Developments of human adrenomedullin-IgG1 Fc fusion proteins

Abstract

Human adrenomedullin (hAM) is a hypotensive peptide hormone that exerts powerful anti-inflammatory effects. However, treatment required continuous administration of hAM, as the half-life of native hAM is quite short in blood. To resolve this problem, we designed two kinds of human IgG1 Fc fusion proteins containing either full-length hAM (IgG1-AM) or hAM residues 6-52 [IgG1-AM (6-52)]. A DNA construct was constructed by connecting DNA sequences encoding hAM and the IgG1 Fc region with a DNA sequence encoding a (GGGGS)3 linker. The molecular weights of IgG1-AM and IgG1-AM (6-52) were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. By protein sequencing, the N-terminal sequence of both recombinant AM-Fc fusions showed the expected human IgG1 sequence. Sufficient concentrations of both AM-Fc fusions were observed in blood 2 days after a single subcutaneous administration. IgG1-AM and IgG1-AM (6-52) stimulated cAMP production in human embryonic kidney-293 cells stably expressing the AM1 receptor. The activity of IgG1-AM (6-52) was higher than that of IgG1-AM. Treatment with IgG1-AM (6-52) inhibited blood pressure increase in spontaneously hypertensive rats. In addition, IgG1-AM (6-52) reduced total inflammation scores in the dextran sulfate sodium colitis model. Therefore, AM-IgG1 Fc fusions represent potential novel therapeutic agents.