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)-(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 Mr 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 MRI(scFv) for EGFRvIII 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 was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR 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.deltaEGFR 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.
Kuan
C T
CT
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Reist
C J
CJ
Foulon
C F
CF
Lorimer
I A
IA
Archer
G
G
Pegram
C N
CN
Pastan
I
I
Zalutsky
M R
MR
Bigner
D D
DD
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000276:immunology* / Q000378:metabolism; D005260:Female; D005910:Glioma / Q000276:immunology / Q000378:metabolism / Q000628:therapy*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D007457:Iodine Radioisotopes; D051379:Mice; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D017510:Protein Folding; D014018:Tissue Distribution
D007135:Immunoglobulin Variable Region; D007457:Iodine Radioisotopes; D066246:ErbB Receptors