Supplementary Material for: Functional analysis of a novel complement C5a receptor 1-blocking monoclonal antibody

Introduction
The complement system anaphylatoxin C5a is a critical player in inflammation. By binding to complement C5a receptor 1 (C5aR1/CD88), C5a regulates many cellular functions, mainly as a potent pro-inflammatory inducer, such as cytokine release, cellular motility, and homeostasis. We describe the generation and selection of a potent antagonistic C5aR1 mouse monoclonal antibody (mAb).

Methods and results
Initial C5aR1 hybridoma clone selection was performed with a cell binding study on human whole blood. The iLite® C5a assay was used to assess C5aR1 inhibition of isolated C5aR1 mAbs, which led to the selection of a particular C5aR1 mAb (clone 18-41-6). Specificity of mAb 18-41-6 for C5aR1 was demonstrated on C5aR1his- and C5aR2his-expressing Flp-In™-CHO cells. C5aR1 mAb 18-41-6 directly inhibited C5a-mediated C5aR1 receptor activation in a PMN calcium flux assay. Full-size and/or F(ab’)2 preparations of mAb 18-41-6 were found to significantly reduce the expression of the activation markers CD11b and CD66b in both a PMN C5a stimulation assay and a whole blood model stimulated with Escherichia coli.

Conclusion
Our results demonstrate that mAb 18-41-6 is a valuable tool for investigating the C5a-C5aR1 axis and a potential therapeutic candidate for inflammatory disease treatment.

## 引言
补体系统的过敏毒素C5a是炎症进程中的关键参与者。C5a通过与补体C5a受体1（Complement C5a Receptor 1, C5aR1/CD88）结合，调控多种细胞功能，其核心作用为强效促炎诱导因子，可介导细胞因子释放、细胞迁移及细胞稳态维持。本研究阐述了一株强效拮抗型C5aR1小鼠单克隆抗体（monoclonal antibody, mAb）的制备与筛选过程。

## 方法与结果
初始C5aR1杂交瘤克隆的筛选通过人全血细胞结合实验完成。本研究采用iLite® C5a检测试剂盒，对分离得到的C5aR1单克隆抗体的C5aR1抑制活性进行评估，最终筛选得到一株特异性C5aR1单克隆抗体（克隆号18-41-6）。通过在表达C5aR1组氨酸融合蛋白（C5aR1his）与C5aR2组氨酸融合蛋白（C5aR2his）的Flp-In™-CHO细胞中开展实验，证实了单克隆抗体18-41-6对C5aR1的特异性。在多形核粒细胞（polymorphonuclear leukocytes, PMN）钙流实验中，该抗体可直接抑制C5a介导的C5aR1受体活化。研究发现，完整全长型或F(ab’)2片段型的单克隆抗体18-41-6制剂，在多形核粒细胞C5a刺激实验以及大肠杆菌（Escherichia coli）刺激的全血模型中，均可显著降低活化标志物CD11b与CD66b的表达水平。

## 结论
本研究结果证实，单克隆抗体18-41-6是研究C5a-C5aR1信号轴的优质工具，同时也是炎症疾病治疗的潜在候选治疗药物。