Abstract 1689: Dynamic monitoring of immune response and reagent efficacy through high throughput label-free impedance-based technology
Authors: Cerignoli Fabio , Xi Biao , Guenther Garret Garret , Muir Lincoln , Zhao Leyna , Abassi Yama 
Published: July 2017

Abstract
In vitro characterization of reagents efficacy in the context of cancer immunotherapy is a necessary step before moving to more expensive animal models and clinical studies. However, current in use in vitro assays like Chromium-51, ATP-based luminescence or flow cytometry are either difficult to implement in high throughput environment or are mainly based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we present the adaptation of an impedance-based platform to monitoring cytotoxic activity of immune cells activated trough different means. Impedance technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. We utilized adherent and B cell leukemia/lymphoma cell lines as well as primary tumor cells as in vitro models for immunotherapy reagents evaluation. We seeded the cells on electrodes coated 96-well plates and monitored cell adhesion and proliferation for 24 hours. The following day effector cells were added at multiple effector:target ratios in presence of BiTEs antibodies and/or anti PD-1/PD-L1 antibodies. Impedance signal was monitored for up to seven days. Control wells were set up with effector cells only or with target plus effector cells but without antibodies. We adapted such adhesion-based technology to monitor non-adherent B-leukemia/lymphoma cells, by developing a strategy where the wells are coated with an anti-CD40 antibody. The coating allows specific adhesion and retention of B cells and measurement of changes in impedance that are proportional to cell number. Using increasing concentrations of EpCAM/CD3 BiTE we demonstrated the suitability of such impedance-based approach to quantitatively monitor the efficacy of immune cells-mediated cancer cell killing both under different effector:target ratios and antibody concentrations. Combination treatments with checkpoint reduced timing and increased amount of killed cancer cells. Similar results were also obtained with engineered Car-T cells against CD19 or NK cell lines, we were able to demonstrate specific killing of tumor B cells at very low effector:target ratios. The results were also confirmed by flow cytometry. Overall, our results demonstrate the value of such approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays. Furthermore, the availability of 384-wells format and minimal sample handling place the technology in an ideal spot for applications in large reagent validation screening or personalized medicine, like therapeutic protocol validation directly on patient samples.
Citation Format: Fabio Cerignoli, Biao Xi, Garret Garret Guenther, Lincoln Muir, Leyna Zhao, Yama Abassi. Dynamic monitoring of immune response and reagent efficacy through high throughput label-free impedance-based technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1689. doi:10.1158/1538-7445.AM2017-1689