Prolonging the circulatory half-life of C1 esterase inhibitor via albumin fusion

Abstract

Hereditary Angioedema (HAE) is an autosomal dominant disease characterized by episodic swelling, arising from genetic deficiency in C1-esterase inhibitor (C1INH), a regulator of several proteases including activated Plasma kallikrein (Pka). Many existing C1INH treatments exhibit short circulatory half-lives, precluding prophylactic use. Hexahistidine-tagged truncated C1INH (trC1INH lacking residues 1-97) with Mutated N-linked Glycosylation Sites N216Q/N231Q/N330Q (H6-trC1INH(MGS)), its murine serum albumin (MSA) fusion variant (H6-trC1INH(MGS)-MSA), and H6-MSA were expressed in Pichia pastoris and purified via nickel-chelate chromatography. Following intravenous injection in mice, the mean terminal half-life of H6-trC1INH(MGS)-MSA was significantly increased versus that of H6-trC1INH(MGS), by 3-fold, while remaining ~35% less than that of H6-MSA. The extended half-life was achieved with minimal, but significant, reduction in the mean second order rate constant of Pka inhibition of H6-trC1INH(MGS)-MSA by 33% relative to that of H6-trC1INH(MGS). Our results validate albumin fusion as a viable strategy for half-life extension of a natural inhibitor and suggest that H6-trC1INH(MGS)-MSA is worthy of investigation in a murine model of HAE.

Fig 1. C1-esterase inhibitor (C1INH) recombinant proteins.

A) Schematic diagram of pdC1INH and recombinant proteins. Numbers relate to C1INH mature protein except as otherwise specified. Proteins are named at right. B) Gel and immunoblot analysis of purified H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA. Total protein amounts of 1 μg were electrophoresed on 10% SDS-PAGE gels that were stained with Coomassie Brilliant Blue (left) or decorated with specific antibodies identified below the panels (left centre, right centre, and right).

Fig 2. Complex characterization of C1INH recombinant proteins.

A) i. Gel-based assays of complex formation. 10% SDS-PAGE gels are shown electrophoresed under reducing conditions and stained with Coomassie Brilliant Blue. Serpins (pdC1INH, H₆-trC1INH(MGS), or H₆-trC1INH(MGS)-MSA) were reacted with Pka at a 5:1 molar ratio for 5-minutes at 37°C. B) Graph of binding assay between pdC1INH:Pka and H₆-trC1INH(MGS)-MSA:Pka at varying ratios (500 ng-1 μg) with PBS-T washing condition of 1M NaCl and 1% Tween-20. The mean and SD of 3 determinations is shown.

Fig 3. Protein clearance of H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA.

A) Logarithmic-linear plot of H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA percentage residual protein in plasma versus time in hours post-injection. B) Linear plot of H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA percentage residual protein in plasma versus time in hours post-injection. C) Percentage residual protein in plasma at 1-hour post-injection. D) Percentage residual protein in plasma at 4-hours post-injection. E) Percentage residual protein in plasma at the times, in hours, specified on the x axis, following injection of H₆-trC1INH(MGS)-MSA (light grey bars) or H₆-MSA (dark grey bars). The mean of 6 determinations ± SD is shown in all cases. *, p < 0.05, ****, p<0.0001 for statistical comparisons indicated by horizontal bars (ns denotes non-significant).