Biology subjects

Van Breedam, W.

Publications and source records attributed to Van Breedam, W..

4 recordsLinked to original sources

bioRxiv2022

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

As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for therapeutic use in various disease indications, and as versatile reagents in research and diagnostics. To further increase the versatility of VHHs, we explored the VHH scaffold in a structure-guided approach to select regions where the introduction of an N-glycosylation N-X-T sequon and its associated glycan should not interfere with protein folding or epitope recognition. We expressed variants of such glycoengineered VHHs in the Pichia pastoris GlycoSwitchM5 strain, allowing us to pinpoint preferred sites at which Man5GlcNAc2-glycans can be introduced at high site occupancy without affecting antigen binding. A VHH carrying predominantly a Man5GlcNAc2 N-glycan at one of these preferred sites showed highly efficient, glycan-dependent uptake by Mf4/4 macrophages in vitro and by alveolar lung macrophages in vivo, illustrating one potential application of glyco-engineered VHHs: a glycan-based targeting approach for lung macrophage endolysosomal system delivery. The set of optimal artificial VHH N-glycosylation sites identified in this study can serve as a blueprint for targeted glyco-engineering of other VHHs, enabling site-specific functionalization through the rapidly expanding toolbox of synthetic glycobiology.

molecular biology↗

bioRxiv2021

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

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.

bioengineering↗

bioRxiv2021

Drug development of an affinity enhanced, broadly neutralizing heavy chain only antibody that restricts SARS-CoV-2 in hamsters

We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and -2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and -2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.

molecular biology↗

bioRxiv2020

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-domain Camelid Antibodies

The pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV-1) and COVID-19 coronavirus (SARS-CoV-2) have all emerged into the human population with devastating consequences. These viruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics to combat these highly pathogenic coronaviruses. Here, we describe the isolation and characterization of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs are capable of potently neutralizing MERS-CoV or SARS-CoV-1 S pseudotyped viruses. The crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs block receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S, and demonstrate that this cross-reactive VHH is capable of neutralizing SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

microbiology↗