Adolescent Health in Myanmar
Support Burnet’s Adolescent Health Programs in Myanmar today.
Support Burnet’s Adolescent Health Programs in Myanmar today.
Support Burnet’s Adolescent Health Programs in Myanmar today.
Support Burnet’s Adolescent Health Programs in Myanmar today.
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. METHODS: 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. RESULTS: Yields of 6 mg/liter induced culture purified protein were achieved. Successful site-specific labeling was demonstrated using a Zn2+ transchelation modification of a pretinning method, which also enabled lower amounts of the reducing agent to be used. The specificity for CEA was retained after labeling. Despite a rapid serum clearance (t(1/2alpha) = 2.8 min), adequate localization to tumor of 5.37% injected dose/g at 4 hr was demonstrated. Moreover, the short-lived t(1/2alpha) of scFv, its early tumor targeting and rapid blood-pool clearance gave tumor-to-blood ratios of 2.07 by 4 hr, enabling early gamma camera imaging. Successful and specific imaging was achieved using LS-174T xenografts in nude mice by 3-6 hr. CONCLUSION: A recombinant MET containing scFv was successfully expressed, purified and labeled with 99Tc. The stable site-specific labeling of 99Tc, combined with the rapid plasma clearance of the scFv, led to successful early in vivo imaging of xenografted mice.