125I-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 V-H-(Gly(4)-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-[I-125]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 EGPRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M-1 by BIAcore analysis, and it was 1.0 x 10(8) + 0.1 x 10(8) M-1 and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70%, The specificity of MR1(scFv) for EGFRvIII,vas demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.Delta 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.Delta EGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [I-125]SIPC-MR1(scFv) and [I-131]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 MRl(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.