Original article

Preclinical evaluation of 89Zr-labeled human antitransferrin receptor monoclonal antibody as a PET probe using a pancreatic cancer mouse model Aya Sugyoa, Atsushi B. Tsujia, Hitomi Sudoa, Kotaro Nagatsub, Mitsuru Koizumic, Yoshinori Ukaid, Gene Kurosawae, Ming-Rong Zhangb, Yoshikazu Kurosawae and Tsuneo Sagaa Objective Pancreatic cancer is aggressive and its prognosis remains poor; thus, effective therapy is urgently needed. Transferrin receptor (TfR) is highly expressed in pancreatic cancer and is considered to be a good candidate for molecular-targeted therapy. We radiolabeled and evaluated fully human anti-TfR monoclonal antibodies as a new PET probe for evaluating the biodistribution of the anti-TfR antibody in pancreatic cancer. Materials and methods TfR expression was evaluated in four human pancreatic cancer (MIAPaCa-2, PANC-1, BxPC-3, and AsPC-1) and murine A4 cell lines. The binding of 125I-labeled anti-TfR antibodies (TSP-A01, TSP-A02, TSP-A03, and TSP-A04) to MIAPaCa-2 cells was compared. 125 I-labeled, 67Ga-labeled, and 89Zr-labeled TSP-A01 were evaluated by cell binding, competitive inhibition, and internalization assays. Biodistribution studies of 125 I-labeled and 89Zr-labeled TSP-A01 were conducted in mice bearing MIAPaCa-2 and A4 tumors. PET imaging with [89Zr]TSP-A01 was carried out. Results MIAPaCa-2 cells showed the highest TfR expression in vitro and in vivo, whereas A4 cells showed no expression. Of the four antibodies, [125I]TSP-A01 showed the highest binding to MIAPaCa-2 cells, but not to A4 cells. The dissociation constant of TSP-A01 was 0.29 nmol/l.

Introduction Pancreatic cancer is a commonly diagnosed cancer and the seventh leading cause of cancer-related death worldwide, accounting for 337 872 of estimated new cancer cases and 330 372 of estimated cancer-related deaths (GLOBOCAN 2012, http://globocan.iarc.fr/). As the symptoms of pancreatic cancer do not appear during its early stage and cancer cells would have often infiltrated the surrounding tissue and organs at the time of diagnosis [1–3], its prognosis is very poor. The 5-year survival rate is less than 5%, the lowest survival rate among major cancers [2]. Therefore, additional effective anticancer therapy is necessary to augment and/or complement the present treatment strategies such as surgery Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.nuclearmedicinecomm.com). 0143-3636 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

Uptake of radiolabeled TSP-A01, especially [89Zr]TSP-A01, was significantly higher in MIAPaCa-2 tumors than in A4 tumors. PET with [89Zr]TSP-A01 clearly visualized MIAPaCa-2 xenografts but not A4 xenografts. Conclusion [89Zr]TSP-A01 is a promising PET probe for evaluating the accumulation of anti-TfR antibody in pancreatic cancer and has the potential to facilitate the selection of appropriate patients who would benefit from anti-TfR antibody therapy. Nucl Med Commun 36:286–294 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Nuclear Medicine Communications 2015, 36:286–294 Keywords: immuno-PET, mouse model, noninvasive imaging, pancreatic cancer, transferrin receptor a Diagnostic Imaging Program, bMolecular Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, cDepartment of Nuclear Medicine, Cancer Institute Hospital, dResearch and Development Division, Perseus Proteomics Inc., Tokyo and eInnovation Center for Advanced Medicine, School of Medicine, Fujita Health University, Aichi, Japan

Correspondence to Atsushi B. Tsuji, PhD, Diagnostic Imaging Program, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Tel: + 81 43 382 3704; fax: + 81 43 206 0818; e-mail: [email protected] Received 8 August 2014 Revised 14 October 2014 Accepted 21 October 2014

and chemo/radiotherapy, especially for patients with advanced/metastatic cancer. Transferrin receptor (TfR), a type II transmembrane glycoprotein found as a homodimer (180 kDa) on the surface of cells, is involved in iron uptake through its interaction with transferrin and the regulation of cell growth [4,5]. Although TfR is expressed at low levels on normal cells, it is expressed at higher levels on cells with a high proliferation rate, such as cancer cells [6–9]. TfR is an attractive molecule for targeted cancer therapy as its expression is upregulated on the cell surface of many cancer types, including pancreatic cancer [8,10,11]. We recently isolated four novel fully human monoclonal antibodies (TSP-A01, TSP-A02, TSP-A03, and TSPA04) against TfR from a large-scale human antibody library constructed using a phage-display system with living pancreatic cancer cells [12,13]. Three of these DOI: 10.1097/MNM.0000000000000245

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Transferrin receptor-targeted immuno-PET Sugyo et al. 287

antibodies induce antibody-dependent cell-mediated cytotoxicity and inhibit cell proliferation of pancreatic cancer cells [12]; they are candidates for anti-TfR therapy of TfR-expressing cancer. Although ∼ 80% of pancreatic cancer shows high TfR expression, some show a low expression [9]. For effective TfR-targeted therapy using an anti-TfR antibody, a noninvasive imaging method for evaluating TfR expression and accumulation of the antibody in each cancer would provide useful information for the selection of appropriate patients who would benefit from the therapy. Although 67Ga-citrate and radiolabeled transferrin are known noninvasive imaging probes for the evaluation of TfR expression [14–18], as the pharmacokinetics of these probes are vastly different than those of antibodies, a radiolabeled antibody with high specificity and affinity to target TfR can be valuable. To our knowledge, there are no reports of imaging with radiolabeled anti-TfR antibody to date. As PET has a high sensitivity and quantification ability, it is a suitable imaging modality for the above-mentioned purpose. In the present study, we radiolabeled anti-TfR antibodies and evaluated the in-vitro and in-vivo properties as a PET probe for evaluating TfR expression and antibody accumulation using a pancreatic cancer xenograft mouse model.

Materials and methods Cell

Human pancreatic cancer cell lines (MIAPaCa-2, PANC-1, BxPC-3, and AsPC-1) were obtained from the American Type Culture Collection (Manassas, Virginia, USA). A4 cells were established from mouse 3T3 cells transfected with a human HER2-expression vector [19]. MIAPaCa-2 and PANC-1 were maintained in D-MEM medium (Sigma, St Louis, Missouri, USA) supplemented with 5% or 10% fetal bovine serum (Sigma) in a humidified incubator maintained at 37°C with 5% CO2. BxPC-3, AsPC-1, and A4 were maintained in RPMI1640 medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum. Subcutaneous tumor mouse model

The animal experimental protocol was approved by the Animal Care and Use Committee of the National Institute of Radiological Sciences, and all animal experiments were conducted in accordance with the institutional guidelines on animal care and handling. BALB/c-nu/nu male mice (5 weeks old; Japan SLC, Shizuoka, Japan) were maintained under specific pathogen-free conditions. To induce a subcutaneous tumor model, mice were inoculated subcutaneously with MIAPaCa-2 (4 × 106) and A4 (1 × 106) cells in the left and right thighs, respectively, under isoflurane anesthesia. As A4 cells grow faster than MIAPaCa-2 cells in mice, the inoculation day of each cell line was adjusted to ensure that tumor xenografts were of almost equal size at the time of the experiments (approximately a 30-day interval between the two inoculations).

TfR protein expression analysis

Western blotting and immunofluorescence staining were conducted as previously described [20,21]. Briefly, for western blotting, cell lysates were resolved by SDS polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Hybond-P; GE Healthcare, Little Chalfont, UK), and reacted with a murine anti-TfR monoclonal antibody (1E6; Abnova, Taipei, Taiwan) overnight at 4°C. The primary antibody was reacted with a horseradish peroxidase-linked antimouse antibody (GE Healthcare) and the membrane was visualized using an ECL Plus kit (GE Healthcare). After images were captured using a LAS-3000 imaging system (Fuji Film, Tokyo, Japan), antibodies on the polyvinylidene difluoride membrane were removed with stripping buffer and then the membrane was used for detecting β-actin expression as a loading control using an anti-β-actin antibody (Sigma). The intensity of staining was measured by Image J (National Institutes of Health, Bethesda, Maryland, USA). For immunofluorescence staining of cultured cells, cells were grown on glass coverslips and fixed in cold methanol for 5 min. Nonspecific binding of the antibody was blocked by applying Block Ace reagent (Dainippon Pharmaceutical, Osaka, Japan) with 10% goat serum for 30 min. Cells were incubated with TSP-A01 as a primary antibody overnight at 4°C. A secondary anti-human antibody conjugated with Cy3 (Jackson Immuno Research Laboratories, West Grove, Pennsylvania, USA) was applied for 30 min at room temperature. Nuclei were stained with DAPI in mounting medium (Vector Laboratories, Burlingame, California, USA). The images were obtained with an exposure time of 2/3 s for detecting TfR using a fluorescence microscope (Olympus, Tokyo, Japan). For immunofluorescence staining of xenograft tumors (MIAPaCa-2 and A4), excised tumors were quickly frozen in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan), from which 30-μm-thick sections were obtained. Dried sections were fixed with 4% paraformaldehyde. Nonspecific binding was blocked by Block Ace reagent with 10% goat serum. The specimens were incubated overnight at 4°C with TSP-A01. A secondary anti-human antibody conjugated with Alexa Fluor 488 (Life Technologies Corporation, Carlsbad, California, USA) was applied for 30 min at room temperature. Nuclei were stained with DAPI in mounting medium. The images were obtained with an exposure time of 2 s for detecting TfR using a fluorescence microscope. Radiolabeling of antibody

Four human anti-TfR antibodies (TSP-A01, TSP-A02, TSP-A03, and TSP-A04) [12] were directly radioiodinated using the chloramine-T method as previously described [22]. Briefly, antibodies (25 μg) in 0.3 mol/l phosphate buffer (pH 7.5) and 18.5 MBq of Na125I (PerkinElmer, Waltham, Massachusetts, USA) were mixed

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and then chloramine-T (Wako Pure Chemical Industries) was added. Radiolabeled antibodies were purified using a Sephadex G-50 (GE Healthcare) spin column (700 × g for 2 min once or twice) after 5 min incubation at room temperature. For 67Ga and 89Zr labeling, TSP-A01 was conjugated with 1-(4-isothiocyanatophenyl)-3-[6,17-dihyroxy-7,10,18,21tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazahepta eicosane]thiourea (desferrioxamine; Macrocyclics, Dallas, Texas, USA) at a desferrioxamine to antibody mixing molar ratio of 3 : 1 as previously described [22,23]. The conjugation ratio of desferrioxamine to antibody was estimated from the 67Ga-desferrioxamine-conjugated antibody to 67 Ga-desferrioxamine ratio determined by size exclusion chromatography with a PD10 column (GE Healthcare). For purification, the desferrioxamine–antibody conjugate was applied to a Sephadex G-50 spin column (700 × g for 2 min once or twice). For 67Ga labeling, the desferrioxamineconjugated antibody (45 μg in 10 μl PBS) was incubated with 600 kBq of 67Ga-chloride (70–80 MBq/ml; Nihon Medi-Physics, Tokyo, Japan) for 1 h at room temperature, and radiolabeled antibodies were purified using a Sephadex G-50 spin column (700 × g for 2 min once or twice). The radiochemical purity was then determined with PD10 column chromatography. For 89Zr labeling, 89Zr was produced by a (p,n) reaction on 89Y (New Metals and Chemicals, Waltham Abbey, UK) in an alumina ceramic vessel (Kyocera, Kyoto, Japan) by vertical irradiation using the NIRS AVF-930 cyclotron and purified with a hydroxamate column using an automated recovery and purification apparatus as previously described [24]. The desferrioxamine-conjugated antibody (100 μg in 20 μl PBS) was incubated with 2.8–5.2 MBq of 89Zr-oxalate (3.7–5.6 GBq/ml in 1 mol/l oxalate, pH 7–8) for 1 h at room temperature, and radiolabeled antibodies were purified using a Sephadex G-50 spin column (700 × g for 2 min once or twice). The radiochemical purity was determined by thin-layer chromatography using 50 mmol/l diethylenetriaminepentaacetic acid (pH 7; Sigma) as the mobile phase.

bound to the cells was counted. Data were analyzed, and the dissociation constant (Kd) was estimated using GraphPad Prism software (Graphpad Software, La Jolla, California, USA). In internalization assays, MIAPaCa-2 cells were preincubated in culture medium with 125 I-labeled and 67Ga-labeled antibody TSP-A01 on ice. After washing, collected cells were further cultured at 37° C or on ice in fresh medium without radiolabeled antibodies. At 0, 1, 3, 6, and 21 h after incubation, the supernatant and the cells were separated by centrifugation. For 125I-labeled antibody, trichloroacetic acid was added to the supernatant on ice and then the nonprotein-bound fraction (supernatant) and protein-bound fraction (pellet) were separated by centrifugation. For 125 I-labeled and 67Ga-labeled antibody, the cells, separated by centrifugation as mentioned above, were washed with acidic buffer and then separated into the membranebound fraction (supernatant) and internalized fraction (pellet) by centrifugation. Biodistribution

When subcutaneous tumors reached a diameter of ∼ 10 mm, the mice were intravenously injected with a mixture of [89Zr]TSP-A01 and [125I]TSP-A01 in PBS (37 kBq each). The specific activities of [89Zr]TSP-A01 and [125I]TSP-A01 were 23 and 233 kBq/μg, respectively, before protein dose adjustment. The total injected protein dose was adjusted to 20 μg per mouse by adding the unlabeled antibody TSP-A01. At 1, 2, 4, and 6 days after injection of radiolabeled antibodies, five mice at each time point were euthanized and blood was obtained from the heart. Tumors and major organs were removed and weighed, and radioactivity counts were measured using a gamma counter (PerkinElmer). The data were expressed as the percentage of injected dose per gram of tissue (%ID/g) normalized to a mouse of 20 g body weight. Tumor uptake data were analyzed by two-way repeatedmeasures analysis of variance, followed by the Student t-test.

In-vitro assay

Cell binding, competitive inhibition, and internalization assays were conducted as previously described [22]. Briefly, in cell binding assays, MIAPaCa-2 or A4 cells in PBS with 1% BSA (Sigma) were incubated with 125 I-labeled antibodies (TSP-A01, TSP-A02, TSP-A03, and TSP-A04), and 67Ga-labeled and 89Zr-labeled TSPA01 on ice. After washing, radioactivity bound to the cells was measured. The immunoreactivity of radiolabeled antibodies was estimated according to the method of Lindmo et al. [25]. Cell binding data of 125I-labeled antibodies were analyzed by analysis of variance, followed by the Student–Newman–Keuls multiple comparison test. In the competitive inhibition assay, 125 I-labeled TSP-A01 was incubated with MIAPaCa-2 cells in the presence of varying concentrations of the unlabeled TSP-A01 on ice. After washing, radioactivity

PET imaging

Mice (n = 2) bearing subcutaneous tumors (MIAPaCa-2 and A4) were injected with ∼ 7.4 MBq of [89Zr]TSP-A01 into a tail vein. The injection protein dose was adjusted to 200 μg per mouse by adding the unlabeled antibody. PET data acquisition was conducted at 15 min (for one mouse) and at 1, 2, 4, and 6 days (for two mice) after administration for 10–20 min using a small-animal PET system (Inveon; Siemens Medical Solutions, Malvern, Pennsylvania, USA) under isoflurane anesthesia. Body temperature was maintained between 36°C and 37°C by means of a lamp and heating pad during the scan. Images were reconstructed using a 3D maximum a posteriori (18 iterations with 16 subsets, β = 0.2) without attenuation correction. The regions of interest were manually drawn over tumors and thigh muscles to calculate the tumor-to-

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Transferrin receptor-targeted immuno-PET Sugyo et al. 289

MIAPaCa-2

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muscle ratios. After imaging, we conducted biodistribution experiments to confirm PET results.

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Results TfR protein expression in pancreatic cancer cells and xenografts

We determined TfR protein expression in the four human pancreatic cancer cell lines (MIAPaCa-2, PANC-1, BxPC-3, and AsPC-1) and the mouse A4 cell line using western blotting and immunofluorescence staining. MIAPaCa-2 showed the highest expression, followed by PANC-1 and AsPC-1 as determined by western blot analysis (Fig. 1a). No expression was detected in BxPC-3 under this condition (Fig. 1a). MIAPaCa-2 also showed the highest expression as determined by immunofluorescence staining. The expression in PANC-1, which was the second highest in western blotting, was lower than that in AsPC-1 (Fig. 1b). Although TfR expression in BxPC-3 was not detected by western blotting, it was detected by immunofluorescence staining (Fig. 1b). TfR expression was not detected in A4 cells by western blotting or immunofluorescence staining. In TfR immunofluorescence staining of MIAPaCa-2 and A4 subcutaneous tumors, whereas the cancer cells of MIAPaCa-2 tumors were intensely stained (Fig. 2, right panel), no staining was detected in A4 tumors (Fig. 2, left panel).

TfR β-Actin Ratio (b)

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Radiolabeling

For 125I labeling of four human anti-TfR monoclonal antibodies (TSP-A01, TSP-A02, TSP-A03, and TSPA04), the radiochemical yields were 33–73%, the radiochemical purities were greater than 96%, and the specific activities were as follows: TSP-A01, 233–385 kBq/μg; TSP-A02, 391 and 423 kBq/μg; TSP-A03, 522 and 485 kBq/μg; and TSP-A04, 430 and 412 kBq/μg. For 67Ga and 89Zr labeling of TSP-A01, it was conjugated with desferrioxamine, and the estimated conjugation ratios ranged from 1.0 to 1.3. The radiochemical yields of 67 Ga-labeled and 89Zr-labeled TSP-A01 were 30–36 and 26–78%, the radiochemical purities were greater than 96 and 92%, and the specific activities were 5–10 and 17–58 kBq/μg, respectively. In-vitro characterization of radiolabeled anti-TfR antibodies

In the cell binding assays of the four 125I-labeled antibodies using MIAPaCa-2 cells, [125I]TSP-A01 bound most highly to MIAPaCa-2 cells; there were significant differences in binding to MIAPaCa-2 cells between [125I] TSP-A01 and the other three antibodies at two cell concentrations tested, except for that between [125I] TSP-A01 and [125I]TSP-A02 at 1.25 × 106 cells (Fig. 3a). All four 125I-labeled antibodies showed negligible binding to A4 cells (Fig. 3b). In the cell binding assays of labeled TSP-A01 with increasing concentrations of MIAPaCa-2 cells, the immunoreactive fractions of [125I]

AsPC-1

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TfR protein expression analysis of pancreatic cancer cell lines (MIAPaCa-2, PANC-1, BxPC-3, and AsPC-1) and a mouse cell line A4. (a) Western blotting analysis of whole-cell lysate using anti-TfR antibody (upper panel) and β-actin (lower panel) of the same PVDF membrane as a loading control. (b) TfR protein expression was determined by immunofluorescence staining with anti-TfR antibody (red). DAPI-stained nuclei (blue). PVDF, polyvinylidene difluoride; TfR, transferrin receptor.

TSP-A01, [67Ga]TSP-A01, and [89Zr]TSP-A01 were estimated to be 0.80, 1.00, and 0.80, respectively (Fig. 4a). The Kd of TSP-A01 was estimated to be 0.29 nmol/l by competitive inhibition assay (Fig. 4b). We examined the temporal change in radioactivity localization of 67Ga-labeled and 125I-labeled TSP-A01 in MIAPaCa-2 cells. The cell membrane-bound fraction rapidly decreased, whereas the internalized and proteinbound fractions in the culture medium rapidly increased for both radiolabeled antibodies (Fig. 4c and d). Internalization seems to have been mostly completed within the first hour of incubation at 37°C, and the change after 1 h was minimal (Fig. 4c and d). Approximately 30% of 67Ga-labeled and 125I-labeled TSP-A01 was internalized after incubation (Fig. 4c and d). When cells were incubated on ice, no fraction changed for at least 3 h (data not shown).

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In-vivo biodistribution of radiolabeled TSP-A01

Biodistribution experiments using 89Zr-labeled and 125 I-labeled TSP-A01 were conducted in nude mice bearing MIAPaCa-2 and A4 xenograft tumors. [89Zr] TSP-A01 uptake in MIAPaCa-2 tumors at day 1 was 9.6 ± 1.5%ID/g, with a peak value of 12.5 ± 2.3%ID/g at day 2 (Fig. 5a). Although the uptake gradually decreased, it was still 11.0 ± 3.1%ID/g at day 6 (Fig. 5a). [89Zr] TSP-A01 uptake in A4 tumors was 7.7 ± 1.2%ID/g at day 1 and decreased with time (Fig. 5a). There were significant differences in [89Zr]TSP-A01 tumor uptake between MIAPaCa-2 and A4 at all time points (P < 0.05). The MIAPaCa-2 to A4 ratio of [89Zr]TSP-A01 was 1.3 ± 0.2 at day 1 and increased with time (3.2 ± 0.4 at day 6). [89Zr]TSP-A01 uptake in normal major organs, including the pancreas, was low and decreased over time except for the liver and the spleen (Fig. 5a). The MIAPaCa-2 tumor to pancreas ratio of [89Zr]TSP-A01 was 6.8 ± 0.9 at day 1, and increased to 13.4 ± 2.4 at day 6. Although [125I]TSP-A01 also accumulated in MIAPaCa-2 tumors, similar to [89Zr]TSP-A01, the peak of tumor uptake was significantly lower than that of [89Zr] TSP-A01 (P < 0.05), and its clearance from tumors was faster than that of [89Zr]TSP-A01 (Fig. 5a and b). The uptake of [125I]TSP-A01 in all normal major organs decreased over time (Fig. 5b). PET imaging of tumor-bearing mice with [89Zr]TSP-A01

To confirm the results of the biodistribution studies, we conducted PET imaging with [89Zr]TSP-A01. Serial PET images in a subcutaneous tumor mouse model bearing MIAPaCa-2 and A4 tumors were obtained at 15 min, and at days 1, 2, 4, and 6 after injection. At

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TfR protein expression analysis of xenografted tumors. TfR expression (green) in MIAPaCa-2 (right) and A4 (left) xenografted tumors determined by immunofluorescence staining of frozen sections (10 μm thick). DAPI-stained nuclei (blue). TfR, transferrin receptor.

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Cell binding assay for 125I-labeled anti-TfR antibodies. Cell binding of 125 I-labeled TSP-A01 (black bars), TSP-A02 (gray bars), TSP-A03 (white bars), and TSP-A04 (dotted bars) to MIAPaCa-2 (a) or A4 (b) cells is shown. Data are expressed as mean ± SD (n = 4). *P < 0.05, **P < 0.01 versus [125I]TSP-A01. NS; not significant; TfR, transferrin receptor.

15 min, radioactivity in the blood pool was very high, whereas that in MIAPaCa-2 and A4 tumors was still low, with no differences between the two (Fig. 6). On day 1, the uptake in the MIAPaCa-2 tumor was markedly increased and higher than that in the A4 tumor, and the background activity started to decrease. On day 2 and later, the high uptake in the MIAPaCa-2 tumor persisted, whereas the background activity continued to decrease, resulting in an increase in the contrast of MIAPaCa-2 tumor over time (Fig. 6). These findings were nearly consistent with those of another mouse (data not shown). The MIAPaCa-2-to-muscle ratios of two mice were 4.6 and 3.7 on day 1 and increased thereafter to 10.7 and 8.8 on day 6. The A4-to-muscle ratios were 2.2 and 2.4 on

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Transferrin receptor-targeted immuno-PET Sugyo et al. 291

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In-vitro assay of anti-TfR antibody TSP-A01 using MIAPaCa-2 cells. (a) Cell binding assay for [89Zr]TSP-A01 (white triangles), [67Ga]TSP-A01 (white circles), and [125I]TSP-A01 (black circles). (b) Competitive inhibition assay using TSP-A01 labeled with and without 125I. Internalization assay for [67Ga]TSP-A01 (c) and [125I]TSP-A01 (d). Changes in percentage of total radioactivity for each fraction are plotted against incubation time at 37°C. These assays were conducted in duplicate. Data represent each replicate. TfR, transferrin receptor.

day 1 and almost unchanged thereafter (2.1 and 2.6 on day 6). Biodistribution data for mice after PET were relatively consistent with the PET images on day 6 (Supplementary Fig. 1, Supplemental digital content, http://links.lww.com/NMC/A37).

Discussion The TfR protein expression of four pancreatic cancer cell lines (MIAPaCa-2, PANC-1, BxPC-3, and AsPC-1) was evaluated by western blotting and immunofluorescence staining to determine a suitable pancreatic cancer cell line to assess radiolabeled anti-TfR antibodies as a PET probe. The MIAPaCa-2 cell line showed the highest expression by both western blotting and immunofluorescence staining. There were some discrepancies in the TfR expression of the other three pancreatic cancer cell lines (PANC-1, BxPC-3, and AsPC-1) by western blotting and immunofluorescence staining. These differences may be caused by

the different antibodies. The commercially available antibody 1E6 for western blotting is not applicable to immunofluorescence staining; the human anti-TfR antibody TSP-A01 for immunofluorescence staining is not applicable to western blotting. TfR expression of a murine A4 cell line was additionally evaluated. The cell line showed no TfR expression as determined by western blotting and immunofluorescence staining. It was confirmed that MIAPaCa-2 cells formed subcutaneous tumors in nude mice and the tumors had high TfR expression, and A4 tumors showed no TfR protein expression as determined by immunofluorescence staining. Therefore, MIAPaCa-2 and A4 cells were selected as a positive control and a negative control, respectively, for the following evaluation. The cell binding potential of four anti-TfR antibodies (TSP-A01, TSP-A02, TSP-A03, and TSP-A04) after 125I

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292 Nuclear Medicine Communications 2015, Vol 36 No 3

Fig. 5

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Biodistribution experiments in nude mice bearing MIAPaCa-2 and A4 xenografts of radiolabeled anti-TfR antibody TSP-A01. Samples were collected and weighed, and radioactivity was measured at days 1 (black bars), 2 (white bars), 4 (gray bars), and 6 (dotted bars) after intravenous injection of 37 kBq each of [89Zr]TSP-A01 (a) and [125I]TSP-A01 (b). Data are expressed as mean ± SD (n = 5). TfR, transferrin receptor.

labeling was evaluated to select a suitable antibody as a PET probe candidate. Three of four antibodies (TSPA01, TSP-A02, and TSP-A03) were highly bound to MIAPaCa-2 cells, but TSP-A04 binding was significantly lower than that of the other three. Although these three antibodies showed cytotoxic effects in vitro and in vivo, TSP-A04 did not [12]. The low binding potential may be a reason why TSP-A04 showed no cytotoxic effects. As TSP-A01 showed the highest binding and its cytotoxic effects were strong [12], TSP-A01 was adopted as the PET probe candidate in the present study. TSP-A01 was labeled with 125I, 67Ga, and 89Zr and the in-vitro properties were evaluated. The cell binding (125I-labeled, 67Ga-labeled, and 89Zr-labeled TSP-A01) and competitive inhibition ([125I]TSP-A01) assays revealed that TSP-A01 specifically bound to MIAPaCa-2 cells with high affinity but not to A4 cells. The

immunoreactive fractions of antibodies labeled with three radionuclides were more than 0.8, indicating that the loss of immunoreactivity by radiolabeling procedures was minimal. The internalization assay (125I-labeled and 67 Ga-labeled TSP-A01) showed that TfR underwent internalization after binding with the specific antibodies, suggesting that labeling with radiometals, such as 67Ga and 89Zr, would be suitable for imaging with this antibody compared with radioiodine. In the present study, 67 Ga was used in place of 89Zr for the internalization assay because of the limited availability of 89Zr. Our assumption is that the result using 68Ga-labeled antibody is considered to be consistent with that using 89 Zr-labeled antibody as the chelate desferrioxamine is common between the two radiometals and these radiometals with the desferrioxamine complex are known to be stable in vitro and in vivo [26–30]. Further, the biodistribution is reported to be similar between 68 Ga-labeled and 89Zr-labeled antibodies [28]. The limitation in the present study is that there is no direct evidence on the fate of the two radiometals after internalization; therefore, a further in-vitro internalization assay using 89Zr-labeled TSP-A01 is needed. From the biodistribution studies, although the uptake of [89Zr]TSP-A01 and [125I]TSP-A01 in MIAPaCa-2 tumors reached the peak at day 2 after injection, that of [89Zr] TSP-A01 at day 2 or later was significantly higher than that of [125I]TSP-A01. It suggests that 89Zr was retained inside the target cells after [89Zr]TSP-A01 internalization, whereas 125I was released from the target cells after [125I] TSP-A01 internalization. These results were consistent with previous studies with other internalizing antibodies [19–21,31]. Several studies have shown that antibodies after internalization are delivered to lysosome for degradation; as a result radiometal-labeled metabolites, such as 111 In-DTPA-lys, are retained in the lysosome [32,33], and radioiodinated catabolites, such as radioiodinated tyrosine and free radioiodine, are released from cells [34]. Uptake of [89Zr]TSP-A01 in normal major organs, except for the liver and spleen, was low and that in the pancreas was very low (< 2%ID/g). The biodistribution in normal organs was relatively consistent with previous reports for 89 Zr-labeled antibodies against other antigens [22,29,30, 35–37]. These results suggest that 89Zr has appropriate characteristics for labeling TSP-A01 as a PET probe to visualize TfR-expressing pancreatic cancer. To confirm the results of biodistribution studies, PET imaging with [89Zr]TSP-A01 was conducted in nude mice bearing MIAPaCa-2 and A4 xenografted tumors. Similar to the biodistribution study, the MIAPaCa-2 tumor was clearly visualized from day 2 to day 6, but the A4 tumor was not. [89Zr]TSP-A01 is a promising PET probe to detect TfR-expressing pancreatic cancer with high sensitivity, and to evaluate the accumulation of antiTfR antibody as a basis for selecting the appropriate patients who would benefit from anti-TfR antibody

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PET imaging with 89Zr-labeled anti-TfR antibody TSP-A01. Serial PET images (maximum intensity projection) of a nude mouse bearing MIAPaCa-2 (yellow arrowhead) and A4 (blue arrowhead) xenografted tumors at 15 min, and at days 1, 2, 4, and 6 after intravenous injection of 3.7 MBq [89Zr] TSP-A01. TfR, transferrin receptor.

therapy. TfR is highly expressed in many other types of cancers, including pancreatic cancer [8–11]. Moreover, the increased expression is reported to be associated with a poor prognosis in several cancers and considered to be caused by increased metastatic rates and refractoriness to treatment [8,11,38–40]. The present study showed that [89Zr]TSP-A01 uptake in major organs was low except in the liver and spleen. [89Zr]TSP-A01 therefore has the potential for detecting TfR-expressing tumors that developed in other organs and for assessing the metastatic potential and treatment resistance of these tumors. Our results in the mouse model may not fully predict the distribution in human patients because TSP-A01 does not bind to murine TfR. Scintigraphy with 67Ga-citrate in patients has shown that TfR expression is limited in normal organs except for the liver [15,17]. This suggests the possibility that [89Zr]TSP-A01 could highly accumulate in the liver of patients. Significant liver uptake could hinder detection of pancreatic cancer located close to the liver. Further clinical studies will be necessary to precisely evaluate normal organ uptake of [89Zr]TSP-A01 and to assess whether [89Zr]TSP-A01 can distinguish between pancreatic cancer and normal organs, especially the liver.

appropriate cancer patients who would benefit from TfRtargeted therapy.

Acknowledgements This work was supported in part by KAKENHI 25861140. The authors thank Yuriko Ogawa for technical assistance, Hisashi Suzuki and Katsuyuki Minegishi for 89 Zr production, Masayuki Hanyu for the hydroxamate column preparation, Hidekatsu Wakizaka for operation and quality control of the PET system, the staff in the Cyclotron Operation section for the NIRS AVF-930 cyclotron operation, and the staff in the Laboratory Animal Sciences section for animal management. Conflicts of interest

There are no conflicts of interest.

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Conclusion Four fully human anti-TfR monoclonal antibodies were evaluated, and TSP-A01 was selected as a PET probe candidate by in-vitro cell binding assay. [89Zr]TSP-A01 was highly accumulated in TfR-expressing tumors but not in non-TfR-expressing tumors. Our findings suggest that PET with [89Zr]TSP-A01 is a promising noninvasive imaging method for providing information on TfR expression and anti-TfR antibody accumulation in pancreatic cancer that could be useful in selecting

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Preclinical evaluation of ⁸⁹Zr-labeled human antitransferrin receptor monoclonal antibody as a PET probe using a pancreatic cancer mouse model.

Pancreatic cancer is aggressive and its prognosis remains poor; thus, effective therapy is urgently needed. Transferrin receptor (TfR) is highly expre...
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