Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody C_H3 domain

Abstract

Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of b-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C H 3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C H 3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C H 3 dimer were similar, but differences were observed. The reduced C H 3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C H 3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C H 3 domain. Hence, these results may have implications for the stability of the intact antibody.Keywords: human antibody; C H 3 domain; disulfide bond; stability; dimerization With the recent advances in heterologous expression of antibodies, more than a dozen monoclonal antibodybased drugs have reached the market. Hundreds more are at various stages of research and development for the treatment of a wide range of diseases, from cancer to inflammation. A number of key scientific and technological advancements, such as the generation of fully human antibody by transgenic mice (Green 1999), have contributed to the successful development of antibody-based therapeutics. Stability and homogeneity of therapeutic antibodies are of prime importance for safety and efficacy. Undesired biochemical, structural, and conformational forms can lead to loss of efficacy and risk of adverse side effects.IgG1 (hereafter referred to as antibody) is a complex molecule, composed of two identical light (L) and heavy Reprint requests to: Masazumi Matsumura,

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“…[43][44][45] We have opined that engineering of the heterodimer such that it retains its thermal stability should function as an indirect indicator that the engineered design is equivalent to the wild type Fc in the interchain pairing and stabilization. In addition, proper folding and stability of the CH3 domain could also contribute to the monomer-dimer equilibrium, 45 thus a stable CH3 heterodimer improves the homogeneity of the engineered antibody.…”

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