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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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64Cu-Labeled anti-c-kit monoclonal antibody 12A8 Fab fragment

[64Cu]12A8 Fab
, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: June 30, 2011.

Chemical name:64Cu-Labeled anti-c-kit monoclonal antibody 12A8 Fab fragment
Abbreviated name:[64Cu]12A8 Fab
Synonym:
Agent Category:Antibody fragment
Target:Proto-oncogene c-Kit; tyrosine-protein kinase kit; mast/stem cell growth factor receptor; CD117
Target Category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:64Cu
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.

Background

[PubMed]

The c-kit (CD 117 antigen or stem cell factor ligand receptor) is a 145-kDa type III glycoprotein receptor tyrosine kinase (TK; encoded by the KIT proto-oncogene) that has a TK at the juxtamembrane position, which is activated when a ligand binds to the extracellular ligand-binding domain of the receptor (1). Almost 70% of gastrointestinal stromal tumors (GISTs) are known to overexpress a c-kit that has a mutation in exon 11 and produces a constitutively activated TK that promotes the survival and proliferation of cells and leads to the development of a malignant phenotype in the cells of the GISTs (1, 2). The biology and mutations of the c-kit gene and the detection, diagnosis, and chemotherapy of cancerous GISTs are discussed in detail elsewhere (3, 4). An immunohistochemical method is commonly used to detect the overexpression of c-kit in GIST cancers, but this invasive technique is known to generate variable results (3, 5). In addition, investigators often use positron emission tomography (PET) with 18F-fluoro-deoxyglucose to detect, monitor, and assess the response of GISTs to chemotherapy with imatinib, but this radiolabeled probe does not distinguish between GISTs and other cancerous tumors that have an epithelial or lymphatic origin (6).

Recently, the 111In-labeled anti–c-kit monoclonal antibody (mAb) 12A8 ([111In]12A8) was developed and evaluated for the detection of GISTs with single-photon emission computed tomography (SPECT) in nude mice bearing xenograft tumors that express c-kit (6). However, with [111In]12A8 the tumors were first visible on the animals at 48 h postinjection (p.i.) and became clearly visible only at 96 h p.i. This indicated that it was necessary to develop a radiolabeled immunoreagent that would have superior pharmacokinetic properties and yield faster results compared to the 111In-labeled mAb. It is well known that mAb fragments such as Fab (~50 kDa) and single-chain antibody fragments (~27 kDa) have superior in vivo pharmacokinetic properties compared to intact mAbs (~150 kDa) (7, 8). In an effort to achieve this goal, a Fab fragment of mAb 12A8 was labeled with 111In (to obtain [111In]12A8 Fab) and with 64Cu (to obtain [64Cu]12A8 Fab), and the two probes were evaluated for the detection of tumors that express c-kit in nude mice (2). This chapter describes PET studies performed with [64Cu]12A8 Fab. The detection of tumors that express c-kit with [111In]12A8 Fab is discussed in a separate chapter of MICAD (www.micad.nih.gov) (9).

Other Sources of Information

c-kit–Related chapters in MICAD

Human c-kit (also known as Hardy-Zuckerman 4 feline sarcoma viral oncogene-like protein (KIT)) protein and mRNA sequence (Source: NCBI GenBank)

Gene (human c-kit; Gene ID: 3815)

c-kit in Online Mendelian Inheritance in Man (OMIM)

Signaling events mediated by c-kit (Source: Pathways Interaction Database)

c-kit–Related clinical trials

Information on c-kit in PubChem Bioassay

Synthesis

[PubMed]

The source of the 12A8 Fab fragment, its conjugation with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and labeling with 64Cu have been described by Yoshida et al. (2). Using cellulose acetate electrophoresis, the DOTA:Fab ratio of this conjugate was estimated to be 1.1. The radiochemical yield and purity of [64Cu]12A8 Fab were reported to be 59.6% and 99.7% (n = 1 experiment), respectively, with a specific activity of 4.5 MBq/μmol (~122 μCi/μmol; n = 1 experiment).

For comparative studies, the 12A8 Fab fragment was also conjugated to N-[(R)-2-amino-3-(p-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (DTPA) and labeled with 111In as described elsewhere (2). The radiochemical yield and purity of [111In]12A8 Fab were reported to be 61.7 ± 15.9% and 98.4 ± 1.3%, respectively, with a specific activity of 1.2 ± 0.3 MBq/μmol (~32.4 ± 8.0 μCi/μmol; n = 3 experiments).

For use as a control, the IgG1 12A8 mAb was also conjugated to DTPA and labeled with 111In to obtain [111In]IgG as described above (2). Using cellulose acetate electrophoresis, the DTPA:mAb ratio of this conjugate was determined to be 1.1. The radiochemical yield and purity of [111In]IgG were reported to be 61.7 ± 15.9% and 98.4 ± 1.3%, respectively, with a specific activity of 3.4 ± 0.9 MBq/μmol (91.8 ± 24.3 μCi/μmol; n = 3 experiments).

For in vitro studies, the 12A8 Fab fragment was labeled with 125I using the chloramine-T method (2). The radiochemical yield and purity of [125I]12A8 Fab were reported to be 84.4% and 98.0%, respectively, with a specific activity of 37.5 MBq/μmol (~1 mCi/μmol; n = 1 experiment).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The in vitro cell binding characterization was performed only for [111In]12A8 IgG, [111In]12A8 Fab, and [125I]12A8 Fab using human small cell lung cancer cells (SY cells) that express high levels of c-kit (confirmed with Western blot analysis) (2). The cell binding specificities of [111In]IgG, [111In]12A8 Fab, and [125I]12A8 Fab were determined to be 50.2%, 37.1%, and 39.2%, respectively. The equilibrium dissociation constants of [111In]IgG, [111In]12A8 Fab, and [125I]12A8 Fab were reported to be 8.0, 18.4, and 18.5 nM, respectively, as determined with a competitive inhibition assay in the presence of increasing concentrations of the unlabeled mAb. The amounts of membrane-bound radioactivity from [111In]12A8 Fab and [125I]12A8 Fab were observed to decrease with time, but only cells exposed to [111In]12A8 Fab showed an increased internalization of the label that reached a maximum of ~15% after incubation for 20 h at 37°C. During the same time, the internalization of [125I]12A8 Fab decreased from ~15% at 1 h after incubation to ~8% at 20 h after incubation, and a corresponding increase in the amount of non–protein-bound radioactivity was observed in the growth medium. From this study, the investigators concluded that either free 125I or some labeled catabolites of [125I]12A8 Fab were released into the cell growth medium during the incubation, which indicated that the radioiodinated mAb fragment was not stable under these study conditions (2).

Animal Studies

Rodents

[PubMed]

The biodistribution patterns of [64Cu]12A8 Fab, [111In]12A8 IgG, and [111In]12A8 Fab were studied in nude mice bearing SY cell tumors and A4 cell tumors (NIH-3T3 cells that are transfected with the human epidermal growth factor receptor gene and do not express human c-kit) on the left and right thighs, respectively (2). To determine the amount of radioactivity that accumulated in the blood, tumor, and other major organs of the animals, the mice (n = 5 animals/group) were administered the 64Cu- or 111In-labeled tracer. Animals administered with [111In]IgG were euthanized at 24, 48, and 96 h p.i., and animals administered with [64Cu]12A8 Fab or [111In]12A8 Fab were euthanized at 1, 6, and 12 h p.i. Data obtained from these studies were standardized to a 20-g body weight mouse and presented as percent of injected dose per gram tissue (% ID/g).

The accumulation values of [64Cu]12A8 Fab in the SY tumors were 2.6 ± 0.7% ID/g, 6.1 ± 0.2% ID/g, and 6.2 ± 2.0% ID/g at 1, 6, and 12 h p.i., respectively, compared to values of 3.9 ± 0.5% ID/g, 4.8 ± 0.8% ID/g, and 2.8 ± 0.3% ID/g in the A4 tumors at the same time points (2). The uptake values of radioactivity in the SY tumors at 6 h and 12 h p.i. were significantly higher (P < 0.01) than those observed with [111In]12A8 Fab at the same time points (see below for details with [111In]12A8 Fab). In addition, the renal uptake of radioactivity from [64Cu]12A8 Fab was significantly lower (P < 0.01) than that observed with the 111In-labeled mAb fragment. The tumor/blood (T/B) ratios for the SY tumors were 0.2, 1.8, and 3.7 at 1, 6, and 12 h p.i. respectively. The T/B ratio at 12 h p.i. was observed to be higher with [111In]12A8 Fab than with [64Cu]12A8 Fab.

In another study, serial PET images were acquired from a mouse injected with [64Cu]12A8 Fab at 1, 6, and 15 h p.i (2). In these images, the SY tumors were not visible at 1 h p.i., but they were clearly visible at 6 h and 15 h p.i., indicating that the tracer was eliminated from the various organs, including blood, by 6 h p.i. Although the imaging data with [111In]12A8 Fab were not presented in the publication, the investigators reported that the PET images obtained with [64Cu]12A8 Fab at 6 h and 15 h p.i. were more clear than the SPECT images acquired with either [111In]12A8 IgG (6) or [111In]12A8 Fab (2).

The radioactivity uptake values for [111In]12A8 IgG in the A4 tumors were 6.0 ± 0.6% ID/g, 6.5 ± 0.7% ID/g, and 5.6 ± 0.8% ID/g at 24, 48, and 96 h p.i., respectively, compared to values of 15.5 ± 1.9% ID/g, 23.3 ± 2.3% ID/g, and 20.3 ± 1.6% ID/g for [111In]12A8 IgG in the SY tumors at the same time points. The T/B ratio for the SY tumors was 1.0 at 24 h p.i. and increased to 2.8 at 96 h p.i. All other organs showed a low uptake of the label that decreased gradually with time.

With [111In]12A8 Fab, the uptake values of the tracer in the A4 tumors were 2.7 ± 0.3% ID/g, 2.1 ± 0.3% ID/g, and 1.3 ± 0.2% ID/g at 1, 6, and 12 h p.i., respectively, compared to 2.7 ± 0.5% ID/g, 4.8 ± 0.8% ID/g, and 4.4 ± 1.2% ID/g at the same time points for the SY tumors. The T/B ratios for the SY tumors were 0.3, 3.3, and 9.7 at 1, 6, and 12 h, respectively. All other organs except the kidneys showed a decrease in radioactivity with time. The accumulation of tracer in the kidneys at 6 h p.i. was observed to be 52.5 ± 5.0% ID/g, and the accumulation decreased to 35.8 ± 4.2% ID/g at 12 h p.i., indicating that the radioactivity was excreted primarily through the renal route.

From these studies, the investigators concluded that both [64Cu]12A8 Fab and [111In]12A8 Fab were suitable to detect GISTs with PET and SPECT, respectively, in rodents (2). However, PET images generated with [64Cu]12A8 Fab were more clear than the SPECT images obtained with either [111In]12A8 IgG or [111In]12A8 Fab.

Other Non-Primate Mammals

[PubMed]

No publications are currently available.

Non-Human Primates

[PubMed]

No publications are currently available.

Human Studies

[PubMed]

No publications are currently available.

Supplemental Information

[Disclaimers]

No information is currently available.

References

1.
Aubin F., Blanke C.D. Metastatic gastrointestinal stromal tumors. Cancer Chemother Pharmacol. 2011;67 Suppl 1:S9–14. [PubMed: 21116628]
2.
Yoshida C., Tsuji A.B., Sudo H., Sugyo A., Sogawa C., Inubushi M., Uehara T., Fukumura T., Koizumi M., Arano Y., Saga T. Development of positron emission tomography probe of 64Cu-labeled anti-C-kit 12A8 Fab to measure protooncogene C-kit expression. Nucl Med Biol. 2011;38(3):331–7. [PubMed: 21492781]
3.
Corless C.L., Heinrich M.C. Molecular pathobiology of gastrointestinal stromal sarcomas. Annu Rev Pathol. 2008;3:557–86. [PubMed: 18039140]
4.
Wang W.L., Conley A., Reynoso D., Nolden L., Lazar A.J., George S., Trent J.C. Mechanisms of resistance to imatinib and sunitinib in gastrointestinal stromal tumor. Cancer Chemother Pharmacol. 2011;67 Suppl 1:S15–24. [PubMed: 21181476]
5.
Miettinen M., Lasota J. KIT (CD117): a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation. Appl Immunohistochem Mol Morphol. 2005;13(3):205–20. [PubMed: 16082245]
6.
Sogawa C., Tsuji A.B., Sudo H., Sugyo A., Yoshida C., Odaka K., Uehara T., Arano Y., Koizumi M., Saga T. C-kit-targeted imaging of gastrointestinal stromal tumor using radiolabeled anti-c-kit monoclonal antibody in a mouse tumor model. Nucl Med Biol. 2010;37(2):179–87. [PubMed: 20152717]
7.
Accardi L., Di Bonito P. Antibodies in single-chain format against tumour-associated antigens: present and future applications. Curr Med Chem. 2010;17(17):1730–55. [PubMed: 20345346]
8.
Marik J., Junutula J.R. Emerging role of immunoPET in receptor targeted cancer therapy. Curr Drug Deliv. 2011;8(1):70–8. [PubMed: 21034420]
9.
Chopra, A., 111In-Labeled DTPA conjugated anti-c-kit monoclonal antibody 12A8 Fab 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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