合成T细胞受体抗原受体，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区，N末端起X个连续氨基酸被缺失，和/或N末端的第X+1位至第18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，其中X为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17或18。
  5. 权利要求1-4中任一项的合成T细胞受体抗原受体，所述第一恒定区衍生自小鼠T细胞受体α链恒定区，且相对于天然小鼠T细胞受体α链恒定区缺失N末端的第1-4位氨基酸，且其中N末端的第5-18位氨基酸被天然人T细胞受体α链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:2。
  6. 权利要求1-5中任一项的合成T细胞受体抗原受体，相对于天然T细胞受体α链恒定区，所述第一恒定区在第48位包含半胱氨酸取代，所述氨基酸编号参考SEQ ID NO:2。
  7. 权利要求1-6中任一项的合成T细胞受体抗原受体，相对于天然T细胞受体α链恒定区，所述第一恒定区在第48位的苏氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:2。
  8. 权利要求1-7中任一项的合成T细胞受体抗原受体，相对于天然T细胞受体α链恒定区，所述第一恒定区包含在跨膜区内的疏水氨基酸取代，所述跨膜区例如包含第111-119位的氨基酸序列，所述氨基酸编号参考SEQ ID NO:2。
  9. 权利要求1-8中任一项的合成T细胞受体抗原受体，相对于天然T细胞受体α链恒定区，所述第一恒定区包含在第112位、114位和/或115位的疏水氨基酸取代，所述氨基酸编号参考SEQ ID NO:2。
  10. 权利要求1-9中任一项的合成T细胞受体抗原受体，相对于天然T细胞受体α链恒定区，所述第一恒定区在第112位的丝氨酸被亮氨酸取代，在114位的甲硫氨酸被 异亮氨酸取代，和/或在115位的甘氨酸被缬氨酸取代，所述氨基酸编号参考SEQ ID NO:2。
  11. 权利要求1-10中任一项的合成T细胞受体抗原受体，所述第一恒定区包含SEQ ID NO:8所示跨膜区。
  12. 权利要求1-11中任一项的合成T细胞受体抗原受体，所述第一恒定区包含选自SEQ ID NO:6-7、9-13和34-36的氨基酸序列。
  13. 权利要求1-12中任一项的合成T细胞受体抗原受体，所述第二恒定区衍生自人T细胞受体β链恒定区、非人灵长类动物T细胞受体β链恒定区、啮齿动物例如小鼠T细胞受体β链恒定区，优选地，所述第一恒定区衍生自小鼠T细胞受体β链恒定区。
  14. 权利要求1-13中任一项的合成T细胞受体抗原受体，所述第二恒定区相对于天然T细胞受体β链恒定区包含N末端25个氨基酸的修饰。
  15. 权利要求1-14中任一项的合成T细胞受体抗原受体，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区，N末端起X个连续氨基酸被缺失，和/或N末端的第X+1位至第25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，其中X为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或25。
  16. 权利要求1-15中任一项的合成T细胞受体抗原受体，所述第二恒定区衍生自小鼠T细胞受体β链恒定区，且相对于天然小鼠T细胞受体β链恒定区缺失N末端的第1-6位氨基酸，且其中N末端的第7-25位氨基酸被天然人T细胞受体β链恒定区的相应氨基酸取代，所述氨基酸编号参考SEQ ID NO:15。
  17. 权利要求1-16中任一项的合成T细胞受体抗原受体，相对于天然T细胞受体β链恒定区，所述第二恒定区在第56位的丝氨酸被突变为半胱氨酸，所述氨基酸编号参考SEQ ID NO:15。
  18. 权利要求1-17中任一项的合成T细胞受体抗原受体，所述第二恒定区包含选自SEQ ID NO:19-21的氨基酸序列。
  19. 权利要求1-18中任一项的合成T细胞受体抗原受体，其中
    i)所述第一恒定区包含SEQ ID NO:34所示氨基酸序列，第二恒定区包含SEQ ID NO:37所示氨基酸序列；
    ii)所述第一恒定区包含SEQ ID NO:7所示氨基酸序列，所述第二恒定区包含SEQ ID NO:20所示氨基酸序列；
    iii)所述第一恒定区包含SEQ ID NO:9所示氨基酸序列，所述所述第二恒定区包含SEQ ID NO:15所示氨基酸序列；
    iv)所述第一恒定区包含SEQ ID NO:11的氨基酸序列，所述第二恒定区包含SEQ ID NO:21所示的氨基酸序列；
    v)所述第一恒定区包含SEQ ID NO:12的氨基酸序列，所述第二恒定区包含SEQ ID NO:37所示氨基酸序列；
    vi)所述第一恒定区包含SEQ ID NO:10的氨基酸序列，第二恒定区包含SEQ ID NO:20所示氨基酸序列；或
    vii)所述第一恒定区包含SEQ ID NO:13的氨基酸序列，且所述第二恒定区包含SEQ ID NO:21所示的氨基酸序列。
  20. 权利要求1-19中任一项的合成T细胞受体抗原受体，所述第一抗原结合区和所述第二抗原结合区各自独立地或组合地特异性结合靶抗原。
  21. 权利要求20的合成T细胞受体抗原受体，其中所述靶抗原是疾病相关抗原，优选是癌症相关抗原，例如选自以下的癌症相关抗原：CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。
  22. 权利要求1-21中任一项的合成T细胞受体抗原受体，所述第一抗原结合区包含特异性结合靶抗原的抗体的重链可变区，且所述第二抗原结合区包含所述抗体的轻链可变区；或者，所述第一抗原结合区包含特异性结合靶抗原的抗体的轻链可变区，且所述第二抗原结合区包含所述抗体的重链可变区。
  23. 权利要求1-22中任一项的合成T细胞受体抗原受体，其中
    所述第一抗原结合区包含SEQ ID NO:22所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:23所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:23所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:22所示氨基酸序列，由此所述STAR特异性结合EGFR；或
    所述第一抗原结合区包含SEQ ID NO:44所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:45所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:45 所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:44所示氨基酸序列，由此所述STAR特异性结合CD19；或
    所述第一抗原结合区包含SEQ ID NO:46所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:47所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:47所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:46所示氨基酸序列，由此所述STAR特异性结合CD20；或
    所述第一抗原结合区包含SEQ ID NO:48所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:49所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:49所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:48所示氨基酸序列，由此所述STAR特异性结合CD22；或
    所述第一抗原结合区包含SEQ ID NO:24所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:25所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:25所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:24所示氨基酸序列，由此所述STAR特异性结合GPC3；或
    所述第一抗原结合区包含特异性结合CD19的抗体的重链可变区，所述重链可变区包含SEQ ID NO:28所示重链CDR1、SEQ ID NO:29所示重链CDR2和SEQ ID NO:30所示重链CDR3，且所述第二抗原结合区包含特异性结合CD19的抗体的轻链可变区，所述轻链可变区包含SEQ ID NO:31所示轻链CDR1、SEQ ID NO:32所示轻链CDR2和SEQ ID NO33所示轻链CDR3；或者，所述第一抗原结合区包含特异性结合CD19的抗体的轻链可变区，所述轻链可变区包含SEQ ID NO:31所示轻链CDR1、SEQ ID NO:32所示轻链CDR2和SEQ ID NO:33所示轻链CDR3，且所述第二抗原结合区特异性结合CD19的抗体的重链可变区，所述重链可变区包含SEQ ID NO:28所示重链CDR1、SEQ ID NO:29所示重链CDR2和SEQ ID NO:30所示重链CDR3，由此所述STAR特异性结合CD19；或
    所述第一抗原结合区包含SEQ ID NO:26所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:27所示氨基酸序列；或者，所述第一抗原结合区包含SEQ ID NO:27所示氨基酸序列，且所述第二抗原结合区包含SEQ ID NO:26所示氨基酸序列，由此所述STAR特异性结合CD19。
  24. 权利要求1-22中任一项的合成T细胞受体抗原受体，所述第一抗原结合区包含特异性结合靶抗原的单链抗体(如scFv)或单域抗体(如骆驼抗体)，和/或所述第二抗原结合区包含特异性结合靶抗原的单链抗体(如scFv)或单域抗体(如骆驼抗体)。
  25. 权利要求24的合成T细胞受体抗原受体，所述第一抗原结合区和所述第二抗原结合区结合相同的靶抗原。
  26. 权利要求24的合成T细胞受体抗原受体，所述第一抗原结合区和所述第二抗原结合区结合相同靶抗原的不同区域(如不同的表位)。
  27. 权利要求24的合成T细胞受体抗原受体，所述第一抗原结合区和所述第二抗 原结合区结合不同的靶抗原，例如，所述两个抗原结合区可以分别结合CD19和CD20，或分别结合CD19和CD22，或分别结合CD38和BCMA，或分别结合PDL1和EGFR。
  28. 一种合成T细胞受体抗原受体(STAR)，其包含α链和β链，所述α链包含第一抗原结合区和第一恒定区，所述β链包含第二抗原结合区和第二恒定区，
    其中第一恒定区是天然T细胞受体α链恒定区或如权利要求1-19任一项中限定的天然T细胞受体α链恒定区变体，其中第二恒定区是天然T细胞受体β链恒定区或如权利要求1-19任一项中限定的天然T细胞受体β链恒定区变体，
    其中第一抗原结合区和第二抗原结合区特异性地结合不同的靶抗原或相同靶抗原的不同区域。
  29. 权利要求28的合成T细胞受体抗原受体，所述第一抗原结合区和/或第二抗原结合区包含特异性结合靶抗原的单链抗体(如scFv)或单域抗体(如骆驼抗体)。
  30. 权利要求29的合成T细胞受体抗原受体，其中
    所述第一抗原结合区或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:22所示氨基酸序列以及SEQ ID NO:23所示氨基酸序列，由此所述第一抗原结合区或所述第二抗原结合区特异性结合EGFR；或
    所述第一抗原结合区或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:44所示氨基酸序列以及SEQ ID NO:45所示氨基酸序列，由此所述第一抗原结合区或所述第二抗原结合区特异性结合CD19；或
    所述第一抗原结合区或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:46所示氨基酸序列以及SEQ ID NO:47所示氨基酸序列，由此所述第一抗原结合区或所述第二抗原结合区特异性结合CD20；或
    所述第一抗原结合区或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:48所示氨基酸序列以及SEQ ID NO:49所示氨基酸序列，由此所述第一抗原结合区或所述第二抗原结合区特异性结合CD22；或
    所述第一抗原结合区或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:24所示氨基酸序列以及SEQ ID NO:25所示氨基酸序列，由此所述第一抗原结合区或所述第二抗原结合区特异性结合GPC3；或
    所述第一抗原结合区或所述第二抗原结合区包含的单链抗体包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:28所示重链CDR1、SEQ ID NO:29所示重链CDR2和SEQ ID NO:30所示重链CDR3，所述轻链可变区包含SEQ ID NO:31所示轻链CDR1、SEQ ID NO:32所示轻链CDR2和SEQ ID NO:33所示轻链CDR3，由此所述第一抗原结合区或所述第二抗原结合区特异性结合CD19；或
    所述第一抗原结合区或所述第二抗原结合区包含的单链抗体包含SEQ ID NO:26所示氨基酸序列以及SEQ ID NO:27所示氨基酸序列，由此所述第一抗原结合区或所述第二抗原结合区特异性结合CD19。
  31. 权利要求29或30的合成T细胞受体抗原受体，所述两个抗原结合区可以分别 结合CD19和CD20，或分别结合CD19和CD22，或分别结合CD38和BCMA，或分别结合PDL1和EGFR。
  32. 一种分离的多核苷酸，其包含编码权利要求1-31中任一项的合成T细胞受体抗原受体(STAR)的α链和/或β链的核苷酸序列。
  33. 权利要求32的多核苷酸，所述多核苷酸包含在同一读码框内的i)编码所述α链的核苷酸序列、ii)编码所述β链的核苷酸序列和iii)位于i)和ii)之间的编码自裂解肽的核苷酸序列。
  34. 权利要求33的多核苷酸，其中所述自裂解肽是2A多肽，例如P2A多肽。
  35. 一种表达载体，其包含与调控序列可操作连接的权利要求32-34中任一项的多核苷酸。
  36. 权利要求35的表达载体，其是病毒载体，例如慢病毒载体。
  37. 一种制备治疗性T细胞的方法，包括在T细胞中表达权利要求1-31中任一项的合成T细胞受体抗原受体(STAR)。
  38. 权利要求37的方法，所述方法包括将权利要求32-34中任一项的多核苷酸或权利要求35或36的表达载体导入T细胞。
  39. 一种治疗性T细胞，其包含权利要求1-31中任一项的合成T细胞受体抗原受体(STAR)，或其通过权利要求37或38的方法获得。
  40. 一种药物组合物，其包含权利要求39的治疗性T细胞，和药物可接受的载体。
  41. 权利要求39的治疗性T细胞或权利要求40的药物组合物在制备用于在对象中治疗疾病例如癌症的药物中的用途。
  42. 权利要求41的用途，其中所述癌症选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤、骨肉瘤、骨癌、胰腺癌、皮肤癌、前列腺癌、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、霍奇金病、非霍奇金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。
PCT/CN2020/103687 2019-12-30 2020-07-23 一种增强型t细胞受体star及其应用 WO2021135178A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
KR1020227026190A KR20220131253A (ko) 2019-12-30 2020-07-23 향상된 t-세포 수용체 star 및 이의 적용
JP2022564686A JP2023508608A (ja) 2019-12-30 2020-07-23 強化されたｔ細胞受容体ｓｔａｒ及びその用途
EP20910062.7A EP4086281A4 (en) 2019-12-30 2020-07-23 IMPROVED T-CELL RECEPTOR STAR AND ITS USE
CN202080091762.0A CN116034113A (zh) 2019-12-30 2020-07-23 一种增强型t细胞受体star及其应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911389706.0 2019-12-30
CN201911389706 2019-12-30

Publications (1)

Publication Number Publication Date
WO2021135178A1 true WO2021135178A1 (zh) 2021-07-08

Family

ID=76686369

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/103687 WO2021135178A1 (zh) 2019-12-30 2020-07-23 一种增强型t细胞受体star及其应用

Country Status (5)

Country Link
EP (1) EP4086281A4 (zh)
JP (1) JP2023508608A (zh)
KR (1) KR20220131253A (zh)
CN (1) CN116034113A (zh)
WO (1) WO2021135178A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023002204A1 (en) * 2021-07-21 2023-01-26 Enara Bio Limited T-cell receptor

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118108833A (zh) * 2024-03-12 2024-05-31 缇纱(合肥)生物技术有限公司 引入外源二硫键的tcr恒定区及其产品和应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014065961A1 (en) * 2012-10-24 2014-05-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services M971 chimeric antigen receptors
US20140255398A1 (en) * 2011-09-30 2014-09-11 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule inducing immune response to target antigen
WO2015143382A1 (en) * 2014-03-21 2015-09-24 Abbvie Inc. Anti-egfr antibodies and antibody drug conjugates
WO2015157252A1 (en) * 2014-04-07 2015-10-15 BROGDON, Jennifer Treatment of cancer using anti-cd19 chimeric antigen receptor
WO2015184228A1 (en) * 2014-05-29 2015-12-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-human papillomavirus 16 e7 t cell receptors
WO2016022400A1 (en) * 2014-08-04 2016-02-11 Fred Hutchinson Cancer Research Center T cell immunotherapy specific for wt-1
CN109790211A (zh) * 2016-08-02 2019-05-21 美国卫生和人力服务部 抗kras-g12d t细胞受体

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2767209C2 (ru) * 2015-10-23 2022-03-16 Еурека Терапьютикс, Инк. Химерные конструкции антитело/т-клеточный рецептор и их применения
AU2017366739B2 (en) * 2016-12-02 2023-11-23 University Of Southern California Synthetic immune receptors and methods of use thereof
EP3579877A4 (en) * 2017-02-09 2020-12-09 The Regents of The University of California CHEMERIC T-LYMPHOCYTE ANTIGENIC RECEPTORS AND METHODS OF USE
MX2020003145A (es) * 2017-09-22 2020-07-29 Wuxi Biologics Ireland Ltd Nuevos complejos de polipeptidos biespecificos.
CN110016074B (zh) * 2018-01-08 2021-03-30 中国科学院广州生物医药与健康研究院 Mage-a3人源化t细胞受体
CN115776991A (zh) * 2020-05-07 2023-03-10 华夏英泰（北京）生物技术有限公司 改进的t细胞受体-共刺激分子嵌合物

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140255398A1 (en) * 2011-09-30 2014-09-11 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule inducing immune response to target antigen
WO2014065961A1 (en) * 2012-10-24 2014-05-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services M971 chimeric antigen receptors
CN104822705A (zh) * 2012-10-24 2015-08-05 美国卫生和人力服务部 M971嵌合抗原受体
WO2015143382A1 (en) * 2014-03-21 2015-09-24 Abbvie Inc. Anti-egfr antibodies and antibody drug conjugates
WO2015157252A1 (en) * 2014-04-07 2015-10-15 BROGDON, Jennifer Treatment of cancer using anti-cd19 chimeric antigen receptor
WO2015184228A1 (en) * 2014-05-29 2015-12-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-human papillomavirus 16 e7 t cell receptors
WO2016022400A1 (en) * 2014-08-04 2016-02-11 Fred Hutchinson Cancer Research Center T cell immunotherapy specific for wt-1
CN109790211A (zh) * 2016-08-02 2019-05-21 美国卫生和人力服务部 抗kras-g12d t细胞受体

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LEWIN, GENES VIII
ROITT ET AL.: "Immunology"
SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL
See also references of EP4086281A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023002204A1 (en) * 2021-07-21 2023-01-26 Enara Bio Limited T-cell receptor

Also Published As

Publication number Publication date
EP4086281A1 (en) 2022-11-09
CN116034113A (zh) 2023-04-28
JP2023508608A (ja) 2023-03-02
EP4086281A4 (en) 2024-02-14
KR20220131253A (ko) 2022-09-27

Similar Documents

Publication Publication Date Title
US11034763B2 (en) Flag tagged CD19-CAR-T cells
Ahmed et al. HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors
WO2017219936A1 (zh) 一种高效稳定表达激活型抗体的car-t细胞及其用途
US11692034B2 (en) CD47-CAR-T cells
WO2020042647A1 (zh) 改进的治疗性t细胞
CN114929751B (zh) Ror1特异性嵌合抗原受体及其治疗应用
WO2021254333A1 (zh) 一种合成t细胞受体抗原受体复合物及其应用
US11332513B2 (en) Chimeric antigen receptors having GITR intracellular domain as co-stimulatory domain
US20240148869A1 (en) Improved T cell receptor-costimulatory molecule chimera
US12030957B2 (en) PLAP-CAR-effector cells
CN111733186A (zh) 靶向cd19的人源化嵌合抗原受体制备及其应用
CN114616337A (zh) 嵌合CD3融合蛋白与基于anti-CD3的双特异性T细胞激活元件的联合表达
WO2021048564A2 (en) Antigen-binding domain
WO2021135178A1 (zh) 一种增强型t细胞受体star及其应用
CN109748968B (zh) Bcma特异性嵌合抗原受体t细胞及其应用
CN112739721A (zh) 一种单域抗体及包含抗体结构的嵌合抗原受体
US20230272341A1 (en) Multifunctional immune effector cell and use thereof
KR102297396B1 (ko) 면역시냅스를 안정화시키는 키메라 항원 수용체(car) t 세포
CN116162168A (zh) 分子开关调控型嵌合抗原受体细胞和抗体的组合及其应用
JP2023527026A (ja) 強化された合成ｔ細胞受容体及び抗原受容体
WO2023274382A1 (zh) 一种多靶点合成t细胞受体抗原/抗体受体及其应用
CN115335405B (zh) 全人源抗人cd22的嵌合抗原受体及其应用
CN109970862A (zh) 一种携带Beacon标签的嵌合抗原受体T细胞改造技术
CN114644716A (zh) 一种抗bxmas1嵌合抗原受体及其修饰的免疫细胞及应用
CN116284435A (zh) EGFRvIII嵌合抗原受体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20910062

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022564686

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020910062

Country of ref document: EP

Effective date: 20220801