An Innovative Single-Cell Approach for Phenotyping and Functional Genotyping of CAR NK Cells

To expedite immunotherapy development, better analysis of treatment efficacy at early in vitro stages is needed. Using a droplet-based microfluidic platform, we have established a method for multi-parameter quantifiable phenotypic and genomic observations of immunotherapies. Chimeric antigen receptor (CAR) NK cells provide treatment of interest in the current immunotherapy landscape and provide an optimal model for evaluating our novel methodology. For this approach, NK cells transduced with a CD19 CAR were compared to non-transduced NK cells in their ability to kill a lymphoma cell line. Using our novel single-cell droplet array platform, we quantified the increase in cytotoxicity and synaptic contact formation of CAR-NK cells over non-transduced NK cells. With our droplet sorter, we separated NK cells based on target cell killing for transcriptomic sequencing. Our data revealed expected improvement in cytotoxicity with the CD19 CAR but more importantly, provided unique insights into the factors involved in the cytotoxic mechanisms of CAR NK cells. This demonstrates a novel, improved system for immunotherapy screening. CAR and non-transduced NK cells were co-encapsulated In droplets in Raji lymphoma cells. Droplets were sorted based on NK cell killing of target cells, and the collected cell populations were submited to Novogene for sequencing.