RECOMBINANT STAPHYLOKINASE VARIANTS WITH ALTERED IMMUNOREACTIVITY .3.SPECIES VARIABILITY OF ANTIBODY-BINDING PATTERNS

Background The ''charged cluster-to-alanine'' substitution variants Sa kSTAR(K35A,E38A,K74A,E75A,R77A) and Sak STAR(K74A,E75A,R77A,E80A,D82A) , previously identified as SakSTAR.M38 and SakSTAR.M89, respectively, induce less antibody formation in patients than wild-type recombinant staphylokinase (SakSTAR), but their specific activities are reduced by 50%. Therefore, the effect of the reversal of one or more of these su bstituted amino acids on the ratio of activity to antigenicity was stu died. Methods and Results Fourteen mutants with one to four ''alanine- to-wild-type'' reversals were expressed in Escherichia coli and highly purified (>95%). In rabbits immunized with wild-type SakSTAR, the com bined K35,E38,K74,E75,R77 or K74,E75,R77,E80,E82 epitope accounted for only 30% of antibody absorption from plasma, and no clear immunodomin ant residue could be identified. In baboons immunized with SakSTAR, th e K35,E38 and K74,E75,R77 epitopes or the K74,E75,R77 and E80,D82 epit opes contributed equally to account for 50% of total antibody binding, but no immunodominant residues were apparent. In pooled plasma from p atients with peripheral arterial occlusion treated with wild-type SakS TAR, about 40% of the antibodies depended on K74 of epitope K74,E75,R7 7 for binding, whereas epitopes K35,E38 and E80,D82 had a negligible c ontribution toward antibody recognition. Conclusions The recognition p attern by SakSTAR variants of antibodies induced with wild-type SakSTA R differs markedly among species. This implies that a systematic evalu ation of re duced antigen recognition and antibody induction in humans will require the development of human or humanized systems. Surprisin gly, SakSTAR(K-74), with a single substitution of Lys74 with Ala, had an intact specific activity but did not absorb 40% of the antibodies i nduced in patients by treatment with wild-type SakSTAR.