基于分泌功能筛选 CAR-T 细胞混合文库

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- (IFN ). 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 -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.