99mTc-labeled divalent and tetravalent CC49 single-chain Fv's: novel imaging agents for rapid in vivo localization of human colon carcinoma.
Radioimmunopharmaceutical agents enabling rapid high-resolution imaging, high tumor-to-background ratios, and minimal immunogenicity are being sought for cancer diagnosis and imaging. Genetic engineering techniques have allowed the design of single-chain Fv's (scFv's) of monoclonal antibodies (mAbs) recognizing tumor-associated antigens. These scFv's show good tumor targeting and biodistribution properties in vivo, indicating their potential as imaging agents when labeled with a suitable radionuclide.


METHODS
Divalent (sc(Fv)(2)) and tetravalent ([sc(Fv)(2)](2)) scFv's of mAb CC49 were evaluated for radioimmunolocalization of LS-174T colon carcinoma xenografts in athymic mice. scFv's were radiolabeled with (99m)Tc by way of the bifunctional chelator succinimidyl-6-hydrazinonicotinate hydrochloride using tricine as the transchelator. The immunoreactivity and in vitro stability of the scFv's were analyzed after radiolabeling. Biodistribution and pharmacokinetic studies were performed to determine the tumor-targeting potential of the radiolabeled scFv's. Whole-mouse autoradiography illustrated the possible application of these (99m)Tc-labeled multivalent scFv's for imaging.


RESULTS
The radiolabeling procedure gave > or =95% radiometal incorporation, with a specific activity of >74 MBq/mg scFv. In solid-phase radioimmunoassay, both sc(Fv)(2) and [sc(Fv)(2)](2) exhibited 75%-85% immunoreactivity, with nonspecific binding between 0.8% and 1.2%. Size-exclusion high-performance liquid chromatography showed sc(Fv)(2) as a 60-kDa protein and [sc(Fv)(2)](2) as a 120-kDa protein. Blood clearance studies showed the elimination half-life of (99m)Tc-labeled sc(Fv)(2) as 144 min and that of [sc(Fv)(2)](2) as 307 min. Whole-body clearance studies confirmed the rapid elimination of scFv's, with half-lives of 184 +/- 19 min for sc(Fv)(2) and 265 +/- 39 min for [sc(Fv)(2)](2) (P < 0.001). At 6 h after administration, the tumor localization was 7.2 +/- 0.7 percentage injected dose per gram of tumor (%ID/g) for (99m)Tc-sc(Fv)(2). (99m)Tc-[sc(Fv)(2)](2) showed a tumor uptake of 19.1 +/- 1.1 %ID/g at the same time; the amount of radioactivity in the tumors was 4-fold higher than in the spleen and kidneys and 2-fold higher than in the liver. Macroautoradiography performed at 6 and 16 h after administration clearly detected the tumor with both scFv's.


CONCLUSIONS
(99m)Tc-labeled multivalent scFv's show good tumor-targeting characteristics and high radiolocalization indices (tumor-to-background ratio). These reagents, therefore, have the potential for use in clinical imaging studies of cancer in the field of nuclear medicine.
Goel
A
A
Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Baranowska-Kortylewicz
J
J
Hinrichs
S H
SH
Wisecarver
J
J
Pavlinkova
G
G
Augustine
S
S
Colcher
D
D
Booth
B J
BJ
Batra
S K
SK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000493:pharmacokinetics; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm / Q000276:immunology; D001345:Autoradiography; D003110:Colonic Neoplasms / Q000000981:diagnostic imaging*; D005818:Genetic Engineering; D018796:Immunoconjugates / Q000493:pharmacokinetics; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D051379:Mice; D008819:Mice, Nude; D016719:Radioimmunodetection; D013667:Technetium / Q000493:pharmacokinetics; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; C091202:B72.3 antibody; D018796:Immunoconjugates; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv; D013667:Technetium