ITGA4 knockout prevents blood-brain-barrier migration of chimeric antigen receptor T cells

Abstract

Chimeric Antigen Receptor (CAR) T cells have shown remarkable anti-tumor activity in various B cell malignancies. However, expanding their use to solid tumors has proven difficult due to the lack of sufficiently tumor-specific target antigens. Restricting CAR T cells from trafficking to certain healthy tissues showing shared expression of an otherwise tumor-specific target antigen may open up new avenues to the treatment of solid tumors with CAR T cells. We developed an electroporation-based CRISPR/Cas9 approach targeting integrin a4 (ITGA4), a key component of the T cell adhesion heterodimer VLA-4 required for blood-brain-barrier migration via the endothelial adhesion protein VCAM-1. We characterize ITGA4 knockout kinetics in human T cells by flow cytometry, determine its effect on T cell adhesion to VCAM-1 using a custom in vitro adhesion assay, determine tissue distribution of ITGA4ko cells in a mouse model and characterize the anti-tumor activity of ITGA4ko CAR T cells using a luminescence-based cytotoxicity assay. Near-complete ITGA4 knockout could be achieved during a standard 12-day CAR T cell production process and significantly decreased adhesion of human T cells to VCAM-1. ITGA4ko T cells appeared to be efficiently redirected from the brain to the bone marrow which was validated by flow cytometry showing almost a complete lack of T cells in the brain after ITGA4 knockout. Importantly, knockout did not significantly change CAR T cell expansion or anti-tumor activity. In conclusion, we show that ITGA4 knockout is feasible, results in efficient exclusion of T cells from the brain without affecting CAR T cell function, representing a promising approach to shape CAR T cell selectivity for the treatment of solid tumors.