Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody
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Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody. / Cyranka, Leon; Mariegaard, Ida; Skjødt, Mikkel Ole; Bayarri-Olmos, Rafael; Mollnes, Tom Eirik; Garred, Peter; Rosbjerg, Anne.
In: Journal of Innate Immunity, Vol. 15, No. 1, 2023, p. 836-849.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody
AU - Cyranka, Leon
AU - Mariegaard, Ida
AU - Skjødt, Mikkel Ole
AU - Bayarri-Olmos, Rafael
AU - Mollnes, Tom Eirik
AU - Garred, Peter
AU - Rosbjerg, Anne
N1 - Publisher Copyright: © 2023 The Author(s).
PY - 2023
Y1 - 2023
N2 - 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. We describe the generation and selection of a potent antagonistic C5aR1 mouse monoclonal antibody (mAb). Methods: 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® 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 Escherichia coli. Results: 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® C5a assay. The mAb 18-41-6 was specific for C5aR1. Full-size and/or F(ab')2 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). 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.
AB - 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. We describe the generation and selection of a potent antagonistic C5aR1 mouse monoclonal antibody (mAb). Methods: 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® 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 Escherichia coli. Results: 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® C5a assay. The mAb 18-41-6 was specific for C5aR1. Full-size and/or F(ab')2 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). 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.
KW - C5a
KW - C5aR1
KW - Complement system
KW - Inflammation
KW - Monoclonal antibody
U2 - 10.1159/000535084
DO - 10.1159/000535084
M3 - Journal article
C2 - 37952515
AN - SCOPUS:85181178791
VL - 15
SP - 836
EP - 849
JO - Journal of Innate Immunity
JF - Journal of Innate Immunity
SN - 1662-811X
IS - 1
ER -
ID: 379715440