Th142 - Chemokine-engineered CAR-T Cells for Autoimmunity: Multiplexed Mrna Technology for Improved Mode of Action and Safety

Abstract Text: Chimeric antigen receptor (CAR)-T cells have recently emerged as a potential cure for systemic autoimmune diseases but their widespread application in rheumatology is limited by costly manufacturing, poor control over biodistribution and pharmacodynamics and fear of long-term side effects. As the immune reset in autoimmunity does not appear to require long-term CAR-T engraftment, mRNA technology bears the promise for transient CAR-T cell treatments with an improved safety and treatment controllability.
Here, we optimized mRNA delivery to primary human T cells using lipid nanoparticles (LNPs) versus electroporation. We developed CAR-T cells co-expressing mRNA-based CD19-CAR or stable CRISPR/Cas9 CAR knock-in (KI) alongside mRNA-encoded chemokine receptors for deeper B-cell depletion in lymphatic tissues (CCR7) and transient targeting of inflammation (CXCR3A).
Both mRNA transfection methods resulted in effective transfection (>80%) and high T cell viability (>90%). In CRISPR/Cas9-engineered CAR-T cells, CAR-KI rate was >20% and cells could be re-transfected with mRNA 6 days post-KI without significant viability loss. In "mRNA-only" CAR-T cells, co-expression of CAR and chemokine receptor mRNAs could be achieved in 80% of cells upon single treatment and lasted ~3d, whereafter cells assumed their original phenotype.
Transfection with CCR7-mRNA strongly boosted the migration of (CAR-)T cells toward CCL21, a marker of secondary lymphoid organs, while CXCR3A-mRNA increased migration toward the inflammatory mediator CXCL11. Chemokine-engineered CAR-T cells outperformed T cells transfected with CAR only in a combined killing and migration in vitro assay, demonstrating a functional benefit of multiplexed CAR-T cells.
Validation in relevant disease models and transition to GMP-compatible manufacturing are ongoing.