Mapping of <scp>C5</scp> ‐Blocking Monoclonal Antibodies Reveals New <scp>C5</scp> Inhibitory Epitopes and Novel Modes of <scp>C5</scp> Inhibition

C5 inhibition is a proven therapeutic target with several monoclonal antibody (mAb) and small molecule drugs in the clinic. To explore alternative modes of inhibition, novel C5-blocking mAbs were generated and compared to therapeutic mAbs eculizumab and crovalimab. Blocking mAbs were selected using complement inhibition in haemolysis assays. C5 binding and competition between mAbs were tested by ELISA and surface plasmon resonance. Impact on C5 cleavage by CVF and native convertases was tested by western blotting of C5 fragments and ELISA for C5a release. Four novel C5-blocking mAbs were identified and shown to inhibit haemolysis with comparable efficacy to crovalimab and eculizumab. Competition assays identified four distinct function-blocking epitopes; mAb 7D4 and eculizumab competed for an α-chain epitope (epitope 1), while 10B6 and crovalimab competed for a β-chain epitope (epitope 2). mAbs 2B11 and 4G2 were not competitive with epitope 1/2 binders or each other, marking two distinct blocking α-chain epitopes (epitopes 3 and 4). Epitope 1 and epitope 2 binders blocked C5 convertase-mediated C5 cleavage and C5a release, while epitope 3 and epitope 4 binders permitted C5 cleavage, demonstrating distinct modes of inhibition. We identified four distinct C5 blocking epitopes; epitope 1 targeted by eculizumab and 7D4, epitope 2 targeted by crovalimab and 10B6 and epitopes 3 and 4 targeted by 2B11 and 4G2 respectively. Although all efficiently blocked haemolysis, epitope 3 and 4 binders permitted C5a release. We reveal (at least) four blocking epitopes on C5 and (at least) two distinct mechanisms of inhibition.