Generation of primary feline chimeric antigen receptor T cells

Abstract OBJECTIVE The purpose of this study was to develop procedures to engineer feline chimeric antigen receptor (CAR) T cells. METHODS 6 healthy cats were used in this study. Blood was collected, and CD3+ primary T cells were enriched by magnetic activated cell sorting, expanded, and used to generate CAR T cells. RESULTS Phorbol myristate acetate plus ionomycin and concanavalin A induced similar early proliferation of CD3-enriched feline CD4+ and CD8+ T cells but phorbol myristate acetate plus ionomycin induced greater expansion over 12 days. Chimeric antigen receptor T cells were engineered by transduction with an FIV-based lentiviral system to express a human CD19 CAR. Feline CD19 CAR T cells demonstrated specific cytotoxicity against human CD19+ target cells. Conditions were developed to polarize the T cells to THelper subsets. CONCLUSIONS We generated functional and specific primary feline CAR T cells and demonstrated conditions to polarize the cells, which may be therapeutically advantageous for CAR T-cell use in a variety of disease contexts. CLINICAL RELEVANCE CAR T therapy has been used with great success for human hematologic malignancies and is under development for use in canines. Our study is the first demonstration of functional feline CAR T cells and describes the procedures for their engineering. These findings lay the foundation for future development of CAR T therapy for multiple feline diseases.