Skip to main content
SpringerLink
Log in

Expression of IgG Monoclonals with Engineered Immune Effector Functions

  • Protocol
  • First Online:
Antibody Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1827))

Abstract

The therapeutic development of monoclonal antibodies requires robust and reliable methods for their recombinant expression and characterization. In this context, an increasingly important aspect in the antibody development process is to determine the contribution of Fc-mediated immune effector functions to therapeutic activity. Here we describe steps for the cloning and mammalian expression of mouse and human IgG monoclonals with reduced immune effector functions, based on mutation of Fc-gamma receptor and complement-binding sites. The resulting antibody preparations contain low levels of endotoxin and are suitable for testing in animal models of disease.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Rouet R, Lowe D, Dudgeon K, Roome B, Schofield P, Langley D, Andrews J, Whitfeld P, Jermutus L, Christ D (2012) Expression of high-affinity human antibody fragments in bacteria. Nat Protoc 7(2):364–373. https://doi.org/10.1038/nprot.2011.448

    Article  PubMed  CAS  Google Scholar 

  2. Simmons LC, Reilly D, Klimowski L, Shantha Raju T, Meng G, Sims P, Hong K, Shields RL, Damico LA, Rancatore P, Yansura DG (2002) Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies. J Immunol Methods 263(1):133–147. https://doi.org/10.1016/S0022-1759(02)00036-4

    Article  PubMed  CAS  Google Scholar 

  3. Wakelin SJ, Sabroe I, Gregory CD, Poxton IR, Forsythe JLR, Garden OJ, Howie SEM (2006) “Dirty little secrets”—endotoxin contamination of recombinant proteins. Immunol Lett 106(1):1–7

    Article  CAS  PubMed  Google Scholar 

  4. Beutler B, Rietschel ET (2003) Innate immune sensing and its roots: the story of endotoxin. Nat Rev Immunol 3(2):169–176

    Article  CAS  Google Scholar 

  5. Copeland S, Warren HS, Lowry SF, Calvano SE, Remick D, Inflammation, the Host Response to Injury I (2005) Acute inflammatory response to endotoxin in mice and humans. Clin Diagn Lab Immunol 12(1):60–67

    PubMed  PubMed Central  CAS  Google Scholar 

  6. Schwarz H, Schmittner M, Duschl A, Horejs-Hoeck J (2014) Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells. PLoS One 9(12):e113840. https://doi.org/10.1371/journal.pone.0113840

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Wolff SM (1973) Biological effects of bacterial endotoxins in man. J Infect Dis 128(Supplement 1):S259–S264

    Article  Google Scholar 

  8. Brooks SA (2006) Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins. Expert Rev Proteomics 3(3):345–359. https://doi.org/10.1586/14789450.3.3.345

    Article  PubMed  CAS  Google Scholar 

  9. Dumont J, Euwart D, Mei B, Estes S, Kshirsagar R (2016) Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol 36(6):1110–1122. https://doi.org/10.3109/07388551.2015.1084266

    Article  PubMed  CAS  Google Scholar 

  10. Chiou HC, Vasu S, Liu CY, Cisneros I, Jones MB, Zmuda JF (2014) Scalable transient protein expression. Methods Mol Biol 1104:35–55. https://doi.org/10.1007/978-1-62703-733-4_4

    Article  PubMed  CAS  Google Scholar 

  11. Liu CY, Spencer V, Kumar S, Liu J, Chiou H, Zmuda JF (2015) Attaining high transient titers in CHO cells. Genet Eng Biotech News 35(17):34–35

    Article  Google Scholar 

  12. Musolino A, Naldi N, Bortesi B, Pezzuolo D, Capelletti M, Missale G, Laccabue D, Zerbini A, Camisa R, Bisagni G (2008) Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu–positive metastatic breast cancer. J Clin Oncol 26(11):1789–1796

    Article  CAS  PubMed  Google Scholar 

  13. Stewart R, Hammond SA, Oberst M, Wilkinson RW (2014) The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer. J ImmunTher Cancer 2(1):29

    Article  Google Scholar 

  14. Moore GL, Chen H, Karki S, Lazar GA Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions. In: MAbs. Taylor & Francis; 2010. pp 181–189

    Google Scholar 

  15. Lee C-H, Romain G, Yan W, Watanabe M, Charab W, Todorova B, Lee J, Triplett K, Donkor M, Lungu OI, Lux A, Marshall N, Lindorfer MA, Goff OR-L, Balbino B, Kang TH, Tanno H, Delidakis G, Alford C, Taylor RP, Nimmerjahn F, Varadarajan N, Bruhns P, Zhang YJ, Georgiou G (2017) IgG fc domains that bind C1q but not effector Fc[gamma] receptors delineate the importance of complement-mediated effector functions. Nat Immunol 18(8):889–898. https://doi.org/10.1038/ni.3770. http://www.nature.com/ni/journal/v18/n8/abs/ni.3770.html - supplementary-information

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Dahan R, Sega E, Engelhardt J, Selby M, Korman AJ, Ravetch JV (2015) FcγRs modulate the anti-tumor activity of antibodies targeting the PD-1/PD-L1 axis. Cancer Cell 28(3):285–295

    Article  CAS  PubMed  Google Scholar 

  17. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, Roddie C, Henry JY, Yagita H, Wolchok JD (2013) Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti–CTLA-4 therapy against melanoma. J Exp Med 210(9):1695–1710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bulliard Y, Jolicoeur R, Windman M, Rue SM, Ettenberg S, Knee DA, Wilson NS, Dranoff G, Brogdon JL (2013) Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies. J Exp Med 210(9):1685–1693

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bulliard Y, Jolicoeur R, Zhang J, Dranoff G, Wilson NS, Brogdon JL (2014) OX40 engagement depletes intratumoral Tregs via activating FcγRs, leading to antitumor efficacy. Immunol Cell Biol 92(6):475–480

    Article  CAS  PubMed  Google Scholar 

  20. Vargas FA, Furness AJS, Solomon I, Joshi K, Mekkaoui L, Lesko MH, Rota EM, Dahan R, Georgiou A, Sledzinska A (2017) Fc-optimized anti-CD25 depletes tumor-infiltrating regulatory T cells and synergizes with PD-1 blockade to eradicate established tumors. Immunity 46(4):577–586

    Article  CAS  Google Scholar 

  21. Vazquez-Lombardi R, Loetsch C, Zinkl D, Jackson J, Schofield P, Deenick EK, King C, Phan TG, Webster KE, Sprent J, Christ D (2017) Potent antitumour activity of interleukin-2-Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells. Nat Commun 8:15373. https://doi.org/10.1038/ncomms15373

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Lee CMY, Iorno N, Sierro F, Christ D (2007) Selection of human antibody fragments by phage display. Nat Protoc 2(11):3001–3008

    Article  CAS  PubMed  Google Scholar 

  23. Fields C, O'Connell D, Xiao S, Lee GU, Billiald P, Muzard J (2013) Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific antibodies. Nat Protoc 8(6):1125–1148

    Article  CAS  PubMed  Google Scholar 

  24. von Boehmer L, Liu C, Ackerman S, Gitlin AD, Wang Q, Gazumyan A, Nussenzweig MC (2016) Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc 11(10):1908–1923

    Article  CAS  Google Scholar 

  25. Malyala P, Singh M (2008) Endotoxin limits in formulations for preclinical research. J Pharm Sci 97(6):2041–2044. https://doi.org/10.1002/jps.21152

    Article  PubMed  CAS  Google Scholar 

  26. Schreier PH, Bothwell A, Mueller-Hill B, Baltimore D (1981) Multiple differences between the nucleic acid sequences of the IgG2aa and IgG2ab alleles of the mouse. Proc Natl Acad Sci 78(7):4495–4499

    Article  CAS  PubMed  Google Scholar 

  27. Smith P, DiLillo DJ, Bournazos S, Li F, Ravetch JV (2012) Mouse model recapitulating human Fcγ receptor structural and functional diversity. Proc Natl Acad Sci 109(16):6181–6186

    Article  PubMed  Google Scholar 

  28. Walsh NC, Kenney LL, Jangalwe S, Aryee K-E, Greiner DL, Brehm MA, Shultz LD (2017) Humanized mouse models of clinical disease. Ann Rev Pathol 12:187–215

    Article  CAS  Google Scholar 

  29. Zeraati M, Langley DB, Schofield P, Moye AL, Rouet R, Hughes WE, Bryan TM, Dinger ME, Christ D (2018) I-motif DNA structures are formed in the nuclei of human cells. Nat Chem 10(6):631–637. https://doi.org/10.1038/s41557-018-0046-3

  30. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345

    Article  CAS  PubMed  Google Scholar 

  31. Vazquez-Lombardi RN, Nevoltris D, Luthra A, Schofield P, Zimmermann C, Christ D (2018) Transient expression of human antibodies in mammalian cells. Nature Protoc 13(1):99–117

    Article  CAS  Google Scholar 

  32. Duncan AR, Woof JM, Partridge LJ, Burton DR, Winter G (1988) Localization of the binding site for the human high-affinity Fc receptor on IgG. Nature

    Google Scholar 

  33. Hezareh M, Hessell AJ, Jensen RC, van de Winkel JGJ, Parren PWHI (2001) Effector function activities of a panel of mutants of a broadly neutralizing antibody against human immunodeficiency virus type 1. J Virol 75(24):12161–12168

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Oganesyan V, Gao C, Shirinian L, Wu H, Dall'Acqua WF (2008) Structural characterization of a human Fc fragment engineered for lack of effector functions. Acta Crystallogr D Biol Crystallogr 64(6):700–704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wines BD, Powell MS, Parren PW, Barnes N, Hogarth PM (2000) The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors FcγRI and FcγRIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A. J Immunol 164(10):5313–5318

    Article  CAS  PubMed  Google Scholar 

  36. Duncan AR, Winter G (1988) The binding site for C1q on IgG. Nature

    Google Scholar 

  37. Idusogie EE, Presta LG, Gazzano-Santoro H, Totpal K, Wong PY, Ultsch M, Meng YG, Mulkerrin MG (2000) Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J Immunol 164(8):4178–4184

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Australian Research Council and the National Health and Medical Research Council.

Author information

Authors and Affiliations

  1. Garvan Institute of Medical Research, Sydney, NSW, Australia

    Rodrigo Vazquez-Lombardi, Romain Rouet & Daniel Christ

  2. Immunology Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

    Damien Nevoltris

  3. Faculty of Medicine, St. Vincent’s Clinical School, University of New South Wales, Sydney, NSW, Australia

    Daniel Christ

Authors
  1. Rodrigo Vazquez-Lombardi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Damien Nevoltris
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Romain Rouet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel Christ
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel Christ .

Editor information

Editors and Affiliations

  1. Immunology Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

    Damien Nevoltris

  2. Institut Paoli-Calmettes, CRCM, Aix Marseille University, CNRS, INSERM, Marseille, France

    Patrick Chames

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Vazquez-Lombardi, R., Nevoltris, D., Rouet, R., Christ, D. (2018). Expression of IgG Monoclonals with Engineered Immune Effector Functions. In: Nevoltris, D., Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 1827. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8648-4_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8648-4_16

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8647-7

  • Online ISBN: 978-1-4939-8648-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation