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PEGylation of Antibody Fragments for Half-Life Extension

  • Simona JevševarEmail author
  • Mateja Kusterle
  • Maja Kenig
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 901)

Abstract

Antibody fragments (Fab’s) represent important structure for creating new therapeutics. Compared to full antibodies Fab’ fragments possess certain advantages, including higher mobility and tissue penetration, ability to bind antigen monovalently and lack of fragment crystallizable (Fc) region-mediated functions such as antibody-dependent cell mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The main drawback for the use of Fab’s in clinical applications is associated with their short half-life in vivo, which is a consequence of no longer having the Fc region. To exert meaningful clinical effects, the half-life of Fab’s need to be extended, which has been achieved by postproduction chemical attachment of polyethylene glycol (PEG) chain to protein using PEGylation technology. The most suitable approach employs PEG-maleimide attachment to cysteines, either to the free hinge cysteine or to C-terminal cysteines involved in interchain disulfide linkage of the heavy and light chain. Hence, protocols for mono-PEGylation of Fab via free cysteine in the hinge region and di-PEGylation of Fab via interchain disulfide bridge are provided in this chapter.

Key words

Fab’ fragment PEGylation Conjugate Half-life extension 

References

  1. 1.
    Liddell JM (2009) Production strategies for antibody fragment therapeutics. BioPharm Int 2:36–42Google Scholar
  2. 2.
    Labrijn AF, Aalberse RC, Schuurman J (2008) When binding is enough: nonactivating antibody formats. Curr Opin Immunol 20:479–485PubMedCrossRefGoogle Scholar
  3. 3.
    Rader C (2009) Overview on concepts and applications of Fab antibody fragments. Curr Protoc Protein Sci. Chapter 6, 6.9.1–6.9.14Google Scholar
  4. 4.
    Chapman AP, Antoniw P, Spitali M et al (1999) Therapeutic antibody fragments with prolonged in vivo half-lives. Nat Biotechnol 17:780–783PubMedCrossRefGoogle Scholar
  5. 5.
    Chen C, Constantinou A, Deonarain M (2011) Modulating antibody pharmacokinetics using hydrophilic polymers. Expert Opin Drug Deliv 8:1221–1236PubMedCrossRefGoogle Scholar
  6. 6.
    Kontermann RE (2009) Strategies to extend plasma half-lives of recombinant antibodies. BioDrugs 23:93–109PubMedCrossRefGoogle Scholar
  7. 7.
    Constantinou A, Epenetos AA, Hreczuk-Hirst D et al (2008) Modulation of antibody pharmacokinetics by chemical polysialylation. Bioconjug Chem 19:643–650PubMedCrossRefGoogle Scholar
  8. 8.
    Jevsevar S, Kunstelj M, Porekar VG (2010) PEGylation of therapeutic proteins. Biotechnol J 5:113–128PubMedCrossRefGoogle Scholar
  9. 9.
    Kinstler O, Molineux G, Treuheit M et al (2002) Mono-N-terminal poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 54:477–485PubMedCrossRefGoogle Scholar
  10. 10.
    Bailon P, Won CY (2009) PEG-modified biopharmaceuticals. Expert Opin Drug Deliv 6:1–16PubMedCrossRefGoogle Scholar
  11. 11.
    Humphreys DP, Heywood SP, Henry A et al (2007) Alternative antibody Fab’ fragment PEGylation strategies: combination of strong reducing agents, disruption of the interchain disulphide bond and disulphide engineering. Protein Eng Des Sel 20:227–234PubMedCrossRefGoogle Scholar
  12. 12.
    Wakefield I, Peters C, Burkly L et al (2010) CDP7657, a monovalent Fab PEG anti-CD40L antibody, inhibits immune responses in both HuSCID mice and non-human primates. Arthritis Rheum 62:1245Google Scholar
  13. 13.
    Vugler A, Sutton D, Marshall D et al (2010) Blockade of CD40L with a monovalent Fab’ PEG monoclonal antibody inhibits disease in the murine collagen-induced arthritis model. Arthritis Rheum 62:1244CrossRefGoogle Scholar
  14. 14.
    Poirier N, Azimzadeh AM, Zhang T et al (2010) Inducing CTLA-4-dependent immune regulation by selective CD28 blockade promotes regulatory T cells in organ transplantation. Sci Transl Med 2:17ra10PubMedCrossRefGoogle Scholar
  15. 15.
    Balan S, Choi JW, Godwin A et al (2007) Site-specific PEGylation of protein disulfide bonds using a three-carbon bridge. Bioconjug Chem 18:61–76PubMedCrossRefGoogle Scholar
  16. 16.
    Shaunak S, Godwin A, Choi JW et al (2006) Site-specific PEGylation of native disulfide bonds in therapeutic proteins. Nat Chem Biol 2:312–313PubMedCrossRefGoogle Scholar
  17. 17.
    Kwong KY, Rader C (2009) E. coli expression and purification of Fab antibody fragments. Curr Protoc Protein Sci. Chapter 6, 6.10Google Scholar
  18. 18.
    Pepinsky RB, Walus L, Shao Z et al (2011) Production of a PEGylated Fab’ of the anti-LINGO-1 Li33 antibody and assessment of its biochemical and functional properties in vitro and in a rat model of remyelination. Bioconjug Chem 22:200–210PubMedCrossRefGoogle Scholar
  19. 19.
    Gach JS, Maurer M, Hahn R et al (2007) High level expression of a promising anti-idiotypic antibody fragment vaccine against HIV-1 in Pichia pastoris. J Biotechnol 128:735–746PubMedCrossRefGoogle Scholar
  20. 20.
    Zhao Y, Gutshall L, Jiang H et al (2009) Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: papain digestion of mAb and transient expression in mammalian cells. Protein Exp Purif 67:182–189CrossRefGoogle Scholar
  21. 21.
    Lu Y, Harding SE, Turner A et al (2008) Effect of PEGylation on the solution conformation of antibody fragments. J Pharm Sci 97:2062–2079PubMedCrossRefGoogle Scholar
  22. 22.
    Leong SR, DeForge L, Presta L et al (2001) Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation. Cytokine 16:106–119PubMedCrossRefGoogle Scholar
  23. 23.
    Kurfurst MM (1992) Detection and molecular-weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl-sulfate polyacrylamide-gel electrophoresis. Anal Biochem 200:244–248PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Sandoz Biopharmaceuticals, Mengeš, Lek Pharmaceuticals d.dMengešSlovenia

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