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.
Highlights
Molecular ON-switches in which a chemical compound induces protein–protein interactions can allow cellular function to be controlled with small molecules
The only molecular ON-switch that has been used in humans in vivo is based on a mutated version of FK506 binding protein (FKBP) 12, which is homodimerized upon administration of the small molecule AP1903 [3]
We present an ON-switch system based on human retinol binding protein 4 and the orally available small molecule A1120
Summary
Molecular ON-switches in which a chemical compound induces protein–protein interactions can allow cellular function to be controlled with small molecules. The ability to control protein–protein interactions with small chemical compounds can open up exciting applications across various fields such as cell biology, immunology, and immunotherapy. Various systems have been introduced that enable such conditional heterodimerization [4,5,6,7,8], including the FRB/FKBP system that is used extensively in vitro Their clinical translation is limited due to unfavorable characteristics of the small molecule or the nonhuman origin of protein components [7, 9,10,11,12]. 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
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