Abstract
Immunoglobulins (Igs) are large proteins of 150 kDa with prolonged residence time in blood. Their half-life is controlled by their ability to interact with the protective neonatal Fc receptor (FcRn, Brambell receptor) present on endothelial cells. Here, we describe a protocol using site-specific mutagenesis of individual residues responsible for this interaction, resulting in engineered antibodies with distinct half-lives. The method is a powerful tool that enables manipulation of half-lives and is applicable to all antibodies and Fc fusion proteins for the development of agents with controlled pharmacokinetic properties. Moreover, the protocol is applicable to any situation where the structure and/or function of engineered proteins are to be studied. The protocol begins with the mutagenesis reaction at the DNA level and proceeds to describe mammalian expression and purification of recombinant proteins, radiolabeling and evaluation in vivo. The time frame for completing the procedure is about 6 months, provided that no complications are encountered.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Adams, G.P. & Weiner, L.M. Monoclonal antibody therapy of cancer. Nat. Biotechnol. 23, 1147–1157 (2005).
Ghetie, V. et al. Increasing the serum persistence of an IgG fragment by random mutagenesis. Nat. Biotechnol. 15, 637–640 (1997).
Dall'Acqua, W.F. et al. Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences. J. Immunol. 169, 5171–5180 (2002).
Hinton, P.R. et al. Engineered human IgG antibodies with longer serum half-lives in primates. J. Biol. Chem. 279, 6213–6216 (2004).
Hinton, P.R. et al. An engineered human IgG1 antibody with longer serum half-life. J. Immunol. 176, 346–356 (2006).
Wu, A.M. & Senter, P.D. Arming antibodies: prospects and challenges for immunoconjugates. Nat. Biotechnol. 23, 1137–1146 (2005).
Kenanova, V. & Wu, A.M. Tailoring antibodies for radionuclide delivery. Expert Opin. Drug Deliv. 3, 53–70 (2006).
Wu, A.M. & Yazaki, P.J. Designer genes: recombinant antibody fragments for biological imaging. Q. J. Nucl. Med. 44, 268–283 (2000).
Slavin-Chiorini, D.C. et al. Biological properties of chimeric domain-deleted anticarcinoma immunoglobulins. Cancer Res. 55, 5957s–5967s (1995).
Kenanova, V. et al. Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments. Cancer Res. 65, 622–631 (2005).
Brambell, F.W. The transmission of immunity from mother to young and the catabolism of immunoglobulins. Lancet 2, 1087–1093 (1966).
Ghetie, V. et al. Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice. Eur. J. Immunol. 26, 690–696 (1996).
Junghans, R.P. & Anderson, C.L. The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor. Proc. Natl. Acad. Sci. USA 93, 5512–5516 (1996).
Israel, E.J. et al. Increased clearance of IgG in mice that lack beta 2-microglobulin: possible protective role of FcRn. Immunology 89, 573–578 (1996).
Rodewald, R. pH-dependent binding of immunoglobulins to intestinal cells of the neonatal rat. J. Cell Biol. 71, 666–669 (1976).
Simister, N.E. & Rees, A.R. Isolation and characterization of an Fc receptor from neonatal rat small intestine. Eur. J. Immunol. 15, 733–738 (1985).
Ober, R.J. et al. Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn. J. Immunol. 172, 2021–2029 (2004).
Tesar, D.B., Tiangco, N.E. & Bjorkman, P.J. Ligand valency affects transcytosis, recycling and intracellular trafficking mediated by the neonatal Fc receptor. Traffic 7, 1127–1142 (2006).
Medesan, C. et al. Delineation of the amino acid residues involved in transcytosis and catabolism of mouse IgG1. J. Immunol. 158, 2211–2217 (1997).
Kim, J.K. et al. Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn. Eur. J. Immunol. 29, 2819–2825 (1999).
Shields, R.L. et al. High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R. J. Biol. Chem. 276, 6591–6604 (2001).
Hornick, J.L. et al. Single amino acid substitution in the Fc region of chimeric TNT-3 antibody accelerates clearance and improves immunoscintigraphy of solid tumors. J. Nucl. Med. 41, 355–362 (2000).
West, A.P., Jr. & Bjorkman, P.J. Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor(,). Biochemistry 39, 9698–9708 (2000).
Martin, W.L., West, A.P., Jr., Gan, L. & Bjorkman, P.J. Crystal structure at 2.8 Å of an FcRn/heterodimeric Fc complex: mechanism of pH-dependent binding. Mol. Cell 7, 867–877 (2001).
Olafsen, T. et al. Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging. Cancer Res. 65, 5907–5916 (2005).
Kunkel, T.A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492 (1985).
Sugimoto, M., Esaki, N., Tanaka, H. & Soda, K. A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide. Anal. Biochem. 179, 309–311 (1989).
Taylor, J.W., Ott, J. & Eckstein, F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 13, 8765–8785 (1985).
Vandeyar, M.A., Weiner, M.P., Hutton, C.J. & Batt, C.A. A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene 65, 129–133 (1988).
Cormack, B.P., Strubin, M., Stargell, L.A. & Struhl, K. Conserved and nonconserved functions of the yeast and human TATA-binding proteins. Genes Dev. 8, 1335–1343 (1994).
Aiyar, A., Xiang, Y. & Leis, J. Site-directed mutagenesis using overlap extension PCR. Methods Mol. Biol. 57, 177–191 (1996).
Ishii, T.M. et al. Site-directed mutagenesis. Methods Enzymol. 293, 53–71 (1998).
Ling, M.M. & Robinson, B.H. Approaches to DNA mutagenesis: an overview. Anal. Biochem. 254, 157–178 (1997).
Cline, J., Braman, J.C. & Hogrefe, H.H. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24, 3546–3551 (1996).
Wallace, R.B. et al. Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res. 6, 3543–3557 (1979).
Cockett, M.I., Bebbington, C.R. & Yarranton, G.T. The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines. Nucleic Acids Res. 19, 319–325 (1991).
Galfre, G. & Milstein, C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 73, 3–46 (1981).
Bebbington, C.R. et al. High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology (NY) 10, 169–175 (1992).
Kozak, M. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125–8148 (1987).
D'Argenio, D.Z. & Schumitzky, A. A program package for simulation and parameter estimation in pharmacokinetic systems. Comput. Programs Biomed. 9, 115–134 (1979).
Zheng, L., Baumann, U. & Reymond, J.L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 32, e115 (2004).
Liu, J. et al. pPIC9-Fc: a vector system for the production of single-chain Fv-Fc fusions in Pichia pastoris as detection reagents in vitro . J. Biochem. (Tokyo) 134, 911–917 (2003).
Powers, D.B. et al. Expression of single-chain Fv-Fc fusions in Pichia pastoris . J. Immunol. Methods 251, 123–135 (2001).
Zhu, Z., Ghose, T., Kralovec, Y. & Yang, C. Immunoreactivity, stability, pharmacokinetics and biodistribution of a monoclonal antibody to human leukemic B cells after three different methods of radioiodination. Nucl. Med. Biol. 21, 873–882 (1994).
Yazaki, P.J. et al. Mammalian expression and hollow fiber bioreactor production of recombinant anti-CEA diabody and minibody for clinical applications. J. Immunol. Methods 253, 195–208 (2001).
Yazaki, P.J. & Wu, A.M. Construction and characterization of minibodies for imaging and therapy of colorectal carcinomas. Methods Mol. Biol. 207, 351–364 (2003).
Gagnon, P. Purification tools for monoclonal antibodies. 1996, 5800 N. Kolb Rd., Suite 5127, Tucson, AZ: Validated Biosystems Inc. 1-150.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Olafsen, T., Kenanova, V. & Wu, A. Tunable pharmacokinetics: modifying the in vivo half-life of antibodies by directed mutagenesis of the Fc fragment. Nat Protoc 1, 2048–2060 (2006). https://doi.org/10.1038/nprot.2006.322
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.322
This article is cited by
-
Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins
Protein & Cell (2018)
-
Saturation mutagenesis on Tyr205 of the cyclic dipeptide C2-prenyltransferase FtmPT1 results in mutants with strongly increased C3-prenylating activity
Applied Microbiology and Biotechnology (2016)
-
Deglycosylation of mAb by EndoS for Improved Molecular Imaging
Molecular Imaging and Biology (2015)
-
Positron Emission Tomography Imaging of Endometrial Cancer Using Engineered Anti-EMP2 Antibody Fragments
Molecular Imaging and Biology (2013)
-
Impact of expression system on the function of the C6.5 diabody PET radiotracer
Tumor Biology (2012)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.