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A conformation-specific ON-switch for controlling CAR T cells with an orally available drug
Charlotte U Zajc1,2, Markus Dobersberger1, Irene Schaffner3
1St. Anna Children's Cancer Research Institute (CCRI), 1090 Vienna, Austria.
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.
Insights
Researchers developed a novel molecular ON-switch using human retinol binding protein 4 (hRBP4) and a small molecule. This system controls cellular functions and shows potential for therapeutic applications in CAR T cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Molecular ON-switches enable cellular function control via small molecules inducing protein-protein interactions.
- Developing ON-switches using clinically relevant compounds and human proteins is crucial for therapeutic advancement.
Purpose of the Study:
- To engineer a novel ON-switch system based on human retinol binding protein 4 (hRBP4) and a small molecule for controlled cellular function.
- To demonstrate the efficacy and specificity of this hRBP4-based ON-switch for potential therapeutic applications.
Main Methods:
- Engineered two protein scaffolds to bind hRBP4 in a manner dependent on the small molecule A1120.
- Determined the crystal structure of the assembled ON-switch to analyze molecular interactions.
- Assessed the affinity of the ON-switch with and without A1120.
- Validated the ON-switch's ability to regulate primary human CAR T cell activity in vitro.
Main Results:
- The engineered binders demonstrated small molecule-dependent interaction with hRBP4.
- The crystal structure revealed specific recognition of A1120-induced conformational changes in hRBP4.
- The ON-switch exhibited an approximately 500-fold increase in affinity upon A1120 addition, confirming high specificity.
- The system successfully modulated the activity of human CAR T cells in vitro.
Conclusions:
- Lipocalin-based molecular ON-switches can be engineered for precise, small molecule-dependent control of protein interactions.
- This hRBP4-A1120 system represents a viable platform for the pharmacological regulation of cellular therapeutics, including CAR T cells.
- The developed ON-switch holds potential for broad application in safe and effective control of cellular therapies.
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