Quantifying the Effect of Antibody N-Glycosylation on Innate Immune Functions: Design Implications for Therapeutic Antibodies

The development and structural manipulation of tumor-specific monoclonal antibodies (mAbs) have primarily focused on their antigen-binding fragment (Fab). This involves improving mAb efficacy to disrupt oncogenic signaling, e.g. via blocking multiple ligand-receptor interactions essential for tumor cell growth and survival. The anti-tumor effectiveness of mAbs, however, has been increasingly attributed to their ability to engage immune effector functions mediated by the constant fragment (Fc) portion of the antibodies. These functions sensitize tumor cells for subsequent killing via innate immune mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). This highlights the potential value of new ways to rationally engineer the Fc domain as a means to optimize the anti-tumor activity of mAbs.

Accumulating evidence suggests that patterns of N-linked glycosylation in the Fc domain of mAbs play a key role in regulating their effector functions. However, a systematic and quantitative understanding of how the complex patterns of Fc glycans control distinct immune effector mechanisms in tumor cells is lacking. A modeling framework, consisting of a system of ordinary differential equations, was developed to model the various combinations of binding of mAbs to the tumor cell antigens and the multiple Fc receptors on immune cells. The developed model recapitulates the effect of N-glycosylation by modifying kinetic binding affinities to Fc receptors. The parameters of the model were estimated using information from the literature as well as fitting to the data from preliminary experiments. Future experiments will test a representative panel of differently glycosylated synthetic cetuximab in a co-culture of colon cancer cell lines and a number of innate immune cells in vitro. This model will be able to predict the effect of N-glycosylation on the immune effector functions and offer engineering insights for enhancing mAb therapeutics.