RECOMBINANT STAPHYLOKINASE VARIANTS WITH ALTERED IMMUNOREACTIVITY .1.CONSTRUCTION AND CHARACTERIZATION

Background Recombinant staphylokinase offers promise for thrombolytic therapy in acute myocardial infarction, but it is immunogenic. Althoug h reduced immunogenicity of heterologous proteinaceous drugs by protei n engineering has not previously been reported, an attempt was made to achieve this in staphylokinase by site-specific mutagenesis. Methods and Results Biospecific interaction analysis of a panel of 17 murine m onoclonal antibodies against recombinant staphylokinase (SakSTAR varia nt) identified three nonoverlapping immunodominant epitopes; two of wh ich could be eliminated by substitution mutagenesis of clusters of two or three char ed amino acids with alanine. Circulating anti-staphylok inase antibodies elicited in patients by treatment with SakSTAR were i ncompletely (<90%) absorbed by these mutants. Therefore, the combinati on variants K35A,E38A,K74A,E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A, E80A,D82A (SakSTAR.M89) were constructed, expressed in Escherichia col i, highly purified by ion-exchange and hydrophobic interaction chromat ography, and characterized. These variants had specific activities tha t were approximately half that of SakSTAR, and they combined the reduc ed reactivity with the panels of monoclonal antibodies of their parent molecules. Absorption of circulating antibodies elicited in patients by treatment with SakSTAR was incomplete in 13 of 16 patients (median values, 68% and 65% with SakSTAR.M38 and SakSTAR.M89, respectively). C onclusions SakSTAR contains three immunodominant epitopes, two of whic h were eliminated by site-directed mutagenesis, yielding combination m utants with relatively maintained specific activities that were not re cognized by a significant fraction of the antibodies elicited in patie nts by treatment with wildtype SakSTAR. These mutants appear to be sui table for more detailed investigation of their thrombolytic and antige nic properties.