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| Chemical name: | Alexa Fluor 750-albumin-binding domain-fused-(ZHER2:342)2 Affibody | |
| Abbreviated name: | Alexa750-ABD-fused-(ZHER2:342)2 Affibody | |
| Synonym: | ||
| Agent category: | Polypeptide | |
| Target: | EGF HER2 receptor | |
| Target category: | Antigen | |
| Method of detection: | Optical, near-infrared (NIR) fluorescence imaging | |
| Source of signal: | Alexa Fluor 750 | |
| Activation: | No | |
| Studies: |
| No structure is available in PubChem. |
[PubMed]
Epidermal growth factor (EGF) is a cytokine 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). The high levels of HER1 and HER2 expression on cancer cells are associated with a poor prognosis (7-10).
Trastuzumab is a humanized immunoglobulin G1 (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 for imaging of human breast cancer (12-16). However, the pharmacokinetics of intact radiolabeled mAbs, 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. A novel class of recombinant affinity ligands (Affibody molecules) for HER2 was constructed based on a Z-domain residue (58 amino acids) from one of the IgG-binding domains of staphylococcal protein A (17). Affibody molecules exhibit high binding affinity to HER2 with Kd values of <50 nM. The ability of various radiolabeled Affibody molecules to image HER2 in tumors has been studied [PubMed]. Albumin-binding domain (ABD)-fused-(ZHER2:342)2 Affibody dimer, a synthetic Affibody molecule, was constructed to be labeled with Alexa Fluor 750 (Alexa750) (18). Alexa750-ABD-(ZHER2:342)2 Affibody has been evaluated in nude mice bearing human SKBR-3 breast adenocarcinoma tumors.
[PubMed]
ABD-(ZHER2:342)2-Cys was incubated with 5 nM neutral tris(2-carboxyethyl)phosphine solution for 20 min at room temperature (18). Immediately after the reduction, the thio-reactive dye Alexa Fluor 750-maleimide was added to the mixture. Alexa750-ABD-(ZHER2:342)2 was purified with column chromatography with estimated labeling efficiencies of 60–80%, assuming a 1:1 conjugation. Alexa480- and Alexa750-ZHER2:342 were prepared similarly.
[PubMed]
Lee et al. (18) performed binding experiments with ZHER2:342 and Alexa750-ZHER2:342 using a Biacore sensor chip immobilized with extracellular domain of chimeric HER2/Fc fusion protein. The Kd value of ZHER2:342 was calculated to be 30 pM for the chimeric HER2/Fc fusion protein; the Kd value of Alexa750-ZHER2:342 was 190 pM. Hence, the binding affinity of the synthetic Affibody molecule Alexa750-ZHER2:342 was approximately five-fold lower than the recombinant Affibody molecule ZHER2:342. In vitro binding specificity tests showed that binding of 5 nM Alexa480-ZHER2:342 to SKOV-3 cells expressing HER2 was receptor-mediated because saturation of receptors by preincubation with non-labeled ZHER2:342 and trastuzumab decreased binding of Alexa480-ZHER2:342 by 97% and 98%, respectively. Confocal microscopy confirmed cellular internalization of both Alexa480-ZHER2:342 and Alexa680-trastuzumab.
[PubMed]
Lee et al. (18) monitored biodistribution using near-infrared optical imaging of HER2-expressing SKBR-3 tumor xenograft-bearing mice (n = 3/group) after intravenous injection of 10 µg Alexa750-ABD-(ZHER2:342)2, 10 µg Alexa750-ZHER2:342, or 50 µg Alexa750-trastuzumab. Tumor fluorescence intensities were measured at 1, 8, 24, and >48 h after tracer injection. Alexa750-ABD-(ZHER2:342)2 showed high signals around the tumor and kidney areas at 8 h after injection. Fluorescence signals in the tumor area peaked at ~24 h (3.6 AU) and decreased gradually to background level at 72 h. The organ with the highest concentration was the kidney (3.7 AU), followed by the liver (1.3 AU), heart (0.6 AU), lung (0.5 AU), and muscle (0.3 AU). Preadministration of ABD-(ZHER2:342)2 (1 mg/mouse) 30 min before Alexa750-ABD-(ZHER2:342)2 injection resulted in decreased uptake in the tumor. On the other hand, Alexa750-trastuzumab exhibited moderate fluorescence intensity in the tumor, which peaked at ~48 h. Alexa750-ZHER2:342 exhibited a high initial accumulation in the tumor with a fast washout. The tumor clearance rates (t½) of Alexa750-ABD-(ZHER2:342)2, Alexa750-ZHER2:342, and Alexa750-trastuzumab were 40.5, 5.7, and 115 h, respectively. The maximum tumor/muscle ratios were 4.5 at 48 h for the dimer, 2.5 at 4 h for the monomer, and 2.5 at 48 h for the intact trastuzumab antibody.
Intramural Research Program, N01-CO-12400, N01-CO-12401
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