Endosialin and VEGF₁₆₅/VEGFR complex as targets for vascular targeting agents.

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Tumor specific markers of angiogenesis are essential for vascular targeting. From the literature, we have selected i) Endosialin (TEM-1) and ii) the complex of VEGF₁₆₅ and VEGFR2 (VEGF₁₆₅-KDR) due to their promising targeting profile. Here we describe the isolation of highly specific human recombinant Fab antibodies (HuCAL GOLD library, MorphoSys AG) to both targets and the results we obtained from further analysis employing these antibodies. i) Endosialin was identified with a monoclonal antibody FB5 as a tumor endothelial specific antigen¹. In contrast, a much broader distribution of the murine homologue was found by others². Panning yielded a broad panel of Fab antibodies to various epitopes of Endosialin. Immunohistochemistry experiments on cryosections of various human tumor tissues revealed staining of stromal components while vessel staining of tumor endothelium was episodic and only detectable in sections of colorectal liver metastases. Analysis of normal foreskin tissue revealed staining of vessels and stromal components. The finding of a broader mRNA distribution for Endosialin² was confirmed by our studies at the protein expression level. For these reasons, we do not consider Endosialin a suitable target for vascular targeting of tumors. ii) The complex of VEGF₁₆₅ and its receptor VEGFR2 has been described as highly specific for the neovasculature of tumors³. Panning on the complex yielded several specific Fab antibodies as shown by ELISA and on cells by FACS experiments. However, the recognized epitope appears to be very sensitive to any modification since none of these antibodies was reactive in immunohistochemistry. Unfortunately, two antibodies with a very low nanomolar affinity suitable for animal testings were species-specific for the complex consisting of the human KDR and human VEGF₁₆₅. Our study shows the feasibility of generating HuCAL GOLD derived Fab antibodies, which are specific for the complex formed by the interaction of VEGF and its receptor KDR. In future panning experiments, we will modify the selection procedure to enrich for cross-reactive antibodies allowing in-vivo testing. We consider this work an important step towards the generation of human VEGF₁₆₅/VEGFR2 directed antibody diagnostics and therapeutics. References: ¹Rettig et al. Proc Natl Acad Sci U S A 89 (22):10832-10836, 1992. ²Opavsky et al. J Biol Chem 276 (42):38795-38807, 2001. ³Brekken et al. Cancer Research 58: 1952-1959,1998.