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[99mTc(CO)3]+-Labeled anti-epidermal growth factor receptor (HER2) affibody ZHER2:342 with a hexa-histidine tag (H6) on the N-terminal.


Chopra A1.


Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.
2010 Dec 16 [updated 2011 Jan 20].

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National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894


Overexpression of the epidermal growth factor receptor type 2 (HER2) is a characteristic feature of a variety of cancers, and HER2 levels in tumors (primary or metastatic) are often used to screen for patients who would benefit from anti-HER2 antibody (Ab) therapy (e.g., for breast cancer), to determine the efficacy of a treatment regimen, or to predict the prognostic outcome for a patient (1). Single-photon emission computed tomography (SPECT) or gamma planar imaging with 99mTc-labeled ligands that target HER2 are often used to detect, diagnose, and develop a treatment regimen for cancers that overexpress this receptor (2). Although many Abs (monoclonal, recombinant, etc.) and their derivatives (single-chain and monovalent or divalent Fab fragments, etc.) have been developed and approved for clinical use for the imaging or radioimmunotherapy of such cancers, these agents have limited efficacy because of their immunogenicity (development of Abs against the anti-HER2 Abs), pharmacokinetic properties (long circulating times), and inability to penetrate deep into tissue (due to the large size of ~150 kDa) (3). Therefore, investigators are constantly developing and evaluating new alternatives to the Ab-based imaging agents (3). Recently, a small (~7 kDa) ZHER2:342 affibody was engineered (4) and labeled with radionuclides such as 99mTc or 111In for the detection of HER2-overexpressing tumors with the use of SPECT (2). Later, the affibody and several of its structural derivatives were labeled with 99mTc and used to detect HER2-expressing cancers under preclinical and clinical conditions (for details on the structural derivatives, see Ekblad et al.) (5, 6). It has been shown that a hexa-histidine tag (H6; also facilitates purification of the H6-bearing compound by immobilized metal ion affinity chromatography) bearing dimeric ZHER2:342 could be labeled with 99mTc-tricarbonyl ([99mTc(CO)3]+) to obtain [99mTc(CO)3]+-H6-(ZHER2:342)2, and that the radiochemical was suitable to visualize the expression of HER2 in tumor-bearing mice (7). However, for the duration of the study, a higher amount of radioactivity was observed to accumulate in the liver compared to the tumors. From these observations the investigators concluded that, because the liver is often the organ to which a cancer metastasizes, H6-(ZHER2:342)2 was suitable only for the imaging of extrahepatic tumors. Other investigators have also shown that an H6 tag located on the N-terminal on a 99mTc-labeled anti-HER2 affibody leads to increased accumulation of label in the hepatic tissue (8, 9). On the basis of a hypothesis that the uptake of the labeled compound by the liver could be reduced by moving the H6 tag from the N-terminal of the ZHER2:342 affibody or by increasing the hydrophilicity of the tag, two new tracers were constructed by Tolmachev et al. (10). In one construct, the H6 tag was moved from the N-terminal of the affibody to the C-terminal to obtain ZHER2:342-H6; in the second construct, the tag located on the N-terminal of the affibody was made more hydrophilic by alternating glutamic acid residues with the histidines to generate HEHEHE ((HE)3) and obtain (HE)3-ZHER2:342. These constructs were subsequently labeled with [99mTc(CO)3]+ to form [99mTc(CO)3]+-ZHER2:342-H6 and [99mTc(CO)3]+-(HE)3-ZHER2:342, respectively. The biodistribution patterns of these radiolabeled affibodies were respectively studied in normal and LS174T (a human colon cancer cell line that has a low expression of HER2) xenograft tumor-bearing nude mice and compared to that of the parent affibody, ZHER2:342 (described previously in (11)), which had an H6 tag on the N-terminal and was labeled with [99mTc(CO)3]+ ([99mTc(CO)3]+-H6-ZHER2:342). This chapter describes the results obtained with [99mTc(CO)3]-H6-ZHER2:342. Results obtained with [99mTc(CO)3]+-ZHER2:342-H6 and [99mTc(CO)3]+-(HE)3-ZHER2:342 are described in separate chapters of MICAD ( and compared to those obtained with [99mTc(CO)3]+-H6-ZHER2:342 (12, 13).

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