Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody
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Published:2023
Issue:1
Volume:15
Page:836-849
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ISSN:1662-811X
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Container-title:Journal of Innate Immunity
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language:en
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Short-container-title:J Innate Immun
Author:
Cyranka Leon,Mariegaard Ida,Skjødt Mikkel-Ole,Bayarri-Olmos Rafael,Mollnes Tom Eirik,Garred Peter,Rosbjerg Anne
Abstract
<b><i>Introduction:</i></b> 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. We describe the generation and selection of a potent antagonistic C5aR1 mouse monoclonal antibody (mAb). <b><i>Methods:</i></b> Initial C5aR1 hybridoma clone selection was performed with a cell-binding study in human whole blood. In-house C5aR1 mAb assessment for C5aR1 inhibition was done via the iLite<sup>®</sup> C5a assay. C5aR1 mAb specificity was investigated on C5aR1his- and C5aR2his-expressing Flp-In™-CHO cells. Physiological C5aR1 inhibition was assessed via a C5a-driven calcium flux assay and stimulation assay based on isolated polymorphonuclear leukocytes (PMNs) and a whole blood model stimulated with <i>Escherichia coli</i>. <b><i>Results:</i></b> The supernatant of hybridoma clones targeting the N-terminal section of C5aR1 displayed efficient binding to C5aR1 in whole blood, which was confirmed for purified mAbs. The C5aR1 mAb 18-41-6 was selected following the assay of in-house C5aR1 mAbs via the iLite<sup>®</sup> C5a assay. The mAb 18-41-6 was specific for C5aR1. Full-size and/or F(ab’)<sub>2</sub> preparations of mAb 18-41-6 were found to efficiently abrogate C5a-induced calcium flux in neutrophils and to significantly reduce the upregulation of the activation markers CD11b (neutrophils, monocytes) and CD66b (neutrophils). <b><i>Conclusion:</i></b> 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.
Subject
Immunology and Allergy