ZBiotech’s N-glycan microarray helps scientists develop novel nanobodies to distinguish IgG Fc glycoforms.

When glycans attach to a protein, they do so in a diversified manner. The same protein might contain different glycan structures at different glycosites, which confers the protein with various distinguishable glycoforms. Antibodies are among the proteins that have glycans attached to the polypeptide chain. Glycoforms of antibodies are especially important. Glycans that attach to the fragment crystallizable domain (Fc) of immunoglobulin G (IgG) fine-tune both complement and FcγR – mediated effector functions. For example, elevated galactosylation of IgG-Fc stimulates C1q binding and complement activity, and the degree of fucosylation determines FcγR binding and downstream antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Because Fc sialyation appears to be negatively correlated with the onset of inflammation and flares of autoimmune disease, targeting sialylation has emerged as a therapeutic option to treat autoimmune diseases. This particular area of study has gained much attention recently, but “robust“ tools to study antibody glycoforms are limited. In the paper published in the PNAS, Kao and colleagues identified a new class of nanobodies that preferentially recognize IgG glycoforms lacking its core-fucose or bearing terminal sialic acids. These nanobodies will be a new tool for studying IgG glycoforms in viro and in vivo.

Circumventing the challenges of animal immunization strategy, the yeast surface display platform allows nanobody discovery within two to three weeks. Harnessing the power of this technology, Kao and colleagues hoped to find IgG glycoform-specific nanobodies by screening a purely synthetic yeast display library with chemoenzymatically glycoengineered IgG glycoforms. They first identified a few parental clones recognizing the afucosylated or sialylated IgG-Fc glycoforms. The continuous affinity maturation process yielded a nanobody called B7, which specifically recognizes the afucosylated IgG with a 1,000-fold improvement in affinity. They further showed that B7 blocked IgG-FcγR interactions in vitro and in vivo with the capability to detect IgG glycoforms on live cells. As certain IgG glycoforms can serve as powerful markers of specific disease states, they assessed the feasibility of adapting B7 to prognostic and diagnostic assays for severe viral infection. B7-adapted biochemical analysis of patient serum samples identified a positive correlation between the level of afucosylated IgG1 and Covid-19 severity.

Unlike lectins specific for glycan residues, the binding specificity of B7 depends on both IgG-Fc backbone and glycan composition. To rule out binding to free glycans, Kao and colleagues evaluated B7 with our N-glycan array. As expected, B7 did not recognize any of the N-glycans regardless of their fucosylation status. As a positive control, lectin Aleuria Aurantia Lectin (AAL) recognized a range of fucosylated N-glycans.

Kao and colleagues used our N-glycan array to verify the binding specificity of nanobody B7. The N-glycan array can help determine antibody specificity to various free N-glycans.