Background

[PubMed]

Epidermal growth factor (EGF) is a cytokine that comprises 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 and 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: 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 and 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 IgG₁ monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 with an affinity constant (K_d) of 0.1 nM (11). Trastuzumab is approved for clinical use for anti-cancer therapies in both Europe and North America. ¹¹¹In-Trastuzumab, Cy5.5-trastuzumab, and ⁶⁸Ga-trastuzumab -F(ab')₂ have been developed for imaging human breast cancer (12-16). However, the pharmacokinetics of the 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. A novel class of recombinant affinity ligands (Affibody molecules) for HER2 based on the Z-domain residues (58 amino acids) from one of the IgG-binding domains of staphylococcal protein A was constructed (17). Affibody molecules exhibit high binding affinity (K_d) to HER2 with K_d values <100 pM. Various radiolabeled Affibody molecules have been studied in terms of their ability to image HER2 in tumors [PubMed]. Mercaptoacetyl-Gly-Gly-Gly (MAG3) was used as a chelating linker to couple ^99mTc to Z_HER2:342 Affibody (^99mTc-MAG3-Z_HER2:342) (18). ^99mTc-MAG3-Z_HER2:342 has been evaluated in nude mice bearing human colon adenocarcinoma tumors.

Synthesis

[PubMed]

MAG3-Z_HER2:342 Affibody was prepared by standard solid-phase peptide synthesis (18). ^99mTc as pertechnetate was added to a solution of MAG3-Z_HER2:342 containing Na/K tartrate and SnCl₂. The mixture was incubated for 60 min at room temperature. The labeling efficiency of ^99mTc incorporation was 82 ± 12% (n = 15) with >98% purity. ^99mTc-MAG3-Z_HER2:342 was purified with size-exclusion chromatography. Specific activities of the preparations were not reported. ^99mTc-MAG3-Z_HER2:342 was found to be stable after incubation for 1 h in murine blood serum at 37°C or in solution containing 300-fold excess cysteine.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Engfeldt et al. (18) performed binding experiments with MAG3-Z_HER2:342 with the use of a Biacore sensor chip immobilized with extracellular domain of HER2 protein. The K_d value of MAG3-Z_HER2:342 was calculated to be 200 pM; the K_d value of Z_HER2:342 was 80 pM. Hence, the binding affinity of the synthetic Affibody molecule MAG3-Z_HER2:342 was 1.5-fold lower than the parent Affibody molecule Z_HER2:342. In vitro binding specificity tests showed that binding of ^99mTc-MAG3-Z_HER2:342 to SKOV-3 cells expressing HER2 was mediated by the receptors because saturation of receptors by preincubation with non-labeled Z_HER2:342 significantly decreased binding of ^99mTc-MAG3-Z_HER2:342. The antigen-binding capacity of ^99mTc-MAG3-Z_HER2:342 was 86%. The cell-bound radioactivity remained at 60–80% of the initially bound activity for up to 24 h when the cells were incubated with ^99mTc-MAG3-Z_HER2:342. In contrast, cells incubated with ¹²⁵I-labeled Z_HER2:342 retained ~35% of the original cell-associated radioactivity at 24 h.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

1.

Carpenter G., Cohen S. Epidermal growth factor. J Biol Chem. 1990;265(14):7709–12. [PubMed: 2186024]

2.

Yarden Y. The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur J Cancer. 2001;37 Suppl 4:S3–8. [PubMed: 11597398]

3.

Rubin I., Yarden Y. The basic biology of HER2. Ann Oncol. 2001;12 Suppl 1:S3–8. [PubMed: 11521719]

4.

Grunwald V., Hidalgo M. Developing inhibitors of the epidermal growth factor receptor for cancer treatment. J Natl Cancer Inst. 2003;95(12):851–67. [PubMed: 12813169]

5.

Mendelsohn J. Anti-epidermal growth factor receptor monoclonal antibodies as potential anti-cancer agents. J Steroid Biochem Mol Biol. 1990;37(6):889–92. [PubMed: 2285602]

6.

Yasui W., Sumiyoshi H., Hata J., Kameda T., Ochiai A., Ito H., Tahara E. Expression of epidermal growth factor receptor in human gastric and colonic carcinomas. Cancer Res. 1988;48(1):137–41. [PubMed: 2446740]

7.

Ang K.K., Berkey B.A., Tu X., Zhang H.Z., Katz R., Hammond E.H., Fu K.K., Milas L. Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res. 2002;62(24):7350–6. [PubMed: 12499279]

8.

Costa S., Stamm H., Almendral A., Ludwig H., Wyss R., Fabbro D., Ernst A., Takahashi A., Eppenberger U. Predictive value of EGF receptor in breast cancer. Lancet. 1988;2(8622):1258. [PubMed: 2903994]

9.

Ethier S.P. Growth factor synthesis and human breast cancer progression. J Natl Cancer Inst. 1995;87(13):964–73. [PubMed: 7629883]

10.

Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology. 2001;61 Suppl 2:1–13. [PubMed: 11694782]

11.

Carter P., Presta L., Gorman C.M., Ridgway J.B., Henner D., Wong W.L., Rowland A.M., Kotts C., Carver M.E., Shepard H.M. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A. 1992;89(10):4285–9. [PMC free article: PMC49066] [PubMed: 1350088]

12.

Perik P.J., Lub-De Hooge M.N., Gietema J.A., van der Graaf W.T., de Korte M.A., Jonkman S., Kosterink J.G., van Veldhuisen D.J., Sleijfer D.T., Jager P.L., de Vries E.G. Indium-111-labeled trastuzumab scintigraphy in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. J Clin Oncol. 2006;24(15):2276–82. [PubMed: 16710024]

13.

Lub-de Hooge M.N., Kosterink J.G., Perik P.J., Nijnuis H., Tran L., Bart J., Suurmeijer A.J., de Jong S., Jager P.L., de Vries E.G. Preclinical characterisation of 111In-DTPA-trastuzumab. Br J Pharmacol. 2004;143(1):99–106. [PMC free article: PMC1575276] [PubMed: 15289297]

14.

Garmestani K., Milenic D.E., Plascjak P.S., Brechbiel M.W. A new and convenient method for purification of 86Y using a Sr(II) selective resin and comparison of biodistribution of 86Y and 111In labeled Herceptin. Nucl Med Biol. 2002;29(5):599–606. [PubMed: 12088731]

15.

Smith-Jones P.M., Solit D., Afroze F., Rosen N., Larson S.M. Early tumor response to Hsp90 therapy using HER2 PET: comparison with 18F-FDG PET. J Nucl Med. 2006;47(5):793–6. [PMC free article: PMC3193602] [PubMed: 16644749]

16.

Smith-Jones P.M., Solit D.B., Akhurst T., Afroze F., Rosen N., Larson S.M. Imaging the pharmacodynamics of HER2 degradation in response to Hsp90 inhibitors. Nat Biotechnol. 2004;22(6):701–6. [PubMed: 15133471]

17.

Wikman M., Steffen A.C., Gunneriusson E., Tolmachev V., Adams G.P., Carlsson J., Stahl S. Selection and characterization of HER2/neu-binding affibody ligands. Protein Eng Des Sel. 2004;17(5):455–62. [PubMed: 15208403]

18.

Engfeldt, T., A. Orlova, T. Tran, A. Bruskin, C. Widstrom, A.E. Karlstrom, and V. Tolmachev, Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the (99m)Tc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence. Eur J Nucl Med Mol Imaging, 2006. [PubMed: 17146656]