Assessment of cytotoxicity of human CAR-NK cells against various canine tumor cell lines.

Abstract

The need for safer, more effective, and off-the-shelf immunotherapies has led to the development of induced pluripotent stem cells (iPSC)-derived natural killer (NK) cells. Previously, we successfully developed human universal potent chimeric antigen receptor-iPSC-derived NK cells (upCAR-NK cells). This study aimed to demonstrate that human upCAR-NK cells exhibit strong cytotoxicity against canine tumor cell lines, including those derived from hematopoietic and solid tumors. Human leukocyte antigen class I and II, programmed cell death protein 1, cytotoxic T lymphocyte-associated protein 4, and T-cell receptor genes were knocked out in iPSCs. Simultaneously, a human cluster of differentiation 19 (CD19)-targeting CAR and CD24 were overexpressed before differentiation into NK cells. To assess the feasibility of xenogeneic application, upCAR-NK and wild-type iPSC-derived NK (WT-NK) cells were co-cultured with canine tumor cell lines, including a canine B-cell leukemia cell line (GL-1), canine osteosarcoma cell line (D17), canine primary mammary tumor cell line (CIPp), and canine metastatic mammary tumor cell line (CIPm), at different effector-to-target ratios, and cytotoxicity was assessed using a lactate dehydrogenase assay. The results showed that upCAR-NK cells exhibited enhanced cytotoxicity against both hematopoietic and solid tumors compared with WT-NK cells. Moreover, upCAR-NK cells exhibited higher cytotoxicity against GL-1 cells than against the other tumor cell lines, whereas WT-NK cells showed no significant difference in cytotoxicity among the tumor cell lines. These findings suggest that a CAR targeting human CD19 may also recognize canine CD19. In conclusion, the future application of upCAR-NK cells for the treatment of canine hematopoietic and solid tumors appears feasible.