WO2024254775A1 - Récepteur antigénique chimérique comprenant un nouveau domaine de costimulation et utilisation associée - Google Patents

Récepteur antigénique chimérique comprenant un nouveau domaine de costimulation et utilisation associée Download PDF

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WO2024254775A1
WO2024254775A1 PCT/CN2023/100067 CN2023100067W WO2024254775A1 WO 2024254775 A1 WO2024254775 A1 WO 2024254775A1 CN 2023100067 W CN2023100067 W CN 2023100067W WO 2024254775 A1 WO2024254775 A1 WO 2024254775A1
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cell
cells
chimeric antigen
antigen receptor
domain
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Chinese (zh)
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周亚丽
汤颖秀
郑冬
任江涛
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Hongkong Bioheng Biotech Ltd
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Hongkong Bioheng Biotech Ltd
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Priority to PCT/CN2023/100067 priority Critical patent/WO2024254775A1/fr
Priority to CN202380098273.1A priority patent/CN121419997A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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 belongs to the field of immunotherapy. More specifically, the present invention relates to a chimeric antigen receptor comprising a novel co-stimulatory domain, and an engineered immune cell comprising such a chimeric antigen receptor and uses thereof.
  • Adoptive cell therapy refers to the in vitro activation and expansion of autologous immune cells, which are then reinfused back into the patient's body to directly kill tumors or stimulate the body's immune response to kill tumor cells.
  • Common adoptive cell therapies include non-specific immunotherapies such as LAK, CIK and NK, and specific immunotherapies such as TIL, CAR-T and TCR-T.
  • CAR-T cell therapy has attracted widespread attention due to its outstanding performance in the field of hematological tumors.
  • CAR-T cell therapy In view of the rapid development of CAR-T cell therapy, a series of new cell therapies with CAR as the core technology have gradually emerged, such as CAR-NK, CAR-NKT, CAR-Treg, CAR- ⁇ T, etc., which have jointly led CAR cell therapy to become a major trend in tumor cell immunotherapy.
  • Chimeric antigen receptor is a cell surface receptor protein that gives immune cells the ability to target specific antigen proteins. Structurally, CAR consists of at least three functional domains: antigen binding domain, transmembrane domain, and intracellular signaling domain. The intracellular signaling domain is composed of co-stimulatory domain and/or primary signaling domain. The design of the CAR structure has gone through the following process: The first-generation CAR has only one intracellular signaling component CD3 ⁇ or Fc ⁇ RI molecule.
  • the second-generation CAR introduces a co-stimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, ICOS, which has greatly improved its function compared with the first-generation CAR, further strengthening CAR -T cell persistence and tumor cell killing ability;
  • the third generation CAR contains two co-stimulatory molecules, CD28 and 4-1BB, CD28 and OX40 are the most common co-stimulatory molecules in the third generation CAR;
  • the fourth generation CAR is mainly designed to overcome the inhibition of the tumor immune microenvironment, and pro-inflammatory cytokines (IL-12, IL-15, IL-18, etc.) and co-stimulatory ligands are introduced during design;
  • the fifth generation CAR-T adds co-stimulatory
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide, which generally includes a ligand binding domain (e.g., an antibody or an antigen binding portion thereof), a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain, each of which is connected by a linker.
  • CAR can redirect the specificity and reactivity of T cells and other immune cells to the selected target in a non-MHC restricted manner using the antigen binding properties of antibodies.
  • Non-MHC restricted antigen recognition gives CAR-expressing immune cells the ability to recognize antigens independent of antigen processing, thereby bypassing the main mechanism of tumor escape.
  • the present invention provides a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, wherein the co-stimulatory domain comprises a mutated CD27 intracellular region.
  • the costimulatory domain is an intracellular functional signal transduction domain from a costimulatory molecule, which includes the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof.”
  • Costimulatory molecule refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (eg, proliferation) of a T cell.
  • Traditional chimeric antigen receptors generally use CD28 and/or 4-1BB as costimulatory domains.
  • the chimeric antigen receptor of the present invention includes a novel costimulatory domain, i.e., a mutated CD27 intracellular region.
  • CD27 is a dimeric transmembrane glycoprotein composed of monomers with a relative molecular weight of 55,000 Da connected by disulfide bonds. It is an important member of the tumor necrosis factor (TNF) superfamily. CD27 is widely expressed on the surface of primitive T cells and memory T cells. CD27 mainly activates the downstream TRAF2 and TRAF5 signaling pathways by binding to its ligand CD70, further inducing the activation of NF- ⁇ B and JNK signaling pathways, and plays an important regulatory role in the activation, proliferation and apoptosis of immune cells, especially T cells.
  • TNF tumor necrosis factor
  • the mutated CD27 intracellular region of the present invention comprises at least one lysine mutation (i.e., a deletion, substitution or insertion of an amino acid), for example, 1 lysine mutation, 2 lysine mutations, 3 lysine mutations.
  • the mutated CD27 intracellular region of the present invention comprises at least one lysine substitution. More preferably, the substitution is to replace lysine with other basic amino acids, such as arginine or histidine.
  • the mutated CD27 intracellular region of the present invention comprises at least 1 (e.g., 1, 2, 3, 4, 5 or 6) amino acid deletions at the 1st to 6th amino acid positions near the C-terminus.
  • the mutated CD27 intracellular region of the present invention comprises at least 1 amino acid (e.g., 1, 2, 3, 4, 5 or 6) continuous deletions at the 1st to 6th amino acid positions near the C-terminus.
  • the mutated CD27 intracellular region of the present invention comprises 4 continuous deletions of amino acids at the 1st to 4th amino acid positions near the C-terminus.
  • the CD27 intracellular region in the present invention has undergone the above-mentioned mutation, which can significantly increase the proliferation level of CAR cells compared to the wild-type CD27 intracellular region, thereby improving the inhibitory effect on tumors.
  • the mutated CD27 intracellular region described in the present invention has the amino acid sequence shown in SEQ ID NO: 2 or 3 or has at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 3.
  • the chimeric antigen receptor of the present invention may also comprise one or more additional costimulatory domains selected from the costimulatory signaling domains of the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM) , CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM or ZAP70, preferably selected from 4-1BB, CD28, CD27, OX40, CD278 and any combination thereof, more preferably Selected from 4-1BB and CD
  • the CAR of the present invention includes a mutated CD27 intracellular region as a costimulatory domain. In some embodiments, the CAR of the present invention includes a mutated CD27 intracellular region and a 4-1BB intracellular region as a costimulatory domain. In some embodiments, the CAR of the present invention includes a mutated CD27 intracellular region and a CD28 intracellular region as a costimulatory domain.
  • the 4-1BB costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17 or 18;
  • the CD28 costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.
  • ligand binding domain refers to any structure or functional variant thereof that can bind to a ligand (e.g., an antigen).
  • the ligand binding domain can be an antibody structure, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, and antigen-binding fragments thereof.
  • the ligand binding domain includes but is not limited to complete antibodies, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, linear antibodies, sdAb (e.g., VH or VL), nanobodies (Nanobody, Nb), recombinant fibronectin domains, anticalins, and DARPINs, etc., preferably selected from Fab, scFv, sdAb, and nanobodies.
  • the ligand binding domain can be monovalent or divalent, and can be a monospecific, bispecific, or multispecific antibody.
  • Single-chain antibodies or “scFvs” are antibodies formed by connecting the variable regions of the heavy chain (VH) and the variable regions of the light chain (VL) of an antibody via a linker.
  • the optimal length and/or amino acid composition of the linker can be selected.
  • the length of the linker significantly affects the folding and interaction of the variable regions of the scFv. In fact, if a shorter linker (e.g., between 5-10 amino acids) is used, intrachain folding can be prevented.
  • a shorter linker e.g., between 5-10 amino acids
  • the scFv may comprise VH and VL linked in any order, such as VH-linker-VL or VL-linker-VH.
  • the linker has the amino acid sequence shown in SEQ ID NO: 30 or 31.
  • telomere sequence a variant that substantially comprises the amino acid sequence of the parent but contains at least one amino acid modification (i.e., substitution, deletion or insertion) compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence.
  • its functional fragment is its antigen-binding portion.
  • a “functional variant” or “functional fragment” has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the parent amino acid sequence, and retains the biological activity of the parent amino acid, such as binding activity.
  • sequence identity refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, the identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity.
  • Those skilled in the art will recognize that some algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and ClustalW.
  • the choice of ligand binding domain depends on the cell surface marker on the target cell associated with the specific disease state to be identified, such as a tumor-specific antigen or a tumor-associated antigen. Therefore, in some embodiments, the ligand binding domain of the present invention is selected from the following One or more targets of: ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD4 0.
  • the ligand binding domain binds to a target selected from the group consisting of CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor ⁇ , CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA, and GPRC5D, more preferably CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, CD7, CD70, and BCMA.
  • a target selected from the group consisting of CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor ⁇ , CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2,
  • the CAR of the present invention can be designed to include a ligand binding domain specific for the antigen.
  • a CD19 antibody can be used as a ligand binding domain of the present invention.
  • the chimeric antigen receptor of the present invention targets CD19.
  • the chimeric antigen receptor of the present invention comprises an anti-CD19 antibody.
  • Antibodies targeting CD19 known in the art can be used in the present invention.
  • the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 10; wherein the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 11.
  • the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:4, CDR2-H as shown in SEQ ID NO:5, and CDR3-H as shown in SEQ ID NO:6, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO:7, and CDR2-L as shown in SEQ ID NO:8 and CDR3-L is shown in SEQ ID NO:9.
  • the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 10, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11.
  • the antibody targeting CD19 in the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 10 and a light chain variable region as shown in SEQ ID NO: 11.
  • the antibody targeting CD19 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12.
  • the antibody targeting CD19 is as shown in SEQ ID NO: 12.
  • transmembrane domain refers to a polypeptide structure that enables chimeric antigen receptors to be expressed on the surface of immune cells (e.g., lymphocytes, NK cells, or NKT cells), and guides immune cells to respond to target cells.
  • the transmembrane domain can be natural or synthetic, or it can be derived from any membrane-bound protein or transmembrane protein. When the chimeric antigen receptor is bound to the target antigen, the transmembrane domain can carry out signal transduction.
  • transmembrane domains in the present invention can be derived from, for example, TCR alpha chains, TCR beta chains, TCR gamma chains, TCR delta chains, CD3 zeta subunits, CD3 epsilon subunits, CD3 gamma subunits, CD3 delta subunits, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof.
  • the transmembrane domain can be synthetic and can mainly include hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from CD8 ⁇ chain or CD28, and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:13-15.
  • intracellular signaling domain refers to a portion of a protein that transduces effector function signals and directs cells to perform a specified function.
  • the intracellular signaling domain is responsible for intracellular signaling after the ligand binding domain binds to the antigen, resulting in the activation of immune cells and immune responses.
  • the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed.
  • the effector function of a T cell can be a cytolytic activity or an auxiliary activity, including the secretion of cytokines.
  • the intracellular signaling domain contained in the chimeric antigen receptor of the present invention can be a cytoplasmic sequence of a T cell receptor and a co-receptor, which works together to initiate signaling after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar functions.
  • the intracellular signaling domain can contain many immunoreceptor tyrosine activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine activation motifs
  • Non-limiting examples of the intracellular signaling domain of the present invention include, but are not limited to, those derived from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the signaling domain of the CAR of the present invention can include a CD3 ⁇ signaling domain, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 19-22.
  • the costimulatory domain and the intracellular signaling domain can be operably linked in any order.
  • the costimulatory domain can be located near the membrane end, while the intracellular signaling domain is located at the distal end, or the stimulatory domain is located at the distal end, while the intracellular signaling domain is located near the membrane end.
  • the costimulatory domain can be located at the membrane end.
  • One or both sides of the signal transduction domain are contained.
  • the chimeric antigen receptor of the present invention may also include a hinge region between the ligand binding domain and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the ligand binding domain. Specifically, the hinge region is used to provide greater flexibility and accessibility for the ligand binding domain.
  • the hinge region may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the hinge region may be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.
  • the hinge region comprises a portion of the hinge region of the CD8 ⁇ chain, Fc ⁇ RIII ⁇ receptor, CD28, IgG4 or IgG1, more preferably a hinge from CD8 ⁇ , CD28 or IgG4, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:23-26.
  • the CAR of the present invention may also include a signal peptide so that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface.
  • the core of the signal peptide may contain a long hydrophobic amino acid segment that has a tendency to form a single ⁇ -helix.
  • At the end of the signal peptide there is usually an amino acid segment that is recognized and cut by a signal peptidase.
  • the signal peptidase can be cut during or after the translocation to produce a free signal peptide and a mature protein.
  • the free signal peptide is then digested by a specific protease.
  • Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from CD8 ⁇ , IgG1, GM-CSFR ⁇ , B2M, etc.
  • the signal peptide that can be used in the present invention has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in any of SEQ ID NO: 27-29.
  • the CAR of the present invention may also include a switch structure to regulate the expression time of CAR.
  • the switch structure may be in the form of a dimerization domain, which causes conformational changes by binding to its corresponding ligand, exposing the extracellular binding domain so that it binds to the targeted antigen, thereby activating the signal transduction pathway.
  • the switch domain may be used to connect the binding domain and the signal transduction domain respectively, and only when the switch domains bind to each other (for example, in the presence of an inducing compound) can the binding domain and the signal transduction domain be connected together through a dimer, thereby activating the signal pathway.
  • the switch structure may also be in the form of a masked peptide.
  • the masking peptide may shield the extracellular binding domain, preventing it from binding to the targeted antigen, and when the masking peptide is cut by, for example, a protease, the extracellular binding domain may be exposed, making it a "normal" CAR structure.
  • Various switch structures known to those skilled in the art may be used in the present invention.
  • the CAR of the present invention may also include a suicide gene, that is, to express a cell death signal that can be induced by an exogenous substance to remove CAR cells when needed (e.g., when serious toxic side effects occur).
  • the suicide gene can be in the form of an inserted epitope, such as a CD20 epitope, RQR8, etc., and when necessary, CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes.
  • the suicide gene may also be herpes simplex virus thymidine kinase (HSV-TK), which can cause cells to die under induced treatment with ganciclovir.
  • HSV-TK herpes simplex virus thymidine kinase
  • the suicide gene may also be iCaspase-9, which can be induced by chemical induction drugs such as AP1903, AP20187, etc. to dimerize iCaspase-9, thereby activating downstream Caspase3 molecules, leading to apoptosis.
  • chemical induction drugs such as AP1903, AP20187, etc.
  • Various suicide genes known to those skilled in the art can be used in the present invention.
  • the present invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor of the present invention.
  • the nucleic acid is DNA or RNA, more preferably DNA.
  • nucleic acid molecule includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in single-stranded and/or double-stranded form, linear or circular, or mixtures thereof (including hybrid molecules). Therefore, nucleic acids according to the present invention include DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA).
  • the nucleic acid is DNA or RNA, more preferably mRNA.
  • Nucleic acids may comprise conventional phosphodiester bonds or unconventional bonds (such as amide bonds, such as those found in peptide nucleic acids (PNA)).
  • the nucleic acids of the present invention may also contain one or more modified bases, such as, for example, trityl bases and uncommon bases (such as inosine). Other modifications are also conceivable, including chemical, enzymatic or metabolic modifications, as long as the multi-chain CAR of the present invention can be expressed from a polynucleotide.
  • Nucleic acids may be provided in an isolated form.
  • nucleic acids may also include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.
  • the nucleic acid sequences of the present invention can be codon optimized for optimal expression in a desired host cell (e.g., immune cell); or for expression in bacteria, yeast, or insect cells. Codon optimization refers to replacing the codons present in the target sequence that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species, and the codons before and after the replacement encode the same amino acid. Therefore, the selection of the optimal codon depends on the codon usage preference of the host genome.
  • the present invention also provides a vector comprising the nucleic acid molecule of the present invention.
  • vector is a nucleic acid molecule used as a medium for transferring (foreign) genetic material into a host cell, where the nucleic acid molecule can, for example, be replicated and/or expressed.
  • Targeting vector is a medium that separates nucleic acid by, for example, homologous recombination or using a hybrid recombinase of a sequence at a specific targeting site to deliver it to the interior of the cell.
  • Expression vector is a vector for the transcription of heterologous nucleic acid sequences (such as those encoding chimeric antigen receptor polypeptides of the present invention) in suitable host cells and the translation of their mRNA. Suitable vectors that can be used for the present invention are known in the art, and many are commercially available.
  • the vector of the present invention includes but is not limited to plasmids, viruses such as retroviruses, slow viruses, adenoviruses, vaccinia viruses, Rous sarcoma viruses (RSV), polyoma viruses and adeno-associated viruses (AAV), phages, phagemids, cosmids and artificial chromosomes (including BAC and YAC).
  • the vector itself is usually a nucleotide sequence, usually a DNA sequence comprising an insert (transgenic) and a larger sequence as a vector "skeleton".
  • the engineered vector is usually also included in the starting point of autonomous replication in the host cell (if stable expression of polynucleotides is required), a selection marker and a restriction enzyme cleavage site (such as a multiple cloning site MCS).
  • the vector may further comprise elements such as a promoter, a polyadenylic acid tail (polyA), a 3'UTR, an enhancer, a terminator, an insulator, an operator, a selection marker, a reporter gene, a targeting sequence and/or a protein purification tag.
  • the vector is an in vitro transcription vector.
  • the present invention provides engineered immune cells comprising a chimeric antigen receptor or a nucleic acid encoding the chimeric antigen receptor.
  • immune cell refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • Immune cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • Immune cells can also be derived from stem cells such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.
  • immune cells can be T cells, macrophages, or Preferably, immune cells are T cells.
  • T cells can be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from T cell lines such as Jurkat, SupT1, etc., cultured in vitro, or T cells obtained from subjects. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can also be concentrated or purified.
  • T cells can be any type of T cells and can be in any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • immune cells are human T cells, NK cells, or NKT cells.
  • Various techniques known to those skilled in the art can be used, such as Ficoll separation to obtain T cells from the blood of a subject.
  • immune cells are engineered to express chimeric antigen receptor polypeptides.
  • the nucleic acid sequence encoding the chimeric antigen receptor polypeptide can be introduced into immune cells by conventional methods known in the art (such as by transduction, transfection, transformation, etc.) to make it express the chimeric antigen receptor polypeptide of the present invention.
  • Transfection is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells.
  • An example is RNA transfection, which is the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells. This term is mainly used for non-viral methods in eukaryotic cells.
  • transduction is generally used to describe the transfer of viral-mediated nucleic acid molecules or polynucleotides.
  • Transfection of animal cells generally involves opening transient holes or "holes" in the cell membrane to allow the uptake of materials.
  • Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to produce liposomes that fuse with the cell membrane and deposit their cargo into the interior.
  • Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA coprecipitation), microinjection, and electroporation.
  • transformation is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, also into non-animal eukaryotic cells (including plant cells). Therefore, transformation is the genetic change of bacteria or non-animal eukaryotic cells, which is produced by direct uptake from its surroundings through the cell membrane and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be achieved by artificial means. In order for transformation to occur, the cell or bacterium must be in a state of competence. For prokaryotic transformation, techniques may include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection and electroporation.
  • the expression of endogenous HLA-I class genes and/or HLA-II class genes of the engineered immune cells of the present invention is not modified. That is, the expression level of any endogenous HLA-I class genes and/or HLA-II class genes is not changed by any artificial intervention method (gene editing or non-gene editing).
  • the expression of at least one endogenous HLA-I class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous HLA-II class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR/CD3 gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR/CD3 gene and at least one endogenous HLA-I class gene of the engineered immune cells of the present invention is suppressed or silenced.
  • the expression of at least one endogenous HLA-I class and HLA-II class gene of the engineered immune cells of the present invention is suppressed or silenced.
  • the expression of at least one endogenous TCR/CD3 gene, at least one endogenous HLA-I class gene and at least one endogenous HLA-II class gene of the engineered immune cells of the present invention is suppressed or silenced.
  • the HLA-I class gene is selected from HLA-A, HLA-B, HLA-C and B2M.
  • the HLA-II class gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK and CIITA, preferably selected from RFX5, RFXAP, RFXANK and CIITA.
  • the TCR/CD3 gene is selected from TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • the expression of one or more endogenous genes selected from the group consisting of CD52, GR, dCK, and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP1 is inhibited or silenced.
  • Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art, including but not limited to, for example, DNA or RNA fragmentation mediated by large-range nucleases, zinc finger nucleases, TALEN, CRISPR/Cas system, base editors, or gene inactivation by antisense oligonucleotides, RNAi, shRNA, transposons, mutations and the like.
  • the present invention also provides a pharmaceutical composition comprising the chimeric antigen receptor, nucleic acid, vector or engineered immune cell of the present invention as an active agent, and one or more pharmaceutically acceptable excipients. Therefore, the present invention also covers the use of the chimeric antigen receptor, nucleic acid, vector or engineered immune cell in the preparation of a pharmaceutical composition or a drug.
  • the term "pharmaceutically acceptable excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of inducing the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, antiadhesives, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delay agents, stabilizers, and tension regulators. It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical composition of the present invention.
  • Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water.
  • the selection of a suitable excipient depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
  • composition according to the present invention can be applied to a variety of routes. Typically, administration is completed parenterally.
  • Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
  • the pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or fluid extracts, or in the form particularly suitable for the desired method of administration.
  • the process known to the present invention for producing drugs may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes.
  • Pharmaceutical compositions comprising, for example, immune cells as described herein are generally provided in solution form, and preferably include a pharmaceutically acceptable buffer.
  • the pharmaceutical composition according to the present invention can also be used in combination with one or more other medicaments suitable for treating and/or preventing the disease to be treated.
  • medicaments suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, sirolimus ...
  • the invention relates to a novel class of drugs comprising the following: oxime, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes and RNA enzymes; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs such as antibody-directed enzyme prodrugs; immunostimulants such as IL-2, chemokines such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof such as anti-CD3 antibodies or fragments thereof, complement activators, foreign protein domain
  • the present invention also provides a method for preparing an engineered immune cell, comprising introducing the chimeric antigen receptor of the present invention or a nucleic acid sequence encoding the chimeric antigen receptor into an immune cell, so that the immune cell expresses the chimeric antigen receptor of the present invention.
  • the immune cell is a human immune cell, more preferably a human T cell, macrophage, dendritic cell, neutrophil, monocyte, NK cell and/or NKT cell.
  • nucleic acid or vector can be introduced into immune cells by physical methods, such as calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc.
  • chemical methods can also be used, such as by colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles, and liposomes.
  • biological methods can also be used to introduce nucleic acid or vector.
  • viral vectors especially retroviral vectors
  • retroviral vectors have become the most commonly used methods for inserting genes into mammals, such as human cells.
  • Other viral vectors can be derived from slow viruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc.
  • nucleic acid or vector After the nucleic acid or vector is introduced into the immune cells, those skilled in the art can amplify and activate the obtained immune cells by conventional techniques.
  • the present invention also provides a method for treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of the chimeric antigen receptor, nucleic acid molecule, vector, engineered immune cell or pharmaceutical composition according to the present invention. Therefore, the present invention also covers the use of the chimeric antigen receptor, nucleic acid molecule, vector or engineered immune cell in the preparation of a drug for treating cancer, infection or autoimmune disease.
  • an effective amount of the immune cells and/or pharmaceutical compositions of the invention are administered directly to a subject.
  • the treatment method of the present invention is an ex vivo treatment.
  • the method comprises the following steps: (a) providing a sample of a subject, wherein the sample comprises immune cells; (b) introducing the chimeric antigen receptor of the present invention into the immune cells in vitro to obtain modified immune cells, and (c) administering the modified immune cells to a subject in need thereof.
  • the immune cells provided in step (a) are selected from T cells, NK cells and/or NKT cells; and the immune cells can be obtained from a sample of the subject (particularly a blood sample) by conventional methods known in the art.
  • immune cells that are capable of expressing the chimeric antigen receptor of the present invention and exerting the desired biological effector functions as described herein may also be used.
  • the immune cells generally selected are compatible with the subject's immune system, i.e., the immune cells preferably do not induce an immunogenic response.
  • "universal receptor cells” may be used, i.e., cells that exert the desired biological effector functions.
  • the generally compatible lymphocytes that can grow and expand in vitro of biological effector functions are required. The use of such cells will not require the acquisition and/or provision of the subject's own lymphocytes.
  • step (c) can be implemented by introducing the nucleic acid or vector described herein into immune cells via electroporation or by infecting immune cells with a viral vector, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector as described above.
  • a viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector as described above.
  • Other conceivable methods include the use of a transfection agent (such as liposomes) or transient RNA transfection.
  • the immune cells are autologous or allogeneic cells, preferably T cells, macrophages, dendritic cells, neutrophils, monocytes, NK cells and/or NKT cells, more preferably T cells, NK cells or NKT cells.
  • autologous refers to any material derived from an individual that will later be reintroduced into that same individual.
  • allogeneic refers to any material derived from a different animal or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are different. In some cases, allogeneic material from individuals of the same species may differ genetically enough to allow antigenic interactions to occur.
  • the term "subject" is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer.
  • the subject is a human.
  • the cancer is a cancer associated with expression of a target to which the ligand binding domain binds.
  • the cancer includes, but is not limited to, brain glioma, blastoma, sarcoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous lung cancer), melanoma, myeloma, neuroblastoma, oral
  • lymphoma including Hodgkin lymphoma and non-Hodgkin lymphoma, such as B-cell lymphoma (including low-grade/follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large mass disease NHL), mantle cell lymphoma, AIDS-
  • the diseases that can be treated with the engineered immune cells or pharmaceutical compositions of the present invention are selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, ovarian cancer, Mesothelioma, breast cancer, lung cancer, prostate cancer, melanoma, myeloma, sarcoma, gastric cancer, etc.
  • the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.
  • the autoimmune diseases include but are not limited to type I diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus.
  • the method further comprises administering one or more additional chemotherapeutic agents, biologics, drugs or treatments to the subject.
  • the chemotherapeutic agent, biologic, drug or treatment is selected from radiation therapy, surgery, antibody reagents and/or small molecules and any combination thereof.
  • Figure 1 scFv expression levels of CAR-T cells determined by flow cytometry.
  • Figure 2 The killing effect of CAR-T cells on target cells.
  • Figure 3 IL-2 release levels after co-culture of CAR-T cells with target cells.
  • Figure 4 Long-term killing effect of CAR-T cells on target cells in vitro.
  • Figure 5 Expansion curve of CAR-T cells after multiple co-culture with tumor cells.
  • CD8 ⁇ signal peptide SEQ ID NO: 28
  • anti-CD19 scFv SEQ ID NO: 12
  • CD8 ⁇ hinge region SEQ ID NO: 24
  • CD8 ⁇ transmembrane region SEQ ID NO: 14
  • CD27 co-stimulatory domain SEQ ID NO: 1
  • CD3 ⁇ intracellular signaling domain SEQ ID NO: 20
  • CAR19-27 plasmid was obtained, and the correct insertion of the target sequence was confirmed by sequencing.
  • CAR19-27(-4) and CAR19-27K4R plasmids which differ from the CAR19-27 plasmid in that 4 amino acids were removed from the intracellular segment (SEQ ID NO: 3) or the fourth amino acid K in the intracellular segment was mutated to R (SEQ ID NO: 2).
  • T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, Cat. No. 40203D) and cultured for 1 day at 37°C and 5% CO2. Then, concentrated lentivirus was added and cultured for 3 days to obtain CAR19-27 T cells, CAR19-27(-4) T cells, and CAR19-27K4R T cells targeting CD19.
  • DynaBeads CD3/CD28 CTSTM Gibco, Cat. No. 40203D
  • concentrated lentivirus was added and cultured for 3 days to obtain CAR19-27 T cells, CAR19-27(-4) T cells, and CAR19-27K4R T cells targeting CD19.
  • CAR T cells containing different mutations in the CD27 intracellular region can effectively express scFv.
  • Example 2 Killing effect of CAR T cells on target cells and cytokine release
  • T cells kill target cells the number of target cells will decrease.
  • T cells are co-cultured with target cells that can express luciferase the number of target cells decreases and the secreted luciferase also decreases.
  • Luciferase can catalyze the conversion of luciferin into oxidized luciferin, and during this oxidation process, bioluminescence will be generated, and the intensity of this luminescence will depend on the level of luciferase expressed by the target cells. Therefore, the detected fluorescence intensity can reflect the ability of T cells to kill target cells.
  • Raji target cells carrying the fluorescein gene were first plated into a 96-well plate at 1 ⁇ 10 4 /well, and then CAR19-27 T cells, CAR19-27(-4)T cells, CAR19-27K4R T cells and NT cells were plated into a 96-well plate for co-culture at different effector-target ratios (i.e., the ratio of effector T cells to target cells).
  • effector-target ratios i.e., the ratio of effector T cells to target cells.
  • the fluorescence value of Raji target cells was measured using an ELISA reader.
  • the killing efficiency was calculated according to the calculation formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean ⁇ 100%, and the results are shown in Figure 2.
  • CAR19-27 T cells, CAR19-27(-4) T cells, and CAR19-27K4R T cells all have the ability to specifically kill target cells, and there is no significant difference.
  • cytokines such as IL-2 and IFN- ⁇
  • ELISA enzyme-linked immunosorbent assay
  • Target cells were plated at 1 ⁇ 10 5 /well in a 96-well plate, and then CAR19-27 T cells, CAR19-27(-4) T cells, CAR19-27K4R T cells, and NT cells were co-cultured with Raji cells at a ratio of 1:1, and the cell co-culture supernatant was collected after 18-24 hours.
  • the 96-well plate was coated with the capture antibody Purified anti-human IL-2Antibody (R&D, Catalog No. DY202) and incubated overnight at 4°C, then the antibody solution was removed, and 250 ⁇ L of PBST (1XPBS containing 0.1% Tween) solution containing 2% BSA (sigma, Catalog No. V900933-1kg) was added and incubated at 37°C for 2 hours. The plate was then washed 3 times with 250 ⁇ L PBST (1XPBS containing 0.1% Tween).
  • CAR19-27 T cells, CAR19-27(-4) T cells and CAR19-27K4R T cells can specifically secrete IL-2, and the IL-2 release levels of CAR19-27(-4) T cells and CAR19-27K4R T cells are significantly higher than those of CAR19-27 T cells. It is worth noting that the IL-2 release level of CAR19-27(-4) T cells is significantly higher than that of CAR19-27 T cells and CAR19-27K4R T cells.
  • Example 3 The ability of CAR T to kill tumor cells in the long term
  • CAR19-27(-4) T cells and CAR19-27K4R T cells are significantly better than those of the CAR19-27 T group, and CAR19-27K4R T cells are better than CAR19-27K4R T cells.
  • the CAR-T cells containing the mutated CD27 intracellular region of the present invention can promote the secretion of cytokines and the expansion of immune cells, and enhance the ability of immune cells to kill tumors in the long term.

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Abstract

La présente invention concerne un récepteur antigénique chimérique, comprenant un domaine de liaison à un ligand, un domaine transmembranaire, un domaine de costimulation et un domaine de signalisation intracellulaire, le domaine de costimulation comprenant une région intracellulaire CD27 mutée. La présente invention concerne également une cellule immunitaire modifiée comprenant le récepteur antigénique chimérique, et son utilisation dans le traitement de maladies, telles que le cancer, les maladies auto-immunes, les infections.
PCT/CN2023/100067 2023-06-14 2023-06-14 Récepteur antigénique chimérique comprenant un nouveau domaine de costimulation et utilisation associée Ceased WO2024254775A1 (fr)

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