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¹²⁵I-labeled Anti-Epidermal Growth Factor Receptor-vIII Single-Chain Fv Exhibits Specific and High-Level Targeting of Glioma Xenografts¹

Departments of Pathology [C-T. K., G. A., C. N. P., M. R. Z., D. D. B.] and Radiology [C. J. R., C. F. F., M. R. Z.], Duke University Medical Center, Durham, North Carolina 27710; Ottawa Regional Cancer Centre, Cancer Research Group, Ottawa, Ontario, Canada K1H 8L6 [I. A. J. L.]; and Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [I. P.]

A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice. The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors. The scFv molecule, designed as V_H-(Gly₄-Ser)₃-V_L, was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer. Size-exclusion chromatography of purified scFv demonstrated a protein monomer of M_r 26,000. Labeling was performed using N-succinimidyl 5-[¹²⁵I]iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5–2.0 mCi/mg, with yields of 35–50% and 45–70%, respectively. The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65–80% when SIPC was used and 50–55% when Iodogen was used. The affinity (K_A) of MR1(scFv) for EGFRvIII was 4.3 x 10⁷ ± 0.1 x 10⁷M^-1 by BIAcore analysis, and it was 1.0 x 10⁸ ± 0.1 x 10⁸M^-1 and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37°C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60–70%. The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.EGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.EGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [¹²⁵I]SIPC-MR1(scFv) and [¹³¹I]SIPC-anti-Tac(scFv) as a control. When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration. Specific:control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.

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