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.