CAR T cell therapy has been an effective treatment option for hematological malignancies. However, the therapeutic potential of CAR T cells can be reduced by several constraints, partly due to immunogenicity and toxicities. The lack of established workflows enabling thorough evaluation of new candidates, limits comprehensive CAR assessment. To improve the selection of lead CAR candidates, we established a stringent, multistep workflow based on specificity assessments, employing multiple assays and technologies. Moreover, we characterized a human FOLR1-directed CAR binding domain. Selection of binding domains was based on extensive specificity assessment by flow cytometry and imaging, to determine on-/off-target and off-tumor reactivity. CAR T cell functionality and specificity were assessed by high-throughput screening and advanced in vitro assays. Our validation strategy highlights that assays comprehensively characterizing CAR functionality and binding specificity complement each other. Thereby, critical specificity considerations can be addressed early in the development process to overcome current limitations for future CAR T cell therapies.
