A conformation-specific ON-switch for controlling CAR T cells with an orally available drug

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug

New publication from our Christian Doppler Laboratory for Next Generation CAR T Cells:
Brey CU., M. Dobersberger, I. Schaffner, G. Mlynek, D. Pühringer, B. Salzer, K. Djinović-Carugo, C. Obinger, W. Holter, MW. Traxlmayr & M. Lehner. A conformation-specific ON-switch for controlling CAR T cells with an orally available drug. PNAS. first published June 17, 2020, https://doi.org/10.1073/pnas.1911154117

Significance

Molecular ON-switches are important tools in chemical biology, enabling protein–protein interactions to be regulated by small molecules. However, currently available ON-switches that induce conditional heterodimerization are suboptimal for therapeutic applications. In this study, we present an ON-switch system based on human retinol binding protein 4 (hRBP4) and the orally available small molecule A1120. Two distinct protein scaffolds, FN3 and rcSso7d, were successfully engineered to bind to hRBP4 in a small molecule-dependent manner, demonstrating the flexibility of the system. The binders specifically associated with the drug-induced conformation of hRBP4. Our study demonstrates that lipocalin-based ON-switches can enable specific regulation of protein heterodimerization and provides proof of concept for potential applications in controlling the activity of human CAR T cells.

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.