Translator Disclaimer
1 May 2010 Cy5.5-labeled Affibody molecule for near-infrared fluorescent optical imaging of epidermal growth factor receptor positive tumors
Author Affiliations +
Affibody protein is an engineered protein scaffold with a three-helical bundle structure. Affibody molecules of small size (7 kD) have great potential for targeting overexpressed cancer biomarkers in vivo. To develop an Affibody-based molecular probe for in vivo optical imaging of epidermal growth factor receptor (EGFR) positive tumors, an anti-EGFR Affibody molecule, Ac-Cys-ZEGFR:1907 (7 kD), is site-specifically conjugated with a near-IR fluorescence dye, Cy5.5-mono-maleimide. Using fluorescent microscopy, the binding specificity of the probe Cy5.5-ZEGFR:1907 is checked by a high-EGFR-expressing A431 cell and low-EGFR-expressing MCF7 cells. The binding affinity of Cy5.5-ZEGFR:1907 (KD) to EGFR is 43.6±8.4 nM, as determined by flow cytometry. For an in vivo imaging study, the probe shows fast tumor targeting and good tumor contrast as early as 0.5 h postinjection (p.i.) for A431 tumors, while MCF7 tumors are barely visible. An ex vivo imaging study also demonstrates that Cy5.5-ZEGFR:1907 has high tumor, liver, and kidney uptakes at 24 h p.i.. In conclusion, Cy5.5-ZEGFR:1907 shows good affinity and high specificity to the EGFR. There is rapid achievement of good tumor-to-normal-tissue contrasts of Cy5.5-ZEGFR:1907, thus demonstrating its potential for EGFR-targeted molecular imaging of cancers.



Epidermal growth factor receptor (EGFR) is a transmembrane protein belonging to ErbB oncogene family. Overexpression of EGFR has been frequently detected in a wide range of human tumors, for example, non-small-cell lung cancer, small-cell carcinoma of the head and neck, esophageal cancer, gastric cancer, gliomas, colon cancer, pancreas cancer, breast cancer, ovarian cancer, bladder cancer, kidney cancer, and prostate cancer.1 Recently, EGFR has emerged as an attractive target for cancer therapy. EGFR-targeted therapies including the use of monoclonal antibodies (MAbs: cetuximab, panitumumab) and tyrosine kinase inhibitors [TKIs: gefitinib (Iressa), erlotinib (Tarceva), lapatinib (Tykerb)] have been developed and extensively studied.2, 3, 4 Meanwhile, molecular probes for EGFR imaging have also been under active investigation.5, 6, 7 It is expected that EGFR-specific molecular probes could potentially be used for early detection of EGFR positive tumors and metastases, prediction of efficacy for EGFR-targeted therapy, and for guidance in surgery etc.

A variety of small-molecule-based EGFR ligands and anti-EGFR antibodies are available for developing molecular probes for different imaging modalities. However, it has been found that small molecules labeled with radionuclides generally showed rapid blood clearance, but very low tumor uptake and poor tumor imaging quality.5, 6, 7 Moreover, these small molecules are relatively sensitive to chemical modifications with large bulky tags, and they may not be a good platform for developing optical imaging probes for EGFR. On the other hand, fluorescent dyes or radiolabeled anti-EGFR MAbs have been prepared and have demonstrated good tumor uptake. However, because of slow tumor targeting ability and slow clearance8, 9, 10 of the MAbs, the tumors can be visualized only at many hours or even days after injection of the probes. Substantial improvement of EGFR molecular probes is still required for preclinical and clinical applications.

Small protein scaffolds have shown great potential for recognizing a variety of biomarkers.11, 12 Among them Affibody molecules are a promising generalizable platform for developing imaging or therapeutic agents for different molecular targets.13, 14, 15 Affibody molecules are small, engineered proteins with only 58 amino acid residues and a three-helix bundle scaffold structure, while they display a binding surface as large as antibodies. High-affinity Affibody proteins (lower nanomolar or even picomolar) against different targets could be obtained through the phage display technique and affinity maturation. Because of the small size (7kDa) and high affinities, Affibody proteins generally show fast tumor targeting (within a half hour), high tumor uptake, and quick clearance from normal tissues. Moreover, Affibody proteins can be chemically synthesized with reasonable yield using conventional solid-phase peptide synthesis. Synthetic Affibodies retain the high binding specificity and affinity. Anti-human epidermal growth factor receptor 2 (HER2) Affibody molecules have been labeled with various radionuclides, organic dyes, and nanoparticles for imaging applications from cell to small animal imaging and even human.16, 17, 18 Dual-dye-labeled Affibody or even tri-labeled Affibody have also been reported.19 Overall, an Affibody protein scaffold has been proven to be an excellent platform for molecular probe development.

An anti-EGFR Affibody protein, ZEGFR:1907 , has shown strong binding (KD=5.4nM) to EGFR with no cross-binding to other growth factor receptors.20 It has been found that Cu64 - and In111 -labeled ZEGFR:1907 display fast and high tumor uptake ( 10% inject dose (ID)/g and 3to6%IDg , respectively, at 4h p.i.), as well as quick clearance from blood pool ( 5%IDg and 1.5to2%IDg , respectively, 4h p.i.) and EGFR nonexpression tissues20, 21, 22, 23, 24 (except kidney). We thus hypothesized that this Affibody could be a good protein for developing optical imaging probes for near-infrared (NIR) fluorescent imaging of EGFR positive tumors. In this research, a cysteine residue was first added to the N-terminus of ZEGFR:1907 . The resulting chemically synthesized protein, Ac-Cys- ZEGFR:1907 (Ac-CVDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK- NH2 ), was site specifically conjugated with a NIR dye, Cy5.5-mono-maleimide. The optical probe Cy5.5- ZEGFR:1907 was further evaluated in the EGFR positive human epithelial carcinoma A431 cell culture and tumor-bearing mice.


Materials and Methods


General Material and Reagent

Cy5.5-mono-maleimide was purchased from GE Healthcare (Piscataway, New Jersey). Dichloromethane, triethylamine, N-hydroxybenzotriazole hydrate (HOBT), diisopropylcarbodiimide (DIC), ethyl acetate, and dithiothriotol (DTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Missouri). Dimethylsulfoxide (DMSO) and ethyl acetate were purchased from Fisher Scientific (Pittsburgh, Pennsylvania). All the other standard reagents were purchased from Sigma-Aldrich Chemical Co. All chemicals were used without further purification.

The Affibody analog, Ac-Cys- ZEGFR:1907 , was prepared by a solid phase peptide synthesis method as previously described.21 Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed on a Dionex Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, California, for analysis and preparation of peptides and bioconjugates) equipped with a photodiode array detector. Five UV wavelengths (218, 254, 280, 500, and 675nm ) were monitored for all the experiments. Analytical RP-HPLC column from Sorbent Tech (Atlanta, Georgia) (Grace Vydac C4, 4.6×250mm ) was used for analysis of the labeled small protein. The mobile phase was solvent A, 0.1% trifluoroacetic acid (TFA)H2O , and solvent B, 0.1%TFA/acetonitrile. The flow rate was 1mLmin , with the mobile phase starting from 80% solvent A and 20% solvent B (0to2min) to 50% solvent A and 50% solvent B at 32min . Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS, model: Perseptive Voyager-DE RP Biospectrometer) (Framingham, Massachusetts) or an electrospray ionization quadrupole mass spectrometer (ESI-MS, model: Micromass ZQ single quadrupole LC-MS) (Milford, Massachusetts) was performed by the Stanford Protein and Nucleic Acid Biotechnology Facility and Stanford Chemistry Department Mass Spectrometry Facility, respectively. The human epithelial carcinoma cancer cell line A431 was obtained from the American Type Tissue Culture Collection (Manassas, Virginia). Female athymic nude mice (nu/nu) were purchased from Charles River Laboratories (Boston, Massachusetts).


Synthesis of Cy5.5- ZEGFR:1907

The general procedure for preparation of Cy5.5- ZEGFR:1907 is as follows. The Affibody molecule Ac-Cys- ZEGFR:1907 was dissolved in freshly degassed phosphate buffer ( 0.1M , pH 7.4) at a concentration of approximately 1mgmL . The NIR dye Cy5.5-mono-maleimide (300μg) in DMSO was then added (1.5 equivalents per equivalent of the Affibody). After vortexing for 2h , the reaction mixture was purified by the HPLC equipped with a Grace Vydac 214TP54-C4 column. Characterization of Cy5.5- ZEGFR:1907 was confirmed using MALDI-TOF-MS. The purity of the bioconjugate was confirmed by the analytical HPLC.

Fluorescence emission of Cy5.5- ZEGFR:1907 was measured on the Fluomax-3 fluorophotometer (HORIBA Jobin Yvon, Edison, New Jersey), and the spectrum was scanned from 650to850nm with an increment of 1nm . The wavelength of excitation light is 635nm . The absorption spectrum of Cy5.5- ZEGFR:1907 was recorded on an Agilent 8453 UV-visible ChemStation (Agilent Technologies, Wilmington, Delaware). The spectrum was scanned from 500to850nm with an increment of 1nm .


Fluorescence Microscopy Study

The A431 and MCF7 cells were cultured in high-glucose Dulbecco modified eagle medium (DMEM) and modified eagle medium (MEM) respectively supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen Life Technologies, Carlsbad, California). The cell line was maintained in a humidified atmosphere of 5% CO2 at 37°C , with the medium changed every other day. A confluent monolayer was detached with trypsin and dissociated into a single cell suspension for further cell culture.

For fluorescence microscopy study, A431 or MCF7 cells (1×104) were cultured on the 35-mm MatTek glass-bottom culture dishes (Ashland, Massachusetts). After 24h , the cells were washed with phosphate-buffered saline (PBS) and then incubated at 37°C with Cy5.5- ZEGFR:1907 (100nM) for 1h in dark. The EGFR-binding specificity of Cy5.5- ZEGFR:1907 in cell culture was verified by incubating the A431 and MCF7 cells with or without large amounts of (blocking dose) nonfluorescent Ac-Cys- ZEGFR:1907 peptide (10μM) . After the incubation period, cells were washed three times with ice-cold PBS. The fluorescence signal of the cells was recorded using an Axiovert 200M fluorescence microscope (Carl Zeiss MicroImaging, Inc., Thornwood, New Jersey) equipped with a Cy5.5 filter set (Exciter, HQ 65020nm ; Emitter, HQ 69535nm ). An AttoArc HBO 100-W microscopic illuminator was used as a light source for fluorescence excitation. Images were taken using a thermoelectrically cooled charge-coupled device (CCD) (Micromax, model RTE/CCD-576, Princeton Instruments Inc., Trenton, New Jersey) and analyzed using MetaMorph Software version 6.2r4 (Molecular Devices Corporation, Downingtown, Pennsylvania).


Binding Affinity Measurement with Flow Cytometry

The flow cytometric analysis was performed as described in the literature with minor modification.23 Samples were illuminated with a sapphire laser at 488nm and a red laser at 635nm on a BD FACSCalibur. The fluorescence and the forward-scattered and side-scattered light from 10,000 cells was detected at a rate of approximately 150eventss . Flow cytometric data were analyzed with FACSDiva Software (BD Biosciences, San Jose, California). Prior to the flow cytometric analysis, A431 cells seeded in 75-cm2 flasks approximately 3days before the experiment were trypsinized [0.05% trypsin to 0.02% ethylenediaminetetraacetic acid (EDTA), 2.5mL , 10min , 37°C ; Lonza Verviers S.P.R.L.] followed by a centrifugation ( 1000g , 3min ). The pellet was subsequently resuspended in the binding buffer [PBS containing 1% bovine serum albumin (BSA) and 0.1% sodium azide (NaAz)]. A431 cells ( 0.2M each, expressing 2×106 receptors/cell) were then distributed in Falcon tubes (15mL) . After centrifugation, the cells were incubated with 5mL of Cy5.5- ZEGFR:1907 at different concentrations (ranging from 0.1to100nM ) for 1h at room temperature in the dark, with continuous rocking. The cells were then centrifuged ( 178g , 4min , 4°C ), washed with the binding buffer twice, resuspended in 500μL of binding buffer, and subjected to flow cytometric analysis. All procedures after the incubation with the probe were performed on ice with ice-cold buffers. A triplicate of the flow cytometric data was analyzed with GraphPad Prism 5 (GraphPad Software, San Diego, California) and a nonlinear regression one-site-specific model was used to calculate the KD of the probe.


Optical Imaging of A431 Tumors in Mice

All animal studies were carried out according to a protocol approved by the Stanford University Administrative Panels on Laboratory Animal Care (APLAC). Female athymic nude mice (nu/nu) at 4to6weeks of age were injected subcutaneously in the shoulder with 5×106 A431 or MCF7 cells suspended in 100μL of PBS. For the MCF7 mice model, 4.7mg of estrogen was planted in the back 2days before inoculation. The tumor-bearing mice were subjected to in vivo optical imaging studies 2to3weeks after implantation.

In vivo fluorescence imaging was performed with an IVIS 200 small animal imaging system (Xenogen, Alameda, California). A Cy5.5 filter set (excitation 615to655nm ; emission 695to770nm ) 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 (pscm2sr) . Images were acquired and analyzed using Living Image 3.0 software (Xenogen, Alameda, California). For the positive control experiment, mice (n=3) were injected via tail vein with 0.5nmol of Cy5.5- ZEGFR:1907 (20nmolkg) and subjected to optical imaging at various time points p.i. For the blocking experiment, mice ( n=3 for each probe also) were injected with the mixture of 300μg of Ac-Cys- ZEGFR:1907 and 0.5nmol of Cy5.5- ZEGFR:1907 . IVIS-200 NIR fluorescent images were acquired using a 1-s exposure time ( fstop=4 , binning=4 ).

To subtract the autofluorescence, optical imaging using an IVIS spectrum small animal imaging system was also performed on mice for ex vivo imaging. The mice injected with Cy5.5- ZEGFR:1907 were euthanized at 24h p.i. and used for ex vivo fluorescence imaging. The tumor and other major tissues were dissected and placed on black papers. A slightly blue shifted excitation filters at 585to620nm was used to measure the tissue autofluorescence. The background filter image was then subtracted from the primary filter image (excitation filter 620to655nm ) of Cy5.5- ZEGFR:1907 fluorescence by image-math. IVIS-spectrum NIR fluorescent images were acquired using a 5-s exposure time ( fstop=4 , binning=4 ) because of the much narrower emission filter range (710to730nm) . The fluorescence images were acquired, and mean fluorescence flux (pscm2sr) for each sample was obtained.


Statistical Analysis

All the data are given as means±SD (standard deviation) of n independent measurements. Statistical analysis was performed using a Student’s t test. Statistical significance was assigned for P values <0.05 . To determine tumor contrast, mean fluorescence intensities of the tumor area (T) at the shoulder of the animal and of the area (N) at the right flank (normal tissue) were calculated by the region-of-interest (ROI) function of Living Image software integrated with Igor (Wavemetrics, Lake Oswego, Oregon). Dividing T by N yielded the contrast between tumor tissue and normal tissue.




Synthesis of Cy5.5- ZEGFR:1907

The Affibody small protein Ac-Cys- ZEGFR:1907 with a cysteine at the N terminal was successfully synthesized using a peptide synthesizer and purified by semipreparative HPLC. The peptide was generally obtained in 10% yield with over 95% purity [Fig. 1 ]. The purified Ac-Cys- ZEGFR:1907 was characterized by MALDI-TOF-MS. The measured molecular weight (MW) was consistent with the expected MW: mz=6690.0 for [M+H]+ (Calculated MW=6688.6 ). Ac-Cys- ZEGFR:1907 was then conjugated with Cy5.5-mono-maleimide in the PBS buffer and purified by HPLC. MS analysis verified the success preparation of the final product Cy5.5- ZEGFR:1907 (the observed MW: mz=7727.2 for [M+H]+ , calculated MW[M+Na]+=7727.4 ). The purity for the final product was over 95% (retention time: 25min ) [Fig. 1], and the recovery yield of the probe was 47%. Spectrum study showed that Cy5.5- ZEGFR:1907 had similar absorption and fluorescence emission characteristics of free Cy5.5 dye, with λab=680nm and λem=696nm [Fig. 1].

Fig. 1

(a) HPLC chromatogram of purified Cy5.5- ZEGFR:1907 and (b) Absorption and emission fluorescence spectra of Cy5.5- ZEGFR:1907 .



Binding Specificity and Affinity of Cy5.5- ZEGFR:1907

To demonstrate the EGFR specificity and subcellular localization of Cy5.5- ZEGFR:1907 , the probe was incubated with A431 tumor cells and fluorescent microscopic imaging was performed. With an 1h incubation at 37°C , intensive fluorescent signal was observed from the A431 cells membrane, and some of probe was also found to be inside of the cells (Fig. 2 ). Furthermore, the fluorescent signal from the cells could be significantly reduced by incubation of the cells with large excess of the Ac-Cys- ZEGFR:1907 (10μM) , demonstrating the binding specificity of Cy5.5- ZEGFR:1907 (Fig. 2). The staining of low EGFR expression MCF7 is barely visible. The flow cytometry study was performed for measurement of the binding affinity of Cy5.5- ZEGFR:1907 . A representative flow cytometric analysis was shown in Fig. 3 . The equilibrium dissociation constant KD was determined to be 43.6±8.4nM using a nonlinear regression one-site-specific model (Fig. 3).

Fig. 2

Cell uptake of Cy5.5- ZEGFR:1907 (100nM) in A431 and MCF7 cells at 37°C incubated with (blocking) or without the presence of the Affibody molecule Ac-Cys- ZEGFR:1907 (10μM) . NIR fluorescent (NIR) and light images (LI) are shown at the top and bottom, respectively.


Fig. 3

(a) Flow cytometric analysis of Cy5.5- ZEGFR:1907 binding to A431 cells at different concentrations (0.1to100nM) and (b) cell population gated against a control blank, where triplicate data at each concentration were fitted with a nonlinear regression one-site- specific model.



In Vivo and Ex Vivo Optical Imaging

Figure 4 shows typical NIR fluorescent images of nude mice bearing subcutaneous A431 and MCF7 tumor after intravenous injection of 0.5nmol of Cy5.5- ZEGFR:1907 . The A431 tumor could be clearly visualized from the surrounding background tissue from 0.5to4h p.i.. Quantification analysis of ROIs was performed and the tumor to normal tissue ratio (TN) as a function of time is depicted in Fig. 4. Cy5.5- ZEGFR:1907 exhibited a fast tumor targeting property in vivo. The tumor-to-normal tissue ratio reached a peak at 2h p.i. The receptor specificity of the probe was verified by the blocking experiment. Unlabeled Ac-Cys- ZEGFR:1907 significantly reduced tumor uptake and tumor contrast at all the time points [Figure 4]. Tumor contrast as quantified by ROI analysis of images indicated that the TN value at 4h p.i. was reduced from 2.9±0.1 to 1.8±0.4 (P<0.05) . At 4h p.i., MCF7 tumor showed much lower TN value (1.54±0.19) .

Fig. 4

(a) In vivo fluorescence IVIS-200 imaging of subcutaneous A431 and MCF7 tumor-bearing nude mice at 10min , 30min , 1h , 2h , and 4h . Cy5.5- ZEGFR:1907 (0.5nmol) with (bottom) or without (top) coinjection of unlabeled Affibody ZEGFR:1907 (300μg) were injected. Arrows indicate the location of tumors. (b) ROI analysis of tumor-to-normal tissue ratios of Cy5.5- ZEGFR:1907 in mice bearing A431 tumor at 10minto4h p.i. (n=3) .


The result for ex vivo tumor mice imaging at 24h p.i., as shown in Fig. 5 . Quantitative analysis showed that there is also large amount of probe trapped inside the kidney without clearance [Fig. 5]. The tumor-to-normal organ (except kidney) ratios are also high [Fig. 5]. For example, the ratios for tumor-to-lung and tumor-to-muscle are 5.6±1.8 and 6.5±0.4 , respectively.

Fig. 5

(a) Ex vivo imaging of tumor and normal tissues of Cy5.5- ZEGFR:1907 after sacrificed the mice at 24h p.i.: Tm, tumor; H, heart; L, lung; Lv, liver; Kn, kidney; Sp, spleen; St, stomach; B, blood; Sk, skin; Ms, Muscle. (B) ROI analysis of fluorescent signal from tumor and normal tissues. (b) Fluorescence intensity ratios of tumor-to-normal tissues based on the ROI analysis. Error bar was calculated as the standard deviation (n=3) .




NIR fluorescent imaging, which uses neither ionizing radiation nor radioactive materials, is emerging as a promising modality that can complement traditional nuclear imaging methods. Optical molecular probes with high sensitivity, stability, fast targeting, and rapid clearance could be prepared.17, 24, 25 Furthermore, optical imaging has very high resolution and shows great potential in clinical translation because of (1) the easy visualization of targeted tissues and (2) its use as supplementary visual aide to provide guidance in surgery.26 Anti-EGFR Affibody, ZEGFR:1907 , was radiolabeled with Cu64 in our recent study, which demonstrated good and fast tumor uptake and long retention.21 It is also of a great interest to us to apply this Affibody molecule for developing an optical imaging probe, since there is a high demand for an EGFR-targeted optical probe with an ideal in vivo imaging profile.

To achieve this goal, Affibody analog Cys- ZEGFR:1907 was synthesized to render site-specific labeling using maleimide chemistry. Conjugation of the small protein with Cy5.5-mono-maleimide was successfully achieved, moreover the absorption and emission fluorescence properties of the probe remain very close to the unconjugated dye (Fig. 1). The probe Cy5.5- ZEGFR:1907 also showed good binding affinity to the A431 cell surface EGFR with a KD value of 43.6±8.4nM , which is considered sufficient for further in vivo evaluation, although target avidity was lower than that of the unmodified Affibody ZEGFR:1907 (5nM) 20 [Fig. 3]. This result implies that the Affibody protein tolerates some N terminal modifications. Fluorescent microscopy of Cy5.5- ZEGFR:1907 showed that the majority of the probe bond to cell surface receptors after 1h incubation, indicating the slow internalization of the Affibody molecule, which is also consistent with previous finding of the radioactive counterpart.20, 21 More importantly, the specificity of the probe was verified with blocking by unlabeled Affibody (Fig. 2). These results warranted the further evaluation of the probe for in vivo EGFR-targeted tumor imaging.

Considering the high sensitivity of in vivo NIR imaging, 0.5nmol of Cy5.5- ZEGFR:1907 was injected into A431 mice, and optical imaging studies were performed using IVIS-200 systems. Consistent with our expectation, good tumor uptakes and imaging qualities were observed [Fig. 4]. The A431 tumor was clearly delineated from normal tissues at as early as 0.5h p.i. Quantification analysis of IVIS-200 images illustrated that tumor uptake reach plateau at 2h [Fig. 4]. These results were attributable to the fast tumor targeting and rapid blood clearance abilities of Affibody molecules. Kidney uptake was also very high even at 24h p.i., which is consisted with the radiometal labeled Affibody probes.20, 21, 27 Further ex vivo imaging study exhibited that tumor-to-blood ratio reached 8.5±3.6 at 24h p.i. Autofluorescence was very obvious for the skin, which increases the background. Ex vivo imaging also revealed that tumor-to-muscle ratio was as high as 6.5±0.4 . These properties are favorable and demonstrate the use of the optical probe for intraoperative optical imaging.

In vivo optical imaging of EGFR has extensively been investigated using monoclonal antibody such as cetuximab (Erbitux) labeled with NIR dyes.8, 9, 28 Cy5.5-labeled cetuximab (abbreviated as Cy5.5-Erbitux) was reported to target EGFR-high-expression MDA-MB-231 tumors and reached a maximum tumor to normal tissue ratio of ca 3 at 24h p.i.. However, because of the slow localization and clearance of the antibody, Cy5.5-Erbitux showed almost no difference between MDA-MB-231 tumor with high EGFR expression and MCF-7 with low expression9 before 6h p.i. In contrast, Cy5.5- ZEGFR:1907 demonstrated fast tumor accumulation ( TN ratio=2.64±0.46 at 1h p.i.) in the A431 model, while accumulation was very low in MCF7 tumor ( TN ratio=1.62±0.22 at 1h p.i., P<0.05 ), which is advantageous for both preclinical and clinical applications. NIR-dye-labeled EGF protein has also been reported for in vivo imaging.29, 30 However, EGF as a natural ligand has some side effects such as stimulation of EGFR phosphorylation, causing vomiting and diarrhea.30, 31 Finally, quantum dot conjugated with reduced EGF (r-Egf) was also reported, but the imaging result was poor and toxicity of quantum dot was a concern even at very low amount.32



Cy5.5- ZEGFR:1907 provided high-sensitivity, receptor-specific molecular imaging of EGFR-positive tumors. It showed good tumor-to-normal tissue ratios, fast tumor targeting ability, and quick blood clearance. Cy5.5- ZEGFR:1907 is a promising probe for further applications in EGFR-targeted optical imaging.


This work was supported, in part, by the California Breast Cancer Research Program 14IB-0091 (Z.C.) and an SNM Pilot Research Grant (to Z.C.). We also would like to thank Steven Jing for the help with Affibody synthesis and purification and Edwin Chang for helpful discussions.



F. X. Real, W. J. Rettig, P. G. Chesa, M. R. Melamed, L. J. Old, and J. Mendelsohn, “Expression of epidermal growth factor receptor in human cultured cells and tissues,” Cancer Res., 46 (9), 4726 –4731 (1986). 0008-5472 Google Scholar


E. Van Cutsem, C. H. Köhne, E. Hitre, J. Zaluski, C. R. Chang Chien, A. Makhson, G. D’Haens, T. Pintér, R. Lim, G. Bodoky, J. K. Roh, G. Folprecht, P. Ruff, C. Stroh, S. Tejpar, M. Schlichting, J. Nippgen, and P. Rougier, “Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer,” N. Engl. J. Med., 360 (14), 1408 –1417 (2009). 0028-4793 Google Scholar


J. G. Paez, P. A. Jänne, J. C. Lee, S. Tracy, H. Greulich, S. Gabriel, P. Herman, F. J. Kaye, N. Lindeman, T. J. Boggon, K. Naoki, H. Sasaki, Y. Fujii, M. J. Eck, W. R. Sellers, B. E. Johnson, and M. Meyerson, “EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy,” Science, 304 (5676), 1497 –1500 (2004). 0036-8075 Google Scholar


M. H. Cohen, J. R. Johnson, Y. F. Chen, R. Sridhara, and R. Pazdur, “FDA drug approval summary: erlotinib (Tarceva) tablets,” Oncologist, 10 461 –466 (2005). 1083-7159 Google Scholar


H. Su, Y. Seimbille, G. Z. Ferl, C. Bodenstein, B. Fueger, K. J. Kim, Y. T. Hsu, S. M. Dubinett, M. E. Phelps, J. Czernin, and W. A. Weber, “Evaluation of [F18]gefitinib as a molecular imaging probe for the assessment of the epidermal growth factor receptor status in malignant tumors,” Eur. J. Nucl. Med. Mol. Imaging, 35 (6), 1089 –1099 (2008). 1619-7070 Google Scholar


A. Pal, A. Glekas, M. Doubrovin, J. Balatoni, T. Beresten, D. Maxwell, S. Soghomonyan, A. Shavrin, L. Ageyeva, R. Finn, S. M. Larson, W. Bornmann, and J. G. Gelovani, “Molecular imaging of EGFR kinase activity in tumors with I124-labeled small molecular tracer and positron emission tomography,” Mol. Imaging Biol., 8 (5), 262 –277 (2006). 1536-1632 Google Scholar


H. Wang, J. Yu, G. Yang, X. Song, X. Sun, S. Zhao, and D. Mu, “Assessment of C11-labeled-4-N-(3-bromoanilino)-6,7-dimethoxyquinazoline as a positron emission tomography agent to monitor epidermal growth factor receptor expression,” Cancer Sci., 98 (9), 1413 –1416 (2007). Google Scholar


J. P. Gleysteen, J. R. Newman, D. Chhieng, A. Frost, K. R. Zinn, and E. L. Rosenthal, “Fluorescent labeled anti-EGFR antibody for identification of regional and distant metastasis in a preclinical xenograft model,” Head Neck, 30 (6), 782 –789 (2008). 1043-3074 Google Scholar


K. Wang, K. Wang, W. Li, T. Huang, R. Li, D. Wang, B. Shen, and X. Chen, “Characterizing breast cancer xenograft epidermal growth factor receptor expression by using near-infrared optical imaging,” Acta Radiol., 11 1 –9 (2009). 0284-1851 Google Scholar


C. R. Divgi, S. Welt, M. Kris, F. X. Real, S. D. J. Yeh, R. Gralla, B. Merchant, S. Schweighart, M. Unger, S. M. Larson, and J. Mendelsohn, “Phase I and imaging trial of indium-111-labeled anti-epidermal growth factor receptor monoclonal antibody 225 in patients with squamous cell lung carcinoma,” J. Natl. Cancer. Inst. (Bethesda), 83 (2), 97 –104 (1991). Google Scholar


R. J. Hosse, A. Rothe, and B. E. Power, “A new generation of protein display scaffolds for molecular recognition,” Protein Sci., 15 14 –27 (2006). 0961-8368 Google Scholar


H. K. Binz, P. Amstutz, and A. Pluckthun, “Engineering novel binding proteins from nonimmunoglobulin domains,” Nat. Biotechnol., 23 (10), 1257 –1268 (2005). 1087-0156 Google Scholar


K. Nord, E. Gunneriusson, J. Ringdahl, S. Ståhl, M. Uhlén, and P. Å. Nygren, “Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain,” Nat. Biotechnol., 15 (8), 772 –777 (1997). 1087-0156 Google Scholar


A. Orlova, V. Tolmachev, R. Pehrson, M. Lindborg, T. Tran, M. Sandström, F. Y. Nilsson, A. Wennborg, L. Abrahmsén, and J. Feldwisch, “Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors,” Cancer Res., 67 (5), 2178 –2186 (2007). 0008-5472 Google Scholar


V. Tolmachev, A. Orlova, R. Pehrson, J. Galli, B. Baastrup, K. Andersson, M. Sandström, D. Rosik, J. Carlsson, H. Lundqvist, A. Wennborg, and F. Y. Nilsson, “Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific Affibody molecule,” Cancer Res., 67 (6), 2773 –2782 (2007). 0008-5472 Google Scholar


Z. Cheng, O. P. De Jesus, M. Namavari, A. De, J. Levi, J. M. Webster, R. Zhang, B. Lee, F. A. Syud, and S. S. Gambhir, “Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules,” J. Nucl. Med., 49 (5), 804 –813 (2008). 0161-5505 Google Scholar


S. B. Lee, M. Hassan, R. Fisher, O. Chertov, V. Chernomordik, G. Kramer-Marek, A. Gandjbakhche, and J. Capala, “Affibody molecules for in vivo characterization of HER2-positive tumors by near-infrared imaging,” Clin. Cancer Res., 14 (12), 3840 –3849 (2008). 1078-0432 Google Scholar


R. P. Baum, A. Orlova, V. Tolmachev, and J. Feldwisch, “Receptor PET/CT and SPECT using an Affibody molecule for targeting and molecular imaging of HER2-positive cancer in animal xenografts and human breast cancer patients [abstract],” J. Nucl. Med., 47 (Supp. l), 108P (2006). 0161-5505 Google Scholar


T. Engfeldt, B. Renberg, H. Brumer, P. Å. Nygren, and A. E. Karlström, “Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein,” ChemBioChem, 6 (6), 1043 –1050 (2005). 1439-4227 Google Scholar


M. Friedman, A. Orlova, E. Johansson, T. L. Eriksson, I. Höidén-Guthenberg, V. Tolmachev, F. Y. Nilsson, and S. Ståhl, “Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding Affibody molecule,” J. Mol. Biol., 376 (5), 1388 –1402 (2008). 0022-2836 Google Scholar


Z. Miao, G. Ren, H. Liu, L. Jiang, and Z. Cheng, “Small-animal PET imaging of human epidermal growth factor receptor positive tumor with a Cu64 labeled affibody protein,” Bioconjugate Chem., 21 (5), 947 –954 (2010). Google Scholar


Z. Miao, G. Ren, H. Liu, L. Jiang, S. S. Gambhir, and Z. Cheng, “A protein scaffold based molecule for EGFR PET imaging,” J. Nucl. Med., 50 (2), 386 (2009). 0161-5505 Google Scholar


V. Tolmachev, M. Friedman, M. Sandström, T. L. Eriksson, D. Rosik, M. Hodik, S. Ståhl, F. Y. Frejd, and A. Orlova, “Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry,” J. Nucl. Med., 50 (2), 274 –283 (2009). 0161-5505 Google Scholar


T. Troy, D. Jekic-McMullen, L. Sambucetti, and B. Rice, “Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models,” Mol. Imaging, 3 (1), 9 –23 (2004). 1535-3508 Google Scholar


Z. Cheng, Y. Wu, Z. Xiong, S. S. Gambhir, and X. Chen, “Near-infrared fluorescent RGD peptides for optical imaging of integrin αvβ3 expression in living mice,” Bioconjugate Chem., 16 (6), 1433 –1441 (2005). 1043-1802 Google Scholar


A. Nakayama, F. del Monte, R. J. Hajjar, and J. V. Frangioni, “Functional near-infrared fluorescence imaging for cardiac surgery and targeted gene therapy,” Mol. Imaging, 1 (4), 365 –77 (2002). 1535-3508 Google Scholar


Z. Cheng, O. P. De Jesus, D. J. Kramer, A. De, J. M. Webster, O. Gheysens, J. Levi, M. Namavari, S. Wang, J. M. Park, R. Zhang, H. Liu, B. Lee, F. A. Syud, and S. S. Gambhir, “Cu64-labeled Affibody molecules for imaging of HER2 expressing tumors,” Mol. Imaging Biol., 12 316 –324 (2009). 1536-1632 Google Scholar


K. P. Withrow, J. R. Newman, J. B. Skipper, J. P. Gleysteen, J. S. Magnuson, K. Zinn, and E. L. Rosenthal, “Assessment of bevacizumab conjugated to Cy5.5 for detection of head and neck cancer xenografts,” Technol. Cancer Res. Treat., 7 (1), 61 –66 (2008). 1533-0346 Google Scholar


S. Ke, X. Wen, M. Gurfinkel, C. Charnsangavej, S. Wallace, E. M. Sevick-Muraca, and C. Li, “Near-infrared optical imaging of epidermal growth factor receptor in breast cancer xenografts,” Cancer Res., 63 (22), 7870 –7875 (2003). 0008-5472 Google Scholar


K. E. Adams, S. Ke, S. Kwon, F. Liang, Z. Fan, Y. Lu, K. Hirschi, M. E. Mawad, M. A. Barry, and E. M. Sevick-Muraca, “Comparison of visible and near-infrared wavelength-excitable fluorescent dyes for molecular imaging of cancer,” J. Biomed. Opt., 12 (2), 024017 (2007). 1083-3668 Google Scholar


A. Cuartero-Plaza, E. Martínez-Miralles, R. Rosell, C. Vadell-Nadal, M. Farré, and F. X. Real, “Radiolocalization of squamous lung carcinoma with I131-labeled epidermal growth factor,” Clin. Cancer Res., 2 (1), 13 –20 (1996). 1078-0432 Google Scholar


P. Diagaradjane, J. M. Orenstein-Cardona, N. E. Colón-Casasnovas, A. Deorukhkar, S. Shentu, N. Kuno, D. L. Schwartz, J. G. Gelovani, and S. Krishnan, “Imaging epidermal growth factor receptor expression in vivo: pharmacokinetic and biodistribution characterization of a bioconjugated quantum dot nanoprobe,” Clin. Cancer Res., 14 (3), 731 –741 (2008). 1078-0432 Google Scholar
©(2010) Society of Photo-Optical Instrumentation Engineers (SPIE)
Zheng Miao, Gang Ren, Hongguang Liu, Lei Jiang, and Zhen Cheng "Cy5.5-labeled Affibody molecule for near-infrared fluorescent optical imaging of epidermal growth factor receptor positive tumors," Journal of Biomedical Optics 15(3), 036007 (1 May 2010).
Published: 1 May 2010

Back to Top