Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
Roche Diagnostics, Penzberg, Germany.
Nat Chem Biol. 2022 Oct;18(10):1125-1134. doi: 10.1038/s41589-022-01095-3. Epub 2022 Aug 8.
Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein.
细胞疗法仍然受到疾病标志物传感器可用性有限的限制。在这里，我们提出了一个集成的靶标到受体的流水线，用于构建可定制的先进模块化双特异性细胞外受体（AMBER），该受体将我们的通用细胞外分子传感器（GEMS）系统与用于生成设计的锚蛋白重复蛋白（DARPins）的高通量平台相结合。为了验证概念，我们选择了具有高度临床相关性的人纤维蛋白降解产物（FDPs）作为标记物，并筛选了 FDP 结合物的 DARPin 文库。我们构建了配备 19 种不同 DARPin 的 AMBER，这些 DARPin 是从 160 个命中物中选择的，其中 4 种作为与已知单链可变片段结合物的异二聚体是功能性的。由验证的 DARPin 组合而成的串联受体也是功能性的。我们通过构建检测病理性 FDP 浓度并通过产生报告蛋白和治疗性抗血栓蛋白做出反应的设计细胞系，在体外和体内证明了这些 AMBER 受体的应用。
