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. 2019 Jul 17;2(3):65-69.
doi: 10.1093/abt/tbz008. eCollection 2019 Jul.

Effect of allotypic variation of human IgG1 on the thermal stability of disulfide-linked knobs-into-holes mutants of the Fc for stable bispecific antibody design

Hiroki Akiba  1 , Reiko Satoh  1 , Satoshi Nagata  1 , Kouhei Tsumoto  1   2   3
Affiliations

Effect of allotypic variation of human IgG1 on the thermal stability of disulfide-linked knobs-into-holes mutants of the Fc for stable bispecific antibody design

Hiroki Akiba et al. Antib Ther. .

Abstract

Background: Disulfide-linked knobs-into-holes (dKiH) mutation is a well-validated antibody engineering technique to force heterodimer formation of different Fcs for efficient production of bispecific antibodies. An artificial disulfide bond is created between mutated cysteine residues in CH3 domain of human IgG1 Fc whose positions are 354 of the "knob" and 349 of the "hole" heavy chains. The disulfide bond is located adjacent to the exposed loop with allotypic variations at positions 356 and 358. Effects of the variation on the biophysical property of the Fc protein with dKiH mutations have not been reported.

Methods: We produced dKiH Fc proteins of high purity by affinity-tag fusion to the hole chain and IdeS treatment, which enabled removal of mispaired side products. Thermal stability was analyzed in a differential scanning calorimetry instrument.

Results: We firstly analyzed the effect of the difference in allotypes of the Fcs on the thermal stability of the heterodimeric Fc. We observed different melting profiles of the two allotypes (G1m1 and nG1m1) showing slightly higher melting temperature of G1m1 than nG1m1. Additionally, we showed different characteristics among heterodimers with different combinations of the allotypes in knob and hole chains.

Conclusion: Allotypic variations affected melting profiles of dKiH Fc proteins possibly with larger contribution of variations adjacent to the disulfide linkage.

Keywords: bispecific antibody; differential scanning calorimetry; disulfide bond; heterodimeric Fc; knobs-into-holes.

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Figures

Figure 1
Figure 1
Features of dKiH mutants of human IgG1 Fc with different allotypes prepared in this study. (A) Structures representing the position of amino acids of allotypic variations of CH3 domain of human IgG1 constant region with mutations introduced in disulfide-linked knobs-into-holes (dKiH) heterodimer. Model structure of dKiH-Fc-k03h03 was made by homology modeling and is superimposed on the crystal structure of dKiH-Fc-k01h01 (PDB ID 5HY9). CH3 heterodimer is viewed by the opposite side from CH2 domains. Green, knob-Fc (G1m1); pale green, knob-Fc (nG1m1); cyan, hole-Fc (G1m1); blue, hole-Fc (nG1m1). Drawn in the PyMOL Molecular Graphics System. Residues specific to the allotypes and dKiH mutants are shown and labeled. (B) A close-up view of the knob chain allotypic variations and artificial disulfide bond introduced in dKiH. (C ,D) Thermal denaturation profiles of the KiH-Fcs with different allotype analyzed in DSC. (C) dKiH-Fc-k01h01 and (D) dKiH-Fc-k03h03. Black, experimental curves; green and blue, fitting curves for the 1st and 2nd denaturation; red, sum of the two fitting curves.
See this image and copyright information in PMC

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