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

ABSTRACT

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.

KEYWORDS:

• Macrophage targeting
• N-glycosylation
• Pichia pastoris GlycoSwitchM5
• Single-domain antibody scaffold
• VHHs

Acknowledgments

We thank Eef Parthoens from the VIB BioImaging Core for technical (live cell) imaging assistance.

Disclosure statement

N.C., B.L., L.v.S., W.V.B., and W.N. are named as inventors on patent application “Glycosylation of variable immunoglobin domains”, WO/2018/206734 A1 and N.C., W.V.B and L.v.S. are named as inventors on patent application “Glycosylated single chain immunoglobulin domains”, WO 2021/116252 A1. The remaining authors declare no competing financial interests. The original manuscript for this article was published as a preprint on bioRxiv.⁴⁸

CRediT Author contributions

Conceptualization: N.C., L.v.S., W.V.B.

Funding acquisition: F.S., L.v.S., N.C., W.V.B., X.S.

Investigation: A.R.M., A.V.H., C.R., B.S., F.S., I.R., K.T., L.v.S., M.F., S.D., S.V., W.N., W.V.B.

Methodology: A.R.M., B.L., B.S., C.R., F.S., I.R., L.v.S., M.F., S.D., W.N., W.V.B.

Supervision: N.C., X.S.

Visualization: C.R., L.v.S.

Writing – original draft: L.v.S, W.V.B.

Writing – review & editing: A.R.M, C.R., M.F., N.C., L.v.S., W.V.B.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2210709.

Additional information

Funding

This work was supported by the European Research Council under ERC Consolidator Grant 616966, “GlycoTarget”; Ghent University Geconcerteerde Onderzoeksacties (GOA); Het Fonds voor Wetenschappelijk Onderzoek - Vlaanderen (FWO) under SBO Grant S007817N, “GlycoDelete” and Regulier Onderzoeksproject Grant G041417N; and Agentschap voor Innovatie door Wetenschap en Technologie (IWT) under Grant 131545.