| Summary: | <p>Immunoglobulins play a key role in humoral immunity and have emerged as an important class of biotherapeutics. While the IgG1 backbone tends to be the conventional choice in these recombinant therapies, several therapeutics that focus on other immunoglobulin isotypes are being developed.</p> <p>With the exception of IgA Fc, the conserved glycan of IgD, IgM, IgG and IgE projects across the surface of an immunoglobulin domain with the two opposing glycans packing within the homodimeric Fc structure. This apparent similarity in structure is largely reflected in shared glycosylation status with most classes containing oligomannose-type glycosylation. However, IgG deviates from this pattern and displays complex-type glycans, albeit with characteristic restriction on glycan branching and the levels of galactosylation. Given that these conserved N-linked glycan sites show divergent processing states despite ostensibly having similar local environments within the Fc domains, we probed the impact of quaternary protein architectures in controlling immunoglobulin glycosylation. We expressed domain fragments of these four related immunoglobulin classes and examined their resulting <em>N</em>-glycosylation status by mass spectrometry and liquid chromatography. We also assess the impact of Fc-based oligomeric architecture on glycosylation. Together, we show that quaternary architecture influences glycosylation in both native and engineered immunoglobulin formats and is an important consideration in the optimization of Fc-based therapeutics.</p> <p>In the second study, we exploited the different cell recruiting properties of IgE and IgG Fc domains by combining them into a single biologic with a tandem Fc format. We developed a panel of engineered antibodies and examined their glycosylation, thermal stability and ability to simultaneously engage multiple receptors. We showed that the Fc domains function independently and are capable of mediating the cellular interactions of both antibody isotypes. Broadening the effector functions of trastuzumab and related antibodies may provide a route to improved therapies against secondary breast cancer.</p> <p>Finally, we also attempted to establish whether the conserved N394-linked glycans could be removed or must be preserved in IgE, especially when utilising this antibody class for oncology indications. We generated three variants of IgE trastuzumab: a wild-type construct carrying the full seven N-linked glycosylation sites, an Fc mutant N394S, and a ‘stabilised’ version of the Fc mutant N394S. We examined their glycosylation, thermal and storage stability, receptor binding properties, and ability to mediate monocytic killing of tumour cells. We showed that removal of the N394-linked glycans greatly compromised the stability and effector functions of trastuzumab IgE. However, the triple mutation Y339S/L359T/V361T could partially restore these features in the trastuzumab IgE N394S mutant.</p> <p>Collectively, such knowledge could be applied to help develop better antibody-based biotherapeutics. </p>
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