Recombinant staphylokinase variants with altered immunoreactivity. I: Construction and characterization

• Published in: Circulation (New York, N.Y.) N.Y.), 1996-07, Vol.94 (2), p.197-206
• Main Authors: COLLEN, D, BERNAERTS, R, DECLERCK, P, DE COCK, F, DEMARSIN, E, JENNE, S, LAROCHE, Y, LIJNEN, H. R, SILENCE, K, VERSTREKEN, M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal; Base Sequence; Biological and medical sciences; Blood. Blood coagulation. Reticuloendothelial system; Chemical Phenomena; Chemistry, Physical; Epitopes - analysis; Fibrinolytic Agents - immunology; Humans; Medical sciences; Metalloendopeptidases - immunology; Metalloendopeptidases - isolation & purification; Molecular Sequence Data; Mutagenesis; Pharmacology. Drug treatments; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification
• ISSN: 0009-7322; 1524-4539
• DOI: 10.1161/01.CIR.94.2.197

Recombinant staphylokinase offers promise for thrombolytic therapy in acute myocardial infarction, but it is immunogenic. Although reduced immunogenicity of heterologous proteinaceous drugs by protein engineering has not previously been reported, an attempt was made to achieve this in staphylokinase by site-specific mutagenesis. Biospecific interaction analysis of a panel of 17 murine monoclonal antibodies against recombinant staphylokinase (SakSTAR variant) identified three nonoverlapping immunodominant epitopes, two of which could be eliminated by substitution mutagenesis of clusters of two or three charged amino acids with alanine. Circulating anti-staphylokinase antibodies elceted in patients by treatment with SakSTAR were incompletely (< 90%) absorbed by these mutants. Therefore, the combination variants K35A,E38A,K74A,E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A,E80A,D82A (SakSTAR.M89) were constructed, expressed in Escherichia coli, highly purified by ion-exchange and hydrophobic interaction chromatography, and characterized. These variants had specific activities that were approximately half that of SakSTAR, and they combined the reduced 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). SakSTAR contains three immunodominant epitopes, two of which were eliminated by site-directed mutagenesis, yielding combination mutants with relatively maintained specific activities that were not recognized by a significant fraction of the antibodies elicited in patients by treatment with wildtype SakSTAR. These mutants appear to be suitable for more detailed investigation of their thrombolytic and antigenic properties.