The C-Trap® provides the world’s first dynamic single-molecule microscope to allow simultaneous manipulation and visualization of single-molecule interactions in real time.
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.
T cells play a pivotal role in tumor immunosurveillance. Multispecific cell engagers (CEs) have been adopted in the field of immuno-oncology to redirect T cells toward cancer cells, thereby unleashing the anti-tumor potential of the patient’s immune system. CE-mediated cell binding induces T cell activation and the formation of an immunological synapse, which is a prerequisite for effective tumor cell lysis.
The strength of the initial binding events between a T cell and a tumor cell dictates the efficiency of the anti-tumor response. Assessing cell avidity, i.e. the total intercellular interaction strength between two cells, gives crucial insights into the efficacy of CEs as anti-tumor therapeutic agents.
Here, we deploy LUMICKS’ high throughput avidity measurement (HTAM) technology to measure CE-induced cell avidity in a high throughput manner. We demonstrate the assay performance characteristics, i.e. specificity, precision, and range, via CE titration experiments in the context of a Jurkat T cell model system. We find that the HTAM CA assay is suitable for candidate screening in high throughput, with high sensitivity and precision.