SHIBATA Yoichi Department of Transfusion Medicine, The University of Tokyo, Professor, 医学部・附属病院, 教授 (30010474)
NAGAWA Hirokazu Department of Surgical Oncology, The University of Tokyo, Professor, 医学部・附属病院, 教授 (80228064)
KITAYAMA Joji Department of Surgical Oncology, The University of Tokyo, Lecturer, 医学部・附属病院, 講師 (20251308)
The aim of our project was to develop monoclonal antibodies reactive with endothelial cells of tumor vasculature. For this purpose, we immunized Balb/c mice with human umbilical vein endothelial cells pre-treated with angiogenic factors. We screened for endothelial cell-reactive monoclonals by the mixed-passive hemagglutination (MPHA) assay, and obtained approximately 500 clones. Among these, we screened for endothelial-specific monoclonals by flow-cytometry, and subsequently by immunostaining of tissue specimens of colon cancer and normal colonic mucosa. By this methodology, we obtained approximately 10 monoclonals with high-reactivity to endothelium of vasculature surrounding colon cancer, but not with vasculature of normal colonic mucosa. In addition, we immunostained sections of normal tissue and tumors of breast, stomach, and brain. Similarly, only the tumor vasculature were stained.
Next, using mass spectrometry, we found that most of these monoclonals were reactive to endoglin (CD105) and one with the integrin aV|33. These antigens are well-known to be predominantly expressed on tumor endothelium, and recently many reports have shown that monoclonals to these antigens are able to suppress tumor growth. Therefore, next we evaluated the effect of these antibodies against endothelial cells by in vitro cytotoxicity assay. Only one of them was able to induce a complement-dependent lysis of endothelial cells. Using a mouse model of subcutaneous implantation of colon cancer, we demonstrated a suppressive effect of these monoclonals on tumor growth.
The next step of our project is to humanize these antibodies for clinical application.