Rafael Bayarri‐Olmos

The complement system is a complex, tightly regulated protein cascade involved in pathogen defense and the pathogenesis of several diseases. Thus, the development of complement modulators has risen as a potential treatment for complement‐driven inflammatory pathologies. The enzymatically inactive MAP‐2 has been reported to inhibit the lectin pathway by competing with its homologous serine protease MASP‐2. The membrane‐bound complement inhibitor CD55 acts on the C3/C5 convertase level. Here, we fused MAP‐2 to the four N‐terminal domains of CD55 generating a targeted chimeric inhibitor to modulate complement activation at two different levels of the complement cascade. Its biological properties were compared in vitro with the parent molecules. While MAP‐2 and CD55 alone showed a minor inhibition of the three complement pathways when co‐incubated with serum (IC50 MAP‐2+CD55 1‐4 = 60.98, 36.10, and 97.01 nM on the classical, lectin, and alternative pathways, respectively), MAP‐2:CD55 1‐4 demonstrated a potent inhibitory activity (IC50 MAP‐2:CD55 1‐4 = 2.94, 1.76, and 12.86 nM, respectively). This inhibitory activity was substantially enhanced when pre‐complexes were formed with the lectin pathway recognition molecule mannose‐binding lectin (IC50 MAP‐2:CD55 1‐4 = 0.14 nM). MAP‐2:CD55 1‐4 was also effective at protecting sensitized sheep erythrocytes in a classical hemolytic assay (CH50 = 13.35 nM). Finally, the chimeric inhibitor reduced neutrophil activation in full blood after stimulation with Aspergillus fumigatus conidia, as well as phagocytosis of conidia by isolated activated neutrophils. Our results demonstrate that MAP‐2:CD55 1‐4 is a potent complement inhibitor reinforcing the idea that engineered fusion proteins are a promising design strategy for identifying and developing drug candidates to treat complement‐mediated diseases.