The transfection of murine hybridomas with the apoptosis suppressor gene bcl‐2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl‐2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl‐2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl‐2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl‐2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl‐2 culture). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:90–98, 1998.

No abstract

Immunotherapeutic targeting of G250/Carbonic anhydrase IX (CA‐IX) represents a promising strategy for treatment of renal cell carcinoma (RCC). The well characterized human‐mouse chimeric G250 (cG250) antibody has been shown in human studies to specifically enrich in CA‐IX positive tumors and was chosen as a carrier for site specific delivery of TNF in form of our IgG‐TNF‐fusion protein (cG250‐TNF) to RCC xenografts. Genetically engineered TNF constructs were designed as CH2/CH3 truncated cG250‐TNF fusion proteins and eucariotic expression was optimized under serum‐free conditions. In‐vitro characterization of cG250‐TNF comprised biochemical analysis and bioactivity assays, alone and in combination with Interferon‐γ (IFNγ). Biodistribution data on radiolabeled [125J] cG250‐TNF and antitumor activity of cG250‐TNF, alone and in combination with IFNγ, were measured on RCC xenografts in BALB/c nu/nu mice. Combined administration of cG250‐TNF and IFNγ caused synergistic biological effects that represent key mechanisms displaying antitumor responses. Biodistribution studies demonstrated specific accumulation and retention of cG250‐TNF at CA‐IX‐positive RCC resulting in growth inhibition of RCC and improved progression free survival and overall survival. Antitumor activity induced by targeted TNF‐based constructs could be enhanced by coadministration of low doses of nontargeted IFNγ without significant increase in side effects. Administration of cG250‐TNF and IFNγ resulted in significant synergistic tumoricidal activity. Considering the poor outcome of renal cancer patients with advanced disease, cG250‐TNF‐based immunotherapeutic approaches warrant clinical evaluation. © 2009 UICC

It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2 transfected cell line exhibited a nearly five fold increase in viable cell number. This finding indicates that under apoptosis-suppressed conditions, shear stress can stimulate cell growth. Batch cultivation of both control and bcl-2 transfected cells in 350 and 400 mOsm media resulted in suppression of cell growth, athough the effect was most marked in the control cell line. Adaptation of control cells to 400 mOsm proved to be impossible to achieve. However, the bcl-2 transfected cells exhibited resistance to the osmotic stress resulting in long term adaptation to a high salt environment. Specific productivity of bcl-2 transfected cells grown in high osmolarity medium was 100% higher than that produced by non-adapted bcl-2 transfected cells grown in normal osmolarity medium. These results demonstrate that bcl-2 has a beneficial effect on hybridoma cultivation under a wide range of culture stresses.