Abstract 5572: T cell receptor modified with EDB-targeting ScFv and CD28 intracellular domain led to complete tumor regression in xenograft model

Abstract

Abstract We explored use of chimeric antigen receptor T cell (CAR-T) for treatment of solid tumors, aiming at efficient killing of tumor cells while minimizing on-target off-tumor toxicities. We designed CAR targeting the extra domain B (EDB)-containing fibronectin, which is an oncofetal antigen widely expressed in tumor stroma and on the surface of cancer cells. Unlike membrane-spanning antigens, EDB-containing fibronectin is embedded in the extracellular matrix, and is enriched in new vasculatures and stromal tissues of tumor tissues. First, we made a second-generation EDB CAR (2G-EDB CAR) that killed EDB+ cells (U87MG, A549, and HUVEC) and showed tumor suppression in a U87MG xenograft tumor model. However, while in vitro the 2G-EDB CAR T cells were sufficiently cytotoxic, the in vivo inhibition of the tumor growth was moderate. Next we designed an EDB CAR that contained CD3ε to create 3ε-EDB CAR. We show that the 3ε-EDB CAR was incorporated into the T cell receptor (TCR) structure. The 3ε-EDB CAR completely displaced the native CD3ε in the TCR based on immune precipitation and Western analysis. The 3ε-EDB CAR was capable of inducing T cell activation and proliferation in the presence of EDB-fibronectin antigen. Further, the 3ε-EDB CAR T cells killed tumor cells containing EDB-fibronectin. In vivo, the 3ε-EDB CAR T cells demonstrated improved inhibition when compared to the 2G-EDB CAR T cells. To further enhance the EDB-targeting CAR, we added intracellular domain of CD28 to the 3ε-EDB CAR to create 3ε28-EDB CAR, and showed the 3ε28-EDB CAR was still incorporated into the TCR complex. The 3ε28-EDB CAR co-opted TCR complex and provided co-stimulatory signal upon binding of EDB antigens. Upon transduction of the 3ε28-EDB CAR into primary T cells, we observed upregulation of T cell activation markers CD25, CD69 and granzyme B in presence of the EDB antigen. We show that 3ε28-EDB CAR triggered the phosphorylation of CD3ζ, suggesting that EDB antigen stimulated co-opted TCR and signaled through the TCR-CD3 complex. T cells transduced with 3ε28-EDB CAR demonstrated stronger proliferation when activated by U87MG cells compared to 2G or 3ε-EDB CARs. Furthermore, 3ε28-EDB CAR T cells inhibited in vivo tumor growth in a dose-dependent manner, and most of the treated mice had no evidence of residual tumors. In conclusion, our results suggest that 3ε28-EDB CAR has great potential for treatment of solid tumors. Citation Format: Zhijie Zhang, Wen Qiu, Chang Liu, Muhan Wang, Yinghua Miao, Qingchao Ji, Rongchen Sun, Hongping Yin, Meijia Yang. T cell receptor modified with EDB-targeting ScFv and CD28 intracellular domain led to complete tumor regression in xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5572.