Pooled CAR-T screening in nonhuman primates identifies designs with enhanced proliferation, trafficking, and persistence

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized treatment for B-cell malignancies, yet over 60% of patients relapse within one year, often due to insufficient CAR-T persistence. While mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. Following ex vivo characterization, we administered pooled autologous CAR-T arrays to three NHPs and tracked CAR abundance longitudinally using a novel digital droplet PCR assay. Ex vivo, CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo, MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study endpoint, unlike other CARs which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T cell activity. Key pointsO_LIWe developed the first pooled CAR-T screening platform in an immunocompetent nonhuman primate model to directly compare CAR designs. C_LIO_LIWe identified MyD88-CD40 costimulatory domain as vastly superior to conventional domains in proliferation, trafficking and persistence. C_LI