M Verstreken

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

BACKGROUND 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. METHODS AND RESULTS 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). CONCLUSIONS 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.

Accelerated Neointima Formation After Vascular Injury in Mice With Stromelysin-3 (MMP-11) Gene Inactivation

The hypothesis that stromelysin-3 (MMP-11), a unique member of the matrix metalloproteinase (MMP) family, plays a role in neointima formation was tested with the use of a vascular injury model in wild-type (MMP-11(+/+)) and MMP-11-deficient (MMP-11(-/-)) mice. Neointima formation 2 to 3 weeks after electric injury of the femoral artery was significantly enhanced in MMP-11(-/-) as compared with MMP-11(+/+) mice, in both mice of a pure 129SV genetic background (0.014 versus 0.0010 mm(2) at 2 weeks, P<0.001) and those of a 50/50 mixed 129SV/BL6 background (0.030 versus 0.013 mm(2) at 3 weeks, P<0.05). The medial areas were comparable, resulting in intima/media ratios that were significantly increased in MMP-11(-/-) as compared with MMP-11(+/+) arteries, in mice of both the 129SV (1. 0 versus 0.18, P<0.001) and mixed (1.5 versus 0.70, P<0.05) backgrounds. Nuclear cell counts in cross-sectional areas of the intima of the injured region were higher in arteries from MMP-11(-/-) mice than in those from MMP-11(+/+) mice (210 versus 48, P<0.001, in pure 129SV mice and 290 versus 150, P<0.01, in mice of the mixed genetic background). Immunocytochemical analysis revealed that alpha-actin-positive and CD45-positive cells were more abundant in intimal sections of MMP-11(-/-) mice. Degradation of the internal elastic lamina was more extensive in arteries of MMP-11(-/-) mice than in those of MMP-11(+/+) mice (39% versus 6.8% at 3 weeks, P<0. 005). The mechanisms by which MMP-11 could impair elastin degradation and cellular migration in this model remain, however, unknown.

Measurement of different forms of plasminogen activator inhibitor 1(PAI-1) using various monoclonal antibody-based enzyme-linked immunosorbent assays

Abstract Six monoclonal antibodies, MA-6H9, MA-7D4, MA-7F5, MA-8H4, MA-12A4 and MA-15H12, were raised against PAI-1. MA-6H9, MA-7D4 and MA-7F5 had an inhibitory effect on PAI-1 activity. Different combinations of these antibodies were used to construct various enzyme-linked immunosorbent assays (ELISA) and allowed the specific measurement of different molecular forms of PAI-1 (latent PAI-1, active PAI-1, t-PAoPAI-1 complex and/or degraded PAI-1). In plasma, obtained from healthy individuals, quantitation of PAI-1 yielded mean values (± SD) between 23 (± 11) and 38 (± 15) ng/ml, depending on the type of ELISA used. In platelets a value of 0.7 fg PAI-1/platelet was found, irrespective of the ELISA used.