Abstract
Therapeutic monoclonal antibodies and Fc-fusion proteins containing antibody Fc fragment may tend to destabilize (e.g. unfold and aggregate), which leads to loss of functions and increase of adverse risks. Although engineering of an additional disulfide bond has been performed in Fc or Fc domains for optimization, the relationships between introduced disulfide bond and alteration of the stability, aggregation propensity and function were still unclear and should be addressed for achievement of better therapeutic outcome. Here, we constructed three human IgG1 Fc mutants including FcCH2-s-s- (one engineered disulfide bond in CH2 domain), FcCH3-s-s- (one engineered disulfide bond in CH3 domain), and FcCH3-s-s-CH2-s-s- (two engineered disulfide bonds in CH2 and CH3 domains, respectively) for evaluation. As expected, each mutated domain shows obviously increased stability during thermo-induced unfolding, and FcCH3-s-s-CH2-s-s- is most thermo-stable among wildtype Fc (wtFc) and three mutants. The order of overall stability against denaturant is FcCH3-s-s-CH2-s-s- > FcCH2-s-s- > FcCH3-s-s- > wtFc. Then the aggregation propensity was compared among these four proteins. Under conditions of incubation at 60 °C, their aggregation resistance is in the order of FcCH3-s-s-CH2-s-s- > FcCH2-s-s- > FcCH3-s-s- ≈ wtFc. In contrast, the order is FcCH3-s-s-CH2-s-s- > FcCH3-s-s- > FcCH2-s-s- ≈ wtFc under acidic conditions. In addition, the Fc-mediated functions are not obviously affected by engineered disulfide bond. Our results give a comprehensive elucidation of structural and functional effects caused by additional disulfide bonds in the Fc fragment, which is important for Fc engineering toward the desired clinical performance.
Highlights
Therapeutic monoclonal antibodies and Fc-fusion proteins containing antibody Fc fragment may tend to destabilize, which leads to loss of functions and increase of adverse risks
Because their theoretic molecular mass calculated by amino acid residues was ϳ24 kDa, and all of them should be glycosylated at Asn297, the size of corresponding bands was correct as shown in SDS-PAGE (Fig. 2A)
CH2 domain did not significantly change the aggregation propensity of FcCH2-s-s- at low pH compared with wildtype Fc (wtFc), it could cooperate with the additional disulfide bond in CH3 domain to further enhance the aggregation resistance (FcCCHH32--ss--ss-- versus FcCH3-s-s-)
Summary
CH2 domain were selected for induction of additional disulfide bonds, which results in various effects on stability and circulation in vivo [11]. The potential change of aggregation propensity of Fc fragment with extra disulfide bonds should be well evaluated. To address these issues, we constructed three Fc mutants with additional disulfide bonds in the CH2 domain, the CH3 domain, and both the CH2 and CH3 domains and expressed them in mammalian cells. A series of experiments were performed for evaluation of the influence of engineered disulfide bonds on structural and functional properties. Our results give straightforward evidence that Fc-based therapeutics could be improved through engineering of disulfide bonds in the Fc fragment
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