I-125-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 epide rmal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, an d evaluated for biodistribution in human glioma-bearing athymic mice. The m utant receptor EGFRvIII has a deletion in its extracellular domain that res ults in the formation of a new, tumor-specific antigen found in glioblastom as, breast carcinomas, and other tumors. The scFv molecule, designed as V-H -(Gly(4)-Ser)(3)-V-L, was expressed in Escherichia coil in inclusion body f orm; recovered scFv fragments were properly refolded in redox-shuffling buf fer. 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 immunoreac tive 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 EGPRv III 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-expres sing 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 M R1(scFv) for EGFRvIII,vas demonstrated in vitro by incubation of radiolabel ed 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. U 87MG.Delta EGFR tumor xenografts, animals received intratumoral or i.v. inf usions 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 hig h tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% inje cted dose per gram of tissue at 24 h following administration. Specific: co ntrol scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated speci fic localization of MRl(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tum or:normal organ ratios.