Confocal microscopy

The success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies has not yet translated into long-term elimination of solid tumors, indicating the need for adequate tuning of CAR T cell functionality. The CAR binding moiety is the critical trigger for CAR T cell signaling. CAR binding affinity alone does not determine T cell effector functions. In a panel of anti-Her2 CARs covering a 4-log affinity range, we observed that rather high affinity and cell avidity above the minimum threshold, combined with elevated tonic signaling, produce adequate T cell capacity for expansion and tumor control. The same scFv mutations increased both antigen-specific affinity, cell avidity, and antigen-independent tonic signaling; above a minimum threshold, raise in affinity translated into cell avidity in a non-linear fashion. In this case, replacement by amino acids of higher hydrophobicity within the scFv coincidentally augmented affinity, non-specific binding, spontaneous CAR clustering, and tonic signaling, all together relating to T cell functionality in an integrated fashion. Data highlight the mechanistic complexity of CAR signaling and suggest inclusion of additional variables, for example, hydrophobic interactions, into the equation when determining the CAR’s antigen-specific and tonic signaling capacities.