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

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.