WO2021147121A1 - 修饰的免疫细胞及其应用 - Google Patents

修饰的免疫细胞及其应用 Download PDF

Info

Publication number
WO2021147121A1
WO2021147121A1 PCT/CN2020/074252 CN2020074252W WO2021147121A1 WO 2021147121 A1 WO2021147121 A1 WO 2021147121A1 CN 2020074252 W CN2020074252 W CN 2020074252W WO 2021147121 A1 WO2021147121 A1 WO 2021147121A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
car
tumor
immune cell
cell
Prior art date
Application number
PCT/CN2020/074252
Other languages
English (en)
French (fr)
Inventor
赵同标
靳利远
朱颖婕
曹磊
曹佳妮
李晓燕
Original Assignee
中国科学院动物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院动物研究所 filed Critical 中国科学院动物研究所
Priority to JP2022567698A priority Critical patent/JP7457899B2/ja
Publication of WO2021147121A1 publication Critical patent/WO2021147121A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464404—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464436—Cytokines
    • A61K39/464442—Chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention belongs to the field of immune cell therapy. Specifically, the present invention relates to genetically modified immune cells to enhance homing and aggregation in solid tumors, thereby enhancing the therapeutic effect of immune cells such as CAR-T cells on solid tumors.
  • ACT adoptive cell therapy
  • CAR-T and TCR-T are important components of adoptive cell therapy, especially CAR-T therapy, which has made remarkable achievements in the treatment of hematological tumors and achieved a high remission rate.
  • a typical CAR structure consists of three parts. It consists of scFv, hinge and transmembrane domains, intracellular costimulatory signals and activation domains that recognize tumor antigens extracellularly.
  • the first-generation CAR does not contain intracellular costimulatory signals, and CAR-T cells have lower killing activity and shorter survival time. Therefore, the second-generation CAR started to add costimulatory signals such as CD28 and 4-1BB, and the characteristics of CAR-T cells using different costimulatory signals were not the same.
  • CD28 enhanced the killing activity of CAR-T cells while 4-1BB It enhances the killing activity of CAR-T cells and prolongs the survival time of CAR-T cells. Subsequently, three-generation CARs that co-express two costimulatory signal domains appeared, but their anti-tumor effects were not as good as the second-generation CAR-T. Therefore, the current clinical applications are mainly second-generation CAR-T cells.
  • CAR-T therapy has not only achieved remarkable results in the treatment of hematological tumors, but also has progressed smoothly in commercialization.
  • the US FDA officially approved the marketing of two CAR-T drugs in 2017.
  • CAR-T cell therapy shines in the treatment of hematological tumors, it does not have a good therapeutic effect for solid tumors.
  • the remission rate is low and toxic side effects such as off-target are prone to occur.
  • the inability of CAR-T cells to effectively accumulate in solid tumors is one of the important factors affecting its efficacy. Although there have been studies using local infusion of CAR-T cells to increase the number of tumor cells and enhance their anti-tumor activity However, this infusion method has a smaller range of use.
  • Patent CN110144325A a targeted T lymphocyte and its preparation method and application, provides a targeted T lymphocyte, including a chimeric antigen receptor CARDR5 targeting DR5 and/or a chimera targeting cMet Antigen receptor CAR c Met, CAR DR5 can specifically target DR5, CAR c Met can specifically target c Met, thereby promoting the expansion of T cells in patients, and killing tumor cells efficiently and specifically , And can effectively inhibit the occurrence of tumor cell escape, and better maintain the vitality and lethality of cells.
  • the chemokine system plays a vital role in the migration of immune cells. For example, the homing of T cells to lymph nodes and the residence of hematopoietic stem cells in the bone marrow all require the participation of the chemokine system.
  • Chemokines are not only related to the migration of immune cells, but also involved in the proliferation and metastasis of tumor cells. Tumor cells often secrete a large number of chemokines to promote their proliferation and metastasis. There are relatively few studies on chemokines to improve T cell targeting.
  • the currently published literature includes "Enhanced tumor traffic of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b, J Immunother, 2010", "T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model, Blood, 2009”, “CXCR1-or CXCR2-modified CAR-T cells co-opt IL-8 for maximum tumor efficiency, antitumor IL-8 for maximum Commun, 2019”, CAR-T cells co-expressing CCR4 or CCR2b or CXCR2 are beneficial to infiltrate into tumor cells expressing CCL17/CCL22 or CCL2 or CXCL8.
  • the present invention provides genetically modified immune cells aimed at enhancing the accumulation of modified immune cells at solid tumors, and aims to solve the defect of low killing activity of modified immune cells such as CAR-T cells in solid tumors.
  • the present invention provides modified immune cells, which include chimeric antigen receptor CAR and chemokine receptor CCR6;
  • the immune cell expresses the chimeric antigen receptor CAR and overexpresses the chemokine receptor CCR6;
  • the immune cells are T cells, NK cells, NKT cells, and macrophages; preferably T cells;
  • the chemokine receptor CCR6 is overexpressed
  • the target gene is transferred to immune cells for expression to obtain chimeric antigen receptor CAR and chemokine receptor CCR6;
  • the methods of transferring the target gene into immune cells include lentivirus, retrovirus, adenovirus, ordinary plasmid vector, episomal vector, nano-delivery system, electrotransduction, transposon, and other genes that can achieve the target gene.
  • the delivery system into the immune cells preferably, the virus is a lentivirus, such as FUW, CSII-EF-MCS or pCDH and other conventional lentiviruses in the art.
  • the chemokine system is CCL20-CCR6.
  • the cytokine secretion of CAR-T cells overexpressing the chemokine receptor CCR6 is not affected.
  • CAR-T cells overexpressing the chemokine receptor CCR6 have no effect on the specific killing ability of tumor cells.
  • CAR-T cells overexpressing the chemokine receptor CCR6 obviously migrate into the tumor secreting CCL20.
  • the CAR includes a leader sequence, an antigen-recognizing scFv, a hinge region and a transmembrane domain, an intracellular costimulatory signal, and an intracellular activation signal.
  • the leader sequence is derived from GM-CSF, CD3, CD4, CD8.
  • the hinge and transmembrane region are derived from CD28, CD3, CD4, CD8, IgG.
  • the intracellular costimulatory signal is derived from CD28, CD134, CD137 (4-1BB), ICOS, NKG2D.
  • the present invention provides the application of the immune cells in medicines for the treatment of related diseases
  • the drug is a drug related to cell therapy
  • the related disease is a tumor, preferably a solid tumor
  • the solid tumor overexpresses CCL20;
  • cervical squamous cell carcinoma and adenocarcinoma colon cancer, esophageal cancer, multiforming glioma, head and neck squamous cell carcinoma, renal clear cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, Pancreatic cancer, rectal cancer, gastric cancer and endometrial cancer;
  • lung adenocarcinoma and lung squamous cell carcinoma More preferably, lung adenocarcinoma and lung squamous cell carcinoma
  • the present invention provides an expression system, which is composed of a CAR expression vector and a CCR6 expression vector; preferably, the vector is a lentiviral vector.
  • the present invention also provides a method for obtaining the immune cells, including:
  • the recombinant plasmids are respectively packaged to obtain the first lentiviral vector and the second lentiviral vector,
  • the two lentiviral vectors are sequentially or co-transfected into immune cells and separated.
  • the CAR coding gene and the CCR6 coding gene are concatenated through 2A sequence or IRES sequence, and then the tandem sequence is inserted into the lentiviral expression plasmid to obtain CAR-2A-CCR6 and CCR6-2A-CAR or CAR-IRES-CCR6 and CCR6 -IRES-CAR.
  • tandem plasmids are respectively packaged to obtain lentiviral vectors
  • the lentiviral vector is stained with immune cells and isolated.
  • the present invention also provides the construction and expression of chimeric antigen receptors.
  • the genes encoding CAR, CCR6, CAR-2A-CCR6, CCR6-2A-CAR, CAR-IRES-CCR, and CCR6-IRES-CAR encoding genes are artificially synthesized, And add the XbaI restriction site at the front end of the sequence, and add the FseI restriction site at the end of the sequence.
  • XbaI and FseI are used to digest the lentiviral expression plasmid and the synthetic gene of interest.
  • the lentiviral expression plasmid and the target synthetic gene after digestion with T4 ligase are used.
  • the T cell culture system is supplemented with 100 UI/mL IL-2 or 10 ng/mL IL7/IL15 combination or contains other factors that are beneficial to T cell culture.
  • the CAR includes a leader sequence, an antigen-recognizing scFv, a hinge and transmembrane domain, an intracellular costimulatory signal, and an intracellular activation signal.
  • the nucleotide sequence of the leader sequence of the CAR is derived from the sequence SEQ ID NO:1.
  • the nucleotide sequence of the scFv targeting the tumor-associated antigen EGFR of the CAR is derived from the sequence SEQ ID NO: 2.
  • nucleotide sequence of the hinge and transmembrane domain of the CAR is derived from the sequence SEQ ID NO: 3.
  • the nucleotide sequence of the intracellular costimulatory signal CD28 of the CAR is derived from the sequence SEQ ID NO: 4.
  • the nucleotide sequence of the intracellular activation signal CD3 ⁇ of the CAR is derived from the sequence SEQ ID NO: 5.
  • the amino acid sequence of the leader sequence of the CAR is derived from the sequence SEQ ID NO: 6.
  • the EGFR-targeting scFv amino acid sequence of the CAR is derived from the sequence SEQ ID NO: 7.
  • amino acid sequence of the hinge and transmembrane domain of the CAR is derived from the sequence SEQ ID NO: 8.
  • amino acid sequence of the intracellular costimulatory signal CD28 of the CAR is derived from the sequence SEQ ID NO: 9.
  • amino acid sequence of the intracellular activation signal CD3 ⁇ of the CAR is derived from the sequence SEQ ID NO: 10.
  • the intracellular costimulatory signal is derived from CD28, CD134, CD137, ICOS.
  • the nucleotide sequence of the chemokine receptor CCR6 is from the sequence SEQ ID NO: 11; the amino acid sequence is from the sequence SEQ ID NO: 12.
  • the tumor-associated antigen can also be selected from AFP, BMCA, CEA, CA-125, CA19-9, CA72-4, CD116, CD117, CD171, CD123, CD16, CD19, CD20, CD22, CD30 , CD61, CD64, CD70, CD71, CD78, CD96, CLL1, CD133, CD138, Her2, GD2, gp36, GPC3, GnTV, EGFRvIII, EpCAM, PSA, PSMA, PAP, NY-ESO-1, LAGA-1a, cMET , P53, Prostein, PCTA-1, MAGE, ROR1, FAP, NKG2D, EMA, ETA, GFAP, MSA, MART-1, NSE, TAG-72, IL13R ⁇ 2, NKR-2, ROR1, VEGFR2, VEGFR, CLD18, EphA2 , Alpha-folateR, CAIX, MG7, LMP1, PD-L1, MUC1, mesothe
  • the beneficial effects achieved by the present invention mainly include but are not limited to the following aspects:
  • the present invention systematically compares the relationship between the chemokine CCL20 and solid tumors, and uses clinical cases of lung cancer and human lung cancer cell lines for verification.
  • the modified CAR-T cells of the present invention significantly promote the accumulation of CAR-T cells in solid tumors, solve the disadvantage that CAR-T cells cannot effectively contact solid tumors, and promote the infiltration of CAR-T cells into solid tumors. Further enhance the anti-tumor activity of CAR-T cells, so that CAR-T cell therapy technology can be applied to treat solid tumors, and overcome the technical defect that CAR-T has poor therapeutic effect on solid tumors in the prior art.
  • the modified CCR6+CAR-T cells of the present invention can be applied to a variety of solid tumors and have wider applicability.
  • the dual-plasmid system is more convenient to combine CCR6 and CAR targeting different antigens, and better expands CCR6+CAR- The scope of application of T.
  • the solid tumors to which the modified CCR6+CAR-T cells of the present invention are applicable include a variety of malignant and highly harmful tumors, including lung cancer, liver cancer, colon cancer, rectal cancer, stomach cancer, kidney cancer, pancreatic cancer, breast cancer, Main organ tumors such as prostate cancer.
  • the modified CCR6+CAR-T cell of the present invention can not only eliminate the primary tumor of the tumor, but also can better track and kill tumor metastases with high expression of CCL20 by using the chemokine system.
  • the cytokine secretion ability and specific killing ability of the modified CCR6+CAR-T cells of the present invention are not affected; however, the ability of homing and aggregation to secreting CCL20 solid tumors is enhanced, and the patient's survival time is prolonged.
  • FIG. 1 Bioinformatics analysis of CCL20 mRNA expression levels in tumor tissues and adjacent normal tissues in the TCGA database.
  • CCL20 is highly expressed in tumor tissues of a variety of solid tumors.
  • Figure 2 ELISA detects the secretion level of CCL20 in the tumor tissues of lung cancer patients and normal tissues adjacent to the cancer. CCL20 is highly expressed in the tumor tissues of most lung cancer patients.
  • Figure 3 ELISA detects the secretion level of human lung cancer cell line CCL20.
  • Normal human lung epithelial cells BEAS-2B hardly secrete CCL20, while H23, A549 and CCL20 overexpression cell lines A549/CCL20 secrete high levels of CCL20.
  • chemokine receptor CCR6 corresponding to CCL20 has low expression levels in the T cells (resting state and activated state) of lung cancer patients, especially in the killer CD8 T cells.
  • FIG. 5 CCR6 and EGFRCAR lentiviral particles infect T cells, and their infection efficiency was analyzed by flow cytometry. The proportion of T cells co-infected with CCR6+EGFRCAR was 23%.
  • FIG. 8 Jurkat T cells overexpressing CCR6 significantly migrate to the tumor cell culture medium containing CCL20.
  • Bioinformatics screens the highly expressed chemokines in solid tumors.
  • TCGA Cancer Genome Atlas
  • Head and neck squamous cell carcinoma, renal clear cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer, endometrial cancer, and uterine sarcoma showed overexpression levels.
  • Follow-up operations were performed to detect the secretion level of CCL20 according to the instructions of the ELISA kit. As shown in Figure 2, CCL20 was overexpressed in the tumor tissues of most patients. Subsequently, the level of CCL20 secretion in human lung cancer cell lines was further tested.
  • CCL20 was detected according to the instructions. As shown in Figure 3, CCL20 was overexpressed in lung cancer cell lines A549, H23 and CCL20 The secretion level in line A549/CCL20 is higher.
  • CCR6 the corresponding receptor of CCL20
  • the lymphocyte separation fluid was used to separate and enrich the peripheral blood PBMCs of lung cancer patients, collect resting and CD3/CD28 beads activated T cells, and then add them.
  • CD4, CD8 and CCR6 flow cytometry antibodies were stained at 4°C and protected from light for 20 minutes, washed once with PBS and transferred to a flow cytometry tube for detection by BD's Aria flow cytometer.
  • CCR6 is low in CD4 T cells and CD8 T cells, especially in CD8 T cells. Therefore, we tried to overexpress CCR6 in T cells to verify whether it can enhance the homing and aggregation of T cells in solid tumors, thereby improving the killing effect.
  • the expression system consists of a vector for expressing CAR and a vector for expressing CCR6; CAR in turn contains leader sequence, antigen-recognizing scFv, hinge and transmembrane domain, intracellular costimulatory signal, intracellular activation signal CD3 ⁇ ; vector is lentiviral vector FUW (See Chimeric antigen receptor T(CAR-T)cells expanded with IL-7/IL-15mediate superior antitumor effects, Protein&Cell, June 2019).
  • the CAR coding gene and CCR6 coding gene were artificially synthesized, and the XbaI restriction site was added to the front end of the sequence, and the FseI restriction site was added to the end of the sequence to be connected to the cloning plasmid; XbaI and FseI were used to cut the lentivirus
  • the expression plasmid and the cloned plasmid containing the target gene are then subjected to agarose gel electrophoresis and gel cutting to recover the linearized lentiviral expression plasmid and target gene fragments; use T4 ligase to ligate the lentiviral expression plasmid and target gene recovered by the cutting gel, And transformed into DH5 ⁇ strain, coated on ampicillin-resistant LB solid plate and cultured overnight; picked out the monoclonal colonies on the plate, inoculated into ampicillin-resistant LB liquid medium and cultivated overnight for 12-14 hours, then small
  • the plasmid was extracted by quantity and verified by
  • the CAR recombinant plasmid and CCR6 recombinant plasmid were extracted without endotoxin in large quantities, and were transfected with psPAX2 and pMD2.G virus packaging plasmids into the virus packaging cells 293T through calcium phosphate at a ratio of 10:9:6, respectively, and collected after 48h
  • the nucleotide sequence of the leader sequence of the CAR is derived from the sequence SEQ ID NO:1.
  • the nucleotide sequence of the scFv targeting the tumor-associated antigen EGFR of the CAR is derived from the sequence SEQ ID NO: 2.
  • nucleotide sequence of the hinge and transmembrane domain of the CAR is derived from the sequence SEQ ID NO: 3.
  • the nucleotide sequence of the intracellular costimulatory signal CD28 of the CAR is derived from the sequence SEQ ID NO: 4.
  • the nucleotide sequence of the intracellular activation signal CD3 ⁇ of the CAR is derived from the sequence SEQ ID NO: 5.
  • the amino acid sequence of the leader sequence of the CAR is derived from the sequence SEQ ID NO: 6.
  • the EGFR-targeting scFv amino acid sequence of the CAR is derived from the sequence SEQ ID NO: 7.
  • amino acid sequence of the hinge and transmembrane domain of the CAR is derived from the sequence SEQ ID NO: 8.
  • amino acid sequence of the intracellular costimulatory signal CD28 of the CAR is derived from the sequence SEQ ID NO: 9.
  • amino acid sequence of the intracellular activation signal CD3 ⁇ of the CAR is derived from the sequence SEQ ID NO: 10.
  • the nucleotide sequence of the CCR6 is from the sequence SEQ ID NO: 11, and the amino acid sequence is from the sequence SEQ ID NO: 12.
  • the lung adenocarcinoma target EGFR and CCL20-CCR6 system were taken as examples, and the EGFR-targeting CAR and chemokine receptor CCR6 lentiviral particles were added at the ratio of infection multiplicity of 10 for infection.
  • the infection efficiency of T cells was detected by flow cytometry. As shown in Figure 5, T cells were successfully infected with CAR and CCR6, and the ratio of CAR and CCR6 double-positive T cells reached 23%.
  • CAR T cells overexpressing CCR6 more effectively killed tumor cells and prolonged the survival time of tumor-bearing mice.

Landscapes

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

Abstract

提供了基因修饰的免疫细胞，该免疫细胞表达嵌合抗原受体并过表达趋化因子受体CCR6，增强了免疫细胞在实体肿瘤处的归巢和聚集以及实体瘤浸润，进而增强了免疫细胞对于实体肿瘤的治疗效果。还提供了修饰免疫细胞的方法和免疫细胞在制备治疗肿瘤的药物中的应用。

Description

修饰的免疫细胞及其应用 技术领域
本发明属于免疫细胞治疗领域。具体而言，本发明涉及通过基因修饰的免疫细胞，使其增强在实体肿瘤内的归巢和聚集，进而增强免疫细胞例如CAR-T细胞对于实体肿瘤的治疗效果。
背景技术
近几年来，肿瘤免疫疗法发展迅速，尤其是过继性细胞疗法(ACT)，即指从病人体内分离出T细胞、NK细胞等免疫细胞，通过体外修饰扩增培养，随后回输入患者体内进行肿瘤治疗的方法。2013年，肿瘤的免疫疗法被Science杂质评为“十大突破”之首。
CAR-T和TCR-T是过继性细胞疗法重要的组成部分，尤其CAR-T疗法，在血液肿瘤的治疗上取得了显著的成就，取得了较高的缓解率，典型的CAR结构由三部分组成，胞外识别肿瘤抗原的scFv、铰链和跨膜结构域、胞内共刺激信号和激活结构域。第一代的CAR并不包含胞内共刺激信号，CAR-T细胞的杀伤活性较低且生存时间较短。因此，第二代CAR开始加入共刺激信号如CD28和4-1BB，采用不同的共刺激信号的CAR-T细胞的特性也不尽相同，CD28增强了CAR-T细胞的杀伤活性而4-1BB则增强CAR-T细胞杀伤活性的同时延长了CAR-T细胞的生存时间。随后，出现了共同表达两个共刺激信号域的三代CAR，然而其抗肿瘤效果并不如二代CAR-T。因此，现在临床应用的主要为二代CAR-T细胞。
CAR-T疗法不仅在血液肿瘤的治疗上成果显著，而且商业化进展顺利，美国FDA于2017年正式批准了两种CAR-T药品上市。尽管CAR-T细胞疗法在血液肿瘤的治疗上大放异彩，而对于实体瘤却并没有较好的治疗效果，缓解率低且容易发生脱靶等毒副作用。CAR-T细胞不能有效的在实体瘤处聚集是影响其疗效的重要因素之一，虽然已有研究采用局部输注CAR-T细胞的方式以增强其在肿瘤细胞的数目进而增强其抗肿瘤活性，然而这种输注方式使用范围较小。因此，仍然需要新的方式诱导CAR-T细胞在实体肿瘤内的归巢聚集。科研人员对CAR-T细胞实体瘤治疗作了大量研究，采用多种方式对CAR-T细胞进行了改良，例如专利CN110157682A,人工靶向修饰的CAR-T细胞及其制备方法与应用,涉及代谢修饰的CAR-T细胞及其制备方法与应用，其通过在CAR-T细胞表面标记偶联有叠氮等的糖类、氨基酸等生物活性分子，使得生物活性分子在活体内可以有效地将CAR-T细胞靶向引导至肿瘤部位；而体内这种靶向与拉近使得CAR-T细胞上特异性CAR分子在肿瘤部位与肿瘤抗原结合，从而刺激CAR-T细胞特异性活化，达到更为高效的肿瘤杀伤能力。专利CN110144325A，一种靶向性T淋巴细胞及其制备方法和应用，提供了一种靶向性T淋巴细胞，包括靶向DR5的嵌合抗原受体CARDR5和/或靶向c Met的嵌合抗原受体CAR c Met，CAR DR5可以专一性的靶向DR5，CAR c Met可以专一性的靶向c Met，从而促进T细胞在患者体内的扩增，高效且特异性的杀伤肿瘤细胞，并且能够有效的抑制肿瘤细胞逃逸的发生，更好的维持细胞的活力和杀伤力。
趋化因子系统对于免疫细胞的迁移起着至关重要的作用。例如，T细胞向淋巴结的归巢、造血干细胞在骨髓中的驻留等过程都需要趋化因子系统的参与。趋化因子不仅与免疫细胞的迁移有关，还参与肿瘤细胞的增殖、转移等过程。肿瘤细胞往往分泌大量的趋化因子促进其增殖和转移。关于趋化因子提高T细胞靶向性的研究相对较少，目前公开的文献有“Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b,J Immunother,2010”，“T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model，Blood,2009”，“CXCR1-or CXCR2-modified CAR-T cells co-opt IL-8for maximal antitumor efficacy in solid tumors，Nat Commun，2019”，CAR-T细胞共表达CCR4或CCR2b或CXCR2有利于向表达CCL17/CCL22或CCL2或CXCL8的肿瘤细胞渗入，然而上述研究适用的实体瘤种类局限，缺乏对趋化因子家族和实体瘤关系的系统性研究，同时趋化因子是否在肿瘤中过表达缺乏临床数据的支持。此外，鉴于细胞因子的多样性以及细胞信号传导途径的复杂性，不同的趋化因子在不同的肿瘤中所起的作用也可能并不相同，理论分析往往和实际研究结果差距较大。
发明内容
本发明提供了基因修饰的免疫细胞，旨在增强经修饰的免疫细胞在实体瘤处聚集的方法，是为了解决经修饰的免疫细胞如CAR-T细胞在实体瘤杀伤活性较低的缺陷。
本发明是通过如下技术方案来实现的：
第一方面，本发明提供了修饰的免疫细胞，所述免疫细胞包括嵌合抗原受体CAR和趋化因子受体CCR6；
进一步地，所述免疫细胞表达嵌合抗原受体CAR，并且过表达趋化因子受体CCR6；
其中，免疫细胞为T细胞，NK细胞，NKT细胞，巨噬细胞；优选T细胞；
可选地，所述趋化因子受体CCR6是过表达的；
可选地，将目的基因转入到免疫细胞进行表达得到嵌合抗原受体CAR和趋化因子受体CCR6；
可选地，将目的基因转入到免疫细胞的方式包括慢病毒、逆转录病毒、腺病毒、普通质粒载体、附加体载体、纳米递送系统、电转导、转座子和其它能够实现目的基因进入免疫细胞的递送系统；优选地，所述病毒为慢病毒，例如FUW、CSII-EF-MCS或pCDH等本领域常规 的慢病毒。
优选地，所述趋化因子系统为CCL20-CCR6。
在本发明中，过表达趋化因子受体CCR6的CAR-T细胞的细胞因子分泌不受影响。
在本发明中，过表达趋化因子受体CCR6的CAR-T细胞对肿瘤细胞的特异性杀伤能力不受影响。
在本发明中，过表达趋化因子受体CCR6的CAR-T细胞明显向分泌CCL20的肿瘤内迁移。
在本发明中，所述CAR包含前导序列，识别抗原的scFv，铰链区和跨膜结构域，胞内共刺激信号，胞内激活信号。
任选地，前导序列来源于GM-CSF，CD3,CD4,CD8。
任选地，铰链和跨膜区来源与CD28,CD3,CD4,CD8,IgG。
优选地，胞内共刺激信号来源于CD28,CD134,CD137(4-1BB),ICOS,NKG2D。
第二方面，本发明提供了所述免疫细胞在治疗相关疾病的药物中应用；
进一步地，所述药物为细胞治疗相关的药物；
所述相关疾病为肿瘤，优选实体肿瘤；
可选地，所述实体肿瘤过表达CCL20；
优选地，宫颈鳞癌和腺癌，结肠癌，食管癌，多形成性胶质细胞瘤，头颈鳞状细胞癌，肾透明细胞癌，肝细胞癌，肺腺癌，肺鳞癌，卵巢癌，胰腺癌，直肠癌，胃癌和子宫内膜癌；
更优选地，肺腺癌和肺鳞癌；
第三方面，本发明提供了一种表达系统，其由表达CAR的载体和表达CCR6的载体组成；优选地，所述载体为慢病毒载体。
第四方面，本发明还提供了一种获得所述免疫细胞的方法，包括：
将CAR的编码基因和CCR6的编码基因分别插入到采用EF-1α启动子的慢病毒表达质粒中，得到CAR重组质粒和CCR6重组质粒；
分别对所述重组质粒进行包装，得到第一慢病毒载体和第二慢病毒载体，
将两种慢病毒载体按顺序或者共转染免疫细胞，分离。
将CAR的编码基因和CCR6的编码基因通过2A序列或IRES序列串联，再将串联序列插入到慢病毒表达质粒中，得到CAR-2A-CCR6和CCR6-2A-CAR或CAR-IRES-CCR6和CCR6-IRES-CAR。
分别对所述串联质粒进行包装，得到慢病毒载体，
将慢病毒载体染免疫细胞，分离。
第五方面，本发明还提供了嵌合抗原受体的构建和表达。
在一些实施方案中，将CAR的编码基因、CCR6的编码基因、CAR-2A-CCR6编码基因、CCR6-2A-CAR、CAR-IRES-CCR和CCR6-IRES-CAR编码基因的编码基因人工合成，并在序列的前端加入XbaI酶切位点，序列的末端加入FseI酶切位点。
在一些实施方案中，使用XbaI和FseI酶切慢病毒表达质粒和目的合成基因。
在一些实施方案中，使用T4连接酶酶切后的慢病毒表达质粒和目的合成基因。
在一些实施方案中，T细胞的培养体系添加100UI/mL的IL-2或10ng/mL的IL7/IL15组合或含有其它有利于T细胞培养的因子。
在一些实施方案中，所述CAR包含前导序列，识别抗原的scFv，铰链和跨膜结构域，胞内共刺激信号，胞内激活信号。
任选地，
所述CAR的前导序列的核苷酸序列来自序列SEQ ID NO：1。
所述CAR的靶向肿瘤相关抗原EGFR的scFv核苷酸序列来自序列SEQ ID NO:2。
所述CAR的铰链和跨膜结构域的核苷酸序列来自序列SEQ ID NO:3。
所述CAR的胞内共刺激信号CD28的核苷酸序列来自序列SEQ ID NO:4。
所述CAR的胞内激活信号CD3ζ的核苷酸序列来自序列SEQ ID NO:5。
所述CAR的前导序列的氨基酸序列来自序列SEQ ID NO：6。
所述CAR的靶向EGFR的scFv氨基酸序列来自序列SEQ ID NO:7。
所述CAR的铰链和跨膜结构域的氨基酸序列来自序列SEQ ID NO:8。
所述CAR的胞内共刺激信号CD28的氨基酸序列来自序列SEQ ID NO:9。
所述CAR的胞内激活信号CD3ζ的氨基酸序列来自序列SEQ ID NO:10。
可选地，胞内的共刺激信号来源于CD28,CD134,CD137,ICOS。
优选地，所述趋化因子受体CCR6的核苷酸序列来自序列SEQ ID NO:11；氨基酸序列来自序列SEQ ID NO:12。
在其他实施方案中，所述肿瘤相关抗原还可选自AFP、BMCA、CEA、CA-125、CA19-9、CA72-4、CD116、CD117、CD171、CD123、CD16、CD19、CD20、CD22、CD30、CD61、CD64、CD70、CD71、CD78、CD96、CLL1、CD133、CD138、Her2、GD2、gp36、GPC3、GnTV、EGFRvIII、EpCAM、PSA、PSMA、PAP、NY-ESO-1、LAGA-1a、cMET、P53、Prostein、PCTA-1、MAGE、ROR1、FAP、NKG2D、EMA、ETA、GFAP、MSA、MART-1、NSE、TAG-72、IL13Rα2、NKR-2、ROR1、VEGFR2、VEGFR、CLD18、EphA2、alpha-folateR、CAIX、MG7、LMP1、PD-L1、MUC1、间皮素、导管上皮粘蛋白、鞘糖脂、肿瘤基质抗原、内皮因子、钙网膜蛋白、酪氨酸酶、胎盘碱性磷酸酶、 甲状腺球蛋白、甲状腺转录因子1、异常ras蛋白、TPSA、FPSA、EB病毒抗原、EBNA、人HPV抗原E6和E7、端粒酶、HBV、gp100、MAGEA3、MAGEA4、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、ELF2M、嗜中性粒细胞蛋白弹性酶、胰岛素生长因子(IGF1)-1、IGF-II和IGFI受体。
与现有技术相比，本发明取得的有益效果主要包括但是并不限于以下几个方面：
本发明系统的比较了趋化因子CCL20和实体肿瘤的关系，并采用肺癌临床病例和人肺癌细胞系进行了验证。
本发明修饰的CAR-T细胞显著的促进了CAR-T细胞在实体肿瘤的聚集，解决了CAR-T细胞不能有效的接触实体肿瘤的劣势，并且促进了CAR-T细胞对实体肿瘤的浸润，进一步增强CAR-T细胞的抗肿瘤活性，使得CAR-T细胞治疗技术能够应用于治疗实体瘤，克服了现有技术中CAR-T对实体瘤治疗效果较差的技术缺陷。
本发明修饰的CCR6+CAR-T细胞可以适用于多种实体肿瘤，具有更广泛的适用性，双质粒系统更加方便的将CCR6和靶向不同抗原的CAR组合，更好的扩展CCR6+CAR-T的适用范围。
本发明修饰的CCR6+CAR-T细胞适用的实体肿瘤包括多种恶行程度和危害性较高的肿瘤，包括肺癌、肝癌、结肠癌、直肠癌、胃癌、肾癌、胰腺癌、乳腺癌、前列腺癌等主要脏器肿瘤。
本发明修饰的CCR6+CAR-T细胞不仅可以消灭肿瘤的原发灶，利用趋化因子系统还可以更好的追踪杀伤CCL20高表达的肿瘤转移灶。
本发明修饰的CCR6+CAR-T细胞的细胞因子分泌能力以及对肿瘤细胞的特异性杀伤能力不受影响；但是增强了向分泌CCL20实体瘤的归巢和聚集的能力，延长患者生存时间。
附图说明
图1 生物信息学分析TCGA数据库中肿瘤组织和癌旁正常组织中CCL20的mRNA表达水平，CCL20在多种实体瘤的肿瘤组织中高表达。
图2 ELISA检测肺癌病人肿瘤组织和癌旁正常组织的CCL20的分泌水平，CCL20在大部分肺癌病人的肿瘤组织中呈现高表达状态。
图3 ELISA检测人肺癌细胞系CCL20的分泌水平，正常人肺上皮细胞BEAS-2B几乎不分泌CCL20，H23、A549和CCL20过表达细胞系A549/CCL20分泌高水平的CCL20。
图4 CCL20对应的趋化因子受体CCR6在肺癌病人的T细胞中(静息状态和激活状态)表达水平较低，尤其在杀伤细胞CD8 T细胞中。
图5 CCR6和EGFRCAR慢病毒颗粒感染T细胞，并通过流式细胞术分析其感染效率，CCR6+EGFRCAR共同感染的T细胞比例为23％。
图6 过表达CCR6并不影响CAR-T细胞IL-2及IFN-γ等细胞因子分泌。
图7过表达CCR6并不影响CAR-T细胞对体外共培养的靶肿瘤细胞的特异性杀伤效果。
图8过表达CCR6的Jurkat T细胞显著的向含有CCL20的肿瘤细胞培养基迁移。
图9过表达CCR6+CAR的Jurkat T细胞显著的在肿瘤细胞(黄色圆圈)处聚集。
图10相同的输注剂量下，与传统的CAR-T细胞相比，CCR6+CAR T细胞拥有更好的抗肿瘤效果，荷瘤小鼠的肿瘤大小显著变小。
图11 CCR6+CAR T输注治疗的小鼠肿瘤中，检测出更多的肿瘤浸润T细胞，表明CCR6促使CAR-T细胞更多的在肿瘤细胞处聚集并进入到肿瘤内部。
图12与传统的CAR-T输注治疗相比，CCR6+CAR T输注治疗显著延长了荷瘤小鼠的生存时间。
具体实施方式
本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的产品及方法已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的产品及方法进行改动或适当变更与组合，来实现和应用本发明技术。为了进一步理解本发明，下面结合实施例对本发明进行详细说明。
实施例1
生物信息学筛选实体肿瘤中高表达的趋化因子。
肿瘤基因组图谱计划(The Cancer Genome Altas,TCGA)由美国NCI发起，旨在通过高通量测序技术加深人类对癌症分子机制的理解。TCGA是全球著名的癌症数据库，包含33种癌症类型，11000多个临床样本。通过GEPIA网站分析工具筛选TCGA中趋化因子CCL20在各种肿瘤中的表达水平，如图1所示，CCL20在宫颈鳞癌和腺癌，结肠癌，食管癌，多形成性胶质细胞瘤，头颈鳞状细胞癌，肾透明细胞癌，肝细胞癌，肺腺癌，肺鳞癌，卵巢癌，胰腺癌，直肠癌，胃癌，子宫内膜癌，子宫肉瘤中呈现过表达水平。
收集肺癌病人的肿瘤组织和癌旁组织，使用预冷的PBS冲洗肿瘤组织后称重，将肿瘤组织剪碎，按照肺癌组织：PBS＝0.1g:10mL的比例冰上充分研磨，5000g 4℃离心10min后取上清分装备用，按照ELISA试剂盒说明书进行后续操作检测CCL20的分泌水平，如图2所示CCL20在大多数病人的肿瘤组织中呈现过表达的状态。随后，进一步检测人肺癌细胞系的 CCL20分泌水平。以4×10 5的密度接种肿瘤细胞于培养皿中，24h后收集培养基上清，按照说明书检测CCL20的分泌水平，如图3所示，CCL20在肺癌细胞系A549,H23和CCL20过表达细胞系A549/CCL20中的分泌水平较高。
同时，通过流式细胞术检测CCL20的对应受体CCR6在T细胞中的表达情况，淋巴细胞分离液分离富集肺癌病人外周血PBMC，收集静息和CD3/CD28 beads激活的T细胞，随后加入CD4，CD8和CCR6流式抗体4℃避光染色20min，PBS清洗1次后转移至流式细胞管中，BD公司的Aria流式细胞仪检测。如图4所示，CCR6在CD4 T细胞和CD8 T细胞中均表达较低，尤其在CD8 T细胞中。因此，我们尝试通过在T细胞中过表达CCR6以验证是否可以增强T细胞在实体瘤的归巢和聚集，进而提高杀伤效果。
实施例2
表达系统的构建。
表达系统由表达CAR的载体和表达CCR6的载体组成；CAR依次包含前导序列，识别抗原的scFv，铰链和跨膜结构域，胞内共刺激信号，胞内激活信号CD3ζ；载体为慢病毒载体FUW(参见Chimeric antigen receptor T(CAR-T)cells expanded with IL-7/IL-15mediate superior antitumor effects,Protein&Cell，June 2019)。
将CAR的编码基因、CCR6的编码基因进行人工合成，并在序列的前端加入XbaI酶切位点，序列的末端加入FseI酶切位点，连入克隆质粒中；使用XbaI和FseI酶切慢病毒表达质粒和含有目的基因的克隆质粒,随后进行琼脂糖凝胶电泳并切胶回收线性化的慢病毒表达质粒和目的基因片段；使用T4连接酶连接切胶回收的慢病毒表达质粒和目的基因，并转化至DH5α菌株中，涂于氨苄抗性的LB固体平板中培养过夜；挑取菌板上的单克隆菌落，接种于氨苄抗性的LB液体培养基中过夜培养12-14小时，随后小量提取质粒并进行酶切验证，分别得到正确连接的CAR重组质粒和CCR6重组质粒。
将CAR重组质粒和CCR6重组质粒进行无内毒素的大量提取，并分别和psPAX2和pMD2.G病毒包装质粒按照10：9：6的比例通过磷酸钙转染至病毒包装细胞293T中，48h后收集含有病毒颗粒的培养基上清，0.45μm的滤器过滤培养基上清并转移至超速离心管中，通过低温超速离心(20000rpm，2h，4℃)浓缩病毒颗粒得到第一慢病毒载体和第二慢病毒载体，随后将两种慢病毒载体共转染免疫细胞。
所述CAR的前导序列的核苷酸序列来自序列SEQ ID NO：1。
所述CAR的靶向肿瘤相关抗原EGFR的scFv核苷酸序列来自序列SEQ ID NO:2。
所述CAR的铰链和跨膜结构域的核苷酸序列来自序列SEQ ID NO:3。
所述CAR的胞内共刺激信号CD28的核苷酸序列来自序列SEQ ID NO:4。
所述CAR的胞内激活信号CD3ζ的核苷酸序列来自序列SEQ ID NO:5。
所述CAR的前导序列的氨基酸序列来自序列SEQ ID NO：6。
所述CAR的靶向EGFR的scFv氨基酸序列来自序列SEQ ID NO:7。
所述CAR的铰链和跨膜结构域的氨基酸序列来自序列SEQ ID NO:8。
所述CAR的胞内共刺激信号CD28的氨基酸序列来自序列SEQ ID NO:9。
所述CAR的胞内激活信号CD3ζ的氨基酸序列来自序列SEQ ID NO:10。
所述CCR6的核苷酸序列来自序列SEQ ID NO:11，氨基酸序列选自序列SEQ ID NO:12。
实施例3
健康人外周血T细胞的分离培养及感染。
采取健康人的外周血，通过密度梯度离心的方法分离得到单个核细胞，随后使用美天旎公司的T细胞富集试剂盒进行T细胞的富集，接种于Lonza的X-VIVO 15培养基中并添加100UI/mL IL-2，随后按说明书加入赛默飞公司的CD3/CD28 Dynabeads进行T细胞的激活扩增。
待T细胞激活36h后，以肺腺癌的靶标EGFR和CCL20-CCR6系统为例，以感染复数为10的比例加入靶向EGFR的CAR和趋化因子受体CCR6的慢病毒颗粒进行感染。1周后，流式细胞仪检测T细胞的感染效率，如图5所示，T细胞成功的感染了CAR和CCR6，并且CAR和CCR6双阳的T细胞比例达到了23％。
实施例4
过表达CCR6不影响CAR-T细胞的因子分泌和细胞杀伤能力
将不同组的2×10 5T细胞和2×10 5肿瘤细胞共孵育24h，随后离心收集上清后，按照赛默飞公司的IL-2和IFN-gama ELISA试剂盒说明书进行后续操作。如图6所示，过表达CCR6并不影响CAR-T细胞的细胞因子分泌能力。随后，我们采用基于荧光素酶的细胞杀伤测定。首先，分别使用GFP-luc病毒颗粒感染靶向肿瘤细胞，得到H1975/luc，H23/luc，A549/luc和A549-CCL20/luc。然后，将不同组的T细胞和肿瘤细胞按照效靶比10：1进行共孵育24h，随后加入荧光素底物测定荧光素值以计算细胞的杀伤。不加T细胞的肿瘤细胞孔为参照，相比与参照孔，降低的荧光值比例即为杀伤效率，公式为：杀伤百分比＝(参照孔荧光值-目的孔荧光值)÷参照孔×100％。如图7所示，CCR6的过表达并不影响CAR-T细胞的杀伤功能。
实施例5
过表达CCR6促使Jurkat T细胞向分泌CCL20的肿瘤细胞迁移
为了验证趋化因子系统引导CAR-T细胞向肿瘤聚集的可行性，我们首先在Jurkat T细胞 系中过表达CCR6和EGFRCAR，随后接种各组细胞于transwell小室中，如图8所示，过表达CCR6的Jurkat细胞明显迁移向含有CCL20的肿瘤培养基上清。随后，接种1×10 6A549/CCL20细胞于小鼠背部皮下，10天后尾静脉输注感染CCR6和CAR病毒的Jurkat/luc细胞，随后与24h,48h和120h检测皮下肿瘤处的荧光强度。如图9所示，相比于EGFR CAR Jurkat细胞，EGFR CAR+CCR6Jurkat细胞明显的在皮下肿瘤处聚集。
实施例6
过表达CCR6的CAR T细胞更有效的杀伤了肿瘤细胞，并延长了荷瘤小鼠的生存时间。
为了进一步验证添加趋化因子受体CAR-T细胞的体内抗肿瘤活性，接种2×10 5H23/luc细胞于小鼠背部皮下，7天后尾静脉输注梯度剂量(1×10 7，3×10 6，1×10 6)的CAR T和CCR6+CAR T细胞，随后小动物成像仪检测荧光素强度来检测肿瘤细胞的大小。结果表明，相同输注剂量的条件下，CCR6+CAR T治疗组与传统CAR-T组相比具有更好的治疗效果(图10)，并且CCR6促进了CAR-T细胞浸润入肿瘤内部(图11)，发挥更好的抗肿瘤效果，并显著延长了荷瘤小鼠的生存时间(图12)。
以上列举的仅是本发明的最佳具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
Figure PCTCN2020074252-appb-000001
Figure PCTCN2020074252-appb-000002
Figure PCTCN2020074252-appb-000003
Figure PCTCN2020074252-appb-000004

Claims (17)

  1. 修饰的免疫细胞，其特征在于，所述免疫细胞表达嵌合抗原受体，并且过表达趋化因子受体CCR6。
  2. 根据权利要求1所述的修饰的免疫细胞，其特征在于，所述免疫细胞未经修饰之前不表达或低表达趋化因子受体CCR6。
  3. 根据权利要求1-2任其一所述的修饰的免疫细胞，其特征在于，所述免疫细胞为T细胞、NK细胞、NKT细胞、巨噬细胞和其它肿瘤杀伤细胞。
  4. 根据权利要求3所述的免疫细胞，其特征在于，所述T细胞为嵌合抗原受体T细胞(CAR-T)、T细胞受体T细胞(TCR-T)和肿瘤浸润T细胞(TIL)。
  5. 根据权利要求1-2任其一所述的免疫细胞，其特征在于，将目的基因转入到所述免疫细胞进行表达嵌合抗原受体和趋化因子受体CCR6。
  6. 根据权利要求5所述的免疫细胞，其特征在于，所述转入方式为：慢病毒、逆转录病毒、普通质粒载体、附加体载体、纳米递送系统、电转导、转座子和其它递送系统。
  7. 根据权利要求1或2或4或6所述的免疫细胞，其特征在于，与未修饰的免疫细胞相比较，所述免疫细胞具有至少a)-e)中的一项性能，
    a)细胞因子分泌能力不受影响；
    b)对肿瘤细胞的特异性杀伤能力不受影响；
    c)增强向分泌CCL20实体瘤的归巢和聚集的能力；
    d)增强在CCL20实体瘤的浸润；以及
    e)延长患者生存时间的能力。
  8. 根据权利要求1或2或4或6所述的免疫细胞，其特征在于，所述嵌合抗原受体包含前导序列，识别肿瘤相关抗原的scFv，铰链区和跨膜结构域，胞内共刺激信号以及胞内激活信号CD3ζ。
  9. 根据权利要求8所述的免疫细胞，其特征在于，所述的肿瘤相关抗原包括AFP、BMCA、CEA、CA-125、CA19-9、CA72-4、CD116、CD117、CD171、CD123、CD16、CD19、CD20、CD22、CD30、CD61、CD64、CD70、CD71、CD78、CD96、CLL1、CD133、CD138、Her2、GD2、gp36、GPC3、GnTV、EGFR、EGFRvIII、EpCAM、PSA、PSMA、PAP、NY-ESO-1、LAGA-1a、cMET、P53、Prostein、PCTA-1、MAGE、ROR1、FAP、NKG2D、EMA、ETA、GFAP、MSA、MART-1、NSE、TAG-72、IL13Rα2、NKR-2、ROR1、VEGFR2、VEGFR、CLD18、EphA2、alpha-folateR、CAIX、MG7、LMP1、PD-L1、MUC1、间皮素、导管上皮粘蛋白、鞘糖脂、肿瘤基质抗原、内皮因子、钙网膜蛋白、酪氨酸酶、胎盘碱性磷酸酶、甲状腺球蛋白、甲状腺转录因子1、异常ras蛋白、TPSA、FPSA、 EB病毒抗原、EBNA、人HPV抗原E6和E7、端粒酶、HBV、gp100、MAGEA3、MAGEA4、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、ELF2M、嗜中性粒细胞蛋白弹性酶、胰岛素生长因子(IGF1)-1、IGF-II和IGFI受体。
  10. 根据权利要求9所述的免疫细胞，其特征在于，所述scFv选自单克隆抗体、嵌合单克隆抗体、人源化单克隆抗体、人抗体、纳米抗体以及合成抗体。
  11. 根据权利要求8所述的免疫细胞，其特征在于，所述嵌合抗原受体包含前导序列，识别EGFR的scFv，CD28或CD8铰链区和跨膜结构域，CD28或CD137(4-1BB)共刺激结构域，以及胞内激活信号CD3ζ。
  12. 权利要求1-11任其一所述免疫细胞在制备治疗肿瘤的药物中应用。
  13. 根据权利要求12所述的应用，其特征在于，所述肿瘤选自实体肿瘤，所述实体肿瘤过表达CCL20。
  14. 根据权利要求13所述的应用，其特征在于，所述实体肿瘤选自宫颈鳞癌，宫颈腺癌，结肠癌，食管癌，多形成性胶质细胞瘤，头颈鳞状细胞癌，肾透明细胞癌，肝细胞癌，肺腺癌，肺鳞癌，卵巢癌，胰腺癌，直肠癌，胃癌以及子宫内膜癌。
  15. 一种基因表达系统，其特征在于，所述基因表达系统包括由表达嵌合抗原受体的第一载体和表达CCR6的第二载体组成；或者包括共表达嵌合抗原受体和CCR6的载体。
  16. 一种修饰免疫细胞的方法，包括：
    将权利要求15所述的基因表达系统转染免疫细胞；
    任选地，体外培养细胞进行扩增。
  17. 根据权利要求16所述的方法，其特征在于，所述免疫细胞为T细胞、NK细胞、NKT细胞、巨噬细胞和其它肿瘤杀伤细胞。
PCT/CN2020/074252 2020-01-20 2020-02-04 修饰的免疫细胞及其应用 WO2021147121A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2022567698A JP7457899B2 (ja) 2020-01-20 2020-02-04 修飾された免疫細胞及びその使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010062327.7A CN110872577B (zh) 2020-01-20 2020-01-20 修饰的免疫细胞及其应用
CN202010062327.7 2020-01-20

Publications (1)

Publication Number Publication Date
WO2021147121A1 true WO2021147121A1 (zh) 2021-07-29

Family

ID=69718514

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/074252 WO2021147121A1 (zh) 2020-01-20 2020-02-04 修饰的免疫细胞及其应用

Country Status (3)

Country Link
JP (1) JP7457899B2 (zh)
CN (1) CN110872577B (zh)
WO (1) WO2021147121A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114456270A (zh) * 2022-03-02 2022-05-10 北京美康基免生物科技有限公司 一种gd2嵌合抗原受体及其应用
CN114645014A (zh) * 2021-12-07 2022-06-21 深圳先进技术研究院 免疫细胞的修饰方法、免疫细胞机器人及系统
CN116063556A (zh) * 2022-08-18 2023-05-05 再少年(北京)生物科技有限公司 Ips来源的car-nk细胞及其治疗癌症的用途

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111733154B (zh) * 2020-04-30 2022-11-04 和也健康科技有限公司 磁场处理的免疫细胞及其用途
CN111944850B (zh) * 2020-08-28 2023-03-31 澳门大学 表达抗cd22嵌合抗原受体和pd-l1阻断蛋白的细胞的制备方法、表达载体及应用
WO2022048314A1 (zh) * 2020-09-03 2022-03-10 上海易慕峰生物科技有限公司 针对循环肿瘤细胞的免疫杀伤细胞在实体瘤治疗中的应用
WO2022061837A1 (en) * 2020-09-27 2022-03-31 Jiangsu Cell Tech Medical Research Institute Co., Ltd. Fibronectin extra domain b (edb) -specific car-t for cancer
CN113005092A (zh) * 2021-01-26 2021-06-22 武汉科技大学 一种敲除pd1的靶向lmp1的car-t细胞的制备方法和应用
CN113621582B (zh) * 2021-09-23 2022-09-02 广州百吉生物制药有限公司 联合表达CCR2b的工程化免疫细胞及其制备和应用
CN115925975A (zh) * 2022-08-03 2023-04-07 山东大学 一种靶向肿瘤干细胞的car-mφ体内外编辑制备方法以及其应用
CN117551199B (zh) * 2023-11-16 2024-04-19 杭州荣谷生物科技有限公司 一种Claudin18.2纳米抗体的制备方法及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015112626A1 (en) * 2014-01-21 2015-07-30 June Carl H Enhanced antigen presenting ability of car t cells by co-introduction of costimulatory molecules
CN108603172A (zh) * 2015-12-16 2018-09-28 Ucl商务股份有限公司 治疗性t细胞
WO2019094847A1 (en) * 2017-11-10 2019-05-16 Jura Bio, Inc. Major histocompatibility complex-based chimeric receptors and uses thereof for treating autoimmune diseases
CN109777784A (zh) * 2019-02-22 2019-05-21 上海尚泰生物技术有限公司 一种增强向肿瘤部位迁移的嵌合抗原受体载体构建方法与应用
WO2019213282A1 (en) * 2018-05-01 2019-11-07 Novartis Ag Biomarkers for evaluating car-t cells to predict clinical outcome

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200054677A1 (en) * 2017-02-21 2020-02-20 The University Of Adelaide T cells expressing chemokine receptors for treating cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015112626A1 (en) * 2014-01-21 2015-07-30 June Carl H Enhanced antigen presenting ability of car t cells by co-introduction of costimulatory molecules
CN108603172A (zh) * 2015-12-16 2018-09-28 Ucl商务股份有限公司 治疗性t细胞
WO2019094847A1 (en) * 2017-11-10 2019-05-16 Jura Bio, Inc. Major histocompatibility complex-based chimeric receptors and uses thereof for treating autoimmune diseases
WO2019213282A1 (en) * 2018-05-01 2019-11-07 Novartis Ag Biomarkers for evaluating car-t cells to predict clinical outcome
CN109777784A (zh) * 2019-02-22 2019-05-21 上海尚泰生物技术有限公司 一种增强向肿瘤部位迁移的嵌合抗原受体载体构建方法与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KADOMOTO SUGURU, IZUMI KOUJI, HIRATSUKA KAORU, NAKANO TAITO, NAITO RENATO, MAKINO TOMOYUKI, IWAMOTO HIROAKI, YAEGASHI HIROSHI, SHI: "Tumor-Associated Macrophages Induce Migration of Renal Cell Carcinoma Cells via Activation of the CCL20-CCR6 Axis", CANCERS, vol. 12, no. 1, 30 December 2019 (2019-12-30), XP055831432, DOI: 10.3390/cancers12010089 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114645014A (zh) * 2021-12-07 2022-06-21 深圳先进技术研究院 免疫细胞的修饰方法、免疫细胞机器人及系统
CN114645014B (zh) * 2021-12-07 2024-04-02 深圳先进技术研究院 免疫细胞的修饰方法、免疫细胞机器人及系统
CN114456270A (zh) * 2022-03-02 2022-05-10 北京美康基免生物科技有限公司 一种gd2嵌合抗原受体及其应用
CN114456270B (zh) * 2022-03-02 2023-12-22 北京美康基免生物科技有限公司 一种gd2嵌合抗原受体及其应用
CN116063556A (zh) * 2022-08-18 2023-05-05 再少年(北京)生物科技有限公司 Ips来源的car-nk细胞及其治疗癌症的用途
CN116063556B (zh) * 2022-08-18 2023-06-16 再少年(北京)生物科技有限公司 Ips来源的car-nk细胞及其治疗癌症的用途

Also Published As

Publication number Publication date
CN110872577A (zh) 2020-03-10
JP7457899B2 (ja) 2024-03-29
CN110872577B (zh) 2020-05-08
JP2023510651A (ja) 2023-03-14

Similar Documents

Publication Publication Date Title
WO2021147121A1 (zh) 修饰的免疫细胞及其应用
US10118964B2 (en) Construction and application of bispecific antibody HER2xCD3
JP6161098B2 (ja) Ｃａｒ発現ベクター及びｃａｒ発現ｔ細胞
Moon et al. Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor
WO2017219934A1 (zh) 一种高效稳定表达抗体的杀伤性细胞及其用途
WO2021120526A1 (zh) 同时靶向间皮素和fap的双靶点嵌合抗原受体及其用途
JP6971986B2 (ja) 免疫療法の抗腫瘍活性を高めるための間葉系幹細胞
CN109803983A (zh) 靶向nkg2dl的特异性嵌合抗原受体t细胞，其制备方法和应用
Wang et al. PiggyBac-engineered T cells expressing a glypican-3-specific chimeric antigen receptor show potent activities against hepatocellular carcinoma
WO2022199125A1 (zh) 一种自分泌IL-15与anti-PD1融合蛋白的免疫细胞
JP7104949B2 (ja) Ｃａｒをコードするヌクレオチド配列、このｃａｒを発現するｒｏｂｏ１ ｃａｒ－ｎｋ細胞、その調製及び使用
Ponterio et al. Identification of targets to redirect CAR T cells in glioblastoma and colorectal cancer: an arduous venture
CN115151635A (zh) 用编码新的嵌合抗原受体的基因转导的基因修饰nk细胞系及其用途
WO2018121679A1 (zh) 表达cxcr4的嵌合抗原受体修饰的淋巴细胞及制备方法和用途
WO2017219933A1 (zh) 一种高效稳定表达抗体的t细胞及其用途
ES2926227T3 (es) Receptor de antígeno quimérico con dominio de activación o bloqueo del receptor de citoquinas
JP2021534770A (ja) 遺伝子修飾免疫細胞の生存及び増加を特異的に刺激するための方法
CN115003700A (zh) 促实体瘤浸润的增强型cart细胞及其制备方法和细胞药物
CN114929863A (zh) 共表达趋化因子受体的免疫效应细胞
CN111732665B (zh) 一种靶向表达癌胚抗原的细胞的嵌合抗原受体
CA3178239A1 (en) Method
Yoon et al. Transfer of Her-2/neu specificity into cytokine-induced killer (CIK) cells with RNA encoding chimeric immune receptor (CIR)
WO2024055339A1 (zh) 用于制备和扩增通用型人源化抗cd19 car-nk细胞的方法及其用途
WO2022171195A1 (zh) 抗cd87抗体与抗pd1抗体联合治疗胃癌
CN114058589B (zh) 具有嵌合抗原受体修饰的免疫细胞、制备方法和药物

Legal Events

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

Ref document number: 20914822

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022567698

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20914822

Country of ref document: EP

Kind code of ref document: A1