Influence of DOTA Chelator Position on Biodistribution and Targeting

Article pubs.acs.org/bc

Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of 111In-Labeled Synthetic Anti-HER2 Affibody Molecules Anna Perols,† Hadis Honarvar,‡ Joanna Strand,‡ Ramkumar Selvaraju,§ Anna Orlova,§ Amelie Eriksson Karlström,† and Vladimir Tolmachev‡,* †

Division of Molecular Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, and §Preclinical PET Platform, Department of Medicinal Chemistry, Uppsala University, Sweden

‡

S Supporting Information *

ABSTRACT: Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumortargeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with 99mTc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule ZHER2:S1 at three different positions: DOTA-A1-ZHER2:S1 (N-terminus), DOTA-K58-ZHER2:S1 (Cterminus), and DOTA-K50-ZHER2:S1 (middle of helix 3). The affinity for HER2 differed slightly among the variants and the KD values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-ZHER2:S1, DOTA-K50-ZHER2:S1, and DOTA-K58-ZHER2:S1, respectively. ZHER2:S1K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-ZHER2:S1 (64 °C) and DOTA-K58-ZHER2:S1 (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with 111In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-ZHER2:S1and DOTA-K58-ZHER2:S1 showed the highest uptake. Overall, DOTA-K58-ZHER2:S1 provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

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malignant cells.3 This overexpression of HER2 is a well-known biomarker in breast cancer4,5 and is linked to poor prognosis.6 It is also linked to other types of cancer such as prostate,7,8 ovarian,9 and colorectal cancer.10 As a result of this association with the malignant behavior of cancer, HER2 has become an interesting target, both for development of HER2-targeting therapeutics and for specific diagnosis. Among the therapeutics directed to HER2-expressing tumors, the best known example is the HER2-binding antibody trastuzumab (Herceptin), which

INTRODUCTION Human epidermal growth receptor type 2 (HER2) is a transmembrane protein which belongs to the ErbB family of receptor tyrosine kinases together with EGFR (HER1), HER3, and HER4. HER2 signaling occurs mainly by dimerization, either with other members of the HER family (heterodimerization) or at high expression levels with HER2 itself (homodimerization). Within the ErbB family, HER2 is the only receptor not having a known ligand.1 The receptor constitutively adopts an active-like conformation prearranged for dimerization, making HER2 the preferred dimerization partner among the HER family members.2 Amplified signaling due to overexpression of HER2 causes cell proliferation and suppression of apoptosis, providing a growth advantage to the © 2012 American Chemical Society

Received: April 28, 2012 Revised: July 4, 2012 Published: July 8, 2012 1661

dx.doi.org/10.1021/bc3002369 | Bioconjugate Chem. 2012, 23, 1661−1670

Bioconjugate Chemistry

Article

were compared with the best available affibody-based imaging tracer, synthetic 111In-DOTA-ZHER2:342 having DOTA conjugated to N-terminal valine. Although the biodistribution profiles of both variants were similar, difference in organ uptake resulted in significant difference in tumor-to-spleen, tumor-tocolon, tumor-to-kidney, and tumor-to-bone ratios.24 This indicated that the placement of the DOTA chelator in different positions on the affibody molecule could influence the biodistribution and targeting properties, which might be a way to maximize the tumor-to-organ ratios for this imaging agent. However, the constructs evaluated by Ahlgren and coworkers24 differed in the production routes, in the chelator conjugation chemistry, and in the sequences of affibody proteins (six different amino acids). It was thus impossible to distinguish the influence of these factors from the influence of the chelator position. To verify the hypothesis that DOTA positioning can be used for modification of targeting properties, a panel of derivatives of the HER2-binding affibody molecule has been synthesized. In the present study, DOTA was conjugated to position A1 (the N-terminus), position K58 (the C-terminus), or position K50 (located in the middle of helix 3) via amide bond. Thus, the proteins in the panel had the same sequence and were conjugated to the chelator in the same way. The binding, biodistribution, and targeting properties of these synthetic affibody molecules were evaluated.

has shown improved survival of treated patients in combination with first-line chemotherapy.11 Patient stratification using noninvasive methods for detection of molecular targets is a promising way to predict a therapeutic outcome and reduce unnecessary toxicities.12 For example, therapies using the monoclonal antibody trastuzumab or the tyrosine kinase inhibitor lapatinib are both targeting HER2expressing breast cancer; however, only 20% of breast cancer patients have HER2-overexpressing tumors.4 Molecular imaging is a promising noninvasive method for investigating the expression of specific tumor-associated molecular therapeutic targets, such as HER2, EGFR, or variants of VEGFR.13 This approach provides information about the molecular and functional properties of a tumor, in contrast to CT or MRI that give anatomical information. In addition, molecular imaging has a substantial advantage over biopsy-based methods, since it allows for repetitive evaluation for monitoring treatment response. Furthermore, the problems with tumor heterogeneity associated with analysis of biopsies can be avoided.13 Radiolabeled antibodies targeting HER2 have been used for imaging of HER2-positive tumors.13 However, the antibodies provide low imaging contrast and are suboptimal imaging agents due to limitations caused by the long circulation time, slow clearance from healthy tissues, and low tumor penetration rate. An alternative to antibodies is smaller affinity proteins, which can be engineered for high affinity. Affibody molecules is a class of affinity proteins, which is derived from the immunoglobulinbinding protein A of Staphylococcus aureus.15 The protein comprises 58 amino acids, which form a triple-helical bundle, with no internal disulfide bonds. Thirteen amino acids on helices 1 and 2 have been randomized for selection of new affinity binders, for example, targeting HER2, EGFR, HER3, IGF1R, or PDGFR beta.14,16 The affibody molecule ZHER2:342, with high affinity to the extracellular part of HER2 (KD = 22 pM), has been generated by affinity maturation of a selected binder.17 Because of the small size, it is possible to produce Affibody molecules by chemical synthesis, which enables sitespecific introduction of functional groups using orthogonal protection strategies.18 The small size, in combination with the high affinity, is a great advantage when using Affibody molecules for in vivo molecular imaging. Affibody molecules show faster clearance and better tissue penetration, which resulted in superior imaging contrast for the Affibody molecules when evaluated side-by-side to other HER2-imaging agents.19,20 Affibody molecules have successfully been labeled with different radionuclides.21 It has been demonstrated in previous studies that labeling-associated modifications of the affibody molecule, such as modification of local charge or lipophilicity due to chelator incorporation, could alter the biodistribution pattern. For example, labeling with 99mTc using peptide-based chelators has shown that even single substitution of the chelating amino acids can alter the biodistribution pattern and shift the excretion from hepatobiliary to renal.22 Differences in biodistribution of [99mTc(CO)3]+-labeled affibody molecules were also seen when the chelating hexahistidine moiety was transferred from the N-terminus to the C-terminus of the protein.23 During the development of site-specific labeling for recombinant anti-HER2 affibody molecules, Ahlgren and coworkers have conjugated maleimido derivatives of DOTA chelator (MMA-DOTA) to a derivative of ZHER2:342 having a Cterminal cysteine, ZHER2:2395-C.24 The targeting properties of the 111In-[MMA-DOTA-C61]-ZHER2:2395 affibody molecule

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MATERIALS AND METHODS Material. Buffers, including 0.1 M phosphate buffered saline (PBS), pH 7.5, 0.2 M ammonium acetate, pH 5.5, and 0.2 M citric acid were prepared using common methods from chemicals supplied by Merck (Darmstadt, Germany). Highquality Milli-Q water (resistance higher than 18 MΩ cm) was used for preparing the solutions. Buffers, which were used for labeling, were purified from metal contamination using Chelex 100 resin (Bio-Rad Laboratories, Richmond, USA). [111In]indium chloride was purchased from Covidien (Hazelwood, U.S.) as a solution in 0.05 M hydrochloric acid. The yield and radiochemical purity of the labeled affibody constructs were analyzed using 150−771 Dark Green, Tec-Control Chromatography strips from Biodex Medical Systems (New York, U.S.). 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. Data on cellular uptake and biodistribution were analyzed by unpaired, two-tailed t-test using GraphPad Prism (v 4.00 for Windows GraphPad Software, San Diego, U.S.) in order to determine any significant differences (P < 0.05). Synthesis of DOTA-Conjugated Affibody Molecules. A synthetic variant of the ZHER2:342 affibody molecule, denoted Z H E R 2 : S 1 , with the sequence AEAKYAKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK-amide was assembled on a 433A Peptide Synthesizer (Applied Biosystems, Foster City, CA) using Fmoc/tBuchemistry in 0.1 mmol scale and Fmoc-rink amide resin with a substitution level of 0.39 mmol/g. Preparation of DOTA-A1ZHER2:S1 (PEP10415), has been described earlier.25 Briefly, the chelator DOTA was conjugated to the deprotected N-terminus of ZHER2:S1, prior to cleavage from solid support. For preparation of DOTA-K50-ZHER2:S1 (PEP11301) and DOTAK58-ZHER2:S1 (PEP11302), an acid-labile side chain protecting 1662



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0.2 M ammonium acetate, pH 5.5, to a concentration of 1 mg/ mL. Aliquots containing 50 μg of each conjugate were stored frozen at −20 °C. For labeling, an aliquot of a conjugate was mixed with predetermined amounts of 111In-chloride solution (ca. 35 MBq at calibration time). The reaction mixtures were incubated at 95 °C. At 10, 20, and 30 min after the start of the incubation, small aliquots (1.4 μL) of reaction mixtures were taken and analyzed by radio-ITLC eluted with 0.2 M citric acid and radiochemical purity was evaluated. In this system, labeled compounds remain at the application point, while free 111In or 111 In-chelate migrates with the solvent front. To cross-validate the ITLC results, an SDS-PAGE analysis on 4−12% bis-tris gel (200 V constant) was performed. To evaluate stability of labeling, the 111In-labeled conjugates were incubated with 500fold excess of EDTA for 4 h at room temperature and then analyzed using ITLC. Distribution of radioactivity along the thin layer chromatography strips and SDS-PAGE gels was measured on a Cyclotrone Storage Phosphor System and analyzed using the OptiQuant image analysis software (Perkin-Elmer, Wellesley, MA, USA). In Vitro Studies. For cell studies, the HER2-expressing ovarian carcinoma SKOV-3, 1.6 × 106 HER2 receptors per cell, and colorectal carcinoma LS174T, 4 × 104 HER2 receptors per cell, cell lines (ATCC, purchased via LGC Promochem, Borås, Sweden) were used. The cell lines were cultured in RPMI medium (Flow Irvine, U.K.) 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 kg, Germany. In vitro specificity test was performed according to the methods described earlier.26 Briefly, a solution of 111In-[DOTAK50]-ZHER2:S1 and 111In-[DOTA-K58]-ZHER2:S1 (1.99 ng/dish, 27 pM) was added to a set of six Petri dishes (ca. 5 × 105 cells in each). For blocking, an excess of nonlabeled recombinant ZHER2:342 (1.99 μg/dish) was added 5 min before 111In-labeled conjugates 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 trypsinEDTA solution (0.25% trypsin, 0.02% EDTA in buffer, Biochrom AG, Berlin, Germany), and the radioactivity in cells and media was measured to calculate percentage of cellbound radioactivity. A comparison of 111In-[DOTA-A1]-ZHER2:S1, 111In-[DOTAK50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 processing by SKOV-3 and LS174T cells was performed according to the method developed and validated by Wållberg and Orlova.26 The cells (ca. 5 × 105 per dish) were incubated with the labeled compounds (1.99 ng/dish, 27 pM) at 37 °C, 5% CO2. At predetermined time points (0.5, 1, 2, 3, 4, 8, and 24 h after incubation start), the medium from a set of three dishes was removed and the cells were washed with ice-cold serum-free medium. The cells were then treated with 0.5 mL 0.2 M glycine buffer containing 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 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. Percent of

group, the 4-methyltrityl (Mtt) group, was used for protection of either Lys50 or Lys58. The Mtt group was removed by TFA/ triisopropylsilane/DCM (1:5:94) treatment for 10 × 2 min. tert-Butyl-protected monoreactive DOTA chelator was manually conjugated to the free ε-amino group of lysine using DOTA (5 equiv), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (5 equiv) and diisopropylethylamine (DIEA) (5 equiv) in NMP at RT overnight, which was repeated until the Ninhydrin test indicated complete conjugation. Removal of side-chain protecting groups and cleavage of the protein from the resin were performed in a single-step treatment for 3 h with TFA/ triisopropylsilane/H2O (95:2.5:2.5) for all variants. Proteins were extracted in tert-butyl methyl ether/water (50:50) three times before the water phase was filtered and lyophilized. Purification and Characterization of DOTA-Conjugated Affibody Molecules. The crude synthetic product was analyzed using RP-HPLC (1200 series, Agilent). For purification, a semipreparative column, Reprosil Gold 10 × 250 mm, 5 μm particle size (Dr Maisch, Ammerbuch, Germany) was used with a flow rate of 3.5 mL/min and a gradient of 25− 45% B (A: 0.1% TFA/H2O and B: 0.1%TFA/CH3CN) over 25 min. Purity analysis was performed with an analytical column, Zorbax CB300-C18 4.6 × 150 mm, 3.5 μm particle size (Agilent), with a flow rate of 1 min/mL using the same gradient as above. Verification of the correct products was performed by ESI-Q-TOF MS (Agilent). Variable temperature measurements (VTM) were performed using a JASCO-810 instrument (Jasco, Tokyo, Japan), in order to determine the melting points for the variants. The DOTAconjugated Affibody molecules were diluted to 0.5 mg/mL in 1 × PBS (pH 7.2) and analyzed in 1 mm quartz cuvette, with the temperature increased by 5 °C/min from 20 to 90 °C and detection at 221 nm. To verify proper refolding after heat treatment, circular dichroism spectra, ranging from 195 to 250 nm, were recorded before and after VTM and presented as the mean value of five accumulated scans. For affinity determination of the DOTA-conjugated proteins, real-time biosensor analysis was performed on a Biacore 3000 instrument (GE Healthcare). The recombinant human extracellular domain of HER2 expressed as an Fc-fusion was diluted to 5 μg/mL in 10 mM NaOAc (pH 4.5), and immobilized on a dextran-coated CM5-chip. NHS/EDC were used for activation and after immobilization, unreacted groups were capped using ethanolamine. For reference, one flow cell was activated and deactivated without ligand immobilization. Pure conjugates were diluted in HBS-EP (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% Tween20, pH 7.2) to concentrations ranging from 0.31 nM to 10 nM and analyzed in duplicate. The concentrations of the proteins were determined by amino acid analysis (Amino Acid Analysis Centre, Uppsala, Sweden). The samples were allowed to interact with the sensor surface during a 5 min association phase followed by a dissociation phase of 10 min, before injection of a short pulse of 25 mM HCl to regenerate the surface. The BIAevaluation 4.1 software was used for the kinetic analysis with a 1:1 Langmuir binding model. Radiolabeling Chemistry and in Vitro Stability. The radioactivity was measured using an automated gamma-counter with a 3 in. NaI(Tl) detector (1480 WIZARD, Wallac OY, Turku, Finland). The data were corrected for background. Before labeling, freeze−dried DOTA-A1-ZHER2:S1, DOTAK50-ZHER2:S1, and DOTA-K58-ZHER2:S1 were reconstituted in 1663



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Table 1. Biophysical Characterization of the DOTA Conjugates variant DOTA-A1-ZHER2:S1 DOTA-K50-ZHER2:S1 DOTA-K58-ZHER2:S1

purity 98% 97% 97%

theor. Mw (Da) 7006.9 7006.9 7006.9

exp. Mw (Da)

ka (M−1·s−1)

kd (s−1)

KD (pM)

Tm (°C)

7007.1 7007.3 7007.0

4.8 × 10 7.2 × 106 7.9 × 106

6.4 × 10−4 7.7 × 10−4 7.4 × 10−4

133 107 94

64 57 62

internalized radioactivity was calculated for each fraction at each time point. In Vivo Studies. The animal experiments were planned and performed in accordance with the national regulation on laboratory animal protection. The animal study plans have been approved by the local Ethics Committee for Animal Research in Uppsala. BALB/C nu/nu mice bearing LS174T colorectal cancer and Ramos lymphoma (as a HER2-negative control) xenografts were used for comparative biodistribution studies. 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 LS174T or Ramos lymphoma cells in Matrigel (BD Bioscience) were implanted in right hind legs. Average tumor weight was 0.54 ± 0.36 g at the time of experiment. Average animal weight was 18.6 ± 0.9 g. The mice were randomized into groups of four. Animals were injected intravenously (tail vein) with 1 μg (30 kBq) of each labeled compound in 100 μL PBS. At four hours after injection, a mixture of Ketalar-Rompun (20 μL of solution per gram body weight; Ketalar: 10 mg/mL; Rompun: 1 mg/mL) was injected intraperitonealy, and the mice were euthanized by a heart puncture using a syringe, prewashed with diluted heparin (5000 IE/mL). Blood and 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 content was used as a measure of hepatobiliary excretion. A set of additional animal studies with a different xenograft model were performed for the conjugate with the best properties, 111In-[DOTA-K58]-ZHER2:S1, to confirm its tumortargeting and imaging capacity. For the biodistribution measurement, 10 × 106 SKOV-3 ovarian carcinoma cells were implanted in right hind legs of BALB/C nu/nu mice. Average tumor weight was 0.30 ± 0.11 g at the time of experiment, and the average animal weight was 18.1 ± 0.6 g. A group of four mice was injected intravenously (tail vein) with 1 μg (30 kBq) 111In-[DOTA-K58]-ZHER2:S1 in 100 μL PBS, and the biodistribution of the radioactivity was measured as described above. One mouse was injected with 3 μg (5 MBq) 111In-[DOTAK58]-ZHER2:S1. At four hours after injection, the animal was euthanized by overdosing of anesthesia, and the cadaver was used in a confirmatory imaging experiment. The SPECT/CT imaging experiment was performed using the Triumph Trimodality system (Gamma Medica, Inc., Northridge, USA), an integrated microSPECT/PET/CT hardware and software platform. A CT acquisition was carried out to place the body of the animal in the camera covering tumor, kidneys, and liver with the following parameters: field of view, 53.82 mm;

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magnification, 2.2; one projection and 512 frames for 2.13 min. SPECT acquisition was performed using two solid-state cadmium zinc telluride (CZT) cameras equipped with a fivepinhole collimator. The following acquisition parameters were used for SPECT: nongated; tomographic; field of view, 53.82 mm; 32 projections/1000 s. SPECT raw data was reconstructed by the FLEX SPECT software, which uses an ordered Subset Expectation Maximization (OSEM) iterative reconstruction algorithm. CT raw files were reconstructed by Filter Back Projection (FBP). SPECT and CT data were fused and analyzed in PMOD (PMOD Technologies Ltd., Zurich, Switzerland). For quantification of uptake, the volumes of interest (VOIs) were defined for tumor and contralateral leg on the CT image, and transferred to the SPECT image.

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RESULTS Design and Synthesis of the DOTA-Conjugated Tracers DOTA-A1-Z HER2:S1 , DOTA-K50-Z HER2:S1 , and DOTA-K58-ZHER2:S1 Affibody Molecules. Conjugation of the DOTA chelator to the ZHER2:S1 affibody molecule via peptide synthesis provided DOTA-A1-ZHER2:S1, DOTA-K50ZHER2:S1, and DOTA-K58-ZHER2:S1, which could be isolated in a final purity of over 97% (Table 1). The identity of all three variants was confirmed by mass spectrometry. According to the surface plasmon resonance measurements, the equilibrium dissociation constants (KD) were 133 pM, 107 pM, and 94 pM for DOTA-A1-ZHER2:S1, DOTA-K50-ZHER2:S1, and DOTA-K58ZHER2:S1, respectively. The melting points for the proteins with DOTA conjugated to either the N- or C-terminus had similar values, 64 and 62 °C, respectively, whereas conjugation of DOTA to the side chain of Lys50 had a certain destabilizing effect on the structure, and decreased the thermal stability of the protein to 57 °C. However, CD analysis showed good refolding properties for all variants after heat treatment to 90 °C. Labeling and Stability of DOTA-Conjugated ZHER2:S1 Affibody Molecules with 111In. Radiochemical yield of all three variants was more than 99% (specific radioactivity of 1.5 MBq/μg) after labeling at 95 °C in all incubation time points. The stability of all three labeled compounds was evaluated by challenge with a 500-fold molar excess of EDTA during 4 h, which demonstrated very high labeling stability (release of free 111 In of less than 1.5%). The radiochemical purity of the treated samples was 96.7 ± 0.43% (average ± SEM), while the purity of the untreated control was 98.1 ± 0.36%, i.e., the difference was within the accuracy of the analytical method. Results of the analysis were confirmed by radioSDS PAGE analysis (Figure 1). In Vitro Studies. A binding specificity test showed that there was a significant decrease of binding of labeled compounds to the HER2 receptors after receptor presaturation by incubation with an access of nonlabeled ZHER2:342 affibody molecules in both SKOV-3, and LS174T cell lines (Figure 2). This indicates that binding of all labeled compounds was HER2 receptor mediated. The level of cell associated radioactivity was lower for nonblocked LS174T than for SKOV-3 cell line, 1664



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In-[DOTA-A1]-ZHER2:S1 and 111In-[DOTA-K50]-ZHER2:S1 with 31 ± 1% and 37 ± 4%, respectively. The cellular processing of 111In-[DOTA-A1]-ZHER2:S1, 111In[DOTA-K50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 by HER2-expressing LS174T cells (presented in Figure 4) show a similar pattern to SKOV-3 cells. However, cell-associated radioactivity increased with a slower rate at early time points in comparison to that of SKOV-3 cells. After 24 h incubation, 35.7 ± 0.1%, 38.5 ± 1%, and 38.5 ± 1% of the cell-associated radioactivity was internalized for 111In-[DOTA-A1]-ZHER2:S1, 111 In-[DOTA-K50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1, respectively. In Vivo Studies. Data concerning targeting specificity of HER2-expressing LS174T xenografts in BALB/C nu/nu mice using 111In-[DOTA-A1]-ZHER2:S1, 111In-[DOTA-K50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 are presented in Figure 5. In order to verify the HER2-specificity of 111In-labeled affibody molecules, their biodistribution at 4 h p.i. was also studied in BALB/C nu/nu mice bearing HER2-negative Ramos lymphoma xenograft (Figure 4). The tumor uptake in HER2-positive xenografts was significantly higher than in HER2-negative, 9.65 ± 1.9 vs 0.17 ± 0.06%ID/g (p < 0.0005), 5.53 ± 0.91 vs 0.25 ± 0.02%ID/g (p < 0.0001), and 11.77 ± 1.94 vs 0.34 ± 0.06%ID/ g (p < 0.0001), for 111In-[DOTA-A1]-ZHER2:S1, 111In-[DOTAK50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1, respectively. This demonstrates specificity of HER2 targeting by all conjugates. Comparison of 111In-[DOTA-A1]-ZHER2:S1, 111In-[DOTAK50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 biodistribution is presented in Figures 6 and 7 and Tables S1 and S2. All radiolabeled conjugates showed rapid clearance from blood and nonexcretory organs. The low radioactivity level in the gastrointestinal tract (with content) indicated that the hepatobiliary pathway played a minor role in the excretion of all radioconjugates. Predominantly renal clearance was accompanied by reabsorption in kidneys. There were apparent differences in the biodistribution of the radioconjugates 111In[DOTA-A1]-ZHER2:S1, 111In-[DOTA-K50]-ZHER2:S1, and 111In[DOTA-K58]-ZHER2:S1. For example, the radioactivity uptake of 111 In-[DOTA-K58]-ZHER2:S1 in lung, liver, and spleen was significantly higher than uptake of two other variants. The bone uptake of 111In-[DOTA-A1]-ZHER2:S1was the highest. Tumor radioactivity accumulation of 111In-[DOTA-K50]-ZHER2:S1 was significantly lower than that of two other variants. The difference between tumor uptakes of 111In-[DOTA-K58]ZHER2:S1 and 111In-[DOTA-A1]-ZHER2:S1 was not significant. Accordingly, high tumor uptake and rapid blood clearance of 111 In-[DOTA-K58]-ZHER2:S1 resulted in significantly higher tumor-to-blood ratio (Figure 7). Low tumor uptake of 111In[DOTA-K50]-ZHER2:S1 resulted in the lowest tumor-to-organ ratios. The biodistribution of the conjugate with the best targeting properties, 111In-[DOTA-K58]-ZHER2:S1, was verified in an additional HER-expressing xenograft model based on the SKOV3 ovarian carcinoma cell line. Comparison between 111In[DOTA-K58]-ZHER2:S1 biodistribution in mice bearing LS174T and SKOV3 xenografts is presented in Figure 8. The biodistribution data were in very good agreement. There was a significant difference (p < 0.05) in the tumor uptake between these two models: 16.3 ± 2.2%ID/g for SKOV3 tumors and 11.7 ± 1.9%ID/g for LS174T. There were also small but

Figure 1. Representative SDS-PAGE analysis of the stability of 111Inlabeled Affibody molecules (111In-[DOTA-K58]-ZHER2:S1) in serum. (1) 111In-[DOTA-K58]-ZHER2:S1 incubated in a 500-fold molar excess of EDTA during 4 h; (2) 111In-acetate used as a marker for lowmolecular-weight compounds. The signal was measured as digital light units (DLU) is proportion to radioactivity in a given point of a lane in the SDS-PAGE gel.

Figure 2. Binding specificity of 111In-[DOTA-K50]-ZHER2:S1, and 111In[DOTA-K58]-ZHER2:S1 to HER2 expressing cell lines SKOV3 (upper row) and LS174T (lower row). Two groups of culture dishes containing test cells were incubated with 27 pM of radiolabeled conjugate. One group of culture dishes was pretreated with saturating amounts of nonlabeled ZHER2:342. Cell-associated radioactivity was calculated as a percentage of total added radioactivity.

reflecting lower expression level of HER2 receptors. Specificity of 111In-[DOTA-A1]-ZHER2:S1 binding to HER2-expressing cells has been established earlier.25 The cellular processing of 111In-[DOTA-A1]-ZHER2:S1, 111In[DOTA-K50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 by HER2-expressing SKOV-3 cells are presented in Figure 3. All labeled compounds demonstrated a similar processing pattern, which is typical for ZHER2:342 and its derivatives. Internalization rate of all three labeled compounds was slow, although internalized fractions increased continuously throughout the experiment. There was some variation between conjugates. After 24 h incubation, the internalization rate of 111In-[DOTAK58]-ZHER2:S1 with 43.6 ± 0.3% was slightly higher than that of 1665



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Figure 3. In vitro cellular processing of 111In-[DOTA-A1]-ZHER2:S1, 111In-[DOTA-K50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 bound to SKOV-3 cells. Cells were incubated with the labeled compounds at 37 °C. Acid wash was used to determine the membrane-bound radioactivity. Cell-bound activity is normalized to the maximum uptake. Error bars might not be seen in some because they, in most cases, are smaller than the symbols.

Figure 4. In vitro cellular processing of 111In-[DOTA-A1]-ZHER2:S1, 111In-[DOTA-K50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 bound to LS174T (moderate HER2 expression) cells. Cells were incubated with the labeled compounds at 37 °C. Acid wash was used to determine the membranebound radioactivity. Cell-bound activity is normalized to the maximum uptake. Error bars might not be seen because they, in most cases, are smaller than the symbols.

influences both biodistribution and blood clearance rate of In-labeled proteins.25 However, not only the composition of a chelator might influence the biodistribution and targeting properties of a targeting peptide, but also its position in the peptide. The position of a chelator in a peptide might influence the binding affinity, by affecting the interaction between the peptide and its molecular target. For example, coupling of DOTA chelators to the C-terminal end of a short α-MSH analogue [Nle4,Asp5,D-Phe7]-α-MSH4−11 (NAPamide) via the ε-amino group of Lys11 instead of the N-terminal α-amino group increased the affinity almost 7-fold, from 1.37 to 0.21 nM.32 Improved affinity translated to twice higher uptake of 111 In-DOTA-NAPamide in melanoma xenografts in mice. At the same time, the blood clearance rate was appreciably slower, 0.12 ± 0.01%ID/g at 4 h p.i. for 111In-DOTA-NAPamide vs 0.03 ± 0.00%ID/g for the protein with DOTA at the Nterminus. Still, such a strong influence of the chelator positioning is not surprising for a short octapeptide, where the molecular weight of the chelator is comparable to the molecular weight of the polypeptide moiety. For the significantly larger affibody molecules, such an influence would be less expected. However, it has been shown that placement of a hexahistidine chelator moiety at either the C- or N-terminus of affibody molecules had a substantial effect on the biodistribution of [99mTc(CO)3]+-labeled affibody molecules.23 It is noteworthy that the placement of the label at the Cterminus reduced hepatic uptake 2-fold, but increased blood radioactivity concentration 4-fold at 4 h pi. That study suggested that positioning of a lipophilic label can modify the biodistribution of affibody molecules.

significant differences in the liver and spleen uptake between the models. MicroSPECT/CT imaging, performed 4 h pi (Figure 9), confirmed the results of the biodistribution experiments. 111In[DOTA-K58]-ZHER2:S1 was capable of high-contrast imaging of the HER2-expressing xenograft. The tumor-to-contralateral leg ratio (calculated as a ratio between average concentrations in the VOIs defined by CT) was 8.2.

111

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DISCUSSION Affibody molecules represent a new class of affinity proteins capable of molecular recognition in vivo. Recently, a clinical study confirmed the feasibility of imaging of HER2-expressing metastases using 111In- and 68Ga-labeled affibody molecules.27 Development of several affibody-based imaging agents for visualization of EGFR,28 HER3,29 IGF1R,14 and PDGFRβ 30 has been reported. It is very likely that affibody molecules will evolve into a novel class of targeting agents for radionuclide molecular imaging. This prompts us to investigate different factors influencing the sensitivity of molecular imaging using affibody molecules. An important parameter here is the ratio of radioactivity concentrations between tumors and healthy tissues, as this ratio determines the imaging contrast. The labeling chemistry (i.e., the chemical and physicochemical properties of the radionuclide and the chelator/linker) is essential, as it can influence the binding affinity and cellular processing of an imaging agent, the cellular retention of a radionuclide, the biodistribution of a targeting peptide, and the excretion pathways of nonbound tracer and radiocatabolites.31 Earlier, we showed that the chemical nature of chelators coupled to the N-terminus of synthetic affibody molecules 1666



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Figure 6. Comparative biodistribution of 111In-[DOTA-A1]-ZHER2:S1, 111 In-[DOTA-K50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 in BALB/C nu/nu mice bearing LS174T xenografts at 4 h after injection. The concentration of radioactivity is expressed as % ID/g, and presented as an average value from 4 animals ± standard deviation. Data for gastrointestinal tract (GI) with content and carcass are presented as % of injected radioactivity per whole sample.

Figure 5. HER2-targeting specificity using 111In-[DOTA-A1]-ZHER2:S1, 111 In-[DOTA-K50]-ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 in BALB/C nu/nu mice. The tumor uptake is expressed as %ID/g, Radioactivity uptake in HER2-positive LS174T xenografts was significantly (p < 0.05) higher than in HER2 negative Ramos xenografts.

Figure 7. Comparison of tumor-to-organ ratios for 111In-[DOTA-A1]Z HER2:S1 , 111 In-[DOTA-K50]-ZHER2:S1, and 111In-[DOTA-K58]ZHER2:S1 in BALB/C nu/nu mice bearing LS174T xenografts at 4 h after injection.

In this study, we investigated the effect of placement of a hydrophilic DOTA chelator in different positions of an affibody molecule. The conjugation chemistry via formation of an amide bond with one of the carboxylic arms of DOTA was the same for all three tested variants. There was a slight influence of the chelator positioning on the affinity (Table 1), with the highest affinity found for [DOTA-K58]-ZHER2:S1. The binding site of affibody molecules comprises amino acids at helices 1 and 2, close to the N-terminus.17,33 NMR studies of a HER2-binding affibody molecule have shown that the N-terminal part of the protein displays a high degree of conformational dynamics,33 suggesting that a chelator conjugated to the flexible N-terminus could affect the affibody binding site. In contrast, placement of the chelator at K58 (C-terminus), at the end of helix 3, provides a large distance between the chelator and the binding site, and it can be speculated that this positioning minimizes interference of the chelator with binding to a molecular target. However, the effect was quite modest, and the difference between the highest and the lowest dissociation constants was only 1.4-fold. There was a noticeable effect of the DOTA position on the melting

point, suggesting a certain destabilizing effect of placement of DOTA at K50, i.e., in the middle of helix 3. All three conjugates were successfully labeled with 111In with high label stability and preserved binding capacity (Figure 2). A study using two different HER2-expressing cell lines suggested a rather small influence of the chelator position on the internalization rate (Figures 3 and 4). However, 111In-[DOTAK58]-ZHER2:S1 still showed the highest degree of internalization in both SKOV-3 and LS174T cells. In vivo, all conjugates could target HER2-expressing LS174T xenografts specifically (Figure 5). However, the tumor uptake of 111In-[DOTA-K50]-ZHER2:S1 was significantly lower than the uptake of the two other conjugates. This cannot be explained by lower bioavailability, as the blood concentration was similar for all variants. It might be that the destabilizing effect of the chelator coupled to helix 3 causes a substantial loss of binding capacity in vivo. The uptake of 111In-[DOTA-K58]-ZHER2:S1 was the highest in lung, liver, and spleen, but the blood concentration decreased more rapidly for this conjugate 1667



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Earlier, a recombinant 111In-[MMA-DOTA-C61]-ZHER2:2395 affibody molecule having DOTA conjugated via maleimide to a C-terminal cysteine has been compared with 111In-DOTAZHER2:342 (with N-terminal DOTA).24 In that study, 111In[MMA-DOTA-C61]-ZHER2:2395 provided equal tumor-to-blood and tumor-to-liver ratios in comparison with 111In-DOTAZHER2:342, but higher tumor-to-spleen and tumor-to-bone ratios. In the present study, 111In-[DOTA-K58]-ZHER2:S1 had not only higher tumor-to-bone ratio compared to 111In-[DOTA-A1]ZHER2:S1, but also higher tumor-to-blood ratio. However, the tumor-to-lung and tumor-to-spleen ratios were lower for 111In[DOTA-K58]-ZHER2:S1. This might be an indication that not only the positioning of chelator, but also the chemistry of its conjugation to affibody molecules influences biodistribution and targeting properties. However, 111In-[MMA-DOTA-C61]ZHER2:2395 and 111In-[DOTA-K58]-ZHER2:S1 differ not only in the way the chelator is conjugated, but also in the amino acid sequence (six different amino acids), which complicates the ability to draw conclusions. We showed earlier that decreased N-terminal lipophilicity in affibody molecules can reduce hepatic uptake and hepatobiliary excretion of radioactivity.23,22 This might further increase the sensitivity of imaging of hepatic metastases. On the basis of these results, it can be suggested that re-engineering of the affibody scaffold aiming to further increase the hydrophilicity of the N-terminus of affibody molecules might be desirable in the case of placement of a chelator at K58. In conclusion, the results of this study suggest that optimization of the labeling chemistry has the potential to further increase the contrast of molecular imaging using affibody molecules. The positioning of the DOTA chelator influences the melting point, the cellular processing of an affibody molecule by cancer cells, and the biodistribution and targeting properties. This creates preconditions for further enhancement of the tumor-to-organ ratios, and thereby, sensitivity of molecular imaging. This is particularly important for visualization of small metastatic lesions, when the partial volume effect causes decrease of the imaging sensitivity.

Figure 8. Comparative biodistribution of 111In-[DOTA-K58]-ZHER2:S1 in BALB/C nu/nu mice bearing LS174T and SKOV3 xenografts at 4 h after injection. The concentration of radioactivity is expressed as %ID/ g, and presented as an average value from 4 animals ± standard deviation. Data for gastrointestinal tract (GI) with content and carcass are presented as % of injected radioactivity per whole sample.

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Figure 9. Imaging of HER2 expression in SKOV3 xenografts in BALB/C nu/nu mice using microSPECT/CT. Images were acquired 4 h after administration of the tracer. Arrows point at tumor (T) and kidneys (K).

ASSOCIATED CONTENT

S Supporting Information *

Tabulated data concerning biodistribution and tumor-to-organ ratios for 111In-[DOTA-A1]-ZHER2:S1, 111In-[DOTA-K50]ZHER2:S1, and 111In-[DOTA-K58]-ZHER2:S1 in female in BALB/ C nu/nu mice bearing LS174T colorectal cancer xenografts 4 h after intravenous injection. This material is available free of charge via the Internet at http://pubs.acs.org.

compared to 111 In-[DOTA-A1]-Z HER2:S1 . Overall, 111In[DOTA-K58]-ZHER2:S1 provided the highest tumor-to-blood and tumor-to-bone ratios, although the tumor-to-liver ratio was the lowest (Figure 7). As blood-borne radioactivity contributes to radioactivity in many organs and tissues, the highest tumorto-blood ratio is an important argument for conjugation of DOTA to K58 in synthetic affibody molecules. Furthermore, 111 In-[DOTA-K58]-ZHER2:S1, as the conjugate with the best in vivo targeting properties, was additionally evaluated in a second xenograft model based on SKOV-3 xenografts. The biodistribution data for both models were in a very good agreement (Figure 8). The higher uptake in SKOV3 xenografts can be explained by the appreciably higher expression of HER2 in this cell line. The small discordance between the models with respect to liver and spleen uptake of radioactivity might be due to certain physiological variability between the animal batches. Furthermore, an imaging study confirmed that 111In-[DOTAK58]-ZHER2:S1 can image SKOV-3 xenografts with high contrast (Figure 9).

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AUTHOR INFORMATION

Corresponding Author

*Phone: +46 18 471 3414. Fax: + 46 18 471 3432. E-mail: [email protected]. Author Contributions

Anna Perols and Hadis Honarvar contributed equally to this study Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from the Swedish Cancer Society (Cancerfonden) and the Swedish Research Council (Vetenskapsrådet). 1668



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Influence of DOTA Chelator Position on Biodistribution and Targeting

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