Department of Clinical Immunology, Laboratory of Molecular Medicine, Section 7631, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark,
Department of Clinical Immunology, Laboratory of Molecular Medicine, Section 7631, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark.
J Innate Immun. 2023;15(1):836-849. doi: 10.1159/000535084. Epub 2023 Nov 10.
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).
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.
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).
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.
补体系统过敏毒素 C5a 是炎症反应的关键因子。通过与补体 C5a 受体 1（C5aR1/CD88）结合，C5a 调节许多细胞功能，主要作为一种有效的促炎诱导剂。我们描述了一种有效的拮抗 C5aR1 鼠单克隆抗体（mAb）的产生和选择。
最初的 C5aR1 杂交瘤克隆选择是在人全血中的细胞结合研究中进行的。使用 iLite® C5a 测定法进行了 C5aR1 mAb 对 C5aR1 抑制的评估。在表达 C5aR1his 和 C5aR2his 的 Flp-In™-CHO 细胞上研究了 C5aR1 mAb 的特异性。通过 C5a 驱动的钙通量测定和基于分离的多形核白细胞（PMN）和用大肠杆菌刺激的全血模型的刺激测定来评估生理 C5aR1 抑制。
针对 C5aR1 N 端部分的杂交瘤克隆的上清液在全血中显示出对 C5aR1 的有效结合，这在纯化的 mAb 中得到了证实。通过 iLite® C5a 测定法对内部 C5aR1 mAb 进行测定后，选择了 C5aR1 mAb 18-41-6。mAb 18-41-6 特异性针对 C5aR1。mAb 18-41-6 的全长和/或 F(ab')2 制剂被发现可有效地消除 C5a 诱导的中性粒细胞中的钙通量，并显著降低激活标志物 CD11b（中性粒细胞、单核细胞）和 CD66b（中性粒细胞）的上调。
我们的结果表明，mAb 18-41-6 是研究 C5a-C5aR1 轴的有价值的工具，也是炎症性疾病治疗的潜在治疗候选物。
