PRECLINICAL CHARACTERIZATION AND IN-VIVO IMAGING STUDIES OF AN ENGINEERED RECOMBINANT TC-99M-LABELED METALLOTHIONEIN-CONTAINING ANTICARCINOEMBRYONIC ANTIGEN SINGLE-CHAIN ANTIBODY

We describe the engineering of a novel single-chain fragment (scFv) me tallothionein (MET) containing anti-carcinoembryonic antigen (CEA) ant ibody (referred to as MET-scFv) for use as a diagnostic imaging agent in colorectal cancer. Methods: Site-directed cloning of annealed oligo nucleotides, containing both the MET and a c-myc tag sequence, into a pUC19-based expression Vector enabled soluble secreted protein express ion from Escherichia coil. Affinity purification was used to purify th e 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 fl uorescence-activated cell sorting analysis on CEA-expressing human col orectal LS-174T cells. Technetium-99m labeling was achieved using a Zn 2+ transchelation step, enabling direct Tc-99m transfer without separa te reduction of MR: In vitro stability was demonstrated by fast protei n liquid chromatography analysis of labeled MET-scFv, incubated with b ovine 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+ transc helation 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(1/2 alpha ) = 2.8 min), adequate localization to tumor of 5.37% injected dose/g at 4 hr was demonstrated. Moreover, the short-lived t(1/2 alpha), of s cFv, its early tumor targeting and rapid blood-pool clearance gave tum or-to-blood ratios of 2.07 by 4 hr, enabling early gamma camera imagin g. Successful and specific imaging was achieved using LS-174T xenograf ts in nude mice by 3-6 hr. Conclusion: A recombinant MET containing sc Fv was successfully expressed, purified and labeled with Tc-99m. The s table site-specific labeling of Tc-99m, combined with the rapid plasma clearance of the scFv, led to successful early in vivo imaging of xen ografted mice.