WO2023207388A1 - 一种cd7基因被敲除的工程化免疫细胞及其用途 - Google Patents

一种cd7基因被敲除的工程化免疫细胞及其用途 Download PDF

Info

Publication number
WO2023207388A1
WO2023207388A1 PCT/CN2023/081600 CN2023081600W WO2023207388A1 WO 2023207388 A1 WO2023207388 A1 WO 2023207388A1 CN 2023081600 W CN2023081600 W CN 2023081600W WO 2023207388 A1 WO2023207388 A1 WO 2023207388A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
domain
engineered immune
gene
sgrna
Prior art date
Application number
PCT/CN2023/081600
Other languages
English (en)
French (fr)
Inventor
周亚丽
陈功
黄洁
张学建
任江涛
Original Assignee
南京北恒生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京北恒生物科技有限公司 filed Critical 南京北恒生物科技有限公司
Publication of WO2023207388A1 publication Critical patent/WO2023207388A1/zh

Links

Classifications

    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention belongs to the field of biomedicine. More specifically, the present invention relates to an engineered immune cell in which the CD7 gene has been knocked out and its use.
  • CAR-T cell immunotherapy involves genetically modifying T cells in vitro so that they can recognize tumor antigens. After amplifying to a certain number, they are reinfused back into the patient's body to kill cancer cells, thereby achieving the purpose of treating tumors.
  • CD7 is a cell surface glycoprotein with a molecular weight of approximately 40kDa and is a member of the immunoglobulin superfamily. CD7 is expressed on most T cells, NK cells, myeloid cells, T-cell acute lymphoblastic leukemia/lymphoma, acute myeloid leukemia and chronic myeloid leukemia. It has been reported that the CD7 molecule acts as a costimulatory signal during T cell activation through binding to its ligand K12/SECTM1.
  • T-ALL T-cell acute lymphoblastic leukemia
  • CD7 itself is also expressed in T cells, in order to avoid killing each other between CAR-T cells, when preparing CAR-T cells targeting CD7, CD7 in T cells needs to be knocked out first.
  • the present invention aims to provide an sgRNA that specifically targets the CD7 gene and an engineered immune cell that uses the sgRNA to knock out the CD7 gene, and its use in disease prevention and/or treatment and/or diagnosis and treatment of cancer, infection or self. Use in immune diseases.
  • the present invention provides an sgRNA comprising the spacer sequence shown in any one of SEQ ID NO: 1, 3, 4, 5, 8, and 9.
  • the spacer sequence is as shown in any one of SEQ ID NO: 1, 3, and 4.
  • the present invention provides nucleic acids encoding the above-mentioned sgRNA and vectors containing the nucleic acids.
  • the present invention provides a method for knocking out the CD7 gene in vitro, including introducing Cas nuclease and the above-mentioned sgRNA into cells.
  • the Cas nuclease is Cas9, Cas12a or Cas13a, in the form of a protein, its encoding nucleic acid or a vector.
  • the Cas nuclease is Cas9.
  • the sgRNA is in the form of RNA, its encoding nucleic acid, or a vector.
  • the cells are immune cells, which are T cells, B cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, etc.
  • the immune cells are T cells, NK cells or NKT cells. More preferably, the T cells are CD4 + CD8 + T cells, CD4 + T cells, CD8 + T cells, memory T cells, naive T cells, ⁇ -T cells and/or ⁇ -T cells.
  • the method further comprises introducing into the immune cell a nucleic acid encoding a chimeric antigen receptor or a T cell receptor or both.
  • the chimeric antigen receptor includes a ligand binding domain, a transmembrane domain, a costimulatory domain, and a primary signaling domain.
  • the ligand binding domain binds the following targets: CD7, TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3 , BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY , CD24, PDGFR- ⁇ , SSEA-4, CD20, Folate receptor ⁇ , ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase,
  • the target is selected from CD7, CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2/neu), MUCl, EGFR, CAIX, WT1, NY -ESO-1, CD79a, CD79b, GPC3, Claudin18.2, NKG2D, or any combination thereof.
  • Those skilled in the art can determine the antigen that needs to be targeted according to the disease to be treated, and thereby design the corresponding chimeric antigen receptor.
  • the ligand binding domain includes an antibody or antigen-binding fragment targeting CD7.
  • the ligand binding domain of the invention comprises a light chain variable region sequence having at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 and A heavy chain variable region sequence having at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 13.
  • the transmembrane domain is selected from the group consisting of TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or any combination thereof.
  • the transmembrane domain is derived from the CD8 alpha chain, which has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, 99% with the amino acid sequence shown in SEQ ID NO:15 Or 100% sequence identity, or the coding sequence of the CD8 ⁇ transmembrane domain has at least 70%, preferably at least 80%, and more preferably at least 90%, 95% with the nucleotide sequence shown in SEQ ID NO:16 , 97%, 99% or 100% sequence identity.
  • the chimeric antigen receptor further comprises a hinge region located between the ligand binding domain and the transmembrane domain.
  • the hinge region includes the hinge region of CD8 ⁇ chain, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably the CD8 ⁇ hinge region has at least 70%, preferably at least 80%, with the amino acid sequence shown in SEQ ID NO: 17, More preferably, there is at least 90%, 95%, 97%, 99% or 100% sequence identity, or the coding sequence of the CD8 alpha hinge has at least 70%, preferably at least 80%, with the nucleotide sequence shown in SEQ ID NO: 18 , more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity.
  • the primary signaling domain is selected from the signaling domain of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, CD66d, or any combination thereof.
  • the primary signaling domain comprises a CD3 ⁇ primary signaling domain, which has at least 70%, preferably at least 80%, and more preferably at least 80% of the amino acid sequence shown in SEQ ID NO: 19 or 21. 90%, 95%, 97%, 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, and more preferably, the nucleotide sequence shown in SEQ ID NO: 20 or 22 At least 90%, 95%, 97%, 99% or 100% sequence identity.
  • the costimulatory domains include, but are not limited to, costimulatory signaling domains derived from 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), CD150(SLAMF1), CD152(CTLA4), CD223(LAG3), CD270(HVEM), CD272(BTLA), CD273(PD-L2), CD274(PD-L1), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, LAT, NKG2C, SLP76, PD1, LIGHT, TRIM, ZAP70, or any combination thereof.
  • costimulatory signaling domains derived from the following proteins: TLR1, TLR2, T
  • the costimulatory domain is from 4-1BB, CD28 or 4-1BB+CD28, more preferably the 4-1BB 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: 23 or the coding sequence of the co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% similarity with the amino acid sequence shown in SEQ ID NO: 24 Sequence identity.
  • the chimeric antigen receptor further includes a signal peptide, which shares at least 70%, preferably at least 80%, more preferably at least 90%, 95% of the amino acid sequence shown in SEQ ID NO: 25. %, 97%, 99% or 100% sequence identity, or the coding sequence of the signal peptide has at least 70%, preferably at least 80%, and more preferably at least 90%, 95% with the amino acid sequence shown in SEQ ID NO: 26 %, 97%, 99% or 100% sequence identity.
  • the T cell receptor targets one or more of HBV, HPV E6, NYESO, mNY-ESO, WT1, MART-1, MAGE-A3, MAGE-A4, P53, Thyroglobulin, Tyrosinase kind.
  • the chimeric antigen receptor or T cell receptor can be introduced into the host cell via a vector.
  • the vector is selected from the group consisting of plasmids, retroviruses, lentiviruses, adenovirus, vaccinia virus, Rous sarcoma virus (RSV), polyomavirus and adeno-associated virus (AAV), phage, phagemid, cosmid Or artificial chromosomes.
  • the vector also contains an origin for autonomous replication in the host cell, a selectable marker, a restriction enzyme cleavage site, a promoter, a poly(A) tail (polyA), a 3'UTR, a 5'UTR, an enhancer elements such as subon, terminator, insulator, operator, selectable marker, reporter gene, targeting sequence and/or protein purification tag.
  • the vector is a plasmid, lentiviral vector, AAV vector, adenoviral vector or retroviral vector.
  • the present invention provides engineered immune cells prepared using the above method of knocking out the CD7 gene.
  • the engineered immune cells further comprise at least one gene selected from the group consisting of GR, dCK, TCR/CD3 genes (e.g., TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ ) whose expression is inhibited or silenced.
  • GR GR
  • dCK TCR/CD3 genes
  • MHC-related genes HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA
  • immunity Checkpoint genes such as 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, TGFBRRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SKI,
  • the engineered immune cells further comprise at least one gene selected from the group consisting of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, whose expression is inhibited or silenced.
  • CD7 and TRAC are knocked out in the engineered immune cells. This inactivation renders the TCR-CD3 complex nonfunctional in the cells. This strategy is particularly useful for avoiding graft-versus-host disease (GvHD).
  • the present invention also provides compositions comprising the above-mentioned engineered immune cells, and the use of the immune cells or compositions in the preparation of medicaments for the treatment/prevention/diagnosis of cancer, infection or autoimmune diseases. .
  • the composition further includes one or more pharmaceutically acceptable excipients.
  • the engineered immune cells or compositions of the invention can also be used in combination with one or more additional chemotherapeutic agents, biologics, drugs, or treatments.
  • the chemotherapeutic agent, biologic, drug, or treatment is selected from radiation therapy, surgery, antibody agents, small molecules, or any combination thereof.
  • the advantage of the present invention is that the screened sgRNA can efficiently knock out the CD7 gene, thereby avoiding mutual killing between CAR-T cells, and preparing universal CAR-T cells by simultaneously knocking out the TRAC gene.
  • Figure 1 shows the knockout situation of TRAC/CD7 gene in TRAC/CD7 double knockout CD7 CAR-T cells (CAR7-37KO).
  • FIG 2 shows the expression levels of CAR in CD7 single knockout CD7 CAR-T cells (CAR7-7KO) and TRAC/CD7 double knockout CD7 CAR-T cells (CAR7-37KO).
  • Figure 3 shows the specific lysis rate of Jurkat target cells by CD7 single knockout CD7 CAR-T cells (CAR7-7KO) and TRAC/CD7 double knockout CD7 CAR-T cells (CAR7-37KO).
  • Figure 4 shows the cytokine IFN ⁇ secretion levels of CD7 single knockout CD7 CAR-T cells (CAR7-7KO) and TRAC/CD7 double knockout CD7 CAR-T cells (CAR7-37KO).
  • the CRISPR/Cas system is a natural immune system of prokaryotes. After being invaded by a virus, certain bacteria can store a small segment of the viral gene into a storage space called CRISPR in their own DNA. When encountering a virus invasion again, the bacteria can identify the virus based on the stored fragments and cut off the virus' DNA to render it ineffective.
  • CRISPR systems include type I, type II and type III. Among them, CRISPR/Cas9 is the most studied type II. In addition to Cas9 nuclease, Cas12a (also known as Cpf1) and Cas13a (also known as C2c2) are also commonly used Cas nucleases. CRISPR/Cas9-mediated gene editing technology can be used to generate transgenic models, regulate transcription, regulate epigenetics, etc.
  • the CRISPR/Cas9 system is mainly composed of Cas9 protein and single-stranded guide RNA (sgRNA).
  • the Cas9 protein has the function of cutting double-stranded DNA, and the sgRNA plays a guiding role.
  • the working principle of this system is that CRISPR RNAs (crRNAs) combine with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex. This complex guides the nuclease Cas9 protein to interact with the spacer sequence in crRNA. Paired sequences target sites to cleave double-stranded DNA.
  • sgRNA also known as “single guide RNA” refers to a guide RNA sequence used to target a specific gene for correction using CRISPR technology, usually consisting of a fusion of crRNA and tracrRNA molecules that are partially complementary to form a complex.
  • crRNA contains a sequence that is complementary to the target sequence so as to hybridize with the target sequence and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to sequence-specifically bind to the target sequence.
  • CRISPR complex Cas9+crRNA+tracrRNA
  • the sgRNA recognizes and cleaves guide RNA-specific DNA targets.
  • the part of the sgRNA responsible for complementary pairing with the target DNA is the spacer sequence it contains.
  • the sgRNA comprises the spacer sequence shown in any one of SEQ ID NO: 1, 3, 4, 5, 8, and 9.
  • sgRNA spacer sequence is generally based on the following principles:
  • the length should generally be about 20nt;
  • the seed sequence (1-12 bases close to the PAM region) should try to avoid ending with more than 4 Ts, and the optimal GC% content should be 40% to 60%;
  • sgRNA spacer needs to consider many factors, such as length, GC content, binding position of the target gene, binding rate to non-target sites, whether it contains SNP, secondary structure, etc.
  • sgRNA can be designed through various online tools.
  • the Cas enzyme can cut any target sequence adjacent to the PAM site, for a specific target gene, the editing efficiency of a large number of sgRNAs designed by online tools is not the same or even very different.
  • the PAM site is 5
  • the editing efficiency of '-NGG-3' is usually higher than that of 5'-NGA-3' or 5'-NAG-3'. Therefore, screening sgRNA with high specificity is crucial to improve the editing efficiency of CRISPR system.
  • nucleic acid may be DNA or RNA.
  • vector is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell where it can be replicated and/or expressed.
  • vectors of the invention include, but are not limited to, linear nucleic acids (e.g., DNA or RNA), plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma viruses (RSV, polypeptides), tumor viruses and adeno-associated viruses (AAV), etc.), phages, phagemids, cosmids and artificial chromosomes (including BAC and YAC).
  • linear nucleic acids e.g., DNA or RNA
  • viruses e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma viruses (RSV, polypeptides), tumor viruses and adeno-associated viruses (AAV), etc.
  • phages emids
  • cosmids and artificial chromosomes including BAC and YAC
  • CAR chimeric antigen receptor
  • CAR includes a ligand-binding domain, a transmembrane domain, a costimulatory domain, and a primary signaling domain.
  • Ligand binding domain refers to any structure or functional variant thereof, including antibodies or antigen-binding fragments, that can specifically bind to a ligand.
  • Transmembrane domain refers to a polypeptide structure that enables chimeric antigen receptors to be expressed on the surface of immune cells (eg, lymphocytes, NK cells, or NKT cells) and directs the immune cells' cellular response against target cells.
  • immune cells eg, lymphocytes, NK cells, or NKT cells
  • Primary signaling domain refers to the portion of the protein that transduces effector functional signals and instructs cells to perform specified functions. It is responsible for primary intracellular signal transmission after the ligand-binding domain binds to the antigen, thereby leading to the development of immune cells and immune responses. activation.
  • Co-stimulatory domain can be an intracellular functional signaling domain derived from a costimulatory molecule; "costimulatory molecule” refers to one that specifically binds to a costimulatory ligand on T cells, thereby mediating costimulation of T cells Homologous binding partner for a reaction (e.g., proliferation).
  • costimulatory molecule refers to one that specifically binds to a costimulatory ligand on T cells, thereby mediating costimulation of T cells Homologous binding partner for a reaction (e.g., proliferation).
  • a "hinge region” connects the transmembrane domain and the ligand-binding domain to provide greater flexibility and accessibility to the ligand-binding domain.
  • the hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the ligand binding domain includes an antibody or antigen-binding fragment targeting CD7.
  • the CAR of the present invention can perform antigen recognition based on antigen-binding specificity. When it binds to a specific antigen, it affects tumor cells, causing them to not grow, to be driven to death, or otherwise affected, and to cause the patient's tumor burden to shrink or be eliminated.
  • the antigen binding domain is fused to an intracellular domain in combination with a 4-1BB costimulatory domain and a CD3 ⁇ signaling domain.
  • antibody includes monoclonal antibodies (including intact antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibodies capable of exhibiting a desired biological activity carrying one or more Antibody fragments or synthetic polypeptides of CDR sequences.
  • a complete antibody consists of two heavy chains and two light chains linked together by disulfide bonds in a "Y"-shaped structure.
  • Each heavy chain and light chain contains a variable region and a constant region.
  • Each of the heavy and light chain variable regions contains three CDRs, and the CDRs are separated by a more conservative framework region (FR).
  • the variable region is responsible for recognition and binding to the antigen, while the constant region mediates the binding of the antibody to host tissue or factors.
  • antigen-binding fragment includes only a portion of an intact antibody, and generally includes an intact antibody.
  • the antigen-binding site of the body and therefore retains the ability to bind antigen.
  • antigen-binding fragments in the present invention include, but are not limited to: Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, disulfide-linked Fv (sdFv), antibody's Heavy chain variable region (VH) or light chain variable region (VL), linear antibodies, "dibodies” with two antigen-binding sites, single domain antibodies, Nanobodies, natural ligands for the antigen, or Its functional fragments, etc.
  • the chimeric antigen receptor of the invention may also comprise a signal peptide such that when expressed in cells such as T cells, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface.
  • the core of the signal peptide may contain long stretches of hydrophobic amino acids, which have a tendency to form a single ⁇ -helix.
  • Signal peptides useful in the present invention are well known to those skilled in the art, such as signal peptides derived from CD8 ⁇ , IgGl, GM-CSFRa, etc.
  • the term "immune cell” refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells.
  • the immune cells are 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, and the like.
  • the immune cells are T cells.
  • the T cells can be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from a T cell line cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including, but not limited to, CD4 + /CD8 + T cells, CD4 + helper T cells (e.g., Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating cells , memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells.
  • T cells can be obtained from the blood of a subject using a variety of techniques known to those skilled in the art, such as Ficoll isolation.
  • the engineered immune cells further comprise at least one gene selected from the group consisting of CD52, GR, dCK, TCR/CD3 genes whose expression is inhibited or silenced (for example, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ ), MHC related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA) and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP
  • the engineered immune cells further comprise at least one gene selected from the group consisting of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, whose expression is inhibited or silenced.
  • the present invention also provides a composition comprising the engineered immune cell body of the present invention.
  • one or more pharmaceutically acceptable excipients are also included.
  • pharmaceutically acceptable excipient means an excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any adverse effects).
  • compositions including but not limited to fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents, glidants, antioxidants, Flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, Stabilizer and tonicity regulator.
  • Various means of administering compositions exist in the art, including, but not limited to, oral, injectable, aerosol, parenteral, and topical administration.
  • compositions of the present invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated.
  • agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer porphyrin sodium II (sorfimersodium photofrin II), temozolomide, topotecan, trimetreateglucuronate, oris Statin E (auristatin E), vincristine, and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase, and RNase; radionuclides, such as iodine-131, Rhenium 186, indium
  • the engineered immune cells and compositions of the present invention can be used as drugs for the treatment/prevention/diagnosis of cancer, infection or autoimmune diseases.
  • cancers that can be treated/prevented/diagnosed with the engineered immune cells or compositions of the invention include non-solid tumors (such as hematological tumors, such as leukemias and lymphomas) and solid tumors.
  • Hematologic neoplasms are cancers of the blood or bone marrow, including, but not limited to, acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma Chikin lymphoma (indolent and high-grade forms), multiple
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid, and can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, pancreatic cancer, ovarian cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, Small bowel cancer, melanoma, kidney cancer, laryngeal cancer, soft tissue cancer, stomach cancer, testicular cancer, colon cancer, esophageal cancer, cervical cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal cancer , eye cancer, intrahepatic cholangiocarcinoma, joint cancer, neck cancer, gallblad
  • cancers that can be treated/prevented/diagnosed with the engineered immune cells or compositions of the present invention include/prevent/diagnose acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and T lymphoblastic lymphoma (T-LBL), early pre-T lymphoblastic leukemia (ETP-ALL), and extranodal NK/T-cell lymphoma.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • T-LBL T lymphoblastic lymphoma
  • ETP-ALL early pre-T lymphoblastic leukemia
  • extranodal NK/T-cell lymphoma extranodal NK/T-cell lymphoma.
  • the spacer sequence targets the exon of the CD7 gene, and the target sequence is unique on the CD7 gene. Fuse the crRNA containing the spacer sequence with tracrRNA to obtain a sequence of about 100 bp, and artificially synthesize the sequence to obtain the sgRNA product.
  • T cells were stimulated with DynaBeads CD3/CD28 CTSTM (Gibco, Cat. No. 40203D) and incubated at 37°C and cultured for 3 days under 5% CO2. Then, 5 ⁇ g of Cas9 protein and 5 ⁇ g of sgRNA were electrotransfected into activated T cells using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX) at 400V and 0.7 ms to obtain CD7 knockout T cells. Immediately after electrotransfection, T cells were placed in 1 ml of pre-warmed medium and cultured in the presence of IL-2 (300 IU/ml) at 37°C and 5% CO2.
  • IL-2 300 IU/ml
  • PE anti-human CD7 Biolegend, Cat. No. 343106
  • the spacer sequences of different sgRNAs and their knockout efficiencies are shown in Table 1.
  • sgRNAs have different knockout efficiencies of the CD7 gene.
  • the knockout efficiencies of SEQ ID NO: 1, 3, 4, 5, 8, and 9 are all higher than 80%
  • the knockout efficiency of 4 is higher than 90%. Based on the knockout efficiency, 7-4 were selected for subsequent experiments.
  • CD8 ⁇ signal peptide SEQ ID NO: 25
  • anti-CD7 scFv SEQ ID NO: 14
  • CD8 ⁇ hinge region SEQ ID NO: 17
  • CD8 ⁇ transmembrane region SEQ ID NO: 15
  • 4-1BB costimulatory domain SEQ ID NO: 23
  • CD3 ⁇ primary signaling domain SEQ ID NO: 19
  • 06366236001 mix immediately, incubate at room temperature for 15 minutes, and then add the plasmid/vector/transfection reagent mixture dropwise to the culture flask of 293T cells. .
  • Viruses were collected at 24 hours and 48 hours, combined, and ultracentrifuged (25000g, 4°C, 2.5 hours) to obtain concentrated lentivirus.
  • T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, Cat. No. 40203D) and cultured at 37°C and 5% CO for 1 day. Then, concentrated lentivirus was added and cultured for 3 days to obtain CD7-targeting CAR-T cells.
  • the target gene is effectively knocked out in CAR7-37KO cells, indicating that the sgRNA selected in the present invention can effectively knock out TRAC and CD7 genes.
  • Jurkat target cells carrying the fluorescein gene were first plated into a 96-well plate at 1 ⁇ 10 4 /well, and then at different effect-target ratios (i.e., effector T cells and (ratio of target cells). The three groups of cells were spread into 96-well plates for co-culture. After 8 hours, the fluorescence value was measured using a microplate reader. According to the calculation formula: (mean fluorescence of target cells - mean fluorescence of sample)/mean fluorescence of target cells ⁇ 100%, the killing efficiency was calculated, and the results are shown in Figure 3.
  • cytokines such as IL2 and IFN- ⁇ are also released.
  • ELISA enzyme-linked immunosorbent assay
  • Jurkat target cells were plated in a 96-well plate at 1 ⁇ 10 5 /well, and then three groups of cells were co-cultured with target cells at a ratio of 1:1. The cell co-culture supernatants were collected after 8 hours.
  • cytokine IFN ⁇ The secretion of cytokine IFN ⁇ in the supernatant was measured by ELISA.
  • Use capture antibody Purified anti-human IFN- ⁇ Antibody Biolegend, Cat. No. 506502 to coat 96-well plates and incubate overnight at 4°C. Then remove the antibody solution and add 250 ⁇ L of PBST (sigma, Cat. No. V900933-1kg) containing 2% BSA (sigma, Cat. No. V900933-1kg).
  • 1XPBS containing 0.1% Tween incubate at 37°C for 2 hours. Then add 50 ⁇ L of cell co-culture supernatant or standard to each well and incubate at 37°C for 1 hour.
  • the reaction was allowed to occur in the dark at room temperature for 30 minutes, after which 50 ⁇ L of 1 mol/L H SO was added to each well to stop the reaction. Within 30 minutes after stopping the reaction, use a microplate reader to detect the absorbance at 450 nm, and calculate the cytokine content based on the standard curve (drawn based on the reading value and concentration of the standard). The results are shown in Figure 4.

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

Abstract

涉及一种CD7基因被敲除的工程化免疫细胞及其用途。设计并合成了特异性靶向CD7基因的sgRNA，其能够精确靶向CD7基因实现基因敲除，且敲除效率高。提供的sgRNA可用于制备靶向CD7的工程化免疫细胞，进一步还可用于制备靶向CD7的通用型CAR-T细胞。

Description

一种CD7基因被敲除的工程化免疫细胞及其用途
相关申请的交叉引用
本发明要求于2022年04月28日提交中国专利局的申请号为CN202210460785.5、名称为“一种CD7基因被敲除的工程化免疫细胞及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明属于生物医药领域。更具体地，本发明涉及一种CD7基因被敲除的工程化免疫细胞及其用途。
背景技术
近几年，癌症免疫治疗技术发展迅速，尤其是嵌合抗原受体T细胞(CAR-T)相关的免疫疗法在肿瘤疾病的治疗上获得了优异的临床效果。CAR-T细胞免疫疗法是将T细胞在体外进行基因改造，使其能够识别肿瘤抗原，在扩增到一定数量后回输至病人体内，进行癌细胞杀伤，从而达到治疗肿瘤的目的。
CD7是一种细胞表面糖蛋白，分子量为约40kDa，属于免疫球蛋白超家族的一员。CD7在大多数的T细胞、NK细胞、髓细胞、T细胞急性淋巴细胞白血病/淋巴瘤、急性粒细胞性白血病和慢性粒细胞白血病中表达。据报道，CD7分子通过与其配体K12/SECTM1的结合在T细胞活化期间起到共刺激信号的作用。此外，据报道，破坏小鼠T祖细胞中的CD7分子依然会产生正常的T细胞发育和体内平衡，表明CD7似乎对T细胞的发育和功能不会产生关键性的影响，这使其成为治疗T细胞急性淋巴细胞白血病(T-ALL)的非常合适的治疗靶点。因此，将CD7作为治疗白血病和淋巴瘤的细胞毒性分子的靶标，运用于CAR-T细胞免疫疗法，将在CD7表达相关的疾病中有重要应用前景。
然而，由于CD7本身也在T细胞中表达，为了避免CAR-T细胞之间的互相杀伤，制备靶向CD7的CAR-T细胞时，需要先敲除T细胞中的CD7。
发明内容
本发明旨在提供一种特异性靶向CD7基因的sgRNA和利用该sgRNA敲除了CD7基因的工程化免疫细胞，及其在疾病预防和/或治疗和/或诊断治疗癌症、感染或自身 免疫性疾病中的用途。
在第一个方面，本发明提供了一种sgRNA，其包含SEQ ID NO：1、3、4、5、8、9任一所示的间隔区序列。优选地，所述间隔区序列如SEQ ID NO：1、3、4任一所示。
在第二个方面，本发明提供了编码上述sgRNA的核酸以及包含所述核酸的载体。
在第三个方面，本发明提供了一种体外敲除CD7基因的方法，包括将Cas核酸酶和上述sgRNA引入细胞。
在一个实施方案中，所述Cas核酸酶是Cas9、Cas12a或Cas13a，以蛋白、其编码核酸或载体的形式存在。优选地，所述Cas核酸酶是Cas9。
在一种实施方案中，所述sgRNA是以RNA、其编码核酸或载体的形式存在。
在一个实施方案中，所述细胞是免疫细胞，所述免疫细胞是T细胞、B细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞等。优选地，所述免疫细胞是T细胞、NK细胞或NKT细胞。更为优选地，所述T细胞是CD4+CD8+T细胞、CD4+T细胞、CD8+T细胞、记忆T细胞、幼稚T细胞、γδ-T细胞和/或αβ-T细胞。
在一个实施方案中，所述方法进一步包括向所述免疫细胞中引入编码嵌合抗原受体或T细胞受体或两者的核酸。
在一个实施方案中，所述嵌合抗原受体包括配体结合结构域、跨膜结构域、共刺激结构域和初级信号传导结构域。
在一个实施方案中，所述配体结合结构域结合以下靶标：CD7、TSHR、CD19、CD123、CD22、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、GPRC5D、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-l lRa、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、豆荚蛋白、HPV E6、E7、MAGE Al、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋 白、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D或它们的任意组合。优选地，所述靶标选自CD7、CD19、CD20、CD22、BAFF-R、CD33、EGFRvIII、BCMA、GPRC5D、PSMA、ROR1、FAP、ERBB2(Her2/neu)、MUC1、EGFR、CAIX、WT1、NY-ESO-1、CD79a、CD79b、GPC3、Claudin18.2、NKG2D或它们的任意组合。本领域技术人员可以根据待治疗的病症确定需要靶向的抗原，从而设计相应的嵌合抗原受体。
在一个实施方案中，所述配体结合结构域包括靶向CD7的抗体或抗原结合片段。优选地，本发明的配体结合结构域包含与SEQ ID NO：12所示的氨基酸序列具有至少90％、95％、97％、99％或100％序列同一性的轻链可变区序列和与SEQ ID NO：13所示的氨基酸序列具有至少90％、95％、97％、99％或100％序列同一性的重链可变区序列。
在一个实施方案中，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、CD154或它们的任意组合。优选地，所述跨膜结构域源自CD8α链，其与SEQ ID NO:15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％、99％或100％的序列同一性，或所述CD8α跨膜结构域的编码序列与SEQ ID NO:16所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％的序列同一性。
在一个实施方案中，所述嵌合抗原受体还包含位于配体结合结构域和跨膜结构域之间的铰链区。优选地，所述铰链区包含CD8α链、FcγRIIIα受体、IgG4或IgG1的铰链区，更优选CD8α铰链区，其与SEQ ID NO：17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％、99％或100％的序列同一性，或者CD8α铰链的编码序列与SEQ ID NO：18所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％的序列同一性。
在一个实施方案中，所述初级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、CD66d或它们的任意组合。优选地，所述初级信号传导结构域包含CD3ζ初级信号传导结构域，该初级信号传导结构域与SEQ ID NO：19或21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％、99％或100％的序列同一性，或其编码序列与SEQ ID NO：20或22所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％、99％或100％的序列同一性。
在一个实施方案中，所述共刺激结构域包括但不限于源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)、CD150(SLAMF1)、CD152(CTLA4)、CD223(LAG3)、CD270(HVEM)、CD272(BTLA)、CD273(PD-L2)、CD274(PD-L1)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、LAT、NKG2C、SLP76、PD1、LIGHT、TRIM、ZAP70或它们的任意组合。优选地，所述共刺激结构域来自4-1BB、CD28或4-1BB+CD28，更优选4-1BB共刺激结构域。所述4-1BB共刺激结构域与SEQ ID NO：23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％、99％或100％的序列同一性，或该共刺激结构域的编码序列与SEQ ID NO：24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％、99％或100％的序列同一性。
在一个实施方案中，所述嵌合抗原受体还包括信号肽，所述信号肽与SEQ ID NO：25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％、99％或100％的序列同一性，或该信号肽的编码序列与SEQ ID NO：26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％、99％或100％的序列同一性。
在一个实施方案中，所述T细胞受体靶向HBV、HPV E6、NYESO、mNY-ESO、WT1、MART-1、MAGE-A3、MAGE-A4、P53、Thyroglobulin、Tyrosinase中的一种或多种。
在一个实施方案中，所述嵌合抗原受体或T细胞受体可以通过载体引入宿主细胞。具体地，所述载体选自质粒、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)、噬菌体、噬菌粒、粘粒或人工染色体。在 一些实施方案中，该载体还包含在宿主细胞中自主复制的起始、选择标记、限制酶切割位点、启动子、多聚腺苷酸尾(polyA)、3’UTR、5’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。具体地，所述载体是质粒、慢病毒载体、AAV载体、腺病毒载体或逆转病毒载体。
在第四个方面，本发明提供了利用上述敲除CD7基因的方法制得的工程化免疫细胞。
在一个实施方案中，所述工程化免疫细胞还包含至少一种选自以下的基因的表达被抑制或沉默：GR、dCK、TCR/CD3基因(例如TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ)、MHC相关基因(HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA)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。优选地，所述工程化免疫细胞还包含至少一种选自以下的基因的表达被抑制或沉默：TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA、PD1、LAG3、TIM3、CTLA4，更优选TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA。更优选地，所述工程化免疫细胞中的CD7和TRAC被敲除，这种失活使得TCR-CD3复合物在细胞中没有功能，该策略对于避免移植物抗宿主病(GvHD)特别有用。
在第五个方面，本发明还提供包含上述工程化免疫细胞的组合物，以及所述免疫细胞或组合物在制备用于治疗/预防/诊断癌症、感染或自身免疫性疾病的药物中的用途。
在一个实施方案中，所述组合物还包含一种或多种药学上可接受的赋形剂。
在一个实施方案中，本发明的工程化免疫细胞或组合物还可以与一种或多种额外的化疗剂、生物制剂、药物或治疗联用。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、小分子或它们的任意组合。
本发明的优点在于，筛选到的sgRNA可以高效敲除CD7基因，从而避免CAR-T细胞之间的互相杀伤，并通过同时敲除TRAC基因来制备通用型CAR-T细胞。
下面将参考附图并结合实施例来详细说明本发明。需要说明的是，本领域的技术人员应该理解本发明的附图及其实施例仅仅是为了举例，并不能对本发明构成任何限制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。
附图说明
图1示出了TRAC/CD7双敲除的CD7CAR-T细胞(CAR7-37KO)中TRAC/CD7基因的敲除情况。
图2示出了CD7单敲除的CD7CAR-T细胞(CAR7-7KO)和TRAC/CD7双敲除的CD7CAR-T细胞(CAR7-37KO)中CAR的表达水平。
图3示出了CD7单敲除的CD7CAR-T细胞(CAR7-7KO)和TRAC/CD7双敲除的CD7CAR-T细胞(CAR7-37KO)对Jurkat靶细胞的特异性裂解率。
图4示出了CD7单敲除的CD7CAR-T细胞(CAR7-7KO)和TRAC/CD7双敲除的CD7CAR-T细胞(CAR7-37KO)的细胞因子IFNγ分泌水平。
具体实施方式
发明详述
除非另有定义，否则本文使用的所有技术和科学术语具有与本申请所属领域的普通技术人员通常理解的相同的含义。为了更容易地理解本申请，下文对某些术语进行了定义。
sgRNA
CRISPR/Cas系统是原核生物的一种天然免疫系统。某些细菌在遭到病毒入侵后，能够把病毒基因的一小段存储到自身的DNA里一个称为CRISPR的存储空间。当再次遇到病毒入侵时，细菌能够根据存写的片段识别病毒，将病毒的DNA切断而使之失效。CRISPR系统有Ⅰ型、Ⅱ型和Ⅲ型，其中CRISPR/Cas9作为Ⅱ型研究最多，除了Cas9核酸酶外，Cas12a(也称Cpf1)和Cas13a(也称C2c2)也是较为常用的Cas核酸酶。CRISPR/Cas9介导的基因编辑技术可用于生成转基因模型、调节转录、调控表观遗传等。
CRISPR/Cas9系统主要是由Cas9蛋白和单链向导RNA(sgRNA)组成，其中Cas9蛋白具有切割DNA双链功能，sgRNA起导向作用。此系统的工作原理是，CRISPR RNAs(crRNAs)通过碱基配对与tracrRNA(trans-activatingRNA)结合形成tracrRNA/crRNA复合物，此复合物引导核酸酶Cas9蛋白在与crRNA中的间隔区序列 配对的序列靶位点剪切双链DNA。
如本文中所用，术语“sgRNA”，又称“单向导RNA”是指用于靶向特定基因从而采用CRISPR技术进行校正的引导RNA序列，通常由部分互补形成复合物的crRNA和tracrRNA分子融合构成，其中crRNA包含与靶序列能够互补以便与该靶序列杂交并且指导CRISPR复合物(Cas9+crRNA+tracrRNA)与该靶序列序列特异性结合的序列。当sgRNA与Cas9核酸酶结合时，识别并结合DNA靶位点、并使DNA靶位点产生切口或切割(引入单链或双链断裂)DNA靶位点。其能够识别并切割向导RNA特异性的DNA靶标。sgRNA中负责与靶标DNA互补配对的部分是其包含的间隔区序列。在一个实施方案中，所述sgRNA包含SEQ ID NO：1、3、4、5、8、9任一所示的间隔区序列。
sgRNA间隔区序列的设计大体依据以下原则：
(1)长度一般应为20nt左右；
(2)碱基组成，种子序列(靠近PAM区的第1-12个碱基)尽量避免以4个以上的T结尾，GC％含量最佳为40％～60％；
(3)种子序列与脱靶位点的匹配数尽可能低；
(4)检查靶向结合位点基因组序列是否存在SNPs；
(5)全基因脱靶效应分析，需考虑脱靶位点最大允许几个错配碱基数等。
可见，sgRNA间隔区的设计需要考虑很多因素，例如长度、GC含量、靶基因的结合位置、与非靶标位点的结合率、是否包含SNP、二级结构等等。目前已经可以通过各种在线工具来设计sgRNA。然而，由于Cas酶可以切割任何邻近PAM位点的靶序列，针对特定的靶基因而言，在线工具设计的大量sgRNA的编辑效率都不尽相同，甚至差异很大，例如，PAM位点是5'-NGG-3'的编辑效率通常就比5'-NGA-3'或5'-NAG-3'的高。因此，筛选特异性高的sgRNA对于CRISPR系统编辑效率的提高至关重要。
核酸、载体
如本文中所用，术语“核酸”可为DNA或RNA。
如本文中所用，术语“载体”是用作将遗传材料转移到细胞中的媒介核酸分子，在细胞中所述核酸分子可以复制和/或表达。
在一个实施方案中，本发明的载体包括但不限于线性核酸(例如DNA或RNA)、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)。
嵌合抗原受体
如本文中所用，术语“嵌合抗原受体”(CAR)包含配体结合结构域、跨膜结构域、共刺激结构域和初级信号传导结构域。
“配体结合结构域”是指可以与配体特异性结合的任何结构或其功能性变体，包括抗体或抗原结合片段。
“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。
“初级信号传导结构域”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，负责在配体结合结构域结合抗原以后的细胞内初级信号传递，从而导致免疫细胞和免疫反应的活化。
“共刺激结构域”可以是来自共刺激分子的细胞内功能性信号传导结构域；“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。
如本文中所用，跨膜结构域和配体结合结构域之间用“铰链区”连接，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。
在一个优选的实施方案中，所述配体结合结构域包括靶向CD7的抗体或抗原结合片段。本发明的CAR当在免疫细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合特定抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。优选地，抗原结合结构域与4-1BB共刺激结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
如本文所用，术语“抗体”包括单克隆抗体(包含完整抗体)、多克隆抗体、多价抗体、多特异性抗体(例如双特异性抗体)、和能够表现期望的生物活性的携带一个或多个CDR序列的抗体片段或合成多肽。通常，完整抗体包括通过二硫键连接在一起的两条重链和两条轻链，呈“Y”形结构。每条重链、轻链均包含可变区和恒定区，重、轻链可变区中各包含三个CDR，CDR被更保守的框架区(FR)隔开。可变区负责与抗原的识别和结合，恒定区则可以介导抗体与宿主组织或因子的结合。
如本文所用，术语“抗原结合片段”仅包含完整抗体的一部分，一般包含完整抗 体的抗原结合位点并因此保留结合抗原的能力。本发明中的抗原结合片段的实例包括但不限于：Fab、Fab'、F(ab')2、Fd片段、Fd′、Fv片段、scFv、二硫键-连接的Fv(sdFv)、抗体的重链可变区(VH)或轻链可变区(VL)、线性抗体、具有两个抗原结合位点的“双体”、单结构域抗体、纳米抗体、所述抗原的天然配体或其功能性片段等。
本发明的嵌合抗原受体还可以包含信号肽，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。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等的信号肽。
工程化免疫细胞
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。例如，免疫细胞可以是T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞。在一个实施方案中，免疫细胞衍生自干细胞，例如成体干细胞、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。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。T细胞也可以被浓缩或纯化。T细胞可以处于任何发育阶段，包括但不限于，CD4+/CD8+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。在一个优选的实施方案中，免疫细胞是人T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。
在一个实施方案中，为减少移植物抗宿主病的风险，所述工程化免疫细胞还包含至少一种选自以下的基因的表达被抑制或沉默：CD52、GR、dCK、TCR/CD3基因(例如TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ)、MHC相关基因(HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA)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。优选地，所述工程化免疫细胞还包含至少一种选自以下的基因的表达被抑制或沉默：TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA、PD1、LAG3、TIM3、CTLA4，更优选TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA。
组合物
本发明还提供一种组合物，其包含本发明所述的工程化免疫细胞体。在一个实施方案中，还包含一种或多种药学上可接受的赋形剂。
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/或赋形剂，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。本领域存在多种施用组合物的方式，包括但不限于口服、注射、气雾剂、肠胃外和局部施用。
本发明的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(sorfimersodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreateglucuronate)、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也可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
治疗/预防/诊断用途
本发明中的工程化免疫细胞和组合物能够用于治疗/预防/诊断癌症、感染或自身免疫性疾病的药物。
在一个实施方案中，可以用本发明的工程化免疫细胞或组合物治疗/预防/诊断的癌症包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)和实体瘤。血液学肿瘤是血液或骨髓的癌症，包括但不限于急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、骨髓增生异常综合征、多毛细胞白血病、伯基特淋巴瘤、弥漫性大细胞淋巴瘤、套细胞淋巴瘤、T淋巴母细胞性白血病/淋巴瘤(T-ALL/LBL)、早期前T淋巴母细胞白血病(ETP-ALL)、结外NK/T细胞淋巴瘤、小淋巴细胞淋巴瘤(SLL)和脊髓发育不良。实体瘤是通常不包含囊肿或液体区的组织的异常肿块，其可以是良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤的实例包括但不限于纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、胰腺癌、卵巢癌，腹膜、大网膜和肠系膜癌、咽癌、前列腺癌、直肠癌、肾癌、皮肤癌、小肠癌、黑素瘤、肾癌，喉癌、软组织癌、胃癌、睾丸癌、结肠癌，食道癌，宫颈癌、腺泡型横纹肌肉瘤、膀胱癌、骨癌、脑癌、乳腺癌、肛门癌、眼癌、肝内胆管癌、关节癌、颈癌、胆囊癌、胸膜癌、鼻癌、中耳癌、口腔癌、外阴癌、甲状腺癌和输尿管癌。优选地，可以用本发明的工程化免疫细胞或组合物治疗/预防/诊断的癌症包括/预防/诊断急性淋巴细胞白血病(ALL)、急性髓细胞白血病(AML)、T淋巴母细胞性淋巴瘤(T-LBL)、早期前T淋巴母细胞白血病(ETP-ALL)和结外NK/T细胞淋巴瘤。
实施例1.设计sgRNA并敲除CD7基因
在CD7基因上选择合适的PAM位点设计sgRNA的间隔区序列。间隔区序列靶向CD7基因的外显子，且在CD7基因上的靶序列是唯一的。将包含间隔区序列的crRNA与tracrRNA融合，获得一条100bp左右的序列，人工合成该序列，得到sgRNA产物。
用DynaBeads CD3/CD28 CTSTM(Gibco,货号40203D)刺激T细胞，并在37℃ 和5％CO2下培养3天。然后，使用BTX Agile Pulse Max电穿孔仪(Harvard Apparatus BTX)，以400V、0.7ms将5μg Cas9蛋白和5μg sgRNA电转染进激活的T细胞内，获得CD7敲除的T细胞。电转染之后，立即将T细胞放入1ml预热的培养基中，并在IL-2(300IU/ml)存在下，在37℃和5％CO2下培养。5天后，使用PE anti-human CD7(Biolegend，货号343106)，通过流式细胞仪检测CD7的表达，从而获得CD7基因的敲除效率。不同sgRNA的间隔区序列及其敲除效率如表1所示。
表1.不同sgRNA的间隔区序列信息
可以看出，不同的sgRNA对CD7基因的敲除效率不同，其中SEQ ID NO：1、3、4、5、8、9的敲除效率均高于80％，SEQ ID NO：1、3、4的敲除效率更是高于90％。依据敲除效率，选取7-4用于后续实验。
实施例2.制备敲除CD7和TRAC/CD7的CAR-T细胞
1.制备CD7CAR-T细胞
合成以下蛋白的编码序列，并将其依次克隆至pGEM-T Easy载体(Promega，货号A1360)：CD8α信号肽(SEQ ID NO：25)、抗CD7 scFv(SEQ ID NO：14)、CD8α铰链区(SEQ ID NO：17)、CD8α跨膜区(SEQ ID NO：15)、4-1BB共刺激结构域(SEQ ID NO：23)、CD3ζ初级信号传导结构域(SEQ ID NO：19)，并通过测序确认目标序列的正确插入。
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120μl 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，获得靶向CD7的CAR-T细胞。
使用BTX Agile Pulse Max电穿孔仪(Harvard Apparatus BTX)，在400V、0.7ms条件下，将10μg Cas9蛋白、5μg CD7 sgRNA(间隔区序列如SEQ ID NO：4所示)或其与5μg TRAC sgRNA(间隔区序列如SEQ ID NO：11所示)的组合电转染进激活的CD7 CAR T细胞内，获得CD7单敲除(CAR7-7KO)和TRAC/CD7双敲除(CAR7-37KO)的CD7 CAR-T细胞。未转染CAR的野生型T细胞用作阴性对照(NT)。
2.编辑效率和CAR水平检测
将CAR7-7KO和CAR7-37KO细胞在37℃和5％CO2下培养11天之后，使用APC-anti human CD3(BD Pharmingen，货号555751)和PE anti-human CD7(Biolegend，货号343106)抗体，通过流式细胞仪检测TRAC和CD7的基因编辑效率，结果如图1所示。
可以看出，CAR7-37KO细胞中目标基因被有效敲除，表明本发明选择的sgRNA可以有效敲除TRAC和CD7基因。
此外，将CAR7-7KO和CAR7-37KO细胞在37℃和5％CO2下培养11天之后，使用Biotin-SP(long spacer)AffiniPure Goat Anti-human IgG,F(ab')Fragment Specific(min X Hu,Bov,Hrs Sr Prot)(jackson immunoresearch，货号109-065-097)作为一抗，APC Streptavidin(BD Pharmingen，货号554067)或PE Streptavidin(BD Pharmingen，货号554061)作为二抗，通过流式细胞仪检测CAR T细胞上的CAR的表达水平，结果如图2所示。
可以看出，CAR7-7KO中的CAR表达水平与CAR7-37KO相当。
实施例3.CAR-T细胞的杀伤效果和细胞因子分泌水平
1.对靶细胞的杀伤效果
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Jurkat靶细胞铺入96孔板中，然后以不同的效靶比(即效应T细胞与靶细胞之比)将三组细胞铺入到96孔板进行共培养，8小时后利用酶标仪测定荧光值。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3所示。
可以看出，与NT相比，CAR7-7KO和CAR7-37KO组细胞都能够有效杀伤靶细胞，且两组杀伤效果无显著性差异。
2.细胞因子的分泌水平
细胞因子的分泌水平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IL2和IFN-γ等。根据以下步骤，使用酶联免疫吸附法(ELISA)来测定CART细胞杀伤靶细胞时细胞因子IFNγ的释放水平。
(1)收集细胞共培养上清液
以1×105/孔将Jurkat靶细胞铺于96孔板中，然后以1:1的比例将三组细胞分别与靶细胞共培养，8小时后收集细胞共培养上清液。
(2)ELISA检测上清中IFNγ的分泌量
用ELISA测定上清液中细胞因子IFNγ分泌量。使用捕获抗体Purified anti-human IFN-γAntibody(Biolegend，货号506502)分别包被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。用250μL PBST(含0.1％吐温的1XPBS)清洗板之后，向各孔分别加入50μL检测抗体Anti-Interferon gamma抗体[MD-1](Biotin)(abcam，货号ab25017)，在37℃孵育1小时。再加入HRP Streptavidin(Biolegend，货号405210)，在37℃孵育30分钟后，弃上清液，用250μL PBST(含0.1％吐温的1XPBS)清洗。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4所示。
可以看出，与NT组相比，CAR7-7KO细胞和CAR7-37KO细胞均有大量的细胞因子分泌。
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 (19)

  1. 一种sgRNA，其包含SEQ ID NO:1、3、4、5、8、9任一所示的间隔区序列。
  2. 编码权利要求1所述的sgRNA的核酸。
  3. 一种载体，其包含根据权利要求2所述的核酸。
  4. 一种体外敲除CD7基因的方法，其包括将Cas核酸酶和sgRNA引入细胞；所述sgRNA是以权利要求1所述RNA、权利要求2所述核酸或权利要求3所述载体的形式存在。
  5. 根据权利要求4所述的方法，所述Cas核酸酶是Cas9、Cas12a或Cas13a。
  6. 根据权利要求4所述的方法，其中所述细胞是免疫细胞，所述免疫细胞是T细胞、B细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。
  7. 根据权利要求6所述的方法，其中所述细胞是CD4+CD8+T细胞、CD4+T细胞、CD8+T细胞、记忆T细胞、幼稚T细胞、γδ-T细胞或αβ-T细胞。
  8. 根据权利要求6所述的方法，其中所述免疫细胞引入了编码嵌合抗原受体和/或T细胞受体的核酸。
  9. 根据权利要求8所述的方法，其中所述嵌合抗原受体包含配体结合结构域、跨膜结构域、共刺激结构域和初级信号传导结构域，所述配体结合结构域靶向CD7、CD19、CD20、CD22、BAFF-R、CD33、EGFRvIII、BCMA、GPRC5D、PSMA、ROR1、FAP、ERBB2、MUC1、EGFR、CAIX、WT1、NY-ESO-1、CD79a、CD79b、GPC3、Claudin18.2、NKG2D中的一种或多种。
  10. 根据权利要求9所述的方法，其中所述配体结合结构域包括靶向CD7的抗体或抗原结合片段。
  11. 根据权利要求10所述的方法，其中所述靶向CD7的抗体或抗原结合片段包含与SEQ ID NO：12所示的氨基酸序列具有至少90％、95％、97％、99％或100％序列同一性的轻链可变区序列和与SEQ ID NO：13所示的氨基酸序列具有至少90％、95％、97％、99％或100％序列同一性的重链可变区序列。
  12. 根据权利要求9所述的方法，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、CD154或它们的任意组合。
  13. 根据权利要求9所述的方法，其中所述共刺激结构域是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、CD27、CD28、CD30、CD40、CD54、CD83、CD134、CD137、CD150、CD152、CD223、CD270、CD272、CD273、CD274、CD276、CD278、CD357、DAP10、LAT、NKG2C、SLP76、PD1、LIGHT、TRIM、ZAP70或它们的任意组合。
  14. 根据权利要求9所述的方法，其中所述初级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、CD66d或它们的任意组合。
  15. 根据权利要求6-14所述的任一项方法获得的工程化免疫细胞，其中CD7基因被敲除。
  16. 根据权利要求15所述的工程化免疫细胞，其中所述工程化免疫细胞还包含至少一种选自以下的基因的表达被抑制或沉默：TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA、PD1、LAG3、TIM3、CTLA4或它们的任意组合。
  17. 根据权利要求16所述的工程化免疫细胞，其中所述工程化免疫细胞中的CD7和TRAC被敲除。
  18. 一种组合物，其包含权利要求15-17任一项所述的工程化免疫细胞。
  19. 权利要求15-17任一项所述的工程化免疫细胞或权利要求18所述的组合物在制备用于治疗/预防/诊断癌症、感染或自身免疫性疾病的药物中的用途。
PCT/CN2023/081600 2022-04-28 2023-03-15 一种cd7基因被敲除的工程化免疫细胞及其用途 WO2023207388A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210460785.5A CN117004603A (zh) 2022-04-28 2022-04-28 一种cd7基因被敲除的工程化免疫细胞及其用途
CN202210460785.5 2022-04-28

Publications (1)

Publication Number Publication Date
WO2023207388A1 true WO2023207388A1 (zh) 2023-11-02

Family

ID=88517267

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/081600 WO2023207388A1 (zh) 2022-04-28 2023-03-15 一种cd7基因被敲除的工程化免疫细胞及其用途

Country Status (2)

Country Link
CN (1) CN117004603A (zh)
WO (1) WO2023207388A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109996811A (zh) * 2016-08-03 2019-07-09 约翰·F·迪帕西奥 用于用嵌合抗原受体治疗t细胞恶性肿瘤的car-t细胞的基因编辑
CN110268049A (zh) * 2016-11-22 2019-09-20 新加坡国立大学 用于t细胞恶性肿瘤免疫疗法的cd7表达阻滞剂和嵌合抗原受体
CN112980800A (zh) * 2021-03-08 2021-06-18 河北森朗生物科技有限公司 Car-t细胞、其构建方法及其应用
CN113383071A (zh) * 2018-11-01 2021-09-10 亘喜生物科技（上海）有限公司 用于t细胞工程化的组合物和方法
CN114144430A (zh) * 2019-11-21 2022-03-04 博生吉医药科技(苏州)有限公司 Cd7-car-t细胞及其制备和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109996811A (zh) * 2016-08-03 2019-07-09 约翰·F·迪帕西奥 用于用嵌合抗原受体治疗t细胞恶性肿瘤的car-t细胞的基因编辑
CN110268049A (zh) * 2016-11-22 2019-09-20 新加坡国立大学 用于t细胞恶性肿瘤免疫疗法的cd7表达阻滞剂和嵌合抗原受体
CN113383071A (zh) * 2018-11-01 2021-09-10 亘喜生物科技（上海）有限公司 用于t细胞工程化的组合物和方法
CN114144430A (zh) * 2019-11-21 2022-03-04 博生吉医药科技(苏州)有限公司 Cd7-car-t细胞及其制备和应用
CN112980800A (zh) * 2021-03-08 2021-06-18 河北森朗生物科技有限公司 Car-t细胞、其构建方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 13 December 1995 (1995-12-13), ANONYMOUS : "sFv antibody, partial [Mus musculus]", XP055928515, retrieved from NCBI Database accession no. AAA83267 *

Also Published As

Publication number Publication date
CN117004603A (zh) 2023-11-07

Similar Documents

Publication Publication Date Title
US20230242877A1 (en) Immune cell comprising chimeric antigen receptor and use thereof
WO2021169977A1 (zh) 新型嵌合抗原受体及其用途
WO2021129559A1 (zh) T细胞受体融合蛋白及其用途
WO2022095803A1 (zh) 靶向cd7的人源化抗体及其用途
WO2020247392A1 (en) Combinations of engineered natural killer cells and engineered t cells for immunotherapy
JP2023534928A (ja) 改変免疫細胞及びその使用
WO2021147929A1 (zh) 多链嵌合抗原受体及其用途
EP4219693A1 (en) Chimeric antigen receptor targeting cd7 and use thereof
US20230248768A1 (en) Engineered immune cell for allotransplantation
EP4273175A1 (en) Novel chimeric antigen receptor and use thereof
WO2022095903A1 (zh) 靶向pd-1h(vista)的抗肿瘤免疫治疗方法
EP4194472A1 (en) Chimeric antigen receptor comprising novel co-stimulatory domain and use thereof
WO2023207388A1 (zh) 一种cd7基因被敲除的工程化免疫细胞及其用途
US20220031747A1 (en) Foxp1-ablated chimeric cells
WO2023207390A1 (zh) 一种ciita基因被敲除的工程化免疫细胞及其用途
WO2023025009A1 (zh) 工程化免疫细胞及其用途
CN111218448B (zh) 工程化免疫细胞的制备及其用途
WO2023016284A1 (zh) 工程化免疫细胞及其用途
WO2022022745A1 (zh) 新型共刺激结构域及其用途
WO2022267983A1 (zh) 工程化免疫细胞及其用途
WO2023083003A1 (zh) 工程化免疫细胞及其用途
CN117430712A (zh) 靶向cxcr6的嵌合抗原受体及其用途

Legal Events

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

Ref document number: 23794824

Country of ref document: EP

Kind code of ref document: A1