Sopko Richelle, Mugford Joshua W, Lehmann Andreas, Shapiro Renée I, Rushe Mia, Kulkarni Abhishek, Worrall Joseph, Amatucci Joseph, Wen Dingyi, Pederson Nels E, Minesinger Brenda K, Arndt Joseph W, Pepinsky Blake
Department of Cell and Protein Sciences, Biogen, Cambridge, MA 02142, USA.
Protein Eng Des Sel. 2017 May 1;30(5):359-372. doi: 10.1093/protein/gzx007.
Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and pharmacokinetics (PK) of the DKK2 polypeptide. Specifically, human serum albumin (HSA) was identified as a highly effective fusion partner. Substitution of selected amino acid residues in DKK2 designed to decrease heparan sulfate binding by HSA-DKK2 variants, further improved the PK properties of the molecule in rodents. The HSA-DKK2 variants were monomeric, as thermally stable as wild type, and active as measured by their ability to bind to and prevent phosphorylation of the Wnt coreceptor LRP6. Our engineering efforts resulted in potent long-lived variants of the canonical Wnt inhibitor DKK2, applicable for Wnt pathway manipulation either by systematic delivery or focused administration at sites of tissue injury.
Wnt信号通路参与从胚胎发育到组织修复与再生等各种各样的生物学过程。Dickkopf-2（DKK2）传统上被定义为一种典型的Wnt抑制剂，不过在急性损伤后的内皮网络形成过程中，它可能在激活非典型Wnt通路方面发挥作用。在此，我们报告了一种DKK2多肽融合伴侣的发现，该融合伴侣显著改善了DKK2多肽的表达、生化特性和药代动力学（PK）。具体而言，人血清白蛋白（HSA）被鉴定为一种高效的融合伴侣。通过HSA-DKK2变体对DKK2中选定氨基酸残基进行取代，旨在减少硫酸乙酰肝素结合，这进一步改善了该分子在啮齿动物中的PK特性。HSA-DKK2变体呈单体形式，热稳定性与野生型一样，并且通过其结合Wnt共受体LRP6并阻止其磷酸化的能力来衡量具有活性。我们通过工程改造得到了典型Wnt抑制剂DKK2的强效长效变体，适用于通过系统给药或在组织损伤部位局部给药来调控Wnt通路。
