Molecular Imaging and Biology

, Volume 16, Issue 4, pp 567–577 | Cite as

Characterization and Evaluation of 64Cu-Labeled A20FMDV2 Conjugates for Imaging the Integrin αvβ6

  • Lina Y. Hu
  • Nadine Bauer
  • Leah M. Knight
  • Zibo Li
  • Shuanglong Liu
  • Carolyn J. Anderson
  • Peter S. Conti
  • Julie L. Sutcliffe
Research Article



The integrin αvβ6 is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for 64Cu radiolabeling of A20FMDV2, an αvβ6 targeting peptide.


Four chelators were conjugated onto PEG28-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4′-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl))bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with 64Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted.


All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37–50 °C for 15 min) with high specific activity (0.58–0.60 Ci/μmol). All radiotracers demonstrated αvβ6-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers 64Cu-CB-TE1A1P-1 and 64Cu-BaBaSar-1 showed improved specificity for the αvβ6 positive tumor in vivo over 64Cu-DOTA-1 and 64Cu-NOTA-1 (+/− tumor uptake ratios—3.82 +/- 0.44, 3.82 ± 0.41, 2.58 ± 0.58, and 1.29 ± 0.14, respectively). Of the four radiotracers, 64Cu-NOTA-1 exhibited the highest liver uptake (10.83 ± 0.1 % ID/g at 4 h).


We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear “best candidate” for the 64Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.

Key words

Integrin αvβ6 Copper-64 PET imaging 

Supplementary material

11307_2013_717_MOESM1_ESM.docx (2.5 mb)
ESM 1(DOCX 2531 kb)


  1. 1.
    Bates RC, Bellovin DI, Brown C et al (2005) Transcriptional activation of integrin β6 during the epithelial–mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma. J Clin Invest 115:339–347PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Bruess JM, Gallo J, DeLisser HM et al (1995) Expression of the β6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J Cell Sci 108:2241–2251Google Scholar
  3. 3.
    Maubant S, Cruet-Hennequart S, Dutoit S et al (2005) Expression of α V-associated integrin β subunits in epithelial ovarian cancer and its relation to prognosis in patients treated with platinum-based regimens. J Mol Histol 36:119–129PubMedCrossRefGoogle Scholar
  4. 4.
    Sipos B, Hahn D, Carceller A et al (2004) Immunohistochemical screening for β6-integrin subunit expression in adenocarcinomas using a novel monoclonal antibody reveals strong up-regulation in pancreatic ductal adenocarcinomas in vivo and in vitro. Histopathology 45:226–236PubMedCrossRefGoogle Scholar
  5. 5.
    Saha A, Ellison D, Thomas GJ et al (2010) High-resolution in vivo imaging of breast cancer by targeting the pro-invasive integrin αvβ6. J Pathol 222:52–63PubMedGoogle Scholar
  6. 6.
    Hazelbag S, Kenter GG, Gorter A et al (2007) Overexpression of the αvβ6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival. J Pathol 212:316–324PubMedCrossRefGoogle Scholar
  7. 7.
    Zhang ZY, Xu KS, Wang JS et al (2008) Integrin ανβ6 acts as a prognostic indicator in gastric carcinoma. Clin Oncol 20:61–66CrossRefGoogle Scholar
  8. 8.
    Li X, Yang Y, Hu Y et al (2003) αvβ6-Fyn signaling promotes oral cancer progression. J Biol Chem 278:41646–41653PubMedCrossRefGoogle Scholar
  9. 9.
    Yang G-Y, Xu K-S, Pan Z-Q et al (2008) Integrin alphavbeta6 mediates the potential for colon cancer cells to colonize in and metastasize to the liver. Cancer Sci 99:879–887PubMedCrossRefGoogle Scholar
  10. 10.
    Elayadi AN, Samli KN, Prudkin L et al (2007) A peptide selected by biopanning identifies the integrin αvβ6 as a prognostic biomarker for nonsmall cell lung cancer. Cancer Res 67:5889–5895PubMedCrossRefGoogle Scholar
  11. 11.
    Gagnon MKJ, Hausner SH, Marik J et al (2009) High-throughput in vivo screening of targeted molecular imaging agents. Proc Natl Acad Sci U S A 106:17904–17909PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Hackel BJ, Kimura RH, Miao Z, et al. (2013) 18F-Fluorobenzoate–Labeled Cystine Knot Peptides for PET Imaging of Integrin αvβ6. J Nucl MedGoogle Scholar
  13. 13.
    Aina OH, Sroka TC, Chen M-L, Lam KS (2002) Therapeutic cancer targeting peptides. Biopolymers 66:184–199PubMedCrossRefGoogle Scholar
  14. 14.
    Hausner SH, DiCara D, Marik J et al (2007) Use of a peptide derived from foot-and-mouth disease virus for the noninvasive imaging of human cancer: generation and evaluation of 4-[18F]fluorobenzoyl A20FMDV2 for in vivo imaging of integrin alphavbeta6 expression with positron emission tomography. Cancer Res 67:7833–7840PubMedCrossRefGoogle Scholar
  15. 15.
    Hausner SH, Abbey CK, Bold RJ et al (2009) Targeted in vivo imaging of integrin alphavbeta6 with an improved radiotracer and its relevance in a pancreatic tumor model. Cancer Res 69:5843–5850PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Hausner SH, Kukis DL, Gagnon MK et al (2009) Evaluation of [64Cu]Cu-DOTA and [64Cu]Cu-CB-TE2A chelates for targeted positron emission tomography with an alphavbeta6-specific peptide. Mol Imaging 8:111–121PubMedGoogle Scholar
  17. 17.
    Blower PJ, Lewis JS, Zweit J (1996) Copper radionuclides and radiopharmaceuticals in nuclear medicine. Nucl Med Biol 23:957–980PubMedCrossRefGoogle Scholar
  18. 18.
    Jiang M, Ferdani R, Shokeen M, Anderson CJ (2013) Comparison of two cross-bridged macrocyclic chelators for the evaluation of 64Cu-labeled-LLP2A, a peptidomimetic ligand targeting VLA-4-positive tumors. Nucl Med Biol 40:245–251PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Cai H, Li Z, Huang CW, et al. (2010) Evaluation of copper-64 labeled AmBaSar conjugated cyclic RGD peptide for improved microPET imaging of integrin αvβ3 expression. Bioconjug Chem 21:1417–1424Google Scholar
  20. 20.
    Liu S, Li Z, Yap LP, Huang CW et al (2011) Efficient preparation and biological evaluation of a novel multivalency bifunctional chelator for 64Cu radiopharmaceuticals. Chemistry 17:10222–10225PubMedCrossRefGoogle Scholar
  21. 21.
    Hausner SH, Carpenter RD, Bauer N, Sutcliffe JL (2013) Evaluation of an integrin alphavbeta6-specific peptide labeled with [18F]fluorine by copper-free, strain-promoted click chemistry. Nucl Med Biol 40:233–239PubMedCrossRefGoogle Scholar
  22. 22.
    Nemeth JA, Nakada MT, Trikha M et al (2007) Alpha-v integrins as therapeutic targets in oncology. Cancer Invest 25:632–646PubMedCrossRefGoogle Scholar
  23. 23.
    Guo Y, Ferdani R, Anderson CJ (2012) Preparation and biological evaluation of (64)Cu labeled Tyr(3)-octreotate using a phosphonic acid-based cross-bridged macrocyclic chelator. Bioconjug Chem 23:1470–1477PubMedCrossRefGoogle Scholar
  24. 24.
    Anderson CJ, Ferdani R (2009) Copper-64 radiopharmaceuticals for PET imaging of cancer: advances in preclinical and clinical research. Cancer Biother Radiopharm 24Google Scholar
  25. 25.
    Anderson CJ (2001) Metabolism of radiometal-labeled proteins and peptides: what are the real radiopharmaceuticals in vivo? Cancer Biother Radiopharm 16:451–455PubMedCrossRefGoogle Scholar
  26. 26.
    Novak-Hofer I, Zimmermann K, Schubiger PA (2001) Peptide linkers lead to modification of liver metabolism and improved tumor targeting of copper-67-labeled antibody fragments. Cancer Biother Radiopharm 16:469–481PubMedCrossRefGoogle Scholar
  27. 27.
    Zeglis BM, Lewis JS (2011) A practical guide to the construction of radiometallated bioconjugates for positron emission tomography. Dalton Trans 40:6168–6195PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Hausner SH, Marik J, Gagnon MKJ, Sutcliffe JL (2008) In vivo positron emission tomography (PET) imaging with an αvβ6 specific peptide radiolabeled using 18F-“click” chemistry: evaluation and comparison with the corresponding 4-[18F]fluorobenzoyl- and 2-[18F]fluoropropionyl-peptides. J Med Chem 51:5901–5904PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    DeNardo SJ, Liu R, Albrecht H et al (2009) 111In-LLP2A-DOTA polyethylene glycol-targeting α4β1 integrin: comparative pharmacokinetics for imaging and therapy of lymphoid malignancies. J Nucl Med 50:625–634PubMedCrossRefGoogle Scholar
  30. 30.
    Haubner R, Decristoforo C (2009) Radiolabelled RGD peptides and peptidomimetics for tumour targeting. Front Biosci 14:872–886CrossRefGoogle Scholar
  31. 31.
    Nguyen K, Parry JJ, Rogers BE, Anderson CJ (2012) Evaluation of copper-64-labeled somatostatin agonists and antagonist in SSTr2-transfected cell lines that are positive and negative for p53: implications for cancer therapy. Nucl Med Biol 39:187–197PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Hausner SH, Bauer N, Sutcliffe JL (2013) In vitro and in vivo evaluation of the effects of aluminum [18F]fluoride radiolabeling on an integrin avb6-specific peptide. Nucl Med Biol 41:43–50PubMedCrossRefGoogle Scholar
  33. 33.
    Bass LA, Lanahan MV, Duncan JR et al (1998) Identification of the soluble in vivo metabolites of indium-111-diethylenetriaminepentaacetic acid-D-Phe1-octreotide. Bioconjug Chem 9:192–200PubMedCrossRefGoogle Scholar
  34. 34.
    Breeman WAP, Kwekkeboom DJ, Kooij PPM et al (1995) Effect of dose and specific activity on tissue distribution of indium-111-pentetreotide in rats. J Nucl Med 36:623–627PubMedGoogle Scholar
  35. 35.
    Lewis JS, Lewis MR, Cutler PD et al (1999) Radiotherapy and dosimetry of 64Cu-TETA-Tyr3-octreotate in a somatostatin receptor-positive, tumor-bearing rat model. Clin Cancer Res 5:3608–3616PubMedGoogle Scholar
  36. 36.
    Smith SV (2004) Molecular imaging with copper-64. J Inorg Biochem 98:1874–1901PubMedCrossRefGoogle Scholar

Copyright information

© World Molecular Imaging Society 2014

Authors and Affiliations

  • Lina Y. Hu
    • 1
  • Nadine Bauer
    • 1
  • Leah M. Knight
    • 1
  • Zibo Li
    • 2
  • Shuanglong Liu
    • 2
  • Carolyn J. Anderson
    • 3
  • Peter S. Conti
    • 2
  • Julie L. Sutcliffe
    • 1
    • 4
    • 5
  1. 1.Department of Biomedical EngineeringUniversity of California DavisDavisUSA
  2. 2.Department of RadiologyUniversity of Southern CaliforniaLos AngelesUSA
  3. 3.Department of RadiologyUniversity of PittsburghPittsburghUSA
  4. 4.Department of Internal Medicine, Division of Hematology/OncologyUniversity of California Davis, School of MedicineSacramentoUSA
  5. 5.Center for Molecular and Genomic ImagingUniversity of California DavisDavisUSA

Personalised recommendations