针对自免疫疾病的新型多聚Fc融合蛋白的优化设计与验证研究

Autoimmune diseases are among the most prevalent diseases affecting human health and are a major source of pain and mortality in China. Indeed, there is a high and presently unmet need for effective and safe medications that target these illnesses with the ability to offer new action mechanisms. Fc-fusion proteins have recently emerged as one of the most promising new classes of protein drugs, exhibiting the highest growth rate in overall sales among all biologics. To improve the effectiveness of these monomeric proteins and expand their capabilities, our group recently pioneered the concept of polymeric Fc-fusions and successfully identified the first general means of linking together any Fc-fusion protein into a complex containing six Fc-fusions (hexa-Fc). We have preliminary data showing that hexa-Fc, without any targeted improvement, is already ~50 times more potent than intravenous immunoglobin (IVIG), the most generally applied medication against autoimmune diseases, in a mouse model of the autoimmune disease, idiopathic thrombocytopenia, providing clear evidence of the significant therapeutic potential for this unique platform..In this project, we will develop hexa-Fc as therapy in two directions: with fused peptides, targeting specific diseases with specific fused peptides, and without fused peptides, as a biosynthetic replacement for IVIG. For the former, we will target two autoimmune diseases that are of particular concern in China, arthritis and type-1 diabetes. Using fused peptides of TNFR2 (a TNF-receptor that functions by binding the inflammatory cytokine TNFα) or GLP-1 (a well-known antihyperglycemic of recent interest as potential type-1 diabetes therapy), we will study the two key, ultimately structural, problems limiting the effectiveness of hexa-Fc for use against specific diseases: (1) how to suppress a general “long distance” inhibitory effect of the fused peptide on the binding to the critical receptor, FcRn (a problem even for monomeric Fc-fusions) and (2) how to maximize simultaneous binding of the fused peptides to their cognate partners. For the latter, towards its development as a replacement for IVIG, we will study the key problem of how to maximize the interaction between hexa-Fc and DC-SIGN, an essential mediator of the effects of IVIG in autoimmune diseases. .Using static and dynamic in silico experiments, atomic force microscopy structural validation, and force spectroscopy, surface plasmon resonance, ELISA, and cell-based assays, we will identify the fundamental design principles for the most maximally functional hexa-Fc complexes, specific and general. At the same time, our findings will optimize these fusion proteins as possible drugs for clinical trials, with the ultimate goal of producing highly effective, lower cost medication. Finally, these results will aid in the design of more effective antibody-based therapy as well as our understanding of antibody structure-function more generally.

Fc融合蛋白被认为是治疗自身免疫疾病最有前景的生物药物。为提高其有效性，我们率先提出了多聚Fc融合蛋白的概念，并通过动物实验初步证明六聚Fc的疗效大于IVIG的50倍。目前六聚Fc蛋白的两大研究方向是连接融合肽治疗特定疾病以及不连接融合肽而直接替代IVIG。针对两类我国高发的自身免疫疾病，类风湿关节炎（TNFR2）和I类糖尿病（GLP-1），我们拟解决构建具有融合肽的Fc多聚体的两个关键问题：如何有效抑制融合肽对Fc与受体相互作用的影响及如何优化多聚化后融合肽与靶蛋白的作用。对于IVIG的直接替代，我们拟系统解析IVIG中起核心作用的DC-SIGN与六聚Fc的相互作用。通过in silico模拟计算、原子力单分子结构分析、单分子力谱及细胞验证等多种手段，我们将系统地建立以功能最大化为目标的多聚Fc融合蛋白的设计原理，为构建更有效的抗体治疗提供基础，并最终实现高疗效低成本的目标。

本项目的总体目标是基于我们原创的Fc模板，结合分子动力学（MD）模拟、原子力显微镜以及生化和细胞学实验，探索获得治疗自身免疫性疾病最有效Fc多聚体的基本设计原理。为了实现上述目标，本项目开展了以下研究：（1）我们通过大量MD计算揭示了单铰链和双铰链Fc融合设计中可采用融合肽的结构范围以及这些结构在Fc结合FcRn受体过程中的潜在影响；（2）通过合作我们筛选制备了一系列突变体，这些突变体可以组装形成比原始Fc六聚体设计更多样化结构和化学计量比的多聚体，从而显著拓展了潜在的治疗应用范围，同时我们还证实了这些突变体与Fc受体（FcR）的结合增强效果，并解析了其中的结构特征；（3）本项目还研究发现表达这些突变体的细胞系对突变体与FcR相互作用的有效性至关重要，这可能是由于糖基化差异所致，由此我们鉴定出一种突变体，其可以以比目前抗血清低十倍的浓度达到同样的抑制乙型流感病毒效果。（4）本项目发展了一种基于生物膜层反射光干涉技术（BLI）有效定量表征多聚Fc复合物与各种配体结合的方法，获得了其相互作用大致自由能图景。（5）本项目通过合作发现我们制备的某些突变体在CNS自身免疫性疾病（慢性炎性脱髓鞘性多发性神经病（CIDP））离体模型中可以抑制抗体/补体介导的脱髓鞘，而且其所需浓度比目前静脉注射免疫球蛋白疗法（IVIG）低200倍。综上所述，本项目的研究结果不仅为设计治疗自身免疫性疾病的有效Fc多聚体复合物提供了基本原则，同时也为下一步研究多聚Fc-抗体复合物提供了可靠的方法。