Abstract
Immunoglobulin G (IgG) can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.
Highlights
Immunoglobulin G (IgG) is the most abundant class of immunoglobulins in human serum
To evaluate the potential stabilizing effects of variable domain glycans, we first analyzed a chimeric anti-TNP antibody produced in our lab, and a vari ant carrying Fab glycans due to introduction of an N-linked glycosylation motif
We confirmed that the transition temperatures (Tm) of three therapeutic antibodies obtained by this analysis with ANS are in the range of previously published Tms [Figure S3 and Table S1 in Supplementary Material [25, 27, 28]]
Summary
Immunoglobulin G (IgG) is the most abundant class of immunoglobulins in human serum. IgGs can contain N-linked glycans in the variable domains, the so-called Fab glycans (Figure 1), in addition to the Fc glycans in the CH2 domains. To investigate the effects of Fab glycans on antibody stability in more detail, we compared the thermostability of adalimumab, a human anti-TNF antibody, with a panel of variants in which we systematically introduced Fab glycosylation sites at several positions across the variable domains corresponding to “progenitor” glycosylation sites as explained earlier. We found that several Fab glycovariants had a higher Tm compared with adalimumab without Fab glycans (NH59, NH82, and NH84, Figures 3B,C; Table S2 in Supplementary Material), which indicates that the Fab glycans introduced in these variants improved antibody stability. For adalimumab variant NH82, the stabilizing effects of the Fab glycans were confirmed using DSC (Figure 3F; Table S4 in Supplementary Material). The thermal unfolding profiles of multiple variants, including adalimumab NL37, where similar Tms were observed, did show a broader transition in comparison with adalimumab (Figures 3D,E; Table S2 in Supplementary Material). Our data indicate that sialic acid is not required for the improved stability, since removal of sialic acid by treatment with neuraminidase (Figure S4A in Supplementary Material) did not affect the Tm for adalimumab NH82 (Figures 3B,C; Table S2 in Supplementary Material)
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