RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Industria Farmacéutica/métodos , Células Madre Embrionarias , Fucosa/química , Inmunoterapia/métodos , Animales , Anticuerpos Monoclonales/química , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Procesos de Crecimiento Celular , Línea Celular , Patos , Fucosa/metabolismo , Ingeniería Genética , Humanos , Mejoramiento de la Calidad , Células Madre/metabolismoRESUMEN
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 x 10(6)), SMPCs (0.5 x 10(6)) and EPCs+SMPCs (0.25 x 10(6)+0.25 x 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<0.001). In addition, capillary and arteriolar densities were enhanced in EPC+SMPC-treated mice compared to SMPC and EPC groups (P<0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficial effect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1-producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activates Tie2-expressing EPCs, resulting in increase of EPC survival and formation of a stable vascular network. Subsequently, the efficiency of EPC- and SMPC-based cotherapy is markedly increased. Therefore, coadministration of different types of vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.
Asunto(s)
Células Endoteliales/trasplante , Miembro Posterior/irrigación sanguínea , Isquemia/terapia , Miocitos del Músculo Liso/trasplante , Neovascularización Fisiológica , Trasplante de Células Madre , Células Madre , Angiotensina I/metabolismo , Animales , Células Endoteliales/metabolismo , Humanos , Isquemia/metabolismo , Masculino , Ratones , Ratones Desnudos , Miocitos del Músculo Liso/citología , Receptor TIE-2/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/metabolismoRESUMEN
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.
Asunto(s)
Células Endoteliales/metabolismo , Células Madre Fetales/metabolismo , Glicoproteínas de Membrana/metabolismo , Neovascularización Fisiológica , Receptor EphB4/metabolismo , Animales , Secuencia de Bases , Adhesión Celular , Células Cultivadas , Cartilla de ADN/genética , Selectina E/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Efrina-B2/metabolismo , Efrina-B2/farmacología , Sangre Fetal/citología , Células Madre Fetales/citología , Células Madre Fetales/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Humanos , Técnicas In Vitro , Isquemia/metabolismo , Isquemia/patología , Isquemia/terapia , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Ratones , Ratones Desnudos , Neovascularización Fisiológica/efectos de los fármacos , Selectina-P/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor EphB4/antagonistas & inhibidores , Receptor EphB4/genéticaRESUMEN
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.