Proteome Capture in Hydrogel Beads for High Resolution Single Cell Analysis

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Summary T cells are among the most diverse immunological cell types and are central to the pathology of a broad range of human diseases. This diversity has largely been defined by their differential expression of cell surface proteins and to a lesser extent, intracellular protein markers, using flow cytometry. Although significant progress has been made, the field is unnecessarily limited by the lack of available antibody reagents. Only 900 of the 20,000 known human proteins have validated flow cytometry reagents, severely limiting the cellular phenotypes and responses that can be measured. Indeed, recent studies using proteomics (mass spectrometry) and genomics (scRNAseq) have identified hundreds of proteins that appear to be differentially expressed within T cell subsets, suggesting that our current definition of T cells using flow cytometry is quite limited. However, other antibody-based methods such as Western blot have antibodies available for more than half the proteome. In our Phase I studies we developed the Exocells method that allows flow cytometry samples to be processed using SDS and heat, similar to Western blot samples. This format makes the flow cytometry samples amenable to staining with antibodies that are validated to work using Western blots. This fundamental advance in flow cytometry sample processing promises to dramatically increase the number of antibodies that can be used in flow cytometry to characterize T cell populations and cell states. In the Phase II studies, we will further optimize the method and perform assay qualification in primary cell samples. Using the optimized method, we will expand the existing repertoire of T cell markers by more than 30%. Finally, using mass cytometry, which is capable of analyzing 40 or more parameters per cell, we will create a T cell staining panel containing 25 known surface markers and 15 of the newly discovered intracellular proteins. This panel will be a powerful tool for identification of novel T cell subsets, for biomarker discovery in various T cell-mediated disease states, and for analysis of immuno-oncology therapeutics. By significantly increasing the number of flow cytometry reagents capable of differentially staining T cells, we seek to drive the discovery of the next generation of T cell subsets and their role in human disease progression and therapeutic efficacy.