111In-Labeled human serum albumin–conjugated Affibody ZHER2:342 that targets the human epidermal growth factor receptor 2 (HER2)


Chopra A.

Publication Details



In vitro



An Affibody molecule is a chain of 58 amino acids (~6.5 kDa) that contains a modified B domain of the staphylococcal protein A and can be obtained by chemical synthesis or produced in bacteria with the use of recombinant technology (1). Because of their small size and high chemical and thermal stability, these molecules are used as radiolabeled probes for the targeted detection and treatment of malignant tumors as discussed elsewhere (1-3). In this regard, the epidermal growth factor receptor 2 (HER2) is considered to be an important Affibody target because it is believed to promote development of the malignant phenotype, it plays a role in the development of resistance to anticancer drugs and radiation therapy, it is overexpressed in several different cancer tumor cells, and it often indicates a poor prognosis for the patient (4). Several radionuclide-labeled Affibodies or their derivatives, such as ZHER2:342, have been evaluated to detect tumors expressing HER2 in preclinical studies as discussed by Tolmachev (5). Although the labeled Affibodies generate high-contrast images of the tumors, a common limitation observed with these tracers is an elevated accumulation of radioactivity in the kidneys, which masks the detection of tumors in adjacent regions of these and other organs (6). In addition, there is also a concern that the high radiation dose(s) produced from radioactivity accumulated in the kidneys can damage these organs in some patients.

Human serum albumin (HSA) is a multifunctional nonimmunogenic circulatory protein (~65 kDa) that has good biocompatible and biodegradation characteristics, and recombinant HSA (rHSA) or its derivative can be used as an excipient to formulate biological drugs (7). In addition, because HSA has a long circulating half-life and is not eliminated through the kidneys, investigators have evaluated the use of rHSA-drug conjugates to improve the efficacy of drugs and to visualize tumors using a radiolabeled integrin receptor-binding peptide coupled to rHSA (6). On the basis of this information, Hoppmann et al. evaluated the ability of a 64Cu- or 111In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraaceticacid (DOTA)-HSA-ZHER2:342 Affibody conjugate to target and visualize xenograft SKOV3 cell tumors in mice with the use of positron emission tomography (PET; for the 64Cu-labeled Affibody) and single-photon emission tomography (SPECT; for the 111In-labeled Affibody), respectively. (6). This chapter describes the investigations performed with [111In]-DOTA-HSA-ZHER2:342. Studies performed with [64Cu]-DOTA-HSA-ZHER2:342 are presented in a separate chapter of MICAD (www.micad.nih.gov ) (8).

Other Sources of Information

Related chapters in MICAD

Other Epidermal growth factor receptor (EGFR) imaging agents in MICAD

EGFR (human) ligands in PubMed.

Human EGFR in OMIM (Online Mendelian Inheritance in Man)

Information on EGFR (human) gene

Protein and nucleotide information regarding EGFR

Anti-EGFR antibody clinical trials

EGFR pathways in Pathways Interaction Database

Production of rHSA in yeast

Protein and mRNA sequence of human serum albumin

HSA clinical trials

Information regarding HSA on Food and Drug Administration web site



The synthesis of DOTA-HSA-ZHER2:342 and its labeling with 111In have been described by Hoppmann et al. (6). Approximately 2 molecules of DOTA and 1–5 molecules of ZHER2:342 were reported to be conjugated to each HSA molecule as determined with matrix-assisted laser desorption/ionization-time of flight-mass spectroscopy. Denaturing polyacrylamide gel electrophoresis of DOTA-HSA-ZHER2:342 revealed a broad band on the gel because different numbers of the Affibody molecule were attached to each molecule of HSA. The radiochemical yield of the labeled Affibody was ~60%, with a specific activity of 14–33 MBq/nmol (0.37–0.89 mCi/nmol). The [111In]-DOTA-HSA-ZHER2:342 was formulated in phosphate-buffered saline (the purification buffer, pH not mentioned), and the stability of the labeled complex was not reported.

In Vitro Studies: Testing in Cells and Tissues


The uptake of [111In]-DOTA-HSA-ZHER2:342 by SKOV3 cells was studied as described by Hoppmann et al. (6). Uptake of the radiochemical was observed to increase from 0.85 ± 0.15% (percent of added radioactivity) at 0.5 h to 1.46 ± 0.2% at 2 h. The uptake of radioactivity by the cells was significantly inhibited (P < 0.05) in the presence of excess (2.12 μM) nonradioactive Affibody. This indicated that the radiolabeled probe bound specifically to the epidermal growth factor receptor (EGFR) on the cells.

Animal Studies



The biodistribution of [111In]-DOTA-HSA-ZHER2:342 was studied in mice bearing SKOV3 cell xenograft tumors as described by Hoppmann et al. (6). The animals (n = 3 mice/group) were injected with 1.5–1.9 MBq of the tracer through the tail vein and euthanized at various time points (varying from 1 h to 48 h postinjection (p.i.)) to determine the amount of radioactivity accumulated in the major organs, including the tumors. Clearance of radioactivity from the blood was slow (~26% injected dose per gram tissue (% ID/g) and ~20% ID/g at 1 h and 4 h p.i., respectively, which decreased to 3.0 ± 0.9% ID/g at 48 h p.i.). A high uptake of the label was observed in the liver (15.6±1.8% ID/g at 4 h p.i.; this decreased to 3.6±2.9% ID/g at 48 h p.i.), but the accumulation was relatively less in the kidneys at these time points (9.9±0.7% ID/g and 5.7±0.9% ID/g at 4 h and 48 h p.i., respectively ). The accumulation of radioactivity in the tumors increased from 4.6±0.5% ID/g at 1 h p.i. to 12.5 ±1.1% ID/g at 4 h p.i. and plateaued at ~16% ID/g from 24 h to 48 h p.i. The tumor/blood ratio increased from 0.15 ± 0.06 at 1 h p.i. to 5.9 ± 2.01 at 48 h p.i., and the tumor/muscle (T/M) ratio increased from 4.09 ± 2.52 at 1 h p.i. to 16.89 ± 4.67 at 48 h p.i. This indicated that the radioactivity from the labeled Affibody was cleared from the system primarily though the hepatobiliary system. To determine the HER2 binding specificity of [111In]-DOTA-HSA-ZHER2:342, the mice were injected with the radiolabeled probe in the presence of excess nonradioactive Affibody (300 μg), and the animals were euthanized at 4 h p.i. A significantly reduced (P < 0.01) uptake of the label by the tumor was observed (6.5% ID/g in blocked versus 12.5% ID/g in the normal animals), and the T/M ratio at this time point was reduced significantly (P < 0.01) to 4.2 ± 1.6 in the blocked mice compared to a T/M ratio of 11.1 ± 1.1 in the normal group.

The tumor-targeting property of the labeled Affibody was confirmed with SPECT imaging of mice (n = 3 animals/time point) at 24 h and 48 h p.i. as detailed by Hoppmann et al. (6). Images acquired at both time points demonstrated that there was a very high accumulation of radioactivity in the tumors. The images also showed that the uptake of radioactivity by the liver was higher than that of the kidneys as observed earlier during the biodistribution studies (see above).

From this study, the investigators concluded that [111In]-DOTA-HSA-ZHER2:342 showed a high specificity for the detection of HER2-expressing tumors in rodents with the use of SPECT imaging (6).

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

Supplemental Information


No information is currently available.

NIH Support

Work reported in this chapter was supported in part by National Cancer Institute (NCI) grant 5R01 CA119053 and NCI In Vivo Cellular Molecular Imaging Center (ICMIC) grant P50 CA114747.


Feldwisch J., Tolmachev V., Lendel C., Herne N., Sjoberg A., Larsson B., Rosik D., Lindqvist E., Fant G., Hoiden-Guthenberg I., Galli J., Jonasson P., Abrahmsen L. Design of an optimized scaffold for affibody molecules. J Mol Biol. 2010;398(2):232–47. [PubMed: 20226194]
Tolmachev V., Orlova A. Influence of labelling methods on biodistribution and imaging properties of radiolabelled peptides for visualisation of molecular therapeutic targets. Curr Med Chem. 2010;17(24):2636–55. [PubMed: 20491631]
Lofblom J., Feldwisch J., Tolmachev V., Carlsson J., Stahl S., Frejd F.Y. Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. 2010;584(12):2670–80. [PubMed: 20388508]
Capala J., Bouchelouche K. Molecular imaging of HER2-positive breast cancer: a step toward an individualized 'image and treat' strategy. Curr Opin Oncol. 2010;22(6):559–66. [PMC free article: PMC3401024] [PubMed: 20842031]
Tolmachev V. Imaging of HER-2 overexpression in tumors for guiding therapy. Curr Pharm Des. 2008;14(28):2999–3019. [PubMed: 18991715]
Hoppmann S., Miao Z., Liu S., Liu H., Ren G., Bao A., Cheng Z. Radiolabeled affibody-albumin bioconjugates for HER2-positive cancer targeting. Bioconjug Chem. 2011;22(3):413–21. [PMC free article: PMC3059402] [PubMed: 21299201]
Chuang V.T., Otagiri M. Recombinant human serum albumin. Drugs Today (Barc) 2007;43(8):547–61. [PubMed: 17925886]
Chopra, A., 64Cu-Labeled human serum albumin conjugated Affibody ZHER2:342 that targets the human epidermal growth factor receptor 2 (HER2). Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -to current. [PubMed: 21656981]