Optimization of canine CD20 chimeric antigen receptor T cell manufacturing and in vitro cytotoxic activity against B‐cell lymphoma

Abstract Canine B‐cell lymphoma is one of the most common haematopoietic neoplasms in veterinary medicine, and it is considered a relevant model for human diffuse large B‐cell lymphoma. Although the standard treatment consisting of multi‐drug chemotherapy is effective in most cases, treatment is often challenging because of relapse and drug resistance. The adoptive transfer of autologous T cells genetically modified to express a CD19‐specific chimeric antigen receptor (CD19 CAR‐T cells) has been shown to be highly effective in human B‐cell malignancies. However, there is no clinically available canine CAR‐T cell therapy. We generated canine second‐generation and third‐generation CAR‐T cells by retroviral gene transduction. Optimization was performed to investigate effective viral transduction protocols and favourable culture conditions for canine CAR‐T cells. The RetroNectin‐bound virus infection method resulted in more than 70% transduction efficiency. The effect of culture conditions on the phenotype of CAR‐T cells was evaluated by the expression of surface markers. in vitro cytotoxicity assays of target cells cultured with CD20 CAR‐transduced cells demonstrated that CD20 CAR‐T cells exhibit cytotoxicity against CD20‐expressing canine B‐cell lymphoma cells and canine CD20‐transduced murine cells, whereas no effect was observed against cells that lacked canine CD20 expression. Our study established virus‐based canine CAR‐T cell generation, providing fundamental data for a better understanding of canine adoptive T‐cell therapy.