title: Construction and evaluation of the tumor imaging properties of 123I-labeled recombinant and enzymatically generated Fab fragments of the TAG-72 monoclonal antibody CC49.
abstract: Tumor-associated glycoprotein-72 (TAG-72) is overexpressed in a high percentage of epithelial cancers and has proven useful as a target for imaging and targeted radiotherapy. Our goal was to express a recombinant Fab (rFab) of the TAG-72 monoclonal antibody CC49 in Pichia pastoris and directly compare its tumor and normal tissue uptake and imaging properties with enzymatically generated Fab (eFab). In this study, the genes coding for CC49 Fab were cloned from hybridoma cells and expressed in P. pastoris. Fab was purified to homogeneity and its immunoreactivity toward bovine submaxillary mucin (TAG-72) confirmed by ELISA. The tumor and normal tissue localization of (123)I-CC49 rFab and eFab were compared in athymic mice bearing s.c. LS174T colon cancer or TAG-72-negative A375 melanoma xenografts. Results showed that pure and immunoreactive rFab of CC49 was produced and labeled with (123)I. At 24 h post i.v. injection (p.i.), tumor uptake for (123)I-rFab in LS174T xenografts was 6.0% ID/g which was 18-fold higher than in A375 tumors. Tumor-to-normal tissue ratios increased between 2 and 24 h and exceeded 5:1 at 24 h p.i. of (123)I-rFab. (123)I-rFab exhibited significantly lower liver uptake at 12 h p.i. and lower kidney uptake at 2 h p.i. than (123)I-eFab. LS174T tumors were imaged as early as 2 h after administration of (123)I-rFab. We conclude that CC49 rFab can be produced in a P. pastoris host system and accumulated at comparable levels as eFab in LS174T colon cancer xenografts in mice. The lower liver uptake of (123)I-rFab as compared with eFab suggests that it may be more useful for imaging liver lesions. No major effect, except for kidneys and liver, was observed on tumor and normal tissue uptake due to introduction of hexahistidine and FLAG affinity tags or peptide linkers in the scaffold of rFab.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
authors.forename: Ying
authors.initials: Y
authors.lastname: Tang
authors.forename: Shaoxian
authors.initials: S
authors.lastname: Yang
authors.forename: Jean
authors.initials: J
authors.lastname: Gariépy
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Scollard
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics*; D000912:Antibodies, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000276:immunology*; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007457:Iodine Radioisotopes; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000000981:diagnostic imaging*; D010843:Pichia / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000737:chemistry / Q000493:pharmacokinetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; C091202:B72.3 antibody; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D007457:Iodine Radioisotopes; D019275:Radiopharmaceuticals; D011994:Recombinant Proteins; C047052:tumor-associated antigen 72