PB0593 - Extending the Circulatory Half-life of an Engineered Kallikrein-Specific Inhibitor

Background: Plasma kallikrein (PK) is a coagulation factor that activates factor XII in the contact pathway and generates vasoactive bradykinin from kininogen. C1-esterase inhibitor (C1INH) controls PK. C1INH deficiency manifests as potentially life-threatening hereditary angioedema (HAE). Several drugs are licensed for HAE treatment and/or prophylaxis, including C1INH concentrates and recombinant Ecallantide (rEcall). rEcall is a small protein comprised of the first Kunitz domain of Tissue Factor Pathway Inhibitor substituted at 7 residues to make it PK-specific. rEcall is licensed for HAE treatment, but its short half-life prevents prophylactic use.

Aims: To compare the inhibition of PK and the in vivo clearance of hexahistidine-tagged rEcall (H6-rEcall) to H6-rEcall genetically fused to human serum albumin (H6-rEcall-HSA).

Methods: H6-rEcall (64 amino acids) and H6-rEcall-HSA (666 amino acids) were expressed in Pichia pastoris, secreted, and purified from conditioned media via nickel-chelate chromatography. PK activity was followed via amidolysis of S2302 and the inhibitory constant (Ki) was determined. Purified proteins were radiolabeled with 125I and injected intravenously into CD1 mice. Acid-precipitable radioactivity in timed blood samples was expressed as a percentage of the peak post-injection value. Values are means ± SD, n = 6. Statistical significance was assessed by Mann-Whitney tests.

Results: Fusion to HSA increased Ki values by 31% (Ki 2.9 ± 0.4 nM for H6-rEcall-HSA vs. 2.2 ± 0.1 nM for H6-rEcall, p = 0.008). 2h post-injection, 26 ± 2 % of H6-rEcall-HSA remained in circulation vs. 6 ± 2 % of that of H6-rEcall (p = 0.002).

Conclusion(s): Fusing HSA to H6-rEcall had minimal effect on PK inhibition. Clearance of H6-rEcall-HSA in vivo was ~4-fold slower than H6-rEcall, an effect less than that observed for similar fusion proteins. H6-rEcall may block determinants on HSA important for recycling via the neonatal Fc receptor. Fusion protein redesign will be required to slow rEcall clearance further.