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99mTc(CO)3-Anti-carcinoembryonic antigen (CEA) humanized CEA5 graft nanobody

99mTc-Humanized CEA5 graft
, PhD
National for Biotechnology Information, NLM, NIH, Bethesda, MD
Corresponding author.

Created: ; Last Update: July 26, 2012.

Chemical name:99mTc(CO)3-Anti-carcinoembryonic antigen antigen (CEA) humanized CEA5 graft nanobody
Abbreviated name:99mTc-Humanized CEA5 graft
Agent category:Antibody fragment, humanized nanobody
Target:Carcinoembryonic antigen (CEA)
Target category:Receptor
Method of detection:Single-photon emission computed tomography (SPECT), gamma planar imaging
Source of signal:99mTc
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about CEA.



Carcinoembryonic antigen (CEA) was first identified from extracts of human colon adenocarcinoma (1) and fetal gut (2). CEA is a β-glycoprotein, and its predominant expression on the cell surface is increased in a variety of carcinomas and in certain inflammatory conditions, such as inflammatory bowel disease (3, 4). CEA has a molecular weight of ~180 kDa, and it can be shed and detected in serum (5). CEA expression is observed in patients with various carcinomas of the colon, lung, thyroid, uterus, ovary, pancreas, and medullary thyroid. Radiolabeled monoclonal antibodies (mAbs) have been developed for both the diagnosis and treatment of tumors (6, 7). However, the pharmacokinetics of intact radiolabeled mAbs, with high liver uptake and slow blood elimination, are generally not ideal for imaging (8, 9). Smaller antibody fragments, such as scFv, Fab, or F(ab')2, have better imaging pharmacokinetics because they are rapidly excreted by the kidneys. Nanobodies are the smallest intact antigen-binding fragments (15 kDa) isolated from heavy-chain camelid antibodies with efficient and specific tumor targeting (10-12). Anti-CEA nanobody CEA5 (NbCEA5, also known as cAb-CEA5) exhibits high binding affinity to CEA (Kd, 0.34 nM) (13). In order to minimize immunogenicity of nanobodies in humans, the antigen-binding loops of NbCEA5 were genetically grafted onto the framework of a humanized nanobody scaffold (h-NBBcII10FGLA) to form humanized CEA5 graft nanobody (14). The three nanobodies (NbCEA5, humanized nanobody scaffold, and humanized CEA5 graft) were radiolabeled with 99mTc using tricarbonyl chemistry for noninvasive in vivo single-photon emission computed tomography (SPECT) imaging of CEA expression in tumors in mice.



NbCEA5, humanized nanobody scaffold, and humanized CEA5 graft were produced as hexahistidine-tagged proteins in Escherichia coli (14). A solution of [99mTc(H2O)3(CO)3]+ and nanobodies was incubated for 90 min at 52°C. 99mTc-Labeled nanobodies were isolated with column chromatography, with a radiochemical purity of >99%. The specific activities, radiochemical yields, and stability of the 99mTc-labeled nanobodies were not reported.

In Vitro Studies: Testing in Cells and Tissues


Flow cytometry analysis showed that NbCEA5 and humanized CEA5 graft bound efficiently to CEA-expressing CHO and human colon adenocarcinoma LS174T cells but not to CEA-negative CHO cells (14). Humanized nanobody scaffold exhibited no binding to either CHO cell types. Binding experiments with the use of a Biacore sensor chip immobilized with CEA protein showed that the Kd values of NbCEA5, humanized nanobody scaffold, and humanized CEA5 graft were 0.34 nM, >10 µM, and 9.88 nM, respectively. 99mTc-NbCEA5 and 99mTc-humanized CEA5 graft exhibited efficient binding to both purified CEA protein and CEA-expressing CHO cells, whereas 99mTc-humanized nanobody scaffold exhibited little binding.

Animal Studies



Vaneycken et al. (14) performed SPECT imaging analysis in nude mice (n = 6) bearing LS174T tumors at 1 h after injection of 45–155 MBq (1.2–4.2 mCi, 0.17 nmol) 99mTc-NbCEA5, 99mTc- humanized CEA5 graft, or 99mTc-humanized nanobody scaffold. Intense radioactivity levels were observed in the kidneys (>87% ID/cm3) and urinary bladder for all 99mTc-nanobodies, which were cleared equally quickly from the blood. Accumulation levels in the liver, lung, and muscle were low (<3.1% ID/cm3). Radioactivity accumulation levels in the tumors for 99mTc-NbCEA5, 99mTc-humanized nanobody scaffold, and 99mTc- humanized CEA5 graft were 7.09 ± 1.36%, 0.21 ± 0.05%, and 6.15 ± 2.33% ID/cm3. The tumor accumulation of 99mTc- humanized CEA5 graft was not significantly different from that of 99mTc-NbCEA5 (P = 0.40). The imaging studies were not confirmed with ex vivo biodistribution studies, and no blocking studies were 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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