Internalizing cancer antibodies from phage libraries selected on tumor cells and yeast-displayed tumor antigens

Abstract

A number of approaches have been utilized to generate antibodies to cancer cell surface receptors that can be used as potential therapeutics. A number of these therapeutic approaches, including antibody-drug conjugates, immunotoxins, and targeted nucleic acid delivery, require antibodies that not only bind receptor but also undergo internalization into the cell upon binding. We previously reported on the ability to generate cancer cell binding and internalizing antibodies directly from human phage antibody libraries selected for internalization into cancer cell lines. While a number of useful antibodies have been generated using this approach, limitations include the inability to direct the selections to specific antigens and to identify the antigen bound by the antibodies. Here we show that these limitations can be overcome by using yeast-displayed antigens known to be associated with a cell type to select the phage antibody output after several rounds of selection on a mammalian cell line. We used this approach to generate several human phage antibodies to yeast-displayed EphA2 and CD44. The antibodies bound both yeast-displayed and mammalian cell surface antigens, and were endocytosed upon binding to mammalian cells. This approach is generalizable to many mammalian cell surface proteins, results in the generation of functional internalizing antibodies, and does not require antigen expression and purification for antibody generation.

Figure 1. Display of antigen domains on the surface of yeast

(a) The extracellular domain (ECD) of receptor EphA2 was displayed on 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.

Figure 2. Recovery of phage antibodies from yeast displayed antigens

(a) Comparison of the recovery of anti-EphA2 phage antibody 2D6 from yeast displaying EphA2 ECD (Y-EphA2) versus yeast displaying an irrelevant protein (Y-CON). A total of 10¹¹ anti-EphA2 phage antibody 2D6 were incubated with each of the yeast displayed antigens. (b) Impact of the elution buffer on the titer of EphA2 phage antibody eluted from the surface of yeast displaying EphA2 ECD. A total of 10¹¹ anti-EphA2 phage antibody 2D6 were incubated with each of the yeast displayed antigen proteins prior to elution. (c) The impact of input phage titer on eluted phage titer. The indicated titer of anti-EphA2 phage antibody 2D6 was incubated with yeast displayed EphA2 ECD or an irrelevant yeast displayed protein and the titer of eluted phage determined.

Figure 3. Strategy for selecting internalizing antigen specific phage antibodies

(a) After two rounds of selection for internalization on the basal breast cancer cell line MDAMB231, the pool of phage antibodies was first incubated with irrelevant control yeast to remove any yeast binding antibodies followed by panning on yeast displaying either EphA2 ECD or CD44 domain 1. (b) The binding signal of the polyclonal phage antibody pool to EphA2 ECD or CD44 domain 1 after 2 rounds of panning was measured by using flow cytometry. The irrelevant control yeast was stained with unselected phage antibody library (R0), round 1 (R1) and round 2 (R2) polyclonal phage. (c) Frequency of antigen specific phage antibodies after one and two rounds of selection on yeast displayed antigen. Binding frequency was determined by analyzing 96 randomly picked phage antibodies for binding to yeast displayed antigen by flow cytometry. The induced yeast cells displaying an irrelevant protein, EphA2-ECD and CD44 domain 1 were stained with un-selected phage library (R0), polyclonal phages from R1 and R2 respectively. The un-induced yeast cells (yeast only), irrelevant phage antibody (phage control) and the un-selected phage antibody library were used as control.

Figure 4. Binding specificity of monoclonal phage antibodies from yeast antigen biopanning

(a) Binding of monoclonal phage antibodies to yeast displayed antigen domains as determined by flow cytometry. The induced yeast cells displaying an irrelevant protein (Y-CON), EphA2-ECD (Y-EphA2 ECD), CD44 link domain (Y-CD44 ECD D1) and CD44 full length ECD (Y-CD44 ECD) were stained with monoclonal phage antibodies isolated from Y-EphA2 and Y-CD44 D1 selections. (b) Binding of monoclonal phage antibodies to MDAMB231 cells as determined by flow cytometry, (c) differential binding of monoclonal phage antibodies to breast cancer cell lines including luminal breast cancer cell lines SUM52PE and MCF7, and basal breast cancer cell line MDAMB231.

Figure 5. Characterization of scFv antibodies by western-blot and flow cytometry

(a) Anti-EphA2 scFv 2D6 or D2-1A7 and anti-CD44 scFv F2-1A6 were used to immunoprecipitate their target antigen from MDAMB231 cells. Antigen was detected by Western blotting using either anti-EphA2 antibody D7 or anti-CD44 antibody Ab-4. (b) EphA2 antibodies D2-1A7 (□), D2-1A9 (◊), and 2D6 (○) compete with ephrin A1 for binding to MDAMB231 cells. Ability of phage antibodies D2-1A7, D2-1A9, and 2D6 binding to MDAMB231 cells in the presence of increasing concentrations of EphA2 ligand, Ephrin A1 was determined by flow cytometry.

Figure 6. Phage antibodies specific to EphA2 and CD44 are endocytosed by MDAMB231 cells

Cultured cells were incubated with irrelevant phage (A), anti-EphA2 phage D2-1A7 (B) and D2-1A9 (C), anti-CD44 phage F2-1A6 (D, E) for 3 hr at 37°C followed by glycine buffer wash. Panels D and E are from the same experiment, but E is imaged at higher magnification. Endocytosis was visulaized by detection of intracellular phage with anti-fd antibody, and analyzing by confocal microscopy.