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

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.