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

Site directed mutagenesis (350 variants) of recombinant staphylokinase (SakSTAR), a potent fibrin-selective thrombolytic agent, was undertaken in order to reduce its antigenicity while maintaining its potency. Variants with K35A, (ie, Lys[K] in position 35 substituted with Ala[A]), E65D or E65Q, K74...

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Bibliographic Details
Published in:Blood 2000-08, Vol.96 (4), p.1425-1432
Main Authors: Laroche, Yves, Heymans, Stephane, Capaert, Sophie, De Cock, Frans, Demarsin, Eddy, Collen, Désiré
Format: Article
Language:English
Subjects:
Antigen Presentation
B-Lymphocytes - immunology
Biological and medical sciences
Blood. Blood coagulation. Reticuloendothelial system
Epitopes, B-Lymphocyte - immunology
Fibrinolytic Agents - adverse effects
Fibrinolytic Agents - immunology
Humans
Medical sciences
Metalloendopeptidases - adverse effects
Metalloendopeptidases - genetics
Metalloendopeptidases - immunology
Mutagenesis, Site-Directed
Pharmacology. Drug treatments
Recombinant Proteins - adverse effects
Recombinant Proteins - genetics
Recombinant Proteins - immunology
Structure-Activity Relationship
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Summary:Site directed mutagenesis (350 variants) of recombinant staphylokinase (SakSTAR), a potent fibrin-selective thrombolytic agent, was undertaken in order to reduce its antigenicity while maintaining its potency. Variants with K35A, (ie, Lys[K] in position 35 substituted with Ala[A]), E65D or E65Q, K74R or K74Q, E80A+D82A, K130T, and K135R displayed increased enzymatic activity or reduced binding of human staphylokinase-specific antibodies. Additive mutagenesis identified 8 variants with intact thrombolytic potencies, which absorbed down to less than a third of SakSTAR-specific antibodies. Intra-arterial administration in 61 patients with peripheral arterial occlusion caused no significant allergic reactions. Median neutralizing antibody titers (with 15 to 85 percentiles), expressed as microgram (μg) compound neutralized per milliliter plasma, were 4.4 (0.3 to 49) for the variants, compared with 12 (4 to 100) in 70 patients given wild-type SakSTAR (P = .002 by Mann-Whitney rank sum test). Overt neutralizing antibody induction (more than 5 μg compound neutralized per milliliter plasma) was observed in 57 of 70 patients (81%) given wild-type SakSTAR, but only in 28 of 60 patients (47%) 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 further clinical development.
ISSN:0006-4971
1528-0020
DOI:10.1182/blood.V96.4.1425