Identification of potent cd19 scfv for car t cells through scfv screening with nk/t‐cell line

Abstract

CD19 is the most promising target for developing chimeric‐antigen receptor (CAR) T cells against B‐cell leukemic cancer. Currently, two CAR‐T‐cell products, Kymriah and Yescarta, are approved for leukemia patients, and various anti‐CD19 CAR T cells are undergoing clinical trial. Most of these anti‐CD19 CAR T cells use FMC63 single‐chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti‐CD19 CAR T cells. We used the KHYG‐1 NK/T‐cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti‐CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti‐ CD19 CAR KHYG‐1 cells. Interestingly, only FMC63 CAR KHYG‐1 and 4G7 CAR KHYG‐1 efficiently lysed CD19‐positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL‐2 when co‐cultured with CD19‐positive cell line, NALM‐6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19‐positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM‐6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B‐cell leukemia patients.