Characterization of CAR T Cells Manufactured Using Genetically Engineered Artificial Antigen Presenting Cells

Objective: Chimeric antigen receptor (CAR) T cell therapy has recently emerged as a promising approach for thetreatment of different types of cancer. Improving CAR T cell manufacturing in terms of costs and product quality is animportant concern for expanding the accessibility of this therapy. One proposed strategy for improving T cell expansionis to use genetically engineered artificial antigen presenting cells (aAPC) expressing a membrane-bound anti-CD3 forT cell activation. The aim of this study was to characterize CAR T cells generated using this aAPC-mediated approachin terms of expansion efficiency, immunophenotype, and cytotoxicity.Materials and Methods: In this experimental study, we generated an aAPC line by engineering K562 cells to expressa membrane-bound anti-CD3 (mOKT3). T cell activation was performed by co-culturing PBMCs with either mitomycinC-treated aAPCs or surface-immobilized anti-CD3 and anti-CD28 antibodies. Untransduced and CD19-CARtransducedT cells were characterized in terms of expansion, activation markers, interferon gamma (IFN-γ) secretion,CD4/CD8 ratio, memory phenotype, and exhaustion markers. Cytotoxicity of CD19-CAR T cells generated by aAPCsand antibodies were also investigated using a bioluminescence-based co-culture assay.Results: Our findings showed that the engineered aAPC line has the potential to expand CAR T cells similar to thatusing the antibody-based method. Although activation with aAPCs leads to a higher ratio of CD8+ and effector memoryT cells in the final product, we did not observe a significant difference in IFN-γ secretion, cytotoxic activity or exhaustionbetween CAR T cells generated with aAPC or antibodies.Conclusion: Our results show that despite the differences in the immunophenotypes of aAPC and antibody-based CAR Tcells, both methods can be used to manufacture potent CAR T cells. These findings are instrumental for the improvementof the CAR T cell manufacturing process and future applications of aAPC-mediated expansion of CAR T cells.