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.
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References
Liddell JM (2009) Production strategies for antibody fragment therapeutics. BioPharm Int 2:36–42
Labrijn AF, Aalberse RC, Schuurman J (2008) When binding is enough: nonactivating antibody formats. Curr Opin Immunol 20:479–485
Rader C (2009) Overview on concepts and applications of Fab antibody fragments. Curr Protoc Protein Sci. Chapter 6, 6.9.1–6.9.14
Chapman AP, Antoniw P, Spitali M et al (1999) Therapeutic antibody fragments with prolonged in vivo half-lives. Nat Biotechnol 17:780–783
Chen C, Constantinou A, Deonarain M (2011) Modulating antibody pharmacokinetics using hydrophilic polymers. Expert Opin Drug Deliv 8:1221–1236
Kontermann RE (2009) Strategies to extend plasma half-lives of recombinant antibodies. BioDrugs 23:93–109
Constantinou A, Epenetos AA, Hreczuk-Hirst D et al (2008) Modulation of antibody pharmacokinetics by chemical polysialylation. Bioconjug Chem 19:643–650
Jevsevar S, Kunstelj M, Porekar VG (2010) PEGylation of therapeutic proteins. Biotechnol J 5:113–128
Kinstler O, Molineux G, Treuheit M et al (2002) Mono-N-terminal poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 54:477–485
Bailon P, Won CY (2009) PEG-modified biopharmaceuticals. Expert Opin Drug Deliv 6:1–16
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 20:227–234
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 62:1245
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 62:1244
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 2:17ra10
Balan S, Choi JW, Godwin A et al (2007) Site-specific PEGylation of protein disulfide bonds using a three-carbon bridge. Bioconjug Chem 18:61–76
Shaunak S, Godwin A, Choi JW et al (2006) Site-specific PEGylation of native disulfide bonds in therapeutic proteins. Nat Chem Biol 2:312–313
Kwong KY, Rader C (2009) E. coli expression and purification of Fab antibody fragments. Curr Protoc Protein Sci. Chapter 6, 6.10
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 22:200–210
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 128:735–746
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 67:182–189
Lu Y, Harding SE, Turner A et al (2008) Effect of PEGylation on the solution conformation of antibody fragments. J Pharm Sci 97:2062–2079
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 16:106–119
Kurfurst MM (1992) Detection and molecular-weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl-sulfate polyacrylamide-gel electrophoresis. Anal Biochem 200:244–248
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Jevševar, S., Kusterle, M., Kenig, M. (2012). PEGylation of Antibody Fragments for Half-Life Extension. In: Proetzel, G., Ebersbach, H. (eds) Antibody Methods and Protocols. Methods in Molecular Biology, vol 901. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-931-0_15
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DOI: https://doi.org/10.1007/978-1-61779-931-0_15
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