UCLA

Adoptive cell therapy has shown great promise for cancer treatment, but existing approaches face significant limitations. For example, chimeric antigen receptor (CAR) T cell therapies have shown clinical efficacy in treating hematologic malignancies, yet challenges remain due to immune suppression, antigen heterogeneity, and severe side effects. To overcome these limitations, alternative strategies, including engineered T cell receptor (TCR) therapies and unconventional T cells, are under active investigation. Among these, unconventional T cells offer distinct advantages for off-the-shelf applications due to their conserved, invariant or semi-variant TCRs. Subsets such as mucosal-associated invariant T (MAIT) cells and invariant natural killer T (iNKT) cells, recognize non-peptide antigens presented by MR1 and CD1d, respectively, and play pivotal roles in immune surveillance, allowing recognition of target cells based on metabolic activity. In chapter 2, we investigate the functional diversity of MAIT and iNKT cells, focusing on the variability in their CDR3 region and the relationship between TCR sequence and function. We develop a nanovial-based functional screening platform that enables high-throughput discovery of TCRs from unconventional T cells based on direct antigen recognition and cytokine secretion. By selectively labeling nanovials, hydrogel microparticles with nanoliter-scale cavities, with MR1 and CD1d molecules displaying their cognate ligands, we achieve dose-dependent capture and activation of MAIT and iNKT cells from complex human PBMC samples comprising tens of millions of cells. Using oligonucleotide barcodes conjugated to nanovials encoding the antigen-presenting molecules and loading cytokine capture antibodies, we perform secretion-encoded single-cell sequencing to link TCR identity, gene expression, antigen specificity, and functional response. Applying this method, we isolate rare reactive T cells, recover their TCRs, and validate five novel MAIT TCRs. All five TCRs, when re-expressed in primary T cells, confer antigen-specific cytokine secretion and cytotoxicity. The top two TCRs were evaluated using an in vivo solid tumor model, demonstrating specific tumor homing and efficacy. This function-first strategy offers a powerful tool to uncover functional TCRs from unconventional T cells, yielding a 100% hit rate when secretion-based validation is included as part of the initial screen, unlocking new opportunities for cell-based immunotherapy.In chapter 3, we present a modular nanovial-based platform for high-throughput, single-cell functional screening of pooled CAR T cell libraries. Nanovials were functionalized with recombinant HER2 antigen and cytokine-capture antibodies to simulate antigen-presenting cells and capture secreted interferon-γ (IFNγ). 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 IFNγ-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 platform’s 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.