Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621.

The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72-96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.

Antitumor effect of vascular endothelial growth factor inhibitor sunitinib in preclinical models of hepatocellular carcinoma.

OBJECTIVE: Tumor recurrence and metastasis is the most common cause of mortality in hepatocellular carcinoma (HCC) patients. Despite positive results with vascular endothelial growth factor (VEGF) inhibitors in preclinical studies using HCC xenograft models, the clinical outcome in HCC patients has been disappointing. So far, only the multitargeted tyrosine kinase inhibitor sorafenib has been shown to significantly improve survival in HCC patients, suggesting that this class of agents could be effective against HCC. Recently, another VEGF inhibitor, sunitinib, showed survival benefits in HCC hepatitis B-positive patients, but failed to improve survival in HCC hepatitis C-positive patients. Obviously, concomitant liver disease, liver function in general, and the local liver environment have a huge impact on treatment outcomes. In this study, we aimed to examine the antiproliferative effect of sunitinib in different HCC cell lines in vitro, and then in xenograft and orthotopic models of HCC in order to assess the effect of the local liver vasculature on drug efficacy. METHODS: Human cancer cell lines Huh7.5, Hep3B, and SK-Hep-1 were used for in-vitro studies. In in-vivo studies, each mouse carried Huh7.5 cells in both the subcutaneous and the intrahepatic compartment; therefore, drug exposure and treatment regimen were identical in both tumors. RESULTS: Sunitinib has the potential to moderately inhibit proliferation in the Huh7.5 cell line, induce p53 in the p53-wild-type cell line SK-hep-1, and to increase the S-phase and the sub-G1 component of the cell cycle in the Hep3B cell line. Diverse responses to sunitinib in HCC cell lines emphasize the heterogeneity of HCC tumors and may further explain the discrepancy between preclinical and clinical results. The in-vivo results show that sunitinib treatment was far less effective against intrahepatic tumors compared with xenografts. Histological data indicate that large solid intrahepatic tumors are severely affected by sunitinib as shown by large areas of necrosis and diminished number of viable tumor cells. CONCLUSION: The real problem when treating intrahepatic tumors with sunitanib and/or other VEGF inhibitors seems to arise from unopposed local growth of the small tumors and perhaps the development of distant micrometastases. Even though both xenograft and orthotopic models have limitations, these models add value to our understanding of tumor biology and help to better design treatment paradigms for patients with HCC. In comparison with xenograft models, the orthotopic HCC model allows for a more realistic assessment of drug efficacy in patients, in particular by enhancing our knowledge of the role that organ vasculature plays in the development of local metastasis and tumor resistance to antiangiogenic treatments.

Development of a new permeability assay using low-efflux MDCKII cells.

Permeability is an important property of drug candidates. The Madin-Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P-glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII-LE (low efflux), has been developed by selecting a subpopulation of low-efflux cells from MDCKII-WT using an iterative fluorescence-activated cell sorting technique with calcein-AM as a Pgp and efflux substrate. MDCKII-LE cells are a subpopulation of MDCKII cells with over 200-fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII-WT. MDCKII-LE cells showed less functional efflux activity than MDCKII-WT based on efflux ratios. Notably, MDCKII-MDR1 showed about 1.5-fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII-LE cells offer clear advantages over the MDCKII-WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII-LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII-LE permeability assay (4-in-1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost-effective.

High-throughput assays for assessing mitochondrial dysfunction caused by compounds that impair mtDNA-encoded protein levels in eukaryotic cells.

Compounds that impair the synthesis of either mitochondrial DNA (mtNDA) or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial ATP production. Toxicity caused by these compounds is seldom identified in 24 to 72 hr cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. Here, we describe three high-throughput screening assays that detect compounds that affect mtDNA-encoded protein levels. All three assays measure the levels of two proteins, one a mtDNA-encoded protein synthesized on mitochondrial ribosomes and the other, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The first assay measures the levels of these two proteins by quantitative image analysis and requires a high-content imaging system. The second assay is an in-cell immunoassay that utilizes infrared dyes for detection of the two proteins and, thus, requires a LI-COR Odyssey system. The third assay is an in-cell immunoassay that utilizes colorimetric detection of the two proteins and requires an absorbance microplate reader.

A high content screening assay for identifying lysosomotropic compounds.

Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable. Hence, we developed a 96-well format, high content screening assay that measures lysosomotropism and cytotoxicity by quantitative image analysis. Forty drugs, including antidepressants, antipsychotics, antiarrhythmics and anticancer agents, were tested for their effects on lysosomotropism and cytotoxicity in H9c2 cells. The assay correctly identified drugs known to cause lysosomotropism and revealed novel information showing that the anticancer drugs, gefitinib, lapatinib, and dasatinib, caused lysosomotropism. Although structurally and pharmacologically diverse, drugs that were lysosomotropic shared certain physicochemical properties, possessing a ClogP>2 and a basic pKa between 6.5 and 11. In contrast, drugs which did not lie in this physicochemical property space were not lysosomotropic. The assay is a robust, rapid screen that can be used to identify lysosomotropic, as well as, cytotoxic compounds, and can be positioned within a screening paradigm to understand the role of lysosomotropism as a contributor to drug-induced toxicity.

High-content screening for compounds that affect mtDNA-encoded protein levels in eukaryotic cells.

Compounds that interfere with the synthesis of either mitochondrial DNA or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial adenosine triphosphate (ATP) production. Toxicity caused by these compounds is seldom identified in 24- to 72-h cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. To address this problem, the authors developed a 96-well format, high-content screening (HCS) assay that measures, in eukaryotic cells, the level of Complex IV-subunit 1, an mtDNA-encoded protein synthesized on mitochondrial ribosomes, and the level of Complex V-alpha subunit, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The effect of several antibiotics and antiretrovirals on these 2 proteins was assessed, in transformed human liver epithelial cells, 6 days after compound treatment. The results confirmed effects of drugs known to reduce mtDNA-encoded protein levels and also revealed novel information showing that several fluoroquinolones and a macrolide, josamycin, impaired expression of mtDNA-encoded proteins. The HCS assay was robust with an average Z' factor of 0.62. The assay enables large-scale screening of compounds to identify those that potentially affect mtDNA-encoded protein levels and can be implemented within a screening paradigm to minimize compound attrition.

Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis.

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.