Abstract Chimeric Antigen Receptor (CAR) T cell therapies have transformed cancer treatment, offering targeted and effective options for patients with hematologic B-cell malignancies, including leukemias, lymphomas and multiple myeloma. Their precision in targeting and eliminating cancer cells has positioned them as a promising approach in cellular immunotherapy. Accurate detection of CAR expression is essential for research, pre-clinical applications, clinical manufacturing, and patient immunomonitoring. To address these needs, we developed CAR Detection Reagents optimized for flow cytometric analysis of CAR-engineered T and NK cells, ensuring exceptional quality and specificity. Our recombinant antigen-based CAR Detection Reagents mimic CAR interactions with tumor cells, targeting key cell surface antigens such as CD19, BCMA, CD22, MSLN, CD123, B7-H3, HER2, and CD33. We also created anti-CAR antibodies for CD19 FMC63 and GD2 14G2a CARs, along with linker-specific antibodies for G4S- and Whitlow-linkers, commonly used in commercial CAR T cell products. All these reagents feature an engineered human IgG1 Fc region to prevent Fcγ receptor binding, ensuring background-free analysis. Available in various conjugates (e.g., fluorophores, biotin), they offer superior performance and flexibility in panel design. Our flow cytometric analysis demonstrated bright and specific staining with low background on primary CAR T/NK cells and model cell lines, crucial for reliable CAR T cell enumeration in product manufacturing and patient monitoring. CAR Detection Reagents are also applicable for magnetic activated cell sorting (MACS). In immunomonitoring post-adoptive cell transfer, rapid isolation of CAR T cells from blood or PBMCs can be critical, as CAR T cells in these samples can be present in very low numbers falling below the detection limit. Magnetic enrichment substantially enhances analytical sensitivity, ensuring long-term monitoring of CAR T cell persistence, vital for assessing treatment efficacy and patient response. Furthermore, MACS-purified CAR T cells can undergo deep phenotyping and functional assays. Detecting CAR T cells in tissue samples is essential for understanding their distribution and activity within the body. This capability allows researchers and clinicians to assess CAR T cell infiltration and therapeutic impact at tumor sites, offering insights into treatment effectiveness and resistance mechanisms. Using fluorescence microscopy, we demonstrate precise detection and enumeration of CD19 FMC63 CAR T cells with a FMC63-targeting anti-CD19 CAR antibody in fresh-frozen and PFA-fixed tissue samples from treated mice. This approach enhances our understanding of CAR T cell dynamics in vivo. In summary, these versatile CAR Detection Reagents are invaluable tools for advancing CAR T cell research and clinical therapies. AI tools were used to improve this abstract. Citation Format: Stephan Wilmes, Desiré Missing, Anja Brauchle, Nadine Borgelt, Johanna Müllers, Monika Winkels, Larissa Möckel, Michaela Niemöller, Dagmar Häußler-Kuballa, Franziska Antonia Bertram, Olga Goldbach, Dominik Poschen, Astrid Imann, Silvia Rüberg, Anijutta Appelshoffer, Fabio El Yassouri, Marsilius Mues, Volker Nölle, Andreas Bosio, Christian Dose, Anne Richter. CAR Detection Reagents - Pioneering advances in CAR T analytics abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3718.
Wilmes et al. (Fri,) studied this question.