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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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124I-Labeled anti-CD20 scFv-sc fragment

[124I]Anti-CD20 scFv-sc DM
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894

Created: ; Last Update: November 24, 2009.

Chemical name:124I-Labeled anti-CD20 scFV-sc fragment
Abbreviated name:[124I]Anti-CD20 ScFv-sc DM
Agent Category:Antibody
Target Category:Antigen
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:124I
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide and gene for information regarding human CD20.



The B cell differentiation antigen (CD20) is a transmembrane, non-glycosylated, hydrophobic protein that is characteristically expressed in more than 90% of non-Hodgkin lymphoma (NHL) tumors. Although the exact cellular functions of CD20 are not known, it is believed to play a role in B cell growth, activation, and maintenance of cellular calcium homeostasis (1). Because of its specific presence in the NHL tumor cells, CD20 is targeted with the antibody (Ab) retuximab, which has been approved by the United States Food and Drug Administration (FDA) for use in monotherapy or in combination with a chemotherapeutic agent for the treatment of this disease (2). For enhanced efficacy, radionuclide-coupled anti-CD20 Abs tositumomab (131I labeled) or ibritumomab (90Y conjugated) were also respectively approved by the FDA for the treatment of NHL and are commercially available in the United States. In addition, several anti-CD20 Abs, alone or in combination with different chemotherapeutic agents, are under evaluation for the treatment of various cancers in clinical trials approved by the FDA. In addition, Abs labeled with different nuclides have been used to detect and monitor cancers using imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography, but, due to long circulation and clearance times of the radiolabeled macromolecules (~150 kDa in size), investigators must wait for awhile before target/background ratios were reduced to suitable levels for imaging purposes (3, 4).

To circumvent the imaging problems encountered with intact radiolabeled Abs, two recombinant retuximab fragments, scFv-CH3 (a minibody; ~80 kDa in size) and a double mutant (DM) scFv-Fc fragment (~105 kDa in size, for more information see (4)), were generated, labeled with 124I, and evaluated by Olafsen et al. for in vivo imaging of human CD20-expressing lymphomas in mice (3). In this chapter, the biodistribution and imaging characteristics of the 124I-labeled scFv-sc fragment ([124I]-scFv-sc DM) are described and compared with those of a 124I-labeled scFv-CH3 minibody ([124I]-scFv-CH3) used under the same experimental conditions. The biodistribution and imaging characteristics of [124I]-scFv-CH3 are described and compared with those of 64Cu-labeled scFv-CH3 in a separate chapter of MICAD ( (5).



The production, purification, and labeling of scFv-sc DM and scFv-CH3 with 124I were described by Olafsen et al. (3). The efficiency of radioiodination of scFv-sc DM and scFv-CH3 was reported to be 84.9% and 80.9%, respectively, as determined with an instant thin-layer chromatography kit. The specific activities of [124I]-scFv-sc DM and [124I]-scFv-CH3 were reported to be 0.066 MBq/μg (1.78 μCi/μg) and 0.069 MBq/μg (1.86 μCi/μg), respectively. The stability and storage conditions used for [124I]-scFv-sc DM and [124I]-scFv-CH3 were not reported.

In Vitro Studies: Testing in Cells and Tissues


The binding of scFv-sc DM to CD20 was confirmed with an indirect immunofluorescence cell-surface staining method using 38C13-huCD20 cells (murine B cell lymphoma 38C13 cells transfected with and expressing the human CD20 gene), as detailed by Olafsen et al. (3) For this study, retuximab was used as a positive control. The binding characteristics of scFv-sc DM were reported to be the same as retuximab.

The immunoreactivity of [124I]-scFv-sc DM was determined by exposing an excess number of 38C13-huCD20 cells (the exact number of cells used for the assay was not reported) as described by Olafsen et al. (3). Immunoreactivity of the radiolabeled minibody was reported to be 32.8% compared with 37.7% reported for [124I]-scFv-CH3. Although immunoreactivity of the radioiodinated Ab fragment was low, the investigators showed that [124I]-scFv-sc could easily target CD20-expressing tumors under in vivo conditions (see below).

Animal Studies



The biodistribution and tumor targeting of [124I]-scFv-sc DM were investigated in mice bearing either 38C13-huCD20 (expressing CD20) or 38C13 (not expressing CD20) cell tumors (n = 4 animals/cell type) (3). The animals were intravenously injected with the radiolabeled Ab fragment and euthanized 4 h and 21 h later (by 21 h no background was apparent in the tumors and organs and only the CD20 positive tumors showed a strong signal) to study biodistribution of the tracer by retrieving the tumors and other major organs. Accumulated radioactivity in the different tissue types was presented as decay-corrected percent injected dose per gram tissue (% ID/g). The average uptake of radioactivity in the CD20-positive tumors at 21 h after the treatment was reported to be 6.0 ± 2.0% ID/g compared with the significantly lower (P = 0.007) accumulation of label in the CD20-nonexpressing tumors (1.6 ± 0.6% ID/g). With [124I]-scFv-CH3, the average uptake of label at 21 h after the treatment was reported to be 12.9 ± 3.4% ID/g in the CD20-positive tumors, and a significantly lower (P = 0.003) amount of label accumulated in the CD20-nonexpressing tumors (1.9 ± 0.5% ID/g). Compared with the CD20-positive tumors, a significantly lower (P = 0.05) uptake of radioactivity (<2.0% ID/g) was observed in the liver, spleen, kidneys, and the lungs. A similar observation was made with the radioiodinated minibody. With [124I]-scFv-sc DM, the radioactivity uptake ratio of the CD20-positive/CD20-negative tumors was reported to be 3.9 ± 0.7 (this ratio was 7.0 ± 3.1 with the minibody), and the CD20-positive tumor/blood ratio was 3.1 ± 1.2 (this ratio was 4.7 ± 1.4 with [124I]-scFv-CH3) at 21 h after injection of the label. No blocking studies were reported with either the labeled Ab fragment or the minibody.

Whole-body imaging of the animals was performed at 4 h and 21 h after injection of the radioiodinated scFv-sc DM (3). On the basis of a region of interest (ROI) analysis of the images, only the CD20-positive tumors were reported to have accumulated radioactivity with the labeled Ab fragment at 21 h after treatment. The ROI analysis of the images also revealed that the CD20-positive/CD20-negative tumor ratios with [124I]-scFv-sc DM were 1.2 ± 0.2 at 4 h and 2.4 ± 0.8 at 21 h. With [124I]-scFv-CH3, these ratios were 2.2 ± 0.8 and 4.0 ± 0.7 at the same time points, indicating that the uptake of label by the CD20-positive tumors was higher with the minibody than with the Ab fragments. With [124I]-scFv-sc DM, the CD20-positive tumor/soft tissue ratio increased from 4.3 ± 1.4 at 4 h to 9.9 ± 4.6 at 21 h, and these ratios followed a similar but higher pattern with [124I]-scFv-CH3 (4.6 ± 0.4 and 17.0 ± 6.7 at 4 h and 21 h, respectively). No blocking studies were reported with either the labeled Ab fragment or the minibody.

With results obtained from these studies, the investigators concluded that the labeled anti-CD20 Ab fragments and the labeled minibody could be used for in vivo imaging in rodents, although the labeled minibody yielded images with a better contrast than the Ab fragments (3).

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


No supplemental information is currently available.

NIH Support

Studies reported in this chapter were supported by National Institutes of Health grants P50 CA107399, P50 CA086306, and CA119367 and a National Cancer Institute grant R24 CA86307.


Cragg M.S., Walshe C.A., Ivanov A.O., Glennie M.J. The biology of CD20 and its potential as a target for mAb therapy. Curr Dir Autoimmun. 2005;8:140–74. [PubMed: 15564720]
Bonavida B. Rituximab-induced inhibition of antiapoptotic cell survival pathways: implications in chemo/immunoresistance, rituximab unresponsiveness, prognostic and novel therapeutic interventions. Oncogene. 2007;26(25):3629–36. [PubMed: 17530016]
Olafsen T., Betting D., Kenanova V.E., Salazar F.B., Clarke P., Said J., Raubitschek A.A., Timmerman J.M., Wu A.M. Recombinant anti-CD20 antibody fragments for small-animal PET imaging of B-cell lymphomas. J Nucl Med. 2009;50(9):1500–8. [PMC free article: PMC2852538] [PubMed: 19690034]
Wu A.M., Olafsen T. Antibodies for molecular imaging of cancer. Cancer J. 2008;14(3):191–7. [PubMed: 18536559]
Chopra, A., [124I/64Cu] Labeled anti-CD20scFv-CH3 minibody [[124I/64Cu]Anti-CD20scFv-CH3]. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​, 2004 -to current.
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