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High-Precision Intrinsic Interactome Elucidation of Chimeric Antigen Receptors via Photocatalytic Micromapping
Sean W Huth1,2, Chenmengxiao Roderick Pan1,2, Philip Raftopoulos1,2
1Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States.
Abstract:
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.
Insights
A new method, μMap-CAR, precisely maps the molecular interactions around Chimeric Antigen Receptor T-cells (CAR-T). This advance aids in engineering better CAR-T therapies for improved cancer treatment efficacy.
Area of Science:
- Immunology
- Biotechnology
- Molecular Biology
Background:
- Chimeric Antigen Receptor T-cell (CAR-T) therapies are effective against hematological cancers but show limited efficacy in solid tumors.
- Improved CAR design is crucial for enhancing CAR-T cell potency, persistence, and safety, especially for broader applications.
- Understanding the CAR molecular microenvironment is essential for CAR engineering but has been limited by a lack of precise characterization methods.
Purpose of the Study:
- To introduce μMap-CAR, a novel high-resolution platform for elucidating the CAR interactome on live T-cell surfaces.
- To enable direct interrogation of CAR interactome differences based on CAR endodomain alterations.
- To define the intrinsic CAR interactome in primary T-cells and identify potential targets for CAR engineering.
Main Methods:
- Development and optimization of a high-resolution photocatalytic proximity labeling platform (μMap-CAR).
- Application of μMap-CAR to a model CAR-T cell system under resting and activated conditions.
- Interrogation of interactome differences upon CAR endodomain alterations and validation of candidates using super-resolution microscopy.
Main Results:
- μMap-CAR successfully elucidated the CAR interactome on live T-cell surfaces with high resolution and sensitivity.
- The platform demonstrated robust performance in model CAR-T cells and enabled linking signaling networks to CAR components.
- The first high-resolution intrinsic CAR interactome in primary T-cells was defined, with candidates validated experimentally.
Conclusions:
- μMap-CAR is a powerful and broadly applicable tool for CAR interactome profiling.
- This platform provides actionable targets for proximity-guided CAR engineering.
- The findings advance the development of more effective CAR-T therapies, particularly for challenging cancers.
