Immunotherapy with CAR-modified T cells is emerging as a powerful therapeutic modality for advanced malignancies. Durable responses have been obtained in a subset of patients with B-cell malignancies after infusion of autologous T cells expressing a CD19-specific CAR, however not all patients respond equally well and the clinical outcome in trials that utilized distinct CD19-CAR construct is inconsistent. CARs are synthetic receptors of modular design, and the rules that govern antigen recognition and signaling by CARs and immune synapse formation between CAR T cells and tumor cells are still poorly defined. We have shown in previous work that CAR affinity and extracellular domain design affect tumor recognition and have established a CAR library to systemically study the influence of these and other parameters on CAR T-cell function. We have an ongoing productive collaboration with Johannes Huppa (Vienna, Austria) and are proposing to utilize a planar lipid-bilayer system and superresolution microscopy to derive more detailed insights into the function of CARs and inform the rational design of receptors with enhanced anti-tumor efficacy and safety. Our group has developed CARs for applications in hematologic and solid tumors incl. receptors specific for the CD19, CS1, BCMA, ROR1 and GD2 antigens, and set up methods for evaluation of CAR T cell function in vitro and in murine tumor models incl. bioluminescence imaging to analyze the engraftment, migration and anti-tumor efficacy of CAR T cells in vivo. Project's objectives are a) the visual and quantitative analysis of antigen binding, sensitivity and signaling through CARs, in comparison to ‘conventional’ TCRs in the same T cell, b) the investigation of the influence of CAR affinity and extracellular domain design on immune synapse formation between CAR T cells and tumor cells and c) the determination of the range (upper/lower threshold) in which superior antigen binding, sensitivity and signaling of CARs translates into enhanced anti-tumor efficacy in vitro and in vivo. We are confident to provide an excellent and inspiring training opportunity in translational oncology, and contribute to the advancement of CAR T-cell therapy to the benefit of our patients.