Interaction analysis of glycoengineered antibodies with CD16a: a native mass spectrometry approach

Minor changes in the quality of biologically manufactured monoclonal antibodies (mAbs) can affect their bioactivity and efficacy. One of the most important variations concerns the N-glycosylation pattern, which directly affects an anti-tumor mechanism called antibody-dependent cell-meditated cytotoxicity (ADCC). Thus, careful engineering of mAbs is expected to enhance both protein-receptor binding and ADCC. The specific aim of this study is to evaluate the influence of terminal carbohydrates within the Fc region on the interaction with the Fc gamma RIIIa/CD16a receptor in native and label-free conditions. The single mAb molecule comprises variants with minimal and maximal galactosylation, as well as alpha 2,3 and alpha 2,6-sialic acid isomers. Here, we apply native electrospray ionization mass spectrometry to determine the solution-phase antibody-receptor equilibria and by using temperature-controlled nanoelectrospray, a thermal stability of the complex is examined. Based on these, we prove that the galactosylation of a fucosylated Fc region increases the binding to CD16a 1.5-fold when compared with the non-galactosylated variant. The alpha 2,6-sialylation has no significant effect on the binding, whereas the alpha 2,3-sialylation decreases it 1.72-fold. In line with expectation, the galactoslylated and alpha 2,6-sialylated mAb:CD16a complex exhibit higher thermal stability when measured in the temperature gradient from 20 to 50 degrees C. The similar binding pattern is observed based on surface plasmon resonance analysis and immunofluorescence staining using natural killer cells. The results of our study provide new insight into N-glycosylation-based interaction of the mAb:CD16a complex.