In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5

Mol Immunol. 1995 Nov;32(16):1183-95. doi: 10.1016/0161-5890(95)00099-2.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / isolation & purification*
  • Antibodies, Monoclonal / pharmacology
  • Base Sequence
  • Blood Pressure / drug effects
  • Complement Activation / drug effects
  • Complement C5 / immunology*
  • Complement C5 / metabolism
  • Heart Rate / drug effects
  • Hemolysis / drug effects
  • Humans
  • Immunoglobulin Variable Region / chemistry
  • Immunoglobulin Variable Region / immunology*
  • Macaca mulatta
  • Mice
  • Molecular Sequence Data
  • Myocardium / immunology*
  • Myocardium / pathology
  • Perfusion
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology

Substances

  • Antibodies, Monoclonal
  • Complement C5
  • Immunoglobulin Variable Region
  • Recombinant Proteins

Associated data

  • GENBANK/L43067