Apoptosis-resistant NS/0 E1B-19K myelomas exhibit increased viability and chimeric antibody productivity under cell cycle modulating conditions

Abstract

Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes (sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAb TM . E1B-19K myelomas cultivated in the presence of NaCl or OptiMAb TM accumulated in the G 1 phase, while those arrested with excess thymidine were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle specific manner. Thus, while all G 1 and most S phase cells quickly underwent apoptosis, G 2 /M cells remained alive and maintained MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAb TM resulted in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement of final MAb yields only in the case of OptiMAb TM . The extension of viability conferred by E1B-19K allowed to further improve the final MAb yield obtained using OptiMAb TM with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control NS/0 cells.

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

“…This has been observed for cells cultivated in batch (Ramirez and Mutharasan, 1990), fed-batch (de Tremblay et al, 1993;Robinson et al, 1994), chemostat (Boraston et al, 1984), and perfusion modes (Al-Rubeai et al, 1992;Banik and Heath, 1995;Batt et al, 1990;de la Broise et al, 1991;Johnson et al, 1996;Hansen et al, 1993;Park and Ryu, 1994;Tokashiki and Takamatsu, 1993;Trampler et al, 1994), as well as in cells treated with cell-cycle blocking agents (Al-Rubeai and Emery, 1990;Jayme, 1991;Mercille and Massie, 1998;Oh et al, 1995;Øyaas et al, 1994a,b;Reddy and Miller, 1994;Suzuki and Ollis, 1990;Takahachi et al, 1994). Indeed, specific antibody production has been shown to be cell cycle dependent and optimum in the G 1 and/or G 2 /M phase (Cazzador and Mariani, 1993;Kromenaker and Srienc, 1991;Linardos et al, 1992;Richieri et al, 1991;Suzuki and Ollis, 1989).…”

“…In perfusion, this clone exhibited a steady-state MAb concentration of 247 ± 15 mg/L throughout the plateau phase, representing more than 2.8-fold what was found in batch culture. In the case of E1B-19K cells, a maximal MAb concentration of 33 mg/L could be found at the end of the control batch culture performed on cells taken out of the perfusion bioreactor (see also Mercille et al, 1999;Mercille and Massie, 1998). In perfusion, the E1B-19K cell line exhibited a steady-state MAb concentration of 231 ± 12 mg/L throughout the plateau phase.…”

“…Although successful in temporarily improving survival, the use of Bcl-2 and Bag-1 did not result in higher MAb yields in the non-proliferating state as compared with normal batch cultures (Simpson et al, 1997;Terada et al, 1997b). Expression of E1B-19K in myeloma cells cultivated in hypertonic medium, in the presence of inhibitors of DNA synthesis or in OptiMAb (Gibco, Grand Island, NY) has lead to an increase in both viability and specific MAb productivity (Mercille and Massie, 1998). However, this increase in specific MAb productivity of E1B-19K cells was translated in an increase in final MAb concentration only in the case of cultures supplemented with OptiMAb (Gibco) (Mercille and Massie, 1998).…”

Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myeloma cells transfected with the E1B-19K adenoviral gene

“…It has been reported that cells exposed to slow growth conditions under adequate nutrient supply will exhibit increased MAb secretion rates (Takahashi et al, 1994). However, arresting cells using cytostatic agents results in a rapid induction of apoptosis in non-protected cells (Singh et al, 1994;Mercille and Massie, 1998). Anti-apoptotic genes such as Bcl-2 or E1B-19K can contribute to a significant increase in cell survival in the presence of these cell-cycle blocking agents (Singh et al, 1996).…”

“…Anti-apoptotic genes such as Bcl-2 or E1B-19K can contribute to a significant increase in cell survival in the presence of these cell-cycle blocking agents (Singh et al, 1996). We have recently observed that under slow growth conditions achieved following supplementation with OptiMabs (Gibco) (Mercille and Massie, 1998) or in perfusion culture mode (Mercille and Massie, 1999), E1B-19K-expressing cells exhibited a greater increase in productivity as compared to nonprotected cells.…”