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bioRxiv · 10.1101/2025.07.02.662376

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

Soemardy, C.·Mei, A.·Castellanos-Rueda, R.·Espinoza, N. G.·Kizerwetter, M.·Spangler, J. B.·Reddy, S. T.·Di Carlo, D.
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Abstract

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 after 3- and 12-hour CAR-specific stimulation. 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 platforms high-throughput capability (>2 million cells screened), compatibility with downstream sequencing, and tunable antigen presentation make it ideal for identifying CAR constructs associated with various time-dependent secretion phenotypes.

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Soemardy, C., Mei, A., Castellanos-Rueda, R., Espinoza, N. G., Kizerwetter, M., Spangler, J. B., Reddy, S. T., Di Carlo, D.. 2025-07-05. Screening of a pooled library of chimeric antigen receptor T cells based on secretory function. https://doi.org/10.1101/2025.07.02.662376

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