Mechanism and engineering of IgG-based monoclonal antibody/receptor interactions

 描述(申请人提供)：片段结晶(Fc)区连接免疫球蛋白G(Ig G)的双重病原体鉴定和破坏特性。病原体调理定位FCS以激活免疫细胞上的促炎Fc受体(FcgRs)。连接在Fc上的天冬酰胺连接的(N)-葡聚糖是低亲和力FcgR产生结合所必需的，尽管目前尚不清楚Fc-N-聚糖如何促进Fc-γR结合，因为N-聚糖不直接接触FcgR。已有研究表明，N-葡聚糖提供了两个Fc结构域的最佳间距，稳定了Fc的四级结构以结合FcγR。来自我们实验室的证据表明，我们的假设不同。我们确定，远离FcγR结合位点的Fc氨基酸突变增加的Fc N-糖链运动与Fc？RIIIa亲和力呈负相关。只有Fc的一个区域，即包含N-糖基化位点的CE多肽环，由于这些Fc突变而受到干扰。这一结果导致了N-葡聚糖通过预组织CE环而影响FcγR亲和力的建议，而不是 通过优化FC区域定向。在这里，我们将通过使用溶液核磁共振光谱测量CE环的结构和运动来直接验证我们的假设，这些环的结构和运动通过多糖或蛋白质工程稳定的多种形式。CE环结构和运动的知识将被应用于重新设计Fc多肽，以产生与FcgRs保持高亲和力的糖基化Fc变异体。从这些研究中发现的免疫系统激活的分子细节对于了解多种疾病的过程将是重要的，并且将是通过工程技术增强治疗性单抗功能以治疗癌症、移植和自身免疫性疾病患者的关键。

 DESCRIPTION (provided by applicant): The fragment crystallizable (Fc) region links the dual pathogen identification and destruction properties of immunoglobulin G (IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fc¿ receptors (FcgRs) on immune cells. Asparagine-linked (N)-glycan attached to Fc is required for productive engagement of the low-affinity FcgRs, though it is not known how the Fc N-glycan contributes to FcγR binding because the N-glycan does not directly contact the FcgRs. It has been suggested that the N-glycan provides optimal spacing of two Fc domains, stabilizing the Fc quaternary structure to bind the FCγR. Evidence from our laboratory points to a different hypothesis. We determined that Fc N-glycan motion, increased by Fc amino acid mutations far from the FcγR binding site, negatively correlated with Fc¿RIIIa affinity. Only a single region of Fc, the CE polypeptide loop that contains the site of N-glycosylation, was perturbed as a result of these Fc mutations. This result led to the proposal that the N-glycan affects FcγR affinity by pre-organizing the CE loop, and not by optimizing Fc domain orientation. Here we will directly test our hypothesis by measuring the structure and motion of the CE loop, in multiple forms stabilized through glycan or protein engineering, using solution nuclear magnetic resonance spectroscopy. The knowledge of CE loop structure and motion will be applied to redesign the Fc polypeptide to generate aglycosylated Fc variants that maintain high affinity for FcgRs. The molecular details of immune system activation that will emerge from these studies will be important to understand the process of multiple diseases and will be critical to enhance therapeutic monoclonal antibody function through engineering to treat cancer, transplant and autoimmune disease patients.