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Tunable pharmacokinetics: modifying the in vivo half-life of antibodies by directed mutagenesis of the Fc fragment

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.

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Figure 1: Schematic presentation of an intact antibody and engineered antibody fragments (scFv and a scFv-Fc) with their molecular weights indicated.
Figure 2
Figure 3: Oligonucleotides and agarose gel electrophoresis.
Figure 4: Overview of the purification and characterization of recombinant antibody fragments.
Figure 5: In vivo blood activity curves of engineered anti-CEA scFv-Fc fragments containing different mutations in the Fc region10.

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References

  1. Adams, G.P. & Weiner, L.M. Monoclonal antibody therapy of cancer. Nat. Biotechnol. 23, 1147–1157 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Ghetie, V. et al. Increasing the serum persistence of an IgG fragment by random mutagenesis. Nat. Biotechnol. 15, 637–640 (1997).

    Article  CAS  PubMed  Google Scholar 

  3. 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).

    Article  PubMed  Google Scholar 

  4. Hinton, P.R. et al. Engineered human IgG antibodies with longer serum half-lives in primates. J. Biol. Chem. 279, 6213–6216 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Hinton, P.R. et al. An engineered human IgG1 antibody with longer serum half-life. J. Immunol. 176, 346–356 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Wu, A.M. & Senter, P.D. Arming antibodies: prospects and challenges for immunoconjugates. Nat. Biotechnol. 23, 1137–1146 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Kenanova, V. & Wu, A.M. Tailoring antibodies for radionuclide delivery. Expert Opin. Drug Deliv. 3, 53–70 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Wu, A.M. & Yazaki, P.J. Designer genes: recombinant antibody fragments for biological imaging. Q. J. Nucl. Med. 44, 268–283 (2000).

    CAS  PubMed  Google Scholar 

  9. Slavin-Chiorini, D.C. et al. Biological properties of chimeric domain-deleted anticarcinoma immunoglobulins. Cancer Res. 55, 5957s–5967s (1995).

    CAS  PubMed  Google Scholar 

  10. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Brambell, F.W. The transmission of immunity from mother to young and the catabolism of immunoglobulins. Lancet 2, 1087–1093 (1966).

    Article  CAS  PubMed  Google Scholar 

  12. Ghetie, V. et al. Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice. Eur. J. Immunol. 26, 690–696 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rodewald, R. pH-dependent binding of immunoglobulins to intestinal cells of the neonatal rat. J. Cell Biol. 71, 666–669 (1976).

    Article  CAS  PubMed  Google Scholar 

  16. 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).

    Article  CAS  PubMed  Google Scholar 

  17. 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).

    Article  CAS  PubMed  Google Scholar 

  18. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Medesan, C. et al. Delineation of the amino acid residues involved in transcytosis and catabolism of mouse IgG1. J. Immunol. 158, 2211–2217 (1997).

    CAS  PubMed  Google Scholar 

  20. 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).

    Article  CAS  PubMed  Google Scholar 

  21. 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).

    Article  CAS  PubMed  Google Scholar 

  22. 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).

    CAS  PubMed  Google Scholar 

  23. 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).

    Article  CAS  PubMed  Google Scholar 

  24. 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).

    Article  CAS  PubMed  Google Scholar 

  25. Olafsen, T. et al. Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging. Cancer Res. 65, 5907–5916 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kunkel, T.A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 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).

    Article  CAS  PubMed  Google Scholar 

  28. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 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).

    Article  CAS  PubMed  Google Scholar 

  30. 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).

    Article  CAS  PubMed  Google Scholar 

  31. Aiyar, A., Xiang, Y. & Leis, J. Site-directed mutagenesis using overlap extension PCR. Methods Mol. Biol. 57, 177–191 (1996).

    CAS  PubMed  Google Scholar 

  32. Ishii, T.M. et al. Site-directed mutagenesis. Methods Enzymol. 293, 53–71 (1998).

    Article  CAS  PubMed  Google Scholar 

  33. Ling, M.M. & Robinson, B.H. Approaches to DNA mutagenesis: an overview. Anal. Biochem. 254, 157–178 (1997).

    Article  CAS  PubMed  Google Scholar 

  34. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Galfre, G. & Milstein, C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 73, 3–46 (1981).

    Article  CAS  PubMed  Google Scholar 

  38. 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).

    CAS  Google Scholar 

  39. Kozak, M. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125–8148 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. D'Argenio, D.Z. & Schumitzky, A. A program package for simulation and parameter estimation in pharmacokinetic systems. Comput. Programs Biomed. 9, 115–134 (1979).

    Article  CAS  PubMed  Google Scholar 

  41. Zheng, L., Baumann, U. & Reymond, J.L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 32, e115 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  42. 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).

    Article  CAS  Google Scholar 

  43. Powers, D.B. et al. Expression of single-chain Fv-Fc fusions in Pichia pastoris . J. Immunol. Methods 251, 123–135 (2001).

    Article  CAS  PubMed  Google Scholar 

  44. 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).

    Article  CAS  PubMed  Google Scholar 

  45. 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).

    Article  CAS  PubMed  Google Scholar 

  46. 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).

    CAS  PubMed  Google Scholar 

  47. Gagnon, P. Purification tools for monoclonal antibodies. 1996, 5800 N. Kolb Rd., Suite 5127, Tucson, AZ: Validated Biosystems Inc. 1-150.

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Correspondence to Tove Olafsen.

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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

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