Recombinant staphylokinase variants with reduced antigenicity due to elimination of B-lymphocyte epitopes

Site directed mutagenesis (350 variants) of recombinant staphylokinase (Sak STAR), a potent fibrin-selective thrombolytic agent, was undertaken in orde r to reduce its antigenicity while maintaining its potency. Variants with K 35A, (ie, Lys[K] in position 35 substituted with Ala[A]), E65D or E65Q, K74 R or K74Q, E80A+D82A, K130T, and K135R displayed increased enzymatic activi ty or reduced binding of human staphylokinase-specific antibodies. Additive mutagenesis identified 8 variants with intact thrombolytic potencies, whic h absorbed down to less than a third of SakSTAR-specific antibodies. Intra- arterial administration in 61 patients with peripheral arterial occlusion c aused no significant allergic reactions. Median neutralizing antibody titer s (with 15 to 85 percentiles), expressed as microgram (mu g) compound neutr alized per milliliter plasma, were 4.4 (0.3 to 49) for the variants, compar ed with 12 (4 to 100) in 70 patients given wild-type SakSTAR (P = .002 by M ann-Whitney rank sum test). Overt neutralizing antibody induction (more tha n 5 mu g compound neutralized per milliliter plasma) was observed in 57 of 70 patients (81%) given wild-type SakSTAR, but only in 28 of 60 patients (4 7%) treated with variants (P < .0001 by Fisher exact test). On the basis of this study, the Variant SakSTAR (K35A, E65Q, K74R, D82A, S84A, T90A, E99D, T101S, E108A, K109A, K130T, K135R) (code SY155) has been selected for furt her clinical development. (Blood. 2000;96:1425-1432) (C) 2000 by The Americ an Society of Hematology.