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[64Cu]-1,4,7,10-Tetra-azacyclododecane-N,N’,N’’,N’’’-tetraacetic acid conjugated knottin 2.5F

[64Cu] Knottin 2.5F
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894

Created: ; Last Update: May 28, 2009.

Chemical name:[64Cu]-1,4,7,10-Tetra-azacyclododecane-N,N’,N’’,N’’’-tetraacetic acid conjugated knottin 2.5F
Abbreviated name:[64Cu]Knottin 2.5F; 64Cu-DOTA-2.5F
Agent Category:Ligand
Target:αvβ3, αvβ5, and α5β1 integrin receptors
Target Category:Receptors
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:64Cu
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.



Integrin receptors that mediate tumor angiogenesis, growth, and metastasis through a complex network of signaling pathways are known to be expressed on the surface of cancerous tumor cells and neovasculature (1-3). Because of their role in tumor development and progression, inhibition of integrin receptor activity is being actively investigated in clinical trials for the treatment and imaging of various cancers (4, 5). Non-invasive imaging probes can be used to determine not only the efficacy of integrin-targeted anti-cancer drugs, but also to monitor disease progression and metastasis (6, 7). Although integrin receptors usually bind through the arginine-glycine-aspartic acid (RGD) motif of the extracellular matrix protein ligands, it is the amino acid residues surrounding the RGD motif that determine the receptor specificity and affinity for the ligand (4, 8, 9). As a result of the small size of the drugs, mainly peptides, that target the integrin receptor, it has been challenging to generate peptides containing the RGD motif that have an improved pharmacokinetic behavior, receptor affinity, and tumor uptake for in vivo imaging purposes. Any modification of the peptide structure has yielded integrin-targeted imaging agents that have only a limited advancement and application in the clinics (10, 11). Only one imaging compound with an RGD motif, a radioactive fluorine-labeled cyclic pentapeptide ([18F]-galacto-RGD), was determined to be suitable to identify integrin-positive tumors and to investigate αvβ3 integrin expression in humans. However, [18F]-galacto-RGD has been shown to have a low tumor uptake, and it generated a high background signal due to accumulation in the liver (12, 13).

In an effort to develop imaging agents for the detection of integrin receptors expressed on tumor cell surface and neovasculature, Kimura et al. (14) used the directed evolution technique (15, 16) to place the integrin-binding RGD motif into a cystine knot peptide (also known as knottin) trypsin inhibitor of the squash plant (Ecballium elaterium) (17). The peptide was reported to have a high affinity for the αvβ3 and αvβ5 or the αvβ3, αvβ5, and α5β1 integrin receptors. In general, knottins have a core structure containing a disulfide bond, are resistant to proteolysis, have a high thermodynamic stability, and are nonimmunogenic (14). The knottin peptide containing an RGD motif (designated as knottin 2.5F) was labeled with radioactive copper (64Cu) to obtain [64Cu]knottin 2.5F (64Cu-1,4,7,10-tetra-azacyclododecane-N,N’,N’’,N’’’-tetraacetic acid-2.5F (64Cu-DOTA-2.5F)) and used to image xenograft tumors in mice with positron emission tomography (PET). It is pertinent to mention here that the amino acid sequence and structure of knottins 2.5D and 2.5F (also studied by Kimura et. al. (14)) are discussed elsewhere (14).



The synthesis of knottin 2.5F was performed with the use of the 9-fluorenylmethylcarbonyl-based solid-phase technique as described by Kimura et al. (14). In addition, two other knottin peptides, FN-RGD2 (for use as positive a control) and FN-RDG2 (negative control) were synthesized using the same technique mentioned above. A third peptide, c(RGDyK), was obtained from commercial sources for use as a second control. The amino acid sequence, structure and properties of the various integrin-binding peptides used for studies described in this chapter are discussed by Kimura et al. (14).

To radiolabel knottin 2.5F, the peptide was mixed with molar excess of an in situ preparation of a sulfosuccinimide ester of DOTA for 1 h at room temperature and then overnight at 4°C (14). By this reaction the chelating agent was attached at the amino terminal of the folded knottin peptide for chelation of the radionuclide (64Cu). The final product, a knottin 2.5F-DOTA conjugate, was purified with reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column as described by Kimura et al. (14). After folding the conjugated peptide as detailed by Kimura et al. (14), the agent was purified with semi-preparative RP-HPLC and freeze-dried for storage at room temperature until required. The peptide purity and molecular mass were confirmed with analytical RP-HPLC and electrospray ionization mass spectrometery. To label with 64Cu, the knottin 2.5F-DOTA conjugate was incubated with 64CuCl2 in 0.1 N sodium acetate buffer (pH 6.3) for 1 h at 45°C. The reaction was terminated by the addition of ethylenediamine tetraacetic acid, and the radiolabeled product was purified with radio-HPLC on a PD-10 column. The purified product was dried, reconstituted in phosphate-buffered saline, and sterilized by filtration. For use as controls, 64Cu-labeled FN-RGD2, FN-RDG2, and c(RGDyK) were also respectively synthesized using the protocol described above. The radiochemical yield of the purified products was usually >80% (at least seven reactions/purifications were performed respectively on different days), and the purity was >95% as determined with HPLC. The specific activity of the various 64Cu-labeled peptides was not reported.

The stability of 64Cu-DOTA-2.5F was determined by exposing it to mouse serum for 1, 4, and 24 h at 37°C (14). Minimal degradation of the radiolabeled peptide was reported at all the time points as determined with HPLC.

Echistatin labeled with radioactive iodine (125I-echistatin), used a s a control in some studies, was obtained from a commercial source (14).

In Vitro Studies: Testing in Cells and Tissues


To determine the relative integrin-binding affinities (reported as 50% inhibitory concentration (IC50)) of the modified and unmodified peptides, including knottin 2.5F, a competition binding assay was performed with 125I-echistatin (a strong antagonist of αvβ3, αvβ5, α5β1 and αiibβ3 integrins (14)) using U-87MG cells (a human glioblastoma cell line that expresses several types of integrins on the cell surface) as described by Kimura et al. (14). The IC50 for DOTA-knottin 2.5F was determined to be 26 ± 5 nmol/L compared with an IC50 values of 590 ± 210 nmol/L and 380 ± 190 nmol/L for the control DOTA-conjugated FN-RGD2 and c(RGDyK) peptides, respectively. The negative control peptide, DOTA-FN-RDG2, did not compete with 125I-echistatin for binding to the U-87MG cells. The DOTA-conjugated peptides (modified) were reported to have IC50 values comparable to the unmodified (not conjugated to DOTA) peptides (14).

The integrin-binding specificity of the DOTA-conjugated knottin peptides was investigated with a competition assay using 125I-echistatin (14). Different concentrations (5 and 50 nmol/L) of the unlabeled DOTA-conjugated knottin peptides were respectively incubated with 125I-echistatin in microtiter plates coated with detergent-solubalized αvβ3, αvβ5, α5β1, and αiibβ3 integrin receptors for 3 h at room temperature. The plates were washed three times to remove unbound radioactivity, and the contents were solubalized in 2 N sodium hydroxide to determine the amount of receptor-bound radioactivity. Compared with the FN-RGD2 and c(RGDyK) peptides, the knottin 2.5F peptide exhibited a higher binding (for details see Figure 2A in reference 14) to the different integrin receptors except αiibβ3, which is expressed mainly on the platelet cells (14).

Animal Studies



The use of 64Cu-DOTA-2.5F as a PET imaging probe was investigated in mice bearing human U-87MG cell xenograft tumors (14). The animals (n = 3 or more for each probe) were injected with the respective 64Cu-DOTA-conjugated peptides through the tail vein and imaged 1 h later for up to 24 h. Calibration of the PET images for quantitation purposes was done as described by Wu et. al (18). At 1 h after administration of the labeled peptides, the tumor uptake of 64Cu-DOTA-2.5F was higher (4.56 ± 0.64% injected dose/gram tissue (% ID/g)) compared with the control 64Cu-DOTA-FN-RGD2 (1.48 ± 0.53% ID/g) and 64Cu-DOTA-c(RGDyK) (2.32 ± 0.55% ID/g) peptides. Also, compared with 64Cu-DOTA-FN-RGD2 (4.19 ± 0.78% ID/g and 3.59 ± 0.87% ID/g at 1 and 4 h after injection, respectively), a lower uptake of 64Cu-DOTA-2.5F (~2% ID/g) was observed in the liver. For the negative control peptide, 64Cu-DOTA-FN-RDG2, the tumor accumulation of radioactivity was reported to be 1.09 ± 0.48% ID/g and 0.76 ± 0.33% ID/g at 1 and 4 h after injection, respectively. No competition studies were reported.

For the in vivo biodistribution studies, animals bearing U-87MG cell xenograft tumors (at least three mice per time point) were injected with 64Cu-knottin 2.5F through the tail vein, and the amount or radioactivity accumulated in the major organs was determined after 0.5 and 4 h (14). At 0.5 h, varying amounts of radioactivity (<0.5–8.0% ID/g) was detected primarily in the tumor, blood, liver, lungs, kidneys, spleen, intestines, skin, and the stomach (for details see Figure 4C in reference 14). By 4 h after administration, the radioactivity was reduced to between <1% ID/g (liver, lung, spleen, intestines, skin, and stomach) and ~2% ID/g (liver and intestines) and ~4% ID/g (kidneys).

From these studies the investigators concluded that the direct evolution–engineered integrin-binding peptides had a good potential for the detection and diagnosis of various cancers (14).

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


NIH Support

  1. Some of the work presented in this chapter was supported by National Cancer Institute grants 5K01 CA104706, 5R25T CA118681, and R25T CA118681 and a National Institutes of Health grant P50 CA114747.


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