Crystal structure at 1. cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation. (formation of tube-like structures in collagen gels) and (matrigel-plug assay in mice) . However, peptides have some pharmacokinetics draw-backs (e.g., short half-life due to proteolytic cleavage) that may limit their use as potential drug candidates. With the Cetylpyridinium Chloride aim of exploring the therapeutic potential of VEGFR-1 blockade in melanoma with a metabolically stable molecule, we produced a mAb (i.e., D16F7) against peptide A4. D16F7 specifically counteracts VEGFR-1 activation and chemotactic response of endothelial, myelomonocytic and melanoma cells to VEGF-A and PlGF without altering ligand binding to the receptor. Therefore, D16F7 is predicted not to interfere with the physiological regulation of VEGF-A activity by sVEGFR-1. Remarkably, in a preclinical murine model D16F7 strongly reduces angiogenesis and melanoma growth. RESULTS Anti-VEGFR-1 D16F7 mAb inhibits human endothelial, melanoma and myelomonocytic cell migration and angiogenesis matrigel plug assay. Angiogenesis was strongly induced five days after injection in C57BL/6 mice flank of matrigel plugs containing VEGF-A or VEGF-A plus control IgG Cetylpyridinium Chloride as stimulus. By contrast, macroscopic analysis of the plugs that included VEGF-A plus D16F7 showed that newly formed blood vessels were not present, as in plugs where VEGF-A was not included (Figure ?(Figure1C,1C, left panel). Macroscopic analysis results were confirmed by quantitative measurement of hemoglobin content in the excised matrigel plugs (Figure ?(Figure1C,1C, right panel). These data demonstrate that D16F7 mAb possesses antiangiogenic activity and is able to cross-react with murine VEGFR-1. Indeed, the A4 peptide derived from human VEGFR-1, which had been used to produce D16F7 mAb, shares ~85% identity with the corresponding murine sequence (amino acids 149 to 161 in human and 150 to 162 in murine VEGFR-1). The down-modulating effect of D16F7 mAb on the migratory response of human melanoma cells to PlGF was analyzed using the CR-Mel cell line, which expresses VEGFR-1 (Figure ?(Figure2A2A and ). Migration of CR-Mel cells exposed to PlGF was strongly down-modulated by D16F7, whereas it was not affected by control mAb (Figure ?(Figure2B2B and ?and2C2C). Open in a separate window Figure 2 D16F7 mAb inhibits the migration of human melanoma and myelomonocytic cells that express VEGFR-1 in response to PlGFThe CR-Mel melanoma cell line (A, B, C) Cetylpyridinium Chloride and HL-60 promyelocytic cell line differentiated with PMA towards monocytic/macrophagic cells (D, E, F) were analyzed for VEGFR-1 expression (A, D) and the effect of D16F7 treatment on the chemotactic response to PlGF (B, C; E, F). VEGFR-1 expression was assessed by RT-PCR utilizing HUVEC and the melanoma cell line M14 as positive and negative controls, respectively. Migration of CR-Mel or differentiated HL-60 cells (2 105 cells/chamber) in response to PlGF (50 ng/ml, 18 h incubation), was evaluated in Boyden chambers equipped with gelatin coated filters, in the presence or absence of 2.5 g/ml D16F7 mAb or control mouse IgG mAb (CTR mAb). NS, non-stimulated cells. Photographs from a representative Rabbit polyclonal to POLB experiment out of three are shown (x200 magnification) (B, E). Histograms represent the arithmetic mean values of migrated cells/microscopic field SD of three independent determinations (C, F). As a model of myelomonocytic cells, HL-60 cell line was induced to differentiate towards the monocytic/macrophage lineage by treatment with phorbol-miristate acetate (PMA). Differentiation of HL-60 cells by PMA was accompanied by VEGFR-1 expression induction (Figure ?(Figure2D)2D) and exposure to D16F7 mAb decreased cell migration triggered by PlGF to background values (Figure ?(Figure2E2E and ?and2F2F). Dose response experiments, aimed at calculating the D16F7 IC50 on PlGF induced cell migration, led to the following results: 0.48 0.08 g/ml for HUV-ST endothelial cells; 0.59 0.17 g/ml for CR-Mel; and 0.12 0.02 g/ml for myelomonocytic HL-60 cells. D16F7 inhibits VEGFR-1 phosphorylation without affecting ligand binding To shed light Cetylpyridinium Chloride on D16F7 mechanism of action, antibody effect on VEGFR-1 ligand binding and TKR activity was evaluated. Inspection of the three-dimensional structure of VEGFR-1 II IgG-like domain, involved in VEGF-A and PlGF binding [31, 32] showed that peptide A4, which had been used as immunogen to produce D16F7 mAb, does not overlap with VEGFR-1.