Autocrine VEGFR1 and VEGFR2 signaling promotes survival in human glioblastoma models in vitro and in vivo.

BACKGROUND: Although the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) system has become a prime target for antiangiogenic treatment, its biological role in glioblastoma beyond angiogenesis has remained controversial. METHODS: Using neutralizing antibodies to VEGF or placental growth factor (PlGF) or the tyrosine kinase inhibitor, cediranib, or lentiviral gene silencing, we delineated autocrine signaling in glioma cell lines. The in vivo effects of VEGFR1 and VEGFR2 depletion were evaluated in orthotopic glioma xenograft models. RESULTS: VEGFR1 and VEGFR2 modulated glioma cell clonogenicity, viability, and invasiveness in vitro in an autocrine, cell-line-specific manner. VEGFR1 silencing promoted mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling, whereas VEGFR2 silencing resulted in cell-type dependent activation of the protein kinase B (PKB)/AKT and MAPK/ERK pathways. These responses may represent specific escape mechanisms from VEGFR inhibition. The survival of orthotopic glioma-bearing mice was prolonged upon VEGFR1 silencing in the LNT-229, LN-308, and U87MG models and upon VEGFR2 silencing in LN-308 and U87MG. Disruption of VEGFR1 and VEGFR2 signaling was associated with decreased tumor size, increased tumor necrosis, or loss of matrix metalloproteinase 9 (MMP9) immunoreactivity. Neutralizing VEGF and PlGF by specific antibodies was superior to either antibody treatment alone in the VEGFR1-dependent LNT-229 model. CONCLUSIONS: Differential dependence on autocrine signaling through VEGFR1 and VEGFR2 suggests a need for biomarker-stratified VEGF(R)-based therapeutic approaches to glioblastoma.

Anti-tumoral, anti-angiogenic and anti-metastatic efficacy of a tetravalent bispecific antibody (TAvi6) targeting VEGF-A and angiopoietin-2.

Vascular endothelial growth factor (VEGF)-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) is a key regulator of blood vessel remodeling and maturation. In tumors, Ang-2 is up-regulated and an unfavorable prognostic factor. Recent data demonstrated that Ang-2 inhibition mediates anti-tumoral effects. We generated a tetravalent bispecific antibody (Ang-2-VEGF-TAvi6) targeting VEGF-A with 2 arms based on bevacizumab (Avastin®), and targeting Ang-2 with 2 arms based on a novel anti-Ang-2 antibody (LC06). The two Ang-2-targeting single-chain variable fragments are disulfide-stabilized and fused to the C-terminus of the heavy chain of bevacizumab. Treatment with Ang-2-VEGF-A-TAvi6 led to a complete abrogation of angiogenesis in the cornea micropocket assay. Metastatic spread and tumor growth of subcutaneous, orthotopic and anti-VEGF-A resistant tumors were also efficiently inhibited. These data further establish Ang-2-VEGF bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer.

Placental growth factor (PlGF)-specific uptake in tumor microenvironment of 89Zr-labeled PlGF antibody RO5323441.

UNLABELLED: Placental growth factor (PlGF) is a member of the proangiogenic vascular endothelial growth factor family, which is upregulated in many tumors. RO5323441, a humanized monoclonal antibody against PlGF, showed antitumor activity in human tumor xenografts. We therefore aimed to radiolabel RO5323441 and preclinically validate this tracer to study drug tumor uptake and organ distribution by PET imaging. (89)Zr-RO5323441 was tested for stability and immunoreactivity in vitro. METHODS: The tumor uptake and organ distribution for 10, 50, and 500 µg of (89)Zr-RO5323441 was assessed in mice bearing human PlGF-expressing hepatocellular cancer (Huh7) xenografts or human renal cell carcinoma (ACHN) xenografts without detectable human PlGF expression. The effect of pretreatment with RO5323441 (20 mg/kg) on (89)Zr-RO5323441 tumor uptake was analyzed in Huh7 xenografts. (111)In-IgG served as a control for nonspecific tumor uptake and organ distribution. Cy5-RO5323441 was injected to study the intratumor distribution of RO5323441 with fluorescence microscopy. RESULTS: (89)Zr-RO5323441 showed a time- and dose-dependent tumor accumulation. Uptake in Huh7 xenografts at 10 µg of (89)Zr-RO5323441 was 8.2% ± 1.7% injected dose (ID)/cm(3) at 144 h after injection, and in ACHN xenografts it was 5.5 ± 0.3 %ID/cm(3) (P = 0.03). RO5323441 pretreatment of Huh7 xenograft-bearing mice reduced (89)Zr-RO5323441 tumor uptake to the level of nonspecific (111)In-IgG uptake. Cy5-RO5323441 was present in the tumors mainly in the microenvironment. CONCLUSION: The findings show that RO5323441 tumor uptake is PlGF-specific and time- and dose-dependent.

A novel angiopoietin-2 selective fully human antibody with potent anti-tumoral and anti-angiogenic efficacy and superior side effect profile compared to Pan-Angiopoietin-1/-2 inhibitors.

There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.