Improved tumor targeting of anti-her2 nanobody through n-succinimidyl 4-guanidinomethyl-3-iodobenzoate radiolabeling

Abstract

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 Nanobodiesmaybe 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 3-125/131I-iodobenzoate (I-SGMIB). Methods: The 5F7GGC Nanobody was radiolabeled using I-SGMIBand, for comparison, withNe-(3-I-iodobenzoyl)-Lys5-Na- maleimido-Gly1-GEEEK (I-IB-Mal-D-GEEEK), another residualizing agent, and by direct radioiodination using IODO-GEN( 125I-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 125I-Nanobody or I-IBMal- D-GEEEK-Nanobody. Peak tumor uptake for I-SGMIBNanobody 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 124I, 123I, and 131I for PET and SPECT imaging and for targeted radiotherapy, respectively. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.