Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv

Mol Immunol. 1996 Dec;33(17-18):1389-401. doi: 10.1016/s0161-5890(96)00078-8.

Abstract

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5G1.1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5G1.1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5G1.1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5G1.1 scFv also effectively blocked C5a generation. Finally, an intact humanized h5G1.1 antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5G1.1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Blocking / genetics
  • Antibodies, Blocking / pharmacology
  • Antibodies, Monoclonal / biosynthesis
  • Antibodies, Monoclonal / immunology
  • Aorta
  • Base Sequence
  • Binding, Competitive / immunology
  • Chickens
  • Complement C5 / immunology*
  • Complement C5a / immunology
  • Complement Inactivator Proteins / pharmacology*
  • Complement Membrane Attack Complex / antagonists & inhibitors
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Erythrocytes / immunology
  • Humans
  • Immunoglobulin Fragments / pharmacology*
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / pharmacology*
  • Molecular Sequence Data
  • Recombinant Fusion Proteins / immunology*
  • Recombinant Fusion Proteins / pharmacology
  • Swine

Substances

  • Antibodies, Blocking
  • Antibodies, Monoclonal
  • Complement C5
  • Complement Inactivator Proteins
  • Complement Membrane Attack Complex
  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Recombinant Fusion Proteins
  • immunoglobulin Fv
  • Complement C5a