Identification of Target and Function Specific Antibodies for Effective Drug Delivery

  • Yu Zhou
  • James D. Marks
Part of the Methods in Molecular Biology™ book series (MIMB, volume 525)


Phage antibody technology is a powerful approach for generating human antibodies to virtually any target antigen. 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 internalized 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 quantitate such that phage antibodies are internalized.

Key words

Phage antibody antibody internalization targeted drug delivery cell selection flow cytometry 



This work was partially supported by NIH grant P50 CA58207.


  1. 1.
    Piccart-Gebhart, M. J., Procter, M., Leyland-Jones, B., Goldhirsch, A., Untch, M., Smith, I., et al. (2005) Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N. Engl. J. Med. 353, 1659–1672.PubMedCrossRefGoogle Scholar
  2. 2.
    Cunningham, D., Humblet, Y., Siena, S., Khayat, D., Bleiberg, H., Santoro, A., et al. (2004) Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N. Engl. J. Med. 351, 337–345.PubMedCrossRefGoogle Scholar
  3. 3.
    Hainsworth, J. D., Burris, H. A., 3rd, Morrissey, L. H., Litchy, S., Scullin, D. C., Jr., Bearden, J. D., 3rd, et al. (2000) Rituximab monoclonal antibody as initial systemic therapy for patients with low-grade non-Hodgkin lymphoma. Blood 95, 3052–3056.PubMedGoogle Scholar
  4. 4.
    Wendtner, C. M., Ritgen, M., Schweighofer, C. D., Fingerle-Rowson, G., Campe, H., Jager, G., et al. (2004) Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission – experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG). Leukemia 18, 1093–1101.PubMedCrossRefGoogle Scholar
  5. 5.
    Kreitman, R. J., Pastan, I. (2006) Immunotoxins in the treatment of hematologic malignancies. Curr. Drug Targets 7, 1301–1311.PubMedCrossRefGoogle Scholar
  6. 6.
    Lazar, G. A., Dang, W., Karki, S., Vafa, O., Peng, J. S., Hyun, L., et al. (2006) Engineered antibody Fc variants with enhanced effector function. Proc. Natl. Acad. Sci. USA 103, 4005–4010.Google Scholar
  7. 7.
    Nielsen, U. B., Kirpotin, D. B., Pickering, E. M., Hong, K., Park, J. W., Refaat Shalaby, M., et al. (2002) Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis. Biochim. Biophys. Acta 1591, 109–118.PubMedCrossRefGoogle Scholar
  8. 8.
    Reichert, J. M., Valge-Archer, V. E. (2007) Development trends for monoclonal antibody cancer therapeutics. Nat. Rev. Drug Discov. 6, 349–356.PubMedCrossRefGoogle Scholar
  9. 9.
    Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D., Winter, G. (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597.PubMedCrossRefGoogle Scholar
  10. 10.
    Sheets, M. D., Amersdorfer, P., Finnern, R., Sargent, P., Lindquist, E., Schier, R., et al. (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, 6157–6162.Google Scholar
  11. 11.
    Andersen, P. S., Stryhn, A., Hansen, B. E., Fugger, L., Engberg, J., Buus, S. (1996) A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells. Proc. Natl. Acad. Sci. USA 93, 1820–1824.Google Scholar
  12. 12.
    Barry, M. A., Dower, W. J., and Johnston, S. A. (1996) Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nat. Med. 2, 299–305.PubMedCrossRefGoogle Scholar
  13. 13.
    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, 6537–6541.Google Scholar
  14. 14.
    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, 3938–3942.Google Scholar
  15. 15.
    Marks, J. D., Ouwehand, W. H., Bye, J. M., Finnern, R., Gorick, B. D., Voak, D., et al. (1993) Human antibody fragments specific for human blood group antigens from a phage display library. Biotechnology (NY) 11, 1145–1149.Google Scholar
  16. 16.
    Hoogenboom, H. R., Lutgerink, J. T., Pelsers, M. M., Rousch, M. J., Coote, J., Van Neer, N., et al. (1999) Selection-dominant and nonaccessible epitopes on cell-surface receptors revealed by cell-panning with a large phage antibody library. Eur. J. Biochem. 260, 774–784.PubMedCrossRefGoogle Scholar
  17. 17.
    Hart, S. L., Knight, A. M., Harbottle, R. P., Mistry, A., Hunger, H. D., Cutler, D. F., et al. (1994) Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly-Asp-containing peptide. J. Biol. Chem. 269, 12468–12474.PubMedGoogle Scholar
  18. 18.
    Becerril, B., Poul, M. A., and Marks, J. D. (1999) Toward selection of internalizing antibodies from phage libraries. Biochem. Biophys. Res. Commun. 255, 386–393.PubMedCrossRefGoogle Scholar
  19. 19.
    Huie, M. A., Cheung, M. C., Muench, M. O., Becerril, B., Kan, Y. W., Marks, J. D. (2001) Antibodies to human fetal erythroid cells from a nonimmune phage antibody library. Proc. Natl. Acad. Sci. USA 98, 2682–2687.Google Scholar
  20. 20.
    O’Connell, D., Becerril, B., Roy-Burman, A., Daws, M., Marks, J. D. (2002) Phage versus phagemid libraries for generation of human monoclonal antibodies. J. Mol. Biol. 321, 49–56.PubMedCrossRefGoogle Scholar
  21. 21.
    Poul, M. A., Becerril, B., Nielsen, U. B., Morisson, P., Marks, J. D. (2000) Selection of tumor-specific internalizing human antibodies from phage libraries. J. Mol. Biol. 301, 1149–1161.PubMedCrossRefGoogle Scholar
  22. 22.
    Heitner, T., Moor, A., Garrison, J. L., Marks, C., Hasan, T., Marks, J. D. (2001) Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library. J. Immunol. Methods 248, 17–30.PubMedCrossRefGoogle Scholar
  23. 23.
    Park, J. W., Hong, K., Kirpotin, D. B., Colbern, G., Shalaby, R., Baselga, J., et al. (2002) Anti-HER2 immunoliposomes: enhanced efficacy attributable to targeted delivery. Clin. Cancer Res. 8, 1172–1181.PubMedGoogle Scholar
  24. 24.
    Zhou, Y., Drummond, D. C., Zou, H., Hayes, M. E., Adams, G. P., Kirpotin, D. B., et al. (2007) Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells. J. Mol. Biol. 371, 934–947.PubMedCrossRefGoogle Scholar
  25. 25.
    Liu, B., Conrad, F., Cooperberg, M. R., Kirpotin, D. B., Marks, J. D. (2004) Mapping tumor epitope space by direct selection of single-chain Fv antibody libraries on prostate cancer cells. Cancer Res. 64, 704–710.PubMedCrossRefGoogle Scholar
  26. 26.
    Goenaga, A. L., Zhou, Y., Legay, C., Bougherara, H., Huang, L., Liu, B., et al. (2007) Identification and characterization of tumor antigens by using antibody phage display and intrabody strategies. Mol. Immunol. 44, 3777–3788.PubMedCrossRefGoogle Scholar
  27. 27.
    Nielsen, U. B., Kirpotin, D. B., Pickering, E. M., Drummond, D. C., Marks, J. D. (2006) A novel assay for monitoring internalization of nanocarrier coupled antibodies. BMC Immunol. 7, 24.PubMedCrossRefGoogle Scholar
  28. 28.
    Schier, R., Marks, J. D., Wolf, E. J., Apell, G., Wong, C., McCartney, J. E., et al. (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, 73–81.PubMedCrossRefGoogle Scholar
  29. 29.
    Liu, B., Huang, L., Sihlbom, C., Burlingame, A., and Marks, J. D. (2002) Towards proteome-wide production of monoclonal antibody by phage display. J. Mol. Biol. 315, 1063–1073.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Yu Zhou
    • 1
  • James D. Marks
    • 1
  1. 1.University of CaliforniaSan FranciscoUSA

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