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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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111In-Labeled anti-CD44v6 chimeric monoclonal antibody U36

111In-cMAb U36
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894, vog.hin.mln.ibcn@dacim

Created: ; Last Update: August 14, 2008.

Chemical name:111In-Labeled anti-CD44v6 chimeric monoclonal antibody U36
Abbreviated name:111In-cMAb U36
Agent Category:Chimeric monoclonal antibody
Target:Adhesion molecule CD44v6
Target Category:Antibody-antigen binding
Method of detection:Single-photon emission computed tomography; planar gamma imaging
Source of Signal/Contrast:111In
  • Checkbox In vitro
  • Checkbox Rodents
Click here for protein and nucleotide information on CD44.



CD44 is a member of the family of cell adhesion molecules that is known to promote cell migration and survival during cancer metastasis (1). The mouse CD44 gene contains ~20 exons, of which 10 are variants that are alternatively spliced for incorporation into the molecule to yield several CD44 variants (CD44v). The different CD44v forms have been reviewed elsewhere (2). The various CD44v molecules are involved in modulating a variety of cellular processes including cell migration and localization in the diverse organs, thereby promoting cancer metastasis (3). Among these variants, the variant containing domain 6 of the exons (CD44v6) has been specifically implicated in tumorigenesis and tumor cell migration (4). In normal tissue CD44v6 is expressed in a subset of epithelial cells, and it is found in most squamous cell carcinomas and some adenocarcinomas, but CD44v6 is seldom observed in tumors that have a nonepithelial origin (4). Also, CD44v6 was reported to have higher expression compared with the epidermal growth factor receptor in samples obtained from head and neck squamous cell carcinoma (HNSCC) tumors of patients (5).

Because of its high expression in HNSCC, CD44v6 has been targeted with a monoclonal antibody (MAb), designated U36, for the diagnosis of this cancer in humans (6, 7). The biodistribution of U36 was investigated after being labeled with radioactive technetium, and it was reported that the labeled MAb could detect all primary tumors of HNSCC, but the micro-tumors or tumor nodes with necrosis, keratin, or fibrin were not visible by imaging (7). This MAb was also evaluated in two clinical trials for the treatment of HNSCC after labeling with radioactive rhenium (186Re), but the 186Re dose was not sufficient to kill the small tumors or the decimated single cells from this cancer (8). U36 was also labeled with radioactive astatine (211At) and iodine (131I) and evaluated for the in vitro treatment of HNSCC cells; the 211At-conjugated MAb was shown to be more lethal to the HNSCC cells than the 131I-labeled homolog (9). In another study, the 211At-labeled MAb was shown to decrease or stabilize the volume of xenograft tumors in nude mice (10). U36 labeled with 124I or 131I has also been evaluated for the detection of xenograft tumors in nude mice, and the selective uptake of both labels by the tumors was confirmed with positron emission tomography (11, 12).

The U36 MAb was recently labeled with radioactive indium (111In), and its in vitro characteristics were investigated with the use of SCC-9 cells derived from HNSCC (13). The use of 111In to label the cMAb was considered because of its longer intracellular retention probably due to the formation of charged catabolites that do not pass through the membrane easily. The biodistribution of the 111In-labeled chimeric MAb (cMAb) was also studied in nude mice bearing xenograft SCC-9 cell tumors (14). Imaging with the mice was also performed with single-photon emission computed tomography 72 h after administration of the 111In-labeled cMAb (14).



The hybridomas that were used to produce the U36 MAb were developed as described by Schrijvers et al. (15). A chimeric version of the MAb (cMAb U36) was prepared as described by Brakenhoff et al. (16). The cMAb U36 was obtained in citrate buffer and separated with size-exclusion chromatography (SEC) on a NAP-5 column. It was then lyophilized and stored at -20°C until required (13).

Sandstrom et al. labeled cMAb U36 with 111In with the use of N-[(R)-2-amino-3-(p-isothiocyanato-pentyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N’,N”,N”-pentaacetic acid (CHX-A”-DTPA) as a chelator (14). The cMAb was mixed with CHX-A”-DTPA in a molar ratio of 5:1 in borate buffer (pH 9.15), and the reaction was allowed to occur for 2 h at 37°C. The conjugated cMAb was then separated from unreacted CHX-A”-DTPA with SEC on a NAP-5 column in acetate buffer (pH 5.5). For labeling, the conjugated cMAb was mixed with 111In, and the mixture was incubated at room temperature for 30 min. The labeled cMAb (111In-cMAb U36) was separated from the reaction components with SEC as described above on a column equilibrated with phosphate-buffered saline. The labeling yield was reported to be 70% to 80%. The specific activity and stability of the labeled cMAb was not reported by the investigators (14).

In Vitro Studies: Testing in Cells and Tissues


The in vitro receptor binding of 111In-cMAb U36 was characterized by using SCC-9 cells (13). The uptake and retention of 111In-cMAb U36 was also studied in the SCC-9 cells at 4° and 37°, C respectively. The dissociation constant for 111In-cMAb U36 was determined to be 1.3 ± 0.3 × 10-8 M. Cellular uptake of the labeled cMAb was observed to plateau at ~4 h after exposure at both 4° and 37°, C respectively. In the presence of unlabeled cMAb, the uptake was minimal and remained constant during this period. To determine cellular retention of the labeled cMAb, the cells were first exposed to the labeled cMAb for 8 h and then washed (wash buffer was not specified) to remove any unbound radioactivity. Complete growth medium was then added to the cells, and the amount of radioactivity retained by the cells was monitored for 16 h. The cells were observed to lose the radioactivity gradually, and ~40% of the initial radioactivity was retained after 8 h of incubation, indicating that the labeled cMAb was internalized by the cells.

Animal Studies



The biodistribution of 111In-cMAb U36 was studied in nude mice bearing xenograft tumors of SCC-9 cells (14). The animals were injected with the labeled cMAb through the tail vein and killed at 6, 24, 48, 72, and 168 h after injection (n = 3 animals/time point). The various organs were harvested, and the incorporated radioactivity was measured. At 48 h after administration, the highest uptake of radioactivity was observed in the liver (13.9 ± 1.47% injected dose/g tissue (% ID/g)), followed by the spleen (10.52 ± 2.81% ID/g), lungs (7.12 ± 4.29% ID/g), skin (5.85 ± 0.64% ID/g), and heart (4.72 ± 0.67% ID/g). The kidneys had the maximum uptake (7.70 ± 0.22% ID/g) at 24 h after injection. The radioactivity decreased in the organs from the peak as a function of time. The tumors had a five-fold higher accumulation of the label compared with blood (54.7 ± 16.6 versus 10.91 ± 1.06) at 168 h after injection of the labeled cMAb. The tumor/blood ratio increased from 0.24 ± 0.10 at 6 h to 4.45 ± 1.35 at 168 h after injection of the radioactivity.

For scintigraphic imaging, two mice bearing SCC-9 xenograft tumors were injected with 111In-cMAb U36, and images were collected after 72 h. From the images, the uptake values were determined to be 7.9% ID/g (mouse 1; tumor weight, 230 mg) and 11.0% ID/g (mouse 2; tumor weight, 60 mg), respectively.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.

Supplemental Information



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Ponta H., Sherman L., Herrlich P.A. CD44: from adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol. 2003;4(1):33–45. [PubMed: 12511867]
Wallach-Dayan S.B., Rubinstein A.M., Hand C., Breuer R., Naor D. DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis. Mol Cancer Ther. 2008;7(6):1615–23. [PubMed: 18566232]
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