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Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.

ABSTRACT: 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, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein-carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan-protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan-protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein-carbohydrate 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.

SUBMITTER: Yu X

PROVIDER: S-EPMC3788586 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.

Yu Xiaojie X Baruah Kavitha K Harvey David J DJ Vasiljevic Snezana S Alonzi Dominic S DS Song Byeong-Doo BD Higgins Matthew K MK Bowden Thomas A TA Scanlan Christopher N CN Crispin Max M

Journal of the American Chemical Society 20130625 26

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, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein-carbohydrate interface (F241A, F243A, V262E, and V264E) increas ...[more]

PMID: 23745692

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Project description:Being able to predict and control concentrated solution properties for solutions of monoclonal antibodies (mAbs) is critical for developing therapeutic formulations. At higher protein concentrations, undesirable solution properties include high viscosities, opalescence, particle formation, and precipitation. The overall aim of this work is to understand the relationship between commonly measured dilute solution parameters, the reduced osmotic second virial coefficient b22 and the diffusion interaction parameter kD and liquid-liquid phase separation, which occurs at higher protein concentrations. For globular proteins such as lysozyme or γB-crystallin, the location of the liquid-liquid coexistence curve is controlled by the net protein-protein interaction, which is related to b22. Because many mAbs undergo reversible self-association due to forming highly directional interactions, it is not known if b22 can be used as a reliable predictor for LLPS since increasing the anisotropy in the interaction potential causes phase separation to occur at much stonger net protein-protein attractions or lower values of b22. Here, we map the coexistence curves for three mAbs, referred to as COE-01, COE-07, and COE-19, in terms of b22 and kD values. The measurements are carried out at a low salt condition near the pI, where protein-protein interactions are expected to be anisotropic due to the presence of electrostatic attractions, and under salting-out conditions at high ammonium sulfate concentrations, which is expected to reduce the anisotropy by screening electrostatic interactions. We also show that deviations from a linear correlation between b22 and kD can be used as an indicator of reversible self-association. Each of the mAbs under salting-out conditions follows the correlation supporting the hypothesis that protein-protein interactions are nonspecific, while deviations from the correlation occur for COE-01 and COE-19 under low salt conditions indicating the mAbs undergo reversible self-association. For five out of the six conditions, the onset of phase separation, as reflected by the reduced virial coefficient at the critical point b22c occurs in a small window -1.6 > b22c > -2.3, which is similar to what has been observed for lysozyme and for bovine γB-crystallin. Under low salt conditions, b22c ≈ -5.1 for COE-19, which we previously showed to self-associate into small oligomers. Our findings suggest that under conditions where mAb interactions are weakly anisotropic, such as occur at high salt conditions, phase separation will begin to occur in a small window of b22. Deviations from the window can occur when mAbs undergo reversible self-association, although this is not always the case and likely depends upon whether or not highly directional interactions are passivated in the oligomer formation. We expect fitting LLPS measurements to simplified interaction models for mAbs will provide additional insight into the nature of the protein-protein interactions and guide their development for calculating concentrated solution properties.

Dataset Information

Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.

Publications

Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.

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