Impact of ligand size and conjugation chemistry on the performance of universal chimeric antigen receptor T-cells for tumor killing

Abstract

All Universal Chimeric Antigen Receptor T-cells (UniCAR T-cells) are T-cells which have been engineered to recognize a haptenated ligand. Due to this feature, UniCAR T-cells have the potential to mediate a potent and selective tumor killing only in the presence of a haptenated tumor ligand, thus avoiding the long-lasting biocidal effects of conventional CAR T-cells. We have used fluorescein-labeled versions of small organic ligands and different antibody formats specific to carbonic anhydrase IX (a tumor-associated antigen) in order to assess whether the killing potential of UniCAR T-cells depended on the molecular features of the haptenated molecule. Both small molecule ligands and larger antibody fragments were potent in mediating tumor cell killing over a broad concentration range. Antibodies could be conveniently used both in IgG format and as smaller diabody fragments. Importantly, the use of site-specific chemical modification strategies for the antibody coupling to fluorescein led to a substantial improvement of tumor cell killing performance, compared to the random modification of primary amino groups on the antibody surface.