Discovery of internalizing antibodies to tumor antigens from phage libraries

Abstract

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.

Figure 1. Phage antibodies are endocytosed into ErbB2 expressing cells

Top panel shows different phage antibody constructs studied. Bottom panel shows immunofluorescent microscopy, staining for phage major coat protein pVIII. Panels A–D phage are displayed in a phagemid vector where there is a single scFv/phage. Panel E phage are displayed in a phage vector with 3–5 copies of scFv/phage. A. Control phage antibody (binds BoNT). B. C6.5 anti-ErbB2 scFv. C. Higher affinity ML3-9 anti-ErbB2 scFv. D. Dimeric C6.5 diabody. E. C6.5 scFv displayed multivalently in true phage vector.

Figure 2. Cartoon of the approach used to select internalizing phage antibodies

A. A phage antibody library is first depleted of common cell surface binders by incubating with a large number of cells lacking the antigens of interest. The depleted phage antibodies are allowed to bind cells at 4°C. B. Cells are then washed to remove unbound phage. C. Cells are then warmed to 37°C to allow endocytosis to occur. D. Surface bound phage are then stripped from the cell surface using low pH buffer. E. Cells containing endocytosed phage are lysed and the lysate used to infect E. coli to produce phage for the next round of selection. F. Selections are repeated for 2–3 rounds

Figure 3. Cartoon of selections to direct internalizing antibodies to a specific target antigen

A. The phage library is selected for two rounds for internalization into a mammalian cell. B. The polyclonal phage from the 2^nd round of selection is applied to yeast displaying the target antigen. C. Unbound phage are removed by washing. D. Bound phage are eluted by incubating yeast with low pH. E. Eluted phage are used to infect E. coli to amplify phage for the next round of selection. F. Selections are repeated for 2–3 rounds.

Figure 4. Display of antigen domains on the surface of yeast

(A) The extracellular domain (ECD) of receptor EphA2 was displayed on the yeast surface and recognized by anti-EphA2 antibody and recombinant mouse Ephrin A1 (R&D) as determined by flow cytometry analysis. (B) The link domain of CD44 (domain 1, or D1) was displayed on the yeast surface and recognized by anti-CD44 rabbit monoclonal antibody as determined by flow cytometry analysis. Both anti-EphA2 and anti-CD44 antibodies did not recognize an irrelevant protein displayed on the yeast surface. The unstained yeast in quadrant 1 represent the parental yeasts that did not induce.