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N(ε)-(3-[131I]Iodobenzoyl)-Lys5-N(α)-maleimido-Gly1-GEEEK-anti-HER2 nanobody 5F7GGC

, PhD
National for Biotechnology Information, NLM, NIH, Bethesda, MD
Corresponding author.

Created: ; Last Update: March 28, 2013.

Chemical name:N(ε)-(3-[131I]Iodobenzoyl)-Lys5-N(α)-maleimido-Gly1-GEEEK-anti-HER2 nanobody 5F7GGC
Abbreviated name:[131I]IB-Mal-D-GEEEK-Nb
Agent category:Antibody fragment, nanobody
Target:Epidermal growth factor receptor (EGFR, HER2)
Target category:Receptor
Method of detection:Single-photon emission computed tomography (SPECT), gamma planar imaging
Source of signal:131I
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about EGF.



Epidermal growth factor (EGF) is a 53-amino-acid growth factor (6.2 kDa) that is secreted by ectodermic cells, monocytes, kidneys, and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF and at least seven other growth factors and their transmembrane receptor kinases play important roles in cell proliferation, survival, adhesion, migration, and differentiation. The EGF receptor (EGFR) family consists of four transmembrane receptors, including EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3), and HER4 (erbB-4) (2). HER1, HER3, and HER4 comprise three major functional domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic tyrosine kinase domain. No ligand has been clearly identified for HER2; however, HER2 can be activated as a result of ligand binding to other HER receptors with the formation of receptor homodimers and/or heterodimers (3). HER1 and HER2 are overexpressed on many solid tumor cells, such as breast, non–small-cell lung, head and neck, and colon cancers (4-6). The high levels of HER1 and HER2 expression on cancer cells are associated with a poor patient prognosis because high levels of expression lead to increased cell proliferation (7-10).

Trastuzumab, a humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 (11), was labeled with 111In (111In-trastuzumab) (12-14) for use with single-photon emission computed tomography (SPECT) to image EGFR expression on solid tumors. However, the pharmacokinetics of the intact radiolabeled mAb, with high liver uptake and slow blood elimination, are generally not ideal for imaging (15, 16). Smaller antibody fragments, such as scFv, Fab, or F(ab')2, have better imaging pharmacokinetics because they are rapidly excreted by the kidneys. Nanobodies are the smallest intact antigen-binding fragments (15 kDa) isolated from heavy-chain camelid antibodies and exhibit efficient and specific tumor targeting (17-19). Anti-HER2 nanobody 5F7GGC was radiolabeled with 131I using N(ε)-(3-[131I]iodobenzoyl)-Lys5-N(α)-maleimido-Gly1-GEEEK ([131I]IB-Mal-D-GEEEK) to form N(ε)-(3-[131I]iodobenzoyl)-Lys5-N(α)-maleimido-Gly1-GEEEK-anti-HER2 nanobody 5F7GGC ([131I]IB-Mal-D-GEEEK-Nb) for use with in vivo SPECT imaging of HER2 expression in tumors in mice (20).



Nanobody 5F7 was produced as a hexahistidine-tagged protein in Escherichia coli. Nanobody 5F7 with a C-terminal cysteine (5F7GGC) was produced in Pichia pastoris (20). A solution of 5F7GGC (6.4 nmol) was treated with 2-iminothiolane (148 nmol) to introduce two sulfhydryl groups per nanobody. The thiol-derivatized 5F7GGC nanobody was labeled with 7.4–59.2 MBq (0.2–1.6 mCi) [131I]IB-Mal-D-GEEEK for 45 min at room temperature. [131I]IB-Mal-D-GEEEK-Nb was purified with PD-10 column chromatography, with 59.6 ± 9.4% (n = 15) labeling efficiency. The specific activity was 0.22–5.28 MBq/nmol (0.06–0.14 mCi/nmol). [131I]IB-Mal-D-GEEEK-Nb was composed of 69.0 ± 5.7% monomer and 31.0 ± 5.7% dimer.

For comparison studies, 5F7GGC (6.4 nmol) was labeled with 11.1–92.5 MBq (0.3–2.5 mCi) Na[125I]iodide using the Iodogen method (20). 125I-Nb was purified with PD-10 column chromatography, with 83.6 ± 5.0% (n = 10) labeling efficiency. The specific activity was 0.55–13.64 MBq/nmol (0.015–0.37 mCi/nmol). 125I-Nb was composed of 63.4 ± 0.6% monomer and 36.6 ± 0.6% dimer.

In Vitro Studies: Testing in Cells and Tissues


In vitro saturation binding assays of [131I]IB-Mal-D-GEEEK-Nb and 125I-Nb to BT474M1 human breast carcinoma cells expressing HER2 receptor were performed. There was a single class of high-affinity binding sites, with Kd values of 3.2 ± 1.0 nM and 1.8 ± 0.6 nM for [131I]IB-Mal-D-GEEEK-Nb and 125I-Nb, respectively. The immunoreactivity values were 75.3 ± 19.6% and 60.5 ± 4.2% for [131I]IB-Mal-D-GEEEK-Nb and 125I-Nb, respectively. Cellular internalization of [131I]IB-Mal-D-GEEEK-Nb was 56.9% at 1 h and 58.3% at 24 h. Cellular internalization of 125I-Nb was 61.4% at 1 h and 32.4% at 24 h.

Animal Studies



Pruszynski et al. (20) performed ex vivo biodistribution studies of 0.15 MBq (4 µCi) [131I]IB-Mal-D-GEEEK-Nb (26 pmol) or 125I-Nb (13 pmol) in nude mice (n = 5/group) bearing BT474M1 xenografts at 1, 2, 4, 8, and 24 h after injection. Tumor accumulation (percent of injected dose/g (% ID/g)) of [131I]IB-Mal-D-GEEEK-Nb (4.7 ± 0.6% ID/g at 8 h and 3.6 ± 1.2% at 24 h) was higher than that of 125I-Nb (2.9 ± 0.2% ID/g at 8 h and 1.2 ± 0.3% at 24 h) at all time points (P < 0.05). For [131I]IB-Mal-D-GEEEK-Nb, the highest radioactivity accumulation at 2 h was observed in the kidneys (179% ID/g), followed by the liver (5.2% ID/g), tumor (4.4% ID/g), lung (2.0% ID/g), spleen (1.3% ID/g), blood (0.9% ID/g), small intestine (0.6% ID/g), bone (0.6% ID/g), heart (0.5% ID/g), stomach (0.4% ID/g), muscle (0.2% ID/g), and thyroid (0.03% ID/organ). 125I-Nb exhibited higher accumulation in most of the normal tissues except the kidneys (12.1% ID/g). The thyroid and stomach accumulation of [131I]IB-Mal-D-GEEEK-Nb was 40- to 50-fold lower than that of 125I-Nb, suggesting that [131I]IB-Mal-D-GEEEK-Nb is less susceptible to deiodination than 125I-Nb. The tumor/blood and tumor/muscle ratios for [131I]IB-Mal-D-GEEEK-Nb were significantly higher than those for 125I-Nb at all time points (P < 0.05). Administration of 1,500-fold molar excess trastuzumab 24 h before the tracers decreased the 2-h tumor accumulation of [131I]IB-Mal-D-GEEEK-Nb and 125I-Nb by 80% and 67%, respectively. Little inhibition was observed in most of the normal tissues.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

CA42324, CA154291, CA14236


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