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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC 2010 March 15.
Published in final edited form as:
PMCID: PMC2833353

Engineered Knottin Peptides: A New Class of Agents for Imaging Integrin Expression in Living Subjects


There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with low nM affinity to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or 64Cu-DOTA to their N-termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. Near-infrared fluorescence and microPET imaging both demonstrated that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 h post injection for two high affinity (IC50 ~20 nM) 64Cu-DOTA-conjugated knottin peptides was 4.47 ± 1.21 and 4.56 ± 0.64 % injected dose/gram (%ID/g), compared to a low affinity knottin peptide (IC50 ~0.4 μM; 1.48 ± 0.53 %ID/g) and c(RGDyK) (IC50 ~1 μM; 2.32 ± 0.55 %ID/g), a low affinity cyclic pentapeptide under clinical development. Furthermore, 64Cu-DOTA-conjugated knottin peptides generated lower levels of non-specific liver uptake (~2 %ID/g) compared to c(RGDyK) (~4 %ID/g) 1 h post injection. MicroPET imaging results were confirmed by in vivo biodistribution studies. 64Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers.

Keywords: protein engineering, positron emission tomography, tumor targeting agents, molecular imaging, integrin binding peptides


Cell surface receptors that are selectively expressed in human malignancies have generated much interest as potential targets for molecular therapeutics and diagnostic agents. Integrins are a family of extracellular matrix adhesion receptors that non-covalently associate into α/β heterodimers with distinct ligand binding specificities (1). Several integrins, including αvβ3, αvβ5, and α5β1, have been shown to be expressed on the surface of cancer cells and the tumor neovasculature (2-5). These integrins have been proposed to mediate angiogenesis, tumor growth, and metastasis (6-8), making them attractive targets for therapeutic intervention. One strategy for inhibiting tumor angiogenesis is to administer agents that will bind to these specific integrin receptors with high affinity and block their function (9-11). This approach highlights a need for non-invasive in vivo imaging probes that can be used to identify patients who will best respond to these integrin-targeted therapies and to monitor disease progression (12-14). In addition, the use of integrins as biomarkers in molecular imaging applications will be important for the early detection of cancer.

Many integrins, including those containing the αv subunit, as well as α5β1 and αiibβ3 integrins, recognize an Arg-Gly-Asp (RGD) motif found in extracellular matrix protein ligands (15). In these ligands, the RGD motif is typically found in solvent-exposed loops, and the structural context of this loop, dictated by the residues flanking the RGD sequence, determines integrin binding affinity and specificity (16). Rational drug design and phage display have generated peptides and peptidomimetics containing the RGD sequence that target αvβ3 (and αvβ5) integrins or α5β1 integrin (4, 10, 15). However, chemical modifications that can be made to these small peptides to improve their receptor binding affinity, tumor uptake, and in vivo pharmacokinetics are limited. Moreover, covalent attachment of imaging probes have affected their integrin binding properties (14). Therefore, despite the prevalence of integrin-binding peptides and peptidomimetics in the literature, suboptimal tumor targeting efficacy and pharmacokinetics have limited their clinical translation as molecular imaging agents (13). Only one compound, a glycosylated cyclic RGD pentapeptide (18F-Galacto-c(RGDfK), has advanced to the clinical level for molecular imaging in human subjects (17, 18). While this agent was able to identify integrin-positive lesions in human subjects and image intensity correlated with αvβ3 integrin expression, its relatively poor tumor uptake and higher background (e.g., liver) indicates there is room for substantial improvements. Moreover, in preclinical studies using a M21 human melanoma mouse xenograft model, 18F-Galacto-c(RGDfK) exhibited low tumor uptake values of 1.6 ± 0.2 %ID/g (19), suggesting that imaging agents that have relatively low tumor contrast in small animal models will likely correlate to low imaging contrast in humans.

To develop a new class of agents to image integrin expression in vivo, we engineered small (~3 kDa), conformationally-constrained peptides that bind to αvβ3vβ5, or αvβ3vβ55β1 integrins with low nM affinity (R.H. Kimura, A.M. Levin, J.R. Cochran, unpublished data). We incorporated RGD-based integrin binding motifs into a surface-exposed loop of the Ecballium elaterium trypsin inhibitor (EETI-II) (20), a cystine knot peptide from the squash family of protease inhibitors (Fig. 1A). This provided a rigid structural framework that enabled us to engineer peptides with low nM integrin binding affinities (IC50 ~10−30 nM) through directed evolution. Cystine knot peptides (also known as knottins) are ideal for in vivo tumor targeting applications as they possess a disulfide-bonded core that confers outstanding proteolytic resistance and thermal stability; moreover, they have been shown to be non-immunogenic (21-24). Due to their small size, knottin peptides can be produced by chemical synthesis which allows for site-specific conjugation of imaging probes, radioisotopes, or chemotherapeutic agents for direct delivery to cancer cells. In addition, knottin peptides can be chemically modified to tailor their in vivo pharmacokinetic properties for diverse clinical applications.

Figure 1
Schematic and sequences of EETI-II knottin peptides. A, Cartoon representation of a knottin peptide, with disulfide bonds between Cys1-Cys4, Cys2-Cy5, and Cys3-Cys6, and an imaging label (star) site-specifically conjugated to the N-terminus. B, Amino ...

In this study, we conjugated optical and PET imaging probes to our engineered integrin-binding knottin peptides to evaluate their potential as in vivo integrin imaging agents. We tested the ability of knottin peptides with varying integrin binding affinities to target tumors in small animal xenograft models using near-infrared fluorescence and microPET imaging. We compared these results to those generated with a knottin peptide containing a scrambled RGD sequence, and a cyclic RGD pentapeptide (c(RGDyK)), another monomeric, unmodified peptide currently under clinical development.

Materials and Methods

Materials, cell lines, and reagents

The U87MG human glioblastoma cell line was obtained from American Type Culture Collection. Detergent-solubilized αvβ3, αvβ5 integrin receptors (both octyl-beta-D-glucopyranoside formulations) and α5β1 integrin (Triton X-100 formulation) were purchased from Millipore, and αiibβ3 (Triton-X100 formulation) was purchased from Enzyme Research Laboratories. 125I-labeled echistatin and c(RGDyK) were purchased from Amersham Biosciences, and Peptides International, respectively. Phosphate buffered saline (PBS) was from Invitrogen. All other chemicals were purchased from Fisher Scientific unless otherwise specified. Integrin binding buffer (IBB) was composed of 25 mM Tris pH 7.4, 150 mM NaCl, 2mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, and 0.1% bovine serum albumin (BSA).

Cell surface integrin receptor competition binding assay

Cell surface competition binding assays were performed as previously described (25). Briefly, 2 × 105 U87MG cells were incubated with 0.06 nM 125I-labeled echistatin and varying concentrations of peptides in IBB at room temperature for 3 h. The cell-bound radioactivity remaining after washing was determined by gamma-counting. Half-maximal inhibitory competition (IC50) values were determined by non-linear regression analysis using Kaleidagraph (Synergy Software), and are reported as the average of experiments performed on three separate days.

Solid phase integrin receptor competition binding assay

Integrin receptor competition binding assays were performed as previously described (26). Briefly, detergent-solubilized αvβ3, αvβ5, α5β1, and αiibβ3 integrin receptors were diluted to a final concentration of 1 μg/mL in IBB. 100 μL aliquots were used to coat wells of Maxisorb plates (NalgeNunc, Fisher Scientific), overnight at 4 °C. The wells were washed and blocked with IBB containing 1% BSA for 2 h at room temperature. 125I-labeled echistatin (0.06 nM) and varying concentrations of unlabeled peptides were incubated in the wells for 3 h at room temperature with gentle rocking, and washed 3 times in IBB. Plate-bound radioactivity was solubilized with 200 μL of boiling 2N NaOH followed by gamma-counting. Each data point represents the average value of triplicate wells.

Cy5.5 chemical conjugation

Cy5.5 monofunctional N-hydroxysuccinimide ester (Amersham Biosciences) was dissolved in a solution of 1 mL of dimethlysulfoxide and 15 μL triethylamine. Purified peptide was added to this Cy5.5 solution, and the reaction was mixed at room temperature in the dark. Cy5.5 conjugation reactions were monitored by absorbance at 675 nm by reversed-phase analytical HPLC. Upon completion, the reaction mixtures were purified by reversed-phase HPLC. Fractions containing Cy5.5-peptide conjugates were collected, lyophilized and redissolved in water. Peptide purity was assessed by analytical reversed-phase HPLC and concentrations were determined by amino acid analysis (AAA Service Laboratory, Damascus, OR). Molecular masses were confirmed with electrospray (ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using Stanford core facilities (Supplemental Table S1).

DOTA chemical conjugation and 64Cu radiolabeling

1,4,7,10-tetradodecane-N, N’, N’’, N’’’–tetraacetic acid (DOTA; Sigma Aldrich) was activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC; Pierce) and N-hydroxysulfonosuccinimide (SNHS; Pierce) in water (pH 5.5) for 40 min at room temperature using a 1:1:1 molar ratio of DOTA:EDC:SNHS. Peptides were dissolved in 300 μL of sodium phosphate buffer (30 mM, pH 8.5), and added to the above in-situ prepared sulfosuccinimidyl ester of DOTA (DOTA-OSSu). A molar excess of DOTA-OSSu was used to drive the conjugation reaction to completion. The reaction was allowed to proceed at room temperature for 1 h and mixed at 4 °C overnight. The resulting DOTA-peptide conjugates were purified by reversed-phase HPLC and stored as a lyophilized solid. The product masses were verified by ESI-MS and MALDI-TOF-MS (Supplemental Table S1) and peptide concentrations were determined by amino acid analysis.

The DOTA-conjugated peptides (25 μg) were radiolabeled with 64Cu by incubating with 2−3 mCi 64CuCl2 (University of Wisconsin-Madison, Madison, WI) in 0.1 N sodium acetate (pH 6.3) for 1 h at 45 °C. The reaction was terminated with the addition of EDTA. The radiolabeled complexes were purified using a PD-10 column (Amersham) or by radio-HPLC using a gamma detector, dried by rotary evaporation, reconstituted in PBS, and passed through a 0.22 μm filter for animal experiments. The radiochemical purity, determined as the ratio of the main product peak to other peaks, was determined by HPLC to be > 95%. The radiochemical yield, determined as the ratio of final activity of the product over the starting activity used for the reaction, was usually over 80%. At least 7 radiolabeling reactions were performed for experiments run on different days.

U87MG glioblastoma xenograft mouse model

U87MG cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 in Dulbecco's modified eagle medium, 10% heat-inactivated fetal bovine serum, and penicillin-streptomycin (all from Invitrogen). Animal procedures were carried out according to a protocol by Stanford University Administrative Panels on Laboratory Animal Care. Female athymic nude mice (nu/nu), obtained at 4−6 weeks of age (Charles River Laboratories, Inc.), were injected subcutaneously in the right or left shoulder with 2 × 107 U87MG glioblastoma cells suspended in 100 μL of PBS. Mice were used for in vivo imaging studies when their tumors reached approximately 8 to 10 millimeters in diameter.

Near-infrared (NIR) fluorescence and microPET imaging of U87MG xenograft tumors

NIR fluorescence imaging was performed with an IVIS 200 (Xenogen) as previously described (25). Briefly, a Cy5.5 filter set was used and fluorescence emission was normalized to photons per second per centimeter squared per steradian (p/s/cm2/sr). Images were acquired and analyzed using Living Image 2.5 (Xenogen). For all experiments, mice (n = 3 for each probe) were injected via tail vein with 1.5 nmol of Cy5.5-labeled peptides in 100 μL PBS and imaged at various times post injection. Tumor contrast was quantified by drawing identically-sized regions of interest (ROI) around the tumor (T) and normal (N) tissue located in the mouse's flank (average radiance, p/s/cm2/sr). Data points represent the average T/N ratio for a group of three animals.

U87MG tumor-bearing mice (n = 3 or more for each probe) were injected with ~100 μCi of 64Cu-DOTA-conjugated peptides via the tail vein and imaged with a microPET R4 rodent model scanner (Siemens Medical) using 3 or 5 min static scans. For blocking experiments, mice were co-injected with 330 μg (~0.5 μmol) of unlabeled c(RGDyK). Images were reconstructed by a two dimensional ordered expectation maximum subset algorithm and calibrated as previously described (27). ROIs were drawn over the tumor on decay-corrected whole body images using ASIPro VM software (Siemens Medical). The mean counts per pixel per minute were obtained from the ROI and converted to counts per milliliter per minute with a calibration constant. ROIs were converted to counts/g/min, and %ID/g values were determined assuming a tissue density of 1 g/mL. No attenuation correction was performed.

In vivo biodistribution studies

Anesthetized nude (nu/nu) mice bearing U87MG tumor xenografts were injected with ~100 μCi of 64Cu-DOTA labeled knottin peptides via the tail vein, and were euthanized at 0.5, 1 and 24 h. Blood, muscle, heart, liver, lungs, kidneys, spleen, brain, intestine, skin, stomach, pancreas, and tumor tissue were removed and weighed, and their radioactivity levels were measured by gamma counting. Results are expressed as the %ID/g of tissue and represent the mean and standard deviation of experiments performed on at least three mice. For each mouse, the activity of tissue samples was calibrated against a known aliquot of the radio-tracer and normalized to the whole bodyweight and to the residual activity present in the tail.

In vivo metabolite analysis

For metabolite analysis, anesthetized nude (nu/nu) mice bearing U87MG tumor xenografts were tail-vein injected with 200−400 μCi of 64Cu-DOTA labeled knottin peptides, and were euthanized at 1, 4, or 24 h. Blood, kidney and tumor tissue were removed and suspended in ~500 μL PBS. Tissues were homogenized with a mortar and pestle and the homogenate was extensively filtered using a Nanosep 10K device (Pall Corporation) to isolate low molecular weight metabolites. The filtrates were analyzed by reversed-phase HPLC under identical conditions used for analyzing the original radiolabeled compound. Eluted fractions were collected in 30 s intervals and a gamma counter was used to determine counts per minute (cpm).

Statistical analysis

All data is presented as the average value ± the standard deviation of n independent measurements. Statistical analysis for animal studies was performed by t-test using Microsoft Excel or Matlab, and significance was assigned for p values < 0.05.


Knottin peptide synthesis and conjugation to Cy5.5 and DOTA

Previously, we used combinatorial methods to engineer EETI-II mutants that bound with low nM affinity (IC50 ~10−30 nM) to integrin receptors that are overexpressed on the tumor vasculature (R.H. Kimura, A.M. Levin, and J.R. Cochran, unpublished data). The sequences of these mutants are shown in Figure 1B. In addition, we also engineered a knottin peptide that binds with weaker integrin binding affinity (FN-RGD2; IC50 ~0.4 μM) by grafting a loop from the extracellular matrix protein fibronectin into EETI-II. A knottin peptide that contains the scrambled sequence RDG and does not bind to integrins was used as a negative control in all studies (FN-RDG2). Knottin peptides were synthesized using Fmoc-based solid phase peptide synthesis, and were purified and folded as described (Supplementary Methods). The N-terminal amine of the folded, purified knottin peptides was used for site-specific attachment of Cy5.5, a NIR fluorochrome, or a DOTA chelating agent and the radionuclide 64Cu. Modified knottin peptides were purified by reversed-phase HPLC and were characterized by mass spectrometry (Supplementary Table S1) and amino acid analysis. Analogous bioconjugation reactions were performed through the ε-amino group of the lysine residue in c(RGDyK), a backbone cyclized pentapeptide which has been extensively characterized for in vivo molecular imaging applications (reviewed in (12, 28)), and is included here for comparison to our knottin peptides.

Binding of Cy5.5 and DOTA-labeled knottin peptides to integrin-expressing tumor cells

The relative binding affinities of DOTA- and Cy5.5-conjugated peptides was tested and compared to unmodified peptides to determine if these modifications disrupt integrin binding interactions. Echistatin is a RGD-containing protein from snake venom that binds to αvβ3 integrin with a KD of 0.36 nM (29). Peptides were tested for their ability to compete for cell surface integrin binding with 125I-labeled echistatin. U87MG glioblastoma cells, which express ~105 αvβ3 integrin receptors per cell (30), were used for these studies. Relative binding affinities for modified and unmodified peptides are reported as IC50 values (Table 1). Knottin peptides 2.5D and 2.5F, which were obtained by directed evolution, were shown to bind to U87MG cells with a significantly stronger affinity (IC50 = 19 ± 6 nM and 26 ± 5 nM, respectively) than both the loop-grafted FN-RGD2 (IC50 = 370 ± 150 nM) and c(RGDyK) (IC50 = 860 ± 400 nM) peptides (Table 1). FN-RDG2 was not able to compete for 125I-echistatin binding to U87MG cells, as expected. Next, DOTA-conjugated peptides were shown to bind to U87MG cells in a dose-dependent manner with affinities that were comparable to the unmodified peptides (Table 1 and Supplemental Fig. S1A). In contrast, Cy5.5-labeled peptides demonstrated stronger affinities (approximately 4−8 fold) to U87MG cells compared to the corresponding unmodified peptides (Table 1 and Supplemental Fig. S1B). This increase in binding could be due to interactions between the hydrophobic dye molecule and cells; however, only occurs with peptides that have affinity for integrin receptors, as the Cy5.5-FN-RDG2 negative control does not exhibit non-specific binding.

Table 1
Summary of U87MG cell binding data for unlabeled, Cy5.5-labeled, and DOTA-conjugated peptides, reported as IC50 values.

Integrin binding specificities of Cy5.5 and DOTA-labeled knottin peptides

Since U87MG cells have been shown to express αvβ5, and α5β1 integrins in addition to αvβ3 integrin (31), we measured integrin binding specificity by competition of 125I-echistatin to detergent-solubilized αvβ3, αvβ5, α5β1, and αiibβ3 integrin receptors coated onto microtiter plates. Unlabeled echistatin, our positive control, bound strongly to all of the tested integrins, in agreement with previous reports (32). All RGD-containing peptides bound to αvβ3 and αvβ5 integrins to some degree, with the knottin peptides 2.5D, and 2.5F showing the strongest levels of binding compared to FN-RGD2 and c(RGDyK) (Fig. 2). DOTA-conjugated FN-RDG2, our negative control, did not bind to any of the integrins used in this study (Fig 2A). The DOTA-conjugated knottin peptide 2.5F bound with strong affinity to α5β1 integrin, while knottin 2.5D exhibited only minimal binding to this receptor. DOTA-labeled peptides did not bind to the αiibβ3 integrin receptor, which is important for in vivo imaging applications, since the αiibβ3 integrin is widely expressed on platelet cells and is involved in mediating the blood clotting process (33). Collectively, the integrin binding specificity of the DOTA-labeled peptides is identical to results obtained with unmodified peptides (data not shown). In contrast, Cy5.5-labeled peptides showed increased binding to all integrin receptors compared to unmodified and DOTA-conjugated peptides (Fig. 2B and data not shown). This was likely due to non-specific binding of the hydrophobic Cy5.5 molecule (i.e. Cy5.5-FN-RGD2 binding to αiibβ3 integrins), but could also be due to increased interactions of Cy5.5-labeled peptides with integrin receptors.

Figure 2
Competition binding of peptides to surface-immobilized integrins. A, DOTA-conjugated peptides. B, Cy5.5-conjugated peptides. To determine integrin binding specificity, 125I-labeled echistatin and 5 nM (black bars) or 50 nM (grey bars) unlabeled polypeptides ...

Knottin peptides as in vivo optical imaging probes

We tested the ability of Cy5.5-labeled knottin peptides to target tumors in small living animals to begin to evaluate their potential as in vivo molecular imaging agents. Whole-body NIR fluorescence imaging of subcutaneous human tumor mouse xenografts was performed and the fluorescence intensity of tumor-to-normal tissue (T/N ratio) was measured as a function of time. Figure 3A shows typical NIR fluorescent images of athymic nude mice bearing subcutaneous U87MG glioblastoma tumors after tail vein injection of 1.5 nmol of Cy5.5-labeled peptides. Cy5.5-labeled knottins 2.5D and 2.5F showed increased T/N ratios compared to signals generated by both FN-RGD2 knottin peptide and c(RGDyK) peptide, which were only slightly higher than the background signal of the FN-RDG2 negative control (Fig. 3B). Since Cy5.5-FN-RDG2 does not appear to bind to U87MG cells (Table 1), the NIR fluorescence signal observed for this peptide in the tumor likely results from the extravasation of the probe from leaky tumor vasculature. Cy5.5-labeled knottins were taken up and retained by the kidneys at all time points tested (Fig. 3A and data not shown).

Figure 3
Near-infrared fluorescence imaging in mouse human tumor xenografts. A, Mice bearing U87MG tumors were intravenously injected with 1.5 nmol of Cy5.5-labeled c(RGDyK) or knottin peptides FN-RDG2, FN-RGD2, 2.5D, or 2.5F. Representative images are shown at ...

Knottin peptides as in vivo PET imaging probes

Next, we tested the potential of engineered knottin peptides for use as PET imaging probes in mice bearing U87MG human tumor xenografts. Higher tumor uptake was observed with 64Cu-DOTA-2.5D and 2.5F knottins relative to 64Cu-DOTA-FN-RGD2 and 64Cu-DOTA-c(RGDyK) (Fig. 4A,B). In microPET imaging, tumor uptake at 1 h post injection for 64Cu-DOTA-2.5D and 64Cu-DOTA-2.5F was 4.47 ± 1.21 %ID/g and 4.56 ± 0.64 %ID/g, respectively, compared to 64Cu-DOTA-FN-RGD2 (1.48 ± 0.53 %ID/g) and c(RGDyK) (2.32 ± 0.55 %ID/g). The knottin-based PET probes exhibited reduced liver uptake (~2 %ID/g) compared to 64Cu-DOTA-c(RGDyK), which showed significantly higher accumulation in the liver (4.19 ± 0.78 %ID/g and 3.59 ± 0.87 %ID/g at 1 and 4 h post injection, respectively). Tumor uptake was blocked by co-injection of 64Cu-DOTA-2.5D with a molar excess (0.5 μmol) of unlabeled c(RGDyK) (1.67 ± 0.28 %ID/g at 1 h post injection) (Fig. 4A,B). The PET signal generated by 64Cu-DOTA-FN-RDG2 reflects the rate of extravasation from the tumor vasculature and subsequent washout, and was found to be 1.09 ± 0.48 %ID/g and 0.76 ± 0.33 %ID/g at 1 and 4 h post injection, respectively.

Figure 4
MicroPET imaging and biodistribution in mouse human tumor xenografts. A, Representative microPET scans (coronal images (top), transverse image (bottom)) of U87MG tumor-bearing athymic mice after injection of 64Cu-DOTA-conjugated peptides or 64Cu-DOTA-2.5D ...

The biodistribution of 64Cu-DOTA-2.5D and 64Cu-DOTA-2.5F in various tissues and organs was determined at 0.5 and 4 h post-injection (Fig. 4C). Both knottin peptides accumulated rapidly in tumors 0.5 h post injection (4.2 ± 1.1 %ID/g for 2.5D and 5.3 ± 0.7 %ID/g for 2.5F). Knottin 2.5F cleared from the tumor at a slower rate than knottin 2.5D (3.4 ± 0.4 %ID/g versus 1.51 ± 0.02 %ID/g 4 h post injection) in agreement with the microPET data. High tumor uptake and rapid blood clearance led to tumor-to-blood ratios of 42.3 ± 8.65 for 64Cu-DOTA-2.5F and 24.93 ± 2.62 for 64Cu-DOTA-2.5D at 4 h post injection (Fig. 4D). In contrast to Cy5.5-labeled knottin peptides, which exhibited high kidney retention, minimal amounts of radioactivity remained in the kidneys after 24 h (1.25 ± 0.11 %ID/g for 64Cu-DOTA-2.5D and 1.09 ± 0.15 %ID/g for 64Cu-DOTA-2.5F, data not shown), indicating that conjugation of different chemical moieties to knottin peptides affected their pharmacokinetic properties. Moderate amounts of radioactivity were observed in the other major organs, including the lungs, skin, spleen, stomach, intestines (1 − 2 %ID/g), and also the liver (2.3 ± 0.8 %ID/g and 2.5 ± 0.2 %ID/g for 2.5D and 2.5F, respectively, 0.5 h post injection). Lower levels of activity (< 1 %ID/g) were present in the blood, heart, bone, brain, and pancreas (Fig. 4C). In addition, there was minimal background signal from muscle tissue (Fig. 4C,D), further demonstrating the potential of knottin peptides as diagnostic agents to detect lesions throughout the body.

Serum stability and metabolite analysis

Finally, we tested the stability of 64Cu-DOTA-2.5D in mouse serum and in the whole mouse blood, tumor, and kidney. First, radio-HPLC analysis was performed 1, 4, and 24 h after peptide incubation in mouse serum at 37 °C. Minimal breakdown products were observed at each time point (Supplementary Fig. S2 and Table S2). Next, the in vivo metabolic stability of 64Cu-DOTA-2.5D in whole mouse blood, tumor tissue, and kidney tissue were determined 1 and 4 h post injection (Fig. 5A-C). Radio-HPLC analysis of solubilized tissue homogenates showed a major elution peak between 19.5 minutes and 20 min, corresponding to the intact radiotracer. Metabolites with retention times between 4 to 6 min could be seen at significant levels in the kidneys after 4 h (Fig. 5C), indicating either breakdown of the probe in the kidneys or metabolites that are generated by various organs in the animal and cleared through renal excretion. Values of % intact tracer isolated from the serum, tumor, and kidneys are summarized in Supplementary Table S2.

Figure 5
In vivo metabolic stability of 64Cu-DOTA-conjugated knottin 2.5D. Homogenized tissues were analyzed by radio-HPLC and gamma counting 1 and 4 h post-injection. A, Mouse blood. B, tumor. C, kidney. The intact radiotracer elutes between 19.5 and 20 min.


There is a critical need for molecular imaging probes that will specifically target integrin receptors and allow noninvasive characterization of tumors for patient-specific cancer treatment and disease management (12, 14, 34). Here, we developed engineered knottin peptides as a new class of agents for imaging integrin-expression in living subjects. We determined that conjugation of Cy5.5 to the knottin peptides slightly increased their integrin binding affinity and decreased their integrin binding specificity, while conjugation of DOTA to the knottin peptides had no effect on integrin binding affinity or specificity (Table 1, Fig. 2, and Supplemental Fig. S1). We also showed that Cy5.5- and 64Cu-DOTA-conjugated FN-RGD2 knottin peptides, which bind to integrins with affinities in the low micromolar range, generated significantly weaker imaging signals compared to knottin peptides 2.5D and 2.5F (Fig. 3 and and4).4). These results strongly suggest that integrin binding affinity influences tumor uptake of knottin peptides, although other factors such as hydrophobicity can also affect tissue biodistribution. Interestingly, in PET studies knottin peptide 2.5F exhibited slower tumor washout compared to 2.5D, resulting in much higher tumor/blood ratios 4 h post injection (Fig 4D). This could be due to the ability of knottin 2.5F to bind more tightly to α5β1 integrins compared to knottin 2.5D (Fig. 2), or potential differences in peptide hydrophobicity, charge, or off-rates of integrin receptor binding. Finally, we demonstrated that knottin peptides were stable in vitro upon prolonged serum incubation, and in vivo in the tumor and blood during the timeframe in which imaging experiments were performed.

To evaluate the use of knottin peptides as molecular imaging agents compared to c(RGDyK), we performed in vivo experiments under identical conditions, as differences in amount of probe injected, image acquisition, and data analysis can influence tumor uptake values. Nevertheless, previously published biodistribution studies with 64Cu-DOTA-c(RGDyK) in a similar U87MG xenograft model showed tumor uptake values of ~2.5 %ID/g at 1 h post injection (35), consistent with our results. In addition to increased tumor uptake, high affinity 64Cu-DOTA-labeled knottin peptides 2.5D and 2.5F demonstrated more favorable tissue distribution as shown by lower liver uptake compared to 64Cu-DOTA-c(RGDyK). Collectively, our data indicate that knottin peptides have potential as diagnostic imaging agents to monitor multiple regions of the body including the chest and abdomen.

Several strategies have been used to improve the in vivo performance of monomeric c(RGDyK) peptides as imaging agents, and can also be applied in future studies to our engineered knottin peptides. Multivalent versions of c(RGDyK) peptides have been synthesized (28), and IC50 values were measured for competition binding of 125I-echistatin with monomeric (203 ± 32 nM), dimeric (103 ± 14 nM), tetrameric (34.6 ± 2.6 nM), and octameric (10.0 ± 1.7 nM) peptides on U87MG cells (36). These multivalent peptides exhibited only slight increases in integrin binding affinity, but resulted in much greater levels of tumor uptake compared to monomeric c(RGDyK). MicroPET imaging of these peptides in U87MG xenograft models indicated tumor uptake values of 9.6 ± 1.4 %ID/g and 10.6 ± 0.7 %ID/g for 64Cu-DOTA-c(RGDyK) tetramer and octamer, respectively, 1 h post injection, with little tumor washout after 20 h (36). One potential drawback of these monovalent or multivalent c(RGDyK) peptides is that they exhibit sustained liver uptake (~2.5−3 %ID/g) (36-38). Kidney uptake was more severely affected by peptide multimerization, with the 64Cu-DOTA-labeled octamer demonstrating greater than 50 %ID/g 1 h post injection (36). PEGylation has been used to improve the pharmacokinetics and tissue distribution of monomeric c(RGDyK) to address undesired liver and kidney uptake (35).

In this study, our goal was to compare the knottin peptides, which are monomeric and unmodified, with the first-generation monomeric, unmodified version of c(RGDyK). In future directions of this work, we are creating PEGylated versions of the knottin peptides, as well as oligomeric knottin proteins that present multiple integrin-binding RGD motifs. We expect these peptides will elicit enhanced tissue distribution and/or tumor uptake compared to unmodified knottin peptides, much like that observed with PEGylated and multivalent c(RGDyK) peptides, respectively. Here, we demonstrate that engineered integrin-binding knottin peptides are promising molecular imaging agents for clinical translation and future development, with potential applications in other imaging modalities including single photon emission computed tomography, targeted ultrasound, and magnetic resonance imaging.

Supplementary Material


We thank Frank V. Cochran for help with peptide synthesis and purification, and Zhe Liu and Zheng Miao for help with radiolabeling for PET experiments. Funded by the NIH/NCI Howard Temin Award 5K01 CA104706 and the Mallinckrodt Faculty Scholar Award (both to J.R.C.), NCI ICMIC P50 CA114747 and NCI 5R25T CA118681 (both to S.S.G.), and a NCI Molecular Imaging Scholars postdoctoral fellowship (to R.H.K.) (R25T CA118681).

Financial Support: NIH/NCI Howard Temin Award 5K01 CA104706 (JRC, RHK) Mallinckrodt Faculty Scholar Award (JRC) The Canary Foundation NCI ICMIC P50 CA114747 (SSG, ZC) NCI 5R25T CA118681 (SSG, RHK)


Potential conflicts of interest. The authors have declared that no competing interests exist.


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