1300

Background. The epidermal growth factor (EGFr/ErbB1/HER1) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Over expression of the EGFr and its ligands have been correlated with aggressiveness and poor prognosis in various tumors such as colon, breast and prostate cancer. Treatments with antagonists to EGFr have been beneficial to some patients. We have previously shown, using mass spectrometry, seven ligand induced EGFr phosphorylation sites (J Am Soc Mass Spectrom. 14:1022-1031) and quantified their phosphate content upon ligand (EGF, TGFα) and inhibitor (AG1478) addition. To test the feasibility and to establish a general protocol for isolating anti phospho-tyrosine (pTyr) specific antibodies we used antibody phage display technology to screen peptides representing the pTyr sites of the EGFr. The single chain phage antibodies were converted to a bivalent format to examine their usefulness in a variety of antibody-based assays including immuno-assay and western blot analysis. Results. After several rounds of panning against 12-mer pTyr peptides representing the pTyr sites in the EGFr, and counter screening against the corresponding non-phosphorylated peptide, 80 unique phage antibody clones were identified for pTyr sites Y992, Y1045, Y 1068, Y1086, Y 1148, and Y1173. Some of these clones (22) cross-reacted with neighbor phospho-peptide. Fifthteen of 58 clones were then converted to a bivalent backbone (one clone, Y1045, was converted to IgG). Antibodies were then tested on EGF-stimulated A431 cells by western analysis and ELISA. Western analysis revealed that 8 of 15 clones tested identified phospho-EGFr bands while 7 of 8 antibodies identified only a single band. The antibodies were then tested in an immunoassay using an ELISA platform. Of 15 clones assayed 10 clones gave a 2-10 fold increase of signal to noise when assayed against EGF-stimulated and unstimulated A431 cells. Functional bivalent antibodies were identified for all of the pTyr sites except for Y1045. The antibodies are also being tested for use in immunohistochemistry and for PhosFlow analysis. Conclusion. We have established a protocol for the isolation of pTyr specific antibodies using phage display technology. Phage clones were isolated by using pTyr peptides representing the pTyr of the EGFr upon ligand induction. The usefulness of these antibodies was demonstrated by western blot analysis and immunoassay after conversion to a bivalent format. These antibodies will allow us to investigate more precisely the activation state of the EGFr by allowing us to interrogate individual sites of Tyr phosphorylation of anti-EGFr.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]