Abstract 3199: Development of a flow cytometry-based assay for measuring specific CAR expression on LGR5-targeting CAR-T cells

Abstract

Abstract The measurement of chimeric antigen receptor (CAR) expression is critical for the development, qualification and quantitation of CAR-T cells for clinical applications. There is a comprehensive range of tools commercially available for specifically measuring expression of the CAR on CD19-targeting CAR-T cells. However, with the rapidly expanding repertoire of CAR-T cells in development targeting a diverse range of tumour antigens, there is a paucity of readily implementable assays that specifically measure CAR expression, which is essential for translation into the clinic. We have developed several CARs targeting the cancer stem cell marker LGR5, one of which (CNA3103) is the lead candidate in an upcoming clinical trial in advanced metastatic colorectal cancer. Previously, the level of CNA3103 expression in this system has been determined indirectly by measuring expression of tEGFR on the CAR-T cell surface, taking advantage of co-expression of the CAR and tEGFR upon transduction with our lentiviral construct. tEGFR expression was found over many CAR-T cell manufacturing batches to report transduction efficiency reproducibly with high sensitivity. However, to specifically quantify expression of the CNA3103, an assay utilising an Fc-tagged recombinant human LGR5 protein, in combination with an anti-human IgG1 Fc-specific secondary antibody was developed. A number of optimisation steps including titration of multiple batches of rhLGR5-Fc, different anti-human IgG1 Fc-specific antibodies and a range of incubation parameters were tested. This resulted in development of an assay that yielded very similar reproducibility and sensitivity to that previously observed with the indirect tEGFR surrogate assay. Overall, the findings highlight the utility of using CAR-targeting recombinant proteins for evaluating CAR expression on CAR-T cells, providing a general flow cytometry-based staining strategy that may be adapted for assessing a diverse repertoire of CARs. Citation Format: Timona Tyllis, Caitlin Abbott, Dylan McPeake, Jade Foeng, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Stuart Mills, Emma Thompson, Lih Tan, Allison Cowin, Claudine Bonder, Timothy Sadlon, Simon Barry, Shaun McColl. Development of a flow cytometry-based assay for measuring specific CAR expression on LGR5-targeting CAR-T cells [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 3199.