ARTICLES Inhibiting Tumor Growth by Targeting Tumor Vasculature with Galectin-1 Antagonist Anginex Conjugated to the Cytotoxic Acylfulvene, 6-Hydroxylpropylacylfulvene Ruud P. M. Dings, † Emily S. Van Laar, ‡ Melissa Loren, † Jeremy Webber, ‡ Yan Zhang, § Stephen J. Waters, ‡ John R. MacDonald, ‡ and Kevin H. Mayo* ,† Departments of Biochemistry, Molecular Biology, and Biophysics, and Biostatistics Core, University of Minnesota, and MGI PHARMA Inc, Bloomington, Minnesota. Received July 2, 2009; Revised Manuscript Received November 11, 2009 Targeted delivery of therapeutic drugs promises to become the norm to treat cancer. Here, we conjugated the cytotoxic agent 6-hydroxypropylacylfulvene (HPAF) to anginex, a peptide that targets galectin-1, which is highly expressed in endothelial cells of tumor vessels. In a human ovarian cancer model in mice, the conjugate inhibited tumor growth better than equivalent doses of either compound alone. Immunofluorescence on tumor tissue demonstrated that the conjugate, like parent anginex, selectively targeted tumor vasculature and inhibited tumor angiogenesis. Increased activity from the conjugate further suggests that HPAF retains at least some of its normal cytotoxic activity when linked to anginex. More importantly perhaps is the observation that the conjugate abrogates apparent systemic toxicity from treatment with HPAF. This work contributes to the development of tumor vascular targeting agents against cancer in the clinic. INTRODUCTION When used at optimal doses, chemotherapeutic agents can result in considerable systemic toxicity. Recently, it was demonstrated in the clinic that chemotherapy at doses below maximally tolerated doses can be very effective when used in combination with the antiangiogenic agent, Avastin (Genentech) (1). In fact, a number of antiangiogenic agents have been developed and used in a similar fashion in various preclinical animal models (2-4). The most promising of these appear to be those that act directly on endothelial cells (EC) (5), because they are effective against a broad spectrum of tumors and less prone to induce drug resistance (5). Moreover, such EC-specific agents are also being used as tumor targeting moieties to increase specificity of chemotherapeutics (6) and to deliver contrast agents for magnetic resonance imaging (MRI) (7). For example, the R v integrin-binding RGD tripeptide has been coupled to the chemotherapeutic drug doxorubicin, and shown in tumor-bearing mice to prolong survival, decrease the optimal effective dose, and reduce toxicity (6). Another EC specific antiangiogenic agent, anginex (8, 9), targets galectin-1 (gal-1) (10), a cell surface glycan binding protein that is highly upregulated in tumor-activated EC (10-12). Differential stromal elevation of gal-1 over the tumor paren- chyma has been reported in several cancers, including cancer of the breast (13), colon (14), prostate (15), and ovary (16). By weakly binding to “carrier protein” plasma fibronectin (17), anginex is transported through the cardiovascular system to the tumor vasculature where the peptide binds gal-1 (10). Anginex binding to gal-1 disrupts tumor EC adhesion and migration, thereby inducing EC apoptosis and resulting in the inhibition of tumor angiogenesis (10, 18). Because gal-1 is likely expressed in most tumor types at relatively high levels, using anginex as a tumor vasculature homing device should be widely applicable. The acylfulvenes are a semisynthetic class of compounds derived from illudin S, a toxin from the Omphalotus illudens mushroom, that demonstrate broad spectrum antitumor activity in studies conducted in vitro and in vivo. Irofulven (MGI-114; 6-hydroxymethylacylfulvene) is a leading member of the acylfulvene analogues and has shown clinical activity against a variety of cancers, either as a monotherapy or in combination with other chemotherapeutic agents (19). Similar to other alkylating agents, dose-limiting toxicities, such as myelosup- pression, neutropenia, thrombocytopenia, nausea, vomiting, and fatigue, are observed when therapeutic concentrations of irof- ulven are used in patients (20, 21). At higher doses, ocular disturbances and symptoms of renal tubular acidosis have been observed (20, 21). Conjugating acylfulvenes to a vascular targeting agent, like anginex, has the potential not only to reduce the chemotherapeutic’s toxicity profile and maintain, or even improve, efficacy in the clinic, but also to be employed against a broader spectrum of tumors. In this report, we conjugated an irofulven analogue, 6-hydroxypropylacylfulvene (HPAF) to anginex and investigated the ability of anginex to act as a tumor vasculature homing device. In human tumor xenograft studies, we found that the conjugate indeed targeted the tumor vascu- * To whom correspondence should be addressed. Dr. K. H. Mayo, Dept. of Biochemistry, 6-155 Jackson Hall, University of Minnesota Health Sciences Center, 321 Church Street, Minneapolis, Minnesota 55455, USA, Phone: 612-625-9968; Fax: 612-624-5121; E-mail: mayox001@tc.umn.edu. † Departments of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota. ‡ MGI PHARMA Inc. § Biostatistics Core, University of Minnesota. Bioconjugate Chem. 2010, 21, 20–27 20 10.1021/bc900287y  2010 American Chemical Society Published on Web 12/18/2009