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WO2019080537A1 - Agent thérapeutique comprenant un virus oncolytique et des cellules tueuses naturelles-récepteur antigénique chimérique, utilisation, kit, et méthode de traitement d'une tumeur et/ou d'un cancer - Google Patents

Agent thérapeutique comprenant un virus oncolytique et des cellules tueuses naturelles-récepteur antigénique chimérique, utilisation, kit, et méthode de traitement d'une tumeur et/ou d'un cancer

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WO2019080537A1
WO2019080537A1 PCT/CN2018/094003 CN2018094003W WO2019080537A1 WO 2019080537 A1 WO2019080537 A1 WO 2019080537A1 CN 2018094003 W CN2018094003 W CN 2018094003W WO 2019080537 A1 WO2019080537 A1 WO 2019080537A1
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cells
cancer
virus
oncolytic
tumor
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PCT/CN2018/094003
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English (en)
Chinese (zh)
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肖�琳
陈璨
胡放
陈霖
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杭州优善生物科技有限公司
杭州康万达医药科技有限公司
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Publication of WO2019080537A1 publication Critical patent/WO2019080537A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention is in the field of biotechnology, and in particular, relates to therapeutic agents and applications comprising oncolytic viruses and CAR-NK cells, kits, methods of treating tumors and/or cancer.
  • Chimeric Antigen Receptor is an artificially engineered receptor, so any specific receptor can be grafted onto immune effector cells.
  • the extracellular specific portion of these receptors used to recognize an antigen is derived from the sequence of the antibody. Because different parts of this receptor have different origins, they are called chimeric receptors.
  • CAR-modified immune cells ie, immune cells expressing the chimeric antigen receptor (CAR) are now the most effective and promising tumor cell immunotherapy products.
  • NK Natural killer cells
  • T cells peripheral blood mononuclear cells
  • NK cells do not produce autocrine growth factors such as IL-2, and they do not increase in value when they encounter specific antigens, and have a short life span. They do not need to be equipped with suicide genes. Limit CAR toxicity.
  • the killing of tumors by NK cells does not depend on the specific antigen presented by MHC. Therefore, in the immunotherapy application of allogeneic NK cells, immune rejection is not produced, and it is a safer immunity than T cells. Treat candidate cells.
  • CAR can avoid the inhibitory signaling pathway in NK cells, while NK cells themselves can express activated receptors and can also utilize antibody-mediated cytotoxicity (ADCC). Therefore, CAR-expressing NK cells are treated in tumors. It is superior to T cells and has a broader application prospect.
  • CAR includes an extracellular portion, a transmembrane region, and an intracellular portion.
  • immune cells carrying CAR the selection of the extracellular and intracellular portions of CAR and its cooperation with immune cell types are extremely important, which is closely related to the specific killing ability of tumors. At present, in the immunotherapy of tumors, it is still necessary to develop a novel and effective CAR-immune cell drug.
  • the way in which the virus treats cancer has developed quite rapidly in the past two decades.
  • One of the biggest advances in viral therapy is the use of tumor cells and normal cells to modify certain viral structures, allowing them to selectively replicate in tumor cells, ultimately achieving the goal of killing tumor cells.
  • These modified viruses are collectively referred to as oncolytic viruses according to their functions, and are derived from adenoviruses, herpes viruses, and poxviruses. It has now been found that certain wild-type viruses also have the function of selectively replicating and inducing tumors in tumor cells.
  • H101 One of the oncolytic virus injections marketed in China is genetically engineered type 5 adenovirus H101, which facilitates replication of the virus in tumor cells.
  • H101 mainly deletes the 55KD and E3 region gene segments of the E1B region of human type 5 adenovirus, and has the characteristic of specifically replicating in tumor cells to finally cause oncolytic.
  • One of the mechanisms is that the 55KD protein encoded by the E1B region of the wild-type adenovirus gene can bind to the p53 protein, thereby inhibiting the clearance of the adenovirus by the p53 gene. Since the virus cannot encode the E1B-55KD protein, the virus cannot replicate in cells normal to p53.
  • the virus In the tumor cells in which the p53 gene is mutated, the virus can be largely replicated because of the inhibition of the p53 gene. In addition, deletion of the E3 region gene fragment allows the virus to be easily recognized and cleared by the immune system (such as NK cells) in vivo, increasing the safety of clinical applications.
  • the current view is that not only the mutation of p53 itself, but also the defect of p53 pathway is beneficial to the selective replication of H101. H101 is administered by intratumoral injection and replicates in a large amount in tumor cells, eventually leading to the dissolution and death of tumor cells.
  • the component of the oncolytic virus drug T-Vec approved by the US FDA is genetically engineered type 1 herpes simplex virus HSV-1.
  • the ICP34.5 and ICP47 genes were deleted in T-Vec and the human immune activator granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was inserted, which replicated and expressed GM-CSF in tumor cells.
  • GM-CSF human immune activator granulocyte-macrophage colony-stimulating factor
  • Direct injection into melanoma lesions can cause tumor cell lysis, thereby rupturing tumor cells, releasing tumor-derived antigens and GM-CSF, and accelerating the anti-tumor immune response.
  • the FDA approved T-Vec as a topical treatment for unresectable lesions in patients with melanoma who relapsed after the first surgery.
  • Poxvirus is a large-sized virus, and the specificity of the oncolytic virus obtained by genetic modification of wild-type poxvirus in tumor cells is greatly improved.
  • some of the oncolytic pox viruses have deleted the gene that expresses thymidine kinase (TK) in the poxvirus DNA, so that the oncolytic virus cannot replicate and proliferate in normal cells.
  • TK thymidine kinase
  • tumor cells synthesize thymidine kinase for replication by oncolytic viruses, so vaccinia virus can replicate in large numbers in tumor cells.
  • some of the oncolytic pox viruses have deleted the gene expressing VGF, which enhances the specific proliferation of the virus in the tumor cells, and finally leads to the dissolution and death of the tumor cells.
  • the oncolytic pox virus removes both the TK gene and the VGF gene.
  • the acne oncolytic virus has not yet been marketed.
  • vaccinia virus has the advantage of administration which can be administered systemically by intravenous injection and reaching tumor targets.
  • the poxvirus genome is large and facilitates genetic modification that contributes to tumor killing.
  • the present invention provides therapeutic agents and uses comprising oncolytic viruses and CAR-NK cells, kits, methods of treating tumors and/or cancer.
  • the present invention provides:
  • a therapeutic agent comprising:
  • a first pharmaceutical composition wherein the first pharmaceutical composition comprises an oncolytic virus in a first pharmaceutically acceptable carrier;
  • the oncolytic virus is capable of selectively replicating in a tumor cell
  • the surface of the NK cell is modified by a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane domain and an intracellular domain, characterized in that
  • the antigen binding domain is derived from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • amino acid sequence of the intracellular domain is selected from amino acids 62 to 113 of DAP12; preferably, the amino acid sequence of the intracellular domain is SEQ ID NO :5 is shown.
  • the second pharmaceutical composition comprises the NK cells in a total dose ranging from 1 ⁇ 10 6 to 1 ⁇ 10 11 per subject per subject.
  • the second pharmaceutical composition comprises the NK cells in a total dose ranging from 6 x 10 7 to 1.2 x 10 10 per subject per subject.
  • the therapeutic agent according to (1), wherein the oncolytic virus is selected from the group consisting of a gene-mutated virus having an oncolysis effect and a wild-type virus having an oncolysis effect.
  • the oncolytic virus is selected from the group consisting of an oncolytic adenovirus, a poxvirus, a herpes simplex virus, a measles virus, a Semliki forest virus, and a vesicular stomatitis Virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, Echo enterovirus, Coxsackie virus, Newcastle disease virus and Maraba virus.
  • NK cells are selected from the group consisting of autologous NK cells and allogeneic NK cells.
  • NK cells are autologous NK cells obtained by in vitro expansion or allogeneic NK cells obtained by in vitro expansion.
  • the therapeutic agent according to (18), wherein the oncolytic effect-containing adenovirus is selected from the group consisting of: Onyx-015, H101, Ad5-yCD/mutTKSR39rep-hIL12, CG0070, DNX-2401, OBP-301, ONCOS-102, ColoAd1, VCN- 01, and / or ProstAtak TM.
  • the active ingredient of the second pharmaceutical composition comprises the NK cells in a total dose ranging from 1 x 10 6 to 1 x 10 11 per subject per subject.
  • the active ingredient of the second pharmaceutical composition comprises the total dose ranging from 6 x 10 7 to 1.2 x 10 10 per NK per patient per subject.
  • the tumor and/or cancer comprises lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterus Cancer, cervical cancer, lymphoma, gastric cancer, esophageal cancer, renal cancer, prostate cancer, pancreatic cancer, leukemia; preferably, the tumor and/or cancer is NKG2D ligand positive, including the tumor and/or cancer The treatment is positive for NKG2D ligand and the tumor and/or cancer is treated to become positive for NKG2D ligand.
  • a kit for a synergistic combination drug for treating tumors and/or cancer comprising: a first container containing an oncolytic virus and a second container containing NK cells, wherein the first container And the second container are independent; and instructions for indicating the timing and mode of administration; wherein the oncolytic virus is capable of selectively replicating in tumor cells; and wherein the surface of the NK cells is chimeric Antigen receptor modification comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane domain and an intracellular domain, wherein the antigen binding domain is from NKG2D A ligand binding domain derived from the intracellular signaling region of DAP12.
  • the second container comprises the NK cells in a total dose ranging from 1 ⁇ 10 6 to 1 ⁇ 10 11 per person per course of treatment.
  • the second container comprises the NK cells in a total dose ranging from 6 x 10 7 to 1.2 x 10 10 per person per course of treatment.
  • the kit according to (26), wherein the oncolytic virus is selected from the group consisting of a gene-mutated virus having an oncolysis effect and a wild-type virus having an oncolysis effect.
  • the oncolytic virus is selected from the group consisting of an oncolytic adenovirus, a poxvirus, a herpes simplex virus, a measles virus, a Semliki forest virus, and a vesicular stomatitis Virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, Echo enterovirus, Coxsackie virus, Newcastle disease virus and Maraba virus.
  • NK cells are selected from the group consisting of autologous NK cells and allogeneic NK cells.
  • NK cells are autologous NK cells obtained by in vitro expansion or allogeneic NK cells obtained by in vitro expansion.
  • the tumor and/or cancer includes lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, Uterine cancer, cervical cancer, lymphoma, gastric cancer, esophageal cancer, renal cancer, prostate cancer, pancreatic cancer, leukemia; preferably, the tumor and/or cancer is NKG2D ligand positive, including the tumor and/or cancer Untreated is NKG2D ligand positive and the tumor and/or cancer is treated to become NKG2D ligand positive.
  • the oncolytic adenovirus is selected from the group consisting of: Onyx-015, H101, Ad5-yCD/mutTKSR39rep-hIL12, CG0070, DNX-2401, OBP-301, ONCOS-102, ColoAd1, VCN- 01, and / or ProstAtak TM.
  • the NK cells were dosed to 1 x 10 10 cells/day.
  • the second container comprises the NK cells in a total dose ranging from 6 x 10 7 to 1.2 x 10 10 per person per course of treatment.
  • a method of treating a tumor and/or cancer comprising the steps of:
  • the surface of the NK cell is modified by a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane domain and an intracellular domain, characterized in that
  • the antigen binding domain is derived from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • the oncolytic virus is selected from the group consisting of an oncolytic adenovirus, a poxvirus, a herpes simplex virus, a measles virus, a Semliki forest virus, and a vesicular stomatitis virus. , poliovirus, retrovirus, reovirus, Seneca Valley virus, Echo enterovirus, Coxsackie virus, Newcastle disease virus and Maraba virus.
  • NK cells are selected from the group consisting of autologous NK cells and allogeneic NK cells.
  • NK cells are autologous NK cells obtained by in vitro expansion or allogeneic NK cells obtained by in vitro expansion.
  • the tumor and/or cancer comprises lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterus Cancer, cervical cancer, lymphoma, gastric cancer, esophageal cancer, renal cancer, prostate cancer, pancreatic cancer, leukemia; preferably, the tumor and/or cancer is NKG2D ligand positive, including the tumor and/or cancer The treatment is positive for NKG2D ligand and the tumor and/or cancer is treated to become positive for NKG2D ligand.
  • oncolytic adenovirus is selected from the group consisting of: Onyx-015, H101, Ad5-yCD/mutTKSR39rep-hIL12, CG0070, DNX-2401, OBP-301, ONCOS -102, ColoAd1, VCN-01, and / or ProstAtak TM.
  • the invention has the following advantages and positive effects:
  • the chimeric antigen receptor of the present invention enables the NK cells modified by it (also referred to as "engineered NKG2D ligand-targeted NK cells”) to have strong and specific targeting to tumors positive for expression of various NKG2D ligands.
  • the killing activity, the preclinical study of the present invention has fully demonstrated that the modified NK cells can significantly reduce or even eliminate the tumor burden in the animal and prolong the survival time of the animal.
  • the engineered NKG2D ligand-targeted NK cells of the invention provide a new choice for treating NKG2D ligand-positive tumor patients, and have good industrial application prospects.
  • the present invention proposes for the first time the concept of combining an oncolytic virus with the chimeric antigen receptor-modified NK cells of the present invention for treating tumors and/or cancer, and the pharmaceutical composition and method provided based on the concept can be fully utilized.
  • the oncolytic virus selectively replicates in tumor cells and kills tumor cells, and further causes subsequent immune responses, while also fully utilizing the function of the NK cells to kill tumor cells, and skillfully utilizing oncolytic
  • the characteristic that the virus selectively replicates in tumor cells makes the tumor cells containing the oncolytic virus a specific target of NK cells, thereby further enhancing the tumor killing effect of the NK cells.
  • oncolytic viruses can stimulate the increase of the expression of various NKG2D ligands on the surface of tumor cells, thereby further cooperating with the chimeric antigen receptor-modified NK cells of the present invention to produce stronger resistance.
  • Tumor synergy The present inventors have found that only the combination of oncolytic virus and the NK cells produces a synergistic effect.
  • the oncolytic virus and the NK cells have the characteristics of recognizing tumor cells, and basically do not cause damage to normal cells, and the combined use of the two has significant advantages in safety and efficacy.
  • the present invention through theoretical exploration and experimental research, enables the respective administration dose, application sequence and application interval of the oncolytic virus and the NK cell to achieve the synergistic effect of the maximum efficiency of the combined application of both, while avoiding both Mutual constraints between them to achieve effective treatment of tumors and / or cancer.
  • FIG. 1 is a schematic diagram showing the construction of a chimeric antigen receptor NKG2D-CD8-DAP12 according to an embodiment of the present invention, wherein “CMV” indicates a CMV promoter sequence, “T7” indicates a T7 promoter sequence, and “5'UTR” indicates a 5'UTR having a Kozak sequence, “SP” indicates a GM-CSF alpha chain signal peptide coding sequence, "NKG2D” indicates a coding sequence of a ligand binding region of NKG2D, and “CD8” indicates a hinge region and a transmembrane region coding sequence of CD8 ⁇ , “DAP12” denotes the intracellular signal region coding sequence of DAP12, “alpha globulin 3'UTR” denotes the 3'UTR of the alpha globulin having a PolyA signal, and “pA 150 " denotes polyA (polyadenylation),
  • Figure 2 is an electropherogram showing the identification fragment of the expression vector pFastbac1-CD8-DAP12 digested with restriction endonucleases SphI and SalI according to one embodiment of the present invention; wherein lane 1 is a DNA molecular weight marker and lane 2 is an identification fragment .
  • Figure 3 is an electropherogram showing the identification fragment of the expression vector pFastbac1-NKG2D-CD8-DAP12 digested with restriction endonucleases SphI and NheI according to an embodiment of the present invention; wherein lane 1 is a DNA molecular weight marker, and lane 2 is Identify the fragment.
  • Figure 4 is a schematic view showing the structure of a recombinant DNA vector pFastbac1-NKG2D-CD8-DAP12 of a chimeric antigen receptor according to an embodiment of the present invention; wherein the clockwise sequence is a forward gene fragment and the counterclockwise is a reverse gene fragment .
  • CMV CMV promoter sequence
  • T7 denotes a T7 promoter sequence
  • 5'UTR denotes a 5'UTR having a Kozak sequence
  • GM-CSF ⁇ denotes a GM-CSF alpha chain signal peptide coding sequence
  • NKG2D The coding sequence of the ligand binding region of NKG2D
  • CD8 indicates the hinge region and transmembrane region coding sequence of CD8 ⁇
  • DAP12 indicates the intracellular signal region coding sequence of DAP12
  • alpha globulin 3'UTR indicates The 3'UTR of the alpha globulin of the PolyA signal.
  • Figure 5 shows the results of analysis of NK cell phenotype by flow cytometry.
  • Figure 5A shows the purity of NK cells when expanded from peripheral blood mononuclear cells for 17 days.
  • the abscissa indicates the CD3 expression intensity, and the ordinate indicates the CD56 expression intensity;
  • FIG. 5B shows the expression intensity of NK cells endogenous NKG2D and CD16 when expanded from peripheral blood mononuclear cells for 17 days, the abscissa indicates the intensity of NKG2D expression, and the ordinate indicates CD16. Expression intensity.
  • Figure 6 shows an electropherogram of a chimeric antigen receptor NKG2D-CD8-DAP12 linearized DNA template synthesized in vitro; Lane 1 is a DNA molecular weight marker and Lane 2 is a linearized DNA template.
  • Figure 7 shows an electropherogram of mRNA of the chimeric antigen receptor NKG2D-CD8-DAP12 synthesized in vitro; Lane 1 is a molecular weight marker and Lane 2 is an mRNA of NKG2D-CD8-DAP12.
  • Figure 8 shows the results of flow cytometry detection of chimeric antigen receptor NKG2D-CD8-DAP12 mRNA electroporation NK cells.
  • Figure 8A shows the intensity of endogenous expression of NKG2D by NK cells (where the left peak is the negative control curve and the right peak is the NK cell NKG2D expression intensity curve);
  • Figure 8B shows the intensity of NKG2D expression by NK cells after electroporation (where The left peak is the negative control curve, and the right peak is the NKG2D expression intensity curve after NK cell electrotransformation of NKG2D-CD8-DAP12 mRNA;
  • Figure 8C shows the comparison of the intensity of NKG2D expression after endogenous expression and electrotransfection of NK cells ( That is, 8A and 8B are combined together) (the left peak is a negative control curve, the middle peak is the NK cell NKG2D expression intensity curve, and the right peak is the NKG2D-CD8-DAP12 mRNA expression level after NK
  • the abscissa indicates the intensity of NKG2D expression, and the ordinate indicates the relative cell number.
  • “NKG2D fluorescence intensity” in the abscissa indicates the fluorescence reading displayed by the flow cytometer when detected with a fluorescent NKG2D antibody.
  • Figure 9 is a graph showing the results of Elispot analysis of IFN- ⁇ release after mixed culture of chimeric antigen receptor NKG2D-CD8-DAP12-modified NK (NKG2D-CAR-NK) cells and human tumor cells in an embodiment of the present invention.
  • mGFP-CAR-NK indicates an mGFP-CAR-modified NK cell group (control group)
  • “NKG2D-CAR-NK” indicates a chimeric antigen receptor NKG2D-CD8-DAP12 (i.e., NKG2D-CAR)-modified NK.
  • the abscissa indicates the tumor cell group
  • the ordinate indicates the relative amount of IFN- ⁇ (expressed as the number of spots per 2.5 ⁇ 10 4 NK cells).
  • FIGS. 10A-G show NKG2D-CAR-NK versus tumor cells HCT116(A), SKOV3(B), Fadu(C), Detroit(D), HepG2(E), MCF7(F), respectively, according to an embodiment of the present invention
  • Figure 11 shows the killing effect of adoptively NKG2D-CAR-NK cells on tumor cells; the dotted line shows the results of the chimeric antigen receptor NKG2D-CD8-DAP12 modified NK cell group (shown as "NKG2D-CAR-NK” (6 injections)"); the solid line shows the results of the NK cell reinfusion group (shown as "PBS control group”).
  • the abscissa indicates the number of days after inoculation of the tumor
  • the ordinate indicates the fluorescence intensity of the tumor cells in the animal recorded by the living imager
  • the radiance shown in the ordinate refers to the number of photons emitted from the surface of the animal per unit time, unit area, and unit radians.
  • Figure 12A-C shows chimeric antigen receptor NKG2D-CD8-DAP12 modified NK cells and chimeric antigen receptor NKG2D-CD8-CD3Z modified NK cells, respectively, for tumor cells SKOV3 (A), Detroit (B), Comparison of the detection results of the killing activity of HCT116(C).
  • mGFP-CAR-NK indicates an mGFP-CAR-modified NK cell group
  • NKG2D-DAP12-CAR-NK indicates an NKG2D-DAP12-CAR-modified NK cell group
  • “NKG2D-CD3Z-CAR-NK” indicates NKG2D-CD3Z-CAR modified NK cell group.
  • the abscissa indicates the ratio of effector cells to tumor cells, and the ordinate indicates the proportion of tumor cells that are lysed after killing.
  • Figure 13 shows the results of flow cytometry detection of the H101-treated tumor cell line Fadu surface NKG2D ligand.
  • the black column in the figure represents tumor cells without H101 treatment
  • the punctate column represents H101-treated tumor cells
  • the abscissa represents different NKG2D ligand staining groups (from left to right, hULBP1, hULBP3, hULBP4, hULBP2/5) /6, MICA/B), the ordinate is the percentage of cells expressing the NKG2D ligand.
  • Figure 14 shows the results of flow cytometry detection of H101G-treated tumor cell line HepG2 surface NKG2D ligand.
  • the black column in the figure represents tumor cells without H101 treatment
  • the punctate column represents H101-treated tumor cells
  • the abscissa represents different NKG2D ligand staining groups (from left to right, hULBP1, hULBP3, hULBP4, hULBP2/5) /6, MICA/B), the ordinate is the percentage of cells expressing the NKG2D ligand.
  • Figure 15 is a graph showing the results of detection of the killing activity of the chimeric antigen receptor NKG2D-CD8-DAP12-modified NK cells against tumor cell Fadu.
  • the black column represents the tumor cells without H101 treatment
  • the dotted pattern column represents the tumor cells after H101 treatment
  • "w/o CAR-NK” means that no CAR-NK cells are added, but NK cells are added
  • CAR-NK Indicates NK cells supplemented with NKG2D-DAP12-CAR modification.
  • the abscissa indicates different experimental groups, and the ordinate indicates the ratio of lysis of tumor cells after killing, and the ratio of effector cells to target cells is 1:1.
  • CAR includes an extracellular portion, a transmembrane region, and an intracellular portion.
  • the extracellular portion in turn includes an antigen binding domain for recognizing and binding an antigen, and a spacer for spacing the antigen binding domain and the transmembrane region;
  • the intracellular portion primarily includes an intracellular domain for signaling.
  • the inventors of the present invention have selected a specific combination of an antigen-binding domain and an intracellular domain for NK cells through theoretical research and experimental exploration, and successfully applied the thus developed CAR to NK cells to make the NK cells play.
  • a strong targeted tumoricidal activity thereby developing a novel and effective engineered NKG2D ligand-targeted NK cell that is available for selection in tumor immunotherapy.
  • the invention provides a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane region and an intracellular domain, Characterized in that the antigen binding domain is from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • NKG2D is an important receptor regulating NK cell killing activity.
  • NKG2D ligand is mainly expressed on the surface of tumor cells and stress cells, and is rarely expressed or even expressed on the surface of normal cells.
  • a large number of tumor cells such as colorectal cancer cells and ovarian cancer Cells, head and neck cancer cells, lymphatic cancer cells, glioma cells, and the like have a large number of ligands for NKG2D expression.
  • the amino acid sequence of the antigen-binding domain is preferably identical to the X-position 216 amino acid sequence of NKG2D, and 73 ⁇ X ⁇ 83, and X is an integer.
  • the amino acid sequence of NKG2D may be the amino acid sequence of NP_031386.2 in Genbank of NCBI (ie, National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov). That is, the amino acid sequence of the antigen-binding domain is preferably selected from amino acids 73-216 of NKG2D and comprises amino acids 83-216.
  • amino acid sequence of the antigen-binding domain is as shown in any one of the following amino acid sequence groups: amino acids 73-216 of NKG2D, amino acids 74-216, amino acids 75-216, 76- 216 amino acids, amino acids 77-216, amino acids 78-216, amino acids 79-216, amino acids 80-216, amino acids 81-216, amino acids 82-216, or 83- 216 amino acids. More preferably, the amino acid sequence of the antigen binding domain is set forth in SEQ ID NO: 3.
  • the intracellular domain acts as a signal to activate NK cells.
  • the intracellular domain of the CAR originally used for T cells has only one signaling molecule, usually the receptor-associated Fc ⁇ RI ⁇ of immunoglobulin E (a subunit of a receptor with high affinity for IgE) or T cell antigen receptor signaling.
  • the underlying conductive molecule DAP12 some intracellular domains comprise a T cell activation domain consisting of one or several T cell activation motifs.
  • the present inventors have found that by combining the antigen-binding domain derived from the ligand binding region of NKG2D described above with the intracellular signal region derived from DAP12, a CAR capable of exerting a strong targeted tumoricidal activity of NK cells can be obtained.
  • the amino acid sequence of the intracellular domain is selected from amino acids 62-113 of DAP12, and the amino acid sequence of the intracellular domain is more preferably as set forth in SEQ ID NO: 5.
  • the amino acid sequence of DAP12 has the Genbank number NP_003323.1.
  • the present invention further selects the spacer and transmembrane regions, thereby obtaining a CAR having a specific combination of antigen binding domain-spacer-transmembrane region-intracellular domain.
  • the spacer junction recognizes and binds to the antigen binding domain and transmembrane region of the antigen.
  • the structure of this region should be flexible so that the antigen binding domain can be adapted to different orientations to facilitate antigen recognition and binding.
  • the simplest form of spacer region is an immunoglobulin hinge region of IgGl (hinge), it may be a portion of an immunoglobulin C H2 C H3 region.
  • the transmembrane region is typically a hydrophobic alpha helix spanning the cell membrane.
  • the spacer is preferably derived from the hinge region of CD8[alpha], which is preferably from the transmembrane region of CD8[alpha].
  • CD8 is a transmembrane glycosylated membrane protein consisting of two subunits, alpha and beta, which interact with T cell surface receptors to bind T cells to specific antigens. CD8 specifically binds to MHC I and mediates cytotoxic T cells. The killing effect.
  • the spacer region and the transmembrane region constitute a spacer transmembrane region, and wherein the amino acid sequence of the spacer transmembrane region is identical to the amino acid sequence of the Yth to the 210th position of CD8 ⁇ , and 118 ⁇ Y ⁇ 128, Y is an integer.
  • the Genbank number of the amino acid sequence of CD8 ⁇ may be NP_001139345.1. That is, the amino acid sequence of the spacer transmembrane region is preferably selected from amino acids 118-210 of CD8 ⁇ and amino acids 128-210.
  • amino acid sequence of the spacer transmembrane region is as shown in any one of the following amino acid sequence groups: amino acids 118-210 of CD8 ⁇ , amino acids 119-210, amino acids 120-210, 121- 210 amino acids, amino acids 122-210, amino acids 123-210, amino acids 124-210, amino acids 125-210, amino acids 126-210, amino acids 127-210, or 128- 210 amino acids.
  • amino acid sequence of the spacer transmembrane region is set forth in SEQ ID NO:4.
  • the antigen-binding domain, the spacer, the transmembrane region and the intracellular domain are sequentially connected in series; between the antigen-binding domain and the spacer, between the spacer and the transmembrane region
  • the transmembrane region and the intracellular domain are operably linked, for example, may be connected by a joint or directly without a joint.
  • a linker is used between the antigen binding domain and the spacer (the linker is, for example, -Ala-Ser-), and the spacer and transmembrane region, the transmembrane region and the cell
  • the internal domains are directly connected without a joint.
  • the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 1.
  • the chimeric antigen receptor has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1;
  • the chimeric antigen receptor has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1.
  • the invention also provides an isolated DNA encoding a chimeric antigen receptor of the invention, the DNA comprising an operably linked, sequentially tandem antigen binding domain coding element, a spacer coding element, a transmembrane a region coding element and an intracellular domain coding element, wherein the antigen binding domain coding element is derived from a ligand binding region encoding DNA of NKG2D, and the intracellular domain coding element is derived from the intracellular signal region encoding DNA of DAP12 .
  • the nucleotide sequence of the antigen-binding domain coding element encodes an amino acid sequence of the antigen-binding domain, preferably, the amino acid sequence of the antigen-binding domain is identical to the X-position 216 amino acid sequence of NKG2D, And 73 ⁇ X ⁇ 83, and X is an integer.
  • amino acid sequence of the antigen-binding domain is as shown in any one of the following amino acid sequence groups: amino acids 73-216 of NKG2D, amino acids 74-216, amino acids 75-216, 76- 216 amino acids, amino acids 77-216, amino acids 78-216, amino acids 79-216, amino acids 80-216, amino acids 81-216, amino acids 82-216, or 83- 216 amino acids.
  • amino acids 73-216 of NKG2D amino acids 74-216, amino acids 75-216, 76- 216 amino acids, amino acids 77-216, amino acids 78-216, amino acids 79-216, amino acids 80-216, amino acids 81-216, amino acids 82-216, or 83- 216 amino acids.
  • amino acids 73-216 of NKG2D amino acids 74-216
  • amino acids 75-216 amino acids 77-216
  • amino acids 78-216 amino acids 79-216
  • amino acids 80-216 amino acids 81-216
  • the nucleotide sequence of the intracellular domain coding element encodes the amino acid sequence of the intracellular domain, preferably, the amino acid sequence of the intracellular domain is selected from positions 62-113 of the intracellular signal region of DAP12 Amino acid.
  • the nucleotide sequence of the intracellular domain coding element is set forth in SEQ ID NO: 8.
  • the spacer coding element is derived from the hinge region encoding DNA of CD8 ⁇
  • the transmembrane region coding element is derived from the transmembrane region encoding DNA of CD8 ⁇ .
  • the spacer coding element and the transmembrane region coding element comprise a spacer transmembrane region coding element, the nucleotide sequence of the spacer transmembrane region coding element encoding an amino acid sequence of the spacer transmembrane region, preferably, The amino acid sequence of the spacer transmembrane region is identical to the amino acid sequence of the Yth position to the 210th position of CD8 ⁇ , and 118 ⁇ Y ⁇ 128, and Y is an integer.
  • amino acid sequence of the spacer transmembrane region is as shown in any one of the following amino acid sequence groups: amino acids 118-210 of CD8 ⁇ , amino acids 119-210, amino acids 120-210, 121- 210 amino acids, amino acids 122-210, amino acids 123-210, amino acids 124-210, amino acids 125-210, amino acids 126-210, amino acids 127-210, or 128- 210 amino acids.
  • amino acids 118-210 of CD8 ⁇ amino acids 119-210
  • amino acids 120-210, 121- 210 amino acids, amino acids 122-210, amino acids 123-210, amino acids 124-210, amino acids 125-210, amino acids 126-210, amino acids 127-210, or 128- 210 amino acids amino acids 118-210 of CD8 ⁇
  • amino acids 119-210 amino acids 120-210, 121- 210 amino acids
  • amino acids 122-210 amino acids 123-210
  • amino acids 124-210 amino acids 125-210
  • the isolated nucleotide sequence encoding the chimeric antigen receptor of the invention is represented by SEQ ID NO: 2.
  • the NKG2D, DAP12 and CD8 ⁇ of the present invention are preferably derived from humans, and their full-length amino acid sequences and nucleotide sequences are known, and can be inquired from public databases commonly used in the art.
  • the present invention also provides an isolated mRNA which is transcribed from the DNA encoding the chimeric antigen receptor of the present invention.
  • the invention also provides a recombinant expression vector comprising a DNA encoding a chimeric antigen receptor according to the invention operably linked to a promoter.
  • the recombinant expression vector comprises a CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, and a GM-CSF alpha chain signal peptide coding sequence in sequence before the DNA encoding the chimeric antigen receptor according to the present invention. And comprising a 3'UTR of an alpha globulin having a polyA signal following the DNA encoding the chimeric antigen receptor according to the invention.
  • the combination of the above-described functional elements of the recombinant expression vector of the present invention can promote transcription and translation of DNA and enhance the stability of mRNA.
  • the present invention also optimizes the structure of each of the above-described action elements to better perform their intended functions.
  • CMV promoter sequence TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCCACCCCATTGACGTCAATGGGAGTTTGTTTGTTTTGGCACCAAAATCAACGGGAC TTTCCAAAATGGAC TTGTTTGTTTTGGCACCAAAATCAA
  • the T7 promoter sequence is TAATACGACTCACTATAG (SEQ ID NO: 17).
  • the T7 promoter functions to initiate transcription of downstream DNA sequences.
  • the sequence of the 5' UTR having the kozak sequence is AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA GCCA CCATG (SEQ ID NO: 18), wherein the sequence underlined is the kozak sequence.
  • the function of the 5'UTR with the kozak sequence is to enhance the translation efficiency of the mRNA.
  • the sequence of the GM-CSF alpha chain signal peptide coding sequence is ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCA (SEQ ID NO: 19), and the amino acid sequence thus obtained is MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 20).
  • the GM-CSF alpha chain signal peptide is a leader sequence that targets the CAR of the present invention to the secretory pathway, and the coding sequence is first translated into a protein together with the CAR in the cell, directing the synthesized protein into the endocrine pathway. The signal peptide has been removed before expression of the CAR on the cell surface.
  • the 3'UTR sequence of the alpha globulin is GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTG AATAAA GCCTGAGTAGGAAGT (SEQ ID NO: 21), wherein the sequence underlined is a polyA signal. Its role is to enhance the stability of mRNA.
  • the basic backbone of the recombinant expression vector is a commercially available pFastbac1 vector into which each of the above elements is inserted.
  • a DNA double-stranded template in which a positive strand carries a PolyA and an inverted strand carries a corresponding PolyT can be synthesized from the recombinant expression vector, for example, using a Tail-PCR technique, such that The instability of the DNA template is reduced, and mRNA can be synthesized in vitro.
  • the range of the number of A in the PolyA carried in the positive chain is 140-170, preferably 150-170, more preferably It is about 150 (for example, 150).
  • the present invention also provides a chimeric antigen receptor-modified NK cell whose surface is modified by the chimeric antigen receptor of the present invention.
  • modification means that an NK cell expresses a chimeric antigen receptor according to the present invention, that is, a transmembrane region of the chimeric antigen receptor is anchored to a cell membrane of the modified NK cell, The antigen binding domain is located on the cell surface and the intracellular domain is located in the cytoplasm.
  • the NK cells may be various types of NK cells known and can be obtained by conventional biological methods.
  • NK cells naturally killer cells
  • the phenotype is CD3 negative CD56.
  • Positive single cells mainly CD16-negative CD56 bright (light) and CD16-positive CD56 dim (dark) two subtypes, respectively, have immunomodulatory and tumor killing in vivo efficacy. Since NK cell action is non-MHC-restricted, there is no need to match the histocompatibility complex of the individual patient in use, that is, NK cells can be used for cell therapy of allogeneic patients, and have wide clinical application value.
  • the invention also provides a method of preparing a chimeric antigen receptor-modified NK cell according to the invention, comprising the steps of:
  • the NK cells described in step 1) can be prepared from peripheral blood mononuclear cells.
  • the purity of the NK cells in the method of the invention may be > 70%, preferably > 80%.
  • NK cell purity refers to the proportion of NK cells in the total cell population.
  • the nucleic acid according to the step 2) is a DNA encoding the chimeric antigen receptor according to the present invention, or an mRNA obtained by transcription of the DNA.
  • the transfection described in step 3) can be carried out by cryoelectroporation techniques or lentiviral vectors.
  • Transfection using cryoelectroporation techniques can be carried out in a manner commonly used in the art, such as the literature "Nakazawa Y, Matsuda K, Kurata T, Sueki A, Tanaka M, Sakashita K, Imai C, Wilson MH, Koike K.
  • Anti- Proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia J Hematol Oncol. 2016 Mar.
  • Transfection using lentiviral vectors can be carried out in a manner commonly used in the art, such as the literature "James N. Kochenderfer, Steven A. Feldman, Yangbing Zhao, Hui Xu, Mary A.
  • the DNA corresponding to amino acids 83-216 of the human NKG2D protein, the DNA corresponding to amino acids 128-210 of human CD8 ⁇ , and the 62nd of human DAP12 are amplified by PCR from the PBMC cDNA library. DNA corresponding to -113 amino acids.
  • the three sequences amplified were ligated and ligated to the pFastbac1 vector by molecular cloning techniques to obtain a recombinant expression vector pFastbac1-NKG2D-CD8-DAP12.
  • the mRNA of the corresponding sequence of NKG2D-CD8-DAP12 was then synthesized.
  • the mRNA was electroporated into the NK cells expanded in vitro by high-efficiency cryoelectroporation to obtain engineered NKG2D ligand-targeted NK cells.
  • Each portion of the chimeric antigen receptor can also be amplified by genomic cDNA of mononuclear cells in venous blood.
  • the invention also provides the use of a chimeric antigen receptor-modified NK cell according to the invention for the preparation of a medicament for the treatment or prevention of a tumor and/or cancer.
  • the tumor and/or cancer is positive for NKG2D ligand, including colorectal cancer, ovarian cancer, head and neck cancer, myeloma, liver cancer, breast cancer, hematoma, cervical cancer, glioma, and the like.
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • the invention also provides the use of a chimeric antigen receptor-modified NK cell according to the invention for the preparation of a medicament for detecting a tumor and/or cancer of a host.
  • the tumor and/or cancer is positive for NKG2D ligand, including colorectal cancer, ovarian cancer, head and neck cancer, myeloma, liver cancer, breast cancer, hematoma, cervical cancer, glioma, and the like.
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • a sample of a tumor and/or a cancer cell taken out from a host may be contacted with a chimeric antigen receptor-modified NK cell of the present invention at a concentration according to the degree of reaction between the two. It can be judged whether the tumor and/or cancer is NKG2D ligand positive or NKG2D ligand negative.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chimeric antigen receptor-modified NK cell according to the present invention as an active ingredient, and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition preferably comprises the chimeric antigen receptor-modified NK cells in a total dose ranging from 1 x 10 6 to 1 x 10 11 per subject per subject; preferably, each treatment lasts for 3 weeks. Days, apply 1-2 times a week.
  • the pharmaceutical composition further preferably comprises the chimeric antigen receptor-modified NK cells in a total dose ranging from 6 x 10 7 to 1.2 x 10 10 per subject per treatment; and preferably, each course of treatment is 3 weeks A total of 21 days, 1-2 times a week.
  • the patient may be treated for one or more courses depending on the actual situation and needs.
  • the pharmaceutical composition can be administered by a suitable route of administration including intravenous administration (for example, intravenous drip administration or intravenous administration) or topical administration (for example, topical administration or local injection). Administration).
  • intravenous administration for example, intravenous drip administration or intravenous administration
  • topical administration for example, topical administration or local injection. Administration.
  • the invention also provides a method of treating a tumor and/or cancer comprising administering to a tumor and/or cancer patient a chimeric antigen receptor modified NK cell according to the invention.
  • the tumor and/or cancer is positive for NKG2D ligand, including colorectal cancer, ovarian cancer, head and neck cancer, myeloma, liver cancer, breast cancer, hematoma, cervical cancer, glioma, and the like.
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • the chimeric antigen receptor-modified NK cells are preferably administered at a dose ranging from 1 ⁇ 10 6 to 1 ⁇ 10 11 cells per patient per course of treatment; preferably, each treatment lasts for 3 weeks for 21 days. Apply 1-2 times a week.
  • the dose of the chimeric antigen receptor-modified NK cells is further preferably a total dose per patient per treatment range of 6 ⁇ 10 7 to 1.2 ⁇ 10 10 cells; and it is also preferred that each treatment has a total of 21 weeks. Days, apply 1-2 times a week.
  • the patient may be treated for one or more courses depending on the actual situation and needs.
  • the chimeric antigen receptor-modified NK cells can be administered by a suitable administration route, such as intravenous administration (for example, intravenous drip administration or intravenous administration) or topical administration (for example, topical drip). Injection or local injection).
  • a suitable administration route such as intravenous administration (for example, intravenous drip administration or intravenous administration) or topical administration (for example, topical drip). Injection or local injection).
  • the invention also provides a tool vector comprising, in turn, an operably linked CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, a GM-CSF alpha chain signal peptide coding sequence, and a polyA signal.
  • a tool vector comprising, in turn, an operably linked CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, a GM-CSF alpha chain signal peptide coding sequence, and a polyA signal.
  • the 3'UTR of alpha globulin comprising, in turn, an operably linked CMV promoter, a T7 promoter, a 5'UTR having a kozak sequence, a GM-CSF alpha chain signal peptide coding sequence, and a polyA signal.
  • tool vector refers to an empty vector for insertion of an exogenous DNA fragment in genetic engineering applications.
  • the foreign DNA fragment When the foreign DNA fragment is inserted, the foreign DNA fragment is inserted between the GM-CSF ⁇ chain signal peptide coding sequence of the tool vector and the 3'UTR of the ⁇ -globulin having a polyA signal (GM-CSF alpha chain signal peptide coding The sequence may have a multiple cloning site between the 3' UTR of the alpha globulin with a polyA signal).
  • the nucleotide sequence of the CMV promoter is set forth in SEQ ID NO: 22, and the nucleotide sequence of the T7 promoter is set forth in SEQ ID NO: 17, the core of the 5'UTR having the kozak sequence.
  • the nucleotide sequence of the GM-CSF alpha chain signal peptide coding sequence is set forth in SEQ ID NO: 19, and the 3'UTR of the alpha globulin having a PolyA signal is shown in SEQ ID NO: 18.
  • the nucleotide sequence is shown in SEQ ID NO:21.
  • the tool vector of the present invention is capable of promoting transcription and translation of the inserted DNA and enhancing the stability of the mRNA.
  • the inventors of the present invention have also proposed a novel combination therapy based on the above-described chimeric antigen receptor-modified NK cells in combination with systematic thinking.
  • the human body is a complex system consisting of ten systems, including breathing, circulation, and digestion. These systems work together to make various complex life activities in the human body work normally. Systematic thinking is to conduct a comprehensive investigation of the interaction and interaction of drug effects, diseases, systems and humans from a holistic perspective.
  • cytotoxic anti-tumor drugs when combined with chemotherapy to treat tumors, patients' long-term survival results are not satisfactory, the reason is the lack of systematic thinking.
  • the body when a tumor occurs, the body can exert anti-tumor effects through various immune effect mechanisms, and the body's anti-tumor mechanism includes both cellular immunity and humoral immunity. They are closely related and interact with each other and involve a variety of immune effector molecules and effector cells. It is generally believed that cellular immunity plays a leading role in the anti-tumor process, and humoral immunity plays a synergistic role in some cases.
  • traditional chemotherapy mainly interferes with RNA or DNA synthesis and mitosis. It is mainly for fast-growing cells. It also attacks the normal immune system of the human body while removing tumor cells. As the body's immunity is destroyed, the tumor cells are bound to rise. ".
  • the present invention believes that other methods for improving the body immunity can be adopted, and various treatment means can be combined by system to maximize the comprehensive therapeutic effect while minimizing the damage to the immune system.
  • the present invention proposes a novel combination therapy for the combination of an oncolytic virus with a chimeric antigen receptor modified NK cell of the invention for the treatment of tumors and/or cancer.
  • the present invention can produce a synergistic effect only by combining an oncolytic virus with the NK cells.
  • the present invention provides a therapeutic agent comprising:
  • a first pharmaceutical composition wherein the first pharmaceutical composition comprises an oncolytic virus in a first pharmaceutically acceptable carrier;
  • the oncolytic virus is capable of selectively replicating in a tumor cell
  • the surface of the NK cell is modified by a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane domain and an intracellular domain, characterized in that
  • the antigen binding domain is derived from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • the therapeutic agent can also be understood as a combination of drugs.
  • the active ingredient of the first pharmaceutical composition is the oncolytic virus, and wherein the active ingredient of the second pharmaceutical composition is the NK cell.
  • the first pharmaceutical composition comprises a therapeutically effective amount of the oncolytic virus, and wherein said second pharmaceutical composition comprises 1 ⁇ 10 5 to 1 ⁇ 10 10 cells / day dose NK cells (for example, the second pharmaceutical composition comprises 1 x 10 7 to 1 x 10 10 cells/day of the NK cells; the second pharmaceutical composition comprises 1 x 10 8 to 5 x 10 9 Cell/day dose of said NK cells; said second pharmaceutical composition comprising 1 x 109 to 4 x 109 cells per day of said NK cells; said second pharmaceutical composition comprising 1 x 10 9 to 3 x 10 9 cells/day of the NK cells).
  • the second pharmaceutical composition comprises the NK cells in a total dose ranging from 1 x 10 6 to 1 x 10 11 per subject per subject. Still preferably, the second pharmaceutical composition comprises the NK cells in a total dose ranging from 6 x 10 7 to 1.2 x 10 10 per subject per subject.
  • the present invention also provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises an oncolytic virus and an NK cell, the oncolytic virus being capable of selectively replicating in a tumor cell, the surface of the NK cell being embedded Modification of an antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane domain and an intracellular domain, wherein the antigen binding domain is derived from The ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • the active ingredient of the pharmaceutical composition consists of the oncolytic virus and the NK cells.
  • the oncolytic virus and the NK cells are each independently present in the pharmaceutical composition without mixing with each other.
  • the oncolytic virus contacts the tumor cells by intratumoral or intravenous administration and the infection enters the tumor cells. Since the oncolytic virus is characterized in that it mainly replicates and proliferates in tumor cells, but does not replicate or replicate in normal cells, a large number of oncolytic viruses appear in infected tumor cells, causing tumor cell lysis and death. . The dissolution of tumor cells releases a large number of tumor antigens and proliferating oncolytic viruses. The antigen further activates the immune system in the body, stimulating NK cells and T cells in the body to continue to attack tumor cells that have not yet died, and the new oncolytic virus will Continue to infect tumor cells that have not yet been infected.
  • NK cells are broad-spectrum immune cells that kill tumor cells, and NK cells can distinguish between tumor cells and normal cells. NK contacts and recognizes tumor cells, recognizes it as an abnormal cell, and then kills it through receptor recognition, antibody-targeted recognition (ADCC), granzyme secretion, perforin secretion, and indirect killing of interferon. The effect of dead tumor cells. In vitro experiments have shown that a healthy NK cell can kill 27 tumor cells in a row during its lifetime.
  • ADCC antibody-targeted recognition
  • NK cells also have antiviral functions.
  • a normal cell is infected with a virus, as the virus replicates a lot, the cell exhibits an aging lesion, and the composition of the protein cluster reflected on the cell membrane changes.
  • the NK cell can recognize the infected patient sharply and efficiently.
  • the cells by the means described above similar to killing tumor cells, kill the infected cells, thereby achieving the purpose of inhibiting viral replication and proliferation. Subsequently, under the action of factors such as antigen stimulation and interferon, other immune cells will continue to act against the virus.
  • the present invention takes into consideration the respective characteristics of oncolytic viruses and NK cells, and uses them in combination.
  • the antiviral mechanism of NK cells is equally applicable to tumor cells infected with oncolytic viruses, and is complementary to its anti-tumor mechanism.
  • the combination also makes tumor cells containing oncolytic virus a specific target of NK cells, thereby enhancing the tumor killing effect of NK cells.
  • the oncolytic virus selectively proliferates in cancer cells, plays a role in killing cancer cells in the cell, and can cause changes in protein receptor clusters on the cancer cell membrane, enhancing the recognition of cancer cells by NK cells, and NK cells outside the cancer cells. Attack, the two together to kill cancer cells, have a better therapeutic effect.
  • oncolytic viruses can stimulate the increase of the expression of various NKG2D ligands on the surface of tumor cells, thereby further cooperating with the chimeric antigen receptor-modified NK cells of the present invention to produce stronger anti-tumor synergy. effect.
  • the wild type virus alone has a mutually restrictive effect on NK cells.
  • the virus can escape the antiviral killing of NK by stimulating the KIR receptor on the surface of NK, thereby evading the activity of NK;
  • NK not only recognizes and kills the cells infected by the virus, but also inhibits the proliferation of the virus. It can also directly secrete interferon and inhibit viral activity.
  • many oncolytic viruses are genetically modified, on the one hand, the specificity of infected tumor cells can be enhanced, and on the other hand, the inhibition of immune cells including NK cells is reduced.
  • the oncolytic virus of the present invention includes a gene-mutated virus having an oncolysis effect and a wild-type virus having an oncolysis effect.
  • the gene-mutated virus having oncolytic effect includes, but is not limited to, an adenovirus, a poxvirus (also known as vaccinia virus), herpes simplex virus (HSV), Measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, and retrovirus; Wild-type viruses that function include (but are not limited to): reovirus, vesicular stomatitis virus, poliovirus, Seneca Valley Virus, Eco type Echo enterovirus, Coxsackie virus, Newcastle disease virus, and maraba virus.
  • the exogenous gene may be integrated into the genome of the oncolytic virus, and the exogenous gene includes an exogenous immunoregulatory gene, an exogenously screened gene, an exogenous reporter gene, and the like.
  • the foreign gene may also not be integrated into the genome of the oncolytic virus.
  • the adenovirus includes, but is not limited to, a human type 5 adenovirus or a human chimeric adenovirus; specifically including, for example: Onyx-015 (available from Onyx Pharmaceuticals), H101 (available from Shanghai three-dimensional organism) Technology Co., Ltd.), Ad5-yCD/mutTKSR39rep-hIL12 (available from Henry Ford Health System), CG0070 (available from Cold Genesys), DNX-2401 (available from DNAtrix), OBP-301 (available) From Oncolys BioPharma), ONCOS-102 (available from Targovax Oy/Oncos Therapeutics), ColoAd1 (available from PsiOxus Therapeutics), VCN-01 (available from VCN Biosciences), ProstAtak TM (available from Advantagene company) and so on.
  • Onyx-015 available from Onyx Pharmaceuticals
  • H101 available from Shanghai three-dimensional organism) Technology Co., Ltd.
  • the adenovirus is H101.
  • the poxvirus may be Wyeth strain, WR strain, Listeria strain or Copenhagen strain.
  • the poxvirus may be functionally deficient in the TK gene, functionally deficient in the VGF gene, and functionally deficient in the TK gene and the VGF gene.
  • the poxvirus may also contain other genetic defects, including but not limited to: HA, F14.5L, F4L.
  • the poxvirus is functionally deficient in the TK gene and the VGF gene.
  • the poxvirus includes, but is not limited to, Pexa-vac (available from Jennerex Biotherapeutics), JX-963 (available from Jennerex Biotherapeutics), JX-929 (available from Jennerex Biotherapeutics), VSC20 (preparation)
  • Pexa-vac available from Jennerex Biotherapeutics
  • JX-963 available from Jennerex Biotherapeutics
  • JX-929 available from Jennerex Biotherapeutics
  • VSC20 preparation
  • the method can be found in the scientific literature: "McCart, JA, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res (2001) 61: 8751-8757.”
  • GL- ONC1 available from Genelux
  • TG6002 available from Transgene
  • the herpes simplex virus includes, but is not limited to, HSV-1, HSV-2 herpes simplex virus; specifically including (for example): (available from Amgen), G207 (available from Medigene), HF10 (available from Takara Bio), Seprehvir (available from Virttu Biologics), OrienX010 (available from Beijing Aoyuan and Lili Bio) , NV1020 (available from Catherax) and so on.
  • the NK cells of the present invention include autologous NK cells and allogeneic NK cells.
  • the NK cells may be NK cells obtained by in vitro expansion. Large-scale in vitro expansion culture techniques for NK cells are known and have been largely mature (see, for example, the following scientific literature: "Somanchi SS, Lee DA. Ex Vivo Expansion of Human NK Cells Using K562 Engineered to Express Membrane Bound IL21 .Methods Mol Biol.2016;1441:175-93.” or "Phan MT, Lee SH, Kim SK, Cho D. Expansion of NK Cells Using Genetically Engineered K562 Feeder Cells. Methods Mol Biol. 2016;1441:167-74 .”). Clinical data confirmed that autologous NK cells, haploidentical NK cells (which belong to allogeneic NK cells), and umbilical cord blood were not toxic and side effects after NK cells were returned to humans, and were not long-term dependent, safe and
  • the purity of the NK cells which can be used for treatment may be: the purity of the autologous NK cells may be 85% or more, and the purity of the allogeneic NK cells may be 90% or more; wherein the proportion of CD3 positive T cells in the allogeneic NK cells is not more than 5 ⁇ 10 5 /kg body weight.
  • the present invention further explores optimization of the respective administration dose, administration sequence and administration interval of the oncolytic virus and NK cells, which are critical, which determine the oncolytic virus Anti-tumor efficacy, anti-tumor efficacy of NK cells, and optimal synergistic killing of both tumor cells.
  • the pharmaceutical composition or therapeutic agent comprises a therapeutically effective amount of the oncolytic virus, and the pharmaceutical composition or therapeutic agent comprises a dose of 1 x 10 5 to 1 x 10 10 cells per day.
  • Said NK cells for example, said pharmaceutical composition or therapeutic agent comprises 1 x 107 to 1 x 10 10 cells/day of said NK cells; said pharmaceutical composition or therapeutic agent comprises 1 x 10 8 to 5 ⁇ 10 9 cells/day of the NK cells; the pharmaceutical composition or therapeutic agent comprising 1 x 10 9 to 4 x 10 9 cells/day of the NK cells; the pharmaceutical composition or treatment
  • the agent comprises 1 x 10 9 to 3 x 10 9 cells/day of the NK cells).
  • the pharmaceutical composition or therapeutic agent comprises a therapeutically effective amount of the oncolytic virus, and the pharmaceutical composition or therapeutic agent comprises a total dose ranging from 1 x 10 6 to 1 x 10 per person per course of treatment. Eleven of said NK cells (for example, said pharmaceutical composition or therapeutic agent comprises a total dose ranging from 6 x 10 7 to 1.2 x 10 10 per NK per subject per patient).
  • different preferred clinical dosage ranges can be employed, such as those described in Table 1.
  • the oncolytic viruses can be administered by their respective modes of administration commonly employed in the art, such as by intratumoral injection or intravenous administration.
  • the NK cells can be administered by administration generally employed in the art, for example, intravenously or topically.
  • the oncolytic virus contained in the pharmaceutical composition or therapeutic agent of the present invention is an oncolytic virus (hereinafter also referred to as "oncolytic adenovirus").
  • oncolytic adenovirus an oncolytic virus
  • the E1 region and/or E3 region of the oncolytic adenovirus is genetically engineered.
  • the oncolytic adenovirus is selected from the group consisting of: Onyx-015, H101, Ad5-yCD/mutTKSR39rep-hIL12, CG0070, DNX-2401, OBP-301, ONCOS-102, ColoAd1, VCN-01 , and / or ProstAtak TM .
  • the active ingredients of the pharmaceutical composition or therapeutic agent of the present invention include 5 ⁇ 10 7 to 5 ⁇ 10 12 VP / day dose of the oncolytic adenovirus (e.g., 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP/day dose of oncolytic adenovirus, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP/day dose of oncolytic adenovirus, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP/day dose of oncolytic adenovirus, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP/day dose of oncolytic adenovirus, etc.) and 1 ⁇ 10 5 to 1 ⁇ 10 10 cells/day of the NK cells (for example, 1 ⁇ 10 7 to 1 ⁇ 10 10 Cell/day dose of said NK cells, 1 x 10 8 to 5 x 10 9 cells/day of said NK cells, 1 x 10 9 to 4 x 10 9 cells/day of said NK cells , 1 ⁇ 10 9 to 3 ⁇ 10 9 cells/day
  • ⁇ 10 9 to 5 ⁇ 10 11 VP / day dose of an oncolytic adenovirus 3 ⁇ 10 10 to 3 ⁇ 10 11 VP / day dose of the oncolytic adenovirus, etc.
  • 1 ⁇ 10 5 1 ⁇ 10 10 cells / day dose of the NK cells e.g., the 1 ⁇ 10 7 to 1 ⁇ 10 10 cells / day dose NK cells, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day
  • the dose consists of the NK cells, 1 x 10 9 to 4 x 10 9 cells/day of the NK cells, 1 x 10 9 to 3 x 10 9 cells/day of the NK cells, and the like.
  • the active ingredients of the pharmaceutical composition or the therapeutic agent of the present invention include 5 ⁇ 10 7 to 5 ⁇ 10 12 VP / day dose of the oncolytic adenovirus (e.g., 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP/day dose of oncolytic adenovirus, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP/day dose of oncolytic adenovirus, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP/day dose of oncolytic adenovirus, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP/day dose of oncolytic adenovirus, etc.) and the total dose per person per treatment range is 1 ⁇ 10 6 - 1 ⁇ 10 11 of said NK cells (for example, each per person)
  • the total dose of the treatment ranges from 6 ⁇ 10 7 to 1.2 ⁇ 10 10 of the NK cells, etc.); preferably, the active ingredient of the pharmaceutical composition or therapeutic agent is from 5 ⁇ 10 7 to 5 ⁇ 10 12 VP/day.
  • Dosage of oncolytic adenovirus eg, 5 x 107 to 1.5 x 10 12 VP/day dose of oncolytic adenovirus, 5 x 108 to 1 x 10 12 VP/day dose of oncolytic adenovirus, 1 x 10 9 to 5 ⁇ 10 11 VP / day dose of oncolytic adenovirus, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP / day dose of oncolytic adenovirus, etc.
  • the total dose range per person per treatment is 1 ⁇ 10 6 - 1 ⁇ 10 11 of said NK cells (for example, the total dose per patient per treatment range is 6 ⁇ 10 7 - 1.2 ⁇ 1 0 10 of the NK cells, etc.).
  • the active ingredients of the pharmaceutical composition or the therapeutic agent of the present invention include 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP / day dose of the oncolytic virus H101 (e.g., 5 ⁇ 10 11 to 1.5 ⁇ 10 12 VP / day dose of oncolytic virus H101, etc.) and 1 x 10 5 to 1 x 10 10 cells/day of the NK cells; preferably, the active ingredient of the pharmaceutical composition or therapeutic agent is 5 x 10 7 Up to 1.5 ⁇ 10 12 VP/day dose of oncolytic virus H101 (eg, 5 ⁇ 10 11 to 1.5 ⁇ 10 12 VP/day dose of oncolytic virus H101, etc.) and 1 ⁇ 10 5 to 1 ⁇ 10 10 cells/ A daily dose of the NK cells (for example, 1 x 107 to 1 x 10 10 cells/day of the NK cells, 1 x 10 8 to 5 x 10 9 cells/day of the NK cells, 1 x 10 9 to 4 x 10 9 cells/day dose of the NK cells, 1 x
  • the active ingredients of the pharmaceutical composition or therapeutic agent of the present invention include 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP / day dose of the oncolytic virus H101 (e.g., 5 ⁇ 10 11 to 1.5 ⁇ 10 12 VP/day dose of oncolytic virus H101, etc.) and the total dose range of 1 ⁇ 10 6 -1 ⁇ 10 11 per NK per patient (for example, the total dose range per person per treatment is 6 ⁇ ) 10 7 - 1.2 ⁇ 10 10 of said NK cells, etc.); preferably, the active ingredient of the pharmaceutical composition or therapeutic agent is from 5 x 10 7 to 1.5 x 10 12 VP/day of the oncolytic virus H101 ( For example, 5 x 10 11 to 1.5 x 10 12 VP/day dose of oncolytic virus H101, etc.) and the total dose per person per treatment range is 1 x 10 6 - 1 x 10 11 of said NK cells (for example, Each dose per patient has a total dose ranging from 6 ⁇ 10 7 to 1.2 ⁇ 10 10 of
  • the oncolytic virus contained in the pharmaceutical composition or therapeutic agent of the present invention is a poxvirus having an oncolysis effect (hereinafter also referred to as "anti-tumor poxvirus").
  • the oncolytic poxvirus is selected from the group consisting of a genetically mutated virus having an oncolysis effect and a wild type virus having an oncolysis effect.
  • the oncolytic poxvirus is functionally deficient in the TK gene and/or the VGF gene.
  • the oncolytic poxvirus is selected from the group consisting of: Pexa-vac, JX-963, JX-929, VSC20, GL-ONC1, and/or TG6002.
  • the active ingredients of the pharmaceutical composition or therapeutic agent of the present invention comprises 1 ⁇ 10 5 to 5 ⁇ 10 9 pfu / day dose oncolytic poxvirus (e.g., 1 ⁇ 10 5 to 3 ⁇ 10 9 a) and 1 ⁇ 10 5 to 1 ⁇ 10 10 cells / day dose pfu / day dose oncolytic poxvirus, 1 ⁇ 10 5 to 1 ⁇ 10 8 pfu / day dose oncolytic poxvirus like NK cells (For example, 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/day of the NK cells, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells/day of the NK cells, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells/day dose of said NK cells, 1 x 10 9 to 3 x 10 9 cells/day of said NK cells, etc.); preferably, the active ingredient of the pharmaceutical composition or therapeutic agent is 1 ⁇ 10 5 to 5 ⁇ 10 9 pfu / day dose of oncolytic pox virus (
  • the active ingredients of the pharmaceutical composition or the therapeutic agent of the present invention comprises 1 ⁇ 10 5 to 5 ⁇ 10 9 pfu / day dose oncolytic poxvirus (e.g., 1 ⁇ 10 5 to 3 ⁇ 10 9 Pfu/day dose of oncolytic pox virus, 1 ⁇ 10 5 to 1 ⁇ 10 8 pfu/day dose of oncolytic pox virus, etc.) and the total dose range per person per treatment is 1 ⁇ 10 6 -1 ⁇ 10 11
  • the NK cells for example, the total dose per subject per treatment range is 6 ⁇ 10 7 -1.2 ⁇ 10 10 of the NK cells, etc.
  • the active ingredient of the pharmaceutical composition or therapeutic agent is 1 ⁇ 10 5 to 5 ⁇ 10 9 pfu / day dose of oncolytic pox virus (for example, 1 ⁇ 10 5 to 3 ⁇ 10 9 pfu / day dose of oncolytic pox virus, 1 ⁇ 10 5 to 1 ⁇ 10 8 pfu / day dose of oncolytic pox
  • the pharmaceutical or therapeutic agents of the present invention may also comprise suitable pharmaceutically acceptable excipients.
  • the pharmaceutical composition or therapeutic of the present invention may further comprise other active ingredients known in the art, such as interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon. - ⁇ (IFN- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ) and the like.
  • IL-2 interleukin-2
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • IFN- ⁇ interferon.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • the pharmaceutical or therapeutic agent of the invention does not comprise bortezomib.
  • a pharmaceutical or therapeutic agent of the invention comprises one or more pharmaceutically acceptable carriers.
  • Pharmaceutical formulations can be prepared by methods known in the art.
  • an active ingredient such as a compound can be formulated with a common excipient, a diluent (for example, phosphate buffer or physiological saline), a tissue culture medium, and a carrier (for example, autologous plasma or human serum albumin) as a suspension.
  • a diluent for example, phosphate buffer or physiological saline
  • tissue culture medium for example, autologous plasma or human serum albumin
  • carrier for example, autologous plasma or human serum albumin
  • Other carriers may include liposomes, micelles, nanocapsules, polymeric nanoparticles, solid lipid particles (see, for example, the literature "E. Koren and V. Torchilin, Life, 63:586-595, 2011").
  • Specific methods of formulating the pharmaceutical or therapeutic agents of the present invention can be found in the scientific literature and in the patent literature, for
  • the invention provides a therapeutic comprising: (a) a first pharmaceutical composition, wherein the first pharmaceutical composition comprises an oncolytic virus in a first pharmaceutically acceptable carrier; and (b) a second pharmaceutical composition, wherein the second pharmaceutical composition comprises NK cells in a second pharmaceutically acceptable carrier; wherein the oncolytic virus is capable of selectively replicating in tumor cells; and wherein the NK cells are
  • the surface is modified by a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane domain and an intracellular domain, characterized in that said antigen binding The domain is derived from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • the first pharmaceutically acceptable carrier and the second pharmaceutically acceptable carrier are the same. In other embodiments, the first pharmaceutically acceptable carrier and the second pharmaceutically acceptable carrier are different.
  • the pharmaceutical composition or therapeutic of the present invention can be used for the treatment of various tumors and/or cancers including, but not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal Cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphoma, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, etc.
  • the tumor and/or cancer is NKG2D ligand positive, including the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated to become NKG2D ligand positive of.
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • the pharmaceutical composition or therapeutic agent of the present invention is administered by first administering the oncolytic virus (for example, oncolytic adenovirus, oncolytic pox virus or oncolytic herpes simplex virus) to a tumor and/or cancer patient, and then, 18-72 hours after administration of the oncolytic virus (eg, 20-70 hours, 22-48 hours, 24-48 hours, 30-48 hours, etc.) to the tumor and/or cancer
  • the oncolytic virus for example, oncolytic adenovirus, oncolytic pox virus or oncolytic herpes simplex virus
  • 18-72 hours after administration of the oncolytic virus eg, 20-70 hours, 22-48 hours, 24-48 hours, 30-48 hours, etc.
  • the tumor and / Or administration of the NK cells by a cancer patient means that the time interval between administration of the first NK cells and the first oncolytic virus administration is 18-72 hours (for example, 20-70 hours, 22-48 hours, 24-48 hours, 30- 48 hours, etc.), or the time interval between administration of the first NK cells and the onset of the oncolytic virus that is most adjacent to it is 18-72 hours (eg, 20-70 hours, 22-48 hours, 24-48) Hours, 30-48 hours, etc.).
  • the time interval between administration of the first NK cell and the onset of the oncolytic virus that is most adjacent to it is 18-72 hours (eg, 20-70 hours, 22-48 hours, 24-48 hours, 30) -48 hours, etc.). Also preferably, the time interval between administration of the first NK cells and administration of the oncolytic virus that is most adjacent to it before is 24-48 hours.
  • the oncolytic virus (for example, oncolytic adenovirus, oncolytic pox virus or oncolytic herpes simplex virus) is administered in a therapeutically effective amount, once a day, continuously administered from 1 to 6
  • the NK cells are administered at a dose ranging from 1 ⁇ 10 6 to 1 ⁇ 10 11 per patient per course (for example, the total dose per patient per treatment range is 6 ⁇ 10 7 -1.2 ⁇ 10) 10 ), administered 1-2 times a week for 3 weeks.
  • the oncolytic virus (for example, oncolytic adenovirus, oncolytic pox virus or oncolytic herpes simplex virus) is administered in a therapeutically effective amount, once every 2 days, continuously.
  • the NK cells are administered at a dose ranging from 1 ⁇ 10 6 to 1 ⁇ 10 11 per patient per treatment (for example, the total dose range per person per treatment is 6 ⁇ 10 7 - 1.2 ⁇ 10 10 ), 1-2 times a week for 3 weeks.
  • the oncolytic virus eg, oncolytic adenovirus, oncolytic pox virus, or oncolytic herpes simplex virus
  • the conditions for administering the NK cells to the tumor and/or cancer patient may be sufficient.
  • administration of the oncolytic virus and administration of the NK cells may be, for example, first administration of the oncolytic virus once a day for 1-6 days, followed by administration of NK cells at intervals of 18-72 hours, 1-2 weekly administration Times, for 3 consecutive weeks.
  • the oncolytic virus is administered first, and the NK cells are administered again 18-72 hours after the oncolytic virus is administered.
  • the oncolytic virus is first administered to a tumor and/or cancer patient, the oncolytic virus is administered in a therapeutically effective amount, administered once; and the oncolytic virus is administered
  • the NK cells are administered to the tumor and/or cancer patient from the 18th hour to the 72th hour thereafter, and the NK cells are administered at a dose ranging from 1 ⁇ 10 6 to 1 ⁇ 10 11 per person per course of treatment. (for example, the total dose per patient per treatment range is 6 ⁇ 10 7 - 1.2 ⁇ 10 10 ), 1-2 times a week for 3 weeks.
  • different preferred clinical dosage ranges can be employed, such as those described in Table 1.
  • Oncolytic viruses are capable of selective replication in tumor or cancer cells and peak over time.
  • the inventors of the present invention found that after a period of replication, the oncolytic virus in the tumor cells promotes killing of tumor cells by NK cells. Therefore, the application interval of the oncolytic virus and NK cells proposed by the present invention achieves a bimodal overlap of the peaks of action of both.
  • the invention also provides the use of a pharmaceutical or therapeutic agent of the invention in the manufacture of a medicament for the treatment of tumors and/or cancer.
  • the tumor and/or cancer includes, but is not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma e.g., head and neck cancer
  • liver cancer e.g., brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma head and neck cancer
  • liver cancer e.g., brain cancer, colorectal cancer
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • the present invention also provides a kit for synergistic combination therapy for treating tumors and/or cancer, comprising a first container containing the oncolytic virus of the present invention and a ovary containing the NK cells of the present invention a second container, wherein said first container and said second container are independent; and instructions for indicating the timing of administration and mode of administration; wherein said oncolytic virus is capable of selectively replicating in tumor cells;
  • the surface of the NK cell is modified by a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, a transmembrane region and an intracellular domain, characterized in that The antigen binding domain is from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • the kit consists of separate containers each containing the oncolytic virus of the invention and the NK cells of the invention, respectively, together with instructions for the timing and mode of administration.
  • the tumor and/or cancer includes, but is not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma e.g., head and neck cancer
  • liver cancer e.g., brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma head and neck cancer
  • liver cancer e.g., brain cancer, colorectal cancer
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • the first container containing the oncolytic virus comprises a therapeutically effective amount of the oncolytic virus
  • the second container containing the NK cells comprises sufficient to provide 1 x 10 5 to 1 x 10 10 cells per day.
  • the dose of the NK cells for example, 1 ⁇ 10 7 - 1 ⁇ 10 10 cells / day dose, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day dose of the NK cells, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells/day dose of the NK cells, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells/day of the NK cells, etc.).
  • the first container containing the oncolytic virus comprises a therapeutically effective amount of the oncolytic virus
  • the second container containing the NK cells comprises sufficient to provide a total dose range of 1 x 10 per person per course of treatment. 6 - 1 ⁇ 10 11 of said NK cells (e.g., the total dose per subject per treatment range is 6 x 10 7 - 1.2 x 10 10 of said NK cells).
  • different preferred clinical dosage ranges can be employed, such as those described in Table 1.
  • the oncolytic viruses can be administered by their respective modes of administration commonly employed in the art, such as by intratumoral injection or intravenous administration.
  • the NK cells can be administered by administration generally employed in the art, for example, by intravenous administration or topical administration.
  • the oncolytic virus is an adenovirus having an oncolytic effect.
  • the E1 region and/or E3 region of the oncolytic adenovirus is genetically engineered.
  • the oncolytic adenovirus is selected from the group consisting of: Onyx-015, H101, Ad5-yCD/mutTKSR39rep-hIL12, CG0070, DNX-2401, OBP-301, ONCOS-102, ColoAd1, VCN-01 , and / or ProstAtak TM .
  • the first container comprises a 5 ⁇ 10 7 to 5 ⁇ 10 12 VP / day dose of the oncolytic adenovirus (e.g., 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP / day Dosage of oncolytic adenovirus, 5 x 108 to 1 x 10 12 VP/day dose of oncolytic adenovirus, 1 x 109 to 5 x 10 11 VP/day dose of oncolytic adenovirus, 3 x 10 10 to 3 ⁇ 10 11 VP/day dose of oncolytic adenovirus, etc.).
  • the oncolytic adenovirus e.g., 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP / day Dosage of oncolytic adenovirus, 5 x 108 to 1 x 10 12 VP/day dose of oncolytic adenovirus, 1 x 109 to 5 x 10 11 VP/day dose of oncolytic adenovirus, 3 x 10 10 to 3 ⁇
  • the first container comprises a 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP / day dose of the oncolytic virus H101 (e.g., 5 ⁇ 10 11 to 1.5 ⁇ 10 12 VP / day dose of an oncolytic virus H101, etc.).
  • the oncolytic virus is an oncolytic virus.
  • the oncolytic poxvirus is selected from the group consisting of a genetically mutated virus having an oncolysis effect and a wild type virus having an oncolysis effect.
  • the oncolytic poxvirus is functionally deficient in the TK gene and/or the VGF gene.
  • the oncolytic poxvirus is selected from the group consisting of: Pexa-vac, JX-963, JX-929, VSC20, GL-ONC1, and/or TG6002.
  • the first container comprises 1 ⁇ 10 5 to 5 ⁇ 10 9 pfu / day dose oncolytic poxvirus (e.g., 1 ⁇ 10 5 to 3 ⁇ 10 9 pfu / day Dosage of oncolytic pox virus, 1 x 10 5 to 1 x 10 8 pfu / day dose of oncolytic pox virus, etc.).
  • oncolytic poxvirus e.g., 1 ⁇ 10 5 to 3 ⁇ 10 9 pfu / day Dosage of oncolytic pox virus, 1 x 10 5 to 1 x 10 8 pfu / day dose of oncolytic pox virus, etc.
  • the invention also provides a method of treating a tumor and/or cancer comprising the steps of:
  • the tumor and / or a cancer patient administering the NK cells of the invention, the surface of which is modified by a chimeric antigen receptor comprising an operably linked, sequentially tandem antigen binding domain, a spacer, Transmembrane and intracellular domains, characterized in that the antigen binding domain is from the ligand binding region of NKG2D, which is derived from the intracellular signaling region of DAP12.
  • 18-72 hours after administration of the oncolytic virus eg, 20-70 hours, 22-48 hours, 24-48 hours, 30-48 hours, etc.
  • the tumor and / Or administration of the NK cells of the present invention to a cancer patient means that the time interval between administration of the first NK cells and the first oncolytic virus administration is 18-72 hours (for example, 20-70 hours, 22-48 hours, 24-48 hours). 30-48 hours, etc., or the time interval between administration of the first NK cell and the onset of the oncolytic virus that is most adjacent to it before the time is 18-72 hours (eg, 20-70 hours, 22-48 hours, 24-48 hours, 30-48 hours, etc.).
  • the time interval between administration of the first NK cell and the onset of the oncolytic virus that is most adjacent to it is 18-72 hours (eg, 20-70 hours, 22-48 hours, 24-48 hours, 30) -48 hours, etc.). Also preferably, the time interval between administration of the first NK cells and administration of the oncolytic virus that is most adjacent to it before is 24-48 hours.
  • the tumor and/or cancer includes, but is not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma e.g., head and neck cancer
  • liver cancer e.g., brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma head and neck cancer
  • liver cancer e.g., brain cancer, colorectal cancer
  • the tumor and/or cancer is positive for NKG2D ligand comprising the tumor and/or cancer being untreated is NKG2D ligand positive and the tumor and/or cancer is treated It became positive for NKG2D ligand.
  • the treatment includes becoming NKG2D ligand positive after treatment with a drug, radiation or biological agent.
  • the oncolytic virus is administered in a therapeutically effective amount once a day for 1-6 days; and the NK cells are administered at a total dose per person per course of treatment.
  • the range is 1 ⁇ 10 6 - 1 ⁇ 10 11 (for example, the total dose per patient per treatment range is 6 ⁇ 10 7 - 1.2 ⁇ 10 10 ), and is administered 1-2 times a week for 3 weeks.
  • the oncolytic virus is administered in a therapeutically effective amount once every 2 days for 2-6 days; and the NK cells are administered at a dose of each person.
  • the total dose range of treatment is 1 ⁇ 10 6 -1 ⁇ 10 11 (for example, the total dose range per patient per treatment is 6 ⁇ 10 7 - 1.2 ⁇ 10 10 ), 1-2 times a week for 3 weeks .
  • administration of the oncolytic virus and administration of the NK cells may be, for example, first administration of the oncolytic virus once a day for 1-6 days, followed by administration of NK cells at intervals of 18-72 hours, 1-2 weekly administration Times, for 3 consecutive weeks).
  • the oncolytic virus is administered first, and the NK cells are administered again 18-72 hours after the oncolytic virus is administered.
  • the oncolytic virus is first administered to a tumor and/or cancer patient, the oncolytic virus is administered in a therapeutically effective amount, administered once; and the oncolytic virus is administered
  • the NK cells are administered to the tumor and/or cancer patient from the 18th hour to the 72th hour thereafter, and the NK cells are administered at a dose ranging from 1 ⁇ 10 6 to 1 ⁇ 10 11 per person per course of treatment. (for example, the total dose per patient per treatment range is 6 ⁇ 10 7 - 1.2 ⁇ 10 10 ), 1-2 times a week for 3 weeks.
  • different preferred clinical dosage ranges can be employed, such as those described in Table 1.
  • the method of treating tumors and/or cancer of the present invention may be performed one or more times on the patient according to actual conditions and needs.
  • the oncolytic viruses can be administered by their respective modes of administration commonly employed in the art, such as by intratumoral injection or intravenous administration.
  • the NK cells can be administered by administration generally employed in the art, for example, by intravenous administration or topical administration.
  • the oncolytic virus is an adenovirus having an oncolytic effect.
  • the E1 region and/or E3 region of the oncolytic adenovirus is genetically engineered.
  • the oncolytic adenovirus is selected from the group consisting of: Onyx-015, H101, Ad5-yCD/mutTKSR39rep-hIL12, CG0070, DNX-2401, OBP-301, ONCOS-102, ColoAd1, VCN-01 , and / or ProstAtak TM .
  • the oncolytic virus is an oncolytic adenovirus, and is administered at a dose of 5 ⁇ 10 7 to 5 ⁇ 10 12 VP / day (e.g., 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP/day, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP/day, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP/day, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP/day, etc.).
  • the oncolytic virus is an oncolytic virus H101, and which is administered at a dose of 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP / day (e.g., 5 ⁇ 10 11 to 1.5 ⁇ 10 12 VP / day, etc. ).
  • the oncolytic virus is an oncolytic virus.
  • the oncolytic poxvirus is selected from the group consisting of a genetically mutated virus having an oncolysis effect and a wild type virus having an oncolysis effect.
  • the oncolytic poxvirus is functionally deficient in the TK gene and/or the VGF gene.
  • the oncolytic poxvirus is selected from the group consisting of: Pexa-vac, JX-963, JX-929, VSC20, GL-ONC1, and/or TG6002.
  • the oncolytic virus is an oncolytic poxvirus and is administered at a dose of from 1 x 10 5 to 5 x 10 9 pfu per day (eg, 1 x 10 5 to 3 x) 10 9 pfu/day, 1 ⁇ 10 5 to 1 ⁇ 10 8 pfu/day, etc.).
  • the percent concentration (%) of each reagent refers to the volume percent concentration (% (v/v)) of the reagent.
  • the H101 oncolytic adenovirus used in the following examples was purchased from Shanghai 3D organism.
  • the PBS used in the following examples was purchased from Lonza under the order number BW17-517Q.
  • VioBright FITC-conjugated anti-hMICA/B antibody used in the following examples was purchased from Miltenyi; PE-conjugated anti-hULBP1 antibody, APC-conjugated anti-hULBP2/5/6 antibody, APC-conjugated anti-hULBP3 antibody, APC-conjugated Anti-hULBP4 antibody was purchased from R&D Systems.
  • the Calcein AM used in the following examples was purchased from Life Technologies.
  • the 24-well cell culture plates used in the following examples (500 ⁇ l per well culture volume) and 96-well cell culture plates (200 ⁇ l per well culture volume) were obtained from Corning.
  • PBMCs venous blood mononuclear cells
  • PBMCs Mononuclear cells
  • RNA extraction kit RNAiso Reagent purchased from Life Technologies
  • the extracted total RNA was reverse transcribed into cDNA of PBMCs using a reverse transcription kit RevertAicTFirst Strand cDNA Synthesis Kit (purchased from Life Technologies), and stored at -20 ° C until use.
  • PCR amplification was carried out with primers P1 (SEQ ID NO: 9) and P2 (SEQ ID NO: 10) to obtain an extracellular domain comprising a NKG2D protein of 402 bp in length.
  • a fragment of the corresponding DNA coding sequence (nucleotide sequence as shown in SEQ ID NO: 6 in the Sequence Listing) has a Sphl and NheI restriction site and a protective base at both ends, respectively.
  • PCR amplification using primers P5 (SEQ ID NO: 13) and P6 (SEQ ID NO: 14) gave an intracellular signal domain containing DAP12 of 156 bp in length (nucleotide sequence such as SEQ ID NO in the sequence listing: A fragment of 8).
  • the PCR amplification reaction system was the same in each step.
  • the PCR reaction conditions were as described in KAPA HiFi Hot Start Ready Mix (2X) (purchased from Kapa Biosystems), and each reaction system (50 ⁇ L) was as follows:
  • Double distilled water 21.5 ⁇ L
  • the above PCR product was separated on a 1% (w/v) agarose gel, and DNA fragment recovery was carried out using an agarose gel DNA recovery kit (purchased from Omega Bio Tek).
  • the fragment containing the extracellular domain DNA of the NKG2D protein of 402 bp in length was subjected to double digestion with Sphl and NheI, and the digested product was subjected to DNA fragment recovery using an agarose gel DNA recovery kit.
  • the commercial vector pFastbac1 (Life Technologies) was added to a CMV promoter, a T7 promoter, a 5'UTR with a Kozak sequence, and a coding sequence for a GM-CSF alpha chain signal peptide (SP in Figure 1). ), and a 3'UTR of alpha globulin with a polyA signal to construct the pFastbac1 basic skeleton vector.
  • the pFastbac1 basic skeleton vector was subjected to double digestion with SphI and SalI, and the digested product was subjected to DNA fragment recovery using an agarose gel DNA recovery kit, and then passed through the CD8 and DAP12 fragments recovered by the previous PCR.
  • lane 1 1000 kb DNA molecular weight marker
  • lane 2 plasmid pFastbac1-CD8-DAP12 digestion fragment, vector backbone 5276 bp, CD8-DAP12 fragment 405 bp.
  • the correct plasmid was sent to AITbiotech for sequencing of the inserted fusion gene fragment, and the correct recombinant plasmid was named pFastbac1-CD8-DAP12.
  • the vector pFastbac1-CD8-DAP12 was digested with SphI and NheI, and the digested product was recovered by DNA fragmentation using an agarose gel DNA recovery kit, and then ligated to the extracellular domain fragment of the previously recovered NKG2D protein by T4 DNA.
  • Enzyme-linked, linked product-transformed One Chemically Competent TOP10 chemically competent cells were cultured at 37 ° C for 18 hours, and then picked up, and cultured at 37 ° C, 250 rpm for 6 hours, and then the plasmid was extracted with a plasmid mini-kit.
  • the extracted plasmid was identified by restriction endonuclease SphI and NheI digestion, and the electrophoresis pattern was identified as shown in Fig. 3; wherein, lane 1: 1000 kb DNA molecular weight marker; lane 2: restriction fragment of plasmid pFastbac1-NKG2D-CD8-DAP12, The vector backbone is 5682 bp and the NKG2D fragment is 402 bp.
  • the correct plasmid was sent to AITbiotech for sequencing of the inserted fusion gene fragment, and the recombinant plasmid with the correct sequencing result was named pFastbac1-NKG2D-CD8-DAP12, and the plasmid map is shown in Figure 4.
  • PBMCs cells with 20 ⁇ 10 6 cells and 2 mL NK cell activator I purchased from Shenzhen Daktronics, DKW35-CYT-NK001
  • NK culture medium was AIM
  • NK cell activator I was uniformly mixed in 400 ml of NK medium, inoculated back into a G-Rex 100 cell culture incubator, and culture was continued for 7 days under the same conditions.
  • NK cells cultured by this method was as high as 90%, as shown in Fig. 5.
  • Fig. 5A indicate that the proportion of CD3-negative and CD56-positive NK cells is 90.2%
  • Fig. 5B indicate that the ratio of NK cell surface receptor NKG2D and CD16 double positive is 96.4%.
  • Tail-PCR technology was used to synthesize large-dose DNA double-stranded templates with PolyA in the positive strand and corresponding PolyT in the reverse strand for in vitro RNA synthesis, which reduced the instability of DNA template.
  • the chimeric antigen receptor NKG2D-CD8-DAP12 coding sequence was subjected to Tail-PCR amplification using the above pFastbac1-NKG2D-CD8-DAP12 vector as a DNA template to synthesize linearization of the chimeric antigen receptor NKG2D-CD8-DAP12 sequence.
  • DNA template, Tail-PCR reaction conditions refer to the instructions of KAPA HiFiHotStartReadyMix (2X), the reaction system (50 ⁇ L) is as follows:
  • pFastbac1-NKG2D-CD8-DAP12 vector DNA template 500ng/ ⁇ L: 0.5 ⁇ L
  • the above PCR product was isolated and identified on a 1% (w/v) agarose gel, as shown in FIG.
  • the correct product was identified for in vitro synthesis of the chimeric antigen receptor NKG2D-CD8-DAP12 mRNA.
  • In vitro mRNA synthesis kit with capped mRNA synthesis kit as mMESSAGEmMACHINE T7 ULTRA Transcription Kit (available from Invitrogen, USA) or mScript TM RNA system (available from the American Epicentre Corporation). According to the instructions of the kit, and using the reagents provided in the kit for synthesis.
  • the in vitro synthesized chimeric antigen receptor NKG2D-CD8-DAP12 mRNA product was isolated and identified on a 1% (w/v) agarose gel, as shown in FIG. The correct mRNA was identified and stored at -80 ° C for storage.
  • NK cells prepared in Preparation Example 2 (1 ⁇ 10 7 cells) and 4 ⁇ g of NKG2D-CD8-DAP12 mRNA were mixed in electroporation P3 (product name "P3 Primary Cell” X Kit L”, Lonza, item number V4XP-3012), placed in a 100 ⁇ l Nucleocuvette TM tube (P3Primary Cell) X Kit L, Lonza, item number V4XP-3012), and frozen in an ice bath for 5 minutes. Then using the 4D-Nucleofector TM electroporation instrument (available from Lonza, Inc., Switzerland), choose their own program NK cell electroporation electroporation.
  • P3 product name "P3 Primary Cell” X Kit L”, Lonza, item number V4XP-3012
  • Test Example 1 Detection of NKG2D-CAR NK cells in vitro killing ability of human tumor cells
  • an ELISPOT assay was performed to detect the secretion of IFN ⁇ , since the secretion of IFN- ⁇ was positively correlated with the anti-tumor activity of NKG2D-CAR NK cells in adoptive cellular immunotherapy.
  • the preparation method of mGFP-CAR NK cells is the same as that of NKG2D-CAR NK cells (NK cells transfected with NKG2D-CD8-DAP12 mRNA), except that the vector is constructed.
  • the outer domain used the mGFP sequence without antigen binding function (its Genbank accession number is YP_002302326.1) as a negative control.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA The preparation of NK cells transfected with NKG2D-CD8-DAP12 mRNA is shown in Preparation 3.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA or mGFP-CD8-DAP12 mRNA were associated with human ovarian cancer cells SKOV3, human colorectal cancer cells HCT116 and SW480, human head and neck cancer cells Detroit, human hepatoma cells HepG2 and human neutrophils
  • the stromal cell U87 was co-cultured on the ELISPOT assay plate, and the ratio of the above NK effector cells to the target cells was 5:1. Each set of experiments was repeated 3 times. After 24 hours of co-cultivation, development and use of the software immunospot for ELISPOT point counting.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA produced more and stronger IFN- ⁇ than NK cells transfected with mGFP-CD8-DAP12 mRNA (detected by ELISPOT assay kit, purchased from Mabtech The product number is 3420-4HST-1).
  • the ELISPOT spots produced by NKG2D-CD8-DAP12 mRNA-transfected NK cells were significantly higher than the control group, as shown in Figure 9.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA or mGFP-CD8-DAP12 mRNA were associated with human colorectal cancer cell HCT116, human ovarian cancer cell SKOV3, human head and neck cancer cells Fadu and Detroit, human hepatoma cell HepG2, human breast cancer
  • the cell MCF7 and the human myeloma cell KG1 were co-cultured in a U-shaped 96-well plate, and the ratio of the above-mentioned NK effector cells to the target cells ranged from 2.5:1 to 10:1. Each set of experiments was repeated 3 times.
  • NKG2D-CAR NK cells were examined using the DELFIA EuTDA cytotoxicity kit (available from PerkinElmer, USA), and the killing effect was calculated by the following formula:
  • % specific lysis ((experimental group release (reading) - blank group release (reading)) / (maximum release (reading) - blank group release (reading))) x 100
  • NKG2D-CAR modified NK cells have broad and strong tumoricidal activity.
  • the killing effect of NKG2D-CAR modified NK cells is At 10:1, it was significantly higher than the killing effect of the control group, as shown in Figure 10.
  • the above results fully demonstrate the universality and high efficiency of NKG2D-CAR NK on tumor killing.
  • Test Example 2 Detection and analysis of NKG2D-CAR NK cells in vivo killing ability of human tumor cells
  • NKG2D-CAR NK cells The in vivo antitumor effect of NKG2D-CAR NK cells was further tested using a mouse model implanted in human tumors.
  • the experimental mice were non-obese diabetic/severe combined immunodeficiency/IL-2R ⁇ cnull (NSG) mice (6-8 weeks, female), and each mouse was implanted with 1 ⁇ 10 7 ovarian cancer cells SKOV3-Luc. .
  • NSG non-obese diabetic/severe combined immunodeficiency/IL-2R ⁇ cnull mice (6-8 weeks, female), and each mouse was implanted with 1 ⁇ 10 7 ovarian cancer cells SKOV3-Luc. .
  • Seven days after tumor implantation tumor growth was observed on the IVIS Spectrum imaging platform using in vivo bioimaging (BLI), and tumor growth was photographed using an imager (available from PerkinElmer, USA).
  • mice with similar BLI intensity were randomly divided into 2 groups: phosphate buffered saline (PBS) group, and NKG2D-CAR NK.
  • PBS phosphate buffered saline
  • NKG2D-CAR NK cell group 5 mice per group.
  • NKG2D-CAR NK cell group NKG2D-CAR-modified NK cells prepared by the method shown in Preparation Example 3 were intraperitoneally injected with 1 ⁇ 10 7 cells per mouse, and each mouse in the PBS group was intraperitoneally injected with a dose of 100 ⁇ L. PBS.
  • the cell injection protocol was: two to five injections per week for a total of three weeks (ie, six injections per group).
  • mice were closely observed and the development of the tumors was recorded by BLI. All light signals and images were analyzed by Xenogen in vivo imaging software v2.5. As shown in Figure 11, on the 28th day after tumor implantation, tumor growth showed that the tumor growth of mice in the PBS group was rapid, the light signal intensity increased about 10 times that of the 7th day, and all 5 of the NKG2D-CAR NK cell group. Tumor growth in mice was not only inhibited, but the original tumors were eliminated and the light signal intensity decreased to about 10% on day 7. Thus, NKG2D-CAR-modified NK cells can effectively kill tumors in vivo.
  • Test Example 3 Comparison of different chimeric antigen receptors
  • the NK cells modified with the chimeric antigen receptor NKG2D-CD8-DAP12 and the NK cells modified with the chimeric antigen receptor NKG2D-CD8-CD3Z were tested on tumor cells to compare the killing viability.
  • NK cells transfected with NKG2D-CD8-DAP12 mRNA, NKG2D-CD8-CD3z mRNA, or mGFP-CD8-DAP12 mRNA were co-cultured with human colorectal cancer cell line HCT116, human ovarian cancer cell line SKOV3, and human head and neck cancer cell line Detroit, respectively.
  • HCT116 human colorectal cancer cell line
  • SKOV3 human ovarian cancer cell line SKOV3
  • human head and neck cancer cell line Detroit respectively.
  • the ratio of the above NK cells to the target cells ranges from 2.5:1 to 10:1.
  • Each set of experiments was repeated 3 times.
  • the ability of NKG2D-CAR NK cells to lyse tumor cells was examined using the DELFIA EuTDA Cytotoxicity Kit (available from PerkinElmer, USA). The killing effect was calculated using the following formula:
  • % specific lysis ((experimental group release (reading) - blank group release (reading)) / (maximum release (reading) - blank group release (reading))) x 100
  • NKG2D-CD8-DAP12 mRNA-modified NK cells had significantly stronger tumoricidal activity than NKG2D-CD8- in the tumoricidal experiments of human colorectal cancer cell HCT116, human ovarian cancer cell SKOV3, and human head and neck cancer cell Detroit.
  • CD3z mRNA modified NK cells The chimeric antigen receptor NKG2D-CD8-DAP12 having the specific constituent unit combination created by the present invention has a remarkable therapeutic effect and a good commercialization prospect.
  • NKG2D-CD8-CD3z modified NK cells were prepared in the same manner as NKG2D-CD8-DAP12 modified NK cells except that the intracellular signal domain was constructed using CD3z (the full-length amino acid sequence of Genbank number: NP_932170). .1) The intracellular signal sequence (shown as SEQ ID NO: 23).
  • Test Example 4 Pretreatment of tumor cells with H101 enhances the in vitro killing ability of NKG2D-CAR NK cells to tumor cells
  • NKG2D ligand on the surface of tumor cells In order to detect the effect of H101 oncolytic adenovirus treatment on the expression of NKG2D ligand on the surface of tumor cells, the expression of NKG2D ligand on human head and neck cancer cell Fadu and human hepatoma cell HepG2 was detected by flow cytometry.
  • the serum-free medium of tumor cells (Fluu medium was purchased from Gibco), HepG2 was cultured and DMEM (purchased from Gibco) was used to dilute H101 virus, Fadu cells were infected at 17 MOI, HepG2 was infected at 34 MOI, and infection was 6 After an hour, discard the virus-containing medium and replace it with standard medium for tumor cells (Fadu medium is RPMI (purchased from Gibco) + 10% FBS (purchased from Sigma), culture of HepG2 and DMEM (purchased from Gibco) + 10% FBS (purchased from Sigma)). Tumor cells without H101 treatment were used as negative controls (the same exchange treatment was performed at the corresponding time points).
  • the culture was continued for 18 hours, and the harvested cells were digested with VioBright FITC-conjugated anti-hMICA/B antibody (Miltenyi), PE-conjugated anti-hULBP1 antibody, APC-conjugated anti-hULBP2/5/6 antibody, and APC-conjugated antibody.
  • hULBP3 antibody, APC-conjugated anti-hULBP4 antibody R&D Systems
  • the expression of NKG2D ligand was detected by flow cytometry (purchased from Novocyte), and each experiment was repeated 3 times. Take the average for statistical analysis.
  • Fadu cell assay shows that compared to the negative control (i.e., the legend is shown as "Fadu"), the H101-infected Fadu cell group (i.e., the legend shown as "H101-treated Fadu”) has a tumor cell surface.
  • NKG2D ligand MICA/B was significantly increased (% of cells expressing MICA/B in the negative control "Fadu” group: 39.1%, percentage of cells expressing MICA/B in the "H101-treated Fadu” group: 50.81%), and ULBP1 was decreased (% of cells expressing ULBP1 in the negative control "Fadu”: 33.4%, percentage of cells expressing ULBP1 in the "H101-treated Fadu” group: 27.11%).
  • the HepG2 cell assay results are shown in Figure 14, which shows that H101 infected HepG2 cells (i.e., the legend shows "H101-treated HepG2") on the surface of tumor cells compared to the negative control (i.e., the legend shows "HepG2").
  • the NKG2D ligand MICA/B was significantly increased (% of cells expressing MICA/B in the negative control "HepG2" group: 8.3%, percentage of cells expressing MICA/B in the "H101-treated HepG2" group: 45.37%).
  • NK cells transfected with oncolytic adenovirus H101 at the corresponding time points but without NKG2D-CD8-DAP12 mRNA were transfected as "w/o CAR-NK+H101"group;
  • a group of NK cells that were neither treated with oncolytic adenovirus H101 nor transfected with NKG2D-CD8-DAP12 mRNA were used as the "w/o CAR-NK” group, which was a negative control group.
  • Each group was subjected to the corresponding liquid exchange operation at the corresponding time; each group of experiments was repeated three times, and the average value was taken for statistical analysis.
  • NKG2D-CAR-modified NK cells had strong tumoricidal activity against Fadu, as shown in Figure 15A (p ⁇ 0.001).
  • NKG2D-CAR-modified NK cells still had strong tumoricidal activity against Fadu, see Figure 15B (p ⁇ 0.001).
  • treatment of Fadu cells with H101 oncolytic adenovirus significantly increased the tumoricidal activity of NKG2D-CAR-modified NK cells against Fadu, as shown in Fig. 15C (Figs. 15A and 15B are shown in Fig. 15C in the same figure). .

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Abstract

L'invention concerne un agent thérapeutique comprenant un virus oncolytique et des cellules tueuses naturelles-récepteur antigénique chimérique, une utilisation, un kit et une méthode de traitement de tumeurs et/ou de cancers. L'agent thérapeutique comprend une première composition pharmaceutique, la première composition pharmaceutique comprenant un virus oncolytique situé dans un premier excipient pharmaceutiquement acceptable ; et une seconde composition pharmaceutique, la seconde composition pharmaceutique comprenant des cellules tueuses naturelles situées dans un second excipient pharmaceutiquement acceptable. Le virus oncolytique peut être répliqué sélectivement dans les cellules tumorales. La surface des cellules tueuses naturelles est modifiée par un récepteur antigénique chimérique, et le récepteur antigénique chimérique comprend un domaine de liaison à l'antigène, une région d'espaceur, une région transmembranaire et un domaine intracellulaire qui sont liés fonctionnellement et dans l'ordre en tandem, le domaine de liaison à l'antigène étant à partir d'une région de liaison à un ligand de NKG2D, le domaine intracellulaire étant à partir d'une région signal intracellulaire de DAP12.
PCT/CN2018/094003 2017-10-27 2018-07-02 Agent thérapeutique comprenant un virus oncolytique et des cellules tueuses naturelles-récepteur antigénique chimérique, utilisation, kit, et méthode de traitement d'une tumeur et/ou d'un cancer WO2019080537A1 (fr)

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