WO2024254775A1 - 包含新型共刺激结构域的嵌合抗原受体及其用途 - Google Patents

包含新型共刺激结构域的嵌合抗原受体及其用途 Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cells
chimeric antigen
antigen receptor
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/100067
Other languages
English (en)
French (fr)
Inventor
周亚丽
汤颖秀
郑冬
任江涛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hongkong Bioheng Biotech Ltd
Original Assignee
Hongkong Bioheng Biotech Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hongkong Bioheng Biotech Ltd filed Critical Hongkong Bioheng Biotech Ltd
Priority to PCT/CN2023/100067 priority Critical patent/WO2024254775A1/zh
Priority to CN202380098273.1A priority patent/CN121419997A/zh
Publication of WO2024254775A1 publication Critical patent/WO2024254775A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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.

Landscapes

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

Abstract

本发明涉及一种嵌合抗原受体，其包含配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述共刺激结构域包含突变的CD27胞内区。本发明还涉及包含此类嵌合抗原受体的工程化免疫细胞及其在治疗疾病，例如癌症、自身免疫性疾病、感染中的用途。

Description

包含新型共刺激结构域的嵌合抗原受体及其用途 技术领域
本发明属于免疫治疗领域。更具体地，本发明涉及包含新型共刺激结构域的嵌合抗原受体，以及包含此类嵌合抗原受体的工程化免疫细胞及其用途。
背景技术
过继性细胞疗法是指通过对自体免疫细胞进行体外激活和扩增，然后将其重新输回至患者体内，从而达到直接杀伤肿瘤或激发机体的免疫应答杀伤肿瘤细胞的目的。常见的过继性细胞疗法包括LAK、CIK和NK等非特异性免疫疗法以及TIL、CAR-T和TCR-T等特异性免疫疗法。其中，CAR-T细胞疗法由于在血液瘤领域的出色表现成功引起了广泛关注。鉴于CAR-T细胞疗法的飞速发展，也逐渐衍生出了CAR-NK、CAR-NKT、CAR-Treg、CAR-γδT等以CAR作为核心技术的一系列新型细胞疗法，共同引领CAR细胞疗法成为肿瘤细胞免疫治疗的一大趋势。
嵌合抗原受体(CAR)是一种细胞表面受体蛋白，它赋予了免疫细胞靶向特定抗原蛋白的能力。从结构上看，CAR至少由抗原结合结构域、跨膜结构域和胞内信号传导结构域三个功能结构域组成，胞内信号传导结构域由共刺激结构域和/或初级信号传导结构域构成。CAR结构的设计经历了以下过程：第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子，由于胞内只有一个活化结构域，因此它只能引起短暂的免疫细胞增殖和较少的细胞因子分泌，而并不能提供长时间的免疫细胞增殖信号和持续的体内抗肿瘤效应，所以并没有取得很好的临床疗效；第二代CAR在原有结构基础上引入一个共刺激分子，如CD28、4-1BB、OX40、ICOS，与第一代CAR相比功能有很大提高，进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力；第三代CAR包含2个共刺激分子，以CD28与4-1BB、CD28与OX40组合作为共刺激分子在第三代CAR中最为常见；第四代CAR主要为了克服肿瘤免疫微环境的抑制，在设计时引入了促炎症细胞因子(IL-12、IL-15、IL-18等)和共刺激配体；第五代CAR-T在第二代的基础上添加了激活其它信号通路的共刺激结构域，比如IL2-2Rβ胞内结合SAAT3/5的结构域等。目前，大多数公司的CAR产品仍以第二代CAR结构为基础，进行探索开发。
研究表明，不同的功能结构域对CAR的功能有重要的影响。例如，国际申请WO2016014789A中，在不改变抗原结合结构域和胞内传导信号结构域的前提下，仅通过改变跨膜结构域，即可大幅度改善CAR的性能，降低CAR-T对非靶细胞的非特异杀伤作用。例如，目前在CAR细胞疗法领域中普遍认为，包含CD28共刺激结构域的CAR细胞显示出更好的细胞增殖活性，包含4-1BB共刺激结构域的CAR细胞显示出更好的持续性。
目前CAR细胞疗法在临床应用中仍存在一些问题，如对实体瘤疗效不佳、持续性差等。因此，有必要开发新的CAR结构，以进一步增强表达CAR细胞疗法的治疗效果。
发明内容
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。
嵌合抗原受体
如本文所用，术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括配体结合结构域(例如抗体或其抗原结合部分)、跨膜结构域、共刺激结构域和胞内信号传导结构域，各个结构域之间通过接头连接。CAR能够利用抗体的抗原结合特性以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。非MHC限制性的抗原识别给予表达CAR的免疫细胞与抗原处理无关的识别抗原的能力，因此绕过了肿瘤逃逸的主要机制。
在第一个方面，本发明提供一种嵌合抗原受体，其包含配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述共刺激结构域包含突变的CD27胞内区。
共刺激结构域是来自共刺激分子的细胞内功能性信号传导结构域，其包含所述共刺激分子的整个细胞内部分，或其功能性片段。“共刺激分子”是指在T细胞上与共刺激配体特异性结合，由此介导T细胞的共刺激反应(例如增殖)的同源结合配偶体。传统的嵌合抗原受体一般使用CD28和/或4-1BB作为共刺激结构域。本发明的嵌合抗原受体包含新型共刺激结构域，即突变的CD27胞内区。
CD27是由二硫键连接的相对分子量为55000Da的单体组成的二聚体跨膜糖蛋白，是肿瘤坏死因子TNF超家族的重要成员。CD27广泛表达于原始T细胞和记忆T细胞表面。CD27主要通过与其配体CD70结合，激活下游的TRAF2和TRAF5信号通路，进一步诱导NF-κB和JNK信号通路的激活，对于免疫细胞，尤其是T细胞的激活、增殖和凋亡发挥重要的调节功能。
与野生型CD27胞内区(SEQ ID NO:1)相比，本发明中所述突变的CD27胞内区包含至少1个赖氨酸突变(即氨基酸的缺失、取代或插入)，例如包含1个赖氨酸突变、2个赖氨酸突变、3个赖氨酸突变。优选地，本发明中所述突变的CD27胞内区包含至少1个赖氨酸的取代。更优选地，所述取代是将赖氨酸替换为其他碱性氨基酸，如精氨酸或组氨酸。
或者，与野生型CD27胞内区(SEQ ID NO:1)相比，本发明中所述突变的CD27胞内区近C-末端的第1-6个氨基酸位点包含至少1个(例如1个、2个、3个、4个、5个或6个)氨基酸缺失。优选地，本发明中所述突变的CD27胞内区近C-末端的第1-6个氨基酸位点包含至少1个氨基酸(例如1个、2个、3个、4个、5个或6个)的连续缺失。更优选地，本发明中所述突变的CD27胞内区包含近C-末端的第1-4个氨基酸位点包含4个氨基酸的连续缺失。
本发明中的CD27胞内区经过上述突变，相比于野生型CD27胞内区，能够显著增加CAR细胞的扩增水平，进而提高对肿瘤的抑制效果。
在一些实施方案中，本发明中所述突变的CD27胞内区具有SEQ ID NO:2或3所示的氨基酸序列或与SEQ ID NO:2或3所示的氨基酸序列具有至少90％、95％、97％或99％或100％的序列同一性。
除了本发明提供的突变的CD27胞内区外，本发明的嵌合抗原受体还可以包含一个或多个额外的共刺激结构域，其选自以下蛋白质的共刺激信号传导结构域：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或ZAP70，优选选自4-1BB、CD28、CD27、OX40、CD278及其任意组合，更优选 选自4-1BB和CD28。
在一些实施方案中，本发明的CAR包含突变的CD27胞内区作为共刺激结构域。在一些实施方案中，本发明的CAR包含突变的CD27胞内区和4-1BB胞内区作为共刺激结构域。在一些实施方案中，本发明的CAR包含突变的CD27胞内区和CD28胞内区作为共刺激结构域。
本领域技术人员已知的4-1BB和CD28共刺激结构域均可用于本发明。例如，4-1BB共刺激结构域与SEQ ID NO:17或18所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；CD28共刺激结构域与SEQ ID NO:16所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
如本文所用，“配体结合结构域”是指可以与配体(例如抗原)结合的任何结构或其功能性变体。配体结合结构域可以是抗体结构，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠源抗体、嵌合抗体及其抗原结合片段。例如，配体结合结构域包括但不限于完整抗体、Fab、Fab’、F(ab’)2、Fv片段、scFv抗体片段、线性抗体、sdAb(例如VH或VL)、纳米抗体(Nanobody，Nb)、重组纤连蛋白结构域、anticalin和DARPIN等，优选选自Fab、scFv、sdAb和纳米抗体。在本发明中，配体结合结构域可以是单价或二价，且可以是单特异性、双特异性或多特异性的抗体。
“单链抗体”或“scFv”是由抗体重链可变区(VH)和轻链可变区(VL)通过接头连接而成的抗体。可以选择接头的最佳长度和/或氨基酸组成。接头的长度会明显影响scFv的可变区折叠和相互作用情况。事实上，如果使用较短的接头(例如在5-10个氨基酸之间)，则可以防止链内折叠。关于接头的大小和组成的选择，参见例如，Hollinger等人,1993Proc Natl Acad.Sci.U.S.A.90:6444-6448；美国专利申请公布号2005/0100543、2005/0175606、2007/0014794；以及PCT公布号WO2006/020258和WO2007/024715，其全文通过引用并入本文。scFv可以包含以任何顺序连接的VH和VL，例如VH-接头-VL或VL-接头-VH。优选地，所述接头具有SEQ ID NO:30或31所示的氨基酸序列。
术语“功能性变体”或“功能性片段”是指基本上包含亲本的氨基酸序列但与该亲本氨基酸序列相比含有至少一个氨基酸修饰(即取代、缺失或插入)的变体，条件是所述变体保留亲本氨基酸序列的生物活性。例如，对于抗体，其功能性片段是其抗原结合部分。优选地，“功能性变体”或“功能性片段”与亲本氨基酸序列具有至少75％，优选至少76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性，并且保留亲本氨基酸的生物活性，例如结合活性。
如本文所用，术语“序列同一性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性。本领域技术人员将认识到，一些算法可以用于使用标准参数来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25：3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215：403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147：195-197)和ClustalW。
配体结合结构域的选择取决于待识别的与具体疾病状态相关的靶细胞上的细胞表面标记，例如肿瘤特异性抗原或肿瘤相关抗原。因此，在一些实施方案中，本发明的配体结合结构域与选自以下 的一个或多个靶标结合：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、CD40、CD44、CD44v6、CD44v7/8、CD47、CD52、CD56、CD57、CD58、CD70、CD72、CD79a、CD79b、CD80、CD81、CD86、CD97、CD123、CD133、CD137、CD138、CD151、CD171、CD179a、CD300LF、CDH16、CSPG4、CS1、Claudin 6、Claudin 18.1、Claudin 18.2、CEA、CEACAM6、CLL1、c-Met、CAIX、CXORF61、CA125、CYP1B1、CS1、ELF2M、EGFR、EPCAM、EGFRvIII、EphA2、ERG/TMPRSS2ETS融合基因、ETV6-AML、EMR2、EGP2，EGP40、FAP、FAR、FBP、FLT3、FOSL1、FCRL5、FCAR、Flt3、Flt4、Frizzled、GD2、GD3、gp100、gp130、GM3、GPC2、GPC3、GPRC5D、GPR20、GloboH、GHRHR、GHR、GITR、Her2、HER3、HER-4、HMWMAA、HAVCR1、HPV E6,E7、HVEM、HIV-1Gag、HLA-A1、HLA-A2、IL6R、IL-11Ra、IL-13Ra、IGF-I受体、LTPR、LIFRP、LRP5、IGLL1、IGF1R、KIT、Kappa Light Chain、KDR、LewisY、LMP2、LY6K、LAGE-1a、legumain、LCK、LAIR1、LILRA2、LY75、MSLN、MUC1、MUC16、MAGE-A1、MAGE3、MAD-CT-1、MelanA/MART1、ML-IAP、MYCN、mut hsp70-2、NCAM、NY-BR-1、NY-ESO-1、NA17、Notch-1-4、nAchR、NKG2D、NKG2D配体、OY-TES1、OR51E2、OX40、PRSS21、PSCA、PD1、PD-L1、PD-L2、PSMA、Prostase、PAP、PDGFR-β、PCTA-1/半乳凝集素8、p53、p53突变体、prostein、PLAC1、PANX3、PAX3、PAX5、PTCH1、RANK、RAGE-1、ROR1、Ras突变体、RhoC、RU1、RU2、Robol、SSEA-4、SSX2、SART3、Sp17、TSHR、Tn Ag、TGS5、TEM1/CD248、TEM7R、TARP、TCRα、TCRβ、TGFBR1、TGFBR2、TNFRSF4、TWEAK-R、TLR7、TLR9、TAG72、TROP-2、Tie 2、TRP-2、TNFR1、TNFR2、TEM1、UPK2VEGFR、WT1、XAGE1、5T4、8H9、αvβ6整合素、CA9、叶酸受体α、肝配蛋白B2、酪氨酸酶、岩藻糖基GM1、邻-乙酰-GD2、叶酸受体β、多聚唾液酸、精子蛋白17、存活蛋白和端粒酶、肉瘤易位断点、人端粒末端逆转录酶/hTERT、雄激素受体、肠羧基酯酶、细胞周期蛋白B1、纤连蛋白、腱生蛋白、肿瘤坏死区的癌胚变体及其任意组合。优选地，所述配体结合结构域与选自以下的靶标结合：CD7、CD19、CD20、CD22、CD30、CD33、CD38、CD70、CD123、CD138、CD171、MUC1、MSLN、AFP、叶酸受体α、CEA、PSCA、PSMA、Her2、EGFR、IL-13Ra、GD2、NKG2D、Claudin 18.2、ROR1、EGFRvIII、CS1、BCMA和GPRC5D，更优选CD19、Claudin 18.2、MSLN、GPRC5D、ROR1、CD7、CD70和BCMA。根据待靶向的抗原，本发明的CAR可以被设计为包括对该抗原具有特异性的配体结合结构域。例如，如果CD19是待靶向的抗原，则CD19抗体可用作本发明的配体结合结构域。
在一些实施方案中，本发明的嵌合抗原受体靶向CD19。优选地，本发明的嵌合抗原受体包含抗CD19抗体。本领域中已知的靶向CD19的抗体均可用于本发明。在一些实施方案中，所述靶向CD19的抗体包含轻链可变区和重链可变区，其中所述重链可变区包含的CDR1-H、CDR2-H和CDR3-H与SEQ ID NO:10所包含的CDR1-H、CDR2-H和CDR3-H相同；其中所述轻链可变区包含的CDR1-L、CDR2-L和CDR3-L与SEQ ID NO:11所包含的CDR1-L、CDR2-L和CDR3-L相同。在一些实施方案中，所述重链可变区包含的CDR1-H如SEQ ID NO:4所示，CDR2-H如SEQ ID NO:5所示，CDR3-H如SEQ ID NO:6所示，所述轻链可变区包含的CDR1-L如SEQ ID NO:7所示，CDR2-L如SEQ ID NO:8 所示，CDR3-L如SEQ ID NO:9所示。
在一些实施方案中，所述靶向CD19的抗体包含轻链可变区和重链可变区，所述重链可变区与SEQ ID NO:10具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，所述轻链可变区与SEQ ID NO:11具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性。优选地，本发明中靶向CD19的抗体包含如SEQ ID NO:10所示的重链可变区和如SEQ ID NO:11所示的轻链可变区。
在一些实施方案中，所述靶向CD19的抗体与SEQ ID NO:12具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性。优选地，所述靶向CD19的抗体如SEQ ID NO:12所示。
如本文所用，术语“跨膜结构域”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。特别适用于本发明中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154及其功能性片段。或者，跨膜结构域可以是合成的并且可以主要地包含疏水性残基如亮氨酸和缬氨酸。优选地，所述跨膜结构域源自CD8α链或CD28，其与SEQ ID NO:13-15任一所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。胞内信号传导结构域负责在配体结合结构域结合抗原以后的细胞内信号传递，从而导致免疫细胞和免疫反应的活化。换言之，胞内信号传导结构域负责活化其中表达CAR的免疫细胞的正常的效应子功能的至少一种。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。
在一些实施方案中，本发明的嵌合抗原受体包含的胞内信号传导结构域可以是T细胞受体和共受体的细胞质序列，其在抗原受体结合以后一同起作用以引发信号传导，以及这些序列的任何衍生物或变体和具有相同或相似功能的任何合成序列。胞内信号传导结构域可以包含许多免疫受体酪氨酸激活基序(ITAM)。本发明的胞内信号传导结构域的非限制性施例包括但不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。在优选的实施方式中，本发明CAR的信号传导结构域可以包含CD3ζ信号传导结构域，该信号传导结构域与SEQ ID NO:19-22任一所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一些实施方案中，共刺激结构域和胞内信号传递结构域可以任何顺序可操作连接。例如，共刺激结构域可以位于近膜端，而胞内信号传导结构域位于远膜端，或者刺激结构域位于远膜端，而胞内信号传导结构域位于近膜端。当含有两个或更多个共刺激结构域时，共刺激结构域可以位于胞 内信号传递结构域的一侧或两侧。
在一些实施方案中，本发明的嵌合抗原受体还可以包含位于配体结合结构域和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至配体结合结构域的任何寡肽或多肽。具体地，铰链区用来为配体结合结构域提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。在优选的实施方式中，所述铰链区包含CD8α链、FcγRIIIα受体、CD28、IgG4或IgG1的铰链区部分，更优选来自CD8α、CD28或IgG4的铰链，其与SEQ ID NO:23-26任一所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一些实施方案中，本发明的CAR还可以包含信号肽，使得当其在细胞例如T细胞中表达时，新生蛋白质被引导至内质网并随后引导至细胞表面。信号肽的核心可以含有长的疏水性氨基酸区段，其具有形成单个α-螺旋的倾向。在信号肽的末端，通常有被信号肽酶识别和切割的氨基酸区段。信号肽酶可以在移位期间或完成后切割，以产生游离信号肽和成熟蛋白。然后，游离信号肽被特定蛋白酶消化。可用于本发明的信号肽是本领域技术人员熟知的，例如衍生自CD8α、IgG1、GM-CSFRα、B2M等的信号肽。在一些实施方案中，可用于本发明的信号肽与SEQ ID NO:27-29任一所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一些实施方案中，本发明的CAR还可以包含开关结构，以调控CAR的表达时间。例如，开关结构可以是二聚化结构域的形式，通过与其相应配体的结合引起构象变化，暴露胞外结合结构域，使其与被靶向抗原结合，从而激活信号传导通路。或者，也可以使用开关结构域分别连接结合结构域和信号传导结构域，仅当开关结构域互相结合(例如在诱导化合物的存在下)时，结合结构域和信号传导结构域才能通过二聚体连接在一起，从而激活信号通路。开关结构还可以是掩蔽肽的形式。掩蔽肽可以遮蔽胞外结合结构域，阻止其与被靶向抗原的结合，当通过例如蛋白酶切割掩蔽肽后，就可以暴露胞外结合结构域，使其成为一个“普通”的CAR结构。本领域技术人员知晓的各种开关结构均可用于本发明。
在一些实施方案中，本发明的CAR还可以包含自杀基因，即，使其表达一个可通过外源物质诱导的细胞死亡信号，以在需要时(例如产生严重的毒副作用时)清除CAR细胞。例如，自杀基因可以是插入的表位的形式，例如CD20表位、RQR8等，当需要时，可以通过加入靶向这些表位的抗体或试剂来消除CAR细胞。自杀基因也可以是单纯疱疹病毒胸苷激酶(HSV-TK)，该基因可使细胞在接受更昔洛韦治疗诱导下死亡。自杀基因还可以是iCaspase-9，可以通过化学诱导药物如AP1903、AP20187等诱导iCaspase-9发生二聚化，从而激活下游的Caspase3分子，导致细胞凋亡。本领域技术人员知晓的各种自杀基因均可用于本发明。
核酸
本发明还提供一种核酸分子，其包含编码本发明的嵌合抗原受体的核酸序列。优选地，所述核酸是DNA或RNA，更优选DNA。
如本文所用，术语“核酸分子”包括核糖核苷酸和脱氧核糖核苷酸的序列，如经修饰的或未经修饰的RNA或DNA，各自为单链和/或双链形式的线性或环状，或它们的混合物(包括杂合分子)。因此，根据本发明的核酸包括DNA(比如dsDNA、ssDNA、cDNA)、RNA(比如dsRNA、ssRNA、mRNA、ivtRNA)，它们的组合或衍生物(比如PNA)。优选地，所述核酸是DNA或RNA，更优选mRNA。
核酸可以包含常规的磷酸二酯键或非常规的键(如酰胺键，比如在肽核酸(PNA)中发现的)。本发明的核酸还可含有一种或多种经修饰的碱基，比如，例如三苯甲基化的碱基和不常见的碱基(比如肌苷)。也可以想到其它修饰，包括化学、酶促或代谢修饰，只要本发明的多链CAR可以从多核苷酸表达即可。核酸可以以分离的形式提供。在一些实施方案中，核酸也可以包括调节序列，比如转录控制元件(包括启动子、增强子、操纵子、抑制子和转录终止信号)、核糖体结合位点、内含子等。
可以对本发明的核酸序列进行密码子优化以在所需的宿主细胞(如，免疫细胞)中进行最佳表达；或者用于在细菌、酵母菌或昆虫细胞中表达。密码子优化是指将目标序列中存在的在给定物种的高度表达的基因中一般罕见的密码子替换为在这类物种的高度表达的基因中一般常见的密码子，而替换前后的密码子编码相同的氨基酸。因此，最佳密码子的选择取决于宿主基因组的密码子使用偏好。
载体
本发明还提供一种载体，包含如本发明所述的核酸分子。
如本文所用，术语“载体”是用作将(外源)遗传材料转移到宿主细胞中的媒介核酸分子，在该宿主细胞中所述核酸分子可以例如复制和/或表达。
载体一般包括靶向载体和表达载体。“靶向载体”是通过例如同源重组或使用特异性靶向位点处序列的杂合重组酶将分离的核酸递送至细胞内部的介质。“表达载体”是用于异源核酸序列(例如编码本发明的嵌合抗原受体多肽的那些序列)在合适的宿主细胞中的转录以及它们的mRNA的翻译的载体。可用于本发明的合适载体是本领域已知的，并且许多可商购获得。在一些实施方案中，本发明的载体包括但不限于质粒、病毒例如逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV)、多瘤病毒和腺相关病毒(AAV)等、噬菌体、噬菌粒、粘粒和人工染色体(包括BAC和YAC)。载体本身通常是核苷酸序列，通常是包含插入物(转基因)的DNA序列和作为载体“骨架”的较大序列。工程化载体通常还包含在宿主细胞中自主复制的起点(如果需要多核苷酸的稳定表达)、选择标记和限制酶切割位点(如多克隆位点MCS)。载体可另外包含启动子、多聚腺苷酸尾(polyA)、3’UTR、增强子、终止子、绝缘子、操纵子、选择标记、报告基因、靶向序列和/或蛋白质纯化标签等元件。在一个具体的实施方案中，所述载体是体外转录的载体。
工程化免疫细胞
本发明提供工程化免疫细胞，其包含嵌合抗原受体或其编码核酸。
如本文所用，术语“免疫细胞”是指免疫系统的具有一种或多种效应子功能(例如，细胞毒性细胞杀伤活性、分泌细胞因子、诱导ADCC和/或CDC)的任何细胞。免疫细胞可以从多种来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织及肿瘤。免疫细胞还可以衍生自成体干细胞、胚胎干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞等干细胞。例如，免疫细胞可以是T细胞、巨噬 细胞、树突状细胞、中性粒细胞、单核细胞、NK细胞和/或NKT细胞。优选地，免疫细胞是T细胞。T细胞可以是任何T细胞，如体外培养的T细胞，例如原代T细胞，或者来自体外培养的T细胞系例如Jurkat、SupT1等的T细胞，或获得自受试者的T细胞。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。T细胞也可以被浓缩或纯化。T细胞可以是任何类型的T细胞并且可以处于任何发育阶段，包括但不限于，CD4+/CD8+双阳性T细胞、CD4+辅助T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞等。优选地，免疫细胞是人T细胞、NK细胞或NKT细胞。可以使用本领域技术人员已知的多种技术，如Ficoll分离从受试者的血液获得T细胞。在本发明中，免疫细胞被工程化以表达嵌合抗原受体多肽。
采用本领域已知的常规方法(如通过转导、转染、转化等)可以将编码嵌合抗原受体多肽的核酸序列引入免疫细胞，使其表达本发明的嵌合抗原受体多肽。“转染”是将核酸分子或多核苷酸(包括载体)引入靶细胞的过程。一个例子是RNA转染，即将RNA(比如体外转录的RNA，ivtRNA)引入宿主细胞的过程。该术语主要用于真核细胞中的非病毒方法。术语“转导”通常用于描述病毒介导的核酸分子或多核苷酸的转移。动物细胞的转染通常涉及在细胞膜中打开瞬时的孔或“洞”，以允许摄取材料。可以使用磷酸钙、通过电穿孔、通过细胞挤压或通过将阳离子脂质与材料混合以产生与细胞膜融合并将它们的运载物沉积入内部的脂质体，进行转染。用于转染真核宿主细胞的示例性技术包括脂质囊泡介导的摄取、热休克介导的摄取、磷酸钙介导的转染(磷酸钙/DNA共沉淀)、显微注射和电穿孔。术语“转化”用于描述核酸分子或多核苷酸(包括载体)向细菌中、也向非动物真核细胞(包括植物细胞)中的非病毒转移。因此，转化是细菌或非动物真核细胞的基因改变，其通过细胞膜从其周围直接摄取并随后并入外源遗传材料(核酸分子)而产生。转化可以通过人工手段实现。为了发生转化，细胞或细菌必须处于感受态的状态。对于原核转化，技术可包括热休克介导的摄取、与完整细胞的细菌原生质体融合、显微注射和电穿孔。
在一些实施方案中，本发明的工程化免疫细胞的内源性HLA-I类基因和/或HLA-II类基因的表达未被修饰。即，没有通过任何人工干预的方法(基因编辑或非基因编辑)来改变任何一种内源性HLA-I类基因和/或HLA-II类基因的表达水平。
在一些实施方案中，本发明的工程化免疫细胞的至少一种内源性HLA-I类基因的表达被抑制或沉默。在一些实施方案中，本发明的工程化免疫细胞的至少一种内源性HLA-II类基因的表达被抑制或沉默。在一些实施方案中，本发明的工程化免疫细胞的至少一种内源性TCR/CD3基因的表达被抑制或沉默。在一些实施方案中，本发明的工程化免疫细胞的至少一种内源性TCR/CD3基因和至少一种内源性HLA-I类基因的表达被抑制或沉默。在一些实施方案中，本发明的工程化免疫细胞的至少一种内源性HLA-I类和HLA-II类基因的表达被抑制或沉默。在一些实施方案中，本发明的工程化免疫细胞的至少一种内源性TCR/CD3基因，至少一种内源性HLA-I类基因和至少一种内源性HLA-II类基因的表达被抑制或沉默。优选地，所述HLA-I类基因选自HLA-A、HLA-B、HLA-C和B2M。优选地，所述HLA-II类基因选自HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK和CIITA，优选选自RFX5、RFXAP、RFXANK和CIITA。优选地，所述 TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε和CD3ζ。
在一些实施方案中，本发明的工程化免疫细胞的一个或多个选自以下内源性基因的表达被抑制或沉默：CD52、GR、dCK和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。
抑制基因表达或使基因沉默的方法是本领域技术人员熟知的，包括但不限于例如通过大范围核酸酶、锌指核酸酶、TALEN、CRISPR/Cas系统、碱基编辑器介导DNA或RNA断裂，或通过反义寡核苷酸、RNAi、shRNA、转座子、突变等技术使基因失活。
药物组合物
在第三个方面，本发明还提供一种药物组合物，其包含本发明所述的嵌合抗原受体、核酸、载体或工程化免疫细胞作为活性剂，和一种或多种药学上可接受的赋型剂。因此，本发明还涵盖所述嵌合抗原受体、核酸、载体或工程化免疫细胞在制备药物组合物或药物中的用途。
如本文所用，术语“药学上可接受的赋型剂”是指在药理学和/或生理学上与受试者和活性成分相容(即，能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用)的载体和/或赋型剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。药学上可接受的赋型剂的实例包括但不限于填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、表面活性剂、稀释剂、润湿剂、防腐剂、乳化剂、包覆剂、等渗剂、吸收延迟剂、稳定剂和张力调节剂。本领域技术人员已知选择合适的赋型剂以制备本发明期望的药物组合物。用于本发明的药物组合物中的示例性赋型剂包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋型剂的选择尤其取决于所使用的活性剂、待治疗的疾病和药物组合物的期望剂型。
根据本发明的药物组合物可适用于多种途径施用。通常，通过胃肠外完成施用。胃肠外递送方法包括局部、动脉内、肌内、皮下、髓内、鞘内、心室内、静脉内、腹膜内、子宫内、阴道内、舌下或鼻内施用。
根据本发明的药物组合物也可以制备成各种形式，如固态、液态、气态或冻干形式，特别可以是软膏、乳膏、透皮贴剂、凝胶、粉末、片剂、溶液、气雾剂、颗粒、丸剂、混悬剂、乳剂、胶囊、糖浆、酏剂、浸膏剂、酊剂或流浸膏提取物的形式，或者是特别适用于所需施用方法的形式。本发明已知的用于生产药物的过程可包括例如常规混合、溶解、制粒、制糖衣、研磨、乳化、包封、包埋或冻干过程。包含例如本文所述的免疫细胞的药物组合物通常以溶液形式提供，并且优选包含药学上可接受的缓冲剂。
根据本发明的药物组合物还可以与一种或多种适用于治疗和/或预防待治疗疾病的其它药剂组合施用。适用于组合的药剂的优选实例包括已知的抗癌药物，比如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立 替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如IL-2，趋化因子比如IL-8、血小板因子4、黑色素瘤生长刺激蛋白等；抗体或其片段，比如抗CD3抗体或其片段，补体活化剂，异种蛋白结构域，同种蛋白结构域，病毒/细菌蛋白结构域和病毒/细菌肽。此外，本发明的药物组合物页可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
制备工程化免疫细胞的方法
在第四个方面，本发明还提供一种制备工程化免疫细胞的方法，包括将本发明的嵌合抗原受体或其编码核酸序列引入免疫细胞，以使所述免疫细胞表达本发明的嵌合抗原受体。
在一些实施方案中，所述免疫细胞是人免疫细胞，更优选人T细胞、巨噬细胞、树突状细胞、中性粒细胞、单核细胞、NK细胞和/或NKT细胞。
将核酸或载体引入免疫细胞并进行表达的方法是本领域已知的。例如，可以通过物理方法，如括磷酸钙沉淀法、脂质转染法、粒子轰击法、显微注射法、电穿孔法等将核酸或载体导入免疫细胞。或者，也可以采用化学方法，如通过胶体分散系统，如大分子复合物、纳米胶囊、微球、珠粒以及基于脂质的系统，包括水包油乳液、胶束、混合胶束及脂质体引入核酸或载体。此外，还可以使用生物方法引入核酸或载体。例如，病毒载体，尤其是逆转录病毒载体等已经成为将基因插入哺乳动物，例如人细胞中的最常用方法。其它病毒载体可以来源于慢病毒、痘病毒、单纯疱疹病毒I、腺病毒及腺相关病毒等。
将核酸或载体引入免疫细胞后，本领域技术人员可以通过常规技术对所得免疫细胞进行扩增和活化。
制药用途/治疗应用
本发明还提供一种治疗患有癌症、感染或自身免疫性疾病的受试者的方法，包括向所述受试者施用有效量的根据本发明所述的嵌合抗原受体、核酸分子、载体、工程化免疫细胞或药物组合物。因此，本发明还涵盖所述嵌合抗原受体、核酸分子、载体或工程化免疫细胞在制备治疗癌症、感染或自身免疫性疾病的药物中的用途。
在一些实施方案中，直接向受试者施用有效量的本发明的免疫细胞和/或药物组合物。
在另一个实施方案中，本发明的治疗方法是离体治疗。具体地，该方法包括以下步骤：(a)提供受试者的样品，所述样品包含免疫细胞；(b)在体外将本发明的嵌合抗原受体引入所述免疫细胞，获得经修饰的免疫细胞，(c)向有此需要的受试者施用所述经修饰的免疫细胞。优选地，步骤(a)中提供的免疫细胞选自T细胞、NK细胞和/或NKT细胞；并且所述免疫细胞可以通过本领域已知的常规方法从受试者的样品(特别是血液样品)中获得。然而，也可以使用能够表达本发明的嵌合抗原受体并发挥如本文所述的所需生物效应功能的其它免疫细胞。此外，通常选择的免疫细胞与受试者的免疫系统相容，即优选所述免疫细胞不引发免疫原性响应。例如，可以使用“通用接受体细胞”，即发挥所 需生物效应功能的普遍相容的可在体外生长和扩增的淋巴细胞。使用此类细胞将不需要获得和/或提供受试者自身淋巴细胞。步骤(c)的离体引入可以通过经由电穿孔将本文所述的核酸或载体引入免疫细胞或通过用病毒载体感染免疫细胞来实施，所述病毒载体为如前所述的慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体。其它可想到的方法包括使用转染试剂(比如脂质体)或瞬时RNA转染。
在一些实施方案中，所述免疫细胞是自体或同种异体的细胞，优选T细胞、巨噬细胞、树突状细胞、中性粒细胞、单核细胞、NK细胞和/或NKT细胞，更优选T细胞、NK细胞或NKT细胞。
如本文所用，术语“自体”是指来源于个体的任何材料稍后将被再引入该相同个体中。
如本文所用，术语“同种异体”是指任何材料来源于与引入该材料的个体相同物种的不同动物或不同患者。当在一个或多个基因座处的基因不同时，认为两个或更多个体彼此为同种异体的。在一些情况下，来自同一物种的各个体的同种异体材料在基因上的不同可能足以发生抗原相互作用。
如本文所用，术语“受试者”是哺乳动物。哺乳动物可以是人、非人灵长类动物、小鼠、大鼠、狗、猫、马或牛，但不限于这些实例。除人以外的哺乳动物可以有利地用作代表癌症动物模型的受试者。优选地，所述受试者是人。
在一些实施方案中，所述癌症是与配体结合结构域结合的靶标表达有关的癌症。例如，所述癌症包括但不限于：脑神经胶质瘤、胚细胞瘤、肉瘤、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌(包括胃肠癌)、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、肝肿瘤、肺癌(例如小细胞肺癌、非小细胞肺癌、腺状肺癌和鳞状肺癌)、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌(例如唇、舌、口和咽)、卵巢癌、胰腺癌、前列腺癌、间皮瘤、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌、Waldenstrom巨球蛋白血症、淋巴瘤(包括霍奇金淋巴瘤和非霍奇金淋巴瘤，例如B细胞淋巴瘤(包括低级/滤泡性非霍奇金淋巴瘤(NHL)、小淋巴细胞性(SL)NHL、中间级/滤泡性NHL、中间级扩散性NHL、高级成免疫细胞性NHL、高级成淋巴细胞性NHL、高级小型非裂化细胞性NHL、大肿块病NHL)、套细胞淋巴瘤、AIDS相关淋巴瘤、伯基特氏淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、MALT淋巴瘤、边缘区淋巴瘤、浆母细胞性淋巴瘤、浆细胞样树突状细胞瘤等)、白血病(包括急性白血病，例如急性淋巴细胞白血病、急性髓细胞白血病、急性非淋巴细胞白血病诸如急性粒细胞白血病(包括未分化型和部分分化型)、急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、红白血病、急性巨核细胞白血病；慢性白血病，例如慢性髓细胞白血病、慢性淋巴细胞白血病、慢性单核细胞白血病；和其他特殊类型的白血病例如毛细胞白血病、幼淋巴细胞白血病、浆细胞白血病、成人T细胞白血病、嗜酸性粒细胞白血病、嗜碱性粒细胞白血病等)、母细胞性浆细胞样树突状细胞瘤、恶性淋巴组织增生疾病、骨髓发育不良、多发性骨髓瘤、骨髓增生异常、以及移植后淋巴细胞增生性紊乱(PTLD)；以及其他与靶标表达有关的疾病。优选地，可以用本发明的工程化免疫细胞或药物组合物治疗的疾病选自：白血病、淋巴瘤、多发性骨髓瘤、脑神经胶质瘤、胰腺癌、卵巢癌、 间皮瘤、乳腺癌、肺癌、前列腺癌、黑色素瘤、骨髓瘤、肉瘤、胃癌等。
在一些实施方案中，所述感染包括但不限于由病毒、细菌、真菌和寄生虫引起的感染。
在一些实施方案中，所述自身免疫性疾病包括但不限于I型糖尿病、腹腔疾病、格雷夫斯病、炎症性肠病、多发性硬化症、银屑病、类风湿性关节炎、艾迪生病、干燥综合征、桥本甲状腺炎、重症肌无力、血管炎、恶性贫血与系统性红斑狼疮等。
在一些实施方案中，所述方法还进一步包括向所述受试者施用一种或多种额外的化疗剂、生物制剂、药物或治疗。在该实施方案中，化疗剂、生物制剂、药物或治疗选自放射疗法、手术、抗体试剂和/或小分子和它们的任意组合。
下面结合具体实施例和附图，进一步阐述本发明。应理解，这些实施例和附图仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
附图说明
图1：通过流式细胞术测定的CAR-T细胞的scFv表达水平。
图2：CAR-T细胞对靶细胞的杀伤效果。
图3：CAR-T细胞分别与靶细胞共培养后的IL-2释放水平。
图4：CAR-T细胞体外长期对靶细胞的杀伤效果。
图5：CAR-T细胞多次与肿瘤细胞共培养后扩增曲线。
具体实施方式
实施例1：构建CAR T细胞
合成以下编码序列，并将其依次克隆至pGEM-T Easy载体：CD8α信号肽(SEQ ID NO:28)、抗CD19 scFv(SEQ ID NO:12)、CD8α铰链区(SEQ ID NO:24)、CD8α跨膜区(SEQ ID NO:14)、CD27共刺激结构域(SEQ ID NO:1)、CD3ζ胞内信号传导结构域(SEQ ID NO:20)，获得CAR19-27质粒，并通过测序确认目标序列的正确插入。用同样的方法获得CAR19-27(-4)、CAR19-27K4R质粒，其与CAR19-27质粒的区别在于，胞内段分别减掉4个氨基酸(SEQ ID NO:3)或将胞内段第四个氨基酸K突变为R(SEQ ID NO:2)。
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)稀释上述质粒后，再根据质粒:病毒包装载体：病毒包膜载体＝4:2:1的比例加入包装载体psPAX2(Addgene，货号12260)和包膜载体pMD2.G(Addgene，货号12259)。然后，加入120ul X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293T细胞的培养瓶中。在24小时和48小时收集病毒，将其合并后，超速离心(25000g，4℃，2.5小时)获得浓缩的慢病毒。
用DynaBeads CD3/CD28 CTSTM(Gibco,货号40203D)激活T细胞，并在37℃和5％CO2下培养1天。然后，加入浓缩的慢病毒，持续培养3天后，分别获得靶向CD19的CAR19-27 T细胞、CAR19-27(-4)T细胞和CAR19-27K4R T细胞。
在37℃和5％CO2下培养12天之后，使用Fluorescein(FITC)AffiniPure Rabbit Anti-Mouse IgG,F(ab')fragment specific(jackson immunoresearch，货号315-095-006)作为一抗，通过流式细胞仪检测CAR T细胞上的scFv的表达水平，结果如图1所示(NT是未经修饰的野生型T细胞)。
可以看出，包含不同的突变的CD27胞内区的CAR T细胞均可以有效表达scFv。
实施例2：CAR T细胞对靶细胞的杀伤效果和细胞因子释放
2.1 CAR T细胞对靶细胞的杀伤效果
当T细胞对靶细胞有杀伤时，靶细胞的数量就会减少。将T细胞和带有可表达荧光素酶的靶细胞共培养后，靶细胞数量减少的同时，分泌的荧光素酶也会随之减少。荧光素酶可以催化荧光素转化为氧化性荧光素，而在此氧化过程中，会产生生物发光，并且这种发光的强度将取决于靶细胞表达的荧光素酶的水平。因此，检测的荧光强度能够反应T细胞对靶细胞的杀伤能力。
为了检测CAR T细胞对靶细胞的杀伤能力，首先以1×104/孔将携带荧光素基因的Raji靶细胞铺入96孔板中，然后以分别以不同的效靶比(即效应T细胞与靶细胞之比)将CAR19-27 T细胞、CAR19-27(-4)T细胞、CAR19-27K4R T细胞和NT细胞铺入到96孔板进行共培养，16-18小时后利用酶标仪测定Raji靶细胞的荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图2所示。
可以看出，与NT组相比，CAR19-27 T细胞、CAR19-27(-4)T细胞、CAR19-27K4R T细胞对靶细胞均有特异性杀伤的能力，且无显著性差异。
2.2 CAR T细胞的细胞因子释放
T细胞杀伤靶细胞时，靶细胞数量减少的同时也会释放细胞因子(例如IL-2和IFN-γ等)。根据以下步骤，使用酶联免疫吸附法(ELISA)来测定CAR T细胞杀伤靶细胞时细胞因子IL-2的释放水平。
(1)收集细胞共培养上清液
以1×105/孔将靶细胞(Raji)铺于96孔板中，然后以1:1的比例将CAR19-27 T细胞、CAR19-27(-4)T细胞、CAR19-27K4R T细胞和NT细胞分别与Raji细胞共培养，18-24小时后收集细胞共培养上清液。
(2)ELISA检测上清中IL-2分泌量
使用捕获抗体Purified anti-humanIL-2Antibody(R&D，货号DY202)包被96孔板4℃孵育过夜，然后移除抗体溶液，加入250μL含有2％BSA(sigma，货号V900933-1kg)的PBST(含0.1％吐温的1XPBS)溶液，37℃孵育2小时。然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。每孔加入50μL细胞共培养上清液或标准品，并在37℃孵育1小时，然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。然后向各孔分别加入50μL检测抗体Anti-IL-2抗体(R&D，货号DY202)，在37℃孵育1小时后，用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。再加入HRP Streptavidin(Biolegend，货号405210)，在37℃孵育30分钟后，弃上清液，加入250μL PBST(含0.1％吐温的1XPBS)，清洗5次。向各孔加入50μL TMB底物溶液。使反应在室温下于暗处发生30分钟，之后向各孔中加入50μL 1mol/L H SO以停止反应。在停止反应的30分钟内，使用酶标仪检测450nm 处吸光度，并根据标准曲线(根据标准品的读值和浓度绘制)计算细胞因子的含量，结果如图3所示。
可以看出，与NT组相比，CAR19-27 T细胞、CAR19-27(-4)T细胞和CAR19-27K4R T均可特异性分泌IL-2，且CAR19-27(-4)T细胞和CAR19-27K4R T细胞IL-2的释放水平均显著高于CAR19-27 T细胞。值得关注的是，CAR19-27(-4)T细胞的IL-2的释放水平相较于CAR19-27 T细胞和CAR19-27K4R T均显著提高。
实施例3 CAR T长期杀伤肿瘤细胞的能力
以5×105/孔将靶细胞(Raji)铺于48孔板中，然后以E:T＝1:2的比例将CAR19-27 T细胞、CAR19-27(-4)T细胞、CAR19-27K4R T细胞与Raji细胞共培养，并分别在培养后D3，D6和D10补加Raji细胞(E:T＝1:1)，每次补加Raji细胞前通过流式检测肿瘤细胞残留评估各组CAR T对肿瘤的杀伤能力，并同时记录各组CAR T的扩增曲线，结果如图4和图5所示。
可以看出，CAR19-27(-4)T细胞、CAR19-27K4R T细胞长期杀伤肿瘤的能力和扩增曲线均显著优于CAR19-27 T组，且CAR19-27K4R T细胞优于CAR19-27K4R T细胞。
以上结果表明，与野生型CD27胞内区相比，本发明中包含突变的CD27胞内区的CAR-T细胞，能够促进细胞因子的分泌和免疫细胞的扩增，增强免疫细胞长期杀伤肿瘤的能力。
需要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (20)

  1. 一种嵌合抗原受体，其包含配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述共刺激结构域包含突变的CD27胞内区；与野生型CD27胞内区相比，所述突变的CD27胞内区包含至少1个赖氨酸突变，或近C-末端的第1-6个氨基酸位点包含至少1个氨基酸缺失。
  2. 权利要求1所述的嵌合抗原受体，其中所述突变的CD27胞内区包含C-末端的第4位赖氨酸残基的缺失或取代，或近C-末端的第1-6个氨基酸位点包含至少1个氨基酸的连续缺失。
  3. 权利要求1或2所述的嵌合抗原受体，其中所述突变的CD27胞内区具有SEQ ID NO:2或3所示的氨基酸序列或与SEQ ID NO:2或3所示的氨基酸序列具有至少90％的序列同一性。
  4. 权利要求1-3任一项所述的嵌合抗原受体，其中所述共刺激结构域进一步包含选自以下蛋白的信号传导结构域：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、ZAP70以及它们的组合。
  5. 权利要求4所述的嵌合抗原受体，其中所述共刺激结构域进一步包含CD28、CD134、CD137或CD278的信号传导结构域或它们的组合。
  6. 权利要求1-5任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域选自以下蛋白的信号传导结构域：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。
  7. 权利要求1-6任一项所述的嵌合抗原受体，其中所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  8. 权利要求1-7任一项所述的嵌合抗原受体，其中所述配体结合结构域是抗体或其抗原结合部分。
  9. 权利要求8所述的嵌合抗原受体，其中所述抗原结合部分选自完整抗体、Fab、Fab'、F(ab')2、Fd、Fd'、Fv、scFv、sdFv、线性抗体、纳米抗体、双体和sdAb。
  10. 权利要求1-9任一项所述的嵌合抗原受体，其中所述配体结合结构域与选自以下的靶标结合：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、CD40、CD44、CD44v6、CD44v7/8、CD47、CD52、CD56、CD57、CD58、CD70、CD72、CD79a、CD79b、CD80、CD81、CD86、CD97、CD123、CD133、CD137、CD138、CD151、CD171、CD179a、CD300LF、CDH16、CSPG4、CS1、Claudin 6、Claudin 18.1、Claudin 18.2、CEA、CEACAM6、CLL1、c-Met、CAIX、CXORF61、CA125、CYP1B1、CS1、ELF2M、EGFR、EPCAM、EGFRvIII、EphA2、ERG/TMPRSS2ETS融合基因、ETV6-AML、EMR2、EGP2，EGP40、FAP、FAR、FBP、FLT3、FOSL1、FCRL5、FCAR、Flt3、Flt4、Frizzled、GD2、GD3、gp100、gp130、GM3、GPC2、GPC3、GPRC5D、GPR20、GloboH、GHRHR、GHR、GITR、Her2、HER3、HER-4、 HMWMAA、HAVCR1、HPV E6,E7、HVEM、HIV-1Gag、HLA-A1、HLA-A2、IL6R、IL-11Ra、IL-13Ra、IGF-I受体、LTPR、LIFRP、LRP5、IGLL1、IGF1R、KIT、Kappa Light Chain、KDR、LewisY、LMP2、LY6K、LAGE-1a、legumain、LCK、LAIR1、LILRA2、LY75、MSLN、MUC1、MUC16、MAGE-A1、MAGE3、MAD-CT-1、MelanA/MART1、ML-IAP、MYCN、mut hsp70-2、NCAM、NY-BR-1、NY-ESO-1、NA17、Notch-1-4、nAchR、NKG2D、NKG2D配体、OY-TES1、OR51E2、OX40、PRSS21、PSCA、PD1、PD-L1、PD-L2、PSMA、Prostase、PAP、PDGFR-β、PCTA-1/半乳凝集素8、p53、p53突变体、prostein、PLAC1、PANX3、PAX3、PAX5、PTCH1、RANK、RAGE-1、ROR1、Ras突变体、RhoC、RU1、RU2、Robol、SSEA-4、SSX2、SART3、Sp17、TSHR、Tn Ag、TGS5、TEM1/CD248、TEM7R、TARP、TCRα、TCRβ、TGFBR1、TGFBR2、TNFRSF4、TWEAK-R、TLR7、TLR9、TAG72、TROP-2、Tie 2、TRP-2、TNFR1、TNFR2、TEM1、UPK2VEGFR、WT1、XAGE1、5T4、8H9、αvβ6整合素、CA9、叶酸受体α、肝配蛋白B2、酪氨酸酶、岩藻糖基GM1、邻-乙酰-GD2、叶酸受体β、多聚唾液酸、精子蛋白17、存活蛋白和端粒酶、肉瘤易位断点、人端粒末端逆转录酶/hTERT、雄激素受体、肠羧基酯酶、细胞周期蛋白B1、纤连蛋白、腱生蛋白、肿瘤坏死区的癌胚变体及其任意组合。
  11. 一种核酸分子，其编码权利要求1-10任一项所述的嵌合抗原受体。
  12. 一种载体，其包含权利要求11所述的核酸分子。
  13. 一种工程化免疫细胞，其包含权利要求1-10任一项所述的嵌合抗原受体或权利要求11所述的核酸分子。
  14. 权利要求13所述的工程化免疫细胞，其中所述免疫细胞选自T细胞、巨噬细胞、树突状细胞、中性粒细胞、单核细胞、NK细胞或NKT细胞。
  15. 权利要求14所述的工程化免疫细胞，其中所述T细胞是CD4+/CD8+双阳性T细胞、CD4+辅助T细胞、CD8+T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞或αβ-T细胞。
  16. 权利要求13-15任一项所述的工程化免疫细胞，其中所述免疫细胞衍生自成体干细胞、胚胎干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞。
  17. 根据权利要求13-16任一项所述的工程化细胞，其中所述工程化细胞中，内源性HLA-I类基因、HLA-II类基因、TCR/CD3基因中的一种或多种基因的表达被抑制或沉默。
  18. 根据权利要求17所述的工程化细胞，其中所述HLA-I类基因选自HLA-A、HLA-B、HLA-C、B2M及其任意组合；所述HLA-II类基因选自HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA及其任意组合；所述TCR/CD3基因选自TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ及其任意组合。
  19. 一种药物组合物，其包含权利要求13-18任一项所述的工程化免疫细胞，和一种或多种药学上可接受的赋型剂。
  20. 权利要求1-10任一项所述的嵌合抗原受体、权利要求11所述的核酸分子、权利要求12所述的载体、权利要求13-18任一项所述的工程化免疫细胞、或权利要求19所述的药物组合物在制备用于治疗癌症、感染或自身免疫性疾病的药物中的用途。
PCT/CN2023/100067 2023-06-14 2023-06-14 包含新型共刺激结构域的嵌合抗原受体及其用途 Ceased WO2024254775A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2023/100067 WO2024254775A1 (zh) 2023-06-14 2023-06-14 包含新型共刺激结构域的嵌合抗原受体及其用途
CN202380098273.1A CN121419997A (zh) 2023-06-14 2023-06-14 包含新型共刺激结构域的嵌合抗原受体及其用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/100067 WO2024254775A1 (zh) 2023-06-14 2023-06-14 包含新型共刺激结构域的嵌合抗原受体及其用途

Publications (1)

Publication Number Publication Date
WO2024254775A1 true WO2024254775A1 (zh) 2024-12-19

Family

ID=93851078

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/100067 Ceased WO2024254775A1 (zh) 2023-06-14 2023-06-14 包含新型共刺激结构域的嵌合抗原受体及其用途

Country Status (2)

Country Link
CN (1) CN121419997A (zh)
WO (1) WO2024254775A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120137041A (zh) * 2025-03-14 2025-06-13 深圳市欧安蒂生物科技有限公司 一种抗gprc5d抗体及其在car-t中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105949325A (zh) * 2016-07-08 2016-09-21 重庆精准生物技术有限公司 包含cd27胞内结构域的嵌合抗原受体、慢病毒载体及其应用
WO2018103734A1 (zh) * 2016-12-09 2018-06-14 北京中捭生物科技有限公司 嵌合抗原受体及其用途和制备方法
CN110526991A (zh) * 2018-05-25 2019-12-03 深圳宾德生物技术有限公司 靶向fap的嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用
CN113272016A (zh) * 2018-10-01 2021-08-17 阿迪塞特生物股份有限公司 关于治疗实体肿瘤的工程化和非工程化γδ-T细胞的组合物和方法
CN114835817A (zh) * 2021-02-01 2022-08-02 四川大学华西医院 一种基于天然配体制备靶向her2/egfr car-t的方法及应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105949325A (zh) * 2016-07-08 2016-09-21 重庆精准生物技术有限公司 包含cd27胞内结构域的嵌合抗原受体、慢病毒载体及其应用
WO2018103734A1 (zh) * 2016-12-09 2018-06-14 北京中捭生物科技有限公司 嵌合抗原受体及其用途和制备方法
CN110526991A (zh) * 2018-05-25 2019-12-03 深圳宾德生物技术有限公司 靶向fap的嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用
CN113272016A (zh) * 2018-10-01 2021-08-17 阿迪塞特生物股份有限公司 关于治疗实体肿瘤的工程化和非工程化γδ-T细胞的组合物和方法
CN114835817A (zh) * 2021-02-01 2022-08-02 四川大学华西医院 一种基于天然配体制备靶向her2/egfr car-t的方法及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN HUANPENG; WEI FENGJIAO; YIN MENG; ZHAO QINGYU; LIU ZHONGHUA; YU BOLAN; HUANG ZHAOFENG: "CD27 enhances the killing effect of CAR T cells targeting trophoblast cell surface antigen 2 in the treatment of solid tumors", CANCER IMMUNOLOGY IMMUNOTHERAPY, SPRINGER, BERLIN/HEIDELBERG, vol. 70, no. 7, 13 January 2021 (2021-01-13), Berlin/Heidelberg , pages 2059 - 2071, XP037479846, ISSN: 0340-7004, DOI: 10.1007/s00262-020-02838-8 *
KEITH SCHUTSKY ET AL.: "Rigorous optimization and validation of potent RNA CAR T cell therapy for the treatment of common epithelial cancers expressing folate receptor", ONCOTARGET., vol. 6, no. 30, 6 October 2015 (2015-10-06), XP055518239, DOI: 10.18632/oncotarget.5029 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120137041A (zh) * 2025-03-14 2025-06-13 深圳市欧安蒂生物科技有限公司 一种抗gprc5d抗体及其在car-t中的应用

Also Published As

Publication number Publication date
CN121419997A (zh) 2026-01-27

Similar Documents

Publication Publication Date Title
JP7462777B2 (ja) 新規キメラ抗原受容体とその使用
CN112029001B (zh) 靶向nk激活性受体的嵌合抗原受体
JP7623020B2 (ja) 同種異系移植のための操作された免疫細胞
JP2024109827A (ja) Ｎｋ阻害性分子を発現する遺伝子改変免疫細胞とその使用
US20240009308A1 (en) Chimeric antigen receptor comprising novel co-stimulatory domain and use thereof
EP4613781A1 (en) Immune rejection-resistant engineered cell
JP2025513626A (ja) Ｃｉｉｔａ遺伝子がノックアウトされた操作された免疫細胞及びその用途
EP4186929A1 (en) Novel co-stimulatory domain and uses thereof
WO2022267983A1 (zh) 工程化免疫细胞及其用途
WO2024254775A1 (zh) 包含新型共刺激结构域的嵌合抗原受体及其用途
CN115785279A (zh) 包含新型共刺激结构域的嵌合抗原受体及其用途
WO2024179620A2 (zh) 耐免疫排斥的工程化细胞
CN117430712A (zh) 靶向cxcr6的嵌合抗原受体及其用途
CA3171344C (en) Engineered immune cell expressing nk inhibitory molecule and use thereof
HK40087333A (zh) 新型共刺激结构域及其用途
WO2023025009A1 (zh) 工程化免疫细胞及其用途
WO2023016284A1 (zh) 工程化免疫细胞及其用途
WO2024192622A1 (zh) 用于同种异体移植的工程化细胞
WO2024165051A1 (zh) 功能增强的细胞疗法
WO2024160180A1 (zh) 用于同种异体移植的组合物及方法
WO2025035310A1 (zh) 工程化免疫细胞及其用途
WO2025035309A1 (zh) 工程化免疫细胞及其用途
WO2024165053A1 (zh) 功能增强的细胞疗法
HK40086008A (zh) 包含新型共刺激结构域的嵌合抗原受体及其用途
HK40084045A (zh) 用於同种异体移植的工程免疫细胞

Legal Events

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

Ref document number: 23941012

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE