Abstract 906: Development of a repeated challenge potency assay for evaluation of immune cell-mediated cytotoxicity in vitro
Authors: Sullivan Denise , Chvatal Stacie , Millard Daniel 
Published: April 2023

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, each representing an important aspect driving clinical efficacy of the cellular product. Importantly, adoptive cell therapies must exhibit potent tumor killing over a prolonged period in vivo. Here, we describe an in vitro potency assay that quantifies immune cell-mediated cytotoxicity in response to repeated challenges with target cells. The repeated challenge assay was performed with two separate protocols, each providing a unique assessment of the effector cell function. In the first method, SKOV3 target cells (5k cells/well) were seeded into a multiwell microplate with embedded electrodes in the substrate. Electrical impedance measurements from the electrodes in each well detected the attachment and proliferation of the target cells. HER-2 specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells at a 1:1 effector to target ratio. Cytolysis of the target cells was calculated by comparing treated wells to no treatment control wells. Additional SKOV3 target cells (5k per well) were added every 24 hours for the repeated challenge conditions, with total tumor cell killing computed from the area under the impedance curve. The repeated challenge condition reached complete killing after the 6th tumor cell challenge (144 hours after first effector addition), whereas the single challenge condition reached complete killing within 96 hours of effector addition. Flow cytometry analysis revealed that the effector cells proliferated significantly more for the repeated challenge condition (4.7 fold increase) than the single challenge condition (1.5 fold increase). In the second method, the CAR T cells were removed after each challenge, counted, and added to a fresh plate of SKOV3 target cells at a 1:1 effector to target cell ratio. Here, the E:T ratio was kept constant for each challenge, allowing a direct comparison of the killing kinetics (KT50) for each subsequent challenge. The KT50 decreased from the first challenge (19.9 hr) to the fourth challenge (83.5 hr). Flow cytometry analysis revealed a reduction in CD8+ cells for the repeated challenge condition, indicating a change in the make-up of the immune cell population. The paradigms described above facilitate a label-free, in vitro workflow to assess potency of immune effector cells across repeated challenges, with the opportunity to incorporate additional assays, like flow cytometry or cytokine release, after each successive challenge.
Citation Format: Denise Sullivan, Stacie Chvatal, Daniel Millard. Development of a repeated challenge potency assay for evaluation of immune cell-mediated cytotoxicity in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 906.