Improved tumor targeting of anti-HER2 nanobody through N-succinimidyl 4-guanidinomethyl-3-iodobenzoate radiolabeling

J Nucl Med. 2014 Apr;55(4):650-6. doi: 10.2967/jnumed.113.127100. Epub 2014 Feb 27.


Nanobodies are approximately 15-kDa proteins based on the smallest functional fragments of naturally occurring heavy chain-only antibodies and represent an attractive platform for the development of molecularly targeted agents for cancer diagnosis and therapy. Because the human epidermal growth factor receptor type 2 (HER2) is overexpressed in breast and ovarian carcinoma, as well as in other malignancies, HER2-specific Nanobodies may be valuable radiodiagnostics and therapeutics for these diseases. The aim of the present study was to evaluate the tumor-targeting potential of anti-HER2 5F7GGC Nanobody after radioiodination with the residualizing agent N-succinimidyl 4-guanidinomethyl 3-(125/131)I-iodobenzoate (*I-SGMIB).

Methods: The 5F7GGC Nanobody was radiolabeled using *I-SGMIB and, for comparison, with N(ε)-(3-*I-iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-GEEEK (*I-IB-Mal-d-GEEEK), another residualizing agent, and by direct radioiodination using IODO-GEN ((125)I-Nanobody). The 3 labeled Nanobodies were evaluated in affinity measurements, and paired-label internalization assays were performed on HER2-expressing BT474M1 breast carcinoma cells and in paired-label tissue distribution measurements in mice bearing subcutaneous BT474M1 xenografts.

Results: *I-SGMIB-Nanobody was produced in 50.4% ± 3.6% radiochemical yield and exhibited a dissociation constant of 1.5 ± 0.5 nM. Internalization assays demonstrated that intracellular retention of radioactivity was up to 1.5-fold higher for *I-SGMIB-Nanobody than for coincubated (125)I-Nanobody or *I-IB-Mal-d-GEEEK-Nanobody. Peak tumor uptake for *I-SGMIB-Nanobody was 24.50% ± 9.89% injected dose/g at 2 h, 2- to 4-fold higher than observed with other labeling methods, and was reduced by 90% with trastuzumab blocking, confirming the HER2 specificity of localization. Moreover, normal-organ clearance was fastest for *I-SGMIB-Nanobody, such that tumor-to-normal-organ ratios greater than 50:1 were reached by 24 h in all tissues except lungs and kidneys, for which the values were 10.4 ± 4.5 and 5.2 ± 1.5, respectively.

Conclusion: Labeling anti-HER2 Nanobody 5F7GGC with *I-SGMIB yields a promising new conjugate for targeting HER2-expressing malignancies. Further research is needed to determine the potential utility of *I-SGMIB-5F7GGC labeled with (124)I, (123)I, and (131)I for PET and SPECT imaging and for targeted radiotherapy, respectively.

Keywords: HER2; Nanobody; SGMIB; breast cancer; radioiodination.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Humanized / pharmacology
  • Antineoplastic Agents / pharmacology
  • Benzoates* / chemical synthesis
  • Breast Neoplasms / diagnostic imaging
  • Breast Neoplasms / metabolism
  • Female
  • Guanidine / analogs & derivatives*
  • Guanidine / chemical synthesis
  • Humans
  • Isotope Labeling
  • Mice
  • Mice, Nude
  • Positron-Emission Tomography
  • Radiopharmaceuticals* / chemical synthesis
  • Receptor, ErbB-2 / metabolism*
  • Single-Domain Antibodies / metabolism*
  • Tissue Distribution
  • Tomography, Emission-Computed, Single-Photon
  • Trastuzumab
  • Xenograft Model Antitumor Assays


  • Antibodies, Monoclonal, Humanized
  • Antineoplastic Agents
  • Benzoates
  • N-succinimidyl 4-guanidinomethyl-3-iodobenzoate
  • Radiopharmaceuticals
  • Single-Domain Antibodies
  • ERBB2 protein, human
  • Receptor, ErbB-2
  • Guanidine
  • Trastuzumab