Abstract LB041: Decoding single-cell protein interactomes of CAR T cells with the Proximity Network Assay

Abstract Proteins on the cell surface are key determinants of the cellular function in the adaptive immune system. The Proximity Network Assay (PNA) is a sequencing-based platform that resolves protein organization at single-cell resolution. Using DNA-barcoded antibodies and proximity-dependent ligation, PNA simultaneously measures the abundance, clustering, and colocalization of 155 immune proteins, generating nanoscale surface maps comprising ∼50,000 molecular positions per cell without the use of optics. This spatially resolved readout enables systematic analysis of the membrane proteome across thousands of cells. We demonstrate the utility of PNA by identifying the proxiome of the CD19 CAR receptor at steady state and revealing dynamic proteomic remodeling during tumor cell encounter, including key phenomena such as trogocytosis and cell-cell conjugate formation that are key for CAR T cell function in vivo. By integrating spatial context with multiplex protein profiling at scale, PNA provides a powerful platform for protein interactomics, biomarker discovery, and mechanistic insights for cellular immunotherapies currently under clinical evaluation. Citation Format: Filip Karlsson, Michele Simonetti, Christina Galonska, Hanna van Ooijen, Tomasz Kallas, Divya Thiagarajan, Maud Schweitzer, Ludvig Larsson, Vincent van Hoef, William Love, Jerell Aguila, Simon Fredriksson. Decoding single-cell protein interactomes of CAR T cells with the Proximity Network Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB041.