Abstract 1782: Functional potency assay predicts CAR-T effectiveness in tumor microenvironment
Authors: Bronevetsky Yelena , Massi Evan , Garcia Candy , Liu Ningchun , Xing Yewei , Czeryba Natalie , Wise Scott , Lim James 
Published: April 2023

Abstract
Chimeric antigen receptor (CAR) T cell therapy holds great promise for the treatment of various cancers, including solid tumors. However, attempts to model the behavior and effectiveness of CAR-T cell therapies for blood cancers and solid tumors have been challenging due to the unique tumor microenvironments in which these cancer cells are found. The tumor microenvironment (TME) is often characterized by hypoxia, increased acidity, and high interstitial fluid pressures, allowing cancer cells to effectively evade immune surveillance. This immunosuppressive TME also contributes to CAR-T cell exhaustion, thereby limiting its antitumor activity and function. To address these concerns, we have developed a proprietary cell-based assay to measure CAR-T cell potency and cytotoxic function in three-dimensional (3D) in vitro cell culture system, human acute B cell lymphoblastic leukemia mouse model, and immunosuppressive tumor microenvironments. Utilizing the AVATAR system, we replicated the oxygen and interstitial fluid pressures found in the vasculature, the bone marrow and solid tumor microenvironments. Tumor cytolysis assays were conducted in these environments to measure cell exhaustion as analyzed by flow cytometry and electrical impedance. In addition, serial tumor challenge assays were performed to examine CAR-T potency and effectiveness in TME. Proof-of-concept experiments were performed using ROR1 CAR-T cells targeting the ovarian adenocarcinoma cell line, SKOV3. CD19 CAR-T were also used targeting the acute lymphoblastic leukemia cell line, NALM6. Defined ratios of effector T cells to tumor cells was assessed to model CAR-T potency in vitro and elevated CD19 CAR-T mediated cytotoxicity was confirmed with the increased ratio of effector T cells to tumor cells in both 2D and 3D culture system. CD19 CAR-T cells also exhibited in vivo dose dependent efficacy against the systemic NALM6-Luc acute lymphoblastic leukemia (ALL) mouse model, quantified by bioluminescence (BLI) image monitoring method. Multiple pressure and oxygen settings were examined to model the cross-section of the bone marrow and solid tumor microenvironments (0 PSI to 5 PSI, 1% to 10% O2). Initial results from these screening experiments show significant decline in ROR1 CAR-T mediated cytotoxicity when performed under TME conditions. However, CD19 CAR-T showed effective cell killing under TME conditions. Interestingly, acclimating and expanding ROR1 CAR-T cells to high pressure and decreased oxygen culture conditions improved potency levels and warrants further investigation. In summary, we observe CAR-T cells comprise the tumor cell killing ability in both in vitro 3D and in vivo animal models. We also describe a physiologically relevant potency assay that incorporates hyperbaric and hypoxic incubation technology to predict the behavior of cell therapies in immunosuppressive tumor microenvironments.
Citation Format: Yelena Bronevetsky, Evan Massi, Candy Garcia, Ningchun Liu, Yewei Xing, Natalie Czeryba, Scott Wise, James Lim. Functional potency assay predicts CAR-T effectiveness in tumor microenvironment [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 1782.