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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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111In-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-AB.Fab4D5

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: November 8, 2007.

Chemical name:111In-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-AB.Fab4D5
Abbreviated name:111In-DOTA-AB.Fab4D5, 111In-AB.Fab4D5
Agent Category:Antibody and peptide
Target:EGF HER2 receptor and albumin
Target Category:Antibody and albumin binding
Method of detection:SPECT, gamma planar
Source of signal:111In
  • Checkbox In vitro
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Click on protein, nucleotide (RefSeq), and gene for more information about HER2.



Epidermal growth factor (EGF) is a cytokine composed of 53 amino acids (6.2 kDa) and is secreted by ectodermic cells, monocytes, kidneys, and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF, along with 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). High levels of HER1 and HER2 expression on cancer cells are associated with a poor prognosis (7-10).

Trastuzumab is a humanized IgG1 monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 with an affinity constant (Kd) of 0.1 nM (11). 111In-Trastuzumab, Cy5.5-trastuzumab, and 68Ga-trastuzumab-F(ab')2 have been developed to image human breast cancer (12-16). However, the pharmacokinetics of intact radiolabeled mAb, with high liver uptake and slow blood elimination, are generally not ideal for imaging. Smaller antibody fragments, such as Fab or F(ab')2, have better imaging pharmacokinetics because they are rapidly excreted by the kidneys. Albumin is known to accumulate in tumors. Nguyen et al. (17) linked an albumin-binding peptide to the carboxyl end of the heavy chain of trastuzumab Fab (Fab4D5) to form a bifunctional AB.Fab4D5, which is able to bind to HER2 and albumin simultaneously. AB.Fab4D5 was conjugated with 111In using 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) as a linker to study its ability for tumor imaging (18).



AB.Fab4D5 was constructed by adding a linker (GGGS) and an albumin-binding peptide (QRLMEDICLPRWGCLWEDDF) to the carboxyl terminus of the heavy chain (18). Fab4D5, AB.Fab4D5, and trastuzumab (10 mg/ml in PBS, 2.5 mM EDTA) were conjugated with 5 mg/ml of DOTA-(N-hydroxysuccinimidyl) ester in N-N'-dimethylacetamide at a 5:1 molar ratio at 30°C for 1 h. Conjugations typically resulted in a ~2 DOTA per protein. 111InCl3 was mixed with DOTA conjugate in ammonium acetate at 25°C for 1 h. Reactions were quenched with 2 mM diethylenetriaminepentaacetic acid, and 111In-DOTA-labeled conjugates were purified using NAP-5 columns. Samples typically contained ~74 MBq/mg protein (2 mCi/mg protein).

In Vitro Studies: Testing in Cells and Tissues


Dennis et al. (18) performed albumin-binding assays with AB.Fab4D5 to displace a biotinylated albumin-binding peptide (SA06b) to prevent binding to rabbit albumin. AB.Fab4D5 and unbiotinylated SA06 displaced SA06b to a similar extent with IC50 values of 107 and 65 nM, respectively, whereas Fab4D5 had no effect in this assay. Binding of AB.Fab4D5 to HER2ecd was comparable with Fab4D5 and was unaffected by the presence of rabbit albumin with IC50 values of 22 and 15 nM, respectively. Affinity of Fab4D5 for HER2ecd was previously reported as 0.1 nM. Ability of AB.Fab4D5 to bind HER2ecd and albumin simultaneously was shown by binding AB.Fab4D5 to immobilized albumin followed by detection with biotinylated HER2ecd. No signal was detected with Fab4D5 in this assay. However, AB.Fab4D5 acts as a bifunctional fusion protein and is able to bind both albumin and HER2ecd simultaneously.

Animal Studies



Dennis et al. (18) monitored biodistribution by integrated single-photon emission computed tomography/computed tomography analysis in nude mice bearing Fo5 mammary tumors after injection of therapeutic doses of 11.1–18.5 MBq (300–500 μCi) 111In-DOTA-labeled conjugates (4 mg/kg). 111In-DOTA-Fab4D5 tumor radioactivity was characterized by rapid but transient accumulation in the tumors at 2 h with little retention, followed by rapid accumulation in the kidney by 6 h. 111In-DOTA-trastuzumab accumulated slowly in tumors and cleared slowly from normal tissues, although significant tumor accumulation was achieved at 24 h. In contrast, 111In-DOTA-AB.Fab4D5 was observed at 2 h in the tumor and gradually leveled at 24 h, similar to 111In-DOTA-trastuzumab. Similar tumor accumulation was achieved at 48 h for both 111In-DOTA-AB.Fab4D5 and 111In-DOTA-trastuzumab (35.9 ± 1.8% and 38.2 ± 3.1% injected dose/g, respectively). However, 111In-DOTA-AB.Fab4D5 accumulated in tumors more rapidly and quickly cleared from blood, leading to a lower overall normal tissue background. Importantly, unlike 111In-DOTA-Fab4D5, 111In-DOTA-AB.Fab4D5 did not accumulate in the kidney, which suggests that association with albumin leads to an altered route of clearance and metabolism. Intravital microscopy at peak tumor accumulation revealed that tumor cell staining by FITC-AB.Fab4D5 was more uniform than for FITC-Fab4D5 or FITC-trastuzumab.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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