Introduction: Anti-angiogenic agents have shown clinical value in combination with chemotherapy by targeting the VEGF pathways. The development of tumor vasculature in human renal cell carcinomas (RCC) is stimulated not only by VEGF but by other angiogenic growth factors such as placental growth factor (PlGF) [1,2]. In the current study, we used a novel fusion protein, sFLT01, that includes human VEGFR1 domain 2 and an IgG1 Fc, which binds to both human VEGF and PlGF. The anti-angiogenic activity of sFLT01 was evaluated in several preclinical models of RCC. Methods: The binding affinity of sFLT01 for PlGF was evaluated in vitro in binding assays quantified by ELISA and Octet and in proliferation assays where endothelial cells are stimulated by PlGF. The ability of sFLT01 to slow tumor growth was evaluated in the 786-O human RCC xenograft model as well as in the mouse RENCA RCC orthotopic and subcutaneous tumor models. sFLT01 was delivered by intraperitoneal injection twice per week at doses ranging from 5–25 mg/kg. Microvessel density (MVD) and other vascular parameters were analyzed in the syngeneic and xenograft tumors by immunohistochemical methods with an antibody against mouse CD31 to identify endothelial cells. Results: sFLT01 bound to human PlGF with great affinity and inhibited HUVEC proliferation with an IC90 of approximately 2.3 nM. Subcutaneous 786-O RCC tumors were inhibited at a dose of 25 mg/kg and median survival time was increased by 33% in the orthotopic RENCA model. sFLT01 reduced MVD in subcutaneous RENCA tumors by approximately 50% and lumen area by approximately 66%. In the 786-O xenograft model, 5 or 25 mg/kg sFLT01 reduced the blood vessel area, lumen size, and vessel perimeter compared to control. Conclusions: sFLT01 is effective at inhibiting blood vessel growth in tumors by binding VEGF and PlGF thereby preventing the development of new vasculature and decreasing existing vasculature. sFLT01 reduces intratumoral blood vessel count, prolongs survival, and delays tumor progression in the RENCA and 786-O renal cell carcinoma models. As an anti-angiogenic agent, sFLT01 may be useful in treating human renal cell carcinomas. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A14.
Introduction: PlGF and VEGF stimulate angiogenesis and promote the growth of tumor vasculature. PlGF is a member of the VEGF family and binds to VEGFR1. sFLT01 is a novel fusion protein comprised of the Fc portion of human IgG1 and the PlGF- and VEGF-binding domain of VEGFR1/Flt-1. The properties of sFLT01 and the potential of sFLT01 as an anti-angiogenic agent to inhibit tumor growth were investigated in several in vitro assays and in multiple xenograft tumor models. Methods: The binding kinetics of sFLT01 for both the human and murine homologues of PlGF and VEGF were assessed by Biacore. The abilities of recombinant human PlGF and VEGF to induce endothelial cell and pericyte proliferation and of sFLT01 to inhibit this stimulation were investigated in cell-based assays. The secretion of human VEGF and PlGF in culture by the HT29 colon carcinoma, H460 lung carcinoma, and A673 sarcoma human cell lines was quantified by ELISA. In efficacy studies, sFLT01 was administered by intraperitoneal injection twice per week to immunodeficient mice bearing HT29, H460, or A673 subcutaneous tumors. Antibodies specific for human IgG and VEGR2 were applied to A673 sarcoma tumor sections from mice treated with sFLT01 to visualize sFLT01 in the tumors and determine VEGFR2 expression in the cellular components. Pericytes and endothelial cells were identified with antibodies against NG2 and CD31. Results: The Biacore results indicated that sFLT01 has high affinity for human and murine PlGF and VEGF. Human recombinant PlGF and VEGF each induced the proliferation of human pericytes and endothelial cells in culture. This stimulation was inhibited by sFLT01. A673 sarcoma, HT29 colon and H460 lung carcinoma cells secreted higher levels of VEGF than PlGF in culture. In vivo, 10 mg/kg sFLT01 was effective at significantly slowing the growth of HT29 colon carcinoma and A673 sarcoma tumors compared to controls. Further analysis of the A673 sarcoma tumors in sFLT01-treated mice by immunohistochemistry revealed that sFLT01 penetrated multiple areas of the tumor. sFLT01 was detected in the vasculature, stroma, necrotic areas, and adjacent to malignant cells. sFLT01 treatment in the A673 model disrupted vessel integrity with a lack of association between endothelial cells and pericytes. A673 sarcoma cells expressed VEGFR2 in vivo. Conclusion: sFLT01 neutralizes the angiogenic activity of multiple vasculogenic VEGF family members in vitro and inhibited the proliferation of cells that form blood vessels, endothelial cells and pericytes. In vivo, sFLT01 treatment resulted in disorganized tumor vasculature thereby slowing the growth of xenografts tumors. The expression of VEGFR2 in A673 sarcoma tumors suggests that VEGF may play a role in autocrine signaling in some malignant cells. sFLT01 has antitumor and antiangiogenic activity in several human tumor xenografts and may offer therapeutic benefit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1388.
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