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Small-molecule control of antibody N-glycosylation in engineered mammalian cells

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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References

  1. 1.

    Weiner, L. M., Murray, J. C. & Shuptrine, C. W. Antibody-based immunotherapy of cancer. Cell 148, 1081–1084 (2012).

  2. 2.

    Jefferis, R. Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action. Trends Pharmacol. Sci. 30, 356–362 (2009).

  3. 3.

    Chiu, M. L. & Gilliland, G. L. Engineering antibody therapeutics. Curr. Opin. Struct. Biol. 38, 163–173 (2016).

  4. 4.

    Liu, L. Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins. J. Pharm. Sci. 104, 1866–1884 (2015).

  5. 5.

    Solá, R. J. & Griebenow, K. A. I. Effects of glycosylation on the stability of protein pharmaceuticals. Biochemistry 98, 1223–1245 (2010).

  6. 6.

    Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M. & Dwek, R. A. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25, 21–50 (2007).

  7. 7.

    Higel, F., Seidl, A., Sörgel, F. & Friess, W. N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins. Eur. J. Pharm. Biopharm. 100, 94–100 (2016).

  8. 8.

    Goetze, A. M. et al. High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology 21, 949–959 (2011).

  9. 9.

    Raju, T. S. & Lang, S. E. Diversity in structure and functions of antibody sialylation in the Fc. Curr. Opin. Biotechnol. 30, 147–152 (2014).

  10. 10.

    Shinkawa, T. et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J. Biol. Chem. 278, 3466–3473 (2003).

  11. 11.

    Reusch, D. & Tejada, M. L. Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 25, 1325–1334 (2015).

  12. 12.

    Hodoniczky, J., Yuan, Z. Z. & James, D. C. Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol. Prog. 21, 1644–1652 (2005).

  13. 13.

    Wang, L. X. & Lomino, J. V. Emerging technologies for making glycan-defined glycoproteins. ACS Chem. Biol. 7, 110–122 (2012).

  14. 14.

    Dekkers, G. et al. Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans. Sci. Rep. 6, 36964 (2016).

  15. 15.

    Tejwani, V., Andersen, M. R., Nam, J. H. & Sharfstein, S. T. Glycoengineering in CHO cells: advances in systems biology. Biotechnol. J. 13, 1700234 (2018).

  16. 16.

    Li, F. et al. Cell culture processes for monoclonal antibody production. MAbs 2, 466–479 (2010).

  17. 17.

    Gramer, M. J. et al. Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol. Bioeng. 108, 1591–1602 (2011).

  18. 18.

    Mori, K. et al. Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA. Biotechnol. Bioeng. 88, 901–908 (2004).

  19. 19.

    Yamane-Ohnuki, N. et al. Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol. Bioeng. 87, 614–622 (2004).

  20. 20.

    Malphettes, L. et al. Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies. Biotechnol. Bioeng. 106, 774–783 (2010).

  21. 21.

    Cristea, S. et al. In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration. Biotechnol. Bioeng. 110, 871–880 (2013).

  22. 22.

    Sun, T. et al. Functional knockout of FUT8 in Chinese hamster ovary cells using CRISPR/Cas9 to produce a defucosylated antibody. Eng. Life Sci. 15, 660–666 (2015).

  23. 23.

    Kanda, Y. et al. Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: a new strategy for generating completely non-fucosylated recombinant therapeutics. J. Biotechnol. 130, 300–310 (2007).

  24. 24.

    Imai-Nishiya, H. et al. Double knockdown of α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC. BMC Biotechnol. 7, 84 (2007).

  25. 25.

    Meuris, L. et al. GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat. Biotechnol. 32, 485–489 (2014).

  26. 26.

    Yang, Z. et al. Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat. Biotechnol. 33, 842–844 (2015).

  27. 27.

    Schulz, M. A. et al. Glycoengineering design options for IgG1 in CHO cells using precise gene editing. Glycobiology 28, 542–549 (2018).

  28. 28.

    Raymond, C. et al. Production of α2,6-sialylated IgG1 in CHO cells. MAbs 7, 571–583 (2015).

  29. 29.

    Umaña, P., Jean-Mairet, J., Moudry, R., Amstutz, H. & Bailey, J. E. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 17, 176–180 (1999).

  30. 30.

    Giddens, J. P., Lomino, J. V., DiLillo, D. J., Ravetch, J. V. & Wang, L.-X. Site-selective chemoenzymatic glycoengineering of Fab and Fc glycans of a therapeutic antibody. Proc. Natl Acad. Sci. USA 115, 12023–12027 (2018).

  31. 31.

    Li, T. et al. Modulating IgG effector function by Fc glycan engineering. Proc. Natl Acad. Sci. USA 114, 3485–3490 (2017).

  32. 32.

    Higel, F., Demelbauer, U., Seidl, A., Friess, W. & Sörgel, F. Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals. Anal. Bioanal. Chem. 405, 2481–2493 (2013).

  33. 33.

    Chen, X. & Flynn, G. C. Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performance liquid chromatography/mass spectrometry. Anal. Biochem. 370, 147–161 (2007).

  34. 34.

    Gaidukov, L. et al. Multi-landing pad DNA integration platform for mammalian cell engineering. Nucleic Acids Res. 46, 4072–4086 (2018).

  35. 35.

    Duportet, X. et al. A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic Acids Res. 42, 13440–13451 (2014).

  36. 36.

    Zong, H. et al. Producing defucosylated antibodies with enhanced in vitro antibody-dependent cellular cytotoxicity via FUT8 knockout CHO-S cells. Eng. Life Sci. 17, 801–808 (2017).

  37. 37.

    Kaneko, Y., Nimmerjahn, F. & Ravetch, J. V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, 670–673 (2006).

  38. 38.

    Washburn, N. et al. Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity. Proc. Natl Acad. Sci. USA 112, E1297–E1306 (2015).

  39. 39.

    Dow, L. E. et al. Conditional reverse tet-transactivator mouse strains for the efficient induction of TRE-regulated transgenes in mice. PLoS One 9, e95236 (2014).

  40. 40.

    Stanton, B. C. et al. Systematic transfer of prokaryotic sensors and circuits to mammalian cells. ACS Synth. Biol. 3, 880–891 (2014).

  41. 41.

    Liang, F., Sen, Ho, W., Q. & Crabtree, G. R. Engineering the ABA plant stress pathway for regulation of induced proximity. Sci. Signal. 4, rs2 (2011).

  42. 42.

    Thomann, M., Reckermann, K., Reusch, D., Prasser, J. & Tejada, M. L. Fc-galactosylation modulates antibody-dependent cellular cytotoxicity of therapeutic antibodies. Mol. Immunol. 73, 69–75 (2016).

  43. 43.

    Liu, S. D. et al. Afucosylated antibodies increase activation of FcγRIIIa-dependent signaling components to intensify processes promoting ADCC. Cancer Immunol. Res. 3, 173–183 (2015).

  44. 44.

    Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553 (2009).

  45. 45.

    Guye, P., Li, Y., Wroblewska, L., Duportet, X. & Weiss, R. Rapid, modular and reliable construction of complex mammalian gene circuits. Nucleic Acids Res. 41, e156 (2013).

  46. 46.

    Cong, L. et al. Multiplex genome engineering using CRISPR/cas systems. Science 339, 819–823 (2013).

  47. 47.

    Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

  48. 48.

    Shang, T. Q. et al. Development and application of a robust N-glycan profiling method for heightened characterization of monoclonal antibodies and related glycoproteins. J. Pharm. Sci. 103, 1967–1978 (2014).

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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.

Author information

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.

Competing interests

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’.

Correspondence to Ron Weiss.

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Supplementary Tables 1–3, Supplementary Figures 1–7

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Fig. 1: Overview of cell engineering for mAb and synthetic gene circuits expression in knockout cell lines.
Fig. 2: FUT8 and β4GALT1 gene deletions abolish mAb fucosylation and galactosylation, and FUT8-C and B4GALT1-C circuits restore them.
Fig. 3: Circuits encoding inducible FUT8 or β4GALT1 gene expression enable tunable levels of fucosylated or galactosylated antibody in FUT8−/− or β4GALT1−/− cells.
Fig. 4: Simultaneous, independent regulation of FUT8 and β4GALT1 gene expression in FUT8−/−/β4GALT1−/− cells integrated with FUT8-ABA and B4GALT1-Dox circuits led to a wide range of fucosylation and galactosylation levels and various levels of binding affinity of mAb to FcγRIIIa.