Next Generation Genetic Discovery Systems in Human Immune Cells

Engineered immune cell therapies offer the promise of more tailored interventions than traditional systemic checkpoint blockades, but so far remain ineffective across the majority of clinical settings, especially solid tumors. To overcome the slew of resistance mechanisms cancers present, various screening modalities have nominated numerous gene knockouts, gene overexpression, and synthetic CAR and TCR sequences to further refine T cell therapies. But among this abundance of gene targets, effective methods to directly compare large numbers of potential clinical constructs to engineer immune cells are lacking. Further, the association of these synthetic genetic sequences with the functional phenotype they impart on an engineered cell is costly, time consuming, and low throughput. Here, we developed Modular Pooled Knockin Screening (ModPoKI), a highly scalable platform to rapidly associate large pools of T cell genetic modifications with high dimensional single cell phenotypes across diverse cancer immunotherapy contexts. We built libraries of transcription factors (TFs) and natural and synthetic surface receptors, including numerous proposed constructs under clinical investigation. Over 20 ModPoKI screens across dozens of healthy human donors in both TCR and CAR T cells in diverse conditions built a high dimensional genotype x phenotype map which benchmarked existing clinical constructs and nominated unique novel cell therapy targets for further clinical development which overcame cancer resistance mechanisms and showed improved in vitro and in vivo performance. Analysis across these diverse functional contexts identified a transcription factor AP4 (TFAP4) construct that enhanced long-term T cell fitness and anti-cancer function in vitro and in vivo. ModPoKI’s modularity further allowed us to generate a ∼10,000-member library of TF combinations, revealing unexpected functional relationships within CAR T cells with synthetic transcriptional programs. Non-viral knockin of a combined BATF-TFAP4 polycistronic construct further enhanced function in vivo. ModPoKI facilitates scalable discovery of complex gene constructs to program and improve engineered immune cell functions tailored to specific cancer resistance mechanisms.