Design and construction of a phosphorylatable chimeric monoclonal antibody with a highly stable phosphate.
A recognition site for the cAMP-dependent protein kinase was introduced into the MAb-chCC49 by site-directed mutation of the coding sequence to make a variant of MAb-chCC49 containing a highly stable phosphate. To design this monoclonal antibody (MAb) without changing its immunoreactivity or biological properties, molecular modeling was used to locate appropriate regions for introduction of the cAMP-dependent phosphorylation site with desirable properties. We selected one position to mutate on the heavy chain based on molecular dynamics study of the solvated antibody. A vector expressing the mutant was constructed and transfected into mouse myeloma NS0 cells that expressed a high level of the resultant MAb-WW5. MAb-WW5 contained the cAMP-dependent phosphorylation site at the hinge region of the heavy chain, could be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity, and retained the phosphate stably. Compared with MAb-chCC49K1, another phosphorylatable variant of MAb-chCC49, the phosphate attached to MAb-WW5 showed much improved stability: about a 10-fold increase in resistance to hydrolysis. MAb-WW5 exhibited the same binding specificity to the TAG-72 antigen on MCF-7 4C10 breast cancer cells as we observed with MAb-chCC49K1. The improved stability of the attached phosphate provides a MAb with potential to be used in diagnosis and therapy of adenocarcinomas.
Wu
Wei
W
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635, USA.
Kerrigan
John E
JE
Yadav
Prem
P
Schwartz
Barbara
B
Izotova
Lara
L
Lavoie
Thomas B
TB
Pestka
Sidney
S
D000230:Adenocarcinoma / Q000175:diagnosis / Q000628:therapy; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000494:pharmacology*; D015195:Drug Design; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009101:Multiple Myeloma / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D017868:Cyclic AMP-Dependent Protein Kinases