Influence of the hinge region on complement activation, C1q binding, and segmental flexibility in chimeric human immunoglobulins.

Abstract

We have characterized a series of genetically engineered chimeric human IgG3 and IgG4 anti-dansyl (DNS) antibodies with identical antibody-combining sites but different hinge region amino acid compositions to determine how the hinge region influences Fab fragment segmental flexibility, Clq binding, and complement activation. Our data support the correlation between "upper hinge" length and Fab segmental flexibility; moreover, we confirm that a hinge region is essential for Clq binding and complement activation. However, the hinge length by itself is not sufficient for complement activity in IgG molecules. We have demonstrated that the IgG4 hinge, which imparts restricted segmental flexibility, reduces the ability of IgG3 molecules to activate complement. We also find that the IgG3 hinge region, which imparts greater segmental motion, is not sufficient to create complement activation activity in IgG4 anti-DNS antibodies. Finally, we conclude that (i) segmental motion is correlated with "upper hinge" length, (ii) hinge length and segmental flexibility is not enough to alter complement binding and activation, and (iMi) segmental flexibility does not correlate with proficiency to activate the complement cascade.

Intrachain disulfide bond in the core hinge region of human IgG4

“…These mutations of CDP571 conferred upon both antibodies the ability to form disulfide bonds between heavy chains, given that only trace amounts of half-IgG molecules assembled into tetramers by noncovalent interactions were observed. A serine to proline substitution in the core hinge region of a chimeric mouse/human IgG4 (Angal et al, 1993) and the substitution of the hinge sequence in an IgG4 human antibody with that from a human IgG3 antibody (Tan et al, 1990) also resulted in a dramatic reduction of the amount of noncovalently associated half-lgG observed. In agreement with studies on a monoclonal human IgG4 (Virella & Parkhouse, 1973), the inter-heavy chain disulfide linkages of CDP571 were shown to be more sensitive to reduction than the heavy-light chain bridge.…”

“…Experiments with recombinant and recombinantlchimeric constructs of monoclonal antibodies have shown that antibodies bearing hinge regions of the y4 subclass (IgG4) are secreted as both disulfide bond-linked tetramers and as half-IgG 407 molecules assembled into tetramers by noncovalent interactions (Morrison et at.. 1988: Colcher et al. 1989: Tan et al. 1990: Canfield & Morrison, 1991: King et at.. 1992: Angal et al, 1993.…”

The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen

“…The upper hinge region (DKTHT) of human IgG1 connects the Fab arms to the core segment and influences Fab-Fab flexibility; N-terminal residues from the upper hinge region are reported to interact with the C H1 domain. [17][18][19] The lower intact antibodies. 14,15 This flexibility enables IgG molecules to bind antigens of a variety of shapes and sizes and also allows the effector domain (Fc) to bind the Fc receptor or complement.…”

Modulation of the Effector Functions of a Human IgG1 through Engineering of Its Hinge Region

“…Conversely, some reports have suggested that a reduction in the flexibility or length of the hinge region directly correlates with an increase in complement activation (2,(7)(8)(9)(10). Finally, other studies have indicated the lack of such a simple correlation (11)(12)(13). Likewise, the nature and properties of the hinge region also influence binding of the Fc to various Fc␥Rs as well as Ab-dependent cell-mediated cytotoxicity (ADCC) activity.…”