Conjugation with Eight-Arm PEG Markedly Improves the In Vitro Activity and Prolongs the Blood Circulation of Staphylokinase

Abstract

Staphylokinase (SAK) is a profibrinolytic protein and can be used for therapy of acute myocardial infarction and coronary thrombosis. However, SAK suffers from a short serum half-life time (∼6 min) that limits its clinical application. PEGylation prolongs the half-life time of SAK, whereas it significantly decreases the bioactivity of SAK for the steric shielding effect of PEG. To improve the bioactivity and prolong the half-life time of SAK, 8-arm PEG maleimide (8-arm PEG) was used for conjugation of multiple SAK molecules in one entity. C terminus of SAK was engineered with cysteine residue, followed by reaction with the maleimide moieties of 8-arm PEG to obtain the conjugate (SAKp-PEG). Conjugation with 8-arm PEG retained the secondary structure of SAK, slightly perturbed the tertiary structure of SAK, and essentially maintained its in vitro bioactivity by the multivalence of SAK. Conjugation with 8-arm PEG increased the hydrodynamic volume and thus significantly prolonged the half-life time of SAK. SAKp-PEG elicited a 1.4-fold increase in the SAK-specific IgG titers as compared with SAK, and rendered no apparent toxicity to the cardiac, liver and renal functions of mice. Thus, multiple conjugation of a protein with 8-arm PEG was an effective strategy to develop a long-acting protein drug with improved bioactivity and prolonged blood circulation.

Structure‐Based Site‐Specific PEGylation of Fibroblast Growth Factor 2 Facilitates Rational Selection of Conjugate Sites

“…Even for protein therapeutics that are already commercially available, such as insulin and growth hormone, frequent administration is often required to achieve the desired efficacy. The most widely recognized and successful approach to improve protein pharmacokinetics is polyethylene glycol modification (PEGylation) . PEGylation is the covalent attachment of biocompatible, nontoxic polyethylene glycol (PEG) chains to bioactive substances .…”

“…The most widely recognized and successful approach to improve protein pharmacokinetics is polyethylene glycol modification (PEGylation). [2][3][4] PEGylation is the covalent attachment of biocompatible, DOI: 10.1002/biot.201900203 nontoxic polyethylene glycol (PEG) chains to bioactive substances. [5] This modification can effectively increase the in vivo circulation half-life by decreasing renal clearance and masking potentially immunogenic epitopes or protease cleavage sites.…”

Production of Long-Acting CNGRC–CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment

“…Although this shielding offers protection of the protein (enzyme) against proteolytic enzymes, it might cause some hindrance on reaching the receptor or the active site. 27 , 28 …”

Structural Biology and Protein Engineering of Thrombolytics

“…After disruption of the identified contacts by combinatorial mutagenesis, the immunogenicity of the best variant decreased to less than 30 % [283,[286], [287], [288], [289]]. Other engineering strategies using PEGylation [[290], [291], [292], [293], [294], [295]], glycosylation [296], protein fusion [288,[297], [298], [299], [300], [301]], and lipidification [302] provided variants of staphylokinase exhibiting higher efficiency, improved half-life, decreased immunogenicity, and a lower risk of reocclusion.…”