WO2019024933A1 - Gpc3-targeted car nk cell - Google Patents

Gpc3-targeted car nk cell Download PDF

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WO2019024933A1
WO2019024933A1 PCT/CN2018/098623 CN2018098623W WO2019024933A1 WO 2019024933 A1 WO2019024933 A1 WO 2019024933A1 CN 2018098623 W CN2018098623 W CN 2018098623W WO 2019024933 A1 WO2019024933 A1 WO 2019024933A1
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cells
cell
cancer
gpc3
domain
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PCT/CN2018/098623
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Chinese (zh)
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李宗海
喻敏
高慧萍
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科济生物医药（上海）有限公司
上海市肿瘤研究所
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    • 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
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • 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
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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
    • A61K2239/53—Liver
    • 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
    • C12N2510/02—Cells for production

Definitions

  • the invention belongs to the field of cellular immunotherapy, and in particular relates to NK cells or cell lines comprising GPC3-CAR.
  • Chimeric antigen receptor (CAR) technology combines single-chain antibodies recognizing tumor-associated antigens with activation motifs of immune cells, and can confer tumor-targeting and stronger killing activity on immune cells through gene transduction. And lasting vitality.
  • This technology was first implemented in T cells and has achieved great success in patients with CD19-positive B-lymphocytic leukemia.
  • CAR-T cells are administered by collecting T cells from patients and being modified, they are prepared in a single preparation, and the treatment cost is high, and a large amount of expansion of T cells in the body usually causes cytokine storms. Therefore there is a risk of security.
  • NK cells can also be targeted by CAR technology to identify and destroy tumor cells.
  • NK cells have multiple mature immortalized cell lines, of which NK-92 can be cultured and expanded in vitro for a long time, the gene phenotype is stable, and the results are reproducible.
  • Some researchers have tried to express CAR in NK-92 cells. (For example, CN201580032729.X), but because tumor tissue has a very complex microenvironment, whether CAR-NK-92 cells can kill tumor cells in vivo will be affected by many factors, which has great uncertainty.
  • CAR-NK research and development is mostly the direct imitation and transformation of CAR-T.
  • the present invention urgently requires an effective CAR-NK cell.
  • the NK cells may be primary NK cells or NK92- cells.
  • the invention provides an NK-92 cell or cell line that expresses a chimeric antigen receptor that specifically recognizes GPC3.
  • Another object of the invention is to provide the use of said NK-92 cells or cell lines for the prevention and/or treatment of cancer, preferably GPC3 expressing cancer.
  • a further object of the invention is to provide the use of the NK-92 cell or cell line as a targeted cell therapeutic and/or for immunotherapy of adoptive cancer.
  • a further object of the present invention is to provide a method for producing the NK-92 cell or cell line, a method for identifying the NK-92 cell or cell line, and an NK-92 cell or cell line obtained or identified by the method. And its use.
  • the present invention provides a genetically engineered NK cell, characterized in that said cell expresses a chimeric receptor that specifically recognizes GPC3, said chimeric receptor comprising an extracellular domain that recognizes GPC3, spans Membrane domains, and/or intracellular domains.
  • the NK cell is an NK-92 cell.
  • the extracellular domain has HCDR1, HCDR2, HCDR3 and/or as shown in SEQ ID NOs: 7, 8, 9 and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 10, 11, and 12. .
  • the extracellular domain has HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 7, 8, and 9, and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 10, 11, and 12.
  • the extracellular domain that specifically recognizes GPC3 comprises the heavy chain variable region set forth in SEQ ID NO: 13 and the light chain variable region set forth in SEQ ID NO: 14.
  • the extracellular domain that specifically recognizes GPC3 has the sequence set forth in SEQ ID NO:1.
  • the chimeric receptor has the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, or 19.
  • the transmembrane domain is selected from the transmembrane domain of the following proteins: alpha, beta or ⁇ chain of a T cell receptor; CD28; CD3 epsilon; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33 CD37;CD64;CD80;CD86;CD134;CD137;CD154;KIRDS2;OX40;CD2;CD27;LFA-1 (CD11a;CD18);ICOS(CD278);4-1BB(CD137);GITR;CD40;BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRF1); CD160; CD19; IL2R ⁇ ; IL2R ⁇ ; IL7R ⁇ ; ITGA1; VLA1; CD49a; ITGA4; IA4; CD49D; ITGA6; VLA-6; CD49f; ITGAD; CD11d; ITGAE; CD103; ITGAL; CD11a; LFA-1
  • SLAMF6 NTB-A, Ly108
  • SLAM SLAMF1, CD150, IPO-3
  • BLAME SLAMF8
  • SELPLG CD162
  • LTBR TBR
  • PAG/Cbp PAG/Cbp
  • NKp44 NKp44
  • NKp30 NKp46
  • NKG2D NKG2D
  • NKG2C NKG2C
  • the intracellular domain comprises: a transcription factor binding domain; a primary signaling domain and/or a costimulatory signaling domain, wherein:
  • the primary signaling domain comprises a functional signal transduction selected from the group consisting of: CD3 ⁇ ; CD3 ⁇ ; CD3 ⁇ ; CD3 ⁇ ; common FcR ⁇ (FCER1G); FcR ⁇ (Fc ⁇ R1b); CD79a; CD79b; Fc ⁇ RIIa; DAP10; Domain, or a combination thereof; and/or
  • the costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27; CD28; 4-1BB (CD137); OX40; CD30; CD40; PD-1; ICOS; Related antigen-1 (LFA-1); CD2; CD7; LIGHT; NKG2C; B7-H3; ligand for specific binding to CD83; CDS; ICAM-1; GITR; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 ( KLRF1);CD160;CD19;CD4;CD8 ⁇ ;CD8 ⁇ ;IL2R ⁇ ;IL2R ⁇ ;IL7R ⁇ ;ITGA4;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ;LFA-1;ITGAM;CD11b;ITGAX;CD11c;ITGB1;CD29;IT
  • the present invention provides the use of the cell of the first aspect of the invention for the preparation of a medicament for preventing and/or treating cancer, the cancer expressing GPC3, preferably from liver cancer, lung cancer, gastric cancer, breast cancer, Melanoma, ovarian cancer, yolk sac tumor, neuroblastoma.
  • the invention provides the use of a cell of the first aspect of the invention for the manufacture of a medicament for use as a targeted cell therapeutic and/or for immunotherapy of adoptive cancer.
  • the prophylactic and/or therapeutic cancer drug, target is described in the instructions for use in the prevention and/or treatment of cancer, a targeted cell therapeutic, or a drug for adoptive cancer immunotherapy.
  • the drug to the cell therapy agent or the immunotherapy for adoptive cancer is administered simultaneously with the chemotherapeutic drug or radiation therapy.
  • the medicament for preventing and/or treating cancer, a targeted cell therapeutic, or a medicament for adoptive cancer immunotherapy further comprises a chemotherapeutic drug.
  • the medicament for preventing and/or treating cancer, a targeted cell therapeutic, or a medicament for adoptive cancer immunotherapy is for treating liver cancer.
  • the chemotherapeutic agent is a chemotherapeutic drug for treating liver cancer; preferably a compound of formula I or formula II:
  • the instructions for the prevention and/or treatment of cancer, a targeted cell therapeutic, or a drug for adoptive cancer immunotherapy describe the individual to be administered prior to administration of the cell. Pretreatment is performed.
  • the pretreatment is to administer a chemotherapeutic drug or radiation therapy to control an increase in tumor burden.
  • the drug for preventing and/or treating cancer, a targeted cell therapeutic agent, or a drug for adoptive cancer immunotherapy further comprises a lymphocyte depleting agent as a pretreatment reagent to consume the Individual lymphocytes.
  • the lymphocyte depleting agent is fludarabine or cyclophosphamide.
  • the present invention provides a method for producing NK cells of the present invention, which comprises: preparing NK cells expressing a chimeric antigen receptor, and further treating the NK cells expressing the chimeric antigen receptor by irradiation; preferably NK cells expressing chimeric antigen receptors were pretreated with gamma irradiation.
  • the NK cell is an NK-92 cell.
  • the present invention provides a method of preventing and/or treating cancer in a subject, the cancer expressing GPC3, comprising administering a cell of the present invention to a subject in need of treatment for cancer.
  • the cancer is preferably selected from the group consisting of liver cancer, lung cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor, neuroblastoma; preferably liver cancer.
  • the method of the invention for preventing and/or treating cancer in a subject comprises simultaneously administering to a subject in need of treatment of cancer a cell of the invention and a chemotherapeutic drug or radiation therapy.
  • the chemotherapeutic agent is a chemotherapeutic agent for treating liver cancer; preferably a compound of formula I or formula II:
  • the method of the invention for preventing and/or treating cancer in a subject comprises pretreating an individual to be administered prior to administration of the cells of the invention.
  • the pretreatment is to administer a chemotherapeutic drug or radiation therapy to control an increase in tumor burden.
  • the pretreatment is to consume lymphocytes of the individual using a lymphocyte depleting agent.
  • the lymphocyte depleting agent is fludarabine or cyclophosphamide.
  • Figure 1 shows the results of flow cytometry of NK-92/9.28.z cells
  • Figure 2A compares the in vitro killing activity of parental NK-92, Mock and NK-92/9.28.z cells;
  • Figure 2B shows the in vitro killing results of NK-92/9.28.z cells at inefficient target ratios;
  • Figure 2C shows the in vitro killing results of NK-92/9.28.z cells under hypoxic conditions;
  • Figure 2D shows the in vitro killing results of NK-92/9.28.z cells in the presence of TGF- ⁇ ;
  • Figure 2E shows serum The effect of horizontal GPC3 on the killing effect of NK-92/9.28.z cells in vitro;
  • Figure 3A shows the release of NK-92/9.28.z cell degranzyme
  • Figure 3B shows the secretion of IFN-gama
  • Figure 3C shows the secretion of NK-92/9.28.z IFN-gamma and the surface of tumor cells GPC3 expression value is proportional;
  • Figure 4A shows the anti-tumor effect of NK-92/9.28.z cells in SK-HEP-1 and SK-HEP-1/GPC3 xenograft models
  • Figure 4B shows NK-92/9.28.z cells infiltrating tumors locally. The results of anti-tumor effects
  • Figure 4C shows the results of local immunohistochemistry of NK-92/9.28.z cells infiltrating tumors;
  • Figure 5 shows the effect of NK-92/9.28.z cell therapy on important organ organs
  • Figure 6A shows the anti-tumor effect of NK-92/9.28.z on endogenous high-expression GPC3 Huh-7 subcutaneous xenografts;
  • Figure 6B shows the anti-tumor effect of NK-92/9.28.z on orthotopic tumors;
  • Figure 6D shows the changes in body weight of mice in the orthotopic transplantation model;
  • Figure 6E shows the NK-92/9.28.z in the orthotopic transplantation model. Infiltration;
  • Figure 7A shows tumor volume changes in the PLC/PRF/5 subcutaneous xenograft model
  • Figure 7B shows tumor weight changes in the PLC/PRF/5 subcutaneous xenograft model
  • Figure 7C shows PLC/PRF/5 subcutaneous xenografts Changes in mouse body weight in the model
  • Figure 8A shows the release of cytokines during killing
  • Figure 8B shows the results of release of IL-6 by monocytes after killing target cells by NK-92/9.28.z;
  • Figure 9A shows in vitro cell killing of PBNK/9.28.z
  • Figure 9B shows interferon release levels of PBNK/9.28.z
  • Figure 10 shows the killing of normal cells by PBNK/9.28.z
  • Figure 11 shows the cell killing of sorafenib in combination with NK-92/9.28.z cells.
  • NK cells are important effector cell types for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance anti-tumor activity, but experience with CAR-engineered NK cells is limited and lacks clinically developed data. . In addition to primary cells, NK cells have many mature cell lines, including NK-92, NKG, YT, NK-YS, HANK-1, YTS, and NKL. In a specific embodiment of the present invention, when the NK-92 cell line or primary NK cells are used, the NK cells modified with the chimeric antigen receptor of the present invention are most effective.
  • CARs chimeric antigen receptors
  • Cellular therapeutic agent particularly “targeted cell therapeutic agent” or “targeted allogeneic cell therapeutic agent” refers to an immune cell suitable for administration to adoptive immunotherapy for adoptive cancer, which is genetically modified to express specificity An antigen-restricting antigen receptor expressed on the surface of a target tumor cell is recognized.
  • “Adoptive, target cell-specific immunotherapy” or “adoptive cancer immunotherapy” or “adoptent cell therapy (ACT)” refers to a form of treatment in which immune cells are transferred to a host carrying a tumor. Immune cells have anti-tumor reactivity and can mediate direct or indirect anti-tumor effects.
  • GPC3 is Glypican-3 (also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1) and is a cell surface protein belonging to sulfuric acid. Heparan glycoprotein family.
  • the GPC3 gene encodes a 70-kDa precursor core protein that can be cleaved by furin to produce a soluble 40-kDa-capable amino-terminal (N-terminal) peptide and 30- A membrane-bound carboxy-terminal (C-terminal) peptide containing two heparan sulfate (HS) sugar chains around kDa.
  • the GPC3 protein is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • chimeric antigen receptor refers to a polypeptide which, when administered in an immune effector cell, provides the cell with a target cell (usually a cancer cell) Specificity, and has intracellular signal production.
  • CAR typically includes at least one extracellular antigen binding domain, a transmembrane domain (also known as a transmembrane or transmembrane domain), and an intracellular domain (also referred to herein as an "intracellular region” or “intracellular domain”).
  • the polypeptide groups are contiguous with each other.
  • a polypeptide group includes a dimerization switch that can couple the polypeptides to each other in the presence of a dimerization molecule, for example, an antigen binding domain can be coupled to an intracellular signaling domain.
  • the "chimeric antigen receptor" is capable of specifically recognizing GPC3.
  • the intracellular domain comprises a primary signaling domain, or a costimulatory signaling domain, or a primary signaling domain and a costimulatory signaling domain.
  • the primary signaling domain may be selected from the group consisting of: CD3 ⁇ ; CD3 ⁇ ; CD3 ⁇ ; CD3 ⁇ ; common FcR ⁇ (FCER1G); FcR ⁇ (Fc ⁇ R1b); CD79a; CD79b; Fc ⁇ RIIa; DAP10; Conduction domain.
  • human CD3 can be selected (amino acid sequence as set forth in SEQ ID NO: 6, nucleotide sequence as set forth in SEQ ID NO: 22).
  • the costimulatory signaling domain can be selected from the signaling domains of proteins: CD27; CD28; 4-1BB (CD137); OX40; CD30; CD40; PD-1; ICOS; Related antigen-1 (LFA-1); CD2; CD7; LIGHT; NKG2C; B7-H3; ligand for specific binding to CD83; CDS; ICAM-1; GITR; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 ( KLRF1);CD160;CD19;CD4;CD8 ⁇ ;CD8 ⁇ ;IL2R ⁇ ;IL2R ⁇ ;IL7R ⁇ ;ITGA4;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ;LFA-1;ITGAM;CD11b;ITGAX;CD11c;ITGB1;CD29;ITGB
  • signaling domain refers to a functional portion of a protein that functions by transmitting information within a cell for regulating cells via a defined signaling pathway by generating a second messenger or by acting as an effector in response to such a messenger. Activity.
  • the intracellular domain of human CD137 amino acid sequence as set forth in SEQ ID NO: 25, nucleotide sequence as set forth in SEQ ID NO: 26
  • human CD28 intracellular domain amino acid sequence, eg, SEQ ID NO: 5
  • the nucleotide sequence is set forth in SEQ ID NO: 21, or a combination of the intracellular domain of human CD137 and the human CD28 intracellular domain.
  • the transmembrane domain is selected from the transmembrane domain of the following proteins: alpha, beta or ⁇ chain of T cell receptor, CD28; CD3 epsilon; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64;CD80;CD86;CD134;CD137;CD154;KIRDS2;OX40;CD2;CD27;LFA-1(CD11a;CD18);ICOS(CD278);4-1BB(CD137);GITR;CD40;BAFFR;HVEM(LIGHTR ;SLAMF7;NKp80(KLRF1);CD160;CD19;IL2R ⁇ ;IL2R ⁇ ;IL7R ⁇ ;ITGA1;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ; LFA-1; ITGAM;
  • transmembrane domain of CD28 amino acid sequence as shown in SEQ ID NO: 4, nucleotide sequence as shown in SEQ ID NO: 20
  • transmembrane domain of CD8 amino acid sequence such as SEQ ID NO: 23
  • nucleotide sequence is as shown in SEQ ID NO:24).
  • the chimeric antigen receptor further comprises a hinge region.
  • the hinge region of CD8 can be selected and the amino acid sequence is set forth in SEQ ID NO:3.
  • the chimeric receptor of the invention has the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, or 19.
  • the GPC3-specific NK-92 cells or primary NK cells of the present invention comprise a lentiviral vector encoding a chimeric antigen receptor comprising a GPC3-specific scFv antibody fragment, a hinge region, and a transmembrane of CD28 And intracellular domains and intracellular domains of CD3 ⁇ .
  • the NK-92 cells or primary NK cells of the invention are characterized by: reduced natural cytotoxicity or no natural cytotoxicity, ie reduced cytotoxicity or absence of GPC3-negative cells compared to GPC3-positive cells Cytotoxicity.
  • the reduced natural cytotoxicity constitutes an important safety feature of the cells or cell lines of the invention, particularly in clinical applications.
  • the NK-92 cells or primary NK cells of the present invention are opposite to unmodified NK-92 cells or primary NK cells - lysing GPC3-expressing tumor cells with high efficiency, but with unmodified NK-92 cells or primary NK Cells are less likely to attack GPC3-negative non-target cells.
  • the GPC3-specific scFv antibody fragment comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.
  • the GPC3-specific CAR used in the present invention comprises:
  • transmembrane domain in a specific embodiment, the transmembrane domain of CD28 or the transmembrane domain of CD8 may be selected, and other transmembrane domains commonly used in the technique of preparing CAR may also be selected.
  • the intracellular domain has an intracellular domain of CD3 ⁇ , and/or a costimulatory signal domain, alternatively, the costimulatory signal domain may be a costimulatory signal domain of CD28 or The costimulatory signal domain of CD137.
  • the CAR-NK-92 cells of the present invention can prevent cell replication by gamma-irradiation as a potential safety measure for clinical applications, while in vitro and in vivo anti-tumor activity is maintained.
  • NK-92 may be tumorigenic.
  • pretreatment of the cells by irradiation, preferably gamma irradiation.
  • the illumination is a safety measure and can be carried out in a variety of conventional manners.
  • NK-92/9.28.z has no enhanced killing effect on GPC3-negative liver cancer cells; when overexpressing GPC3, these cells are more sensitive to NK-92/9.28.z, reflecting NK-92/9.28.z cells.
  • the role of targeted killing One obstacle to the treatment of solid tumors by cells is that too few effector cells enter the local microenvironment, failing to achieve an effective target-to-kill ratio. When the target ratio is greatly reduced, if the incubation time is extended, it can reach a very large Good specific killing; hypoxia and TGF- ⁇ in the microenvironment are factors that restrict the effect of cell therapy, and the present invention shows that NK-92/9.28.z still has a good specificity in the presence of both.
  • NK-92/9.28.z cells sexual killing effect; there is secretory GPC3 in the blood, but it does not affect the toxic effect of NK-92/9.28.z cells on tumor cells in the present invention.
  • Our data indicate that NK-92 cells encoding GPC3-CAR can tolerate a variety of adverse conditions and are clinically useful for the treatment of various GPC3-positive malignancies.
  • CD107a is a protein on lysosome. It can indicate the fusion of lysosome and cell membrane when releasing granzyme, which is on NK-92 cells.
  • An activation indicator We can reveal the targeting of CAR from the NK cell activation level.
  • CAR-NK mediates the lysis of target cells caused by a variety of endogenous cytotoxic receptors, and tumors with heterogeneous expression of antigen may be more advantageous than CAR-T.
  • Allogeneic infusion of NK cells does not cause graft-versus-host disease, and its KIR-MHC mismatch-based allogeneic model can also avoid or reduce KIR-mediated inhibitory signals, making it possible to commercialize universal and efficient "NK blood products" .
  • NK-92/9.28.z also has an antigen-dependent antitumor effect in vivo, which acts by inducing tumor cell apoptosis and inhibiting proliferation; and does not infiltrate important organ tissue-inducing cells. Toxic effects; does not cause side effects associated with weight loss in treatment.
  • NK-92 cells are a tumor-derived killer cell line, which has the risk of tumor formation in the body. It is often necessary to infuse after irradiation.
  • This animal experiment established a set of irradiation of NK-92/9.28.z cells. The results suggest that the effect is basically the same as that of the NK-92/9.28.z in the non-irradiated group, providing an animal experimental basis for its clinical safety application.
  • NK-92/9.28.z cells retain target cell specificity in vivo and are capable of penetrating tissue and homing to distant tumor sites.
  • the in vivo anti-tumor activity of irradiated NK-92/9.28.z cells was identical to that of non-irradiated cells. This may be of interest for future clinical applications of NK-92/9.28.z, where irradiation of cells can be performed as in previous phase I clinical trials of unmodified NK-92 cells (Arai et al., 2008; Tonn Etc., 2013) included as a security measure.
  • Immune cells in tumor patients are often functionally impaired due to the immunosuppressive activity of cancer. Therefore, for adoptive cancer immunotherapy with NK cells, donor-derived allogeneic cells are preferred because they do not recognize tumor cells as "self", thereby circumventing inhibitory signals (Geller and Miller; 2011) . We have shown that this advantage can be extended to CAR-engineered NK-92 cells. Such cells are clinically useful for the treatment of various GPC3-positive malignancies.
  • the primary NK cells obtained from peripheral blood culture can specifically kill GPC3-positive hepatoma cell line secreting IFN- ⁇ after CAR modification, while Mock and untransfected NK cells have weaker killing effect. The expression of IFN- ⁇ was also lower. In vitro toxicity experiments indicated that the modified primary NK cells had no obvious toxic effects on normal tissue cells.
  • this example selects antibody AB1 (the sequence of the scFv as shown in SEQ ID NO: 1) that targets GPC3 as the extracellular domain of the chimeric antigen receptor.
  • the pre-prepared CAR has the sequence shown in SEQ ID NO: 17.
  • PRRLSIN-cPPT.EF-1 ⁇ a lentiviral plasmid expressing the second-generation chimeric antigen receptor of antibody AB1, PRRLSIN-cPPT.EF-1 ⁇ -AB1-28Z.
  • the AB1-28Z sequence consists of the CD8 ⁇ signal peptide (SEQ ID NO: 2), the scFv of AB1 (SEQ ID NO: 30), the CD8hinge (SEQ ID NO: 15), the CD28 transmembrane region (SEQ ID NO: 20), and the intracellular signal.
  • the conduction domain (SEQ ID NO: 21) and the intracellular domain CD3 (CD ID NO: 22) of CD3 consist.
  • the target gene plasmids PRRLSIN-cPPT.EF-1 ⁇ -EGFP (Mock) and PRRLSIN-cPPT.EF-1 ⁇ -AB1-28Z 5.4 ⁇ g and packaging plasmid pRsv-REV 6.2 ⁇ g, RRE-PMDLg 6.2 ⁇ g, Vsvg 2.4 ⁇ g was dissolved in 800 ⁇ L of blank DMEM medium and mixed for 5 min at room temperature; 60 ⁇ g of PEI (1 ⁇ g/ ⁇ l) was dissolved in 800 ⁇ l of serum-free DMEM medium, gently mixed (or vortexed at 1000 rpm for 5 seconds); The mixed solution was added to the PEI mixture, gently added after the addition, and incubated at room temperature for 20 min to obtain a transfection complex;
  • the virus titers after concentration were: lentivirus AB1-28Z: 2.4 ⁇ 10 8 U/ml, lentivirus Mock: 2 ⁇ 10 8 U/ml.
  • Retronectin coated 24 well plates 380 ⁇ l of 5 ⁇ g/ml retronectin solution (PBS) was added to each well, and after overnight incubation at 4 degrees, the retronectin solution (PBS) in a 24-well plate was discarded, and washed twice with PBS;
  • Amplification culture The infected cells were passaged every other day at a density of 5 ⁇ 10 5 /mL, and a recombinant human IL-2 having a final concentration of 500 U/mL was supplemented in the lymphocyte culture solution.
  • Mock NK-92 cells (designated Mock) expressing empty vector or NK-92 cells expressing CAR (designated NK-92/9.28.z) were obtained.
  • Control cells were incubated with 50 ⁇ l of PE-SA (1:200 dilution) antibody for 45 min on ice, washed twice with PBS (2% NBS), and resuspended as a control;
  • Test group cells 50 ⁇ l 1:50 diluted biotin-goat anti-human IgG, F(ab') 2 antibody, incubated on ice for 45 min; PBS (2% NBS) twice; add 50 ⁇ l PE-SA (1 :200 dilution) antibody incubation on ice for 45 min;
  • the cells received by the sorting receiving tube are placed in a 37-degree, 5% CO 2 incubator for cultivation;
  • NK-92/9.28.z The positive rate of NK-92/9.28.z was detected by flow cytometry. The results are shown in Figure 1.
  • the flow-sorted NK-92/9.28.z cells were compared with the Mock group and the Parental NK-92 group.
  • the surface has a significantly enhanced expression of CAR.
  • the Mock group was NK-92 cells transfected with Mock plasmid
  • Parental NK-92 was the original NK-92 cells.
  • Cytotoxicity was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit (Promega). The specific method is described in the instruction manual of CytoTox 96 non-radioactive cytotoxicity test kit, and the detection time is 6h.
  • Target cells 50 ⁇ L of 2 ⁇ 10 5 /mL HepG2; Huh-7; Hep3B; PLC/PRF/5; SK-HEP-1; SK-HEP-1/GPC3; SMMC7721; SMMC7721/GPC3 cells in 96well plate (GPC3-positive liver cancer cell lines: HepG2, Huh-7, SK-HEP-1/GPC3, PLC/PRF/5, SMMC7721/GPC3, GPC3-negative liver cancer cell lines: SK-HEP-1, SMMC7721).
  • Effector cells Mock; NK-92 and NK-92/9.28.z cells as described in Example 1 were added at a target ratio of 3:1, 1.5:1 or 0.75:1.
  • NK-92/9.28.z For GPC3-positive liver cancer cells, the in vitro toxicity of NK-92/9.28.z was significantly stronger than that of the Mock group and the Parental NK-92 group.
  • NK-92 /9.28.z has no enhanced killing effect; when overexpressing GPC3, these cells are more sensitive to NK-92/9.28.z, reflecting the targeted killing effect of NK-92/9.28.z cells.
  • NK-92/9.28.z To further confirm the effect of tumor microenvironment on NK-92/9.28.z, the cytotoxic effect of NK-92/9.28.z on hepatoma cell lines under different conditions and conditions was also examined.
  • the hypoxic incubator is used to simulate the hypoxic environment, with Huh-7 as the target cell.
  • the in vitro toxicity of different effective target ratios of NK-92/9.28.z was measured under different oxygen conditions (1% oxygen concentration in low oxygen environment and 20% oxygen concentration in normal oxygen environment). The results are shown in Fig. 2C. As shown, the killing function of NK-92/9.28.z was not significantly affected in a low oxygen environment.
  • TGF- ⁇ is another important factor that restricts the effect of cell therapy.
  • Huh-7 as the target cell
  • the results shown in Figure 2D TGF- ⁇ can significantly inhibit the killing function of CAR-T/9.28.z, while the NK-92/9.28.z cell surface for TGF- ⁇ It exhibited a considerable degree of resistance and its killing function was not significantly affected by TGF- ⁇ .
  • CAR-T/9.28.z is a T cell expressing the CAR of SEQ ID NO: 17, which is derived from peripheral blood, and which is infected with lentivirus AB1-28Z by T cells in a molecular biology routine in the art.
  • CAR-T/9.28.z is a T cell expressing the CAR of SEQ ID NO: 17, which is derived from peripheral blood, and which is infected with lentivirus AB1-28Z by T cells in a molecular biology routine in the art.
  • CN104140974A, CN106397593A and the like refer to CN104140974A, CN106397593A and the like.
  • GPC3 is a cell surface marker, but the N-terminal peptide of GPC3 protein can be cleaved into blood (GPC3N), and can also be cleaved by the noumase at the GPI site to form a soluble GPC3 protein (GPC3 ⁇ GPI). ) released into the blood. Serum levels of GPC3 may compete with GPC3 on the surface of liver cancer cells, affecting the killing effect of NK-92/9.28.z.
  • We expressed GPC3N and GPC3 ⁇ GPI proteins by protein recombination method, and added different concentrations of the above soluble GPC3 protein in the killing system, using Huh-7 as the target cell, and incubating for 6 hours under the condition of 3:1 effective target ratio. The results are shown in Figure 2E, and the two did not significantly affect the killing function of NK-92/9.28.z cells at each concentration.
  • NK cells have a rapid toxic killing effect depending on lysosome-releasing granzyme
  • a protein on CD107a lysosome can indicate the fusion of lysosome and cell membrane when releasing granzyme, so we detected NK- Degranulation of 92/9.28.z and Parental NK-92 cells co-incubated with tumors.
  • NK-92/9.28.z cells and control group NK-92 cells were co-cultured with SK-HEP-1, SK-HEP-1/GPC3 and K562 cells in different proportions by flow cytometry.
  • CD107a expression The medium was used as a negative control and PMA + IONO stimulation was used as a positive control.
  • NK-92/9.28.z cells were seeded into 24-well plates, and 2 ⁇ 10 5 target cells were inoculated at a final volume of 400 ul. No exogenous IL-2 was added, and culture was carried out. The suspension cells were collected at 6 hours in the culture of the box; the expression of CD107a was detected by flow cytometry, and the results are shown in Fig.
  • IFN- ⁇ is one of the most important cytokines secreted by NK cells.
  • NK-92/9.28.z we also compared the secretion of NK-92/9.28.z and NK-92 cells in the common hepatocellular carcinoma cell line at a target-to-target ratio of 1.5:1.
  • IFN- ⁇ levels The details are as follows: 1) 5 ⁇ 10 4 NK-92/9.28.z cells were seeded into a 24-well plate, and 5 ⁇ 10 4 target cells were inoculated at a final volume of 400 ul without exogenous IL-2. The supernatant was collected in an incubator for 24 hours; 2) the expression of IFN-gama was detected by ELISA;
  • Example 4 Therapeutic effect of NK-92/9.28.z cells on SK-HEP-1 and SK-HEP-1/GPC3 xenografts
  • mice were inoculated with SK-HEP-1 and SK-HEP-1/GPC3 xenografts
  • the above cells were inoculated subcutaneously, 2 ⁇ 10 6 /piece, and the tumor volume reached 30-50 mm 3 after about 15 days;
  • mice On day d17, the transplanted tumor volume of Nod-Scid mice was measured and grouped. The mice were divided into 3 groups, including: saline control group, Mock control group, NK-92/9.28.z group, 6-8 mice in each group;
  • Nod-Scid mouse cells were subjected to adoptive immunotherapy. 200ul normal saline or 200ul NK-92, Mock and NK-92/9.28.z cells were injected through the tail vein once every 5 days;
  • the volume of SK-HEP-1 and SK-HEP-1/GPC3 xenografts was measured every 3-4 days, the changes in tumor volume of each group of mice were recorded, and the growth curve of tumor volume with time was plotted.
  • NK-92/9.28.z cell infiltration was detected by flow cytometry.
  • NK-92/9.28.z infiltration and detection of tumor tissue proliferation were detected by immunohistochemistry.
  • Apoptosis index; important organs of SK-HEP-1/GPC3 transplanted tumor mice were taken for HE and immunohistochemical staining.
  • NK-92/9.28.z had no significant effect with the control group; whereas for SK-HEP-1/GPC3, NK-92/9.28.
  • the tumor in the treatment group was significantly smaller than the control group, suggesting that NK-92/9.28.z also has an antigen-dependent antitumor effect in vivo.
  • Infiltration was detected by flow cytometry in the animal model SK-HEP-1/GPC3, and the results were detected in the SK-HEP-1/GPC3 tumor model treated with NK-92/9.28.z as shown in Fig. 4B.
  • SK-HEP-1/GPC3 transplanted tumor mice were taken for HE staining. The results are shown in Fig. 5. There was no significant difference between the groups and no pathological changes were observed. This cell is safe to treat and does not infiltrate important organ tissues to induce cytotoxicity.
  • Example 5 Therapeutic effect of NK-92/9.28.z on endogenous high GPC3 Huh-7 subcutaneous and orthotopic transplantation tumors
  • Huh-7 subcutaneous and orthotopic transplantation tumors were inoculated with NOD/SCID mice.
  • the subcutaneous xenograft model was established: subcutaneously inoculated Huh-7 cells, 2 ⁇ 10 6 /piece, and the tumor volume reached 30-50 mm 3 after about 14 days, and a mouse subcutaneous xenograft model was obtained.
  • In situ animal models were more able to simulate the real situation in patients, and an animal model of in situ Huh-7 liver cancer was established by inoculation, and four mice in each group were randomly assigned.
  • Adoptive NK-92/9.28.z and Parental NK-92 cells were used for adoptive immunotherapy of subcutaneous and orthotopic xenografts by subcutaneous and orthotopic transplantation of intraperitoneal cyclophosphamide at 12 and 13 days, respectively. 100mg/kg).
  • Nod-Scid mouse xenograft volume was measured and grouped on day D15.
  • Subcutaneous xenograft mice were divided into three groups, including: saline control group, NK-92 group, NK-92/9.28.z group, each group of mice 6-8; in situ tumor group was divided into two groups Including: NK-92 group and NK-92/9.28.z group, 4 mice per group. D15 and 17 days, adoptive immunotherapy for grouped NOD/SCID mouse cells. 200ul normal saline or 200ul NK-92, Mock and NK-92/9.28.z cells were injected through the tail vein once every 5 days;
  • the volume of Huh-7 subcutaneous xenografts was measured every 3-4 days, the changes in tumor volume of each group of mice were recorded, and the growth curve of tumor volume with time was plotted. The results are shown in Fig. 6A. The growth of Huh-7 subcutaneous xenografts was shown. The tumor volume and tumor weight of the NK-92/9.28.z treatment group were significantly lower than the other two groups.
  • Fig. 6D The growth of liver tumors in situ was measured by Luciferase every other week, and the results are shown in Fig. 6D.
  • the liver was taken for immunohistochemical detection, and the results are shown in Fig. 6E.
  • Figure 6D shows an increase in the body weight of each group of mice, suggesting that NK-92/9.28.z does not cause treatment-related weight loss side effects; while residual tissue is detected, the results are shown in Figure 6E, found in tumor tissue
  • the infiltrating NK-92/9.28.z was significantly higher than the control group, but its expression was not detected in the liver, suggesting that NK-92/9.28.z does not cause liver infiltration, indicating NK-92/9.28.z safety. it is good.
  • Example 6 Therapeutic effect of NK-92/9.28.z cells on endogenous low expression of GPC3PLC/PRF/5 subcutaneous xenografts
  • NOD/SCID mice were inoculated with PLC/PRF/5 subcutaneous xenografts, and the above cells were inoculated subcutaneously, 2 ⁇ 10 6 /piece, and the tumor volume reached 30-50 mm 3 after about 15 days;
  • Subcutaneous xenografts were injected intraperitoneal cyclophosphamide (100 mg/kg) on days 12 and 13 respectively.
  • mice On day d17, the transplanted tumor volume of Nod-Scid mice was measured and grouped. The mice were divided into 3 groups, including: saline control group, Mock control group, NK-92/9.28.z group and NK-92/9.28.z irradiation group, 6-8 mice in each group;
  • Fig. 7 The experimental results are shown in Fig. 7, and Figs. 7A and 7B suggest that they also have a good antitumor effect in vivo.
  • Figure 7C suggests that each mouse is well-balanced, suggesting that NK-92/9.28.z treatment does not cause weight loss side effects; NK-92 cells are a tumor-derived killer cell line that has a risk of tumor formation in the body and is often clinically needed. After irradiation, the infusion was performed. In this animal experiment, a group of NK-92/9.28.z cells irradiation group (Irradiated NK-92/9.28.z) was established. The results showed that the effect was the same as that of the non-irradiated group. 92/9.28.z is basically consistent, providing an animal test basis for its clinical safety applications.
  • CRS is a common complication of CAR-T cell therapy and may even be life-threatening. There may be multiple mechanisms of cross-reactivity and activation of CRS. It is reported that IL6, IL-1 ⁇ and IL-2 are also involved in causing or expanding cells. An important cytokine of factor release syndrome.
  • NK-92/9.28.z NK-92 cells during the killing of SK-HEP-1 and SK-HEP-1/GPC3 at a target-to-target ratio of 1.5:1.
  • the secretion of IL6, IL-1 ⁇ and IL-2 by NK-92/9.28.z was detected.
  • NK-92/9.28.z cells did not release IL-1 ⁇ , IL during the killing process. -2 and IL-6.
  • NK-92/9.28.z can induce IL-6 release from monocytes after killing target cells.
  • NK-92/9.28.z and CAR-T/9.28.z were incubated with tumor cells Huh-7 and monocytes for 36 h, and then detected the secretion of IL-6 in the culture system by ELISA. Specifically, 5 ⁇ 10 3 monocytes were seeded into 96-well plates, and 3 ⁇ 10 4 NK-92/9.28.z or CAR-T/9.28.z cells were inoculated simultaneously, and 2 ⁇ 10 4 target cells Huh were added.
  • NK-92/9.28.z did not stimulate the release of IL-6 by monocytes in the process of killing tumor cells compared with CAR-T cells, and is a very safe effector cell.
  • Example 8 CAR modification enhances anti-tumor effect of primary NK cells
  • PBMCs were co-cultured with K562-mbIL21 cells (exposed with membrane bound interleukin-21 and IL-2 on K562 cells, and NK cells were expanded in vitro, magnetic beads ( Sorted from Meitian), the primary NK cells were obtained and named PBNK cells.
  • PBNK cells were infected with a lentivirus of AB1-28Z to obtain a primary NK cell expressing CAR, designated PBNK/9.28.z.
  • PBNK cells were infected with an empty plasmid to obtain Mock' cells.
  • Fig. 9A The secretion level of IFN- ⁇ was examined, and the results are shown in Fig. 9B.
  • the normal PBMC was used as the target cell to detect the cell killing ability of Mock' cells and PBNK/9.28.z. As shown in Fig. 10, it was shown that the modified primary NK cells had no obvious toxic effects on normal tissue cells.
  • Example 9 Targeting GPC3 chimeric antigen receptor-modified NK cells in combination with sorafenib for hepatocellular carcinoma
  • Hepatoma Huh-7 cells (target cells) were inoculated. After 24 h, different concentrations of sorafenib (2 ⁇ M, 5 ⁇ M and 10 ⁇ M, respectively) or DMSO were added, with or without different target ratios of NK-92/9.28.z cells. (1:1, 1:5, and 1:10), after incubation for 24 h, cytotoxicity was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit. The results of the experiment are shown in Table 1. NK-92/9.28.z cells have significant killing effect on Huh-7 and are effective in target-dependent ratio. The greater the target ratio, the more killing effect of NK-92/9.28.z cells.
  • the cell killing toxicity was 69.2% ⁇ 3.5%, 36.8% ⁇ 6.5%, 22.2% ⁇ 4.6% for the strong and effective target ratios of 1:1, 1:5, and 1:10; the sorafenib concentration was 10 ⁇ M, 5 ⁇ M.
  • the cell killing toxicity was 63.3.3 ⁇ 7.9%, 45.6 ⁇ 5.6% and 25.3 ⁇ 4.6%, respectively; the cytotoxicity of the combination group was 88.1% ⁇ 8.9%, 56.2% ⁇ 5.8% and 38.3%, respectively.
  • SK-HEP-1 and SK-HEP-1/GPC3 were mixed 50% each to prepare SK-HEP-1/GPC3 (50%) heterogeneous mixed cells.
  • the cytotoxicity of sorafenib, NK-92/9.28.z cells, or a combination thereof was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit. The results of the experiment are shown in Figure 11.
  • the solanin 5 ⁇ M for 48h has approximately the same killing effect on SK-HEP-1/GPC3 and SK-HEP-1/GPC3 (50%) heterogeneous mixed cells, and the killing rate is respectively It was 54.5% ⁇ 6.7% and 56.8% ⁇ 5.6%.
  • NK-92/9.28.z cells alone had significant killing effect on SK-HEP-1/GPC3, and the effective target ratio was 33.2% killing rate of 73.2% ⁇ 9.2%.
  • NK-92/9.28.z cells were used alone, and the killing rate was 39.8% ⁇ at a target-to-target ratio of 3:1. 5.6%.
  • the inhibitory effect of sorafenib combined with NK-92/9.28.z cells on liver cancer was significantly enhanced, up to 93.6% ⁇ 10.2%, which was 1.28 times that of NK-92/9.28.z group.
  • the Fini single-agent group was 1.72 times; the latter heterogeneous mixed cells, the combined toxicity of the two was up to 88.9% ⁇ 7.8%, which was 2.23 times that of the NK-92/9.28.z group.
  • the Rafini single drug group was 1.56 times. This result shows that the combination of sorafenib and NK-92/9.28.z cells can increase the killing rate of heterogeneous cell populations to a greater extent.

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Abstract

Provided is a chimeric antigen receptor NK cell expressing specific recognition for GPC3 and preparation method therefor, as well as a use of the cell in prevention and/or treatment of cancers, especially a GPC3-expression cancer. Also provided is a use of the NK cell as a targeting cell therapeutic agent and/or in adoptive immunotherapy of cancers.

Description

靶向GPC3的CAR NK细胞CAR NK cells targeting GPC3 技术领域Technical field
本发明属于细胞免疫治疗领域，具体涉及包含GPC3-CAR的NK细胞或细胞系。The invention belongs to the field of cellular immunotherapy, and in particular relates to NK cells or cell lines comprising GPC3-CAR.
背景技术Background technique
嵌合抗原受体(Chimeric antigen receptor，CAR)技术将识别肿瘤相关抗原的单链抗体和免疫细胞的活化基序相结合，通过基因转导可以赋予免疫细胞肿瘤靶向性、更强的杀伤活性和持久的生命力。该项技术最先在T细胞中实施并在CD19阳性的B淋巴细胞性白血病患者取得了巨大的成功。但由于CAR-T细胞是通过采集患者的T细胞，并进行修饰后给药的，为单人单次制备，治疗成本较高，并且，T细胞在体内的大量扩增通常引发细胞因子风暴，因此存在安全性的风险。Chimeric antigen receptor (CAR) technology combines single-chain antibodies recognizing tumor-associated antigens with activation motifs of immune cells, and can confer tumor-targeting and stronger killing activity on immune cells through gene transduction. And lasting vitality. This technology was first implemented in T cells and has achieved great success in patients with CD19-positive B-lymphocytic leukemia. However, since CAR-T cells are administered by collecting T cells from patients and being modified, they are prepared in a single preparation, and the treatment cost is high, and a large amount of expansion of T cells in the body usually causes cytokine storms. Therefore there is a risk of security.
NK细胞作为重要的广谱强力的肿瘤免疫细胞，也能够通过CAR技术改造赋予其靶向识别并摧毁肿瘤细胞的作用。NK细胞有多个成熟的永生化的细胞系，其中NK-92可在体外长期培养扩增，基因表型稳定，研究结果具有可重复性，已有一些研究人员尝试将NK-92细胞表达CAR(例如CN201580032729.X)，但由于肿瘤组织具有极复杂的微环境，CAR-NK-92细胞能否在体内实现对肿瘤细胞的杀伤会受到多种因素的影响，具有极大的不确定性。并且由于前期CAR-T研发积累了大量的设计策略和构建方法方面的经验，CAR-NK的研发大多是对CAR-T的直接模仿和改造。As an important broad-spectrum and powerful tumor immune cell, NK cells can also be targeted by CAR technology to identify and destroy tumor cells. NK cells have multiple mature immortalized cell lines, of which NK-92 can be cultured and expanded in vitro for a long time, the gene phenotype is stable, and the results are reproducible. Some researchers have tried to express CAR in NK-92 cells. (For example, CN201580032729.X), but because tumor tissue has a very complex microenvironment, whether CAR-NK-92 cells can kill tumor cells in vivo will be affected by many factors, which has great uncertainty. And because the previous CAR-T research and development accumulated a large number of design strategies and construction methods, CAR-NK research and development is mostly the direct imitation and transformation of CAR-T.
因此，本发明急需有效的CAR-NK细胞。Therefore, the present invention urgently requires an effective CAR-NK cell.
发明内容Summary of the invention
本发明的目的在于提供一种表达特异性识别GPC3的嵌合抗原受体的NK细胞。It is an object of the present invention to provide an NK cell which expresses a chimeric antigen receptor which specifically recognizes GPC3.
在具体的实施方式中，NK细胞可以是原代的NK细胞，也可以是NK92-细胞。例如，本发明提供一种表达特异性识别GPC3的嵌合抗原受体的NK-92细胞或细胞系。In a specific embodiment, the NK cells may be primary NK cells or NK92- cells. For example, the invention provides an NK-92 cell or cell line that expresses a chimeric antigen receptor that specifically recognizes GPC3.
本发明的另一目的在于提供所述NK-92细胞或细胞系在预防和/或治疗癌症，优选GPC3表达癌症中的用途。Another object of the invention is to provide the use of said NK-92 cells or cell lines for the prevention and/or treatment of cancer, preferably GPC3 expressing cancer.
本发明还有的目的在于提供所述NK-92细胞或细胞系作为靶向细胞治疗剂和/或用于过继性癌症免疫治疗的用途。A further object of the invention is to provide the use of the NK-92 cell or cell line as a targeted cell therapeutic and/or for immunotherapy of adoptive cancer.
本发明还有的目的在于提供用于产生该NK-92细胞或细胞系的方法、用于鉴别该NK-92细胞或细胞系的方法以及通过该方法获得或鉴别的NK-92细胞或细胞系及其用途。A further object of the present invention is to provide a method for producing the NK-92 cell or cell line, a method for identifying the NK-92 cell or cell line, and an NK-92 cell or cell line obtained or identified by the method. And its use.
在第一方面，本发明提供一种基因工程改造的NK细胞，其特征在于，所述细胞表达特异性识别GPC3的嵌合受体，所述的嵌合受体包含识别GPC3的胞外域、跨膜域、和/或细胞内结构域。In a first aspect, the present invention provides a genetically engineered NK cell, characterized in that said cell expresses a chimeric receptor that specifically recognizes GPC3, said chimeric receptor comprising an extracellular domain that recognizes GPC3, spans Membrane domains, and/or intracellular domains.
在具体的实施方式中，所述NK细胞是NK-92细胞。In a specific embodiment, the NK cell is an NK-92 cell.
在优选的实施方式中，所述胞外域具有SEQ ID NO：7、8、9所示的HCDR1、HCDR2、HCDR3和/或及SEQ ID NO：10、11、12所示的LCDR1、LCDR2、LCDR3。In a preferred embodiment, the extracellular domain has HCDR1, HCDR2, HCDR3 and/or as shown in SEQ ID NOs: 7, 8, 9 and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 10, 11, and 12. .
在具体的实施方式中，所述胞外域具有SEQ ID NO：7、8、9所示的HCDR1、HCDR2、HCDR3，以及SEQ ID NO：10、11、12所示的LCDR1、LCDR2、LCDR3。In a specific embodiment, the extracellular domain has HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 7, 8, and 9, and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 10, 11, and 12.
在具体的实施方式中，所述特异性识别GPC3的胞外域含有SEQ ID NO：13所示的重链可变区和SEQ ID NO：14所示的轻链可变区。In a specific embodiment, the extracellular domain that specifically recognizes GPC3 comprises the heavy chain variable region set forth in SEQ ID NO: 13 and the light chain variable region set forth in SEQ ID NO: 14.
在具体的实施方式中，所述特异性识别GPC3的胞外域具有SEQ ID NO：1所示的序列。In a specific embodiment, the extracellular domain that specifically recognizes GPC3 has the sequence set forth in SEQ ID NO:1.
在具体的实施方式中，所述嵌合受体具有SEQ ID NO：16、17、18、或19所示的氨基酸序列。In a specific embodiment, the chimeric receptor has the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, or 19.
在具体的实施方式中，In a specific embodiment,
(i)所述的跨膜域选自以下蛋白的跨膜结构域：T细胞受体的α、β或ζ链；CD28；CD3ε；CD45；CD4；CD5；CD8；CD9；CD16；CD22；CD33；CD37；CD64；CD80；CD86；CD134；CD137；CD154；KIRDS2；OX40；CD2；CD27；LFA-1(CD11a；CD18)；ICOS(CD278)；4-1BB(CD137)；GITR；CD40；BAFFR；HVEM(LIGHTR)；SLAMF7；NKp80(KLRF1)；CD160；CD19；IL2Rβ；IL2Rγ；IL7Rα；ITGA1；VLA1；CD49a；ITGA4；IA4；CD49D；ITGA6；VLA-6；CD49f；ITGAD；CD11d；ITGAE；CD103；ITGAL；CD11a；LFA-1；ITGAM；CD11b；ITGAX；CD11c；ITGB1；CD29；ITGB2；CD18；LFA-1；ITGB7；TNFR2；DNAM1(CD226)；SLAMF4(CD244、2B4)；CD84；CD96(Tactile)；CEACAM1；CRTAM；Ly9(CD229)；CD160(BY55)；PSGL1；CD100(SEMA4D)；(i) The transmembrane domain is selected from the transmembrane domain of the following proteins: alpha, beta or ζ chain of a T cell receptor; CD28; CD3 epsilon; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33 CD37;CD64;CD80;CD86;CD134;CD137;CD154;KIRDS2;OX40;CD2;CD27;LFA-1 (CD11a;CD18);ICOS(CD278);4-1BB(CD137);GITR;CD40;BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRF1); CD160; CD19; IL2Rβ; IL2Rγ; IL7Rα; ITGA1; VLA1; CD49a; ITGA4; IA4; CD49D; ITGA6; VLA-6; CD49f; ITGAD; CD11d; ITGAE; CD103; ITGAL; CD11a; LFA-1; ITGAM; CD11b; ITGAX; CD11c; ITGB1; CD29; ITGB2; CD18; LFA-1; ITGB7; TNFR2; DNAM1 (CD226); SLAMF4 (CD244, 2B4); CD84; CD96 (Tactile) ; CEACAM1; CRTAM; Ly9 (CD229); CD160 (BY55); PSGL1; CD100 (SEMA4D);
SLAMF6(NTB-A、Ly108)；SLAM(SLAMF1、CD150、IPO-3)；BLAME(SLAMF8)；SELPLG(CD162)；LTBR；PAG/Cbp；NKp44；NKp30；NKp46；NKG2D；和NKG2C；和/或SLAMF6 (NTB-A, Ly108); SLAM (SLAMF1, CD150, IPO-3); BLAME (SLAMF8); SELPLG (CD162); LTBR; PAG/Cbp; NKp44; NKp30; NKp46; NKG2D; and NKG2C;
(ii)所述的细胞内结构域包括：转录因子结合域；一级信号传导结构域和/或共刺激信号传导结构域，其中：(ii) The intracellular domain comprises: a transcription factor binding domain; a primary signaling domain and/or a costimulatory signaling domain, wherein:
(1)所述一级信号传导结构域包含选自：CD3ζ；CD3γ；CD3δ；CD3ε；常见FcRγ(FCER1G)；FcRβ(FcεR1b)；CD79a；CD79b；FcγRIIa；DAP10；和DAP12的蛋白质的功能信号传导结构域，或其组合；和/或(1) The primary signaling domain comprises a functional signal transduction selected from the group consisting of: CD3ζ; CD3γ; CD3δ; CD3ε; common FcRγ (FCER1G); FcRβ (FcεR1b); CD79a; CD79b; FcγRIIa; DAP10; Domain, or a combination thereof; and/or
(2)所述共刺激信号传导结构域包含选自如下的蛋白质的功能信号传导结构域：CD27；CD28；4-1BB(CD137)；OX40；CD30；CD40；PD-1；ICOS；淋巴细 胞功能相关的抗原-1(LFA-1)；CD2；CD7；LIGHT；NKG2C；B7-H3；特异性结合CD83的配体；CDS；ICAM-1；GITR；BAFFR；HVEM(LIGHTR)；SLAMF7；NKp80(KLRF1)；CD160；CD19；CD4；CD8α；CD8β；IL2Rβ；IL2Rγ；IL7Rα；ITGA4；VLA1；CD49a；ITGA4；IA4；CD49D；ITGA6；VLA-6；CD49f；ITGAD；CD11d；ITGAE；CD103；ITGAL；CD11a；LFA-1；ITGAM；CD11b；ITGAX；CD11c；ITGB1；CD29；ITGB2；CD18；LFA-1；ITGB7；TNFR2；TRANCE/RANKL；DNAM1(CD226)；SLAMF4(CD244；2B4)；CD84；CD96(Tactile)；CEACAM1；CRTAM；Ly9(CD229)；CD160(BY55)；PSGL1；CD100(SEMA4D)；CD69；SLAMF6(NTB-A；Ly108)；SLAM(SLAMF1；CD150；IPO-3)；BLAME(SLAMF8)；SELPLG(CD162)；LTBR；LAT；GADS；SLP-76；PAG/Cbp；NKp44；NKp30；NKp46；和NKG2D，或其组合。(2) The costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27; CD28; 4-1BB (CD137); OX40; CD30; CD40; PD-1; ICOS; Related antigen-1 (LFA-1); CD2; CD7; LIGHT; NKG2C; B7-H3; ligand for specific binding to CD83; CDS; ICAM-1; GITR; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 ( KLRF1);CD160;CD19;CD4;CD8α;CD8β;IL2Rβ;IL2Rγ;IL7Rα;ITGA4;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ;LFA-1;ITGAM;CD11b;ITGAX;CD11c;ITGB1;CD29;ITGB2;CD18;LFA-1;ITGB7;TNFR2;TRANCE/RANKL;DNAM1(CD226);SLAMF4(CD244;2B4);CD84;CD96(Tactile CEACAM1; CRTAM; Ly9 (CD229); CD160 (BY55); PSGL1; CD100 (SEMA4D); CD69; SLAMF6 (NTB-A; Ly108); SLAM (SLAMF1; CD150; IPO-3); BLAME (SLAMF8); SELPLG (CD162); LTBR; LAT; GADS; SLP-76; PAG/Cbp; NKp44; NKp30; NKp46; and NKG2D, or a combination thereof.
在第二方面，本发明提供本发明第一方面所述的细胞用于制备预防和/或治疗癌症药物的用途，所述癌症表达GPC3，所述癌症优选自肝癌，肺癌，胃癌、乳腺癌、黑色素瘤、卵巢癌、卵黄囊瘤、神经母细胞瘤。In a second aspect, the present invention provides the use of the cell of the first aspect of the invention for the preparation of a medicament for preventing and/or treating cancer, the cancer expressing GPC3, preferably from liver cancer, lung cancer, gastric cancer, breast cancer, Melanoma, ovarian cancer, yolk sac tumor, neuroblastoma.
在第三方面，本发明提供本发明第一方面所述的细胞在制备用作靶向细胞治疗剂和/或用于过继性癌症免疫治疗的药物中的用途。In a third aspect, the invention provides the use of a cell of the first aspect of the invention for the manufacture of a medicament for use as a targeted cell therapeutic and/or for immunotherapy of adoptive cancer.
在具体的实施方式中，所述预防和/或治疗癌症的药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物的使用说明书中记载了所述预防和/或治疗癌症药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物与化疗药物或放射治疗同时给予。In a specific embodiment, the prophylactic and/or therapeutic cancer drug, target is described in the instructions for use in the prevention and/or treatment of cancer, a targeted cell therapeutic, or a drug for adoptive cancer immunotherapy. The drug to the cell therapy agent or the immunotherapy for adoptive cancer is administered simultaneously with the chemotherapeutic drug or radiation therapy.
在优选的实施方式中，所述预防和/或治疗癌症的药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物还包含化疗药物。In a preferred embodiment, the medicament for preventing and/or treating cancer, a targeted cell therapeutic, or a medicament for adoptive cancer immunotherapy further comprises a chemotherapeutic drug.
在具体的实施方式中，所述预防和/或治疗癌症的药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物用于治疗肝癌。In a specific embodiment, the medicament for preventing and/or treating cancer, a targeted cell therapeutic, or a medicament for adoptive cancer immunotherapy is for treating liver cancer.
在具体的实施方式中，所述化疗药物是治疗肝癌的化疗药物；优选式I或者式II所示的化合物：In a specific embodiment, the chemotherapeutic agent is a chemotherapeutic drug for treating liver cancer; preferably a compound of formula I or formula II:
Figure PCTCN2018098623-appb-000001
Figure PCTCN2018098623-appb-000001
在具体的实施方式中，所述预防和/或治疗癌症的药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物的使用说明书中记载了在给予细胞之前，对要给药的个体进行预处理。In a specific embodiment, the instructions for the prevention and/or treatment of cancer, a targeted cell therapeutic, or a drug for adoptive cancer immunotherapy describe the individual to be administered prior to administration of the cell. Pretreatment is performed.
在优选的实施方式中，所述预处理为给予化疗药物或者放射治疗，控制肿瘤负荷的增加。In a preferred embodiment, the pretreatment is to administer a chemotherapeutic drug or radiation therapy to control an increase in tumor burden.
在具体的实施方式中，所述预防和/或治疗癌症的药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物还包含淋巴细胞消耗剂作为进行预处理的试剂，以便消耗所述个体的淋巴细胞。In a specific embodiment, the drug for preventing and/or treating cancer, a targeted cell therapeutic agent, or a drug for adoptive cancer immunotherapy further comprises a lymphocyte depleting agent as a pretreatment reagent to consume the Individual lymphocytes.
在具体的实施方式中，所述淋巴细胞消耗剂为氟达拉滨、环磷酰胺。In a specific embodiment, the lymphocyte depleting agent is fludarabine or cyclophosphamide.
在第四方面，本发明提供本发明的NK细胞的制备方法，包括：制备得到表达嵌合抗原受体的NK细胞，对表达嵌合抗原受体的NK细胞采用照射的方法进一步处理；优选地，采用γ射线照射预处理表达嵌合抗原受体的NK细胞。In a fourth aspect, the present invention provides a method for producing NK cells of the present invention, which comprises: preparing NK cells expressing a chimeric antigen receptor, and further treating the NK cells expressing the chimeric antigen receptor by irradiation; preferably NK cells expressing chimeric antigen receptors were pretreated with gamma irradiation.
在具体的实施方式中，所述NK细胞是NK-92细胞。In a specific embodiment, the NK cell is an NK-92 cell.
在第五方面，本发明提供预防和/或治疗对象的癌症的方法，所述癌症表达GPC3，包括将本发明的细胞给予需要治疗癌症的对象。In a fifth aspect, the present invention provides a method of preventing and/or treating cancer in a subject, the cancer expressing GPC3, comprising administering a cell of the present invention to a subject in need of treatment for cancer.
在优选的实施方式中，所述癌症优选自肝癌、肺癌、胃癌、乳腺癌、黑色素瘤、卵巢癌、卵黄囊瘤、神经母细胞瘤；优选肝癌。In a preferred embodiment, the cancer is preferably selected from the group consisting of liver cancer, lung cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor, neuroblastoma; preferably liver cancer.
在优选的实施方式中，本发明的预防和/或治疗对象的癌症的方法包括同时给予需要治疗癌症的对象本发明的细胞与化疗药物或放射治疗。In a preferred embodiment, the method of the invention for preventing and/or treating cancer in a subject comprises simultaneously administering to a subject in need of treatment of cancer a cell of the invention and a chemotherapeutic drug or radiation therapy.
在优选的实施方式中，所述化疗药物是治疗肝癌的化疗药物；优选式I或者式II所示的化合物：In a preferred embodiment, the chemotherapeutic agent is a chemotherapeutic agent for treating liver cancer; preferably a compound of formula I or formula II:
Figure PCTCN2018098623-appb-000002
Figure PCTCN2018098623-appb-000002
在优选的实施方式中，本发明的预防和/或治疗对象的癌症的方法包括在给予本发明的细胞之前，对要给药的个体进行预处理。In a preferred embodiment, the method of the invention for preventing and/or treating cancer in a subject comprises pretreating an individual to be administered prior to administration of the cells of the invention.
在优选的实施方式中，所述预处理为给予化疗药物或者放射治疗，控制肿瘤负荷的增加。In a preferred embodiment, the pretreatment is to administer a chemotherapeutic drug or radiation therapy to control an increase in tumor burden.
在优选的实施方式中，所述预处理为利用淋巴细胞消耗剂消耗所述个体的淋巴细胞。In a preferred embodiment, the pretreatment is to consume lymphocytes of the individual using a lymphocyte depleting agent.
在优选的实施方式中，所述淋巴细胞消耗剂为氟达拉滨、环磷酰胺。In a preferred embodiment, the lymphocyte depleting agent is fludarabine or cyclophosphamide.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to form a new or preferred technical solution. Due to space limitations, we will not repeat them here.
附图说明DRAWINGS
图1是NK-92/9.28.z细胞的流式检测结果；Figure 1 shows the results of flow cytometry of NK-92/9.28.z cells;
图2A比较了亲代(Parental)NK-92、Mock和NK-92/9.28.z细胞的体外杀伤活性；图2B显示了在低效靶比下NK-92/9.28.z细胞的体外杀伤结果；图2C显示了在缺氧状态下NK-92/9.28.z细胞的体外杀伤结果；图2D显示了在TGF-β存在下NK-92/9.28.z细胞的体外杀伤结果；图2E显示了血清水平的GPC3对NK-92/9.28.z细胞的体外杀伤影响结果；Figure 2A compares the in vitro killing activity of parental NK-92, Mock and NK-92/9.28.z cells; Figure 2B shows the in vitro killing results of NK-92/9.28.z cells at inefficient target ratios; Figure 2C shows the in vitro killing results of NK-92/9.28.z cells under hypoxic conditions; Figure 2D shows the in vitro killing results of NK-92/9.28.z cells in the presence of TGF-β; Figure 2E shows serum The effect of horizontal GPC3 on the killing effect of NK-92/9.28.z cells in vitro;
图3A显示了NK-92/9.28.z细胞脱颗粒酶的释放情况；图3B显示了IFN-gama的分泌结果；图3C显示了NK-92/9.28.z IFN-gamma分泌量与肿瘤细胞表面GPC3表达值呈正比；Figure 3A shows the release of NK-92/9.28.z cell degranzyme; Figure 3B shows the secretion of IFN-gama; Figure 3C shows the secretion of NK-92/9.28.z IFN-gamma and the surface of tumor cells GPC3 expression value is proportional;
图4A显示了SK-HEP-1和SK-HEP-1/GPC3移植瘤模型中，NK-92/9.28.z细胞的抑瘤作用；图4B显示了NK-92/9.28.z细胞浸润肿瘤局部发挥抗肿瘤作用的结果；图4C显示了NK-92/9.28.z细胞浸润肿瘤局部的免疫组化结果；Figure 4A shows the anti-tumor effect of NK-92/9.28.z cells in SK-HEP-1 and SK-HEP-1/GPC3 xenograft models; Figure 4B shows NK-92/9.28.z cells infiltrating tumors locally. The results of anti-tumor effects; Figure 4C shows the results of local immunohistochemistry of NK-92/9.28.z cells infiltrating tumors;
图5显示了NK-92/9.28.z细胞治疗对重要脏器器官的影响；Figure 5 shows the effect of NK-92/9.28.z cell therapy on important organ organs;
图6A显示了NK-92/9.28.z对内源性中高表达GPC3Huh-7皮下移植瘤的抑瘤情况；图6B显示了NK-92/9.28.z对原位肿瘤的抑瘤情况；图6C为Huh-7原位移植瘤的生长情况统计图；图6D显示了原位移植瘤模型中各组小鼠体重变化情况；图6E显示了原位移植瘤模型中NK-92/9.28.z的浸润情况；Figure 6A shows the anti-tumor effect of NK-92/9.28.z on endogenous high-expression GPC3 Huh-7 subcutaneous xenografts; Figure 6B shows the anti-tumor effect of NK-92/9.28.z on orthotopic tumors; Statistical analysis of the growth of Huh-7 orthotopic tumors; Figure 6D shows the changes in body weight of mice in the orthotopic transplantation model; Figure 6E shows the NK-92/9.28.z in the orthotopic transplantation model. Infiltration;
图7A显示了PLC/PRF/5皮下移植瘤模型的肿瘤体积变化情况；图7B显示了PLC/PRF/5皮下移植瘤模型的肿瘤重量变化情况；图7C显示了PLC/PRF/5皮下移植瘤模型中小鼠体重变化情况；Figure 7A shows tumor volume changes in the PLC/PRF/5 subcutaneous xenograft model; Figure 7B shows tumor weight changes in the PLC/PRF/5 subcutaneous xenograft model; Figure 7C shows PLC/PRF/5 subcutaneous xenografts Changes in mouse body weight in the model;
图8A显示了在杀伤过程中细胞因子的释放情况；图8B显示了NK-92/9.28.z杀伤靶细胞后单核细胞释放IL-6的结果；Figure 8A shows the release of cytokines during killing; Figure 8B shows the results of release of IL-6 by monocytes after killing target cells by NK-92/9.28.z;
图9A显示了PBNK/9.28.z的体外细胞杀伤情况，图9B显示了PBNK/9.28.z的干扰素释放水平；Figure 9A shows in vitro cell killing of PBNK/9.28.z, and Figure 9B shows interferon release levels of PBNK/9.28.z;
图10显示了PBNK/9.28.z对正常细胞的杀伤情况；Figure 10 shows the killing of normal cells by PBNK/9.28.z;
图11显示了索拉菲尼和NK-92/9.28.z细胞联用的细胞杀伤情况。Figure 11 shows the cell killing of sorafenib in combination with NK-92/9.28.z cells.
具体实施方式Detailed ways
在下面更详细地描述本发明之前，应理解本发明不限于本文中描述的特定的方法、方案和试剂，因为这些是可以变化的。还应理解，本文中使用的方法是仅用于描述特定实施方式的目的，且不意图限制本发明的范围，其仅由所附权利要求限定。除非另外说明，本文中使用的所有科学和技术术语具有本领域技术人员通常理解的含义。Before the present invention is described in more detail below, it is to be understood that the invention is not limited to the specific methods, protocols, and reagents described herein, as these may vary. It is also to be understood that the methods used herein are for the purpose of describing the particular embodiments, and are not intended to limit the scope of the invention. Unless otherwise stated, all scientific and technical terms used herein have the meaning commonly understood by one of ordinary skill in the art.
术语the term
所述的“天然杀伤(NK)细胞”，是用于过继性癌症免疫治疗的重要效应细胞类型。与T细胞类似，NK细胞可以被修饰以表达嵌合抗原受体(CAR)来增强抗肿瘤活性，但关于CAR-工程化NK细胞的经验是有限的，且缺乏临床开发的数据。。除了原代细胞之外，NK细胞有许多成熟的细胞系，包括NK-92、NKG、YT、NK-YS、HANK-1、YTS和NKL等等。在本发明的具体实施方式中，采用NK-92细胞系或者原代NK细胞时，用本发明的嵌合抗原受体修饰的NK细胞的效果最佳。The "natural killer (NK) cells" are important effector cell types for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance anti-tumor activity, but experience with CAR-engineered NK cells is limited and lacks clinically developed data. . In addition to primary cells, NK cells have many mature cell lines, including NK-92, NKG, YT, NK-YS, HANK-1, YTS, and NKL. In a specific embodiment of the present invention, when the NK-92 cell line or primary NK cells are used, the NK cells modified with the chimeric antigen receptor of the present invention are most effective.
“细胞治疗剂”，特别是"靶向细胞治疗剂"或"靶向同种异基因细胞治疗剂"是指适合施用以用于过继性癌症免疫治疗的免疫细胞，其经遗传修饰以表达特异性识别在靶肿瘤细胞的表面上表达的限定抗原的抗原受体。“过继性、靶细胞特异性免疫治疗”或“过继性癌症免疫治疗”或“过继性细胞治疗(ACT)”是指其中免疫细胞转移到携带肿瘤的宿主的治疗形式。免疫细胞具有抗肿瘤反应性且可以介导直接或间接的抗肿瘤作用。"Cellular therapeutic agent", particularly "targeted cell therapeutic agent" or "targeted allogeneic cell therapeutic agent" refers to an immune cell suitable for administration to adoptive immunotherapy for adoptive cancer, which is genetically modified to express specificity An antigen-restricting antigen receptor expressed on the surface of a target tumor cell is recognized. "Adoptive, target cell-specific immunotherapy" or "adoptive cancer immunotherapy" or "adoptent cell therapy (ACT)" refers to a form of treatment in which immune cells are transferred to a host carrying a tumor. Immune cells have anti-tumor reactivity and can mediate direct or indirect anti-tumor effects.
术语“GPC3”为磷脂酰肌醇蛋白多糖-3(Glypican-3，又称DGSX，GTR2-2，MXR7，OCI-5，SDYS，SGB，SGBS或SGBS1)，是一种细胞表面蛋白，属于硫酸乙酰肝素蛋白多糖家族。GPC3基因编码产生70-kDa左右的前体核心蛋白，该前体蛋白能够被弗林蛋白酶(furin)剪切产生40-kDa左右的可溶性的能够进入血液的氨基端(N末端)肽和30-kDa左右含有2个硫酸乙酰肝素(HS)糖链的膜结合的羧基端(C末端)肽。GPC3蛋白通过糖基磷脂酰肌醇(GPI)锚依附在细胞膜上。The term "GPC3" is Glypican-3 (also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1) and is a cell surface protein belonging to sulfuric acid. Heparan glycoprotein family. The GPC3 gene encodes a 70-kDa precursor core protein that can be cleaved by furin to produce a soluble 40-kDa-capable amino-terminal (N-terminal) peptide and 30- A membrane-bound carboxy-terminal (C-terminal) peptide containing two heparan sulfate (HS) sugar chains around kDa. The GPC3 protein is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor.
本文所用的“嵌合抗原受体”、“嵌合受体”或“CAR”是指这样的多肽，当其在免疫效应细胞中时，给所述的细胞提供针对靶细胞(通常是癌细胞)的特异性，并且具有细胞内信号产生。CAR通常包括至少一个细胞外抗原结合结构域、跨膜结构域(也称跨膜区或跨膜域)和细胞内结构域(本文中也称为“胞内区”或“胞内域”)。在某些方面，多肽组彼此邻接。多肽组包括在存在二聚化分子时可以使多肽彼此偶联的二聚化开关，例如，可以使抗原结合结构域偶联至胞内信号传导结构域。As used herein, "chimeric antigen receptor", "chimeric receptor" or "CAR" refers to a polypeptide which, when administered in an immune effector cell, provides the cell with a target cell (usually a cancer cell) Specificity, and has intracellular signal production. CAR typically includes at least one extracellular antigen binding domain, a transmembrane domain (also known as a transmembrane or transmembrane domain), and an intracellular domain (also referred to herein as an "intracellular region" or "intracellular domain"). . In certain aspects, the polypeptide groups are contiguous with each other. A polypeptide group includes a dimerization switch that can couple the polypeptides to each other in the presence of a dimerization molecule, for example, an antigen binding domain can be coupled to an intracellular signaling domain.
所述的“嵌合抗原受体”能够特异性识别GPC3。The "chimeric antigen receptor" is capable of specifically recognizing GPC3.
在具体实施方式中，所述的细胞内结构域包括一级信号传导结构域、或共刺激信号传导结构域、或者一级信号传导结构域和共刺激信号传导结构域。In a specific embodiment, the intracellular domain comprises a primary signaling domain, or a costimulatory signaling domain, or a primary signaling domain and a costimulatory signaling domain.
在具体实施例中，一级信号传导结构域可以选自：CD3ζ；CD3γ；CD3δ；CD3ε；常见FcRγ(FCER1G)；FcRβ(FcεR1b)；CD79a；CD79b；FcγRIIa；DAP10；和DAP12的蛋白质的功能信号传导结构域。在一实施例中，可以选择人CD3ζ(氨基酸序列如SEQ ID NO:6所示，核苷酸序列如SEQ ID NO:22所示)。In a particular embodiment, the primary signaling domain may be selected from the group consisting of: CD3ζ; CD3γ; CD3δ; CD3ε; common FcRγ (FCER1G); FcRβ (FcεR1b); CD79a; CD79b; FcγRIIa; DAP10; Conduction domain. In one embodiment, human CD3 can be selected (amino acid sequence as set forth in SEQ ID NO: 6, nucleotide sequence as set forth in SEQ ID NO: 22).
在具体实施例中，共刺激信号传导结构域可以选自如下的蛋白质的信号传导结构域：CD27；CD28；4-1BB(CD137)；OX40；CD30；CD40；PD-1；ICOS；淋巴细胞功能相关的抗原-1(LFA-1)；CD2；CD7；LIGHT；NKG2C；B7-H3；特异性结合CD83的配体；CDS；ICAM-1；GITR；BAFFR；HVEM(LIGHTR)；SLAMF7；NKp80(KLRF1)；CD160；CD19；CD4；CD8α；CD8β；IL2Rβ；IL2Rγ；IL7Rα；ITGA4；VLA1；CD49a；ITGA4；IA4；CD49D；ITGA6；VLA-6；CD49f；ITGAD；CD11d；ITGAE；CD103；ITGAL；CD11a；LFA-1；ITGAM；CD11b；ITGAX；CD11c；ITGB1；CD29；ITGB2；CD18；LFA-1；ITGB7；TNFR2；TRANCE/RANKL；DNAM1(CD226)；SLAMF4(CD244；2B4)；CD84；CD96(Tactile)；CEACAM1；CRTAM；Ly9(CD229)；CD160(BY55)；PSGL1；CD100(SEMA4D)；CD69；SLAMF6(NTB-A；Ly108)；SLAM(SLAMF1；CD150；IPO-3)；BLAME(SLAMF8)；SELPLG(CD162)；LTBR；LAT；GADS；SLP-76；PAG/Cbp；NKp44；NKp30；NKp46；和NKG2D，或其组合。In a particular embodiment, the costimulatory signaling domain can be selected from the signaling domains of proteins: CD27; CD28; 4-1BB (CD137); OX40; CD30; CD40; PD-1; ICOS; Related antigen-1 (LFA-1); CD2; CD7; LIGHT; NKG2C; B7-H3; ligand for specific binding to CD83; CDS; ICAM-1; GITR; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 ( KLRF1);CD160;CD19;CD4;CD8α;CD8β;IL2Rβ;IL2Rγ;IL7Rα;ITGA4;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ;LFA-1;ITGAM;CD11b;ITGAX;CD11c;ITGB1;CD29;ITGB2;CD18;LFA-1;ITGB7;TNFR2;TRANCE/RANKL;DNAM1(CD226);SLAMF4(CD244;2B4);CD84;CD96(Tactile CEACAM1; CRTAM; Ly9 (CD229); CD160 (BY55); PSGL1; CD100 (SEMA4D); CD69; SLAMF6 (NTB-A; Ly108); SLAM (SLAMF1; CD150; IPO-3); BLAME (SLAMF8); SELPLG (CD162); LTBR; LAT; GADS; SLP-76; PAG/Cbp; NKp44; NKp30; NKp46; and NKG2D, or a combination thereof.
术语“信号传导结构域”是指通过在细胞内传递信息而起作用的蛋白质的功能性部分，用来通过产生第二信使或通过响应这样的信使起效应物作用经由确定的信号传导途径调节细胞的活性。在一实施例中，可以选择人CD137的胞内域(氨基酸序列如SEQ ID NO：25所示，核苷酸序列如SEQ ID NO：26所示)、或者人CD28胞内域(氨基酸序列如SEQ ID NO：5所示，核苷酸序列如SEQ ID NO：21所示)，或者人CD137的胞内域和人CD28胞内域的组合。The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell for regulating cells via a defined signaling pathway by generating a second messenger or by acting as an effector in response to such a messenger. Activity. In one embodiment, the intracellular domain of human CD137 (amino acid sequence as set forth in SEQ ID NO: 25, nucleotide sequence as set forth in SEQ ID NO: 26), or human CD28 intracellular domain (amino acid sequence, eg, SEQ ID NO: 5, the nucleotide sequence is set forth in SEQ ID NO: 21, or a combination of the intracellular domain of human CD137 and the human CD28 intracellular domain.
所述的跨膜域选自以下蛋白的跨膜结构域：T细胞受体的α、β或ζ链、CD28；CD3ε；CD45；CD4；CD5；CD8；CD9；CD16；CD22；CD33；CD37；CD64；CD80；CD86；CD134；CD137；CD154；KIRDS2；OX40；CD2；CD27；LFA-1(CD11a；CD18)；ICOS(CD278)；4-1BB(CD137)；GITR；CD40；BAFFR；HVEM(LIGHTR)；SLAMF7；NKp80(KLRF1)；CD160；CD19；IL2Rβ；IL2Rγ；IL7Rα；ITGA1；VLA1；CD49a；ITGA4；IA4；CD49D；ITGA6；VLA-6；CD49f；ITGAD；CD11d；ITGAE；CD103；ITGAL；CD11a；LFA-1；ITGAM；CD11b；ITGAX；CD11c；ITGB1；CD29；ITGB2；CD18；LFA-1；ITGB7；TNFR2；DNAM1(CD226)；SLAMF4(CD244；2B4)；CD84；CD96(Tactile)；CEACAM1；CRTAM；Ly9(CD229)；CD160(BY55)；PSGL1；CD100(SEMA4D)；SLAMF6(NTB-A；Ly108)；SLAM(SLAMF1；CD150；IPO-3)；BLAME(SLAMF8)；SELPLG(CD162)；LTBR；PAG/Cbp；NKp44；NKp30；NKp46；NKG2D；和NKG2C；所述的细胞内结构域包括：转录因子结合域；一级信号传导结构域和/或共刺激信号传导结构域，其中：(1)所述一级信号传导结构域包含选自：CD3ζ；CD3γ；CD3δ；CD3ε；常见 FcRγ(FCER1G)；FcRβ(FcεR1b)；CD79a；CD79b；FcγRIIa；DAP10；和DAP12的蛋白质的功能信号传导结构域，或其组合；和/或(2)所述共刺激信号传导结构域包含选自如下的蛋白质的功能信号传导结构域：CD27；CD28；4-1BB(CD137)；OX40；CD30；CD40；PD-1；ICOS；淋巴细胞功能相关的抗原-1(LFA-1)；CD2；CD7；LIGHT；NKG2C；B7-H3；特异性结合CD83的配体；CDS；ICAM-1；GITR；BAFFR；HVEM(LIGHTR)；SLAMF7；NKp80(KLRF1)；CD160；CD19；CD4；CD8α；CD8β；IL2Rβ；IL2Rγ；IL7Rα；ITGA4；VLA1；CD49a；ITGA4；IA4；CD49D；ITGA6；VLA-6；CD49f；ITGAD；CD11d；ITGAE；CD103；ITGAL；CD11a；LFA-1；ITGAM；CD11b；ITGAX；CD11c；ITGB1；CD29；ITGB2；CD18；LFA-1；ITGB7；TNFR2；TRANCE/RANKL；DNAM1(CD226)；SLAMF4(CD244；2B4)；CD84；CD96(Tactile)；CEACAM1；CRTAM；Ly9(CD229)；CD160(BY55)；PSGL1；CD100(SEMA4D)；CD69；SLAMF6(NTB-A；Ly108)；SLAM(SLAMF1；CD150；IPO-3)；BLAME(SLAMF8)；SELPLG(CD162)；LTBR；LAT；GADS；SLP-76；PAG/Cbp；NKp44；NKp30；NKp46；和NKG2D，或其组合。作为示例，可以选择CD28的跨膜域(氨基酸序列如SEQ ID NO:4所示，核苷酸序列如SEQ ID NO:20所示)、CD8的跨膜域(氨基酸序列如SEQ ID NO:23所示，核苷酸序列如SEQ ID NO:24所示)。The transmembrane domain is selected from the transmembrane domain of the following proteins: alpha, beta or ζ chain of T cell receptor, CD28; CD3 epsilon; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64;CD80;CD86;CD134;CD137;CD154;KIRDS2;OX40;CD2;CD27;LFA-1(CD11a;CD18);ICOS(CD278);4-1BB(CD137);GITR;CD40;BAFFR;HVEM(LIGHTR ;SLAMF7;NKp80(KLRF1);CD160;CD19;IL2Rβ;IL2Rγ;IL7Rα;ITGA1;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ; LFA-1; ITGAM; CD11b; ITGAX; CD11c; ITGB1; CD29; ITGB2; CD18; LFA-1; ITGB7; TNFR2; DNAM1 (CD226); SLAMF4 (CD244; 2B4); CD84; CD96 (Tactile); CEACAM1; CRTAM; Ly9 (CD229); CD160 (BY55); PSGL1; CD100 (SEMA4D); SLAMF6 (NTB-A; Ly108); SLAM (SLAMF1; CD150; IPO-3); BLAME (SLAMF8); SELPLG (CD162); LTBR ;PAG/Cbp; NKp44; NKp30; NKp46; NKG2D; and NKG2C; the intracellular domain comprises: a transcription factor binding domain; a primary signaling domain and/or a costimulatory signaling junction a domain, wherein: (1) the primary signaling domain comprises a protein selected from the group consisting of: CD3ζ; CD3γ; CD3δ; CD3ε; common FcRγ (FCER1G); FcRβ (FcεR1b); CD79a; CD79b; FcγRIIa; DAP10; a functional signaling domain, or a combination thereof; and/or (2) the costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27; CD28; 4-1BB (CD137); OX40 CD30; CD40; PD-1; ICOS; lymphocyte function-associated antigen-1 (LFA-1); CD2; CD7; LIGHT; NKG2C; B7-H3; ligand for specific binding to CD83; CDS; ICAM-1 ;GITR;BAFFR;HVEM(LIGHTR);SLAMF7;NKp80(KLRF1);CD160;CD19;CD4;CD8α;CD8β;IL2Rβ;IL2Rγ;IL7Rα;ITGA4;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6 ;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a;LFA-1;ITGAM;CD11b;ITGAX;CD11c;ITGB1;CD29;ITGB2;CD18;LFA-1;ITGB7;TNFR2;TRANCE/RANKL;DNAM1(CD226 SLAMF4 (CD244; 2B4); CD84; CD96 (Tactile); CEACAM1; CRTAM; Ly9 (CD229); CD160 (BY55); PSGL1; D100 (SEMA4D); CD69; SLAMF6 (NTB-A; Ly108); SLAM (SLAMF1; CD150; IPO-3); BLAME (SLAMF8); SELPLG (CD162); LTBR; LAT; GADS; SLP-76; PAG/Cbp ; NKp44; NKp30; NKp46; and NKG2D, or a combination thereof. As an example, the transmembrane domain of CD28 (amino acid sequence as shown in SEQ ID NO: 4, nucleotide sequence as shown in SEQ ID NO: 20), transmembrane domain of CD8 (amino acid sequence such as SEQ ID NO: 23) can be selected. As shown, the nucleotide sequence is as shown in SEQ ID NO:24).
在具体实施方式中，嵌合抗原受体还含有铰链区。在一实施例中，可以选择CD8的铰链区，氨基酸序列如SEQ ID NO:3所示。In a specific embodiment, the chimeric antigen receptor further comprises a hinge region. In one embodiment, the hinge region of CD8 can be selected and the amino acid sequence is set forth in SEQ ID NO:3.
在具体的实施方式中，本发明的嵌合受体具有SEQ ID NO:16、17、18、或19所示的氨基酸序列。In a specific embodiment, the chimeric receptor of the invention has the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, or 19.
GPC3-特异性NK-92细胞或原代NK细胞GPC3-specific NK-92 cells or primary NK cells
本发明的GPC3-特异性NK-92细胞或原代NK细胞含有编码嵌合抗原受体的慢病毒载体，该嵌合抗原受体包含GPC3-特异性scFv抗体片段、铰链区、CD28的跨膜和细胞内结构域及CD3ζ的细胞内结构域。The GPC3-specific NK-92 cells or primary NK cells of the present invention comprise a lentiviral vector encoding a chimeric antigen receptor comprising a GPC3-specific scFv antibody fragment, a hinge region, and a transmembrane of CD28 And intracellular domains and intracellular domains of CD3ζ.
本发明的NK-92细胞或原代NK细胞特征在于：显示降低的天然细胞毒性或不显示天然细胞毒性，即：与GPC3阳性的细胞相比，对GPC3-阴性细胞具有降低的细胞毒性或没有细胞毒性。The NK-92 cells or primary NK cells of the invention are characterized by: reduced natural cytotoxicity or no natural cytotoxicity, ie reduced cytotoxicity or absence of GPC3-negative cells compared to GPC3-positive cells Cytotoxicity.
所述降低的天然细胞毒性构成本发明的细胞或细胞系的重要安全性特征，特别是在临床应用方面。The reduced natural cytotoxicity constitutes an important safety feature of the cells or cell lines of the invention, particularly in clinical applications.
本发明的NK-92细胞或或原代NK细胞与未修饰的NK-92细胞或原代NK细胞相反-以高效率裂解GPC3-表达肿瘤细胞，但与未修饰NK-92细胞或原代NK细胞相比较少可能攻击GPC3-阴性非靶细胞。The NK-92 cells or primary NK cells of the present invention are opposite to unmodified NK-92 cells or primary NK cells - lysing GPC3-expressing tumor cells with high efficiency, but with unmodified NK-92 cells or primary NK Cells are less likely to attack GPC3-negative non-target cells.
进一步的特征：在一个实施方式中，GPC3-特异性scFv抗体片段包含SEQ ID NO:1所示的氨基酸序列或由其组成。Further features: In one embodiment, the GPC3-specific scFv antibody fragment comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.
嵌合抗原受体(CAR)是本领域中已知的。本发明中使用的GPC3-特异性CAR包含：Chimeric antigen receptors (CAR) are known in the art. The GPC3-specific CAR used in the present invention comprises:
(i)GPC3-特异性scFv抗体片段，(i) a GPC3-specific scFv antibody fragment,
(ii)铰链区，(ii) the hinge area,
(iii)跨膜域，在一具体实施方式中，可选择CD28的跨膜域或者CD8的跨膜域，也可以选择制备CAR的技术中，常用的其他的跨膜域。(iii) Transmembrane domain, in a specific embodiment, the transmembrane domain of CD28 or the transmembrane domain of CD8 may be selected, and other transmembrane domains commonly used in the technique of preparing CAR may also be selected.
(iv)胞内域，在一具体实施方式中，胞内域具有CD3ζ的细胞内结构域，和/或共刺激信号域，可选的，共刺激信号域可以为CD28的共刺激信号域或者CD137的共刺激信号域。本发明的CAR-NK-92细胞，可以采用γ-照射阻止细胞的复制，以作为对于临床应用的潜在安全性措施，而体外和体内抗肿瘤活性被保持。(iv) an intracellular domain, in a specific embodiment, the intracellular domain has an intracellular domain of CD3ζ, and/or a costimulatory signal domain, alternatively, the costimulatory signal domain may be a costimulatory signal domain of CD28 or The costimulatory signal domain of CD137. The CAR-NK-92 cells of the present invention can prevent cell replication by gamma-irradiation as a potential safety measure for clinical applications, while in vitro and in vivo anti-tumor activity is maintained.
考虑到NK-92可能存在致瘤性。在利用本发明的CAR NK-92细胞进行治疗时，优选包括通过照射，优选γ-照射进行细胞的预处理。Considering that NK-92 may be tumorigenic. In the treatment with the CAR NK-92 cells of the present invention, it is preferred to include pretreatment of the cells by irradiation, preferably gamma irradiation.
本领域技术人员应理解，所述照射是一种安全性措施，可采用各种常规的方式进行。Those skilled in the art will appreciate that the illumination is a safety measure and can be carried out in a variety of conventional manners.
靶向GPC3NK-92/9.28.z细胞的细胞毒性分析Cytotoxicity analysis targeting GPC3NK-92/9.28.z cells
对于GPC3阴性的肝癌细胞，NK-92/9.28.z没有增强的杀伤作用；当过表达GPC3后，这些细胞对NK-92/9.28.z敏感性增强，体现了NK-92/9.28.z细胞靶向性杀伤的作用。细胞治疗实体瘤的一个障碍是进入局部微环境的效应细胞太少，达不到一个有效的效靶比去杀伤细胞，当大幅度降低效靶比，如果延长共孵时间，也能够达到一个很好的特异性杀伤；微环境中低氧和TGF-β是制约细胞治疗发挥效应的因素，而本发明显示在二者存在的情况下，NK-92/9.28.z也还是存在很好的特异性杀伤效果；血液存在分泌性GPC3，但在本发明中并不会影响NK-92/9.28.z细胞对肿瘤细胞的毒性作用。我们的数据表明编码GPC3-CAR的NK-92细胞可以耐受多种恶劣条件发挥作用，对于各种GPC3-阳性恶性肿瘤的治疗是临床上可用的。NK-92/9.28.z has no enhanced killing effect on GPC3-negative liver cancer cells; when overexpressing GPC3, these cells are more sensitive to NK-92/9.28.z, reflecting NK-92/9.28.z cells. The role of targeted killing. One obstacle to the treatment of solid tumors by cells is that too few effector cells enter the local microenvironment, failing to achieve an effective target-to-kill ratio. When the target ratio is greatly reduced, if the incubation time is extended, it can reach a very large Good specific killing; hypoxia and TGF-β in the microenvironment are factors that restrict the effect of cell therapy, and the present invention shows that NK-92/9.28.z still has a good specificity in the presence of both. Sexual killing effect; there is secretory GPC3 in the blood, but it does not affect the toxic effect of NK-92/9.28.z cells on tumor cells in the present invention. Our data indicate that NK-92 cells encoding GPC3-CAR can tolerate a variety of adverse conditions and are clinically useful for the treatment of various GPC3-positive malignancies.
NK-92/9.28.z细胞脱颗粒和细胞因子分泌情况NK-92/9.28.z cell degranulation and cytokine secretion
NK细胞起快速的毒性杀伤作用依赖于溶酶体释放颗粒酶，CD107a是溶酶体上的一个蛋白，在释放颗粒酶的时候能指示溶酶体与细胞膜的融合情况，是NK-92细胞上的一个活化指标。我们可以从NK细胞活化层面揭示了CAR的靶向性，有实施例显示NK-92/9.28.z与GPC3阳性的肝癌细胞共孵有显著增强的CD107a表达，而与GPC3阴性的肝癌细胞共孵则没有观察到这个现象；对NK本身敏感的K562细胞，两组的CD107a表达一致，说明CAR修饰不会影响NK细胞内源性杀伤功能；NK-92/9.28.z和对照组细胞分别与肿瘤细胞共孵育时，NK-92/9/28.z组明显分泌更多的IFN-gama，并且NK-92/9.28.z组IFN-gama分泌量与肿瘤细胞 表面GPC3表达值呈正比，因此，本发明从细胞因子层面提示了CAR的靶向特性。The rapid toxic killing effect of NK cells depends on lysosome-releasing granzyme. CD107a is a protein on lysosome. It can indicate the fusion of lysosome and cell membrane when releasing granzyme, which is on NK-92 cells. An activation indicator. We can reveal the targeting of CAR from the NK cell activation level. Some examples show that NK-92/9.28.z co-hatching with GPC3-positive liver cancer cells has significantly enhanced CD107a expression, while co-incubation with GPC3-negative liver cancer cells. This phenomenon was not observed; the K107 cells sensitive to NK itself, the expression of CD107a in both groups was consistent, indicating that CAR modification did not affect the endogenous killing function of NK cells; NK-92/9.28.z and control cells and tumors, respectively When the cells were co-incubated, NK-92/9/28.z group secreted more IFN-gama, and NK-92/9.28.z group IFN-gama secretion was proportional to the expression of GPC3 on the surface of tumor cells. The present invention suggests the targeting properties of CAR from the cytokine level.
NK-92/9.28.z细胞对移植瘤的治疗效果Therapeutic effect of NK-92/9.28.z cells on transplanted tumor
CAR-NK介导多种内源性细胞毒受体引起的靶细胞裂解，面对抗原不均一表达的肿瘤可能比CAR-T更有优势。NK细胞异体输注不会引起移植物抗宿主病，其基于KIR-MHC错配的异体模式还可以避免或减少KIR介导的抑制性信号，使得商业化通用型高效“NK血液制品”成为可能。CAR-NK mediates the lysis of target cells caused by a variety of endogenous cytotoxic receptors, and tumors with heterogeneous expression of antigen may be more advantageous than CAR-T. Allogeneic infusion of NK cells does not cause graft-versus-host disease, and its KIR-MHC mismatch-based allogeneic model can also avoid or reduce KIR-mediated inhibitory signals, making it possible to commercialize universal and efficient "NK blood products" .
本发明的实施例表明NK-92/9.28.z在体内也存在抗原依赖性地抑瘤作用，通过诱导肿瘤细胞凋亡和抑制增殖来发挥作用的；且不会浸润重要的脏器组织诱导细胞毒性作用；不会引起治疗相关体重下降副作用。The examples of the present invention indicate that NK-92/9.28.z also has an antigen-dependent antitumor effect in vivo, which acts by inducing tumor cell apoptosis and inhibiting proliferation; and does not infiltrate important organ tissue-inducing cells. Toxic effects; does not cause side effects associated with weight loss in treatment.
NK-92细胞是一个肿瘤来源的杀伤细胞系，输入体内有成瘤风险，临床上往往需要辐照后再进行输注，本动物实验设立了一组NK-92/9.28.z细胞的辐照组，结果提示其效果跟未辐照组的NK-92/9.28.z基本一致，为其临床安全应用提供了动物实验依据。NK-92 cells are a tumor-derived killer cell line, which has the risk of tumor formation in the body. It is often necessary to infuse after irradiation. This animal experiment established a set of irradiation of NK-92/9.28.z cells. The results suggest that the effect is basically the same as that of the NK-92/9.28.z in the non-irradiated group, providing an animal experimental basis for its clinical safety application.
本发明证明NK-92/9.28.z细胞在体内保留靶细胞特异性，且能够穿透组织和归巢到远处肿瘤位点。重要地，经照射的NK-92/9.28.z细胞的体内抗肿瘤活性与非照射的细胞相同。这对于NK-92/9.28.z的未来临床应用可能是有意义的，其中细胞的照射可以如之前在未修饰的NK-92细胞的I期临床实验中所进行的(Arai等,2008；Tonn等,2013)作为安全性措施包括。The present invention demonstrates that NK-92/9.28.z cells retain target cell specificity in vivo and are capable of penetrating tissue and homing to distant tumor sites. Importantly, the in vivo anti-tumor activity of irradiated NK-92/9.28.z cells was identical to that of non-irradiated cells. This may be of interest for future clinical applications of NK-92/9.28.z, where irradiation of cells can be performed as in previous phase I clinical trials of unmodified NK-92 cells (Arai et al., 2008; Tonn Etc., 2013) included as a security measure.
肿瘤患者中的免疫细胞通常由于癌症的免疫抑制活性而功能上受损。因此，对于采用NK细胞的过继性癌症免疫治疗，供体来源的同种异基因细胞是优选的，因为它们不识别肿瘤细胞为“自身的”，从而规避抑制性信号(Geller和Miller；2011)。我们已经证明，这一优势可以扩展到CAR-工程化的NK-92细胞。这样的细胞对于各种GPC3-阳性恶性肿瘤的治疗是临床上可用的。Immune cells in tumor patients are often functionally impaired due to the immunosuppressive activity of cancer. Therefore, for adoptive cancer immunotherapy with NK cells, donor-derived allogeneic cells are preferred because they do not recognize tumor cells as "self", thereby circumventing inhibitory signals (Geller and Miller; 2011) . We have shown that this advantage can be extended to CAR-engineered NK-92 cells. Such cells are clinically useful for the treatment of various GPC3-positive malignancies.
CAR修饰增强原代NK细胞抗肿瘤效果CAR modification enhances the anti-tumor effect of primary NK cells
来自外周血培养得到的原代NK细胞(PBNK细胞)经CAR修饰后，能够特异性杀伤GPC3阳性的肝癌细胞系分泌IFN-γ，而Mock和未转染的NK细胞明显杀伤效应更弱，胞内IFN-γ的表达也更低，体外毒性实验提示，修饰后的原代NK细胞对正常的组织细胞没有明显的毒性作用。The primary NK cells (PBNK cells) obtained from peripheral blood culture can specifically kill GPC3-positive hepatoma cell line secreting IFN-γ after CAR modification, while Mock and untransfected NK cells have weaker killing effect. The expression of IFN-γ was also lower. In vitro toxicity experiments indicated that the modified primary NK cells had no obvious toxic effects on normal tissue cells.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions.
实施例1.CAR-NK92细胞的制备Example 1. Preparation of CAR-NK92 cells
作为示例性的，本实施例选择靶向GPC3的抗体AB1(scFv的序列如SEQ ID NO：1所示)作为嵌合抗原受体的胞外域。预制备的CAR具有SEQ ID NO：17所示的序列。Illustratively, this example selects antibody AB1 (the sequence of the scFv as shown in SEQ ID NO: 1) that targets GPC3 as the extracellular domain of the chimeric antigen receptor. The pre-prepared CAR has the sequence shown in SEQ ID NO: 17.
1、质粒构建1, plasmid construction
以PRRLSIN-cPPT.EF-1α为载体，构建了表达抗体AB1的二代嵌合抗原受体的慢病毒质粒，PRRLSIN-cPPT.EF-1α-AB1-28Z。AB1-28Z序列由CD8α信号肽(SEQ ID NO：2)、AB1的scFv(SEQ ID NO：30)、CD8hinge(SEQ ID NO：15)、CD28跨膜区(SEQID NO：20)和胞内信号传导结构域(SEQ ID NO：21)以及CD3的胞内段CD3ξ(SEQ ID NO：22)组成。Using PRRLSIN-cPPT.EF-1α as a vector, a lentiviral plasmid expressing the second-generation chimeric antigen receptor of antibody AB1, PRRLSIN-cPPT.EF-1α-AB1-28Z, was constructed. The AB1-28Z sequence consists of the CD8α signal peptide (SEQ ID NO: 2), the scFv of AB1 (SEQ ID NO: 30), the CD8hinge (SEQ ID NO: 15), the CD28 transmembrane region (SEQ ID NO: 20), and the intracellular signal. The conduction domain (SEQ ID NO: 21) and the intracellular domain CD3 (CD ID NO: 22) of CD3 consist.
2、慢病毒包装、病毒浓缩及滴度测定2. Lentiviral packaging, virus concentration and titer determination
a.慢病毒包装a. Lentiviral packaging
1)以5×10 6的密度接种培养至第6～10代的293T细胞于10cm培养皿中，37℃，5％CO 2培养过夜，培养基为含10％胎牛血清(Gibico)的DMEM； 1) Seeded 293T cells cultured to the 6th to 10th generations at a density of 5 × 10 6 in a 10 cm culture dish, and cultured overnight at 37 ° C, 5% CO 2 , and the medium was DMEM containing 10% fetal bovine serum (Gibico). ;
2)分别将目的基因质粒PRRLSIN-cPPT.EF-1α-EGFP(Mock)和PRRLSIN-cPPT.EF-1α-AB1-28Z 5.4μg与包装质粒pRsv-REV 6.2μg、RRE-PMDLg6.2μg、Vsvg 2.4μg溶入800μL空白DMEM培养液，混匀室温孵育5min；将60μg PEI(1μg/μl)溶解于800μl的无血清DMEM培养液中，轻轻混匀(或1000rpm涡旋5秒钟)；将质粒混合液加入PEI混合液中，加入后轻轻混匀，室温下孵育20min，得到转染复合物；2) The target gene plasmids PRRLSIN-cPPT.EF-1α-EGFP (Mock) and PRRLSIN-cPPT.EF-1α-AB1-28Z 5.4 μg and packaging plasmid pRsv-REV 6.2 μg, RRE-PMDLg 6.2 μg, Vsvg 2.4 Μg was dissolved in 800 μL of blank DMEM medium and mixed for 5 min at room temperature; 60 μg of PEI (1 μg/μl) was dissolved in 800 μl of serum-free DMEM medium, gently mixed (or vortexed at 1000 rpm for 5 seconds); The mixed solution was added to the PEI mixture, gently added after the addition, and incubated at room temperature for 20 min to obtain a transfection complex;
3)将转染复合物1.6ml滴加入含11ml DMEM培养基的10cm培养皿中培养，得到慢病毒AB1-28Z，收集病毒液上清，进行浓缩，并测定滴度。3) 1.6 ml of the transfection complex was added dropwise to a 10 cm culture dish containing 11 ml of DMEM medium to obtain lentivirus AB1-28Z, and the supernatant of the virus solution was collected, concentrated, and the titer was measured.
浓缩后的病毒滴度分别为：慢病毒AB1-28Z：2.4×10 8U/ml，慢病毒Mock：2×10 8U/ml。 The virus titers after concentration were: lentivirus AB1-28Z: 2.4 × 10 8 U/ml, lentivirus Mock: 2 × 10 8 U/ml.
3、CAR-NK-92细胞系的制备3. Preparation of CAR-NK-92 cell line
1)Retronectin包被24孔板：每孔加入380μl 5μg/ml的retronectin溶液(PBS)，4度孵育过夜后弃去24孔板中的retronectin溶液(PBS)，PBS洗两次；1) Retronectin coated 24 well plates: 380 μl of 5 μg/ml retronectin solution (PBS) was added to each well, and after overnight incubation at 4 degrees, the retronectin solution (PBS) in a 24-well plate was discarded, and washed twice with PBS;
2)将NK-92细胞接种于包被了retronectin的24孔板内，每孔细胞数目5×10 5，培养液体积500μl；按照MOI＝30在PBMC细胞中加入浓缩后的慢病毒(慢病毒AB1-28Z或者慢病毒Mock)，32度，1800rpm，离心40min后，转移至细胞培养箱中； 2) Inoculate NK-92 cells into a 24-well plate coated with retronectin, the number of cells per well is 5 × 10 5 , and the volume of the culture solution is 500 μl; the concentrated lentivirus (llow virus) is added to the PBMC cells according to MOI=30. AB1-28Z or lentivirus Mock), 32 degrees, 1800 rpm, after centrifugation for 40 min, transfer to a cell culture incubator;
3)扩增培养：感染后的细胞每隔一天采用5×10 5/mL的密度进行传代，同时在淋巴细胞培养液中补加终浓度500U/mL的重组人IL-2。 3) Amplification culture: The infected cells were passaged every other day at a density of 5 × 10 5 /mL, and a recombinant human IL-2 having a final concentration of 500 U/mL was supplemented in the lymphocyte culture solution.
得到表达空载体的Mock NK-92细胞(命名为Mock)或者表达CAR的NK-92细胞(命名为NK-92/9.28.z)。Mock NK-92 cells (designated Mock) expressing empty vector or NK-92 cells expressing CAR (designated NK-92/9.28.z) were obtained.
4、流式分选仪FACSAriaII分选CAR阳性表达的NK-92细胞系(NK-92/9.28.z)1)准备1×10 7-2×10 7个细胞，4度，5000rpm，离心5min，弃上清，PBS洗两次； 4. Flow sorter FACSAriaII sorted CAR-positive NK-92 cell line (NK-92/9.28.z) 1) Prepared 1 × 10 7 - 2 × 10 7 cells, 4 degrees, 5000 rpm, centrifuged for 5 min Discard the supernatant and wash twice with PBS;
2)对照组细胞加入50μl PE-SA(1:200稀释)抗体冰上孵育45min，PBS(2％NBS)洗两次，重悬后作为对照；2) Control cells were incubated with 50 μl of PE-SA (1:200 dilution) antibody for 45 min on ice, washed twice with PBS (2% NBS), and resuspended as a control;
3)检测组细胞+50μl 1:50稀释的生物素-山羊抗人IgG，F(ab’) 2抗体，冰上孵育45min；PBS(2％NBS)洗两次；加入50μl PE-SA(1:200稀释)抗体冰上孵育45min； 3) Test group cells + 50 μl 1:50 diluted biotin-goat anti-human IgG, F(ab') 2 antibody, incubated on ice for 45 min; PBS (2% NBS) twice; add 50 μl PE-SA (1 :200 dilution) antibody incubation on ice for 45 min;
4)重悬于1ml PBS(10％FBS+1％青链霉素)；4) Resuspend in 1ml PBS (10% FBS + 1% streptomycin);
5)充分混匀细胞后分选取300ul细胞悬液进行细胞计数和流式分选仪FACSAriaII机器调试；5) After thoroughly mixing the cells, select 300 ul of cell suspension for cell counting and flow sorter FACSAria II machine debugging;
6)调节机器电压、设置门的位置，控制分选进样速率为4000-5000个细胞/s，分选出实验组PE阳性的细胞；6) Adjust the machine voltage, set the position of the door, control the sorting injection rate to 4000-5000 cells/s, and sort out the PE positive cells of the experimental group;
7)分选接收管接收的细胞置于37度，5％CO 2孵箱进行培养； 7) The cells received by the sorting receiving tube are placed in a 37-degree, 5% CO 2 incubator for cultivation;
流式细胞仪检测NK-92/9.28.z的阳性率，结果如图1所示；经过流式分选的NK-92/9.28.z细胞，相对于Mock组和Parental NK-92组，其表面有显著增强的CAR的表达。其中，Mock组为转染了Mock质粒的NK-92细胞，Parental NK-92就是原始的NK-92细胞。The positive rate of NK-92/9.28.z was detected by flow cytometry. The results are shown in Figure 1. The flow-sorted NK-92/9.28.z cells were compared with the Mock group and the Parental NK-92 group. The surface has a significantly enhanced expression of CAR. Among them, the Mock group was NK-92 cells transfected with Mock plasmid, and Parental NK-92 was the original NK-92 cells.
实施例2.靶向GPC3NK-92/9.28.z细胞的细胞毒性测定Example 2. Cytotoxicity assay targeting GPC3NK-92/9.28.z cells
采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)测定细胞毒性。具体方法参照CytoTox 96非放射性细胞毒性检测试剂盒说明书，检测时间为第6h。Cytotoxicity was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit (Promega). The specific method is described in the instruction manual of CytoTox 96 non-radioactive cytotoxicity test kit, and the detection time is 6h.
靶细胞：分别接种50μL 2×10 5/mL的HepG2；Huh-7；Hep3B；PLC/PRF/5；SK-HEP-1；SK-HEP-1/GPC3；SMMC7721；SMMC7721/GPC3细胞于96well板(GPC3阳性的肝癌细胞系：HepG2、Huh-7、SK-HEP-1/GPC3、PLC/PRF/5、SMMC7721/GPC3，GPC3阴性的肝癌细胞系：SK-HEP-1、SMMC7721)。 Target cells: 50 μL of 2×10 5 /mL HepG2; Huh-7; Hep3B; PLC/PRF/5; SK-HEP-1; SK-HEP-1/GPC3; SMMC7721; SMMC7721/GPC3 cells in 96well plate (GPC3-positive liver cancer cell lines: HepG2, Huh-7, SK-HEP-1/GPC3, PLC/PRF/5, SMMC7721/GPC3, GPC3-negative liver cancer cell lines: SK-HEP-1, SMMC7721).
效应细胞:按效靶比3:1、1.5:1或0.75:1加实施例1所述的Mock；NK-92及NK-92/9.28.z细胞。Effector cells: Mock; NK-92 and NK-92/9.28.z cells as described in Example 1 were added at a target ratio of 3:1, 1.5:1 or 0.75:1.
实验结果如图2A所示，对于GPC3阳性的肝癌细胞，相比Mock组和Parental NK-92组，NK-92/9.28.z的体外毒性明显更强，对于GPC3阴性的肝癌细胞，NK-92/9.28.z没有增强的杀伤作用；当过表达GPC3后，这些细胞对NK-92/9.28.z敏感性增强，体现了NK-92/9.28.z细胞靶向性杀伤的作用。The results of the experiment are shown in Fig. 2A. For GPC3-positive liver cancer cells, the in vitro toxicity of NK-92/9.28.z was significantly stronger than that of the Mock group and the Parental NK-92 group. For GPC3-negative liver cancer cells, NK-92 /9.28.z has no enhanced killing effect; when overexpressing GPC3, these cells are more sensitive to NK-92/9.28.z, reflecting the targeted killing effect of NK-92/9.28.z cells.
为了进一步确认肿瘤微环境对NK-92/9.28.z的影响，还检测了NK-92/9.28.z在不同状态和条件下对肝癌细胞系的毒性作用。To further confirm the effect of tumor microenvironment on NK-92/9.28.z, the cytotoxic effect of NK-92/9.28.z on hepatoma cell lines under different conditions and conditions was also examined.
(1)、要想达到良好的抗肿瘤效果，往往需要足够数量的效应细胞去杀伤肿瘤 细胞。然而，细胞治疗实体瘤的一个障碍就是进入局部微环境的效应细胞太少，往往达不到有效的效靶比杀伤肿瘤。为了模拟这种体内环境，我们以Huh-7作为靶细胞，降低效靶比后，共孵24小时，进行检测，结果如图2B所示，均能够达到很好对杀伤。(1) In order to achieve good anti-tumor effects, a sufficient number of effector cells are often required to kill tumor cells. However, one of the obstacles to the treatment of solid tumors by cells is that too few effector cells enter the local microenvironment, often failing to achieve effective efficacy targets than killing tumors. In order to simulate this in vivo environment, we used Huh-7 as a target cell, and after reducing the effective target ratio, we incubated for 24 hours and tested it. The results are as shown in Fig. 2B, and all of them can achieve good killing.
(2)、由于绝大多数实体瘤内存在缺氧微环境，缺氧微环境是重要的抑制免疫治疗效果的因素，因此利用缺氧培养箱模拟缺氧环境，以Huh-7作为靶细胞，检测了在不同含氧状态下(低氧环境的氧气浓度为1％，正常含氧环境的氧气浓度为20％)不同效靶比NK-92/9.28.z的体外毒性情况，结果如图2C所示，在低氧环境下NK-92/9.28.z的杀伤功能并未被显著影响。(2) Because most of the solid tumors have an oxygen-deficient microenvironment, the hypoxic microenvironment is an important factor to suppress the effect of immunotherapy. Therefore, the hypoxic incubator is used to simulate the hypoxic environment, with Huh-7 as the target cell. The in vitro toxicity of different effective target ratios of NK-92/9.28.z was measured under different oxygen conditions (1% oxygen concentration in low oxygen environment and 20% oxygen concentration in normal oxygen environment). The results are shown in Fig. 2C. As shown, the killing function of NK-92/9.28.z was not significantly affected in a low oxygen environment.
(3)、TGF-β是制约细胞治疗发挥效应的另一个重要因素，以Huh-7作为靶细胞，我们在杀伤体系中加入了5-20ng/mL的TGF-β，在效靶比为3:1的条件下，孵育24h，结果如图2D所示，TGF-β能够相对明显地抑制CAR-T/9.28.z的杀伤功能，而NK-92/9.28.z细胞表对TGF-β却表现了相当程度的抵抗，其杀伤功能没有明显地被TGF-β影响。(3), TGF-β is another important factor that restricts the effect of cell therapy. With Huh-7 as the target cell, we added 5-20 ng/mL of TGF-β to the killing system, and the effective target ratio is 3. Incubation for 24 h under the conditions of 1:1, the results shown in Figure 2D, TGF-β can significantly inhibit the killing function of CAR-T/9.28.z, while the NK-92/9.28.z cell surface for TGF-β It exhibited a considerable degree of resistance and its killing function was not significantly affected by TGF-β.
其中，CAR-T/9.28.z为表达SEQ ID NO：17所示的CAR的T细胞，该T细胞来自外周血，经本领域常规的分子生物学，将慢病毒AB1-28Z感染T细胞得到，具体制备方法可参考CN104140974A、CN106397593A等所示。Wherein CAR-T/9.28.z is a T cell expressing the CAR of SEQ ID NO: 17, which is derived from peripheral blood, and which is infected with lentivirus AB1-28Z by T cells in a molecular biology routine in the art. For specific preparation methods, refer to CN104140974A, CN106397593A and the like.
(4)、GPC3是一种细胞表面标记物，但GPC3蛋白N端的肽段可被切断入血(GPC3N)，还可以被notum酶在GPI位点剪切形成一个可溶性的GPC3蛋白(GPC3△GPI)释放入血。血清水平的GPC3可能与肝癌细胞膜表面的GPC3竞争，影响NK-92/9.28.z的杀伤效果。我们通过蛋白重组方法表达了GPC3N和GPC3△GPI蛋白，在杀伤体系中加入不同浓度的上述可溶性GPC3蛋白，以Huh-7作为靶细胞，在效靶比为3:1的条件下，孵育6h，结果如图2E所示，二者在各个浓度下均不会显著影响NK-92/9.28.z细胞的杀伤功能。(4) GPC3 is a cell surface marker, but the N-terminal peptide of GPC3 protein can be cleaved into blood (GPC3N), and can also be cleaved by the noumase at the GPI site to form a soluble GPC3 protein (GPC3△GPI). ) released into the blood. Serum levels of GPC3 may compete with GPC3 on the surface of liver cancer cells, affecting the killing effect of NK-92/9.28.z. We expressed GPC3N and GPC3△GPI proteins by protein recombination method, and added different concentrations of the above soluble GPC3 protein in the killing system, using Huh-7 as the target cell, and incubating for 6 hours under the condition of 3:1 effective target ratio. The results are shown in Figure 2E, and the two did not significantly affect the killing function of NK-92/9.28.z cells at each concentration.
实施例3.NK-92/9.28.z细胞脱颗粒和细胞因子分泌情况Example 3. Degranulation and cytokine secretion of NK-92/9.28.z cells
由于NK细胞起快速的毒性杀伤作用依赖于溶酶体释放颗粒酶，CD107a溶酶体上的一个蛋白，在释放颗粒酶的时候能指示溶酶体与细胞膜的融合情况，因此我们检测了NK-92/9.28.z和Parental NK-92细胞与肿瘤共孵育时脱颗粒情况。通过流式细胞技术，把NK-92/9.28.z细胞与对照组NK-92细胞与SK-HEP-1、SK-HEP-1/GPC3与K562细胞按照不同比例共培养后检测NK细胞表面的CD107a表达情况。培养基作为阴性对照，PMA+IONO刺激作为阳性对照。具体如下：将3×10 5的NK-92/9.28.z细胞接种到24孔板，同时接种2×10 5的靶细胞，终体积为400ul，不添加外源性的IL-2，放培养箱培养6小时候收集悬浮细胞；采用流式方式检测表明CD107a的表达，结果如图3A所示，相比对照组(Parental NK-92)，NK-92/9.28.z与GPC3阳性的肝癌细胞(SK-HEP-1/GPC3)共孵有显著增强的 CD107a表达，而对GPC3阴性的肝癌细胞没有观察到这个变化，从NK细胞活化层面揭示了CAR的靶向性。对NK本身敏感的K562细胞，两组的CD107a表达一致，说明CAR修饰不会影响NK细胞内源性杀伤功能。PMA+IONO可以非特异性激活免疫细胞，PMA+IONO作用后，NK-92和NK-92/9.28.z上CD107a表达均显著升高，差别无统计学差异，提示嵌合抗原受体修饰NK-92细胞不会影响细胞内源性的杀伤功能。 Since NK cells have a rapid toxic killing effect depending on lysosome-releasing granzyme, a protein on CD107a lysosome can indicate the fusion of lysosome and cell membrane when releasing granzyme, so we detected NK- Degranulation of 92/9.28.z and Parental NK-92 cells co-incubated with tumors. NK-92/9.28.z cells and control group NK-92 cells were co-cultured with SK-HEP-1, SK-HEP-1/GPC3 and K562 cells in different proportions by flow cytometry. CD107a expression. The medium was used as a negative control and PMA + IONO stimulation was used as a positive control. The details are as follows: 3×10 5 NK-92/9.28.z cells were seeded into 24-well plates, and 2×10 5 target cells were inoculated at a final volume of 400 ul. No exogenous IL-2 was added, and culture was carried out. The suspension cells were collected at 6 hours in the culture of the box; the expression of CD107a was detected by flow cytometry, and the results are shown in Fig. 3A, compared with the control group (Parental NK-92), NK-92/9.28.z and GPC3-positive liver cancer cells ( SK-HEP-1/GPC3) co-incubation significantly enhanced CD107a expression, whereas this change was not observed in GPC3-negative liver cancer cells, revealing the targeting of CAR from the NK cell activation level. The expression of CD107a in K562 cells sensitive to NK itself was consistent, indicating that CAR modification did not affect the endogenous killing function of NK cells. PMA+IONO can non-specifically activate immune cells. After PMA+IONO treatment, the expression of CD107a on NK-92 and NK-92/9.28.z was significantly increased, and the difference was not statistically different, suggesting that chimeric antigen receptor modified NK- 92 cells do not affect the endogenous killing function of the cells.
IFN-γ是NK细胞分泌的最重要细胞因子之一，我们还比较了在效靶比1.5:1的情况下，NK-92/9.28.z和NK-92细胞杀伤常见肝癌细胞系过程中分泌的IFN-γ水平。具体如下：1)将5×10 4的NK-92/9.28.z细胞接种到24孔板，同时接种5×10 4的靶细胞，终体积为400ul，不添加外源性的IL-2，放培养箱培养24小时候收集上清；2)采用ELISA的方法检测IFN-gama的表达； IFN-γ is one of the most important cytokines secreted by NK cells. We also compared the secretion of NK-92/9.28.z and NK-92 cells in the common hepatocellular carcinoma cell line at a target-to-target ratio of 1.5:1. IFN-γ levels. The details are as follows: 1) 5×10 4 NK-92/9.28.z cells were seeded into a 24-well plate, and 5×10 4 target cells were inoculated at a final volume of 400 ul without exogenous IL-2. The supernatant was collected in an incubator for 24 hours; 2) the expression of IFN-gama was detected by ELISA;
结果如图3B和3C所示，，图3B显示NK-92/9/28.z明显分泌更多的IFN-gama；图3C提示NK-92/9.28.z IFN-gama分泌量与肿瘤细胞表面GPC3表达值呈正比，从细胞因子层面提示了CAR的靶向特性。The results are shown in Figures 3B and 3C, Figure 3B shows that NK-92/9/28.z clearly secretes more IFN-gama; Figure 3C suggests that NK-92/9.28.z IFN-gama secretion and tumor cell surface The expression of GPC3 is directly proportional, suggesting the targeting properties of CAR from the cytokine level.
实施例4.NK-92/9.28.z细胞对SK-HEP-1和SK-HEP-1/GPC3移植瘤的治疗效果Example 4. Therapeutic effect of NK-92/9.28.z cells on SK-HEP-1 and SK-HEP-1/GPC3 xenografts
a.NOD/SCID小鼠接种SK-HEP-1和SK-HEP-1/GPC3移植瘤a.NOD/SCID mice were inoculated with SK-HEP-1 and SK-HEP-1/GPC3 xenografts
通过皮下接种上述细胞，2×10 6/只，约15天后瘤体积达到30-50mm 3； The above cells were inoculated subcutaneously, 2×10 6 /piece, and the tumor volume reached 30-50 mm 3 after about 15 days;
b.扩增Mock、NK-92/9.28.z和Parental NK-92细胞；b. Amplification of Mock, NK-92/9.28.z and Parental NK-92 cells;
c.对SK-HEP-1和SK-HEP-1/GPC3移植瘤进行NK-92/9.28.z细胞过继免疫治疗c. NK-92/9.28.z cell adoptive immunotherapy for SK-HEP-1 and SK-HEP-1/GPC3 xenografts
1)d16天，对Nod-Scid小鼠通过腹腔注射环磷酰胺(100mg/kg)；1) On day d16, Nod-Scid mice were injected intraperitoneally with cyclophosphamide (100 mg/kg);
2)d17天，测量Nod-Scid小鼠移植瘤体积并进行分组。小鼠共分为3组，包括：生理盐水对照组、Mock对照组、NK-92/9.28.z组，每组小鼠6-8只；2) On day d17, the transplanted tumor volume of Nod-Scid mice was measured and grouped. The mice were divided into 3 groups, including: saline control group, Mock control group, NK-92/9.28.z group, 6-8 mice in each group;
3)d17天，对分组后的Nod-Scid小鼠细胞过继免疫治疗。通过尾静脉注射200ul生理盐水或200ul NK-92、Mock和NK-92/9.28.z细胞，每5天一次；3) On day d17, the grouped Nod-Scid mouse cells were subjected to adoptive immunotherapy. 200ul normal saline or 200ul NK-92, Mock and NK-92/9.28.z cells were injected through the tail vein once every 5 days;
每隔3-4天测量SK-HEP-1和SK-HEP-1/GPC3移植瘤体积的大小，记录每组小鼠瘤体积的变化，并绘制瘤体积随时间的生长曲线。The volume of SK-HEP-1 and SK-HEP-1/GPC3 xenografts was measured every 3-4 days, the changes in tumor volume of each group of mice were recorded, and the growth curve of tumor volume with time was plotted.
取SK-HEP-1/GPC3移植瘤小鼠肿瘤组织，通过流式方式检测NK-92/9.28.z细胞浸润，通过免疫组化方式检测NK-92/9.28.z浸润和检测肿瘤组织增殖及凋亡指标；取SK-HEP-1/GPC3移植瘤小鼠重要脏器，做HE及免疫组化染色。The tumor tissues of SK-HEP-1/GPC3 transplanted tumor mice were obtained, and NK-92/9.28.z cell infiltration was detected by flow cytometry. NK-92/9.28.z infiltration and detection of tumor tissue proliferation were detected by immunohistochemistry. Apoptosis index; important organs of SK-HEP-1/GPC3 transplanted tumor mice were taken for HE and immunohistochemical staining.
结果是如图4A所示，对于GPC3阴性表达的SK-HEP-1，NK-92/9.28.z与对照组均没有明显效果；而对SK-HEP-1/GPC3，NK-92/9.28.z治疗组肿瘤要明显小于对照组，提示NK-92/9.28.z在体内也存在抗原依赖性地抑瘤作用。在SK-HEP-1/GPC3这个动物模型中通过流式方法检测了浸润，结果如4B图所示， 在接受NK-92/9.28.z治疗的SK-HEP-1/GPC3肿瘤模型中检测到了强荧光，而接受NK-92治疗的SK-HEP-1/GPC3模型和接受以上两种治疗的SK-HEP-1肿瘤模型组都没有检测到明显增强的荧光，提示NK-92/9.28.z可以靶向性浸润GPC3阳性肿瘤；取SK-HEP-1/GPC3模型残余肿瘤组织进行免疫组化染色，结果如图4C所示，CD56在NK-92/9.28.z治疗后切片中弥漫存在，而在NK-92治疗后切片中CD56表达含量很低，从免疫组化层面提示了NK-92/9.28.z靶向性浸润GPC3阳性肿瘤的作用。免疫组化结果进一步提示，相比NK-92细胞，SK-HEP-1/GPC3动物模型在接受NK-92/9.28.z治疗后的肿瘤组织切片Ki67显著降低而剪切型Caspase-3明显升高，提示NK-92/9.28.z是通过抑制肿瘤细胞增殖及促进肿瘤细胞凋亡起效。As a result, as shown in Fig. 4A, for SK-HEP-1 negatively expressed by GPC3, NK-92/9.28.z had no significant effect with the control group; whereas for SK-HEP-1/GPC3, NK-92/9.28. The tumor in the treatment group was significantly smaller than the control group, suggesting that NK-92/9.28.z also has an antigen-dependent antitumor effect in vivo. Infiltration was detected by flow cytometry in the animal model SK-HEP-1/GPC3, and the results were detected in the SK-HEP-1/GPC3 tumor model treated with NK-92/9.28.z as shown in Fig. 4B. Strong fluorescence, and the SK-HEP-1/GPC3 model treated with NK-92 and the SK-HEP-1 tumor model group receiving both treatments did not detect significantly enhanced fluorescence, suggesting NK-92/9.28.z Targeted infiltration of GPC3-positive tumors; residual tissue of SK-HEP-1/GPC3 model was used for immunohistochemical staining. As shown in Figure 4C, CD56 was diffusely present in the NK-92/9.28.z treated sections. The expression of CD56 in the sections after NK-92 treatment was very low, suggesting the role of NK-92/9.28.z in invasive GPC3-positive tumors from the immunohistochemical level. The results of immunohistochemistry further indicated that compared with NK-92 cells, the SK-HEP-1/GPC3 animal model showed a significant decrease in Ki67 and a significant increase in the shear-type Caspase-3 after treatment with NK-92/9.28.z. High, suggesting that NK-92/9.28.z is effective by inhibiting tumor cell proliferation and promoting tumor cell apoptosis.
取了SK-HEP-1/GPC3移植瘤小鼠心脏、肝脏、肺脏、肾脏、胰脏，进行HE染色，结果如图5所示，各组无明显区别且均没有观察到病理性改变；提示这个细胞治疗安全性高，不会浸润重要的脏器组织诱导细胞毒性作用。The heart, liver, lung, kidney and pancreas of SK-HEP-1/GPC3 transplanted tumor mice were taken for HE staining. The results are shown in Fig. 5. There was no significant difference between the groups and no pathological changes were observed. This cell is safe to treat and does not infiltrate important organ tissues to induce cytotoxicity.
实施例5.NK-92/9.28.z对内源性中高GPC3Huh-7皮下和原位移植瘤的治疗效果Example 5. Therapeutic effect of NK-92/9.28.z on endogenous high GPC3 Huh-7 subcutaneous and orthotopic transplantation tumors
采用NOD/SCID小鼠接种Huh-7皮下和原位移植瘤。Huh-7 subcutaneous and orthotopic transplantation tumors were inoculated with NOD/SCID mice.
皮下移植瘤模型的建立：通过皮下接种Huh-7细胞，2×10 6/只，约14天后瘤体积达到30-50mm 3，得到小鼠皮下移植瘤模型。 The subcutaneous xenograft model was established: subcutaneously inoculated Huh-7 cells, 2×10 6 /piece, and the tumor volume reached 30-50 mm 3 after about 14 days, and a mouse subcutaneous xenograft model was obtained.
原位动物模型更能模拟病人体内真实情况，接种建立了原位Huh-7肝癌的动物模型，通过随机分组方式，每组四只小鼠。In situ animal models were more able to simulate the real situation in patients, and an animal model of in situ Huh-7 liver cancer was established by inoculation, and four mice in each group were randomly assigned.
采用NK-92/9.28.z和Parental NK-92细胞，对皮下和原位移植瘤进行过继免疫治疗，具体方法为：皮下和原位移植瘤分别在12、13天进行腹腔环磷酰胺注射(100mg/kg)。D15天，测量Nod-Scid小鼠移植瘤体积并进行分组。皮下移植瘤小鼠共分为3组，包括：生理盐水对照组、NK-92组、NK-92/9.28.z组，每组小鼠6-8只；原位肿瘤组共分为两组，包括：NK-92组和NK-92/9.28.z组，每组4只小鼠。D15和17天，对分组后的NOD/SCID小鼠细胞过继免疫治疗。通过尾静脉注射200ul生理盐水或200ul NK-92、Mock和NK-92/9.28.z细胞，每5天一次；Adoptive NK-92/9.28.z and Parental NK-92 cells were used for adoptive immunotherapy of subcutaneous and orthotopic xenografts by subcutaneous and orthotopic transplantation of intraperitoneal cyclophosphamide at 12 and 13 days, respectively. 100mg/kg). Nod-Scid mouse xenograft volume was measured and grouped on day D15. Subcutaneous xenograft mice were divided into three groups, including: saline control group, NK-92 group, NK-92/9.28.z group, each group of mice 6-8; in situ tumor group was divided into two groups Including: NK-92 group and NK-92/9.28.z group, 4 mice per group. D15 and 17 days, adoptive immunotherapy for grouped NOD/SCID mouse cells. 200ul normal saline or 200ul NK-92, Mock and NK-92/9.28.z cells were injected through the tail vein once every 5 days;
每隔3-4天测量Huh-7皮下移植瘤体积的大小，记录每组小鼠瘤体积的变化，并绘制瘤体积随时间的生长曲线，结果如图6A所示。显示了Huh-7皮下移植瘤的生长情况，NK-92/9.28.z治疗组肿瘤体积和肿瘤重量均明显低于其他两组。The volume of Huh-7 subcutaneous xenografts was measured every 3-4 days, the changes in tumor volume of each group of mice were recorded, and the growth curve of tumor volume with time was plotted. The results are shown in Fig. 6A. The growth of Huh-7 subcutaneous xenografts was shown. The tumor volume and tumor weight of the NK-92/9.28.z treatment group were significantly lower than the other two groups.
对于原位移植瘤，开始进行NK-92/9.28.z或NK-92细胞治疗后，每周进行动物成像，研究结果如图6B所示，NK-92/9.28.z治疗组小鼠肿瘤荧光强度明显弱于NK-92组，说明NK-92/9.28.z对Huh-7原位移植瘤也具有显著疗效，图6C显示了Huh-7原位移植瘤的生长情况，通过总生物体荧光强度进行分析，与parental NK-92细胞治疗组相比，NK-92/9.28.z对Huh-7原位移植瘤也有很好的抑瘤作用 (***p<0.001)。For orthotopic xenografts, after NK-92/9.28.z or NK-92 cells were started, the animals were imaged weekly. The results of the study are shown in Figure 6B. The tumor fluorescence of the mice in the NK-92/9.28.z treatment group. The intensity was significantly weaker than that of the NK-92 group, indicating that NK-92/9.28.z also had a significant effect on Huh-7 orthotopic transplantation tumors. Figure 6C shows the growth of Huh-7 orthotopically transplanted tumors by total bioluminescence. Intensity was analyzed. Compared with the parental NK-92 cell treatment group, NK-92/9.28.z also had a good antitumor effect on Huh-7 orthotopic transplantation (***p<0.001).
每隔一周通过Luciferase检测肝脏原位肿瘤的生长情况，结果如图6D所示。实验结束时取其肝脏进行免疫组化检测，结果如图6E所示。图6D显示了各组小鼠体重呈可比性的增加趋势，提示NK-92/9.28.z不会引起治疗相关体重下降副作用；同时残余组织进行检测，结果如6E所示，发现了肿瘤组织中浸润型NK-92/9.28.z明显高于对照组，而在肝脏中检测不到其表达，提示NK-92/9.28.z不会引起肝脏的浸润，显示NK-92/9.28.z安全性好。The growth of liver tumors in situ was measured by Luciferase every other week, and the results are shown in Fig. 6D. At the end of the experiment, the liver was taken for immunohistochemical detection, and the results are shown in Fig. 6E. Figure 6D shows an increase in the body weight of each group of mice, suggesting that NK-92/9.28.z does not cause treatment-related weight loss side effects; while residual tissue is detected, the results are shown in Figure 6E, found in tumor tissue The infiltrating NK-92/9.28.z was significantly higher than the control group, but its expression was not detected in the liver, suggesting that NK-92/9.28.z does not cause liver infiltration, indicating NK-92/9.28.z safety. it is good.
实施例6.NK-92/9.28.z细胞对内源性低表达GPC3PLC/PRF/5皮下移植瘤的治疗效果Example 6. Therapeutic effect of NK-92/9.28.z cells on endogenous low expression of GPC3PLC/PRF/5 subcutaneous xenografts
NOD/SCID小鼠接种PLC/PRF/5皮下移植瘤，通过皮下接种上述细胞，2×10 6/只，约15天后瘤体积达到30-50mm 3； NOD/SCID mice were inoculated with PLC/PRF/5 subcutaneous xenografts, and the above cells were inoculated subcutaneously, 2×10 6 /piece, and the tumor volume reached 30-50 mm 3 after about 15 days;
皮下移植瘤分别在12、13天进行腹腔环磷酰胺注射(100mg/kg)；Subcutaneous xenografts were injected intraperitoneal cyclophosphamide (100 mg/kg) on days 12 and 13 respectively.
8)d17天，测量Nod-Scid小鼠移植瘤体积并进行分组。小鼠共分为3组，包括：生理盐水对照组、Mock对照组、NK-92/9.28.z组和NK-92/9.28.z辐照组，每组小鼠6-8只；8) On day d17, the transplanted tumor volume of Nod-Scid mice was measured and grouped. The mice were divided into 3 groups, including: saline control group, Mock control group, NK-92/9.28.z group and NK-92/9.28.z irradiation group, 6-8 mice in each group;
9)d17天，对分组后的Nod-Scid小鼠细胞过继免疫治疗。通过尾静脉注射200ul生理盐水或200ul NK-92、Mock和NK-92/9.28.z细胞，每5天一次。9) On day d17, the grouped Nod-Scid mouse cells were subjected to adoptive immunotherapy. 200 ul of normal saline or 200 ul of NK-92, Mock and NK-92/9.28.z cells were injected through the tail vein once every 5 days.
实验结果如图7所示，图7A和7B提示其也有很好的体内抑瘤作用。图7C提示各小鼠体重均衡可比，提示NK-92/9.28.z治疗不会引起体重下降副作用；NK-92细胞是一个肿瘤来源的杀伤细胞系，输入体内有成瘤风险，临床上往往需要辐照后再进行输注，本动物实验设立了一组NK-92/9.28.z细胞的辐照组(Irradiated NK-92/9.28.z)，结果提示其效果跟未辐照组的NK-92/9.28.z基本一致，为其临床安全应用提供了动物实验依据。The experimental results are shown in Fig. 7, and Figs. 7A and 7B suggest that they also have a good antitumor effect in vivo. Figure 7C suggests that each mouse is well-balanced, suggesting that NK-92/9.28.z treatment does not cause weight loss side effects; NK-92 cells are a tumor-derived killer cell line that has a risk of tumor formation in the body and is often clinically needed. After irradiation, the infusion was performed. In this animal experiment, a group of NK-92/9.28.z cells irradiation group (Irradiated NK-92/9.28.z) was established. The results showed that the effect was the same as that of the non-irradiated group. 92/9.28.z is basically consistent, providing an animal test basis for its clinical safety applications.
实施例7.NK-92/9.28.z在杀伤靶细胞过程中的细胞因子释放Example 7. Cytokine release during killing of target cells by NK-92/9.28.z
CRS是CAR-T细胞治疗的常见并发症，甚至可危及生命，CRS的发生可能存在多种机制交叉反应和激活，据报道，IL6、IL-1β、IL-2也都是参与引起或扩大细胞因子释放综合征的重要细胞因子。CRS is a common complication of CAR-T cell therapy and may even be life-threatening. There may be multiple mechanisms of cross-reactivity and activation of CRS. It is reported that IL6, IL-1β and IL-2 are also involved in causing or expanding cells. An important cytokine of factor release syndrome.
因此，我们比较了在效靶比1.5:1的情况下，NK-92/9.28.z和NK-92细胞杀伤SK-HEP-1和SK-HEP-1/GPC3过程中分泌的细胞因子水平，检测了NK-92/9.28.z分泌IL6、IL-1β、IL-2的情况，结果如图8A所示，在杀伤过程中，NK-92/9.28.z细胞不会释放IL-1β、IL-2和IL-6。Therefore, we compared the levels of cytokines secreted by NK-92/9.28.z and NK-92 cells during the killing of SK-HEP-1 and SK-HEP-1/GPC3 at a target-to-target ratio of 1.5:1. The secretion of IL6, IL-1β and IL-2 by NK-92/9.28.z was detected. As shown in Fig. 8A, NK-92/9.28.z cells did not release IL-1β, IL during the killing process. -2 and IL-6.
为了进一步分析NK-92/9.28.z诱发细胞因子风暴的风险，分析了NK-92/9.28.z杀伤靶细胞后能否诱导单核细胞释放IL-6。我们将NK-92/9.28.z和CAR-T/9.28.z和肿瘤细胞Huh-7和单核细胞共孵育36h后，通过ELISA检测培养体系中IL-6 的分泌情况。具体的，将用5×10 3单核细胞接种到96孔板，同时接种3×10 4NK-92/9.28.z或CAR-T/9.28.z细胞，再加入2×10 4靶细胞Huh-7，终体积为400μL，不添加外源性的IL-2，放培养箱培养36h后收集上清。对照组包括任意一种细胞和细胞之间两两共培养组，收集上清后通过ELISA方法检测IL-6分泌情况。结果如图8B所示(*p<0.05,**p<0.01，***p<0.001)，只有CAR-T/9.28.z与Huh-7和单核细胞共存的情况下，IL-6的释放量明显增加，而NK-92/9.28.z与Huh-7共存下，不会引起单核细胞释放IL-6。说明与CAR-T细胞相比，NK-92/9.28.z在杀伤肿瘤细胞的过程中不会刺激单核细胞释放IL-6，是一个非常安全的效应细胞。 To further analyze the risk of NK-92/9.28.z-induced cytokine storms, it was analyzed whether NK-92/9.28.z can induce IL-6 release from monocytes after killing target cells. We incubated NK-92/9.28.z and CAR-T/9.28.z with tumor cells Huh-7 and monocytes for 36 h, and then detected the secretion of IL-6 in the culture system by ELISA. Specifically, 5×10 3 monocytes were seeded into 96-well plates, and 3×10 4 NK-92/9.28.z or CAR-T/9.28.z cells were inoculated simultaneously, and 2×10 4 target cells Huh were added. -7, the final volume was 400 μL, no exogenous IL-2 was added, and the supernatant was collected after incubating in an incubator for 36 hours. The control group consisted of any two cells co-cultured between cells and cells. The supernatant was collected and the IL-6 secretion was detected by ELISA. The results are shown in Fig. 8B (*p<0.05, **p<0.01, ***p<0.001), and only CAR-T/9.28.z coexists with Huh-7 and monocytes, IL-6. The release of NK-92/9.28.z and Huh-7 did not cause IL-6 release from monocytes. This indicates that NK-92/9.28.z does not stimulate the release of IL-6 by monocytes in the process of killing tumor cells compared with CAR-T cells, and is a very safe effector cell.
实施例8.CAR修饰增强原代NK细胞抗肿瘤效果Example 8. CAR modification enhances anti-tumor effect of primary NK cells
采用本领域常规的细胞培养技术，取PBMC，与K562-mbIL21细胞(在K562细胞表面过表达白介素-21(membrane bound interleukin-21)和IL-2共培养、体外扩增NK细胞，磁珠(购自美天旎)分选，得到原代NK细胞，命名为PBNK细胞。Using conventional cell culture techniques in the art, PBMCs were co-cultured with K562-mbIL21 cells (exposed with membrane bound interleukin-21 and IL-2 on K562 cells, and NK cells were expanded in vitro, magnetic beads ( Sorted from Meitian), the primary NK cells were obtained and named PBNK cells.
参照实施例1的操作，采用AB1-28Z的慢病毒感染PBNK细胞，得到表达CAR的原代NK细胞，命名为PBNK/9.28.z。采用空质粒感染PBNK细胞，得到Mock’细胞。Following the procedure of Example 1, PBNK cells were infected with a lentivirus of AB1-28Z to obtain a primary NK cell expressing CAR, designated PBNK/9.28.z. PBNK cells were infected with an empty plasmid to obtain Mock' cells.
参照实施例2的操作，以Huh-7细胞和HepG2细胞为靶细胞，采用LDH试剂盒，进行体外细胞毒性测定，不同效靶比下孵育24h，结果如图9A所示。检测IFN-γ的分泌水平，结果如图9B所示。Referring to the procedure of Example 2, Huh-7 cells and HepG2 cells were used as target cells, and NKH kit was used for in vitro cytotoxicity assay, and the different effect targets were incubated for 24 hours, and the results are shown in Fig. 9A. The secretion level of IFN-γ was examined, and the results are shown in Fig. 9B.
以正常的PBMC为靶细胞，检测Mock’细胞和PBNK/9.28.z的细胞杀伤能力，结果如图10所示，显示修饰后的原代NK细胞对正常的组织细胞没有明显的毒性作用。The normal PBMC was used as the target cell to detect the cell killing ability of Mock' cells and PBNK/9.28.z. As shown in Fig. 10, it was shown that the modified primary NK cells had no obvious toxic effects on normal tissue cells.
实施例9.靶向GPC3嵌合抗原受体修饰NK细胞与索拉菲尼联合用于肝细胞肝癌Example 9. Targeting GPC3 chimeric antigen receptor-modified NK cells in combination with sorafenib for hepatocellular carcinoma
接种肝癌Huh-7细胞(靶细胞)，24h后加入不同浓度索拉菲尼(浓度分别为2μM、5μM和10μM)或DMSO，加或不加不同效靶比的NK-92/9.28.z细胞(1:1、1:5和1:10)，孵育24h后，采用CytoTox 96非放射性细胞毒性检测试剂盒测定细胞毒性。实验结果如表1所示，NK-92/9.28.z细胞对Huh-7有显著的杀伤，并呈效靶比依赖性，效靶比越大，NK-92/9.28.z细胞杀伤效应越强，效靶比1:1、1:5、1:10时，细胞杀伤毒性分别为69.2％±3.5％、36.8％±6.5％、22.2％±4.6％；索拉菲尼浓度为10μM、5μM和2μM时，细胞杀伤毒性分别为63.3.3±7.9％、45.6±5.6％和25.3±4.6％；而二者联合组的细胞毒性分别为88.1％±8.9％、56.2％±5.8％和38.3％±2.4％，三组的协同指数分别为：0.42、0.54和0.36，提示索拉菲尼和NK-92/9.28.z细胞联合在一定浓度和效靶比可以协同杀伤肝癌细胞。Hepatoma Huh-7 cells (target cells) were inoculated. After 24 h, different concentrations of sorafenib (2 μM, 5 μM and 10 μM, respectively) or DMSO were added, with or without different target ratios of NK-92/9.28.z cells. (1:1, 1:5, and 1:10), after incubation for 24 h, cytotoxicity was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit. The results of the experiment are shown in Table 1. NK-92/9.28.z cells have significant killing effect on Huh-7 and are effective in target-dependent ratio. The greater the target ratio, the more killing effect of NK-92/9.28.z cells. The cell killing toxicity was 69.2%±3.5%, 36.8%±6.5%, 22.2%±4.6% for the strong and effective target ratios of 1:1, 1:5, and 1:10; the sorafenib concentration was 10μM, 5μM. At 2 μM, the cell killing toxicity was 63.3.3±7.9%, 45.6±5.6% and 25.3±4.6%, respectively; the cytotoxicity of the combination group was 88.1%±8.9%, 56.2%±5.8% and 38.3%, respectively. ±2.4%, the synergistic indices of the three groups were: 0.42, 0.54 and 0.36, respectively, suggesting that the combination of sorafenib and NK-92/9.28.z cells can synergistically kill liver cancer cells at a certain concentration and effective target ratio.
表1.NK-92/9.28.z细胞与索拉菲尼联合对肝癌细胞Huh-7杀伤率比较Table 1. Comparison of killing rate of Huh-7 in hepatoma cells by NK-92/9.28.z cells combined with sorafenib
Figure PCTCN2018098623-appb-000003
Figure PCTCN2018098623-appb-000003
备注：与单一给予NK‐92/9.28.z或者索拉菲尼的组别相比，具有显著性差异，*p<0.05or^p<0.05。Remarks: Significant differences compared to the group given NK-92/9.28.z or sorafenib alone, *p<0.05or^p<0.05.
为模拟抗原异质性，将SK-HEP-1和SK-HEP-1/GPC3混合各50％，制备SK-HEP-1/GPC3(50％)群异质性混合细胞。采用CytoTox 96非放射性细胞毒性检测试剂盒测定索拉菲尼、NK-92/9.28.z细胞、或者其组合的细胞毒性。实验结果如图11所示，索拉菲尼5μM作用48h对SK-HEP-1/GPC3和SK-HEP-1/GPC3(50％)异质性混合细胞有大致相同的杀伤作用，杀伤率分别为54.5％±6.7％和56.8％±5.6％。单用NK-92/9.28.z细胞对SK-HEP-1/GPC3有显著的杀伤，效靶比3:1杀伤率为73.2％±9.2％。对50％SK-HEP-1+50％SK-HEP-1/GPC3这群异质性混合细胞，单用NK-92/9.28.z细胞，效靶比3:1时杀伤率为39.8％±5.6％。对前者细胞，索拉菲尼联合NK-92/9.28.z细胞后对肝癌的抑制作用明显增强，最高达93.6％±10.2％，是NK-92/9.28.z组的1.28倍，是索拉菲尼单药组的1.72倍；对后者异质性混合细胞，二者联用后产生的杀伤毒性最高达88.9％±7.8％，是NK-92/9.28.z组的2.23倍，是索拉菲尼单药组的1.56倍。这一结果显示索拉菲尼和NK-92/9.28.z细胞联用可以更大程度增加异质性细胞群体的杀伤率。To mimic antigen heterogeneity, SK-HEP-1 and SK-HEP-1/GPC3 were mixed 50% each to prepare SK-HEP-1/GPC3 (50%) heterogeneous mixed cells. The cytotoxicity of sorafenib, NK-92/9.28.z cells, or a combination thereof was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit. The results of the experiment are shown in Figure 11. The solanin 5μM for 48h has approximately the same killing effect on SK-HEP-1/GPC3 and SK-HEP-1/GPC3 (50%) heterogeneous mixed cells, and the killing rate is respectively It was 54.5% ± 6.7% and 56.8% ± 5.6%. NK-92/9.28.z cells alone had significant killing effect on SK-HEP-1/GPC3, and the effective target ratio was 33.2% killing rate of 73.2%±9.2%. For 50% SK-HEP-1+50%SK-HEP-1/GPC3 heterogeneous mixed cells, NK-92/9.28.z cells were used alone, and the killing rate was 39.8%± at a target-to-target ratio of 3:1. 5.6%. For the former cells, the inhibitory effect of sorafenib combined with NK-92/9.28.z cells on liver cancer was significantly enhanced, up to 93.6%±10.2%, which was 1.28 times that of NK-92/9.28.z group. The Fini single-agent group was 1.72 times; the latter heterogeneous mixed cells, the combined toxicity of the two was up to 88.9%±7.8%, which was 2.23 times that of the NK-92/9.28.z group. The Rafini single drug group was 1.56 times. This result shows that the combination of sorafenib and NK-92/9.28.z cells can increase the killing rate of heterogeneous cell populations to a greater extent.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the present invention.
本发明所用的序列信息总结如下：The sequence information used in the present invention is summarized as follows:
Figure PCTCN2018098623-appb-000004
Figure PCTCN2018098623-appb-000004
Figure PCTCN2018098623-appb-000005
Figure PCTCN2018098623-appb-000005
Figure PCTCN2018098623-appb-000006
Figure PCTCN2018098623-appb-000006

Claims (17)

  1. 一种基因工程改造的NK细胞，其特征在于，所述细胞表达特异性识别GPC3的嵌合受体，所述的嵌合受体包含识别GPC3的胞外域、跨膜域、和/或细胞内结构域。A genetically engineered NK cell, characterized in that said cell expresses a chimeric receptor that specifically recognizes GPC3, said chimeric receptor comprising an extracellular domain, a transmembrane domain, and/or an intracellular domain that recognizes GPC3 Domain.
  2. 如权利要求1所述的基因工程改造的NK细胞，其特征在于，所述NK细胞是NK92细胞。The genetically engineered NK cell of claim 1, wherein the NK cell is an NK92 cell.
  3. 根据权利要求1或2所述的细胞，其特征在于，所述胞外域为识别GPC3的抗体，具有SEQ ID NO：7、8、9所示的HCDR1、HCDR2、HCDR3，以及SEQ ID NO：10、11、12所示的LCDR1、LCDR2、LCDR3。The cell according to claim 1 or 2, wherein the extracellular domain is an antibody recognizing GPC3, having HCDR1, HCDR2, HCDR3 and SEQ ID NO: 10 as shown in SEQ ID NOs: 7, 8, and 9. LCDR1, LCDR2, and LCDR3 shown in 11, 12 and 12.
  4. 根据权利要求3所述的细胞，其特征在于，所述抗体含有SEQ ID NO：13所示的重链可变区和SEQ ID NO：14所示的轻链可变区。The cell according to claim 3, wherein the antibody comprises the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 14.
  5. 根据权利要求4所述的细胞，其特征在于，所述抗体具有SEQ ID NO：1所示的序列。The cell according to claim 4, wherein the antibody has the sequence of SEQ ID NO: 1.
  6. 根据权利要求5所述的细胞，其特征在于，所述嵌合受体具有SEQ ID NO：16、17、18、或19所示的氨基酸序列。The cell according to claim 5, wherein the chimeric receptor has the amino acid sequence shown in SEQ ID NO: 16, 17, 18, or 19.
  7. 根据权利要求1或2所述的细胞，其特征在于，The cell according to claim 1 or 2, characterized in that
    (i)所述的跨膜域选自以下蛋白的跨膜结构域：T细胞受体的α、β或ζ链；CD28；CD3ε；CD45；CD4；CD5；CD8；CD9；CD16；CD22；CD33；CD37；CD64；CD80；CD86；CD134；CD137；CD154；KIRDS2；OX40；CD2；CD27；LFA-1(CD11a；CD18)；ICOS(CD278)；4-1BB(CD137)；GITR；CD40；BAFFR；HVEM(LIGHTR)；SLAMF7；NKp80(KLRF1)；CD160；CD19；IL2Rβ；IL2Rγ；IL7Rα；ITGA1；VLA1；CD49a；ITGA4；IA4；CD49D；ITGA6；VLA-6；CD49f；ITGAD；CD11d；ITGAE；CD103；ITGAL；CD11a；LFA-1；ITGAM；CD11b；ITGAX；CD11c；ITGB1；CD29；ITGB2；CD18；LFA-1；ITGB7；TNFR2；DNAM1(CD226)；SLAMF4(CD244、2B4)；CD84；CD96(Tactile)；CEACAM1；CRTAM；Ly9(CD229)；CD160(BY55)；PSGL1；CD100(SEMA4D)；SLAMF6(NTB-A、Ly108)；SLAM(SLAMF1、CD150、IPO-3)；BLAME(SLAMF8)；SELPLG(CD162)；LTBR；PAG/Cbp；NKp44；NKp30；NKp46；NKG2D；和NKG2C；和/或(i) The transmembrane domain is selected from the transmembrane domain of the following proteins: alpha, beta or ζ chain of a T cell receptor; CD28; CD3 epsilon; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33 CD37;CD64;CD80;CD86;CD134;CD137;CD154;KIRDS2;OX40;CD2;CD27;LFA-1 (CD11a;CD18);ICOS(CD278);4-1BB(CD137);GITR;CD40;BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRF1); CD160; CD19; IL2Rβ; IL2Rγ; IL7Rα; ITGA1; VLA1; CD49a; ITGA4; IA4; CD49D; ITGA6; VLA-6; CD49f; ITGAD; CD11d; ITGAE; CD103; ITGAL; CD11a; LFA-1; ITGAM; CD11b; ITGAX; CD11c; ITGB1; CD29; ITGB2; CD18; LFA-1; ITGB7; TNFR2; DNAM1 (CD226); SLAMF4 (CD244, 2B4); CD84; CD96 (Tactile) ; CEACAM1; CRTAM; Ly9 (CD229); CD160 (BY55); PSGL1; CD100 (SEMA4D); SLAMF6 (NTB-A, Ly108); SLAM (SLAMF1, CD150, IPO-3); BLAME (SLAMF8); SELPLG (CD162) LTBR; PAG/Cbp; NKp44; NKp30; NKp46; NKG2D; and NKG2C; and/or
    (ii)所述的细胞内结构域包括一级信号传导结构域和/或共刺激信号传导结构域，其中：(ii) The intracellular domain comprises a primary signaling domain and/or a costimulatory signaling domain, wherein:
    (1)所述一级信号传导结构域包含选自：CD3ζ；CD3γ；CD3δ；CD3ε；常见FcRγ(FCER1G)；FcRβ(FcεR1b)；CD79a；CD79b；FcγRIIa；DAP10；和DAP12的蛋白质的功能信号传导结构域，或其组合；和/或(1) The primary signaling domain comprises a functional signal transduction selected from the group consisting of: CD3ζ; CD3γ; CD3δ; CD3ε; common FcRγ (FCER1G); FcRβ (FcεR1b); CD79a; CD79b; FcγRIIa; DAP10; Domain, or a combination thereof; and/or
    (2)所述共刺激信号传导结构域包含选自如下的蛋白质的功能信号传导结构域：CD27；CD28；4-1BB(CD137)；OX40；CD30；CD40；PD-1；ICOS；淋巴细胞功能相关的抗原-1(LFA-1)；CD2；CD7；LIGHT；NKG2C；B7-H3；特异性结合CD83的 配体；CDS；ICAM-1；GITR；BAFFR；HVEM(LIGHTR)；SLAMF7；NKp80(KLRF1)；CD160；CD19；CD4；CD8α；CD8β；IL2Rβ；IL2Rγ；IL7Rα；ITGA4；VLA1；CD49a；ITGA4；IA4；CD49D；ITGA6；VLA-6；CD49f；ITGAD；CD11d；ITGAE；CD103；ITGAL；CD11a；LFA-1；ITGAM；CD11b；ITGAX；CD11c；ITGB1；CD29；ITGB2；CD18；LFA-1；ITGB7；TNFR2；TRANCE/RANKL；DNAM1(CD226)；SLAMF4(CD244；2B4)；CD84；CD96(Tactile)；CEACAM1；CRTAM；Ly9(CD229)；CD160(BY55)；PSGL1；CD100(SEMA4D)；CD69；SLAMF6(NTB-A；Ly108)；SLAM(SLAMF1；CD150；IPO-3)；BLAME(SLAMF8)；SELPLG(CD162)；LTBR；LAT；GADS；SLP-76；PAG/Cbp；NKp44；NKp30；NKp46；和NKG2D，或其组合。(2) The costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27; CD28; 4-1BB (CD137); OX40; CD30; CD40; PD-1; ICOS; Related antigen-1 (LFA-1); CD2; CD7; LIGHT; NKG2C; B7-H3; ligand for specific binding to CD83; CDS; ICAM-1; GITR; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 ( KLRF1);CD160;CD19;CD4;CD8α;CD8β;IL2Rβ;IL2Rγ;IL7Rα;ITGA4;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ;LFA-1;ITGAM;CD11b;ITGAX;CD11c;ITGB1;CD29;ITGB2;CD18;LFA-1;ITGB7;TNFR2;TRANCE/RANKL;DNAM1(CD226);SLAMF4(CD244;2B4);CD84;CD96(Tactile CEACAM1; CRTAM; Ly9 (CD229); CD160 (BY55); PSGL1; CD100 (SEMA4D); CD69; SLAMF6 (NTB-A; Ly108); SLAM (SLAMF1; CD150; IPO-3); BLAME (SLAMF8); SELPLG (CD162); LTBR; LAT; GADS; SLP-76; PAG/Cbp; NKp44; NKp30; NKp46; and NKG2D, or a combination thereof.
  8. 根据权利要求1-7任一项的细胞用于制备预防和/或治疗癌症的药物的用途，所述癌症表达GPC3，所述癌症优选自肝癌，肺癌，胃癌、乳腺癌、黑色素瘤、卵巢癌、卵黄囊瘤、神经母细胞瘤。Use of a cell according to any one of claims 1 to 7 for the preparation of a medicament for the prevention and/or treatment of cancer, said cancer expressing GPC3, preferably from liver cancer, lung cancer, gastric cancer, breast cancer, melanoma, ovarian cancer , yolk sac tumor, neuroblastoma.
  9. 根据权利要求1-7任一项的细胞在制备用作靶向细胞治疗剂和/或用于过继性癌症免疫治疗的药物中的用途。Use of a cell according to any one of claims 1 to 7 for the preparation of a medicament for use as a targeted cell therapeutic and/or for immunotherapy of adoptive cancer.
  10. 根据权利要求8或9所述的用途，其特征在于，所述预防和/或治疗癌症的药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物的使用说明书中记载了所述预防和/或治疗癌症药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物与化疗药物或放射治疗同时给予。The use according to claim 8 or 9, wherein the prevention is described in the instruction manual for the drug for preventing and/or treating cancer, the targeted cell therapeutic agent or the drug for adoptive cancer immunotherapy And/or a cancer treatment drug, a targeted cell therapy agent, or a drug for adoptive cancer immunotherapy is administered concurrently with a chemotherapeutic drug or radiation therapy.
  11. 根据权利要求10所述的用途，其特征在于，所述预防和/或治疗癌症的药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物用于治疗肝癌。The use according to claim 10, characterized in that the medicament for preventing and/or treating cancer, a targeted cell therapeutic agent or a medicament for adoptive cancer immunotherapy is for treating liver cancer.
  12. 根据权利要求11所述的用途，其特征在于，所述化疗药物是治疗肝癌的化疗药物；优选式I或者式II所示的化合物：The use according to claim 11, wherein the chemotherapeutic drug is a chemotherapeutic drug for treating liver cancer; preferably a compound of formula I or formula II:
    Figure PCTCN2018098623-appb-100001
    Figure PCTCN2018098623-appb-100001
  13. 根据权利要求8或9所述的用途，其特征在于，所述预防和/或治疗癌症的药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物的使用说明书中记载了在给予细胞之前，对要给药的个体进行预处理。The use according to claim 8 or 9, wherein the administration of the drug for the prevention and/or treatment of cancer, the targeted cell therapeutic agent or the drug for adoptive cancer immunotherapy is described in the administration of the cell Previously, the individual to be administered was pretreated.
  14. 根据权利要求13所述的用途，其特征在于，所述预防和/或治疗癌症的药物、靶向细胞治疗剂或用于过继性癌症免疫治疗的药物还包含淋巴细胞消耗剂作为进行 预处理的试剂，以便消耗所述个体的淋巴细胞。The use according to claim 13, wherein the drug for preventing and/or treating cancer, a cell-targeting therapeutic agent or a drug for adoptive cancer immunotherapy further comprises a lymphocyte depleting agent as a pretreatment Reagents to consume lymphocytes from the individual.
  15. 根据权利要求14所述的用途，其特征在于，所述淋巴细胞消耗剂为氟达拉滨、环磷酰胺。The use according to claim 14, wherein the lymphocyte depleting agent is fludarabine or cyclophosphamide.
  16. 如权利要求1所述的NK细胞的制备方法，包括：制备得到表达嵌合抗原受体的NK细胞，对表达嵌合抗原受体的NK细胞采用照射的方法进一步处理；优选地，采用γ射线照射预处理表达嵌合抗原受体的NK细胞。The method for producing NK cells according to claim 1, comprising: preparing NK cells expressing a chimeric antigen receptor, and further treating the NK cells expressing the chimeric antigen receptor by irradiation; preferably, using γ-rays The NK cells expressing the chimeric antigen receptor are pretreated by irradiation.
  17. 如权利要求16所述的NK细胞的制备方法，其特征在于，所述NK细胞是NK-92细胞。The method of producing NK cells according to claim 16, wherein the NK cells are NK-92 cells.
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