ABSTRACT
BACKGROUND: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. OBJECTIVE: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. METHODS: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. RESULTS: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. CONCLUSIONS: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.
Subject(s)
Antibodies, Monoclonal/pharmacology , Complement C2/antagonists & inhibitors , Complement Inactivating Agents/pharmacology , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/pharmacokinetics , Calcium , Complement Activation/drug effects , Complement C2/analysis , Complement C2/metabolism , Complement Inactivating Agents/blood , Complement Inactivating Agents/pharmacokinetics , Epitope Mapping , Female , Humans , Hydrogen-Ion Concentration , Macaca fascicularis , MaleABSTRACT
We describe a method for high-throughput, parallel purification of secreted proteins to analyse large numbers of protein samples in cell-based assays for the discovery of protein therapeutics. The procedure is generic and capable of 96 parallel purifications and compatible, in both yield and purity, with a wide assay range. By optimising expression and purification steps as well as using novel hardware, in particular a chromatography press capable to purify target proteins from viscous media, we exemplify the process for the generation of single-chain Fv antibody fragments (scFv) and the purification of full-length IgG. The described process can operate robustly with a throughput of over 2,000 samples per month.