Specific Cytotoxicity Of NK Cells Against Human Hepatocellular Carcinoma And Construction Of CAR-GPC3 Expression Vector

At present,the incidence and mortality of hepatocellular carcinoma(HCC)remain high.Traditional treatment methods can not significantly prolong the survival of HCC patients,coupled with a high recurrence rate of HCC,so it is urgently needed to find new treatments for HCC.Tumor immunotherapy clearing tumors by restoring anti-tumor immune functions of the body,including immune checkpoint inhibitors,therapeutic antibodies,tumor vaccines,adoptive cell therapy,and so on.In recent years,immunotherapy has shown strong anti-tumor activity in the treatment of various solid tumors,such as melanoma treated by BCG Vaccine or interferon,non-small cell lung cancer by anti-PD1/PD-L1 antibodies and kidney cancer by IL2.Multiple immunotherapy drugs have been approved by food and drug administration(FDA)for clinical application.Immunotherapy has opened up a new direction for cancer treatment and has a broad prospect.At present,there have been many immune cells used for tumors'clinical studies,including natural killer cells(NK),dendritic cells(DC),and cytokine induced killer cells(cytokine induced killer cells(CIK),T cells and so on.NK are a group of lymphocytes that have cytotoxicity against multiple tumor cells.Their ability to recognize and kill tumors mainly depends on the cross-regulation of activating and inhibitory receptors.The recognition of target antigens by NK cells is not restricted to the patient's HLA phenotype,thus it can be used to treat different HLA patients and kill different tumors carrying the same antigen while making it possible to make NK cell banks.With the rise of chimeric antigen receptor T cells(CAR-T),the construction of CAR modified NK cells has also received social attention.Based on the biological characteristics of NK cells,CAR-NK cells have some innovations and advantages:they have higher cytotoxicity,broader-spectrum cytotoxicity against different tumor cells,economical,can be made into NK bank,and without cause factor storms.The inability to proliferate in vivo shows better safety.CAR-NK cells targeting hematologic tumors and solid tumor antigens have achieved significant effects in animal models and vitro experiments.The cytotoxicity of primary CAR-NK cells targeting CD19 antigen against hematological malignancies has entered preclinical studies,and studies targeting GD2 antigens against breast cancer stem cells are underway.CAR-NK-92cells successfully targeted CD138,CS1 or other antigens to kill multiple myeloma.However,current CAR technology remains a huge challenge in the solid tumors'studies.In addition to complex microenvironment,lacking specific antigens and expressing inhibitory ligands are the main reasons.In response to these problems,we intend to use GPC3 protein as a specific antigen targeting HCC,and use NK-92 cells lacking inhibitory receptors as effector cells to avoid the interference of inhibition ligands expressed by HCC.In methods,we verified the cytotoxicity of NK-92 cells against HCC,constructed CAR-GPC3 vector targeting HCC.We hope to construct CAR-NK cells targeting GPC3 to achieve the objection of enhancing cytotoxicity against HCC and improve the treatment to HCC.This study includes three chapters:Chapter 1:Establishment and optimization of induced amplified methods for NK cells from different sourcesEstablish differentiated and expanded methods inducing from cord blood mononuclear cells and peripheral blood mononuclear cells into NK cells and obtain NK cells from different sources in vitro.NK cells cluster growth and singal cell is bright and round under an optical inverted microscope.After umbilical cord blood derived mononuclear cells were induced for 17 days,the proportion of typical NK cells of CD3~-/CD56~+can reach 65.9%;peripheral blood mononuclear cells-derived NK cells can reach 32.2%after 14 days induction.Methods for culturing NK-92 cells were established and optimized.99.4%cells had CD3~-/CD56~+phenotypic characteristics.This chapter provides sufficient cells sources to clarify the cytotoxicity of NK cells against HCC.Chapter 2:Cytotoxicity of natural killer cells against Hepatocellular Carcinoma.Since NK-92 cells are immortalized cell lines and lack of inhibitory receptors,it has become preferred cell for establishing cytotoxicity assays.The experimental results show that it has a good cytotoxicity against HCC both in vitro and in vivo.Enzyme linked Immunosorbent assay(ELISA)found that NK-92 cells release more IFN-?when co-cultured with five kinds of hepatoma cells.The activity of lactate dehydrogenase cytotoxicity assay(LDH)in the supernatant of hepatoma cells co-cultured with NK cells was increased.When the effector targetor ratio was 20:1,cytotoxicity to Huh7.5.1,Hep3B,Hep G2,SK-Hep1,and SMMC7721 could reach20%.These results demonstrate the cytotoxicity of NK-92 cells against HCC in vitro.Subsequently,subcutaneous(sc)xenografts HepG2 and SMMC7721 models of HCC was established.when HepG2 cells tumor-bearing mice were euthanized,transplanted tumor volumes of NK-92 cell treatment group and PBS control group were 180mm~3,320mm~3 respectively and transplanted tumor weight were 0.223;0.145 respectively.Transplanted tumor volume were reduced and weight loss.In the euthanasia of SMMC7721 tumor-bearing mice,the volumes of transplanted tumor in the two groups were 440mm~3;780mm~3,respectively and the weight were 0.3163;0.418,respectively.The volume and weight was reduced.The results verify the cytotoxicity of NK-92 cells against HCC in vivo,providing a theoretical basis for further verification cytotoxicity of CAR-NK cells.Chapter 3:Construction and preliminary validation of specific CAR vectors targeting Hepatocellular Carcinoma.The key to constructing CAR-NK cells specifically killing HCC is the selection of HCC target.The immunohistochemistry and immunofluorescence results of tumor-bearing mice with HCC showed that the proportion of GPC3~+HCC decreased after NK-92 cells treatment,suggesting that GPC3 may be a potential target for HCC.Western blot and flow experiments showed that GPC3 was high expressed on HCC.Combining relevant literature reports,we finally selected GPC3 as target antigen for HCC,then analyzed and determined two kinds of protein sequence in CAR scFv region.On this basis,we successfully constructed third generation CAR-GPC3lentiviral vector and verified the expression of CAR genes in 293T cells from RNA level and protein level.PCR experiments used CAR gene as a primer and specific bands were observed in 293T cells transfected with CAR gene by electrophoresis.Western blot experiments showed that specific bands were seen in the same cells using the intracellular CD3?of CAR as target protein.The next step is to construct CAR-NK-92 cells specificly killing HCC.In this study,NK cells were induced and expanded from different sources in vitro.The cytotoxicity of NK-92 cells to HCC was verified by a series of experiments in vitro and animal models.It is pointed out that NK cells are expected to be an effective treatment for HCC and provide new strategies for the clinical treatment of HCC.The successful construction of CAR-GPC3 lentivirus expression vector targeting HCC lays the foundation for the construction of CAR-NK cells in the next work.This strategy has important theoretical significance in tumor immunotherapy and broad application prospects in the field of individualized regenerative medicine.The researchers hope that CAR-NK cells can overcome the bottleneck of the current CAR technology in solid tumors.The development of CAR technology depends on the transformation of NK cells and the depth study of CAR structure.Only in this way can CAR-NK cells go further on the anti-tumor path.In the next work,we will continue to construct NK cells expressing CAR-GPC3.Based on its enhanced cytotoxicity against HCC,specific Immunotherapy products for HCC will be made,and this strategy is expected to improve the therapeutic level of HCC.