Development of a liquid tumor potency assay for evaluation of immune cell-mediated cytotoxicity in vitro

Abstract

Abstract The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune (effector) cells and cancer (target) cells—to evaluate the function, specificity, and sensitivity of a product. A variety of in vitro assays are used to characterize the proliferation, cytokine release, and cell-mediated cytotoxicity of engineered immune cells, such as chimeric antigen receptor (CAR) T cells, with each representing an important aspect driving clinical efficacy of the cellular product. Previous work has shown that impedance-based potency assays provide a real-time, label-free measure of effector cell-mediated cytotoxicity for adherent target cells. Here, we describe an in vitro potency assay that quantifies effector cell-mediated cytotoxicity of liquid tumor target cells. First, the wells were coated with antibody to tether the liquid tumor target cells (Raji = anti-CD40, K562 = anti-CD71, Daudi = anti-CD40). Tethering the liquid tumor target cells significantly increased the impedance measurement, as compared to untethered controls. CD19-specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells across a range of effector to target ratios. Cytolysis of the target cells was calculated by comparing the impedance in treated wells to no treatment control wells. As expected, the CD19-specific CAR T cells produced significantly higher cytolysis in the Daudi (54.3%) and Raji (70.4%) cells, which express CD19, as compared to the K562 cells (30.5%). These results support a real-time, label-free potency assay with liquid tumor target cells.