Vanderschueren S, Stassen J M, Collen D
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
J Cardiovasc Pharmacol. 1996 Jun;27(6):809-15. doi: 10.1097/00005344-199606000-00007.
Staphylokinase, a bacterial plasminogen activator, is a potent, highly fibrin-specific but antigenic thrombolytic agent in humans. In an effort to attenuate the antigenicity of wild-type staphylokinase (SakSTAR variant), 2 of its 3 immunodominant epitopes were altered by substituting clusters of 2 or 3 charged amino acids with alanine, yielding the mutant SakSTAR.M38 (K35A, E38A, K74A, E75A, R77A), which was less antigenic in inbred New Zealand White rabbits. In the present study, groups of 6 baboons (Papio hamadryas) were randomized to SakSTAR (group 1) or SakSTAR.M38 (group 2). The thrombolytic potencies of 50 micrograms/kg compound at baseline, assessed in an extracorporeal thrombosis model, were similar: 77 +/- 2.9% (mean +/- SEM) clot lysis in group 1 and 83 +/- 3.6% in group 2. Groups 1 and 2 were immunized subcutaneously at 2, 3, and 5 weeks with 500 micrograms SakSTAR or SakSTAR.M38, respectively. From 6 weeks, group 1 developed significantly more antibody-related neutralizing activity than group 2 (maximal titer at 8 weeks of 100 +/- 23 micrograms SakSTAR and of 22 +/- 7.1 micrograms SakSTAR.M38 neutralized per milliliter of plasma, respectively). Neutralizing activities subsequently decreased gradually to 10-20% of peak values at 18 weeks. At 6 weeks, both groups were resistant to thrombolysis with 50 micrograms/kg of either compound. Rechallenge at 18 weeks with 250 micrograms/kg of the immunizing compound showed a significantly better recovery of the thrombolytic potency of SakSTAR.M38 (68 +/- 4.5% clot lysis) than of SakSTAR (39 +/- 5.3% clot lysis). Neither agent degraded fibrinogen or depleted alpha 2-antiplasmin. Therefore, SakSTAR.M38 is comparably active and fibrin-specific but less antigenic than wild-type SakSTAR. These findings in outbred primates confirm and extend earlier observations in inbred rabbits and provide a basis for the further development of staphylokinase variants with reduced antigenicity in humans.
葡萄球菌激酶是一种细菌纤溶酶原激活剂，在人类中是一种强效、高度纤维蛋白特异性但具有抗原性的溶栓剂。为了减弱野生型葡萄球菌激酶（SakSTAR变体）的抗原性，通过用丙氨酸取代2个或3个带电荷氨基酸的簇，改变了其3个免疫显性表位中的2个，产生了突变体SakSTAR.M38（K35A、E38A、K74A、E75A、R77A），其在近交新西兰白兔中抗原性较低。在本研究中，将6只狒狒（阿拉伯狒狒）分为一组随机分为SakSTAR组（第1组）或SakSTAR.M38组（第2组）。在体外血栓形成模型中评估，50微克/千克化合物在基线时的溶栓效力相似：第1组的血栓溶解率为77±2.9%（平均值±标准误），第2组为83±3.6%。第1组和第2组分别在第2、3和5周皮下注射500微克SakSTAR或SakSTAR.M38进行免疫。从第6周开始，第1组产生的抗体相关中和活性明显高于第2组（第8周时最大滴度分别为每毫升血浆中100±23微克SakSTAR中和和22±7.1微克SakSTAR.M38中和）。中和活性随后逐渐下降至18周时峰值的10 - 20%。在第6周时，两组对50微克/千克的任何一种化合物溶栓均有抗性。在第18周用250微克/千克的免疫化合物再次激发时，SakSTAR.M38的溶栓效力恢复情况（血栓溶解率68±4.5%）明显优于SakSTAR（39±5.3%）。两种药物均未降解纤维蛋白原或消耗α2 - 抗纤溶酶。因此，SakSTAR.M38具有相当的活性和纤维蛋白特异性，但抗原性低于野生型SakSTAR。在远交灵长类动物中的这些发现证实并扩展了早期在近交兔中的观察结果，并为进一步开发在人类中抗原性降低的葡萄球菌激酶变体提供了基础。
