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125I-Labeled mouse anti-human carbonic anhydrase IX monoclonal antibody

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

Created: ; Last Update: May 30, 2012.

Chemical name:125I-Labeled mouse anti-human carbonic anhydrase IX monoclonal antibody
Abbreviated name:125I-MAb
Agent Category:Antibodies
Target:Carbonic anhydrase IX (CA IX)
Target Category:Enzymes
Method of detection:Single-photon emission computed tomography (SPECT) or planar imaging
Source of signal / contrast:125I
  • Checkbox In vitro
  • Checkbox Rodents
No structure is available.



125I-Labeled mouse anti-human carbonic anhydrase IX (CA IX) monoclonal antibody (MAb), abbreviated as 125I-MAb, is a probe developed by Li et al. for molecular imaging of tumor hypoxia (1).

CA IX is a membrane-associated glycoprotein that consists of an extracellular catalytic domain, a transmembrane anchor, and a short C-terminal cytoplasmic tail (2, 3). The expression of CA IX is largely restricted to the epithelial cells of the gastrointestinal tract, especially the gastric epithelial cells where CA IX catalyzes the hydration of carbon dioxide to bicarbonate and protons (1, 4). CA IX is one of the two CA isoforms (IX and XII) in the CA family that are overexpressed in various solid tumors (3). The CA IX overexpression is closely related to the activation of hypoxia-inducible factor-1 (HIF-1) and the inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene due to hypoxia or the mutation of the VHL gene (5, 6). In this process, HIF-1 upregulates the expression of CA IX while the VHL protein downregulates the expression of CA IX by involving in the degradation of HIF-1 α-subunits. In some renal cell carcinomas, CA IX could be significantly overexpressed (up to 150-fold) because of the VHL mutation and constitutive HIF-1 activation (2, 3, 6).

The direct outcome of CA IX overexpression is the acidification of the tumor microenvironment, leading to extracellular pH values of ~6.5 in contrast to 7.4 in normal tissues (4). The acidic microenvironment has been shown to be associated with tumorigenic transformation, chromosomal rearrangements, extracellular matrix breakdown, and tumor cell migration, invasion, and resistance to radio/chemotherapy (2, 3, 5). CA IX may also play a role in tumor cell growth by providing bicarbonate to the synthesis of pyrimidine nucleotides.

Because of these facts, CA IX has been investigated as a marker for cellular hypoxia and tumor prognosis and as a target for tumor imaging and therapy (7, 8). For these purposes, a large set of chemical inhibitors and several MAb have been developed (7-9). Ahlskog et al. described the generation and characterization of two high-affinity human MAb (A3 and CC7) specific to the extracellular domain of human CA IX (7). The radionuclide-labeled chimeric antibody cG250 and its Fab and F(ab')2 fragments have been tested by Divgi et al. as molecular probes for imaging clear-cell renal carcinomas and HT29 human colorectal tumor xenografts (10). Li et al. investigated the biodistribution of 125I-MAb in animals bearing HT29 tumor xenografts (1). The results from these studies suggest that anti-CA IX MAb can selectively recognize CA IX in tumor cells and preferentially localize at sites of hypoxia in tumors after intravenous administration. This chapter summarizes the data obtained with 125I-MAb.



Mouse anti-human CA IX MAb (the clone information not reported) is commercially available. Li et al. labeled the antibody with no-carrier-added Na125I using the Iodogen method (1). The time of labeling reaction was 10 min, and the radiochemical purity of 125I-MAb was 98%. 125I-MAb was stable in new-born calf serum and phosphate-buffered saline for 96 h without obvious decrease in its radiochemical purity (>90%). The radiochemical yield and specific activity were not reported.

In Vitro Studies: Testing in Cells and Tissues


The expression of CA IX in HT-29 tumor xenografts was analyzed with immunohistochemistry of tumor sections, which showed that CA IX was predominantly expressed in the areas adjacent to necrosis but not in the areas of necrosis (1).

Animal Studies



Biodistribution studies were performed in athymic male mice bearing HT-29 xenografts after tail vein injection of 1.295 MBq (35 µCi) 125I-MAb (n = 5 mice/time point) (1). During the first 12 h, a relatively high uptake of 125I-MAb was observed in the tumors as well as in the liver, kidney, spleen, lung, and intestine. At 24 h, the uptake of 125I-MAb in the normal organs decreased, whereas it reached the highest level (4.9 ± 1.2% injected dose per gram tissue (ID/g)) in tumors. The uptake value was significantly higher in tumors than in normal tissues (P < 0.05). However, the highest tumor/muscle ratio (8.16) was observed at 12 h after injection. The uptake in the thyroid was 0.4 ± 0.1% ID/g at 12 h and increased to 1.4 ± 0.04% ID/g at 96 h after injection, indicating metabolic de-iodination of the 125I-MAb.

Binding of 125I-MAb with whole blood cells in vivo was evaluated at 24 h after injection of 3.7 MBq (100 µCi) 125I-MAb (0.1 ml) into an athymic mouse via the tail vein (1). The mouse was euthanized and blood cells were collected and washed completely to remove the unbound 125I-MAb. The radioactivity in the blood was higher than in tumors and other organs (see Figure 2 in Li et al. (1)) and this value in the fraction of blood cells was 5.5% of the blood activity at 24 h after injection, indicating that a small fraction of 125I-MAb diffused into blood cells.

Planar imaging was performed at 48 h after injection of 5.55 MBq (150 µCi) 125I-MAb (0.1 ml) via the tail vein (1). Preferential accumulation of 125I-MAb in the tumors was detected. A medium accumulation of radioactivity was also detected in the thyroid. Blocking studies were performed with injection of 100-fold molar excess of unlabeled antibody at 24 h before injection of 125I-MAb. The results showed that unlabeled MAb effectively blocked the accumulation of 125I-MAb in the tumor. The radioactivity in the thyroid was also decreased significantly, but no explanation for the mechanism of the decrease.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


Li J., Shi L., Wang C., Zhang X., Jia L., Li X., Zhou W., Qi Y., Zhang L. Preliminary biological evaluation of (1)(2)I-labeled anti-carbonic anhydrase IX monoclonal antibody in the mice bearing HT-29 tumors. Nucl Med Commun. 2011;32(12):1190–3. [PubMed: 21968435]
Winum J.Y., Rami M., Scozzafava A., Montero J.L., Supuran C. Carbonic anhydrase IX: a new druggable target for the design of antitumor agents. Med Res Rev. 2008;28(3):445–63. [PubMed: 17880011]
Guler O.O., De Simone G., Supuran C.T. Drug design studies of the novel antitumor targets carbonic anhydrase IX and XII. Curr Med Chem. 2010;17(15):1516–26. [PubMed: 20166929]
Pastorekova S., Zatovicova M., Pastorek J. Cancer-associated carbonic anhydrases and their inhibition. Curr Pharm Des. 2008;14(7):685–98. [PubMed: 18336315]
Kaluz S., Kaluzova M., Liao S.Y., Lerman M., Stanbridge E.J. Transcriptional control of the tumor- and hypoxia-marker carbonic anhydrase 9: A one transcription factor (HIF-1) show? Biochim Biophys Acta. 2009;1795(2):162–72. [PMC free article: PMC2670353] [PubMed: 19344680]
Arjumand W., Sultana S. Role of VHL gene mutation in human renal cell carcinoma. Tumour Biol. 2012;33(1):9–16. [PubMed: 22125026]
Ahlskog J.K., Schliemann C., Marlind J., Qureshi U., Ammar A., Pedley R.B., Neri D. Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours. Br J Cancer. 2009;101(4):645–57. [PMC free article: PMC2736829] [PubMed: 19623173]
Stillebroer A.B., Oosterwijk E., Oyen W.J., Mulders P.F., Boerman O.C. Radiolabeled antibodies in renal cell carcinoma. Cancer Imaging. 2007;7:179–88. [PMC free article: PMC2151324] [PubMed: 18055291]
Rami M., Cecchi A., Montero J.L., Innocenti A., Vullo D., Scozzafava A., Winum J.Y., Supuran C.T. Carbonic anhydrase inhibitors: design of membrane-impermeant copper(II) complexes of DTPA-, DOTA-, and TETA-tailed sulfonamides targeting the tumor-associated transmembrane isoform IX. ChemMedChem. 2008;3(11):1780–8. [PubMed: 18956406]
Divgi C.R., Pandit-Taskar N., Jungbluth A.A., Reuter V.E., Gonen M., Ruan S., Pierre C., Nagel A., Pryma D.A., Humm J., Larson S.M., Old L.J., Russo P. Preoperative characterisation of clear-cell renal carcinoma using iodine-124-labelled antibody chimeric G250 (124I-cG250) and PET in patients with renal masses: a phase I trial. Lancet Oncol. 2007;8(4):304–10. [PubMed: 17395103]


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