PASylated Coversin, a C5-Specific Complement Inhibitor with Extended Pharmacokinetics, Shows Enhanced Anti-Hemolytic Activity in Vitro.
The Ornithodoros moubata Complement Inhibitor (OmCI) binds complement component 5 (C5) with high affinity and, thus, selectively prevents proteolytic activation of the terminal lytic complement pathway. A recombinant version of OmCI (also known as Coversin and rEV576) has proven efficacious in several animal models of complement-mediated diseases and successfully completed a phase Ia clinical trial. Coversin is a small 17 kDa lipocalin protein which has a very short plasma half-life if not bound to C5; therefore, the drug requires frequent dosing. We have improved the pharmacokinetics of Coversin by N-terminal translational conjugation with a 600 residue polypeptide composed of Pro, Ala, and Ser (PAS) residues. To this end, PAS-Coversin as well as the unmodified Coversin were functionally expressed in the cytoplasm of E. coli and purified to homogeneity. Both versions showed identical affinity to human C5, as determined by surface plasmon resonance measurements, and revealed similar complement inhibitory activity, as measured in ELISAs with human serum. In line with the PEG-like biophysical properties, PASylation dramatically prolonged the plasma half-life of uncomplexed Coversin by a factor ≥50 in mice. In a clinically relevant in vitro model of the complement-mediated disease paroxysmal nocturnal hemoglobinuria (PNH) both versions of Coversin effectively reduced erythrocyte lysis. Unexpectedly, while the IC50values were comparable, PAS-Coversin reached a substantially lower plateau of residual lysis at saturating inhibitor concentrations. Taken together, our data demonstrate two clinically relevant improvements of PASylated Coversin: markedly increased plasma half-life and considerably reduced background hemolysis of erythrocytes with PNH-induced phenotype.
Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München , Emil-Erlenmeyer-Forum 5, 85354 Freising (Weihenstephan), Germany.
Nadine
N
Kuhn
Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ulm University , Helmholtzstrasse 20, 89081 Ulm, Germany.
Christoph Q
CQ
Schmidt
Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München , Emil-Erlenmeyer-Forum 5, 85354 Freising (Weihenstephan), Germany.|XL-protein GmbH , Lise-Meitner-Strasse 30, 85354 Freising, Germany.
Martin
M
Schlapschy
Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München , Emil-Erlenmeyer-Forum 5, 85354 Freising (Weihenstephan), Germany.|XL-protein GmbH , Lise-Meitner-Strasse 30, 85354 Freising, Germany.
Arne
A
Skerra
D000818:Animals; D002942:Circular Dichroism; D003182:Complement C5 / Q000037:antagonists & inhibitors; D051056:Complement Inactivating Agents / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects; D005260:Female; D006457:Hemoglobinuria, Paroxysmal / Q000188:drug therapy; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D020349:Surface Plasmon Resonance
D003182:Complement C5; D051056:Complement Inactivating Agents; D011994:Recombinant Proteins