Bevacizumab reduces tumor targeting of antiepidermal growth factor and anti‐insulin‐like growth factor 1 receptor antibodies

Bevacizumab (antivascular endothelial growth factor [anti‐VEGF]) and cetuximab (antiepidermal growth factor receptor [anti‐EGFR]) are approved antibodies for treatment of cancer. However, in advanced colorectal cancer, the combination fails to improve survival. As the reason for the lack of activity is unknown, our study aims to determine the effect of bevacizumab on targeting of anti‐EGFR and insulin‐like growth factor 1 receptor (IGF‐1R) antibodies in tumors with single‐photon emission computed tomography (SPECT)/CT imaging. Mice with subcutaneous EGFR and IGF‐1R‐expressing SUM149 xenografts received a single dose of bevacizumab (10 mg/kg) or saline. After 4 days, mice were injected with radiolabeled cetuximab or R1507, an anti‐IGF‐1R antibody. A control group received a radiolabeled irrelevant IgG (hLL2). Three days later, SPECT/CT images were acquired and mice were dissected to determine the concentration of antibodies in the tissues. Tumors were analyzed immunohistochemically to determine vascular density (CD34), VEGF, EGFR and IGF‐1R expression. SPECT/CT imaging revealed that bevacizumab treatment significantly reduced tumor targeting of radiolabeled cetuximab by 40% from 33.1 ± 1.1 %ID/g to 19.8 ± 5.7 %ID/g (p = 0.009) for untreated and bevacizumab‐treated tumors, respectively. A similar effect was found for ¹¹¹In‐R1507: tumor targeting of R1507 decreased by 35%. No significant differences in tumor uptake were observed in mice that received an irrelevant IgG. Uptake in normal organs was not altered by bevacizumab. Immunohistochemical analysis showed that vascular density decreased with 43%, whereas EGFR and IGF‐1R expression was unaltered. In conclusion, bevacizumab treatment significantly reduces tumor targeting of anti‐EGFR and anti‐IGF‐1R antibodies. This emphasizes the importance of timing and sequencing of bevacizumab in combination with other antibodies.     

Anti‐angiogenic and anti‐tumor effects of TAK‐593, a potent and selective inhibitor of vascular endothelial growth factor and platelet‐derived growth factor receptor tyrosine kinase

We recently reported that TAK‐593, a novel imidazo[1,2‐b]pyridazine derivative, is a highly potent and selective inhibitor of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor tyrosine kinase families. Moreover, TAK‐593 exhibits a uniquely long‐acting inhibitory profile towards VEGF receptor 2 (VEGFR2) and PDGF receptor β (PDGFRβ). In this study, we demonstrated that TAK‐593 potently inhibits VEGF‐ and PDGF‐stimulated cellular phosphorylation and proliferation of human umbilical vein endothelial cells and human coronary artery smooth muscle cells. TAK‐593 also potently inhibits VEGF‐induced tube formation of endothelial cells co‐cultured with fibroblasts. Oral administration of TAK‐593 exhibited strong anti‐tumor effects against various human cancer xenografts along with good tolerability despite a low level of plasma exposure. Even after the blood and tissue concentrations of TAK‐593 decreased below the detectable limit, a pharmacodynamic marker (phospho VEGFR2) was almost completely suppressed, indicating that its long duration of enzyme inhibition might contribute to the potent activity of TAK‐593. Immunohistochemical staining indicated that TAK‐593 showed anti‐proliferative and pro‐apoptotic effects on tumors along with a decrease of vessel density and inhibition of pericyte recruitment to microvessels in vivo. Furthermore, dynamic contrast‐enhanced magnetic resonance imaging revealed that TAK‐593 reduced tumor vessel permeability prior to the onset of anti‐tumor activity. In conclusion, TAK‐593 is an extremely potent VEGFR/PDGFR kinase inhibitor whose potent anti‐angiogenic activity suggests therapeutic potential for the treatment of solid tumors.     

Phase I clinical trial of human vascular endothelial growth factor receptor 1 peptide vaccines for patients with metastatic renal cell carcinoma

Background:

It is well known that renal cell carcinoma (RCC) represents one of the most immune-responsive cancers. Although the lack of defined antigens in RCC has hindered more specific vaccine development, research regarding vaccination therapy has been of special interest for the treatment of RCC for more than 30 years.

Methods:

To evaluate the safety of the vascular endothelial growth factor receptor 1 (VEGFR1) peptide vaccination and its clinical outcomes, data from 18 metastatic RCC (mRCC) patients treated with VEGFR1 vaccine were collected. Toxicity assessments were performed. Clinical outcomes included assessment using CT scanning, magnetic resonance imaging or X-ray examination in accordance with the WHO Response Evaluation Criteria in Solid Tumors.

Results:

No patient showed any toxicities of grade 3 or greater. Of the 18 patients, 2 patients showed a partial response during treatment. Stable disease for more than 5 months was observed in eight patients with a median duration of 16.5 months (4–32 months). At the time of the analysis in this study, six patients were alive with a median follow-up of 30 months (26–36 months).

Conclusion:

These results suggest that VEGFR1 peptide vaccine is safe and is recommended for further trials for patients with mRCC.     

Phase I clinical trial using peptide vaccine for human vascular endothelial growth factor receptor 2 in combination with gemcitabine for patients with advanced pancreatic cancer

Vascular endothelial growth factor receptor 2 (VEGFR2) is an essential factor in tumor angiogenesis and in the growth of pancreatic cancer. Immunotherapy using epitope peptide for VEGFR2 (VEGFR2‐169) that we identified previously is expected to improve the clinical outcome. Therefore, a phase I clinical trial combining of VEGFR2‐169 with gemcitabine was conducted for patients with advanced pancreatic cancer. Patients with metastatic and unresectable pancreatic cancer were eligible for the trial. Gemcitabine was administered at a dose of 1000 mg/m² on days 1, 8, and 15 in a 28‐day cycle. The VEGFR2‐169 peptide was subcutaneously injected weekly in a dose‐escalation manner (doses of 0.5, 1, and 2 mg/body, six patients/one cohort). Safety and immunological parameters were assessed. No severe adverse effect of grade 4 or higher was observed. Of the 18 patients who completed at least one course of the treatment, 15 (83%) developed immunological reactions at the injection sites. Specific cytotoxic T lymphocytes (CTL) reacting to the VEGFR2‐169 peptide were induced in 11 (61%) of the 18 patients. The disease control rate was 67%, and the median overall survival time was 8.7 months. This combination therapy for pancreatic cancer patients was tolerable at all doses. Peptide‐specific CTL could be induced by the VEGFR2‐169 peptide vaccine at a high rate, even in combination with gemcitabine. From an immunological point of view, the optimal dose for further clinical trials might be 2 mg/body or higher. This trial was registered with ClinicalTrial.gov (no. NCT 00622622). (Cancer Sci 2009)     

An angiogenic switch in breast cancer involves estrogen and soluble vascular endothelial growth factor receptor 1

Estrogen is involved in breast tumorigenesis, but the precise mechanisms for its oncogenic and angiogenic actions are poorly understood. Angiogenesis is regulated, in part, by these critical components: vascular endothelial growth factor (VEGF) and its two receptors (VEGFR-1 and VEGFR-2). VEGFR-2 is a positive angiogenic signal transducer, whereas VEGFR-1, especially its soluble form (soluble VEGFR-1), is a negative regulator of VEGF availability. We found that breast epithelial cells express soluble VEGFR-1 and hypothesized that because estrogen can regulate expression of members of the VEGF family, it might stimulate angiogenesis in breast cancer by decreasing expression of soluble VEGFR-1. Soluble VEGFR-1 expression decreased in estrogen receptor (ER)-positive but not in ER-negative breast cancer cell lines treated with estrogen. Pretreatment of the cells with the ER antagonist ICI 182,780 blocked the effect. The estrogen-mediated decrease in soluble VEGFR-1 expression was accompanied by a statistically significant increase in angiogenesis in vivo. Our data suggest that inhibition of soluble VEGFR-1 expression represents a novel mechanism--an estrogen-driven angiogenic switch--possibly responsible for breast carcinoma progression.     

Clinical phase I study of elpamotide,a peptide vaccine for vascular endothelial growth factor receptor 2, in patients with advanced solid tumors

Targeting of tumor angiogenesis with vaccines is a potentially valuable approach to cancer treatment. Elpamotide is an immunogenic peptide derived from vascular endothelial growth factor receptor 2, which is expressed at a high level in vascular endothelial cells. We have now carried out a phase I study to evaluate safety, the maximum tolerated dose, and potential pharmacodynamic biomarkers for this vaccine. Ten HLA‐A*24:02‐positive patients with advanced refractory solid tumors received elpamotide s.c. at dose levels of 0.5, 1.0, or 2.0 mg once a week on a 28‐day cycle. Five patients experienced an injection site reaction of grade 1 and 2, which was the most frequent adverse event. In the 1.0 mg cohort, one patient experienced proteinuria of grade 1 and another patient developed both hypertension and proteinuria of grade 1. No adverse events of grade 3 or higher were observed, and the maximum tolerated dose was therefore not achieved. The serum concentration of soluble vascular endothelial growth factor receptor 2 decreased significantly after elpamotide vaccination. Microarray analysis of gene expression in PBMCs indicated that several pathways related to T cell function and angiogenesis were affected by elpamotide vaccination, supporting the notion that this peptide induces an immune response that targets angiogenesis in the clinical setting. In conclusion, elpamotide is well tolerated and our biomarker analysis indicates that this anti‐angiogenic vaccine is biologically active. Clinical trial registration no. UMIN000008336.     

Inhibition of tumor growth with antiangiogenic cancer vaccine using epitope peptides derived from human vascular endothelial growth factor receptor 1.

PURPOSE: Antiangiogenic therapy is now considered to be one of promising approaches to treat various types of cancer. In this study, we examined the possibility of developing antiangiogenic cancer vaccine targeting vascular endothelial growth factor receptor 1 (VEGFR1) overexpressed on endothelial cells of newly formed vessels in the tumor. EXPERIMENTAL DESIGN: Epitope-candidate peptides were predicted from the amino acid sequence of VEGFR1 based on their theoretical binding affinities to the corresponding HLAs. The A2/Kb transgenic mice, which express the alpha1 and alpha2 domains of human HLA-A*0201, were immunized with the epitope candidates to examine their effects. We also examined whether these peptides could induce human CTLs specific to the target cells in vitro. RESULTS: The CTL responses in A2/Kb transgenic mice were induced with vaccination using identified epitope peptides restricted to HLA-A*0201. Peptide-specific CTL clones were also induced in vitro with these identified epitope peptides from peripheral blood mononuclear cells donated by healthy volunteers with HLA-A*0201. We established CTL clones in vitro from human peripheral blood mononuclear cells with HLA-A*2402 as well. These CTL clones were shown to have potent cytotoxicities in a HLA class I-restricted manner not only against peptide-pulsed target cells but also against target cells endogenously expressing VEGFR1. Furthermore, immunization of A2/Kb transgenic mice with identified epitope peptides restricted to HLA-A*0201 was associated with significant suppression of tumor-induced angiogenesis and tumor growth without showing apparent adverse effects. CONCLUSIONS: These results strongly suggest that VEGFR1 is a promising target for antiangiogenic cancer vaccine and warrants further clinical development of this strategy.     

Anti-flt1 peptide, a vascular endothelial growth factor receptor 1-specific hexapeptide, inhibits tumor growth and metastasis.

PURPOSE: The purpose of this study was to develop antagonists specific for the vascular endothelial growth factor receptor 1 (VEGFR1) and to investigate the effects of the antagonists on the VEGF-induced endothelial cell functions and tumor progression. EXPERIMENTAL DESIGN: Hexapeptides that inhibit binding of VEGFR1 and VEGF were identified through screening of synthetic peptide library. A selected peptide, anti-Flt1, was investigated for binding specificity with various receptors and ligand peptides. Effects of the peptide on proliferation, cell migration, and fibrin gel-based angiogenesis of endothelial cells were also investigated. The activity of anti-Flt1, in vivo, was evaluated for inhibition of tumor growth and metastasis in VEGF-secreting cancer cell-implanted mice by s.c. injections of the peptide. RESULTS: Here, we report on a short peptide that binds to VEGFR1 and prevents binding of VEGF. A hexapeptide, anti-Flt1 (Gly-Asn-Gln-Trp-Phe-Ile or GNQWFI), was identified from peptide libraries. The anti-Flt1 peptide shows specificity toward binding to VEGFR1 and it inhibits binding of VEGF, placental growth factor (PlGF), and VEGF/PlGF heterodimer to VEGFR1. This peptide does not inhibit the proliferation of endothelial cells induced by VEGF and VEGF/PlGF heterodimer but it effectively blocks VEGF-induced migration of endothelial cells and their capacity to form capillary-like structures on fibrin gel-based in vitro angiogenesis system. Furthermore, growth and metastasis of VEGF-secreting tumor cells were also significantly inhibited by s.c. injections of anti-Flt1 peptide in nude mice. Accordingly, VEGF-induced migration and capillary formation are mediated through VEGFR1, and these processes may play an important role in the growth and metastasis of VEGF-secreting tumors. CONCLUSIONS: We show that a peptide (anti-Flt1) specific for VEGFR1 inhibits growth and metastasis of tumor that secretes VEGF. The effects on endothelial cell functions, in vitro, indicate that the anticancer activity of anti-Flt1 peptide with reduced blood vessel density could also be due to the blocking of VEGFR1-mediated endothelial cell migration and tube formation. Although the effects of anti-Flt1 peptide still remain to be further characterized, the receptor 1-specific peptide antagonist, anti-Flt1, has potential as a therapeutic agent for various angiogenesis-related diseases, especially cancer.     

It takes two to tango: Combinations of conventional cytotoxics with compounds targeting the vascular endothelial growth factor–vascular endothelial growth factor receptor pathway in patients with solid malignancies

Through advances in molecular biology, insight into the mechanisms driving malignancies has improved immensely and as a result, various factors playing an essential role in the biology of numerous tumor types have been revealed. By using compounds that specifically block the function of a single factor being crucial for tumor pathogenesis, it was hoped to exert antitumor activity while avoiding toxicities characteristic for conventional chemotherapy. One of the processes of crucial importance in the development of cancer, and consequently an attractive target, is angiogenesis. In recent years, several key factors for angiogenesis have been identified, including ligands, receptors, and transduction signaling factors. Of these, the vascular endothelial growth factor (VEGF) pathway has been found to be activated in numerous tumor types and considered one of the main drivers of angiogenesis. Roughly, VEGF-mediated angiogenesis can be inhibited by two approaches: either by monoclonal antibodies directed towards VEGF or its corresponding receptors, or by kinase inhibitors targeting the signal transduction of the VEGF receptors. As monotherapy, several kinase inhibitors exert antitumor activity in tumor types such as renal cell carcinoma. However, in most tumor types, the antitumor activity of compounds targeting the VEGF pathway is limited. In recent years, evidence is mounting that the paradigm of one single factor that drives malignant behavior applies rarely and is an oversimplification for most tumors in which there are multiple driving pathways. Consequently, multitargeting rather than single-targeting approaches are required. One of the means is by combining targeted agents with conventional cytotoxics. As the VEGF pathway also affects the sensitivity of tumor cells to chemotherapeutics, combinations of compounds targeting this pathway and conventional cytotoxics have been explored. This review addresses such combinations. ( Cancer Sci 2009; 00: 000–000)     

血管内皮生长因子及其受体与骨肉瘤关系的研究进展

血管内皮生长因子（ＶＥＧＦ）是一种序列高度保守、高度特异性的促血管内皮细胞生长因子,广泛分布于人和动物体内的大脑、肾脏、肝脏、脾脏、胰腺和骨骼等组织中,对内皮细胞具有强烈的促有丝分裂作用,刺激血管内皮细胞增殖和血管通透性增加,促进新生血管形成。ＶＥＧＦ通过与血管内皮细胞表面受体（ＶＥＧＦＲ）特异性结合发挥生物学效应。抑制ＶＥＧＦ及ＶＥＧＦＲ的活性可以减缓或阻滞骨肉瘤侵袭和转移。研究表明,ＶＥＧＦ及ＶＥＧＦＲ对肿瘤血管及淋巴管的生成及肿瘤侵袭和转移起重要作用。本文对ＶＥＧＦ及ＶＥＧＦＲ与骨肉瘤血管与淋巴管生成及其侵袭与转移的关系作一综述。     

Mapping of angiogenic markers for targeting of vectors to tumor vascular endothelial cells

The vasculature of mouse breast tumor spheroids grown on mammary fat pad tissue in an intravital microscopy (IVM) viewing chamber was shown to derive from infiltrating angiogenic mammary vessels. The receptors tissue factor (TF), alpha V beta 3 integrin and Tie-2 were expressed on the vascular endothelium in the periphery but not in the center of the tumor spheroids nor in the mammary tissue nor in smooth muscle tissue, whereas Tie-1 and PCAM-1 were expressed extensively in the entire tumor and in the vascular endothelium of the entire tumor nodule and in normal mammary tissue. TF is a specific target for adenoviral vector-mediated cancer immunotherapy. Subcutaneous injection of the AdfVII/IgG(1)Fc vector leads to the release into the system circulation of a fVII/IgG(1)Fc immunoconjugate molecule that binds specifically and tightly to TF on vascular endothelial cells and tumor cells, activating a cytolytic immune response against the targeted cells. We show that a single administration of the AdfVII/IgG(1)Fc vector destroys the peripheral but not the central vasculature of a tumor spheroid, causing partial tumor regression; additional administrations prevent regeneration of the peripheral vasculature and regrowth of the tumor. These findings indicate that a critical parameter for optimizing tumor damage is the schedule for successive administrations of the AdfVII/IgG(1)Fc, which should coincide with the regeneration of the peripheral vasculature and continue until the tumor is destroyed.     

The clinical toxicity profile of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors; a review

Clinical experience with vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors is rapidly increasing, and some compounds have already been approved for regular anticancer treatment.Apart from their activity, much attention has been focussed on the clinical toxicity profile of these compounds.This review describes the most frequently occurring side-effects of both antibodies and tyrosine kinase inhibitors and discusses some of the underlying mechanisms. Some practical guidelines for treatment of the side-effects are given.     

Antitumor treatment efficacy by targeting epidermal growth factor receptor and vascular endothelial growth factor receptor-2 in an orthotopic human glioblastoma model

Using an orthotopic intracerebral model from our established HM55-BGIV-101 tumor line, we investigated the antitumor effect on the angiogenesis and growth of human glioblastoma after treatment with monoclonal antibody DC101 against the vascular endothelial growth factor receptor-2 and monoclonal antibody C225 against the epidermal growth factor receptor. Nude mice bearing intracerebral glioblastoma xenografts were treated intraperitoneally with DC101 and C225 either alone or in combination. Histopathological analysis of solid tumor volume, satellite tumor number, microvessel density, tumor cell proliferation, and apoptosis was performed. In the DC101-treated group, solid tumor volume and microvessel density were reduced by 59.7 and 64%, respectively; tumor cell proliferative activity was reduced by 53.2% and the apoptotic index (AI) was increased by 66.7%; satellite tumor number was enhanced by 84.4%. C225 alone reduced satellite tumor number by 43.3%, but had no effect on solid tumor volume, microvessel density, tumor cell proliferation, and apoptosis. C225 combined with DC101 not only reduced solid tumor volume, microvessel density, tumor cell proliferative activity, and increased AI, but also reduced satellite tumor number. Inhibition of angiogenesis achieved by DC101 can cause increased tumor cell invasiveness. In our studies this increased tumor cell invasiveness was inhibited simultaneously by C225, which provides a theoretical basis for treatment of glioblastoma by the method of combining drugs with different pharmacological activity.     

Dual targeting of vascular endothelial growth factor and bone morphogenetic protein‐9/10 impairs tumor growth through inhibition of angiogenesis

Clinical development of anti‐angiogenic agents has been a major landmark in cancer therapy for several types of cancers. Signals mediated by both vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)‐9 and 10 have been implicated in tumor angiogenesis. However, previous studies have shown that targeting the individual signals was not sufficiently effective in retarding tumor growth in certain preclinical and clinical conditions. In the present study, we developed a novel decoy chimeric receptor that traps both VEGF and BMP‐9/10. Single targeting of either VEGF or BMP‐9/10 signals significantly reduced the formation of tumor vessels in a mouse xenograft model of human pancreatic cancer; however, it did not show significant therapeutic effects on tumor growth. In contrast, dual targeting of the angiogenic signals resulted in more significant inhibition of tumor angiogenesis, leading to delay of tumor growth. Our findings suggest that simultaneous blockade of VEGF and BMP‐9/10 signals is a promising therapeutic strategy for the cancers that are resistant to anti‐VEGF and BMP‐9/10 therapies.     

A strategy for antitumor vascular therapy by targeting the vascular endothelial growth factor: receptor complex

Vascular endothelial growth factor (VEGF) is produced by cancer cells in response to hypoxia and is the primary stimulant of vascularization in solid tumors. Endothelial cells lining the blood vessels of these tumors have a high concentration of receptor-bound VEGF on their surface, providing a target for antibody- directed cancer therapy. To obtain a cloned antibody to this target when bound to its receptor on tumor endothelium, we used phage display technology to create a single-chain Fv (sFv) antibody library from mice immunized with the 165-amino acid isoform of human VEGF-A. We selected, purified, and characterized LL4, an anti-VEGF sFv that was shown to react with receptor-bound VEGF. LL4 bound selectively to blood vessel endothelium, as shown by immunohistochemistry on tissue sections of human tumors. Furthermore, using autoradiography and grain counting of histological sections, systemically administered LL4 was shown to localize selectively to the endothelial lining of tumor blood vessels in human colorectal carcinoma xenografts in vivo. This study demonstrates the feasibility of targeting tumor vasculature using recombinant antibodies to the VEGF:receptor complex.     

Selective targeting of angiogenic tumor vasculature by vascular endothelial-cadherin antibody inhibits tumor growth without affecting vascular permeability

Vascular endothelial-cadherin (VE-cadherin) is an endothelial cell-specific adhesion molecule that is localized exclusively at cell-cell contacts referred to as adherens junctions. VE-cadherin-mediated adhesion is crucial for proper assembly of vascular structures during angiogenesis as well as for maintenance of a normal vascular integrity. We have shown previously that a monoclonal antibody (BV13) to VE-cadherin not only inhibits the formation of vascular tubes during tumor angiogenesis but also disrupts adherens junctions of normal vasculature with a concomitant increase in vascular permeability. The goal of the current studies was to block VE-cadherin function during angiogenesis without disrupting existing junctions on normal endothelium. Using in vitro screening assays to test for functional blocking of adherens junction formation and in vivo assays to detect antibody effects on vascular permeability in normal tissues, we have identified a novel blocking antibody (E4G10) that inhibits VE-cadherin function during angiogenesis but does not disrupt existing adherens junctions on normal vasculature. E4G10 inhibited formation of vascular tubes in vivo in the Matrigel plug and corneal micropocket assays. E4G10 also inhibited tumor growth in three models of mouse and human tumors via an antiangiogenic mechanism. Examination of normal mouse and tumor tissues showed that E4G10 bound to endothelial cells in a subset of tumor vasculature but not to normal vasculature. Bromodeoxyuridine labeling experiments showed that E4G10 specifically targeted a subset of tumor endothelium that is undergoing active cell proliferation, which likely reflects the activated, angiogenic endothelium. These findings indicate that VE-cadherin can be selectively targeted during states of pathological angiogenesis, despite its ubiquitous distribution throughout the entire vasculature. Our data also suggest that antibody E4G10 recognizes VE-cadherin epitopes that are only accessible on endothelial cells forming new adherens junctions, such as in angiogenic tumor vasculature.     

miR-296 regulates growth factor receptor overexpression in angiogenic endothelial cells

A key step in angiogenesis is the upregulation of growth factor receptors on endothelial cells. Here, we demonstrate that a small regulatory microRNA, miR-296, has a major role in this process. Glioma cells and angiogenic growth factors elevate the level of miR-296 in primary human brain microvascular endothelial cells in culture. The miR-296 level is also elevated in primary tumor endothelial cells isolated from human brain tumors compared to normal brain endothelial cells. Growth factor-induced miR-296 contributes significantly to angiogenesis by directly targeting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFRbeta. Furthermore, inhibition of miR-296 with antagomirs reduces angiogenesis in tumor xenografts in vivo.