Impact of Ligand Size and Conjugation Chemistry on the Performance of Universal Chimeric Antigen Receptor T-Cells for Tumor Killing
Christian Pellegrino, Nicholas Favalli, Michael T. Sandholzer, Laura Volta, Gabriele Bassi, Jacopo Millul, Samuele Cazzamalli, Mattia Matasci, Alessandra Villa, Renier Myburgh, Markus G. Manz, Dario Neri
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.