Abstract 898: Generation and validation of anti-linker monoclonal antibodies for the detection of surface expressed scFv-based CARs

Abstract

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of cells with greater persistence, there is a need in multiple phases of the CAR-T development pipeline for highly specific detection reagents that can be leveraged to monitor the expression of CARs on the cell surface. Many commercially available CAR detection reagents, however, either lack specificity or are not versatile in their ability to detect CARs of differing antigen specificity. Here, we report on the generation and validation of rabbit monoclonal antibodies raised against two linker sequences that are commonly integrated into single-chain variable fragment (scFv)-based CARs. These antibodies serve as versatile detection reagents that can be used to interrogate the surface expression of CARs. Methods: The monoclonal antibodies, E7O2V and E3U7Q, were generated by rabbits immunized with peptide sequences most commonly used to construct the linker region of scFv based CARs, Gly4Ser and Whitlow, respectively. E7O2V and E3U7Q were validated for specificity and versatility using flow cytometric analysis of non-transduced versus CAR-transduced cell lines and primary human T cells. Results: Flow cytometric analysis of live Jurkat cells and primary human T cells transduced with CAR constructs revealed that E7O2V and E3U7Q could detect surface expressed CARs containing the appropriate linker sequence, independently of scFv specificity. No specific staining was observed on non-transduced cells. Conclusions: In a flow cytometry assay, E7O2V and E3U7Q specifically detect surface expressed scFv-based CARs containing either a Gly4Ser linker or a Whitlow linker, respectively. Furthermore, these monoclonal antibodies are versatile in that they can also detect their respective linker sequence independently of scFv specificity. The potential exists to leverage these antibodies for CAR-T cell enrichment and for incorporation into multiparametric flow cytometry panels used to phenotype CAR-T cells during the discovery, manufacturing, and clinical phases of the development pipeline. Citation Format: Amrik Singh, Sarah L'Heureux, Ryan Sinapius, Michael Kvorjak, Emma Wade, Jason Lohmueller, Jeremy Fisher, Reginaldo Prioli. Generation and validation of anti-linker monoclonal antibodies for the detection of surface expressed scFv-based CARs [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 898.