Abstract
An Mr 57,000 single-chain chimeric plasminogen activator, K12G0S32, consisting of a variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 and of a 33-kDa (amino acids Ala132 to Leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-PA-33k) was studied. K12G0S32, secreted by infected Spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography and gel filtration. It was obtained essentially as a single-chain molecule with a Ka = 5.5 x 10(9) M-1 for immobilized fragment D-dimer, similar to that of MA-15C5. The specific activity of both its single-chain and two-chain forms on fibrin plates was 100,000 IU/mg of urokinase-type plasminogen activator (u-PA) equivalent. Activation of plasminogen by two-chain K12G0S32 obeyed Michaelis-Menten kinetics with Km = 2.9 +/- 0.6 microM and a k2 = 3.7 +/- 0.6 s-1 (mean +/- S.D.; n = 3), as compared to Km = 12 microM and k2 = 4.8 s-1 for rtcu-PA-32k (recombinant low Mr two-chain u-PA consisting of amino acids Leu144 to Leu411). Single-chain K12G0S32 induced a dose- and time-dependent lysis of a 125I-fibrin-labeled human plasma clot immersed in citrated human plasma; 50% lysis in 2 h was obtained with 0.70 +/- 0.07 micrograms/ml (mean +/- S.D.; n = 5), as compared with 8.8 +/- 0.1 micrograms/ml for rscu-PA-32k (recombinant low Mr single-chain u-PA consisting of amino acids Leu144 to Leu411) (mean +/- S.D.; n = 3). With two-chain K12G0S32, 50% clot lysis in 2 h required 0.25 +/- 0.03 micrograms/ml (mean +/- S.D.; n = 3), as compared with only 0.62 +/- 0.04 micrograms/ml (mean +/- S.D.; n = 2) for rtcu-PA-32k. These results indicate that low Mr single-chain u-PA can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form.
Highlights
Characterization of a Chimeric Plasminogen Activator Consisting of a Single-chain Fv FragmentDerived from a Fibrin FragmentD-Dimerspecific Antibody and a Truncated Single-chain Urokinase*
We describe the construction and characterization of a single-chain chimericplasminogenactivator, K12GoS32,2inwhich the fibrin binding capacity of scFv-K12G0, was linked to recombinant single-chain u-PA (rscu-PA)-33k
Culture supernatants and purified proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) on 10-25% gradient gels according to Laemmli [27]
Summary
Characterization of a Chimeric Plasminogen Activator Consisting of a Single-chain Fv FragmentDerived from a Fibrin FragmentD-Dimerspecific Antibody and a Truncated Single-chain Urokinase*. Conjugation of KlzGoSsz, secreted by infected Spodopterafrugiperdu insect cells at a rate of 1.6 pg/106cells/48 h, was purified to homogeneity by ion-exchange chromatography andgel filtration. It was obtained essentiallyas a single-chain molecule with a K,= 5.6 X loQM" for immobilized fragment D-dimer, similar to thaotf MAl6C6. Thespecific activity of both its single-chain and single-chain or low M,two-chain urokinase with Fab' fragments of these antibodies [2] indicated that only univalent antibody binding is required for the observed antibody-mediated targeting
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