High-Precision Intrinsic Interactome Elucidation of Chimeric Antigen Receptors via Photocatalytic Micromapping (μMap-CAR).

Chimeric Antigen Receptor T-cell (CAR-T) therapies represent a powerful modality for treating a variety of hematological cancers. However, limited efficacy in broader disease contexts, particularly solid tumors, underscores the need for improved CAR design to enhance potency, persistence, and safety. Key design features of CARs have been profoundly informed by extensive knowledge of the T-cell receptor (TCR) and its interactome. To advance CAR-T therapies, new functionally relevant proteins are needed to support engineering efforts. Until now, the actual molecular microenvironment surrounding CARs has remained poorly defined, chiefly due to the lack of a characterization method with the required precision and sensitivity. Herein, we introduce μMap-CAR, a high-resolution photocatalytic proximity labeling platform featuring key methodological optimizations that enable direct elucidation of the CAR interactome on live T-cell surfaces. The platform performs robustly in a model CAR-T cell system under both resting and simulated activation conditions. The high sensitivity of μMap-CAR allows interrogation of interactome differences upon CAR endodomain alterations, linking perturbed signaling networks directly to CAR components. We further deliver the first high-resolution intrinsic CAR interactome in primary T-cells, defined by shared interactors across donors, and validate candidates via super-resolution microscopy and CAR-T activation perturbation. This platform constitutes a powerful, broadly applicable tool for CAR interactome profiling while also providing actionable targets for proximity-guided CAR engineering applications.