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131I-Labeled anti-α folate receptor human antibody fragment AFRA-DMF5.3 dimer

, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: December 26, 2011.

Chemical name:131I-Labeled anti-α folate receptor human antibody fragment AFRA-DMF5.3 dimer
Abbreviated name:[131I]-AFRA-DFM5.3
Agent Category:Antibody
Target:α Folate receptor
Target Category:Receptor
Method of detection:Single-photon emission computed tomography (SPECT); gamma planar imaging
Source of signal / contrast:131I
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.



Folic acid (FA; also known as folate or vitamin B9) is a water-soluble vitamin that is essential for the synthesis and repair of cellular DNA. FA also acts as a cofactor for many biological reactions and has an important role in cell maintenance and proliferation. The folate molecule is transported into the cell by the folate receptor (FR; there are two types, α and β), which is a well-investigated member of the FR family. The FRα is a ~40-kDa molecule that is fastened to the cell membrane surface via a glycophosphatidylinositol linkage and facilitates the transport of folate through endocytosis (1). Most tissues have little to no expression of the FRα, but it is expressed at measurable levels in the epithelial cells of the lungs, kidneys, and choroid plexus of the brain. However, the malignant tumors of these tissues overexpress the FRα in order to meet the increased demand for FA, which is required to synthesize cellular DNA due to the increased rate of cell proliferation (1). Therefore, the FRα is perceived to be an excellent target for the imaging and therapy of the various cancers, including that of the ovaries (2).

Most cancers tend to metastasize and are often treated with antibody (Ab)-based therapies (such as immunotherapy and/or radioimmunotherapy) that target a specific receptor (see Table 1 in Julien et al. (3) for details), but the main limitation of this therapeutic approach is that labeled Abs are not able to penetrate deep into solid tumors due to their large size and charge characteristics (4). In contrast, ovarian cancer cells are found mainly in the peritoneal cavity, and the disease is usually metastasized within the abdominal cavity (5). Studies have shown that intraperitoneal immuno- or radioimmunotherapy directed against the FRα can be used to treat this ailment, but these therapies are not considered mainstream treatments for this cancer. This is because the 131I used to label the Abs for radioimmunotherapy has a half-life of ~8 days, creates a radiation safety challenge to family and caretakers because of the high energy photons and the patients can develop human anti-mouse Abs, both with immuno- or radioimmunotherapy, thereby limiting the use of this therapeutic regimen (3, 6).

Recently, a completely human Fab fragment directed against the FRα, designated C4, was prepared by using a phage display library followed by an epitope imprinting selection. C4 was shown to have the same binding specificity as the parent Ab (7). It was hypothesized that a chemical dimer of the C4 fragment generated by molecular and chemical transformation of the Fab would be more suitable for the treatment of ovarian cancer because it would have a small size and would easily penetrate tumors (7). The Fab dimer, designated AFRA-DFM5.3, was prepared, labeled with 131I, and shown to accumulate primarily in the subcutaneous FR-expressing tumors in athymic mice. In another study, the label from [131I]-AFRA-DFM5.3 was reported to rapidly accumulate in free-floating clumps of ovarian cancer cells and solid tumors in the peritoneal cavity (8).



The expression and purification of the AFRA Fab fragment from Escherichia coli and its chemical dimerization to obtain AFRA-DFM5.3 has been described by Figini et al. (7). The dimerized Fab fragment was labeled with 131I using a commercially available kit (with Iodogen-precoated iodination tubes), and the radiolabeling efficiency of the reaction was 86 ± 1.5% (n = 4 experiments). The labeled dimer was purified on a PD-10 desalting column and formulated in 100 mM sodium phosphate (pH 7.4), 150 mM NaCl, and 2% human serum albumin. The formulated preparation was filtered through a 0.22-μm membrane and evaluated for sterility and pyrogenicity. The radiochemical yield and radiochemical purity of the final preparation were not reported. The specific activity of the final radiolabeled product was 196 ± 25.9 MBq/mg (5.3 ± 0.7 mCi/mg; n = 4 experiments).

In Vitro Studies: Testing in Cells and Tissues


[131I]-AFRA-DFM5.3 was reported to have an immunoreactivity of 68 ± 5.2% (n = 4 experiments), as determined with A431FR (A431 cells transfected with the FR) and OVCAR3 cell-binding assays (7).

The labeled dimeric Fab was stable in buffer or fresh human serum for at least 24 h at 4°C or 37°C, respectively, as determined with direct binding on fixed cells and SDS-PAGE, respectively (7).

Using A431FR cells, the IC50 of non-radioactive AFRA-DFM5.3 was determined to be 15.3 nM (7).

Animal Studies



The biodistribution of [131I]-AFRA-DFM5.3 was studied in nu/nu mice bearing subcutaneous tumors of A431FR cells (7). Control mice bore A431 cell tumors. The animals (n = 4 mice/time point) were given an intravenous injection of 1–1.5 MBq/mouse (27–40.5 μCi/mouse) 131I-AFRA-DFM5.3 and euthanized at various time points varying from 1 h to 24 h postinjection (p.i.) to determine the amount of label accumulated in the tumors and the major organs. Data obtained from the study were presented as percent of injected dose per gram tissue (% ID/g).

Table: Uptake of Radioactivity from [131I]-AFRA-DFM5.3 in Tumors and Select Organs from A431 Cell (Control) or A431FR Cell Tumor-Bearing Mice. Data Obtained from Figini et al. (7).

Control Mice (A431 Cell Tumors)A431FR Tumor-Bearing Mice
Time post-injection (hours)
Blood (B)*7.27 ± 1.883.41 ± 1.421.53 ±
0.13 ±
12.37 ± 3.175.11 ±
2.10 ±
0.20 ±
Tumor (T)3.77 ±
2.84 ± 1.041.89 ±
0.18 ±
5.35 ±
6.29 ±
4.56 ±
1.77 ±
Kidney30.67 ± 4.944.01 ± 1.012.18 ±
0.32 ±
30.95 ± 4.6812.69 ± 9.853.54 ±
0.46 ±
Liver3.43 ±
1.44 ± 0.640.72 ±
0.13 ±
6.31 ±
3.07 ±
3.54 ±
0.23 ±
Muscle1.03 ±
0.69 ± 0.370.36 ±
0.04 ±
1.08 ±
0.93 ±
0.39 ±
0.05 ±
T/B Ratio0.520.831.231.410.431.232.179.09

* Data presented as % ID/g.

From the above Table, it is evident that radioactivity from [131I]-AFRA-DFM5.3 was cleared rapidly through the urinary route of the animals. The tumor/blood radioactivity (T/B) ratio in both groups of animals increased with time, but at 6 h p.i. and 24 h p.i. the ratios in the A431FR tumor-bearing mice were approximately two-fold or six-fold higher, respectively, compared with the ratios in the control animals. No blocking studies were reported.

In another study, the uptake of radioactivity from [131I]-AFRA-DFM5.3 was evaluated after intraperitoneal injection of the labeled Ab dimer in nude mice bearing two different types of intraperitoneal ovarian carcinoma xenografts (from A431FR and A431MK cells, respectively) (8). Animals bearing the A431MK cell tumors served as controls for this study. Mice (n = 3–4 animals) were injected with the radiolabeled Ab dimer and euthanized at different time points (1 h p.i. to 15 h p.i.) as detailed above to obtain the various organs and the acsites fluid. The circulating half-life of [131I]-AFRA-DFM5.3 was longer than that of a low or non-binding control Ab ([131I]-AFRA-DFM6.1; circulation times were 4.4 ± 0.6 h versus 3.3 ± 0.2 h, respectively). With [131I]-AFRA-DFM5.3, rapid and high accumulation of radioactivity was observed in the tumor cells present in the ascites at 1 h p.i., but the solid tumors showed accumulation only after 3 h p.i. A significant difference (P < 0.0001) in the accumulation of label from [131I]-AFRA-DFM5.3 was apparent between the tumors of mice that developed only solid tumors and those that developed the solid tumors and/or the ascites. In animals with the ascites, the floating tumor cells showed a high uptake of the tracer compared to a very low uptake of radioactivity from the control labeled Ab (25.11 ± 6.94% ID/g versus 0.23 ± 0.05% ID/g, respectively). In addition, although [131I]-AFRA-DFM5.3 had a longer circulating half-life compared with the control Ab, both labeled Abs showed a similar pattern of uptake in the normal tissues or organs. No blocking studies were reported.

The efficacy of an intraperitoneal injection of [131I]-AFRA-DFM5.3 was evaluated for the locoregional radioimmunotherapy of animals (n = 4–8 mice) showing intraperitoneal IGROV-1 or IVCAR3 cell growth (8). All animals given the treatment had a statistically longer survival time (P = 0.0347 to <0.0001) compared to the control animals. Similar results were obtained with a single treatment of [131I]-AFRA-DFM5.3 independent of the dose used (37 MBq or 55 MBq/mouse (1 mCi or 1.5 mCi/mouse)). A necropsy of all animals that survived showed that the mice were tumor-free compared with control animals, which showed the presence of both the solid tumors and the ascites.

From these studies, the investigators concluded that [131I]-AFRA-DFM5.3 was suitable for the locoregional treatment of ovarian cancer in mice.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

Supplemental Information


No information is currently available.


Basal E., Eghbali-Fatourechi G.Z., Kalli K.R., Hartmann L.C., Goodman K.M., Goode E.L., Kamen B.A., Low P.S., Knutson K.L. Functional folate receptor alpha is elevated in the blood of ovarian cancer patients. PLoS One. 2009;4(7):e6292. [PMC free article: PMC2707611] [PubMed: 19617914]
van Dam G.M., Themelis G., Crane L.M., Harlaar N.J., Pleijhuis R.G., Kelder W., Sarantopoulos A., de Jong J.S., Arts H.J., van der Zee A.G., Bart J., Low P.S., Ntziachristos V. Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: first in-human results. Nat Med. 2011;17(10):1315–9. [PubMed: 21926976]
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Figini M., Martin F., Ferri R., Luison E., Ripamonti E., Zacchetti A., Mortarino M., Di Cioccio V., Maurizi G., Allegretti M., Canevari S. Conversion of murine antibodies to human antibodies and their optimization for ovarian cancer therapy targeted to the folate receptor. Cancer Immunol Immunother. 2009;58(4):531–46. [PubMed: 18704410]
Zacchetti A., Martin F., Luison E., Coliva A., Bombardieri E., Allegretti M., Figini M., Canevari S. Antitumor Effects of a Human Dimeric Antibody Fragment 131I-AFRA-DFM5.3 in a Mouse Model for Ovarian Cancer. J Nucl Med. 2011;52(12):1938–46. [PubMed: 22068897]


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