Evaluation of an anti-p185(HER2) (scFv-C(H)2-C(H)3)2 fragment following radioiodination using two different residualizing labels: SGMIB and IB-Mal-D-GEEEK

Nucl Med Biol. 2009 Aug;36(6):671-80. doi: 10.1016/j.nucmedbio.2009.04.002.


Introduction: A 105-kDa double mutant single-chain Fv-Fc fragment (scFv-Fc DM) derived from the anti-p185(HER2) hu4D5v8 antibody (trastuzumab; Herceptin) has been described recently. The goal of this study was to investigate whether improved tumor targeting could be achieved with this fragment through the use of residualizing radioiodination methods.

Methods: The scFv-Fc DM fragment was radioiodinated using N-succinimidyl 4-guanidinomethyl 3-[(131)I]iodobenzoate ([(131)I]SGMIB) and N(epsilon)-(3-[(131)I]iodobenzoyl)-Lys(5)-N(alpha)- maleimido-Gly(1)-GEEEK ([(131)I]IB-Mal-D-GEEEK), two residualizing radioiodination agents that have been used successfully with intact antibodies. Paired-label internalization assays of the labeled fragments were performed in vitro using MCF7 human breast cancer cells transfected to express HER2 (MCF7-HER2); comparisons were made to scFv-Fc DM directly radioiodinated using Iodogen. The tissue distribution of the scFv-Fc DM labeled with [(125)I]IB-Mal-d-GEEEK and [(131)I]SGMIB was compared in athymic mice bearing MCF7-HER2 xenografts.

Results: The scFv-Fc DM fragment was labeled with [(131)I]SGMIB and [(131)I]IB-Mal-d-GEEEK in conjugation yields of 53% and 25%, respectively, with preservation of immunoreactivity for HER2. Internalization assays indicated that labeling via SGMIB resulted in a 1.6- to 3.5-fold higher (P<.05) retention of radioactivity, compared to that from the directly labeled fragment, in HER2-expressing cells during a 24-h observation period. Likewise, the amount of radioactivity retained in cells from the IB-Mal-d-GEEEK-labeled fragment was 1.4- to 3.3-fold higher (P<.05). Tumor uptake of radioiodine activity in athymic mice bearing MCF7-HER2 xenografts in vivo was significantly higher for the [(125)I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment compared with that of the [(131)I]SGMIB-labeled fragment, particularly at later time points. The uptake of (125)I was threefold (3.6+/-1.1 %ID/g vs. 1.2+/-0.4 %ID/g) and fourfold (3.1+/-1.7 %ID/g vs. 0.8+/-0.4 %ID/g) higher than that for (131)I at 24 and 48 h, respectively. However, the [(125)I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment also exhibited considerably higher levels of radioiodine activity in liver, spleen and kidney.

Conclusions: The overall results further demonstrate the potential utility of these two prosthetic groups for the radiohalogenation of internalizing monoclonal antibodies and their fragments. Specifically, the trastuzumab-derived double mutant fragment in combination with these residualizing agents warrants further evaluation for imaging and possibly treatment of HER2 expressing malignancies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Monoclonal / metabolism
  • Benzoates / chemistry*
  • Biological Transport
  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism
  • Cell Line, Tumor
  • Female
  • Gene Expression Regulation, Neoplastic
  • Guanidine / analogs & derivatives*
  • Guanidine / chemistry
  • Humans
  • Immunoglobulin Fc Fragments / chemistry
  • Immunoglobulin Fc Fragments / genetics
  • Immunoglobulin Fc Fragments / immunology
  • Immunoglobulin Fc Fragments / metabolism
  • Immunoglobulin Variable Region / chemistry*
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / immunology*
  • Immunoglobulin Variable Region / metabolism
  • Iodine Radioisotopes / chemistry
  • Mice
  • Mutation
  • Oligopeptides / chemistry*
  • Receptor, ErbB-2 / genetics
  • Receptor, ErbB-2 / immunology*
  • Tissue Distribution
  • Transplantation, Heterologous


  • Antibodies, Monoclonal
  • Benzoates
  • Immunoglobulin Fc Fragments
  • Immunoglobulin Variable Region
  • Iodine Radioisotopes
  • N(epsilon)-(3-iodobenzoyl)-Lys(5)-Nalpha-maleimido-Gly(1)-glycyl-glutamyl-glutamyl-gluamyl-lysine
  • N-succinimidyl 4-guanidinomethyl-3-iodobenzoate
  • Oligopeptides
  • Receptor, ErbB-2
  • Guanidine