Alpha1-Antichymotrypsin Present in Therapeutic C1-Inhibitor Products Competes with Selectin - Sialyl LewisX Interaction
Authors: Engel Ruchira , Nunez Laura Delvasto , Roem Dorina , van Mierlo Gerard , Holst Stephanie , Van Oers Marinus H.J. , Wuhrer Manfred , Wouters Diana , Zeerleder Sacha 
Published: October 2019

BACKGROUND: The plasma protein C1-inhibitor (C1-inh), belongs to the serpin superfamily and is the major inhibitor of the proteases of the complement and contact phase pathways. Hereditary or acquired deficiency of functional C1-inh results in angioedema episodes in affected individuals due to uncontrolled contact pathway activation and therapeutic C1-inh products are effective treatment for these patients. Therapeutic C1-inh products have also been shown to attenuate neutrophil activation and infiltration in various inflammatory conditions. This 'novel' anti-inflammatory effect of C1-inh is attributed to its non-serpin N-terminal domain. This domain is thought to express the tetrasaccharide, sialyl Lewisx (SLeX), through which C1-inh can interact with selectins on inflamed endothelium and prevent neutrophil rolling. However, C1-inh products contain small but significant amounts of co-purified proteins, the major one being the glycoprotein α1- antichymotrypsin (ACT), which is also an anti-inflammatory serpin. The potential influence of the glycans of ACT on SLeX - selectin interactions is not clear.

METHOD: We investigated the presence of SLeX -like epitopes on C1-inh and ACT from commercially available therapeutic C1-inh preparations using western blotting and mass-spectrometry. The influence of the products and separated C1-inh and ACT on SLeX -selectin interaction was investigated in an a model system where SLeX -beads were rolled on immobilized E-selectin molecules.

RESULT: We do not find any evidence of SLeX on C1-inh using either western blotting with anti-SLeX antibodies or by mass spectrometric analysis of C1-inh N- glycans. C1-inh products show modest but significant interference in SLeX -selectin interaction but surprisingly this is not observed for 'pure C1-inh' obtained from gel-filtration of the commercial product. On the contrary, ACT, also from the C1-inh product, shows the presence of SLeX -like epitopes, as detected by the antibody HECA-452 on western blot. In addition, at concentrations present in C1-inh products (20 -150 μg ACT/ mg active C1-inh), ACT can interfere with SLeX -selectin interactions, in a sialic acid dependent manner. These concentrations of ACT can be achieved in vivo with a dose of as low as 2000 U of a C1-inh product, suggesting that ACT can contribute to the anti-inflammatory effects observed in studies with C1-inh products.

CONCLUSION: We conclude that the 'novel' anti-inflammatory effects of C1-inh are unlikely due to SLeX and can in fact be partly due to ACT. This fresh evidence challenges a long held assumption and paves the way for development of ACT, alone or in combination with C1-inh, as a new anti-inflammatory therapeutic.

Disclosures

Engel: ViroPharma Inc.: Research Funding. Nunez:ViroPharma Inc.: Research Funding. Roem:ViroPharma Inc.: Research Funding. van Mierlo:ViroPharma Inc.: Research Funding. Wouters:ViroPharma: Research Funding. Zeerleder:ViroPharma: Other: Receives an unrestricted grant from Viropharma.