COMPARISON OF THE PRODUCTION OF A HUMAN MONOCLONAL-ANTIBODY AGAINST HIV-1 BY HETEROHYBRIDOMA CELLS AND RECOMBINANT CHO CELLS - A FLOW CYTOMETRIC STUDY

The production of human monoclonal antibodies for therapeutic use is o f increasing importance for treatment of viral infections such as AIDS . As human x mouse heterohybridomas rarely reach the growth rates and cell specific production rates of mouse hybridomas the transfection of standard cell lines, such as CHO or BHK, is a promising alternative. This has the additional advantage that the IgG subtype can be changed to suit the desired application. However, the use of a cell line that has not originally developed to produce antibodies, as lymphocytes and myeloma cells have, might have unrecognised drawbacks. This will be e specially significant in the case of antibodies as each molecule consi sts of 4 chains linked by disulphide bonds which require specific intr acellular factors to be properly folded and processed (Heavy chain bin ding protein, Protein Disulfide Isomerase a.o.). In this study we have therefore compared two cell lines: a human x mouse heterohybridoma pr oducing IAM-2F5, a human IgG(3) antibody specific for HIV-1 with neutr alising properties and a Chinese Hamster Ovary cell transfected with d ihydrofolate reductase and with the heavy and light chain genes of IAM -2F5 modified to IgG(1). From each cell line three subclones were sele cted with low, medium and high specific production rates. Batch cultur es were performed and the following cellular parameters analysed by fl ow cytometry; 1) total RNA content (translational activity); 2) total protein content; 3) cell cycle phase distribution; 4) concentration of light and heavy chains; 5) concentration of helper proteins such as B iP and PDI. The production rate of heterohybridoma cells was best refl ected in the intracellular concentration of kappa chain, while the gam ma chain concentration was comparable for all three subclones. In the CHO cells the gamma chain expression and thus gene copy number appeare d to be the limiting factor. The GRP78/BiP concentration in CHO remain ed unchanged in spite of a 5-fold higher concentration of gamma chain in the high producing subclone. The PDI concentration in CHO cells was much lower compared to the heterohybridoma cells, irrespective of pro duction rates.