WO2023274382A1 - Récepteur de cellules t synthétique multicible et récepteur antigène/anticorps et application correspondante - Google Patents

Récepteur de cellules t synthétique multicible et récepteur antigène/anticorps et application correspondante Download PDF

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WO2023274382A1
WO2023274382A1 PCT/CN2022/103054 CN2022103054W WO2023274382A1 WO 2023274382 A1 WO2023274382 A1 WO 2023274382A1 CN 2022103054 W CN2022103054 W CN 2022103054W WO 2023274382 A1 WO2023274382 A1 WO 2023274382A1
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constant region
chain
antibody
cells
domain
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PCT/CN2022/103054
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Chinese (zh)
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林欣
周志霄
芮魏
赵学强
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华夏英泰（北京）生物技术有限公司
清华大学
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Priority to CN202280059340.4A priority Critical patent/CN117897411A/zh
Publication of WO2023274382A1 publication Critical patent/WO2023274382A1/fr

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    • 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
    • A61K39/461—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
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/464411—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
    • A61K39/464416—Receptors for cytokines
    • A61K39/464419—Receptors for interleukins [IL]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedicine, in particular to a multi-targets Synthetic TCR and Antigen/Antibody Receptor (M-STA/AR) and its application.
  • M-STA/AR multi-targets Synthetic TCR and Antigen/Antibody Receptor
  • the present invention discloses a multi-targets synthetic T cell receptor antigen/antibody receptor (Multi-targets Synthetic TCR and Antigen/Antibody Receptor, M-STA/AR), comprising the multi-targets synthetic TCR Cell receptor antigen/antibody receptor immune cells and their uses.
  • CAR-T chimeric antigen receptor T cell
  • CAR-T therapy is based on the expression of CAR molecules in T cells.
  • the CAR molecule consists of three parts: the extracellular region is derived from the antigen recognition domain of the antibody, which is responsible for recognizing the target antigen; the transmembrane region; and the intracellular region is the signaling molecule and co-stimulatory signal molecule derived from the T cell receptor, which is responsible for the recognition of the target antigen. Transmits T cell activation signals after receiving stimulation. When the CAR molecule binds to its corresponding antigen, the CAR molecule will aggregate, activate the effector function of T cells, and kill the target tumor cells.
  • TCR-T therapy is based on the T cell receptor (TCR).
  • TCR is the identity characteristic of T cells.
  • T cells can be divided into ⁇ T cells and ⁇ T cells.
  • T precursor cells will first undergo VDJ rearrangement of TCR ⁇ and TCR ⁇ chains. If the rearrangement is successful, they will develop into ⁇ T cells. If the rearrangement fails, the precursor cells will undergo VDJ rearrangement of TCR ⁇ and TCR ⁇ chains, and then develop into ⁇ T cells.
  • ⁇ T cells account for 90%-95% of peripheral blood T cells, while ⁇ T cells account for 5%-10% of peripheral blood T cells.
  • Two types of T cells recognize antigens in an MHC-restricted and MHC-unrestricted manner, and play an important role in immunity to pathogens and tumors.
  • T cell receptor (TCR) complex molecule contains multiple chains, TCR ⁇ chain and TCR ⁇ chain (or TCR ⁇ chain and TCR ⁇ chain) are responsible for recognizing MHC-polypeptide molecules, the other 6 CD3 subunits and TCR ⁇ / ⁇ chain (or TCR ⁇ / ⁇ chain) combined with the function of signal transduction.
  • the natural TCR complex contains a total of 10 ITAM signal sequences, which can theoretically transmit stronger signals than CAR.
  • Using the signal transduction function of natural TCR it will be possible to construct a new type of receptor to alleviate the incapacity of T cells, so that they can better play the role of anti-solid tumors.
  • TCR variable region sequence can be replaced with the antibody variable region sequence to obtain a synthetic T cell receptor antigen receptor (Synthetic 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 the activation of T cells. So far, no one has constructed such a new receptor for the treatment of diseases.
  • variable region of the TCR is replaced by the active region of the antigen protein
  • another new type of synthetic T cell receptor antigen/antibody receptor Synthetic TCR and Antigen/Antibody Receptor, STA/AR
  • T cells can specifically bind antibodies or cell surface receptors. If the variable region of TCR is replaced with the active region of self-antigen protein, it can theoretically clear the B cells against the antigen in the body, thereby preventing the corresponding autoantibodies generation. So far, no one has constructed a new type of receptor from it for the treatment of diseases, let alone constructed such a new type of receptor.
  • the pathogenesis of a disease often involves multiple targets, or the diagnosis and treatment of a disease must be carried out systematically for different targets. Therefore, on the basis of single-target CAR, STAR and STA/AR, more and more Antibodies recognize regions, thereby targeting more targets. More systematic treatment, on the one hand, can be applied to some patients who do not respond to some drugs due to mutations and other reasons, on the other hand, it can kill tumor cells more comprehensively, thereby reducing the possibility of disease recurrence.
  • M-STA/AR-T derived from natural TCR lacks co-stimulatory signals in T cell activation during activation, and its proliferation and activation capabilities are often affected. Therefore, there remains a need in the art for improved TCRs and corresponding TCR-T therapies.
  • the term “and/or” covers all combinations of the items connected by the term, and each combination should be deemed to have been individually listed herein.
  • “A and/or B” includes “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 consist of said sequence, or may have additional amino acids or core elements at one or both ends of said protein or nucleic acid. Nucleotides, but still have the activity described in the present invention. In addition, those skilled in the art know that the methionine encoded by the start codon at the N-terminal of the polypeptide may be retained in some practical cases (such as when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide.
  • 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 EMBL-EBI's online alignment tool (https://www.ebi.ac.uk/Tools/psa/), where the two sequences can use the Needleman-Wunsch algorithm with default parameters to align.
  • the amino acid in the polypeptide can also be described herein It is "the alanine at the 48th position of the polypeptide, and the amino acid position refers to SEQ ID NO: x".
  • SEQ ID NO: 1 or 3 for the amino acid positions related to the constant region of the ⁇ chain.
  • SEQ ID NO: 2 or 4 for the amino acid positions related to the constant region of the ⁇ chain.
  • the present invention provides a multi-targets synthetic T cell receptor antigen/antibody receptor (Multi-targets Synthetic TCR and Antigen/Antibody Receptor, M-STA/AR), the multi-targets synthetic TCR Cell receptor antigen/antibody receptor is a multi-target T cell receptor antigen/antibody receptor M-STA/AR complex, which contains
  • the constant region on the ⁇ chain is the first constant region, and the constant region on the ⁇ chain is the second constant region; the N-terminal of the first constant region and/or the second constant region is connected to the molecular structure,
  • the molecular structure includes a nucleic acid or its analogue or a polypeptide structure, and the polypeptide structure includes a protein domain.
  • the molecular structure includes two or more in series;
  • gamma chain and delta chain of TCR the native variable region of said gamma chain and/or delta chain is deleted
  • the constant region on the ⁇ chain is the first constant region, and the constant region on the ⁇ chain is the second constant region; the N-terminal of the first constant region and/or the second constant region is connected to the molecular structure,
  • the molecular structure includes a nucleic acid or its analogue or a polypeptide structure, and the polypeptide structure includes a protein domain.
  • the molecular structure includes two or more in series.
  • the first constant region is a variant from the constant region of the natural T cell receptor ⁇ chain or ⁇ chain, and the variant includes one or more of the following groups: a. N-terminal modification; b. cysteine substitution ; and/or c. hydrophobic amino acid substitutions;
  • the second constant region is a variant from the constant region of the natural T cell receptor beta chain or delta chain, the variant comprising one or more of the following: a. N-terminal modification; and/or b. cysteine substitution .
  • the M-STA/AR further comprises one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ ; wherein at least one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ is connected to its C-terminal At least one functional domain; or, wherein at least one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ has at least one functional domain connected to its C-terminus.
  • N-terminal modification means that the N-terminus of the mentioned amino acid sequence (polypeptide or protein) contains one or more amino acid substitutions, deletions and/or additions, and the term may also include the mentioned Deletion of one or more consecutive amino acids from the N-terminus of the amino acid sequence (polypeptide or protein).
  • the first constant region comprises a modification of the N-terminal 18 amino acids relative to the constant region of the natural T cell receptor ⁇ chain.
  • the first constant region is derived from the constant region of the alpha chain of the mouse T cell receptor, and relative to the constant region of the natural mouse T cell receptor alpha chain, X consecutive amino acids from the N-terminus are replaced by natural human Corresponding amino acid substitutions in the constant region of the T cell receptor ⁇ chain, and/or deletion of amino acids from position X+1 to position 18 of the N-terminus, where X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.
  • the second constant region comprises an N-terminal 25 amino acid modification relative to the native T cell receptor beta chain constant region.
  • the second constant region is derived from the mouse T cell receptor beta chain constant region, and relative to the natural mouse T cell receptor beta chain constant region, X consecutive amino acids from the N-terminus are replaced by natural human Corresponding amino acid substitutions in the constant region of the T cell receptor beta chain, and/or deletion of at least one amino acid from position X+1 to position 25 of the N-terminus, 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.
  • cyste substitution or “hydrophobic amino acid substitution” means that the original amino acid in the mentioned amino acid sequence (polypeptide or protein) is replaced by cysteine or hydrophobic amino acid.
  • hydrophobic amino acid substitution may be a hydrophilic amino acid being replaced by a hydrophobic amino acid, or an amino acid having a low hydrophobicity may be replaced by an amino acid having a high hydrophobicity.
  • the second constant region is derived from the constant region of the beta chain of the mouse T cell receptor, and relative to the constant region of the natural mouse T cell receptor beta chain, the 17th, 21-25 amino acids at the N-terminal are deleted, and wherein the N-terminal Amino acids at positions 1-16 are substituted by corresponding amino acids in the constant region of the natural human T-cell receptor beta chain, and the amino acid at position 56, such as serine, is mutated into cysteine.
  • the first constant region is a native TCR ⁇ chain constant region, for example, a native human TCR ⁇ chain constant region (an exemplary human TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 1) or a native mouse TCR ⁇ chain constant region (an exemplary mouse TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 3); or the first constant region is a natural TCR ⁇ chain constant region, for example, a natural human TCR ⁇ chain constant region (an exemplary human TCR ⁇ chain constant region chain constant region amino acid sequence is shown in SEQ ID NO:50) or native mouse TCR gamma chain constant region (an exemplary mouse TCR gamma chain constant region amino acid sequence is shown in SEQ ID NO:51).
  • a native human TCR ⁇ chain constant region an exemplary human TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 1
  • a native mouse TCR ⁇ chain constant region an exemplary mouse TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 3
  • the first constant region is a modified TCR alpha chain constant region or a modified TCR gamma chain constant region.
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which includes one or more of the following modifications relative to the wild-type mouse TCR ⁇ chain constant region:
  • amino acid at position 6 is replaced by aspartic acid D,
  • amino acid at position 13 is replaced by arginine R,
  • the amino acid at position 48 is mutated to cysteine C
  • the amino acid at position 112 is mutated to leucine L
  • amino acid at position 114 is mutated to isoleucine I
  • amino acid at position 115 is mutated to valine V, and/or,
  • Lysine K in the intracellular region of the constant region was mutated to arginine R.
  • the amino acid at position 6 is glutamic acid E
  • the amino acid at position 13 is lysine K
  • the amino acid at position 48 is threonine T
  • the amino acid at position 112 is Serine S
  • the amino acid at position 114 is methionine M
  • the amino acid at position 115 is glycine G.
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, and relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 48, such as threonine T, is mutated to Cysteine C.
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, relative to the wild-type mouse TCR ⁇ chain constant region, the 112th amino acid such as serine S is changed to leucine acid L, the amino acid at position 114 such as methionine M is changed to isoleucine I, and the amino acid at position 115 such as glycine G is changed to queline V.
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, and relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 6, such as glutamic acid E, is replaced by asparagus Amino acid D is substituted, the 13th amino acid such as lysine K is replaced by arginine R, and the 15th-18th amino acid is deleted.
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, and relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 48, such as threonine T, is mutated to Cysteine C, amino acid at position 112 such as serine S is changed to leucine L, amino acid at position 114 such as methionine M is changed to isoleucine I, amino acid at position 115 such as Glycine G is changed to valine V.
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, and relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 6, such as glutamic acid E, is replaced by asparagus Amino acid D substitution, amino acid at position 13 such as lysine K is replaced by arginine R, amino acid at position 15-18 is deleted, amino acid at position 48 such as threonine T is mutated to cysteine C , the amino acid at position 112 such as serine S is changed to leucine L, the amino acid at position 114 such as methionine M is changed to isoleucine I, and the amino acid at position 115 such as glycine G is changed Propine V.
  • the amino acid at position 6 such as glutamic acid E
  • amino acid at position 13 such as lysine K
  • arginine R amino acid at position 15-18 is deleted
  • amino acid at position 48 such as threonine T is mutated to cysteine C
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which, relative to the wild-type mouse TCR ⁇ chain constant region, lacks the natural intracellular region of the constant region, such as deletion of 136- 137th amino acid.
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, relative to the wild-type mouse TCR ⁇ chain constant region, the lysine K in the intracellular region of the constant region is mutated to sperm amino acid R.
  • the first constant region comprises the amino acid sequence shown in one of SEQ ID NO: 1, 3, 5, 7, 8, 26, 41, 42, and 48.
  • the second constant region is a native TCR beta chain constant region, for example, a native human TCR beta chain constant region (an exemplary human TCR beta chain constant region amino acid sequence is shown in SEQ ID NO: 2) or a native mouse TCR beta chain constant region (an exemplary mouse TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 4); or wherein the second constant region is a natural TCR ⁇ chain constant region, for example, a natural human TCR ⁇ chain constant region (an exemplary human TCR ⁇ chain constant region (exemplary human The TCR delta chain constant region amino acid sequence is shown in SEQ ID NO:52) or native mouse TCR delta chain constant region (an exemplary mouse TCR delta chain constant region amino acid sequence is shown in SEQ ID NO:53).
  • a native human TCR beta chain constant region an exemplary human TCR beta chain constant region amino acid sequence is shown in SEQ ID NO: 2
  • a native mouse TCR beta chain constant region an exemplary mouse TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID
  • the second constant region is a modified TCR beta chain constant region; or the second constant region is a modified TCR delta chain constant region.
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which includes one or more of the following modifications relative to the wild-type mouse TCR ⁇ chain constant region:
  • amino acid at position 3 is substituted by lysine K
  • amino acid at position 6 is substituted by phenylalanine F,
  • amino acid at position 9 is replaced by glutamic acid E,
  • amino acid at position 11 is replaced by alanine A
  • amino acid at position 12 is substituted by valine V,
  • the amino acid at position 56 is mutated to cysteine C
  • the amino acid at position 3 is arginine R
  • the amino acid at position 6 is threonine T
  • the amino acid at position 9 is lysine K
  • the amino acid at position 11 is serine S
  • the amino acid at position 12 is The amino acid at position 1 is leucine L
  • the amino acid at position 17 and 21-25 is deleted
  • the amino acid at position 56 is serine S.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, and its amino acid at position 56, such as serine S, is mutated to a cysteine relative to the wild-type mouse TCR ⁇ chain constant region. amino acid C.
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region, and its third amino acid such as arginine R is replaced by lysine Acid K substitution, amino acid at position 6 such as threonine T is replaced by phenylalanine F, amino acid at position 9 such as lysine K is replaced by glutamic acid E, amino acid at position 11 such as serine S is replaced by alanine Acid A is substituted, the 12th amino acid such as leucine L is replaced by valine V, and the 17th, 21-25 amino acid is deleted.
  • arginine R is replaced by lysine Acid K substitution
  • amino acid at position 6 such as threonine T is replaced by phenylalanine F
  • amino acid at position 9 such as lysine K is replaced by glutamic acid E
  • amino acid at position 11 such as serine S is replaced by alanine Acid A is substituted
  • the modified TCR beta chain constant region is derived from the mouse TCR beta chain constant region, relative to the wild-type mouse TCR beta chain constant region, the 56th amino acid such as serine S is mutated to cysteine Acid C, the amino acid at position 3 such as arginine R is replaced by lysine K, the amino acid at position 6 such as threonine T is replaced by phenylalanine F, and the amino acid at position 9 such as lysine K is replaced by gluten Amino acid E is substituted, the 11th amino acid such as serine S is replaced by alanine A, the 12th amino acid such as leucine L is substituted by valine V, and the 17th, 21-25 amino acids are deleted.
  • the 56th amino acid such as serine S is mutated to cysteine Acid C
  • the amino acid at position 3 such as arginine R is replaced by lysine K
  • the amino acid at position 6 such as threonine T is replaced by phenylalanine F
  • the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region, and the lysine K in the transmembrane region of the constant region is mutated to sperm amino acid R.
  • the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which, relative to the wild-type mouse TCR ⁇ chain constant region, lacks the natural intracellular region of the constant region, such as deletion of 167- 172 amino acids.
  • the modified TCR beta chain constant region comprises the amino acid sequence shown in one of SEQ ID NO:2, 4, 6, 9, 27, 43 and 49.
  • the M-STA/AR in which the first constant region comes from natural T cell receptor ⁇ chain and the second constant region comes from natural T cell receptor ⁇ chain is called ⁇ multi-target M-STA/AR complex.
  • the M-STA/AR in which the first constant region comes from the natural T cell receptor ⁇ chain and the second constant region comes from the natural T cell receptor ⁇ chain is called the ⁇ multi-target M-STA/AR complex.
  • the native intracellular region of at least one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ multi-target M-STA/AR complex is deleted, or wherein all The native intracellular region of at least one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ in the ⁇ M-STA/AR complex is deleted.
  • the native intracellular region of the TCR ⁇ chain and/or TCR ⁇ chain in the ⁇ multi-target M-STA/AR complex is deleted, or wherein the TCR ⁇ chain and/or TCR ⁇ chain in the ⁇ TCR The native intracellular domain is deleted.
  • the functional domain is connected directly or through a linker to the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , in which the natural intracellular region is deleted.
  • CD3 ⁇ , CD3 ⁇ and CD3 ⁇ at least one of the C-terminus.
  • the functional domain is connected directly or through a linker to the C-terminus of the TCR ⁇ chain and/or TCR ⁇ chain in which the natural intracellular region is deleted.
  • the functional domain is connected directly or through a linker to the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ chain whose native intracellular region is deleted , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ at least one of the C-terminus.
  • the functional domain is connected directly or through a linker to the C-terminus of the TCR ⁇ chain and/or TCR ⁇ chain in which the native intracellular region is deleted.
  • the linker is a (G 4 S)n linker, wherein n represents an integer from 1-10.
  • one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ in the ⁇ multi-target M-STA/AR complex has at least one functional domain linked to its C-terminus.
  • the TCR ⁇ chain and/or the TCR ⁇ chain in the ⁇ TCR has at least one functional domain connected to its C-terminus.
  • one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ in the ⁇ multi-target M-STA/AR complex has at least one functional domain linked to its C-terminus.
  • the TCR ⁇ chain and/or TCR ⁇ chain in the ⁇ TCR has at least one functional domain connected to its C-terminus.
  • none of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the multi-target M-STA/AR complex comprises at least one functional domain additionally linked to its C-terminus.
  • the TCR ⁇ chain in the ⁇ multi-target M-STA/AR complex has at least one functional domain connected to its C-terminus.
  • the TCR ⁇ chain in the ⁇ TCR has at least one functional domain connected to its C-terminus.
  • the TCR ⁇ chain in the ⁇ multi-target M-STA/AR complex has at least one functional domain connected to its C-terminus.
  • the TCR ⁇ chain in the ⁇ TCR has at least one functional domain connected to its C-terminus.
  • the TCR ⁇ chain in the ⁇ multi-target M-STA/AR complex has at least one functional domain connected to its C-terminus.
  • the TCR ⁇ chain in the ⁇ TCR is connected with at least one functional domain at its C-terminus.
  • the TCR ⁇ chain in the ⁇ multi-target M-STA/AR complex has at least one functional domain connected to its C-terminus.
  • the TCR ⁇ chain in the ⁇ TCR has at least one functional domain connected to its C-terminus.
  • two of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ in the ⁇ multi-target M-STA/AR complex have at least one functional domain linked to their C-terminus.
  • two of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ in the ⁇ multi-target M-STA/AR complex have at least one functional domain linked to their C-terminus.
  • each of the TCR ⁇ chain and the TCR ⁇ chain in the ⁇ multi-target M-STA/AR complex has at least one functional domain connected to its C-terminus.
  • each of the TCR ⁇ chain and the TCR ⁇ chain in the ⁇ TCR has at least one functional domain linked to its C-terminus.
  • TCR alpha chain and TCR beta chain are deleted.
  • the functional domain is linked directly or via a linker to the C-terminal of the TCR ⁇ chain and TCR ⁇ chain in which the native intracellular region is deleted.
  • each of the TCR ⁇ chain and the TCR ⁇ chain in the ⁇ multi-target M-STA/AR complex has at least one functional domain connected to its C-terminus.
  • each of the TCR ⁇ chain and the TCR ⁇ chain in the ⁇ TCR has at least one functional domain connected to its C-terminus.
  • TCR gamma chain and TCR delta chain are deleted.
  • the functional domain is directly or via a linker connected to the C-terminal of the TCR ⁇ chain and TCR ⁇ chain in which the native intracellular region is deleted.
  • two or more of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ multi-target M-STA/AR complex may be linked to the same or different functions domain.
  • the TCR ⁇ chain and/or the TCR ⁇ chain in the ⁇ TCR may be linked to the same or different functional domains.
  • two or more of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ multi-target M-STA/AR complex may be linked to the same or different functions domain.
  • the TCR ⁇ chain and/or the TCR ⁇ chain in the ⁇ TCR may be linked to the same or different functional domains.
  • At least one of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ in the ⁇ multi-target M-STA/AR complex is linked with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains.
  • the TCR ⁇ chain and/or TCR ⁇ chain in the ⁇ TCR has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains connected to its C-terminus .
  • At least one of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ multi-target M-STA/AR complex has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains.
  • the TCR ⁇ chain and/or TCR ⁇ chain in the ⁇ TCR has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains connected to its C-terminus .
  • 1, 2, 3, 4, 5 or 6 of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ in the complex have at least one functional domain connected to their C-terminus, For example intracellular domains of co-stimulatory molecules.
  • one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ in the complex has at least one functional domain connected to its C-terminus, such as costimulatory molecular intracellular domain.
  • the TCR ⁇ chain in the complex is linked to at least one functional domain at its C-terminus, such as an intracellular domain of a co-stimulatory molecule.
  • the intracellular domain of the costimulatory molecule is OX40 or ICOS.
  • the TCR beta chain, CD3delta, CD3gamma, CD3epsilon, and CD3zeta do not comprise at least one functional domain, such as a co-stimulatory molecular intracellular domain, additionally linked to their C-terminus.
  • CD3 ⁇ in the complex has at least one functional domain linked to its C-terminus, such as an intracellular domain of costimulatory molecules.
  • TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ do not comprise at least one functional domain, such as a co-stimulatory molecular intracellular domain, additionally linked to their C-terminus.
  • two of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the complex have at least one functional domain connected to their C-terminus, such as an intracellular domain of costimulatory molecules.
  • each of the TCR ⁇ chain and the TCR ⁇ chain in the complex has at least one functional domain connected to its C-terminus, such as an intracellular domain of a co-stimulatory molecule.
  • the intracellular domain of the costimulatory molecule is OX40 or ICOS.
  • CD3delta, CD3gamma, CD3epsilon, and CD3zeta do not comprise at least one functional domain, such as a co-stimulatory molecular intracellular domain, additionally linked to their C-terminus.
  • the functional domain is an exogenous functional domain.
  • the functional domain is an exogenous intracellular domain, eg, a domain that functions in intracellular signaling.
  • exogenous means a protein or nucleic acid sequence from a foreign species, or, if from the same species, a protein or protein whose composition and/or location has been significantly altered from its native form by deliberate human intervention. nucleic acid sequence.
  • a "functional domain” may be an intracellular domain of a co-stimulatory molecule such as CD40, OX40, ICOS, CD28, 4-1BB, CD27 or CD137; it may also be an intracellular domain of a co-inhibitory molecule.
  • a co-stimulatory molecule such as CD40, OX40, ICOS, CD28, 4-1BB, CD27 or CD137; it may also be an intracellular domain of a co-inhibitory molecule.
  • Domains such as the intracellular domain of TIM3, PD1, CTLA4, or LAG3; can also be cytokine receptors such as interleukin receptors (eg, IL-2 ⁇ receptor, IL-7 ⁇ receptor, or IL-21 receptor) , interferon receptors, tumor necrosis factor superfamily receptors, colony stimulating factor receptors, chemokine receptors, growth factor receptors, or intracellular domains of other membrane proteins; or domains of intracellular proteins such as NIK.
  • interleukin receptors eg, IL-2 ⁇ receptor, IL-7 ⁇ receptor, or IL-21 receptor
  • interferon receptors eg, tumor necrosis factor superfamily receptors, colony stimulating factor receptors, chemokine receptors, growth factor receptors, or intracellular domains of other membrane proteins
  • chemokine receptors chemokine receptors
  • growth factor receptors or intracellular domains of other membrane proteins
  • intracellular proteins such as NIK.
  • the functional domain can also be a cytokine receptor intracellular domain directly or through a linker (eg (G 4 S)n linker, wherein n represents an integer of 1-10) and a human STAT5 activation module (the amino acid sequence is shown in SEQ ID NO :35) fusion.
  • a linker eg (G 4 S)n linker, wherein n represents an integer of 1-10) and a human STAT5 activation module (the amino acid sequence is shown in SEQ ID NO :35) fusion.
  • the functional domain is an intracellular domain of a co-stimulatory molecule, preferably an intracellular domain of OX40 or ICOS, more preferably an intracellular domain of OX40.
  • An exemplary CD40 intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 10.
  • An exemplary OX40 intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 11.
  • An exemplary ICOS intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 12.
  • An exemplary CD28 intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 13.
  • An exemplary 4-1BB intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 14.
  • An exemplary CD27 intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 15.
  • An exemplary IL-2 ⁇ receptor intracellular domain comprises the amino acid sequence shown in SEQ ID NO:32.
  • An exemplary IL-17 ⁇ receptor intracellular domain comprises the amino acid sequence shown in SEQ ID NO:33.
  • An exemplary IL-21 receptor intracellular domain comprises the amino acid sequence shown in SEQ ID NO:34.
  • the fusion amino acid sequence of an exemplary IL-2 ⁇ receptor intracellular domain and a human STAT5 activation module is shown in SEQ ID NO:36.
  • the fusion amino acid sequence of an exemplary IL-17 ⁇ receptor intracellular domain and a human STAT5 activation module is shown in SEQ ID NO:37.
  • Protein domain refers to the protein domain located at the N-terminus of the first constant region and/or the N-terminus of the second constant region of the multi-target M-STA/AR, which is composed of more than one structural domain of the recombinant protein in series.
  • the structural domain includes the active structural region of the protein, and the recombinant proteins are the same or different, as shown in Figure 9 and Figure 10 .
  • the tandem recombination domain recombinant protein is directly connected to the N-terminus of the constant region.
  • the tandem protein domain is directly connected to the N-terminus of the constant region or connected by a linker, so
  • the linker LAMA or (G 4 S)n linker wherein n represents an integer of 1-10, preferably, n is 1 or 3, and the sequence of the linker LAMA is shown in EPKIPQPQPKPQPQPQPQPQPKPQPKPEP (SEQ ID NO: 54).
  • tandem protein domains are directly connected; in some embodiments, the tandem protein domains are connected through a linker, the linker LAMA, (EA 3 K)n or (G 4 S)n linker, wherein n represents an integer of 1-10, preferably, n is 1 or 3; in some embodiments, there are both direct connection and linker between the protein domains in series
  • the linker LAMA, (EA 3 K)n or (G 4 S)n linker wherein n represents an integer of 1-10, preferably, n is 1 or 3, the sequence of the linker LAMA is as follows EPKIPQPQPKPQPQPQPQPKPQPKPEP (SEQ ID NO: 54).
  • the recombinant protein is an antibody targeting a specific ligand or antigen, and the antigen is preferably an autoantigen.
  • the protein domain is derived from the domain of receptor or antibody, for example, the protein domain is antibody, antibody variable region, single chain antibody (ScFv), multimer of single chain antibody, single domain antibody (SdFv ) and/or multimers of single domain antibodies.
  • the recombinant protein is an antigen targeting a specific receptor or antibody
  • the protein domain is composed of the domain of the antigen, which may be a monomer or a multimer.
  • the protein domain is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or hexamer of a specific ligand or antigen or domain thereof. nonamers and/or decamers.
  • the single domain antibody also known as Nanobody, is selected from NbLILRB4, Nb22-2, Nb22-5, Nb22-6, Nb22-67, Nb22, Nb123, Nb19, Nb20-2, Nb20-3 and/or Nb33.
  • the single-chain antibody is selected from FMC63, OFA and/or 334, and the 334 is an anti-CD19 antibody whose sequence comprises a light chain variable region sequence such as SEQ ID NO: 46 and a sequence such as SEQ ID NO: 46.
  • the sequence of the heavy chain variable region shown in ID NO: 47 is derived from the prior art (CN112210007A), which is cited in its entirety in this application as a reference.
  • the recombinant protein is not a known antibody or antigen.
  • the recombinant protein includes both antibodies and antigens, or proteins other than non-antigen antibodies;
  • a selectable tag is attached to the N-terminus of the tandem protein domain, and the tag is selected from short-chain polypeptides; in a specific embodiment, the tag is cmyc.
  • the protein domain refers to the first tandem protein domain or the second tandem protein domain, and the first tandem protein domain is connected to the N-terminal of the first constant region, or the second The two tandem protein domains are linked at the N-terminus of the second constant region.
  • the protein domain refers to a first tandem protein domain and a second tandem protein domain, the first tandem protein domain is connected to the N-terminal of the first constant region, and, the A second tandem protein domain is linked N-terminally to the second constant region.
  • the protein domain only includes the first tandem protein domain, and the first tandem recombination domain is formed by tandem of Nb22 antibody and cmvc.
  • the protein domain only includes the first tandem protein domain, and the first tandem recombination domain is formed by tandem of Nb123 antibody and cmvc.
  • said protein domain only comprises the second tandem protein domain, and said second tandem recombination domain comprises only Nb123 antibody.
  • said protein domain only comprises the second tandem protein domain, and said second tandem recombination domain comprises only NbLILRB4 antibody.
  • the protein domain comprises a first tandem protein domain and a second tandem protein domain
  • the first tandem recombination domain is composed of a first single-chain antibody (ScFv) and/or a first
  • the single domain antibody (SdFv) is formed in series
  • the second series recombination domain is formed by another second single chain antibody (ScFv) and/or the second single domain antibody (SdFv) in series.
  • the single-chain antibody is located at the N-terminal of the single-chain antibody, more preferably, the single-chain antibody and the constant region are directly connected, and further preferably, the single-chain antibody and the single-domain antibody are connected through a linker.
  • the first single-chain antibody is OFA
  • the second single-chain antibody is FMC
  • the first single-domain antibody is Nb123
  • the second single-chain antibody is Nb22 .
  • the protein domain comprises a first tandem protein domain and a second tandem protein domain
  • the first tandem recombination domain is composed of a first single domain antibody (SdFv)
  • the second tandem The recombination domain is composed of another second single domain antibody (SdFv).
  • both the single domain antibody and the constant region are directly linked.
  • the first single domain antibody is Nb123
  • the second single domain antibody is NbLILRB4.
  • the first single domain antibody is NbLILRB4, and the second single domain antibody is Nb123.
  • the protein domain only includes the first tandem protein domain
  • the first tandem recombination domain is formed by Nb123, Nb22 and cmvc in series
  • Nb123 is directly connected to the N-terminal of the constant region
  • Nb22 and Nb123 are connected by a linker LAMA
  • cmyc is directly connected to the N-terminus of Nb22.
  • the sequence of the linker LAMA is shown in EPKIPQPQPKPQPQPQPQPQPKPQPKPEP (SEQ ID NO: 54).
  • the protein domain only includes the first tandem protein domain
  • the first tandem recombination domain is formed by Nb123, Nb22 and cmvc in series
  • Nb123 is directly connected to the N-terminal of the constant region
  • Nb22 and Nb123 are connected by a linker (G 4 S)n, wherein n represents an integer of 1-10, preferably, n is 1 or 3, and cmyc is directly connected to the N-terminal of Nb22.
  • the protein domain only includes the first tandem protein domain
  • the first tandem recombination domain is formed by Nb22, Nb123 and cmvc in series
  • Nb22 is directly connected to the N-terminal of the constant region
  • Nb22 and Nb123 are connected by linker LAMA
  • cmyc is directly connected to the N-terminal of Nb123.
  • the sequence of the linker LAMA is shown in EPKIPQPQPKPQPQPQPQPQPKPQPKPEP (SEQ ID NO: 54).
  • the protein domain only includes the first tandem protein domain
  • the first tandem recombination domain is formed by Nb22, Nb123 and cmvc in series
  • Nb22 is directly connected to the N-terminal of the constant region
  • Nb22 and Nb123 are connected through a linker (G 4 S)n, wherein n represents an integer of 1-10, preferably, n is 1 or 3, and cmyc is directly connected to the N-terminal of Nb123.
  • the protein domain only includes the second tandem protein domain
  • the second tandem recombination domain is formed by tandem Nb123 and NbLILRB4
  • Nb123 is directly connected to the N-terminal of the constant region
  • NbLILRB4 and Nb123 are connected by a linker (G 4 S)n, wherein n represents an integer of 1-10, preferably, n is 1 or 3.
  • the protein domain only includes the second tandem protein domain
  • the second tandem recombination domain is formed by tandem Nb123 and NbLILRB4
  • Nb123 is directly connected to the N-terminal of the constant region
  • NbLILRB4 and Nb123 are connected by linker (EA 3 K)n, wherein n represents an integer of 1-10, preferably, n is 1 or 3.
  • the protein domain only includes the second tandem protein domain
  • the second tandem recombination domain is formed by Nb123 and NbLILRB4 in series
  • NbLILRB4 is directly connected to the N-terminal of the constant region
  • NbLILRB4 and Nb123 are connected by a linker (G 4 S)n, wherein n represents an integer of 1-10, preferably, n is 1 or 3.
  • the protein domain only includes the second tandem protein domain
  • the second tandem recombination domain is formed by Nb123 and NbLILRB4 in series
  • NbLILRB4 is directly connected to the N-terminal of the constant region
  • NbLILRB4 and Nb123 are connected by linker (EA 3 K)n, wherein n represents an integer of 1-10, preferably, n is 1 or 3.
  • the sequence of the Nb22 antibody is as shown in SEQ ID NO:38 or as shown in SEQ ID NO:39; the sequence of the FMC antibody is as shown in SEQ ID NO:44, and the sequence of OFA is as shown in SEQ ID NO:45 As shown, the sequence of 334 is shown in SEQ ID NO:46 and SEQ ID NO:47, the sequence of Nb123 antibody is shown in SEQ ID NO:55, and the sequence of NbLILRB4 antibody is shown in SEQ ID NO:56.
  • the CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and/or CD3 ⁇ are of human origin.
  • the human CD3 ⁇ comprises the amino acid sequence shown in SEQ ID NO:28.
  • the human CD3 ⁇ comprises the amino acid sequence shown in SEQ ID NO:29.
  • the human CD3 ⁇ comprises the amino acid sequence shown in SEQ ID NO:30.
  • the human CD3 ⁇ comprises the amino acid sequence shown in SEQ ID NO: 31.
  • the present invention provides an isolated therapeutic immune cell comprising any of the above-mentioned multi-target synthetic T cell receptor antigen/antibody receptors.
  • the immune cells are T cells. In other embodiments, the immune cells are NK cells.
  • the present invention provides an isolated polynucleotide comprising a nucleus encoding at least one of TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ as defined above in the present invention
  • Nucleotide sequence, at least one of the TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ has at least one exogenous intracellular functional domain connected to its C-terminus.
  • the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding any one of the multi-target synthetic T cell receptor antigen/antibody receptors of the present invention as defined above.
  • the isolated polynucleotide comprises a nucleotide sequence encoding a TCR ⁇ chain and/or a TCR ⁇ chain having at least one co-stimulatory molecule linked to its C-terminus. domain.
  • the polynucleotide comprises i) the nucleotide sequence encoding the ⁇ chain, ii) the nucleotide sequence encoding the ⁇ chain and iii) the nucleotide sequence located in i) within the same reading frame.
  • the nucleotide sequence encoding the ⁇ chain may be located at the 5' end or 3' end of the nucleotide sequence encoding the ⁇ chain.
  • the isolated polynucleotide comprises a nucleotide sequence encoding a TCR ⁇ chain and/or a TCR ⁇ chain having at least one co-stimulatory molecule linked to its C-terminus. domain.
  • the polynucleotide comprises i) the nucleotide sequence encoding the gamma chain, ii) the nucleotide sequence encoding the delta chain and iii) within the same reading frame located in i) The nucleotide sequence encoding the self-cleaving peptide between and ii).
  • the nucleotide sequence encoding the ⁇ chain may be located at the 5' end or 3' end of the nucleotide sequence encoding the ⁇ chain.
  • Self-cleaving peptide as used herein means a peptide that can undergo self-cleavage within a cell.
  • the self-cleaving peptide may contain a protease recognition site, thereby being recognized and specifically cleaved by intracellular proteases.
  • the self-cleaving peptide may be a 2A polypeptide.
  • 2A polypeptides are a class of short peptides from viruses whose self-cleavage occurs during translation. When the 2A polypeptide is used to connect two different target proteins and express them in the same reading frame, the two target proteins are produced in a ratio of almost 1:1.
  • Commonly used 2A polypeptides can be P2A from porcine techovirus-1, T2A from Thosea asigna virus, and E2A from equine rhinitis A virus. and F2A from foot-and-mouth disease virus. Among them, P2A has the highest cleavage efficiency and is therefore preferred.
  • a variety of functional variants of these 2A polypeptides are also known in the art and may also be used in the present invention.
  • the invention provides an expression vector comprising a 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 an RNA capable of translation (such as mRNA).
  • the expression vector is a viral vector, such as a lentiviral vector.
  • regulatory sequence and “regulatory element” are used interchangeably to refer to a sequence that is located upstream (5' non-coding sequences), midway or downstream (3' non-coding sequences) of a coding sequence and that affects the transcription, RNA processing or Stable or translated nucleotide sequences.
  • An expression regulatory 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 means that a regulatory element (such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.) is linked to a nucleic acid sequence (such as a coding sequence or an open reading frame) such that the nucleotide Transcription of the sequences is controlled and regulated by said transcriptional regulatory elements.
  • a regulatory element such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.
  • nucleic acid sequence such as a coding sequence or an open reading frame
  • the present invention provides a method for preparing the therapeutic immune cell of the present invention, comprising introducing the polynucleotide of the present invention or the expression vector of the present invention into the immune cell.
  • the immune cells of the invention can be obtained by various non-limiting methods from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion , spleen tissue and tumors.
  • cells may be derived from healthy donors or from patients diagnosed with cancer.
  • a 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), and then activated and expanded with specific antibodies.
  • PBMC peripheral blood mononuclear cells
  • the immune cells are derived from autologous cells of the subject being treated.
  • autologous means that a cell, cell line or population of cells used to treat a subject is derived from said subject.
  • the immune cells, eg, T cells are derived from allogeneic cells, eg, from a donor compatible with the human leukocyte antigen (HLA) of the subject. The cells from the donor can be converted to non-aloreactive cells using standard protocols and replicated as necessary to generate cells that can be administered to one or more patients.
  • HLA human leukocyte antigen
  • the present invention provides a use of the above-mentioned immune cells in the preparation of a pharmaceutical composition.
  • the present invention provides a pharmaceutical composition, which comprises the therapeutic immune cells of the present invention, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the present invention provides a use of a therapeutic immune cell of the present invention or a pharmaceutical composition of the present invention in the manufacture of a medicament for treating a disease, such as cancer, in a subject.
  • a "subject” as used herein refers to an organism suffering from or susceptible to a disease (eg, cancer) that can be treated by the cells, methods, or pharmaceutical compositions of the invention.
  • a disease eg, 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 a therapeutic immune cell of the present invention or a 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. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder.
  • an "effective amount" of a cell or pharmaceutical composition of the invention preferably results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of injury or disability resulting from disease affliction.
  • an "effective amount" of cells or pharmaceutical compositions of the present invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably, relative to untreated subjects. 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 animal model systems predictive of efficacy against human tumors. Alternatively, it can also be assessed by examining the ability to inhibit tumor cell growth, which can be determined in vitro by assays well known to those skilled in the art.
  • dosage levels of the cells in the pharmaceutical compositions of the invention may be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without toxicity to the patient.
  • the selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the particular Other drugs, compounds and/or materials with which the composition is used in conjunction, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.
  • Administration of therapeutic immune cells or pharmaceutical compositions or medicaments according to the invention may be performed in any convenient manner, 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 "tumor” mentioned in the present invention includes but not limited to lymphoma, brain cancer, non-small cell lung cancer, cervical cancer, esophageal cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer , rectal cancer, stomach cancer, bladder cancer, lung cancer, bronchus cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophagus cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma , astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma.
  • the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia;
  • the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma , T-cell lymphoma, and Waldenstrom macroglobulinemia;
  • said sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma
  • the present invention uses the signal transduction function of TCR and its antigen-binding region to transform the antigen-binding region into a multi-target protein domain.
  • the modified synthetic TCR (M-STA/AR) can be used to combine two or more TCRs at one time. More than two active proteins or polypeptides, the active proteins or polypeptides may be antigens, antibodies or antibody fragments, cell surface receptors, etc. If the targeted proteins or polypeptides are linked in the pathogenic mechanism of the disease, it will be possible to establish New therapeutic strategies for related diseases.
  • the modified M-STA/AR can be used as a tool for drug screening, which can specifically bind Polypeptides or protein molecules with multiple protein domains, said polypeptides or protein molecules may be key polypeptides or proteins for disease treatment.
  • the M-STA/AR constructed by the present invention will have a powerful screening function of multi-dimensional superposition.
  • the present invention uses recombinant technology to modify the constant region of TCR, which is expected to make the genetically modified M-STA/AR obtain better membrane stability, significantly reduce the ability of ⁇ / ⁇ chain pairing, and interact with endogenous TCR.
  • the mismatch rate is also reduced, it is easier to introduce T cells, and the signal transduction ability is stronger.
  • the present invention uses recombinant technology to modify the constant region of TCR, so that the genetically modified M-STA/AR can better respond to the stimulation of external information and activate the function of T cells.
  • Figure 1 Schematic diagram of optimization of M-STA/AR by constant region cysteine modification, transmembrane and intracellular region modification.
  • Figure 2 Schematic representation of optimization of M-STA/AR by addition of co-stimulatory molecule receptor intracellular domains to ⁇ and/or ⁇ chains.
  • Figure 3 Schematic diagram of the optimization of M-STA/AR by adding co-stimulatory molecule receptor intracellular domains directly or via linkers after deletion of the intracellular domains of the ⁇ and/or ⁇ chains.
  • Figure 4 Schematic representation of optimization of M-STA/AR by adding co-stimulatory molecule receptor intracellular domain to CD3 subunit.
  • Figure 5 Schematic representation of optimization of M-STA/AR by addition of cytokine receptor intracellular signaling domains to ⁇ and/or ⁇ chains.
  • Figure 6 Schematic diagram of optimization of M-STA/AR by modification of cysteine in the constant region, modification of the transmembrane region and N-terminal rearrangement.
  • Figure 7 Example of the attachment position of the co-stimulatory molecule molecular domain to the M-STA/AR structure. Only the linkage to the alpha chain is shown.
  • Figure 8 Example of an M-STA/AR structure containing a co-stimulatory molecular domain.
  • Figure 10 An example of the M-STA/AR structure in which the first constant region and the second constant region are connected in series.
  • Figure 12 The killing ability of different M-STA/AR-T on target cells, where the histogram on each title on the abscissa represents rfp, Z13, DS, Loop-car from left to right.
  • Figure 13 The killing ability of different M-STA/AR-T on target cells, where the histogram on each title on the abscissa represents RFP, Z13, Dual-Star, Loop-car from left to right.
  • Figure 14 The killing ability of different M-STA/AR-T on target cells, where the histogram on each title on the abscissa represents RFP, Z3, Z4, Z13 from left to right.
  • Figure 15 The killing ability of different M-STA/AR-T on target cells, where the histogram on each title on the abscissa represents RFP, Z3, Z4, Z13 from left to right.
  • Figure 16 Construction examples of Z112, Z113, Z117, Z122, Z123, Z124, Z125.
  • Figure 19 The killing ability of different M-STA/AR-T on target cells, where the histogram on each title on the abscissa represents RFP, Z104, Z106, Z112, Z113, Z117, Z122, Z123 from left to right , Z124, Z125.
  • M-STA/AR multi-target synthetic T cell receptor antigen/antibody receptor
  • the M-STA/AR molecule has two chains, the first chain is the fusion of the first protein domain and the constant region (C ⁇ ) of the T cell receptor alpha chain (TCR ⁇ ), and the second chain is the second protein The domain is fused to the constant region (C ⁇ ) of the T cell receptor beta chain (TCR ⁇ ).
  • the two protein domains and the constant region domains can be arranged and combined to form multiple constructs with different configurations but similar functions.
  • Immunoreceptor Tyrosine-based Activation Motif is a motif that plays a role in signal transduction in TCR molecules, and its conserved sequence is YxxL/V.
  • the intracellular region of CD3 ⁇ , ⁇ , ⁇ , ⁇ chain contains 1 ITAM sequence
  • the intracellular region of CD3 ⁇ chain contains 3 ITAM sequences, so a complete M-STA/AR complex contains a total of 10 ITAM sequences.
  • the ITAM sequence in the cell will be phosphorylated successively, thereby activating downstream signaling pathways and activating transcription factors such as NF- ⁇ , NFAT and AP-1, Initiate the activation of T cells and produce effector functions.
  • M-STA/AR is able to better activate T cells and has significantly lower background activation in the absence of antigenic stimulation compared to conventional chimeric antigen receptor CARs, thus having significant advantages (See Chinese invention patent application number: 201810898720.2). However, further improvements to M-STA/AR are still desired.
  • mutant M-STA/AR mutant M-STA/AR
  • modified M-STA/AR ub-M-STA/AR
  • the M-STA/AR prototype was designed using the constant region sequences of human-derived TCR ⁇ / ⁇ chains (or TCR ⁇ and ⁇ chains) (wild-type human TCR ⁇ constant region, SEQ ID NO:1; wild-type human TCR ⁇ constant region, SEQ ID NO:1) ID NO: 2). Due to the high functional conservation of the constant region sequences (mouse TCRaC-WT, SEQ ID NO:3; mouse TCRbC-WT, SEQ ID NO:4) of human, primate and mouse TCR ⁇ / ⁇ chains, And the key amino acid sequences are the same, so they can be replaced with each other.
  • the inventors replaced the constant region of the M-STA/AR molecule with a mouse sequence to enhance the function of the M-STA/AR molecule after being transferred into human T cells.
  • the inventors carried out a cysteine point mutation to the M-STA/AR molecule to introduce an intermolecular disulfide bond to enhance the bond between the two chains of the M-STA/AR molecule. Pair with each other to reduce mismatch with endogenous TCR.
  • the 48th threonine T is mutated into cysteine C (mouse TCRaC-Cys, SEQ ID NO: 5) in the constant region of the TCR ⁇ chain
  • the 56th serine S is mutated into cysteine in the TCR ⁇ chain constant region C (mouse TCRbC-Cys, SEQ ID NO: 6).
  • three amino acid mutations are carried out in the transmembrane region of the constant region of the TCR ⁇ chain from 111 to 119: change serine S at position 112 to leucine L, and methionine M at position 114 to mutate Leucine I, Glycine G at position 115 are changed to valine V.
  • the overall amino acid sequence of this region is changed from LSVMGLRIL to LLVIVLRIL. This modification is called mouse TCRaC-TM9, and the resulting constant region sequence is SEQ ID NO:7.
  • the ubiquitin activating enzyme, ubiquitin conjugating enzyme and ubiquitin ligase undergo a series of ubiquitinating reactions to ubiquitinize the lysine at the intracellular end of the TCR molecule and the transmembrane region. This will lead to the occurrence of T cell endocytosis, leading to the endocytosis of TCR molecules into the cell, and further degradation by lysosomes, thereby reducing the concentration of TCR molecules on the surface of T cell membranes, resulting in a continuous decline in the activation effect of T cells.
  • the inventors modified the amino acids of the transmembrane regions or intracellular regions of the ⁇ and ⁇ chains in the above-mentioned mut-M-STA/AR molecule, including the intracellular region of the ⁇ chain constant region of the M-STA/AR molecule, and the ⁇ chain
  • the lysine in the transmembrane region of the constant region is mutated into arginine, and the constant region sequence is Mouse TCR ⁇ C-Arg mut (SEQ ID NO: 8) and the constant region sequence is Mouse TCR ⁇ C-Arg mut (SEQ ID NO: 9) respectively.
  • the inventors designed a new structure to carry out the mut-M-STA/AR complex
  • the enhanced mut-M-STA/AR cells can be customized according to the needs, so as to improve the clinical response rate of TCR-T and achieve lasting curative effect.
  • TCR is a specific marker on the surface of all T cells, which are divided into two types: ⁇ TCR and ⁇ TCR, and the corresponding T cells are ⁇ T cells and ⁇ T cells, respectively.
  • the present inventors modified the costimulatory signals of ⁇ -M-STA/AR and ⁇ -M-STA/AR to enhance the performance of ⁇ T cells and ⁇ T cells, respectively.
  • the TCR of ⁇ T cells consists of two chains, TCR ⁇ and TCR ⁇ , accounting for 90%-95% of the total T cells.
  • ⁇ TCR is composed of variable region and constant region, in which the variable region is extremely diverse and plays the role of antigen recognition and binding, while the constant region domain plays the role of structural interaction and signal transduction.
  • the present invention introduces the intracellular end sequences of human co-stimulatory molecule receptors into the C-terminus of the ⁇ -M-STA/AR constant region (see Figure 2 for a schematic diagram of its structure), Explore its effect on M-STA/AR T cell function.
  • the M-STA/AR constant region described in the present invention includes an unmodified WT-M-STA/AR constant region, a cys-M-STA/AR constant region containing additional intermolecular disulfide bonds, a murine Thm-M - the constant region of STA/AR and mut-M-STA/AR incorporating the three modifications described in subsection 1.
  • the co-stimulatory signal transduction structure comprises the intracellular signal transduction domain of CD40, OX40, ICOS, CD28, 4-1BB or CD27, and the sequences are respectively SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 , SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15.
  • the co-stimulatory intracellular domain can be tandem at the C-terminal of the TCR ⁇ chain, or the C-terminal of the ⁇ chain, or the C-terminal of both ⁇ and ⁇ chains (co-M-STA/AR).
  • the costimulatory molecular domain and the C-terminus of the TCR constant region can be connected directly or through G 4 S/(G 4 S)n (G 4 S linker sequences are respectively SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25), or Remove the intracellular end sequence of the TCR molecule and then connect (mouse TCR ⁇ chain constant region (mouse TCRaC-del mut, SEQ ID NO: 26) with cysteine substitution and hydrophobic region modification removed from the intracellular end, removed TCR ⁇ chain constant region (mouse TCR ⁇ C-del
  • the TCR of ⁇ T cells is composed of two chains, TCR ⁇ and TCR ⁇ . According to the type of TCR ⁇ chain, ⁇ T cells can be divided into three subgroups, ⁇ 1, ⁇ 2 and ⁇ 3. Different subgroups have different distributions in the human body. ⁇ T cells primarily recognize non-peptide antigens in a non-MHC-restricted manner and play an important role in pathogen and tumor surveillance. Experiments have proved that co-stimulatory signals such as CD28 or 4-1BB play a very important role in the activation and proliferation of ⁇ T cells. The present inventors introduced the intracellular end sequences of human co-stimulatory molecule receptors into the C-terminals of TCR ⁇ and TCR ⁇ respectively, so as to improve the performance of ⁇ T cells.
  • CD3 subunits include ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain, which form a T cell receptor complex with TCR molecules, and transmit signals from extracellular to intracellular, thereby regulating the state of cells and responding to stimuli.
  • the inventors modified the CD3 molecule and introduced the intracellular domain of the human co-stimulatory molecule receptor into the CD3 ⁇ chain (SEQ ID NO:28), the C-terminus of the ⁇ chain (SEQ ID NO:29), the ⁇ chain (SEQ ID NO:30) and the ⁇ chain (SEQ ID NO:31) (see Figure 4 for a schematic diagram of its structure).
  • the engineered CD3 molecule was expressed in mut-M-STA/AR T cells to improve its function.
  • Cytokines play an important role in T cell proliferation, anti-tumor, differentiation and other functions. Different cytokines combine with their receptors to transmit signals from extracellular to intracellular, thereby regulating the state of cells and responding to stimuli. In addition, studies have shown that the downstream molecule STAT5 (SEQ ID NO: 35) is activated through a cascade reaction at the intracellular end of the IL-2 receptor, thereby enhancing the transcription of T cell proliferation-related molecules and enhancing the proliferation ability of CAR-T cells.
  • STAT5 SEQ ID NO: 35
  • the inventors modified the M-STA/AR molecules to transduce intracellular signals from human cytokine receptors Regions (such as: IL-2 ⁇ receptor intracellular end IL2Rb, SEQ ID NO: 32; IL-7 ⁇ receptor intracellular end; SEQ ID NO: 33; IL-21 receptor intracellular end, SEQ ID NO: 34, etc.
  • human cytokine receptors Regions such as: IL-2 ⁇ receptor intracellular end IL2Rb, SEQ ID NO: 32; IL-7 ⁇ receptor intracellular end; SEQ ID NO: 33; IL-21 receptor intracellular end, SEQ ID NO: 34, etc.
  • the STAT5 activation module can be further connected to the IL-2 ⁇ or IL-7R ⁇ receptor cell through G 4 S Region end (IL-2RbQ, SEQ ID NO: 36; IL-7RbQ, SEQ ID NO: 37) (see Figure 5 for a schematic diagram of its structure).
  • the vectors used in the present invention including viral vectors, plasmid vectors, etc., are purchased from commercial companies or synthesized by commercial companies, and the full-length sequences of these vectors are obtained, and the definite enzyme cutting sites are known.
  • the TCR mentioned in the present invention can be any functional TCR, including WT-M-STA/AR, mut-M-STA/AR, ub-M-STA/AR, co-M-STA used in the present invention /AR, co-linker-M-STA/AR, CK-M-STA/AR, co-CD3-M-STA/AR, etc.
  • gene fragments such as the variable region of TCR, the constant region of TCR, the intracellular region of co-stimulatory molecule receptors, the intracellular signal transduction region of cytokine receptors, the tag sequence and the linker (linker) etc. are all derived from commercial Chemical company synthesis. These gene fragments were connected by means of PCR.
  • the protein structure domain comprising SEQ ID NO:38 (structure domain derived from Nb22), SEQ ID NO:39 (structure domain derived from Nb22), SEQ ID NO:44 (structure derived from FMC) was used. domain) and/or the protein domain shown in SEQ ID NO:45 (domain derived from OFA), and a blank control group expressing only RFP protein shown in SEQ ID NO:40 (Red Fluorescent Protein, RFP) Mock compares experiments.
  • the lentiviral vector used in this statement is pHAGE-EF1 ⁇ -IRES-RFP, the linear vector is obtained by restriction endonuclease Not I/Nhe I, the gene fragment is obtained by synthesis and PCR method, and the complete vector is obtained by homologous recombination method.
  • pHAGE can stably insert the target gene into the genome of the target cell, 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. By stably expressing luciferase in the target cells, the number of target cells can be indicated after adding the substrate, thereby reflecting the response of functional cells to effect on target cells.
  • the PHAGE-EF1A vector carries restriction endonuclease NotI/ClaI cutting sites. Use these two enzymes to cut the vector, use NCBI to obtain luciferase and GFP sequences, and synthesize the fragments through the commercial company Ruiboxingke.
  • the Overlap PCR method first combines the luciferase gene and the GFP gene, and then connects the luciferase-GFP fragment into the pHAGE vector by homologous recombination.
  • the lentivirus was packaged by Lenti-X-293T, the lentiviral liquid was concentrated by the PEG8000 concentration method, and the virus titer was determined by the gradient dilution method, and then infected with the lymphoma cell line Raji, 72 hours after infection, observe whether there are GFP-positive cells by fluorescence microscope, and then sort GFP-positive cells by flow cytometry, select single clones, and build a library for storage.
  • the target cells were co-incubated with the luciferase substrate to detect the expression and detection level of luciferase to determine the expression level.
  • TCR ⁇ - ⁇ -Jurkat cell line was constructed.
  • Obtain the exon sequences of the constant regions of the TCR ⁇ chain and ⁇ chain respectively at NCBI submit the exon 1 sequences of the constant regions of the ⁇ chain and ⁇ chain to the tools.genome-engineering.org website to design guide sequences, synthesize oligo sequences according to the results, and then construct sgRNA-LentiCRISPR lentiviral vector (purchased from Aidi Gene).
  • the guide sequence of the ⁇ chain is linked to LentiCRISPR-puro
  • the guide sequence of the ⁇ chain is linked to LentiCRISPR-BSD.
  • Packaging of sgRNA-LentiCRISPR lentivirus Spread HEK-293T on a 10cm culture dish in advance, and when the cells grow to 80%-90%, add the transfection system to HEK-293T, put the cells back into the 37-degree incubator, and continue nourish. Count as 0 hour; after 12 hours after transfection, change into fresh 10% FBS-DMEM. The virus can be harvested 48 hours and 72 hours after transfection. Centrifuge and filter the medium containing the virus, add PEG8000 to mix, let it stand at 4 degrees for more than 12 hours, then centrifuge at 3500rpm for 30min, discard the supernatant, and resuspend the pellet with an appropriate volume of medium. Freeze at -80°C or use directly.
  • TCR ⁇ - ⁇ -Jurkat cell bank Surviving cells were aspirated, centrifuged and cultured with complete medium to obtain TCR ⁇ - ⁇ -Jurkat cell bank.
  • the TCR ⁇ - ⁇ -Jurkat cell bank was sorted into a 96-well plate by flow cytometry Aria. After two weeks of culture, the single clones that were successfully sorted and cultured were aspirated and expanded for culture.
  • the monoclonal cell lines were identified with antibodies against TCR ⁇ chain and ⁇ chain respectively, and the cell lines with defects in both chains were amplified to obtain endogenous TCR knockout Jurkat-T cell lines.
  • 1640 complete medium for 10-fold dilution If it is a virus stock solution, the virus volume in the first well is 100 ⁇ L, and if it is a concentrated solution, the virus volume in the first well is 1 ⁇ L.
  • the diluted cells were added to the virus wells, 100 ⁇ L/well, mixed, 32°C, 1500rpm, centrifuged for 90min, and cultured in a 37°C, 5% CO 2 incubator for 72h.
  • the cells on the 96-well flat-bottom plate were sucked into the round-bottom 96-well plate, centrifuged at 1800 rpm for 5 min at 4°C, and the supernatant was discarded. After adding 200uL 1 ⁇ PBS, centrifuge at 1800rpm for 5min at 4°C and discard the supernatant. Add 200uL of 4% neutral formaldehyde tissue fixative, store in the dark, and use flow cytometry to detect the infection efficiency.
  • Viruses with the following plasmids packaged by the above method pHAGE-EF1A-IRES-RFP, WT-M-STA/AR, mut-M-STA/AR, co-WT-M-STA/AR( ⁇ -4-1BB- WT, ⁇ -CD27-WT, ⁇ -CD28-WT, ⁇ -ICOS-WT, ⁇ -OX40-WT, ⁇ -OX40-WT, ⁇ -OX40-WT), co-M-STA/AR( ⁇ -4 -1BB, ⁇ -CD27, ⁇ -CD28, ⁇ -ICOS, ⁇ -OX40, ⁇ -OX40, ⁇ -OX40), co-CD3-M-STA/AR(CD3 ⁇ -4-1BB, CD3 ⁇ -CD28, CD3 ⁇ - ICOS, CD3 ⁇ -OX40, CD3 ⁇ -4-1BB, CD3 ⁇ -CD28, CD3 ⁇ -ICOS, CD3 ⁇ -OX40, CD3
  • the Jurkat T cell line was cultured with RPMI1640 medium containing 10% FBS at a culture density of 3 ⁇ 10 5 /ml, the highest not exceeding 3 ⁇ 10 6 /ml, and subcultured every 1-2 days. After cell counting, take the required amount of cells, supplement the medium to adjust to the above density, and place them in a CO 2 incubator for culture.
  • RPMI 1640 medium containing 10% FBS For cell counting, take 1 ⁇ 10 6 /ml cells and replace them by centrifugation, resuspend in 1ml RPMI 1640 medium containing 10% FBS, add to a 24-well plate, add appropriate amount of virus solution, centrifuge at 1500rpm for 90min, and culture in CO 2 box culture. After 12 hours of infection, the medium was completely changed to fresh RPMI 1640 medium containing 10% FBS, and the positive rate was detected within 72 hours.
  • the primary T cells were cultured in X-VIVO medium containing 10% FBS and 100IU/ml IL-2.
  • the initial culture density was 1 ⁇ 10 6 /ml, and CD3 and RetroNectin r-Fibronectin ( The final concentration is 5ug/ml) in pre-coated well plates.
  • the culture density is 5 ⁇ 10 5 /ml, the highest not exceeding 3 ⁇ 10 6 /ml, and subcultured every 1-2 days.
  • the cells were blown evenly and counted, centrifuged at 5 ⁇ 10 5 /ml, discarded the supernatant, the staining solution was PBS+2%FBS+2mM EDTA, added the corresponding antibody for incubation, incubated for 30min, then added PBS to wash twice, On-board testing.
  • the target cells are anti-Cq1B cells (Raji-luciferase), anti-Dsg1B cells, anti-Dsg3B cells, anti-MPO B cells, anti-PR3B cells and primary T cells as suspension cells.
  • the target cell culture medium can be mixed and centrifuged.
  • the specific steps are: use the packaged and purified WT-M-STA/AR and mut-M-STA/AR T viruses to infect primary T cells, use flow cytometry to detect the infection efficiency the day before the co-cultivation, and determine the functional cells and target cells For the ratio, the ratio of 1:1 is generally used, and the total number of T cells is calculated according to the infection efficiency.
  • the target cells are generally used at a rate of 1 ⁇ 10 5 /well (96-well plate).
  • the target antibody of the present invention is generally a cell surface receptor, which can be used to activate M-STA/AR-T cells to detect the function of T cells. Usually 1 ⁇ 10 5 /well positive T cells are added, centrifuged, and activated for 24 hours to collect T cells or target cells to detect T cell function.
  • Target cell Raji-luciferase and primary T cells are suspension cells. When co-incubating, take the corresponding number of cells and use the target cell medium to mix and centrifuge. The specific steps are: use the packaged and purified co-M-STA/AR and mut-M-STA/AR T viruses to infect primary T cells, use flow cytometry to detect the infection efficiency the day before the co-cultivation, and determine the functional cells and target cells Ratio, generally use the ratio of 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8 for co-incubation, in addition to the co-incubation difference detection in time, the general detection time Points are 6h, 12h, 24h, 36h, 48h.
  • the target cell Raji-luciferase was incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation and IL-2 secretion, and after 7 days by flow cytometry
  • the positive T cells were obtained by cytometer sorting, and after resting for two days without antigen stimulation, they were co-incubated with target cells for 24 hours to detect the killing of T cells.
  • the amount of target cells generally used was 1 ⁇ 105 /well (96-well plate ).
  • the target antibody of the present invention is generally a cell surface receptor, which can be used to activate M-STA/AR-T cells to detect the function of T cells, usually add 1 ⁇ 10 5 /well positive T cells, centrifuge, and activate after 24h Collect cell suspension or culture supernatant to detect T cell function, or detect T cell killing function after activation for 6h, 12h, 24h, 36h, 48h or 7 days.
  • 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 (Cat. No. 88-7025, 88-7346, 88-7316 respectively) .
  • the specific steps are: dilute 10X Coating Buffer to 1X with ddH 2 O, add coating antibody (250X), mix well and add to 96-well plate, 100 ⁇ l/well. After sealing with plastic wrap, place at 4°C overnight, wash 3 times with 1X PBST (0.05% Tween 20 in 1X PBS), 260 ⁇ l/well each time, dilute 5XELISA/ELISPOT Diluent to 1X with ddH2O, add to 96-well plate, 200 ⁇ l/well , stand at room temperature for 1h. Wash once with PBST, dilute the standard song (range: 2-250, 4-500, 4-500), and dilute the sample 20-50 times with 1xDiluent.
  • T cells During the activation of T cells, a large number of cytokines are released to help T cells kill target cells or promote the expansion of T cells themselves.
  • the mut-M-STA/AR-T cells and Raji-luciferase cells of each structure were initially co-cultured according to the effect-to-target ratio of 1:3, which was recorded as day 0, and then the cells were collected on day 1 and day 7 respectively Perform flow analysis.
  • the medium used was 1640 complete medium without IL2, and the initial TCR T cells were 1 ⁇ 10 5 cells.
  • the samples at each time point were incubated independently, and the remaining co-incubated samples were half-changed every other day. , supplemented with target cells.
  • the cells used for flow cytometric analysis were first stained with anti-human CD3 antibody, and the specified volume of cells was collected and recorded on the machine, and the number and proportion of T cells in the system were obtained through conversion.
  • the proliferation of mut-M-STA/AR in which the intracellular domain of OX40 is separately connected in series with the C-terminus of TCR ⁇ chain ( ⁇ -OX40) or ⁇ chain ( ⁇ -OX40).
  • T cells where the intracellular domain of the co-stimulatory molecule OX40 is linked to mut-M-STA/AR at effector-to-target ratios of 1:2 and 1:4.
  • Target cell Raji-luciferase and primary T cells are suspension cells. When co-incubating, take the corresponding number of cells and use the target cell medium to mix and centrifuge. The specific steps are: use the packaged and purified co-M-STA/AR and mut-M-STA/AR T viruses to infect primary T cells, use flow cytometry to detect the infection efficiency the day before the co-cultivation, and determine the functional cells and target cells Ratio, generally use 1:1 or 2:1 ratio for co-incubation, in addition, the target cell Raji-luciferase was co-incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation. The total number of T cells is calculated according to the infection efficiency, and the amount of target cells generally used is 1 ⁇ 10 5 /well (96-well plate).
  • the target antibody of the present invention is generally a cell surface receptor, which can be directly used for the activation of T cells to detect the function of T cells. Usually, 1 ⁇ 10 5 /well positive T cells are added, centrifuged, and after 24 hours of activation, the cell suspension or culture supernatant is collected to detect the function of T cells.
  • the results of the T cell killing function test by luciferase detection method showed that the costimulatory intracellular domain was connected in series at the C-terminus of the CD3 ⁇ or CD3 ⁇ or CD3 ⁇ or CD3 ⁇ chain, and was co-expressed on mut-M-STA/ART.
  • the effect-to-target ratio of cells and target cells is 1:1.
  • T cells During the activation of T cells, a large number of cytokines are released to help T cells kill target cells or promote the expansion of T cells themselves.
  • the mut-M-STA/AR-T cells and Raji-luciferase cells of each structure were initially co-cultured according to the effect-to-target ratio of 1:3, which was recorded as day 0, and then the cells were collected on day 1 and day 7 respectively Perform flow analysis.
  • the medium used was 1640 complete medium without IL2, and the initial TCR T cells were 1 ⁇ 10 5 cells.
  • the samples at each time point were incubated independently, and the remaining co-incubated samples were half-changed every other day. , supplemented with target cells.
  • the cells used for flow cytometric analysis were first stained with anti-human CD3 antibody, and the specified volume of cells was collected and recorded on the machine, and the number and proportion of T cells in the system were obtained through conversion.
  • Target cell Raji-luciferase and primary T cells are suspension cells. When co-incubating, take the corresponding number of cells and use the target cell medium to mix and centrifuge. The specific steps are: use the packaged and purified co-M-STA/AR and mut-M-STA/AR T viruses to infect primary T cells, use flow cytometry to detect the infection efficiency one day before co-cultivation, and determine the functional cells and target cells Ratio, generally use 1:1 or 2:1 ratio for co-incubation, in addition, the target cell Raji-luciferase was co-incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation. The total number of T cells is calculated according to the infection efficiency, and the amount of target cells generally used is 1 ⁇ 10 5 /well (96-well plate).
  • the target antibody of the present invention is generally a cell surface receptor, which can be directly used for the activation of T cells to detect the function of T cells, usually add 1 ⁇ 10 5 /well of positive T cells, centrifuge, and collect the cell suspension after 24 hours of activation Or culture supernatant to detect T cell function.
  • T cell killing function test was detected by luciferase detection method.
  • 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 (Cat. No. 88-7025, 88-7346, 88-7316 respectively) .
  • 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. After sealing with plastic wrap, place at 4°C overnight, 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
  • T cells During the activation of T cells, a large number of cytokines and other chemokines are released, and the signals are transduced into the nucleus through cytokine or chemokine receptors to regulate the changes of T cell differentiation.
  • the differentiation of T cells is from naive T cells (naive) to central memory T cells (Tcm) to effector memory T cells (Tem) and finally to effector T cells (Teff).
  • the proliferation and persistence of T cells in vivo are affected by the number of T cells that differentiate into central memory T cells (Tcm) and effector memory T cells (Tem).
  • Tcm central memory T cells
  • Tem effector memory T cells
  • the classification of memory T cells is roughly divided into T cells in a stem cell state, central memory T cells, and effector memory T cells.
  • the proportion of central memory T cell differentiation affects the persistent killing effect of T cells in vivo.
  • the ratio of naive T cells to effector T cells affects the killing effect and persistence of T cells on tumors in vivo.
  • the differentiation of T cells can be known by detecting the expression of CD45RA and CCR7 on the surface of T cells by flow cytometry. After co-incubating T cells with target cells for 7 days, centrifuge, stain T cells with anti-human anti-human-CD45RA-Percp-cy5.5 and anti-human anti-human-CCR7-APC flow antibodies for 30 minutes, centrifuge, After washing once with PBS, fix with 4% paraformaldehyde solution, and detect T cell differentiation by flow cytometry.
  • Target cell Raji-luciferase and primary T cells are suspension cells. When co-incubating, take the corresponding number of cells and use the target cell medium to mix and centrifuge. The specific steps are: use the packaged and purified co-M-STA/AR and mut-M-STA/AR T viruses to infect primary T cells, use flow cytometry to detect the infection efficiency the day before the co-cultivation, and determine the functional cells and target cells Ratio, generally use 1:1 or 2:1 ratio for co-incubation, in addition, the target cell Raji-luciferase was co-incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation. The total number of T cells is calculated according to the infection efficiency, and the amount of target cells generally used is 1 ⁇ 10 5 /well (96-well plate).
  • the target of the present invention is generally cell surface receptors, which can be directly used for the activation of T cells to detect the function of T cells, usually add 1 ⁇ 10 5 /well of positive T cells, centrifuge, and collect the cell suspension after activation for 24 hours Or culture supernatant to detect T cell function.
  • T cells During the activation of T cells, a large number of cytokines 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.
  • T cells After T cells are stimulated with target cells or antigens, T cells are collected, centrifuged, and the supernatant is taken.
  • the IFN- ⁇ and IL-2 ELISA kits used are Human IL-2 Uncoated ELISA and Human IFN- ⁇ Uncoated ELISA (catalogue numbers 88-7025, 88-7346, and 88-7316, respectively).
  • 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. After sealing with plastic wrap, place at 4°C overnight, 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
  • T cells During the activation of T cells, a large number of cytokines and other chemokines are released, and the signals are transduced into the nucleus through cytokine or chemokine receptors to regulate the changes of T cell differentiation.
  • the proliferation and persistence of T cells in vivo is affected by the number of T cells differentiated into memory T cell types.
  • the classification of memory T cells is roughly divided into T cells in a stem cell state, central memory T cells, and effector memory T cells.
  • the differentiation of T cells can be known by detecting the expression of CD45RA and CCR7 on the surface of T cells by flow cytometry.
  • T cells After co-incubating T cells with target cells for 7 days, centrifuge, stain T cells with anti-human anti-human-CD45RA-Percp-cy5.5 and anti-human anti-human-CCR7-APC flow antibodies for 30 minutes, centrifuge, After washing once with PBS, fix with 4% paraformaldehyde solution, and detect T cell differentiation by flow cytometry.
  • Target cell Raji-luciferase and primary T cells are suspension cells. When co-incubating, take the corresponding number of cells and use the target cell medium to mix and centrifuge. The specific steps are: use the packaged and purified co-M-STA/AR and mut-M-STA/AR T viruses to infect primary T cells, use flow cytometry to detect the infection efficiency the day before the co-cultivation, and determine the functional cells and target cells Ratio, generally use 1:1 or 2:1 ratio for co-incubation, in addition, the target cell Raji-luciferase was co-incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation. The total number of T cells is calculated according to the infection efficiency, and the amount of target cells generally used is 1 ⁇ 10 5 /well (96-well plate).
  • the target of the present invention is generally cell surface receptors, which can be directly used for the activation of T cells to detect the function of T cells, usually add 1 ⁇ 10 5 /well of positive T cells, centrifuge, and collect the cell suspension after activation for 24 hours Or culture supernatant to detect T cell function.
  • T cells During the activation of T cells, a large number of cytokines 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.
  • T cells After T cells are stimulated with target cells or antigens, T cells are collected, centrifuged, and the supernatant is taken.
  • the IFN- ⁇ and IL-2 ELISA kits used are Human IL-2 Uncoated ELISA and Human IFN- ⁇ Uncoated ELISA (catalogue numbers 88-7025, 88-7346, and 88-7316, respectively).
  • 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. After sealing with plastic wrap, keep 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 ddH 2 O, Add to 96-well plate, 200 ⁇ l/well, and let stand at room temperature for 1h.
  • 1X PBST also known as Wash Buffer, 0.05% Tween 20 in 1X PBS
  • T cells During the activation of T cells, a large number of cytokines and other chemokines are released, and the signals are transduced into the nucleus through cytokine or chemokine receptors to regulate the changes of T cell differentiation.
  • the proliferation and persistence of T cells in vivo is affected by the number of T cells differentiated into memory T cell types.
  • the classification of memory T cells is roughly divided into T cells in a stem cell state, central memory T cells, and effector memory T cells.
  • the differentiation of T cells can be known by detecting the expression of CD45RA and CCR7 on the surface of T cells by flow cytometry.
  • T cells After co-incubating T cells with target cells for 7 days, centrifuge, stain T cells with anti-human anti-human-CD45RA-Percp-cy5.5 and anti-human anti-human-CCR7-APC flow antibodies for 30 minutes, centrifuge, After washing once with PBS, fix with 4% paraformaldehyde solution, and detect T cell differentiation by flow cytometry.
  • hM-STA/AR refers to M-STA/AR comprising a human TCR constant region.
  • hmct M-STA/AR refers to M-STA/AR comprising the cysteine substitution shown in Example 1 and the constant region modification of the transmembrane region.
  • the constant region of the mouse TCRa chain is hmct M-STA/AR TCRa (SEQ ID NO:41), and the constant region of the mouse TCRb chain is hmct M-STA/AR TCRb (SEQ ID NO:6).
  • the rearrangement scheme of the 25 amino acids at the N-terminal of the constant region of the TCR ⁇ chain (the mouse sequence is DLRNVTPPKVSLFEPSKAEIANKQK), through the analysis of amino acid properties, it was found that the mouse and human sequences only belonged to homogeneous amino acid substitutions at the R3K and L12V sites, while at T6F, K9E
  • the , S11A, K17E, A21S, N22H and K23T sites are all substitutions of different amino acid properties, so it can be considered that the protein properties near this site are not conserved, and can be modified without affecting the function.
  • the 1-16 amino acid sequence was retained and humanized, and the 17, 21-25 amino acids were deleted.
  • the TCRa chain constant region obtained through N-terminal rearrangement is Nrec M-STA/AR TCRa (SEQ ID NO: 42), and the TCRb chain constant region is Nrec M-STA/AR TCRb (SEQ ID NO: 43).
  • the original unoptimized M-STA/AR structure (human TCR ⁇ / ⁇ M-STA/AR, hM-STA/AR) was selected, and the modified hmct M-STA/AR with murineization of C region, Cystine modification and transmembrane modification, and Nrec M-STA/AR with N-terminal modification on hmct M-STA/AR.
  • the present invention introduces the intracellular end sequences of human co-stimulatory molecule receptors into the C-terminus of the M-STA/AR constant region (the structure schematic diagram is shown in Figure 7), to explore its effect on M-STA/AR-T cell function.
  • the M-STA/AR constant region structure described in the present invention selects the above-mentioned M-STA/AR structure (human TCR ⁇ / ⁇ M-STA/AR, hM-STA/AR) that was originally not optimized, and on this basis includes Modified hmct M-STA/AR, and Nrec M-STA/AR with N-terminal modification on hmct M-STA/AR.
  • the co-stimulatory signal transduction structure includes intracellular signal transduction domains of CD40, OX40, ICOS, CD28, 4-1BB, and CD27.
  • modification can occur in TCR ⁇ chain, ⁇ chain, and ⁇ ⁇ chain.
  • co-stimulatory Molecular modification occurs on the TCR ⁇ chain, and its structural schematic diagram is shown in Figure 8.
  • a sequence such as Nb22 (anti-CD22-9) of SEQ ID NO:39 and a sequence such as OFA (anti-CD20) of SEQ ID NO:45 were selected as part of the selection of the first protein domain, such as Nb22 of SEQ ID NO:38 ( Anti-CD22) and FMC63 (anti-CD19) shown in SEQ ID NO: 44 as part of the second protein domain, wherein OFA is directly connected to the first constant region, FMC63 is directly connected to the second constant region, OFA and Nb123 through a linker (G 4 S)n connection, FMC63 and Nb22 are connected through a linker (G 4 S)n, wherein n is 1, 2 or 3.
  • M-STA/AR contains two polypeptide chains, and the Nb123-OFA domain is fused with the TCRbC chain of hM-STA/AR/hmct M-STA/AR/Nrec M-STA/AR structure as the first polypeptide segment, or The Nb22-FMC63 domain is fused with the TCRaC chain of hM-STA/AR hmct M-STA/AR Nrec M-STA/AR as the second polypeptide segment. Both use GM-CSFR signal peptide signal peptide.
  • the two chain gene sequences of hM-STA/AR/hmct M-STA/AR/Nrec M-STA/AR are linked by the Furin-SGSG-p2A protease cleavage site polypeptide segment, and the two polypeptide chains will be transcribed and translated together The protein is reached, and then it is cleaved by the proteases corresponding to furin and p2A, and finally becomes two independent protein chains.
  • the entire gene was inserted into the lentiviral expression vector pHAGE through the restriction endonuclease sites NheI and NotI. This vector carries ampicillin resistance, EF1 ⁇ promoter and IRES-RFP fluorescent reporter gene.
  • the following plasmids were obtained by cloning assembly, transformation, sequencing, and plasmid extraction of gene fragments: Z13-hM-STA/AR, Z13-hmct M-STA/AR, Z13-Nrec M-STA/AR.
  • M-STA/AR vectors containing co-stimulatory factors in three M-STA/AR vectors targeting anti-C1q antibodies Z13-hM-STA/AR, Z13-hmct M-STA/AR, Z13-Nrec M- On the basis of STA/AR, costimulatory factors CD40, OX40, ICOS, CD28, 41BB, and CD27 were constructed, and the above sequences were obtained by gene synthesis.
  • connection mode of the co-stimulatory factor of the present invention is as follows: Add the same co-stimulatory factors to TCR ⁇ and ⁇ chains at the same time.
  • Z13-hM-STA/AR-CD40, Z13-hM-STA/AR-OX40, Z13-hM-STA/AR-ICOS, Z13-hM-STA/AR-CD28, Z13-hM-STA/AR were constructed -41BB, Z13-hM-STA/AR-CD27, Z13-hmct M-STA/AR-CD40, Z13-hmct M-STA/AR-OX40, Z13-hmct M-STA/AR-ICOS, Z13-hmct M -STA/AR-CD28, Z13-hmct M-STA/AR-41BB, Z13-hmct M-STA/AR-CD27, Z13-Nrec M-STA/AR-CD40, Z13-Nrec M-STA/AR-OX40 , Z13-Nrec M-STA/AR-ICOS, Z13-Nrec M-STA/AR
  • Uninfected T cells NC group
  • the number of T cells adjusted according to the positive rate of RFP was 4 ⁇ 10 5
  • the number of RAJI-luc cells was 4 ⁇ 10 5
  • the number of RAJI-luc cells was 4 ⁇ 10 5
  • the results showed that hM-STA/AR was significantly lower than hmct M-STA/AR and Nrec M-STA/AR, and Nrec M-STA/AR had the highest killing efficiency.
  • the method of the present application constructs M-STA/AR targeting CD22 (Z3, see the left side of Figure 11 for its structure example), wherein Nb22 is directly connected to the first constant region, and cmyc is directly connected to the N-terminal of Nb22;
  • the method of the present application constructs M-STA/AR targeting CD123 (Z4, see Figure 11 for an example of its structure), wherein Nb123 is directly connected to the first constant region, and cmyc is directly connected to the N-terminal of Nb123;
  • the constructed cells are Z3-T, Z4-T, Z13-T, dual-STAR-T, loop-CAR-T, respectively.
  • FIG. 12 illustrates the LDH index detected to characterize the cell killing effect after co-incubation with target cells. It can be seen that Z13, DS, and Loop-car all exhibit a certain killing effect; Figure 13 shows that after co-incubation with target cells, the detected LDH index is used to characterize the cell killing effect.
  • Z13, DS, and Loop-car all exhibit a certain killing effect
  • Figure 14 shows that after co-incubation with target cells, the detected LDH index is used to characterize the cell killing effect. It can be seen that Z13, Z3, and Z4 all exhibit a certain killing effect
  • Figure 15 shows that after co-incubating with target cells, the detected LDH index is used to characterize the cell killing effect. It can be seen that Z13, Z3, and Z4 have shown certain killing effects.
  • NSG immunodeficient mice were used as the model.
  • the mouse genotype is NOD-Prkdcem26Il2rgem26/Nju, lacks T cells, B cells, NK cells, and its macrophages and dendritic cells are also defective.
  • NSG mice are currently the most complete immunodeficiency mouse strains, because they will not produce rejection reactions to transplanted tumors and T cells, and thus are widely used in preclinical research on T cell therapy.
  • female NSG mice aged 6-8 weeks were used, and the weight difference of mice in each batch of experiments was controlled within 2 g.
  • Mice were housed in individually ventilated cages within specific pathogen-free (SPF) clean-grade barriers, provided with a normal diet and drinking water with an acidic pH to prevent pathogen contamination.
  • SPPF pathogen-free
  • T cells cause side effects on experimental animals. Whether the reinfused T cells have significant toxicity can be evaluated by observing the behavioral state of the mice, conducting pathological analysis of the mice, and analyzing slices of the vital organs of the mice. At the same time, by analyzing the infiltration of T cells in 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 level of cytokines in the blood of mice, such as IL-2, IFN- ⁇ , TNF ⁇ or IL-6, etc., it can be judged whether T cells will cause a systemic cytokine storm.
  • cytokines such as IL-2, IFN- ⁇ , TNF ⁇ or IL-6, etc.
  • Example 2 M-STA/AR targeting LILRB4 and CD123 (Z112, Z113, Z117, Z122, Z123, Z124, and Z125 structure examples are shown in Figure 16), wherein, Nb123 (anti-CD123) and NbLILRB4 (anti-LILRB4)
  • the connection between is through G 4 S/(G 4 S)n connection or through EA 3 K/(EA 3 K)n connection;
  • the method of the present application constructs M-STA/AR targeting CD123 (Z104, see Figure 17 for a structural example diagram), wherein Nb123 is directly connected to the second constant region;
  • the method of the present application constructs M-STA/AR targeting LILRB4 (Z106, see Figure 18 for an example of its structure), wherein NbLILRB4 is directly connected to the second constant region; wherein, the sequence of the Nb123 antibody is as shown in SEQ ID NO:55 Shown, the sequence of NbLILRB4 antibody is shown in SEQ ID NO:56.
  • the constructed cells were Z104-T, Z106-T, Z112-T, Z113-T, Z117-T, Z122-T, Z123-T, Z124-T, Z125-T.
  • step 2 The cells in step 2 were co-incubated with THP1, CD123KO-THP1 (CD123 was knocked out by CRISPR in THP1 cells), and RB4KO-THP1 cells (LILRB4 was knocked out by CRISPR in THP1 cells) to evaluate the constructed T cell killing ability.
  • THP1, CD123KO-THP1 CD123 was knocked out by CRISPR in THP1 cells
  • RB4KO-THP1 cells LILRB4 was knocked out by CRISPR in THP1 cells
  • Figure 19 illustrates that after co-incubation with target cells, the index of detected luciferase expression is used to characterize the cell killing effect. It can be seen that all constructs displayed killing ability when co-incubated with THP1 cells. When co-incubated with THP1 lacking CD123, Z104-T single-targeting CD123 had no killing ability; when co-incubating with THP1 lacking LILRB4, Z106-T single-targeting LILRB4 also had no killing ability. While other constructs expressing dual targets showed killing ability.
  • NSG immunodeficient mice were used as the model.
  • the mouse genotype is NOD-Prkdcem26Il2rgem26/Nju, lacks T cells, B cells, NK cells, and its macrophages and dendritic cells are also defective.
  • NSG mice are currently the most complete immunodeficiency mouse strains, because they will not produce rejection reactions to transplanted tumors and T cells, and thus are widely used in preclinical research on T cell therapy.
  • female NSG mice aged 6-8 weeks were used, and the weight difference of mice in each batch of experiments was controlled within 2 g.
  • Mice were housed in individually ventilated cages within specific pathogen-free (SPF) clean-grade barriers, provided with a normal diet and drinking water with an acidic pH to prevent pathogen contamination.
  • SPPF pathogen-free
  • T cells cause side effects on experimental animals. Whether the reinfused T cells have significant toxicity can be evaluated by observing the behavioral state of the mice, conducting pathological analysis of the mice, and analyzing slices of the vital organs of the mice. At the same time, by analyzing the infiltration of T cells in 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 mouse blood, such as IL-2, IFN- ⁇ , TNF ⁇ or IL-6, etc., it can be judged whether T cells will cause a systemic cytokine storm.
  • cytokines in the mouse blood such as IL-2, IFN- ⁇ , TNF ⁇ or IL-6, etc.
  • Figure 20 shows the effect of RFP-T, Z104-T, Z106-T, Z112-T, Z113-T, Z117-T, Z122-T, Z123-T, Z124-T, Z125-T on tumors in NSG mice Ability to kill and inhibit.
  • RFP-T as a control, the other groups all showed tumor inhibitory effects, and the effect of dual-target T was also significantly better than that of single-target. Further comparison shows that Z124-T and Z125-T have relatively better tumor inhibitory effect.

Abstract

La présente invention concerne un récepteur de cellules T (TCR) synthétique multicible et un récepteur antigène/anticorps (M-STA/AR), une cellule T contenant la M-STA/AR et l'utilisation de la M-STA/AR et de la cellule T.
PCT/CN2022/103054 2021-06-30 2022-06-30 Récepteur de cellules t synthétique multicible et récepteur antigène/anticorps et application correspondante WO2023274382A1 (fr)

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