A DNA vaccine targeting angiomotin inhibits angiogenesis and suppresses tumor growth Lars Holmgren*, Elena Ambrosino†, Olivier Birot*, Carl Tullus*, Niina Veitonma¨ ki*, Tetyana Levchenko*, Lena-Maria Carlson*, Piero Musiani‡, Manuela Iezzi‡, Claudia Curcio†, Guido Forni†§, Federica Cavallo†¶, and Rolf Kiessling* *Department of Oncology and Pathology, Cancer Centre Karolinska, Karolinska Institutet, SE171 76 Stockholm, Sweden; †Department of Clinical and Biological Sciences, University of Turin, I-10043 Orbassano, Italy; ‡Aging Research Center, ‘‘Gabriele d’Annunzio’’ University Foundation, I-66013 Chieti, Italy; and §Molecular Biotechnology Center, University of Turin, I-10123 Turino, Italy Communicated by Judah Folkman, Harvard Medical School, Boston, MA, April 20, 2006 (received for review September 28, 2005) Endogenous angiogenesis inhibitors have shown promise in pre- terization of the angiostatin receptor angiomotin (Amot) that is clinical trials, but clinical use has been hindered by low half-life in expressed in tumor and placental endothelium (15). Other recep- circulation and high production costs. Here, we describe a strategy tors for angiostatin have also been identified: for example, ATP that targets the angiostatin receptor angiomotin (Amot) by DNA synthase, the integrin ␣v␤3, and c-Met (16). Amot is a membrane- vaccination. The vaccination procedure generated antibodies that associated protein that mediates angiostatin inhibition of endothe- detected Amot on the endothelial cell surface. Purified Ig bound to lial migration and tube formation in vitro (17–20). A role of Amot the endothelial cell membrane and inhibited endothelial cell mi- in cell motility is also indicated by the finding that Amot-deficient gration. In vivo, DNA vaccination blocked angiogenesis in the mouse embryos exhibit a migratory defect in the anterior visceral matrigel plug assay and prevented growth of transplanted tumors endoderm at embryonic day 7.5 (21). High Amot mRNA levels also for up to 150 days. We further demonstrate that a combination of have been correlated to poor survival in breast cancer patients (22). DNA vaccines encoding Amot and the extracellular and transmem- Therefore, the functional role of Amot as an angiostatin receptor brane domains of the human EGF receptor 2 (Her-2)͞neu oncogene and its expression in angiogenic vessels makes it a possible target for inhibited breast cancer progression and impaired tumor vascular- antiangiogenic therapy. ization in Her-2͞neu transgenic mice. No toxicity or impairment of The development of active immunotherapy of cancer has been normal blood vessels could be detected. This work shows that DNA hampered by limited success in the clinic, related to difficulties of vaccination targeting Amot may be used to mimic the effect of breaking tolerance against weak self-antigens on tumor cells and angiostatin. the genetic variability of tumor cells resulting in immunologic escape variants. This limited clinical efficacy has spurred the cancer vaccines ͉ neoplasia ͉ neovascularization ͉ breast cancer ͉ development of combination approaches in which tumor vaccines angiostatin are combined with cancer therapies that target the tumor stroma, which is more genetically stable. Recent evidence has shown the he expansion of the circulatory system by the mechanism of feasibility of targeting molecules that are expressed by angiogenic Tangiogenesis is a driving force behind diseases such as cancer, endothelium [for example, VEGF-R2 (23, 24)] and that synergy macular degeneration, and atherosclerosis (1). Inhibition of the between tumor immunotherapy and antiangiogenic therapy can be signaling pathways underlying pathological angiogenesis therefore achieved (25). Here, we have used DNA vaccination to break offers a possible way of intervening with the progress of the disease. tolerance and invoke an immune response against Amot. Our Indeed, recent evidence has shown that treatment with antibodies approach generates Amot-specific Ig, resulting in inhibition of targeting VEGF in combination with chemotherapy prolongs life in angiogenesis and tumor growth without detectable toxicity and thus patients with metastatic colon, breast, and lung cancer (2, 3). At circumventing the problems experienced with endogenous angio- least in the case of tumor growth, inhibition of one signaling genesis inhibitors. pathway may not be sufficient, because compensatory pathways Results may be activated when VEGF signaling is inhibited (4, 5). Tumor angiogenesis may therefore involve a coordinate expression of a Amot Is Expressed in Endothelial Cells During Angiogenesis. For DNA variety of angiogenic factors, such as IL-8, basic fibroblast growth vaccination, we used cDNA encoding the human p80 isoform of factor (bFGF), and others (6). The implication of these findings is Amot inserted into the pcDNA3 vector (Fig. 1A). Transient trans- that therapies that target more than one pathway or target endo- fection of this vector into HeLa cells yielded an expected 80-kDa thelial functions downstream of these pathways could improve the band similar to that of endogenous p80 Amot in bovine capillary efficacy of antiangiogenic therapies. endothelial cells (Fig. 1B). To verify that the target protein was The induction of tumor angiogenesis is likely regulated by a present in endothelial cells during angiogenesis, we analyzed Amot balance between endogenous proangiogenic and antiangiogenic levels in lobular mammary carcinomas that spontaneously arise in BALB͞c mice that are transgenic for the human EGF receptor 2 factors (7). At least 16 endogenous angiogenesis inhibitors have ͞ been isolated that exhibit antiangiogenic and tumor suppressive (Her-2) neu oncogene (BALB-neuT mice) (26). p80 Amot was activity (8, 9). One of the earliest inhibitors to be reported, clearly detectable by Western blot analysis of two individual car- angiostatin, was shown to be specific for endothelial cells and could cinomas from 22- and 24-week-old BALB-neuT mice. However, maintain dormancy of established metastases in vivo (10, 11). Amot was not detectable in normal breast tissue from a virgin However, the serum half-life of angiostatin in patients is Ϸ3h, making it necessary to administer high amounts of angiostatin at Conflict of interest statement: L.H. and R.K. are supported by grants from BioInvent frequent intervals (12). Thus, the pharmacodynamics of angiostatin International (Lund, Sweden). and other inhibitors such as endostatin constitute a major obstacle Abbreviations: Amot, angiomotin; bFGF, basic fibroblast growth factor; Her-2, human EGF for the use of these agents in cancer patients (13, 14). To design receptor 2; KO, knockout; MAE, mouse aortic endothelial cell; PECAM, platelet endothelial alternative molecules more suited for antiangiogenic therapy, it is cell adhesion molecule. of importance to identify the receptors that mediate the antiangio- ¶To whom correspondence should be addressed. E-mail: [email protected]. genic effect. We have previously reported the cloning and charac- © 2006 by The National Academy of Sciences of the USA 9208–9213 ͉ PNAS ͉ June 13, 2006 ͉ vol. 103 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603110103 Downloaded by guest on October 1, 2021 Fig. 1. Expression of Amot in angiogenic vessels. (A) The human p80 isoform of Amot was cloned into the pCDNA3 vector, which was then used for DNA vaccination in mice. (B) Western blot analysis of Amot protein levels. Bovine capillary endothelial cells (BCE) expressed both p80 and p130 isoforms of Amot. Transient transfection with the pcDNA3-Amot construct into HeLa cells yielded a band corresponding to 80 kDa (Amot), whereas vector control was negative. The TUBO mouse breast cancer cell line is derived from BALB-neuT transgenic mice and did not express detectable levels of Amot. In contrast, Fig. 2. Amot DNA vaccination inhibits tumor growth. (A) WT BALB͞c mice were total lysates from tumors harvested at 22 and 24 weeks after birth from vaccinated twice with either Amot or control vector pcDNA3 21 and 7 days before BALB-neuT transgenic mice showed detectable expression of p80 Amot (BALB- s.c. challenge with the TUBO mouse breast cancer cell line. In three independent neuT). Breast tissue harvested from a normal virgin female mouse was below experiments, Amot vaccination suppressed tumor growth for Ͼ150 days in 12 of detection (Br). (C) The top row shows a vessel positive for Amot and for the 18 treated mice; in two mice, tumors recurred 150 days after challenge. (B)To endothelial marker PECAM in a tumor derived from a BALB-neuT mouse. The determine whether the induction of the antitumor activities depended on CD4ϩ two middle rows show angiogenic vessels activated by bFGF in the in vivo T cells, we conducted an in vivo T cell depletion of this subset during DNA matrigel plug assay that were strongly positive for Amot as shown by immu- vaccination. Mice were treated with either anti-CD4 or control IgG before and nofluorescence analysis using polyclonal anti-Amot antibodies. Functional during vaccinations with Amot or empty vector. The antitumor effect of vacci- vessels were visualized by i.v. FITC-dextran injection before euthanization of nation against Amot was abrogated in the CD4-depleted mice. The size of tumors the animal. The bottom row shows vessels of the normal stroma that did not in Amot-vaccinated mice receiving control IgG or anti-CD4 Ig was significantly stain positively for Amot. (Scale bars, 100 ␮m.) different between 7 and 35 days after challenge (n ϭ