Engineering Hydrophobic Protein鈥揅arbohydrate Interactions to Fine-Tune Monoclonal Antibodies

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• 作者：Xiaojie Yu ; Kavitha Baruah ; David J. Harvey ; Snezana Vasiljevic ; Dominic S. Alonzi ; Byeong-Doo Song ; Matthew K. Higgins ; Thomas A. Bowden ; Christopher N. Scanlan ; Max Crispin
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：July 3, 2013
• 年：2013
• 卷：135
• 期：26
• 页码：9723-9732
• 全文大小：782K
• 年卷期：v.135,no.26(July 3, 2013)
• ISSN：1520-5126

文摘

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, Fc纬RIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein鈥揷arbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for Fc纬RIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 脜, which revealed localized destabilization of this glycan鈥損rotein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on Fc纬RIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to Fc纬RIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man₅GlcNAc₂) glycoform, the individual effect of each mutation on Fc纬RIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (Fc纬RI, Fc纬RIIA, Fc纬RIIB, and Fc纬RIIIB). These data indicate that destabilization of the glycan鈥損rotein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for Fc纬R binding. Engineering of the protein鈥揷arbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.