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

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 VH-(Gly4Ser)3-VL, 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 Mr 26,000. Labeling was performed using N-succinimidyl 5-[I]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 (KA) of MR1(scFv) for EGFRvIII was 4.3 3 10 6 0.1 3 10 M by BIAcore analysis, and it was 1.0 3 10 6 0.1 3 10 M 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.DEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or antiEGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.DEGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [I]SIPC-MR1(scFv) and [I]SIPC-antiTac(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.