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Construction of phosphorylatable monoclonal antibody to a tumor-associated antigen
1Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635, USA.
A phosphorylation site was introduced into chimeric monoclonal antibody B72.3 (MAb-chB72.3) by site-specific mutation of the coding sequence. The phosphorylation site for the cAMP-dependent protein kinase was positioned at the carboxyl terminus of the heavy-chain constant region of 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 32P-labeled MAb-chB72.3-P protein bound to cells expressing the tumor-associated glycoprotein 72 antigen. The introduction of phosphorylation sites into MAbs provides a new type of MAb for the diagnosis and treatment of cancers.
A phosphorylation site was introduced into chimeric monoclonal antibody B72.3 (MAb-chB72.3) by site-specific mutation of the coding sequence. The phosphorylation site for the cAMP-dependent protein kinase was positioned at the carboxyl terminus of the heavy-chain constant region of 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 32P-labeled MAb-chB72.3-P protein bound to cells expressing the tumor-associated glycoprotein 72 antigen. The introduction of phosphorylation sites into MAbs provides a new type of MAb for the diagnosis and treatment of cancers.