Abstract B009: CAR-T-cell monitoring using specific multimers: a fast and specific method allowing uniform evaluation

Abstract Background Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary new pillar in cancer treatment. In research, CAR-T-cells are often detected through tags added to the CAR construct. However, these methods lack sensitivity and often do not address CAR receptors’ recognition of their target. Detection methods using the targeted ligand exist but they are indirect and laborious. In this study we developed a flexible platform to manufacture direct CAR-T cells detecting multimer reagents (CAR Dextramer®) allowing detection of CAR-T cells of different specificities and affinities. Methods Different CAR Dextramer® prototype reagents were generated. The reagents were composed of PE fluorescent multimer and target proteins of different specificities (BCMA, Mesothelin…) and in different stochiometric ratios. CAR Dextramer® functionality and specificity were evaluated in a flow cytometry bead assay. Cells expressing specific CAR constructs were directly stained with CAR Dextramer® reagents or a control, washed and acquired on FACS to evaluate CAR Dextramer® CAR-T detection ability. Results The CAR Dextramer® reagents specifically detected cognate antibodies displayed on beads and not irrelevant antibodies, demonstrating that target proteins displayed on multimer had the correct conformation and the ability to detect CAR receptor antibodies. The different CAR Dextramer® prototypes, however, gave different signal-to-noise ratios and did not work equally well, highlighting the importance of multimer:target protein ratio. All CAR Dextramer® reagents specifically detected CAR-T cells expressing cognate CAR constructs but did not work equally well. Conclusions We have established a CAR Dextramer® manufacturing platform that allows fast development of new CAR Dextramer® reagents. Critical parameters influencing CAR Dextramer® performance like target protein (i) quality (ii) stoichiometry and (iii) functionality are determined during this development phase leading to optimized reagent manufacturing.This new platform allows simple and quick manufacturing of reliable CAR detection reagents. These reagents open new possibilities for (i) directly detecting and monitoring CAR-T-cells of different specificities without being dependent on tags (ii) demonstrating cell surface expression of a functional target binding CAR of CAR T cells, which a tag doesn´t reveal. This technology has been proven to work on transduced cells and have the potential to measure persistence of CAR-T cells in patient blood samples. Citation Format: Elizabeth Epps, Kevin Lenogue, Liselotte Brix. CAR-T-cell monitoring using specific multimers: a fast and specific method allowing uniform evaluation abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B009.