Chemical coupling of a monoclonal antisurfactant protein-B antibody to human urokinase for targeting surfactant-incorporating alveolar fibrin.
Intraalveolar fibrin formation is a common histopathological finding in acute inflammatory and chronic interstitial lung diseases. Incorporation of hydrophobic surfactant components into polymerizing fibrin results in a severe loss of surface activity, altered mechanical and structural clot properties, and a reduced susceptibility toward fibrinolytic degradation. Such events have been implicated in atelectasis formation, impairment of gas exchange, and provocation of fibroproliferative changes. In an effort to address the unique features of alveolar fibrin, we designed a hybrid molecule consisting of a monoclonal antibody against surfactant protein SP-B (8B5E) and the catalytic domain of urokinase (B-chain), which was termed MABUC. The urokinase B-chain was prepared by limited reduction of human two-chain-urokinase and subsequent affinity purification and coupled to the antibody using a heterobifunctional cross-linker. Purification of the chimeric protein included gel filtration chromatography and affinity chromatography. An ELISA-like microtiter plate assay, based on the immunological detection of the SP-B moiety and the fibrinolytic activity of the u-PA domain, was developed for the detection of the hybrid molecule. Chromogenic substrate assays, (125)I-based fibrin plate assays, and active site titration were performed to analyze the specific fibrinolytic activity of the conjugate. MABUC was found to fully retain the ability of SP-B binding and the fibrinolytic activity of u-PA. In addition, MABUC was noted to be 1.5-2-fold more effective in the dissolution of surfactant embedding clots and to be approximately 3-fold more resistant against PAI-1, the predominant fibrinolysis inhibitor in the alveolar compartment, as compared to the native u-PA. The superiority of MABUC was particularly prominent (>5-fold efficacy) when investigating clot material incorporating both PAI-1 and surfactant, as a mimicry of alveolar fibrin. We conclude that urokinase and 8B5E can be cross-linked chemically, thus yielding a fibrinolytic enzyme with enhanced substrate specifity for surfactant-containing clots and higher PAI-1 resistance as compared to native u-PA.
Ruppert
Clemens
C
Department of Internal Medicine, Justus-Liebig-University Giessen, Giessen, Germany.
Schmidt
Reinhold
R
Grimminger
Friedrich
F
Suzuki
Yasuhiro
Y
Seeger
Werner
W
Lehr
Claus-Michael
CM
Günther
Andreas
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry; D016503:Drug Delivery Systems; D005337:Fibrin / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000737:chemistry*; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000494:pharmacology*; D008168:Lung / Q000378:metabolism; D011014:Pneumonia / Q000188:drug therapy; D011498:Protein Precursors / Q000276:immunology*; D011510:Proteolipids / Q000276:immunology*; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D005343:Fibrinolytic Agents; D018796:Immunoconjugates; D011498:Protein Precursors; D011510:Proteolipids; C105511:surfactant protein B propeptide; D005337:Fibrin; D014568:Urokinase-Type Plasminogen Activator