Stable recombinant expression of the anti HIV-1 monoclonal antibody 2F5 after IgG3/IgG1 subclass switch in CHO cells.

The human monoclonal antibody (humAb) 2F5 is a potent candidate for immunotherapy of HIV-1. The xenohybridoma derived humAb 2F5 is of IgG3 kappa isotype. Generally, IgG1 isotype has a longer half-life (beta-clearance) in humans than IgG3. Therefore the isotype was switched to an IgG1 by ligation of the 2F5 heavy chain variable region to an IgG1 constant region and expressed as IgG1 kappa in CHO cells. CHO clones have been established, which stably express humAb 2F5 at high levels. The specificity, affinity and in vitro function of both isotypes were identical.

Changes during subclone development and ageing of human antibody-producing recombinant CHO cells.

Some of the problems encountered with human or human-mouse heterohybridomas, such as low growth rates and high serum requirements, have led to the increased use of recombinant cell lines for production of human antibodies. To evaluate the suitability of such alternative cell lines for the production of human antibodies we have analysed several subclones with differing specific production rates of a recombinant CHO cell line. Gene copy number and site of chromosomal integration for the light and heavy chain and the dhfr gene were determined by in-situ hybridisation. Specific mRNA content was analysed by Northern blot. In addition the intracellular content in light and heavy chain was measured by flow cytometry and the specific secretion rates were determined. The stability of gene expression was followed in the highest producing subclone for over a year. As previously seen in heterohybridoma cells a high expression rate of light chain is beneficial in speeding up secretion rates of whole antibody. When grown in the presence of G418 and methotrexate the amplified gene copies in the genome of recombinant CHO cells were stable over more than 100 passages. However, the expression of light chain, and with it the secretion rate, decreased with time. The low intracellular concentration of light chain resulted in accumulation of heavy chain in the endoplasmic reticulum due to retention by chaperones. The specific secretion rate decreased by 50% after 100 passages. When no G418 or methotrexate were present 75% of the gene copies were lost after 100 passages.

Analysis of changes during subclone development and ageing of human antibody-producing heterohybridoma cells by northern blot and flow cytometry.

The economic importance of obtaining high-producing subclones for large scale production of pharmaceutical proteins is self-evident. However, few papers have studied the changes that occur during subclone development. This information would be important for further improvement of screening and subcloning protocols. We have therefore compared subclones of a human-mouse heterohybridoma cell line producing a human antibody againt HIV-1. Three subclones with low, medium and high specific production rates were selected for this study and their light and heavy chain mRNA content, the intracellular content of light and heavy chain and the specific secretion rates compared. In addition the long time stability of antibody expression in the highest producing subclone was analysed for one year. For the three subclones a correlation between the intracellular content in light chain and the secretion rate was found, while the intracellular content in heavy chain was the same for all three subclones. These results indicate that the assembly in the endoplasmic reticulum (ER) is one of the major rate limiting factors in antibody production. During long time cultivation of the heterohybridoma cell line a continuous decrease in light and heavy chain production was seen without the appearance of a non producing sub-population.

Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization.

Electrofusion and EBV transformation were studied by immortalizing human PBLs from blood of HIV-1-positive volunteers. A panel of 33 cell lines producing human monoclonal antibodies (Hu-MAbs) against HIV-1 was established by cell fusion or EBV transformation. For the first fusion experiments the source of B lymphocytes was peripheral blood of HIV-1-infected donors in CDC stages II or III with CD4 cell counts higher than 500/mm3. Later on, from these patients only, those with high anti-HIV titers were chosen as blood donors. By that means the yield of stable specific hybridomas was increased twofold. In our experiments electrofusion turned out to be a more efficient immortalization method than EBV transformation, due to a high and constant immortalization rate. The hybridomas were stable after intensive subcloning and could be cultivated over a period of 8 months without loss in monoclonal antibody production. Immunoglobulin class, subtype, reactivity against HIV-1 proteins, Western blot patterns, immunofluorescence, and epitopes were characterized. The subtype of all antibodies was IgG1 or IgG3. The light chain was predominantly kappa. All antibodies showed reactivity against HIV-1 envelope or core protein. All hybridomas were stable and suited for mass production. Several Hu-MAbs are becoming an important tool in the field of diagnosis, research, and immunotherapy.