Construction of phosphorylatable chimeric monoclonal antibody CC49 with a tyrosine srC kinase recognition site.
A phosphorylation site for a tyrosine kinase was introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (Tyr) encoding one tyrosine kinase phosphorylation site into an expression vector. The phosphorylation site was created by incorporating the predicted consensus sequences for phosphorylation by the tyrosine kinase at the carboxyl terminus of the heavy chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49Tyr) was expressed and purified. The MAb-chCC49Tyr protein can be phosphorylated by the tyrosine Src kinase with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49Tyr protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into monoclonal antibodies (MAb) provides a new reagent for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, a tyrosine phosphorylation site can also be used to introduce phosphorylation sites into proteins.
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
Schlom
J
J
Pestka
S
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D019061:src-Family Kinases