Abstract 2911: Identification of novel CAR transmembrane and endodomains for improved persistence and cytotoxicity of NK cell therapies

Natural killer cells offer a promising treatment strategy for cancer due to their efficacy and favorable safety profile. However, CAR NK cells in solid tumors have had limited success and considerable efforts are being undertaken to improve their efficacy and persistence. Designing optimal CAR transmembrane and intracellular endodomains may increase expression, strength of activation, co-localization within synapses, and improve functional outcomes. First generation NK CARs had CD8ɑ or CD28 transmembrane domains, similar to CAR T cells. They also included endodomain combinations of CD3ζ, CD28, and 4-1BB, while subsequent generations included combinations of NKG2D, DNAM-1, and 2B4 endodomains, with the intent of incorporating NK-specific activation signals. To identify optimal design sequences for NK-based CARs, we developed an in vitro screening platform based on the functional attributes of NK cells, cytotoxicity and persistence. We designed and synthesized two CAR libraries comprising ~2,700 and ~1,300 endodomains drawn from pairwise combinations of 52 and 36 co-stimulatory motifs, respectively. Through a pooled screening experiment, we identified multiple signaling domains that improved the persistence of NK cells in low cytokine conditions throughout two weeks of re-stimulation. Isolated lines of NK cells expressing the top hits demonstrated higher cytotoxicity both immediately after transfection and after two weeks of re-stimulation with tumor cell lines, validating their improved therapeutic potential. We additionally trained a machine learning model using the counts of signaling domain-binding motifs as features to predict the cytotoxicity and persistence of the screened endodomains. This discriminator model and the feature importance can be used to recommend novel co-stimulatory motif combinations for future screens. Top hits in these screens included co-stimulatory domains known to enhance proliferation, cytotoxicity, and tumor clearance in animal models and commonly used in CAR T cell therapies. We frequently observed these domains in novel combinations or in combination with other endodomains not presently used in CAR T therapies. Additionally, we identified transmembrane domains derived from NK surface receptors that increased the presentation of CAR on the NK cell surface. We subsequently integrated these transmembrane domains with hits from our endodomain screen and again used re-stimulation and cytotoxicity assays to functionally test potent