title: Bacterial expression of a kemptide fusion protein facilitates 32P labeling of a humanized, anti-carcinoembryonic antigen (hMN-14) antibody fragment.
abstract: Despite the potential advantages of 32P over other isotopes for radioimmunotherapy, its development as a therapeutic has been hindered by the difficulty of the labeling chemistry. Recently, a heptapeptide [Kemptide (KPT)] has been chemically conjugated to antibodies, and the conjugates have successfully been labeled with 32P enzymatically by using bovine protein kinase. By using genetic engineering, we have produced a chimera (Fab.KPT) consisting of the Fab' moiety of the complementarity-determining region-grafted anti-carcinoembryonic antigen-monoclonal antibody, MN14, and a heptapeptide derivative of KPT (Trp-Arg-Arg-Ala-Ser-Leu-Gly). The recombinant protein was expressed in Escherichia coli as a soluble secretory product. The presence of the KPT derivative downstream of the COOH terminus of the hinge region did not impair the binding affinity of the antibody fragment. The Fab.KPT was enzymatically phosphorylated with 32P by bovine protein kinase, without significant effect on the resultant immunoreactivity; 100% of the 32P-labeled Fab.KPT was complexed with liquid carcinoembryonic antigen. The 32P-labeled humanized MN-14 Fab.KPT is expected to have longer blood circulation half-life, allowing for an improved therapeutic efficacy in radioimmunotherapy.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002272:Carcinoembryonic Antigen / Q000276:immunology; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
chemical_list: D002272:Carcinoembryonic Antigen; D007140:Immunoglobulin Fab Fragments; D009842:Oligopeptides; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; C037642:kemptide