The Construction of ROR1 Targeting Chimeric Antigen Receptor Modified T Cells and Its Killing Effect for ROR1-positive Tumor Cells

ObjectiveTo test the killing effect of type Ⅰ receptor tyrosine kinase-like orphan receptor（ROR1） chimeric antigen receptor T cell （CAR-T） on several ROR1-expressing tumor cells in vitro. MethodsThe CAR gene was designed and synthesized by constructing the lentiviral vector plasmid,and BamHⅠ/EcoRⅠ was used to identify the plasmid. The expression levels of ROR1 among a variety of tumor cell lines were compared using flow cytometry (FCM). The killing effect of CAR-T on positive cells was detected by FCM,the LDH assay and ELISA. ResultsThe double enzyme digestion identified CAR gene was successfully constructed to the lentivirus vector plasmid. FCM detection showed that the efficiency of CAR-T infection was about 47.23%. Multiple tumor cells expressed ROR1 in varying degrees. The FCM and the LDH assay indicated that CAR-T specifically killed ROR1-positive tumor cells. On positive target cells,more interferonI-γ (FN-γ) could be released during the CAR-T killing process than control T (P<0.05). ConclusionWe successfully constructed ROR1 CAR-T. CAR-T can specifically kill ROR1-positive tumor cells and cause the release of large amounts of IFN-γ,providing an experimental basis for clinical application.