5708

VEGF is a key factor to promote (tumor) angiogenesis and is released by tumor cells and host cells including platelets. It has been demonstrated that platelets contribute to angiogenesis, mainly by release of VEGF from their α-granules upon activation. Evidence is also accumulating that platelets are involved in tumor induced angiogenesis and metastasis formation. In addition, it is known that platelets contain and take up plasma proteins in their α-granules, including immunoglobulins. In this study we report that bevacizumab, a humanized antibody against VEGF, with clinical activity in a variety of cancer types, is taken up by platelets in a dose dependent manner (0–20ug/ml). We measured the uptake of bevacizumab by platelets in vitro after labeling the antibody with fluorescence or biotin by FACS analysis or Western blot respectively. Upon activation by thrombin the preincubated platelets released bevacizumab. In addition, the incubation of platelets with bevacizumab caused a dose dependent decrease in platelet derived free-VEGF as measured by ELISA (R*D). Fluorescence microscopy revealed a granular pattern of FITC-labeled bevacizumab in platelets after incubation. These results are indicative of colocalization of bevacizumab in the platelet α-granules. Subsequently, we found that the proangiogenic activity of platelets is significantly impaired by the uptake of bevacizumab as reflected by inhibition of endothelial cell proliferation in vitro (85% inhibition over IgG control, P<0.01). To explore whether platelet uptake of bevacizumab also occurs in vivo, we administered 10 mg/kg of FITC-labeled bevacizumab or FITC-labeled control IgG intravenously in rabbits and we were able to confirm the uptake of bevacizumab and IgG by platelets. In these experiments, the half life time of FITC-labeled bevacizumab in platelets was more than two times longer than in plasma. Taken together, these findings indicate that the distribution of bevacizumab in cancer patients is partly mediated by transport in platelets and its release affected by platelet aggregation. Therefore, these results may provide an explanation for the observed abnormalities in wound healing and coagulation in cancer patients treated with bevacizumab. In addition, based on our studies and the existing data on the role of platelets in tumor angiogenesis, we hypothesize that the uptake of bevacizumab and the consequently impaired platelet angiogenic activity may play a role in its antiangiogenic and antitumor effect in patients with cancer.

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