Construction And Identification Of Chimeric Antigen Receptor Modified NK-92 Cells Targeting The Folate Receptor ? In Ovarian Cancer

Background:The mortality of ovarian cancer ranks first in all gynecological malignant tumors and its morbidity is also very high.Locating deeply in the pelvic cavity and lacking of early symptoms,70%patients of ovarian cancer are diagnosed at the advanced stage when they first visit a gynecologist.In recent decades,the treatments of ovarian cancer which include cytoreductive surgery,chemotherapy,radiotherapy and targeted therapy have been largely improved,but the five-year survival rate hasn't been on the rise,still keeping around 30%.So seeking for a sensitive early diagnostic indicator or a therapeutic target will be of great significance.A chimeric antigen receptor(CAR)is a type of genetically engineered receptor.The structure of CARs is typically composed of a single-chain variable fragment(scFv)from an antigen-specific mAb which recognizes and binds tumor-related antigens independent of major histocompatibility complex(MHC)restriction,and an intracellular signaling tail including CD3? immunoreceptor tyrosine-based activation motif domain and/or one/two costimulatory receptor(CD28,4-1BB,or OX40)from lymphocyte antigen-activated receptor.It is reported that the up-regulation of aFR is seen in 90%of epithelial ovarian cancers while in normal tissues its expression is extremely low or nearly zero.In early studies,certain groups constructed the first generation CAR-T cells and demonstrated its targeted cytotoxicity against aFR-positive ovarian cancer cells,but in its clinical trials,the first generation anti-aFR CAR-T cells had failed due to lack of proliferating ability in vivo And recently,many researchers suggested that NK cells might be better CAR drivers because of their relatively limited survival time,multiple cell sources and different cytokine release profiles with T cells.Furthermore,NK-92 is the most studied cell source.At present,there is no publication about CAR-NK cells adoptive therapy in treating ovarian cancer Therefore,our group plan to construct the third generation CAR-NK-92 cells in this experiment and identify its special cytotoxicity against aFR-positive ovarian cancerMethods:1.We constructed the third generation CAR which composed of a single chain variable fragment from anti-aFR monoclonal antibody C4,an IgGl Fc,a CD8a hinge,a CD28 transmembrane,intracellular CD28 and CD137 costimulatory domains and CD3?component and ligated into the lentiviral expression vector pLenti-aFR-CAR,then viral particles were packaged to infect NK-92 cells2.The expression of anti-?FR CAR on NK-92 cells was determined by real-time PCR,Western Blot and flow cytometry3.After coincubation of effector cells and target cells,we used real time cell analysis(RTCA),LDH assay,CD107a staining,and ELISA to monitor the special cytotoxicity of the CAR-NK-92 cells against aFR-positive ovarian cancer cells in vitroResults:1.We successfully designed the third anti-aFR-CAR,and through transformation of competent cells,preparation of plasmids,DNA gel electrophoresis and DNA sequencing,we verified the correctness of the CAR sequence.And then we packaged the corresponding lentiviral particles,infected and selected NK-92 cells,and finally obtained high purity of NK-92-aFR-CAR cells?2.Remarkable inhibition of proliferation of aFR-positive SKOV3 cells was seen by real time cell analysis(RTCA)when co-cultured with NK-92-?FR-CAR cells while no obvious inhibitory effect of ?FR-negative A-431 cells3.The direct killing effect of NK-92-?FR-CAR on SKOV3 cells was determined by LDH release assay4.ELISA assay suggested high level of IFN-?,demonstrating that NK-92-?FR-CAR cells effectively secreted cytokine when co-cultured with SKOV3 cells5.Flow cytometry measured high level of CD 107a,indicating improved degranulation of NK-92-?FR-CAR cells when co-cultured with aFR-positive SKOV3 cellsConclusions:Our results showed that the NK-92-aFR-CAR cells can secret high level of IFN-y,TNF-? and CD 107a and selectively and effectively kill aFR-positive ovarian cancer cells in vitro,which lays a solid foundation for future preclinical and clinical studies on NK-92-aFR-CAR cells.