Construction Of Novel HER2 Chimeric Antigen Receptors And In Vitro Cytotoxicity Of CAR-NK Cells

HER2 protein belongs to epidermal growth factor receptor family,which leads to activation of downsteam signal transduction pathways and gene expression upon interacting with epidermal growth factor to form homodimer.HER2 is highly expressed on the surface of various malignant tumor cells such as breast cancer.HER2 positive breast cancer is highly malignant and has a poor prognosis.Therefore,HER2 is considered as an important target for anti-tumor therapy.Affibody is a novel scaffold protein that has been artificially screened and modified.Affibody is called‘artificial antibody'because of its binding to antigens which is similar to antigen-antibody binding.Affibody has low molecular weight,strong specificity,high affinity,so it is often used as a molecular probe for bioimaging or as a carrier for therapeutic drugs for cancer.In the few decades,tumor immunotherapy has been attracted more and more attention in tumor therapy field.Among them,as a new immunotherapy method,chimeric antigen receptor?CAR?-modified NK cells?CAR-NK?technology has initially begun commercial application.The innovation of this article was to construct a plasmid(Z_HER2-28?-pLVX-Puro)using Z_HER2,an affibody which specifically recognizes HER2,as a chimeric antigen receptor extracellular domain.To our knowledge,this is the first paper that affibody was used as the CAR extracellular domain.In addition,in this study we also constructed a second-generation CAR-modified NK-92MI cell stabilizing cell line with anti-HER2 humanized scFv as the extracellular domain.Firstly,we constructed three recombinant anti-HER2 lentiviral expression plasmids in vitro.Secondly,we used 293TX cells to package lentivirus particles and determined viral titer,and then concentrated lentivirus particles using an ultrafiltration centrifuge tubes or ultracentrifugation.After performing transfection of 293TX cells and screening of puromycin,Western Blot proved the expression of three kinds of CAR proteins in protein level,while in cell level,fluorescence imaging and immunofluorescence mirrored the expression of CAR strutures.In addition,we transfected NK-92MI cell line with anti-HER2 scFv-28?CAR lentivirus particles and screened CAR-NK cells with puromycin and following fluorescence imaging evaluated the the expression of CAR strutures.Finally,LDH release assay was used to evaluate its specific cytotoxicity against HER2 high-expression breast cancer cell lines,providing experimental basis for anti-HER2 CAR-NK cells treatments of breast cancer.