Todd Juan

Cloning and characterization of a specific receptor for mouse oncostatin M.

ABSTRACT Oncostatin M (OSM) is a member of a family of cytokines that includes ciliary neurotrophic factor, interleukin-6, interleukin-11, cardiotrophin-1, and leukemia inhibitory factor (LIF). The receptors for these cytokines consist of a common signaling subunit, gp130, to which other subunits are added to modify ligand specificity. We report here the isolation and characterization of a cDNA encoding a subunit of the mouse OSM receptor. In NIH 3T3 cells (which endogenously express gp130, LIF receptor β [LIFRβ], and the protein product, c12, of the cDNA described here), mouse LIF, human LIF, and human OSM signaled through receptors containing the LIFRβ and gp130 but not through the mouse OSM receptor. Mouse OSM, however, signaled only through a c12-gp130 complex; it did not use the LIF receptor. Binding studies demonstrated that mouse OSM associated directly with either the c12 protein or gp130. These data highlight the species-specific differences in receptor utilization and signal transduction between mouse and human OSM. In mouse cells, only mouse OSM is capable of activating the mouse OSM receptor; human OSM instead activates the LIF receptor. Therefore, these data suggest that all previous studies with human OSM in mouse systems did not elucidate the biology of OSM but, rather, reflected the biological actions of LIF.

Biochemical Characterization of AMG 102: A Neutralizing, Fully Human Monoclonal Antibody to Human and Nonhuman Primate Hepatocyte Growth Factor

AMG 102 is a fully human monoclonal antibody that selectively targets and neutralizes hepatocyte growth factor/scatter factor (HGF/SF). A detailed biochemical and functional characterization of AMG 102 was done to support its clinical development for the treatment of cancers dependent on signaling through the HGF/SF:c-Met pathway. In competitive equilibrium binding experiments, AMG 102 bound to human and cynomolgus monkey HGF with affinities of approximately 19 pmol/L and 41 pmol/L, respectively. However, AMG 102 did not detect mouse or rabbit HGF on immunoblots. Immunoprecipitation experiments showed that AMG 102 preferentially bound to the mature, active form of HGF, and incubation of AMG 102/HGF complexes with kallikrein protease indicated that AMG 102 had no apparent effect on proteolytic processing of the inactive HGF precursor. AMG 102 inhibited human and cynomolgus monkey HGF-induced c-Met autophosphorylation in PC3 cells with IC50 values of 0.12 nmol/L and 0.24 nmol/L, respectively. AMG 102 also inhibited cynomolgus monkey HGF-induced migration of human MDA-MB-435 cells but not rat HGF-induced migration of mouse 4T1 cells. Epitope-mapping studies of recombinant HGF molecules comprising human/mouse chimeras and human-to-mouse amino acid substitutions showed that amino acid residues near the NH2-terminus of the β-chain are critical for AMG 102 binding. Bound AMG 102 protected one trypsin protease cleavage site near the NH2-terminus of the β-chain of human HGF, further substantiating the importance of this region for AMG 102 binding. Currently, AMG 102 is in phase II clinical trials in a variety of solid tumor indications. Mol Cancer Ther; 9(2); 400–9

Activity of panitumumab alone or with chemotherapy in non-small cell lung carcinoma cell lines expressing mutant epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) kinase domain mutations cause hyperresponsiveness to ligand and hypersensitivity to small-molecule tyrosine kinase inhibitors. However, little is known about how these mutations respond to antibodies against EGFR. We investigated the activity of panitumumab, a fully human anti-EGFR monoclonal antibody, in vitro in mutant EGFR-expressing non-small cell lung carcinoma (NSCLC) cells and in vivo with chemotherapy in xenograft models. Mutant EGFR-expressing NSCLC cells (NCI-H1975 [L858R+T790M] and NCI-H1650 [Δ746-750]) and CHO cells were treated with panitumumab before EGF stimulation to assess the inhibition of EGFR autophosphorylation. Established tumors were treated with panitumumab (25, 100, or 500 μg/mouse twice a week) alone or with docetaxel (10 or 20 mg/kg once a week) or cisplatin (7.5 mg/kg once a week). Antitumor activity and levels of proliferation markers were analyzed. Treatment of mutant EGFR-expressing CHO and NSCLC cells with panitumumab inhibited ligand-dependent autophosphorylation. In NCI-H1975 and NCI-H1650 xenografts, treatment with panitumumab alone or with cisplatin inhibited tumor growth compared with control (P < 0.0003). With panitumumab plus docetaxel, enhanced antitumor activity was seen in both xenografts versus panitumumab alone. Panitumumab treatment alone decreased Ki-67 and phospho- mitogen-activated protein kinase (pMAPK) staining in both xenografts compared with control. Docetaxel enhanced panitumumab activity in NCI-H1650 xenografts (decreased Ki-67 and pMAPK staining by >60%) when compared with either agent alone. Panitumumab inhibits ligand-induced EGFR phosphorylation, tumor growth, and markers of proliferation alone or with docetaxel in NSCLC cell lines that express clinically observed EGFR kinase domain mutations, including the small-molecule tyrosine kinase inhibitor-resistant T790M mutation. [Mol Cancer Ther 2009;8(6):1536–46]

DNA binding by C/EBP proteins correlates with hepatocyte proliferation

SummaryThe leucine zipper transcription factors C/EBPα and C/EBPβ exhibit growth-related variations of expression and DNA binding during liver regeneration. We examined the expression of C/EBP proteins in relation to hepatocyte proliferation by studying their DNA-binding activity in primary mouse hepatocytesin vitro. Mouse hepatocytes were dissociated by collagenase perfusion and cultured in a serum-free, defined medium containing a variety of growth factors and hormones. Cell protein extracts were collected every 24 h for up to 10 d and examined for DNA-binding activity by gel retardation analysis using a C/EBP consensus sequence oligomer (bZIP). C/EBPα is the major bZIP-binding protein present in the dissociated cells prior to plating. With the culture conditions we employed, little or no binding of C/EBP proteins was observed in the first 24 to 48 h of cultivation. After 48 h, C/EBPβ binding activity was elevated relative to the level seen in freshly dissociated cells. In contrast, C/EBPα binding continued to be greatly reduced and no C/EBPδ binding was observed. C/EBPβ binding remained elevated for the duration of the experiment. Additional growth factor treatment (EGF, FGF, TGFα, and HGF) of the hepatocytes did not appreciably alter the pattern of C/EBP binding. However, TGFβ treatment, known to decrease hepatocyte proliferation, increased C/EBPβ binding activity earlier and more actively than in control cells. This study confirms a negative correlation between DNA binding by the C/EBP transactivator proteins and the proliferation of primary mouse hepatocytesin vitro.