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WO2023274386A1 - 靶向egfr的通用型car-t细胞及其制备方法 - Google Patents

靶向egfr的通用型car-t细胞及其制备方法 Download PDF

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WO2023274386A1
WO2023274386A1 PCT/CN2022/103092 CN2022103092W WO2023274386A1 WO 2023274386 A1 WO2023274386 A1 WO 2023274386A1 CN 2022103092 W CN2022103092 W CN 2022103092W WO 2023274386 A1 WO2023274386 A1 WO 2023274386A1
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cells
hla
seq
gene
antigen receptor
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PCT/CN2022/103092
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English (en)
French (fr)
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尚小云
王丹
李甲璐
蒋海娟
马少文
高兴旺
马丽
沈慧
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宁波茂行生物医药科技有限公司
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Priority to CN202280046725.7A priority Critical patent/CN117940138A/zh
Publication of WO2023274386A1 publication Critical patent/WO2023274386A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/418—Antigens related to induction of tolerance to non-self
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4204—Epidermal growth factor receptors [EGFR]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4221—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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    • 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
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    • 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
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    • 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
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/50—Cellular immunotherapy characterised by the use of allogeneic cells

Definitions

  • This application relates to the field of biomedicine, in particular to a universal CAR-T cell targeting EGFR and its preparation method and application.
  • EGFR belongs to the ErbB family of receptor tyrosine kinases and plays a key role in epithelial cell physiology. Ligand-induced homodimerization or heterodimerization of EGFR will lead to autophosphorylation of its tyrosine kinase domain, which will recruit a series of downstream signaling pathways, such as PI3K/AKT and Ras/ Faf/MEK/ERK. EGFR is frequently mutated or overexpressed in different types of cancer and is a target used in clinical practice for several cancer therapies. In pathological states, mainly in lung cancer, breast cancer and glioblastoma, EGFR is a driver of tumorigenesis.
  • EGFR Abnormal activation of EGFR in cancer is mainly due to amplification and point mutations at genomic loci, but transcriptional upregulation or ligand overproduction due to autocrine/paracrine mechanisms has also been found. Furthermore, EGFR is increasingly considered as a biomarker of tumor drug resistance due to the discovery that amplification or secondary mutation of EGFR occurs under drug stress. EGFR is overexpressed in 63% of primary and 10% of secondary glioblastomas. EGFR affects the migration of neural stem cells during development and promotes cell proliferation by activating MAPK and PI3K/Akt signaling pathways.
  • CAR-T cell technology is a cell-based therapy that has produced excellent results in tumor immunotherapy, especially in the treatment of blood tumors.
  • CAR-T immunotherapy uses genetically modified T cells that can specifically recognize and kill tumor cells expressing specific antigens without being restricted by MHC.
  • CAR-T immunotherapy has achieved good results in the treatment of various B cell malignancies, such as CAR-T cells targeting CD19 in the treatment of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma ( NHL).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • NHL non-Hodgkin's lymphoma
  • the purpose of the present invention is to prepare a universal CAR-T cell targeting EGFR, recognize tumor cell surface antigens, and knock out the TCR and HLA-A genes expressed by the cells, thereby reducing the immune rejection caused by allogeneic CAR-T therapy Response, prolong cell survival time, improve anti-tumor effect.
  • One of the purposes of the present application is to provide a chimeric antigen receptor targeting EGFR.
  • the details are as follows: the anti-EGFR scFv is used as the targeting part, and is connected to the intracellular signal transduction domain through the hinge region and the transmembrane region of the CD8 molecule. Composition of the signal domain.
  • Another purpose of the present application is to provide a UCAR-T cell with higher safety.
  • the source of the donor is based on the HLA-B homozygote in the population.
  • One of the alleles of the patient’s HLA-B is consistent with the homozygote of the donor. Cells from these donors can cover a high proportion of the patient population. Reduce the rejection caused by HLA-B.
  • HLA-B mainly selects B*40 homozygote, B*15 homozygote, B*46 homozygote, B*13 homozygote, B*51 homozygote, B*58 homozygote, B*07 homozygote with high frequency in the population Homozygote, B*35 homozygote, B*44 homozygote, B*52 homozygote, B*57 homozygote, B*54 homozygote, B*55 homozygote.
  • the knockout strategy targets HLA-A molecules that are highly related to TRAC and rejection, while retaining other HLA-I molecules, which not only reduces the rejection of allogeneic cells, but also avoids the complete knockout of HLA molecules being cleared by NK cells
  • the occurrence of allogeneic CAR-T cells greatly prolongs the half-life of allogeneic CAR-T cells in vivo. Simultaneously reduced GVHD response induced by allogeneic cell therapy.
  • TCR gene selects gene TRAC encoding TCR ⁇ chain
  • HLA-A selects A*02 homozygote, A*11 homozygote, A*02/A11 heterozygote and A*24 homozygote with high frequency in the population.
  • the transfection methods of CRISPR/Cas9 gene editing mainly include plasmid method, mRNA method and RNP method.
  • the RNP method refers to the assembly of spcas9 ribonucleoprotein (RNP) and sgRNA into an RNP complex in vitro, and the complex is introduced into cells by electroporation. Compared with the other two methods, it has the advantages of fast degradation of spCas9 protein and low off-target risk, and is very The electrotransfer efficiency is greatly improved, thereby improving the editing efficiency of the target gene.
  • the double knockout efficiency can reach more than 90%.
  • Another object of the present application is to provide a method for preparing universal CAR-T cells targeting EGFR.
  • the chimeric antigen receptor comprises a targeting moiety comprising HCDR3 comprising the amino acid sequence shown in SEQ ID NO:8.
  • the targeting moiety comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:6.
  • the targeting moiety comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:4.
  • the targeting moiety comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region set forth in SEQ ID NO: 1.
  • the targeting moiety comprises HCDR1, HCDR2, HCDR3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:8; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4.
  • the targeting moiety comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 comprises SEQ ID NO:3 Amino acid sequence shown.
  • the targeting moiety comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5 .
  • the targeting moiety comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:7 .
  • the targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO:9 Amino acid sequence shown.
  • the targeting moiety comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 3; the H- FR2 comprises the amino acid sequence shown in SEQ ID NO:5; the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:7; and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:9.
  • the targeting moiety comprises a VH comprising the amino acid sequence shown in SEQ ID NO:1.
  • the chimeric antigen receptor comprises a targeting moiety comprising LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 17.
  • the targeting moiety comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 15.
  • the targeting moiety comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 13.
  • the targeting moiety comprises LCDR1, LCDR2 and LCDR3 in the light chain variable region set forth in SEQ ID NO: 10.
  • the targeting moiety comprises LCDR1, LCDR2, LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 17; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:13.
  • the targeting moiety comprises L-FR1
  • the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1
  • the L-FR1 comprises SEQ ID NO: 12 Amino acid sequence shown.
  • the targeting moiety comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:14 .
  • the targeting moiety comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:16 .
  • the targeting moiety comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO: 18 Amino acid sequence shown.
  • the targeting moiety comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12; the L- FR2 comprises the amino acid sequence shown in SEQ ID NO:14; said L-FR3 comprises the amino acid sequence shown in SEQ ID NO:16; and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO:18.
  • the targeting moiety comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 10.
  • the targeting moiety comprises an antibody or antigen-binding fragment.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the targeting moiety comprises a scFv.
  • the scFv targets EGFR.
  • the targeting moiety comprises the amino acid sequence shown in SEQ ID NO: 198.
  • the chimeric antigen receptor comprises a transmembrane domain comprising a transmembrane domain derived from a protein selected from the group consisting of: CD8A, CD8B, CD28, CD3e, CD3 ⁇ , 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM , DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, CD154, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, and SLAM.
  • a protein selected from the group consisting of: CD8A, CD8B, CD28, CD3e, CD3 ⁇ , 4-1BB, CD4, CD27, CD7, PD-1, TRAC
  • said transmembrane domain of said chimeric antigen receptor comprises a transmembrane domain derived from CD8A or CD8B.
  • the transmembrane domain of the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:69.
  • the chimeric antigen receptor comprises a co-stimulatory signaling domain comprising a co-stimulatory signaling domain derived from a protein selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, and MyD88.
  • a protein selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, F
  • the co-stimulatory signaling domain in the chimeric antigen receptor comprises a co-stimulatory signaling domain derived from 4-1BB.
  • the co-stimulatory signaling domain in the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:102.
  • the N-terminal of the co-stimulatory signaling domain in the chimeric antigen receptor is directly or indirectly linked to the C-terminal of the transmembrane domain.
  • the chimeric antigen receptor comprises an intracellular signal transduction domain comprising an intracellular signal transduction domain derived from a protein selected from or Its combination: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP-12 and at least one Domains of ITAMs.
  • the intracellular signaling domain of the chimeric antigen receptor comprises an intracellular signaling domain derived from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , or CD3 ⁇ .
  • the intracellular signal transduction domain in the chimeric antigen receptor comprises SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:103 to The amino acid sequence shown in any one of SEQ ID NO:113.
  • the N-terminus of the intracellular signal transduction domain in the chimeric antigen receptor is directly or indirectly linked to the C-terminus of the co-stimulatory signal transduction domain.
  • the chimeric antigen receptor includes a hinge region between the targeting moiety and the transmembrane domain, the hinge region comprising a hinge region derived from a protein selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, TIM1, SLAM, CD30, and LIGHT.
  • a protein selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, TIM1, SLAM, CD30, and LIGHT.
  • the hinge region comprises a hinge region derived from CD8.
  • the hinge region comprises the amino acid sequence shown in any one of SEQ ID NO: 114 to SEQ ID NO: 135.
  • the chimeric antigen receptor comprises a non-targeting portion
  • the non-targeting portion of the chimeric antigen receptor comprises the transmembrane domain of CD8A, the hinge region of CD8, the intracellular Costimulatory signaling domain and CD3 ⁇ intracellular signaling domain.
  • the non-targeting portion comprises the amino acid sequence shown in SEQ ID NO:19.
  • the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 199.
  • the present application provides one or more isolated nucleic acid molecules encoding said chimeric antigen receptor.
  • the present application provides one or more vectors comprising said isolated nucleic acid molecule.
  • the vector comprises a viral vector.
  • the vector comprises a lentiviral vector.
  • the present application provides one or more immune cells comprising and/or expressing said isolated nucleic acid molecule or said vector, and/or said chimeric antigen receptor.
  • the immune cells are of human origin.
  • the immune cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, ⁇ T cells, monocytes, dendritic cells, granulocytes, lymphocytes cells, leukocytes, and/or peripheral blood mononuclear cells.
  • NK cells natural killer cells
  • macrophages include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, ⁇ T cells, monocytes, dendritic cells, granulocytes, lymphocytes cells, leukocytes, and/or peripheral blood mononuclear cells.
  • the immune cells include T cells.
  • the immune cells include modified immune cells.
  • the modified immune cells include cells that reduce immune rejection from allogeneic cell therapy.
  • TCR T cell antigen receptor
  • MHCI major histocompatibility complex
  • the modification in the immune cells includes down-regulation of the expression and/or activity of one or more genes related to immune rejection.
  • the gene related to immune rejection is selected from one or more genes in the group consisting of TRAC, TRBC, HLA-A, HLA-B, B2M and CIITA.
  • the expression and/or activity of the TRAC gene and the HLA-A gene are down-regulated in the modified immune cells compared with the unmodified corresponding cells in the immune cells.
  • the expression and/or activity of the CIITA gene is not down-regulated in the modified immune cells compared to the corresponding cells without the modification.
  • the expression and/or activity of the B2M gene is not down-regulated in the modified immune cells compared to the corresponding cells without the modification.
  • the expression and/or activity of the TRAC gene and the HLA-A gene are down-regulated in the modified immune cell compared with the corresponding wild-type cell.
  • the expression and/or activity of the B2M gene is not down-regulated in the modified immune cells compared to the corresponding wild-type cells.
  • the expression and/or activity of the CIITA gene is not down-regulated in the modified immune cells compared to the corresponding wild-type cells.
  • down-regulating the expression level and/or activity of the gene comprises down-regulating the expression and/or activity of a nucleic acid molecule encoding the gene; and/or down-regulating the expression and/or activity of a protein product encoded by the gene. / or the activity is downregulated.
  • the modification includes: gene knockout, gene mutation and/or gene silencing.
  • said modification comprises administering to said immune cells one or more agents selected from the group consisting of antisense RNA, siRNA, shRNA and sgRNA.
  • the modification further comprises administering to the immune cells a sgRNA targeting an exon portion of the TRAC gene.
  • the sgRNA targeting the exon portion of the TRAC gene comprises the nucleotide sequence shown in any one of SEQ ID NO:138 to SEQ ID NO:152.
  • the modification comprises administering to the immune cells a sgRNA targeting an exon portion of the HLA-A gene.
  • the sgRNA targeting the exon portion of the HLA-A gene comprises the nucleotide sequence shown in any one of SEQ ID NO:153 to SEQ ID NO:193.
  • said modifying further comprises administering a Cas enzyme to said cell.
  • the Cas enzyme includes a Cas9 protein.
  • the modification comprises administering to the cell an antisense RNA comprising the nucleotide sequence shown in any one of SEQ ID NO: 194 to SEQ ID NO: 197.
  • the immune cells are HLA-B homozygous cells.
  • the HLA-B homozygotes include HLA-B*40 homozygotes, HLA-B*15 homozygotes, HLA-B*46 homozygotes, HLA-B*13 homozygotes, HLA-B homozygotes *51 homozygote, HLA-B*58 homozygote, HLA-B*07 homozygote, HLA-B*35 homozygote, HLA-B*44 homozygote, HLA-B*52 homozygote, HLA-B*57 Homozygous, HLA-B*54 homozygous or HLA-B*55 homozygous.
  • the immune cells are HLA-A homozygous or heterozygous cells.
  • the HLA-A homozygote or heterozygote includes HLA-A*02 homozygote, HLA-A*11 homozygote, HLA-A*02/A*11 heterozygote or HLA-A* 24 homozygotes.
  • the present application provides a method for preparing immune cells, which includes introducing the nucleic acid molecule and/or the carrier into the immune cells.
  • the method further includes: before/after introducing the nucleic acid molecule and/or the vector into the immune cells, modifying the immune cells, the modification includes immune rejection-related The expression and/or activity of one or more of the genes is downregulated.
  • the gene associated with immune rejection is selected from one or more genes in the group consisting of TRAC, TRBC, HLA-A, HLA-B, B2M and CIITA.
  • the expression and/or activity of the TRAC gene and the HLA-A gene in the immune cells are down-regulated compared to the immune cells without the modification.
  • the expression and/or activity of the CIITA gene in the immune cells is not down-regulated compared to the immune cells without the modification.
  • the expression and/or activity of the B2M gene in the immune cells is not down-regulated compared to the immune cells without the modification.
  • the expression and/or activity of the TRAC gene and the HLA-A gene are down-regulated in the immune cells compared to wild-type cells.
  • the expression and/or activity of the CIITA gene is not down-regulated in the immune cells compared to wild-type cells.
  • the expression and/or activity of the B2M gene is not down-regulated in the immune cells compared to wild-type cells.
  • the down-regulation of the expression level and/or activity of the gene in the method includes down-regulation of the expression and/or activity of a nucleic acid molecule encoding the gene; and/or down-regulation of the gene encoded by the gene The expression and/or activity of the protein product is downregulated.
  • the modification in the method includes: gene knockout, gene mutation and/or gene silencing.
  • said modifying in said method comprises administering to said immune cells one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA and sgRNA.
  • said modifying in said method comprises administering to said immune cells a sgRNA targeting an exon portion of said TRAC gene.
  • the sgRNA targeting the exon portion of the TRAC gene in the method comprises the nucleotide sequence shown in any one of SEQ ID NO:138 to SEQ ID NO:152.
  • said modifying in said method comprises administering to said immune cells a sgRNA targeting an exon portion of said HLA-A gene.
  • the sgRNA targeting the exon portion of the HLA-A gene in the method comprises the nucleotides shown in any one of SEQ ID NO:153 to SEQ ID NO:193 sequence.
  • said modifying in said method further comprises administering a Cas enzyme to said cell.
  • the Cas enzyme in the method includes a Cas9 protein.
  • the modification comprises administering to the cell an antisense RNA comprising the nucleotide sequence shown in any one of SEQ ID NO: 194 to SEQ ID NO: 197.
  • the immune cells of the method are derived from humans.
  • the immune cells in the method include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, ⁇ T cells, monocytes, dendritic cells, Granulocytes, lymphocytes, leukocytes and/or peripheral blood mononuclear cells.
  • NK cells natural killer cells
  • NKT cells NKT cells
  • ⁇ T cells monocytes, dendritic cells, Granulocytes, lymphocytes, leukocytes and/or peripheral blood mononuclear cells.
  • the immune cells of the method comprise T cells.
  • the immune cells in the method are HLA-B homozygous cells.
  • the HLA-B homozygotes in the method include HLA-B*40 homozygotes, HLA-B*15 homozygotes, HLA-B*46 homozygotes, HLA-B*13 homozygotes Zygote, HLA-B*51 homozygous, HLA-B*58 homozygous, HLA-B*07 homozygous, HLA-B*35 homozygous, HLA-B*44 homozygous, HLA-B*52 homozygous, HLA-B*57 homozygote, HLA-B*54 homozygote or HLA-B*55 homozygote.
  • the immune cells in the method are HLA-A homozygous or heterozygous cells.
  • the HLA-A homozygote or heterozygote in the method includes HLA-A*02 homozygote, HLA-A*11 homozygote, HLA-A*02/A*11 heterozygote Or HLA-A*24 homozygous.
  • the present application provides the application of the chimeric antigen receptor, the isolated nucleic acid molecule, the vector, and/or the immune cells in the preparation of CAR-T cells.
  • the present application provides one or more pharmaceutical compositions, which comprise the chimeric antigen receptor, the isolated nucleic acid molecule, the carrier, and/or the immune cell, and optionally a pharmaceutically acceptable carrier.
  • the present application provides one or more of the antigen chimeric receptors, the isolated nucleic acid molecules, the vectors, the immune cells, and/or the pharmaceutical composition , for use in the treatment of a disease or condition associated with the expression of EGFR.
  • the diseases or disorders associated with the expression of EGFR include diseases or disorders associated with up-regulated expression of EGFR.
  • the disease or condition associated with expression of EGFR comprises a tumor.
  • the tumor comprises an EGFR positive tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a hematoma.
  • the tumor comprises glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, pancreatic cancer, eye cancer, mesothelioma, biliary tract cancer, prostate cancer, Liver cancer, fibrosarcoma, colon and/or stomach cancer.
  • the tumor comprises fibrosarcoma.
  • the present application provides the chimeric antigen receptor, the isolated nucleic acid molecule, the carrier, the immune cell, and/or the pharmaceutical composition in the preparation of medicine Use, the medicine is used for treating diseases or diseases related to the expression of EGFR.
  • the diseases or disorders associated with the expression of EGFR include diseases or disorders associated with up-regulated expression of EGFR.
  • the disease or condition associated with expression of EGFR comprises a tumor.
  • the tumor comprises an EGFR positive tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a hematoma.
  • the tumor comprises glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, pancreatic cancer, eye cancer, mesothelioma, biliary tract cancer, prostate cancer, Liver cancer, fibrosarcoma, colon and/or stomach cancer.
  • the tumor comprises fibrosarcoma.
  • the present application provides a method for preventing and/or treating diseases or conditions related to the expression of EGFR, which comprises administering an effective amount of the chimeric antigen receptor to a subject in need, wherein The isolated nucleic acid molecule, the carrier, the immune cell, and/or the pharmaceutical composition.
  • the diseases or disorders associated with the expression of EGFR include diseases or disorders associated with up-regulated expression of EGFR.
  • the disease or condition associated with expression of EGFR comprises a tumor.
  • the tumor comprises an EGFR positive tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a hematoma.
  • the tumor comprises glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, pancreatic cancer, eye cancer, mesothelioma, biliary tract cancer, prostate cancer, Liver cancer, fibrosarcoma, colon and/or stomach cancer.
  • the tumor comprises fibrosarcoma.
  • Figure 1 shows the exemplary anti-EGFR CAR gene lentiviral expression vector described in this application.
  • Figure 2 shows the construction strategy of anti-EGFR UCAR-T cells described in this application.
  • Figures 3A-3D show the phenotype detection results of anti-EGFR UCAR-T cells described in this application (knockout efficiency, transfection efficiency, amplification factor, memory T cell ratio).
  • Figure 4 shows the results of killing target cells by anti-EGFR UCAR-T cells described in the present application.
  • Figures 5A-5C show the detection results of cytokine secretion in the co-culture of anti-EGFR UCAR-T cells and target cells described in this application.
  • Figure 6 shows the anti-tumor effect of the anti-EGFR UCAR-T cells described in this application in vivo.
  • Figure 7 shows the results of the in vivo half-life detection of anti-EGFR UCAR-T cells described in this application.
  • Figures 8A-8B show the results of in vivo rejection of the anti-EGFR UCAR-T cells described in this application.
  • Figure 9 shows the off-target analysis of targeting anti-EGFR UCAR-T cells described in this application.
  • Figure 10 shows the chromosomal translocation analysis of targeting anti-EGFR UCAR-T cells described in this application.
  • Figure 11 shows the karyotype analysis of the targeting anti-EGFR UCAR-T cells described in this application.
  • Figure 12 shows the Cas9 residue analysis of targeting anti-EGFR UCAR-T cells described in this application.
  • FIG. 13 shows the results of Sanger sequencing of the TRAC gene in this application after Sg9RNA editing.
  • Figure 14 shows the results of TA clone detection of the TRAC gene in this application after Sg9RNA editing.
  • Figure 15 shows the results of flow cytometry detection of the TRAC gene in this application after Sg9RNA editing.
  • Figure 16 shows the results of Sanger sequencing of the HLA-A02 gene in this application after Sg2RNA editing.
  • Figure 17 shows the results of Sanger sequencing of the HLA-A02 gene in this application after Sg5RNA editing.
  • Figure 18 shows the results of Sanger sequencing of the HLA-A11 gene in this application after Sg21 RNA editing.
  • Figure 19 shows the results of Sanger sequencing of the HLA-A11 gene in this application after editing by Rsg2 RNA.
  • 20A-20B show the results of simultaneous knockout of HLA-A02 and TRAC in the modified immune effector cells of the present application.
  • 21A-21B show the protein levels of HLA-A02 and TRAC in the modified immune effector cells of the present application.
  • Figure 22 shows the mRNA levels of TRAC, HLA-A, B2M and CIITA in the modified immune effector cells of the present application.
  • 23A-23B show the protein levels of B2M and CIITA in the modified immune effector cells of the present application.
  • 24A-24D show the protein levels of TRAC, HLA-A, B2M and CIITA in the modified immune effector cells of the present application.
  • Figures 25A-25B show the knockout of TRAC and HLA-A mRNA levels in the modified immune effector cells of the present application.
  • 26A-26B show the protein levels of CD69 and CD137 in the modified immune effector cells of the present application.
  • Figure 27 shows the co-culture of the modified immune effector cells and NK cells of the present application.
  • Figure 28 shows the expression level of IFN- ⁇ by the modified immune effector cells of the present application.
  • 29A-29D show the protein levels of TRAC, HLA-A, B2M and CIITA in the modified immune effector cells of the present application.
  • Figure 30 shows the infection efficiency of the modified immune effector cells of the present application to CAR.
  • Figure 31 shows the expansion factor of the modified immune effector cells of the present application.
  • Figure 32 shows the killing effect of the modified immune effector cells of the present application on CD19 positive target cells.
  • Figure 33 shows the dosing regimen for administering the modified immune effector cells of the present application.
  • Figure 34 shows the killing effect of the modified immune effector cells of the present application on tumors in mice.
  • chimeric antigen receptor generally refers to a group of polypeptides, usually two in the simplest embodiment, which, when in an immune cell, provide the cell with respect to a target cell ( Usually specific for cancer cells) and generate intracellular signals.
  • a CAR comprises at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain”) comprising Functional signaling domains of defined stimulatory and/or co-stimulatory molecules.
  • the set of polypeptides are in the same polypeptide chain (eg, comprising chimeric fusion proteins).
  • the set of polypeptides is discontinuous from each other, eg, in different polypeptide chains.
  • the set of polypeptides includes a dimerization switch that, in the presence of a dimerization molecule, can couple the polypeptides to each other, eg, can couple an antigen binding domain to an intracellular signaling domain.
  • the stimulatory molecule of the CAR is the zeta chain associated with the T cell receptor complex.
  • the cytoplasmic signaling domain comprises a primary signaling domain (eg, the primary signaling domain of CD3-zeta).
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below.
  • the co-stimulatory molecule may be selected from 4-1BB (ie 4-1BB), CD27, ICOS and/or CD28.
  • a CAR comprises a chimeric fusion protein that may comprise an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule.
  • a CAR comprises a chimeric fusion protein that may comprise an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule domains and functional signaling domains derived from stimulatory molecules.
  • the CAR comprises a chimeric fusion protein which may comprise an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain comprising a protein derived from one or more co-stimulatory molecules.
  • Functional signaling domains and functional signaling domains derived from stimulatory molecules may comprise an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain comprising a protein derived from one or more co-stimulatory molecules.
  • the CAR comprises a chimeric fusion protein which may comprise an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain comprising at least two derived from one or more common A functional signaling domain of a stimulatory molecule and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g. scFv) during cellular processing and localizes the CAR to the cell membrane.
  • EGFR protein generally belongs to the ErbB family of receptor tyrosine kinases, which play a key role in the physiology of epithelial cells.
  • the amino acid sequence of the human EGFR protein can be found at UniProt/Swiss-Prot Accession No. P00533.
  • the isolated antigen-binding fragment can bind to EGFR protein.
  • the terms "EGFR protein”, “EGFR antigen” and “EGFR-Fc recombinant protein” are used interchangeably and include any variant or isoform thereof naturally expressed by cells.
  • CDR complementarity determining region
  • CDRs Chothia CDRs
  • Padlan FASEB J.9:133-139 (1995)
  • MacCallum J Mol Biol 262(5):732-45 (1996).
  • CDR boundaries for CDR may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, although they can be shortened according to predictions or experimental findings that specific residues or groups of residues or even CDRs as a whole do not significantly affect antigen binding. or extended. In this application, the IMGT numbering system is used.
  • FR generally refers to the more highly conserved portions of antibody variable domains, known as the framework regions.
  • the variable domains of native heavy and light chains may each comprise four FR regions, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL. (L-FR1, L-FR2, L-FR3 and L-FR4).
  • Framework region generally refers to the art-recognized portion of an antibody variable region that exists between the more divergent (ie hypervariable) CDRs.
  • Framework regions are typically referred to as Frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and provide the backbone for representing the six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space, to Forms the antigen-binding surface.
  • single chain antibody or “scFv” generally refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a linker.
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • the 2 domains, VL and VH, are encoded by separate genes, they can be joined using recombinant methods, through a synthetic linker, making them a single protein chain, where the VL and VH regions pair to form what is known as a single-chain Fv (scFv). monovalent molecule.
  • the term "antibody” when referring to fragments includes said single chain antibodies which can be produced by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.
  • homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • a homologue will comprise the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues having similar chemical properties/functions), or can be expressed in terms of sequence identity.
  • a sequence having a percentage identity of any one of the SEQ ID NOs of the mentioned amino acid sequence or nucleotide sequence means having said percentage identity over the entire length of the mentioned SEQ ID NO the sequence of.
  • sequence alignment can be performed by various means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment across the full-length sequences being compared.
  • KD is used interchangeably with “KD”, and generally refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, and the unit is M (mol/L).
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length, isolated from their natural environment or artificially synthesized .
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having various functions as described above.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing an appropriate host cell containing the vector.
  • the term "host cell” generally refers to an individual cell, cell line or cell culture that can or has contained a vector comprising the isolated nucleic acid molecule described herein, or is capable of expressing the isolated antigen-binding fragment described herein things.
  • the host cells may include progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cells, but they only need to be able to express the isolated antigen-binding fragments described in this application.
  • the host cells can be obtained by using the vectors described in this application to transfect cells in vitro.
  • the host cell can be a prokaryotic cell (such as Escherichia coli), or a eukaryotic cell (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells).
  • the host cells may be E. coli cells.
  • the host cell may be a yeast cell.
  • the host cell can be a mammalian cell.
  • the mammalian cells may be CHO-K1 cells.
  • immune effector cells generally refers to immune cells that participate in the immune response and perform effector functions.
  • the exercising effector functions may include clearing foreign antigens or promoting immune effector responses and the like.
  • Immune cells may include plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • the immune effector cells of the present application may be autologous/autogeneic ("own") or non-autologous ("non-self", eg allogeneic, syngeneic or allogeneic).
  • autologous generally refers to cells from the same subject.
  • Allogeneic generally refers to cells that are of the same species but are genetically different from those to which they are being compared.
  • Isgeneic generally refers to cells of a different subject that are genetically identical to the cells being compared.
  • Allogeneic generally refers to a cell of a different species than the compared cell.
  • the cells of the present application are autologous or allogeneic.
  • the term "modify” generally refers to altering the state or structure of a cell and/or changing the state or structure of a cell.
  • the change is usually compared with the state or structure of the corresponding unmodified cell, and the change may include a change in the expression level or function of an endogenous gene, such as down-regulating the expression level of an endogenous gene in a cell by means of genetic engineering, Up-regulation or non-expression, the genetic engineering means may include homologous recombination, CRISPR/Cas9 system gene editing, etc.; the change may also include changes in cellular protein expression, structure or function, such as through the endogenous gene expression level or Changes in protein expression, changes in structure or function achieved by changes in function, such as changes in protein expression, changes in structure or function achieved by regulating protein translation, post-translational modification; the changes may also include introducing exogenous Genes, expression of foreign proteins, etc.
  • T cell receptor alpha cons-stant T cell receptor alpha cons-stant
  • T cell receptor T cell receptor
  • MHC major histocompatibility complex
  • ⁇ T cells account for about 95% of the total T cells, and ⁇ T cells account for about 5% of the total T cells. This ratio varies during ontogeny and in disease states (such as leukemia) and also varies between species. different.
  • Each chain that makes up the TCR contains a variable region and a constant region.
  • the gene encoding the ⁇ chain (TRA, such as the information shown in HGNC: 12027) is located on chromosome 14 and consists of multiple gene segments, including variable Segment (V), connecting segment (J) and constant region (C), TRAC gene usually refers to the gene sequence (for example, the information shown in HGNC:12029) encoding T cell receptor ⁇ chain constant region (C), which is located in Chromosome 14 (14q11.2;14:22,547,505-22,552,131).
  • variable segment (V) genes encoding the N-segment antigen recognition domain is rearranged with one of the junction segment (J) to generate a functional V region exon that is transcribed and spliced together with the
  • the constant regions (C) are linked to form the T cell receptor alpha chain coding sequence.
  • MHC major histocompatibility complex antigen
  • HLA human leukocyte antigen
  • HLA class I antigens (A, B, C in humans) allow each cell to be recognized as "self", while HLA class II antigens (DR, DP and DQ in humans) participate in the communication between lymphocytes and antigen-presenting cells reaction between. Both have been implicated in the rejection of transplanted organs.
  • An important aspect of the HLA gene system is its polymorphism. Different alleles exist for each gene, MHC class I (A, B, and C) and MHC class II (DP, DQ, and DR). HLA alleles are indicated by numbers and subscripts. For example, two unrelated individuals may carry the class I HLA-B genes B5 and Bw41, respectively. Allelic products differ in one or more amino acids in the alpha and/or beta domains.
  • HLA-A MHC class I and II proteins
  • HLA-B HLA-DR
  • the HLA genes are clustered in a "superlocus" present on chromosome position 6p21, which encodes six classical Transplantation of HLA genes and at least 132 protein-coding genes. The complete locus measures roughly 3.6Mb, with at least 224 loci.
  • haplotype a set of alleles present on a single chromosome, inherited from one parent, that tends to be inherited as a group.
  • the set of alleles inherited from each parent forms a haplotype, some of which tend to be associated together. Identifying a patient's haplotype can help predict the probability of finding a matching donor and help develop a search strategy because some alleles and haplotypes are more common than others and they are more common in different races and ethnicities The frequencies in the distribution are different.
  • HLA-A generally refers to a type of human leukocyte antigen polypeptide chain, encoded by the HLA-A gene located on human chromosome 6p21.3 (for example, the information shown in HGNC:4931).
  • HLA-A is one of three major polypeptide types that make up MHC class I molecules on the surface of human cells, the others including HLA-B and HLA-C.
  • the heterodimer composed of the ⁇ chain encoded by the HLA-A gene and the ⁇ chain ( ⁇ 2-microglobulin) encoded by the B2M gene is the HLA-A class MHC I molecule.
  • the ⁇ chain encoded by the HLA-A gene may comprise an ⁇ 1 domain, an ⁇ 2 domain, an ⁇ 3 domain, a transmembrane region, and a cytoplasmic region, wherein the ⁇ 1 domain and the ⁇ 2 domain may be combined with peptides to be activated by MHC I Molecules (eg HLA-A class) present the peptides to cells of the immune lineage.
  • MHC I Molecules eg HLA-A class
  • the ⁇ chain of the MHC I molecule is polymorphic, and its primary structure has many changes. As of December 2013, there are 2432 known HLA-A alleles , encoding 1740 active proteins and 117 inactive proteins.
  • HLA-A alleles may include those named by the WHO HLA Factor Nomenclature Committee included in the IMGT/HLA database version 3.38.0 (https://www.ebi.ac.uk/ipd/imgt/hla/) Sequence information of the different HLA-A alleles.
  • HLA-B generally refers to a part of the gene family of the human leukocyte antigen (HLA) complex.
  • HLA is the human version of the major histocompatibility complex (MHC), a family of genes present in many species. The genes in this complex are divided into three basic groups: class I, class II and class III.
  • MHC major histocompatibility complex
  • HLA-B gene and two related genes, HLA-A and HLA-C are the major MHC class I genes.
  • the HLA-B gene is located in band 21.3 of the short (p) arm of chromosome 6, from base pairs 31,353,871 to 31,357,211.
  • HLA-B is one of the three main HLAs that should be matched between donor and recipient.
  • HLA-A HLA-B
  • HLA-DR MHC class II
  • HLA-matched refers to a donor-recipient pair in which there is no mismatch in HLA antigens between the donor and recipient, such as providing hematopoietic stem cell transplantation therapy to a recipient in need of A donor for a stem cell transplant.
  • HLA-matched (i.e., in which all 6 alleles are matched) donor-recipient pairs have a reduced risk of graft rejection because endogenous T cells and NK cells are less likely to enter the graft recognized as foreign and thus less likely to mount an immune response against the graft.
  • HLA-mismatched refers to a donor- A recipient pair, such as a donor who provides a hematopoietic stem cell transplant to a recipient in need of hematopoietic stem cell transplantation therapy.
  • one haplotype is matched while the other is not.
  • HLA-mismatched donor-recipient pairs may have an increased risk of graft rejection relative to HLA-matched donor-recipient pairs because endogenous Sexual T cells and NK cells are more likely to recognize an incoming graft as foreign, and such T cells and NK cells are therefore more likely to mount an immune response against the graft.
  • B2M generally refers to ⁇ 2-microglobulin ( ⁇ 2-microglobulin), which is one of the components of MHC class I molecules.
  • ⁇ 2 microglobulin also known as ⁇ chain
  • B2M is normally expressed in all nucleated cells.
  • ⁇ 2 microglobulin is encoded by the B2M gene located at 15q21.1 (for example, the information shown in HGNC:914).
  • CIITA generally refers to the transactivator of major histocompatibility complex class II (MHC II).
  • the transactivator may be a protein having an acidic transcription activation domain, 4 LRRs (leucine rich repeats) and a GTP binding domain.
  • the protein can be localized in the nucleus and acts as a positive regulator of the transcription of major histocompatibility complex class II (MHC II) genes, known as the "master control factor” for the expression of these genes.
  • MHC II major histocompatibility complex class II
  • the protein also binds GTP and uses the binding to GTP to transport itself into the nucleus where it normally acts in a coactivator-like manner through acetyltransferase (AT) activity.
  • the protein is encoded by a gene located at 16p13.13 (for example the information shown at HGNC:7067), enabling the generation of several transcript variants encoding different isoforms.
  • wild-type cell generally refers to a naturally occurring or naturally derived cell.
  • T cells generally refers to thymus-derived cells that participate in various cell-mediated immune responses.
  • nucleic acid or “polynucleotide” or “nucleic acid molecule” generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless specifically limited, the term may include nucleic acids that contain analogs of natural nucleotides that have similar binding properties to a reference nucleic acid (for example, for which sequence information is shown) and in a manner similar to naturally occurring nucleotides metabolism. Unless otherwise stated, the sequence of a nucleic acid may include conservatively modified variants thereof, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated.
  • expression generally refers to the transcription and/or translation of a specific nucleotide sequence.
  • gene mutation generally refers to changes in the composition or sequence of base pairs in the structure of a gene. For example, point mutations caused by single base changes, or deletions, duplications, and insertions of multiple bases.
  • the term "gene silencing” generally refers to preventing the expression of certain genes by regulatory mechanisms. It can mainly include two types: one is transcriptional gene silencing (TGS) at the transcriptional level caused by factors such as DNA methylation, heterochromatinization, and position effects, and the other is post-transcriptional gene silencing (post -transcriptional gene silencing (PTGS), that is, at the post-transcriptional level of the gene, it affects the expression of the gene by specifically interfering with the target RNA.
  • TGS transcriptional gene silencing
  • PTGS post-transcriptional gene silencing
  • the expression of the corresponding gene is downregulated/decreased.
  • Gene silencing is generally considered to be a gene knockdown mechanism, and methods commonly used to silence genes can be RNAi, etc.
  • endogenous refers to any substance derived from or produced within an organism, cell, tissue or system.
  • exogenous refers to any substance introduced from or produced outside of an organism, cell, tissue or system.
  • antisense RNA generally refers to a single-stranded RNA that is complementary to the transcript mRNA (messenger RNA). Antisense RNA can inhibit gene expression by binding to mRNA. For example, the combination of antisense RNA and target mRNA increases the sensitivity of the double-stranded RNA molecule to RNase III and degrades it; for example, antisense RNA binds to the upstream non-coding region of mRNA, thereby directly inhibiting the translation of target mRNA .
  • siRNA generally refers to the abbreviation of Small interfering RNA (small interfering RNA) or short in-terfering RNA (short interfering RNA).
  • siRNA is a type of double-stranded non-coding RNA molecule with a length of about 18-28 base pairs, which can cause mRNA degradation through complementary binding to mRNA, thereby interfering with the expression of specific genes.
  • siRNA may be a product obtained by treating long double-stranded RNA or shRNA with Dicer enzyme.
  • the siRNA enters the cell and forms an RNA-induced silencing complex (RISC) with other proteins, the sense strand is degraded, and the antisense strand can bind to a complementary targeting sequence, thereby achieving gene silencing.
  • RISC RNA-induced silencing complex
  • shRNA generally refers to the abbreviation of short hairpin RNA, namely “short hairpin RNA”.
  • shRNA usually includes two short inverted repeat sequences separated by a stem-loop sequence to form a hairpin structure.
  • 5-6 T bases can also be included as the transcription terminator of RNA polymerase III.
  • shRNA can enter cells through viral vectors or plasmids, and be transcribed under the action of polymerase II or polymerase III, and the transcripts are exported from the nucleus (usually through Exportin 5) and then transported after being treated by Dicer To RISC, the sense strand is degraded, and the antisense strand can bind to a complementary targeting sequence, thereby achieving gene silencing.
  • CRISPR/Cas system generally refers to a group of molecules comprising an RNA-guided nuclease or other effector molecule and a gRNA molecule capable of directing and implementing the RNA-guided nuclease or other effector molecule Nucleic acid is modified at a target sequence, eg, causing degradation of the target sequence.
  • a CRISPR system comprises a gRNA and a Cas protein, e.g., a Cas9 protein.
  • Cas9 systems systems comprising Cas9 or functional mutants thereof are referred to herein as “Cas9 systems” or "CRISPR/Cas9 systems”.
  • the gRNA molecule and the Cas molecule can complex to form a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • gRNA molecule or “guide RNA”, “guide RNA”, “guide RNA”, “guide RNA molecule”, “gRNA” are used interchangeably and generally refer to Nucleases or other effector molecules (generally complexed with gRNA molecules) to nucleic acid molecules on the target sequence. In certain embodiments, this is accomplished by hybridizing a portion of the gRNA to DNA (e.g., via a gRNA guidance domain) and by binding a portion of the gRNA molecule to an RNA-guided nuclease or other effector molecule (e.g., at least via gRNAtracr). described guide.
  • a gRNA molecule consists of a single contiguous polynucleotide molecule, referred to herein as a "single guide RNA” or “sgRNA” or the like.
  • a gRNA molecule consists of multiple (eg, two) polynucleotide molecules that are themselves capable of associating (typically by hybridization), referred to herein as “dual guide RNA” or “dgRNA” and the like.
  • Cas protein generally refers to the enzyme responsible for cutting DNA in the CRISPR/Cas system. Enzymes from Type I, II, and III CRISPR/Cas systems may be included. For example, Cas3, Cas9, Cas10.
  • Cas9 protein generally refers to the enzyme from the bacterial type II CRISPR/Cas system responsible for cutting DNA. Cas9 can include the wild-type protein and its functional mutants.
  • allele generally refers to the different variations that the gene sequence at a locus may have.
  • a genetic locus also known as a gene locus or site, refers to a fixed location on a chromosome, such as where a certain gene is located. The arrangement of loci in the genome is called a genetic map.
  • homozygous generally refers to a genotyped individual whose two alleles on the same locus of the homologous chromosome are the same.
  • a pair of relative genes can have individuals of both genotypes AA and aa.
  • heterozygote generally refers to a diploid individual whose two alleles at the same site on the homologous chromosome are different, such as Aa. Heterozygous genotypes generally have higher fitness than homozygous dominant or homozygous recessive genotypes, a phenomenon known as heterozygous dominance.
  • the term "pharmaceutically acceptable” generally refers to a drug that is commensurate with a reasonable benefit/risk ratio, suitable within the scope of sound medical judgment for use in contact with human and animal tissues without undue toxicity, irritation, Those compounds, materials, compositions and/or dosage forms for allergic reactions or other problems or complications.
  • the term "pharmaceutically acceptable carrier” generally refers to any of those conventionally used, and is subject only to physico-chemical considerations such as solubility and reactivity with active binding agents. lack of) and is limited by the route of administration.
  • the pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public.
  • a pharmaceutically acceptable carrier is one that is chemically inert to the active ingredients of the pharmaceutical composition and that exhibits no adverse side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce an adverse, allergic or other inappropriate reaction when administered to an animal or a human.
  • compositions are free of pyrogens and other impurities that could be harmful to humans or animals.
  • Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like; their use is well known in the art.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphates, citrates, or other organic acids; antioxidants; , such as ascorbic acid; low-molecular-weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine amino acids; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or Or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).
  • buffers such as phosphates, citrates,
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides a chimeric antigen receptor (CAR), which may comprise a targeting moiety, which may comprise at least one CDR in the heavy chain variable region VH.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the HCDR of the isolated antigen-binding protein can be divided in any form, as long as the VH is identical to the amino acid sequence shown in SEQ ID NO: 1, the HCDR obtained by any form of division can fall under the protection of this application within range.
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • amino acids For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the isolated antigen binding proteins described herein are defined by the IMGT coding system.
  • the targeting moiety may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8.
  • the HCDR3 of the targeting moiety can be defined by the IMGT coding system.
  • the targeting moiety may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the HCDR2 of the targeting moiety can be defined by the IMGT coding system.
  • the targeting moiety may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4.
  • HCDR1 of the targeting moiety can be defined by the IMGT coding system.
  • the targeting moiety may comprise HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:1.
  • the targeting moiety may comprise HCDR1, HCDR2, HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; And the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the antigen-binding protein may comprise antibody A1 or an antigen-binding fragment (eg, scFv) having the same HCDR3 as it (eg, having the same HCDR1-3 as it).
  • the targeting moiety may comprise H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the H-FR1 may comprise SEQ ID NO:3 Amino acid sequence shown.
  • the targeting moiety may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5 .
  • the targeting moiety may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:7 .
  • the targeting moiety may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 may comprise SEQ ID NO:9 Amino acid sequence shown.
  • the targeting moiety may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 3; the H- FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:7; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:9 .
  • the antigen-binding protein may comprise antibody A1 or an antigen-binding fragment (eg, scFv) having the same H-FR1-4 therewith.
  • the targeting moiety in the chimeric antigen receptor may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the nucleotide sequence encoding the VH can be SEQ ID NO:2.
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the LCDR of the isolated antigen-binding protein can be divided in any form, as long as the VL is identical to the amino acid sequence shown in SEQ ID NO: 10, the LCDR obtained by dividing in any form can fall under the protection of this application within range.
  • the antigen binding protein may comprise a light chain variable region VL, and the VL may comprise at least one, two or three of LCDR1, LCDR2 and LCDR3.
  • the targeting moiety in the chimeric antigen receptor may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:17.
  • the LCDR3 of the targeting moiety can be defined by the IMGT coding system.
  • the targeting moiety in the chimeric antigen receptor may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:15.
  • the LCDR2 of the targeting moiety can be defined by the IMGT coding system.
  • the targeting moiety in the chimeric antigen receptor may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:13.
  • LCDR1 of the targeting moiety can be defined by the IMGT coding system.
  • the targeting moiety in the chimeric antigen receptor may comprise LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:10.
  • the targeting moiety in the chimeric antigen receptor may comprise LCDR1, LCDR2, LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 17; the LCDR2 may comprise SEQ ID The amino acid sequence shown in NO:15; And the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO:13.
  • the antigen-binding protein may comprise antibody A1 or an antigen-binding fragment (eg, scFv) that has the same LCDR3 as it (eg, has the same LCDR1-3 as it).
  • the targeting moiety in the chimeric antigen receptor may comprise L-FR1
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1
  • the L-FR1 -FR1 may comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the targeting moiety in the chimeric antigen receptor may comprise L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise SEQ Amino acid sequence shown in ID NO:14.
  • the targeting moiety in the chimeric antigen receptor may comprise L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise SEQ Amino acid sequence shown in ID NO:16.
  • the targeting moiety in the chimeric antigen receptor may comprise L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 -FR4 may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the targeting moiety may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 12; the L- FR2 may comprise the amino acid sequence shown in SEQ ID NO:14; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:16; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:18 .
  • the antigen-binding protein may comprise antibody A1 or an antigen-binding fragment (eg, scFv) having the same L-FR1-4 therewith.
  • the targeting moiety in the chimeric antigen receptor may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the nucleotide sequence encoding the VL is SEQ ID NO: 11.
  • the targeting moiety in the chimeric antigen receptor may comprise HCDR1-3 and LCDR1-3.
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4; the The LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:17; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:15; and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:13.
  • the antigen-binding protein may comprise antibody A1 or an antigen-binding fragment (eg, scFv) that has the same LCDR3 and HCDR3 as it (eg, has the same LCDR1-3 and HCDR1-3 as it).
  • an antigen-binding fragment eg, scFv
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:13;
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:15;
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:17.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:3; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:7
  • the amino acid sequence shown; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:9; the L-FR1 can comprise the amino acid sequence of SEQ ID NO:12; the L-FR2 can comprise the amino acid sequence of SEQ ID NO The amino acid sequence shown in: 14;
  • the L-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 16;
  • the L-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the antigen binding protein may include antibody A1 or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 10.
  • the antigen binding protein may comprise antibody A1 or an antigen binding protein having the same light chain variable region as it.
  • the targeting moiety in the chimeric antigen receptor may include an antibody or an antigen-binding fragment.
  • the antigen-binding fragment in the chimeric antigen receptor can be selected from the following group: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di- scFv, VHH and/or dAb.
  • the targeting moiety in the chimeric antigen receptor may include scFv.
  • the scFv can target EGFR.
  • the C-terminus of the VH may be directly or indirectly linked to the N-terminus of the VL.
  • the C-terminus of the VL may be directly or indirectly linked to the N-terminus of the VH.
  • transmembrane domain which may comprise a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8A, CD8B, CD28, CD3e, CD3 ⁇ , 4- 1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, CD154, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, and SLAM.
  • proteins selected from the group consisting of: CD8A, CD8B, CD28, CD3e, CD3 ⁇ , 4- 1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CTLA-4, L
  • the transmembrane domain may comprise a transmembrane domain derived from CD8A or CD8B.
  • transmembrane domain may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:69.
  • an intracellular co-stimulatory signaling domain which may comprise intracellular co-stimulatory signaling derived from one or more proteins selected from the group consisting of Domains: CD28, 4-1BB, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM , DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, and MyD88.
  • Domains CD28, 4-1BB, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR
  • the intracellular co-stimulatory signaling domain may include a co-stimulatory signaling domain derived from 4-1BB.
  • intracellular co-stimulatory signaling domain may comprise the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:102.
  • an intracellular signal transduction domain which may comprise an intracellular signal transduction domain derived from one or more proteins selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP-12 and structures containing at least one ITAM area.
  • an intracellular signal transduction domain derived from one or more proteins selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP-12 and structures
  • the intracellular signal transduction domain may comprise a signal transduction domain derived from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ or CD3 ⁇ .
  • the intracellular signal transduction domain may comprise any one of SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:103 to SEQ ID NO:113 Amino acid sequence shown.
  • a hinge region between the targeting moiety and the transmembrane domain may comprise a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4 , IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, TIM1, SLAM, CD30, and LIGHT.
  • the hinge region may comprise a hinge region derived from CD8.
  • the hinge region may comprise the amino acid sequence shown in any one of SEQ ID NO:114 to SEQ ID NO:135.
  • the chimeric antigen receptor may include a signal peptide.
  • the signal peptide may comprise the amino acid sequence shown in SEQ ID NO:136.
  • the nucleic acid sequence of the signal peptide may comprise the nucleotide sequence of SEQ ID NO:137.
  • the non-targeting portion of the chimeric antigen receptor may include a hinge region, a transmembrane domain, an intracellular co-stimulatory signal transduction domain and an intracellular signal transduction domain.
  • the chimeric antigen receptor uses an anti-EGFR single-chain antibody (scFv) as a targeting moiety, and is connected to an intracellular signal transduction domain through a hinge region and a transmembrane domain, and the intracellular signal transduction domain is composed of The co-stimulatory signaling domain is composed of an intracellular signal transduction domain.
  • scFv anti-EGFR single-chain antibody
  • the non-targeting portion of the chimeric antigen receptor may comprise the transmembrane domain of the CD8A molecule, the hinge region of CD8, the intracellular co-stimulatory signaling domain of 4-1BB, and the intracellular signal transduction domain of CD3 ⁇ . guiding domain.
  • the chimeric antigen receptor uses an anti-EGFR single-chain antibody (scFv) as a targeting moiety, and is connected to the intracellular signal transduction domain through the hinge region and transmembrane domain of the CD8 molecule, and the intracellular signal transduction structure
  • the domain consists of the 4-1BB intracellular co-stimulatory signaling domain and the CD3 ⁇ intracellular signaling domain.
  • the non-targeting portion of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 19.
  • the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 199.
  • the present application provides one or more isolated nucleic acid molecules encoding the aforementioned chimeric antigen receptors.
  • the isolated nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO:200.
  • the present application provides a vector, which may comprise the aforementioned isolated nucleic acid molecule.
  • the vector may include a viral vector or a non-viral vector.
  • the non-viral vector may include Sleeping Beauty system or piggybac system.
  • the vector may include a lentiviral vector.
  • the present application provides a cell, which may comprise the aforementioned chimeric antigen receptor, the aforementioned isolated nucleic acid molecule, and/or the aforementioned vector.
  • the present application provides the aforementioned chimeric antigen receptor, the aforementioned isolated nucleic acid molecule, the aforementioned vector, the aforementioned cell, or the application of the aforementioned immune cell in the preparation of CAR-T cells.
  • the present application provides an immune cell, which may comprise the aforementioned nucleic acid molecule or the aforementioned vector, and/or express the aforementioned CAR.
  • the immune cells may include human cells.
  • the immune cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, white blood cells and/or or peripheral blood mononuclear cells.
  • the immune cells mentioned above may include T cells.
  • the immune cells may include modified immune cells.
  • the modified immune cells may include cells that reduce immune rejection caused by allogeneic cell therapy.
  • TCR T cell antigen receptor
  • MHCI major histocompatibility complex
  • the modification may include down-regulation of the expression and/or activity of one or more genes related to immune rejection.
  • the gene related to immune rejection can be selected from one or more genes in the following group: TRAC, TRBC, HLA-A, HLA-B, B2M and CIITA.
  • the gene related to immune rejection can be selected from one or more genes in the following group: TRAC, TRBC, HLA-A and HLA-B.
  • the gene related to immune rejection can be selected from one or more genes in the following group: TRAC, TRBC and HLA-A.
  • the gene related to immune rejection can be selected from one or more genes in the following group: TRAC and HLA-A.
  • the expression and/or activity of TRAC gene and HLA-A gene can be down-regulated in the modified immune cells.
  • the expression and/or activity of the CIITA gene is not down-regulated in the modified immune cells compared with the corresponding cells without the modification.
  • the expression and/or activity of the B2M gene is not down-regulated in the modified immune cells compared with the corresponding cells without the modification.
  • the expression and/or activity of TRAC gene and HLA-A gene are down-regulated in the modified immune cells.
  • the expression and/or activity of the B2M gene is not down-regulated in the modified immune cells.
  • the expression and/or activity of the CIITA gene is not down-regulated in the modified immune cells compared with the corresponding wild-type cells.
  • the expression level and/or activity of the gene is down-regulated includes down-regulating the expression and/or activity of the nucleic acid molecule encoding the gene; and/or down-regulating the expression and/or activity of the protein product encoded by the gene or the activity is downregulated.
  • the modification may include: gene knockout, gene mutation and/or gene silencing.
  • the modification may include the knockout of any one of the two TRAC alleles and the knockout of any one of the two HLA-A alleles in the immune cells.
  • the modification may include the knockout of two TRAC alleles and the knockout of any one of the two HLA-A alleles in the immune cells.
  • the modification may include knocking out the exons of the TRAC gene and knocking out the exons of the HLA-A gene in the immune cells.
  • the modification may include administering one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA and CRISPR/Cas9 system to the immune cells.
  • the modification may include administering the CRISPR/Cas9 system to the immune cells.
  • the modification may further include administering sgRNA targeting the exon part of the TRAC gene to the immune cells.
  • the sgRNA targeting the exon part of the TRAC gene may comprise the nucleotide sequence shown in any one of SEQ ID NO:138 to SEQ ID NO:152.
  • the modification may include administering to the immune cells sgRNA targeting the exon portion of the HLA-A gene.
  • the sgRNA targeting the exon part of the HLA-A gene may comprise the nucleotide sequence shown in any one of SEQ ID NO:153 to SEQ ID NO:193.
  • the modification may also include administering a Cas enzyme to the cell.
  • Cas enzymes may include Cas9 protein.
  • the modification may include applying antisense RNA to the cell, and the antisense RNA may comprise the nucleotide sequence shown in any one of SEQ ID NO:194 to SEQ ID NO:197.
  • the immune cells may be HLA-B homozygous cells.
  • the HLA-B homozygous may include HLA-B*40 homozygous, HLA-B*15 homozygous, HLA-B*46 homozygous, HLA-B*13 homozygous, HLA-B *51 homozygote, HLA-B*58 homozygote, HLA-B*07 homozygote, HLA-B*35 homozygote, HLA-B*44 homozygote, HLA-B*52 homozygote, HLA-B*57 Homozygous, HLA-B*54 homozygous, HLA-B*55 homozygous.
  • the immune cells may be HLA-A homozygous or heterozygous cells.
  • the HLA-A homozygote or heterozygote may include HLA-A*02 homozygote, HLA-A*11 homozygote, HLA-A*02/A*11 heterozygote or HLA-A* 24 homozygotes.
  • the present application provides a method for preparing immune cells, which may include introducing the aforementioned nucleic acid molecules or the aforementioned vectors into the immune cells.
  • the method may further include: before/after introducing the aforementioned nucleic acid molecule or the aforementioned vector into the immune cells, modifying the immune cells, the modification including one or The expression and/or activity of multiples were downregulated.
  • the method may include: after introducing the aforementioned nucleic acid molecule or the aforementioned vector into the immune cells, modifying the immune cells, the modification including one or more genes related to immune rejection Expression and/or activity is downregulated.
  • the method for preparing immune cells may include:
  • the modification includes down-regulating the expression and/or activity of one or more genes related to immune rejection.
  • the method for preparing immune cells may include:
  • CD3 magnetic beads were added in proportion to collect CD3-T cells (cells not bound to magnetic beads).
  • the gene related to immune rejection is selected from one or more genes in the following group: TRAC, TRBC, HLA-A, HLA-B, B2M and CIITA.
  • the expression and/or activity of the TRAC gene and the HLA-A gene in the immune cells are down-regulated compared with the expression and/or activity of the corresponding genes in the corresponding cells without the modification.
  • the expression and/or activity of the CIITA gene is not down-regulated compared to the expression and/or activity of the corresponding gene in the corresponding cell without said modification.
  • the expression and/or activity of the B2M gene is not down-regulated compared to the expression and/or activity of the corresponding gene in the corresponding cell without said modification.
  • the expression and/or activity of the TRAC gene and the HLA-A gene of the immune cells are down-regulated.
  • the expression and/or activity of the CIITA gene was not down-regulated compared to the corresponding wild-type cells.
  • the expression and/or activity of B2M genes was not down-regulated compared to corresponding wild-type cells.
  • the expression level and/or activity of the gene is down-regulated, which may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the gene; and/or down-regulating the expression and/or activity of the protein product encoded by the gene. / or the activity is downregulated.
  • the modification includes: gene knockout, gene mutation and/or gene silencing.
  • the modification may include the knockout of any one of the two TRAC alleles and the knockout of any one of the two HLA-A alleles in the immune cells.
  • the modification may include the knockout of two TRAC alleles and the knockout of any one of the two HLA-A alleles in the immune cells.
  • the modification may include knocking out the exons of the TRAC gene and knocking out the exons of the HLA-A gene in the immune cells.
  • the modification may include administering one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA and CRISPR/Cas9 system to the immune cells.
  • the modification may include administering the CRISPR/Cas9 system to the immune cells.
  • the modification may include administering sgRNA targeting the exon part of the TRAC gene to the immune cells.
  • the sgRNA targeting the exon part of the TRAC gene may comprise the nucleotide sequence shown in any one of SEQ ID NO:138 to SEQ ID NO:152.
  • the modification may include administering to the immune cells sgRNA targeting the exon portion of the HLA-A gene.
  • the sgRNA targeting the exon part of the HLA-A gene may comprise the nucleotide sequence shown in any one of SEQ ID NO:153 to SEQ ID NO:193.
  • the modification may also include administering Cas enzyme to the cells.
  • Cas enzymes may include Cas9 protein.
  • the modification may include applying antisense RNA to the cell, and the antisense RNA may comprise the nucleotide sequence shown in any one of SEQ ID NO:194 to SEQ ID NO:197.
  • the immune cells may include human cells.
  • the immune cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, white blood cells and/or or peripheral blood mononuclear cells.
  • the immune cells may include T cells.
  • the cells may be HLA-B homozygous cells.
  • the HLA-B homozygous may include HLA-B*40 homozygous, HLA-B*15 homozygous, HLA-B*46 homozygous, HLA-B*13 homozygous, HLA-B *51 homozygote, HLA-B*58 homozygote, HLA-B*07 homozygote, HLA-B*35 homozygote, HLA-B*44 homozygote, HLA-B*52 homozygote, HLA-B*57 Homozygous, HLA-B*54 homozygous, HLA-B*55 homozygous.
  • the cells may be HLA-A homozygous or heterozygous cells.
  • the HLA-A homozygote or heterozygote may include HLA-A*02 homozygote, HLA-A*11 homozygote, HLA-A*02/A*11 heterozygote or HLA-A* 24 homozygotes.
  • the method for preparing immune cells may include:
  • CD3 magnetic beads were added in proportion to collect CD3-T cells (cells not bound to magnetic beads).
  • the present application provides a pharmaceutical composition, which may comprise the aforementioned chimeric antigen receptor, the aforementioned isolated nucleic acid molecule, the aforementioned carrier, the aforementioned cell, and/or the aforementioned immune cell, and optionally A pharmaceutically acceptable carrier.
  • the pharmaceutical composition may include the aforementioned immune cells and optionally a pharmaceutically acceptable carrier.
  • the present application provides the aforementioned antigen chimeric receptor, the aforementioned isolated nucleic acid molecule, the aforementioned carrier, the aforementioned cell, the aforementioned immune cell, and/or the aforementioned pharmaceutical composition, which can be used to treat Expression-related diseases or conditions.
  • the diseases or disorders associated with the expression of EGFR may include diseases or disorders associated with up-regulation of the expression of EGFR.
  • the diseases or conditions associated with the expression of EGFR may include EGFR-positive tumors.
  • EGFR-positive tumors compared with normal cells, the protein expression of EGFR on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
  • the tumor may include glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, pancreatic cancer, eye cancer, mesothelioma, biliary tract cancer, prostate cancer, liver cancer, fibrosarcoma , colon and/or stomach cancer.
  • the tumor can include fibrosarcoma.
  • the present application provides the aforementioned chimeric antigen receptor, the aforementioned isolated nucleic acid molecule, the aforementioned vector, the aforementioned cell, the aforementioned immune cell, and/or the aforementioned pharmaceutical composition in the preparation of medicines,
  • the medicament can be used to treat diseases or conditions associated with the expression of EGFR.
  • the diseases or disorders associated with the expression of EGFR may include diseases or disorders associated with up-regulation of the expression of EGFR.
  • the diseases or disorders associated with the expression of EGFR may include diseases or disorders associated with up-regulation of the expression of EGFR.
  • the diseases or conditions associated with the expression of EGFR may include EGFR-positive tumors.
  • EGFR-positive tumors compared with normal cells, the protein expression of EGFR on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
  • the tumor may include glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, pancreatic cancer, eye cancer, mesothelioma, biliary tract cancer, prostate cancer, liver cancer, fibrosarcoma , colon and/or stomach cancer.
  • the tumor can include fibrosarcoma.
  • the present application provides a method for preventing or treating a disease or disorder related to the expression of EGFR, which may include administering to a subject in need an effective amount of the aforementioned chimeric antigen receptor, the aforementioned isolated nucleic acid molecule , the aforementioned carrier, the aforementioned cell, the aforementioned immune cell, and/or the aforementioned pharmaceutical composition.
  • the disease or disorder associated with expression of EGFR may include a disease or disorder associated with up-regulated expression of EGFR.
  • the diseases or disorders associated with the expression of EGFR may include diseases or disorders associated with up-regulation of the expression of EGFR.
  • the diseases or conditions associated with the expression of EGFR may include EGFR-positive tumors.
  • EGFR-positive tumors compared with normal cells, the protein expression of EGFR on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
  • the tumor may include glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, pancreatic cancer, eye cancer, mesothelioma, biliary tract cancer, prostate cancer, liver cancer, fibrosarcoma , colon and/or stomach cancer.
  • the tumor can include fibrosarcoma.
  • the anti-EGFR CAR structure includes: an EGFR antigen-binding region (derived from an anti-EGFR scFv whose amino acid sequence is shown in SEQ ID NO: 198), a CD8A extracellular hinge region, a CD8A transmembrane region, and a 4-1BB cell co-stimulatory domain and a CD3 ⁇ activation signaling domain.
  • the amino acid sequence of the non-antigen binding domain of EGFR CAR is shown in SEQ ID NO:19, and the nucleotide sequence is shown in SEQ ID NO:20.
  • the EGFR CAR lentiviral expression vector constructs the EGFR CAR lentiviral expression vector, and the schematic diagram of the vector (see Figure 1). Optimization: select the commercial lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP as the backbone, and carry out element transformation on the basis of this vector.
  • the ampicillin resistance gene ⁇ -lactamase of the vector was replaced with the aminoglycoside phosphotransferase derived from Tn5, so that the vector had kanamycin resistance.
  • the CMV promoter and its adjacent downstream multiple cloning sites which are potentially threatening for in vivo applications.
  • the copGFP gene expressed by the EF1 promoter in the original vector was deleted, the SalI restriction site was retained, and the SmaI restriction site was added at the 5' end of SalI for vector construction to form the final destination vector.
  • the added SmaI restriction site is a single restriction site for the final destination vector, and other sequence parts of the vector do not have this restriction site.
  • the non-limiting design principle of the sgRNA of the CRISPR/Cas9 system used in this example is: 5'-NNN(20)-NGG-3', NGG is called protospacer adjacent motif (PAM), wherein , N represents A, T, C or G.
  • PAM protospacer adjacent motif
  • sgRNAs Since many sgRNAs can be designed on the same exon, and sgRNAs consisting of 20 nucleotide sequences may appear repeatedly in the genome, use the website http://crispr.cos.uni-heidelberg.de to Carry out the design and evaluation of sgRNA, paste the exon sequence to this website, the website designs sgRNA and conducts prediction evaluation, the higher the score in the evaluation, it means that there may be higher editing efficiency and lower off-target risk, from which Select the sgRNA with higher score to test.
  • the sgRNA targeting the TRAC gene is shown in SEQ ID NO:138 to SEQ ID NO:152
  • the sgRNA targeting the HLA-A02 gene is shown in SEQ ID NO:153 to SEQ ID NO:174
  • the targeting HLA-A11 gene The sgRNA of the gene is shown in SEQ ID NO:175 to SEQ ID NO:185
  • the sgRNA targeting the HLA-A24 gene is shown in SEQ ID NO:186 to SEQ ID NO:193, which were synthesized by GenScript Biotechnology Company.
  • HLA-B typing of the recipient select HLA-B homozygotes that match the HLA-B typing of the recipient.
  • the source of the donor is based on the HLA-B homozygote in the population.
  • One of the alleles of the patient's HLA-B is consistent with the donor's HLA-B homozygote.
  • Cells from these donors can cover a high number of patient populations . Reduce the rejection caused by the inconsistency of HLA-B subtypes.
  • HLA-B mainly selects B*40 homozygote, B*15 homozygote, B*46 homozygote, B*13 homozygote, B*51 homozygote, B*58 homozygote, B*07 homozygote with high frequency in the population Homozygote, B*35 homozygote, B*44 homozygote, B*52 homozygote, B*57 homozygote, B*54 homozygote, B*55 homozygote.
  • HLA-A selects A*02 homozygotes, A*11 homozygotes and A*02/A11 heterozygotes with higher frequencies in the population.
  • Peripheral blood was collected from healthy donors and diluted 1:1 with PBS buffer.
  • cell separation solution Ficoll
  • blood cell dilution solution very slowly along the tube wall, and centrifuge at 800g for 20min at room temperature (set the centrifuge to increase speed 1, reduce speed 0).
  • the liquid in the centrifuge tube is divided into PBS and serum layer, white blood cell layer, lymphocyte separation solution, and red blood cell layer from top to bottom.
  • remove the PBS and serum layer move the white blood cell layer to a new 50ml centrifuge tube, add PBS to 40ml to wash the cells, and centrifuge at 450g for 10min. After centrifugation, the supernatant was discarded to obtain peripheral blood mononuclear cells. Cell counts were performed after the cells were resuspended.
  • T cells in peripheral blood mononuclear cells were extracted using EasySep TM Human T Cell Separation Kit (purchased from StemCell Technologies, catalog number: 17951). Adjust the PBMC density to 5 ⁇ 10 7 cells/ml, and add PBS buffer in the range of 0.25-2ml; first add cocktail to mix well, then add isolation cocktail at 50 ⁇ l/ml, mix well and place at room temperature for 5 minutes; shake RapidSpheres by vortex After vortexing for 30s, add 40 ⁇ l/ml to the cells and mix well; add buffer to the multiple of 2.5ml, and gently blow up and down 2-3 times; add 2.5ml to each tube respectively, and put Place the cryovial on the magnetic stand, and let it stand at room temperature for 3 minutes; gently open the cap of the cryovial, carefully hold the two sides to pick up the magnetic stand, keep it upside down for 2-3 seconds, and pour the cell solution into a new centrifuge tube at one time; use 10 - After resuspending the cells in 20ml buffer (dependured
  • CD3+T cells were obtained according to the method of Example 2 (D0 day), and activated with CD3/CD28 antibody magnetic beads, and transfected with a lentiviral vector (the EGFR CAR lentiviral expression vector prepared in Example 1) on D1 day after activation, On D2, the lentiviral vector was washed away, and on D3, electroporation was performed.
  • a lentiviral vector the EGFR CAR lentiviral expression vector prepared in Example 1
  • TRAC sgRNA AGAGTCTCTCAGCTGGTACA (SEQ ID NO: 138), A02 sgRNA: CTGACCATGAAGCCACCCCTG (SEQ ID NO: 155), A11 sgRNA: GGCCCCTCCTGCTCTATCCA (SEQ ID NO: 185).
  • Sorting CD3-negative T cells centrifuging after counting cells, discarding supernatant; resuspending cells with buffer and mixing, adding CD3 magnetic beads to 20ul CD3 magnetic beads/ 107 cells, mixing evenly, and incubating in a 4-degree refrigerator.
  • Cell harvesting Collect cells in a centrifuge tube and discard after centrifugation, wash the cells again with normal saline, centrifuge, prepare a cryopreservation solution, resuspend the centrifuged cells in the cryopreservation solution, and draw the cell suspension into the final product with a cell freezer In the storage bag, label the cell cryopreservation bag for the next step of freezing.
  • Knockout efficiency (AB)/A ⁇ 100%; A is the positive expression rate of the control group; B is the positive expression rate of the knockout group.
  • EGFR target cells HT1080-Luciferase-GFP; adjust the state of the target cells to the logarithmic growth phase, and need to be continuously passaged twice before the experiment;
  • the target cells were resuspended in 1640+10% FBS, and three 24-well plates were taken for each target site, and the target cells were inoculated according to the amount of 2x10 ⁇ 5/well. (Both target cells and effector cells were seeded at a density of 2x10 ⁇ 6/ml). Then add effector cells according to the E/T (effect-to-target ratio, effector cells: target cells) ratio. Replenish each well to the maximum volume (such as 600ul). The control was inoculated with the same number of target cells without adding effector cells (600ul). The well plate was placed in a 5% CO 2 , 37° C. incubator and incubated for 24 hours. E/T: 1:2, 1:1, 2:1, 5:1, 10:1 plank, repeat three times.
  • anit-EGFR UCAR-T has a significant killing effect on HT1080-Luciferase-GFP cells, and the killing efficiency can reach more than 90% when the effect-to-target ratio is 1:2 (see Figure 4).
  • Anti-EGFR UCAR-T cells co-cultured with target cells to detect cytokine secretion
  • Example 5 anti-tumor effect of anti-EGFR UCAR-T cells in vivo
  • mice 8-10-week-old NSG mice were subcutaneously injected with tumor cell HT1080-Luciferase-GFP (5x10 ⁇ 6). The mice were divided into three groups with 5 mice in each group. The tumor formation time was generally 2-4 weeks. Anti-EGFR UCAR-T cells, anti-EGFR CAR-T cells, and non-gene-knockout T cells 5E6 were injected intratumorally into each group of mice, with a single point injection and an injection volume of 50ul. Tumor regression in mice was monitored by luciferase (see Figure 5).
  • mice Prepare 15 humanized immune system mice (hHSC-NCG). Divide into 3 groups. Preparation of cells, experimental group anti-EGFR UCAR-T cells (knockout TRAC+HLA-A02); control group 1: anti-EGFR CAR-T; control group 2: anti-EGFR UCAR-T cells (knockout TRAC+B2M ); each mouse was injected with 1x10 ⁇ 7 cells, and blood was collected at different time points D0, 2h, D3, D7, D14, D21, D28, D35, D42, D49, D56, D60. Genomes were extracted from blood samples at different time points, and the copy/ng genome DNA was calculated by QPCR absolute quantification method. The positive control used UCAR-T cells harvested on the 14th day, and the negative control used DEPC water.
  • GVHD response Prepare TRAC, HLA-A double-knockout T cells, T cells without gene knockout, irradiate allogeneic PBMC, stimulate the two groups of cells prepared respectively, and detect the level of IFN-r.
  • TRAC HLA-A double knockout T cell group IFN- ⁇ secretion level is very low, indicating that TRAC knockout reduces the GVHD response.
  • Allogeneic reaction Allogeneic PBMC stimulated the cells of the two groups after irradiation, and detected the level of IFN-r.
  • GVHD response prepare TRAC, HLA-A double knockout T cells, and T cells without gene knockout. 8-10 weeks NSG mice were injected with 1x10 ⁇ 7 respectively, and the graft-versus-host reaction was observed through clinical indicators: survival rate, fur texture and skin integrity, etc. Cytokine detection: Peripheral blood serum was collected to detect the levels of cytokines such as IL6, IL-2, TNF- ⁇ , IFN- ⁇ , etc. Blood collection time: before reinfusion, 24h, D3, D7, D14, D28, 2M. Detection of organ lesions: At the end of the observation period (about 2 months), the spleen, liver, skin, gastrointestinal tract, lung, and kidney of the mice were taken for HE section staining analysis.
  • mice injected with untreated T cells developed lethal graft-versus-host disease (GVHD) within 2 months of injection. None of the mice receiving TRAC, HLA-A double knockout cells developed GVHD; the TRAC, HLA-A double knockout T cell group secreted very low levels of cytokines IL6, IL-2, TNF- ⁇ , IFN- ⁇ ; and The morphology of different organs of the mice was normal. It shows that TRAC, HLA-A double-knockout T cell group greatly reduces the GVHD response.
  • GVHD lethal graft-versus-host disease
  • Allogeneic reaction preparation of TRAC, HLA-A double knockout CAR-T cells, and co-injection of 1x10 ⁇ 7 TCR-HLA-A-double knockout CAR-T cells and 2x10 ⁇ 6 allogeneic T cells into NSG mice in vivo.
  • Control group Inject 1x10 ⁇ 7 TCR-CAR-T cells into NSG mice.
  • Cytokine detection Peripheral blood serum was collected to detect the levels of cytokines such as IL6, IL-2, TNF- ⁇ , IFN- ⁇ , etc. Time points: D1, D5, D7, D10, D14.
  • Control group transfer to CAS9+ODN label
  • On-target and off-target-WGS (Whole genome sequencing): 1 ⁇ 10 ⁇ 6 T cells without gene knockout, TRAC, and HLA-A double-knockout T cells were taken respectively and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for detection .
  • Result analysis double-knockout T cells (TRAC+HLA-A) detected whether chromosomal translocation occurred on D14 (harvest).
  • the detection results the detection values of both translocation methods were close to zero detection value, indicating that no rearrangement of the loci occurred ( See Figure 10).
  • Residual Cas9 protein During cell preparation, 1 ⁇ 10 ⁇ 6 cells at each of the three time points before knockout, after knockout, and before harvest were lysed, and then protein quantification kit (NOVATEINBIO, Cat. No. NB-E1372PR ) for quantification, the samples in each group were adjusted to the same loading volume of 2 ⁇ g, and the CRISPR/Cas9 protein ELISA kit was used for detection according to the instructions.
  • the Cas9 protein in the sample is firmly and stably placed on the test paper well. Then use the detection antibody to recognize the bound Cas9 protein, and then develop with the developer.
  • the Cas9 ratio is proportional to the absorbance, and the absolute amount of Cas9 protein is quantified by comparing with the Cas9 control.
  • Double knockout T cells detected the residue of spCas9 at four time points before electroporation (D3), before electroporation (D5), D9, and D14 (harvest). Trace residues were detected before solution (D5), but not detected at the other three time points. (See Figure 12).
  • the RNP complex was transferred into the activated T cells prepared in Example 2 by electroporation using an electroporation kit (purchased from LONZA, product number V4XXP-3024). Preheat the medium (X-VIVO15 medium + 10% FBS + IL2 (200 U/ml) + IL7 (10 ng/ml) + IL15 (5 ng/ml)) in the well plate 30 minutes in advance.
  • the sgRNA sequence of TRAC is sg9 (as shown in SEQ ID NO: 138)
  • the sgRNA sequence of HLA-A is HLA-A02 Sg2 (as shown in SEQ ID NO: 154) or HLA-A02Sg5 (as shown in SEQ ID NO: 154) ID NO: 155) or HLA-A11 sg21 (as shown in SEQ ID NO: 185) or HLA-A11Rsg2 (as shown in SEQ ID NO: 184)
  • Example 2 Count the activated T cells cultured in Example 2, centrifuge at 300g for 8min, discard the supernatant, add PBS to resuspend the cells, absorb 1E7 cells and centrifuge again at 300g for 8min, discard the supernatant, and resuspend the cells with 100 ⁇ l of prepared electroporation buffer .
  • Add the preheated medium into the electro-cup then transfer the cells into the preheated medium in the well plate with a matching pipette, and then place them in a 37°C, 5% CO 2 incubator for culture.
  • Knockout efficiency (AB)/A ⁇ 100%; A is the positive expression rate of the control group; B is the positive expression rate of the knockout group.
  • the three detection results of TRAC single gene knockout are shown in Figure 13 to Figure 15, and the knockout efficiency calculation results are shown in Table 1.
  • the three detection methods are basically the same, and only the Sanger sequencing method was used to detect the editing efficiency in subsequent experiments.
  • the RNP complex was transferred into the activated T cells prepared in Example 2 by electroporation using an electroporation kit (purchased from LONZA, product number: V4XXP-3024). Preheat the medium (X-VIVO15 medium + 10% FBS + IL2 (200 U/ml) + IL7 (10 ng/ml) + IL15 (5 ng/ml)) in the well plate 30 minutes in advance.
  • TRAC Sg9 20 ⁇ g TRAC sgRNA (TRAC Sg9), 20 ⁇ g HLA-A sgRNA (HLA-A02 Sg2 or HLA-A02 Sg5 or HLA-A11 sg21 or targeting HLA-A*24:02:01, HLA-A A*30:01:01:01, HLA-A*33:01:01:01, HLA-A*03:01:01:01, HLA-A*01:01:01:01, or HLA-A* 26:01:01:01:01 sgRNA) into PCR tubes (no RNA), and then add 10 ⁇ g Cas9 protein (purchased from thermo, product number A36499) respectively, mix gently, and incubate at room temperature for 12 minutes.
  • Cas9 protein purchased from thermo, product number A36499
  • Example 2 Count the activated T cells cultured in Example 2, centrifuge at 300g for 8min, discard the supernatant, add PBS to resuspend the cells, absorb 1E7 cells and centrifuge again at 300g for 8min, discard the supernatant, and resuspend the cells with 100 ⁇ l of prepared electroporation buffer .
  • Add the preheated medium into the electro-cup then transfer the cells into the preheated medium in the well plate with a matching pipette, and then place them in a 37°C, 5% CO 2 incubator for culture.
  • the double-gene knockout efficiency is detected by sequencing, and TRAC-negative and HLA-A-negative T cells with a double-gene knockout efficiency of not less than 80% can be obtained.
  • the results are shown in Figure 20-21.
  • Figure 20A shows the results of using HLA-A02 Sg5 to knock out HLA-A02
  • the upper line shows the results of the control group (that is, the results of knocking out HLA-A02 Sg5 are not used)
  • the next line shows the simultaneous knockout of HLA -The results of A02 and TRAC
  • Figure 20B shows the results of knocking out TRAC using TRAC Sg9, where the upper line shows the results of the control group (that is, no knocking out with TRAC Sg9); the next line shows simultaneous knockout HLA-A02 and TRAC results.
  • Figure 21A-21B shows the knockout situation of knockout HLA-A02 and TRAC protein level, wherein NEG refers to the negative control, WT refers to the situation without any knockout treatment, TRAC+HLA-A double knockout refers to the simultaneous knockout of HLA- Results of A02 and TRAC.
  • Example 13 Differences in the expression of TRAC gene, HLA-A gene, B2M gene and CIITA gene in T cells with double gene knockout and corresponding genes in corresponding cells
  • Example 2 The activated T cells prepared in Example 2 were divided into two groups, one group was used as a control, and the other group prepared TRAC gene and HLA-A gene double gene knockout T cells according to the method in Example 5, according to Sanger sequencing was performed in the manner of step (1) of Example 4. According to the sequencing results, the TRAC and HLA-A double gene knockout cells were obtained.
  • the prepared double gene knockout T cells are incubated with corresponding TRAC and HLA-A antibodies, and the double gene knockout cell lines can be obtained by flow sorting or magnetic bead sorting.
  • RNA extraction kit purchased from QIAGEN, catalog number: 74004
  • reverse transcription kit purchased from Applied Biosystems, catalog number: 4368814
  • FIG. 22-23 shows the results.
  • Fig. 22 shows the mRNA level determination of gene expression, and wherein Fig. 22 shows the mRNA level of TRAC, HLA-A, B2M and CIITA;
  • WT refers to the situation without any knockout treatment
  • double knockout group refers to TRAC gene and HLA-A gene double knockout T cells results.
  • Figure 23 shows the protein level determination of gene expression, wherein Figure 23A-23B shows the protein expression levels of B2M and CIITA respectively; wherein NEG refers to the negative control, WT refers to the situation without any knockout treatment, TRAC+HLA-A Double knockout refers to the result of T cells with double gene knockout of TRAC gene and HLA-A gene.
  • Example 14 Preparation of T cells knocked out of the TRAC gene, HLA-A/B2M gene and CIITA gene and verification of the expression changes of the corresponding three genes
  • TRAC, HLA-A and CIITA genes in TRAC, HLA-A and CIITA gene knockout T cells were down-regulated; compared with control cells, TRAC, B2M and CIITA triple gene knockout The protein expressions of TRAC, HLA-A and CIITA genes were down-regulated in T cells.
  • FIGS 24A-24D show the knockout status of TRAC, HLA-A and B2M protein levels in sequence.
  • WT refers to the situation without any knockout treatment
  • TRAC+HLA-A double knockout refers to the result of T cells with double gene knockout of TRAC gene and HLA-A gene
  • TRAC+HLA-A+CIITA triple knockout refers to TRAC, The result of the T cells of HLA-A and CIITA triple gene knockout
  • TRAC+B2M+CIITA triple knockout refers to the result of B2M, CIITA and TRAC triple gene knockout of T cells
  • TRAC+HLA-A knockdown refers to Example 16 Results of preparation of TRAC and HLA-A knockdown T cells.
  • Figure 24D shows the knockdown of CIITA protein levels.
  • Embodiment 15 designs antisense RNA sequence
  • RNA sequences of the corresponding genes (TRAC gene and HLA-A gene) through the database https://www.ncbi.nlm.nih.gov/ or www.ensembl.org/, and design siRNA with reference to the following principles:
  • the antisense RNA sequence that design obtains comprises HLA-A-homo-551 (it comprises the nucleotide sequence shown in SEQ ID NO:194); HLA-A-homo-NEG (it comprises the nucleotide sequence shown in SEQ ID NO:195) nucleotide sequence); TRAC-homo-375 (which comprises the nucleotide sequence shown in SEQ ID NO: 196); TRAC-homo-NEG (which comprises the nucleotide sequence shown in SEQ ID NO: 197).
  • Double gene knockdown was performed using the antisense RNA designed by Example 15.
  • the company prepares lentivirus (Gimma) with antisense RNA sequences of TRAC gene and HLA-A gene.
  • CD3 + T cells were prepared according to the method in Example 2 (D0 days), and activated with CD3/CD28 antibody magnetic beads, and the activated T cells were transfected with lentiviruses carrying the antisense RNA sequences of the TRAC gene and the HLA-A gene (D1 day), wash off the lentiviral vector on D2, and continue to culture until D5.
  • the T cells cultured to D5 days were collected, and the gene knockdown efficiency was detected by quantitative PCR or Western Blot and other methods.
  • T cells with TRAC gene and HLA-A gene knockdown can be obtained by flow sorting or magnetic bead sorting.
  • the results showed that the mRNA and protein expression levels of TRAC and HLA-A were down-regulated in the TRAC and HLA-A gene knockdown group.
  • Figures 25A-25B sequentially show the knockout status of TRAC and HLA-A mRNA levels.
  • WT refers to the situation without any knockout treatment
  • TRAC+HLA-A double knockout refers to the result of T cells with double gene knockout of TRAC gene and HLA-A gene.
  • the knockout levels of TRAC and HLA-A protein levels can be referred to the results in FIG. 24 .
  • T cells without gene knockout, double gene knockout, three gene knockouts and double gene knockdown in Examples 2, 12, 14 and 16 compare the cell counts of several T cell activities in each group and take them respectively 1*10 6 cells were inoculated in a 24-well plate, and PHA (0.3 ⁇ g/ml) (ionomycin +) or 5 ng/ml PMA and 50 ng/ml ionomycin were added to the cells in each well, and after continuing to culture for 5 hours, use CD69 (early activation) (purchased from BD Biosciences, catalog number: FN50) and CD137 (late stage) (purchased from BD Biosciences, catalog number: 4B4-1) antibodies were used to detect the activation status of cells by flow cytometry. The results showed that the activity of T cells with double gene knockout and double gene knockout was better than that of triple gene knockout T cells.
  • TRAC+HLA-A double knockout refers to the result of T cells with double gene knockout of TRAC gene and HLA-A gene
  • TRAC+HLA-A+CIITA triple knockout refers to TRAC
  • the result of the T cells of HLA-A and CIITA triple gene knockout wherein TRAC+B2M+CIITA triple knockout refers to the result of B2M, CIITA and TRAC triple gene knockout of T cells
  • TRAC+HLA-A knockdown refers to Example 16 Results of preparation of TRAC and HLA-A knockdown T cells.
  • T cells without gene knockout, double gene knockout, three gene knockouts and double gene knockdown in Examples 2, 12, 14 and 16 were labeled with CFSE (invitrogen, C34554), and the cell counts were taken as 1 *10 6 cells were co-cultured with NK cells (NK92MI) at a ratio of 1:1. After 24 hours, the co-cultured cells were collected, and the ratio of CFSE-positive cells in the mixed cells was detected by flow cytometry.
  • NK+T refers to the situation in which NK cells are co-cultured with T cells without any knockout treatment
  • NK+TRAC+HLA-A knockdown refers to the combination of NK cells with the TRAC gene and HLA-A gene prepared in Example 16
  • the results of the knockdown T cells co-culture NK+TRAC+HLA-A double knockout refers to the co-culture of NK cells and T cells with TRAC gene and HLA-A gene double gene knockout
  • NK+TRAC+HLA -A+CIITA triple knockout refers to the situation where NK cells are co-cultured with TRAC, HLA-A and CIITA triple knockout T cells
  • NK+TRAC+B2M+CIITA triple knockout refers to the situation where NK cells are combined with B2M
  • No gene knockout, double gene knockout, three gene knockout and double gene knockdown T cells in Examples 2, 12, 14 and 16 were prepared from peripheral blood from donor 1.
  • CD3 + T cells were prepared from peripheral blood from donor 2.
  • Each group of cells prepared from the peripheral blood of donor 1 was mixed with the CD3 + T cells prepared from the peripheral blood of donor 2 in equal proportions, and the expression level of IFN- ⁇ in the cell mixed system was detected 24 hours later. The results showed that the expression level of IFN- ⁇ in the double gene knockout T cell group was lower than that in the triple gene knockout T cell group.
  • TRAC+HLA-A double knockout refers to the result of T cells with double gene knockout of TRAC gene and HLA-A gene
  • TRAC+HLA-A+CIITA Triple knockout refers to the result of T cells knocked out by three genes of TRAC, HLA-A and CIITA
  • TRAC+B2M+CIITA triple knockout refers to the result of T cells knocked out by three genes of B2M, CIITA and TRAC
  • TRAC+HLA-A knockout Low refers to the result of TRAC gene and HLA-A gene knockdown T cells prepared in Example 16.
  • Example 20 Preparation of CAR-T cells with double gene knockout of TRAC gene and HLA-A gene, CAR-T cells with triple gene knockout of TRAC gene, HLA-A gene and CIITA gene, and knockout of TRAC gene, B2M gene and CIITA gene Removed CAR-T cells
  • TRAC gene and HLA-A gene double gene knockout cells according to the methods in Example 12 and Example 14 respectively, TRAC gene, HLA-A gene and CIITA gene as well as TRAC gene, B2M gene and CIITA gene knockout CAR-T cells.
  • the double-gene knockout and triple-gene knockout CAR-T cells can be obtained through flow cytometry detection, and the yield of double-gene knockout CAR-T cells is higher than that of triple-gene knockout CAR-T cells.
  • FIGS 29A-29D show the knockout status of TRAC, HLA-A and B2M protein levels in sequence.
  • Figure 29D shows the knockdown of CIITA protein levels.
  • WT refers to the situation without any knockout treatment
  • TRAC+HLA-A double knockout refers to the result of CAR-T cells with double gene knockout of TRAC gene and HLA-A gene
  • TRAC+HLA-A+CIITA triple knockout refers to The results of CAR-T cells with triple gene knockout of TRAC, HLA-A and CIITA
  • TRAC+B2M+CIITA triple knockout refers to the results of CAR-T cells with triple gene knockout of B2M, CIITA and TRAC.
  • the transfection efficiency of CD19CAR is shown in Figure 30A-30B.
  • CAR30%+ represents the transfection efficiency of CD19 CAR.
  • Figure 31 shows the expansion factor of different cells. Among them, CAR-T cells with double gene knockout of TRAC gene and HLA-A gene had the highest amplification factor.
  • Figure 32 shows the killing effect of Raji-Luciferase on CD19 target cells, among which the killing effect of CAR-T cells with double knockout of TRAC gene and HLA-A gene is the most significant.
  • each E/T ratio is the result corresponding to the legend of A-D from left to right.
  • NSG mice were injected with tumor cells intravenously. After the tumor was successfully established, CAR-T cells with double gene knockout of TRAC gene and HLA-A gene, CAR-T cells with triple gene knockout, or CAR-T cells without gene knockout were reinfused into the mice. T cells, monitoring tumor volume in mice.
  • the tumor growth rate was significantly slowed down in mice transfused with double-gene knockout CAR-T cells.
  • Figure 33 shows the administration method to mice, i.v. means intravenous injection, and CAR-T cells represent double-gene knockout CAR-T cells expressing CD19 CAR and triple-gene knockout CAR-T cells.
  • Figure 32 shows the volume of tumors in mice after administration of CAR-T cells. Among them, Figure 32 from left to right shows the three genes of CD19 CAR-T cells, TRAC, HLA-A and CIITA respectively administered with normal saline, unmodified T cells, TRAC gene and HLA-A gene knockout. Tumor volume in mice after knockout CD19 CAR-T cells, B2M, CIITA and TRAC knockout CD19 CAR-T cells. The results showed that the growth rate of tumors was significantly slowed down in mice transfused with CAR-T cells with double gene knockout of TRAC gene and HLA-A gene.
  • the present application prepares a chimeric antigen receptor targeting EGFR.
  • the antigen-binding domain (targeting part) of the recombinant receptor is derived from scFv, which has the characteristics of stable structure.
  • the present application provides a lentiviral expression vector.
  • the ampicillin resistance gene ⁇ -lactamase of the vector was replaced with aminoglycoside phosphotransferase derived from Tn5, so that the vector had kanamycin resistance;
  • the potentially threatening CMV promoter and its adjacent downstream multiple cloning sites in in vivo applications; delete the copGFP gene expressed by the EF1 promoter in the original vector, retain the SalI restriction site, and add it at the 5' end of SalI
  • the SmaI restriction site is used for vector construction to form the final destination vector.
  • the added SmaI restriction site is a single restriction site for the final destination vector, and other sequence parts of the vector do not have this restriction site.
  • This application optimizes the protein-RNA complex electrotransfection technology. A double gene knockout efficiency of more than 90% in primary T cells was obtained.
  • the source of donors for this application is based on the high frequency of HLA-B homozygotes in the population.
  • One allele of the patient’s HLA-B is consistent with the donor’s homozygosity.
  • Cells from these donors can cover a high number of patients population, and can reduce the rejection caused by HLA-B.
  • This application screened out the HLA-A molecules that are highly related to rejection and knocked them out, while retaining other HLA-I molecules, which not only reduced the rejection of allogeneic cells, but also avoided the complete knockout of HLA molecules being NK
  • the occurrence of cell clearance greatly prolongs the half-life of allogeneic CAR-T cells in vivo.
  • This application is the first to construct high-efficiency double-knockout TCR, HLA-A EGFR-UCAR-T cells, to achieve a safe shelf-type ready-to-use therapeutic agent, improve anti-tumor effect, and be used for breast cancer and other diseases the treatment.

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Abstract

一种靶向EGFR的通用型CAR-T细胞及其制备方法，所述通用型CAR-T细胞包含嵌合抗原受体，所述嵌合抗原受体包含靶向部分，所述靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:8所示的氨基酸序列。所述通用型CAR-T细胞在识别肿瘤抗原的同时，还能降低异体CAR-T治疗引起的免疫排斥反应，延长细胞存活时间，提高抗肿瘤效果。

Description

靶向EGFR的通用型CAR-T细胞及其制备方法 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向EGFR的通用型CAR-T细胞及其制备方法和应用。
背景技术
EGFR属于受体酪氨酸激酶的ErbB家族，在上皮细胞生理机理能中发挥关键作用。通过配体诱导EGFR的同源二聚化或异源二聚化将导致其酪氨酸激酶结构域的自体磷酸化，这种磷酸化会招募一系列下游信号通路，如PI3K/AKT和Ras/Faf/MEK/ERK。EGFR经常在不同类型的癌症中发生突变或过表达，并且是多种癌症疗法临床实践中使用的靶标。在病理状态下，主要是在肺癌、乳腺癌和胶质母细胞瘤中，EGFR是肿瘤发生的驱动因素。EGFR在癌症中的非正常活化主要是由于基因组基因座处的扩增和点突变引起的，但是也发现转录上调或由于自分泌/旁分泌机制导致的配体过量产生。此外，由于发现在药物压力下产生EGFR的扩增或继发突变，EGFR被越来越多地视为肿瘤耐药性的生物标记。EGFR在63％的原发性和10％的继发性胶质母细胞瘤中过表达。EGFR在发育过程中影响神经干细胞的迁移，并通过激活MAPK和PI3K/Akt信号通路促进细胞增殖。
随着肿瘤免疫理论的发展与技术的进步，细胞免疫治疗肿瘤吸引越来越多的关注。CAR-T细胞技术是一种基于细胞的治疗手段，已经在肿瘤免疫治疗尤其是血液肿瘤的治疗中产生极佳的效果。CAR-T免疫治疗使用基因改造的T细胞，可特异性地识别并杀死表达特定抗原的肿瘤细胞，而不受MHC限制性影响。CAR-T免疫疗法在各种B细胞恶性肿瘤的治疗中取得良好效果，如靶向CD19的CAR-T细胞治疗急性淋巴白血病(ALL)，慢性淋巴白血病(CLL)和非霍奇金淋巴瘤(NHL)。同时，CAR-T细胞对胶质瘤的临床应用正在进行中，并显示出令人鼓舞的结果。然而，病人自体T细胞在体外扩增困难或功能降低，导致制备的CAR-T细胞产品数量不足或质量差。通用型CAR T细胞是从健康供体分离获得T细胞，制备的CAR-T细胞不但扩增效率高、活力强，而且感染阳性率也有提高，但是通用型CAR-T也面临着移植物抗宿主疾病(GVHD)和免疫排斥的问题。
因此，亟需靶向EGFR的通用型CAR-T细胞，在识别肿瘤细胞表面抗原的同时，还能降低异体CAR-T治疗引起的免疫排斥反应，延长细胞存活时间，提高抗肿瘤效果。
发明内容
本发明的目的在于制备一种靶向EGFR的通用型CAR-T细胞，识别肿瘤细胞表面抗原的同时，敲除细胞表达的TCR和HLA-A基因，从而降低异体CAR-T治疗引起的免疫排斥反应，延长细胞存活时间，提高抗肿瘤效果。
本申请的目的之一在于提供一种靶向EGFR的嵌合抗原受体。具体如下：以抗EGFR scFv为靶向部分，通过CD8分子铰链区和跨膜区与胞内信号转导结构域相连接，胞内信号转导结构域由4-1BB共刺激信号与CD3ζ胞内信号域组成。
本申请的另一个目的在于提供一种安全性更高的UCAR-T细胞。首先供者来源基于人群中的HLA-B纯合子，患者HLA-B其中一个等位基因和供者纯合子一致即可，来源于这些供者的细胞能覆盖高比例的患者人群。降低HLA-B引起的排异反应。HLA-B主要选择人群中频率较高的B*40纯合子，B*15纯合子，B*46纯合子，B*13纯合子，B*51纯合子，B*58纯合子，B*07纯合子，B*35纯合子，B*44纯合子，B*52纯合子，B*57纯合子，B*54纯合子，B*55纯合子。敲除策略针对TRAC和排异高度相关的HLA-A分子进行敲除，而保留了其他HLA-I类分子，既减少了异体细胞的排异，也避免了HLA分子完全敲除被NK细胞清除的发生，大大延长了同种异体CAR-T细胞在体内的半衰期。同时降低同种异体细胞治疗引起的GVHD反应。TCR基因选择编码TCRα链的基因TRAC，HLA-A选择人群中频率较高的A*02纯合子，A*11纯合子，A*02/A11杂合子及A*24纯合子。
本申请的另一个目的在于提供一种高效率敲除TCR，HLA-A的基因编辑方法。CRISPR/Cas9基因编辑的转染方法主要有质粒法，mRNA法以及RNP法。RNP法指体外将spcas9核糖核蛋白(RNP)和sgRNA组装成RNP复合物，通过电转将复合物导入细胞中，和其他两种方法比，具有spCas9蛋白降解快，脱靶风险低的优势，并且很大程度上提高了电转效率，从而提高目的基因的编辑效率。采用优化后RNP方法双敲TRAC和HLA-A基因，双敲效率可达90％以上。
本申请的另一个目的在于提供一种靶向EGFR通用型CAR-T细胞的制备方法。
在某些实施方式中，所述嵌合抗原受体包含靶向部分，所述靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含HCDR2，所述HCDR2包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含HCDR1，所述HCDR1包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含SEQ ID NO:1所示的重链可变区中的HCDR1、HCDR2和HCDR3。
在某些实施方式中，所述靶向部分包含HCDR1、HCDR2、HCDR3，所述HCDR3包含SEQ ID NO:8所示的氨基酸序列；所述HCDR2包含SEQ ID NO:6所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2包含SEQ ID NO:5所示的氨基酸序列；所述H-FR3包含SEQ ID NO:7所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含VH，所述VH包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包含靶向部分，所述靶向部分包含LCDR3，所述LCDR3包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含LCDR2，所述LCDR2包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含LCDR1，所述LCDR1包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含SEQ ID NO:10所示的轻链可变区中的LCDR1、LCDR2和LCDR3。
在某些实施方式中，所述靶向部分包含LCDR1、LCDR2、LCDR3，所述LCDR3包含SEQ ID NO:17所示的氨基酸序列；所述LCDR2包含SEQ ID NO:15所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接地相连，且所述L-FR1包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端直接或间接地相连，且所述L-FR4包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:12所示的氨基酸序列；所述L-FR2包含SEQ ID NO:14所示的氨基酸序列；所述L-FR3包含SEQ ID NO:16所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述靶向部分包含VL，所述VL包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述靶向部分包括抗体或抗原结合片段。
在某些实施方式中，所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb。
在某些实施方式中，所述靶向部分包括scFv。
在某些实施方式中，所述scFv靶向EGFR。
在某些实施方式中，所述靶向部分包含SEQ ID NO:198所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体包括跨膜域，所述跨膜域包含源自选自下述蛋白的跨膜域或其组合：CD8A、CD8B、CD28、CD3e、CD3ε、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、CD154、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64和SLAM。
在某些实施方式中，所述的嵌合抗原受体中所述跨膜域包含源自CD8A或CD8B的跨膜域。
在某些实施方式中，所述的嵌合抗原受体中所述跨膜域包含SEQ ID NO:21至SEQ ID NO:69中任一项所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体包括共刺激信号传导结构域，所述共刺激信号传导结构域包含源自选自下述蛋白的共刺激信号传导结构域或其组合：CD28、4-1BB、CD27、CD2、CD7、CD8A、CD8B、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD40和MyD88。
在某些实施方式中，所述的嵌合抗原受体中所述共刺激信号传导结构域包括源自4-1BB的共刺激信号传导结构域。
在某些实施方式中，所述的嵌合抗原受体中所述共刺激信号传导结构域包含SEQ ID NO:70至SEQ ID NO:102中任一项所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体中所述共刺激信号传导结构域的N端与所述跨膜域的C端直接或间接地连接。
在某些实施方式中，所述的嵌合抗原受体包括胞内信号转导结构域，所述胞内信号转导结构域包含源自选自下述蛋白的胞内信号转导结构域或其组合：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述的嵌合抗原受体中所述胞内信号转导结构域包含源自CD3ζ、CD3δ、CD3γ或CD3ε的胞内信号转导结构域。
在某些实施方式中，所述的嵌合抗原受体中所述胞内信号转导结构域包含SEQ ID NO:86、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:103至SEQ ID NO:113中任一项所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体中所述胞内信号转导结构域的N端与所述共刺激信号传导结构域的C端直接或间接地连接。
在某些实施方式中，所述的嵌合抗原受体在靶向部分和跨膜域之间包括铰链区，所述铰链区包含源自选自下述蛋白的铰链区或其组合：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、TIM1、SLAM、CD30和LIGHT。
在某些实施方式中，所述铰链区包含源自CD8的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:114至SEQ ID NO:135中任一项所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包含非靶向部分，所述嵌合抗原受体的非靶向部分包含CD8A的跨膜域、CD8的铰链区、4-1BB的胞内共刺激信号传导结构域和CD3ζ胞内信号转导结构域。
在某些实施方式中，所述的非靶向部分包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体包含SEQ ID NO:199所示的氨基酸序列。
另一方面，本申请提供了一种或多种分离的核酸分子，其编码所述的嵌合抗原受体。
另一方面，本申请提供了一种或多种载体，其包含所述的分离的核酸分子。
在某些实施方式中，所述的载体包括病毒载体。
在某些实施方式中，所述的载体包括慢病毒载体。
另一方面，本申请提供了一种或多种免疫细胞，其包含和/或表达所述的分离的核酸分子或所述的载体，和/或所述的嵌合抗原受体。
在某些实施方式中，所述的免疫细胞来源于人。
在某些实施方式中，所述的免疫细胞包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、γδT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在某些实施方式中，所述的免疫细胞包括T细胞。
在某些实施方式中，所述的免疫细胞包括经修饰的免疫细胞。
在某些实施方式中，所述经修饰的免疫细胞包括降低同种异体细胞治疗引起的免疫排斥反应的细胞。
在某些实施方式中，所述经修饰的免疫细胞中的T细胞抗原受体(TCR)和主要组织相容性复合体(MHCI，MHCII)在T细胞中的功能受到抑制。
在某些实施方式中，所述的免疫细胞中所述修饰包括与免疫排斥相关基因中的一个或多个的表达和/或活性被下调。
在某些实施方式中，其中所述与免疫排斥相关基因选自下组中的一种或多种基因：TRAC、TRBC、HLA-A、HLA-B、B2M和CIITA。
在某些实施方式中，所述的免疫细胞中所述经修饰的免疫细胞与未经修饰的相应细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调。
在某些实施方式中，所述经修饰的免疫细胞与未经所述修饰的相应细胞相比，CIITA基因的表达和/或活性未被下调。
在某些实施方式中，所述经修饰的免疫细胞与未经所述修饰的相应细胞相比，B2M基因的表达和/或活性未被下调。
在某些实施方式中，所述经修饰的免疫细胞与相应的野生型细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调。
在某些实施方式中，所述经修饰的免疫细胞与相应的野生型细胞相比，B2M基因的表达和/或活性未被下调。
在某些实施方式中，所述经修饰的免疫细胞与相应的野生型细胞相比，CIITA基因的表达和/或活性未被下调。
在某些实施方式中，所述基因的表达水平和/或活性被下调包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
在某些实施方式中，所述修饰包括：基因敲除、基因突变和/或基因沉默。
在某些实施方式中，所述修饰包括向所述免疫细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和sgRNA。
在某些实施方式中，所述修饰还包括向所述免疫细胞施用靶向所述TRAC基因外显子部分的sgRNA。
在某些实施方式中，所述靶向所述TRAC基因外显子部分的sgRNA包含SEQ ID NO:138至SEQ ID NO:152中任一项所示的核苷酸序列。
在某些实施方式中，所述修饰包括向所述免疫细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
在某些实施方式中，所述靶向所述HLA-A基因外显子部分的sgRNA包含SEQ ID NO:153至SEQ ID NO:193中任一项所示的核苷酸序列。
在某些实施方式中，所述修饰还包括向所述细胞施用Cas酶。
在某些实施方式中，Cas酶包括Cas9蛋白。
在某些实施方式中，所述修饰包括向所述细胞施用反义RNA，所述反义RNA包含SEQ ID NO:194至SEQ ID NO:197中任一项所示的核苷酸序列。
在某些实施方式中，所述免疫细胞为HLA-B纯合子细胞。
在某些实施方式中，所述HLA-B纯合子包括HLA-B*40纯合子，HLA-B*15纯合子，HLA-B*46纯合子，HLA-B*13纯合子，HLA-B*51纯合子，HLA-B*58纯合子，HLA-B*07纯合子，HLA-B*35纯合子，HLA-B*44纯合子，HLA-B*52纯合子，HLA-B*57纯合子，HLA-B*54纯合子或HLA-B*55纯合子。
在某些实施方式中，所述免疫细胞为HLA-A纯合子或杂合子细胞。
在某些实施方式中，所述HLA-A纯合子或杂合子包括HLA-A*02纯合子，HLA-A*11纯合子，HLA-A*02/A*11杂合子或HLA-A*24纯合子。
另一方面，本申请提供了一种制备免疫细胞的方法，其包括向免疫细胞中引入所述的核酸分子和/或所述的载体。
在某些实施方式中，所述的方法还包括：在向免疫细胞中引入所述的核酸分子和/或所述的载体之前/之后，修饰所述免疫细胞，所述修饰包括与免疫排斥相关基因中的一个或多个的表达和/或活性被下调。
在某些实施方式中，所述与免疫排斥相关基因选自下组中的一种或多种基因：TRAC、TRBC、HLA-A、HLA-B、B2M和CIITA。
在某些实施方式中，在所述的方法中，与未经所述修饰的免疫细胞相比，使所述免疫细胞中TRAC基因和HLA-A基因的表达和/或活性下调。
在某些实施方式中，在所述的方法中，与未经所述修饰的免疫细胞相比，所述免疫细胞中的CIITA基因的表达和/或活性未被下调。
在某些实施方式中，在所述的方法中，与未经所述修饰的免疫细胞相比，所述免疫细胞中的B2M基因的表达和/或活性未被下调。
在某些实施方式中，在所述的方法中，与野生型细胞相比，使所述免疫细胞中的TRAC基因和HLA-A基因的表达和/或活性被下调。
在某些实施方式中，在所述的方法中，与野生型细胞相比，所述免疫细胞中的CIITA基因的表达和/或活性未被下调。
在某些实施方式中，在所述的方法中，与野生型细胞相比，所述免疫细胞中的B2M基因的表达和/或活性未被下调。
在某些实施方式中，所述的方法中所述基因的表达水平和/或活性被下调包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
在某些实施方式中，所述的方法中所述修饰包括：基因敲除、基因突变和/或基因沉默。
在某些实施方式中，所述的方法中所述修饰包括向所述免疫细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和sgRNA。
在某些实施方式中，所述的方法中所述修饰包括向所述免疫细胞施用靶向所述TRAC基因外显子部分的sgRNA。
在某些实施方式中，所述的方法中所述靶向所述TRAC基因外显子部分的sgRNA包含SEQ ID NO:138至SEQ ID NO:152中任一项所示的核苷酸序列。
在某些实施方式中，所述的方法中所述修饰包括向所述免疫细胞施用靶向所述HLA-A 基因外显子部分的sgRNA。
在某些实施方式中，所述的方法中所述靶向所述HLA-A基因外显子部分的sgRNA包含SEQ ID NO:153至SEQ ID NO:193中任一项所示的核苷酸序列。
在某些实施方式中，所述的方法中所述修饰还包括向所述细胞施用Cas酶。
在某些实施方式中，所述的方法中Cas酶包括Cas9蛋白。
在某些实施方式中，所述修饰包括向所述细胞施用反义RNA，所述反义RNA包含SEQ ID NO:194至SEQ ID NO:197中任一项所示的核苷酸序列。
在某些实施方式中，所述的方法中所述免疫细胞来源于人。
在某些实施方式中，所述的方法中所述免疫细胞包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、γδT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在某些实施方式中，所述的方法中所述免疫细胞包括T细胞。
在某些实施方式中，所述的方法中所述免疫细胞为HLA-B纯合子细胞。
在某些实施方式中，所述的方法中所述HLA-B纯合子包括HLA-B*40纯合子，HLA-B*15纯合子，HLA-B*46纯合子，HLA-B*13纯合子，HLA-B*51纯合子，HLA-B*58纯合子，HLA-B*07纯合子，HLA-B*35纯合子，HLA-B*44纯合子，HLA-B*52纯合子，HLA-B*57纯合子，HLA-B*54纯合子或HLA-B*55纯合子。
在某些实施方式中，所述的方法中所述免疫细胞为HLA-A纯合子或杂合子细胞。
在某些实施方式中，所述的方法中所述HLA-A纯合子或杂合子包括HLA-A*02纯合子，HLA-A*11纯合子，HLA-A*02/A*11杂合子或HLA-A*24纯合子。
另一方面，本申请提供了所述的嵌合抗原受体，所述的分离的核酸分子，所述的载体，和/或所述的免疫细胞在制备CAR-T细胞中的应用。
另一方面，本申请提供了一种或多种药物组合物，其包含所述的嵌合抗原受体，所述的分离的核酸分子，所述的载体，和/或所述的免疫细胞，以及任选地药学上可接受的载剂。
另一方面，本申请提供了一种或多种所述的抗原嵌合受体，所述的分离的核酸分子，所述的载体，所述的免疫细胞，和/或所述的药物组合物，其用于治疗与EGFR的表达相关的疾病或病症。
在某些实施方式中，所述与EGFR的表达相关的疾病或病症包括与EGFR的表达上调相关的疾病或病症。
在某些实施方式中，所述与EGFR的表达相关的疾病或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括EGFR阳性的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括血液瘤。
在某些实施方式中，所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、宫颈癌、食管癌、胰腺癌、眼癌、间皮瘤、胆道癌、前列腺癌、肝癌、纤维肉瘤、结肠癌和/或胃癌。
在某些实施方式中，所述肿瘤包括纤维肉瘤。
另一方面，本申请提供了所述的嵌合抗原受体，所述的分离的核酸分子，所述的载体，所述的免疫细胞，和/或所述的药物组合物在制备药物中的用途，所述药物用于治疗与EGFR的表达相关的疾病或病症。
在某些实施方式中，所述与EGFR的表达相关的疾病或病症包括与EGFR的表达上调相关的疾病或病症。
在某些实施方式中，所述与EGFR的表达相关的疾病或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括EGFR阳性的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括血液瘤。
在某些实施方式中，所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、宫颈癌、食管癌、胰腺癌、眼癌、间皮瘤、胆道癌、前列腺癌、肝癌、纤维肉瘤、结肠癌和/或胃癌。
在某些实施方式中，所述肿瘤包括纤维肉瘤。
另一方面，本申请提供了一种预防和/或治疗与EGFR的表达相关的疾病或病症的方法，其包括向有需要的受试者施用有效量的所述的嵌合抗原受体，所述的分离的核酸分子，所述的载体，所述的免疫细胞，和/或所述的药物组合物。
在某些实施方式中，所述与EGFR的表达相关的疾病或病症包括与EGFR的表达上调相关的疾病或病症。
在某些实施方式中，所述与EGFR的表达相关的疾病或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括EGFR阳性的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括血液瘤。
在某些实施方式中，所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、宫颈癌、食管癌、胰腺癌、眼癌、间皮瘤、胆道癌、前列腺癌、肝癌、纤维肉瘤、结肠癌和/或胃癌。
在某些实施方式中，所述肿瘤包括纤维肉瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是本申请所述示例性的anti-EGFR CAR基因慢病毒表达载体。
图2显示的是本申请所述anti-EGFR UCAR-T细胞的构建策略。
图3A-3D显示的是本申请所述anti-EGFR UCAR-T细胞表型检测结果(敲除效率，转染效率，扩增倍数，记忆T细胞比例)。
图4显示的是本申请所述anti-EGFR UCAR-T细胞对靶细胞的杀伤结果。
图5A-5C显示的是本申请所述anti-EGFR UCAR-T细胞与靶细胞共培养细胞因子分泌检测结果。
图6显示的是本申请所述显示的是本申请所述anti-EGFR UCAR-T细胞体内抗肿瘤效果。
图7显示的是本申请所述anti-EGFR UCAR-T细胞体内半衰期检测结果。
图8A-8B显示的是本申请所述anti-EGFR UCAR-T细胞体内排异反应结果。
图9显示的是本申请所述靶向anti-EGFR UCAR-T细胞脱靶分析。
图10显示的是本申请所述靶向anti-EGFR UCAR-T细胞染色体易位分析。
图11显示的是本申请所述靶向anti-EGFR UCAR-T细胞核型分析。
图12显示的是本申请所述靶向anti-EGFR UCAR-T细胞Cas9残留分析。
图13显示的是本申请中TRAC基因在Sg9RNA编辑后Sanger测序的结果。
图14显示的是本申请中TRAC基因在Sg9RNA编辑后TA克隆检测的结果。
图15显示的是本申请中TRAC基因在Sg9RNA编辑后流式细胞检测的结果。
图16显示的是本申请中HLA-A02基因在Sg2RNA编辑后Sanger测序的结果。
图17显示的是本申请中HLA-A02基因在Sg5RNA编辑后Sanger测序的结果。
图18显示的是本申请中HLA-A11基因在Sg21RNA编辑后Sanger测序的结果。
图19显示的是本申请中HLA-A11基因在Rsg2RNA编辑后Sanger测序的结果。
图20A-20B显示的是本申请的经修饰的免疫效应细胞中同时敲除HLA-A02和TRAC的结果。
图21A-21B显示的是本申请的经修饰的免疫效应细胞中HLA-A02和TRAC的蛋白水平。
图22显示的是本申请的经修饰的免疫效应细胞中TRAC、HLA-A、B2M和CIITA的mRNA水平。
图23A-23B显示的是本申请的经修饰的免疫效应细胞中B2M和CIITA的蛋白水平。
图24A-24D显示的是本申请的经修饰的免疫效应细胞中TRAC、HLA-A、B2M和CIITA的蛋白水平。
图25A-25B显示的是本申请的经修饰的免疫效应细胞中TRAC和HLA-A mRNA水平的敲除情况。
图26A-26B显示的是本申请的经修饰的免疫效应细胞中CD69和CD137的蛋白水平。
图27显示的是本申请的经修饰的免疫效应细胞与NK细胞共培养的情况。
图28显示的是本申请的经修饰的免疫效应细胞表达IFN-γ的水平。
图29A-29D显示的是本申请的经修饰的免疫效应细胞中TRAC、HLA-A、B2M和CIITA的蛋白水平。
图30显示的是本申请的经修饰的免疫效应细胞对CAR的感染效率。
图31显示的是本申请的经修饰的免疫效应细胞的扩增倍数。
图32显示的是本申请的经修饰的免疫效应细胞对CD19阳性靶细胞的杀伤效果。
图33显示的是施用本申请的经修饰的免疫效应细胞的给药方案。
图34显示的是本申请的经修饰的免疫效应细胞对小鼠体内肿瘤的杀伤效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“嵌合抗原受体”或“CAR”通常是指一组多肽，在最简单的实施方案中 通常有两种，其当在免疫细胞中时，提供细胞对靶细胞(通常为癌细胞)的特异性，并产生细胞内信号。在一些实施方案中，CAR包含至少一个细胞外抗原结合结构域，跨膜域和胞质信号传导结构域(本文中也称为“胞内信号转导结构域”)，其包含衍生自如下所定义的刺激分子和/或共刺激分子的功能性信号传导结构域。在一些实施方案中，该组多肽在相同的多肽链中(例如，包含嵌合融合蛋白)。在一些实施方案中，该组多肽彼此不连续，例如在不同的多肽链中。在一些方面，该组多肽包括二聚化开关，其在二聚化分子的存在下可将多肽彼此偶联，例如可将抗原结合结构域偶联至胞内信号转导结构域。一方面，CAR的刺激分子是与T细胞受体复合物相关的ζ链。在一个方面，细胞质信号传导结构域包含一级信号传导结构域(例如，CD3-ζ的一级信号传导结构域)。在一个方面，细胞质信号传导结构域还包含一个或多个衍生自如下定义的至少一种共刺激分子的功能性信号传导结构域。一方面，共刺激分子可以选自4-1BB(即4-1BB)，CD27，ICOS和/或CD28。一方面，CAR包含嵌合融合蛋白，其可以包含细胞外抗原识别结构域，跨膜域和包含衍生自刺激分子的功能性信号传导结构域的细胞内信号转导结构域。一方面，CAR包含嵌合融合蛋白，其可以包含细胞外抗原识别结构域，跨膜域和细胞内信号转导结构域，细胞内信号转导结构域包含衍生自共刺激分子的功能性信号传导结构域和衍生自刺激分子的功能性信号传导结构域。一方面，CAR包含嵌合融合蛋白，其可以包含细胞外抗原识别结构域，跨膜域和细胞内信号转导结构域，细胞内信号转导结构域包含衍生自一个或多个共刺激分子的功能性信号传导结构域和衍生自刺激分子的功能性信号传导结构域。一方面，CAR包括嵌合融合蛋白，其可以包含细胞外抗原识别结构域，跨膜域和细胞内信号转导结构域，细胞内信号转导结构域包含至少两个衍生自一个或多个共刺激分子的功能性信号传导结构域和衍生自刺激分子的功能性信号传导结构域。在一个方面，CAR包含CAR融合蛋白的氨基末端(N-ter)上任选的前导序列。在一个方面，CAR进一步包含在细胞外抗原识别结构域的N末端的前导序列，其中前导序列任选地在细胞加工过程中从抗原识别结构域(例如scFv)切除，并将CAR定位于细胞膜。
在本申请中，术语“EGFR蛋白”通常属于受体酪氨酸激酶的ErbB家族，在上皮细胞生理机理能中发挥关键作用。人EGFR蛋白的氨基酸序列可以见于UniProt/Swiss-Prot登录号P00533。本申请中，所述分离的抗原结合片段可以结合EGFR蛋白。本申请中，术语“EGFR蛋白”、“EGFR抗原”和“EGFR-Fc重组蛋白”可以互换使用，并且包括由细胞天然表达的其任何变体或同种型。
在本申请中，术语“互补决定区”(CDR)通常是指在抗原结合片段可变区内的互补性决定区。在本申请中，所述重链可变区存在3个CDRs，所述CDRs对于每个可变区命名为 HCDR1、HCDR2和HCDR3。这些CDRs的确切边界已根据不同系统不同地限定。由Kabat(Kabat等人，Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987)和(1991))描述的系统，不仅提供了可应用于抗原结合片段的任何可变区的明确残基编号系统，还提供了限定3个CDRs的精确残基边界。这些CDRs可以被称为Kabat CDRs。Chothia和同事(Chothia和Lesk，J.Mol.Biol.196：901-917(1987)以及Chothia等人，Nature 342：877-883(1989))发现尽管在氨基酸序列水平上具有大的多样性，但是Kabat CDRs内的某些亚部分采取几乎相同的肽主链构象。这些亚部分命名为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别指轻链和重链区域。这些区域可以被称为Chothia CDRs，所述Chothia CDRs具有与Kabat CDRs重叠的边界。与Kabat CDRs重叠的限定CDRs的其他边界已由Padlan(FASEB J.9：133-139(1995))和MacCallum(J Mol Biol 262(5)：732-45(1996))描述。另外，其他的CDR边界定义可能不严格地遵循上述系统之一，但仍将与Kabat CDRs重叠，尽管按照特定残基或残基组或甚至整个CDRs并不显著影响抗原结合的预测或实验发现，它们可以缩短或加长。在本申请中，使用的是IMGT编号系统。
在本申请中，术语“FR”通常是指抗体可变结构域的更高度保守的部分，其被称为框架区。例如，天然重链和轻链的可变结构域各自可以包含四个FR区，即在VH中四个(H-FR1，H-FR2，H-FR3和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3和L-FR4)。“框架区”通常是指本领域识别的抗体可变区中存在于分歧性更高的(即高变)CDR之间的部分。此类框架区典型地称为框架1至4(FR1、FR2、FR3和FR4)且提供用于在三维空间中呈现六个CDR(三个来自重链且三个来自轻链)的骨架，以形成抗原结合表面。
在本申请中，术语“单链抗体”或“scFv”通常是指包含通过连接体连接的抗体重链可变区(VH)和抗体轻链可变区(VL)的分子。尽管2个结构域VL和VH由分隔的基因编码，可使用重组方法将它们连接，通过合成的接头使它们成为单个蛋白质链，其中VL和VH区配对形成被称为单链Fv(scFv)的单价分子。见，例如Bird等人，Science 242：423-426(1988)；和Huston等人，Proc.Natl.Acad.Sci.USA，85：5879-5883(1988)。当涉及术语“抗体”片段时包括所述单链抗体，通过重组技术或完整抗体的酶促或化学切割可制备所述单链抗体。
在本申请中，术语“同源性”通常可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中 的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。
为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
在本申请中，术语“KD”可与“KD”互换使用，通常是指特定的抗体-抗原相互作用的解离平衡常数，单位为M(mol/L)。KD可通过物质AB和其解离得到的物质A和物质B的浓度来计算：KD＝c(A)*c(B)/c(AB)。由该公式可知，KD值越大，说明解离越多，代表物质A、B之间的亲和力越弱；反之，KD值越小，说明解离越少，代表物质A、B之间的亲和力越强。
在本申请中，术语“分离的核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“宿主细胞”通常是指可以或已经含有包括本申请所述分离的核酸分子的载体，或者能够表达本申请所述分离的抗原结合片段的个体细胞，细胞系或细胞培养物。所述宿主细胞可以包括单个宿主细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述分离的抗原结合片段即可。所述宿主细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述宿主细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。例如，所述的宿主细胞可以是大肠杆菌细胞。例如，所述的宿主细胞可以是酵母细胞。例如，所述的宿主细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。
在本申请中，术语“免疫效应细胞”通常是指参与免疫应答，行使效应功能的免疫细胞。 例如所述行使效应功能可以包括清除异物抗原或促进免疫效应子应答等。免疫细胞可以包括浆细胞、T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞。
本申请的免疫效应细胞可以是自体/自身的(autologous/autogeneic)(“自己的”)或非自体的(“非自己的”，例如同种异体的、同基因的或异基因的)。在本申请中，术语“自体的”通常是指来自相同受试者的细胞。“同种异体的”通常是指与相比较的为相同物种但在遗传上不同的细胞。“同基因的”通常是指在遗传上与相比较的细胞相同的不同受试者的细胞。“异基因的”通常是指物种与相比较的细胞不同的细胞。在一些实施方式中，本申请的细胞是自体的或同种异体的。
在本申请中，术语“修饰”通常是指改变细胞的状态或结构和/或细胞的状态或结构的改变。所述改变通常是与相应未经所述修饰的细胞的状态或结构相比，所述改变可以包括内源基因表达水平或功能的变化，例如通过基因工程手段使得细胞内源基因表达水平下调、上调或不表达，所述基因工程手段可以包括同源重组、CRISPR/Cas9系统基因编辑等；所述改变还可以包括细胞蛋白质表达、结构或功能的变化，例如通过所述内源基因表达水平或功能的变化而实现的相应蛋白质表达的变化、结构或功能的变化，例如通过调节蛋白质翻译、翻译后修饰而实现的蛋白质表达的变化、结构或功能的变化；所述改变还可以包括引入外源基因、表达外源蛋白质等。
在本申请中，术语“TRAC”通常是指T细胞受体α链恒定区(T cell receptor alpha con-stant)。T细胞受体(TCR)通常是指位于T细胞表面的特异性受体，能够识别与主要组织相容性复合物(MHC)分子结合的抗原。TCR通常由两条不同的蛋白质链组成(即异源二聚体)。在人类中，多数T细胞中的TCR由一条α链和一个β链(分别由TRA和TRB编码)组成，这一类T细胞被称为αβT细胞，少数的的T细胞中，TCR由γ链和δ链(分别由TRG和TRD编码)组成，这一类T细胞被称为γδT细胞。通常情况下，αβT细胞约占T细胞总数的95％，γδT细胞约占T细胞总数的5％，该比率在个体发育过程中和患病状态(例如白血病)中发生变化，物种之间也有所不同。组成TCR的每一条链都含有可变区与恒定区，在人类中，编码α链的基因(TRA，例如HGNC:12027所示的信息)位于14号染色体，由多基因片段构成，包括可变段(V)、连接段(J)以及恒定区(C)，TRAC基因通常是指编码T细胞受体α链恒定区(C)的基因序列(例如HGNC:12029所示的信息)，其位于14号染色体(14q11.2；14：22,547,505-22,552,131)。通常编码N段抗原识别域的可变段(V)基因中的1个与连接段(J)中的一个重排产生一个功能性V区外显子，该外显子被转录并通过剪接与 恒定区(C)连接，从而形成T细胞受体α链编码序列。
在本申请中，术语“主要组织相容性复合物抗原”(“MHC”，在人类的情况下也称为“人类白细胞抗原”(“HLA”))通常是指在细胞表面上表达的赋予细胞独特抗原身份的蛋白质。MHC/HLA抗原是被T细胞和NK细胞识别为源自于与免疫细胞相同的造血干细胞来源(“自身”)或识别为源自于另一种造血重建细胞来源(“非自身”)的靶分子。识别了两种主要类别的HLA抗原：HLA I类和HLA II类。HLA I类抗原(人类中的A、B、C)使每个细胞都可被识别为“自身”，而HLA II类抗原(人类中的DR、DP和DQ)参与淋巴细胞和抗原呈递细胞之间的反应。两者都已经与移植器官的排斥有牵涉。HLA基因系统的一个重要方面是其多态性。每个基因，MHC I类(A、B和C)和MHC II类(DP、DQ和DR)存在着不同的等位基因。HLA等位基因由数字和下标表示。例如，两个不相关的个体可能分别携带I类HLA-B基因B5和Bw41。等位基因产物在α和/或β结构域的一个或更多个氨基酸中有差异。大量的特异性抗体或核酸试剂被用于使用表达I类和II类分子的白细胞对个体的HLA单倍型进行分型。通常用于HLA分型的基因是六种MHC I类和II类蛋白，即HLA-A；HLA-B和HLA-DR各自有两个等位基因。HLA基因簇集在存在于染色体位置6p21上的“超级基因座”中，所述“超级基因座”编码在免疫系统以及一些其他的基本分子和细胞过程的调控中具有重要作用的6个经典的移植HLA基因和至少132个蛋白质编码基因。完整的基因座粗略度量为3.6Mb，具有至少224个基因座。这种簇集的一个效果是“单倍型”，即存在于单条染色体上的一组等位基因，是从一个亲本遗传来的，倾向于作为一组进行遗传。从每个亲本遗传来的一组等位基因形成一个单倍型，其中一些等位基因倾向于关联在一起。鉴定患者的单倍型可以帮助预测找到匹配供体的概率，并且帮助制定搜索策略，因为一些等位基因和单倍型比其他等位基因和单倍型更常见，而且它们在不同种族和民族中分布的频率不同。
在本申请中，“HLA-A”通常是指一类人类白细胞抗原(human leukocyte antigens)多肽链，由位于人类染色体6p21.3的HLA-A基因(例如HGNC:4931所示的信息)编码。HLA-A是构成人类细胞表面I类MHC分子的三种主要多肽类型之一，其他还包括HLA-B和HLA-C。由HLA-A基因编码的α链和B2M基因编码的β链(β2-微球蛋白)组成的异二聚体即为HLA-A类MHC I分子。所述由HLA-A基因编码的α链可以包含α1结构域、α2结构域、α3结构域、跨膜区以及胞质区，其中α1结构域、α2结构域可以与肽段结合从而由MHC I分子(例如HLA-A类)将所述肽段呈递给免疫系细胞。在人类中，与大多数哺乳动物相似，MHC I分子的α链为多态性的，其一级结构有较多变化，截至2013年12月，共有2432个已知的HLA-A等位基因，编码1740个活性蛋白和117个无效蛋白。在本申请中，HLA-A等位基因可以 包括IMGT/HLA数据库3.38.0版(https://www.ebi.ac.uk/ipd/imgt/hla/)收录的由WHO HLA因子命名委员会命名的不同HLA-A等位基因的序列信息。
在本申请中，术语“HLA-B”通常是指人类白细胞抗原(HLA)复合物的基因家族的一部分。HLA是主要组织相容性复合体(MHC)的人类版本，MHC是一个存在于许多物种中的基因家族。在这个复杂的基因被分为三个基本组：I类，II类和III类。在人类中，HLA-B基因和两个相关基因HLA-A和HLA-C是MHC I类的主要基因。HLA-B基因位于6号染色体短(p)臂的细胞带21.3，从碱基对31,353,871到31,357,211。HLA-B是供者和受者之间应匹配的三个主要HLA之一。它们是HLA-A、HLA-B(均为I类MHC)和HLA-DR(II类MHC)。如果这两种组织具有编码这三种HLA的相同基因，那么排斥的可能性和严重程度就会降到最低。HLA-B的数百个版本(等位基因)是已知的，每个版本都有一个特定的编号(例如HLA-B27)。密切相关的等位基因被归类在一起；例如，至少有28个非常相似的等位基因是HLA-B27的亚型。这些亚型被指定为HLA-B*2701至HLA-B*2728。
在本申请中，术语“HLA匹配的”是指其中供体和受体之间HLA抗原没有不匹配的供体-受体对，所述供体诸如向需要造血干细胞移植疗法的受体提供造血干细胞移植物的供体。HLA匹配的(即其中所有6个等位基因都是匹配的)供体-受体对具有降低的移植物排斥的风险，原因是内源性T细胞和NK细胞不太可能将进入的移植物识别为外来的，并且因此不太可能产生针对移植物的免疫应答。
在本申请中，术语“HLA不匹配的”是指其中供体和受体之间至少一种HLA抗原(特别是对于HLA-A、HLA-B和HLA-DR)是不匹配的供体-受体对，所述供体诸如向需要造血干细胞移植疗法的受体提供造血干细胞移植物的供体。在一些实施方案中，一个单倍型是匹配的，而另一个是不匹配的。HLA不匹配的供体-受体对相对于HLA匹配的供体-受体对可能具有增加的移植物排斥的风险，原因是在HLA不匹配的供体-受体对的情况下，内源性T细胞和NK细胞更可能将进入的移植物识别为外来的，并且这样的T细胞和NK细胞因此更可能产生针对移植物的免疫应答。
在本申请中，术语“B2M”通常是指是β2微球蛋白(β2-microglobulin)，是MHC I类分子的组成部分之一。β2微球蛋白(也称为β链)可以与HLA编码的α链组成MHC I类的分子。B2M通常在所有有核的细胞中都有表达。在人类中，β2微球蛋白由位于15q21.1的B2M基因(例如HGNC:914所示的信息)所编码。
在本申请中，术语“CIITA”通常是指Ⅱ类主要组织相容性复合体(MHCⅡ)的反式激活因子。所述反式激活因子可以是具有酸性转录激活结构域、4个LRR(富含亮氨酸的重复序列) 和GTP结合结构域的蛋白质。所述蛋白质可位于细胞核中，作为II类主要组织相容性复合体(MHCⅡ)基因转录的正向调节剂，被称为表达这些基因的“主控制因子”。该蛋白质还可结合GTP，并利用与GTP结合来使其自身转运到细胞核中，在细胞核中，其通常使通过乙酰转移酶(AT)活性以类似共激活剂的方式起作用。在人类中，所述蛋白质由位于16p13.13的基因(例如HGNC:7067所示的信息)编码，能够产生几种编码不同同工型的转录物变体。
在本申请中，术语“野生型细胞”通常是指自然存在的或者自然来源的细胞。
在本申请中，术语“T细胞”通常是指胸腺衍生的细胞，其参与各种细胞介导的免疫反应。
在本申请中，术语“核酸”或“多核苷酸”或“核酸分子”通常时指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其单链形式或双链形式的聚合物。除非特别限定，否则该术语可以包括含天然核苷酸的类似物的核酸，所述核酸具有与参考核酸(例如示出了序列信息)相似的结合特性并且按照与天然存在核苷酸相似的方式代谢。除非另外说明，核酸的序列可以包括其保守方式修饰的变体，例如简并密码子置换、等位基因、直向同源物、SNP和互补序列，以及明确指出的序列。
在本申请中，术语“表达”通常是指特定核苷酸序列的转录和/或翻译。
在本申请中，术语“基因突变”通常是指基因在结构上发生的碱基对组成或排列顺序的改变。例如单个碱基改变所引起的点突变，或多个碱基的缺失、重复和插入等。
在本申请中，术语“基因沉默”通常是指通过调节机制阻止某些基因的表达。主要可以包括两种：一种是由于DNA甲基化、异染色质化以及位置效应等因素引起的转录水平上的基因沉默(transcriptional gene silencing,TGS)，另一种是转录后基因沉默(post-transcriptional gene silencing,PTGS)，即在基因转录后的水平上通过对靶标RNA进行特异性干预而影响基因的表达。通常情况下当基因沉默时，相应基因表达下调/减少。而当基因被敲除时则通常表现为不表达，例如在细胞中，某种特定基因的所有等位基因均被敲除后则表现为该基因的表达消失。基因沉默通常被认为是一种基因敲低机制，通用于沉默基因的方法可以如RNAi等。
在本申请中，术语“内源”指来自生物、细胞、组织或系统或在其内部产生的任何物质。
在本申请中，术语“外源”指从生物、细胞、组织或系统外部引入或在其外部产生的任何物质。
在本申请中，术语“反义RNA”通常是指一种与转录产物mRNA(信使RNA)互补的单链RNA。反义RNA可通过与mRNA的结合抑制基因的表达。例如，反义RNA与靶mRNA结合后引起该双链RNA分子对RNA酶Ⅲ的敏感性增加，使其降解；例如，反义RNA与mRNA的上游非编码区结合，从而直接抑制靶mRNA的翻译。
在本申请中，术语“siRNA”通常是指Small interfering RNA(小干扰RNA)或short in-terfering RNA(短干扰RNA)的缩写。siRNA是一类双链非编码RNA分子，长度约为18-28个碱基对，可通过与mRNA的互补结合引起mRNA的降解从而干扰特定基因的表达。在某些实施方式中，siRNA可以是长双链RNA或shRNA经Dicer酶处理得到的产物。在某些实施方式中，siRNA进入细胞与其他蛋白质形成RNA诱导沉默复合体(RISC)，有义链发生降解，反义链可与互补的靶向序列结合，从而实现基因沉默。
在本申请中，术语“shRNA”通常是指short hairpin RNA的缩写，即“短发夹RNA”。shRNA通常包括两个短反向重复序列，中间由一茎环(loop)序列分隔，组成发夹结构。通常还可以包括5-6个T碱基作为RNA聚合酶Ⅲ的转录终止子。在某些实施方式中，shRNA可经由病毒载体或质粒进入细胞中，在聚合酶Ⅱ或聚合酶Ⅲ的作用下进行转录，转录产物自细胞核输出(通常可经由Exportin 5)后经Dicer处理后输送至RISC，有义链发生降解，反义链可与互补的靶向序列结合，从而实现基因沉默。
在本申请中，术语“CRISPR/Cas系统”通常是指包含RNA引导的核酸酶或其他效应分子和gRNA分子的一组分子，所述分子能够指引和实现由RNA引导的核酸酶或其他效应分子在靶序列处修饰核酸，例如引起靶序列降解。在某些实施方式中，CRISPR系统包含gRNA和Cas蛋白，例如，Cas9蛋白。包含Cas9或其功能性突变体的系统在本申请中称作“Cas9系统”或“CRISPR/Cas9系统”。在某些实施方式中，gRNA分子和Cas分子可以复合，以形成核糖核蛋白(RNP)复合体。
在本申请中，术语“gRNA分子”或“向导RNA”、“指导RNA”、“指引RNA”、“向导RNA分子”、“gRNA”可互换使用，通常是指能够促进特异性指引RNA引导的核酸酶或其他效应分子(一般与gRNA分子复合)至靶序列上的核酸分子。在某些实施方案中，通过gRNA的一部分与DNA(例如，通过gRNA导引结构域)杂交并且通过gRNA分子的一部分与RNA指导的核酸酶或其他效应分子结合(例如，至少通过gRNAtracr)实现所述引导。在某些实施方案中，gRNA分子由单一的连续多核苷酸分子组成，在本文中称作“单一向导RNA”或“sgRNA”等。在其他实施方案中，gRNA分子由本身能够缔合(一般通过杂交)的多个(例如二个)多核苷酸分子组成，在本文中称作“双重向导RNA”或“dgRNA”等。
在本申请中，术语“Cas蛋白”通常是指CRISPR/Cas系统中负责剪切DNA的酶。可以包括来自Ⅰ、Ⅱ、Ⅲ型CRISPR/Cas系统的酶。例如，Cas3、Cas9、Cas10。
在本申请中，术语“Cas9蛋白”通常是指负责剪切DNA的来自细菌II型CRISPR/Cas系统的酶。Cas9可以包括野生型蛋白及其有功能性突变体。
在本申请中，“等位基因”通常是指基因座上的基因序列可能具有的不同变化的形式。基因座也称作基因位点或位点，是指染色体上的固定位置，例如某个基因所在。基因座在基因组中的排列位置称为基因图谱(genetic map)。
在本申请中，术语“纯合子”通常是指同源染色体在同一基因座上的两个等位基因相同的基因型个体。一对相对基因可以有AA和aa两种基因型的个体。
在本申请中，术语“杂合子”通常是指二倍体中同源染色体同一位点上的两个等位基因不相同的基因型个体，如Aa。杂合基因型一般比纯合显性或纯合隐性基因型的适应性都要高，这种现象被称为杂合子优势。
在本申请中，术语“药学上可接受的”通常是指代与合理的益处/风险比相称、在合理医学判断范围内适合用于与人类和动物的组织接触而不具有过度毒性、刺激、过敏应答或其它问题或并发症的那些化合物、材料、组合物和/或剂型。
在本申请中，术语“药学上可接受的载剂”通常是指常规使用的那些载剂中的任一种，并且仅受到物理-化学考虑因素(如溶解性和与活性结合剂的反应性的缺乏)限制，并且受给药途径限制。本文所描述的药学上可接受的载剂，例如媒剂、佐剂、赋形剂和稀释剂为所属领域的技术人员所熟知并且公众可容易获得。在一个方面中，药学上可接受的载剂是对医药组合物的活性成分具有化学惰性的载剂，并且是在使用条件下不具有不利的副作用或毒性的载剂。在一些实施例中，当向动物或人类给予时，载剂不产生不良、过敏或其它不适当的反应。在一些方面中，医药组合物不含热原质以及会对人类或动物有害的其它杂质。药学上可接受的载剂包括任何和所有溶剂、分散介质、涂料、抗细菌剂和抗真菌剂、等张剂和吸收延迟剂等等；其用途在所属领域中是众所周知。
可接受的载剂、赋形剂或稳定剂对接受者无毒性并且优选在所采用的剂量和浓度下是惰性的，并且包括缓冲夜，如磷酸盐、柠檬酸盐或其它有机酸；抗氧化剂，如抗坏血酸；低分子量多肽；蛋白质，如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，如甘氨酸、谷氨酰胺、天冬酰胺、精氨酸或赖氨酸；单糖、双糖和其它碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；糖醇，如甘露糖醇或山梨糖醇；盐形成抗衡离子，如钠；和/或非离子表面活性剂，如Tween、Pluronics或聚乙二醇(PEG)。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、 6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
嵌合抗原受体
一方面，本申请提供了一种嵌合抗原受体(CAR)，其可包含靶向部分，所述靶向部分可包含重链可变区VH中的至少一个CDR。例如，所述VH可以包含SEQ ID NO:1所示的氨基酸序列。在本申请中，所述分离的抗原结合蛋白的HCDR可以通过任何形式划分，只要VH与SEQ ID NO:1所示的氨基酸序列相同，以任何形式划分得到的HCDR都可落入本申请的保护范围内。
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、AbM、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，本申请所述分离的抗原结合蛋白通过IMGT编码系统定义。
在本申请中，所述靶向部分可包含HCDR3，所述HCDR3可包含SEQ ID NO:8所示的氨基酸序列。例如，所述靶向部分的HCDR3可通过IMGT编码系统定义。
在本申请中，所述靶向部分可包含HCDR2，所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列。例如，所述靶向部分的HCDR2可通过IMGT编码系统定义。
在本申请中，所述靶向部分可包含HCDR1，所述HCDR1可包含SEQ ID NO:4所示的氨基酸序列。例如，所述靶向部分的HCDR1可通过IMGT编码系统定义。
在本申请中，所述靶向部分可包含SEQ ID NO:1所示的重链可变区中的HCDR1、HCDR2和HCDR3。
在本申请中，所述靶向部分可包含HCDR1、HCDR2、HCDR3，所述HCDR3可包含SEQ  ID NO:8所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；以及所述HCDR1可包含SEQ ID NO:4所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体A1或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合片段(例如，scFv)。
在本申请中，所述靶向部分可包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1可包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述靶向部分可包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可包含SEQ ID NO:5所示的氨基酸序列。
在本申请中，所述靶向部分可包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可包含SEQ ID NO:7所示的氨基酸序列。
在本申请中，所述靶向部分可包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4可包含SEQ ID NO:9所示的氨基酸序列。
在本申请中，所述靶向部分可包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:5所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:7所示的氨基酸序列；以及所述H-FR4可包含SEQ ID NO:9所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体A1或与其具有相同H-FR1-4的抗原结合片段(例如，scFv)。
在本申请中，所述嵌合抗原受体中所述靶向部分可包含重链可变区VH，所述VH可包含SEQ ID NO:1所示的氨基酸序列。例如，编码所述VH的核苷酸序列可为SEQ ID NO:2。
在本申请中，所述VL可以包含SEQ ID NO:10所示的氨基酸序列。在本申请中，所述分离的抗原结合蛋白的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:10所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。
在本申请中，所述抗原结合蛋白可包含轻链可变区VL，所述VL可包含LCDR1、LCDR2和LCDR3中的至少一个、两个或三个。
在本申请中，所述嵌合抗原受体中所述靶向部分可包含LCDR3，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列。例如，所述靶向部分的LCDR3可通过IMGT编码系统定义。
在本申请中，所述嵌合抗原受体中所述靶向部分可包含LCDR2，所述LCDR2可包含SEQ ID NO:15所示的氨基酸序列。例如，所述靶向部分的LCDR2可通过IMGT编码系统定义。
在本申请中，所述的嵌合抗原受体中所述靶向部分可包含LCDR1，所述LCDR1可包含SEQ ID NO:13所示的氨基酸序列。例如，所述靶向部分的LCDR1可通过IMGT编码系统定义。
在本申请中，所述的嵌合抗原受体中所述靶向部分可包含SEQ ID NO:10所示的轻链可变区中的LCDR1、LCDR2和LCDR3。
在本申请中，所述的嵌合抗原受体中所述靶向部分可包含LCDR1、LCDR2、LCDR3，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:15所示的氨基酸序列；以及所述LCDR1可包含SEQ ID NO:13所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体A1或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗原结合片段(例如，scFv)。
在本申请中，所述的嵌合抗原受体中所述靶向部分可包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接地相连，且所述L-FR1可包含SEQ ID NO:12所示的氨基酸序列。
在本申请中，所述的嵌合抗原受体中所述靶向部分可包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2可包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述的嵌合抗原受体中所述靶向部分可包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3可包含SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述的嵌合抗原受体中所述靶向部分可包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端直接或间接地相连，且所述L-FR4可包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述靶向部分可包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1可包含SEQ ID NO:12所示的氨基酸序列；所述L-FR2可包含SEQ ID NO:14所示的氨基酸序列；所述L-FR3可包含SEQ ID NO:16所示的氨基酸序列；以及所述L-FR4可包含SEQ ID NO:18所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体A1或与其具有相同L-FR1-4的抗原结合片段(例如，scFv)。
在本申请中，所述嵌合抗原受体中所述靶向部分可包含轻链可变区VL，所述VL可包含SEQ ID NO:10所示的氨基酸序列。例如，编码所述VL的核苷酸序列为SEQ ID NO:11。
在本申请中，所述嵌合抗原受体中所述靶向部分可包含HCDR1-3以及LCDR1-3。所述HCDR3可包含SEQ ID NO:8所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；以及所述HCDR1可包含SEQ ID NO:4所示的氨基酸序列；所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:15所示的氨基酸序列；以及所述LCDR1可包含SEQ ID NO:13所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体A1或与其具有相同LCDR3及HCDR3(例如，与其具有相同LCDR1-3及HCDR1- 3)的抗原结合片段(例如，scFv)。
在本申请中，所述抗原结合蛋白可包含重链可变区和轻链可变区。所述抗原结合蛋白的重链可变区可包含HCDR1-3以及H-FR1-4。所述抗原结合蛋白的轻链可变区可包含LCDR1-3以及L-FR1-4。例如，所述HCDR1可包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:8所示的氨基酸序列；所述LCDR1可包括SEQ ID NO:13所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:15所示的氨基酸序列；所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列。例如，所述H-FR1可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:5所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:7所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:9所示的氨基酸序列；所述L-FR1可包括SEQ ID NO:12的氨基酸序列；所述L-FR2可包含SEQ ID NO:14所示的氨基酸序列；所述L-FR3可包含SEQ ID NO:16所示的氨基酸序列；所述L-FR4可包含SEQ ID NO:18所示的氨基酸序列。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:1所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体A1或与其具有相同重链可变区的抗原结合蛋白。例如，所述抗原结合蛋白的轻链可变区可包含SEQ ID NO:10所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体A1或与其具有相同轻链可变区的抗原结合蛋白。
在本申请中，所述的嵌合抗原受体中所述靶向部分可包括抗体或抗原结合片段。
在本申请中，所述的嵌合抗原受体中所述抗原结合片段可选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb。
在本申请中，所述的嵌合抗原受体中所述靶向部分可包括scFv。例如，所述scFv可靶向EGFR。例如，在所述scFv中，所述VH的C端可与所述VL的N端直接或间接地连接。例如，在所述scFv中，所述VL的C端可与所述VH的N端直接或间接地连接。
在本申请中，其可包括跨膜域，所述跨膜域可包含源自选自下组中的一种或多种蛋白的跨膜域：CD8A、CD8B、CD28、CD3e、CD3ε、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、CD154、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64和SLAM。
在本申请中，其中所述跨膜域可包含源自CD8A或CD8B的跨膜域。
在本申请中，其中所述跨膜域可包含SEQ ID NO:21至SEQ ID NO:69中任一项所示的氨基酸序列。
在本申请中，其可包括胞内共刺激信号传导结构域，所述胞内共刺激信号传导结构域可包含源自选自下组中的一种或多种蛋白的胞内共刺激信号传导结构域：CD28、4-1BB、CD27、CD2、CD7、CD8A、CD8B、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD40和MyD88。
在本申请中，其中所述胞内共刺激信号传导结构域可包括源自4-1BB的共刺激信号传导结构域。
在本申请中，其中所述胞内共刺激信号传导结构域可包含SEQ ID NO:70至SEQ ID NO:102中任一项所示的氨基酸序列。
在本申请中，其可包括胞内信号转导结构域，所述胞内信号转导结构域可包含源自选自下组中的一种或多种蛋白的胞内信号转导结构域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、DAP10、DAP-12和至少包含一个ITAM的结构域。
在本申请中，其中所述胞内信号转导结构域可包含源自CD3ζ、CD3δ、CD3γ或CD3ε的信号传导结构域。
在本申请中，其中所述胞内信号转导结构域可包含SEQ ID NO:86、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:103至SEQ ID NO:113中任一项所示的氨基酸序列。
在本申请中，其在靶向部分和跨膜域之间可包括铰链区，所述铰链区可包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、TIM1、SLAM、CD30和LIGHT。
在本申请中，所述铰链区可包含源自CD8的铰链区。
在本申请中，所述铰链区可包含SEQ ID NO:114至SEQ ID NO:135中任一项所示的氨基酸序列。
在本申请中，所述嵌合抗原受体可包括信号肽。所述信号肽可包含SEQ ID NO:136所示的氨基酸序列。例如，所述信号肽的核酸序列可包含SEQ ID NO:137的核苷酸序列。
在本申请中，所述嵌合抗原受体的非靶向部分可包括铰链区，跨膜域，胞内共刺激信号传导结构域与胞内信号转导结构域。
例如，所述嵌合抗原受体以抗EGFR单链抗体(scFv)为靶向部分，通过铰链区和跨膜域与胞内信号转导结构域相连接，胞内信号转导结构域由胞内共刺激信号传导结构域与胞内 信号转导结构域组成。
在某些实施方式中，所述嵌合抗原受体的非靶向部分可包含CD8A分子跨膜域、CD8的铰链区、4-1BB的胞内共刺激信号传导结构域和CD3ζ胞内信号转导结构域。
又例如，所述嵌合抗原受体以抗EGFR单链抗体(scFv)为靶向部分，通过CD8分子铰链区和跨膜域与胞内信号转导结构域相连接，胞内信号转导结构域由4-1BB胞内共刺激信号传导结构域与CD3ζ胞内信号转导结构域组成。
在某些实施方式中，所述嵌合抗原受体的非靶向部分包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体包含SEQ ID NO:199所示的氨基酸序列。
核酸分子、载体、细胞
另一方面，本申请提供一种或多种分离的核酸分子，其可编码前述的嵌合抗原受体。
在本申请中，所述的分离的核酸分子可包含SEQ ID NO:200所示的核苷酸序列。
另一方面，本申请提供一种载体，其可包含前述的分离的核酸分子。
在本申请中，所述的载体可包括病毒载体或非病毒载体。
在本申请中，所述非病毒载体可包括睡美人系统或piggybac系统。
在本申请中，所述的载体可包括慢病毒载体。
另一方面，本申请提供一种细胞，其可包含前述的嵌合抗原受体，前述的分离的核酸分子，和/或前述的载体。
另一方面，本申请提供前述的嵌合抗原受体，前述的分离的核酸分子，前述的载体，前述的细胞，或前述的免疫细胞在制备CAR-T细胞中的应用。
免疫细胞
另一方面，本申请提供一种免疫细胞，其可包含前述的所述的核酸分子或前述的载体，和/或表达前述的CAR。
在本申请中，所述的免疫细胞可包括人细胞。
在本申请中，所述免疫细胞可包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在本申请中，述的免疫细胞可包括T细胞。
在本申请中，所述的免疫细胞可包括经修饰的免疫细胞。
在本申请中，其中所述经修饰的免疫细胞可包括降低同种异体细胞治疗引起的免疫排斥反应的细胞。
在本申请中，其中所述经修饰的免疫细胞中的T细胞抗原受体(TCR)和主要组织相容性复合体(MHCI，MHCII)在T细胞中的功能受到抑制。
在本申请中，其中所述修饰可包括与免疫排斥相关基因中的一个或多个的表达和/或活性被下调。
在本申请中，其中所述与免疫排斥相关基因可选自下组中的一种或多种基因：TRAC、TRBC、HLA-A、HLA-B、B2M和CIITA。
在本申请中，其中所述与免疫排斥相关基因可选自下组中的一种或多种基因：TRAC、TRBC、HLA-A和HLA-B。
在本申请中，其中所述与免疫排斥相关基因可选自下组中的一种或多种基因：TRAC、TRBC和HLA-A。
在本申请中，其中所述与免疫排斥相关基因可选自下组中的一种或多种基因：TRAC和HLA-A。
在本申请中，所述经修饰的免疫细胞与未经修饰的相应细胞相比，TRAC基因和HLA-A基因的表达和/或活性可被下调。
在本申请中，其中所述经修饰的免疫细胞与未经所述修饰的相应细胞相比，CIITA基因的表达和/或活性未被下调。
在本申请中，其中所述经修饰的免疫细胞与未经所述修饰的相应细胞相比，B2M基因的表达和/或活性未被下调。
在本申请中，其中所述经修饰的免疫细胞与相应的野生型细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调。
在本申请中，其中所述经修饰的免疫细胞与相应的野生型细胞相比，B2M基因的表达和/或活性未被下调。
在本申请中，其中所述经修饰的免疫细胞与相应的野生型细胞相比，CIITA基因的表达和/或活性未被下调。
在本申请中，其中所述基因的表达水平和/或活性被下调包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
在本申请中，其中所述修饰可包括：基因敲除、基因突变和/或基因沉默。
在本申请中，所述修饰可包括所述免疫细胞中两个TRAC等位基因中的任意一个被敲除并且两个HLA-A等位基因中的任意一个被敲除。
在本申请中，所述修饰可包括所述免疫细胞中两个TRAC等位基因被敲除并且两个HLA- A等位基因中的任意一个被敲除。
在本申请中，所述修饰可包括所述免疫细胞中TRAC基因外显子被敲除并且HLA-A基因外显子被敲除。
在本申请中，其中所述修饰可包括向所述免疫细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和CRISPR/Cas9系统。
在本申请中，其中所述修饰可包括向所述免疫细胞施用CRISPR/Cas9系统。
在本申请中，其中所述修饰还可包括向所述免疫细胞施用靶向所述TRAC基因外显子部分的sgRNA。
在本申请中，其中所述靶向所述TRAC基因外显子部分的sgRNA可包含SEQ ID NO:138至SEQ ID NO:152中任一项所示的核苷酸序列。
在本申请中，其中所述修饰可包括向所述免疫细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
在本申请中，所述靶向所述HLA-A基因外显子部分的sgRNA可包含SEQ ID NO:153至SEQ ID NO:193中任一项所示的核苷酸序列。
在本申请中，所述修饰还可包括向所述细胞施用Cas酶。
在本申请中，Cas酶可包括Cas9蛋白。
在本申请中，所述修饰可包括向所述细胞施加反义RNA，所述反义RNA可包含SEQ ID NO:194至SEQ ID NO:197中任一项所示的核苷酸序列。
在本申请中，所述免疫细胞可为HLA-B纯合子细胞。
在本申请中，其中所述HLA-B纯合子可包括HLA-B*40纯合子，HLA-B*15纯合子，HLA-B*46纯合子，HLA-B*13纯合子，HLA-B*51纯合子，HLA-B*58纯合子，HLA-B*07纯合子，HLA-B*35纯合子，HLA-B*44纯合子，HLA-B*52纯合子，HLA-B*57纯合子，HLA-B*54纯合子，HLA-B*55纯合子。
在本申请中，其中所述免疫细胞可为HLA-A纯合子或杂合子细胞。
在本申请中，其中所述HLA-A纯合子或杂合子可包括HLA-A*02纯合子，HLA-A*11纯合子，HLA-A*02/A*11杂合子或HLA-A*24纯合子。
另一方面，本申请提供一种制备免疫细胞的方法，其可包括向免疫细胞中引入前述的核酸分子或前述的载体。
在本申请中，所述的方法还可包括：在向免疫细胞中引入前述的核酸分子或前述的载体之前/之后，修饰所述免疫细胞，所述修饰包括与免疫排斥相关基因中的一个或多个的表达和 /或活性被下调。
在本申请中，所述的方法可包括：在向免疫细胞中引入前述的核酸分子或前述的载体之后，修饰所述免疫细胞，所述修饰包括与免疫排斥相关基因中的一个或多个的表达和/或活性被下调。
例如，所述制备免疫细胞的方法可以包括：
(1)向免疫细胞中引入前述的核酸分子或前述的载体；
(2)修饰所述免疫细胞，所述修饰包括与免疫排斥相关基因中的一个或多个的表达和/或活性被下调。
例如，所述制备免疫细胞的方法可以包括：
(1)采集健康人的外周血，进行PBMC的分离，按照比例加入CD3磁珠孵育，进行CD3+T细胞分选；将CD3/CD28抗体偶联磁珠混匀，按计算的量取出适量磁珠悬液加入到T细胞培养体系中，激活T细胞，过夜培养；
(2)根据EGFR CAR病毒的滴度感染T细胞；
(3)同时敲除TRAC和HLA-A基因；
(4)CD3阴性T细胞分选：按照比例加入CD3磁珠，收集CD3-T细胞(磁珠未结合的细胞)。
在本申请中，其中所述与免疫排斥相关基因选自下组中的一种或多种基因：TRAC、TRBC、HLA-A、HLA-B、B2M和CIITA。
在本申请中，与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，下调所述免疫细胞中TRAC基因和HLA-A基因的表达和/或活性。
在本申请中，与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，CIITA基因的表达和/或活性未被下调。
在本申请中，与未经所述修饰的相应细胞中相应基因的表达和/或活性相比，B2M基因的表达和/或活性未被下调。
在本申请中，与相应的野生型细胞相比，所述免疫细胞的TRAC基因和HLA-A基因的表达和/或活性被下调。
在本申请中，与相应的野生型细胞相比，CIITA基因的表达和/或活性未被下调。
在本申请中，与相应的野生型细胞相比，B2M基因的表达和/或活性未被下调。
在本申请中，其中所述基因的表达水平和/或活性被下调可包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
在本申请中，其中所述修饰包括：基因敲除、基因突变和/或基因沉默。
在本申请中，所述修饰可包括所述免疫细胞中两个TRAC等位基因中的任意一个被敲除并且两个HLA-A等位基因中的任意一个被敲除。
在本申请中，所述修饰可包括所述免疫细胞中两个TRAC等位基因被敲除并且两个HLA-A等位基因中的任意一个被敲除。
在本申请中，所述修饰可包括所述免疫细胞中TRAC基因外显子被敲除并且HLA-A基因外显子被敲除。
在本申请中，所述修饰可包括向所述免疫细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和CRISPR/Cas9系统。
在本申请中，其中所述修饰可包括向所述免疫细胞施用CRISPR/Cas9系统。
在本申请中，其中所述修饰可包括向所述免疫细胞施用靶向所述TRAC基因外显子部分的sgRNA。
在本申请中，所述靶向所述TRAC基因外显子部分的sgRNA可包含SEQ ID NO:138至SEQ ID NO:152中任一项所示的核苷酸序列。
在本申请中，所述修饰可包括向所述免疫细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
在本申请中，所述靶向所述HLA-A基因外显子部分的sgRNA可包含SEQ ID NO:153至SEQ ID NO:193中任一项所示的核苷酸序列。
在本申请中，其中所述修饰还可包括向所述细胞施用Cas酶。
在本申请中，Cas酶可包括Cas9蛋白。
在本申请中，所述修饰可包括向所述细胞施加反义RNA，所述反义RNA可包含SEQ ID NO:194至SEQ ID NO:197中任一项所示的核苷酸序列。
在本申请中，其中所述免疫细胞可包括人细胞。
在本申请中，所述免疫细胞可包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在本申请中，所述免疫细胞可包括T细胞。
在本申请中，其中所述细胞可为HLA-B纯合子细胞。
在本申请中，其中所述HLA-B纯合子可包括HLA-B*40纯合子，HLA-B*15纯合子，HLA-B*46纯合子，HLA-B*13纯合子，HLA-B*51纯合子，HLA-B*58纯合子，HLA-B*07纯合子，HLA-B*35纯合子，HLA-B*44纯合子，HLA-B*52纯合子，HLA-B*57纯合子，HLA- B*54纯合子，HLA-B*55纯合子。
在本申请中，其中所述细胞可为HLA-A纯合子或杂合子细胞。
在本申请中，其中所述HLA-A纯合子或杂合子可包括HLA-A*02纯合子，HLA-A*11纯合子，HLA-A*02/A*11杂合子或HLA-A*24纯合子。
例如，所述制备免疫细胞的方法可以包括：
(1)采集健康人的外周血，进行HLA分型检测，选取符合我们需要的分型，进行PBMC的分离，按照比例加入CD3磁珠孵育，进行CD3+T细胞分选；将CD3/CD28抗体偶联磁珠混匀，按计算的量取出适量磁珠悬液加入到T细胞培养体系中，激活T细胞，过夜培养；
(2)根据EGFR CAR病毒的滴度感染T细胞；
(3)同时敲除TRAC和HLA-A基因；
(4)CD3阴性T细胞分选：按照比例加入CD3磁珠，收集CD3-T细胞(磁珠未结合的细胞)。
用途、药物组合物与治疗方法
另一方面，本申请提供一种药物组合物，其可包含前述的嵌合抗原受体，前述的分离的核酸分子，前述的载体，前述的细胞，和/或前述的免疫细胞，以及任选地药学上可接受的载剂。
例如，所述药物组合物可包括前述的免疫细胞以及任选地药学上可接受的载剂。
另一方面，本申请提供前述的抗原嵌合受体，前述的分离的核酸分子，前述的载体，前述的细胞，前述的免疫细胞，和/或前述的药物组合物，其可用于治疗与EGFR的表达相关的疾病或病症。
在本申请中，其中所述与EGFR的表达相关的疾病或病症可包括与EGFR的表达上调相关的疾病或病症。
在本申请中，其中所述与EGFR的表达相关的疾病或病症可包括EGFR阳性的肿瘤。在EGFR阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的EGFR的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。例如，所述肿瘤可包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、宫颈癌、食管癌、胰腺癌、眼癌、间皮瘤、胆道癌、前列腺癌、肝癌、纤维肉瘤、结肠癌和/或胃癌。例如，所述肿瘤可包括纤维肉瘤。
另一方面，本申请提供前述的嵌合抗原受体，前述的分离的核酸分子，前述的载体，前述的细胞，前述的免疫细胞，和/或前述的药物组合物在制备药物中的用途，所述药物可用于 治疗与EGFR的表达相关的疾病或病症。
在本申请中，其中所述与EGFR的表达相关的疾病或病症可包括与EGFR的表达上调相关的疾病或病症。
在本申请中，其中所述与EGFR的表达相关的疾病或病症可包括与EGFR的表达上调相关的疾病或病症。
在本申请中，其中所述与EGFR的表达相关的疾病或病症可包括EGFR阳性的肿瘤。在EGFR阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的EGFR的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。例如，所述肿瘤可包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、宫颈癌、食管癌、胰腺癌、眼癌、间皮瘤、胆道癌、前列腺癌、肝癌、纤维肉瘤、结肠癌和/或胃癌。例如，所述肿瘤可包括纤维肉瘤。
另一方面，本申请提供预防或治疗与EGFR的表达相关的疾病或病症的方法，其可包括向有需要的受试者施用有效量的前述的嵌合抗原受体，前述的分离的核酸分子，前述的载体，前述的细胞，前述的免疫细胞，和/或前的药物组合物。
在某些实施方式中，其中所述与EGFR的表达相关的疾病或病症可包括与EGFR的表达上调相关的疾病或病症。
在本申请中，其中所述与EGFR的表达相关的疾病或病症可包括与EGFR的表达上调相关的疾病或病症。
在本申请中，其中所述与EGFR的表达相关的疾病或病症可包括EGFR阳性的肿瘤。在EGFR阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的EGFR的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。例如，所述肿瘤可包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、宫颈癌、食管癌、胰腺癌、眼癌、间皮瘤、胆道癌、前列腺癌、肝癌、纤维肉瘤、结肠癌和/或胃癌。例如，所述肿瘤可包括纤维肉瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1
1.1抗EGFR嵌合抗原受体(CAR)的设计
抗EGFR CAR结构包括:一个EGFR抗原结合区(来源于抗EGFR的scFv，其氨基酸序列如SEQ ID NO:198所示)，一个CD8A胞外铰链区，一个CD8A跨膜区，一个4-1BB胞内共刺激域和一个CD3ζ激活信号域。EGFR CAR的非抗原结合域的氨基酸序列如SEQ ID NO:19所示，核苷酸序列如SEQ ID NO:20所示。
1.2 EGFR CAR的慢病毒载体构建
根据EGFR序列信息及CAR载体结构，构建EGFR CAR慢病毒表达载体，载体示意图(见图1)。进行优化：选择商业化慢病毒表达载体pCDH-CMV-MCS-EF1-copGFP为骨架，在此载体基础上进行元件改造。首先，将载体的氨苄抗性基因β-内酰胺酶替换为源自Tn5的氨基糖苷磷酸转移酶，使载体具有卡那霉素抗性。其次，我们删除了在体内应用中具有潜在威胁性的CMV启动子及其临近的下游多克隆位点。最后，将原载体中由EF1启动子启动表达的copGFP基因删除，保留SalI酶切位点，并在SalI 5’端加入SmaI酶切位点供载体构建用，形成最终目的载体。加入的SmaI酶切位点是最终目的载体的单一酶切位点，载体的其他序列部分不具有该酶切位点。优化后构建嵌合抗原受体慢病毒表达载体，经sanger测序确证序列无误后，进行慢病毒包装。
1.3设计导向RNA
通过网站https://www.ncbi.nlm.nih.gov/，查找并下载相应基因序列，使用SnapGene软件打开基因序列，可在目的基因的不同外显子上设计sgRNA。在本实施例中采用的CRISPR/Cas9系统的sgRNA非限制性的设计原则为：5’-NNN(20)-NGG-3’，NGG被称为原间隔子相邻基序(PAM)，其中，N表示A、T、C或G。由于在同一外显子上可以设计出较多sgRNA，并且由20个核苷酸序列组成的sgRNA可能会在基因组中重复出现，所以利用网站http://crispr.cos.uni-heidelberg.de来进行sgRNA的设计与评估，将外显子序列粘贴至该网站，网站设计出sgRNA并进行预测评估，在评估中得分越高，则说明可能存在较高的编辑效率和较低的脱靶风险，从中选择得分较高的sgRNA进行试验。靶向TRAC基因的sgRNA如SEQ  ID NO:138至SEQ ID NO:152所示，靶向HLA-A02基因的sgRNA如SEQ ID NO:153至SEQ ID NO:174所示，靶向HLA-A11基因的sgRNA如SEQ ID NO:175至SEQ ID NO:185所示，靶向HLA-A24基因的sgRNA如SEQ ID NO:186至SEQ ID NO:193所示，由金斯瑞生物科技公司合成。
实施例2
2.1供体筛选
根据受体的HLA-B分型，选择与受体HLA-B分型匹配的HLA-B纯合子。
首先供者来源基于人群中的HLA-B纯合子，患者HLA-B的其中一个等位基因和供者HLA-B纯合子一致即可，来源于这些供者的细胞能覆盖高数量的患者人群。降低HLA-B亚型不一致引起的排异反应。HLA-B主要选择人群中频率较高的B*40纯合子，B*15纯合子，B*46纯合子，B*13纯合子，B*51纯合子，B*58纯合子，B*07纯合子，B*35纯合子，B*44纯合子，B*52纯合子，B*57纯合子，B*54纯合子，B*55纯合子。HLA-A选择人群中频率较高的A*02纯合子，A*11纯合子及A*02/A11杂合子。
2.2 CD3+T细胞制备
(1)从外周血中分离PBMC
从健康捐献者中采集外周血，用PBS缓冲液按照1：1进行外周血稀释。在新的50mL离心管中先加入稀释后血量1/3的细胞分离液(Ficoll)，然后沿着管壁非常缓慢加入血细胞稀释液，800g常温离心20min(离心机设置升速1、降速0)。离心后离心管中的液体自上而下分为PBS与血清层、白细胞层、淋巴细胞分离液、红细胞层。去除PBS与血清层，将白细胞层移至新的50ml离心管中，加入PBS至40ml清洗细胞，450g离心10min。离心后弃上清，即得到外周血单个核细胞。细胞重悬后进行细胞计数。
(2)复苏冻存的健康人PBMC
将冻存的健康人PBMC细胞在37℃水浴锅中进行复苏，完全融化后将细胞吸到含有10ml含10％FBS的X-VIVO15培养基(购自LONZA)的15ml离心管中，400g离心8min；去上清，加入2ml X-VIVO15培养基(含10％FBS和终浓度100μg/ml的DNase I)室温孵育15min，孵育的过程中不断振荡；将孵育结束后的溶液用40μm的滤网进行过滤，并吸取10ml PBS缓冲液重悬底部的细胞再加到滤网上，过虑后400g离心8min，离心后弃上清，细胞重悬后进 行细胞计数。
(3)CD3 +T细胞分选
使用EasySep TM人T细胞分选试剂盒(购自StemCell Technologies，货号：17951)提取外周血单个核细胞(PBMC)中的T细胞。将PBMC密度调整至5×10 7细胞/ml，PBS缓冲液的添加范围在0.25-2ml；先加cocktail混匀再按照50μl/ml加入isolation cocktail，混匀后室温放置5min；将RapidSpheres用旋涡振荡仪涡旋30s后加按照40μl/ml加入至细胞中混匀；补加缓冲液至2.5ml的倍数，上下轻轻吹打2-3次；按照每管2.5ml分别加到冻存管中，将冻存管置于磁力架上，室温放置3min；轻轻打开冻存管盖，小心持两边拿起磁力架，倒置保持2-3s，将细胞液一次性倒入新的离心管中；用10-20ml缓冲液(视细胞量)重悬细胞后，300g离心10min，弃掉上清，得到CD3 +T细胞。
(4)T细胞激活
激活试剂按培养基:Transact＝99:1体积比进行配置，培养基为X-VIVO15培养基(含5％FBS、200U/ml IL2、10ng/ml IL7和5ng/ml IL15)、Transact购自美天旎。T细胞按每1×106个细胞用1ml激活试剂(含有10μl Transact)充分重悬后，放置于37℃、5％CO 2培养箱中孵育1天。
实施例3
3.1转病毒
按照实施例2的方式获得CD3+T细胞(D0天)，并用CD3/CD28抗体磁珠激活，激活后于D1天进行慢病毒载体(实施例1制备的EGFR CAR慢病毒表达载体)转染，D2天洗去慢病毒载体，D3天进行电转。
3.2基因敲除
使用电转试剂盒(购自LONZA，货号V4XXP-3024)通过电转方式将RNP复合体转入3.1制备的激活后T细胞(取D3天的CAR-T细胞为初始细胞)。取样计数后收集细胞离心，用PBS重悬细胞沉淀。提前30min在孔板中预热培养基(X-VIVO15培养基+10％FBS+IL2(200U/ml)+IL7(10n g/ml)+IL15(5ng/ml))。准备电转缓冲液：Nucleofector Solution：Supplement按照82:18进行配置；根据每个电转体系使用1x10 7的细胞进行分配RNP复合物(Cas9:sgRNA＝2:1)，先将10μg sgRNA加入到PCR管(无RNA酶)中，再加 入20μg Cas9蛋白(购自thermo，货号A36499)，轻轻混匀后，室温孵育12min。将上述细胞进行计数，300g离心8min弃上清，加入PBS重悬细胞，吸取1E7个细胞重新300g离心8min，弃上清后用100μl配置好的电转缓冲液重悬细胞。将孵育好的RNP复合体加入上述细胞悬液中，轻柔混匀，然后将混合物轻轻地转移到电转杯中。将电转杯放在Lonza-4D电转仪上，选用EO-115电转程序进行电转。往电转杯中加入预热的培养基，然后用配套吸管将细胞转入孔板中预热的培养基中，然后放置于37℃、5％CO 2培养箱中培养48小时后收细胞，sanger测序检测编辑效率，同时收细胞FACS检测敲除效率。
其中sgRNA序列TRAC sgRNA：AGAGTCTCTCAGCTGGTACA(SEQ ID NO:138)，A02sgRNA：CTGACCATGAAGCCACCCTG(SEQ ID NO:155)，A11 sgRNA：GGCCCCTCCTGCTCTATCCA(SEQ ID NO:185)。
3.3 CD3阴性T细胞分选
进行CD3阴性T细胞分选，细胞计数后离心，弃上清；用buffer重悬细胞并混匀，按20ul CD3磁珠/10 7的细胞加CD3磁珠，混合均匀后放4度冰箱孵育，加buffer洗涤细胞离心后进行磁珠分离，首先将column放在磁极上，下面对应放离心管，用buffer浸润column(LD)，将细胞加到column上，不要产生气泡，用buffer清洗column 2次，收集清洗下来的液体(CD3-T)于15ml离心管中，取部分细胞进行细胞计数。
3.4细胞培养
镜下观察细胞状态，取细胞进行稀释计数，补充全培养基维持细胞密度在3x10^5-1x10^6个/ml，中间补/换液，放入37℃、5％CO 2培养。细胞收获：收集到细胞离心管中离心后弃上，用生理盐水再次洗涤细胞，离心，配制冻存液，冻存液重悬离心后的细胞，用注射器吸取细胞悬液至终制品用细胞冻存袋中，在细胞冻存袋上贴好标签，进行下一步冻存。
3.5基因敲除效率检测
(1)Sanger测序检测
细胞计数，取3～5×10 4细胞，2000r/min离心5min，尽量去干净上清，然后每管加20μl DE裂解液，细胞裂解后加到PCR管中，瞬时离心后上PCR仪，上机条件：65℃30min，4℃30s、95℃2min、16℃无限。使用引物对TRAC-For/TRAC-Rev，或HLA-A For/HLA-A Rev， 将裂解产物作为模板进行PCR，将PCR产物交送金唯智进行Sanger测序。得到sanger测序结果后，使用网站：https://moriaritylab.shinyapps.io/editr_v10/中的EditR编辑器预测编辑发生的位置以及编辑效率。
(2)流式检测细胞计数
取10E5至10E8细胞，2000rpm离心5min，去上清，然后每管加100μl的PBS缓冲液重悬细胞，再加入anti-human AB TCR-APC(购自eBioscience)抗体5μl，HLA-A12 Monoclonal Antibody(BB7.2)，APC，eBioscince TM(购自invitrogen)抗体5μl，混合均匀后于室温孵育10min。2000rpm离心5min后再以PBS缓冲液洗2遍，重悬细胞并通过BD FACSAria流式细胞仪进行检测，可得细胞表面TCR、HLA-A12表达阳性率。敲除效率＝(A-B)/A×100％；A为对照组表达阳性率；B为敲除组表达阳性率。
结果如图3A-3D所示，anti-EGFR UCAR-T细胞CAR阳性率可达34％以上(图3A)，anti-EGFR UCAR-T细胞中心记忆比例达45％左右(图3B)，anti-EGFR UCAR-T细胞双敲效率高达95％以上(图3C)，平均扩增倍数100X以上(图3D)。
实施例4 anti-EGFR UCAR-T细胞体外细胞毒性分析
4.1 anti-EGFR UCAR-T细胞对靶细胞的杀伤
(1)EGFR靶细胞：HT1080-Luciferase-GFP；调整靶细胞状态至对数生长期，在进行实验前需连续传代2次；
(2)制备anti-EGFR UCAR-T细胞及对照组anti-EGFR CAR-T，T细胞，流式检测敲除效率，转染效率，CD3-T分选效率及记忆T细胞的比例，统计扩增倍数(见图2)；
(3)离心收集制备的几组细胞，每组6x10^6的细胞；
(4)将靶细胞重悬于1640+10％FBS中，每个靶点取3块24孔板，按照2x10^5/孔的量接种靶细胞。(靶细胞和效应细胞都是2x10^6/ml的密度接种)。然后按照E/T(效靶比，效应细胞：靶细胞)比例，加入效应细胞。每孔补液到最大体积(如600ul)。对照接种同样数量的靶细胞，不加效应细胞(600ul)。将孔板置于5％CO 2、37℃培养箱中，培养24小时。E/T：1:2，1:1，2:1，5:1，10:1铺板，重复三次。
(5)培养24小时，将孔板从培养箱中取出，收集200ul上清。然后通过检测Luciferase活性反映重组CAR-T细胞对靶细胞的裂解能力。
靶细胞裂解百分数计算公式：
Figure PCTCN2022103092-appb-000001
结果分析：anit-EGFR UCAR-T对HT1080-Luciferase-GFP细胞有显著的杀伤作用，在效靶比1:2时即可达到90％以上的杀伤效率(见图4)。
4.2 anti-EGFR UCAR-T细胞与靶细胞共培养细胞因子分泌检测
收集上述共培养体系的上清，检测细胞因子分泌水平。结果分析(图5A-5C)：anti-EGFR UCAR-T显著激活，大量分泌IL-2，IFN-γ和TNF-α细胞因子。
实施例5 anti-EGFR UCAR-T细胞体内抗肿瘤效果
8-10周大的NSG小鼠皮下注射肿瘤细胞HT1080-Luciferase-GFP(5x10^6)，小鼠分成三组，每组5只，成瘤时间一般2-4周。分别向每组小鼠瘤内注射anti-EGFR UCAR-T细胞，anti-EGFR CAR-T细胞，无基因敲除的T细胞5E6，单点注射，注射体积50ul。通过荧光素酶监测小鼠肿瘤消退情况(见图5)。
结果分析(图6)：回输anti-EGFR UCAR-T细胞的小鼠，肿瘤生长速度明显减缓，anti-EGFR UCAR-T细胞表现出卓越的抗肿瘤效果。
实施例6靶向EGFR UCAR细胞体内半衰期检测
准备人源化免疫系统小鼠(hHSC-NCG)15只。分成3组。制备细胞，实验组anti-EGFR UCAR-T细胞(敲除TRAC+HLA-A02)；对照组1：anti-EGFR CAR-T；对照组2：anti-EGFR UCAR-T细胞(敲除TRAC+B2M)；每只小鼠注射1x10^7细胞，不同的时间点采血D0，2h,D3,D7,D14,D21,D28,D35,D42,D49,D56,D60。提取不同时间点血样中基因组，用QPCR绝对定量的方法计算copy/ng genome DNA，阳性对照用第14天收获的UCAR-T细胞，阴性对照用DEPC水。
结果分析(图7)：anti-EGFR UCAR-T细胞(敲除TRAC+HLA-A02)在小鼠体内存活时间约70天，并在week7-week9进行二次扩增。
实施例7通用型T细胞的体外安全性验证
(1)GVHD反应：制备TRAC，HLA-A双敲T细胞，无基因敲除的T细胞，辐照同种异体PBMC，分别刺激制备的2组细胞，检测IFN-r的水平。
结果分析：TRAC，HLA-A双敲T细胞组IFN-γ分泌水平很低，表明TRAC敲除降低了 GVHD反应。
(2)同种异体反应：同种异体PBMC刺激辐照后的2组细胞，检测IFN-r的水平。
结果分析：TRAC，HLA-A双敲T细胞组IFN-γ分泌水平很低，表明HLA-A的敲除降低了同种异体反应。
实施例8通用型T细胞的体内安全性验证
(1)GVHD反应：制备TRAC，HLA-A双敲T细胞，无基因敲除的T细胞。取8-10周NSG小鼠分别注射1x10^7，通过临床指标：存活率，皮毛纹理和皮肤完整性等，观察移植物抗宿主反应。细胞因子检测：取外周血血清，检测IL6、IL-2、TNF-α、IFN-γ等细胞因子的水平。取血时间点：回输前，24h，D3，D7，D14，D28，2M。脏器病变检测：观察期结束时(2个月左右)，取小鼠的脾脏，肝脏，皮肤、胃肠道，肺，肾脏进行HE切片染色分析。
结果分析：注射未经处理的T细胞的5只小鼠中，有4只在注入后2个月内发生了致死性异种移植物抗宿主病(GVHD)。而接受TRAC，HLA-A双敲细胞的小鼠中没有一个出现GVHD；TRAC，HLA-A双敲T细胞组细胞因子IL6、IL-2、TNF-α、IFN-γ分泌水平很低；并且小鼠不同器官形态正常。说明TRAC，HLA-A双敲T细胞组很大程度上降低了GVHD反应。
(2)同种异体反应：制备TRAC，HLA-A双敲CAR-T细胞，共同注射1x10^7TCR-HLA-A-双敲CAR-T细胞和2x10^6同种异型T细胞到NSG小鼠体内。对照组：注射1x10^7TCR-CAR-T细胞到NSG小鼠体内。
不同时间点取血测CAR拷贝数。比较两组CAR的拷贝数变化。时间点：D1，D5，D7，D10，D14。
细胞因子检测：取外周血血清，检测IL6、IL-2、TNF-α、IFN-γ等细胞因子的水平。时间点：D1，D5，D7，D10，D14。
结论：D13天，对照组小鼠排异反应明显，基本检测不到拷贝数；而实验组拷贝数仍处在较高水平，说明排异反应明显减弱，实验组细胞在小鼠体内存活时间延长；说明TRAC，HLA-A双敲CAR-T细胞组很大程度上降低了排异反应(见图8A-8B)。
实施例9基因编辑的安全性分析
制备TRAC，HLA-A双敲T细胞，无基因敲除的T细胞，检测敲除效率后，进行以下分析：
(1)脱靶：
对照组：转CAS9+ODN标签
实验组：转CAS9+sgRNA(TRAC+HLA-A)+ODN标签
On-target and off-target-WGS:(Whole genome sequencing)：分别取无基因敲除的T细胞、TRAC，HLA-A双敲的T细胞各1×10^6送苏州金维智生物科技有限公司检测。
结果分析：实验组脱靶率很低，而且脱靶主要集中在基因之间和内含子上，对基因功能的影响不大(见图9)。
(2)染色体易位：采用qPCR方法对同时编辑TRAC和HLA基因座时可能发生的重排进行了定量。两个易位被标记为TRAC:HLA，HLA:TRAC。合成的模板质粒中的阳性参考样品作为检测对照进行评估。以HLA基因组靶区两侧的扩增片段作为内对照。提取基因组DNA进行实时定量PCR，根据标准曲线和Cq值计算基因组DNA的基因拷贝数。
结果分析：双敲T细胞(TRAC+HLA-A)在D14(收获)检测是否发生染色体易位，检测结果：两种易位方式检测值都接近零检值，提示基因座没有发生重排(见图10)。
(3)核型分型：分别取铺满度在70-80％的无基因敲除的T细胞、TRAC，HLA-A双敲的T细胞各1×10^6装于2个T25瓶中，加满培养液，盖上全密封盖子，缠上封口膜，寄送至浙江如耀生物科技有限公司检测。
结果分析：和对照组比，实验组核型正常(见11)。
(4)Cas9蛋白残留：细胞制备时，取敲除前、敲除后以及收获前三个时间点的细胞各1×10^6进行裂解，随后用蛋白定量试剂盒(NOVATEINBIO，货号NB-E1372PR)进行定量，将各组样品调整至相同上样量2μg，根据说明书用CRISPR/Cas9蛋白ELISA试剂盒进行检测。样品中的Cas9蛋白被牢固稳定地点在试纸孔上。然后使用检测抗体识别被绑定的Cas9蛋白，然后显影剂进行显影。Cas9比值与吸光度成正比，通过与Cas9对照品比较来定量Cas9蛋白的绝对量。
结果分析：双敲T细胞(TRAC+HLA-A)分别在电转前(D3)、电转后换液前(D5)、D9、D14(收获)四个时间点检测spCas9的残留，除了电转后换液前(D5)检测到微量残留，其余三个时间点均未检出。(见图12)。
实施例10制备单基因敲除的T细胞
使用电转试剂盒(购自LONZA，货号V4XXP-3024)通过电转方式将RNP复合体转入实施例2制备的激活后T细胞。提前30min在孔板中预热培养基(X-VIVO15培养基+10％FBS+IL2(200U/ml)+IL7(10n g/ml)+IL15(5ng/ml))。电转缓冲液按照Nucleofector Solution：Supplement＝82:18进行配置。准备RNP复合体：TRAC的sgRNA序列为sg9(如SEQ ID NO: 138所示)，HLA-A的sgRNA序列为HLA-A02 Sg2(如SEQ ID NO:154所示)或HLA-A02Sg5(如SEQ ID NO:155所示)或HLA-A11 sg21(如SEQ ID NO:185所示)或HLA-A11Rsg2(如SEQ ID NO:184所示)，先将20μg sgRNA加入到PCR管(无RNA酶)中，再加入10μg Cas9蛋白(购自thermo，货号A36499)，轻轻混匀后，室温孵育12min。将实施例2培养的激活后T细胞进行计数，300g离心8min弃上清，加入PBS重悬细胞，吸取1E7个细胞重新300g离心8min，弃上清后用100μl配置好的电转缓冲液重悬细胞。将孵育好的RNP复合体加入上述细胞悬液中，轻柔混匀，然后将混合物轻轻地转移到电转杯中。将电转杯放在Lonza-4D电转仪上，选用EO-115电转程序进行电转。往电转杯中加入预热的培养基，然后用配套吸管将细胞转入孔板中预热的培养基中，然后放置于37℃、5％CO 2培养箱中培养。
实施例11基因敲除效率检测方法的比较
(1)Sanger测序检测
细胞计数，取3～5×104细胞，2000r/min离心5min，尽量去干净上清，然后每管加20μl DE裂解液，细胞裂解后加到PCR管中，瞬时离心后上PCR仪，上机条件：65℃30min，4℃30s、95℃2min、16℃无限。使用引物对TRAC-For/TRAC-Rev，或HLA-A For/HLA-A Rev，将裂解产物作为模板进行PCR，将PCR产物交送金唯智进行Sanger测序。得到sanger测序结果后，使用网站：https://moriaritylab.shinyapps.io/editr_v10/中的EditR编辑器预测编辑发生的位置以及编辑效率。
(2)TA克隆测序检测
利用AxyPrepTM PCR产物清洁试剂盒(购自AXYGEN)，将PCR产物纯化，随后用试剂盒(DNA A-Tailing Kit，购自TaKaRa)将纯化后的PCR产物加粘性末端，通过DNA Ligation Kit Ver2.1(购自TaKaRa)将产物连接至T载体(pMDTM19-T Vector Cloning Kit购自TaKaRa)，连接产物转化感受态细胞(DH5alpha)，然后涂布于含氨苄抗性的LB板，在37℃培养箱培养约12小时后挑单菌落，并将单菌落菌液交由金维智进行测序。敲除效率＝突变克隆数/总克隆数。
(3)流式检测细胞计数
取10E5至10E8细胞，2000rpm离心5min，去上清，然后每管加100μl的PBS缓冲液重悬细胞，再加入anti-human AB TCR-APC(购自eBioscience)抗体5μl，HLA-A02 Monoclonal Antibody(BB7.2)，APC，eBioscince TM(购自invitrogen)抗体5μl，混合均匀后于室温孵育10min。2000rpm离心5min后再以PBS缓冲液洗2遍，重悬细胞并通过BD FACSAria流式细胞仪进行检测，可得细胞表面TCR、HLA-A02表达阳性率。敲除效率＝(A-B)/A×100％；A为 对照组表达阳性率；B为敲除组表达阳性率。
TRAC单基因敲除的三种检测结果如图13至图15所示，敲除效率计算结果如表1所示，三种检测方法基本相同，后续试验仅采用Sanger测序方法检测编辑效率。
表1基因敲除效率检测方法结果
Figure PCTCN2022103092-appb-000002
针对HLA-A02基因编辑的Sanger测序方法结果如图16-17所示，编辑效率均为90％；针对HLA-A11基因编辑的Sanger测序方法结果如图18-19所示。
实施例12制备TRAC基因和HLA-A基因双基因敲除的T细胞
使用电转试剂盒(购自LONZA，货号：V4XXP-3024)通过电转方式将RNP复合体转入实施例2制备的激活后T细胞。提前30min在孔板中预热培养基(X-VIVO15培养基+10％FBS+IL2(200U/ml)+IL7(10ng/ml)+IL15(5ng/ml))。电转缓冲液按照Nucleofector Solution：Supplement＝82:18进行配置。准备RNP复合体：分别将20μg TRAC sgRNA(TRAC Sg9)，20μg HLA-A sgRNA(HLA-A02 Sg2或HLA-A02 Sg5或HLA-A11 sg21或者靶向HLA-A*24:02:01、HLA-A*30:01:01:01、HLA-A*33:01:01:01、HLA-A*03:01:01:01、HLA-A*01:01:01:01或HLA-A*26:01:01:01的sgRNA)加入到PCR管(无RNA)中，再各自加入10μg Cas9蛋白(购自thermo，货号A36499)，轻轻混匀后，室温孵育12min。将实施例2培养的激活后T细胞进行计数，300g离心8min弃上清，加入PBS重悬细胞，吸取1E7个细胞重新300g离心8min，弃上清后用100μl配置好的电转缓冲液重悬细胞。将孵育好的TRAC与HLA-A的RNP复合体加入上述细胞悬液中，轻柔混匀，然后将混合物轻轻地转移到电转杯中。将电转杯放在Lonza-4D电转仪上，选用EO-115电转程序进行电转。往电转杯中加入预热的培养基，然后用配套吸管将细胞转入孔板中预热的培养基中，然后放置于37℃、5％CO 2培养箱中培养。
通过测序检测双基因敲除效率，可得到双基因敲除效率均不低于80％的TRAC阴性、HLA-A阴性的T细胞。结果如图20-21所示。其中图20A显示的是利用HLA-A02 Sg5敲除HLA-A02的结果，其中上一行显示的是对照组结果(即没有使用HLA-A02 Sg5进行敲除)； 下一行显示的是同时敲除HLA-A02和TRAC的结果；其中图20B显示的是利用TRAC Sg9敲除TRAC的结果，其中上一行显示的是对照组结果(即没有使用TRAC Sg9进行敲除)；下一行显示的是同时敲除HLA-A02和TRAC的结果。图21A-21B显示了敲除HLA-A02和TRAC蛋白水平的敲除情况，其中NEG指阴性对照，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指同时敲除HLA-A02和TRAC的结果。
实施例13双基因敲除的T细胞中TRAC基因，HLA-A基因，B2M基因和CIITA基因与相应细胞中的相应基因的表达区别
(1)利用实施例2制备的激活后T细胞，分为两组，一组作为对照，另一组按照实施例5的方法制备TRAC基因和HLA-A基因双基因敲除的T细胞，按照实施例4步骤(1)的方式进行Sanger测序。依据测序结果获得TRAC和HLA-A双基因敲除的细胞。将制备的双基因敲除T细胞与相应TRAC和HLA-A抗体孵育，通过流式分选或磁珠分选，可以得到双基因敲除的细胞株。
(2)检测双基因敲除的T细胞与对照组相比mRNA表达水平的变化。使用RNA提取试剂盒(购自QIAGEN，货号：74004)提取RNA，使用逆转录试剂盒(购自Applied Biosystems，货号：4368814)对RNA进行逆转录得到cDNA，以cDNA为模板进行定量PCR检测。
(3)检测双基因敲除的T细胞与对照组相比蛋白表达水平的变化。使用全蛋白提取试剂(购自Thermo Scientific，货号：87787)提取蛋白，通过Western Blot方法或流式方法检测蛋白表达水平，使用的抗体分别为TRAC抗体(购自eBioscience货号：17-9986-42)、HLA-A抗体(购自Merck货号：17-9876-41)、B2M抗体(购自Invitrogen货号：A15770)和CIITA抗体(购自OriGene货号：CF812200)。
Sanger测序检测双基因敲除的T细胞中TRAC和/或HLA-A基因的核苷酸序列相对于对照组发生了变化；定量PCR显示双基因敲除的T细胞中TRAC和/或HLA-A基因mRNA表达量下调，而B2M和/或CIITA基因的mRNA表达量没有下调。FACS和Western Blot结果显示双基因敲除的T细胞中蛋白表量下调，B2M和/或CIITA蛋白表达量没有下调。
结果如图22-23所示。其中，图22显示的是基因表达的mRNA水平测定，其中图22显示了TRAC、HLA-A、B2M和CIITA的mRNA水平；其中WT指没有经任何敲除处理的情况，双敲组指TRAC基因和HLA-A基因双基因敲除的T细胞的结果。图23显示的是基因表达的蛋白水平测定，其中图23A-23B分别显示了B2M和CIITA的蛋白表达水平；其中NEG指阴性对照，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的T细胞的结果。
实施例14.制备TRAC基因、HLA-A/B2M基因和CIITA基因三基因敲除的T细胞并验证其中相应三种基因的表达变化
(1)按照实施例13步骤(1)的方式准备对照组和TRAC基因、HLA-A基因和CIITA基因三基因敲除的细胞，以及TRAC基因、B2M基因和CIITA基因三基因敲除的细胞。
(2)按照实施例13步骤(3)的方式通过FACS和Western Blot方法检测蛋白表达水平变化。
相对于对照组细胞，TRAC、HLA-A和CIITA三基因敲除的T细胞中TRAC、HLA-A和CIITA基因的蛋白表达量下调；相对于对照组细胞，TRAC、B2M和CIITA三基因敲除的T细胞中TRAC、HLA-A和CIITA基因的蛋白表达量下调。
(3)使用TRAC(购自eBioscience，货号：17-9986-42)、HLA-A(购自Merck，货号：17-9876-41)、B2M(购自：Invitrogen，货号：A15770)抗体通过流式细胞术检测实施例13中的双基因敲除细胞和本实施例中的两种三基因敲除细胞的敲除效率，结果显示在单细胞水平同时实现多基因敲除的效率，双基因敲除明显高于三基因敲除。
结果如图24A-24D所示。其中图24A-24C依次为TRAC、HLA-A和B2M蛋白质水平的敲除情况。其中，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的T细胞的结果；TRAC+HLA-A+CIITA三敲指TRAC、HLA-A和CIITA三基因敲除的T细胞的结果；其中TRAC+B2M+CIITA三敲指B2M、CIITA和TRAC三基因敲除的T细胞的结果；TRAC+HLA-A敲低指实施例16制备的TRAC基因和HLA-A基因敲低的T细胞的结果。图24D显示了CIITA蛋白质水平的敲除情况。
图24的结果显示，和WT对照组相比，TRAC、HLA-A、CIITA和B2M的蛋白质水平下调。同时，与TRAC+HLA-A+CIITA三敲或TRAC+B2M+CIITA三敲相比，TRAC+HLA-A双敲的敲除效率更高。
实施例15设计反义RNA序列
通过数据库https://www.ncbi.nlm.nih.gov/或www.ensembl.org/，获得相应基因(TRAC基因和HLA-A基因)的转录RNA序列，参考如下原则设计siRNA：
尽量避免起始密码子下游50-100个核苷酸与终止密码子上游100个核苷酸的序列；选择长度小于30个核苷酸的序列；避免4个或以上的连续相同碱基；避免内含子区域；避免重复序列；避免单核苷酸多态性(SNP)位点；序列GC含量30％-60％之间，优先选择序列模式AA(N<sub>19)、NA(N<sub>21)或NAR(N<sub>17)YNN，A为腺苷酸；T为胸腺苷酸；R为 腺苷酸或鸟苷酸(嘌呤类)；Y为胸腺苷酸或胞苷酸(嘧啶类)；N为腺苷酸、胸腺苷酸、鸟苷酸或胞苷酸；对选择的序列进行同源性比较分析，避免反义RNA与其他基因或序列具有显著的同源性，由此造成脱靶效应。同源性分析利用NCBI Blast tool:Nucleotide-nucleotide BLAST(blastn)，UCSC Blat tool或Ensembl Blast进行。
设计获得的反义RNA序列包括HLA-A-homo-551(其包含SEQ ID NO:194所示的核苷酸序列)；HLA-A-homo-NEG(其包含SEQ ID NO:195所示的核苷酸序列)；TRAC-homo-375(其包含SEQ ID NO:196所示的核苷酸序列)；TRAC-homo-NEG(其包含SEQ ID NO:197所示的核苷酸序列)。
实施例16制备TRAC基因和HLA-A基因敲低的T细胞
利用通过实施例15设计的反义RNA进行双基因敲低。公司制备TRAC基因和HLA-A基因反义RNA序列的慢病毒(吉玛)。按照实施例2的方式制备CD3 +T细胞(D0天)，并用CD3/CD28抗体磁珠激活，将携带TRAC基因和HLA-A基因的反义RNA序列的慢病毒转染激活的T细胞(D1天)，D2天洗去慢病毒载体，继续培养至D5天。收集培养至D5天的T细胞，通过定量PCR或Western Blot等方法对基因敲低效率进行检测。对获得的T细胞进行相应TRAC和HLA-A抗体标记，通过流式分选或磁珠分选的方式可以得到TRAC基因和HLA-A基因敲低的T细胞。结果显示，TRAC和HLA-A基因敲低组中TRAC和HLA-A的mRNA和蛋白表达水平均下调。其中，图25A-25B依次为TRAC和HLA-A mRNA水平的敲除情况。其中，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的T细胞的结果。其中，为TRAC和HLA-A蛋白质水平的敲除水平可以参见图24的结果。
实施例17不同T细胞活性的区别
制备实施例2、12、14和16中的无基因敲除、双基因敲除、三种基因敲除和双基因敲低的T细胞，比较几种T细胞活性各组细胞计数并分别各取1*10 6细胞接种24孔板中，每孔加入PHA(0.3μg/ml)(离子霉素+)或5ng/ml的PMA和50ng/ml的ionomycin于细胞中，继续培养5小时后，使用CD69(早期活化)(购自BD Biosciences，货号：FN50)、CD137(偏晚期)(购自BD Biosciences，货号：4B4-1)抗体，流式检测细胞的活化状态。结果表明，双基因敲除、双基因敲低的T细胞活性优于三基因敲除的T细胞。
CD69和CD137的蛋白质水平表达情况分别参见图26A-26B。其中，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的T细胞的结 果；TRAC+HLA-A+CIITA三敲指TRAC、HLA-A和CIITA三基因敲除的T细胞的结果；其中TRAC+B2M+CIITA三敲指B2M、CIITA和TRAC三基因敲除的T细胞的结果；TRAC+HLA-A敲低指实施例16制备的TRAC基因和HLA-A基因敲低的T细胞的结果。
实施例18不同T细胞对异体NK细胞反应性的区别
对实施例2、12、14和16中的无基因敲除、双基因敲除、三种基因敲除和双基因敲低的T细胞进行CFSE(invitrogen，C34554)标记，细胞计数，分别取1*10 6细胞并以1：1比例与NK细胞(NK92MI)进行共培养，24小时后收集共培养的各组细胞，流式细胞检测混合细胞中CFSE阳性细胞的比率。
结果表明，NK细胞对双基因敲除、双基因敲低的T细胞的杀伤毒性低于三基因敲除的T细胞。结果如图27所示。其中，NK+T指将NK细胞与没有经任何敲除处理的T细胞共培养的情况；NK+TRAC+HLA-A敲低指将NK细胞与实施例16制备的TRAC基因和HLA-A基因敲低的T细胞的结果共培养的情况；NK+TRAC+HLA-A双敲指将NK细胞与TRAC基因和HLA-A基因双基因敲除的T细胞共培养的情况；NK+TRAC+HLA-A+CIITA三敲指将NK细胞与TRAC、HLA-A和CIITA三基因敲除的T细胞共培养的情况；NK+TRAC+B2M+CIITA三敲指将NK细胞与B2M、CIITA和TRAC三基因敲除的T细胞共培养的情况。
实施例19不同的T细胞异体免疫排斥反应的区别
供者1来源的外周血制备实施例2、12、14和16中的无基因敲除、双基因敲除、三种基因敲除和双基因敲低的T细胞。供者2来源的外周血制备CD3 +T细胞。将供者1外周血制备的各组细胞分别与供者2外周血按照实施例2制备的CD3 +T细胞等比例混合，24小时后检测细胞混合体系中的IFN-γ的表达水平。结果显示，双基因敲除的T细胞组IFN-γ的表达水平低于三基因敲除的T细胞组。
结果如图28所示，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的T细胞的结果；TRAC+HLA-A+CIITA三敲指TRAC、HLA-A和CIITA三基因敲除的T细胞的结果；其中TRAC+B2M+CIITA三敲指B2M、CIITA和TRAC三基因敲除的T细胞的结果；TRAC+HLA-A敲低指实施例16制备的TRAC基因和HLA-A基因敲低的T细胞的结果。
实施例20制备TRAC基因和HLA-A基因双基因敲除的CAR-T细胞，TRAC基因，HLA-A基因和CIITA基因三基因敲除的CAR-T细胞以及TRAC基因，B2M基因和CIITA基因敲除的CAR-T细胞
(1)按照实施例2的方式获得CD3 +T细胞(D0天)，并用CD3/CD28抗体磁珠激活，激活后于D1天进行慢病毒载体(包含CD19-CAR、CD20-CAR或BCMA-CAR等的慢病毒)转染，D2天洗去慢病毒载体，D3天对CAR阳性的T细胞进行分选并继续培养至D5天。
(2)取D5天的CAR-T细胞为初始细胞，分别按照实施例12和实施例14中的方式制备TRAC基因和HLA-A基因双基因基因敲除的细胞，TRAC基因、HLA-A基因和CIITA基因以及TRAC基因、B2M基因和CIITA基因三基因敲除的CAR-T细胞。
(3)通过流式细胞技术检测可得到上述双基因敲除和三基因敲除的CAR-T细胞，其中双基因敲除CAR-T细胞的得率高于三基因敲除CAR-T细胞。
结果如图29A-29D所示。其中，图17A-17C依次为TRAC、HLA-A和B2M蛋白质水平的敲除情况。图29D显示了CIITA蛋白质水平的敲除情况。其中，WT指没有经任何敲除处理的情况，TRAC+HLA-A双敲指TRAC基因和HLA-A基因双基因敲除的CAR-T细胞的结果；TRAC+HLA-A+CIITA三敲指TRAC、HLA-A和CIITA三基因敲除的CAR-T细胞的结果；其中TRAC+B2M+CIITA三敲指B2M、CIITA和TRAC三基因敲除的CAR-T细胞的结果。
其中，CD19CAR的转染效率如图30A-30B所示。其中，CAR30％+即代表CD19 CAR的转染效率。
图31显示了不同细胞的扩增倍数。其中，TRAC基因和HLA-A基因双基因基因敲除的CAR-T细胞扩增倍数最高。
实施例21 TRAC基因和HLA-A基因双基因敲除的CAR-T细胞抗肿瘤效果
制备实施例21中的TRAC基因和HLA-A基因双敲除的CAR-T细胞(靶向CD19、CD20或BCMA)，接种表达荧光素酶基因的靶细胞(靶基因阳性的白血病或淋巴瘤细胞系，如Raji、Jurkat、MM1S等)至孔板中，再分别以不同效靶比(1∶2.5，1∶1，5：1，10：1)加入双基因敲除的CAR-T细胞、三基因敲除的CAR-T细胞或无基因敲除的T细胞，共培养24小时后将细胞转移至检测孔板中，加入荧光素酶底物，酶标仪检测荧光值。杀伤效率＝1-靶细胞T细胞共培养荧光值/单独培养的靶细胞荧光值。
结果显示TRAC基因和HLA-A基因双敲除的CAR-T细胞对肿瘤细胞有显著的杀伤效 果。
图32显示了对CD19靶细胞Raji-Luciferase的杀伤效果，其中TRAC基因和HLA-A基因双敲除的CAR-T细胞的杀伤效果最为显著。其中每个E/T比下从左到右依次是A-D的图注所对应的结果。
实施例22 TRAC基因和HLA-A基因双基因敲除的CAR-T细胞抗肿瘤效果
NSG小鼠静脉注射肿瘤细胞，肿瘤成功建立后向小鼠体内回输TRAC基因和HLA-A基因双基因敲除的CAR-T细胞、三基因敲除的CAR-T细胞或无基因敲除的T细胞，监测小鼠肿瘤体积。
回输双基因敲除CAR-T细胞的小鼠，肿瘤生长速度明显减缓。
结果如图33-34所示。其中，图33显示了对小鼠的给药方式，i.v.表示静脉注射，CAR-T细胞代表表达CD19 CAR的双基因敲除的CAR-T细胞、三基因敲除的CAR-T细胞。图32显示了小鼠在施用CAR-T细胞后体内肿瘤的体积情况。其中，图32从左至右列依次显示了分别施用生理盐水、未改造的T细胞、TRAC基因和HLA-A基因双基因敲除的CD19 CAR-T细胞、TRAC、HLA-A和CIITA三基因敲除的CD19 CAR-T细胞、B2M、CIITA和TRAC三基因敲除的CD19 CAR-T细胞后，小鼠体内肿瘤的体积情况。结果发现回输TRAC基因和HLA-A基因双基因敲除的CAR-T细胞的小鼠，肿瘤生长的速度明显减缓。
综上，
1.本申请制备了一种靶向EGFR的嵌合抗原受体，该重组受体的抗原结合域(靶向部分)来自scFv，具有结构稳定的特点。
2.本申请提供了一种慢病毒表达载体。以pCDH-CMV-MCS-EF1-copGFP为骨架，将载体的氨苄抗性基因β-内酰胺酶替换为源自Tn5的氨基糖苷磷酸转移酶，使载体具有卡那霉素抗性；删除了在体内应用中具有潜在威胁性的CMV启动子及其临近的下游多克隆位点；将原载体中由EF1启动子启动表达的copGFP基因删除，保留SalI酶切位点，并在SalI 5’端加入SmaI酶切位点供载体构建用，形成最终目的载体。加入的SmaI酶切位点是最终目的载体的单一酶切位点，载体的其他序列部分不具有该酶切位点。
3.本申请优化了蛋白质RNA复合体电转染技术。获得了原代T细胞中90％以上的双基因敲除效率。
4.本申请供者来源基于人群中高频出现的HLA-B纯合子，患者HLA-B其中一个等位基因和供者纯合子一致即可，来源于这些供者的细胞能覆盖高数量的患者人群，且能够降低HLA-B引起的排异反应。
5.本申请筛选出了与排异高度相关的HLA-A分子进行敲除，而保留了其他HLA-I类分子，既减少了异体细胞的排异，也避免了HLA分子完全敲除被NK细胞清除的发生，大大延长了同种异体CAR-T细胞在体内的半衰期。
6.本申请首次构建了高效率双敲除TCR，HLA-A的EGFR-UCAR-T细胞，做到一种安全型货架式即用型治疗剂，提高抗肿瘤效果，用于乳腺癌等疾病的治疗。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (129)

  1. 嵌合抗原受体(CAR)，其包含靶向部分，所述靶向部分包含HCDR3，所述HCDR3包含SEQ ID NO:8所示的氨基酸序列。
  2. 根据权利要求1所述的嵌合抗原受体，其中所述靶向部分包含HCDR2，所述HCDR2包含SEQ ID NO:6所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的嵌合抗原受体，其中所述靶向部分包含HCDR1，所述HCDR1包含SEQ ID NO:4所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:1所示的重链可变区中的HCDR1、HCDR2和HCDR3。
  5. 根据权利要求1-4中任一项所述的嵌合抗原受体，其中所述靶向部分包含HCDR1、HCDR2、HCDR3，所述HCDR3包含SEQ ID NO:8所示的氨基酸序列；所述HCDR2包含SEQ ID NO:6所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:4所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:3所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:5所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:7所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:9所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的嵌合抗原受体，其中所述靶向部分包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:3所示的氨基酸序列；所述H-FR2包含SEQ ID NO:5所示的氨基酸序列；所述H-FR3包含SEQ ID NO:7所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO:9所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的嵌合抗原受体，其中所述靶向部分包含VH，所述VH包含SEQ ID NO:1所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的嵌合抗原受体，其中所述靶向部分包含LCDR3，所述 LCDR3包含SEQ ID NO:17所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的嵌合抗原受体，其中所述靶向部分包含LCDR2，所述LCDR2包含SEQ ID NO:15所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的嵌合抗原受体，其中所述靶向部分包含LCDR1，所述LCDR1包含SEQ ID NO:13所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:10所示的轻链可变区中的LCDR1、LCDR2和LCDR3。
  16. 根据权利要求1-15中任一项所述的嵌合抗原受体，其中所述靶向部分包含LCDR1、LCDR2、LCDR3，所述LCDR3包含SEQ ID NO:17所示的氨基酸序列；所述LCDR2包含SEQ ID NO:15所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:13所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的嵌合抗原受体，其中所述靶向部分包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接地相连，且所述L-FR1包含SEQ ID NO:12所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的嵌合抗原受体，其中所述靶向部分包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:14所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的嵌合抗原受体，其中所述靶向部分包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:16所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的嵌合抗原受体，其中所述靶向部分包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端直接或间接地相连，且所述L-FR4包含SEQ ID NO:18所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的嵌合抗原受体，其中所述靶向部分包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:12所示的氨基酸序列；所述L-FR2包含SEQ ID NO:14所示的氨基酸序列；所述L-FR3包含SEQ ID NO:16所示的氨基酸序列；以及所述L-FR4包含SEQ ID NO:18所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的嵌合抗原受体，其中所述靶向部分包含VL，所述VL包含SEQ ID NO:10所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的嵌合抗原受体，其中所述靶向部分包括抗体或抗原结合片段。
  24. 根据权利要求23所述的嵌合抗原受体，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb。
  25. 根据权利要求1-24中任一项所述的嵌合抗原受体，其中所述靶向部分包括scFv。
  26. 根据权利要求25所述的嵌合抗原受体，其中所述scFv靶向EGFR。
  27. 根据权利要求1-26中任一项所述的嵌合抗原受体，其中所述靶向部分包含SEQ ID NO:198所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的嵌合抗原受体，其包括跨膜域，所述跨膜域包含源自选自下述蛋白的跨膜域或其组合：CD8A、CD8B、CD28、CD3e、CD3ε、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、CD154、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64和SLAM。
  29. 根据权利要求28所述的嵌合抗原受体，其中所述跨膜域包含源自CD8A或CD8B的跨膜域。
  30. 根据权利要求28-29中任一项所述的嵌合抗原受体，其中所述跨膜域包含SEQ ID NO:21至SEQ ID NO:69中任一项所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的嵌合抗原受体，其包括共刺激信号传导结构域，所述共刺激信号传导结构域包含源自选自下述蛋白的共刺激信号传导结构域或其组合：CD28、4-1BB、CD27、CD2、CD7、CD8A、CD8B、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD40和MyD88。
  32. 根据权利要求31所述的嵌合抗原受体，其中所述共刺激信号传导结构域包括源自4-1BB的共刺激信号传导结构域。
  33. 根据权利要求31-32中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域包含SEQ ID NO:70至SEQ ID NO:102中任一项所示的氨基酸序列。
  34. 根据权利要求31-33中任一项所述的嵌合抗原受体，其中所述共刺激信号传导结构域的N端与所述跨膜域的C端直接或间接地连接。
  35. 根据权利要求1-34中任一项所述的嵌合抗原受体，其包括胞内信号转导结构域，所述胞内信号转导结构域包含源自选自下述蛋白的胞内信号转导结构域或其组合：CD3ζ、 CD3δ、CD3γ、CD3ε、CD79a、CD79b、FceRIγ、FceRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、DAP10、DAP-12和至少包含一个ITAM的结构域。
  36. 根据权利要求35所述的嵌合抗原受体，其中所述胞内信号转导结构域包含源自CD3ζ、CD3δ、CD3γ或CD3ε的胞内信号转导结构域。
  37. 根据权利要求35-36中任一项所述的嵌合抗原受体，其中所述胞内信号转导结构域包含SEQ ID NO:86、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:103至SEQ ID NO:113中任一项所示的氨基酸序列。
  38. 根据权利要求35-37中任一项所述的嵌合抗原受体，其中所述胞内信号转导结构域的N端与所述共刺激信号传导结构域的C端直接或间接地连接。
  39. 根据权利要求1-38中任一项所述的嵌合抗原受体，其在靶向部分和跨膜域之间包括铰链区，所述铰链区包含源自选自下述蛋白的铰链区或其组合：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、TIM1、SLAM、CD30和LIGHT。
  40. 根据权利要求39所述的嵌合抗原受体，所述铰链区包含源自CD8的铰链区。
  41. 根据权利要求39-40中任一项所述的嵌合抗原受体，所述铰链区包含SEQ ID NO:114至SEQ ID NO:135中任一项所示的氨基酸序列。
  42. 根据权利要求1-41中任一项所述的嵌合抗原受体，所述嵌合抗原受体包含非靶向部分，所述嵌合抗原受体的非靶向部分包含CD8A的跨膜域、CD8的铰链区、4-1BB的胞内共刺激信号传导结构域和CD3ζ胞内信号转导结构域。
  43. 根据权利要求42所述的嵌合抗原受体，所述嵌合抗原受体的非靶向部分包含SEQ ID NO:19中任一项所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的嵌合抗原受体，其包含SEQ ID NO:199所示的氨基酸序列。
  45. 分离的核酸分子，其编码权利要求1-44中任一项所述的嵌合抗原受体。
  46. 载体，其包含权利要求45所述的分离的核酸分子。
  47. 根据权利要求46所述的载体，其包括病毒载体。
  48. 根据权利要求46-47中任一项所述的载体，其包括慢病毒载体。
  49. 免疫细胞，其包含和/或表达权利要求45所述的分离的核酸分子或权利要求46-48中任一项所述的载体，和/或权利要求1-44中任一项所述的嵌合抗原受体。
  50. 根据权利要求49所述的免疫细胞，其中所述的免疫细胞来源于人。
  51. 根据权利要求49-50中任一项所述的免疫细胞，其中所述的免疫细胞包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、γδT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
  52. 根据权利要求49-51中任一项所述的免疫细胞，其包括T细胞。
  53. 根据权利要求49-52中任一项所述的免疫细胞，其中所述的免疫细胞包括经修饰的免疫细胞。
  54. 根据权利要求53所述的免疫细胞，其中所述经修饰的免疫细胞包括降低同种异体细胞治疗引起的免疫排斥反应的细胞。
  55. 根据权利要求53-54中任一项所述的免疫细胞，其中所述经修饰的免疫细胞中的T细胞抗原受体(TCR)和主要组织相容性复合体(MHCI，MHCII)在T细胞中的功能受到抑制。
  56. 根据权利要求53-55中任一项所述的免疫细胞，其中所述修饰包括与免疫排斥相关基因中的一个或多个的表达和/或活性被下调。
  57. 根据权利要求56所述的免疫细胞，其中所述与免疫排斥相关基因选自下组中的一种或多种基因：TRAC、TRBC、HLA-A、HLA-B、B2M和CIITA。
  58. 根据权利要求53-57中任一项所述的免疫细胞，其中所述经修饰的免疫细胞与未经修饰的相应细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调。
  59. 根据权利要求53-58中任一项所述的免疫细胞，其中所述经修饰的免疫细胞与未经所述修饰的相应细胞相比，CIITA基因的表达和/或活性未被下调。
  60. 根据权利要求53-59中任一项所述的免疫细胞，其中所述经修饰的免疫细胞与未经所述修饰的相应细胞相比，B2M基因的表达和/或活性未被下调。
  61. 根据权利要求53-60中任一项所述的免疫细胞，其中所述经修饰的免疫细胞与相应的野生型细胞相比，TRAC基因和HLA-A基因的表达和/或活性被下调。
  62. 根据权利要求53-61中任一项所述的免疫细胞，其中所述经修饰的免疫细胞与相应的野生型细胞相比，B2M基因的表达和/或活性未被下调。
  63. 根据权利要求53-62中任一项所述的免疫细胞，其中所述经修饰的免疫细胞与相应的野生型细胞相比，CIITA基因的表达和/或活性未被下调。
  64. 根据权利要求56-63中任一项所述的免疫细胞，其中所述基因的表达水平和/或活性被下调包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
  65. 根据权利要求53-64中任一项所述的免疫细胞，其中所述修饰包括：基因敲除、基因突变和/或基因沉默。
  66. 根据权利要求53-65中任一项所述的免疫细胞，其中所述修饰包括向所述免疫细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和sgRNA。
  67. 根据权利要求53-66中任一项所述的免疫细胞，其中所述修饰还包括向所述免疫细胞施用靶向所述TRAC基因外显子部分的sgRNA。
  68. 根据权利要求67所述的免疫细胞，其中所述靶向所述TRAC基因外显子部分的sgRNA包含SEQ ID NO:138至SEQ ID NO:152中任一项所示的核苷酸序列。
  69. 根据权利要求53-68中任一项所述的免疫细胞，其中所述修饰包括向所述免疫细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
  70. 根据权利要求69所述的免疫细胞，其中所述靶向所述HLA-A基因外显子部分的sgRNA包含SEQ ID NO:153至SEQ ID NO:193中任一项所示的核苷酸序列。
  71. 根据权利要求53-70中任一项所述的免疫细胞，其中所述修饰还包括向所述细胞施用Cas酶。
  72. 根据权利要求71所述的免疫细胞，其中Cas酶包括Cas9蛋白。
  73. 根据权利要求53-72中任一项所述的免疫细胞，其中所述修饰包括向所述细胞施用反义RNA，所述反义RNA包含SEQ ID NO:194至SEQ ID NO:197中任一项所示的核苷酸序列。
  74. 根据权利要求49-73中任一项所述的免疫细胞，其中所述免疫细胞为HLA-B纯合子细胞。
  75. 根据权利要求74所述的免疫细胞，其中所述HLA-B纯合子包括HLA-B*40纯合子，HLA-B*15纯合子，HLA-B*46纯合子，HLA-B*13纯合子，HLA-B*51纯合子，HLA-B*58纯合子，HLA-B*07纯合子，HLA-B*35纯合子，HLA-B*44纯合子，HLA-B*52纯合子，HLA-B*57纯合子，HLA-B*54纯合子或HLA-B*55纯合子。
  76. 根据权利要求49-75中任一项所述的免疫细胞，其中所述免疫细胞为HLA-A纯合子或杂合子细胞。
  77. 根据权利要求76所述的免疫细胞，其中所述HLA-A纯合子或杂合子包括HLA-A*02纯合子，HLA-A*11纯合子，HLA-A*02/A*11杂合子或HLA-A*24纯合子。
  78. 一种制备免疫细胞的方法，其包括向免疫细胞中引入权利要求45所述的核酸分子和/或权利要求46-48中任一项所述的载体。
  79. 根据权利要求78所述的方法，其还包括：在向免疫细胞中引入权利要求45所述的核酸分子和/或权利要求46-48中任一项所述的载体之前/之后，修饰所述免疫细胞，所述修饰包括与免疫排斥相关基因中的一个或多个的表达和/或活性被下调。
  80. 根据权利要求79所述的方法，其中所述与免疫排斥相关基因选自下组中的一种或多种基因：TRAC、TRBC、HLA-A、HLA-B、B2M和CIITA。
  81. 根据权利要求78-80中任一项所述的方法，与未经所述修饰的免疫细胞相比，使所述免疫细胞中TRAC基因和HLA-A基因的表达和/或活性下调。
  82. 根据权利要求78-81中任一项所述的方法，与未经所述修饰的免疫细胞相比，所述免疫细胞中的CIITA基因的表达和/或活性未被下调。
  83. 根据权利要求78-82中任一项所述的方法，与未经所述修饰的免疫细胞相比，所述免疫细胞中的B2M基因的表达和/或活性未被下调。
  84. 根据权利要求78-83中任一项所述的方法，与野生型细胞相比，使所述免疫细胞中的TRAC基因和HLA-A基因的表达和/或活性被下调。
  85. 根据权利要求78-84中任一项所述的方法，与野生型细胞相比，所述免疫细胞中的CIITA基因的表达和/或活性未被下调。
  86. 根据权利要求78-85中任一项所述的方法，与野生型细胞相比，所述免疫细胞中的B2M基因的表达和/或活性未被下调。
  87. 根据权利要求79-86中任一项所述的方法，其中所述基因的表达水平和/或活性被下调包括使编码所述基因的核酸分子的表达和/或活性下调；和/或使所述基因编码的蛋白质产物的表达和/或活性被下调。
  88. 根据权利要求79-87中任一项所述的方法，其中所述修饰包括：基因敲除、基因突变和/或基因沉默。
  89. 根据权利要求79-88中任一项所述的方法，其中所述修饰包括向所述免疫细胞施用一种或多种选自下组的物质：反义RNA、siRNA、shRNA和sgRNA。
  90. 根据权利要求79-89中任一项所述的方法，其中所述修饰包括向所述免疫细胞施用靶向所述TRAC基因外显子部分的sgRNA。
  91. 根据权利要求90所述的方法，其中所述靶向所述TRAC基因外显子部分的sgRNA包含SEQ ID NO:138至SEQ ID NO:152中任一项所示的核苷酸序列。
  92. 根据权利要求79-91中任一项所述的方法，其中所述修饰包括向所述免疫细胞施用靶向所述HLA-A基因外显子部分的sgRNA。
  93. 根据权利要求92所述的方法，其中所述靶向所述HLA-A基因外显子部分的sgRNA包含SEQ ID NO:153至SEQ ID NO:193中任一项所示的核苷酸序列。
  94. 根据权利要求79-93任一项所述的方法，其中所述修饰还包括向所述细胞施用Cas酶。
  95. 根据权利要求94所述的方法，其中Cas酶包括Cas9蛋白。
  96. 根据权利要求79-95中任一项所述的方法，其中所述修饰包括向所述细胞施用反义RNA，所述反义RNA包含SEQ ID NO:194至SEQ ID NO:197中任一项所示的核苷酸序列。
  97. 根据权利要求78-96中任一项所述的方法，其中所述免疫细胞来源于人。
  98. 根据权利要求78-97中任一项所述的方法，所述免疫细胞包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、γδT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
  99. 根据权利要求78-98中任一项所述的方法，其中所述免疫细胞包括T细胞。
  100. 根据权利要求78-99中任一项所述的方法，其中所述免疫细胞为HLA-B纯合子细胞。
  101. 根据权利要求100所述的方法，其中所述HLA-B纯合子包括HLA-B*40纯合子，HLA-B*15纯合子，HLA-B*46纯合子，HLA-B*13纯合子，HLA-B*51纯合子，HLA-B*58纯合子，HLA-B*07纯合子，HLA-B*35纯合子，HLA-B*44纯合子，HLA-B*52纯合子，HLA-B*57纯合子，HLA-B*54纯合子或HLA-B*55纯合子。
  102. 根据权利要求78-101中任一项所述的方法，其中所述免疫细胞为HLA-A纯合子或杂合子细胞。
  103. 根据权利要求102所述的方法，其中所述HLA-A纯合子或杂合子包括HLA-A*02纯合子，HLA-A*11纯合子，HLA-A*02/A*11杂合子或HLA-A*24纯合子。
  104. 权利要求1-44中任一项所述的嵌合抗原受体，权利要求45所述的分离的核酸分子，权利要求46-48中任一项所述的载体，和/或权利要求49-77中任一项所述的免疫细胞在制备CAR-T细胞中的应用。
  105. 药物组合物，其包含权利要求1-44中任一项所述的嵌合抗原受体，权利要求45所述的分离的核酸分子，权利要求46-48中任一项所述的载体，和/或权利要求49-77中任一项所述的免疫细胞，以及任选地药学上可接受的载剂。
  106. 权利要求1-44中任一项所述的抗原嵌合受体，权利要求45所述的分离的核酸分子，权利要求46-48中任一项所述的载体，权利要求49-77中任一项所述的免疫细胞，和/或权利要求105所述的药物组合物，其用于治疗与EGFR的表达相关的疾病或病症。
  107. 根据权利要求106所述的用途，其中所述与EGFR的表达相关的疾病或病症包括与EGFR的表达上调相关的疾病或病症。
  108. 根据权利要求106-107中任一项所述的用途，其中所述与EGFR的表达相关的疾病或病症包括肿瘤。
  109. 根据权利要求108所述的用途，其中所述肿瘤包括EGFR阳性的肿瘤。
  110. 根据权利要求108-109中任一项所述的用途，其中所述肿瘤包括实体瘤。
  111. 根据权利要求108-110中任一项所述的用途，其中所述肿瘤包括血液瘤。
  112. 根据权利要求108-111中任一项所述的用途，其中所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、宫颈癌、食管癌、胰腺癌、眼癌、间皮瘤、胆道癌、前列腺癌、肝癌、纤维肉瘤、结肠癌和/或胃癌。
  113. 根据权利要求108-112中任一项所述的用途，其中所述肿瘤包括纤维肉瘤。
  114. 权利要求1-44中任一项所述的嵌合抗原受体，权利要求45所述的分离的核酸分子，权利要求46-48中任一项所述的载体，权利要求49-77中任一项所述的免疫细胞，和/或权利要求105所述的药物组合物在制备药物中的用途，所述药物用于治疗与EGFR的表达相关的疾病或病症。
  115. 根据权利要求114所述的用途，其中所述与EGFR的表达相关的疾病或病症包括与EGFR的表达上调相关的疾病或病症。
  116. 根据权利要求114-115中任一项所述的用途，其中所述与EGFR的表达相关的疾病或病症包括肿瘤。
  117. 根据权利要求116所述的用途，其中所述肿瘤包括EGFR阳性的肿瘤。
  118. 根据权利要求116-117中任一项所述的用途，其中所述肿瘤包括实体瘤。
  119. 根据权利要求116-118中任一项所述的用途，其中所述肿瘤包括血液瘤。
  120. 根据权利要求116-119中任一项所述的用途，其中所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、宫颈癌、食管癌、胰腺癌、眼癌、间皮瘤、胆道癌、前列腺癌、肝癌、纤维肉瘤、结肠癌和/或胃癌。
  121. 根据权利要求116-120中任一项所述的用途，其中所述肿瘤包括纤维肉瘤。
  122. 预防和/或治疗与EGFR的表达相关的疾病或病症的方法，其包括向有需要的受试者施用有效量的权利要求1-44中任一项所述的嵌合抗原受体，权利要求45所述的分离的核酸分子，权利要求46-48中任一项所述的载体，权利要求49-77中任一项所述的免疫细胞，和/或权利要求105所述的药物组合物。
  123. 根据权利要求122所述的方法，其中所述与EGFR的表达相关的疾病或病症包括与EGFR的表达上调相关的疾病或病症。
  124. 根据权利要求122-123中任一项所述的方法，其中所述与EGFR的表达相关的疾病或病症包括肿瘤。
  125. 根据权利要求124所述的方法，其中所述肿瘤包括EGFR阳性的肿瘤。
  126. 根据权利要求124-125中任一项所述的方法，其中所述肿瘤包括实体瘤。
  127. 根据权利要求124-126中任一项所述的方法，其中所述肿瘤包括血液瘤。
  128. 根据权利要求124-127中任一项所述的方法，其中所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、宫颈癌、食管癌、胰腺癌、眼癌、间皮瘤、胆道癌、前列腺癌、肝癌、纤维肉瘤、结肠癌和/或胃癌。
  129. 根据权利要求124-128中任一项所述的方法，其中所述肿瘤包括纤维肉瘤。
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