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. Author manuscript; available in PMC: 2016 May 5.
Published in final edited form as: Nucl Med Biol. 2012 Nov 12;40(2):190–196. doi: 10.1016/j.nucmedbio.2012.10.003

Initial characterization of a dually radiolabeled peptide for simultaneous monitoring of protein targets and enzymatic activity

Efrem Mebrahtu 1, Alexander Zheleznyak 1, Minjun A Hur 1, Richard Laforest 1, Suzanne E Lapi 1,*
PMCID: PMC4857598  NIHMSID: NIHMS781543  PMID: 23154178

Abstract

Objective

The goal of this study was to develop dually radiolabeled peptides for simultaneous imaging of cancer cell localization by targeting the αvβ3 integrin and their pathophysiology by targeting the activity of the proteolytic enzyme MMP2, involved in the metastatic process.

Methods

A hybrid peptide c(RGDfE)K(DOTA)PLGVRY containing a RGD motif for binding to the αvβ3 integrin, a metal chelator (DOTA) for radiolabeling with [64Cu], and the MMP2 substrate cleavage sequence PLGVRY with terminal tyrosine for labeling with [123I] was synthesized, labeled with [64Cu] and [123I], and evaluated in vitro as a potential imaging agent.

Results

The peptide was synthesized and labeled with [64Cu] and [123I] with 300 and 40 μCi/μg (542 and 72.2 mCi/μmol) specific activities, respectively, and radiochemical purity of>98%.c(RGDfE)K(DOTA)PLGVRY demonstrated high affinity for αvβ3 integrins(Kd = 83.4 ± 13.2 nM) in both substrate competition and cell binding assays. c(RGDfE)K(DOTA)PLGVRY peptide, but not the scrambled version, c(RGDfE)K(DOTA)GRPLVY was specifically cleaved by MMP2.

Conclusions

These results demonstrate the feasibility of developing dually radiolabeled peptides for the simultaneous imaging of cancer cells and their pathophysiologic activity.

Keywords: PET, SPECT, MMP, Integrin

Introduction

Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are important imaging methods widely used in the detection and staging of cancers [1]. The unique advantage of PET and SPECT imaging techniques is their potential for detecting disease related biochemical and physiologic abnormalities prior to the appearance of anatomical changes which can be visualized by conventional imaging techniques such as CT and MRI. Additionally, PET and SPECT imaging can assist in drug discovery and development[2]. High sensitivity and specificity are the strengths of PET and SPECT modalities, but both of these techniques suffer from comparatively low resolution when compared to other imaging modalities such as CT and MRI.

PET and SPECT imaging can be used for the assessment of treatment, estimation of prognosis, staging of cancer and for imaging angiogenesis, apoptosis, hypoxia, and various receptors such as the epidermal growth factor receptors (EGFR). However, there are limitations to PET and/or SPECT applications using probes that would be activated with the onset of cancer associated enzymatic activity.

Integrins are a family of hetero-dimericadhesion receptors expressed on the cell surface that mediate cell-cell and cell-extracellular matrix (ECM) interactions associated with normal growth and adhesion, tumorigenesis and neo-angiogenesis. αvβ3 integrins are highly over expressed in a number of cancers such as ovarian cancer[3], neuroblastoma[4], breast cancer[5], and melanoma[6, 7]. Expression of αvβ3 integrins on cancer cells potentiates metastasis by facilitating invasion and movement across blood vessels [8]. It is well established that RGD expressing proteins bind specifically to αvβ3 integrins and this property has been exploited for the development of imaging probes labeled with various radionuclides[9–11].

One of the challenging targets of cancer imaging is metastatic potential. This process is facilitated by the active basal membrane degrading action of matrix metalloproteinases (MMPs) [12, 13] along with other enzymes, such as urokinase plasminogen activators[14], and cathepsins[15]. Many MMPs are overexpressed in cancers, and their expression level has been shown to correlate with tumor stage[16], invasiveness [17, 18]metastasis[19] and neo-angiogenesis [20]. MMP2 is a member of the gelatinase group of enzymes and is one of the most widely studied MMPs, among the many known MMPs [21], MMP2 is known to target the PLGVR sequence and cleaves it between glycine and valine[22]. Integrins regulate expression and activation of MMPs, and participate in guiding MMPs to their targets by simultaneous binding of MMPs and extra cellular matrix (ECM) molecules. The integrin αvβ3 iscolocalized with MMP2 in glioma and breast cancer [5, 23]. Thus, there is a need to study the interactions and colocalization of these two important proteins in cancer cells. To achieve this, we have designed a peptide which incorporates both the αvβ3 integrin targeting and the MMP2 substrate moieties c(RGDfE)K(DOTA)PLGVRY (Figure. 1). [64Cu] (half-life = 12.7 h; β+655 keV [17.4%]; β−573 keV [39%]) conjugation of the peptide through the DOTA yields a PET agent, while radio-halogenation of the only tyrosine in the peptide with [123I] (half-life = 13.2 h; EC 159 keV [83.3%]) will give a SPECT signal. Conceptually, soon after the injection of the [64Cu] and [123I] double radiolabeled peptide, the PET and SPECT images will be colocalized. With time, as the MMPs secreted from the cancer cells cleave the peptide between glycine (G) and valine (V), the SPECT images will be delocalized from the PET image. This colocalization and delocalization will serve as a measure of the enzymatic activity of the MMPs.

Figure 1.

Figure 1

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a) Peptide concept to study protein targeting and enzymatic activity (b) structure of c(RGDfE)K(DOTA)PLGVRY.

Here, we report the design and characterization of a dually radiolabeled peptide [64Cu]c(RGDfE)K(DOTA)PLGVRY[123I] for targeting cancer cells and enzymatic activity of MMP2 associated with them, in vitro. In these proof of principle experiments, we illustrate that radiolabeled peptides could be used for targeting proteins and studying enzymatic activity simultaneously. To the best of our knowledge this is the first time that radioisotopes have been used to monitor enzymatic activity in the form of substrate cleavage. This principle has the potential to be translated into clinical applications, and may be a tool used in the diagnosis and treatment of cancer.

Materials and methods

Unless otherwise specified, chemicals were purchased from Sigma-Aldrich. [64Cu] was produced on a CS-15 biomedical cyclotron at Washington University School of Medicine cyclotron facility according to published procedures [24]. [123I] was obtained from Nordion (Kanata, ON, Canada) in Na123I form in 0.1M NaOH. Recombinant human integrinsαvβ3, MMP2 and MMP9 were obtained from R&D Systems (Minneapolis, MN). Human plasma vitronectin purified protein was obtained from Millipore (Billerica, MA). All peptides were purchased from CPC scientific, San Jose, CA. M21 (αvβ3 positive) and M21L (αvβ3 negative) cell lines were obtained from ATCC. All solutions were prepared using ultrapure 18 MΩ-cm water. Radioactive samples were counted using a 1480 automatic gamma counter (PerkinElmer, Massachusetts, MA). EZ-Link sulfo-NHS biotin was obtained from Fisher Scientific, (Pittsburgh, PA). Pierce pre-coated iodination tubes were obtained from Thermo Fisher Scientific Inc., (Rockford, IL). Abbreviations:BSA (Bovine serum albumin); HBSS (Hank’s balanced salt solution); HBSS salt solution (HBSS containing 2mM MgCl2, 2 mM CaCl2, 0.1 mM MnCl2, 2% BSA, HBSS); HPLC (High Pressure Liquid Chromatography).

PET/SPECT imaging of phantoms

One of the technical challenges with this approach is the generation of overlaid PET and SPECT images which may be acquired on different instruments and the amount of “cross-talk” between the two modalities and the impact on resolution. To this end we have performed a study in which a phantom containing both PET and SPECT radionuclides in separate compartments was imaged sequentially on small animal SPECT and PET scanners. This phantom also contained a 22Na source to measure the resolution in the PET scanner.

A phantom consisting of a water-filled cylinder containing two 1 ml tubes was sequentially imaged in the NanoSPECT/CT scanner (Bioscan Inc.) and in the microPET-Inveon-MM (Siemens Preclinical Solutions). One 1 mL tube contained 64Cu in the concentration of 100uCi/mL in water at the time of imaging. The other tube contained 1.2 mCi/mL of 123I in water. A 7 μCi Na-22 point source was located in between the two tubes. The purpose of the study was to determine if the 511 keV annihilation photons would affect the quantitative accuracy of the 123I data on the nanoSPECT system. Vice versa, this experiment will also indicate if 123I activity would influence PET accuracy and spatial resolution. The difference concentrations of activity reflect the difference in sensitivity between the PET and SPECT scanners

SPECT imaging was performed with the standard protocol of spiral SPECT using 24 camera positions/360 degrees with 30 second exposure at each position with 9-pinhole (1.4 mm each) collimators. A 10% energy window was employed. Images were reconstructed with the manufacturer provided MLEM (maximum-likelihood estimation maximization) algorithm with resolution recovery through modeling of system response of the detector and the pinhole collimators. A manufacturer provided low-level noise suppression was applied and 6 MLEM iterations were used. Images were reconstructed with isotropic 0.3 mm voxel size. Absolute camera calibration was performed by scanning a syringe containing 1 mL of a preparation with a known concentration of 123I.

The phantom was then moved to the small animal Inveon PET/CT scanner (Siemens Preclinical Solutions, Knoxville, TN) where data was acquired for 10 minutes in 3 dimensions. Listmode data was sorted into a single 3D sinogram and images were reconstructed with the manufacturer provided 2D-FBP algorithm with attenuation correction, scatter and normalization on a 256×256 matrix with a reconstruction zoom of 3. Images were reconstructed with a 0.13 mm pixel size. Attenuation correction was created by forward projection of the X-ray CT images of the phantom.

PET images were registered to the SPECT/CT data using IRW (Inveon Research Workplace) software (Siemens Preclinical Solutions, Knoxville, TN). Average activity concentration was measured by drawing free form VOIs inside the compartments or either 64Cu or 123I activity on the CT images. The spatial resolution was measured by fitting a Gaussian distribution to a profile transverse profile traced through the Na-22 point source activity. The FWHM of the point spread function was derived from the fitted width of the Gaussian distribution.

Radiolabeling of c(RGDfE)K(DOTA)PLGVRY with[64Cu]

64CuCl2 in NH4OAc (1.47 mCi, 15 μl) was added to c(RGDfE)K(DOTA)PLGVRY (5 μg) (or the fragmented peptide c(RGDfE)K(DOTA)PLG)) in 0.1M NH4OAC (pH 7.0) and incubated at 40 °C for 45–60 minutes with agitation. Radiochemical purity was assessed by analytical radio-HPLC (100% A to 100% B in 20 minutes, 1 mL/min; A = 0.1% TFA in H2O, B = 0.1 % TFA in acetonitrile). Chromatographic media was a Biobasic-18 BIO column (Fisher Scientific, Pittsburgh, PA) with 5 μm bead diameter and 4.6 × 150 mm column dimensions. Specific activity and radiochemical purity were calculated on the basis of radio-HPLC.

Radiolabeling of c(RGDfE)K(DOTA)PLGVRY with [123I]

Na123I in PBS(6 μl, 0.313 mCi) was combined with10 μg c(RGDfE)K(DOTA)PLGVRY (or fragmented peptides PLGVRY, GVRY and VRY) in iodination reagent coated iodogen tubes (Thermo Scientific, Rockford IL). The mixture was incubated for 10–15 minutes, at room temperature with occasional agitation. Specific activity and radiochemical purity were assessed with HPLC as described above.

αvβ3 integrin binding assay

The affinity of the peptide for αvβ3 integrins was determined using the method of Haubner et al [25, 26]. 96 well plates were coated with 100 μl of human integrin αvβ3 (1μg/ml) in coating buffer (20 mMTris, pH 7.4, 150 mMNaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) for 16 hours at 4 °C. Non-specific sites were blocked with blocking buffer (3% BSA in coating buffer, 200 μl per well) for 1 hour at 4 °C. After washing the plates three times with washing buffer (0.1% BSA in coating buffer), serially diluted c(RGDfE)K(DOTA)PLGVRY in binding buffer (1% BSA in coating buffer) was added with 14 nM biotinylated vitronectin solution, as previously described [27]. The plates were incubated at 37 °C for 3 hours and were washed three times with binding buffer. Bound biotinylated vitronectin was measured by adding ExtrAvidin alkaline phosphatase conjugate (Sigma) at 1/35,000 dilution for 1h at room temperature using the p-nitrophenyl phosphate substrate solution as the chromogen for 30 minutes at room temperature in the dark. Absorbance was measured at 405 nm. Each concentration was done in triplicate, and non-specific binding was subtracted from each data point. Nonlinear regression was used to fit binding curves (Prism, 5.0; Graph Pad).

Cell binding studies with αvβ3 positive M21 and negative M21L cells

1×106 M21 or M21L cells were seeded in T-25 flasks overnight at 37 °C in humidified atmosphere supplemented with 5% CO2. The cells were washed with washing buffer (0.1% BSA in HBSS 2 mM MgCl2, 2 mM CaCl2, 0.1mM MnCl2) and blocked with blocking buffer (3% BSA in HBSS, 2 mL) for 1 hour. Subsequently, the cells were washed twice with washing buffer and incubated with [64Cu]c(RGDfE)K(DOTA)PLGVRY (3 μg, 14 μCi) in binding buffer (1% BSA in HBSS, 2 mL) at 37 °C for 1 hour. The cells were washed twice with washing buffer, and collected using 0.05% Trypsin-EDTA in PBS. The radioactivity associated with the cells was measured by gamma-counter.

MMP2 peptide cleavage assays

MMP2 was activated by adding APMA (4-aminophenylmercuricacetate), as previously described [28]. MMP2 (5 μl, 0.5 μg in PBS) was added to either[64Cu]c(RGDfE)K(DOTA)PLGVRY or the scrambled peptide [64Cu]c(RGDfE)K(DOTA)GRPLVY(2 μg in 20 μl of NH4OAc, 0.1M, pH 7.0) in 100 μl of 50 mMTris, pH 7.5, 10 mMCaCl2, 150 mMNaCl, 0.05% Brij35 (TCNB). The mixture was incubated at 37 °C with agitation. Reaction progress was determined by HPLC 100% H2O/0.1% TFA to 100% acetonitrile/0.1% TFA in 20 minutes.

Ilomastat inhibition of MMP2 activity

Ilomastat (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) is an MMP2 inhibitor [29, 30]. MMP2 (1.4 μg, in 18 μl TCNB) was added to varying concentrations of ilomastat (0 nM to 910 μM), in TCNB (90 μl). [64Cu]c(RGDfE)K(DOTA)PLGVRY (15 μl, 1 μg, 160 μCi) was added to each of the reaction mixtures and the solution was incubated at 37 °C for 24 hours. The enzymatic activity of MMP2 was monitored by HPLC determination of the fragmented peptide.

Serum study of [64Cu]c(RGDfE)K(DOTA)PLGVRY and c(RGDfE)K(DOTA)PLGVRY[123I]

The serum stability of [64Cu]c(RGDfE)K(DOTA)PLGVRY and c(RGDfE)K(DOTA)PLGVRY-[123I] was studied by incubating rat serum (135 μl) with [64Cu]c(RGDfE)K(DOTA)PLGVRY (20 μl, 2.8 μg) or c(RGDfE)K(DOTA)PLGVRY[123I] (20 μl, 2.4 μg) at 37 °C. When studying for ilomastat effect 45 μl of ilomastat (2.5 mM in DMSO) was added to 90 μl of rat serum; to which was added 20 μl of the radiolabeled peptide. The stability of the peptide was determined by HPLC at 1, 2, 4, 14 and 24 hours. HPLC conditions were from 100% A to 100% B in 20 minutes at 1 mL/min flow rate where A = 0.1% TFA in H20, B = 0.1% TFA in acetonitrile. The chromatographic media was Biobasic-18 BIO column (Fisher Scientific) with 5 μm bead diameter, and 4.6 × 150 mm column size.

Results

PET/SPECT imaging of Phantoms

Although an intense background (due to the 511 keV gamma ray penetrating the 6 mm thick Tungsten collimators) were observed in the projection sinogram data in the SPECT imaging, this background turned out to be effectively removed by the noise-suppression algorithm and resulted in very low contamination of the 123I data by the 64Cu activity (1.8%) in the final image. The 123I activity was accurately recovered and a very small amount is observed in the sphere containing 64Cu. From the PET dataset, we observe a negligible amount of counts in the 123I sphere as could be expected, since the energy of these gamma rays is well below the energy threshold for PET and also are not measured in coincidences. The 159 keV photons from the decay of the 123I are effectively discarded by the lower level discriminator of the energy acceptance window of the PET camera which is set at 350 keV on our Inveon system. The PET/SPECT/CT tri-modality image obtained in this experiment is shown in Figure 2. Table 1 contains the ROI data obtained for each sphere and each data set. The measurement of the 22Na point source yielded a PET resolution of 1.58 mm. This value is in agreement with reported measurement of spatial resolution for the Inveon Scanner of 1.62 mm[31].

Figure 2.

Figure 2

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64Cu PET/CT images (Left), 123ISPECT/CT images (Right) of the 2-tube phantom.

Table 1.

Measured activity concentrations of the two radionuclides in each sphere. The amount radioactivity detected as a percentage of the true value is given.

Compartment 123I activity (SPECT) 64Cu activity (PET)
123I Tube 100% 0.02%
64Cu Tube 1.8% 100 %
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[64Cu] and [123I] Radiolabeling

After incubation of the peptide with [64Cu] for 45 minutes or with [123I] for 10 minutes, 5 μl of the reaction mixture was injected onto the HPLC. The free [64Cu] and [123I] retention times were 2.4 and 2.2 minutes respectively, while the [64Cu] or [123I] radiolabeled peptide had retention times of 8.6 and 9.1 minutes, respectively. c(RGDfE)K(DOTA)PLGVRY was successfully labeled with [64Cu] and [123I] with 300 and 40 μCi/μg specific activities, respectively (Figure 3). The [64Cu] and [123I] labeling efficiency was greater than 95% and no further purification was required. Thus both radionuclides demonstrate facile labeling of c(GRDfE)K(DOTA)PLGVRY.

Figure 3.

Figure 3

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HPLC traces of a) c(RGDfE)K(DOTA)PLGVRY b) [64Cu]c(RGDfE)K(DOTA)PLGVRY c)c(RGDfE)K(DOTA)PLGVRY[123I]. HPLC eluent from 100% water (0.1% TFA) to 100% acetonitrile (0.1%TFA) over 20 minutes. [a)UV-Visible trace b) and c) radiationtraces]

In addition, the fragmented peptidec(RGDfE)K(DOTA)PLG was labeled with [64Cu], yielding [64Cu]c(RGDfE)K(DOTA)PLG with a retention time of 8.1 minutes. The fragmented peptides PLGVRY, GVRY and VRY were also labeled with [123I] efficiently giving PLGVRY[123I], GVRY[123I] and VRY[123I] with retention times of 9.2, 8.5 and 8.0 minutes, respectively.

c(RGDfE)K(DOTA)PLGVRY affinity to cellularαvβ3 integrins

To evaluate the binding affinity of c(RGDfE)K(DOTA)PLGVRY to αvβ3 a solid-phase competitive binding assay was used. The peptide was tested for its ability to compete for immobilized αvβ3 with biotinylated vitronectin. As shown in Figure 4, when various concentrations of the peptide were added to the reaction mixture, a concentration-dependent binding to αvβ3 integrin was demonstrated, with KD value of 83.4 ± 13.2 nM. This result demonstrated high affinity of c(RGDfE)K(DOTA)PLGVRY to αvβ3 integrins in competitive binding assay.

Figure 4.

Figure 4

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c(RGDfE)K(DOTA)PLGVRY competitive binding assay with αvβ3, in the presence of vitronectin (14nM). (Kd = 83.4 ± 13.2 nM)

Cell binding of c(RGDfE)K(DOTA)PLGVRY

To examine the in vitro receptor-binding specificity of c(RGDfE)K(DOTA)PLGVRY to αvβ3, cell binding studies using the αvβ3 positive M21 and αvβ3 negative M21L[32] human melanoma cells were conducted as described in the Methods section. Assessment of the whole-cell associated activity showed tenfold higher specific binding to the αvβ3-positive M21 than to the αvβ3-negative M21L cells (Figure 5).

Figure 5.

Figure 5

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[64Cu]c(RGDfE)K(DOTA)PLGVRY shows increased binding to M21 (αvβ3 positive) over M21L (αvβ3 negative) cells.

MMP2 cleavage of the c(RGDfE)K(DOTA)PLGVRY

The peptide [64Cu]c(RGDfE)K(DOTA)PLGVRY contains a PLGVR sequence that is recognized and cleaved by MMP2[22]. The cleavage was confirmed by HPLC: with time the radiolabeled peptide parent peak at 8.7 minutes diminished and was replaced with a new peak at 8.1 minutes (Fig. 6.a). This new peak is due to the fragmented peptide [64Cu]c(RGDfE)K(DOTA)PLG which was confirmed as by analyses of the radiolabeled fragment [64Cu]c(RGDfE)K(DOTA)PLG (8.1 minutes, Table 1). The specificity of the cleavage was further confirmed by inhibition of the peptide cleavage in the presence of 910 μM of ilomastat, an MMP2 inhibitor. Serially diluted concentrations of ilomastat were added to the activated MMP2 and were incubated with the [64Cu]c(RGDfE)K(DOTA)PLGVRY. Fig. 6.b shows inhibition of the MMP2 cleavage-activity with increasing concentrations of ilomastat, from 1 nM to 1000 μM. With increasing concentration of ilomastat the peptide was increasingly protected from cleavage.

Figure 6.

Figure 6

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MMP2 cleaving action and its inhibition by ilomastat a) [64Cu]c(RGDfE)K(DOTA)PLGVRY with MMP2 (red) and peptide only (blue) after 24 hr incubation at 37°C. b) MMP2 inhibition with increasing concentration of ilomastat.

MMP2 cleavage of the c(RGDfE)K(DOTA)GRPLVY

Previous work by Bremer et al [22] has shown that MMP2 targets the PLGVR sequence and cleaves between glycine and valine. In order to study the sequence specificity of MMP2 cleaving action on the peptide [64Cu]c(RGDfE)K(DOTA)PLGVRY, a scrambled peptide[64Cu]c(RGDfE)K(DOTA)GRPLVY was used. MMP2 cleaved [64Cu]c(RGDfE)K(DOTA)PLGVRY, while the scrambled peptide [64Cu]c(RGDfE)K(DOTA)GRPLVY was not affected. As illustrated in Figure 7, while the percentage of [64Cu]c(RGDfE)K(DOTA)PLGVRY decreased over time due to the cleaving action, the scrambled peptide [64Cu]c(RGDfE)K(DOTA)GRPLVY was 100% intact even after 24 hours.

Figure 7.

Figure 7

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Cleaving action over time of MMP on [64Cu]c(RGDfE)K(DOTA)GRPLVY (square) and [64Cu]c(RGDfE)K(DOTA)PLGVRY (circle).

In addition, the peptide fraction c(RGDfE)K(DOTA)PLG was labeled with [64Cu] and its retention time was same as that of the MMP2 cleavage product of [64Cu]c(RGDfE)K(DOTA)PLGVRY (8.1 minutes), proving the expected cleavage of the peptide by MMP2 at the previously specified sequence position. In a similar way, VRY[123I] had the same retention time as the cleavage product of c(RGDfE)K(DOTA)PLGVRY[123I] (8.0 minutes, Table 2).

Table 2.

HPLC retention times (in minutes) of: a)64Cu and 123I labeling of the peptides. b)Cleavage of the radiolabeled peptides. HPLC condition was from 100% water (0.1% TFA) to 100% acetonitrile (0.1% TFA).

Table 2.a
Compound Retention time 64Cu labeled retention time 123I labeled retentiontime
c(RGDfE)K(DOTA)PLGVRY 8.2 8.6 9.1 & 9.4
c(RGDfE)K(DOTA)PLG 7.8 8.1 NA
c(RGDfE)K(DOTA)(PEG)2PLGVRY 8.8 8.9 ND
c(RGDfE)K(DOTA)GRPLVY 8.3 8.6 ND
PLGVRY 7.9 NA 9.2 & 9.7
GVRY 6.7 NA 8.5 & 9.0
VRY 6.5 NA 8.0 & 8.5
Table 2.b
Compound Retention time Cleavage product, retention time
[64Cu]c(RGDfE)K(DOTA)PLGVRY 8.6 8.1
c(RGDfE)K(DOTA)PLGVRY[123I] 9.1 8.0
[64Cu ]c(RGDfE)K(DOTA)PLG 8.1 No change
[64Cu]c(RGDfE)K(DOTA)(PEG)2PLGVRY 8.9 8.3
c(RGDfE)K(DOTA)(PEG)2PLGVRY[123I] 9.1 8.0
[64Cu]c(RGDfE)K(DOTA)GRPLVY 8.7 No change
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NA = Not applicable; ND = Not determined.

Serum stability of [64Cu]c(RGDfE)K(DOTA)PLGVRY and c(RGDfE)K(DOTA)PLGVRY[123I]

After 24 hours incubation with rat serum, 55% c(RGDfE)K(DOTA)PLGVRY[123I]was intact. Degradation due to both the cleaving action of MMPs as well as other peptidases in rat serum and dehalogenation of the iodine was observed, new peaks for degraded products (3.8 minutes (1%), 6.3 minutes (5%) and 7.0 minutes (15%)) and free [123I] (2.6 minutes, 20%) appeared. In the presence of ilomastat (0.73 mM), degradation was mainly due to dehalogenation of the [123I] (2.6 minutes, 16 %) but no other degradation products were detected. The peptide [64Cu]-c(RGDfE)K(DOTA)PLGVRY was also partially degraded as new peaks appeared when incubated in serum due to protease action, 8.0 minutes (23%) and 8.5 minutes (18%) (48 hours). (Data not shown) This was confirmed as the addition of ilomastat (0.73 mM) to the reaction mixture increased the stability of the peptide to 94% (Figure 8a, 123Iproduct and 8b, 64Cu product).

Figure 8.

Figure 8

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Serum study of a) c(RGDfE)K(DOTA)PLGVRY[123I] and b) [64Cu]c(RGDfE)K(DOTA)PLGVRY in rat serum at 37 °C. (Circle = labeled peptide only, no ilomastat; Square = labeled peptide with ilomastat added.)

Discussion

RGD peptides have been known to target αvβ3 integrins[33, 34]. In the last decade a number of cyclic RGD containing pentapeptides have been studied for their binding to different tumors expressing αvβ3 integrins. c(RGDfV) is one of the best selective αvβ3 antagonists with an IC50 value in the lower nanomolar range[35]. Previous studies have established that while the identity of the 5th amino acid does not affect affinity, the sequence has a higher affinity if the 4th amino acid is hydrophobic (tyrosine or phenylalanine)[36]. Hence, we opted to use the sequence-c(RGDfE), having the hydrophobic D-phenylalanine at the 4th position and glutamic acid at the 5th position and phenylalanine in place of tyrosine, so only one tyrosine is present at the right end of the peptide c(RGDfE)K(DOTA)PLGVRY to avoid multiple halogenation sites during radiolabeling with[123I]. Having glutamic acid instead of lysine at the 5th position offers a carboxylic group side chain for conjugation with the N-terminal of the DOTA conjugated lysine. Though a large side chain (-K(DOTA)PLGVRY) has been incorporated, the IC50 value of c(RGDfE)K(DOTA)PLGVRY (83.4 ± 13.2nM) is in the nanomolar range similar to that of many other published pentapeptides: c(rGDFV) 25 ± 4 μM; c(RGdFV) 18.1 ± 14.8 μM; c(RGDfV) 50 ± 24 nM; c(RGDFv) 0.4 ± 0.1 μM;[35]c(RGDsV) 21 nM; c(RGDkV) 29 nM; c(RGDpV) 580 nM; c(RGDfV); c(RGDfK) 4.2 nM; c(RGDfS) 12 nM; c(RGDfG) 1.6 nM; c(RGDfA) 20 nMetc[36]. The peptide c(ERGDf) with the same amino acids in the cyclic pentapeptide had an IC50 value of 0.28 ± 0.004 and 3.2 ± 0.16 μM by fluorescence polarization and cell adhesion, respectively [37]. The fact that [64Cu]c(RGDfE)K(DOTA)PLGVRY showed much higher binding to the αvβ3 positive M21 melanoma cells than the αvβ3 negative M21L cells establishes the selective binding of the peptide.

Here we report proof of principle studies which for the first time use radioactivity in detecting enzymatic activities in vitro. MMP2 has been shown to cleave the substrate peptide c(RGDfE)K(DOTA)PLGVRY between the glycine and valine. The inability to cleave the scrambled peptide c(RGDfE)K(DOTA)GRPLVY illustrates the specificity of the cleaving action. This nuclear approach may have certain advantages over other imaging approaches: namely sensitivity and depth. Radioactive probes can help detect protein targets and potentially monitor biochemical transformations at concentrations as low as nanomolar to picomolar and deep into the body. The use of radioactive probes for monitoring enzymatic activity could be extended to other enzymes besides the proteinases such as cathepsin, and serum-protease urokinases. In the meantime, work is ongoing to realize this application in vivo. Overall using a dually ([64Cu] and [123I]) radiolabeled c(RGDfE)K(DOTA)PLGVRY we have shown that radioactivity can serve in the detection of enzymatic activity of MMP2 in vitro. The same principle may be used for the study of other enzymes involved in cancer progression and metastasis as well as in other biological processes.

Acknowledgments

The authors would like to thank Nicole Fettig, Amanda Roth, Ann Stroncek, Lori Strong, Paul Eisenbeis, Tom Voller and Paul C. Carey for technical help. This research was supported by DOE BER(US DOE DESC0002114).

Footnotes

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