Abstract LB-381: Mitigating on-target off-tumor cytotoxicity of EpCAM CAR-T by affinity tuning

Chimeric antigen receptor (CAR)-T cell therapy has shown robust anti-cancer responses in hematologic malignancies. However, application of this therapeutic approach to solid tumors has been hindered by multiple challenges, one of which is on-target/off-tumor cytotoxicity to normal tissues. Tumor-specific antigens exclusively present on tumor cells are rare. Most CAR-T cells are designed to target tumor associated antigens (TAAs) expressed in high levels on tumor cells. Yet, normal tissues express these antigens as well, albeit at much lower densities.To mitigate the on-target/off-tumor cytotoxicity, we developed a strategy for fine tuning the affinity of CARs to selectively target tumor cells. Epithelial cell adhesion molecule (EpCAM) is highly expressed in epithelial cells and overexpressed in tumor cells in a variety of epithelial carcinomas. High-affinity (nM range) EpCAM-targeting CAR-T cells kill both normal human epithelial cells and EpCAM-high tumor cells in vitro. To develop CAR-T cells specific to EpCAM-high cancers, we identified low-affinity scFv variants by rational design of amino acid substitutions. The affinities of these scFvs were measured by Surface Plasmon Resonance (SPR). CAR-T cells equipped with low-affinity scFvs showed antigen-dependent activation, proliferation and Th1-like cytokine secretion when co-cultured with target cells having varied levels of EpCAM. Importantly, low-affinity CAR-T cells still lysed EpCAM-high tumor cells but spared normal human epithelial cells in vitro. Treatment of MKN28 xenograft mice with low-affinity CAR-T cells resulted in tumor regression and prolonged survival. Moreover, transcriptomic profiling of CAR-T cells revealed significant differences in gene expression levels between high- and low- affinity CARs, particularly as they relate to the functional state of these CAR-T cells, and their resistance to exhaustion. Our results show that rational design of scFv can aid in the identification of CARs that are minimally reactive toward normal tissues while effectively eliminating tumors. Furthermore, affinity-tuned CARs demonstrate better overall fitness and antitumor activity in vivo. This affinity fine-tuning approach shows promise as a general strategy for identifying a therapeutic window for CAR-T cells targeting novel TAAs that may have been overlooked because of basal expression levels in normal tissues. Citation Format: Huan Yang, Janusz Puc, Yanping Yang, Jaclyn E. McCloskey, Yogindra Vedvyas, Hongtao Li, Irene M. Min, Moonsoo M. Jin, Eric v. Hofe. Mitigating on-target off-tumor cytotoxicity of EpCAM CAR-T by affinity tuning [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-381.