PMID: 9443738Jan 27, 1998Paper

Preclinical characterization and in vivo imaging studies of an engineered recombinant technetium-99m-labeled metallothionein-containing anti-carcinoembryonic antigen single-chain antibody

Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine
G A PieterszK A Chester

Abstract

We describe the engineering of a novel single-chain fragment (scFv) metallothionein (MET) containing anti-carcinoembryonic antigen (CEA) antibody (referred to as MET-scFv) for use as a diagnostic imaging agent in colorectal cancer. Site-directed cloning of annealed oligonucleotides, containing both the MET and a c-myc tag sequence, into a pUC19-based expression vector enabled soluble secreted protein expression from Escherichia coli. Affinity purification was used to purify the protein using an anti-c-myc affinity column. The specificity of both the unlabeled and labeled MET-scFv for CEA was demonstrated by solid-phase enzyme-linked immunosorbent assay and radioimmunoassay and by fluorescence-activated cell sorting analysis on CEA-expressing human colorectal LS-174T cells. Technetium-99m labeling was achieved using a Zn2+ transchelation step, enabling direct 99mTc transfer without separate reduction of MET. In vitro stability was demonstrated by fast protein liquid chromatography analysis of labeled MET-scFv, incubated with bovine serum albumin (BSA), transferrin and mouse serum. Last, in vivo pharmacokinetics, biodistribution and imaging were performed. Yields of 6 mg/liter induced culture purified protein were achieved. Succe...Continue Reading

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