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^99mTc-Labeled Divalent and Tetravalent CC49 Single-Chain Fv’s: Novel Imaging Agents for Rapid In Vivo Localization of Human Colon Carcinoma

Department of Biochemistry and Molecular Biology, Department of Radiation Oncology, and Department of Pathology and Microbiology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska; and Coulter Pharmaceutical Inc., San Francisco, California

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 ^99mTc 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 ^99mTc-labeled multivalent scFv’s for imaging. Results: The radiolabeling procedure gave 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 ^99mTc-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 ^99mTc-sc(Fv)₂. ^99mTc-[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. Conclusion: ^99mTc-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.

Key Words: single-chain Fv • multivalency • ^99mTc • biodistribution • macroautoradiography

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