Bioconjugate Chem. 2008, 19, 235–243
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Evaluation of Maleimide Derivative of DOTA for Site-Specific Labeling of Recombinant Affibody Molecules Sara Ahlgren,† Anna Orlova,‡,§ Daniel Rosik,‡ Mattias Sandström,| Anna Sjöberg,‡ Barbro Baastrup,‡ Olof Widmark,‡ Gunilla Fant,‡ Joachim Feldwisch,‡,§ and Vladimir Tolmachev*,†,‡,§ Division of Nuclear Medicine, Department of Medical Sciences, Uppsala University, Uppsala, Sweden, Affibody AB, Bromma, Sweden, Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden, Hospital Physics, Department of Oncology, Uppsala University Hospital, Uppsala, Sweden. Received August 15, 2007; Revised Manuscript Received October 4, 2007
Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in ViVo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10maleimidoethylacetamide (MMA-DOTA) for 111In-labeling of anti-HER2 Affibody molecules His6-ZHER2:342Cys and ZHER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His6-ZHER2:342-Cys and 27 pM for ZHER2:2395-Cys, comparable with 22 pM for the parental ZHER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111In-[MMA-DOTA-Cys61]-ZHER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tagcontaining counterpart. In mice bearing HER2-expressing LS174T xenografts, 111In-[MMA-DOTA-Cys61]ZHER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 ( 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 ( 8. Xenografts were clearly visualized already 1 h p.i.
INTRODUCTION A growing number of cellular genotype and phenotype changes associated with the malignant transformation of cells have been identified. This knowledge is utilized for development of drugs that target particular alterations in gene expression. Due to the diversity of cancer phenotypes, only a fraction of all patients may benefit from a given targeted therapeutic drug. Therefore, targeting medicines need to be personalized, i.e., adapted to the cancer phenotype of the individual patient. For example, the HER2 tyrosine kinase receptor is the target for the humanized monoclonal antibody trastuzumab (Herceptin), which is approved for the clinical treatment of breast cancer. However, only 25–30% of all breast cancer patients display tumor disease overexpressing HER2, and in ViVo detection of HER2 overexpression is therefore necessary (1, 2). Radionuclide molecular imaging of HER2 expression in tumors may help to avoid problems associated with biopsies, such as sampling errors and discordance of HER2 expression in primary tumors and metastases. Initial attempts for radionuclide imaging were done using 111In-labeled trastuzumab to identify potential responders to trastuzumab therapy (3). However, the sensitivity of detection was low (4). Generally speaking, low sensitivity is a problem * Communicating author. Vladimir Tolmachev, Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 81 Uppsala, Sweden. Phone: +46 18 471 3414, Fax: + 46 18 471 3432,
[email protected]. † Department of Medical Sciences, Uppsala University. ‡ Affibody AB. § Rudbeck Laboratory, Uppsala University. | Department of Oncology, Uppsala University Hospital.
when using full-size IgG antibodies as targeting agents for imaging, since their long residence time in blood causes high background and reduces contrast of imaging. The use of smaller targeting proteins, e.g., (Fab′)2, Fab, and scFv antibody fragments, with more rapid tumor localization and more rapid clearance is considered a promising way to increase the radionuclide imaging contrast. One class of promising candidates for in ViVo targeting agents are Affibody molecules, small (7 kDa) proteins based on a modified scaffold of the B-domain of protein A (5). Affibody molecule libraries are constructed by randomization of 13 amino acids in helix 1 and 2 of the modified domain B. Selection is performed, e.g., by phage display and enables development of high-affinity binders to protein targets. The selection of the Affibody molecule ZHER2:342, binding the extracellular domain of HER2 with an affinity of 22 pM, was described earlier (6), and high-contrast imaging of HER2-expressing xenografts after indirect radioiodination was demonstrated. However, indirect radioiodination is a two-step labeling procedure followed by purification, which might complicate its translation into hospital radiopharmacy and delay introduction into routine clinical use. A coupling of suitable chelators and the use of radioactive metals, such as, e.g., 111In (T1/2 ) 2.8 days), as labels enables kit formulation and simplifies logistics of radiopharmaceutical distribution and preparation. The use of benzyl isothiocyanate derivatives of DTPA (7) and DOTA (8) has been evaluated for labeling of recombinant ZHER2:342 with 111In. Both types of chelator could be used to make conjugates capable of specific imaging of HER2 in ViVo, but 111In-benzyl-DTPA-ZHER2:342 (further referred to as 111In-Bz-DTPA-Z342) provided better tumor-to-blood ratio (50 ( 14 vs 23 ( 5) and tumor-to-liver
10.1021/bc700307y CCC: $40.75 2008 American Chemical Society Published on Web 12/29/2007
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ratio (3.2 ( 0.6 vs 1.5 ( 0.1) than 111In-Bz-DOTA-Z342 in a direct head-to-head comparison 4 h p.i. in mice bearing HER2expressing xenografts (8). Isothiocyanate reacts to amino groups. The ZHER2:342 Affibody molecule contains 7 amino groups, 1 at the N-terminus and 6 on lysine residue side chains. As expected, coupling with isothiocyanate produced a heterogeneous mixture of conjugates, containing different numbers of chelators, attached randomly at different positions. Clearly, a controlled sitespecific attachment of chelator is more desirable, since it provides a homogeneous product with high batch-to-batch reproducibility. The spontaneously folding cysteine-independent structure of Affibody molecules enables a complete peptide synthesis of this protein and site-specific incorporation of different functional groups (9). Synthetic Affibody molecules ZHER2:342 with chelators incorporated at the N-terminus have been prepared and characterized in ViVo (10-12). DOTA has been used for labeling with 111In (10) and mercaptoacetylcontaining peptide-based chelators maGGG and maSSS for labeling with 99mTc (11, 12). In ViVo molecular imaging using synthetic 111In-DOTA-ZHER2:342 (further referred to as 111InDOTA-Z342) resulted in specific and high-contrast targeting of HER2-expressing xenografts in mice (tumor-to-blood ratio of 7 already 1 h p.i.). Moreover, the clinical utility of this tracer in SPECT and PET has been confirmed, following labeling with 111 In or 68Ga, respectively (13, 14). Chemical production is only suitable for production of small proteinaceous agents such as monovalent Affibody molecules. Larger molecules, including dimeric or bispecific Affibody molecules, have to be produced recombinantly. Also, in this case, site-specific labeling is desirable. The absence of cysteines in the Affibody molecule provides the possibility to obtain a unique attachment site by introduction of a single cysteine, e.g., as a C-terminal residue. The use of a bifunctional chelator, linker molecule, or other reporter with a thiol-reactive moiety, e.g., maleimide or iodoacetamide, enables directed conjugation with this particular cysteine. This has been used previously for coupling of, e.g., Oregon Green 488 or horseradish peroxidase (HRP) to the dimeric Affibody molecule (ZHER2:477)2-Cys (15) for in Vitro detection of HER2. The use of the ((4-hydroxyphenyl)ethyl)maleimide (HPEM) linker molecule enabled sitespecific labeling of a dimeric form of a previous generation of the HER2-binding Affibody molecule with radiobromine (16) and radioiodine (17). The goal of the present study was to evaluate site-specific labeling of cysteine-containing Affibody molecules with 111In using the chelator maleimide-monoamide-DOTA (MMADOTA; 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid10-maleimidoethylacetamide). For this purpose, several cysteinecontaining variants of Affibody molecules were produced, conjugated with MMA-DOTA, and labeled with 111In. In a comparative biodistribution, a His-tag containing variant, 111In[MMA-DOTA-Cys61]-His6-ZHER2:342-Cys (further referred to as 111 In-DOTA-H6-Z342-C), was compared with non-His-tagged 111 In-[MMA-DOTA-Cys61]-ZHER2:2395-Cys (further referred to as 111In-DOTA-Z2395-C) and synthetic 111In-DOTA-Z342 variants. The tumor-targeting properties of the best recombinant cysteine-containing site-specifically labeled variant, 111In-DOTAZ2395-C, were compared in tumor-bearing mice with the synthetic 111 In-DOTA-Z342 and the best previous recombinantly produced conjugate with non-site-specific coupling of the chelator, 111InBz-DTPA-Z342.
MATERIALS AND METHODS Materials. MMA-DOTA was purchased from Macrocyclics (Dallas, TX, USA). A synthetic DOTA-ZHER2:342 was made by standard Fmoc peptide synthesis in a single chemical process by Bachem (Bubendorf, Switzerland) and labeled as described
Ahlgren et al.
by Orlova et al. (10). A recombinant 111In-Bz-DTPA-Z342 was produced and labeled according to Tolmachev et al. (6). Buffers, such as 0.1 M phosphate buffered saline (PBS), pH 7.5, and 0.2 M ammonium acetate, pH 5.5, were prepared using common methods from chemicals supplied by Merck (Darmstadt, Germany). High-quality Milli-Q water (resistance higher than 18 MΩ/cm) was used for preparing solutions. Buffers, which were used for conjugation and labeling, were purified from metal contamination using Chelex 100 resin (Bio-Rad Laboratories, Richmond, CA, USA). PD-10 and NAP-5 size exclusion columns were from Amersham Biosciences, Uppsala, Sweden. [111In]indium chloride was purchased from Tyco. Silica gel impregnated glass fiber sheets for instant thin-layer chromatography (ITLC SG) were from Gelman Sciences Inc. Cells in the in Vitro experiments were detached using trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer, Biochrom AG, Berlin, Germany). Ketalar (50 mg/mL, Pfizer, NY, USA), Rompun (20 mg/mL, Bayer, Leverkusen, Germany), and Heparin (5000 IE/mL, Leo Pharma, Köpenhamn, Danmark) were obtained commercially. Data on cellular uptake, biodistribution, and gamma camera images were analyzed by unpaired, two-tailed t-test using GraphPad Prism (version 4.00 for Windows GraphPad Software, San Diego, California, USA) in order to determine any significant differences (P < 0.05). Instrumentation. The radioactivity was measured using an automated gamma counter with 3 in. NaI(Tl) detector (1480 WIZARD, Wallac Oy, Turku, Finland). 111In was measured with the use of both photopeaks and summation peak (energy setting from 140 to 507 keV). Distribution of radioactivity along the ITLC strips and SDS-PAGE gels was measured on a Cyclone Storage Phosphor System (further referred to as PhosphorImager) and analyzed using the OptiQuant image analysis software. Cells were counted using electronic cell counter (Beckman Coulter). The Affibody molecule were analyzed by high-performance liquid chromatography and online mass spectrometry (HPLC-MS) using an Agilent 1100 HPLC/MSD. The mass spectrometer was equipped with electrospray ionization (ESI) and single quadropol. The software used for analysis and evaluation was Chemstation ReV. B.02.01. (Agilent). For HPLC-MS analysis, 60 µL of diluted sample (1:1 dilution with solution A: 0.1% TFA in MQ-water) was loaded onto a Zorbax 300SB-C18 (4.6 × 150, 3.5 µm) RPC column equilibrated with solution A with 15% solution B (0.1% TFA in acetonitrile) at a flow rate of 1 mL/min. After 2 min with 15% B solution, the proteins were eluted using a linear gradient, 15–65% solution B in 22 min. The mass spectrometer was run according to the manufacturer’s recommendations. SDS-PAGE analysis was performed using NuPAGE 4–12% Bis-Tris Gel (Invitrogen) in MES buffer (200 V constant). Affinity of Affibody molecules to HER2 was analyzed using a Biacore 2000 instrument (Biacore, Uppsala, Sweden) according to the method described earlier (10). Melting point analysis was performed using a JASCO J-810 spectropolarimeter (JASCO, Tokyo, Japan) as described earlier (11). Protein Expression and Purification. A C-terminal cysteine was introduced into Affibody molecules according to a method described earlier (16). Escherichia coli BL21(DE3)/pAY01662 was run in an 800 mL batch fermentor cultivation (System Greta, Belach Bioteknik AB, Sweden) at 37 °C using TSB+YEmedium (30 g/L Tryptic Soy Broth (Merck), 5 g/L Yeast Extract (Merck)) supplemented with 50 mg/mL Kanamycin. The culture was automatically induced at an OD (600 nm) of 1 by the addition of IPTG to a concentration of 0.5 mM. Five hours after induction, the culture was automatically cooled to
