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 Fcgamma receptor I.
The properties of IgG and its subcomponents are being exploited to generate new therapeutics with selected biological activities. In this study, a series of truncated, humanized IgG1 antibodies was expressed in Chinese hamster ovary cells, to evaluate the contribution of structural components to glycosylation and function. The series includes L243 IgG1 (alpha-MHC Class II) lacking a CH3 domain pair (DeltaCH3-IgG1), single-chain Fv fusion proteins with Fc or a hinge-CH2 domain, Fc with/out a hinge, and a single CH2 domain. Glycosylation of IgG Fc is important for recognition by effector ligands such as Fcgamma receptors. HPLC analysis of released and pyridylaminated oligosaccharides indicates that intact IgG1 and scFvFc antibodies are galactosylated and sialylated to levels similar to those observed previously for normal human IgG1. The truncated forms express increased levels of digalactosylated (30-83%) or sialylated (9-21%) oligosaccharide chains with the highest levels observed for the single CH2 domain. These data show which architectural components influence IgG glycosylation processing and that the (CH3)2 pair is particularly influential. When MHC Class II bearing (JY) cells were sensitized with L243 DeltaCH3-IgG1, scFvFc, or scFvhCH2 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 scFvhCH2 greatly decreased its capacity to trigger superoxide production. Thus, the L243 scFvhCH2 homo-dimer constitutes the minimal truncated form that binds the MHC Class II antigen and triggers superoxide production through FcgammaRI.
Lund
J
J
Department of Immunology, The Medical School, Birmingham, UK. J.Lund@bham.ac.uk
Takahashi
N
N
Popplewell
A
A
Goodall
M
M
Pound
J D
JD
Tyler
R
R
King
D J
DJ
Jefferis
R
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002240:Carbohydrate Sequence; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D013481:Superoxides / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009844:Oligosaccharides; D013481:Superoxides