Th149 - Single Cell protein Interactomics using Proximity Networks help unravel membrane protein architecture of CD19 CAR T cells at rest and during tumor interaction

Abstract Text: Diseases are caused by aberrant cell function. These functions are executed by proteins interacting with other proteins and clustering. This dynamic interactome represents a vast and unstudied dimension in translational research with massive potential as novel biomarkers, drug targets, and discoveries of new therapeutic avenues. Protein interactomics by Proximity Networks delivers this new exciting omics dimension. Herein, we present the Proximity Network Assay, where 155 target proteins are positioned in a DNA-based spatial network of >40,000 nodes from single cells without optics. Barcoded antibodies bound to target proteins are subjected to in situ rolling circle amplification, generating multiple barcode copies that subsequently serve as spatial network nodes to several other proximal neighbors via the hybridization of a linker-oligonucleotide. Pairs of proximal barcodes are inserted into the two ends of the linker by gap-fill ligation, readable by DNA sequencing. We utilized the Proximity Network Assay to comprehensively analyze the membrane protein organization of CD19 CAR T cells in both resting condition and during coculture with CD19-expressing tumor cells. Proximity Network Assay provided critical insights into the nano-organization and functional dynamics of CAR T cell membranes, demonstrating the utility of the Proximity Network Assay in uncovering the mechanistic underpinnings of CAR T cell biology. This knowledge paves the way for optimizing CAR T cell therapies to enhance their efficacy and persistence in clinical settings.
At nanoscale resolution in 3D, Proximity Networks will be applicable to high throughput protein interactomics studies in immuno oncology, hematology, and cell therapy research.