A conformation-specific ON-switch for controlling CAR T cells with an orally available drug
Charlotte U. Zajc, Markus Dobersberger, Irene Schaffner, Georg Mlynek, Dominic Pühringer, Benjamin Salzer, Kristina Djinović‐Carugo, Peter Steinberger, Annika De Sousa Linhares, Nicole Yang, Christian Obinger, Wolfgang Holter, Michael W. Traxlmayr, Manfred Lehner
Abstract
Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.