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68Ga-1,4,7-Triazacyclononane-1,4,7-triacetic acid-c(RGDfK)-human serum albumin-tissue inhibitor of matrix metalloproteinase 2 fusion protein

68Ga-NOTA-RGD-HSA-TIMP2
, PhD
National for Biotechnology Information, NLM, NIH, Bethesda, MD
Corresponding author.

Created: ; Last Update: May 24, 2012.

Chemical name:68Ga-1,4,7-Triazacyclononane-1,4,7-triacetic acid-c(RGDfK)-human serum albumin-tissue inhibitor of matrix metalloproteinase 2 fusion protein
Abbreviated name:68Ga-NOTA-RGD-HSA-TIMP2
Synonym:
Agent category:Protein
Target: αvβ3 integrin receptor
Target category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal/contrast:68Ga
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
No structure is currently available in PubChem.

Background

[PubMed]

Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (1). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). Tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degrading the basement membrane and ECM (3).

Several families of MMPs are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastases (4-7). MMP expression is highly regulated in normal cells, such as trophoblasts, osteoclasts, neutrophils, and macrophages. Elevated levels of MMPs have been found in tumors associated with a poor prognosis for cancer patients (8). There are four members of endogenous tissue inhibitors of metalloproteinases (TIMPs), which regulate the activity of MMPs leading to inhibition of tumor growth and metastasis (9, 10). TIMP-2 (TIMP2) is a bifunctional inhibitor of angiogenesis by inhibition of proteinase activity of MMPs and endothelial cell proliferation via binding to α3β1 (the N-terminal domain) and by MMP-independent anti-angiogenic activity (the C-terminal domain) (11, 12). Kang et al. (13) fused the N-terminal domain of TIMP2 to the C-terminus of human serum albumin (HSA) to form HSA/TIMP2 fusion protein (HSA-TIMP2), which is readily secreted by the transfected yeast Saccharomyces cerevisiae. HSA-TIMP2 retains its anti-angiogenic activity at the C-terminal domain with little MMP inhibitory activity at the N-terminal domain. Lee et al. (14) have evaluated Cy5.5-HSA/TIMP2 (Cy5.5-HSA-TIMP2) for in vivo near-infrared (NIR) fluorescence imaging of rat prostate MLL tumors in nude mice showing maximum tumor accumulation at 2 d after injection.

Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (15). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The αvβ3 integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (16-21). Expression of the αvβ3 integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. The peptide sequence Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including αvβ3. The αvβ3 antagonists are being studied as anti-tumor and anti-angiogenic agents (18, 22, 23). Various radiolabeled RGD peptides (antagonists) have been introduced for imaging of tumors and tumor angiogenesis (24). Choi et al. (25) conjugated multiple c(RGDfK) peptides to HSA-TIMP2 to enhance the binding capacity of the protein (RGD-HSA-TIMP2) to tumors and their vasculatures. 68Ga-NOTA-RGD-HSA-TIMP2 and 68Ga-NOTA-HSA-TIMP2 have been studied as potential positron emission tomography (PET) probes for imaging αvβ3 integrin receptors in nude mice bearing human glioblastoma U87MG tumors.

Synthesis

[PubMed]

HSA-TIMP2 and c[RGDfK(COCH2SH)] was incubated with N-succinimidyl iodoacetate to form a thioether linkage between the RGD peptide and HSA-TIMP2 protein (25). RGD-HSA-TIMP2 was purified with column chromatography. The molecular weights of RGD-HSA-TIMP2 and HSA-TIMP2 were calculated to be 88.42 kDa and 92.78 kDa with mass spectroscopy, respectively. There were 6 RGD molecules per RGD-HSA-TIMP2. Addition of NOTA groups to RGD-HSA-TIMP2 and HSA-TIMP2 was performed by mixing 0.05 µmol protein with 2.2 µmol SCN-Bz-NOTA in 0.1 M sodium carbonate buffer (pH 9.5) for 20 h at room temperature. The NOTA-conjugated proteins were purified with column chromatography. The number of NOTA moieties per NOTA-RGD-HSA-TIMP2 and NOTA-HSA-TIMP2 were calculated to be 6.6 and 5.1, respectively. For 68Ga labeling, a solution of 74 MBq (2 mCi) 68GaCl3 and NOTA-RGD-HSA-TIMP2 or NOTA-HSA-TIMP2 was incubated for 20 min at room temperature. 68Ga-NOTA-RGD-HSA-TIMP2 and 68Ga-NOTA-HSA-TIMP2 was purified with column chromatography, with yields of 30%–35% and total synthesis time of 30 min. Their specific activities and radiochemical purities were not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Flow cytometry analysis showed that 45% and 91% of U89MG cells were positive after incubation with HSA-TIMP2-FITC and RGD-HSA-TIMP2-FITC for 24 h at 4°C (25), respectively. Confocal microscopy showed internalization of fluorescence activity inside the cells incubated with 1.6 nM RGD-HSA-TIMP2-FITC for 3 h at 37°C but not with HSA-TIMP2-FITC. No blocking studies were performed with unlabeled RGD-HSA-TIMP2.

In vitro cell viability of U87MG was assessed after incubation with various concentrations (0.11–3.2 nM) of RGD, HSA-TIMP2, and RGD-HSA-TIMP2 for 24 h at 37°C (25). The cell viability values with 3.2 nM RGD, HSA-TIMP2, and RGD-HSA-TIMP2 were 92.5 ± 4.0%, 73.5 ± 7.9% (P < 0.05 versus RGD), and 66.7 ± 0.6% (P < 0.01 versus RGD), respectively.

Cellular accumulation of 68Ga-NOTA-RGD-HSA-TIMP2 and 68Ga-NOTA-HSA-TIMP2 was compared in human glioblastoma U87MG (positive for αvβ3 integrin) and rat glioblastoma C6 (negative for αvβ3 integrin) cells in culture (25). The radioactivity levels of 68Ga-NOTA-HSA-TIMP2 were low in C6 (0.23 ± 0.01% incubation dose (ID)) and U87MG (0.49 ± 0.01% ID) cells at 3 h after incubation. However, the radioactivity level of 68Ga-NOTA-RGD-HSA-TIMP2 was higher in U87MG (3.87 ± 0.49% ID) than in C6 (0.71 ± 0.01% ID) cells. Co-incubation of 68Ga-NOTA-RGD-HSA-TIMP2 with excess c(RGDyK) significant inhibited the radioactivity level in U89MG cells by 70% (P < 0.01). No affinity constants were reported for the three constructs.

Animal Studies

Rodents

[PubMed]

Choi et al. (25) performed ex vivo biodistribution studies of 3.7 MBq (0.01 mCi) 68Ga-NOTA-RGD-HSA-TIMP2 and 68Ga-NOTA-HSA-TIMP2 in nude mice (n = 3/group) bearing U87MG xenografts at 3 h after injection. The tumor accumulation of radioactivity was 1.1 ± 0.2% ID/g and 0.4 ± 0.1% ID/g for 68Ga-NOTA-RGD-HSA-TIMP2 and 68Ga-NOTA-HSA-TIMP2 (P < 0.01), respectively. The organ with the highest accumulation was the liver for both radiolabeled proteins with ~40% ID/g. The kidney and spleen accumulation values were 3% ID/g and 8% ID/g, respectively. The blood concentrations were approximately four-fold higher than the tumor accumulation, suggesting that more time is needed to have optimal accumulation in the tumors. No blocking studies were performed.

Choi et al. (25) performed whole-body PET/CT imaging in nude mice (the number of mice was not reported) bearing U87MG tumors at 1 h and 3 h after intravenous injection of 7.2 MBq (0.2 mCi) 68Ga-NOTA-RGD-HSA-TIMP2 and 68Ga-NOTA-HSA-TIMP2. Little tumor accumulation of 68Ga-NOTA-HSA-TIMP2 was observed at 1 h (0.56 ± 0.08% ID/g) and 3 h (0.69 ± 0.09% ID/g). The tumor accumulation of 68Ga-NOTA-RGD-HSA-TIMP2 was 0.77 ± 0.07% ID/g and 1.95 ± 0.50% ID/g at 1 h and 3 h, respectively. High liver accumulation was observed with both tracers. Immunofluorescence staining of tumor tissue sections showed an increase in CD31 (endothelial cell biomarker) expression in the tumor tissue. No blocking studies were performed.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

References

1.
Bosman F.T., Stamenkovic I. Functional structure and composition of the extracellular matrix. J Pathol. 2003;200(4):423–8. [PubMed: 12845610]
2.
Jiang W.G., Puntis M.C., Hallett M.B. Molecular and cellular basis of cancer invasion and metastasis: implications for treatment. Br J Surg. 1994;81(11):1576–90. [PubMed: 7827878]
3.
Albelda S.M. Role of integrins and other cell adhesion molecules in tumor progression and metastasis. Lab Invest. 1993;68(1):4–17. [PubMed: 8423675]
4.
Keppler D., Sameni M., Moin K., Mikkelsen T., Diglio C.A., Sloane B.F. Tumor progression and angiogenesis: cathepsin B & Co. Biochem Cell Biol. 1996;74(6):799–810. [PubMed: 9164649]
5.
Liu J., Sukhova G.K., Sun J.S., Xu W.H., Libby P., Shi G.P. Lysosomal cysteine proteases in atherosclerosis. Arterioscler Thromb Vasc Biol. 2004;24(8):1359–66. [PubMed: 15178558]
6.
Berchem G., Glondu M., Gleizes M., Brouillet J.P., Vignon F., Garcia M., Liaudet-Coopman E. Cathepsin-D affects multiple tumor progression steps in vivo: proliferation, angiogenesis and apoptosis. Oncogene. 2002;21(38):5951–5. [PubMed: 12185597]
7.
Brix, K., A. Dunkhorst, K. Mayer, and S. Jordans, Cysteine cathepsins: Cellular roadmap to different functions. Biochimie, 2007. [PubMed: 17825974]
8.
Deryugina E.I., Quigley J.P. Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev. 2006;25(1):9–34. [PubMed: 16680569]
9.
Baker A.H., Edwards D.R., Murphy G. Metalloproteinase inhibitors: biological actions and therapeutic opportunities. J Cell Sci. 2002;115(Pt 19):3719–27. [PubMed: 12235282]
10.
Jiang Y., Goldberg I.D., Shi Y.E. Complex roles of tissue inhibitors of metalloproteinases in cancer. Oncogene. 2002;21(14):2245–52. [PubMed: 11948407]
11.
Fernandez C.A., Butterfield C., Jackson G., Moses M.A. Structural and functional uncoupling of the enzymatic and angiogenic inhibitory activities of tissue inhibitor of metalloproteinase-2 (TIMP-2): loop 6 is a novel angiogenesis inhibitor. J Biol Chem. 2003;278(42):40989–95. [PubMed: 12900406]
12.
Seo D.W., Li H., Guedez L., Wingfield P.T., Diaz T., Salloum R., Wei B.Y., Stetler-Stevenson W.G. TIMP-2 mediated inhibition of angiogenesis: an MMP-independent mechanism. Cell. 2003;114(2):171–80. [PubMed: 12887919]
13.
Kang W.K., Park E.K., Lee H.S., Park B.Y., Chang J.Y., Kim M.Y., Kang H.A., Kim J.Y. A biologically active angiogenesis inhibitor, human serum albumin-TIMP-2 fusion protein, secreted from Saccharomyces cerevisiae. Protein Expr Purif. 2007;53(2):331–8. [PubMed: 17368046]
14.
Lee M.S., Kim Y.H., Kim Y.J., Kwon S.H., Bang J.K., Lee S.M., Song Y.S., Hahm D.H., Shim I., Han D., Her S. Pharmacokinetics and biodistribution of human serum albumin-TIMP-2 fusion protein using near-infrared optical imaging. J Pharm Pharm Sci. 2011;14(3):368–77. [PubMed: 21962154]
15.
Hynes R.O. A reevaluation of integrins as regulators of angiogenesis. Nat Med. 2002;8(9):918–21. [PubMed: 12205444]
16.
Grzesik W.J. Integrins and bone--cell adhesion and beyond. Arch Immunol Ther Exp (Warsz) 1997;45(4):271–5. [PubMed: 9523000]
17.
Jin H., Varner J. Integrins: roles in cancer development and as treatment targets. Br J Cancer. 2004;90(3):561–5. [PMC free article: PMC2410157] [PubMed: 14760364]
18.
Kumar C.C. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets. 2003;4(2):123–31. [PubMed: 12558065]
19.
Ruegg C., Dormond O., Foletti A. Suppression of tumor angiogenesis through the inhibition of integrin function and signaling in endothelial cells: which side to target? Endothelium. 2002;9(3):151–60. [PubMed: 12380640]
20.
Varner J.A., Cheresh D.A. Integrins and cancer. Curr Opin Cell Biol. 1996;8(5):724–30. [PubMed: 8939661]
21.
Wilder R.L. Integrin alpha V beta 3 as a target for treatment of rheumatoid arthritis and related rheumatic diseases. Ann Rheum Dis. 2002;61 Suppl 2:ii96–9. [PMC free article: PMC1766704] [PubMed: 12379637]
22.
Kerr J.S., Mousa S.A., Slee A.M. Alpha(v)beta(3) integrin in angiogenesis and restenosis. Drug News Perspect. 2001;14(3):143–50. [PubMed: 12819820]
23.
Mousa S.A. alphav Vitronectin receptors in vascular-mediated disorders. Med Res Rev. 2003;23(2):190–9. [PubMed: 12500288]
24.
Haubner R., Beer A.J., Wang H., Chen X. Positron emission tomography tracers for imaging angiogenesis. Eur J Nucl Med Mol Imaging. 2010;37 Suppl 1:S86–103. [PMC free article: PMC3629959] [PubMed: 20559632]
25.
Choi N., Kim S.M., Hong K.S., Cho G., Cho J.H., Lee C., Ryu E.K. The use of the fusion protein RGD-HSA-TIMP2 as a tumor targeting imaging probe for SPECT and PET. Biomaterials. 2011;32(29):7151–8. [PubMed: 21719102]

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