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

ABSTRACT

OBJECTIVE: To 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. METHODS: The 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. RESULTS: The 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). CONCLUSION: We 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.