Isolation of human antibodies to tumor-associated endothelial cell markers by in vitro human endothelial cell selection with phage display libraries

Abstract

Human antibodies selectively targeting angiogenic vessels hold great promise for the immunotherapy of human malignancies and can help to elucidate the molecular mechanisms regulating angiogenesis. By selecting a large antibody phage display library on proliferating stimulated HUVEC, we have isolated 15 human antibodies that bind to HUVEC in flow cytometric analysis, 11 of which target the vasculature of colorectal carcinomas as demonstrated by immunohistochemical analysis. The four most specific antibodies, TEM-A, TEM-C, TEM-M and TEM-Q, also stain the vasculature of a series of carcinomas derived from liver, ovary, kidney, bladder, lung and breast, and either react weakly or not all with the vasculature of corresponding normal tissues. All four antibodies react with the vasculature of endometrium, but only TEM-M and TEM-Q react with the vasculature of placenta. As shown by non-reducing western blot analysis, 9/15 of the antibodies recognize either one or two distinct bands on HUVEC cell lysates, with molecular weights of 175 and/or 110–125 kDa. Antibodies identified by this approach may be used for the identification of new markers of angiogenesis and/or tumor vasculature. The selected antibodies may prove useful as new prognostic markers, for in vivo tumor imaging purposes and for further development of targeted therapies.