Evaluation of a Maleimido Derivative of NOTA for Site-Specific

Mar 29, 2011 - Section of Medical Physics, Department of Oncology, Uppsala University Hospital, Uppsala, Sweden. §. Division of Molecular Biotechnolo...
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Evaluation of a Maleimido Derivative of NOTA for Site-Specific Labeling of Affibody Molecules Vladimir Tolmachev,*,‡ Mohamed Altai,‡ Mattias Sandstr€om,† Anna Perols,§ Amelie Eriksson Karlstr€om,§ Frederic Boschetti,|| and Anna Orlova‡ ‡

Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Sweden Section of Medical Physics, Department of Oncology, Uppsala University Hospital, Uppsala, Sweden § Division of Molecular Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden CheMatech, Djion, France

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†

ABSTRACT: Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMANOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule ZHER2:2395 resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys61]ZHER2:2395 with 111In gave a yield of >95% after 20 min at 60 °C. In vitro cell tests demonstrated specific binding of [111InMMA-NOTA-Cys61]-ZHER2:2395 to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [111In-MMA-NOTA-Cys61]-ZHER2:2395 was 8.2 ( 0.9% IA/g and the tumor-to-blood ratio was 31 ( 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [111In-MMA-NOTA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [111In-MMANOTA-Cys61]-ZHER2:2395 due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.

’ INTRODUCTION Increasing specificity of cancer treatment is currently based on molecular targeting of malignancy-associated molecular alterations in cancer cells. Both patient stratification for therapy and monitoring of therapy response require accurate detection of the expression level of a molecular target at the onset of therapy and during treatment.1,2 Radionuclide imaging of molecular targets might be a valuable clinical tool, since it is less sensitive to heterogeneity of target expression and noninvasive permitting serial investigations, which is essential for pharmacodynamic studies.1,2 A new category of promising radionuclide imaging probes, scaffold-based proteins, has emerged during recent years.3 The scaffold is a protein framework that can carry altered amino acids providing new binding specificities. Affibody molecules are a successful example of the application of the scaffold principle for the development of imaging agents.4 The small size (ca. 7 kDa) and high affinity (low nanomolar to picomolar level) of Affibody molecules provide rapid extravasation and diffusion inside r 2011 American Chemical Society

tumors, stable binding to molecular targets, and rapid clearance of nonbound probe, making high-contrast imaging possible. The robust disulfide-independent structure and rapid high-fidelity refolding of Affibody molecules in the physiological environment permit their labeling in harsh conditions.5 Site-specific labeling is important to provide chemically uniform radioconjugates with well-defined in vivo properties. For short peptides, such site-specificity is easy to achieve by incorporation of a chelator during peptide synthesis. This approach has been successfully applied to Affibody molecules as well.5 It would be attractive, however, to be able also to label sitespecifically recombinant proteins, since efficient peptide synthesis of, e.g. multimeric constructs or fusion proteins, is problematic. The feasibility of site-specific labeling of recombinant Received: October 26, 2010 Revised: March 9, 2011 Published: March 29, 2011 894

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Scheme 1

Affibody molecules would provide high flexibility for production even of monomeric forms. The absence of cysteines in Affibody molecules enables us to solve this problem by the introduction of a unique cysteine residue in a desirable position and the use of thiol-directed chemistry for labeling or chelator coupling.5 This approach has been used for labeling of Affibody molecules with radiobromine,6 radioiodine,7 18F,8,9 99mTc,10 111In,1113 57Co,14 and 64Cu.15,16 A maleimido derivative of the macrocyclic chelator DOTA was used for labeling of Affibody molecules with 111In, 57Co, and 64 Cu. The use of DOTA provides stable coupling of many nuclides to targeting proteins.17 However, DOTA is not always the chelator of choice. For example, DOTA is a suboptimal chelator for copper isotopes due to in vivo instability.18 The use of NOTA19 or cross-bridged chelators20,21 for labeling with radiocopper provides more stable labeling than the use of DOTA, as well as lower liver uptake. Moreover, different chelators influence in different ways the affinity of an imaging peptide, the cellular processing and retention of a radionuclide, the biodistribution of a targeting peptide, and the excretion pathways of a nonbound tracer and radiocatabolites.22 This influences the sensitivity and specificity of the imaging. Therefore, several chelators should be evaluated for each targeting protein to select the most suitable for each particular application. NOTA (2,20 ,200 -(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid) is a promising chelator for labeling of targeting agents with a number of radiometals. NOTA provides more stable complexes with gallium and indium than DOTA.23,24 Several NOTA derivatives have been used for labeling of targeting peptides with Ga isotopes2527 and 111In.25 Biodistribution data indicate adequate in vivo stability of the conjugates. The feasibility of 68 Ga-labeling of NOTA-conjugated peptides at room temperature within a reasonable time (10 min) has been shown.28 Recently, a very elegant method for labeling of peptides with 18 F using the formation of [18F]aluminum fluoride and its complexation with NOTA has been proposed.29 Taken together, derivatives of NOTA are very promising chelators for radiolabeling of targeting proteins and peptides.

The goal of this study was to develop a maleimido derivative of NOTA (maleimide monoamide NOTA; MMA-NOTA), couple it to the HER2-binding Affibody molecule ZHER2:2395 ([Ala1, Glu2,Thr23,Val59,Asp60,Cys61]ZHER2:342) having a C-terminal cysteine, and evaluate the 111In-labeled conjugate in vitro and in vivo. 111In is a widely used nuclide for single-photon emission tomography (SPECT). Indium forms stable hexacoordinate complexes with NOTA in aqueous solutions.30 Although one carboxylic arm of NOTA participates in an amide bond in the case of MMA-NOTA, it was demonstrated earlier that the amide carbonyl oxygen can contribute to stable chelation of indium.31

’ MATERIALS AND METHODS Material. ZHER2:2395, a variant of ZHER2:342 having a C-terminal cysteine, was produced according to Ahlgren and coworkers.11 The commercially available (4,7-bis-tert-butoxycarbonylmethyl-[1,4,7]triazonan-1-yl)-acetic acid (NOTA(tBu)2) was provided by CheMatech (Dijon, France). MMA-DOTA was purchased from Macrocyclics (Dallas, TX, USA). The purity and identity of all macromolecules and their conjugates was confirmed using high-performance liquid chromatography with on-line mass spectrometric detection, as described below. All purified proteins demonstrated a single peak with a mass coinciding with the theoretical molecular weight calculated with an accuracy of (1.5 Da, which is within the accuracy of the instrument. Buffers, such as 0.1 M phosphate buffered saline (PBS), pH 7.5, and 0.2 M ammonium acetate, pH 6.5, were prepared using common methods from chemicals supplied by Merck (Darmstadt, Germany). High-quality Milli-Q water (resistance higher than 18 MΩ/cm) was used for preparing solutions. Buffers, which were used for conjugation and labeling, were purified from metal contamination using 23 g Chelex 100 resin per liter (Bio-Rad Laboratories, Richmond, CA, USA). NAP-5 size exclusion columns were from GE Healthcare BioSciences (Uppsala, Sweden). [111In]indium chloride was purchased from Covidien. The yield and radiochemical purity of the labeled Affibody constructs were analyzed using 150771 dark 895

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Scheme 2

green, Tec-Control chromatography strips from Biodex Medical Systems (New York, USA). The accuracy of radio-ITLC analysis was cross-validated by SDS-PAGE. Ketalar [ketamine] (50 mg/mL, Pfizer, NY, USA), Rompun [xylazin] (20 mg/mL, Bayer, Leverkusen, Germany), and Heparin (5000 IE/mL, Leo Pharma, Copenhagen, Denmark) were obtained commercially. The radioactivity was measured using an automated gamma counter with a 3-inch NaI(Tl) detector (1480 WIZARD, Wallac Oy, Turku, Finland). Distribution of radioactivity along the thin layer chromatography strips was measured on a Cyclone Storage Phosphor System and analyzed using the OptiQuant image analysis software. Cells were counted using an electronic cell counter (Beckman Coulter). Data on cellular uptake and biodistribution were analyzed by unpaired, two-tailed t-test using GraphPad Prism (version 4.00 for Windows GraphPad Software, San Diego, USA) in order to determine any significant differences (P < 0.05). Synthesis and Characterization of ((4-Carboxymethyl7-{[2-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-ethylcarbamoyl]methyl}-[1,4,7]triazonan-1-yl)-acetic acid) (MMA-NOTA, compound 3) (Scheme 1). Mass spectra were recorded on a Bruker Micro ToFQ Instrument in electrospray ionization (ESI) mode. NMR spectra were recorded on a 300 MHz BRUKER Avance spectrometer. Measurements were made using the “Plateforme d'Analyse Chimique et de Synthese Moleculaire de l'Universite de Bourgogne” (PACSMUB). N,N-Diisopropylethylamine (DIPEA, 63 mg, 48 mmol, Acros, Geel, Belgium) was added to a solution of 100 mg (0.24 mmol) of NOTA(tBu)2 (1), 97 mg (0.25 mmol) of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, (HBTU, Iris Biotech, Maktrewitz, Germany), 39 mg (0.29 mmol) of hydroxybenzotriazole (HOBt) (Sigma-Aldrich, Saint Quentin Fallavier, France), and 67 mg (0.25 mmol) of N-(2-aminoethyl)maleimide (Sigma-Aldrich, Saint Quentin Fallavier, France) in 12 mL of CH2Cl2 (Sigma-Aldrich, Saint Quentin Fallavier, France). The mixture was stirred at room temperature for 12 h. The solution was then diluted with 100 mL of CH2Cl2 and washed with 30 mL of 2.2 mM citric acid, 30 mL of an aqueous solution of NaHCO3 (5%), and 50 mL of water. The organic phase was dried on MgSO4 (Acros, Geel, Belgium), and the solvent was removed under vacuum. The residue, a yellow oil, was dissolved in 50 mL of diethyl ether (Sigma-Aldrich, Saint Quentin Fallavier, France). The white precipitate formed after filtration, and diethyl ether was removed under vacuum. The remaining oil was purified by chromatography (SiO2, CH2Cl2, then EtOH/CH2Cl2 5/95 to 10/90). Compound 2 was obtained as a pale yellow oil (m = 80 mg, yield = 62%). NMR 1H (CDCl3, 298 K, 300 MHz): δ 1.45 (s, 18H), 2.60 (br, 4H), 2.83 (br, 8H), 3.19 (s, 2H), 3.32 (S, 4H), 3.45 (m, 2H), 3.61 (m, 2H), 6.67 (s, 2H), 9.15 (s, 1H). ESI-MS m/z: calcd [MH]þ, 537.32; found 537.33. Anal. (C26H43N5O7) C,H,N:

calcd C, 58.08; H, 8.06; N, 13.03; found C, 57.83; H, 7.88, N, 12.77. For deprotection, trifluoroacetic acid (TFA, 290 μL) was added to a solution of 38 mg of 2 in 192 μL of CH2Cl2. The mixture was stirred at room temperature for 24 h. The solvents were removed by heating under vacuum at maximum 35 °C. When the evaporation was almost complete, 10 mL of diethyl ether was added. A white precipitate was formed, filtered, and washed several times with diethyl ether. Compound 3 was obtained as a white hygroscopic powder (m = 10 mg). NMR 1H (D2O, 298 K, 300 MHz): δ 3.07 (t, 4H, J = 5.6 Hz), 3.25 (t, 4H, J = 5.6 Hz), 3.32 (s, 4H), 3.42 (m, 2H), 3.59 (s, 2H), 3.64 (m, 2H), 3.92 (s, 4H). ESI-MS m/z: calcd [MH]þ 426.20; found 426.19. Anal. (C18H27N5O7, 1.5TFA) calcd C, 50.82; H, 6.40; N, 16.40; found C, 50.61; H, 6.21; N, 16.19. Coupling of MMA-NOTA (3) to Affibody Molecules and Characterization of Conjugate (Scheme 2). Conjugation of MMA-NOTA (3) to ZHER2:2395 was performed similarly to the coupling of MMA-DOTA described earlier.11 Briefly, ZHER2:2395 (stock solution, 5 mg/mL) was incubated during 2 h in 30 mM DTT at 40 °C to reduce any spontaneously formed disulfide bonds. After incubation, the reduced ZHER2:2395 was purified using a NAP-5 column pre-equilibrated and eluted with degassed 0.2 M ammonium acetate, pH 6.5. The solution of freshly reduced Affibody molecules was mixed with a precalculated amount of a freshly prepared solution of MMA-NOTA in 0.2 M ammonium acetate, pH 6.5, (1 mg/mL) to obtain a chelator-to-protein ratio of 1:1 or 3:1. The vial was filled with argon gas, and the mixture was incubated at 40 °C for 2 h. The coupling efficiency was determined using analytical RPHPLC (Agilent 1200 series) with a Zorbax 300SB-C18 4.6  150 mm column with 3.5 μm particle size (Agilent, Santa Clara, CA, USA). A flow rate of 1 mL/min and a gradient of 2545% B over 25 min (solvent A = 0.1%TFA-H2O and solvent B = 0.1%TFACH3CN) were used for analysis. The Affibody molecule conjugated with a chelator-to-protein ratio of 3:1 was purified for further experiments by semipreparative RP-HPLC, using a Reprosil Gold 300 C18 10  250 mm column with 5 μm particle size (Dr. Maisch, HPLC, GmbH, Ammerbuch, Germany). The flow rate was 2.5 mL/min and the gradient the same as above. The purified fraction was then analyzed by analytical RP-HPLC, as described above. The degree of conjugation as well as the final purity of the purified conjugate were determined by integrating the area of the peaks in the HPLC elution profiles. The molecular weights were determined by ESI-Q-TOF (Agilent). The binding kinetics of the purified [MMA-NOTA-Cys61]ZHER2:2395 Affibody molecule were investigated by real-time biospecific interaction analysis, using BIAcore 3000 (GE Healthcare, Uppsala, Sweden). The binding kinetics of [MMA-DOTACys61]-ZHER2:2395 and parental ZHER2:342 were investigated at 896

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NOTA-Cys61]-ZHER2:2395 (0.015 ng protein per dish, 2 nM) was added to six Petri dishes (ca. 106 cells in each). For blocking, an excess of nonlabeled recombinant ZHER2:342 (2.5 μg) was added 10 min before [111In-MMA-NOTA-Cys61]-ZHER2:2395 to saturate the receptors. The cells were incubated during one hour in a humidified incubator at 37 °C. Thereafter, the media was collected, the cells were detached by trypsin-EDTA solution, and the radioactivity in cells and media was measured to calculate a percentage of cell-bound radioactivity. A comparison of [111In-MMA-NOTA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 internalization by DU-145 cells was performed according to the method developed  and Orlova.33 The cells (ca. 106 per and validated by Wallberg dish) were incubated with the labeled compounds (0.015 ng protein per dish, 2 nM) at 37 °C, 5% CO2. At predetermined time points (1, 2, 3, 4, and 8 h after incubation start), the media from a set of three dishes was removed and the cells were washed three times with ice-cold serum-free medium. The cells were then treated with 0.5 mL 0.2 M glycine buffer with 4 M urea, pH 2.5, for 5 min on ice. The acidic solution was collected and the cells were washed additionally with 0.5 mL glycine buffer, which was collected and pooled with the first fraction. The radioactivity in the acid wash fractions was considered as membrane-bound radioactivity. After addition of 0.5 mL 1 M NaOH, cells were incubated at 37 °C for 0.5 h before the alkali containing cell debris was collected. The cell dishes were washed with an additional 0.5 mL NaOH, and the alkaline fractions were pooled. The radioactivity in the alkaline fractions was considered internalized. A percent of internalized radioactivity was calculated for each time point. Animal Studies. The animal experiments were planned and performed in accordance with the national regulation on laboratory animals' protection. The animal study plans have been approved by the local Ethics Committee for Animal Research in Uppsala. Nude male NMRI nu/nu mice bearing DU-145 prostate cancer xenografts were used. The mice were kept using standard sterile diet, bedding, and environment with free access to food and water. The mice were acclimatized for one week at the Rudbeck Laboratory animal facility before any experimental procedures. For tumor implantation, 5  106 DU-145 cells in Matrigel (BD Bioscience) were implanted on the right flank. Average tumor weight was 0.67 ( 0.26 g at the time of experiment. Average animal weight was 30 ( 2 g. The mice were randomized into groups of four. Animals were injected intravenously (tail vein) with 0.3 μg conjugate (50 kBq) in 100 μL PBS. At four hours after injection, a mixture of KetalarRompun (20 μL of solution per gram body weight; Ketalar, 10 mg/mL; Rompun, 1 mg/mL) was intraperitonealy injected and the mice were euthanized by a heart puncture using a 1 mL syringe, prewashed with diluted heparin (5000 IE/mL). Blood as well as lung, liver, spleen, kidneys, tumor, samples of muscle and bone, gastrointestinal tract (with its content), and remaining carcass were collected in preweighed plastic vials. Organs and tissue samples were weighed and measured for radioactivity using an automatic gamma counter. The tissue uptake values were calculated as percent injected activity per gram tissue (% ID/g), except for the gastrointestinal tract and the carcass, which were calculated as % ID per whole sample. The radioactivity in the gastrointestinal tract with its contents was used as a measure of hepatobiliary excretion. To confirm that the xenograft accumulation of [111In-MMANOTA-Cys61]-ZHER2:2395 was HER2-specific, the receptors in one

the same time, to avoid intra-assay variability. Human HER2ECD (R&D systems, Minneapolis, MN, USA), recombinantly produced as an Fc-chimera, was diluted to 5 μg/mL in 10 mM NaOAc, pH 4.5, and immobilized by EDC/NHS-mediated coupling on a carboxymethylated dextran-coated CM5 chip. A flow cell activated with EDC/NHS and deactivated with ethanolamine was used as reference. All samples were diluted in HBSEP buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% Tween20, pH 7.2) and analyzed in duplicate in 6 concentrations ranging from 0.3 nM to 10 nM at a flow rate of 50 μL/min with a 5 min association phase and a 10 min dissociation phase. After each cycle. 15 mM HCl was injected to regenerate the surface. The final response was calculated by subtracting the reference surface, and the kinetic analysis was performed using BIAevaluation 4.1 software using a 1:1 Langmuir model. In order to measure the melting point of the [MMA-NOTACys61]-ZHER2:2395 conjugate, variable temperature measurements were performed using a JASCO J-810 instrument (JASCO, Tokyo, Japan). The sample was diluted to 0.5 mg/ mL in PBS (pH 7.2) and measured at 221 nm in a 1 mm optical cuvette. The temperature was increased by 5 °C/min ranging from 20 to 90 °C. The secondary structure was determined at 20 °C before and after melting point measurements using circular dichroism (CD) with wavelengths ranging from 195 to 250 nm. Labeling [MMA-NOTA-Cys61]-ZHER2:2395 with 111In. To establish the optimum labeling temperature, a solution of [MMA-NOTA-Cys61]-ZHER2:2395 (30 μg, 4 nmol) in 54 μL 0.2 M ammonium acetate, pH 5.5, was mixed with 40 μL 111In chloride solution (15 MBq at calibration time). The mixture was incubated at room temperature, 40 or 60 °C. At 10, 20, and 30 min after the start of the incubation, a small aliquot (0.8 μL) of reaction mixture was taken and analyzed by radio-ITLC eluted with 0.2 M citric acid, pH 2.0. In this system, radiolabeled Affibody molecules remain at the application point while free 111In or 111In-chelates migrate with the solvent front. The analytical system was verified using a blank experiment, where no Affibody molecule was added to the reaction mixture. Blank experiments showed that less than 0.5% of radioactivity remained at the application point of ITLC. The experiments were performed at least in duplicate. For further experiments (stability tests, in vitro and in vivo studies), labeling at 60 °C during 30 min was selected. An EDTA-challenge test was performed to evaluate the labeling stability of [111In-MMA-NOTA-Cys61]-ZHER2:2395. Two samples were diluted with a 1000-fold molar excess of the disodium salt of EDTA solution in water, while two control samples were diluted with equal amount of PBS. After four-hour incubation at room temperature, all samples were analyzed using radio-ITLC. Labeling of [MMA-DOTA-Cys61]-ZHER2:2395 with 111In for comparative biodistribution experiments was performed as described by Ahlgren and co-workers.11 In Vitro Studies. For cell studies, the HER2-expressing ovarian carcinoma SKOV-3 (1.2  106 HER2 receptors per cell32) and prostate carcinoma DU-145 cell line (∼5  104 HER2 receptors per cell, A. Orlova, unpublished results)  Sweden) were (ATCC, purchased via LGC Promochem, Boras, used. The cell lines were cultured in RPMI medium (Flow Irvine, UK) supplemented with 10% fetal calf serum (Sigma, USA), 2 mM L-glutamine, and PEST (penicillin 100 IU/mL and 100 μg/mL streptomycin), all from Biokrom AG, Germany. An in vitro specificity test was performed according to the methods described earlier.33 Briefly, a solution of [111In-MMA897

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Figure 2. Cell-binding specificity of [111In-MMA-NOTA-Cys61]ZHER2:2395 labeled at 60 °C during 30 min. The test was performed with ovarian cancer cell line SKOV-3 and prostate cancer cell line DU145. In the blocking experiment, binding sites were saturated by adding to the cells an excess of nonlabeled ZHER2:342. Data are presented as mean values for three samples ( maximum errors.

Figure 1. Labeling yield of [MMA-NOTA-Cys61]-ZHER2:2395 with 111 In in 0.2 M ammonium acetate buffer, pH 5.5, as a function of time at different temperatures. Data are presented as mean values of the results of two to eight experiments ( standard deviations.

group of mice were presaturated by subcutaneous injection of 500 μg of unlabeled ZHER2:342 60 min prior to the injection of radiolabeled Affibody molecule, while another group was injected with [111In-MMA-NOTA-Cys61]-ZHER2:2395 without preblocking. For comparison, the biodistribution of [111In-MMA-DOTACys61]-ZHER2:2395 was studied in DU-145 xenograft-bearing mice. For gamma camera imaging, two DU-145 xenograft-bearing mice were injected with 2 MBq 111In-[MMA-NOTA-Cys61]ZHER2:2395 (amount of protein 1 μg). Four hours post-injection, the animals were sacrificed by overdosing Ketalar/Rompun followed by cervical dislocation. After euthanasia, the urine bladders were excised and cadavers were stored on ice until imaging. Imaging was performed at the department of Nuclear Medicine at Uppsala University Hospital using a GE Infinia gamma camera equipped with a medium energy general purpose (MEGP) collimator. Static images (40 min), obtained with a zoom factor of 2, were digitally stored in a 256  256 matrix. The evaluation of the images was performed using the Osiris 4.19 software (University Hospital of Geneva, Switzerland). In each animal, a region of interest (ROI) was drawn around the tumor. The same region was copied to a contralateral thigh. Tumor-to-contralateral thigh ratios were calculated based on total counts in the ROIs.

The secondary structure determined by CD spectra showed R-helical content both before and after variable temperature measurements, indicating that the molecule refolds after heat treatment. The melting point of [MMA-DOTA-Cys61]ZHER2:2395 was determined to be 68 °C. Labeling [MMA-NOTA-Cys61]-ZHER2:2395 with 111In. The kinetics of [111In-MMA-NOTA-Cys61]-ZHER2:2395 formation at different temperatures is presented in Figure 1. The labeling at room temperature was inefficient. Increasing the reaction temperature to 40 °C improved the yield; however, the analytical radiochemical yield was only 62 ( 7% (n = 3) after 30 min incubation. Further temperature increase to 60 °C resulted in an increase of the radiochemical yield to 97.5 ( 0.5% (n = 12) within 30 min. For further experiments, the [MMA-NOTA-Cys61]-ZHER2:2395 was labeled at 60 °C and used without further purification. Conjugates with a specific radioactivity of 14.5 GBq/μmol (2 MBq/μg) were obtained with the yield of more than 95% (labeling at 60 °C), when indium-111 was used for labeling three days after production. The stability of [111In-MMA-NOTA-Cys61]-ZHER2:2395 was evaluated by challenge with a 1000-fold molar excess of EDTA over 4 h. The EDTA challenge demonstrated very high labeling stability. The radiochemical purity of the treated samples was 96.2 ( 0.6% (average ( SEM), while the purity of the untreated control was 98.7 ( 0.1%, i.e., the difference was within the accuracy of the analytical method. In Vitro Studies. Binding specificity tests demonstrated that the binding of [111In-MMA-NOTA-Cys61]-ZHER2:2395 to living HER2-expressing cells was receptor-mediated, because saturation of the receptors by preincubation with nonlabeled ZHER2:342 significantly decreased the binding of the radiolabeled Affibody molecule (for all studied cell-lines, p < 0.0001) (Figure 2). The level of cell-associated radioactivity was appreciably lower for the nonblocked DU-145 cell line than for the SKOV-3 cell line, reflecting the lower expression level of HER2. A comparison of the internalization of [111In-MMA-NOTACys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 by DU-145 prostate cancer cells is presented in Figure 3. The internalization of both compounds was relatively slow, although internalized fractions increased continuously throughout the experiment. The internalization rate of [111In-MMA-NOTACys61]-ZHER2:2395 was slightly higher than the rate of [111InMMA-DOTA-Cys61]-ZHER2:2395. After 8 h incubation, 27 ( 1% and 21.5 ( 0.6% of the cell-associated radioactivity was internalized for 111In-MMA-NOTA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395, respectively.

’ RESULTS Coupling of MMA-NOTA and Characterization of Conjugate. The degree of MMA-coupling to the Affibody molecules

was determined by RP-HPLC analysis. Conjugation of MMANOTA to ZHER2:2395-Cys using a chelator-to-protein ratio of 1:1 resulted in 49% conjugated protein, 3% nonconjugated disulfide-linked dimeric protein and the remaining found as nonconjugated monomeric protein. Increasing the chelator-to-protein molar ratio to 3:1 resulted in 80% of MMA-NOTA-conjugated ZHER2:2395, while 17% was found as dimeric nonconjugated protein. The purification of [MMA-NOTA-Cys61]-ZHER2:2395 by RP-HPLC provided a product with 96% purity. This protein was used for further analysis. Binding analyses of the conjugated molecules were performed using SPR-based biosensor interaction. The dissociation constant (KD) for the interaction between the Affibody conjugate and the extracellular domain of the HER2 receptor (recombinantly produced as an Fc-chimera) was calculated to be 72 pM from the association rate constant (ka) and the dissociation rate constant (kd). In the same experiment, the dissociation constants for [MMA-DOTA-Cys61]-ZHER2:2395 and parental ZHER2:342 were determined to be 74 pM and 78 pM, respectively. 898

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uptake was reduced from 8.3 ( 1.0% to 0.53 ( 0.5% IA/g (p < 0.0001), demonstrating specificity of HER2 targeting. A comparison of [111In-MMA-NOTA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 biodistribution is presented in Figure 5. Tumor targeting was equally efficient for both variants, and there was no significant difference in the tumor radioactivity accumulation. Both conjugates demonstrated rapid clearance from blood and the majority of normal organs and tissues. The renal excretion was dominating, which resulted in a low radioactivity accumulation in the gastrointestinal tract but high accumulation in kidneys due to reabsorption. There were, however, certain differences in biodistribution of [111In-MMANOTA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]ZHER2:2395. Radioactivity cleared more rapidly from blood in the case of [111In-MMA-NOTA-Cys61]-ZHER2:2395. Significantly (p < 0.005) lower radioactivity concentration was also found in lung, liver, spleen, kidney, and bone (Figure 5a). Accordingly, [111In-MMA-NOTA-Cys61]-ZHER2:2395 provides significantly higher tumor-to-blood, tumor-to-lung, tumor-to-spleen, tumorto-muscle, and tumor-to-bone ratios (Figure 5b).

Animal Studies. In order to verify the specificity of [111In-

MMA-NOTA-Cys61]-ZHER2:2395 accumulation in HER2-expressing DU-145 xenografts, its biodistribution (4 h pi) was studied after saturating the binding epitope of HER2 by preinjection of a large excess of nonlabeled ZHER2:342 (Figure 4). The tumor

Figure 3. Internalization of [111In-MMA-NOTA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 by HER2-expressing DU145 cell line. Cells were incubated with radiolabeled Affibody molecules at 37 °C. Data presented as a percentage of internalized radioactivity (an average of three samples with standard deviations); error bars might be not seen because they are smaller than the symbols.

Figure 4. Specificity of [111In-MMA-NOTA-Cys61]-ZHER2:2395 uptake in DU-145 xenografts 4 h after injection. To saturate HER2 in tumors, a group of animals in the blocking experiment was preinjected with 500 μg of nonlabeled ZHER2:342 60 min before injection of radiolabeled conjugate (designated as blocked). All animals were injected with 0.3 μg of labeled Affibody molecules. Data are expressed as %ID/g and are presented as mean ( SD for 4 animals. * Data for GI tract (with content) and carcass are presented as %ID per whole sample.

Figure 6. Anterior gamma camera images of nude mice bearing DU-145 xenografts 4 h after injection of [111In-MMA-NOTA-Cys61]-ZHER2:2395. Contours derived from a digital photograph were superimposed over gamma camera images to facilitate interpretation. Arrows point at tumors (T) and kidneys (K).

Figure 5. Comparison of targeting of DU-145 xenografts using [111In-MMA-NOTA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395. Data are presented as an average from four animals ( SD. Left panel: Biodistribution 4 h p.i. Data are expressed as % ID/g. *Data for the gastrointestinal tract (with its content) and for carcass are presented as % ID per whole sample. Right panel: Tumor-to-organ ratios 4 h p.i. 899

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ARTICLE

Images acquired four hours after the i.v. injection of the [111InMMA-NOTA-Cys61]-ZHER2:2395 into mice bearing DU-145 xenografts confirmed the capacity of this conjugate to visualize HER2-expression (Figure 6). In agreement with the biodistribution data, a prominent radioactivity uptake was observed in the kidneys. No radioactivity accumulation in other healthy organs and tissues was observed. Tumors were clearly visualized already at 4 h p.i. (tumor-to-contralateral side ratio 8.3 ( 0.8).

conjugates.7 To provide site-specific chelator coupling, a cysteine was engineered at the C-terminus of anti-HER2 Affibody molecule ZHER2:342, providing an Affibody molecule ZHER2:2395 with a unique binding site for thiol-directed coupling of chelators.11 To use this approach for NOTA coupling, a maleimido derivate of NOTA was developed in this study. The synthesis of MMA-NOTA is based on a commercially available precursor NOTA(tBu)2. It is straightforward and provides a reasonable overall yield of ∼20%. MMA-NOTA was efficiently coupled to prereduced ZHER2:2395. Importantly, coupling of this chelator did not cause any measurable decrease of ZHER2:2395 affinity to HER2 (the observed difference in KD for [MMA-NOTA-Cys61]-ZHER2:2395, [MMA-DOTA-Cys61]ZHER2:2395, and parental ZHER2:342 was within the accuracy of the SPR method). This preservation of affinity is essential for targeting of cancer with low level of HER2 overexpression, such as prostate cancer. CD spectra have demonstrated that the coupling of MMA-NOTA does not prevent accurate refolding of [MMA-NOTA-Cys61]-ZHER2:2395 after heat treatment, permitting the use of elevated temperatures for the labeling reaction. This was essential, since efficient labeling with 111In required some moderate (up to 60 °C) warming (Figure 1). Literature data suggest that warming (up to 70 °C) is required also for labeling of NOTA with copper radioisotopes.19 In agreement with CD data, [111In-MMA-NOTA-Cys61]-ZHER2:2395 was capable of specific binding to HER2-expressing cells after labeling at 60 °C (Figure 2). It has been shown earlier that different chelators have different influence on the internalization rate of radiolabeled peptides, e.g., somatostatin analogues, by cancer cells.4446 Previous data indicated that internalization of ZHER2:342 and its derivatives, including ZHER2:2395, by HER2-expressing cancer cell lines is slow and that the influence of a chelator on the internalization rate is small.10,11,33 However, our latest data suggested that internalization of [99mTc(CO)3]þ-labeled ZHER2:342 can depend on the nature of histidine-containing chelators.47 The current study is another demonstration of the influence of the chelator on the internalization rate of Affibody molecules. Though the overall internalization rate of [111In-MMA-NOTA-Cys61]-ZHER2:2395 was still slow (27 ( 1% of cell-bound radioactivity was internalized at 8 h), it was higher than the internalization of [111InMMA-DOTA-Cys61]-ZHER2:2395 (21.5 ( 0.6% internalized at 8 h) (Figure 3). The internalization of tracers labeled using residualizing radiometals is considered an important and useful factor for the accumulation of radiotracer in a cancer cell,48 so the increased internalization rate is a positive influence of maleimidoNOTA on the targeting properties of ZHER2:2395. The excellent binding properties of [111In-MMA-NOTACys61]-ZHER2:2395 enables specific targeting of DU-145 prostate cancer xenografts, despite the relatively low HER2 expression of this cell line (Figure 5). Gamma camera experiments confirmed the results of the biodistribution studies demonstrating clear visualization of DU-145 xenografts 4 h p.i. (Figure 6). This was achieved despite location of the xenografts close to the kidneys, an organ with a high level of radioactivity accumulation. The targeting properties of [111In-MMA-NOTA-Cys61]ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 were compared in nude mice bearing DU-145 prostate cancer xenografts (Figure 5). There was no significant difference between these conjugates in accumulation of radioactivity in tumors. However, [111In-MMA-NOTA-Cys61]-ZHER2:2395 cleared more rapidly from blood, lung, liver, and spleen. Bone uptake of

’ DISCUSSION Currently, a major approach for the treatment of disseminated cancer is based on identification of molecular abnormalities causing malignant transformation of cells and molecular recognition of such abnormalities for selective treatment of cancer while sparing healthy tissues. One such abnormality is the overexpression of the transmembrane tyrosine kinase HER2, which is widely used as a target for different therapeutic strategies mainly in breast cancer.34,35 Expression of HER2 has also been suggested as one of the possible molecular pathways to androgen independence of prostate cancer.36 Preclinical data suggest that HER2-targeting therapies can be efficient against androgen-independent prostate cancer.37,38 However, treatment of prostate cancer with the HER2-targeting monoclonal antibody trastuzumab or the tyrosine kinase inhibitor lapatinib has failed to demonstrate efficacy in clinical trials.3941 The failure of these trials was attributed to the absence of patient stratification according to the HER2 expression level.39 Since only a fraction of tumors express HER2, the patients should be stratified according to expression levels to select those who would most likely benefit from HER2targeting therapy. This would make cancer treatment more personalized and, thus, more efficient. Radionuclide molecular imaging is a promising way to detect the HER2 expression level, since it is noninvasive and may overcome issues associated with heterogeneity of HER2 expression in primary tumors and metastases.42 However, due to the relatively low level of HER2 expression in prostate cancer (Human Protein Atlas, http:// www.proteinatlas.org/), further perfection of the radionuclide imaging probes is desirable. Comparison of preclinical literature data suggests that small targeting agents provide higher tumor-to-organ ratios of radioactivity concentration and, for this reason, higher sensitivity of HER2 detection.42 Particularly, Affibody molecules have demonstrated superior imaging properties in comparison with monoclonal antibodies and their fragments due to small size (7 kDa) and picomolar affinity. Direct head-to-head comparison of the 124 I-labeled anti-HER2 Affibody molecule ZHER2:342 and the anti-HER2 antibody trastuzumab confirmed conclusions derived from literature data.43 However, further improvement of imaging sensitivity is required, particularly for visualization of tumors with relatively low HER2 expression level, such as in castrationindependent prostate cancer. Besides selection of optimal targeting proteins, optimization of labeling chemistry might improve the sensitivity of radionuclide molecular imaging, as labeling chemistry can influence the targeting properties of an imaging agent.22 NOTA and its derivatives seem to be attractive chelators for labeling of targeting proteins because of the versatility and high stability of the chelates with a number of nuclides suitable for imaging. The use of site-specific labeling is essential, since this provides uniform biodistribution properties of the radiolabeled 900

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ARTICLE  (10) Ahlgren, S., Wallberg, H., Tran, T. A., Widstr€om, C., Hjertman, M., Abrahmsen, L., Berndorff, D., Dinkelborg, L. M., Cyr, J. E., Feldwisch, J., Orlova, A., and Tolmachev, V. (2009) Targeting of HER2expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine. J. Nucl. Med. 50, 781–789. (11) Ahlgren, S., Orlova, A., Rosik, D., Sandstr€om, M., Sj€ oberg, A., Baastrup, B., Widmark, O., Fant, G., Feldwisch, J., and Tolmachev, V. (2008) Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant Affibody molecules. Bioconjugate Chem. 19, 235–243.  (12) Tolmachev, V., Xu, H., Wallberg, H., Ahlgren, S., Hjertman, M., Sj€ oberg, A., Sandstr€ om, M., Abrahmsen, L., Brechbiel, M. W., and Orlova, A. (2008) Evaluation of a maleimido derivative of CHX-A00 DTPA for site-specific labeling of affibody molecules. Bioconjugate Chem. 19, 1579–1587.  (13) Tolmachev, V., Rosik, D., Wallberg, H., Sj€oberg, A., Sandstr€om, M., Hansson, M., Wennborg, A., and Orlova, A. (2010) Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR 111In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount. Eur. J. Nucl. Med. Mol. Imaging 37, 613–622.  (14) Wallberg, H., Ahlgren, S., Widstr€om, C., and Orlova, A. (2010) Evaluation of the radiocobalt-labeled [MMA-DOTA-Cys61]Z HER2:2395(-Cys) affibody molecule for targeting of HER2-expressing tumors. Mol. Imaging Biol. 12, 54–62. (15) Miao, Z., Ren, G., Liu, H., Jiang, L., and Cheng, Z. (2010) Smallanimal PET imaging of human epidermal growth factor receptor positive tumor with a 64Cu labeled affibody protein. Bioconjugate Chem. 21, 947–954. (16) Cheng, Z., De Jesus, O. P., Kramer, D. J., De, A., Webster, J. M., Gheysens, O., Levi, J., Namavari, M., Wang, S., Park, J. M., Zhang, R., Liu, H., Lee, B., Syud, F. A., and Gambhir, S. S. (2010) 64Cu-labeled affibody molecules for imaging of HER2 expressing tumors. Mol. Imaging Biol. 12, 316–324. (17) De Le on-Rodríguez, L. M., and Kovacs, Z. (2008) The synthesis and chelation chemistry of DOTA-peptide conjugates. Bioconjugate Chem. 19, 391–402. (18) Anderson, C. J., Wadas, T. J., Wong, E. H., and Weisman, G. R. (2008) Cross-bridged macrocyclic chelators for stable complexation of copper radionuclides for PET imaging. Q. J. Nucl. Med. Mol. Imaging 52, 185–192. (19) Prasanphanich, A. F., Nanda, P. K., Rold, T. L., Ma, L., Lewis, M. R., Garrison, J. C., Hoffman, T. J., Sieckman, G. L., Figueroa, S. D., and Smith, C. J. (2007) [64Cu-NOTA-8-Aoc-BBN(714)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues. Proc. Natl. Acad. Sci. U.S.A. 104, 12462–12467. (20) Garrison, J. C., Rold, T. L., Sieckman, G. L., Figueroa, S. D., Volkert, W. A., Jurisson, S. S., and Hoffman, T. J. (2007) In vivo evaluation and small-animal PET/CT of a prostate cancer mouse model using 64Cu bombesin analogs: side-by-side comparison of the CB-TE2A and DOTA chelation systems. J. Nucl. Med. 48, 1327–1337. (21) Hausner, S. H., Kukis, D. L., Gagnon, M. K., Stanecki, C. E., Ferdani, R., Marshall, J. F., Anderson, C. J., and Sutcliffe, J. L. (2009) Evaluation of [(64)Cu]Cu-DOTA and [(64)Cu]Cu-CB-TE2A chelates for targeted positron emission tomography with an alpha(v)beta (6)-specific peptide. Mol. Imaging 8, 111–121. (22) Tolmachev, V., and Orlova, A. (2010) Influence of labelling methods on biodistribution and imaging properties of radiolabelled peptides for visualisation of molecular therapeutic targets. Curr. Med. Chem. 17, 2636–2655. (23) Clarke, E., and Martell, A, E. (1991) Stabilities of the Fe(III), Ga(III) and In(III) chelates of N,N’,N’’-triazacyclononanetriacetic acid. Inorg. Chim. Acta 181, 273–280. (24) Clarke, E. T., and Martell, A. E. (1992) Stabilities of trivalent metal ion complexes of the tetraacetate derivatives of 12-, 13-, and 14-membered tetraazamacrocycles. Inorg. Chim. Acta 190, 37–46.

radioactivity was also lower for this conjugate. This is important in the case of prostate cancer, where bones are the major metastatic site. This resulted in significantly higher tumor-toorgan ratios in the case of NOTA-conjugated Affibody molecules. This should provide better contrast on HER2 imaging and, hopefully, better sensitivity in clinical settings. Apparently, the use of MMA-NOTA is not limited to sitespecific labeling of Affibody molecules. Advantages of this chelator, such as high complex stability and thiol-directed sitespecific labeling, can be used for development of a variety of targeting agents, such as scaffold-based affinity proteins and Fab0 antibody fragments. In conclusion, MMA-NOTA is a very promising bifunctional chelator for thiol-directed site-specific labeling of Affibody molecules and other targeting proteins for in vivo radionuclide molecular imaging.

’ AUTHOR INFORMATION Corresponding Author

*Vladimir Tolmachev, Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 81 Uppsala, Sweden. Phone: þ46 18 471 3414. Fax: þ46 18 471 3432. E-mail: [email protected].

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