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Radiolabeling of TETA- and CB-TE2A-conjugated peptides with copper-64

Nature Protocols volume 1, pages 3062–3068 (2006)Cite this article

Abstract

The number of radiopharmaceuticals containing copper radionuclides for diagnostic imaging and targeted radiotherapy has grown considerably over the past few decades. This expansion has created the need for protocols allowing for the efficient chelation of 64Cu to peptide-chelator conjugates. Step 1A of this protocol describes a 64Cu-radiolabeling procedure for 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA)-conjugated peptides. This reaction is facile and requires the incubation of 64CuCl2 in 0.1 M ammonium acetate buffer with the TETA-peptide for 30 min at room temperature (20–23 °C). Step 1B of this protocol describes the radiolabeling procedure for 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A)-conjugated peptides. The CB-TE2A-peptide can be labeled with 64Cu in 0.1 M ammonium acetate buffer in 2 h at 95 °C. In both cases, the conjugates can be radiolabeled with 64Cu at greater than 95% purity and with specific activities of 37–111 MBq μg−1 (1–3 mCi μg−1). Both protocols are straightforward and can be completed within 3 h.

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Figure 1: Structures of the molecules discussed in the protocol.
Figure 2: A representative chromatogram of a [64Cu]TETA-D-Hep III using the Waters HPLC system previously described, a mobile phase consisting of 0.1% TFA in H2O (solution A) and acetonitrile (0.1% TFA)/water (0.1% TFA), 9:1 (solution B) and a gradient consisting of 22% B to 32% B in 20 min (1.0 ml min−1 flow rate).
Figure 3: A representative chromatogram of a [64Cu]CB-TE2A-c(RGDyK) using the Waters HPLC system previously described, a mobile phase consisting of 0.1% TFA in H2O (solution A) and 0.1% TFA in CH3CN (solution B), and a gradient consisting of 1% B to 70% B in 20 min.

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References

  1. Wadas, T.J., Wong, E.H., Weisman, G.R. & Anderson, C.J. Copper chelation chemistry and its role in copper radiopharmaceuticals. Curr. Pharm. Des. (in the press).

  2. Smith, S.V. Molecular imaging with copper-64. J. Inorg. Biochem. 98, 1874–1901 (2004).

    Article  CAS  Google Scholar 

  3. Mountz John, D. et al. Molecular imaging: new applications for biochemistry. J. Cell. Biochem. Suppl. 39, 162–171 (2002).

    Article  CAS  Google Scholar 

  4. Rowland, D.J., Lewis, J.S. & Welch, M.J. Molecular imaging: the application of small animal positron emission tomography. J. Cell. Biochem. Suppl. 39, 110–115 (2002).

    Article  Google Scholar 

  5. Blok, D., Feitsma, R.I.J., Vermeij, P. & Pauwels, E.J.K. Peptide radiopharmaceuticals in nuclear medicine. Eur. J. Nucl. Med. 26, 1511–1519 (1999).

    Article  CAS  Google Scholar 

  6. Okarvi, S.M. Recent progress in fluorine-18 labelled peptide radiopharmaceuticals. Eur. J. Nucl. Med. 28, 928–938 (2001).

    Article  Google Scholar 

  7. Signore, A. et al. Peptide radiopharmaceuticals for diagnosis and therapy. Eur. J. Nucl. Med. 28, 1555–1565 (2001).

    Article  CAS  Google Scholar 

  8. Sun, X. & Anderson, C.J. Production and applications of copper-64 radiopharmaceuticals. Meth. Enzymol. 386, 237–261 (2004).

    Article  CAS  Google Scholar 

  9. Anderson, C.J. & Welch, M.J. Radiometal-labeled agents (non-technetium) for diagnostic imaging. Chem. Rev. 99, 2219–2234 (1999).

    Article  CAS  Google Scholar 

  10. Blower, P.J., Lewis, J.S. & Zweit, J. Copper radionuclides and radiopharmaceuticals in nuclear medicine. Nucl. Med. Biol. 23, 957–980 (1996).

    Article  CAS  Google Scholar 

  11. Hnatowich, D.J., Layne, W.W., Childs, R.L., Lanteigne, D. & Davis, M.A. Radioactive labeling of antibody: a simple and efficient method. Science 220, 613–615 (1983).

    Article  CAS  Google Scholar 

  12. Parker, D., Morphy, J.R., Jankowski, K. & Cox, J. Implementation of macrocycle-conjugated antibodies for tumor-targeting. Pure Appl. Chem. 61 (9): 1637–1641 (1990).

    Article  Google Scholar 

  13. Cole, W.C. et al. Serum stability of 67Cu chelates: comparison with 111In and 57Co. Nucl. Med. Biol. 13, 363–368 (1986).

    CAS  Google Scholar 

  14. Yeh, S.M., Sherman, D.G. & Meares, C.F. A new route to 'bifunctional' chelating agents: conversion of amino acids to analogs of ethylenedinitrilotetraacetic acid. Anal. Biochem. 100, 152–159 (1979).

    Article  CAS  Google Scholar 

  15. Meares, C.F. & Wensel, T.G. Metal chelates as probes of biological systems. Acc. Chem. Res. 17, 202–209 (1984).

    Article  CAS  Google Scholar 

  16. Moi, M.K., Meares, C.F., McCall, M.J., Cole, W.C. & DeNardo, S.J. Copper chelates as probes of biological systems: stable copper complexes with a macrocyclic bifunctional chelating agent. Anal. Biochem. 148, 249–253 (1985).

    Article  CAS  Google Scholar 

  17. Cole, W.C. et al. Comparative serum stability of radiochelates for antibody radiopharmaceuticals. J. Nucl. Med. 28, 83–90 (1987).

    CAS  PubMed  Google Scholar 

  18. Meares, C.F. et al. Macrocyclic chelates of radiometals for diagnosis and therapy. Br. J. Cancer Suppl. 10, 21–26 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Kukis, D.L., Li, M. & Meares, C.F. Selectivity of antibody-chelate conjugates for binding copper in the presence of competing metals. Inorg. Chem. 32, 3981–3982 (1993).

    Article  CAS  Google Scholar 

  20. Wu, Y. et al. MicroPET imaging of glioma integrin αvβ3 expression using (64)Cu-labeled tetrameric RGD peptide. J. Nucl. Med. 46, 1707–1718 (2005).

    CAS  PubMed  Google Scholar 

  21. Li, W.P., Lewis, J.S., Srinivasan, A., Schmidt, M.A. & Anderson, C.J. Copper-64/61 and iodine-125-labeled DOTA-D-TYR1-octreotate: a new somatostatin analog for labeling with metals and halogens. J. Labelled Comp. Radiopharm. 44 (suppl 1): S948–S950 (2001).

    Article  Google Scholar 

  22. McQuade, P. et al. Imaging of melanoma using 64Cu- and 86Y-DOTA-ReCCMSH(Arg11), a cyclized peptide analogue of α-MSH. J. Med. Chem. 48, 2985–2992 (2005).

    Article  CAS  Google Scholar 

  23. Chen, X. et al. Integrin αvβ3-targeted imaging of lung cancer. Neoplasia 7, 271–279 (2005).

    Article  CAS  Google Scholar 

  24. Chen, X. et al. MicroPET and autoradiographic imaging of breast cancer αvβ3 integrin expression using F-18 and Cu-64-labeled RGD peptides. Bioconjug. Chem. 15, 41–49 (2004).

    Article  Google Scholar 

  25. Chen, X. et al. Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor αvβ3-integrin expression. J. Nucl. Med. 45, 1776–1783 (2004).

    CAS  PubMed  Google Scholar 

  26. Anderson, C.J. et al. Preparation, biodistribution and dosimetry of copper-64-labeled anti-colorectal carcinoma monoclonal antibody fragments 1A3-F(ab′)2 . J. Nucl. Med. 36, 850–858 (1995).

    CAS  PubMed  Google Scholar 

  27. Anderson, C.J. et al. Radiotherapy, toxicity and dosimetry of copper-64-TETA-octreotide in tumor-bearing rats. J. Nucl. Med. 39, 1944–1951 (1998).

    CAS  PubMed  Google Scholar 

  28. Anderson, C.J. et al. Copper-64-TETA-octreotide as a PET imaging agent for patients with neuroendocrine tumors. J. Nucl. Med. 42, 213–221 (2001).

    CAS  PubMed  Google Scholar 

  29. Lewis, J.S., Srinivasan, A., Schmidt, M.A. & Anderson, C.J. In vitro and in vivo evaluation of 64Cu-TETA-Tyr3-octreotate. A new somatostatin analog with improved target tissue uptake. Nucl. Med. Biol. 26, 267–273 (1998).

    Article  Google Scholar 

  30. Lewis, J.S. et al. Comparison of four 64Cu-labeled somatostatin analogs in vitro and in a tumor-bearing rat model: evaluation of new derivatives for PET imaging and targeted radiotherapy. J. Med. Chem. 42, 1341–1347 (1999).

    Article  CAS  Google Scholar 

  31. Lewis, M.R. et al. Conjugation of monoclonal antibodies with TETA using activated esters: biological comparison of 64Cu-TETA-1A3 with 64Cu-BAT-2IT-1A3. Cancer Biother. Radiopharm. 16, 483–494 (2001).

    Article  CAS  Google Scholar 

  32. Cutler, P.D. et al. Dosimetry of copper-64-labeled monoclonal antibody 1A3 as determined by PET imaging of the torso. J. Nucl. Med. 36, 2363–2371 (1995).

    CAS  PubMed  Google Scholar 

  33. Wang, M. et al. Subcellular localization of somatostatin analogues: implications for targeted radiotherapy of cancer. Cancer Res. 63, 6864–6869 (2003).

    CAS  PubMed  Google Scholar 

  34. Edwards, W.B., Anderson, C.J., Fields, G.B. & Welch, M.J. Evaluation of type IV collagen fragments as potential tumor imaging agents. Bioconjug. Chem. 12, 1057–1065 (2001).

    Article  CAS  Google Scholar 

  35. Weisman, G.R., Rogers, M.E., Wong, E.H., Jasinski, J.P. & Paight, E.S. Cross-bridged cyclam. Protonation and Li+ complexation in a diamond-lattice cleft. J. Am. Chem. Soc. 112, 8604–8605 (1990).

    Article  CAS  Google Scholar 

  36. Weisman, G.R. et al. Synthesis and transition-metal complexes of new cross-bridged tetraamine ligands. J. Chem. Soc. Chem. Commun., 947–948.

  37. Niu, W. et al. Copper(II) and zinc(II) complexes of amide pendant-armed cross-bridged tetraamine ligands. Polyhedron 23, 1019–1025 (2004).

    Article  CAS  Google Scholar 

  38. Wong, E.H. et al. Synthesis and characterization of cross-bridged cyclams and pendant-armed derivatives and structural studies of their copper(II) complexes. J. Am. Chem. Soc. 122, 10561–10572 (2000).

    Article  CAS  Google Scholar 

  39. Woodin, K.S. et al. Kinetic inertness and electrochemical behavior of copper(II) tetraazamacrocyclic complexes: possible implications for in vivo stability. Eur. J. Inorg. Chem. 23, 4829–4833 (2005).

    Article  Google Scholar 

  40. Boswell, C.A. et al. Comparative in vivo stability of copper-64-labeled cross-bridged and conventional tetraazamacrocyclic complexes. J. Med. Chem. 47, 1465–1474 (2004).

    Article  CAS  Google Scholar 

  41. Sun, X. et al. Radiolabeling and in vivo behavior of copper-64-labeled cross-bridged cyclam ligands. J. Med. Chem. 45, 469–477 (2002).

    Article  CAS  Google Scholar 

  42. Sprague, J.E. et al. Preparation and biological evaluation of copper-64-labeled Tyr3-octreotate using a cross-bridged macrocyclic chelator. Clin. Cancer Res. 10, 8674–8682 (2004).

    Article  CAS  Google Scholar 

  43. Wadas, T.J., Wong, E.H., Weisman, G.R. & Anderson, C.J. Copper chelation chemistry and its role in copper radiopharmaceuticals. Curr. Pharm. Design 13, 3–16 (2007).

    Article  CAS  Google Scholar 

  44. Edwards, W.B. Cancer Imaging with PET. Ph.D. Thesis, Washington University in St. Louis (1999).

    Google Scholar 

  45. Mant, C.T. & Hodges, R.S. High-Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis, and Conformation (CRC Press, Boca Raton, Florida, 2000).

    Google Scholar 

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Acknowledgements

The authors gratefully acknowledge NIH grants R01 CA93375 (C.J.A.), R01 CA064475 (C.J.A.) and F32 CA115148 (T.J.W.). The production of 64Cu at Washington University School of Medicine has been supported by the NCI grant R24 CA86307.

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  1. Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 South Kingshighway Boulevard, Campus Box 8225, St. Louis, 63110, Missouri, USA

    Thaddeus J Wadas & Carolyn J Anderson

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Correspondence to Carolyn J Anderson.

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Wadas, T., Anderson, C. Radiolabeling of TETA- and CB-TE2A-conjugated peptides with copper-64. Nat Protoc 1, 3062–3068 (2006). https://doi.org/10.1038/nprot.2006.431

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