Spatial profiling of CAR protein organization reveals in vivo remodeling during CAR-T therapy

Chimeric antigen receptor (CAR) T cell therapy utilizes genetically engineered patient-derived T cells to target cancer cells. Despite its clinical successes in multiple cancer types, the underlying molecular mechanisms by which molecules on CAR-T cells and surrounding cells interact with other proteins and collectively determine treatment efficacy remain elusive. Most previous studies have relied on transcriptome profiling, which does not fully reflect protein-level organization and interactions. In this study, we developed an antibody-oligonucleotide conjugate targeting the FMC63 region of CAR and integrated it into molecular pixelation (MPX). This approach enabled profiling of the dynamics of CAR molecules on cell surfaces as well as their colocalization with other proteins at the single-cell level. By applying MPX to longitudinal samples from three patients undergoing CAR-T cell therapy, we characterized the dynamic changes in CAR-associated protein organization in both pre-infusion CAR products and post-infusion peripheral blood. While CAR protein abundance and polarization showed limited variation across clinical courses, remodeling of a CAR-centered co-localization network was observed over time, including different retentions of specific molecular associations between patients with different clinical outcomes. Although derived from a limited cohort, our study identifies insights from this methodological framework beyond those gained by conventional omics analyses and offers results of a systematic investigation to predict and enhance CAR therapeutic outcomes. Key pointsO_LIMolecular pixelation was applied for chimeric antigen receptor (CAR) profiling at single-molecule and single-cell resolutions. C_LIO_LIProtein and transcriptome analyses of the CAR molecule showed dynamic remodeling during CAR-T therapy in patients with non-Hodgkin lymphoma. C_LI