CONSTRUCTION OF PHOSPHORYLATABLE CHIMERIC MONOCLONAL-ANTIBODY CC49

Phosphorylation sites were introduced into chimeric monoclonal antibod y CC49 (MAb-chCC49) by inserting synthetic fragments encoding two and six phosphorylation sites into an expression vector, pdHL7. The phosph orylation sites were created by using the predicted consensus sequence s for phosphorylation by the cAMP-dependent protein kinase to the carb oxyl terminus of the heavy chain constant region of the MAb-chCC49. Th e resultant modified antibodies (MAb-chCC49K1 and MAb-chCC49-6P) were expressed in NSO cells and purified. The MAb-chCC49K1 protein contains two phosphorylation sites per heavy chain whereas the MAb-chCC49-6P p rotein contains six sites per heavy chain. Both MAb-chCC49K1 and MAb-c hCC49-6P proteins can be phosphorylated by the catalytic subunit of cA MP-dependent protein kinase with [gamma-P-32]ATP to high specific acti vity. The P-32-labeled MAb-chCC49K1 and MAb-chCC49-6P proteins bind to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into a monoclonal antibody provides a reagent for the diagnosis and treatment of cancer. The use of multiple phosphorylation sites pr ovides antibodies with very high specific radioactivity and demonstrat es that cassettes of phosphorylation sites can be introduced into prot eins without altering their functional activity.