Chemical Coupling of a Monoclonal Antisurfactant Protein-B Antibody to Human Urokinase for Targeting Surfactant-Incorporating Alveolar Fibrin

Abstract

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.

Chemical crosslinking of urokinase to pulmonary surfactant protein B for targeting alveolar fibrin

“…And fourthly, when analyzing the sensitivity towards inhibition by PAI-1, the predominant alveolar urokinase antagonist, SPUC turned out to be more resistant than the native molecule, and ~ 5-fold higher concentrations of PAI-1 were necessary to gain the same degree of inhibition. Thus, SPUC was proven not only to be superior to urokinase but also to the urokinase-anti-SP-B antibody conjugate as recently described (51).…”

“…Similarly, care had to be taken not to impair the catalytic property of u-PA during the limited reduction of urokinase necessary for generation and isolation of the B-chain. For this purpose, the enzyme was stabilized by the presence of benzamidine, a known competitive inhibitor targeting the active moiety of u-PA during the reduction process as recently suggested (51,52). Isolation of the important disulfide bonds in close proximity to the catalytic triad.…”

Advanced Therapeutic Strategies for Chronic Lung Disease Using Nanoparticle-Based Drug Delivery

“…Traditional pharmacotherapy for chronic lung diseases can be classified into a few categories according to types of therapeutic agents. A variety of chemical drugs, peptides, antibodies, and genetic molecules (e.g., siRNA, shRNA, and miRNA) have been employed to treat the chronic lung diseases [ 7 , 8 , 9 , 10 ]. Unfortunately, most of chronic lung disease cannot be completely cured by pharmacotherapy alone.…”

Targeted delivery of proteins by nanosized carriers

“…Almost one century later, the generation of murine monoclonal antibodies (mAbs) made possible the use of mAbs in clinical research. 33,34 In most cases, antibodies and fragments or variants thereof, oligopeptides, carbohydrates, glycolipids, and folic acid were selected for targeting different organs and tissues, including brain capillaries, 35 central nervous system, [36][37][38][39][40] choroidal neovascularization, 41 skeletal muscles, 42 cancer cells, [43][44][45][46][47][48][49][50][51] intestines, 52 liver, [53][54][55][56][57] lung, 58,59 lymphocytes, 60 and the vascular endothelium. [61][62][63] The preparation of targeted drug delivery systems is largely based on random chemical conjugation of drug carriers directly to targeting proteins.…”