Abstract 4806: CAR binders affect CAR T cell tonic signaling, durability, and sensitivity to target

Patients can develop human anti mouse immune responses against CD19- specific CAR T cells (CARTs) due to the use of a murine scFv to redirect T cells. To mitigate immunogenicity, a humanization process of the FMC63- based binder was undertaken that resulted in a CAR (119) that when introduced into T cells showed improved activity in a humanized mouse model. To further characterize how binding domains affect CART function, we screened a yeast display library to identify an array of fully human CD19 scFv binders. We saw significant differences in the ability of CARs employing these CD19 binders to be expressed on the cell surface, induce tonic signaling, redirect T cell function, mediate tumor killing, recognize lower levels of CD19 antigen, and maintain function upon continuous antigen exposure. From this in vitro analysis, CARTs using binders 42 and 52 were selected to be evaluated in a humanized mouse model. Here, we observed that CARTs using both 42 and 52 binders controlled the initial tumor burden and prevented tumor establishment upon rechallenge. We advanced a CAR construct using binder 42 for more advanced preclinical testing due to its greater similarity to FMC63 based binders and ability to robustly respond to tumors expressing lower levels of CD19. We found that this binder uniquely bound CD19 using distinct contact residues than FMC63 with ∼40-fold lower affinity. Combined with their potential for less immunogenicity and comparable in vivo activity, CARTs using the 119 or 42 binder should be considered for clinical use. Citation Format: Divanshu Shukla, Khatuna Gabunia, Shannon E. McGettigan, Prachi R. Patel, Shannon Christensen, Ting Jia Fan, Decheng Song, Yanping Luo, Yanling Wang, Huaishan Wang, Regina M. Young, Carl H. June, John Scholler, James L. Riley. CAR binders affect CAR T cell tonic signaling, durability, and sensitivity to target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4806.