A fusion protein of the two tick-derived C5 inhibitors OmCI and RaCI: complete C5 inhibition despite strong...

The complement system must be tightly regulated to prevent tissue damage and disease, yet current C5 inhibitors like the anti-C5 monoclonal antibody Eculizumab - though clinically approved (e.g., for paroxysmal nocturnal haemoglobinuria, PNH) - fail to achieve complete terminal pathway blockade under conditions of strong complement activation. To address this limitation, we investigated tick-derived C5 inhibitors and discovered that combinatorial targeting of non-overlapping C5 epitopes enables full inhibition. Leveraging this insight, we engineered a bifunctional fusion protein RaCI-gly-OmCI (R-g-O), recombinantly merging OmCI (Ornithodoros moubata Complement Inhibitor) and RaCI (Rhipicephalus appendiculatus Complement Inhibitor), which bind distinct sites on C5. Surface plasmon resonance experiments demonstrate subnanomolar binding of the building blocks and the fusion protein to C5. In contrast to the building blocks OmCI and RaCI, R-g-O achieves complete C5 inhibition under strong complement activation conditions in an alternative pathway haemolysis experiment. In classical pathway haemolysis settings, OmCI and R-g-O completely protected from lysis while RaCI only achieved about 50% protection. The fusion protein R-g-O represents a highly effective tool for blocking terminal pathway activity in research, surpassing the limitations of single-agent C5 inhibitors.