Pooled CAR-T screening in pig-tailed macaques identifies designs with enhanced proliferation, trafficking, and...

Area of Science:

• Immunology
• Cell Therapy
• Oncology

Background:

• Chimeric antigen receptor T (CAR-T) cell therapy shows promise for B-cell malignancies but suffers from high relapse rates due to poor CAR-T cell persistence.
• Current preclinical models often fail to predict clinical CAR-T cell efficacy, necessitating more translationally relevant models.

Purpose of the Study:

• To systematically evaluate CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model.
• To identify novel CAR designs with improved persistence and anti-tumor activity for B-cell malignancies.

Main Methods:

• Engineered 20 CD20-targeted CARs with varying hinge, transmembrane, and costimulatory domains.
• Administered pooled CAR-T cell arrays to NHPs and monitored CAR abundance using digital droplet PCR.
• Assessed CAR-T cell function ex vivo and in vivo, including expansion, cytokine secretion, and tissue trafficking.

Main Results:

• CAR-T cells with the MyD88-CD40 costimulatory domain exhibited enhanced activation, unique cytokine profiles, and resistance to exhaustion ex vivo.
• In vivo, MyD88-CD40 CARs demonstrated dramatic expansion, outperforming CD28- and 4-1BB-based CARs, and achieved robust B-cell depletion.
• MyD88-CD40 CARs, especially with CD28 hinge/transmembrane domains, showed superior lymphoid tissue trafficking and prolonged persistence compared to other CAR designs.

Conclusions:

• NHP models are valuable for screening CAR designs and optimizing CAR-T cell therapy.
• MyD88-CD40 CARs represent a potent new class of CARs with enhanced functional attributes and superior in vivo performance.
• These findings offer critical insights into CAR features that drive improved CAR-T cell activity and may overcome clinical relapse challenges.