Selection of Phage Antibody Libraries for Binding and Internalization into Mammalian Cells
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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.
KeywordsWash Cell Immobilize Metal Affinity Chromatography scFv Antibody Antibody Library Phage Antibody
- 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
- 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
- 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