Membrane-anchored ephrinB2 and its receptor EphB4 are involved in the formation of blood and lymphatic vessels in normal and pathologic conditions. Eph/ephrin activation requires cell-cell interactions and leads to bidirectional signaling pathways in both ligand- and receptor-expressing cells. To investigate the functional consequences of blocking ephrinB2 activity, 2 highly specific human single-chain Fv (scFv) Ab fragments against ephrinB2 were generated and characterized. Both Ab fragments suppressed endothelial cell migration and tube formation in vitro in response to VEGF and provoked abnormal cell motility and actin cytoskeleton alterations in isolated endothelial cells. As only one of them (B11) competed for binding of ephrinB2 to EphB4, these data suggest an EphB-receptor–independent blocking mechanism. Anti-ephrinB2 therapy reduced VEGF-induced neovascularization in a mouse Matrigel plug assay. Moreover, systemic administration of ephrinB2-blocking Abs caused a drastic reduction in the number of blood and lymphatic vessels in xenografted mice and a concomitant reduction in tumor growth. Our results show for the first time that specific Ab-based ephrinB2 targeting may represent an effective therapeutic strategy to be used as an alternative or in combination with existing antiangiogenic drugs for treating patients with cancer and other angiogenesis-related diseases.

Melanoma is the most aggressive skin cancer once metastasis begins; therefore, it is important to characterize the molecular players involved in melanoma dissemination. The chemokine receptor CXCR4 and the membrane-bound metalloproteinase MT1-MMP are expressed on melanoma cells and represent candidate molecules for the control of metastasis. Using human melanoma transfectants that either overexpress or silence CXCR4 or MT1-MMP, or that have a combination of overexpression and interference of these proteins, we show that CXCR4 and MT1-MMP coordinate their activities at different steps along melanoma cell metastasis into the lungs. Results from in vivo xenograft mouse models of melanoma lung colonization and mice survival and short-term, homing nested polymerase chain reaction experiments from lung samples indicated that CXCR4 is required at early phases of melanoma cell arrival in the lungs. In contrast, MT1-MMP is not needed for these initial steps but promotes subsequent invasion and dissemination of the tumor with CXCR4. Investigation of potential cross talk between CXCR4 and MT1-MMP revealed that MT1-MMP accumulates intracellularly after melanoma cell stimulation with the CXCR4 ligand CXCL12, and that this process involves the activation of the Rac-Erk1/2 pathway. Subsequent to cell contact with specific basement membrane proteins, MT1-MMP redistributes to the cell membrane in a phosphatidylinositol 3-kinase-dependent manner. These results suggest that combination therapies that target CXCR4 and MT1-MMP should improve the limitations of the current therapies for metastatic melanoma.

Human single-chain Fv directed against fibroblast growth factor receptor 3 (FGFR3) have been shown to block proliferation of RT112 bladder carcinoma cells in vitro. Here, we examined the ability of the recombinant gelonin toxin (rGel) to enhance this inhibitory effect in vitro and in vivo on the bladder cancer cell line RT112 and the corresponding xenografts. Immunotoxins were genetically engineered by fusing FGFR3-specific Fv fragments (3C) to the NH₂ terminus of rGel and expressed as a soluble protein in Escherichia coli. The 3C/rGel fusion construct showed an IC₅₀ of 200 nmol/L against log-phase RT112 cells compared with 1,500 nmol/L for free rGel. Immunofluorescence studies showed that the 3C/rGel construct internalized rapidly into the cytoplasm of RT112 cells within 1 h of exposure. The mechanism of immunotoxin-induced cell death was found to be mediated by apoptosis. RT112 tumor xenografts in severe combined immunodeficient mice treated with 50 mg/kg 3C/rGel exhibited considerable growth delay relative to control tumors and a significant reduction of 55% to 70% in mean tumor size. Immunohistochemical analysis showed that tumors from mice treated with 3C/rGel displayed considerable apoptotic damage compared with control groups. Subcellular location of FGFR3 in immunotoxin-treated tumors indicated a translocation of FGFR3 to the nuclear membrane in contrast to tumors from saline-treated controls. These results show that FGFR3-driven immunotoxins may be an effective therapeutic agent against human bladder and other tumor types overexpressing FGFR3. [Mol Cancer Ther 2008;7(4):862–73]

Pulmonary drug delivery offers a minimally invasive and efficient method for treating lung conditions, leveraging the lungs’ extensive surface area and blood flow for rapid drug absorption. Nebulized therapies aim to deliver drugs directly to the lung tissue. This study investigates the histological impact of nebulized tocilizumab—a monoclonal antibody targeting IL-6, traditionally administered intravenously for rheumatoid arthritis and severe COVID-19—on a murine model. Thirty BALB/c mice were nebulized with tocilizumab (10 mg, 5 mg, and 2.5 mg) and six controls were nebulized with saline solution. They were euthanized 48 h later, and their organs (lungs, nasal mucosa, and liver) were analyzed by a microscopic histological evaluation. The results indicate that all the mice survived the 48 h post-nebulization period without systemic compromise. The macroscopic examination showed no abnormalities, and the histopathological analysis revealed greater lung vascular changes in the control group than in the nebulized animals, which is attributable to the euthanasia with carbon dioxide. Additionally, increased alveolar macrophages were observed in the nebulized groups compared to controls. No significant histological changes were observed in the liver, indicating the safety of nebulized tocilizumab. In conclusion, these findings suggest the potential of nebulized tocilizumab for treating pulmonary inflammation, warranting further research to establish its efficacy and safety in clinical settings.