Programmable DARPin-based receptors for the detection of thrombotic markers

Area of Science:

• Biotechnology and Synthetic Biology
• Molecular and Cellular Biology
• Biomedical Engineering

Background:

• Cellular therapies are limited by the lack of specific sensors for disease markers.
• Developing novel biosensors is crucial for advancing disease detection and therapeutic interventions.

Purpose of the Study:

• To create a customizable platform for generating novel extracellular receptors for disease marker detection.
• To demonstrate the utility of the developed system in detecting clinically relevant markers and enabling therapeutic responses.

Main Methods:

• Developed an integrated pipeline combining the generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins).
• Screened a DARPin library for binders against human fibrin degradation products (FDPs).
• Constructed and validated advanced modular bispecific extracellular receptors (AMBERs) using selected DARPins, including heterodimers and tandem receptors.

Main Results:

• Successfully screened 160 potential binders and selected 19 DARPins for constructing AMBERs.
• Identified 4 functional DARPins that formed functional heterodimers with a known single-chain variable fragments binder.
• Demonstrated that tandem receptors composed of validated DARPins were also functional.
• Engineered designer cell lines capable of detecting pathological FDP concentrations in vitro and in vivo.

Conclusions:

• The developed AMBER system provides a customizable and efficient method for generating novel extracellular sensors.
• Functional AMBER receptors were successfully created and validated for detecting FDPs, a marker of clinical relevance.
• Designer cells equipped with AMBER receptors can detect disease markers and trigger specific therapeutic protein production.