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

[99mTc(CO)3]+-H6-ZHER2:342
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894

Created: ; Last Update: January 20, 2011.

Chemical name:[99mTc(CO)3]+-Labeled anti-epidermal growth factor receptor (HER2) affibody ZHER2:342 with a hexa-histidine tag (H6) on the N-terminal
Abbreviated name:[99mTc(CO)3]+-H6-ZHER2:342
Synonym:
Agent Category:Affibody (antibody)
Target:Epidermal growth factor receptor (HER2)
Target Category:Receptor
Method of detection:Single-photon emission computed tomography (SPECT); gamma planar imaging
Source of signal / contrast:99mTc
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
No structure is available in PubChem.

Background

[PubMed]

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 (www.micad.nih.gov) and compared to those obtained with [99mTc(CO)3]+-H6-ZHER2:342 (12, 13).

Other Sources of Information

Affibody ZHER2:342 chapters in MICAD

Other 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

Synthesis

[PubMed]

The production and purification of H6-ZHER2:342 were performed as described by Tolmachev et al. (10). The purity of H6-ZHER2:342 was reported to be 98%, and the agent had a molecular weight of 8.2 kDa. Labeling of the histidine-tagged affibody with [99mTc(CO)3]+ is also described by Tolmachev et al. (10). The radiochemical yield and purity of the tracer were 80% and >95%, respectively. The specific activity of the labeled affibody was 14–15.8 GBq/μmol (378–426 mCi/μmol). Radio-colloid formation in the final preparation was <1.5% as determined with reversed-phase high-performance liquid chromatography.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

[99mTc(CO)3]+-H6-ZHER2:342 was stable in phosphate-buffered saline for at least 24 h at room temperature (23°C), and 97.2 ± 0.2% of the radiolabeled affibody was intact at the end of incubation as determined with instant thin-layer chromatography (10). A challenge with 500- or 1,000-fold excess histidine for 1 h at 37°C showed that >98% of the labeled compound was intact at the end of the incubation period. The association equilibrium constant (KD), the association rate constant (Ka), and the dissociation rate constant (Kd) of the radiochemical were 23 pM, 4.0 × 106 M-1s-1, and 8.79 × 10-5 s-1, respectively, as determined with biosensor analysis.

The binding specificities of [99mTc(CO)3]+-H6-ZHER2:342 were reported to be 48.9 ± 0.8%, 6.1 ± 0.2%, and 6.7 ± 0.1% for the SKOV-3, DU-145, and PC-3 cells, respectively, which was significantly (P < 0.0005) reduced to 2.3 ± 0.5%, 3.0 ± 0.3%, and 3.6 ± 0.1% after pretreatment of the cells with 1,000-fold excess unlabeled H6-ZHER2:342 (10). Cellular internalization of the radiolabeled affibody by the SKOV-3 cells after 24 h incubation at 37°C was determined to be 80.1 ± 0.5%. The antigen binding capacity of the labeled conjugate in the presence of 100-fold excess receptor concentration (using intact SKOV-3 cells) was reported to be 79.6 ± 0.6%.

Animal Studies

Rodents

[PubMed]

The biodistribution of [99mTc(CO)3]+-H6-ZHER2:342 was studied in normal male Naval Medical Research Institute (NMRI) mice (n = 4 animals/time point) after intravenous injection of the tracer as described by Tolmachev et al. (10). Accumulated radioactivity in various organs of the animals was determined at 4 h and 24 h postinjection (p.i.) and was presented as percent of injected dose per gram tissue (% ID/g). At 4 h p.i., 92.0 ± 23.0% ID/g radioactivity was detected in the kidneys and the accumulated radioactivity was reduced to 45.0 ± 6.0% ID/g by 24 h p.i. In the liver, accumulation of the tracer was 10.0 ± 3.0% ID/g at 4 h p.i. and decreased to 7.0 ± 1.0% ID/g at 24 h p.i. The spleen showed an uptake of 1.3 ± 0.3% ID/g at 4 h p.i., which decreased to 0.8 ± 0.1% ID/g by 24 h p.i. All other organs showed <1.0% ID/g radioactivity at both time points. These observations indicated that the label had a rapid clearance from the blood and was excreted primarily through the kidneys.

In another study, investigators studied the biodistribution of [99mTc(CO)3]+-H6-ZHER2:342 in the various organs of NMRI nude mice (n = 4 animals) bearing LS174T cell xenograft tumors as detailed above (10). The accumulation of radioactivity in these mice was reported to follow the same pattern as that observed with the normal animals. At 4 h p.i., the accumulation of radioactivity from this tracer was 82.0 ± 9.0% ID/g, 8.1 ± 0.8% ID/g, and 2.2 ± 0.3% ID/g in the kidneys, liver, and the xenograft tumors, respectively. The tumor/blood, tumor/liver, tumor/kidney, and the tumor/muscle ratios for this tracer were 4.5 ± 0.1, 0.29 ± 0.04, 0.028 ± 0.002, and 21.0 ± 0.2, respectively.

Gamma planar imaging of a tumor-bearing mouse showed that the animal had a similar pattern of label accumulation as described above (10).

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

Supplemental Information

[Disclaimers]

No information is currently available

References

1.
Tolmachev V., Rosik D., Wallberg H., Sjoberg A., Sandstrom M., Hansson M., Wennborg A., Orlova A. Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR 111In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount. Eur J Nucl Med Mol Imaging. 2010;37(3):613–22. [PubMed: 19838701]
2.
Orlova A., Feldwisch J., Abrahmsen L., Tolmachev V. Update: affibody molecules for molecular imaging and therapy for cancer. Cancer Biother Radiopharm. 2007;22(5):573–84. [PubMed: 17979560]
3.
Holliger P., Hudson P.J. Engineered antibody fragments and the rise of single domains. Nat Biotechnol. 2005;23(9):1126–36. [PubMed: 16151406]
4.
Wahlberg E., Lendel C., Helgstrand M., Allard P., Dincbas-Renqvist V., Hedqvist A., Berglund H., Nygren P.A., Hard T. An affibody in complex with a target protein: structure and coupled folding. Proc Natl Acad Sci U S A. 2003;100(6):3185–90. [PMC free article: PMC152267] [PubMed: 12594333]
5.
Tolmachev V., Friedman M., Sandstrom M., Eriksson T.L., Rosik D., Hodik M., Stahl S., Frejd F.Y., Orlova A. Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry. J Nucl Med. 2009;50(2):274–83. [PubMed: 19164241]
6.
Ekblad T., Orlova A., Feldwisch J., Wennborg A., Karlstrom A.E., Tolmachev V. Positioning of 99mTc-chelators influences radiolabeling, stability and biodistribution of Affibody molecules. Bioorg Med Chem Lett. 2009;19(14):3912–4. [PubMed: 19364646]
7.
Orlova A., Nilsson F.Y., Wikman M., Widstrom C., Stahl S., Carlsson J., Tolmachev V. Comparative in vivo evaluation of technetium and iodine labels on an anti-HER2 affibody for single-photon imaging of HER2 expression in tumors. J Nucl Med. 2006;47(3):512–9. [PubMed: 16513621]
8.
Ahlgren S., Orlova A., Rosik D., Sandstrom M., Sjoberg A., Baastrup B., Widmark O., Fant G., Feldwisch J., Tolmachev V. Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules. Bioconjug Chem. 2008;19(1):235–43. [PubMed: 18163536]
9.
Ahlgren S., Wallberg H., Tran T.A., Widstrom C., Hjertman M., Abrahmsen L., Berndorff D., Dinkelborg L.M., Cyr J.E., Feldwisch J., Orlova A., Tolmachev V. Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine. J Nucl Med. 2009;50(5):781–9. [PubMed: 19372467]
10.
Tolmachev V., Hofstrom C., Malmberg J., Ahlgren S., Hosseinimehr S.J., Sandstrom M., Abrahmsen L., Orlova A., Graslund T. HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [(m)Tc(CO)](+), and shows improved biodistribution with reduced hepatic radioactivity accumulation. Bioconjug Chem. 2010;21(11):2013–22. [PubMed: 20964447]
11.
Orlova A., Magnusson M., Eriksson T.L., Nilsson M., Larsson B., Hoiden-Guthenberg I., Widstrom C., Carlsson J., Tolmachev V., Stahl S., Nilsson F.Y. Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer Res. 2006;66(8):4339–48. [PubMed: 16618759]
12.
Chopra, A., [99mTc(CO)3]+-Labeled anti-epidermal growth factor receptor (HER2) affibody ZHER2:342 with a hexa-histidine tag (H6) on the N-terminal. 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: 21290629]
13.
Chopra, A., [99mTc(CO)3]+-Labeled anti-epidermal growth factor receptor (HER2) affibody ZHER2:342 with a hexa-histidine tag (H6) on the C-terminal. 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: 21290634]

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