Conjugation of staphylokinase with the arabinogalactan-PEG conjugate: Study on the immunogenicity, in vitro bioactivity and pharmacokinetics

Abstract

Staphylokinase (SAK) is a bacterial protein with profibrinolytic activity. However, SAK suffers from short serum half-life and high immunogenicity. PEGylation with high Mw (20 kDa or 40 kDa) could decrease the immunogenicity and prolong the serum half-life of the proteins. However, the PEGylated protein could induce the anti-PEG antibodies and its bioactivity was significantly decreased. Arabinogalactan (AG) is a health-promoting substance with numerous biological activities. Conjugation of AG is an alternative strategy to solve the above-mentioned problems. However, conjugation with AG significantly decreased the bioactivity of a protein by shielding the bioactive domain. Here, AG conjugation and PEGylation were combined to improve the therapeutic efficacy of SAK. PEG with low Mw (2 kDa or 5 kDa) acted as a linker to conjugate AG from Larix. As compared with SAK-AG (22.3%), the conjugates (SAK-P2K-AG and SAK-P5K-AG) largely maintained the bioactivity of SAK (73.8% and 62.9%). The two conjugates both showed an 8-fold decrease in the SAK-specific IgG titers and a prolonged serum half-life. Moreover, the conjugates did not render any apparent toxicity to the heart, liver and renal functions of mice. Thus, our conjugation strategy is promising for the development of an effective long-acting therapeutic protein.

Introduction

Staphylokinase (SAK) is a bacterial protein with high profibrinolytic activity [1,2]. SAK has been potentially used for the therapy of coronary thrombosis or acute myocardial infarction [3,4]. However, SAK is an extrinsic protein with high immunogenicity and can strongly elicit the SAK-specific neutralizing antibodies, which lowers the therapeutic efficacy of SAK [5]. Besides, SAK suffers from short half-life and the repeated administrations are required to maintain the physiological effect of SAK. This renders great pain and mental stress to the patients. Thus, a long-acting SAK drug is highly desired to circumvent these problems.

PEGylation, chemical conjugation with polyethylene glycol (PEG), is a successful approach to decrease the toxicity, immunogenicity and the dosing frequency of a protein, along with prolonging its serum half-life [6,7]. PEGylation with high Mw PEG (20 kDa or 40 kDa) can possibly elicit PEG-specific immunogenicity and significantly lower the bioactivity of the protein [8]. For instance, PEG-asparaginase (Ovation, USA) and PEG-uricase (Savient, USA) can induce high level of anti-PEG antibodies and lead to the immune-mediated side effect [9,10]. The anti-PEG antibodies also accelerate the clearance of the PEGylated proteins [11].

Conjugation with human serum albumin, Fc domain of IgG and polysaccharides (PS) has been used as alternative strategies to solve this problem [[12], [13], [14]]. In particular, arabinogalactan is considered a health-promoting PS with a large variety of biological activities such as antivirus, hypoglycemic, antioxidant, and hypolipidemic effects [[15], [16], [17]]. Conjugation with PS enhanced the hydrophilic property, heat stability and hydrodynamic volume of a protein [18]. Conjugation with PS also prolonged the half-life time by avoiding renal clearance and reduced the immunogenicity of a protein by shielding the antigenic epitopes [18,19]. However, the bulky PS may drastically reduce the bioactivity of the protein by shielding its bioactive domain. Thus, a strategy is highly desired to overcome the opposite effect of prolonged plasma half-life and possessing low bioactivity of the PS-protein conjugate.

Recently, PEG with low Mw has been used as a linker to improve the immunogenicity of the meningococcal PS conjugate vaccine, where the distance between the meningococcal PS antigen and the carrier protein was elongated by the PEG linker [20]. This reduced the steric shielding effect of carrier protein on the epitopes of PS antigen. Recent studies suggested that the anti-PEG immune response depended on the immunogenicity of proteins, the extent of PEGylation, and the Mw of methoxyl PEG [8]. Due to its low Mw (2 kDa) and absence of methoxyl moiety, the PEG linker elicited very weak anti-PEG immune response. On one hand, the PEG linker could minimize the steric shielding effect of PS on SAK. On the other hand, the steric shielding effect of PEG was much lower than that of AG and could prolong the serum circulation of SAK.

In order to improve the therapeutic efficacy of SAK, arabinogalactan (AG) from Larix and PEG with low Mw (2 kDa or 5 kDa) were synergistically used for conjugation of SAK in the present study. A recombinant SAK was connected with Gly-Gly-Cys at C-terminus, which lacked the first 10 amino acids of native SAK. Heterobifunctional PEG (PEG2K or PEG5K) reagents were conjugated with AG, followed by covalent linkage with SAK. The C-terminus of SAK was far from the bioactive domain and used for site-specific conjugation with AG-PEG. The PEG linker could elongate the steric distance between AG and SAK, which decreased the steric shielding effect of AG. The structure, bioactivity, immunogenicity, toxicity and pharmacokinetics of the resultant SAK-containing samples were investigated in details. Our conjugation strategy is promising for development of an effective long-acting therapeutic protein.