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Blood, 15 August 2000, Vol. 96, No. 4, pp. 1425-1432
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Recombinant staphylokinase variants with reduced antigenicity due
to elimination of B-lymphocyte epitopes
Yves Laroche,
Stephane Heymans,
Sophie Capaert,
Frans De Cock,
Eddy Demarsin, and
Désiré Collen
From the Center for Transgene Technology and Gene
Therapy, Flanders Interuniversity Institute for Biotechnology, KU
Leuven, Belgium.
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.
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