| Background and purpose:Colorectal cancer is a common malignant tumor.In our country,its morbidity and mortality are ranked 5th.Approximately 190,000 people died from colorectal cancer each year.In recent years,minimally invasive surgery combined with accelerated rehabilitation surgery and targeted drugs have made certain progress in the treatment of colorectal cancer,but these treatment methods have not yet met the clinical treatment needs of colorectal cancer.In recent years,immune cell therapy has received widespread attention,and it is expected to become a new breakthrough direction in the treatment of malignant tumors following surgery,radiotherapy,and anti-tumor chemotherapy.In particular,Chimeric Antigen Receptor(CAR)modified immune cell therapy has attracted special attention.At present,the more mature research is CAR-T cell therapy,but it has been found in the clinical treatment of CAR-T cell that there are serious complications such as cytokine release syndrome,off-target effect,neurotoxicity,etc.,which limits its wide use to a certain extent.CAR-NK cell therapy hardly causes the above-mentioned serious complications,and the safety is significantly improved.Moreover,CAR-NK cells do not have allogeneic rejection,and general immune cell therapy products can be prepared.At present,NK cells used in CAR-NK therapy are mainly derived from peripheral blood(PB),umbilical cord blood(UCB)and NK92 cell lines,all of which have their own defects:NK cells derived from peripheral blood and umbilical cord blood are not convenient for long-term storage,and the donor variability is large.The total amount of NK cells is unstable;the NK92 cell line must be inactivated by irradiation before treatment in vivo,but irradiation will greatly reduce the tumor-killing ability of CARNK92 cells.Pluripotent stem cells(Pluripotent stem cells,PSCs)are induced to differentiate in vitro to obtain fully functional NK cells,providing a new source of cells.At the same time,gene editing can be carried out at the seed cell stage,and pluripotent stem cells modified by CAR genes are used to induce differentiation to obtain a large number of Stable NK cell therapy products,which can solve the shortcomings of NK cells derived from peripheral blood and umbilical cord blood and NK92 cell lines.Carcinoembryonic antigen(CEA)is expressed on the surface of most advanced colorectal cancer cells.It is currently the most widely used gastrointestinal tumor marker.CEA can be recognized and specifically bound by CAR-NK cells.It is an ideal target antigen for CAR-NK immune cell therapy against colorectal cancers.is.Therefore,in this project,by establishing a CAR carrier-modified pluripotent stem cell line targeting carcinoembryonic antigen(CEA),and further inducing it to differentiate into NK cells,a universal and "off-the-shelf”CAR-CEA-NK cells for the treatment of colorectal cancer were obtained,which are expected to provide a new and effective clinical treatment for colorectal cancer.Methods:1.Construction of a pluripotent stem cell line modified by C AR-CEA transfection.The Carcinoembryonic antigen(CEA)was selected as the target antigen for the treatment of colorectal cancer,and a CEA-targeted lentiviral expression CAR vector was constructed,which was then transfected into pluripotent stem cells to obtain pluripotent stem cell lines stably expressing CAR genes.2.Large-scale preparation of CAR-NK cells derived from pluripotent stem cells.CAR gene-modified pluripotent stem cells were efficiently induced to obtain hematopoietic stem/progenitor cells by spin EB method in vitro,and then were efficiently induced to CAR-NK cells,co-culture with OP9-DL1 cells to provide an induced microenvironment.3.Establishment of NK cells induction methods from umbilical cord blood and NK92 cell line culture.Methods for inducing NK cells from umbilical cord blood and an optimized culture system for NK92 cell lines were established.NK cells derived from cord blood and NK92 cell lines were used as the research control group.4.Verification of the anti-tumor activity of CAR-CEA-NK cells against colorectal cancer in vitro.Three colorectal cancer cell lines were selected:SW1116(CEA high expression),SW837(CEA moderate expression,and Caco-2(CEA negative).Lactate Dehydrogenase(LDH)Cytotoxicity test and enzyme-linked immunosorbent assay of interferon(IFN-y)release were used to verify the killing function of NK cells.5.Verification of the anti-tumor activity and safety of CAR-CEA-NK cells against colorectal cancer in vivo.The CEA high-expressing cell line SW1116 was used to construct a NOD SCID murine xenograft model to verify the tumor killing function and safety of the pluripotent stem cell-derived CAR-CEA-NK cells.Result1.Using carcinoembryonic antigen(CEA)as the target antigen,construct a CEAtargeted CAR vector and transfect pluripotent stem cells to obtain pluripotent stem cell lines that stably express CAR genes and have good differentiation potential.2.Establish a technical method for inducing differentiation of multiple hESCs into NK cells in vitro and use CAR gene-modified hESC as a technical method for NK cells to prepare seed cells in vitro,and finally obtain the target CAR CEA-NK cells.3.CAR hESC-NK cells are similar to UCB-NK cells and NK92 cells in cell morphology and cell phenotype.4.hESC-NK,UCB-NK and NK92 have certain killing effects against colorectal cancer cell lines in vitro,and there is no statistical difference between the groups,while CAR-CEA hESC-NK cells have significant enhanced killing effects on CEA positive colorectal cancer in vitro,but no such effect for CEA-negative colorectal cancer.5.CAR-CEA hESC-NK cells have a significant killing effect in vivo in xenotransplantation mouse models of colorectal cancer,and their anti-tumor activity is significantly stronger than the Untreated group,hESC-NK treatment group,UCB-NK treatment group and NK92 treatment group.And CAR-CEA hESC-NK cells have good biological safety in vivo.Conclusion1.Choose CEA as the target antigen to construct a CAR-CEA-modified pluripotent stem cell line for colorectal cancer.Pluripotent stem cells can be used as seed cells for CAR-NK research.Carrying out CAR modification on them at the beginning of the research can obtain pluripotent stem cell lines stably expressing CAR genes.2.Establish a technical method for the differentiation of pluripotent stem cells into NK cells.Pluripotent stem cells can be used as an ideal source of NK cells for CARNK research.3.Establish CAR gene modified pluripotent stem cells as NK cells seed cells to prepare and obtain NK cell products for colorectal cancer in vitro.The CAR modified pluripotent stem cell line maintains the potential to induce differentiation into NK cells and can stably express the CAR gene.4.Tumor killing function verification and biosafety evaluation of NK cells derived from CAR-CEA modified pluripotent stem cells in vitro and in vivo.NK cells derived from CAR-CEA modified pluripotent stem cells can be used as an effective treatment against colorectal cancer.In summary,it is finally confirmed that NK cells derived from chimeric antigenmodified pluripotent stem cells have an ideal tumor-killing effect and good safety in the treatment of colorectal cancer,which provides reliable theoretical support for its next clinical application and promotion. |
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