Abstract #LB-163: A novel antiangiogenic feature of a dual targeting antibody (DMS3008) against EGFR and VEGF

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO An antibody simultaneously targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), two major tumor growth-driving machineries, may provide a novel therapeutic strategy. Human domain antibodies selected against EGFR and VEGF were formatted into a fully human dual-targeting IgG (DMS3008), to target both antigens in a single molecule. We previously reported that DMS3008 inhibited the activation of EGFR as effectively as cetuximab, neutralized VEGF as effectively as bevacizumab in vitro, and suppressed xenografted lung and head and neck cancer tumor growth. In this study, we describe a novel feature of this dual antibody in Tu212 head and neck squamous cell carcinoma cells expressing high levels of EGFR and able to undergo antibody-VEGF-EGFR endocytosis. Immunofluorescence revealed that, similar to cetuximab, DMS3008 was able to induce EGFR internalization. In contrast to bevacizumab or cetuximab, when Tu212 cells were exposed to VEGF-488 (0.5 \#956;g/ml) and DMS3008 (1 µg/ml), the level of internalized VEGF-488 measured by flow cytometry increased with time and could be completely diminished by an excess of VEGF. When Tu212 cells were pre-exposed to EGF (100 ng/ml) for 1 hr, however, VEGF-488 internalization was significantly abrogated, indicating that internalization was dependent on EGFR. Confocal Live Ultraview ERS imaging revealed a dynamic process of VEGF internalization and apoptosis after Tu212 cells were exposed to DMS3008 and VEGF-488 for up to 4 hrs. In addition, internalized VEGF-488 was shown by immunofluorescence to be partially co-localized with the early endosome marker EAA1 and the late endosome marker CD63. The antitumor activity of the dual antibody was examined in mice bearing Tu212 xenografts. Animals received 10 mg/kg body weight of bevacizumab, cetuximab, a combination of bevacizumab and cetuximab, DMS3008, isotopic IgG, or PBS i.p. twice weekly for 4 weeks. DMS3008 suppressed tumor growth as effectively as cetuximab or bevacizumab alone or in combination, when compared with PBS or IgG controls (p<0.05 for all treatment vs. control groups). DMS3008 also induced a similar degree of apoptosis as the bevacizumab and cetuximab combination, as shown by TUNEL staining. Quantification of CD34 staining revealed that treatment with DMS3008 completely diminished tumor microvessel formation, as did cetuximab or bevacizumab alone or combined, when compared with IgG or PBS (p=0.002 for all treatment vs. control groups). In conclusion, our current studies demonstrate a novel mechanism of antiangiogenesis through DMS3008-mediated EGFR-dependent internalization of VEGF. Our findings suggest that the dual-targeting antibody DMS3008 represents a novel class of agents with broad and versatile functional implications. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-163.