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|Title:||Preclinical characterization and in vivo imaging studies of an engineered recombinant technetium-99m-labeled metallothionein-containing anti-carcinoembryonic antigen single-chain antibody.|
|Authors:||Pietersz, Geoffrey A;Patrick, M R;Chester, K A|
|Affiliation:||Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia.|
|Citation:||Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine; 39(1): 47-56|
|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. 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.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.|
|Internal ID Number:||9443738|
Antibodies, Monoclonal.diagnostic use.pharmacokinetics
Colorectal Neoplasms.radionuclide imaging
Mice, Inbred BALB C
Recombinant Proteins.diagnostic use.pharmacokinetics
|Appears in Collections:||Journal articles|
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