Expression and characterization of truncated forms of humanized L243 IgG1 - Architectural features can influence synthesis of its oligosaccharide chains and affect superoxide production triggered through human Fc gamma receptor I

The properties of IgG and its subcomponents are being exploited to generate new therapeutics with selected biological activities. In this study, a ser ies of truncated, humanized IgG1 antibodies was expressed in Chinese hamste r ovary cells, to evaluate the contribution of structural components to gly cosylation and function. The series includes L243 IgG1 (alpha -MHC Class II ) lacking a C(H)3 domain pair (DeltaC(H)3-IgG1), single-chain Fv fusion pro teins with Fc or a hinge-C(H)2 domain, Fc with/out a hinge, and a single C( H)2 domain. Glycosylation of IgG Fc is important for recognition by effecto r ligands such as Fc gamma receptors. HPLC analysis of released and pyridyl aminated oligosaccharides indicates that intact IgG1 and scFvFc antibodies are galactosylated and sialylated to levels similar to those observed previ ously for normal human IgG1. The truncated forms express increased levels o f digalactosylated (30-83%) or sialylated (9-21%) oligosaccharide chains wi th the highest levels observed for the single C(H)2 domain. These data show which architectural components influence IgG glycosylation processing and that the (C(H)3)(2) pair is particularly influential. When MHC Class II bea ring (JY) cells were sensitized with L243 DeltaC(H)3-IgG1, scFvFc, or scFvh C(H)2 they elicited superoxide production, from U937 cells, at levels of 35 -45% relative to that obtained for intact L243 IgG1 (100%). Mild reduction and alkylation of the hinge disulphide bonds of scFvhC(H)2 greatly decrease d its capacity to trigger superoxide production. Thus, the L243 scFvhC(H)2 homo-dimer constitutes the minimal truncated form that binds the MHC Class II antigen and triggers superoxide production through Fc gamma RI.