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64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-anti-prostate-specific membrane antigen 3/A12 monoclonal antibody

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD
Corresponding author.

Created: ; Last Update: September 16, 2009.

Chemical name:64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-anti-prostate-specific membrane antigen 3/A12 monoclonal antibody
Abbreviated name:64Cu-DOTA-3/A12
Agent category:Antibody
Target:Prostate-specific membrane antigen (PSMA), or N-acetyl α-linked acidic dipeptidase (NAALADase)
Target category:Antigen
Method of detection:PET
Source of signal:64Cu
  • Checkbox In vitro
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Click on protein, nucleotide (RefSeq), and gene for more information about PSMA.



Prostate-specific membrane antigen (PSMA) is a cell-surface glycoprotein with a molecular weight of ~100 kDa. It is a unique, type II, transmembrane-bound glycoprotein that is overexpressed on prostate tumor cells and in the neovasculature of most solid prostate tumors, but not in the vasculature of normal tissues (1, 2). It has also been detected in other tissues such as the kidneys, the proximal small intestine, and the salivary glands (2). PSMA was found to have N-acetyl α-linked acidic dipeptidase or glutamate carboxypeptidase II activity (3). PSMA may play an important role in the progression of prostate cancer and glutamatergic neurotransmission, and in the absorption of folate (4). In the central nervous system, PSMA metabolizes N-acetyl-aspartyl-glutamate, and in the proximal small intestine it removes γ-linked glutamates from poly-γ-glutamate folate and folate hydrolase (2). PSMA can be used as a marker for the detection of metastatic cancers with imaging agents. Although a commercially available monoclonal antibody (111In-labeled Capromomab pendetide (111In-CYT-356)) is in clinical use for the detection of prostate cancer, the results obtained with this antibody are not entirely reliable (5). In addition, the antibody has limited access to tumors and may produce low signal/noise ratios because the target is the intracellular domain of PSMA (6, 7). Monoclonal antibody (mAb) 3/A12 was found to be reactive with purified PSMA; 3/A12 also showed a strong and specific binding to LNCaP human prostate cancer cells and PSMA-transfected cells in culture (8). 64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-3/A12 mAb (64Cu-DOTA-3/A12) has been studied for use in positron emission tomography (PET) to image PSMA expression in nude mice bearing human cancer xenografts (9).



DOTA-3/A12 was incubated with 64CuCl2 for 40 min at 40°C (9). 64Cu-DOTA-3/A12 was purified with high-performance liquid chromatography. The radiochemical purity was 95% with a specific radioactivity of 67.3 MBq/nmol (1.8 mCi/nmol). 64Cu-DOTA-3/A12 was stable in saline buffer and mouse serum at 37°C for up to 24 h. The number of DOTA molecules per antibody was not reported.

In Vitro Studies: Testing in Cells and Tissues


The binding affinity of 3/A12 and DOTA-3/A12 for PSMA was determined with the use of PSMA-expressing C4-2 prostate cancer cells (9). In this assay, phycoerythrin-labeled ant-IgG was used as a secondary antibody for cytofluorometric analysis. The concentrations that reached a 50% saturation of PSMA sites were 10 nM for 3/A12 and 15 nM for DOTA-3/A12.

Animal Studies



Elsasser-Beile et al. (9) performed ex vivo biodistribution studies of 64Cu-DOTA-3/A12 (~0.15 nmol) in nude mice bearing PSMA-positive C4-2 tumor xenografts (n = 8 mice) or PSMA-negative DU 145 tumor (n = 7 mice) xenografts. The C4-2 tumor uptake was 35.1 ± 8.0% injected dose per gram (ID/g) at 48 h after injection. The tumor/muscle and tumor/blood ratios were 27 and 2, respectively. On the other hand, the radioactivity level in DU 145 tumors was 12.8 ± 2.7% ID/g. The organ with the highest accumulation was the liver (10% ID/g), followed by the kidneys (6% ID/g), heart (5% ID/g), colon (3% ID/g), and stomach (2% ID/g). The whole-body distribution of 64Cu-DOTA-3/A12 was also assessed with PET imaging at 3, 24, and 48 h after injection. The tumor/muscle ratios for the C4-2 tumors were 3.3, 7.8, and 9.6 at these time points, respectively. The C4-2 tumor exhibited a two-fold higher radioactivity than the DU 145 tumor at 24 h after injection. The highest radioactivity levels were visualized in the liver and the C4-2 tumor; the DU 145 tumors were barely visualized. Pretreatment with 5 nmol mAb 3/A12 3 h before 64Cu-DOTA-3/A12 injection in mice bearing the C4-2 tumors inhibited the radioactivity accumulation nearly to the background level of the DU 145 tumors.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


Feneley M.R., Jan H., Granowska M., Mather S.J., Ellison D., Glass J., Coptcoat M., Kirby R.S., Ogden C., Oliver R.T., Badenoch D.F., Chinegwundoh F.I., Nargund V.H., Paris A.M., Britton K.E. Imaging with prostate-specific membrane antigen (PSMA) in prostate cancer. Prostate Cancer Prostatic Dis. 2000;3(1):47–52. [PubMed: 12497162]
Ghosh A., Heston W.D. Tumor target prostate specific membrane antigen (PSMA) and its regulation in prostate cancer. J Cell Biochem. 2004;91(3):528–39. [PubMed: 14755683]
Luthi-Carter R., Barczak A.K., Speno H., Coyle J.T. Molecular characterization of human brain N-acetylated alpha-linked acidic dipeptidase (NAALADase). J Pharmacol Exp Ther. 1998;286(2):1020–5. [PubMed: 9694964]
O'Keefe, D., D. Bachich, and W.D. Heston, Prostate specific membrane antigen. Prostate cancer, biology, genetics, and the new therapeutics., ed. L. Chung, W. Issacs, and J. Simons. 2001, New Jersey: Humana Press. 307-326.
Ponsky L.E., Cherullo E.E., Starkey R., Nelson D., Neumann D., Zippe C.D. Evaluation of preoperative ProstaScint scans in the prediction of nodal disease. Prostate Cancer Prostatic Dis. 2002;5(2):132–5. [PubMed: 12497003]
Sundaresan G., Yazaki P.J., Shively J.E., Finn R.D., Larson S.M., Raubitschek A.A., Williams L.E., Chatziioannou A.F., Gambhir S.S., Wu A.M. 124I-labeled engineered anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice. J Nucl Med. 2003;44(12):1962–9. [PMC free article: PMC4167879] [PubMed: 14660722]
Jain R.K. Transport of molecules, particles, and cells in solid tumors. Annu Rev Biomed Eng. 1999;1:241–63. [PubMed: 11701489]
Elsasser-Beile U., Wolf P., Gierschner D., Buhler P., Schultze-Seemann W., Wetterauer U. A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and therapeutic targeting of prostate cancer. Prostate. 2006;66(13):1359–70. [PubMed: 16894535]
Elsasser-Beile U., Reischl G., Wiehr S., Buhler P., Wolf P., Alt K., Shively J., Judenhofer M.S., Machulla H.J., Pichler B.J. PET Imaging of Prostate Cancer Xenografts with a Highly Specific Antibody against the Prostate-Specific Membrane Antigen. J Nucl Med. 2009;50(4):606–611. [PubMed: 19289418]


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