Research ArticleBasic Science Investigations

Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with 18F-Labeled Affibody Molecule ZHER2:2395 in a Mouse Model for Ovarian Cancer

Sandra Heskamp, Peter Laverman, Daniel Rosik, Frederic Boschetti, Winette T.A. van der Graaf, Wim J.G. Oyen, Hanneke W.M. van Laarhoven, Vladimir Tolmachev and Otto C. Boerman
Journal of Nuclear Medicine January 2012, 53 (1) 146-153; DOI: https://doi.org/10.2967/jnumed.111.093047

Abstract

Affibody molecules are small (7 kDa) proteins with subnanomolar targeting affinity. Previous SPECT studies in xenografts have shown that the Affibody molecule 111In-DOTA-ZHER2:2395 can discriminate between high and low human epidermal growth factor receptor type 2 (HER2)–expressing tumors, indicating that radiolabeled Affibody molecules have potential for patient selection for HER2-targeted therapy. Compared with SPECT, PET with positron-emitting radionuclides, such as 18F, may improve imaging of HER2 expression because of higher sensitivity and improved quantification of PET. The aim of the present study was to determine whether the 18F-labeled NOTA-conjugated Affibody molecule ZHER2:2395 is a suitable agent for imaging of HER2 expression. The tumor-targeting properties of 18F-labeled ZHER2:2395 were compared with 111In- and 68Ga-labeled ZHER2:2395 in mice with HER2-expressing SK-OV-3 xenografts. Methods: ZHER2:2395 was conjugated with NOTA and radiolabeled with 18F, 68Ga, and 111In. Radiolabeling with 18F was based on the complexation of Al18F by NOTA. The 50% inhibitory concentration values for NOTA-ZHER2:2395 labeled with 19F, 69Ga, and 115In were determined in a competitive cell-binding assay using SK-OV-3 cells. Mice bearing subcutaneous SK-OV-3 xenografts were injected intravenously with radiolabeled NOTA-ZHER2:2395. One and 4 h after injection, PET/CT or SPECT/CT images were acquired, and the biodistribution was determined by ex vivo measurement. Results: The 50% inhibitory concentration values for 19F-, 69Ga-, and 115In-NOTA-ZHER2:2395 were 5.0, 6.3, and 5.3 nM, respectively. One hour after injection, tumor uptake was 4.4 ± 0.8 percentage injected dose per gram (%ID/g), 5.6 ± 1.6 %ID/g, and 7.1 ± 1.4 %ID/g for 18F-, 68Ga-, and 111In-NOTA-ZHER2:2395, respectively, and the respective tumor-to-blood ratios were 7.4 ± 1.8, 8.0 ± 1.3, and 4.8 ± 1.3. Tumor uptake was specific, because uptake could be blocked efficiently by coinjection of an excess of unlabeled ZHER2:2395. PET/CT and SPECT/CT images clearly visualized HER2-expressing SK-OV-3 xenografts. Conclusion: This study showed that 18F-NOTA-ZHER2:2395 is a promising new imaging agent for HER2 expression in tumors. Affibody molecules were successfully labeled with 18F within 30 min, based on the complexation of Al18F by NOTA. Further research is needed to determine whether this technique can be used for patient selection for HER2-targeted therapy.

Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family. Dimerization of HER2 with other members of the epidermal growth factor receptor family leads to activation of several downstream pathways, including PI3K-AKT and MAPK, resulting in increased tumor cell proliferation, cell motility, and survival (1). HER2 is overexpressed in 18%–25% of all breast carcinomas and in subsets of ovarian, lung, prostate, and gastric cancers (1,2). Breast cancers that overexpress HER2 have been associated with aggressive tumor growth, high relapse, and poor prognosis (2). Furthermore, HER2-overexpressing tumors may be more resistant to endocrine therapy and chemotherapy (3).

Different therapeutic strategies have been developed to target HER2, including the monoclonal antibody trastuzumab and tyrosine kinase inhibitor lapatinib. Trastuzumab targets the extracellular domain of HER2, inhibits intracellular signaling pathways, and causes antibody-dependent cell-mediated cytotoxicity (1). Trastuzumab significantly improves survival of patients with HER2-positive advanced breast and gastric cancer, when combined with chemotherapy(4–6). Lapatinib targets the intracellular domain of both HER2 and epidermal growth factor receptor (1). The addition of lapatinib to capecitabine prolongs progression-free survival of patients with advanced HER2-positive breast cancer (7).

Currently, trastuzumab treatment is recommended only for breast and gastric cancer patients with HER2-overexpressing tumors (8). Therefore, accurate assessment of HER2 expression is essential for patient selection for HER2-targeted therapy. Nowadays, HER2 expression is determined on tumor biopsies by immunohistochemical staining of HER2 protein expression or fluorescence in situ hybridization of HER2 messenger RNA expression. Up to 20% of the current HER2 assessments may be inaccurate (9,10). Furthermore, HER2 expression can differ between the primary tumor and metastases (11,12). Therefore, in vivo imaging of HER2 expression may result in more accurate detection of HER2 expression, avoiding misinterpretation due to intratumoral and interlesional heterogeneity. Moreover, such an imaging method would allow monitoring of HER2 expression during the course of disease, without the need of repetitive invasive biopsies.

Several approaches have been used for PET and SPECT of HER2 expression, including radiolabeled antibodies trastuzumab and pertuzumab (HER2 dimerization inhibitor) and radiolabeled trastuzumab fragments (13–16). These radiotracers were able to visualize HER2 expression, but their large size resulted in slow tumor accumulation and slow clearance from the circulation. A new class of targeting proteins are Affibody (Affibody AB) molecules. These are based on a 58-amino-acid (7 kDa) scaffold and can be selected to bind with high affinity to various tumor-associated antigens. Their small size allows rapid extravasation in tumors and rapid clearance from the blood, resulting in high-contrast imaging within several hours after injection (17,18). The HER2-targeting Affibody molecule ZHER2:342 and its derivates have been radiolabeled with several radionuclides and have shown specific targeting of HER2 (17). Previous studies in xenografts have shown that the Affibody molecule 111In-DOTA-ZHER2:2395 can discriminate between tumors with high and low HER2 expression (19). A clinical pilot study with 111In- and 68Ga-DOTA-ZHER2:342 has shown that it is feasible to visualize HER2-expressing tumors in patients with metastatic breast cancer (20). Therefore, Affibody molecules have the potential for patient selection for HER2-targeted therapy.

Recently, ZHER2:2395, a variant of ZHER2:342 with a C-terminal cysteine, was coupled to maleimide monoamide NOTA (MMA-NOTA) and was radiolabeled with 111In. This site-specifically labeled conjugate allowed high-contrast imaging of HER2-expressing xenografts (21). Imaging of HER2 expression with Affibody molecules can be improved by the use positron-emitting radionuclides such as 18F, because of the higher sensitivity and better quantification of PET than SPECT. Kramer-Marek et al. have conjugated ZHER2:342 with N-2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM) (22). 18F-FBEM-ZHER2:342 could visualize HER2-expressing tumors. However, this labeling reaction required a long synthesis time. Lately, a new method was described for labeling of NOTA-conjugated peptides with 18F, which is based on the formation of aluminum18F-fluoride (Al18F) and its complexation by NOTA (23,24). This method allows rapid and efficient labeling of peptides and proteins with 18F.

The aim of the present study was to determine whether the 18F-labeled Affibody molecule ZHER2:2395 is a suitable imaging agent for HER2 expression in mice with SK-OV-3 xenografts. For this purpose, NOTA-conjugated ZHER2:2395 was radiolabeled with 18F, based on the complexation of Al18F by NOTA. 18F-NOTA-ZHER2:2395 was compared with 111In- and 68Ga-labeled ZHER2:2395.

MATERIALS AND METHODS

Cell Line and Affibody Molecule

The HER2-overexpressing ovarian cancer cell line SK-OV-3 was cultured and maintained as a monolayer in RPMI 1640 medium (Gibco, BRL Life Sciences Technologies) supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL). The HER2-negative breast cancer cell line SUM149 (Asterand) was cultured and maintained as monolayer in Ham F12 medium (Gibco, BRL Life Sciences Technologies) supplemented with 5% fetal calf serum, 10 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), hydrocortisone (1 μg/mL), and insulin (5 μg/mL) at 37°C in a humidified atmosphere with 5% CO2. ZHER2:2395 was produced as described previously (25).

Affibody Conjugation with MMA-NOTA

The production of MMA-NOTA and coupling of MMA-NOTA to ZHER2:2395 has been described elsewhere (21,25). In short, reduced ZHER2:2395 was incubated for 1 h at 37°C with a freshly prepared solution of MMA-NOTA in 0.2 M ammonium acetate, pH 5.5 (1 mg/mL), at a chelator-to-protein ratio of 3:1. The NOTA-conjugated Affibody molecules were purified by semipreparative reversed-phase high-performance liquid chromatography (HPLC), using a 300SB C18 column (9.4 × 250 mm; Zorbax) at a flow rate of 8 mL/min, with the following buffer system: buffer A, 0.1% trifluoroacetic acid (TFA) in water; buffer B, 0.1% TFA in acetonitrile; and a gradient of 25%–40% buffer B over 11 min.

Radiolabeling

18F Labeling

Radiolabeling with 18F was based on the complexation of Al18F by NOTA as described elsewhere (23,24). Briefly, a Chromafix PS-HCO3 cartridge (ABX) with 2–6 GBq of 18F (BV Cyclotron VU) was washed with 3 mL of metal-free water. 18F was eluted from the cartridge with 100 μL of 0.9% NaCl. Al18F was prepared by adding 2 mM AlCl3 in 0.1 M sodium acetate, pH 4 (8.5 μL of AlCl3 per GBq of 18F). NOTA-ZHER2:2395 (250 μg) was dissolved in 25 μL of 0.5 M sodium acetate, pH 4. To the dissolved Affibody molecule, 25 μL of acetonitrile were added. After adding 50 μL of Al18F (1.16–1.27 GBq), the reaction mixture was incubated for 15 min at 90°C. Subsequently, the reaction mixture was applied on a 1-mL Oasis HLB cartridge (30 mg; Waters) to remove unincorporated Al18F. The cartridge was washed with 3 mL of H2O, and 18F-NOTA-ZHER2:2395 was eluted with 300 μL of ethanol. Finally, 18F-NOTA-ZHER2:2395 was applied on a NAP-5 column (GE Healthcare Life Sciences), preequilibrated with phosphate-buffered saline (PBS), to change the buffer from ethanol to PBS. Before injection, 18F-NOTA-ZHER2:2395 was diluted in PBS containing 0.5% bovine serum albumin (BSA). Labeling efficiency and radiochemical purity were determined with instant-thin layer chromatography (ITLC) and HPLC.

68Ga Labeling

NOTA-ZHER2:2395 was labeled with 68Ga eluted from a TiO2-based 1,850-MBq 68Ge/68Ga generator (IGG-100; Eckert and Ziegler) using 0.1 M HCl (Ultrapure; J.T. Baker). NOTA-ZHER2:2395 was dissolved in 0.25 M ammonium acetate, pH 5.4. Radiolabeling with 68Ga was performed by adding 120 μL of 2.5 M HEPES, pH 5.6, and 1 mL of 68Ga eluate (165–192 MBq) to NOTA-ZHER2:2395 (18–21 μg). The final pH of the labeling reaction was 3.5. The reaction mixture was incubated for 15 min at 90°C. After the labeling reaction, 50 mM ethylenediaminetetraacetic acid (EDTA) was added to a final concentration of 5 mM to complex nonincorporated 68Ga3+. Subsequently, the labeling mixture was purified on a PD10 column (GE Healthcare Life Sciences), eluted with PBS containing 0.5% BSA. Before injection, 68Ga-NOTA-ZHER2:2395 was diluted in PBS containing 0.5% BSA. Labeling efficiency, colloid, and radiochemical purity were determined with ITLC and HPLC.

111In Labeling

NOTA-ZHER2:2395 was dissolved in 0.25 M ammonium acetate, pH 5.4. For radiolabeling, 70 μg of NOTA-ZHER2:2395 was incubated with 260 MBq of 111In (Covidien BV) in 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer, pH 5.4 (twice the volume of 111In), for 15 min at 90°C. After incubation, 50 mM EDTA was added to a final concentration of 5 mM. Subsequently, the reaction mixture was purified on a PD10 column, eluted with PBS containing 0.5% BSA. 111In-NOTA-ZHER2:2395 was diluted in PBS containing 0.5% BSA. Labeling efficiency and radiochemical purity were determined with ITLC and HPLC.

Quality Control

HPLC

Labeling efficiency was analyzed by reversed-phase high-performance liquid chromatography on an Agilent 1200 system (Agilent Technologies). A monolithic C18 column (Onyx, 4.6 × 100 mm; Phenomenex) was used at a flow rate of 1 mL/min, with the following buffer system: buffer A, 0.1% v/v TFA in water; buffer B, 0.1% v/v TFA in acetonitrile; and a gradient of 97% buffer A to 0% buffer A at 5–15 min. The radioactivity of the eluate was monitored using an in-line NaI radiodetector (Raytest GmbH). Elution profiles were analyzed using Gina-star software (version 2.18; Raytest GmbH).

ITLC

Radiochemical purity was determined using ITLC on silica gel chromatography strips (Agilent Technologies), with 0.1 M ammonium acetate containing 0.1 M EDTA, pH 5.5, as the mobile phase. The presence of colloid was analyzed using methanol:0.5 M HEPES, pH 3.5 (1:1) as the mobile phase.

In Vitro Studies

In Vitro Binding of Radiolabeled NOTA-ZHER2:2395

Labeling with 18F requires incubation of the NOTA-conjugated Affibody molecule at 90°C in 25% acetonitrile. To determine whether HER2 binding was affected by these labeling conditions, an in vitro binding experiment was performed. NOTA-ZHER2:2395 was radiolabeled with 18F. Unlabeled NOTA-ZHER2:2395 was incubated for 15 min at room temperature without acetonitrile or at 90°C in the presence of 25% acetonitrile.

SK-OV-3 and SUM149 cells were cultured to confluency in 6-well plates. Cells were washed with PBS and incubated for 2 h at 4°C with 4 kBq of 18F-NOTA-ZHER2:2395, without unlabeled Affibody molecules or in the presence of 50 nM unlabeled NOTA-ZHER2:2395 (preincubated at room temperature or 90°C) in a total volume of 1 mL of binding buffer (SK-OV-3: RPMI 1640 containing 0.5% BSA; SUM149: Ham F12, 10 mM HEPES, containing 0.5% BSA). Separate wells were incubated with 4 kBq of 18F-NOTA-ZHER2:2395 and 50 nM trastuzumab to study whether trastuzumab interfered with Affibody molecule binding. After incubation, cells were washed with PBS, and the cell-associated activity was measured in a shielded well-type γ-counter (Perkin-Elmer).

Scatchard Analysis

Scatchard analysis was performed to determine the dissociation constant (Kd) and the number of binding sites for 18F-NOTA-ZHER2:2395 on SK-OV-3 cells. Cells were cultured to confluency in 6-well plates and were incubated for 2 h at 4°C with increasing concentrations of 18F-NOTA-ZHER2:2395 (0.03–30 nM) in 1 mL of binding buffer. Nonspecific binding was determined by coincubation with an excess of 1 μM unlabeled NOTA-ZHER2:2395. After incubation, cells were washed with PBS, and the cell-associated activity was measured in a shielded well-type γ-counter.

50% Inhibitory Concentration (IC50) Determination

The IC50 for binding HER2 on SK-OV-3 was determined in a competitive binding assay using 19F-, 69Ga-, or 115In-NOTA-ZHER2:2395 to compete for binding with 111In-NOTA-ZHER2:2395.

NOTA-ZHER2:2395 was dissolved in 0.25 M ammonium acetate, pH 5.4. 19F-NOTA-ZHER2:2395 was prepared by adding a 2.5-fold molar excess of AlCl3 and a 5-fold molar excess of 19F to 15 μg of NOTA-ZHER2:2395 in 0.5 M sodium acetate, pH 4. 115In-NOTA-ZHER2:2395 was prepared by adding a 3-fold molar excess of 115InCl3 to 15 μg of NOTA-ZHER2:2395 in 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer, pH 5.4. 69Ga-NOTA-ZHER2:2395 was formed by adding a 3-fold molar excess of 69Ga nitrate to 15 μg of NOTA-ZHER2:2395 in 2.5 M HEPES buffer, pH 7.0. All labeling mixtures were incubated for 15 min at 90°C. EDTA (50 mM) was added to a final concentration of 5 mM.

NOTA-ZHER2:2395 (10 μg) was radiolabeled with 1.9 MBq of 111In. Labeling efficiency, determined using ITLC, was 95%, and no further purification was required.

SK-OV-3 cells were cultured to confluency in 6-well plates. To determine the apparent IC50, cells were washed with PBS and incubated for 2 h at 4°C in 2 mL of binding buffer with a trace amount of 1 kBq of 111In-NOTA-ZHER2:2395 and increasing concentrations of 19F-, 69Ga-, or 115In-NOTA-ZHER2:2395 (0.001–30 nM). After incubation, cells were washed with PBS, and the cell-associated activity was measured in a γ-counter. The IC50 was defined as the Affibody molecule concentration at which 50% of binding without competitor was reached. IC50 values were calculated using Prism software (version 5.03 for Windows [Microsoft]; GraphPad Software).

Serum Stability

18F-NOTA-ZHER2:2395 (3.7 MBq) was incubated for 4 h at 37°C in 500 μL of human and mouse serum. After incubation, stability was analyzed using ITLC.

Animal Studies

Animal experiments were performed in female BALB/c nude mice (Janvier SAS) and were conducted in accordance with the principles laid out by the revised Dutch Act on Animal Experimentation (1997) and approved by the institutional Animal Welfare Committee of the Radboud University Nijmegen. At 6–8 wk of age, mice were inoculated subcutaneously with 5 × 106 SK-OV-3 cells (mixed 2:1 with Matrigel [BD Biosciences]). Experiments were initiated when the tumors reached approximately 0.1 cm3.

Biodistribution Studies

Five groups (n = 6) of mice were injected intravenously with 10 μg of 18F-NOTA-ZHER2:2395 (1 MBq), 68Ga-NOTA-ZHER2:2395 (5 MBq), or 111In-NOTA-ZHER2:2395 (0.4 MBq). Separate groups (n = 3) of mice were coinjected with an excess of unlabeled ZHER2:2395 (500 μg). One and 4 h (18F and 111In only) after injection, mice were euthanized by CO2/O2 asphyxiation. Tumor, blood, muscle, lung, heart, spleen, pancreas, intestine, kidney, liver, small intestine, and bone were dissected and weighed. Activity was measured in a γ-counter. To calculate the uptake of radiolabeled Affibody molecules in each sample as a fraction of the injected dose, aliquots of the injected dose were counted simultaneously. The results were expressed as percentage of the injected dose per gram (%ID/g).

PET/CT

Mice were injected intravenously with 10 μg of 18F-NOTA-ZHER2:2395 (10 MBq) or 10 μg of 68Ga-NOTA-ZHER2:2395 (11 MBq). One and 4 h (18F only) after injection, mice were scanned on an animal PET/CT scanner (Inveon; Siemens Preclinical Solutions) with an intrinsic spatial resolution of 1.5 mm (26). First, a CT scan (spatial resolution, 113 μm; 80 kV; 500 μA) was acquired for anatomic reference, followed by a PET emission scan of 30 min (1 h after injection) or 60 min (4 h after injection). Before the last scan, mice were euthanized, and after acquisition the biodistribution of radiolabeled Affibody molecules was measured ex vivo. Scans were reconstructed with Inveon Acquisition Workplace software (version 1.5; Siemens Preclinical Solutions), using an ordered-set expectation maximization 3-dimensional maximum a posteriori algorithm with the following parameters: matrix, 256 × 256 × 159; pixel size, 0.43 × 0.43 × 0.8 mm; and β-value of 1.5 with uniform variance. Representative cross sections located approximately in the center of the tumor were displayed. Tumor-to-liver ratios were calculated with the Inveon Research Workplace software (IRW, version 3.0).

SPECT/CT

Mice were injected with 10 μg of 111In-NOTA-ZHER2:2395 (18 MBq). One and 4 h after injection, mice were scanned on an animal SPECT/CT device (U-SPECT-II; MILabs), using a 1.0-mm-diameter pinhole rat collimator cylinder (27). SPECT scans were acquired for 45 min (1 h after injection) or 60 min (4 h after injection), followed by CT scans (spatial resolution, 160 μm; 40 kV; 612 μA) for anatomic reference. Before the last scan, mice were euthanized, and after acquisition the biodistribution of radiolabeled Affibody molecules was measured ex vivo. Scans were reconstructed with MILabs reconstruction software, which uses an ordered-subset expectation maximization algorithm, with a voxel size of 0.375 mm. Tumor-to-liver ratios were calculated with the Inveon Research Workplace software (IRW, version 3.0).

Statistical Analysis

Statistical analyses were performed using SPSS software (version 16.0) and GraphPad Prism (version 5.03) for Windows. Differences in uptake of radiolabeled Affibody molecules were tested for significance using the nonparametric Mann–Whitney test. A P value below 0.05 was considered significant.

RESULTS

Radiolabeling

The radiolabeling yield for 18F, 68Ga, and 111In was 21.0% ± 5.7%, 84.6% ± 0.9%, and 94.0%, respectively. After purification on an HLB cartridge and NAP-5 column (18F) or PD10 column (68Ga and 111In), ITLC indicated that radiochemical purity exceeded 95%. No colloid (<1%) was formed during the labeling reactions. Specific activities of 7,700 ± 3,000, 39,600 ± 500, and 22,400 GBq/mmol were obtained for 18F-, 68Ga-, and 111In-NOTA-ZHER2:2395, respectively (end of synthesis).

In Vitro Binding Assays

In vitro binding assays showed that incubation at 90°C in 25% acetonitrile did not affect binding of NOTA-ZHER2:2395 to HER2 (Fig. 1). Both samples (preincubated at room temperature and 90°C in acetonitrile) were equally effective in blocking the binding of 18F-NOTA-ZHER2:2395. Furthermore, 18F-NOTA-ZHER2:2395 did not show specific binding to the HER2-negative cell line SUM149, and in agreement with previous studies, trastuzumab did not interfere with Affibody molecule binding to SK-OV-3 cells.

FIGURE 1.
FIGURE 1.

In vitro binding of 18F-NOTA-ZHER2:2395 to SK-OV-3 and SUM149 cells. Cells were incubated with 18F-NOTA-ZHER2:2395 in presence of unlabeled NOTA-ZHER2:2395, which was preincubated for 15 min at room temperature without acetonitrile, at 90°C in 25% acetonitrile (MeCN), or in presence of trastuzumab. RT = room temperature.

Scatchard analysis revealed that the Kd of 18F-NOTA-ZHER2:2395 was 6.5 ± 1.2 nM, and the number of binding sites on SK-OV-3 cells was 1.3 × 106 ± 0.4 × 106 per cell. The binding curves of the IC50 determination are shown in Figure 2. The IC50 of 19F-NOTA-ZHER2:2395 was 5.0 ± 0.1 nM, which was similar to 69Ga- and 115In-NOTA-ZHER2:2395 (6.3 ± 0.1 nM and 5.1 ± 0.04 nM, respectively).

FIGURE 2.
FIGURE 2.

Competitive binding curves of IC50 determination of 19F-, 69F-, and 115In-NOTA-ZHER2:2395 on SK-OV-3 cells. 111In-NOTA-ZHER2:2395 was used as radioactive tracer.

Stability studies showed that 18F-NOTA-ZHER2:2395 did not release Al18F after incubation in human or mouse serum at 37°C for 4 h, indicating excellent stability.

Biodistribution Studies

The results of the biodistribution studies are summarized in Figure 3. Mean tumor weight was 112 ± 54 mg. Tumor uptake at 1 h after injection of 18F-NOTA-ZHER2:2395 was 4.4% ± 0.8 %ID/g, compared with 5.6% ± 1.6 %ID/g and 7.1% ± 1.4 %ID/g for 68Ga- and 111In-NOTA-ZHER2:2395, respectively. Coinjection of an excess of unlabeled ZHER2:2395 resulted in a significant (P < 0.05) reduction in tumor uptake to 2.9% ± 0.3 %ID/g, 1.7% ± 0.5 %ID/g, and 3.0% ± 0.5 %ID/g for 18F-, 68Ga-, and 111In-NOTA-ZHER2:2395, respectively. Uptake in other organs was not affected by the coinjection of an excess of unlabeled ZHER2:2395. All radiolabeled Affibody molecules showed rapid clearance from the blood and normal organs, resulting in tumor-to-blood ratios of 7.4 ± 1.8 and 8.0 ± 1.3 for 18F- and 68Ga-NOTA-ZHER2:2395, respectively (Fig. 4). In contrast, 111In-NOTA-ZHER2:2395 cleared from the blood more slowly, resulting in a significantly lower tumor-to-blood ratio of 4.8 ± 1.3 (P < 0.05). 18F-NOTA-ZHER2:2395 showed good tumor retention: 4 h after injection, tumor uptake was 4.9% ± 0.7 %ID/g, which was comparable to the uptake at 1 h after injection. Tumor uptake of 111In-NOTA-ZHER2:2395 was 7.7% ± 2.6 %ID/g at 4 h after injection. Because of faster blood clearance, the tumor-to-blood ratio of 18F-NOTA-ZHER2:2395 was significantly higher than that of 111In-NOTA-ZHER2:2395 (145 ± 24 vs. 42 ± 13). All 3 radiolabeled Affibody molecules were excreted and reabsorbed by the kidneys, resulting in high kidney accumulation. The bone uptake was low, indicating stable complexation of 18F by the NOTA chelator.

FIGURE 3.
FIGURE 3.

Biodistribution of 18F-NOTA-ZHER2:2395, 68Ga-NOTA-ZHER2:239, and 111In-NOTA-ZHER2:2395 in mice bearing subcutaneous SK-OV-3 xenografts at 1 h (A) and 4 h (B) after injection.

FIGURE 4.
FIGURE 4.

Tumor-to-blood ratios of 18F-NOTA-ZHER2:2395, 68Ga-NOTA-ZHER2:2395, and 111In-NOTA-ZHER2:2395 in mice bearing subcutaneous SK-OV-3 xenografts.

PET/CT and SPECT/CT

PET/CT and SPECT/CT images acquired at 1 and 4 h after injection are shown in Figure 5. 18F-NOTA-ZHER2:2395 clearly visualized HER2-expressing SK-OV-3 xenografts, with good contrast to normal tissue. 68Ga- and 111In-NOTA-ZHER2:2395 also visualized SK-OV-3 xenografts with PET/CT and SPECT/CT, respectively. High accumulation of radiolabeled Affibody molecules in the kidneys was observed, with activity mainly localized in the renal cortex. Tumor-to-liver ratios were derived from the PET and SPECT images and ranged between 4.9 and 7.1, 1.6 and 5.3, and 2.6 and 5.8 for 18F-, 68Ga-, and 111In-NOTA-ZHER2:2395, respectively. These ratios correlated significantly with the tumor-to-liver ratio measured in the biodistribution study (r = 0.87, P = 0.023).

FIGURE 5.
FIGURE 5.

PET/CT and SPECT/CT images of mice bearing subcutaneous SK-OV-3 xenografts after injection of radiolabeled NOTA-ZHER2:2395.

DISCUSSION

The preclinical studies described in this paper show that 18F-NOTA-ZHER2:2395 is a promising radiotracer for HER2-expressing xenografts. This is the first time, to our knowledge, that Affibody molecules were radiolabeled with 18F, based on the complexation of Al18F by NOTA, which is a fast (30 min) labeling reaction. 18F-NOTA-ZHER2:2395 has a high affinity for HER2 (Kd = 6.5 nM) and showed efficient and specific accumulation in HER2-expressing tumors. PET images revealed high tumor–to–normal tissue contrast.

The in vitro affinity of cold labeled NOTA- ZHER2:2395 and the in vivo biodistribution of 18F-NOTA-ZHER2:2395 were compared with 68Ga- and 111In-labeled NOTA-ZHER2:2395. In vitro, all 3 cold labeled compounds showed similar IC50 in the nanomolar range, indicating that the affinity was not affected by the radionuclide that was chelated by NOTA. The comparative biodistribution study showed HER2-specific accumulation in the tumor of all radiolabeled Affibody molecules, with the highest tumor uptake for 111In-NOTA-ZHER2:2395. However, 18F- and 68Ga-labeled Affibody molecules cleared more rapidly from the blood, resulting in higher tumor-to-blood ratios than 111In-labeled Affibody molecules. Radiolabeled Affibody molecules showed good tumor retention, tumor uptake remained high 4 h after injection, and tumor-to-blood ratios increased rapidly because of the ongoing clearance from the blood. Both PET/CT and SPECT/CT images clearly visualized HER2-expressing xenografts, with high contrast to normal tissues. Radiolabeled Affibody molecules were reabsorbed by the kidneys, resulting in high kidney uptake, which was mainly localized in the cortex. PET/CT did not reveal any bone uptake, indicating that the 18F was stably complexed by NOTA.

Overall, all 3 radiolabeled Affibody molecules were able to target and visualize HER2-expressing tumors. However, the use of positron-emitting radionuclides such as 18F and 68Ga is preferred over 111In because of the higher sensitivity and better quantification of PET than of SPECT. Moreover, clinical PET/CT cameras have a better resolution than SPECT. An advantage of 18F over 68Ga is the longer half-life (110 vs. 68 min). Therefore, 18F-NOTA-ZHER2:2395 allows imaging at several hours after injection, which is preferred because of the higher tumor–to–normal tissue ratios. Moreover, imaging at several hours after injection is practical in the clinical situation. Furthermore, the positron range of 18F (2.3 mm in water) is shorter than that of 68Ga (8.9 mm in water), which can improve image quality, especially in the preclinical setting (28).

Several other approaches for imaging of HER2 expression have been described. ZHER2:342 or its derivates have been radiolabeled with different radionuclides and showed specific targeting of HER2-expressing xenografts (17). Kramer-Marek et al. have conjugated ZHER2:342 with 18F-FBEM (22). 18F-FBEM-ZHER2:342, compared with our 18F-labeled Affibody molecule, was able to bind HER2 with good affinity and showed comparable uptake in SK-OV-3 xenografts. The specific activities of 18F-NOTA-ZHER2:2395 and 18F-FBEM-ZHER2:342 were comparable at the end of synthesis. However, the preparation of 18F-FBEM-ZHER2:342 is based on the synthesis of a fluorinated synthon, which is subsequently reacted with the Affibody molecule. This process requires a long synthesis time of approximately 2 h. We were able to radiolabel Affibody molecules with 18F within 30 min using a 2-step, 1-pot reaction. In addition, in future studies the specific activity of 18F-NOTA-ZHER2:2395 may be improved by using different chelators and solvents (29,30).

Besides radiolabeled Affibody molecules, other radiotracers have been developed. For example, radiolabeled trastuzumab and pertuzumab have been used for SPECT and PET of HER2 expression (13,14,16,31). However, these large proteins are characterized by slow tumor penetration and clearance from the circulation. A direct comparison of 124I-labeled ZHER2:342 and trastuzumab showed that Affibody molecules have superior imaging properties (32). Radiolabeled trastuzumab resulted in higher absolute tumor uptake, but tumor–to–normal tissue ratios were considerably higher for Affibody molecules. Moreover, high-contrast images with Affibody molecules could be obtained within 6 h after injection, in contrast to radiolabeled trastuzumab, for which the highest tumor–to–normal tissue ratios were obtained several days after injection. A disadvantage of trastuzumab-based tracers is that they cannot be used to monitor changes in HER2 expression during trastuzumab treatment, because of competition of the therapeutic antibody with the tracer molecule for the same epitope on HER2. In contrast, in agreement with previously published data (22), trastuzumab did not interfere with the binding of NOTA-ZHER2:2395 to HER2, because of the binding of trastuzumab to different domains. Therefore, Affibody molecules can be used to study HER2 expression during trastuzumab treatment, which offers exciting opportunities to get more insight into the dynamics of the HER2 receptor during trastuzumab (combination) therapy.

Translation of HER2 imaging from the preclinical setting to patients may improve patient selection for HER2-targeted therapy, because in vivo imaging avoids misinterpretation due to intratumoral and interlesional tumor heterogeneity. Furthermore, HER2 imaging can be used to monitor expression during the course of disease or during trastuzumab treatment, without the need of repetitive invasive biopsies. Recently, a clinical pilot study showed that it is feasible to visualize HER2-expressing tumors in patients with metastatic breast cancer, using radiolabeled Affibody molecules (20).

Despite the advantages of imaging HER2 expression with Affibody molecules, a few potential limitations have to be considered. First, HER2-overexpressing tumors can release HER2 in the circulation. Circulating HER2 can bind radiolabeled Affibody molecules and may therefore interfere with tumor targeting. However, a first pilot study showed that HER2-overexpressing tumors could be visualized, even in the presence of shed HER2 (20). A second potential limitation is the reabsorption of Affibody molecules by the kidneys, resulting in high kidney accumulation. This may interfere with the visualization of tumor lesions located in the kidney—not a major problem as far as breast and gastric cancers are concerned, because they are not likely to metastasize to the kidneys. Still, there are indications, such as the visualization of lower vertebral lesions, for which HER2 imaging with 18F-NOTA-ZHER2:2395 could be improved by developing strategies to minimize the reabsorption of Affibody molecules by the kidney. Potential ways of reducing kidney uptake are the use of positively charged amino acids, gelofusin, or albumin fragments (33–35).

CONCLUSION

This study showed that 18F-NOTA-ZHER2:2395 is a promising new imaging agent for HER2 expression in tumors. Affibody molecules were successfully labeled with 18F within 30 min, based on the complexation of Al18F by NOTA. Further research is needed to determine whether this technique can be used for patient selection for HER2-targeted therapy.

DISCLOSURE STATEMENT

The costs of publication of this article were defrayed in part by the payment of page charges. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

We thank Bianca Lemmers–de Weem, Kitty Lemmens–Hermans, and Henk Arnts (Central Animal Facility, Radboud University Nijmegen) for technical assistance. This study was financially supported by a personal research grant from the Dutch Research Council (016.096.010) and financial support by the Swedish Cancer Society (Cancerfonden) and the Swedish Research Council (Vetenskapsrådet). The patented AlF labeling method was generously made available by Immunomedics, Inc. (Morris Plains, NJ). No other potential conflict of interest relevant to this article was reported.

Footnotes

  • Published online Dec. 15, 2011.

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  • Received for publication May 13, 2011.
  • Accepted for publication August 30, 2011.
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