BUILDING HIGH-AFFINITY HUMAN-ANTIBODIES BY ALTERING THE GLYCOSYLATIONON THE LIGHT-CHAIN VARIABLE REGION IN N-ACETYLGLUCOSAMINE-SUPPLEMENTED HYBRIDOMA CULTURES

We attempted to improve antibody affinity by varying glycosylation on the light chain variable region. The human hybridoma line HB4C5 produc es an antibody reactive to lung adenocarcinoma, which possess a N-glyc osylated carbohydrate chain on the light chain hypervariable region. I t has been shown that altering this carbohydrate structure can be acco mplished by varying the level of N-acetylglucosamine in glucose free m edium, a change in the carbohydrate chain could be induced which resul ted in modifying antigen binding. By culturing the cells in media cont aining more than 20 mM N-acetylglucosamine, cells produced antibody wi th 10 fold improved affinity as compared with antibody produced in 20 mM glucose-containing medium. A newly induced light chain glycoform pr oduced in the N-acetylglucosamine-containing medium was shown to be re sponsible for this antigen binding enhancement. Addition of glucose in the N-acetylglucosamine-containing media led to decreased antibody af finity and slightly inhibited production of a new light chain in a dos e-dependent manner. Combination of 20 mM N-acetylglucosamine and 0.5 m M glucose gave a higher antibody production without the decrease of th e antigen binding. These results indicate that optimization of N-glyco sylation on the light chain, which leads to higher antigen binding, ca n be accomplished by adjusting a ratio of glucose and N-acetylglucosam ine in the culture medium.