Construction of Phosphorylatable Monoclonal Antibodies to Tumor Associated Antigens. The mouse monoclonal antibody B72.3 recognizes the tumor associated antigen TAG-72 that is expressed on the surface of human adenocarcinomas. This monoclonal antibody has begun to be used in clinical research protocols for the diagnosis and therapy of adenocarcinomas. To develop more effective methods for labeling this monoclonal antibody we introduced a phosphorylation site into chimeric monoclonal antibody B72.3 (MAb-chB72.3) by site-specific mutation of the coding sequence. The chimeric molecule contains the mouse variable region fused to the human IgG1 constant region. The phosphorylation site for the cAMP-dependent protein kinase was positioned at the carboxyl terminus of the heavy chain constant region of the MAb chB72.3. The resultant modified MAb chB72.3-P was expressed in 293 cells and purified. The MAb-chB72.3-P protein was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high radiospecific activity. The P-labeled MAb-chB72.3-P protein bound to cells expressing the TAG-72 antigen and was stable in serum. To improve the monoclonal antibody for use in humans, a second generation monoclonal antibody with greater affinity, CC49, was used and a chimeric version, chCC49, with the human IgG1 constant region was used. Phosphorylation sites were introduced into this chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting three synthetic fragments encoding two phosphorylation sites into an expression vector, pdHL7. The resultant modified antibody, MAb-chCC49-6P, containing six phosphorylation sites per heavy chain, was expressed in NS0 cells and purified. The MAb-chCC49-6P protein can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to exceptionally high specific activity. The 32P-labeled MAb-chCC49-6P protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into monoclonal antibodies (MAb) provides a procedure for labeling MAb for the diagnosis and treatment of cancers. Additional work in this area will focus on evaluating the introduction of new protein kinase recognition sites and their characterization to find the most effective ones for use in diagnosis and therapy of cancer.