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177Lu-Benzyl-diethylenetriamine pentaacetic acid-anti-carbonic anhydrase IX small immunoprotein A3

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

Created: ; Last Update: January 12, 2010.

Chemical name:177Lu-Benzyl-diethylenetriamine pentaacetic acid-anti-carbonic anhydrase IX small immunoprotein A3
Abbreviated name:177Lu-Bn-DTPA-SIP-A3
Agent category:Antibody fragment, small immunoprotein (SIP)
Target:Carbonic anhydrase IX, hypoxia
Target category:Enzyme
Method of detection:Single-photon emission computed tomography (SPECT), gamma planar
Source of signal:177Lu
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about human carbonic anhydrase IX.



In a variety of solid tumors, hypoxia has been found to lead to tumor progression and to the resistance to chemotherapy and radiotherapy (1-3). Tumor oxygenation is heterogeneously distributed within human tumors (4). Hypoxia in malignant tumors is thought to be a major factor limiting the efficacy of chemotherapy and radiotherapy. It would be beneficial to assess tumor oxygenation before and after therapy to provide an evaluation of tumor response to treatment and an insight into new therapeutic treatments (5). Tumor oxygenation is measured invasively using computerized, polarographic, oxygen-sensitive electrodes, which is regarded as the gold standard (6). Functional and non-invasive imaging of intratumoral hypoxia has been demonstrated to be feasible for the measurement of tumor oxygenation (7). This has led to the research and development of hypoxia-targeted, non-invasive markers of tumor hypoxia.

Chapman proposed the use of 2-nitroimidazole compounds for hypoxia imaging (8). 2-Nitroimidazole compounds are postulated to undergo reduction in hypoxic conditions, forming highly reactive oxygen radicals that subsequently bind covalently to macromolecules inside the cells (9). [18F]Fluoromisonidazole ([18F]FMISO) is the most widely used positron emission tomography (PET) tracer for imaging tumor hypoxia (7). Carbonic anhydrase (CA) IX is one of the most highly overexpressed genes in cells under hypoxic conditions (10). It is a transmembrane glycoprotein with CA activity in the extracellular domain, and it is found to be overexpressed in renal cell, cervical, lung, and colorectal tumors. Monoclonal antibodies have been developed for in vitro and in vivo localization of CA IX on cells (11). Recently, a bispecific A3 monoclonal antibody to CA IX was constructed by the fusion of two A3 scFv fragments to form a divalent scFv small immunoprotein (SIP-A3; molecular weight, ~80 kDa) (12). SIP-A3 was labeled with 177Lu via isothiocyanate-benzyl-diethylenetriamine pentaacetic acid (SCN-Bn-DTPA) to form 177Lu-Bn-DTPA-SIP-A3 for use in single-photon emission computed tomography (SPECT) imaging of CA IX in nude mice bearing human solid tumors.



Isothiocyanate-benzyl-DTPA and SIP-A3 (50:1 molar ratio) were incubated in 0.1 M sodium bicarbonate buffer (pH 8.2) for 1 h at room temperature (12). Benzyl-DTPA-SIP-A3 was isolated from the incubation mixture with a PD-10 column. Mass spectroscopy analysis showed ~2 DTPA molecules per SIP-A3 molecule. Benzyl-DTPA-SIP-A3 in 125 mM ammonium acetate buffer (pH 5.5) was mixed with a predetermined amount of 177Lu. The mixture was incubated for 1 h at room temperature with a labeling efficiency of >95%. 177Lu-Bn-DTPA-SIP-A3 was purified with column chromatography. The specific activity of 177Lu-Bn-DTPA-SIP-A3 was not reported.

In Vitro Studies: Testing in Cells and Tissues


Ahlskog et al. (12) performed binding experiments with scFv-A3 using a Biacore sensor chip immobilized with extracellular domain of CA IX. The dissociation constant (Kd ) was 2.4 nM. Cytometry analysis showed a strong binding of SIP-A3 to LS174T colorectal tumor cells and a moderate binding to SK-RC-52 renal carcinoma cells. Immunofluorescence staining of various tumor sections revealed a strong binding of SIP-A3 to LS174T tumors, HT-28 colorectal carcinomas, SW 1222 colorectal carcinomas, U87 gliomas, and SK-RC-52 tumors, but not of MCF-7 breast carcinomas and Ramos lymphomas.

Animal Studies



Ahlskog et al. (12) studied the biodistribution of 177Lu-Bn-DTPA-SIP-A3 in mice (n = 4) bearing LS174T tumors at 24 h after injection. The tracer accumulation in the LS174T tumors was 2.41 ± 0.19% injected dose per gram (ID/g). The organ with the highest radioactivity was the kidneys (85.7% ID/g), followed by the liver (11.8% ID/g), spleen (5.2% ID/g), and lung (1.0% ID/g). The tumor/blood ratio was 16.7. 177Lu-Bn-DTPA-SIP-HyNEL10 (anti-lysozyme) showed tumor accumulation of 1.13 ± 0.08% ID/g with a tumor/blood ratio of 12.9. The tumor accumulation of 177Lu-Bn-DTPA-SIP-A3 was significantly higher than that of 177Lu-Bn-DTPA-SIP-HyNEL10 (P < 0.001). Ex vivo immunohistochemical staining of tumors and tissues at 6 h after injection of SIP-A3 and 0.5 h after injection of pimonidazole (hypoxia staining) showed that a higher fluorescence signal was detected in the tumor lesions than in the normal tissues. There was an overlap between pimonidazole staining and SIP-A3 fluorescence signal in the tumor sections. No blocking experiment was performed.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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