Construction of phosphorylatable chimeric monoclonal antibody CC49.
Phosphorylation sites were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting synthetic fragments encoding two and six phosphorylation sites into an expression vector, pdHL7. The phosphorylation sites were created by using the predicted consensus sequences for phosphorylation by the cAMP-dependent protein kinase to the carboxyl terminus of the heavy chain constant region of the MAb-chCC49. The resultant modified antibodies (MAb-chCC49K1 and MAb-chCC49-6P) were expressed in NS0 cells and purified. The MAb-chCC49K1 protein contains two phosphorylation sites per heavy chain whereas the MAb-chCC49-6P protein contains six sites per heavy chain. Both MAb-chCC49K1 and MAb-chCC49-6P proteins can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity. The 32P-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 provides antibodies with very high specific radioactivity and demonstrates that cassettes of phosphorylation sites can be introduced into proteins without altering their functional activity.
Lin
L
L
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA.
Gillies
S D
SD
Lan
Y
Y
Izotova
L
L
Wu
W
W
Schlom
J
J
Pestka
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000912:Antibodies, Neoplasm / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000276:immunology*; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; C091202:B72.3 antibody; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; C047052:tumor-associated antigen 72