Comparative antigenicity of recombinant wild-type staphylokinase (SakSTAR) and a selected mutant (SakSTAR.M38) in a baboon thrombolysis model.
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.
Vanderschueren
S
S
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Stassen
J M
JM
Collen
D
D
D000818:Animals; D000939:Epitopes / Q000276:immunology; D005343:Fibrinolytic Agents / Q000276:immunology* / Q000494:pharmacology*; D006439:Hemodynamics / Q000187:drug effects; D008666:Metalloendopeptidases / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D010215:Papio; D011994:Recombinant Proteins / Q000276:immunology; D015912:Thrombolytic Therapy; D013997:Time Factors
D000939:Epitopes; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus