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July 5th, 2025
Version: 1
University of California, Los Angeles
bioengineering
biorxiv

Screening of a pooled library of chimeric antigen receptor T cells based on secretory function

Soemardy, C.Open in Google Scholar•Mei, A.Open in Google Scholar•Castellanos-Rueda, R.Open in Google Scholar•Espinoza, N. G.Open in Google Scholar•Kizerwetter, M.Open in Google Scholar•Spangler, J. B.Open in Google Scholar•Reddy, S. T.Open in Google Scholar•Di Carlo, D.Open in Google Scholar

Chimeric antigen receptor (CAR) T cell therapies have shown promise in treating hematologic malignancies, but challenges remain due to immune suppression, antigen heterogeneity, and insufficient functional screening platforms. Here, we present a modular nanovial-based platform for high-throughput, single-cell functional screening of pooled CAR T cell libraries. Nanovials, hydrogel microparticles with nanoliter-scale cavities, were functionalized with recombinant HER2 antigen and cytokine-capture antibodies to simulate antigen-presenting cells and capture secreted interferon-{gamma} (IFN{gamma}). This system enabled the selective capture, activation, and functional profiling of CAR T cells based on antigen engagement and cytokine secretion. We screened a 32-variant CAR library with diverse intracellular signaling domains, using nanovials to isolate IFN{gamma}-secreting cells at 3- and 12-hour timepoints. IL15RA-containing CARs, particularly IL15RA-CD28, were preferentially enriched in the sorted T cells after 3 hours of stimulation, consistent with early effector activation profiles. By 12 hours, IL15RA-containing constructs remained enriched while other CD40-containing domains showed delayed but substantial enrichment, suggesting prolonged signaling dynamics. The platform's high-throughput capacity (>2 million cells screened), compatibility with downstream sequencing, and tunable antigen presentation make it ideal of identifying CAR constructs associated with various time-dependent secretion phenotypes.

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