Abstract
N-linked glycosylation in monoclonal antibodies (mAbs) is crucial for structural and functional properties of mAb therapeutics, including stability, pharmacokinetics, safety and clinical efficacy. The biopharmaceutical industry currently lacks tools to precisely control N-glycosylation levels during mAb production. In this study, we engineered Chinese hamster ovary cells with synthetic genetic circuits to tune N-glycosylation of a stably expressed IgG. We knocked out two key glycosyltransferase genes, α-1,6-fucosyltransferase (FUT8) and β-1,4-galactosyltransferase (β4GALT1), genomically integrated circuits expressing synthetic glycosyltransferase genes under constitutive or inducible promoters and generated antibodies with concurrently desired fucosylation (0–97%) and galactosylation (0–87%) levels. Simultaneous and independent control of FUT8 and β4GALT1 expression was achieved using orthogonal small molecule inducers. Effector function studies confirmed that glycosylation profile changes affected antibody binding to a cell surface receptor. Precise and rational modification of N-glycosylation will allow new recombinant protein therapeutics with tailored in vitro and in vivo effects for various biotechnological and biomedical applications.
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Data availability
The authors declare that all relevant data supporting the findings of this study are available within the paper and its Supplementary Information. Biological materials generated in this study are available on Addgene or from the corresponding author upon reasonable request. Circuits FUT8-Dox, FUT8-ABA, B4GALT1-Dox and B4GALT1-ABA are available as Addgene plasmid numbers 124631, 124632, 124633 and 124639, respectively.
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Acknowledgements
We thank F. Lee for help with PCR analysis, K. Jagtap and S. Mamo for help with mammalian cell culture, and B. Teague for critical reading of the manuscript. This work was supported by the Pfizer-MIT PTM collaboration.
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M.M.C., L.G., G.J., J.J.S., R.C., J.K.M., B.C.M., B.F., D.A.L., N.M.S., T.K.L. and R.W. conceived and designed the study. M.M.C., L.G., G.J. and W.A.T. designed genetic circuits. M.M.C., L.G., G.J. and J.L.L. constructed genetic circuits. M.M.C., L.G. and G.J. constructed cell lines. M.M.C. and G.J. performed fed-batch cultures. A.-H.A.C., K.C., B.T. and J.K.M. performed glycan analysis. S.D., D.A.L. and M.S. conceived and performed computational analysis. P.S. performed SPR analysis. M.M.C., L.G. and G.J. wrote the manuscript. All authors commented on and approved the manuscript.
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A US patent concerning the technology described in this paper has been filed by Pfizer, Inc. and Massachusetts Institute of Technology entitled ‘Mammalian Synthetic Biology Approaches for the Precise Control of Protein N-Linked Glycosylation’.
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Chang, M.M., Gaidukov, L., Jung, G. et al. Small-molecule control of antibody N-glycosylation in engineered mammalian cells. Nat Chem Biol 15, 730–736 (2019). https://doi.org/10.1038/s41589-019-0288-4
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DOI: https://doi.org/10.1038/s41589-019-0288-4
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