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Monoclonal antibodies (mAbs) represent the fastest growing class of therapeutic proteins. The increasing demand for mAb manufacturing and the associated high production costs call for the pharmaceutical industry to improve its current production processes or develop more efficient alternative production platforms. The experimental control of IgG fucosylation to enhance antibody dependent cell cytotoxicity (ADCC) activity constitutes one of the promising strategies to improve the efficacy of monoclonal antibodies and to potentially reduce the therapeutic cost. We report here that the EB66 cell line derived from duck embryonic stem cells can be efficiently genetically engineered to produce mAbs at yields beyond a 1 g/L, as suspension cells grown in serum-free culture media. EB66 cells display additional attractive grown characteristics such as a very short population doubling time of 12 to 14 hours, a capacity to reach very high cell density (> 30 million cells/mL) and a unique metabolic profile resulting in low ammonium and lactate accumulation and low glutamine consumption, even at high cell densities. Furthermore, mAbs produced on EB66 cells display a naturally reduced fucose content resulting in strongly enhanced ADCC activity. The EB66 cells have therefore the potential to evolve as a novel cellular platform for the production of high potency therapeutic antibodies.

Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemic tissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and is required for the formation of a mature and functional vascular network. Therefore, we assessed whether coadministration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiency of cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or without intravenous injection of EPCs (0.5×10 6 ), SMPCs (0.5×10 6 ) and EPCs+SMPCs (0.25×10 6 +0.25×10 6 ). Vessel density and foot perfusion were increased in mice treated with EPCs+SMPCs compared to animals receiving EPCs alone or SMPCs alone ( P P

HB-GAM/Pleiotrophin and Midkine (MK) are developmentally-regulated proteins with putative functions during cell growth and differentiation. Using the P19 cell which is a model to study the events associated with early development, we examined the expression and cellular localization of HB-GAM and MK during neural differentiation of P19 cells induced by retinoic acid (RA). The temporal expressions of HB-GAM and MK transcripts and both the levels and cellular localizations of the corresponding proteins appeared dramatically different. MK mRNA, already expressed in untreated P19 cells, was transiently increased by exposure to RA and then largely down regulated. More interestingly, HB-GAM which was not detected in untreated P19 cells was strongly expressed after 2 days of RA treatment and this expression persists throughout the duration of the culture suggesting that it could be involved in different aspects of this differentiation process.

Pleiotrophin is a heparin-binding growth factor involved in the differentiation and proliferation of neuronal tissue during embryogenesis, and also secreted by melanoma and breast carcinoma cells. Pleiotrophin exhibits mitogenic and angiogenic properties and has been shown to influence the vascular supply, expansion and metastasis of tumour cells. Our aim was to study the serum and plasma concentrations of pleiotrophin and the classical angiogenic growth factor vascular endothelial growth factor. Using a specific ELISA-test we studied patients with small cell lung cancer (n=63), and patients with non-small cell lung cancer (n=22) in comparison to healthy control subjects (n=41). In most of the lung cancer patients (81%), we found serum levels of pleiotrophin above those of control subjects (P

Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.

Anaplastic lymphoma kinase (ALK) is a novel neuronal orphan receptor tyrosine kinase that is essentially and transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. To determine whether ALK could play a role in neuronal differentiation, we established a model system that allowed us to mimic the normal activation of this receptor. We expressed, in PC12 cells, a chimeric protein in which the extracellular domain of the receptor was replaced by the mouse IgG 2b Fc domain. The Fc domain induced the dimerization and oligomerization of the chimeric protein leading to receptor phosphorylation and activation, thus mimicking the effect of ligand binding, whereas the wild type ALK remained as a monomeric nonphosphorylated protein. Expression of the chimera, but not that of the wild type ALK or of a kinase inactive form of the chimera, induced the differentiation of PC12 cells. Analysis of the signaling pathways involved in this process pointed to an essential role of the mitogen-activated protein kinase cascade. These results are consistent with a role for ALK in neuronal differentiation.

Pleiotrophin (PTN) is a developmentally regulated protein that has been shown to be involved in tumor growth and metastasis presumably by activating tumor angiogenesis. To clarify the potential angiogenic activity of PTN and to analyze the signaling pathways involved in this process, we used an in vitro model of Human Umbilical Vein Endothelial Cells (HUVEC). We show that PTN was mitogenic toward a variety of endothelial cells including HUVEC, stimulated HUVEC migration across a reconstituted basement membrane and induced the formation of capillary-like structures by HUVEC grown as 3D-cultures in Matrigel or collagen. The signaling pathways triggered following endothelial cell stimulation by PTN were studied by using pharmacological inhibitors of the Phosphoinositide-3 kinase (PI3K) and endothelial Nitric Oxide Synthase (eNOS), two enzymes that have been shown to be crucial in the angiogenic response to Vascular Endothelial Growth Factor (VEGF). Whereas wortmannin (a PI3K inhibitor) and L-NAME (an eNOS inhibitor) dramatically reduced HUVEC growth induced by VEGF, only the former inhibitor reduced the growth induced by PTN and to a lesser extent that stimulated by basic Fibroblast Growth Factor. Thus, our results indicate that PTN induces angiogenesis and utilizes PI3K- but not eNOS-dependent pathways for its angiogenic activity.

Pleiotrophin (PTN) is a developmentally regulated protein which exhibits neurite-outgrowth, mitogenic, and angiogenic properties. It has also been shown to be involved in tumor growth and metastasis. Here we used primary BEL (bovine epithelial lens) cells to investigate the signal transduction pathways involved in the mitogenic activity of recombinant PTN. PTN was purified from conditioned media of SW-13 cells transfected with the human PTN cDNA. We show that inhibitors of tyrosine kinase, mitogen-activated protein kinase, or phosphoinositide (PI) 3-kinase inhibit DNA synthesis stimulated by PTN. Analysis of tyrosine-phosphorylated proteins following PTN stimulation showed phosphorylation of two novel 190- and 215-kDa proteins in addition to SHC, ERK1, and ERK2. A mobility shift of phosphorylated ERK1 and ERK2 was detected with a panERK antibody confirming the phosphorylation of the two ERKs. Furthermore, in vitroimmunocomplex kinase assay with Akt1, a natural substrate of PI 3-kinase, showed an activation of the kinase following PTN stimulation and a reversal by the PI 3-kinase inhibitor wortmannin. We conclude that the mitogenic activity of PTN is dependent on tyrosine kinase activation and utilizes the mitogen-activated protein kinase and the PI 3-kinase pathways to transduce a mitogenic signal.

Three 125I-iodinated estrogen derivatives; 16α-[125I]iodo-11β-methoxy-17β-estradiol ([125I]IME2), E-17α-[125I]iodovinylestradiol ([125I]IVE2) and E-17α-[125I]iodovinyl-11β-methoxyestradiol ([125I]MIME2) were synthesized with high specific activities and their binding characteristics and biological effects were analyzed in vitro using the estrogen-receptor-positive human breast cancer cell line MCF-7. Direct and competitive bindings in a whole cell assay indicated that the three derivatives bound to the estrogen receptor (ER) with high affinity. The dissociation constants of the three compounds were higher than the one found for tritiated E2 suggesting that the presence of the methoxy and the vinyl groups decreased the binding affinity. The decreasing affinity order is: [ 3 H ] E 2 ⩾ [ 125 I ] IVE 2 > [ 125 I ] IVE 2 > [ 125 I ] MIVE 2 . The three unlabeled derivatives as well as E2 have been found to activate the vitellogenin promoter fused to the chloramphenicol acetyl transferase reporter gene transfected into MCF-7 cells. This assay showed that the ER is activated in the presence of the three compounds. Unlabeled IME2 produced stimulatory effects similar to those of E2 on MCF-7 growth in vitro. Unlabeled IVE2 and MIVE2 at concentrations lower than 10−8 enhanced MCF-7 growth but a lower level that E2 and IME2 did. Our results clearly indicate that the three derivatives behave like estrogens and that they would be excellent candidates for imaging or monitoring ER-containing breast tumors. Because its affinity for the ER is greatest, IME2 would be the best-suited of the three as a radiotracer.

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2–Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2–Fc–stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.

Background: Growth factors produced by tumor cells are essential for tumor expansion and may be useful in monitoring tumor progression or therapeutic efficacy if the factors are released into the circulation. In this study, we measured serum levels of pleiotrophin, a secreted heparin-binding growth and angiogenesis factor, in mice bearing human tumor xenografts to determine whether these levels reflected overall tumor burden, and we examined the relationship between tumor expression of pleiotrophin and serum levels of this factor in patients with cancer. Methods: Pleiotrophin in serum from mice and humans was measured by use of a highly sensitive enzyme-linked immunosorbent assay. For the clinical studies, serum specimens were obtained from 193 patients with various cancers of the gastrointestinal tract and from 28 healthy control subjects. In a subset of 64 cancer patients, serum levels of pleiotrophin were measured at the time of surgery, and tumor expression of this factor was detected immunohistochemically. All P values are two-sided. Results: In mice, serum pleiotrophin levels were found to increase as a function of tumor size. In humans, elevated serum pleiotrophin levels were found in patients with pancreatic cancer (n = 41; P