Structure-based antigenic epitope and PEGylation improve the efficacy of staphylokinase

Abstract

Staphylokinase (Sak) holds promise for use in thrombolytic therapy for acute myocardial infarction. However, its immunogenicity is a major disadvantage under clinical conditions. PEGylation has become a sophisticated method to decrease that immunogenicity. In this report, according predicted epitope from the active center, five residues, including Gly79, Leu82, Lys84, Ala97, and Arg104 have been mutant as cysteine for mono PEGylation, respectively. According to the relative immunogenicity of Sak or its PEGylation derivatives, the amount of specific anti-Sak IgG antibodies elicited by PEGylation proteins, including C79G, C82L, C84K, C97A, and C104R in BALB/c mice decreased by approximately 15–75% each. PEGylated Sak derivatives showed a decrease of up to 75% in the immune reactivity in PEG-Sak-C104R. Thrombelastography experiments showed that two PEG-conjugated derivatives, PEG-Sak-C97A (Ly30, 68.14 ± 2.51%) and PEG-Sak-C104R (Ly30, 66.49 ± 5.97%), the LY30 of PEG-Sak-C97A, and PEG-Sak-C104R produced values very similar to those of wild-type Sak. The fibrin plate assays showed the bioactivity of PEG-Sak-C104R to exhibit the most activity approximately as much as urokinase (diameter of halo pattern, 18.6 ± 1.06 mm) and tPA (diameter of halo pattern, 17.2 ± 0.49 mm). The Sak PEGylation derivative PEG-Sak-C104R was also selected for further in vivo activity experimentation. The thrombolytic ability of PEG-Sak-C104R is a little lower than wild-type Sak, whereas, this PEGylated protein retained high activity suitable for thrombolytic therapy. Collectively, with the in vivo and in vitro experiments, the present study suggests that site mutant PEGylation, PEG-Sak-C104R, is a suitable type of PEGylation for clinical applications. Further optimization would help maintain the bioactivity and decrease the immunogenicity of staphylokinase.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer

“…The mono-PEGylation strategy ensures drug homogeneity and assures the quality of the drug [ 36 , 37 , 38 , 39 , 40 ]. BCA-M was genetically modified to replace a serine with a cysteine residue (Ser 161 → Cys 161 ) that would subsequently be used for the coupling of a 20 kDa PEG-maleimide.…”

Designs of Antigen Structure and Composition for Improved Protein-Based Vaccine Efficacy

“…However, chemical modification, such as PEGylation, is largely non-specific, thus resulting in unpredictable outcomes for vaccines. For this reason, it is mainly employed to reduce therapeutic protein antigenicity (141)(142)(143)(144). Ideally, immunogen surface should be considered for each individual vaccine in order to simultaneously direct antibody-mediated immune response toward important epitopes and prevent pre-existing antibody recognition of carrier-specific immunodominant regions.…”

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.…”

“…Modification with covalently linked DNA or RNA allows disruption of interactions between t-PA and LRP1 receptor with concurrent regulation of enzymatic activity [340,341]. PEGylation can reduce immunogenicity and increase the half-life of prokaryotic plasminogen activators [239,292]. Newly established methods for targeted delivery will be used to target the action of plasminogen activators into the thrombus with minimized side-effects.…”