Generation and Monitoring of Cell Lines Producing Humanized Antibodies

• Published in: Clinical cancer research Vol. 5; no. 10; pp. 3101s - 3105s
• Main Authors: Losman, M J, Qu, Z, Krishnan, I S, Wang, J, Hansen, H J, Goldenberg, D M, Leung, S O
• Format: Journal Article
• Language: English
• Published: United States American Association for Cancer Research 01.10.1999
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Blotting, Western; Cell Line; DNA, Complementary - analysis; Humans; Methotrexate - pharmacology; Mice
• ISSN: 1078-0432; 1557-3265

Antibody humanization has eliminated or reduced the human antimouse antibody response associated with the administration of murine antibodies. We have successfully humanized three different antibodies: ( a ) hMN-3 (granulocyte targeting); ( b ) hMu-9 (colorectal cancer targeting); and ( c ) hWI2 (anti-idiotype to the anti-carcinoembryonic antigen antibody MN-14). All humanized antibodies demonstrated immunoreactivities comparable to their parent counterparts. Previously, we reported the generation of high productivity cell lines for hMN-14 and hLL2 using the amplifiable vector pdHL2. Through amplification, selection, and cloning procedures, cell lines capable of large scale production were established, and further enhancement of production was achieved by a fed-perfusion bioreactor process. Using a similar and improved approach, we have enhanced the production of the above-mentioned humanized antibodies by gene amplification induced by a stepwise increase in the concentration of methotrexate in the culture media. A reliable IgG determination method is essential to monitor amplification, especially at the final cloning stage, for the selection of the subclones with the highest productivity. We found that measurement of humanized IgG concentration in culture media supplemented with more than 1 µm methotrexate by a standard ELISA assay could be unreliable and misleading. Whereas the determination of antibody by adsorption/elution on protein A from a 100-ml culture is accurate and reproducible, the method is time-consuming, tedious, and labor intensive. We have recently developed a Western blot assay that enables us to monitor the productivity of the cultures. The assay is simple and sensitive, and it makes simultaneous determinations of relative antibody production from individual clones at the 96-well stage feasible. With this method, amplification, cloning, and adaptation to serum-free conditions of multiple cell lines can be monitored in an efficient manner.