Reactive disulfide bonds in immunoglobulin G. A unique feature in serum proteins of different species

124

We report here the engineering of a humanized anti-human EphA2 mAb (mAb 12G3H11) in an effort to explore the relationship between the hinge of a human IgG1 and its effector functions. mAb 12G3H11, used here as a model, is directed against the human receptor tyrosine kinase EphA2, which is an actively investigated target for cancer therapy due to its up-regulation in many cancer cells. Various rational modifications were introduced into the hinge region of mAb 12G3H11. These mutations were predicted to modulate the hinge’s length, flexibility, and/or biochemical properties. We show that the upper and middle hinge both play important, although functionally distinct roles. In particular, middle hinge modifications predicted to decrease its rigidity or length as well as eliminating either one of its two cysteine residues had a strong negative impact on C1q binding and complement-dependent cytotoxicity. Disruption of covalent bonds between both H chains may account in part for these effects. We also describe middle hinge mutants with a significantly decreased ability to bind FcγRIIIA and trigger Ab-dependent cell-mediated cytotoxicity. Conversely, we also generated upper hinge mutants exhibiting an increase in C1q binding and complement-dependent cytotoxicity activity. Therefore, this approach represents a novel strategy to fine-tune the biological activity of a given human IgG1. We also define, for the first time in such a systematic fashion, the relationship between various characteristics of the middle and upper hinge and the corresponding effector functions.

Section: Discussionsupporting

confidence: 80%

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

Dall’Acqua¹,

Cook²,

Damschroder³

et al. 2006

124

“…These four Cys residues were assumed to be involved in forming inter-H chain disulfide bonds (24,25) although no definitive data have been presented demonstrating that this is indeed the case. In fact, human IgG2 has been reported to contain free SH-group(s) although the position of the free Cys has not been elucidated (26). Therefore, it is possible that not all four Cys form inter-H chain disulfide bonds and/or that some of the inter-H chain disulfide bonds are unstable, as is the case for human IgG4 (6, 7).…”

Section: Discussionmentioning

confidence: 99%

Human IgG2 Can Form Covalent Dimers

Yoo

Wims

Chan

et al. 2003

122

105

Unlike IgA and IgM, IgG has not yet been shown to form covalent polymers. However in the presence of specific Ag, murine IgG3 has been shown to polymerize through noncovalent interactions. In contrast to the noncovalent oligomers found with murine IgG3, we have detected covalent dimers in three different recombinant human IgG2 Abs produced in myeloma cells. Both IgG2,κ and IgG2,λ can form dimers. In addition, analysis of pooled human γ globulin and several normal sera revealed the presence of IgG2 dimers. The IgG2 dimers are in contrast to the noncovalent IgG dimers found in pooled sera of multiple donors resulting from idiotype/anti-idiotype (Id/anti-Id) interactions. Cyanogen bromide cleavage analysis suggests that one or more Cys residues in the γ2 hinge are involved in dimer assembly. The potential role of IgG2 dimers in immunity against carbohydrate Ags is discussed.

“…IgG1 appears to be the most susceptible isotype. Human IgG1 has been shown to have a reactive interchain disulfide that readily undergoes SH-catalyzed disulfide interchange (17), and this may facilitate the cleavage of the interchain disulfides. In addition, it has been shown that in human IgG1, the hinge peptide is fully exposed to solvent (18).…”

Section: Discussionmentioning

confidence: 99%

Influence of the Isotype of the Light Chain on the Properties of IgG

Montano

Morrison

2002

It is widely appreciated that the isotype of the H chain of the Ab molecule influences its functional properties. We have now investigated the contribution of the isotype of the L chain to the structural and functional properties of the Ab molecule. In these studies, the L chain variable region of a murine anti-dansyl Ab was joined to either human κ or λ constant region domains and expressed with mouse-human chimeric H chains of the four human IgG isotypes. The resulting Abs were secreted as fully assembled molecules although, as has been previously observed, IgG4 with either κ or λ L chains was also secreted as HL half-molecules. However, the isotype of the L chain can influence the kinetics of intracellular assembly with IgG1λ, IgG2λ, and IgG4λ assembling more slowly than their κ counterparts. The isotype of the L chain also influenced the susceptibility of the interchain disulfide bonds to attack by reducing agents with variable effects, depending on the isotype of the H chains. For IgG2, but not for IgG1, -3, and -4, the isotype of the L chain influenced the rate of clearance in mice, with IgG2λ having a shorter in vivo half-life than IgG2κ. Only slight differences were also observed between λ and κ molecules in their kinetics of binding to and dissociation from the hapten dansyl. These studies demonstrate that the isotype of the L chain has only a slight impact on the structural and functional properties of variable region identical Abs.