99mTc(CO)3-Anti-epidermal growth factor receptor nanobody 8B6

99mTc(CO)3-8B6

Leung K.

Publication Details

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Table

In vitro Rodents

Background

[PubMed]

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 as well as 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 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 as 111In-trastuzumab (12-14). C225, an anti-EGFR (HER1), mouse-human chimeric, monoclonal IgG1 antibody, also known as erbitux or cetuximab, was labeled as 99mTc-EC-C225 (15, 16) for imaging EGFR expression on solid tumors using single-photon emission computed tomography (SPECT). However, the pharmacokinetics of the intact radiolabeled mAb, with high liver uptake and slow blood elimination, are generally not ideal for imaging (17, 18). 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 (19-21). Anti-HER1 nanobody 8B6 was radiolabeled with 99mTc using tricarbonyl chemistry (99mTc-8B6) for noninvasive in vivo SPECT imaging of HER1 expression in tumors in mice (22).

Synthesis

[PubMed]

Nanobody 8B6 was produced as a hexahistidine-tagged protein in Escherichia coli (22). A solution of [99mTc(H2O)3(CO)3]+ and 8B6 (pH 7–8) was incubated for 90 min at 60°C with a radiochemical yield of >90%. The specific activity, radiochemical purity, and stability of 99mTc-8B6 were not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Flow cytometry analysis showed that 8B6 binding was correlated to HER1 expression in DU145 human prostate carcinoma (200,000 sites/cell) and A431 human epidermoid carcinoma (2,000,000 sites/cell) cell lines (22). The binding of 99mTc-8B6 (10 pmol) to A431 cells was inhibited by >90% with 10 nmol unlabeled 8B6.

Animal Studies

Rodents

[PubMed]

Huang et al. (22) performed ex vivo biodistribution studies of 35 MBq (1 mCi) 99mTc-8B6 (0.67 nmol/mouse) in normal mice (n = 6) at 3 h after injection. The tissue with the highest radioactivity accumulation (percent injected dose/g (% ID/g)) was the kidney (141.3% ID/g), followed by the liver (19.6% ID/g), spleen (5.1% ID/g), lung (3.3% ID/g), small intestine (1.2% ID/g), heart (1.1% ID/g), muscle (0.8% ID/g), and brain (0.1% ID/g). The blood circulation half-life of 99mTc-8B6 was ~ 1.5 h.

SPECT imaging analysis was performed in nude mice bearing A431 (n = 6) or DU145 (n = 5) tumors after injection of 116 MBq (3.1 mCi) (0.67 nmol)) 99mTc-8B6 at 3 h after injection (22). The A431tumors (5.2 ± 0.1% ID/cm3) were clearly visualized (P < 0.001), along with the kidneys (53.6 ± 8.3% ID/cm3) and liver (15.6 ± 1.9% ID/cm3), but the DU145 tumors (1.8 ± 0.3% ID/cm3) were not. Tumor/background ratios were 7.4 and 3.2 for A431 and DU145 tumors, respectively. No blocking studies were performed with unlabeled 8B6.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

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