Doiron Kailyn E, Way Jeffrey C, Silver Pamela A
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA.
bioRxiv. 2025 Aug 26:2025.04.10.648227. doi: 10.1101/2025.04.10.648227.
Erythropoietin (EPO) suppresses apoptosis and promotes survival by signaling through EPO-R/EPO-R on hematopoietic progenitors or EPO-R/CD131 on non-hematopoietic cells. However, EPO signaling through EPO-R/CD131 is controversial and there is no solved structure of a complex. Here, we constructed a structural model of EPO-R/CD131 and designed several anti-EPO-R, anti-CD131 bispecific proteins that selectively activate EPO-R/CD131. Treatment with these fusion proteins is sufficient to activate STAT5 phosphorylation downstream of EPO-R/CD131 without engaging EPO-R/EPO-R. We demonstrated that proteins with a tandem scFv or bispecific antibody format activate EPO-R/CD131, in contrast to an equimolar mixture of the individual scFvs. Finally, we explored the effect of modifications to binding domain arrangement and linker length and found results consistent with our structural model of an EPO-R/CD131 complex. These findings highlight the utility of bispecific scaffolds in the development of cytokine receptor agonists and provide a foundation for the study of EPO-R/CD131 biology and future clinical development.
促红细胞生成素（EPO）通过造血祖细胞上的EPO-R/EPO-R或非造血细胞上的EPO-R/CD131信号传导来抑制细胞凋亡并促进存活。然而，EPO通过EPO-R/CD131的信号传导存在争议，且尚无复合物的解析结构。在此，我们构建了EPO-R/CD131的结构模型，并设计了几种选择性激活EPO-R/CD131的抗EPO-R、抗CD131双特异性蛋白。用这些融合蛋白进行处理足以激活EPO-R/CD131下游的STAT5磷酸化，而无需激活EPO-R/EPO-R。我们证明，与单个单链抗体片段（scFv）的等摩尔混合物相比，具有串联scFv或双特异性抗体形式的蛋白可激活EPO-R/CD131。最后，我们探究了结合域排列和接头长度修饰的影响，发现结果与我们的EPO-R/CD131复合物结构模型一致。这些发现突出了双特异性支架在细胞因子受体激动剂开发中的实用性，并为EPO-R/CD131生物学研究和未来临床开发奠定了基础。
