Background

[PubMed]

Positron emission tomography (PET) is a common technique used in conjunction with fluorodeoxyglucose labeled with radioactive fluorine ([¹⁸F]FDG) to investigate metabolism and to detect and evaluate the efficacy of cancer therapy (1, 2). Although a variety of ¹⁸F-labeled small molecule compounds, their derivatives, and antibodies (including those that were bioengineered) have been developed and evaluated in addition to [¹⁸F]FDG for PET imaging, the short physical half-life of this isotope (110 min) and the requirement of specialized equipment and personnel for the synthesis of ¹⁸F derivatives are major limitations for its use in the clinic (3). Investigators have also evaluated the use of radioactive copper (⁶⁴Cu) in an effort to develop PET imaging agents that have a longer half-life (12.7 h); however, biodistribution studies in rats have revealed that transchelation of ⁶⁴Cu to copper binding proteins such as ceruloplasmin and superoxide dismutase in the liver and albumin results in the high background observed with this isotope during imaging (4). In this regard, investigators have been in search of chelating agents that can be used to produce ⁶⁴Cu complexes with an extended stability under in vivo conditions, have good labeling properties, and are easily conjugated to molecules or antibodies with specific targets for PET imaging (5, 6).

Disialogangliosides (GD2) are ubiquitous and abundant surface glycolipid antigens found on tumors of neuroectodermal origin, including melanoma and neuroblastoma tumors. Therefore, these antigens are considered ideal targets for the immunotherapy of these diseases (7, 8). The use of an anti-GD2 monoclonal antibody (mAb) is one of the options available for the treatment of these cancers, and several clinical trials have been approved by the United States Food and Drug Administration for the use of this antibody to treat children and adults. The status of some of the clinical trials being conducted with the anit-GD2 antibody has been reviewed by Modak et al. (7).

Voss et al. developed a novel chelating agent, 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine (SarAr), that they used to chelate ⁶⁴Cu to an anti-GD2 mAb. The authors evaluated SarAr under in vivo conditions for the imaging of human neuroblastoma and melanoma tumors in athymic mice (6).

Synthesis

[PubMed]

A murine anti-GD2 antibody, 14.G2a, was purchased commercially, and a chimeric derivative of this antibody, ch14.18, was engineered as detailed elsewhere (9). The ch14.18 mAb was used to perform the studies detailed in this chapter. The two antibodies have a similar murine variable region sequence and an identical antigen-binding Fv region, and they were reported to contain distinct constant region sequences (6).

Synthesis of the SarAr chelator was described by Di Bartolo et al. (10). Using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), the SarAr ligand was conjugated through its primary aromatic group to the carboxyl groups of the glutamate and aspartate residues of the 14.G2a and ch14.18 mAbs (6). To achieve this, EDC was added to the mAb in a ratio of 250:1 for SarAr:IgG as described elsewhere using sodium acetate buffer (pH 5.0) at 37°C for 30 min (11). Unbound SarAr was removed from the immunoconjugates with high-performance liquid chromatography (HPLC), and the SarAr-conjugated mAbs were concentrated by use of spin filters for storage at -80°C. Four SarAr molecules were estimated to be bound to each mAb molecule as determined by serial dilution experiments (6).

The SarAr-conjugated antibodies were radiolabeled using [⁶⁴Cu]copper chloride in sodium acetate buffer (pH 5.0) at 37°C for 30 min (6). The investigators reported the same labeling efficiency could also be obtained at 21°C for 10 min. The labeling efficiency was routinely 99% as determined by HPLC and thin-layer chromatography (TLC); the Rf values for TLC were not provided. Specific activity of [⁶⁴Cu]SarAr mAb was reported to be 10 μCi/6.6 nmol (370 kBq/6.6 nmol). An investigation of ⁶⁴Cu-labeled ch14.18 by radioimmunoassay and cell-binding studies showed that the labeled mAb had immunoreactivity similar to the unlabeled mAb (6).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

No publications are currently available.

Animal Studies

Human Studies

[PubMed]

No publications are currently available.

Supplemental Information

[Disclaimers]

References

1.

Kumar R. , Basu S. , Torigian D. , Anand V. , Zhuang H. , Alavi A. Role of modern imaging techniques for diagnosis of infection in the era of 18F-fluorodeoxyglucose positron emission tomography. Clin Microbiol Rev. 2008; 21 (1):209–24. [PMC free article: PMC2223836] [PubMed: 18202443]

2.

Juweid M.E. 18F-FDG PET as a routine test for posttherapy assessment of Hodgkin's disease and aggressive non-Hodgkin's lymphoma: where is the evidence? J Nucl Med. 2008; 49 (1):9–12. [PubMed: 18077522]

3.

Smith S.V. Molecular imaging with copper-64. J Inorg Biochem. 2004; 98 (11):1874–901. [PubMed: 15522415]

4.

Boswell C.A. , Sun X. , Niu W. , Weisman G.R. , Wong E.H. , Rheingold A.L. , Anderson C.J. Comparative in vivo stability of copper-64-labeled cross-bridged and conventional tetraazamacrocyclic complexes. J Med Chem. 2004; 47 (6):1465–74. [PubMed: 14998334]

5.

Sprague J.E. , Peng Y. , Fiamengo A.L. , Woodin K.S. , Southwick E.A. , Weisman G.R. , Wong E.H. , Golen J.A. , Rheingold A.L. , Anderson C.J. Synthesis, characterization and in vivo studies of Cu(II)-64-labeled cross-bridged tetraazamacrocycle-amide complexes as models of peptide conjugate imaging agents. J Med Chem. 2007; 50 (10):2527–35. [PubMed: 17458949]

6.

Voss S.D. , Smith S.V. , DiBartolo N. , McIntosh L.J. , Cyr E.M. , Bonab A.A. , Dearling J.L. , Carter E.A. , Fischman A.J. , Treves S.T. , Gillies S.D. , Sargeson A.M. , Huston J.S. , Packard A.B. Positron emission tomography (PET) imaging of neuroblastoma and melanoma with 64Cu-SarAr immunoconjugates. Proc Natl Acad Sci U S A. 2007; 104 (44):17489–93. [PMC free article: PMC2077283] [PubMed: 17954911]

7.

Modak S. , Cheung N.K. Disialoganglioside directed immunotherapy of neuroblastoma. Cancer Invest. 2007; 25 (1):67–77. [PubMed: 17364560]

8.

Riemer A.B. , Forster-Waldl E. , Bramswig K.H. , Pollak A. , Zielinski C.C. , Pehamberger H. , Lode H.N. , Scheiner O. , Jensen-Jarolim E. Induction of IgG antibodies against the GD2 carbohydrate tumor antigen by vaccination with peptide mimotopes. Eur J Immunol. 2006; 36 (5):1267–74. [PubMed: 16568495]

9.

Gillies S.D. , Lo K.M. , Wesolowski J. High-level expression of chimeric antibodies using adapted cDNA variable region cassettes. J Immunol Methods. 1989; 125 (1-2):191–202. [PubMed: 2514231]

10.

Di Bartolo N.M. , Sargenson A.M. , Donlevy T.M. , Smith S.V. Synthesis of a new cage ligand, SarAr, and its complexation with select transition metal ions for potential use in radioimaging. J Chem Soc Dalton Trans. 2001;(15):2303–2309.

11.

Di Bartolo N.M. , Sargenson A.M. , Smith S.V. New 64Cu PET imaging agents for personalized medicine and drug development using the hexa-aza, SarAr. Organic and Biomolecular Chemistry. 2006; 4 :335–3357. [PubMed: 17036125]

12.

Mueller B.M. , Reisfeld R.A. , Gillies S.D. Serum half-life and tumor localization of a chimeric antibody deleted of the CH2 domain and directed against the disialoganglioside GD2. Proc Natl Acad Sci U S A. 1990; 87 (15):5702–5. [PMC free article: PMC54395] [PubMed: 2198570]

13.

Cheung N.K. , Neely J.E. , Landmeier B. , Nelson D. , Miraldi F. Targeting of ganglioside GD2 monoclonal antibody to neuroblastoma. J Nucl Med. 1987; 28 (10):1577–83. [PubMed: 3655911]