Abstract 4419: Use of luciferase-labeled target cells to explore immune cell killing in high throughput format in 2D and 3D co-cultures
Authors: Castro Carla , Metzger Philipp , Feger Daniel , Ulrich Sarah , Siedentopf Oliver , Ehlert Jan E. , Weber Holger 
Published: April 2023

Abstract
With the development and approval of cutting-edge immune-based approaches for cancer treatment in recent years, the global immune-oncological (IO) pipelines have dramatically increased. To overcome the challenges of rising treatment resistance, new strategies are being developed pointing towards enhancing and sustaining T cell-mediated cytotoxicity, exploiting NK, monocytes and γδT cell killing capacity, and combinatorial therapies, among others.
Examples of such strategies are the bispecific antibodies, which are mainly T-cell (BiTE) engagers, but also NK-cell engagers. In addition to the FDA approved immune checkpoint inhibitors, monoclonal antibodies against tumor antigens are being developed to enhance the cytotoxic response of NK and macrophages/monocytes through antibody-induced cell cytotoxicity (ADCC)
To facilitate the efficacy analysis of BiTEs, we developed a 384-well format assay system based on luciferase-labeled tumor cells (advantages: high-throughput capability, non-radioactive, non-toxic, relatively low cost, and easily adaptable to automation). As only the tumor cells are labeled, the specific detection of luciferase exclusively correlates to the number of viable tumor cells, allowing for co-cultures with high excess of effector cells without the effector cells causing interference. Due to high sensitivity, few tumor cells are required for a sufficient signal/noise ratio, keeping the overall need for primary effector cells to a minimum, even at high effector/target cell ratios. This is of great advantage when working with rare cytotoxic subpopulations (e.g. NK cells), precious samples from patients, or simply saves resources when performing large exploratory studies. In addition, combination of low 384-well format volumes with nanodrop agent dispensing technology minimizes the amounts of expensive antibodies and other agents.
Applying this technology, we evaluated the combinatorial effects of Blinatumomab with other clinically relevant agents on the luciferase-labeled GCB-like DLBCL cell line OCI-LY1. Depending on compound combination, we observe synergistic (e.g. Lenalidomide) but also antagonistic effects (e.g. MEK-inhibitor, Selumetinib, and MCL-1-inhibitor, S63845). We also developed CD3-stimulated T cell killer assays either using target cells grown in monolayer or spheroids, with the intent to study checkpoint inhibitors or other T cell activation modulators. In contrast to target cells grown in monolayers, immune cytotoxicity of spheroids by anti-CD3 stimulated T cells was incomplete, showing the additional challenge for effector cells when using 3D cultures as targets. Moreover, we show an ADCC example using NK as cytotoxic cells. Our data supports the outstanding usefulness of this methodological approach for the exploration of the overall efficiency of a therapy to enhance tumor cell killing by immune effector cells.
Citation Format: Carla Castro, Philipp Metzger, Daniel Feger, Sarah Ulrich, Oliver Siedentopf, Jan E. Ehlert, Holger Weber. Use of luciferase-labeled target cells to explore immune cell killing in high throughput format in 2D and 3D co-cultures. [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 4419.