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.