Abstract 4802: CAR-T cell therapy workflow for discovery and development
Authors: David Cobeta Lopez, Benita Quist, Sara Compte Sancerni, Sophie Vermond, Namrata Jayanth, Monika Hiller, Lauren Schewitz-Bowers, Daniel Rocca, Robert Nunan, Louise Brackenbury, Rhiannon Griffiths, Mark Aspinall O’Dea, Eva Oswald, Julia Schueler, Renny Feldman, Sabrina de Munnik, Gemma Moiset
Journal: Cancer Research (2025)

Engineered T cell therapies such as Chimeric Antigen Receptor (CAR) T cells and T cell receptor (TCR)-engineered T cells have emerged as a promising therapy. To date, seven CAR-T cell products have been approved by the FDA for the treatment of hematological malignancies and many more products are currently being explored for other therapeutic areas such as autoimmune diseases. Due to its innovative nature and the complex biology involved, CAR-T cell therapy product development can face many challenges. While most CAR programs rely on precursor molecules (scFv or VHH) affinity screening, this has proven to not always correlate best to the derived CAR-T activity observed in vivo. However, screening many CAR constructs in primary T cells is associated with lengthy timelines and high costs for their production. To overcome current limitations and streamline the lead selection, Charles River Laboratories offers alternative screening methodologies. Using Jurkat cells as a reporter cell line, plasmids encoding any number of CAR constructs can be transfected and transiently expressed. Afterwards, the CAR-Jurkat cells activation can be measured by the increased expression of CD69 upon co-culture with cells bearing the target of interest. This approach better mimics the cell-to-cell interactions critical for the CAR-T cell therapy function and allow for a more physiologically relevant ranking of the CAR constructs, aiding the selection of the lead candidate with higher success chances. Furthermore, the lack of pharmacologically relevant animal models emphasizes the need to evaluate the safety of CAR-T cell therapy products in vitro. An important step is the identification of potential off-target binders, where the Retrogenix cell microarray technology can be used for the selection and characterization of leads. Additionally, we have developed in vitro co-culture assays using a wide variety of primary or iPSC-derived human cells (representing various tissues including vital organs) to assess potential off-target and on-target/off-tumor effects. A different safety concern to take into consideration is to determine the CAR vector copy number (VCN) and insertional sites and to identify potential insertional mutagenesis effects after introducing the CAR cassette. Lastly, the cells can be tested in vivo to assess their efficacy in a more representative system and gain insights in their bio-distribution, homing sites, prevalence, and differentiation. In conclusion, we have developed a complete workflow to screen CAR candidates in a surrogate model reducing the costs and timelines for CAR-T cell therapies development funneling down the number of CARs to be tested in the in vitro safety and in vivo studies, that would support Investigational New Drug application in accordance with the regulatory agencies’ requirements. Citation Format: David Cobeta Lopez, Benita Quist, Sara Compte Sancerni, Sophie Vermond, Namrata Jayanth, Monika Hiller, Lauren Schewitz-Bowers, Daniel Rocca, Robert Nunan, Louise Brackenbury, Rhiannon Griffiths, Mark Aspinall O’Dea, Eva Oswald, Julia Schueler, Renny Feldman, Sabrina de Munnik, Gemma Moiset. CAR-T cell therapy workflow for discovery and development [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 4802.