Selection of Phage Antibody Libraries for Binding and Internalization into Mammalian Cells

  • Yu Zhou
  • James D. MarksEmail author
Part of the Springer Protocols Handbooks book series (SPH)

Phage antibody technology is a powerful approach for generating human antibodies to target antigens. For many therapeutic applications, it is useful to generate antibodies that bind to cell surface receptors in a manner where binding results in internalization of the antibody. This allows use of the antibody to deliver toxic payloads intracellularly to achieve a therapeutic effect. Here we describe how phage antibody libraries can be directly selected on tumor cell lines to generate antibodies binding cell surface receptors and which are rapidly internalize upon binding. Protocols are provided showing how to: 1) directly select internalizing antibodies from phage antibody libraries; 2) screen phage antibodies in a high throughput flow cytometry assay for binding to the tumor cell line used for selection; 3) identify the antigen bound by the phage antibody using immunoprecipitation and mass spectrometry; and 4) verify and quanttiate that phage antibodies are internalized.


Wash Cell Immobilize Metal Affinity Chromatography scFv Antibody Antibody Library Phage Antibody 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Andersen PS, Stryhn A, Hansen BE, Fugger L, Engberg J, Buus S (1996) A recombinant antibody with the antigen-specific, major histocompatability complex-restricted specificity of T-cells. Proc Natl Acad Sci USA 93(2):1820–1824PubMedCrossRefGoogle Scholar
  2. Barry MA, Dower WJ, Johnston SA (1996) Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nat Med 2(3):299–305PubMedCrossRefGoogle Scholar
  3. Becerril B, Poul MA, Marks JD (1999) Toward selection of internalizing antibodies from phage libraries. Biochem Biophys Res Commun 255(2):386–393PubMedCrossRefGoogle Scholar
  4. Cai X, Garen A (1995) Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries. Proc Natl Acad Sci USA 92(14):6537–6541PubMedCrossRefGoogle Scholar
  5. de Kruif J, Terstappen L, Boel E, Logtenberg T (1995) Rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library. Proc Natl Acad Sci USA 92(6):3938–3942PubMedCrossRefGoogle Scholar
  6. Goenaga AL, Zhou Y, Legay C, Bougherara H, Huang L, Liu B, Drummond DC, Kirpotin DB, Auclair C, Marks JD, Poul MA (2007) Identification and characterization of tumor antigens by using antibody phage display and intrabody strategies. Mol Immunol 44(15):3777–3788PubMedCrossRefGoogle Scholar
  7. Hart SL, Knight AM, Harbottle RP, Mistry A, Hunger H-D, Cutler DF, Williamson R, Coutelle C (1994) Cell binding and internalization by filamentous phage displaying a cyclic arg-gly-asp-containing peptide. J Biol Chem 269:12468–12474PubMedGoogle Scholar
  8. Heitner T, Moor A, Garrison JL, Marks C, Hasan T, Marks JD (2001) Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library. J Immunol Methods 248(1–2):17–30PubMedCrossRefGoogle Scholar
  9. Hoogenboom HR, Lutgerink JT, Pelsers MM, Rousch MJ, Coote J, Van Neer N, De Bruine A, Van Nieuwenhoven FA, Glatz JF, Arends JW (1999) Selection-dominant and nonaccessible epitopes on cell-surface receptors revealed by cell-panning with a large phage antibody library. Eur J Biochem 260(3):774–784PubMedCrossRefGoogle Scholar
  10. Liu B, Huang L, Sihlbom C, Burlingame A, Marks JD (2002) Towards proteome-wide production of monoclonal antibody by phage display. J Mol Biol 315(5):1063–1073PubMedCrossRefGoogle Scholar
  11. Liu B, Conrad F, Cooperberg MR, Kirpotin DB, Marks JD (2004) Mapping tumor epitope space by direct selection of single-chain Fv antibody libraries on prostate cancer cells. Cancer Res 64(2):704–710PubMedCrossRefGoogle Scholar
  12. Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (1991) By-passing immunization: Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222(3):581–597PubMedCrossRefGoogle Scholar
  13. Marks JD, Ouwehand WH, Bye JM, Finnern R, Gorick BD, Voak D, Thorpe S, Hughes-Jones NC, Winter G (1993) Human antibody fragments specific for blood group antigens from a phage display library. Biotechnology 11(10):1145–1149PubMedCrossRefGoogle Scholar
  14. Nielsen UB, Kirpotin DB, Pickering EM, Hong K, Park JW, Refaat Shalaby M, Shao Y, Benz CC, Marks JD (2002) Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis. Biochim Biophys Acta 1591(1–3):109–118PubMedCrossRefGoogle Scholar
  15. Nielsen UB, Kirpotin DB, Pickering EM, Drummond DC, Marks JD (2006) A novel assay for monitoring internalization of nanocarrier coupled antibodies. BMC Immunol 7:24PubMedCrossRefGoogle Scholar
  16. O’Connell D, Becerril B, Roy-Burman A, Daws M, Marks JD (2002) Phage versus phagemid libraries for generation of human monoclonal antibodies. J Mol Biol 321(1):49–56PubMedCrossRefGoogle Scholar
  17. Poul MA, Becerril B, Nielsen UB, Morisson P, Marks JD (2000) Selection of tumor-specific internalizing human antibodies from phage libraries. J Mol Biol 301(5):1149–1161PubMedCrossRefGoogle Scholar
  18. Schier R, Marks JD, Wolf EJ, Apell G, Wong C, McCartney JE, Bookman MA, Huston JS, Houston LL, Weiner LM, Adams GP (1995) In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library. Immunotechnology 1(1):73–81PubMedCrossRefGoogle Scholar
  19. Sheets MD, Amersdorfer P, Finnern R, Sargent P, Lindquist E, Schier R, Hemingsen G, Wong C, Gerhart JC, Marks JD, Lindqvist E (1998) Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci USA 95(11):6157–6162PubMedCrossRefGoogle Scholar
  20. Zhou Y, Drummond DC, Zou H, Hayes ME, Adams GP, Kirpotin DB, Marks JD (2007) Impact of Single-chain Fv Antibody Fragment Affinity on Nanoparticle Targeting of Epidermal Growth Factor Receptor-expressing Tumor Cells. J Mol Biol 371(4):934–947PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Departments of Anesthesia and Pharmaceutical ChemistryUniversity of CaliforniaSan FranciscoUSA

Personalised recommendations