A matched set of chimeric IgG1 and IgG4 antibodies were used to investigate the role of the IgG hinge in binding to Ag with differing space between the epitopes. Antibodies bearing identical V regions and either IgG1 or IgG4 C regions were engineered with and without hinges. We measured the binding of these antibodies to the peptide CYYYEEEEY and to CYYYEEEEY-BSA conjugates with decreasing numbers of peptides per BSA molecule. We earlier showed that V region differences in antibodies could affect Ag binding patterns in solid-phase but not solution-phase assays; however, both types of assay yielded similar results for the hinge-deleted antibodies. Binding of CYYYEEEEY-BSA by hinge-deleted and intact IgG1 was similar, but intact IgG1 bound free peptide better than did hinge-deleted IgG1. Intact IgG4 antibody bound less well to CYYYEEEEY and CYYYEEEEY-BSA than did IgG1 but, surprisingly, hinge-deleted IgG4 showed better binding than did intact IgG4 and was more like the IgG1 antibodies in binding affinity. Thus, the IgG4 hinge may impart a structural constraint that prevents high affinity binding to Ag. The hinge-deleted IgG4 antibody did not activate C, although it bound Ag similarly to IgG1. This study is the first to address the effect of the IgG hinge on Ag binding by using well defined Ag with different epitope densities. Our results may provide an explanation for the apparent low affinity of IgG4 antibodies.
Chimeric antibodies to the synthetic polypeptide (Tyr, Glu)-Ala-Lys ((T,G)-A-L) were used to examine C activation by human IgG1. Two IgG1 antibodies, which contained mouse L chains and H chains with mouse V domains and human C domains, differed only in their VH domain. Ag binding and C activation by these antibodies were analyzed by ELISA. When limiting amounts of Ag were used in the assays, the antibodies required different quantities of Ag for optimal binding, suggesting that the antibodies bind to different epitopes on the (T,G)-A-L molecule. However, when competitive inhibition assays were performed with an optimal concentration of Ag, there were no differences in relative binding affinities for (T,G)-A-L or dissociation characteristics of the antibodies. C activation was examined at optimal Ag concentration to ensure equivalent binding of two IgG1 antibodies to Ag. After combination with immobilized Ag, these two antibodies bearing different V regions exhibited marked differences in the binding of C components C1q and C3d. When present in equal amounts in the assay, antibody 10B activated C and bound more C1q and C3d than antibody B11. These results indicate that V region differences can affect C activation by IgG.
Activation of C by immune complexes (IC) in tissues and the inflammatory consequences are major determinants in the pathogenesis of many autoimmune disorders. To assess the factors involved in C activation by such IC, we examined the binding of C components by chimeric IgG1 antibodies bound to immobilized Ag. We previously reported that alterations in the H chain V regions can affect the binding of first component of C (C1q) and a major breakdown product of the third C component (C3b) when otherwise identical antibodies were bound to immobilized (Tyr, Glu)-Ala-Lys. To evaluate C activation of these antibodies in well defined IC, we utilized a 9-amino acid peptide conjugated to BSA as Ag. The peptide:BSA conjugate was bound similarly by the two IgG1 antibodies which differed mainly in the CDR3 regions, but also in 9 other amino acids in the H chain V region. When soluble IC were prepared with the two antibodies, they activated C similarly. However, C activation by solid phase Ag:antibody complexes differed; we found that antibody 10B bound more C1q and C3b than antibody B11 did, unless the Ag was present at high density on the plates. These data suggest that the variable region differences affect C activation by these antibody when they are bound to immobilized Ag. Furthermore, these results underscore the differences in C activation by the same antibody depending upon whether the IC are free in solution or immobilized.
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