GlyConnect: a glycan-based conjugation extension of the GlycoDelete technology

Abstract

Recently, our lab developed GlycoDelete, a technology suite that allows a radical simplification of eukaryotic N-glycosylation. The technology allows to produce glycoproteins that carry single GlcNAc, LacNAc, or LacNAc-Sia type glycans on their N-linked glycosylation sequons. GlycoDelete-type N-glycans are uniquely suited for glycan-based conjugation purposes, as these provide a short, homogeneous and hydrophilic link to the protein backbone. Targeting GlycoDelete-glycans allows for highly site-specific conjugation at sites in the protein which are normally occupied by bulky glycans, thus ensuring minimal interference with protein structure and function. The current manuscript describes the evaluation and optimization of both chemical and chemo-enzymatic conjugation of molecules onto the GlycoDelete-type glycans of a limited set of benchmark proteins

GlycoVHH: Introducing N-glycans on the camelid VHH antibody scaffold - Optimal sites and use for macrophage delivery

“…Glycan-based conjugation strategies have been successfully used in the production of antibody-drug conjugates (ADCs), and resulted in a more homogeneous product profile (Van Delft et al, 2014; Van Delft et al, 2015a; Van Delft et al, 2015b). We are reporting on this application of glyco-VHHs elsewhere (Van Breedam et al, 2021).…”

“…This (CDR) glycosylation is not preferred in terms of easy developability of the VHH, as glycan structures are often heterogeneous and different across expression systems, leading to heterogeneous antigen binding behavior. However, the introduction of N-glycans on the scaffold rather than in the CDR could enable glycan structure synthetic biology and glycan-targeted coupling chemistry according to emerging approaches (Baskin and Bertozzi, 2007; Jacobs et al, 2009; Meuris et al, 2014; Van Breedam et al, 2021) to import novel functionality to VHHs, without affecting antigen binding.…”