GlaxoSmithKline, Structure Function Characterization, CMCA, Collegeville, Pennsylvania, USA.
GlaxoSmithKline, Structure Function Characterization, CMCA, Collegeville, Pennsylvania, USA.
J Biol Chem. 2021 Oct;297(4):101136. doi: 10.1016/j.jbc.2021.101136. Epub 2021 Aug 28.
Lack of simple and robust methods to determine complement activation in human serum induced by antigen-antibody complexes is a major hurdle for monitoring therapeutic antibody drug quality and stability. Dezamizumab is a humanized IgG1 monoclonal antibody that binds to serum amyloid P component (SAP) for potential treatment of systemic amyloidosis. The mechanism of action of Dezamizumab includes the binding of SAP, complement activation through classical pathway, and phagocytosis; however, the steps in this process cannot be easily monitored. We developed two novel methods to determine Dezamizumab-SAP complex-induced complement activation. Complement component 3 (C3) depletion was detected by homogeneous time-resolved fluorescence (HTRF), and C3a desArg fragment, formed after the cleavage of C3 to yield C3a followed by removal of its C-terminal arginine residue, was determined using Meso Scale Discovery (MSD) technology. We found that the presence of both Dezamizumab and SAP was required for complement activation via both methods. The optimal molar ratio of Dezamizumab:SAP was 6:1 in order to obtain maximal complement activation. The relative potency from both methods showed a good correlation to Dezamizumab-SAP-dependent complement component 1q (C1q) binding activity in Dezamizumab thermal-stressed samples. Both SAP and C1q binding, as determined by surface plasmon resonance and the two complement activation potency methods described here, reflect the mechanism of action of Dezamizumab. We conclude that these methods can be used to monitor Dezamizumab quality for drug release and stability testing, and the novel potency methods reported here can be potentially used to evaluate complement activity induced by other antigen-antibody complexes.
缺乏简单而稳健的方法来测定人血清中由抗原-抗体复合物诱导的补体激活，这是监测治疗性抗体药物质量和稳定性的主要障碍。Dezamizumab 是一种人源化 IgG1 单克隆抗体，与血清淀粉样蛋白 P 成分（SAP）结合，用于潜在治疗系统性淀粉样变性。Dezamizumab 的作用机制包括 SAP 的结合、经典途径的补体激活和吞噬作用；然而，这个过程中的步骤不易监测。我们开发了两种新的方法来确定 Dezamizumab-SAP 复合物诱导的补体激活。通过均相时间分辨荧光（HTRF）检测补体成分 3（C3）的耗竭，并用 Meso Scale Discovery（MSD）技术测定 C3 裂解后形成的 C3a desArg 片段，该片段随后去除其 C 末端精氨酸残基。我们发现，两种方法均需要同时存在 Dezamizumab 和 SAP 才能通过补体激活。为了获得最大的补体激活，Dezamizumab:SAP 的最佳摩尔比为 6:1。两种方法的相对效力与 Dezamizumab 热应激样品中依赖于 Dezamizumab-SAP 的补体成分 1q（C1q）结合活性具有良好的相关性。通过表面等离子体共振和这里描述的两种补体激活效力方法测定的 SAP 和 C1q 结合反映了 Dezamizumab 的作用机制。我们得出结论，这些方法可用于监测用于药物释放和稳定性测试的 Dezamizumab 质量，并且这里报道的新型效力方法可潜在地用于评估其他抗原-抗体复合物诱导的补体活性。
