SEQUENCES OF ANTIGENIC EPITOPES OF STREPTOKINASE IDENTIFIED VIA RANDOM PEPTIDE LIBRARIES DISPLAYED ON PHAGE

Though streptokinase (SK) is widely used to treat humans with thrombot ic disease, it is antigenic and anti-SK antibody causes allergic react ions and neutralizes SK's therapeutic effects. To pinpoint the fine st ructure of two immunodominant, continuous epitopes in SK, we used unco nstrained 15 and 6-mer random peptide libraries displayed on phage (th eoretical complexity of 3.2 x 10(19) and 0.64 x 10(8) unique sequences ). The first epitope, recognized by both human Ab and murine monoclona l (m)Abs, was previously localized to the amino terminus of SK. Repeat ed panning and selection experiments against a 15-mer peptide phage li brary, using a representative mAb (A2.5) to this epitope, identified a dominant structural motif (GP[R/L]WL) corresponding to amino acids 3 to 7 of native SK, which was consistent with previous epitope mapping. These findings were further confirmed by: (1) the fact that a synthet ic peptide spanning the epitope of A2.5 (AGPEWLL) specifically inhibit ed the binding of A2.5 to SK and (2) the finding that mAb 9D10, which competes with mAb A2.5 for binding to SK, independently selected, from a different random hexamer Library, an epitope sequence spanning resi dues 4 to 9 that overlaps the A2.5 epitope. Similar studies of the sec ond epitope in SK, which is immunodominant for murine but not human an tibodies, identified a consensus sequence KS(K/L)P(F/Y) corresponding to amino acids 59 to 63 of SK; this was confirmed by epitope peptide b inding experiments. This epitope is cleaved and destroyed when SK reac ts with human but not murine plasminogen. Thus, pinpointing the sequen ces of antigenic epitopes of SK: (1) provides a potential explanation for species differences in SK's antigenicity, (2) demonstrates the ove rlapping fine structure of epitopes recognized by competitive mAbs, (3 ) confirms previous epitope mapping studies and (4) has the potential to identify antigenic sequences that lead to allergic reactions in pat ients treated with SK. (C) 1997 Academic Press Limited.