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

Abstract

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.

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

“…2 Thus, studies that attempt to explain this disparity have focused on the hinge region, where the sequence variation is highest. 2,[4][5][6][7][8][9][10][11][12][13][14] The unique lower hinge residues from an IgG2 have been placed into both an IgG1 and IgG3 antibody background 4,11,12,15 and assayed for binding to Fc receptors. The IgG1 mutant showed reduced binding to all tested Fc gamma receptors, however, the reduction observed was well below that of the wild-type IgG2/Fc receptors affinities.…”

“…Mutations outside of defined contact residues in the upper and middle hinge, as well as CH2, also have been shown to impact Fc receptor binding. 6,12 This has been attributed to either the length and flexibility of the hinge or interactions of the Fab region with the Fc portion of the antibody. 3,6 Although the crystal structure of a full-length human IgG1 antibody has been solved, a complete crystal structure of an IgG2 antibody does not yet exist.…”

“…6,12 This has been attributed to either the length and flexibility of the hinge or interactions of the Fab region with the Fc portion of the antibody. 3,6 Although the crystal structure of a full-length human IgG1 antibody has been solved, a complete crystal structure of an IgG2 antibody does not yet exist. 16 Models of IgG2 antibodies have been made using the IgG1 structure as a guide as well as the disulfide bond arrangements of IgG2 antibodies determined more than 30 years ago.…”

Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis

“…The interchain S S bonds are located in the hinge and the upper C H 2 domain that seems more flexible than other domains in the protein conformation (12,13). Therefore, the interchain S S bonds are much more sensitive to cleavage by S-sulfonation or by mild reduction followed by S-alkylation (reduction/alkylation) than are the intrachain S S bonds (14,15).…”

“…The interchain S S bonds help maintain the conformation of the hinge and the C H 2 domain of IgG1 Abs, which makes contact with FcgRs (12,13,16). Therefore, S-sulfonation and reduction/alkylation of mAbs are expected to modify their effector functions.…”