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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/64Cu-Labeled anti-CD20 minibody

[124I/64Cu]Anti-CD20 ScFv-CH3 dimer
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894

Created: ; Last Update: November 24, 2009.

Chemical name:[124I/64Cu]Anti-CD20 minibody
Abbreviated name:[124I/64Cu]Anti-CD20 ScFv-CH3 dimer
Synonym:
Agent Category:Antibody
Target:CD-20
Target Category:Antigen
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:124I/64Cu
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide and gene for information regarding human CD20.

Background

[PubMed]

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 modified scFv-Fc fragment (~105 kDa in size), 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 minibody ([124I]-scFv-CH3) are described and compared with those of a 64Cu-labeled scFv-CH3 minibody ([64Cu]-scFv-CH3) used under the same experimental conditions. The biodistribution and imaging characteristics of the 124I-labeled scFv-Fc fragment ([124I]-scFv-Fc) are described and compared with those of [124I]-scFv-CH3 in a separate chapter of MICAD (www.micad.nih.gov) (5).

Synthesis

[PubMed]

The production, purification, and labeling of scFv-CH3 with 124I were done as described by Olafsen et al. (3). The efficiency of the minibody labeling reaction was reported to be 80.9% as determined with an instant thin-layer chromatography (ITLC) kit. The specific activity of [124I]-scFv-CH3 was reported to be 0.069 MBq/μg (1.86 μCi/μg). The stability and storage conditions used for [124I]-scFv-CH3 were not reported.

The minibody was also labeled with 64Cu as described by Olafsen et al. (3). The labeling efficiency of the reaction was reported to be 65.4% as determined with an ITLC kit. The specific activity of [64Cu]-scFv-CH3 was reported to be 0.042 MBq/μg (1.13 μCi/μg). The stability and storage conditions used for [64Cu]-scFv-CH3 were not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The binding of scFv-CH3 to CD20 was confirmed using 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 immunoreactivity of [124I]-scFv-CH3 was determined by exposing an excess number of 38C13-huCD20 cells (exact cell number not reported) as described by Olafsen et al. (3). Immunoreactivity of the radiolabeled minibody was reported to be 37.7%. Although immunoreactivity of the radioiodinated minibody was low, the investigators showed that [124I]-scFv-CH3 could easily target CD20-expressing tumors under in vivo conditions (see below). The immunoreactivity of [64Cu]-scFv-CH3 was not reported.

Animal Studies

Rodents

[PubMed]

The biodistribution and tumor targeting of [124I]-scFv-CH3 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 minibody and euthanized 4 h and 21 h later 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 12.9 ± 3.4% ID/g compared with the significantly lower (P = 0.003) accumulation of label in the CD20-nonexpressing tumors (1.9 ± 0.5% ID/g). Each of the major organs (liver, spleen, kidneys, and the lungs) were also reported to have a significantly lower (P = 0.05) uptake (<2.0% ID/g) compared with the CD20-positive tumors. The radioactivity uptake ratio of the CD20-positive/CD20-negative tumors was 7.0 ± 3.1, and the CD20-positive tumor/blood ratio was 4.7 ± 1.4 at 21 h after injection of the label. No blocking studies were reported.

Whole-body imaging of the animals was performed at 4 h and 21 h after injection of the radioiodinated minibody (3). An analysis of the images revealed that only the CD20-positive tumors had accumulated radioactivity 21 h after treatment with the labeled minibody. A region of interest (ROI) analysis of the images revealed that the CD20-positive/CD20-negative tumor ratios were 2.2 ± 0.8 at 4 h and 4.0 ± 0.7 at 21 h. In addition, the CD20-positive tumor/soft tissue ratio was observed to increase from 4.6 ± 0.4 at 4 h to 17.0 ± 6.7 at 21 h. No blocking studies were reported.

The biodistribution of [64Cu]-scFv-CH3 was also studied in mice bearing 38C13-huCD20 (n = 2 animals) or 38C13 (n = 3 animals) cell tumors for comparison with the biodistribution of [124I]-scFv-CH3 (3). The average uptake of radioactivity by the 38C13-huCD20 and 38C13 cell tumors with the 64Cu-labeled minibody at 19 h after the treatment was reported to be 6.5 ± 3.8% ID/g and 4.7 ± 1.7% ID/g, respectively, generating a ratio of 1.4 between the radioactivity accumulated in the 38C13-huCD20 and 38C13 cell tumors (the same ratio with the radioiodinated minibody was 7.0 at 21 h after the treatment). No blocking studies with [64Cu]scFv-CH3 were reported. An ROI analysis of images taken at 4 h and 19 h after treatment with the 64Cu-labeled minibody revealed that the CD20-positive/CD20-negative tumor ratios were 2.3 and 1.9, respectively, at these time points. These results suggested that, because of a high background, the use of [64Cu]-scFv-CH3 was less favorable than [124I]-scFv-CH3 for imaging purposes (3).

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

Supplemental Information

[Disclaimers]

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.

References

1.
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]
2.
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]
3.
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]
4.
Wu A.M., Olafsen T. Antibodies for molecular imaging of cancer. Cancer J. 2008;14(3):191–7. [PubMed: 18536559]
5.
Chopra, A., [124I] Labeled anti-CD20 modified scFv-Fc fragment [[124I]Anti-CD20 modified scFv-Fc fragment]. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -to current.
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