Isolation of Antibody Binders to MISIIR from a Phage Display Library by Sorting

  • Andy Qingan YuanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2108)


Cell surface antigens represent the most common targets for antibody-based cancer therapy. Isolation of lead antibodies to these membrane targets from antibody repertoires, such as immunized or naïve phage display libraries, has been a challenging task, which is an outstanding issue when soluble portion of the target(s) is not available, and/or a naïve phage display library is used. Common cell-based panning methods often encounter numerous difficulties, including high background and loss of cells during repeated washes. Here we described a novel FACS sorter-based protocol to isolate single-chain Fv molecules specific for defined antigen MSIIR expressed on stably transformed mammalian cells, and screening of unique binders to the tumor target.

Key words

Phage display Biopanning Antibody Sorting Single-chain Fv (scFv) 


  1. 1.
    Carter P, Smith L, Ryan M (2004) Identification and validation of cell surface antigens for antibody targeting in oncology. Endocr Relat Cancer 11:659–687CrossRefGoogle Scholar
  2. 2.
    Ross JS, Schenkein DP, Pietrusko R, Rolfe M, Linette GP, Stec J, Stagliano NE, Ginsburg GS, Symmans WF, Pusztai L et al (2004) Targeted therapies for cancer 2004. Am J Clin Pathol 122:598–609CrossRefGoogle Scholar
  3. 3.
    Adams GP, Weiner LM (2005) Monoclonal antibody therapy of cancer. Nat Biotechnol 23:1147–1157CrossRefGoogle Scholar
  4. 4.
    Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making antibodies by phage display technology. Annu Rev Immunol 12:433–455CrossRefGoogle Scholar
  5. 5.
    Coomber DW (2002) Panning of antibody phage-display libraries. Standard protocols. Methods Mol Biol 178:133–145PubMedGoogle Scholar
  6. 6.
    Nagy P, Friedlander E, Tanner M, Kapanen AI, Carraway KL, Isola J, Jovin TM (2005) Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line. Cancer Res 65:473–482PubMedGoogle Scholar
  7. 7.
    Krag DN, Shukla GS, Shen GP, Pero S, Ashikaga T, Fuller S, Weaver DL, BurdetteRadoux S, Thomas C (2006) Selection of tumor-binding ligands in cancer patients with phage display libraries. Cancer Res 66:7724–7733CrossRefGoogle Scholar
  8. 8.
    Rajotte D, Arap W, Hagedorn M, Koivunen E, Pasqualini R, Ruoslahti E (1998) Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display. J Clin Invest 102:430–437CrossRefGoogle Scholar
  9. 9.
    Poul MA, Becerril B, Nielsen UB, Morisson P, Marks JD (2000) Selection of tumor specific internalizing human antibodies from phage libraries. J Mol Biol 301:1149–1161CrossRefGoogle Scholar
  10. 10.
    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 248:17–30CrossRefGoogle Scholar
  11. 11.
    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 260:774784CrossRefGoogle Scholar
  12. 12.
    Watters JM, Telleman P, Junghans RP (1997) An optimized method for cell-based phage display panning. Immunotechnology 3:21–29CrossRefGoogle Scholar
  13. 13.
    Nef S, Parada LF (2000) Hormones in male sexual development. Genes Dev 14:3075–3086CrossRefGoogle Scholar
  14. 14.
    Masiakos PT, MacLaughlin DT, Maheswaran S, Teixeira J, Fuller AF Jr, Shah PC, Kehas DJ, Kenneally MK, Dombkowski DM, Ha TU et al (1999) Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS. Clin Cancer Res 5:3488–3499PubMedGoogle Scholar
  15. 15.
    He WW, Gustafson ML, Hirobe S, Donahoe PK (1993) Developmental expression of four novel serine/threonine kinase receptors homologous to the activin/transforming growth factor-beta type II receptor family. Dev Dyn 196:133–142CrossRefGoogle Scholar
  16. 16.
    Baarends WM, van Helmond MJ, Post M, van der Schoot PJ, Hoogerbrugge JW, de Winter JP, Uilenbroek JT, Karels B, Wilming LG, Meijers JH et al (1994) A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the mullerian duct. Development 120:189–197PubMedGoogle Scholar
  17. 17.
    Teixeira J, He WW, Shah PC, Morikawa N, Lee MM, Catlin EA, Hudson PL, Wing J, Maclaughlin DT, Donahoe PK (1996) Developmental expression of a candidate mullerian inhibiting substance type II receptor. Endocrinology 137:160–165CrossRefGoogle Scholar
  18. 18.
    Schier R, Marks JD, Wolf EJ, Apell G, Wong C, McCartney JE, Bookman MA, Huston JS, Houston LL, Weiner LM 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 1:73–81CrossRefGoogle Scholar
  19. 19.
    Gould LH, Sui J, Foellmer H, Oliphant T, Wang T, Ledizet M, Murakami A, Noonan K, Lambeth C, Kar K et al (2005) Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus. J Virol 79:14606–14613CrossRefGoogle Scholar
  20. 20.
    Yuan QA, Simmons HH, Robinson MK, Russeva M, Marasco WA, Adams GP (2006) Development of engineered antibodies specific for the Mullerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer. Mol Cancer Ther 5:2096–2105CrossRefGoogle Scholar
  21. 21.
    Kingsbury GA, Junghans RP (1995) Screening of phage display immunoglobulin libraries by anti-M13 ELISA and whole phage PCR. Nucleic Acids Res 23:2563–2564CrossRefGoogle Scholar
  22. 22.
    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 222:581–597CrossRefGoogle Scholar
  23. 23.
    Adams GP, Schier R, Marshall K, Wolf EJ, McCall AM, Marks JD, Weiner LM (1998) Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies. Cancer Res 58:485–490PubMedGoogle Scholar
  24. 24.
    Matson CF (1965) Polyacrylamide gel electrophoresis. A simple system using gel columns. Anal Biochem 13:294–304CrossRefGoogle Scholar
  25. 25.
    Rothe C, Urlinger S, L­hning C, Prassler J, Stark Y, Jðger U, Hubner B, Bardroff M, Pradel I, Boss M, Bittlingmaier R, Bataa T, Frisch C, Brocks B, Honegger A, Urban M (2008) The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. J Mol Biol 376(4):1182–1200CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Antibody TechnologyEXCYTE LLCRockvilleUSA

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