In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5
| Autor: | Mark J. Evans, John P. Mueller, Steven H. Nye, Russell P. Rother, Stephen P. Squinto, Allen J. Norin, James A. Wilkins, Galo A Grijalva, Dennis W. Wolff, Denise M Therrien, Scott A. Rollins |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
medicine.drug_class
Molecular Sequence Data Immunology Immunoglobulin Variable Region Blood Pressure chemical and pharmacologic phenomena Monoclonal antibody Hemolysis law.invention Pathogenesis Mice Heart Rate law In vivo medicine Animals Humans Amino Acid Sequence Complement Activation Molecular Biology Base Sequence biology Chemistry Myocardium Antibodies Monoclonal Complement C5 medicine.disease Macaca mulatta Molecular biology Recombinant Proteins In vitro Complement system Perfusion Recombinant DNA biology.protein Antibody |
| Zdroj: | Molecular Immunology. 32:1183-1195 |
| ISSN: | 0161-5890 |
| DOI: | 10.1016/0161-5890(95)00099-2 |
| Popis: | Complement activation has been implicated in the pathogenesis of several human diseases. Recently, a monoclonal antibody, (N19-8) that recognizes the human complement protein C5 has been shown to effectively block the cleavage of C5 into C5a and C5b, thereby blocking terminal complement activation. In this study, a recombinant N19-8 scFv antibody fragment was constructed from the N19-8 variable regions, and produced in both mammalian and bacterial cells. The N19-8 scFv bound human C5 and was as potent as the N19-8 monoclonal antibody at inhibiting human C5b-9-mediated hemolysis of chicken erythrocytes. In contrast, the N19-8 scFv only partially retained the ability of the N19-8 monoclonal antibody to inhibit C5a generation. To investigate the ability of the N19-8 scFv to inhibit complement-mediated tissue damage, complement-dependent myocardial injury was induced in isolated mouse hearts by perfusion with Krebs-Henseleit buffer containing 6% human plasma. The perfused hearts sustained extensive deposition of human C3 and C5b-9, resulting in increased coronary artery perfusion pressure, end-diastolic pressure, and a decrease in heart rate until the hearts ceased beating approximately 10 min after addition of plasma. Hearts treated with human plasma supplemented with either the N19-8 monoclonal antibody or the N19-8 scFv did not show any detectable changes in cardiac performance for at least 1 hr following the addition of plasma. Hearts treated with human plasma alone showed extensive deposition of C3 and C5b-9, while hearts treated with human plasma containing N19-8 scFv showed extensive deposition of C3, but no detectable deposition of C5b-9. Administration of a 100 mg bolus dose of N19-8 scFv to rhesus monkeys inhibited the serum hemolytic activity by at least 50% for up to 2 hr. Pharmacokinetic analysis of N19-8 scFv serum levels suggested a two-compartment model with a T1/2 alpha of 27 min. Together, these data suggest the recombinant N19-8 scFv is a potent inhibitor of the terminal complement cascade and may have potential in vivo applications where short duration inhibition of terminal complement activity is desirable. |
| Databáze: | OpenAIRE |
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