Effects of Lysine-Containing Mercaptoacetyl-Based Chelators on the

Nov 26, 2008 - and organ samples were collected and measured. Gamma Camera Imaging with 99mTc-maSKS-ZHER2:342. Two mice were intravenously ...
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TECHNICAL NOTES Effects of Lysine-Containing Mercaptoacetyl-Based Chelators on the Biodistribution of 99mTc-Labeled Anti-HER2 Affibody Molecules Thuy A. Tran,*,† Torun Ekblad,‡ Anna Orlova,†,§ Mattias Sandstro¨m,| Joachim Feldwisch,§ Anders Wennborg,§ Lars Abrahmse´n,§ Vladimir Tolmachev,†,§,⊥ and Amelie Eriksson Karlstro¨m‡ Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Medical Radiation Physics, Uppsala University Hospital, and Department of Medical Sciences, Uppsala University, Uppsala, Sweden, School of Biotechnology, Division of Molecular Biotechnology, Royal Institute of Technology, Stockholm, Sweden, and Affibody AB, Bromma, Sweden. Received June 18, 2008; Revised Manuscript Received November 4, 2008

The effects of polar (mercaptoacetyl-triseryl) and negatively charged (mercaptoacetyl-triglumatyl) chelators on the biodistribution of 99mTc-labeled anti-HER2 Affibody molecules were previously investigated. With glycine, serine, and glutamate, we demonstrated that substitution with a single amino acid in the chelator can significantly influence the biodistribution properties and the excretion pathways. Here, we have taken this investigation further, by analyzing the effects of introduction of a positive amino acid residue on the in vivo properties of the 99mTclabeled Affibody molecule. The Affibody molecules with mercaptoacetyl-seryl-lysyl-seryl (maSKS) and mercaptoacetyl-trilysyl (maKKK) extensions were produced by peptide synthesis and labeled with 99mTc in alkaline conditions. A comparative biodistribution was performed in normal mice to evaluate the excretion pathway. A shift toward renal excretion was obtained when serine was substituted with lysine in the chelating sequence. The radioactivity in the gastrointestinal tract was reduced 3-fold for the 99mTc-maSKS-ZHER2:342 and 99mTc-maKKKZHER2:342 in comparison with the 99mTc-maSSS- ZHER2:342 conjugate 4 h post injection (p.i.). The radioactivity in the liver was elevated when a triple substitution of positively charged lysine was used. The tumor targeting properties of 99mTc-maSKS-ZHER2:342 were further investigated in SKOV-3 xenografts. The tumor uptake of 99mTc-maSKSZHER2:342 was 17 ( 7% IA/g 4 h p.i. Tumor xenografts were well-visualized by gamma scintigraphy. In conclusion, the substitution with one single lysine in the chelator results in better tumor imaging properties of the Affibody molecule ZHER2:342 and is favorable for imaging of tumors and metastases in the abdominal area. Multiple lysine residues in the chelator are, however, undesirable due to elevated uptake both in the liver and kidneys.

INTRODUCTION The overexpression or gene amplification of the human epidermal growth factor type 2, HER2 receptor (also known as ErbB2), is found in about 20% of all breast cancers (1, 2). HER2 expression also occurs in a number of other cancer types such as lung, colorectal, urothelial, ovary, and pancreas cancer (3, 4). The first therapeutic agent that targets HER2-positive breast cancer, approved for clinical use, was the humanized antibody trastuzumab (Herceptin, Genentech Inc.). Pertuzumab (Omnitarg, Genentech) is currently in phase III studies for treatment of HER2-positive breast cancer. Another type of targeting agent that is entering the clinics is the tyrosine kinase inhibitor, lapatinib (Tykerb, GlaxoSmithKline), that blocks several down* Corresponding author. Thuy Tran, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, S-751 85, Uppsala, Sweden. Phone: +46 18 471 3829. Fax: + 46 18 471 34 32. Email: [email protected]. † Rudbeck Laboratory, Uppsala University. ‡ Royal Institute of Technology. § Affibody AB. | Uppsala University Hospital, Uppsala University. ⊥ Department of Medical Sciences, Uppsala University.

stream signaling pathways of EGFR and HER2 receptors, involved in cell proliferation, invasion, and apoptosis (5). Determination of HER2 status in breast cancer diagnostics is critically important for therapeutic decision making. The accuracy, precision, and reproducibility of a diagnostic method are highly essential in order to identify patients who would most likely benefit from anti-HER2 treatment. At present, immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH) of biopsy samples are clinically used for evaluating HER2 status (6). However, some limitations (6, 7) associated with these tests might cause false-negative results. Recently, the American Society of Clinical Oncology and the College of American Pathologist expert panel (8) reported that approximately 20% of current HER2 testing may be inaccurate. As an alternative, radionuclide imaging may be applied to visualize HER2 expression in both whole primary tumors and metastatic lesions, including sites where biopsies may be difficult to obtain. HER2 has no known natural ligands, and HER2-targeted diagnostic imaging has been investigated using panels of radiolabeled agents, including the intact antibodies trastuzumab and pertuzumab, Fab and F(ab′)2 fragment of trastuzumab, minibody and scFv-Fc of trastuzumab, and ICR 12 (an antiHER2 monoclonal antibody) (9). Recently, a new class of targeting agents, Affibody molecules, has been extensively studied for radionuclide therapy and imaging (10-12). Affibody

10.1021/bc800244b CCC: $40.75  2008 American Chemical Society Published on Web 11/26/2008

Technical Notes

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Table 1. Biophysical Characterization Data protein

synthetic yield (%)

theoretical MW (Da)

experimental MW (Da)

KD (pM)

Tm (°C)

ZHER2:342 maSSS-ZHER2:342 maKKK-ZHER2:342 maSKS-ZHER2:342

21 14 12 14

6718 7054 7177 7095

6718 7053 7177 7094

80 400 270 310

64 65 68 65

molecules are small (7 kDa) and cysteine-free three-helix bundle proteins, based on the 58 amino acid B-domain of staphylococcal protein A. By randomization of 13 solvent-exposed residues, a library of 3 × 109 variants has been generated and used for selection of affinity ligands to a variety of proteins (13). The 7 kDa Affibody molecules are much smaller than antibodies and well-suited for in vivo imaging purposes. For HER2 targeting, the Affibody molecule ZHER2:342 with an affinity of 22 pM (14) has been developed and labeled with a number of radioisotopes, 125I (14), 111In (15, 16), 90Y (17), 68Ga, 177Lu (10), and 18F (18, 19). All conjugates demonstrated good targeting of HER2-expressing SKOV-3 xenografts in mice (14, 16). The radionuclide 99mTc with a photon energy of 140 keV and a half-life of 6 h has also been investigated as a label for ZHER2: 342 because it is a readily available and cost-effective radionuclide for diagnostics. Earlier, the synthetic Affibody molecule ZHER2:342 with the chelator maGGG (mercaptoacetyltriglycyl) site-specifically introduced at the N-terminus has been labeled with technetium-99m (20). Even though imaging of HER2 expression in tumors in mice was feasible with 99mTc-maGGGZHER2:342, a high hepatobiliary clearance leading to high accumulation in the intestine was seen in the biodistribution studies. The hepatobiliary excretion can be reduced by reducing the lipophilicity of a peptide tracer (21, 22). Substitution of the chelator sequences with more hydrophilic amino acids in Affibody molecules, such as maSSS (mercaptoacetyl-triseryl) (20) or maEEE (mercaptoacetyl-triglutamyl) (23), results in suppressed hepatobiliary excretion, enabling abdominal detection. The use of negatively charged side chains, mercaptoacetyltriglutamyl (maEEE), has proven to be efficient in reducing the hepatobiliary excretion (23). The combination of two serine and one glutamate in the chelator, as in mercaptoacetyl-glutamylseryl-glutamyl (maESE), shows favorable properties for imaging (24). In this study, we aimed to investigate the influence of the positively charged hydrophilic amino acid lysine on the biodistribution properties. For this purpose, the two novel Affibody molecules maSKS-ZHER2:342 (mercaptoacetyl-seryl-lysyl-seryl) and maKKK-ZHER2:342 (mercaptoacetyl-trilysyl) have been synthesized and labeled with 99mTc. The chelating sequences maSKS and maKKK were incorporated in the N-terminus during the peptide synthesis. These two conjugates were compared with each other and with maSSS-ZHER2:342 (mercaptoacetyl-triseryl) with respect to the HER2-binding, cellular processing, and biodistribution properties. The labeled conjugates were not analyzed for their isomeric identities. They were used as a racemic mixture in all experiments.

EXPERIMENTAL PROCEDURES Peptide Synthesis. The ZHER2:342 sequence was assembled using Fmoc/tBu solid-phase peptide synthesis on a Fmoc-amide resin with a substitution of 0.67 mmol/g (Applied Biosystems) as previously described (20). 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. After synthesis of the full-length Affibody, the Tc-chelating peptide sequences Ser-Lys-Ser (SKS) and Lys-Lys-Lys (KKK) were introduced to the N-terminus of the ZHER2:342-sequence

by coupling the corresponding protected amino acids (FmocSer(tBu)-OH and/or Fmoc-Lys(Boc)-OH) one residue at the time in a stepwise manner. In the manual coupling reactions, 5 mol equiv (Eq) of Fmoc-protected amino acid were coupled with 5 Eq HBTU, 5 Eq HOBt, and 10 Eq N-ethyldiisopropylamine (DIEA, from Lancaster Synthesis, Morecambe, England). Ninhydrin tests were performed to evaluate the efficiency of the coupling reactions. Fmoc-deprotection was achieved by 20 min incubation in 20% piperidine-N-methylpyrrolidone (NMP, from VWR). Finally, the mercaptoacetyl moiety of the chelator was introduced by coupling S-trityl-protected mercaptoacetic acid (AnaSpec Inc., San Jose, CA) to the N-terminal amino group of the resin-bound peptide. The protecting groups were then deprotected and the full-length peptides with appended maSKS and maKKK chelators were released from the solid support 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 (Merck International)/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 20-60% B (B: 0.1% TFAacetonitrile (CH3CN; Merck International), 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 25-45% B. Biosensor Analysis. A Biacore 2000 instrument (Biacore AB, Uppsala, Sweden) was used for real-time biospecific interaction analysis. The extracellular domain of HER2 (Fox Chase Cancer Center, Philadelphia, USA) and human serum albumin, HSA (KabiVitrum, Sweden) were immobilized on a CM5 sensor chip by EDC/NHS coupling. HER2 and HSA were diluted to 20 µg/ mL in 20 mM NaOAc pH 4.5 and the immobilization levels reached 500 and 900 response units (RU), respectively. The different Affibody molecules were diluted to concentrations varying from 0.5 to 10 nM in HBS (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% Surfactant P20, pH 7.4), and the binding kinetics were studied at a flow rate of 50 µL/min in HBS as running buffer in a 5 min association phase and a 10 min dissociation phase. After each injection of analyte, the sensor surface was regenerated by injection with 20 mM HCl in HBS. The specific response was obtained by subtracting the signal from the control surface (HSA) from the signal from binding to the HER2 surface. Mass transfer limitations were avoided by keeping a high flow rate and a low immobilization level of HER2 protein on the sensor surface. Melting Point Analysis. Variable temperature measurements were performed using a JASCO J-810 spectropolarimeter (JASCO, Tokyo, Japan). Samples were diluted to a concentration of 50 µM in PBS pH 7.4. A cell with an optical path length of 1 mm was used. CD spectra from 250 to 195 nm were obtained at 20 °C before and after melting tests. For melting point measurements, the absorbance was measured at 221 nm and the temperature was increased by 5 °C/min from 20 to 90 °C. 99m Tc-Labeling. For labeling, 20 µL of maSKS-ZHER2:342 or maKKK-ZHER2:342 (1 mg/mL in deionized, degassed water) was mixed with 20 µL 0.15 M NaOH to obtain a final pH of about 11. Stannous chloride (10 µL, 1 mg of SnCl2 · 2H2O in 1 mL 0.01 M HCl) was added to the mixture, followed by 100-200 µL (about 50 MBq) of freshly eluted pertechnetate solution. The mixture was slightly vortexed and incubated at room temperature for 60 min. After labeling,99mTc-maSKS-ZHER2:342 and 99mTc-maKKK-ZHER2:342 were isolated from unreacted technetium and other low-molecular-weight components using size-exclusion chromatography on disposable NAP-5 columns

2570 Bioconjugate Chem., Vol. 19, No. 12, 2008 Table 2. Characterization Data of

99m

Tran et al.

Tc-Labeled Conjugates

conjugate labeling yield, % isolated yield, % radiochemical purity, % antigen binding capacity, % stability during cysteine challenge, %

99m

Tc-maSSS-ZHER2:342 97 ( 1 82 ( 5 99 ( 1 55 ( 15 78.6 ( 0.6

(Amersham Pharmacia Biotech AB, Uppsala, Sweden) preequilibrated with PBS. The purity was assessed by analysis with ITLC strips (Pall Life Sciences, Ann Arbor, USA), 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 a Cyclone Storage Phosphor System (http:// www.perkinelmer.com/) (Perkin-Elmer, Wellesley, MA). 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. To validate the radiochemical purity obtained with ITLC, an analysis was ¨ KTAprime LC system (GE Healthcare, performed on an A Uppsala Sweden) with the UV detector set to 280 nm. The analysis was run on a prepacked Superdex Peptide 10/300 GL column (GE Healthcare, Uppsala Sweden), with a flow rate of 0.5 mL/min using a 50 mM phosphate buffer with 150 mM NaCl (pH 7.0) as running buffer. Fractions were collected and radioactivity was measured on a gamma counter. 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, 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, eluted in PBS. Plasma Stability Test. Serum samples were prepared as described in ref 20. Shortly, a serum sample (240 µL) was mixed with freshly labeled 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 (25), 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, 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. (26). Internalization and Retention Study. The internalization rates of the labeled conjugates on SKOV-3 cells were determined as

99m

Tc-maKKK-ZHER2:342 95 ( 1 79 ( 6 99 ( 1 66.4 ( 0.6 91 ( 4

99m

Tc-maSKS-ZHER2:342 98 ( 2 80 ( 3 99.7 ( 0.3 64 ( 2 94.7 ( 0.9

described in Ahlgren et al. (27). Shortly, cultured SKOV-3 cells were incubated for 3 h at 4 °C using a 5:1 molar ratio of 99mTclabeled conjugates to HER2 receptors. All procedures were performed on ice. Thereafter, the incubation medium was discarded, and the cells were washed three times using ice-cold serum-free medium. After addition of 1 mL complete medium, the cells were incubated further at 37 °C. At designated time points, up to 24 h, one group of three dishes was analyzed for cell-associated radioactivity. Medium was collected; cells were washed three times with ice-cold serum-free medium (these two steps were omitted for the time point 0 h) and treated with 0.5 mL of 4 M urea solution in 0.2 M glycine buffer, pH 2.5, for 5 min on ice. The acid fraction was collected, and cells were washed with additional 0.5 mL acid solution. The radioactivity in the acid wash fraction was considered membrane-bound radioactivity. After addition of 0.5 mL of 1 M NaOH, the cells were incubated at 37 °C for at 30 min, and the basic solution was collected. The cell dishes were washed with additional 0.5 mL of basic solution. The radioactivity in the alkaline fractions was considered internalized radioactivity. The radioactivity content of the samples was measured as mentioned above. Antigen Binding Capacity Study. Assessment of antigen binding capacity of 99mTc-labeled conjugates was performed using SKOV-3 cells according to Engfeldt et al. (26). In Vivo Animal Studies. The animal study was approved by the Local Ethics Committee for Animal Research. In comparative biodistribution studies, non-tumor-bearing NMRI mice were used. In tumor targeting and imaging experiments with 99mTc-maSKS-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 (5000 IE/mL, Leo Pharma, Copenhagen, Denmark). Blood and organ samples were taken and weighed, and their radioactivity was 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. Twelve mice were randomly divided into 3 groups with 4 animals each. The three groups were subcutaneously injected with 99mTc-maSSS-ZHER2:342, 99m Tc-maSKS-ZHER2:342, and 99mTc-maKKK-ZHER2:342, respectively (1 µg Affibody ligand, ∼100 kBq in 100 µL of PBS). The mice were euthanized at 4 h p.i.; organ samples were collected and measured. In previous studies, we have validated that the biodistribution of radiolabeled Affibody molecules 4 h p.i. is the same for both i.v. and s.c. injection and independent of the label (28, 29). Subcutaneous injections were mainly chosen since they are more reproducible (smaller errors) when injecting into a large number of animals (24). Biodistribution in Mice with SKOV-3 Xenografts. The tumor targeting properties of 99mTc-maSKS-ZHER2:342, which demonstrated the best biodistribution properties in normal mice, were evaluated in female BALB/c nu/nu nude mice bearing SKOV-3 xenografts. The mice were intravenously injected with 99mTc-

Technical Notes

Bioconjugate Chem., Vol. 19, No. 12, 2008 2571

Figure 1. Chelator structures. Dashed lines indicate the border to the Affibody molecule ZHER2:342.

maSKS-ZHER2:342 (1 µg Affibody ligand, ∼ 100 kBq in 100 µL of PBS). After 0.5, 1, 4, 8, and 24 h p.i., the mice were euthanized as described earlier. In the binding specificity study, one group of mice was preinjected with a 1000-fold molar excess of nonlabeled Affibody molecules prior to injection of 99m Tc-maSKS-ZHER2:342. At 4 h p.i., the mice were euthanized and organ samples were collected and measured. Gamma Camera Imaging with 99mTc-maSKS-ZHER2:342. Two mice were intravenously injected through the tail vein with 3 MBq of 99mTc-maSKS-ZHER2:342 (10 µg in 100 µL of PBS) and imaged at 4 h p.i. Imaging was performed using a Siemens e.Cam gamma camera equipped with a LEHR collimator at the Department of Nuclear Medicine of Uppsala University Hospital. Static images were collected during 10 min and evaluation of the images was performed with Osiris v 4.19 software (Digital Imaging Unit, University Hospital of Geneva, Switzerland). Data Treatment. In vitro and in vivo data were processed with Microsoft Excel 2003 (Microsoft, Redmond, WA) and all graphs were drawn with GraphPad Prism v 4 (GraphPad Software, San Diego; CA). Statistical analysis was performed using Student’s t test.

RESULTS Peptide Synthesis and Characterization. The Fmoc solidphase synthesis of ZHER2:342 resulted in a yield of 21% of the full-length peptide, as determined by analytical RP-HPLC. The 99m Tc-chelating sequences maSKS or maKKK were introduced N-terminally by manual synthesis, resulting in total synthetic yields of 14% and 12%, respectively (Table 1). The HPLC retention times were 13.9, 14.3, and 14.4 min for maKKK-ZHER2: 342, maSKS-ZHER2:342, and maSSS-ZHER2:342, respectively. Following purification by RP-HPLC, the purity was higher than 90%. Data of maSSS-ZHER2:342 (20) are included for comparison. 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. The binding kinetics of the different Tc-chelating peptides were compared in a biospecific interaction analysis on a Biacore 2000 instrument. As indicated in Table 1, both conjugates showed similar subnanomolar affinities to surface-immobilized HER2 receptors: the dissociation constants (KD) were 310 pM for maSKS-ZHER2:342 and 270 pM for maKKK-ZHER2:342 (Figure 2A). The melting points were 64-68 °C for the conjugates, which are close to the previously reported values of ZHER2:342 (see Table 1). The CD spectra of the proteins showed that the structures of the Affibody molecules were similar and refolded after heating to 90 °C (Figure 2B,C). 99m Tc-Labeling and Stability Studies. The radiolabeling yields were above 90% for both 99mTc-maSKS-ZHER2:342 and 99m Tc-maKKK-ZHER2:342, as shown in Table 2. The isolated yield after size-exclusion chromatography using NAP-5 columns was about 80%, and the radiochemical purity of the purified products

Figure 2. Illustrative Biacore sensorgram of maKKK-ZHER2:342 and maSKS-ZHER2:342, both at 5 nM (A) and CD spectra before and after heating to 90 °C of maKKK-ZHER2:342 (B) and maSKS-ZHER2:342 (C), respectively.

was typically over 97% for both lysine-containing conjugates. Size-exclusion chromatography confirmed that the retention volume of radiolabeled conjugates corresponded to the retention volume of monomeric form of Affibody molecules (see Figure 3). A typical specific activity of 18 GBq/µmol was obtained for both 99mTc-labeled Affibody molecules. The presence of reduced hydrolyzed technetium was less than 2% in all experiments. As presented in Table 2, the stability of the labeled conjugates was evaluated in a cysteine challenge test and the results showed high stability of the labeled conjugates, with over 90% radioactivity still attached to the conjugates. After incubation in mouse serum, the stability of 99mTc-maSKS-ZHER2:342 and 99m Tc-maKKK-ZHER2:342 was good. No release of free pertech-

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serine with one lysine in the chelator (maSSS vs maSKS), the kidney uptake was doubled from 18 ( 4% to 33 ( 2% IA/g (p ) 0.001), while the accumulation in intestines with content was significantly reduced from 11 ( 2% to 4.3 ( 0.5% IA/whole sample (p ) 0.0006). Substitution of all serines with lysines (maSSS to maKKK) significantly increased the kidney uptake 7-fold (p ) 5.7 × 10-7) while the intestine uptake was decreased 2-fold (p ) 0.0004), reaching the same level as for maSKS. On the other hand, the radioactivity of 99mTc-maKKK-ZHER2: 342 was found to be significantly higher in other organs such as blood, spleen, stomach and salivary glands, thyroid, muscle, and liver (p < 0.05). The liver uptake was estimated to be about 5 times higher for 99mTc-maKKK-ZHER2:342 than for 99mTcmaSKS-ZHER2:342 (6.8 ( 2.5% vs 0.49 ( 0.28% IA/g, p ) 0.002). Figure 3. Size-exclusion chromatography analysis of radiolabeled conjugates was performed using a Superdex Peptide 10/300 GL column as described in the Experimental Procedures section. Radioactivity of the fractions was measured off-line.

netate could be detected (data not shown). However, there is a peak, corresponding to a higher molecular weight, of approximately 19% of the radioactivity. This could indicate some binding of the labeled conjugate to blood proteins. In Vitro Cell Studies. Binding Specificity and Antigen Binding Capacity. The in vitro binding specificity of 99mTcmaKKK-ZHER2:342 and 99mTc-maSKS-ZHER2:342 to SKOV-3 cells showed specific binding to HER2 receptors, since it could be blocked with receptor saturation by 100-fold excess of unlabeled ZHER2:342. The cell-binding radioactivity was reduced from 33 ( 2% to 5.1 ( 0.3% (p < 0.0005) between the nonsaturated and saturated groups for 99mTc-maSKS-ZHER2:342 and from 35 ( 5% to 5.4 ( 0.04% (p < 0.0005) for 99mTc-maKKK-ZHER2: 342. The antigen binding capacity was estimated to be around 60% for the 99mTc-labeled ZHER2:342, indicating that the labeling procedure had a minor effect on the conjugates. Cellular Retention and Internalization. The cellular processing of 99mTc-labeled ZHER2:342 Affibody molecules is illustrated in Figure 4. The conjugates displayed a high retention of radioactivity on SKOV-3 cells, with a slow decrease over time. After 24 h, the cell-associated radioactivity retained by the SKOV-3 cells was about 60-70%. The internalization of the labeled conjugates was slow over time, with about 12% of radioactivity internalized 4 h after interrupted incubation. Both conjugates showed a similar internalization pattern within the error of the experiment. In Vivo Animal Studies. Biodistribution in Normal Mice. The comparative biodistribution in NMRI mice (Figure 5) showed that, by replacing one

Biodistribution in Mice Bearing SKOV-3 Xenografts. Data from the comparative biodistribution suggested that 99mTcmaSKS-ZHER2:342 was the most suitable conjugate of the two new tested variants, with low radioactivity accumulated in the intestines and an intermediate level of kidney uptake. Thus, this conjugate was further evaluated in HER2-expressing SKOV-3 xenografts. The distribution of 99mTc-maSKS-ZHER2:342 was examined at 0.5, 1, 2, 4, 8, and 24 h p.i. as presented in Figure 6. The tumor accumulation was high, reaching 17 ( 8% IA/g at 4 h p.i., and the radioactivity was cleared rapidly from the blood circulation, with 0.35 ( 0.13% IA/g at 4 h after administration. The tumor-to-blood ratio was 47 at this time point. Other organs showed low uptake, and the clearance from these organs was rapid over time. A blocking experiment demonstrates that 99mTc-maSKS-ZHER2:342 bound specifically to HER2 receptors in SKOV-3 xenografts (Table 4). The tumor uptake in the blocked group with excess of nonlabeled Affibody molecules was significantly lower (p < 0.001). Much smaller but significant (p < 0.05) reduction of uptake was found also in blood, lung, liver, and spleen and in intestines of animals in the blocking group. A possible explanation is that some part of radiolabeled 99mTc-maSKS-ZHER2:342 should dissociate continuously from the receptors in the xenografts, drain from the tumors, and re-enter the blood circulation. Such a depot is much smaller when HER2 receptors are blocked by a nonradioactive Affibody molecule. Gamma Camera Imaging with 99mTc-maSKS-ZHER2:342. The gamma camera image of SKOV-3 xenografts 4 h after administration of 99mTc-maSKS-ZHER2:342 is shown in Figure 7. The tumors located on the right hind legs accumulated a large amount of radioactivity and were clearly visualized. A high accumulation of radioactivity was also seen in the kidneys, while no or very little uptake was seen in other organs. The ROI

Figure 4. Cellular retention and internalization after interrupted incubation on HER2-expressing SKOV-3 cells of (A) 99mTc-maSKS-ZHER2:342 and (B) 99mTc-maKKK-ZHER2:342. The graphs show mean values from three cell dishes ( SD.

Technical Notes

Bioconjugate Chem., Vol. 19, No. 12, 2008 2573

Table 3. Comparison of Biodistribution Data of 99m

99m

Tc-maSSS-ZHER2:342

blood kidneys liver intestinea T/B ratio

0.14 ( 0.07 18 ( 4 0.5 ( 0.3 11 ( 2 76 ( 8

99m

Tc-Labeled ZHER2:342 Containing Different Chelators in Mice 4 h p.i.

Tc-maSKS-ZHER2:342

99m

0.15 ( 0.02 33 ( 2 0.6 ( 0.2 4.3 ( 0.5 47 ( 23

Tc-maKKK-ZHER2:342 0.23 ( 0.01 127 ( 9 7(2 4.0 ( 0.3 NDb

99m

Tc-maESE-ZHER2:342 0.12 ( 0.02 22 ( 3 0.19 ( 0.01 1.7 ( 0.4 58 ( 6

99m

Tc-maEEE-ZHER2:342 0.09 ( 0.02 95 ( 23 0.21 ( 0.02 3(2 38 ( 7

a

Uptake is presented as % IA/g organ, except for the intestine, where the uptake is for the whole sample. Data of 99mTc-maSSS-ZHER2:342, Tc-maESE-ZHER2:342, and 99mTc-maEEE-ZHER2:342 are taken from refs 20, 23, 24, respectively. The tumor-to-blood ratios (T/B ratio) were obtained in SKOV-3 xenografts 4 h pi. b ND: not determined in xenograft model. 99m

Table 4. In Vivo Binding Specificity of SKOV-3 Xenografts, 4 h p.i.a blood tumor lung liver spleen stomach kidney salivary glands

99m

Tc-maSKS-ZHER2:342

blocked

nonblocked

0.16 ( 0.02 2.5 ( 0.64 0.24 ( 0.04 0.44 ( 0.04 0.17 ( 0.03 0.5 ( 0.2 45 ( 3 0.6 ( 0.03

0.25 ( 0.05** 14 ( 3.1* 0.41 ( 0.08** 1.1 ( 0.4** 0.26 ( 0.04** 0.56 ( 0.03 47 ( 5 0.8 ( 0.3

a The blocked group (n ) 3) was intravenously pre-injected with an excess amount of nonlabeled ZHER2:342. Results are presented as percentage of injected activity per gram of tissue (% IA/g). * Statistical significance according to Student’s t test gave p < 0.001. ** Statistical significance according to Student’s t test gave p < 0.05.

Figure 5. Comparative biodistribution of ZHER2:342 labeled with 99mTc using maSSS, maSK,S and maKKK chelators in NMRI normal mice, 4 h p.i. Data are presented as an average % IA/g of four animals ( SD. *Data for intestine are presented as % IA per whole sample. Historical data of 99mTc-maSSS-ZHER2:342 was taken from ref 20.

analysis showed that the ratio of the tumor to the contralateral thigh was (average ( standard deviation) 43 ( 2.

DISCUSSION In this study, the HER2-targeting Affibody molecule, ZHER2: with the chelators mercaptoacetyl-seryl-lysyl-seryl (maSKS) and mercaptoacetyl-trilysyl (maKKK) attached to the N-terminus were synthesized in order to investigate how the incorporation of the hydrophilic positively charged amino acid lysine in the chelator sequence would affect the biodistribution properties in mice for imaging of HER2 expression in vivo. These two conjugates, maSKS-ZHER2:342 and maKKK-ZHER2:342, were labeled with technetium-99m and were compared with a formerly studied conjugate, 99mTc-maSSS-ZHER2:342. The radioisotope 99m Tc was chosen because of its advantages of low cost and availability, a reasonably long half-life (T1/2 ) 6 h), and suitable photon energy for SPECT cameras.

342,

The 99mTc-labeling of maSKS-ZHER2:342 and maKKK-ZHER2: were efficient and resulted in yields of more than 94%. Preserved HER2 binding capacity on SKOV-3 cells was demonstrated for all conjugates. In vitro, both 99mTc-maSKSZHER2:342 and 99mTc-maKKK-ZHER2:342 were found to be quite stable after 2 h incubation in mouse serum. Approximately 80% of the conjugates remained intact, and the rest seemed to bind to serum proteins (data not shown). In comparison to 99mTcmaSSS-ZHER2:342, which did not demonstrate such binding under the same conditions, this level of protein binding (20% of the radioactivity) does not seem to affect clearance from the blood and other organs (see Table 3). The in vitro cell studies showed that the labeled Affibody molecules had a slow internalization rate in SKOV-3 cells over time, which is consistent with previous results obtained for 111Inlabeled Affibody molecules (27). Other investigations have indicated that slow internalization of antibodies is advantageous for tumor targeting due to better penetration and retention in tumors (30). The retention of 99mTc-maSKS-ZHER2:342 and 99mTcmaKKK-ZHER2:342 was high, with about 60-70% of radioactivity retained in the cells 24 h following incubation. This level of retention is similar to what was previously determined for other 99m Tc-labeled synthetic conjugates (20, 23). The in vivo results showed that a single substitution of one serine with one lysine in the chelator (maSSS to maSKS) decreased the hepatobiliary excretion 2-fold and that the kidney uptake was increased. This shift might be explained by the overall higher hydrophilicity of the lysine in the chelator. However, substitution of all three serines with lysines (maSSS to maKKK) did not improve the hepatobiliary excretion further (intestine uptake 4 ( 0.3% IA/sample for labeled maKKKZHER2:342 compared to 4.3 ( 0.5% IA/sample for maSKS-ZHER2: 342, p ) 0.34), while the kidney accumulation was significantly higher. The radioactivity of 99mTc-maKKK-ZHER2:342 was also found to be higher in other organs including the liver, spleen, stomach, and salivary glands, compared with what was seen with labeled maSSS-ZHER2:342 or maSKS-ZHER2:342. An interesting finding from the biodistribution study was the elevated liver uptake for the maKKK conjugate compared with the maSKS or maSSS conjugates. It has previously been reported that positively charged substances preferentially accumulate in liver (31), and it has been suggested that cationization of proteins by chemical modification could be a way to achieve liver-specific targeting of a protein drug (32). Furthermore, the accumulation of radioactivity in the liver was higher for an antibody radioiodinated via a strongly positively charged D-amino acid tag (KRYRR) than for the same antibody radioiodinated through an N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) reagent (33), demonstrating a dramatic influence by a minor modification of the protein. Similarly, a previous study of Affibody molecules demonstrated that the use of multiple copies of the amino acid histidine (as in His-tagged Affibody molecules) also caused a high accumulation of radioactivity in the liver (27). In the present study, it was therefore not completely unexpected that using multiple lysine residues for 99mTc-labeling could promote elevated liver ac342

2574 Bioconjugate Chem., Vol. 19, No. 12, 2008

Tran et al.

Figure 6. Biodistribution of 99mTc-maSKS-ZHER2:342 in BALB/c nu/nu mice bearing SKOV-3 xenografts at 0.5, 1, 4, 8, and 24 h p.i. Data are presented as an average % IA/g of four animals ( SD.

Figure 7. Gamma camera imaging of Balb/c nu/nu mice, bearing SKOV-3 xenografts on the right hind legs. The mice were imaged 4 h after administration of 99mTc-maSKS-ZHER2:342. The tumors are indicated by arrows, while the hot spots visualized in the images are the kidneys.

cumulation. However, the detailed mechanism behind this phenomenon is still unclear. Out of the three conjugates, 99mTc-maSKS-ZHER2:342 showed the most promising biodistribution properties, with intermediate accumulation of radioactivity in the kidney and low accumulation in the liver. This conjugate was therefore further evaluated in mice with SKOV-3 xenografts. The in vivo evaluation in mice bearing tumors showed that 99mTc-maSKS-ZHER2:3 led to specific and efficient accumulation in the SKOV-3 tumors (Table 4 and Figure 6). Four hours after injection, 17% IA/g was

detected in the tumors, while the blood clearance was rapid; resulting in good tumor-to-blood and tumor-to-nontumor ratios for the gamma scintigraphy. The images acquired in gamma camera after an i.v. injection of 99mTc-maSKS-ZHER2:3 clearly visualized the tumors on the right hind legs of nude mice (Figure 7). Since small-sized (Mw < 60 kDa) substances are rapidly cleared through renal filtration, a high amount of radioactivity was also seen in the kidneys. Our previous results showed that an increase of the chelator hydrophilicityimprovestheexcretionpropertiesofZHER2:342 (20,23). It is therefore interesting to compare the biodistribution properties of the new positively charged chelators with those of previously studied neutral and negatively charged chelators. As illustrated in Table 3, 99mTc-maSSS-ZHER2:342 was used as the starting conjugate for the development of a suitable HER2 imaging agent. 99mTc-maSSS-ZHER2:342 provided good tumorto-organ ratios for imaging (20), but an elevated hepatobiliary excretion was seen (intestine uptake of 11 ( 2% of injected activity per whole sample), which would complicate detection of metastases in the abdominal area. The use of multiple negatively charged glutamic acids (99mTc-maEEE-ZHER2:342) successfully switched the hepatobiliary to renal excretion (23), but the substitution of serine with multiple positively charged lysine (99mTc-maKKK-ZHER2:342) remarkably increased the liver uptake (0.21% IA/g for maEEE vs 7% IA/g for maKKK). This excludes 99mTc-maKKK-ZHER2:342 from further evaluation in tumor-bearing mice, since its clinical application would be restricted for detection of tumors in the extrahepatic sites. On the other hand, the substitution with one single lysine, as in 99m Tc-maSKS-ZHER2:342, seemed superior to 99mTc-maEEEZHER2:342 in terms of renal uptake (see Table 3). Along these lines, the substitution with two glutamic acids instead of three in the chelator sequence has been evaluated. The resulting combination of serine and glutamic acid (99mTc-maESE-ZHER2: 342) was shown to provide imaging properties as good as for 99m Tc-maSKS-ZHER2:342 in terms of reduced renal uptake and

Technical Notes

low blood, liver, and intestine accumulation, while maintaining the tumor localization capacity for imaging (24). In conclusion, we have shown that substitution with serine for lysine in the chelators has a clear effect on the biodistribution of radioactivity. The introduction of multiple lysines gives rise to a high liver accumulation. However, substitution of one serine with one single lysine in the mercaptoacetyl-based chelator of Affibody molecules reduced the hepatobiliary excretion 2-fold, and the 99mTc-maSKS-ZHER2:342 conjugate is thus favorable for imaging. We believe that our observation on the influence of different amino acid residues in the mercaptoacetyl-based chelators would be useful for design of; not only Affibody molecules, but also other small-sized peptide or scaffold-protein based tracers.

ACKNOWLEDGMENT This work was financially supported by the Swedish Cancer Society (Cancerfonden) and the Swedish Research Council (Vetenskapsrådet). The authors thank Ewa Jerwanska at the Department of Nuclear Medicine, Akademiska sjukhuset, for the technetium eluate. We thank Daniel Rosik at Affibody AB, Bromma, for the LC analysis.

LITERATURE CITED (1) Pegram, M. D., Pauletti, G., and Slamon, D. J. (1998) HER2/neu as a predictive marker of response to breast cancer therapy. Breast Cancer Res. Treat. 52, 65–77. (2) Yarden, Y. (2001) Biology of HER2 and its importance in breast cancer. Oncology 61, 1–13, Suppl 2. (3) Stern, D. F., Heffernan, P. A., and Weinberg, R. A. (1986) p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity. Mol. Cell. Biol. 6, 1729–40. (4) Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and McGuire, W. L. (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235, 177–82. (5) Moy, B., and Goss, P. E. (2006) Lapatinib: current status and future directions in breast cancer. Oncologist 11, 1047–57. (6) Bast, R. C., Jr., Ravdin, P., Hayes, D. F., Bates, S., Fritsche, H., Jr., Jessup, J. M., Kemeny, N., Locker, G. Y., Mennel, R. G., and Somerfield, M. R. (2001) 2000 update of recommendations for the use of tumor markers in breast and colorectal cancer: clinical practice guidelines of the American Society of Clinical Oncology. J. Clin. Oncol. 19, 1865–78. (7) Hicks, D. G., and Kulkarni, S. (2008) HER2+ breast cancer: review of biologic relevance and optimal use of diagnostic tools. Am. J. Clin. Pathol. 129, 263–73. (8) Wolff, A. C., Hammond, M. E., Schwartz, J. N., Hagerty, K. L., Allred, D. C., Cote, R. J., Dowsett, M., Fitzgibbons, P. L., Hanna, W. M., Langer, A., McShane, L. M., Paik, S., Pegram, M. D., Perez, E. A., Press, M. F., Rhodes, A., Sturgeon, C., Taube, S. E., Tubbs, R., Vance, G. H., van de Vijver, M., Wheeler, T. M., and Hayes, D. F. (2007) American Society of Clinical Oncology/ College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch. Pathol. Lab. Med. 131, 18. (9) Tolmachev, V. (2008) Imaging of HER-2 overexpression in tumors for guiding therapy. Curr. Pharm. Des. 14, 2999–3019. (10) Tolmachev, V., Orlova, A., Nilsson, F. Y., Feldwisch, J., Wennborg, A., and Abrahmsen, L. (2007) Affibody molecules: potential for in vivo imaging of molecular targets for cancer therapy. Expert Opin. Biol. Ther. 7, 555–68. (11) Nilsson, F. Y., and Tolmachev, V. (2007) Affibody molecules: new protein domains for molecular imaging and targeted tumor therapy. Curr. Opin. Drug DiscoVery DeV. 10, 167–75. (12) Cai, W., Niu, G., and Chen, X. (2008) Multimodality imaging of the HER-kinase axis in cancer. Eur. J. Nucl. Med. Mol.

Bioconjugate Chem., Vol. 19, No. 12, 2008 2575 Imaging 35, 186–208. (13) Nord, K., Gunneriusson, E., Ringdahl, J., Stahl, S., Uhlen, M., and Nygren, P. A. (1997) Binding proteins selected from combinatorial libraries of an R-helical bacterial receptor domain. Nat. Biotechnol. 15, 772–7. (14) Orlova, A., Magnusson, M., Eriksson, T. L., Nilsson, M., Larsson, B., Hoiden-Guthenberg, I., Widstrom, C., Carlsson, J., Tolmachev, V., Stahl, S., and Nilsson, F. Y. (2006) Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer Res. 66, 4339–48. (15) Orlova, A., Tran, T., Widstro¨m, C., Engfeldt, T., Karlstrom, A. E., and Tolmachev, V. (2007) Pre-clinical evaluation of [111In]-benzyl-DOTA-ZHER2:342, a potential agent for imaging of HER2 expression in malignant tumors. Int. J. Mol. Med. 20, 397–404. (16) Tolmachev, V., Nilsson, F. Y., Widstrom, C., Andersson, K., Rosik, D., Gedda, L., Wennborg, A., and Orlova, A. (2006) 111Inbenzyl-DTPA-ZHER2:342, an affibody-based conjugate for in vivo imaging of HER2 expression in malignant tumors. J. Nucl. Med. 47, 846–53. (17) Fortin, M. A., Orlova, A., Malmstrom, P. U., and Tolmachev, V. (2007) Labelling chemistry and characterization of [90Y/ 177 Lu]-DOTA-ZHER2:342-3 Affibody molecule, a candidate agent for locoregional treatment of urinary bladder carcinoma. Int. J. Mol. Med. 19, 285–91. (18) Kramer-Marek, G., Kiesewetter, D. O., Martiniova, L., Jagoda, E., Lee, S. B., and Capala, J. (2008) [(18)F]FBEM-Z(HER2:342)Affibody molecule-a new molecular tracer for in vivo monitoring of HER2 expression by positron emission tomography. Eur. J. Nucl. Med. Mol. Imaging 35, 1008–18. (19) Namavari, M., Padilla De Jesus, O., Cheng, Z., De, A., Kovacs, E., Levi, J., Zhang, R., Hoerner, J. K., Grade, H., Syud, F. A., and Gambhir, S. S. (2008) Direct site-specific radiolabeling of an affibody protein with 4-[(18)F]fluorobenzaldehyde via oxime chemistry. Mol. Imaging Biol. 10, 177–81. (20) Engfeldt, T., Tran, T., Orlova, A., Widstro¨m, C., Feldwisch, J., Abrahmse´n, L., Wennborg, A., Eriksson, A. K., and Tolmachev, V. (2007) 99mTc-chelator engineering to improve tumour targeting properties of a HER2-specific Affibody. Eur. J. Nucl. Med. Mol. Imaging 34, 1843–53. (21) Zhu, Z., Wang, Y., Zhang, Y., Liu, G., Liu, N., Rusckowski, M., and Hnatowich, D. J. (2001) A novel and simplified route to the synthesis of N3S chelators for 99mTc labeling. Nucl. Med. Biol. 28, 703–8. (22) Decristoforo, C., and Mather, S. J. (2002) The influence of chelator on the pharmacokinetics of 99mTc-labelled peptides. Q. J. Nucl. Med. 46, 195–205. (23) Tran, T., Engfeldt, T., Orlova, A., Sandstrom, M., Feldwisch, J., Abrahmsen, L., Wennborg, A., Tolmachev, V., and Karlstrom, A. E. (2007) 99mTc-maEEE-ZHER2:342, an affibody molecule-based tracer for the detection of HER2 expression in malignant tumors. Bioconjugate Chem. 18, 1956–64. (24) Ekblad, T., Tran, T., Orlova, A., Widstro¨m, C., Feldwisch, J., Wennborg, A., Abrahmse´n, L., Karlstro¨m Eriksson, A., and Vladimir, T. (2008) Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake. Eur. J. Nucl. Med. Mol. Imaging, In press. (25) Persson, M., Tolmachev, V., Andersson, K., Gedda, L., Sandstrom, M., and Carlsson, J. (2005) [(177)Lu]pertuzumab: experimental studies on targeting of HER-2 positive tumour cells. Eur. J. Nucl. Med. Mol. Imaging 32, 1457–62. (26) Engfeldt, T., Orlova, A., Tran, T., Bruskin, A., Widstrom, C., Karlstrom, A. E., and Tolmachev, V. (2006) Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycylglycyl (MAG3) sequence. Eur. J. Nucl. Med. Mol. Imaging 34, 722–733. (27) Ahlgren, S., Orlova, A., Rosik, D., Sandstrom, M., Sjoberg, 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 mol-

2576 Bioconjugate Chem., Vol. 19, No. 12, 2008 ecules. Bioconjugate Chem. 19, 235–43. (28) Tran, T., Engfeldt, T., Orlova, A., Widstrom, C., Bruskin, A., Tolmachev, V., and Karlstrom, A. E. (2007) In vivo evaluation of cysteine-based chelators for attachment of 99mTc to tumortargeting affibody molecules. Bioconjugate Chem. 18, 549–558. (29) Orlova, A., Rosik, D., Sandstrom, M., Lundqvist, H., Einarsson, L., and Tolmachev, V. (2007) Evaluation of [(111/ 114m)In]CHX-A′′-DTPA-Z(HER2:342), an affibody ligand conjugate for targeting of HER2-expressing malignant tumors. Q. J. Nucl. Med. Mol. Imaging 51, 314–23. (30) Schmidt, M. M., Thurber, G. M., and Wittrup, K. D. (2008) Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability, Cancer Immunol. Immunother. PMID:18408925.

Tran et al. (31) Nishikawa, M., Hasegawa, S., Yamashita, F., Takakura, Y., and Hashida, M. (2002) Electrical charge on protein regulates its absorption from the rat small intestine. Am. J. Physiol. Gastrointest. LiVer Physiol. 282, G711-9. (32) Ma, S. F., Nishikawa, M., Katsumi, H., Yamashita, F., and Hashida, M. (2005) Cationic charge-dependent hepatic delivery of amidated serum albumin. J. Controlled Release 102, 583–94. (33) Foulon, C. F., Welsh, P. C., Bigner, D. D., and Zalutsky, M. R. (2001) Positively charged templates for labeling internalizing antibodies: comparison of N-succinimidyl 5-iodo-3-pyridinecarboxylate and the D-amino acid peptide KRYRR. Nucl. Med. Biol. 28, 769–77. BC800244B