Pooled CAR-T screening in pig-tailed macaques identifies designs with enhanced proliferation, trafficking, and persistence
Abstract
Abstract Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of B-cell malignancies; however, >60% of patients relapse within 1 year, often due to insufficient CAR-T persistence. Although 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, to our knowledge, 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. After ex vivo characterization, we administered pooled autologous CAR-T arrays to 3 NHPs and tracked CAR abundance longitudinally using a novel digital droplet polymerase chain reaction 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 CAR-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 end point, 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 activity.
Article Details
Authors (23)
Lucy H. Maynard
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Eric J. Cavanaugh
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Haiying Zhu
3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA
Carly E. Starke
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Sarah M. Doherty
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Teresa Einhaus
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Ailyn C. Pérez-Osorio
3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA
Laurence Stensland
3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA
Cameron Blair
4Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
Aoife M. Roche
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia
John K. Everett
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia
Robert D. Murnane
3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA
Michelle Hoffman
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Veronica Nelson
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Sarah Herrin
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Chad Littlewood
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Kaycee Camou
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Erica Wilson
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Christopher Wessel
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
Frederic D. Bushman
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia
Keith R. Jerome
3Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA
Hans-Peter Kiem
Christopher W. Peterson
1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA