Universal high-sensitivity CAR T-cell monitoring by targeting linker sequences

Introduction Chimeric antigen receptor (CAR) T-cell therapy has become a standard-of-care in oncology, yet standardized monitoring of circulating CAR T cells remains a major challenge due to diverse CAR constructs and limited availability of detection reagents. The antibody domain of the CAR commonly consists of the heavy and light chain connected through a linker, typically either 4x glycine and 1x serine (G4S) or a defined amino acid sequence (Whitlow/218). Here, we evaluated the novel monoclonal antibodies (mAbs) targeting the linker sequence as a universal tool for CAR detection. Methods Using flow cytometry, we compared anti-linker mAbs with conventional reagents, including anti-idiotype CD19.FMC63 mAb, CD19 and BCMA antigen-based detection reagents (Ag), and anti-F(ab′) 2 mAb. Analyses were performed on commercial CD19- and BCMA-directed CAR T-cell products and an investigational Claudin-6 (CLDN6) CAR T-cell product. Performance was further assessed across diverse experimental platforms for clinical monitoring and in a murine model to evaluate sensitivity and translational relevance. Results Linker-mAbs detected all tested CAR constructs with high specificity and sensitivity, matching target-specific binding with conventional Ag reagents. Anti-linker mAbs demonstrated minimal background and low limits of quantification both comparable to Ag reagents. Longitudinal monitoring in lymphoma and myeloma patients revealed consistent CAR T-cell kinetics between anti-linker mAbs and Ag reagents. High performance of linker-based CAR-detection was demonstrated in high-dimensional, multi-parameter flow cytometry, in immunofluorescence imaging and in a murine model of anti-CD19 CAR T cells. Conclusion These findings establish anti-Whitlow/218 and anti-G4S mAbs as sensitive, specific, and universal reagents for CAR detection across multiple targets, constructs, and species, providing a standardized platform for harmonization of CAR T-cell monitoring in preclinical, clinical trial, and diagnostic settings.