Development of optimized cytotoxicity assays for assessing the antitumor potential of CAR-T cells

CAR-T cells are T cells expressing a chimeric antigen receptor (CAR) rendering them capable of killing tumor cells after recognition of a target antigen. CD19 CAR-T cells have revolutionized the treatment of hematological malignancies. Their function is typically assessed by cytotoxicity assays using human allogeneic cell lines expressing the target antigen CD19 such as Nalm-6. However, an alloreactive reaction is observed with these cells, leading to a CD19-independent killing. To address this issue, we developed a fluorescence microscopy-based potency assay using murine target cells to provide an optimized cytotoxicity assay with enhanced specificity towards CD19. Murine NIH/3T3 (3T3) fibroblast-derived cell line and EL4 T-cell lymphoma-derived cell line were used as targets (no xenoreactivity was observed after coculture with human T cells). 3T3 and EL4 cells were engineered to express eGFP (enhanced Green Fluorescent Protein) and CD19 or CD22 using retroviral vectors. CD19 CAR-T cells and non-transduced (NT) control T cells were produced from several donors. After 4 h or 24 h, alloreactive cytotoxicity against CD19⁺ Nalm-6-GFP cells and CD19^- Jurkat-GFP cells was observed with NT or CAR-T cells. In the same conditions, CAR-T but not NT cells specifically killed CD19⁺ but not CD19^- 3T3-GFP or EL4-GFP cells. Both microscope- and flow cytometry-based assays revealed as sensitive as impedance-based assay. Using flow cytometry, we could further determine that CAR-T cells had mostly a stem cell-like memory phenotype after contact with EL4 target cells. Therefore, CD19⁺ 3T3-GFP or EL4-GFP cells and fluorescence microscopy- or flow cytometry-based assays provide convenient, sensitive and specific tools to evaluate CAR-T cell function with no alloreactivity.