1956
Bioconjugate Chem. 2007, 18, 1956–1964
99m
Tc-maEEE-ZHER2:342, an Affibody Molecule-Based Tracer for the Detection of HER2 Expression in Malignant Tumors Thuy Tran,† Torun Engfeldt,‡ Anna Orlova,†,§ Mattias Sandström,| Joachim Feldwisch,†,§ Lars Abrahmsén,§ Anders Wennborg,§ Vladimir Tolmachev,†,§,⊥ and Amelie Eriksson Karlström*,‡ Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Sweden, School of Biotechnology, Division of Molecular Biotechnology, Royal Institute of Technology, Stockholm, Sweden, Affibody AB, Bromma, Sweden, Medical Radiation Physics, Uppsala University Hospital, Uppsala University, Uppsala, Sweden, and Department of Medical Sciences, Uppsala University, Uppsala, Sweden. Received July 13, 2007; Revised Manuscript Received September 10, 2007
Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of 99mTc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule ZHER2:342 with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in Vitro stability were investigated. The biodistribution of 99mTc-maGEG-ZHER2:342 and 99mTc-maEEE-ZHER2:342 was compared with 99mTc-maGGG-ZHER2:342 in normal mice, and the tumor targeting properties of 99mTc-maEEEZHER2:342 were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in Vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for 99mTc-labeling of ZHER2:342 reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. 99m Tc-maEEE-ZHER2:342 showed a receptor-specific tumor uptake of 7.9 ( 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with 99mTc-maEEE-ZHER2:342 could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for 99mTc labeling is an efficient way to modify the in ViVo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making 99mTc-maEEE-ZHER2:342 a promising tracer for clinical imaging of HER2 overexpression in tumors.
INTRODUCTION Malignant transformation of cells may cause altered expression of distinctive molecular features relative to the surrounding tissues. Overexpression of a certain protein is often correlated with tumor progression and/or with tumor response to a certain treatment. Molecular imaging of such protein targets provide important diagnostic information that can influence patient management (1). One such target that has been extensively studied is the HER2/neu receptor (also known as erbB-2), a member of the receptor tyrosine kinase superfamily. HER2 is overexpressed in 20–30% of all breast cancers and in a variety of other tumors of epithelial origin (2, 3). Treatment with the humanized antibody trastuzumab (Herceptin, Genentech) improves the survival of patients with HER2-positive breast cancers, likely by the prevention of receptor dimerization and disruption of signaling pathways (4). Other anti-HER2 therapeutic agents are under clinical evaluation, including the monoclonal antibody pertuzumab (Omnitarg) (5), the heat-shock * Corresponding author. Amelie Eriksson Karlström, School of Biotechnology, Division of Molecular Biotechnology, Royal Institute of Technology, AlbaNova University Center, Roslagsvägen 30B, S-106 91 Stockholm, Sweden. Tel: +46-8-5537 8333. Fax: +46-8-5537 8481. E-mail:
[email protected]. † Division of Biomedical Radiation Sciences, Uppsala University. ‡ Royal Institute of Technology. § Affibody AB. | Medical Radiation Physics, Uppsala University. ⊥ Department of Medical Sciences, Uppsala University.
protein 90 inhibitor 17-AAG (6), and the tyrosine kinase inhibitor lapatinib (7). An accurate determination of HER2 status is required in order to select breast cancer patients for Herceptin therapy. Currently, both the American Association of Clinical Oncology (8) and the European Group on Tumor Markers (9), recommend that HER2 expression should be evaluated on every primary breast cancer in order to identify patients who may benefit from Herceptin treatment. The most widely used methods for the detection of HER2 are immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH) of biopsy samples (10). However, these tests have some limitations (8), including inability to detect expression heterogeneity within a tumor lesion (11, 12) and discordance in HER2 expression between the primary tumor and the metastases (13). There is also subjectivity in evaluating the staining score (11). In contrast, radionuclide imaging may be applied to visualize HER2 expression in both whole primary tumors and metastatic lesions. HER2 has no natural ligands (14), and monoclonal antibodies have been evaluated as targeting agents for radionuclide imaging (15). The general issues associated with the large size of intact IgG (Mw ∼150 kDa) are slow blood clearance and slow tumor penetration, leading to low contrast of images (16). Reduction of the size of targeting proteins is considered a promising approach to improve the image contrast (17) and has generated a number of anti-HER2 antibody fragments such as F(ab′)2 (18), Fab (19), single chain Fv (25–55k Da) (20), and diabody (21). Our approach to improve the radionuclide imaging contrast is to create a relatively small HER2-targeting protein by using
10.1021/bc7002617 CCC: $37.00 2007 American Chemical Society Published on Web 10/19/2007
Tumor Targeting with Affibody Molecules
Affibody technology, on the basis of the 58 amino acid B-domain of staphylococcal protein A. Randomization of 13 solvent-exposed residues has generated an Affibody library (3 × 109 variants), which has been used for the selection of affinity ligands to a variety of proteins (22). Affibody molecules are low molecular weight (7 kDa) and cysteine-free three-helix bundle proteins. The anti-HER2 Affibody molecule ZHER2:342 binds to the extracellular domain of HER2 with an affinity of 22 pM (23). Recombinant ZHER2:342, labeled with 125I and 111In, demonstrated clear visualization of HER2-expressing SKOV-3 xenografts in mice (23, 24). However, the use of peptide synthesis for the production of targeting agents offers the advantage of site-specific incorporation of chelators. ZHER2:342 has been produced by peptide synthesis with concomitant coupling of a DOTA chelator to the N-terminus of the protein (25). The labeling of this protein with 111In and 68Ga also provided high quality imaging of HER2-expressing tumors in patients (26). One of the most commonly used radionuclides for diagnostics is 99mTc, as it has favorable photon energy (140 keV), is relatively inexpensive, and readily available. Its physical halflife (t1/2 ) 6 h) is compatible with the rapid kinetics of Affibody molecules. Previously, the synthetic Affibody molecule ZHER2:342 has been produced with the chelator maGGG (mercaptoacetyltriglycyl) site-specifically introduced at the N-terminus for the attachment of technetium-99m (27). The 99mTc-maGGG-ZHER2:342 specifically targets HER2-expressing tumors in mice, resulting in high tumor-to-nontumor ratios for imaging (27). However, this conjugate had predominant hepatobiliary clearance that caused a high radioactivity accumulation in the intestine (intestine plus content contained 30% of the injected activity at 4 h pi). This would restrict its use for the detection of HER2 tumors and metastases in the abdominal area on the day of injection (27). The excretion pathway can be shifted from hepatobiliary to renal if the lipophilicity of the radiolabeled conjugates is reduced (28, 29). For example, the use of the aspartic acidcontaining chelators MADG and MAD2, for chelation of 99mTc has clearly increased the renal and decreased the hepatobiliary excretion of 99mTc-labeled chemotactic peptide conjugates (30). Glutamic acid (E) is a polar, negatively charged, and very hydrophilic amino acid (31). Moreover, glutamic acid is more suitable for peptide synthesis than aspartic acid since a commonly occurring side reaction under synthetic conditions is the formation of aspartimide by cyclization of aspartyl residues (32). We hypothesized that the use of glutamic acid instead of glycine in the chelating sequence of ZHER2:342 would decrease the lipophilicity of Affibody molecules enough to suppress hepatobiliary excretion. The goal of this study was to investigate whether the use of mercaptoacetyl-triglutamyl chelator would provide better biodistribution properties of Affibody molecules, resulting in a tracer suitable for imaging on the day of injection. Here, we compare the biodistribution of 99mTc-labeled mercaptoacetyl-Gly-Gly-Gly-ZHER2:342 (99mTc-maGGG-ZHER2:342), mercaptoacetyl-Gly-Glu-Gly-ZHER2:342 (99mTc-maGEG-ZHER2:342), and mercaptoacetyl-Glu-Glu-Glu-ZHER2:342 (99mTc-maEEEZHER2:342) and describe the in ViVo tumor targeting properties of the best conjugate, 99mTc-maEEE-ZHER2:342.
MATERIALS AND METHODS Peptide Synthesis. The ZHER2:342-sequence was assembled using Fmoc/tBu solid phase peptide synthesis on an Fmoc-amide resin with a substitution of 0.67 mmol/g (Applied Biosystems) as previously described (27). 1-Hydroxybenzotriazole (HOBt) and 2-(1H -benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (Advanced ChemTech, Louisville, KY) were used to activate equal molar equivalents of Fmoc-protected amino acid.
Bioconjugate Chem., Vol. 18, No. 6, 2007 1957
The Tc-chelating sequences were introduced by step-wise manual synthesis on the N-terminus of the ZHER2:342-sequence. Twenty percent piperidine-N-methylpyrrolidone (NMP, from VWR) was used for Fmoc deprotection. Acylations were performed with 5 molar equivs of amino acid, HBTU, and HOBt and 10 equivs of N-ethyldiisopropylamine (DIEA, from Lancaster Synthesis, Morecambe, England). S-trityl-mercaptoacetic acid was from AnaSpec Inc. (San Jose, CA). Peptides were released from the solid support and permanent protection groups by incubation in trifluoroacetic acid (TFA, Merck international)/H2O/ethanedithiol (EDT, Aldrich chemical company Inc.)/triisopropylsilane (TIS, Merck International) (94:2.5:2.5:1) for 2 h. The proteins were extracted in tert-butyl methyl ether/H2O (50:50). Analytical RP-HPLC was run on the crude synthetic product using a 4.5 × 150 mm polystyrene/divinylbenzene matrix column with 5 µm particles (Amersham Biosciences, Sweden) and a 20 min elution gradient from 20 to 60% B (B, 0.1% TFACH3CN; A, 0.1% TFA-H2O). The molecular weights of the modified Affibody molecules were confirmed by mass spectrometry, and the proteins were purified on the same column in a 20 min gradient from 25 to 45% B. Biosensor Analysis. EDC/NHS chemistry was used to immobilize the extracellular domain of HER2 (Fox Chase Cancer Center, Philadelphia, PA) and Human Serum Albumin, HSA, (KabiVitrum, Sweden) on a CM5 sensor chip (Biacore AB, Sweden). HER2 and HSA were diluted to 20 µg/mL in 20 mM NaOAc at pH 4.5, and the immobilization levels reached 500 and 900 response units (RU), respectively. The different Affibody conjugates were diluted to concentrations varying from 0.5 to 10 nM in HBS (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20 at pH 7.4), and the binding kinetics were studied in a 5 min association phase and a 10 min dissociation phase, followed by regeneration with 20 mM HCl in HBS. 99m Tc Labeling. For labeling, 20 µL of maGEG-ZHER2:342 or maEEE-ZHER2:342 (1 mg/mL in deionized degassed water) was mixed with 20 µL of 0.15 M NaOH to obtain a final pH of about 11. A solution of 10 µL of SnCl2 · 2 H2O in 0.01 M HCl (1 mg/mL) was added to the mixture, followed by 100–200 µL of freshly eluted pertechnetate solution. The mixture was slightly vortexed and incubated at room temperature for 60 min. After labeling, 99mTc-maGEG-ZHER2:342 and 99mTc-maEEE-ZHER2:342 were isolated from unreacted technetium and other lowmolecular-weight components using size-exclusion chromatography on disposable NAP-5 columns (Amersham Pharmacia Biotech AB, Uppsala, Sweden) pre-equilibrated with PBS. The purity was assessed by analysis with ITLC SG (Gelman Sciences Inc., Ann Arbor), eluted in PBS. Validation experiments demonstrated that in this system, pertechnetate and cysteine complexes of 99mTc migrate with the eluent front, while labeled Affibody molecules under these conditions remain at the origin. The labeling yield and the stability were determined using ITLC strips, eluted with PBS, and analyzed with Cyclone Storage Phosphor System. To determine the presence of reduced hydrolyzed technetium, ITLC was eluted with pyridine/acetic acid/water (5:3:1.5). In this eluent, the technetium colloids remained at the origin, while the radiolabeled Affibody molecules, pertechnetate and other complexes of 99mTc, migrated with the solvent front. Stability Studies. Cysteine Challenge. The stability of labeled Affibody conjugates was tested in 300 molar excess of cysteine. A fresh solution of cysteine was prepared (1 mg/mL in PBS at pH 7.0). The radiolabeled conjugates were added to a final molar ratio of cysteine to peptide of 300:1. The test tubes were incubated for 2 h at 37 °C, and the radiochemical purity was analyzed using ITLC.
1958 Bioconjugate Chem., Vol. 18, No. 6, 2007
Plasma Stability Test. Serum samples were prepared as described in ref 27. Shortly, a serum sample (240 µL) was mixed with freshly labeled 99mTc-maEEE-ZHER2:342 (10 µL) to obtain an Affibody molecule concentration similar to the concentration in blood at the moment of injection. The sample was incubated for 1 h at 37 °C. After incubation, the sample was analyzed on NuPAGE 4–12% Bis-Tris Gel (Invitrogen) in MES buffer (200 V constant). A sample of pertechnetate, not treated in blood serum, was used as reference and run in parallel with the test sample on the same gel. After electrophoresis, the radioactivity distribution along the gel was evaluated using a Cyclone Storage Phosphor System. In Vitro Cell Studies. The HER2-expressing ovarian carcinoma cell line SKOV-3, displaying approximately 1.2 × 106 HER2 receptors per cell (33), was used in this study. The cell line was cultured in McCoy’s medium (Flow Irvine, UK). The medium was 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. The cells were cultured at 37 °C in a humidified incubator with 5% CO2 and trypsinized using trypsin–EDTA solution (0.25% trypsin and 0.02% EDTA) from Biokrom Kg, Germany. SKOV-3 cells were cultivated on Petri dishes with a diameter of 3.5 cm to a cell density of 2–5 × 105 cells per dish. Binding Specificity Study. To test the binding specificity of the labeled conjugates, one set of SKOV-3 cells was presaturated with a 100-fold excess of nonlabeled Affibody molecules 10 min before the labeled conjugate was added and measured according to Engfeldt et al. (27). Cellular Retention Study. Labeled conjugate (0.8 ng/105 cells) was added in 1 mL complete medium to SKOV-3 cells and incubated for 2 h at 37 °C. The incubation medium was removed, and the cells were washed six times with ice-cold serum-free medium, and after the addition of 1 mL of complete medium, the cell dishes were incubated at 37 °C and 5% CO2 at predetermined time points. The incubation medium was removed, and the dishes were washed six times with 1 mL serum-free medium. The cells were detached with 0.5 mL of trypsin–EDTA for 10 min in 37 °C and resuspended with 0.5 mL of complete medium. The media were collected, and the radioactivity was measured on an automated gamma counter. Antigen Binding Capacity Study. An assessment of the antigen binding capacity of 99mTc-labeled conjugates was performed using SKOV-3 cells according to Engfeldt et al. (27). In ViWo Animal Studies. The animal study was approved by the Local Ethics Committee for Animal Research. In comparative biodistribution studies, nontumor-bearing NMRI mice were used. In tumor-targeting and imaging experiments with 99mTc-maEEE-ZHER2:342, female BALB/c nu/nu nude mice were used. The animals were acclimatized for one week at the Rudbeck laboratory animal facility before tumor implantation. The tumors were grafted by subcutaneous injection of ∼107 SKOV-3 cells in the right hind leg. Xenografts were allowed to develop during 6 weeks. In all biodistribution studies, the mice were euthanized at predetermined time points with an intraperitoneal injection of Ketalar-Rompun solution (20 µL of solution per gram of body weight: Ketalar [ketamine], 10 mg/ mL; Rompun [xylazin], 1 mg/mL). The mice were heartpunctured with a 1 mL syringe rinsed with diluted heparin (5,000 IE/mL, Leo Pharma, Copenhagen, Denmark). Blood and organ samples were taken, weighed, and their radioactivity measured using an automatic gamma counter. The organ uptake values were calculated as percent injected activity per gram tissue (% IA/g). Biodistribution in NMRI Mice. Eight mice were randomly divided into two groups with four animals each. The two groups were subcutaneously injected with 99mTc-maGEG-ZHER2:342 and
Tran et al. Table 1. Peptide Synthesis and Characterization Data protein
synthetic yield (%)
theoretical Mw (Da)
experimental Mw (Da)
KD (pM)
ZHER2:342 maGGG-ZHER2:342 maGEG-ZHER2:342 maEEE-ZHER2:342
21 16 17 13
6718 7021 7036 7180
6718 7020 7035 7179
80 200 360 410
Table 2. In Witro Data of
99m
Tc-labeled Conjugates 99m
conjugate labeling yield, % isolated yield, % radiochemical purity, % antigen binding capacity, % stability during cysteine challenge, %
Tc-ma GGGZHER2:342
99m
Tc-ma GEGZHER2:342
99m
Tc-ma EEE-
ZHER2:342
82 ( 12 76 ( 8 78 ( 1
93 ( 6 76 ( 3 97 ( 3
90 ( 2 76 ( 4 98 ( 2
86 ( 1
68.7 ( 0.8
66.7 ( 0.3
88 ( 2
94 ( 3
93 ( 3
Tc-maEEE-ZHER2:342 (1µg Affibody ligand, ∼100 kBq in 100 µL of PBS). The mice were euthanized at 4 h pi; organ samples were collected and measured. Biodistribution in Mice with SKOV-3 Xenografts. The tumortargeting properties of 99mTc-maEEE-ZHER2:342 were evaluated in female BALB/c nu/nu nude mice bearing SKOV-3 xenografts. The mice were subcutaneously co-injected with 99mTc-maEEEZHER2:342 and 125I-PIB-ZHER2:342 (total of 1 µg Affibody ligand and ∼100 kBq in 100 µL of PBS). In previous studies, we have validated that the biodistribution of radiolabeled Affibody molecules 4 h pi is the same for both i.v. and s.c. injection and is independent of the label (34, 35). However, subcutaneous injections are generally more reproducible (smaller errors), which is important for comparative biodistribution studies. After 1, 4, and 6 h pi, the mice were euthanized as described earlier. In the binding specificity study, one group of mice was preinjected with 1,000fold molar excess of nonlabeled Affibody molecules prior to injection of 99mTc-maEEE-ZHER2:342. At 4 h pi, the mice were sacrificed, and organ samples were collected and measured. Gamma Camera Imaging with 99mTc-maEEE-ZHER2:342. One group of mice was preinjected through the tail vein with 1000fold excess of nonlabeled Affibody molecules in order to block the receptors and was imaged after 5 h pi. The other three groups were intravenously injected through the tail vein with 3 MBq of 99mTc-maEEE-ZHER2:342 (10 µg in 100 µL of PBS) and imaged at 1, 2, and 5 h pi. Imaging was performed using a Siemens gamma camera equipped with a LEHR collimator at the Department of Nuclear Medicine of Uppsala University Hospital. Static images were collected for 10 min, and evaluation of the images was performed with a Hermes system (Hermes Medical Solutions). 99m
RESULTS Peptide Synthesis. The Fmoc solid-phase synthesis of ZHER2:342 resulted in a yield of 21% of the full-length peptide, as determined by analytical RP-HPLC. The 99mTc-chelating sequences of maGEG or maEEE were introduced N-terminally by manual synthesis, resulting in total synthetic yields of 17% and 13%, respectively (Table 1). Following purification by RPHPLC, the purity was higher than 90%. The molecular weights of the different conjugates were determined by mass spectrometry and correlated well with the theoretically calculated masses, as shown in Table 1. Biosensor Analysis. The binding kinetics of the different Tcchelating peptides were compared in a biospecific interaction analysis on a Biacore 2000 instrument. As indicated in Table
Tumor Targeting with Affibody Molecules
Bioconjugate Chem., Vol. 18, No. 6, 2007 1959
Figure 1. Chelator structures.
Figure 3. Cellular retention of labeled conjugates in HER2-expressing ovarian carcinoma cells, SKOV-3 cells. 125I-PIB-ZHER2:342 (23) was included for a comparison.
Figure 4. Biodistribution of labeled conjugates in NMRI normal mice at 4 h pi. Data are presented as an average from four animals ( SEM. Uptake in thyroid and muscle were less than 0.08%IA per whole sample and 0.09%IA/g, respectively. Figure 2. Stability of (A) 99mTc-maGEG-ZHER2:342 and (B) 99mTcmaEEE-ZHER2:342 in mouse serum at 1 h and 37 °C was analyzed using SDS–PAGE. The left peaks (1) represent intact 99mTc-labeled ZHER2: 342 (two samples were analyzed). The right peak (2) indicates the reference sample of free pertechnetate.
1, both conjugates showed similar subnanomolar affinities to surface-immobilized HER2 receptors: the dissociation constants (KD) were 410 pM and 360 pM for maEEE-ZHER2:342 and maGEG-ZHER2:342, respectively. 99m Tc Labeling. The radiolabeling efficiencies were over 90% for both 99mTc-maEEE-ZHER2:342 and 99mTc-maGEG-ZHER2:342, as shown in Table 2. The isolated yield from size-exclusion chromatography using NAP-5 columns was about 76%, and the radiochemical purity of purified products was typically over 95% for both conjugates. The presence of reduced hydrolyzed technetium was less than 2% in all experiments. Stability Studies. The in Vitro stability of the labeled conjugates was evaluated in a cysteine challenge test. Incubation of the labeled Affibody molecules for 2 h in the presence of a 300-fold molar excess of cysteine demonstrated high stability, with 93% radioactivity still attached to the conjugate (Table 2). The in Vitro stability of 99mTc-maGEGZHER2:342 and 99mTc-maEEE-ZHER2:342 in mouse serum is shown
in Figure 2. The radioactivity in the gel showed a single peak of the 99mTc-labeled ZHER2:342 (two samples were analyzed on the same gel), indicating that no detectable transchelation to serum proteins or release of pertechnetate occurred. This experiment also confirmed that Affibody molecules were stable against degradation by blood plasma proteases since no fragments with lower molecular weights were detected. In Vitro Cell Studies. Binding Specificity and Antigen Binding Capacity. The binding specificity of 99mTc-maEEEZHER2:342 and 99mTc-maGEG-ZHER2:342 was evaluated in the HER2-expressing ovarian cancer cell line SKOV-3. The binding was specific since it could be blocked with receptor saturation by a 100-fold excess of unlabeled ZHER2:342. The antigen binding capacity was estimated to be around 60% for the 99mTc-maEEEZHER2:342, indicating that the labeling procedure had a minor effect on the conjugates. Cellular Retention. The cellular retention of 99mTc-labeled ZHER2:342 and a radioiodinated Affibody molecule, 125I-PIBZHER2:342, is illustrated in Figure 3. The radioiodinated Affibody molecule was described in earlier studies (23) and included here for comparison. 99mTc-maEEE-ZHER2:342 and the radioiodinated conjugate displayed a similar pattern in the beginning, but after 4 h, the iodine label continued to decrease, while the technetium
1960 Bioconjugate Chem., Vol. 18, No. 6, 2007
Figure 5. Biodistribution of 99mTc-maEEE-ZHER2:342 in BALB/c nu/nu mice with HER2-expressing SKOV-3 xenografts at 1, 4, and 6 h pi. Data are presented as an average from four animals ( SEM. The uptake in thyroid was less than 0.09%IA per whole sample.
Figure 6. Dot plot of tumor uptake (%IA/g) in BALB/c nu/nu mice with SKOV-3 xenografts. Blocked mice received an excess of nonlabeled Affibody molecule prior to the injection of 99mTcmaEEE-ZHER2:342. Student’s t-test gave p < 0.0001.
label showed a discrete slow increase in radioactivity over time. After 24 h, the cell-associated radioactivity retained in the SKOV-3 cells was about 70% for 99mTc-maEEE-ZHER2:342 and 40% for the counterpart, 125I-PIB-ZHER2:342 (p < 0.001). 99mTcmaGEG-ZHER2:342 possessed higher cell-associated radioactivity retention than 99mTc-maEEE-ZHER2:342 in the first hours but decreased in the same way as that of 125I-PIB-ZHER2:342, with 50% of the cell-associated radioactivity retained after 24 h. In ViWo Animal Studies. Biodistribution in Normal Mice. Figure 4 presents the comparative biodistribution of 99mTcmaGEG- and 99mTc-maEEE-ZHER2:342 in normal NMRI mice at 4 h pi compared to that of 99mTc-maGGG-ZHER2:342. It was shown that the radioactivity uptake was low in all organs, except in the kidneys and intestines. The high uptake in the kidneys is explained by the renal clearance and tubular reabsorption of the small-sized Affibody molecules. A significant difference between the conjugates was observed in the kidneys and intestines. 99mTc-maEEE-ZHER2:342 had 3 times lower radioactivity in the intestines compared to that of 99mTc-maGEGZHER2:342 (3.1 ( 1.9%IA/g vs 8.7 ( 0.8%IA/g; p < 0.001), while the kidney uptake was 10 times higher (95 ( 23 vs 8.9 ( 0.8 %IA/g; p < 0.001). The uptake in the liver, stomach, and salivary glands of 99mTc-maEEE-ZHER2:342 was significantly lower (p < 0.05) compared with that of 99mTc-maGEG-ZHER2:342 and 99mTcmaGGG-ZHER2:342 (Figure 4). Biodistribution in Mice with SKOV-3 Xenografts. The in ViVo tumor-targeting properties of 99mTc-maEEE-ZHER2:342 were evaluated in BALB/c nu/nu mice bearing HER2-expressing SKOV-3 xenografts (Figure 5). The results showed that the
Tran et al.
radioactivity was cleared rapidly from blood circulation, reaching 0.15 ( 0.05 %IA/g at 6 h pi. The radioactivity clearance from other organs followed the same pattern. Tumor uptake was 9.1 ( 1.4 %IA/g at 1 h pi, 7.9 ( 1.0 %IA/g at 4 h pi, and 6.1 ( 0.4 %IA/g at 6 h pi, indicating a decrease over time. In the receptor specificity study, one group of mice was preinjected with 1,000-fold molar excess of nonlabeled ZHER2:342 in order to saturate the receptors before the administration of 99mTcmaEEE-ZHER2:342. The tumor uptake (Figure 6) in the blocked group was 0.3 ( 0.07 %IA/g and 7.9 ( 1.0 %IA/g in the nonblocked group (p < 0.0001), demonstrating that tumor uptake was HER2-specific. The radioiodinated Affibody molecule 125I-PIB-ZHER2:342 (23) visualizes HER2-expressing xenografts with high contrast and was therefore co-injected with 99mTc-maEEE-ZHER2:342 as an internal reference for comparison. The tumor-to-organ ratios are listed in Table 3, showing that there was a significant (p < 0.05) difference in most organs between the two labels. Especially, the blood radioactivity concentration was much lower for the 99m Tc-label than for the 125I-label at all time points (p < 0.001), while the uptake in liver and stomach was lower for the 125Ilabel. In general, the use of the technetium label provided better tumor-to-organ ratios than the iodine label. Gamma Camera Imaging with 99mTc-maEEE-ZHER2:342. The tumor-targeting capacity of 99mTc-maEEE-ZHER2:342 was evaluated in BALB/c nu/nu mice bearing SKOV-3 tumors. The gamma camera images were acquired 1, 2, and 5 h after the administration of 99mTc-maEEE-ZHER2:342. The tumors could be detected as early as 1 h pi, and the image contrast became better with time (Figure 7A and B). Tumor-to-nontumor ratios for the contralateral thigh were (average ( maximum error) 8.3 ( 0.3, 19 ( 2.1, and 35 ( 0.6 at 1, 2, and 5 h pi, respectively. Two mice were preinjected with nonlabeled Affibody molecules in order to block the receptors, and it was shown clearly that the binding of 99mTc-maEEE-ZHER2:342 was receptor-specific in ViVo because the tumors were not seen, while the tumors in the nonblocked mice were clearly visualized (Figure 7B). Some radioactivity accumulation is seen in the salivary glands at 1 h pi, which might indicate some release of free pertechnetate. No uptake in any other organs was seen, except in the kidneys, which corresponded well with the biodistribution data. Tumorto-nontumor ratios for the contralateral thigh were 35 ( 0.6 for the nonblocked mice and 3.6 ( 1.4 for the blocked mice.
DISCUSSION Affibody molecules are a new class of small targeting proteins displaying optimal biodistribution properties for imaging purposes (24, 27). Pilot patient investigations with 68Ga- and 111 In-labeled Affibody molecules have demonstrated their utility for the imaging of HER2 expression in metastatic breast cancer (26). However, for SPECT imaging, technetium-99m might be the most ideal radioisotope for the labeling of Affibody molecules because of convenient logistics, dosimetric properties, and particularly because the physical half-life (t1/2 ∼ 6 h) matches the quick blood clearance of Affibody molecules. Earlier studies on a 99mTc-labeled Affibody molecule, containing a mercaptoacetyltriglycyl chelator (27), showed favorable tumor-targeting properties (high tumor-to-blood ratios and excellent binding specificity) of the conjugate. However, this conjugate exhibited high hepatobiliary clearance, making scintigraphic detection of tumors in the abdominal area difficult. It has previously been reported that it is possible to shift the excretion pathway from hepatobiliary to renal by substituting the chelating amino acids with more hydrophilic characters. For this reason, substitution with more hydrophilic chelating residues in the mercaptoacetyl-containing Affibody molecule ZHER2:342 was investigated as a strategy for the reduction of abdominal
Tumor Targeting with Affibody Molecules Table 3. Tumor-to-Organ Ratios of
99m
Bioconjugate Chem., Vol. 18, No. 6, 2007 1961 Tc-maEEE-ZHER2:342 and
125
I-PIB-ZHER2:342 in BALB/c nu/nu Mice Bearing SKOV-3 Xenografts
1h tumor/organ ratios T/blood T/lung T/kidney T/liver T/stomach a
99m
6.1 ( 0.7 6 ( 1a 0.050 ( 0.004a 8 ( 1a 3(1
Significance comparison between
Tc- and
I
2.5 ( 0.3 2.5 ( 0.2 0.30 ( 0.02 2.3 ( 0.2 4.5 ( 0.6
a
99m
4h 125
Tc
125
99m
Tc
38 ( 7 14 ( 12 0.08 ( 0.01a 15 ( 2a 6 ( 2a a
6h 125
I
10 ( 0.8 10 ( 3.6 1.1 ( 0.3 12 ( 0.7 15 ( 6
99m
125
Tc
43 ( 12 18 ( 15 0.08 ( 0.01a 19 ( 3a 8 ( 3a a
I
18 ( 2 27 ( 2 2.2 ( 0.1 23 ( 3 53 ( 23
I-label using Student’s paired t-test; p < 0.05.
accumulation. We have found that in the case of the 99mTclabeled Affibody molecule ZHER2:342, a substitution of the maGGG-chelator with maSSS (mercaptoacetyl-triseryl chelator) caused a clear shift from hepatobiliary to renal excretion (36). Still, this conjugate was not optimal for detecting tumors in the abdomen area on the day of injection, thus additional improvement was necessary. One way to further increase the hydrophilicity is to use amino acids with charged side chains such as glutamic acids. The results from the Biacore experiments showed that the ZHER2:342 with the N-terminally incorporated maGEG or maEEE by peptide synthesis bound with high affinity to HER2. Both maGEG-ZHER2:342 and maEEE-ZHER2:342 were successfully 99mTclabeled with a labeling yield of over 90% and a radiochemical purity of more than 95%. Both 99mTc-maGEG- and 99mTcmaEEE-ZHER2:342 were stable in Vitro during incubation in 300fold molar excess of cysteine for 2 h at 37 °C. Furthermore, no evidence of the release of radioactivity from both conjugates or technetium transchelation to blood plasma proteins could be detected after incubation in mouse serum for 1 h, signifying its suitability for in ViVo use. The labeled conjugates also preserved specific binding to HER2 receptors in SKOV-3 ovarian carcinoma cells in Vitro. The cellular retention of 99mTc-maGEGZHER2:342 in SKOV-3 cells was higher compared with the radioiodinated counterpart, 125I-PIB-ZHER2:342. The cell-associated radioactivity of 99mTc-maEEE-ZHER2:342 decreased rapidly in the first 4 h after interrupted incubation and had an appearance similar to that of the radioiodinated one. Surprisingly, the cellassociated radioactivity increased slowly at later time points, indicating some residualizing properties of this conjugate. One possible explanation is that the dissociated conjugate was reabsorbed and internalized, causing the slow increase in the curve. The same phenomenon has been observed earlier with 111 In-labelled Affibody molecules (24). A possible method to discriminate between the internalized and membrane-bound fractions would be very helpful for a more reliable interpretation of the results of the cellular retention experiment. However, as we have reported earlier (24), common methods of internalization assays are inefficient with ZHER2:342 because of strong binding to cells, even in an acidic environment. The substitution of a single glycine with glutamic acid (maGGG vs maGEG) in the chelator showed a clear effect on the excretion pattern in normal mice, with a significant 3-fold reduction of the radioactivity accumulation in the intestines (hepatobiliary excretion). The hepatobiliary clearance was even more reduced when all three glycines in the chelator were replaced by glutamic acids (maGGG to maEEE), resulting in a 10-fold decrease in intestine accumulation. The reduced hepatobiliary excretion, accompanied by the increased kidney uptake, which was especially pronounced in the case of a triple substitution, verified that a clear shift from hepatobiliary to renal excretion occurred. Interestingly, reduction of the radioactivity level in the intestines was not directly translated into an increase of renal accumulation, and the use of the maEEE moiety as chelator caused a higher reabsorption of radioactivity in the kidneys. This effect could be considered as negative per se, but the overall effect on the changed excretion pattern is positive
Figure 7. Gamma-camera imaging with 99mTc-maEEE-ZHER2:342 in BALB/c nu/nu mice bearing SKOV-3 xenografts. (A) Tumors were well visualized on the right hind legs already at 1 and 2 h pi. (B) The image contrast was better at 5 h pi, and the tumors were blocked with a preinjection of excess of nonlabeled Affibody molecules.
since the position of the kidneys is well-defined, and the use of SPECT/CT makes it possible to distinguish the high radioactivity accumulation in the kidneys from other organs. In contrast, radioactivity in the gastrointestinal tract is more problematic, both by elevated background level and by the occasional formation of hot spots. One concern is the high renal radioactivity accumulation of 99mTc-maEEE-ZHER2:342. This high kidney accumulation could potentially be dose-limiting when using radiolabeled proteins for therapy. In the case of diagnostic applications, the conjugates are labeled with gamma-emitting nuclides, which generate low local doses. The only particulate
1962 Bioconjugate Chem., Vol. 18, No. 6, 2007 Table 4. Biodistribution Data of 99m
blood kidneys liver intestineb
Tc-maGGGZHER2:342
0.30 ( 0.03 6.6 ( 0.4 0.9 ( 0.3 32 ( 4
99m
Tran et al.
Tc-labeled ZHER2:342, Containing Different Chelators at 4 h pia
99m
Tc-CGGZHER2:342
0.55 ( 0.02 51 ( 4 0.86 ( 0.09 12.6 ( 0.7
99m
Tc-CGGGZHER2:342
0.52 ( 0.05 47 ( 7 0.69 ( 0.09 12.0 ( 0.6
99m
Tc-maGSGZHER2:342
0.10 ( 0.02 8(2 0.2 ( 0.1 17 ( 2
99m
Tc-maSSSZHER2:342
0.14 ( 0.07 18 ( 4 0.5 ( 0.3 11 ( 2
99m
Tc-maGEGZHER2:342
0.15 ( 0.03 8.9 ( 0.8 0.3 ( 0.1 8.7 ( 0.8
99m
Tc-maEEEZHER2:342
0.09 ( 0.02 95 ( 23 0.21 ( 0.02 3(2
a Data on the chelators maGGG, CGG (cysteinyl-diglycyl), CGGG (cysteinyl-triglycyl) maGSG (mercaptoacetyl-glycyl-seryl-glycyl) and maSSS (mercaptoacetyl-triseryl) are historical controls, taken from refs 34 and 35. b Uptake is presented as %IA/g organ, except for the intestine, where the uptake is for the whole sample.
radiations of such nuclides (99mTc or 111In) are the Auger and conversion electrons, which are inefficient in causing renal damage. For example, the use of 111In-labeled Octreoscan for therapy demonstrated that renal doses as high as 45 Gy did not cause negative side effects (37). Apparently, renal doses in the case of diagnostic application will be much lower. Another interesting observation is that uptake in the liver, stomach, and salivary glands was significantly lower for 99m Tc-maEEE-ZHER2:342 compared with that for 99mTc-maGEGZHER2:342 and 99mTc-maGGG-ZHER2:342 (Figure 4). This indicates that the glycine-to-glutamic acid substitution improves the overall in ViVo stability of the chelates to reoxidation, as these organs are well known to accumulate free pertechnetate. This stabilizing effect on the conjugate is probably contributed from the side chains of the glutamic acid residues in the chelator. Earlier, the maGGG chelator has been compared with the cysteine-based chelators, cysteine-diglycyl (CGG) and cysteinetriglycyl (CGGG) (34). As summarized in Table 4, a comparison with historical data showed a 3-fold reduction of hepatobiliary excretion and a 2-fold increase of renal excretion in the case of the maEEE chelator in comparison with those of cysteine-based chelators. This shift indicates the higher hydrophilicity of the maEEE compared to that of the cysteine-based chelator. A comparison with our previous data (36) shows also that the incorporation of glutamic acid in meracaptoacetyl-based chelators shifts the excretion pathway more efficiently to renal than does the incorporation of serine. 99m Tc-maEEE-ZHER2:342 was the obvious choice for further evaluation in SKOV-3 xenografted mice because this conjugate showed the best pharmacokinetics properties in normal mice (a low degree of hepatobiliary excretion and the lowest uptake in liver, salivary glands, and stomach). Tumor targeting was efficient, with the highest value of 9.1 ( 1.4 %IA/g reaching 1 h pi. Tumor uptake was HER2-specific since the administration of an excess amount of nonlabeled Affibody molecules could saturate HER2-receptors in the tumors and significantly reduce the uptake of 99mTc-maEEE-ZHER2:342 in ViVo. The receptor saturation led to a 24-fold decrease in tumor uptake at 4 h pi, from 7.9 ( 1.0 %IA/g to 0.33 ( 0.07 %IA/g (p < 0.0001). The accumulated radioactivity in the tumor uptake decreased slowly over time. At the same time, radioactivity in the kidneys and other organs also declined rapidly, resulting in a tumor-to-blood (T/B) ratio of 38 at 4 h pi and 43 at 6 h pi, respectively. It is worthwhile to mention that 99mTc-maEEE-ZHER2:342 seems to be superior in comparison with antibody-based HER2 imaging agents that are under preclinical investigation. For example, the T/B ratio was 3 at 24 h pi for 99m Tc-HYNIC-trastuzumab (19), 1.6 at 24 h pi for 111In-DTPAtrastuzumab (38), 2.3 at 6 h pi for 111In-T84.66 minibody (39), 0.7 at 6 h pi for the 111In-DOTA 10H8 hinge-minibody (40), 2.6 at 4 h pi for the 125I-labeled sFv (21), and 1.3 at 4 h pi for the 125 I-labeled (sFv′)2 (41). Gamma camera imaging showed that 99mTc-maEEE-ZHER2:342 accumulated specifically in SKOV-3 tumors since the tumors were blocked with a preinjection of nonlabeled Affibody molecules (right group, Figure 7B). Most interestingly, the tumors were clearly
visualized at 1 h pi, and the contrast in the image at 2 h pi did not differ significantly, enabling early detection of HER2-tumors. The clinical potential of an imaging agent relies on a number of parameters, such as high affinity and selectivity, low nonspecific uptake, and high specific tumor accumulation. Early detection is preferable in patient management, and high tumorto-organ ratios are crucial. This can be achieved by rapid blood clearance and favorable excretion kinetics (renal clearance being preferred) (42). The current study presents an imaging agent that fulfilled these requirements, suggesting a very high potential of 99mTc-maEEE-ZHER2:342 for the detection of HER2 expression. To our knowledge, this is the first time mercaptoacetyltriglutamyl is being characterized as a chelator for 99mTc. The results have demonstrated that this chelator decreases the hepatobiliary excretion of peptides and provides a more stable technetium attachment than the mercaptoacetyl-triglycyl chelator.
CONCLUSIONS The use of peptide synthesis for the coupling of chelator sequences to Affibody molecules for 99mTc-labeling is an efficient way to modify in ViVo kinetics of Affibody molecules. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelates, resulted in minimal abdominal accumulation, enabling the rapid detection of HER2positive tumors and their metastases in the abdominal area. Thus, 99m Tc-maEEE-ZHER2:342 appears as a promising tracer for clinical imaging of HER2-overexpression in tumors and metastases.
ACKNOWLEDGMENT This work was supported by the Swedish Cancer Society (Cancerfonden) and the Swedish Research Council (Vetenskapsrådet).
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