Abstract A28: Establishment of an ex vivo tissue culture platform as a preclinical model to assess the mechanism of action of Bicycle® tumor-targeted immune cell agonists in NSCLC
Authors: Shah Chinmayee , Campbell Carly , Cohen Heather , Hurov Kristen , Brandish Phil , Keen Nicolas , Bueno Raphael , Innocenti Simona , Barlow Julianne , Gustafson Corinne 
Published: December 2022

Abstract
The translation of drug discovery to bedside for cancer therapy is low with only 5% of anti-cancer compounds getting past phase III clinical trials. Since the tumor microenvironment is complex, there is a need for a relevant preclinical model to bridge the gap between tumor behavior and clinical therapeutic response for novel therapies. We have developed a novel class of modular synthetic drugs, termed Bicycle® tumor-targeted immune cell agonists (Bicycle® TICAs), which are multifunctional molecules comprised of constrained bicyclic peptides [1]. BT7480, a Nectin-4-dependent CD137 agonist, is the first molecule of this class and entered clinical evaluation in 2021. Preclinical data in syngeneic mouse models showed that BT7480 led to a profound reprogramming of the tumor immune microenvironment including early myeloid cell activation that preceded T cell infiltration and upregulation of cytotoxicity-related genes [2]. We are also developing CD137 Bicycle® TICAs that are targeted to the EphA2 receptor. The tumor antigens Nectin-4 and EphA2 have both been reported to be overexpressed in lung cancer [3-4] and our data using immunohistochemistry (IHC) of lung tumor microarrays (TMAs) supports these findings [5-6]. To investigate the mechanism of action of Bicycle® TICAs in a human model system, here we describe the development and optimization of an ex vivo organotypic histoculture model that uses freshly resected human non-small cell lung cancer (NSCLC) tumor tissue and preserves the tumor microenvironment and heterogeneity. NSCLC was chosen as the tumor type to study based on evidence of co-expression and co-localization of Nectin-4 and CD137, the targets of BT7480, in TMAs using spatial proteomic profiling. We found that NSCLC tumor explants maintain tissue integrity and tumor cell viability up to 72 hours in culture. The baseline tumor tissue was also phenotypically characterized for tumor and immune cell content using flow cytometry and IHC by monitoring the tumor cell marker PanCK, the tumor antigens of interest Nectin-4 and EphA2, and several immune cell markers, including CD45, CD3, CD8, and CD137. Importantly, we found that the tumor explants were infiltrated with viable immune cells in a subpopulation that expressed CD137. We were also able to elucidate a T-cell specific cytokine response within 48 hours by subjecting the explants to anti-CD3 and anti-CD28 stimulation, which supports the proposition that our histoculture platform contains live and functional tumor infiltrating lymphocytes (TILs). Overall, we demonstrate a rapid and reproducible ex vivo histoculture platform that is potentially poised to evaluate functional proof-of-concept of Bicycle TICA™ molecules in a human model system. References: [1] Upadhyaya P, et al., JITC 2021;9:e001762 [2] Hurov K, et al., JITC 2021;9:e002883 [3] Challita-Eid PM, et al., Cancer Res 2016;76:3003 [4] Kinch MS, et al., Clin Cancer Res 2003;9:613 [5] Campbell C, et al., Cancer Res 2020;80:5300 [6] Campbell C, et al., Cancer Res 2021;81:1197
Citation Format: Chinmayee Shah, Carly Campbell, Heather Cohen, Kristen Hurov, Phil Brandish, Nicolas Keen, Raphael Bueno, Simona Innocenti, Julianne Barlow, Corinne Gustafson. Establishment of an ex vivo tissue culture platform as a preclinical model to assess the mechanism of action of Bicycle® tumor-targeted immune cell agonists in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A28.