Discovery of Internalizing Antibodies to Tumor Antigens from Phage Libraries

Phage antibody technology can be used to generate human antibodies to essentially any antigen. Many therapeutic target antigens are cell surface receptors, which can be challenging targets for antibody generation. In addition, for many therapeutic applications, one needs antibodies that not only bind the cell surface receptor but also are internalized into the cell upon binding. This allows use of the antibody to deliver a range of payloads into the cell to achieve a therapeutic effect. In this chapter, we describe how human phage antibody libraries can be selected directly on tumor cell lines to generate antibodies that bind cell surface receptors and which upon binding are rapidly internalized into the cell. Specific protocols show how to (1) directly select cell binding and internalizing antibodies from human phage antibody libraries, (2) screen the 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) direct cell binding and internalizing selections to a specific tumor antigen by sequential selection on a tumor cell line followed by selection on yeast displaying the target tumor antigen on the yeast surface.