Transfection monoclonal antibody

Expression levels of the mutant thrombin receptors were determined in transiently transfected COS cells by assaying the level of surface expressed thrombin receptors by FACS analysis, usually for mutant receptors that showed a significant diminution of function. COS cells were transfected by calcium phosphate-DNA co-precipitation30 with 10 ng of DNA. The transfected cells were analyzed by FACS 48 h after transfection. A monoclonal antibody specific for the amino terminal region was used as the primary antibody to detect thrombin receptors present on the surface of the cells.31 A FITC conjugated goat anti-mouse IgG was used as a secondary antibody. Expression levels of the mutant receptors were determined relative to expression levels of wild-type controls. Expression-level data (% of control) for some of the mutant receptors are listed in Table 2 (wild-type thrombin... 

An example of results from a tissue cross-reactivity study comparing binding of a monoclonal antibody therapeutic to human, cynomolgus monkey, and mouse tissues is shown in Table 9.2. A cell line that did not express the target was used as negative control tissue the same cell line transfected with the... 

Sequential Transfection. A simple approach to boosting expression is to repeat the transfection on previously transfected cells but with selectable markers not used in the first transfection. Xoma (Berkeley, CA), Sunol Molecular (Miramar, FL), and ICOS (Bothell, WA) have used this approach successfully. Fivefold or greater improvements in expression can be achieved in a single sequential transfection. ICOS reports the added advantage of balancing heavy and light chain ratios to improve the secretion and expression of recombinant monoclonal antibodies. 

Klohe EP, Watts R, Bahl M, Alber C. Yu W-Y, Anderson R, Silver J, Gregersen PK, Karr RW (1988) Analysis of the molecular specificities of anti-class II monoclonal antibodies by using L cell transfectants expressing HLA class II molecules. J Immunol 141 2458—2164. 

The mechanisms underlying instabihty in recombinant cell hnes are poorly understood. Barnes et al. [37, 38] determined that there may be molecular features of transfectants that predicate instabihty. These authors studied a series of GS-NSO cell lines making an anti-CD38 monoclonal antibody. Although copy number remained constant in these cell lines, there was a loss in expression of mRNA during prolonged culture. This did not result in loss of productivity in all of the cell lines. It seems that productivity was not influenced provided that levels of antibody mRNA remained above a critical threshold value. 

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