Raucci Luisa, Perrone Cosimo Damiano, Barbera Stefano, de Boer Laurens Julius, Tosi Gian Marco, Brunetti Jlenia, Bracci Luisa, Pozzi Cecilia, Galvagni Federico, Orlandini Maurizio
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena 53100, Italy.
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena 53100, Italy; Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Rudbeck Laboratory, Uppsala SE-75185, Sweden.
Int J Biol Macromol. 2025 May;309(Pt 4):143118. doi: 10.1016/j.ijbiomac.2025.143118. Epub 2025 Apr 12.
CD93 is a receptor predominantly expressed on the surface of endothelial cells, where it plays a pivotal role in angiogenesis through its interaction with the extracellular matrix. In our previous studies, we identified the monoclonal antibody 4E1 as a potent inhibitor of angiogenesis by targeting the CD93-Multimerin-2 axis. Here, we report the development of 4E1 as a recombinant whole immunoglobulin and a single-chain variable fragment, designated sc-4E. Both formats retained the binding properties of the parental monoclonal antibody and exhibited comparable inhibitory effects on endothelial cell migration and differentiation. To elucidate the molecular basis of the 4E1-CD93 interaction, we initially employed machine learning-based modeling and docking analyses of the variable heavy and light domains of 4E1. Subsequent crystallographic analysis of sc-4E provided high-resolution structural insights, confirming and validating the predicted model. Further docking experiments and molecular dynamics simulations using the crystallographic structures of CD93 and sc-4E revealed that the interaction is primarily mediated by the CDR-H3 and CDR-L2 loops. Notably, these regions engage with the sushi-like domain of CD93, which is critical for its interaction with Multimerin-2. This comprehensive structural and functional characterization of 4E1 and sc-4E underscores their potential as anti-angiogenic agents. By effectively inhibiting endothelial cell migration and differentiation, 4E1 derivatives represent promising therapeutic candidates for the treatment of ocular vascular diseases driven by pathological angiogenesis.
CD93是一种主要在内皮细胞表面表达的受体，它通过与细胞外基质相互作用在血管生成中发挥关键作用。在我们之前的研究中，我们鉴定出单克隆抗体4E1是一种通过靶向CD93 - 多聚体蛋白-2轴来有效抑制血管生成的物质。在此，我们报告了4E1作为重组全免疫球蛋白和单链可变片段（命名为sc-4E）的研发情况。这两种形式都保留了亲本单克隆抗体的结合特性，并对内皮细胞迁移和分化表现出相当的抑制作用。为了阐明4E1与CD93相互作用的分子基础，我们最初采用基于机器学习的4E1重链和轻链可变区建模与对接分析。随后对sc-4E的晶体学分析提供了高分辨率的结构见解，证实并验证了预测模型。使用CD93和sc-4E的晶体结构进行的进一步对接实验和分子动力学模拟表明，这种相互作用主要由互补决定区H3（CDR-H3）和互补决定区L2（CDR-L2）环介导。值得注意的是，这些区域与CD93的寿司样结构域相互作用，这对其与多聚体蛋白-2的相互作用至关重要。对4E1和sc-4E的这种全面的结构和功能表征突出了它们作为抗血管生成剂的潜力。通过有效抑制内皮细胞迁移和分化，4E1衍生物代表了治疗由病理性血管生成驱动的眼部血管疾病的有前景的治疗候选物。
