In Vitro Validation of Cancer Immunotherapy Reagents Using an Impedance‐Based Platform

Abstract

Immunotherapy is emerging as one of the most promising approaches in cancer treatment, allowing specific targeting of cancer cells from immune cells, mediated by surface marker expression. In vitro characterization of the efficacy and potency of cancer immunotherapy reagents is a necessary step before moving to animal models and Phase I and II clinical studies. In this study we report the validation and optimization of protocols for analyzing the efficacy of reagents designed to stimulate the immune response against cancer cells using an impedance‐based and label‐free platform. The platform allows detection of cell adhesion and cell death trough monitoring the change in conductance of microelectrodes embedded in the bottom of 96‐wells cell culture plates. Because cell adhesion alters the conductivity of the electrodes, any change in cell morphology, cell adhesion or cell number is detected as variation of an impedance‐related parameter named Cell index (Ci). The platform avoids the use of any chemical label or sample processing, allowing continuous monitoring of cell activity over prolonged time periods. Furthermore, the untouched cells are still available for secondary assays that can better clarify the biological mechanism involved in the response.To mimic tumors that are target of immunotherapy efforts we utilized several carcinoma cell lines whit different level of expression of the EpCAM membrane protein. Such antigen is overexpressed in many tumors and is currently under evaluation in clinical trials. In PC3 prostate cancer cells we measured cell cytotoxicity of Primary Blood Mononuclear Cells (PBMCs) mediated by an EpCAM/CD3 Bispecific T cell Engager (BiTE) antibody. The use of the impedance‐based platform allowed measuring the full kinetic of the cytotoxic activity under different effector:target ratios and BiTE concentrations. Apoptosis of tumor cells was confirmed trough flow cytometer analysis. We also demonstrated the relation between amount of PBMCs cytotoxic activity and EpCAM surface expression trough testing MCF7 (high EpCAM expressor) and HeLa (low EpCAM expressor).To evaluate cancer immunotherapy protocols in lymphomas and leukemia, we developed a protocol where lymphomas cells are immobilized on electrodes‐coated plates and the T‐Lymphoblast cell line TALL‐104 is used as cytotoxic effector.Taken together our results demonstrate high sensitivity of the impedance‐based approach in measuring cytotoxic activity in sophisticated high throughput set up. Furthermore, the availability of continuous recording of the Cell Index over the temporal scale allows better characterization, comparing to alternative end point assays, of construct efficacy.