Evaluation of Four Affibody-Based Near-Infrared Fluorescent Probes

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Evaluation of Four Affibody-Based Near-Infrared Fluorescent Probes for Optical Imaging of Epidermal Growth Factor Receptor Positive Tumors Shibo Qi,†,‡ Zheng Miao,† Hongguang Liu,† Yingding Xu,† Yaqing Feng,‡ and Zhen Cheng*,† †

Molecular Imaging Program at Stanford (MIPS), Department of Radiology, and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California, 94305-5344 ‡ School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China ABSTRACT: The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity for EGFR, ZEGFR:1907, has been reported. We are interested in translating Affibody molecules to potential clinical optical imaging of EGFR positive cancers. In this study, four anti-EGFR Affibody based near-infrared (NIR) fluorescent probes were thus prepared, and their in vivo performance was evaluated in the mice bearing EGFR positive subcutaneous A431 tumors. Methods: The Affibody analogue, Ac-Cys-ZEGFR:1907, was synthesized using solid-phase peptide synthesis method. The purified small protein was then site-specifically conjugated with four NIR fluorescent dyes, Cy5.5-monomaleimide, Alex-Fluor-680-maleimide, SRfluor680-maleimide, or IRDye-800CW-maleimide, to produce four optical probesCy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, SR680ZEGFR:1907, and 800CW-ZEGFR:1907. The EGFR binding property and specificity of the four NIR fluorescent Affibody probes were studied by fluorescence microscopy using high EGFR expressing A431 cells and low expressing MCF7 cells. The binding affinities of the probes (KD) to EGFR were further determined by flow cytometry. In vivo optical imaging of the four probes was performed in the mice bearing subcutaneous A431 tumors. Results: The four NIR optical probes were prepared in high purity. In vitro cell imaging studies demonstrated that all of them could specifically bind to EGFR positive A431 cells while showing minimum uptake in low EGFR expressing MCF7 cells. Flow cytometry showed that Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 possessed high binding affinity in low nanomolar range (43.6 ± 8.4 and 28.3 ± 4.9, respectively). In vivo optical imaging of the four probes revealed that they all showed fast tumor targeting ability and good tumor-to-normal tissue contrast as early as 0.5 h postinjection (p.i.). The tumor-to-normal tissue ratio reached a peak at 2 to 4 h p.i. by regional of interest (ROI) analysis of images. Ex vivo studies further demonstrated that the four probes had high tumor uptakes. Particularly, Cy5.5-ZEGFR:1907 and Alex680-ZEGFR:1907 displayed higher tumor-to-normal tissue ratios than those of the other two probes. Conclusion: This work demonstrates that Affibody proteins can be modified with different NIR fluorescent dyes and used for imaging of EGFR expressing tumors. Different NIR fluorescent dyes have variable impact on the in vitro binding and in vivo performance of the resulting Affibody based probes. Therefore, selection of an appropriate NIRF label is important for optical probe development. The probes developed are promising for further tumor imaging applications and clinical translation. Particularly, Alex680-ZEGFR:1907 and Cy5.5-ZEGFR:1907 are excellent candidates as EGFR-targeted probes for optical imaging.

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INTRODUCTION Epidermal growth factor receptor (EGFR) is a very important tumor target and has been found to be overexpressed in a wide range of human tumors such as nonsmall cell lung cancer, small cell carcinoma of head and neck, colon cancer, breast cancer, ovary cancer, prostate cancer, and so forth.1 Many biopharmaceuticals and drugs including Cetuximab, Lapatinib, Gefitinib, and Erlotinib have been developed and extensively studied for EGFR targeted therapies.2−5 Novel molecular probes for EGFR imaging have also been under active investigation.6,7 The EGFR specific molecular probes are expected to be very useful in many applications including early detection of EGFR positive tumors and metastases, surgical guidance, and prediction of EGFR targeted therapy efficacy. Small protein scaffolds have recently shown great potential for molecular recognition.8,9 The Affibody protein scaffold is a great example that is based on the Z-domain scaffold derived © 2012 American Chemical Society

from one of the IgG-binding domains of staphylococcal protein A (SPA) and has been demonstrated to be a generalizable platform for developing imaging and therapeutic agents.10 Affibody molecules are composed of 58-amino-acid residues and form a three-helix bundle scaffold structure. High-affinity Affibody proteins against different targets could be readily constructed through randomization of the 13 amino acid residues in helices 1 and 2.11−13 Because of the small size and high affinities, Affibody proteins have the advantages of fast tumor targeting, high tumor uptake, and quick clearance from normal tissues. With reasonable yield, Affibody proteins can be obtained using conventional solid-phase peptide synthesis; importantly, high binding affinity and specificity are retained Received: November 10, 2011 Revised: April 20, 2012 Published: May 23, 2012 1149

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weight = 1231) was purchased from Molecular Targeting Technologies Inc. (West Chester, PA), and IRDye-800CWmalelimide was purchased from LI-COR Biosciences (Lincoln, NE). Dichloromethane, triethylamine, N-hydroxybenzotriazole hydrate (HOBT), and ethyl acetate were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific (Pittsburgh, PA). All the other standard reagents were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). The purification of the crude product was carried out on a semipreparative reversed-phase high-performance liquid chromatography (HPLC) system equipped with a 170U 5-Channel UV−vis absorbance detector (Dionex Summit HPLC systme, Dionex Corporation, Sunnyvale, CA) using a Vydac protein and peptide column (218TP510, 5 μm 250 × 10 mm). The mobile phase was solvent A, 0.1% trifluoroacetic acid (TFA)/ H2O, and solvent B, 0.1% TFA/acetonitrile. The flow rate was 1 mL/min, with the mobile phase starting from 65% solvent A and 35% solvent B (0−2 min) to 55% solvent A and 45% solvent B at 32 min for Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, and 800CW-ZEGFR:1907. While for SR680-ZEGFR:1907, the mobile phase started from 60% solvent A and 40% solvent B (0−2 min) to 50% solvent A and 50% solvent B at 32 min. Matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, model: Perspective Voyager-DE RP Biospectrometer) (Framingham, MA) was performed by the Stanford Protein and Nucleic Acid Biotechnology Facility. The human epithelial carcinoma cancer cell line A431 was obtained from the American Type Tissue Culture Collection (Manassas, VA). Female athymic nude mice (nu/nu) were purchased from Charles River Laboratories (Boston, MA). Synthesis of Ac-Cys-ZEGFR:1907 and Conjugation of NIRF Dyes with Ac-Cys-ZEGFR:1907. The Affibody analogue, Ac-Cys-ZEGFR:1907, was prepared by a solid-phase peptide synthesis method as previously described.19,25 The peptide was purified by a RP-HPLC on a C-4 column. The target product was collected and lyophilized. Characterization of the Affibody analogue was confirmed using MALDI-TOF-MS. The purity of the product was confirmed by the analytical HPLC. The general procedure for the conjugation of each of the four monomaleimide-NIRF dye with Ac-Cys-ZEGFR:1907 is as follows. The Affibody molecule was dissolved in freshly degassed phosphate buffer (0.1 M, pH 7.4) at concentration of approximately 1 mg/mL. The NIRF dye in DMSO was then irrespectively added (2.5 equiv of the Affibody). After vortexing for 3 h at room temperature, the crude product was purified by a RP-HPLC on a C-4 column. The fractions containing the probes were collected and lyophilized. Characterization of the four probes was also carried out using MALDI-TOF-MS. The purities of the four probes were confirmed by the analytical HPLC. Fluorescence emission of the four probes was measured on the Fluomax-3 fluorophotometer (HORIBA, Jobin Yvon, Edison, NJ), and the spectrum was scanned from 700 to 850 nm (for Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, and SR680ZEGFR:1907) or 780 to 850 nm (for 800CW-ZEGFR:1907) with increments of 5 nm. The absorption spectra of the four probes were recorded on an Agilent 8453 UV−visible ChemStation (Agilent Technologies, Wilmington, DE). The spectrum was scanned from 450 to 850 nm with increments of 1 nm. Fluorescence Microcopy Studies. The A431 cells and MCF7 cells were cultured in high-glucose Dulbecco modified Eagle's medium (DMEM) and modified Eagle's medium

through the synthetic process. In the past few years, Affibody molecules with high affinities and specificities against a variety of targets including EGFR, human epidermal growth factor receptor 2 (HER2), platelet derived growth factor receptor (PDGFR), and others have been constructed. Additionally, Affibody proteins have been labeled with various radionuclides, organic dyes, and nanoparticles for imaging applications.14−17 These studies have demonstrated that Affibody molecules are a promising platform for tumor targeting and therapy. An anti-EGFR Affibody protein, ZEGFR:1907, has shown strong binding affinity to EGFR with good specificity.18 64Cu and 111In labeled ZEGFR:1907 display fast tumor targeting and excellent imaging contrast.19,20 Of note, this Affibody molecule possesses a good binding affinity to human EGFR expressed in A431 cells and a good tumor uptake in murine liver, spleen, and colon, all of which express a high level of EGFR.18 Additionally, binding of the native ligand EGF to the murine organs can be saturated, and thus, both pieces of evidence suggest that ZEGFR:1907 possesses cross-reactivity with both human and murine EGFR.21 We thus hypothesized that this Affibody protein could be utilized to develop probes for near-infrared (NIR) fluorescent (NIRF) imaging of EGFR positive tumors using murine models. In this research, a cysteine residue was first added to the N-terminus of Ac-Cys-ZEGFR:1907. The resulting chemically synthesized protein, Ac-CVDNKFNKEMWAAW E E I R NL P N L N G W Q M T A F I A S L V D D P S QS A N L LAEAKKLNDAQAPK-NH2, was then site specifically labeled with four different NIR dyes, Cy5.5-monomaleimide, AlexaFluor-680-maleimide, SRfluor680-maleimide (SR-1004, a farred emitting dye which belongs to the squaraine rotaxane family of dyes),22−24 or IRDye-800CW-malelimide, to generate optical probe, Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, SR680-ZEGFR:1907, 800CW-ZEGFR:1907, respectively (Figure 1). All of the four NIR

Figure 1. Schematic structure of anti-EGFR Affibody protein based NIR fluorescent probes.

dyes are commercially available and have demonstrated both chemical and photochemical stabilities. The four Affibodybased NIR fluorescent probes were further evaluated in EGFR positive human epithelial carcinoma A431 cell culture and tumor bearing mice with the goal to systematically compare in vitro and in vivo characteristics of the four NIR dyes in the context of ZEGFR:1907 analogues, and with the additional goal of discovering a good Affibody based optical imaging probe for EGFR positive tumors.

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MATERIALS AND METHODS General. All commercially available chemical reagents were used without further purification. Cy5.5-monomaleimide was purchased from GE Healthcare (Piscataway, NJ). Alexa-Fluor680-C2 maleimide was purchased from Invitrogen Co. (Carlsbad, CA), SRfluor680-maleimide (SR-1004, molecular 1150

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The tumor-bearing mice were subjected to in vivo optical imaging studies 3 weeks after inoculation. In vivo fluorescence imaging was performed with an IVIS 200 small animal imaging system (Caliper, Alameda, CA). A Cy5.5 filter set (excitation 615 to 655 nm; emission 695 to 770 nm) was used for acquiring Cy5.5-ZEGFR:1907 fluorescence in vivo. Identical illumination settings (lamp voltage, filters, f/stop, fields of view, binning) were used to acquire all images, 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 3.0 software (Caliper, Alameda, CA). For the experiment, mice (n = 5) were injected via tail vein with 0.5 nmol of each probe (20 nmol/kg) and subjected to optical imaging at various time points postinjection (p.i.). IVIS-200 NIRF images were acquired using 1 s exposure time (f/stop = 4, binning = 4). The mice in the experiment were then euthanized at 24 h p.i. The tumor and major tissues and organs were dissected, wet weighted, and placed on black paper. The ex vivo fluorescence images were acquired, and mean fluorescence flux (p/s/cm2/sr) for each sample was obtained, and the tumor to normal tissue ratios were then calculated. Statistical Analysis. All the data are given as mean ± SD (standard deviation) of independent measurements. Statistical analysis was performed using a Student’s t test. Statistical significance was assigned for p values 0.05). In contrast, 800CW-ZEGFR:1907 exhibited much lower tumor to normal organ ratios in general (Figure 5B, p < 0.05).

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DISCUSSION NIRF imaging is emerging as a powerful tool for noninvasively imaging diseases in preclinical studies, and it also has a great potential in clinical applications such as providing real-time surgical information, as well as functional/molecular information of the disease process. Optical molecular probes with high sensitivity, stability, fast targeting, and rapid clearance can usually be readily prepared.15,26 EGFR is an important tumor biomarker. In vivo optical imaging of EGFR has been extensively investigated using monoclonal antibody such as cetuximab (Erbitux) labeled with NIR dyes.27,28 However, Cy5.5-labeled cetuximab suffered from slow localization and clearance. NIR dye labeled EGF protein has also been studied for in vivo imaging.29 However, EGF as a native ligand has significant side effects such as vomiting and diarrhea, as consequences of stimulation of EGFR.30 Furthermore, NIR nanoparticles such as quantum dots have also been used to conjugate with reduced EGF(r-Egf) for imaging. However, this method was reported to suffer from poor imaging quality and significant toxicity.31 Therefore, new strategies for development of optical probes for EGFR are highly desired. Affibody proteins have been demonstrated to be great platforms for molecular probe development.13,14 HER2 and EGFR specific Affibody molecule based optical probes have been studied and successfully used for cell imaging and in vivo tumor imaging.19,20,29,30,32,33 In one of our recent studies, the 1153

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Figure 4. (A) In vivo fluorescence imaging of subcutaneous A431 tumor-bearing nude mice at 0.25, 0.5, 1, 2, 4, and 12 h after injection of 0.5 nmol Cy5.5-ZEGFR:1907 (1), Alexa680-ZEGFR:1907 (2), SR680ZEGFR:1907 (3), or 800CW-ZEGFR:1907 (4). Arrows in blue color and ovals in green color indicate the location of tumors and kidneys, respectively. (B) ROI analysis of tumor-to-normal tissue ratios of the four probes in mice bearing A431 tumor at different time points p.i. (n = 5).

Figure 5. (A) Representative ex vivo NIRF images of dissected organs of mice bearing A431 tumor sacrificed 24 h after intravenous injection of Cy5.5-ZEGFR:1907 (Probe 1), Alexa680-ZEGFR:1907 (Probe 2), SR680ZEGFR:1907 (Probe 3), or 800CW-ZEGFR:1907 (Probe 4) at a dose of 0.5 nmol per mouse. The numeric label for each organ is as follows: 1: Blood; 2: Heart; 3: Lung; 4: Liver; 5: Spleen; 6: Pancreas; 7: Stomach; 8: Intestines; 9: Kidneys; 10: Skin; 11: Muscle; 12: Bone; 13: Tumor; 14: Brain. (B) Fluorescence intensity ratios of tumor-to-normal tissues based on the ROI analysis. Error bar was calculated as the standard deviation (n = 5).

absorption and emission fluorescence properties of the probes remained very close to the unconjugated dyes (Figure 2B). Using FACS analysis, it was found that Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 exhibited high binding affinity, while SR680-ZEGFR:1907 had much lower binding affinity (Table 1), suggesting that different fluorescent dyes could affect the bioactivity of the resulting probes. The cell uptake and binding specificity of the four NIR fluorescent Affibody probes were studied with high-EGFR-expressing A431 cells as well as lowEGFR-expressing MCF7 cells using fluorescence microscopy. The results showed that with all four fluorescent Affibody analogues the majority of the probes bound to cell surface receptors at 1 h incubation, indicating slow internalization of the Affibody molecules. Importantly, the specificity of all the probes was verified by both blocking with unlabeled Affibody and their low uptakes in low EGFR expressing MCF7 cells (Figure 3). Interestingly, it was also found that SR680ZEGFR:1907 displayed low signals in A431 cells with a high background. This could be caused by its relatively low binding affinity than that of Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907. In general, the reasonable tumor cell binding affinity and high specificity warranted further evaluations and characterizations of the four probes with in vivo tumor imaging study, particularly

Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907, given their high binding affinity. Whole-body optical imaging of subcutaneous tumor xenograft mice was then performed by the IVIS-200 system. As shown in Figure 4A, the subcutaneous A431 tumor could be clearly distinguished from the surrounding tissue from 0.25 h up to 12 h p.i. of 0.5 nmol of each probe. It was found that all probes showed fast tumor targeting and good T/N contrast as early as 0.25 h p.i.; the T/N ratio reached the peak at 2 to 4 h p.i. by ROI analysis of images (Figure 4B) before the probes were slowly washed out over time. The favorable in vivo imaging results of these probes are attributable at least partially to fast tumor targeting and rapid blood clearance abilities of Affibody molecules. Although all four probes displayed favorable in vitro and in vivo characteristics, there did exist some significant differences. In particular, Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 showed higher T/N ratios than other two, though it is not significant, 1154

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ACKNOWLEDGMENTS This work was supported, in part, by a Startup fund from Department of Radiology, Stanford University (to Z.C.), and a fellowship from China Scholarship Council (to S. Q.).

which is consistent with the fact that these two probes possess high binding affinity (Figure 4B). More importantly, compared to Cy5.5-ZEGFR:1907 and 800CW-ZEGFR:1907, both Alexa680ZEGFR:1907 and SR680-ZEGFR:1907 were found to show much lower uptakes in kidneys especially at later time points (Figure 4A and Figure 5A). Their distribution patterns in liver, which is characterized by EGFR overexpression, were also variable. While Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, and SR680ZEGFR:1907 all showed expectedly high liver uptakes that were comparable to corresponding tumor uptakes from 0.25 to 12 h p.i., 800CW-ZEGFR:1907 showed significantly lower liver uptake than its tumor uptake during the same time period (Figure 4A, p < 0.05). At 24 h p.i., however, Figure 5 showed that for all probes except 800CW-ZEGFR:1907 tumor-to-liver ratios reached upward of 3-to-1, which indicated a more rapid washout from liver when compared with that of tumor for these 3 probes. The only exception is 800CW-ZEGFR:1907, for which tumor-to-liver ratio approximated 1-to-1 at 24 h p.i.. This can be explained by a high washout rate from tumor and a relatively low washout rate from liver. Unfortunately, we were not able to characterize 800CW-ZEGFR:1907 further regarding its binding affinity, but from in vitro fluorescent microscopy, we know that this probe displays inferior cell uptake, particularly when compared with Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907. The ex vivo imaging results also demonstrated that 800CW-ZEGFR:1907 had the lowest tumor-to-normal organ ratio. It should also be noted here that there is one caveat when using OI to image liver. Liver, similar to spleen and kidneys, is notable for the dark color as well as a high concentration of hemoglobin, both of which can lead to a significant absorption of luminescent signals. Therefore, special considerations should be taken when interpreting liver uptake results from OI studies, particularly when one would like to compare these with nuclear imaging studies. Collectively, these data suggest that different NIRF dyes have variable impact on the in vivo behavior of the resulting Affibody-based probes. It is highly important to investigate the effects of different dyes on the in vivo profile of a biomolecule. Because of the high affinity and contrast, for further applications, Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 are the most promising among all the probes tested in this research.

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ABBREVIATIONS: EGFR, Epidermal growth factor receptor 1; NIR, near-infrared; PET, positron emission tomography; HPLC, high-performance liquid chromatography; p.i., postinjection

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REFERENCES

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CONCLUSION All anti-EGFR Affibody based NIRF probes studied in this research show good tumor imaging quality, tumor-to-normal tissue ratios, and fast tumor targeting ability, which demonstrates that the Affibody molecule Ac-Cys-ZEGFR:1907 can be used for tumor EGFR optical imaging. Especially, Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 are shown to be the most promising probes for further tumor imaging applications and clinical translations. Furthermore, it also demonstrates that different NIRF dyes have different impact on the in vitro binding and in vivo performance of the resulting Affibody based probes. Therefore, selection of an appropriate NIRF label is important for optical probe development.

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AUTHOR INFORMATION

Corresponding Author

*650-723-7866 (Voice), 650-736-7925 (Fax), E-mail: zcheng@ stanford.edu. Notes

The authors declare no competing financial interest. 1155

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dx.doi.org/10.1021/bc200596a | Bioconjugate Chem. 2012, 23, 1149−1156