64Cu-DOTA-Anti-CTLA-4 mAb Enabled PET Visualization of CTLA-4 on the T-Cell Infiltrating Tumor Tissues

Kei Higashikawa; Katsuharu Yagi; Keiko Watanabe; Shinichiro Kamino; Masashi Ueda; Makoto Hiromura; Shuichi Enomoto

doi:10.1371/journal.pone.0109866

Abstract

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) targeted therapy by anti-CTLA-4 monoclonal antibody (mAb) is highly effective in cancer patients. However, it is extremely expensive and potentially produces autoimmune-related adverse effects. Therefore, the development of a method to evaluate CTLA-4 expression prior to CTLA-4-targeted therapy is expected to open doors to evidence-based and cost-efficient medical care and to avoid adverse effects brought about by ineffective therapy. In this study, we aimed to develop a molecular imaging probe for CTLA-4 visualization in tumor. First, we examined CTLA-4 expression in normal colon tissues, cultured CT26 cells, and CT26 tumor tissues from tumor-bearing BALB/c mice and BALB/c nude mice by reverse transcription polymerase chain reaction (RT-PCR) analysis and confirmed whether CTLA-4 is strongly expressed in CT26 tumor tissues. Second, we newly synthesized ⁶⁴Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-anti-mouse CTLA-4 mAb (⁶⁴Cu-DOTA-anti-CTLA-4 mAb) and evaluated its usefulness in positron emission tomography (PET) and ex-vivo biodistribution analysis in CT26-bearing BALB/c mice. High CTLA-4 expression was confirmed in the CT26 tumor tissues of tumor-bearing BALB/c mice. However, CTLA-4 expression was extremely low in the cultured CT26 cells and the CT26 tumor tissues of tumor-bearing BALB/c nude mice. The results suggested that T cells were responsible for the high CTLA-4 expression. Furthermore, ⁶⁴Cu-DOTA-anti-CTLA-4 mAb displayed significantly high accumulation in the CT26 tumor, thereby realizing non-invasive CTLA-4 visualization in the tumor. Together, the results indicate that ⁶⁴Cu-DOTA-anti-CTLA-4 mAb would be useful for the evaluation of CTLA-4 expression in tumor.

Citation: Higashikawa K, Yagi K, Watanabe K, Kamino S, Ueda M, Hiromura M, et al. (2014) ⁶⁴Cu-DOTA-Anti-CTLA-4 mAb Enabled PET Visualization of CTLA-4 on the T-Cell Infiltrating Tumor Tissues. PLoS ONE 9(11): e109866. https://doi.org/10.1371/journal.pone.0109866

Editor: Gabriele Multhoff, Technische Universitaet Muenchen, Germany

Received: May 20, 2014; Accepted: September 3, 2014; Published: November 3, 2014

Copyright: © 2014 Higashikawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by a grant-in-aid for COE projects by MEXT, Japan, titled “Center of excellence for molecular and gene targeting therapies with micro-doze molecular imaging modalities.”, a grant from Japan Society for the Promotion of Science (grant number: 12J06885, URL: http://www.jsps.go.jp/english/index.html), and a grant form the Ministry of Education, Culture, Sports, Science and Technology (URL: http://www.pref.okayama.jp/page/287673.html). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Cancer is a complex mixture of host and tumor cells. Whereas the human body has the ability to produce an anti-tumor immune response, cancers develop multiple strategies to evade the host immune system [1]. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), also known as cluster of differentiation 152 (CD152), is one of the most important molecules that are involved in the downregulation of the immune system and the anti-tumor response. CTLA-4 is expressed predominantly on the surface of two major subsets of CD4⁺ T cells: regulatory T cells (Tregs) and activated CD4⁺ effector cells, and activated CD8⁺ effector T cells [2], [3]. In addition, recent research showed that various tumor cells also express CTLA-4 [4].

CTLA-4 targeted therapy augments endogenous response to tumor cells, thereby leading to tumor cell death when utilized on its own or with other therapeutic interventions [3]. It is for this reason that CTLA-4 has attracted attention as a target molecule of cancer immunotherapy [5]. Fully human anti-CTLA-4 monoclonal antibodies (mAbs), ipilimumab and tremelimumab, were developed for the treatment of cancer patients. Ipilimumab is the first drug to demonstrate survival benefits in metastatic melanoma patients, and was approved by the US Food and Drug Administration (FDA) for the treatment of advanced melanoma in 2011. Pre-clinical and clinical trials of anti-CTLA-4 mAbs have been conducted for the treatment of other cancers, including colon, breast, lung, ovarian, and prostate cancers [3], [6].

Although CTLA-4-targeted therapy is an attractive method for the treatment of various cancers, the therapy is beset by several problems. First, the enhanced T cell response by the CTLA-4 blockade frequently produces autoimmune-related adverse effects, such as rash, diarrhea, colitis, hepatitis, and hypophysitis [7], [8]. A superagonist antibody for CD28 (TGN1412), which directly stimulates T cells, caused life-threatening inflammatory reactions in a London clinical trial [9]. Extreme precaution must be taken when CTLA-4-targeted antibodies are used for the treatment because CTLA-4 is an antagonist of CD28–ligand interactions [10]. Second, antibody drugs are extremely expensive. One treatment course of ipilimumab in the United States consists of four doses at US$30,000 per dose [2], [11]. Clearly, there is an urgent need to develop a method to screen patients for sensitivity to the CTLA-4-targeted therapy, to eliminate adverse effects brought about by ineffective therapy and reduce unnecessary financial burden in non-sensitive patients. The identification of CTLA-4 expression in tumor prior to molecular targeted therapy would lead to evidence-based and cost-efficient medical care.

Biopsy is principally conducted to evaluate the expression of molecules of interest. However, it is an invasive and stressful procedure. Moreover, biopsy evaluates the expression of target molecules only in a localized region of the tumor. Thus, it is difficult to acquire information of a patient's sensitivity to a molecular targeted drug for tumors existing in whole body.

Molecular imaging can provide molecular information of the whole body in a noninvasive manner and be used for the determination of sensitivity to antibody drugs. Tumor imaging probes for human epidermal growth factor receptor 2 (HER2) [12]–[14], epidermal growth factor receptor (EGFR) [15]–[18], and vascular endothelial growth factor (VEGF) [19], [20], which are the target molecules of trastuzumab, cetuximab/panitumumab, and bevacizumab, respectively, have been developed. The expression of those molecules in tumor was detected with their respective probes by positron emission tomography (PET) or single photon emission computed tomography (SPECT). However, to our knowledge, a molecular imaging probe that targets CTLA-4 has yet to be developed.

In this study, we aimed to develop a molecular imaging probe for CTLA-4 visualization in tumor. First, CTLA-4 expression was examined in CT26 tumor tissues and cultured CT26 cells by reverse transcription polymerase chain reaction (RT-PCR) analysis. Second, we newly developed ⁶⁴Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA)-anti-mouse CTLA-4 mAb by introducing DOTA groups to anti-mouse CTLA-4 mAb and subsequent radiolabeling with ⁶⁴Cu. The utility of ⁶⁴Cu-DOTA-anti-CTLA-4 mAb as an imaging probe was assessed by PET imaging and ex-vivo biodistribution analysis. We prepared tumor-bearing mice by syngeneic implantation of CT26 cells (mouse colon tumor cell line) to BALB/c mice for PET imaging. Immune-deprived mice bearing human tumor cell lines were not used because T cells might be responsible for the CTLA-4 expression in the tumor tissues.

Materials and Methods

Cell culture

CT26 was purchased from American Type Culture Collection and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 10 U/mL penicillin, and 10 mg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.

Preparation of subcutaneous tumor model mice

Female BALB/c and BALB/c (nu/nu) nude mice (4–6 weeks old) were purchased from CLEA Japan Inc. Tumor-bearing BALB/c and BALB/c nude mice were prepared by subcutaneously implanting CT26 cells (1–4×10⁶ cells). Investigations were initiated after receiving approval from the committee on animal experiments of Okayama University.

RT-PCR analysis

RNA extraction and cDNA synthesis were conducted by using the same methods as our previous report [21]. Total RNA was isolated from cultured cells and tissues with TRIZOL reagent (Life Technologies Co., Ltd.) and a PureLink RNA Mini Kit (Life Technologies Co., Ltd.). One microgram of total RNA was used as the template for single-strand cDNA synthesis with a Transcriptor First Strand cDNA Synthesis Kit (Roche Co., Ltd.). Analysis of mRNA expression levels was carried out with RT-PCR using TaKaRa Ex Taq (TaKaRa Co., Ltd.). The amplification of β-actin is shown as internal control. Primer sequences are listed in Table S1. The amplicons were separated on agarose gel (AGAROSE I, Amresco, Inc.), stained with ethidium bromide, and visualized with a Benchtop 2UV Transilluminator (UVP, Inc.).

⁶⁴Cu-DOTA-anti-CTLA-4 mAb production

Anti-mouse CTLA-4 mAb (200–500 µg) (R&D Systems, Inc.) was conjugated to DOTA-mono-N-hydroxysuccinimide ester (DOTA-mono-NHS ester; Macrocyclics, Inc.) in phosphate-buffered saline without calcium and magnesium (pH 7.5) (PBS (−)), by using a 100-fold molar excess of DOTA-mono-NHS ester. The mixture was stirred at room temperature (RT) for three hours to give the DOTA-anti-CTLA-4 antibody. The DOTA-anti-CTLA-4 antibody was purified with a PD-10 column (GE Healthcare Co., Ltd.) and an Amicon-Ultra 50 K device (Millipore Co., Ltd.). The DOTA-anti-CTLA-4 antibody was analyzed by size-exclusion high-performance liquid chromatography (SE-HPLC) using TSK-GEL Super SW3000 (Tosoh Co., Ltd.). The mobile phase of 10 mM PBS (−) containing 0.3 M NaCl was used and the flow rate was 0.35 mL/min.

⁶⁴Cu was produced by irradiating a 99.6% ⁶⁴Ni-enriched nickel target with 12 MeV protons using a cyclotron (CYPRIS-HM12, Sumitomo Heavy Industries, Ltd.). Then, ⁶⁴Cu was purified with a Muromac column (Muromachi Technos Co., Ltd.). The buffer solution of DOTA-anti-CTLA-4 mAb was replaced with 0.1 M acetate buffer (pH 6.5) three times by using an Amicon-Ultra 50 K device (Millipore Co., Ltd.). DOTA-anti-CTLA-4 mAb was radiolabeled with ⁶⁴Cu by incubating at 40°C for one hour. To remove excess ⁶⁴Cu, the buffer was replaced with 0.2 M glycine buffer by using the Amicon-Ultra 50 K device. Buffer of the purified antibody solution was replaced with PBS (−) by using the Amicon-Ultra 50 K device. The resultant solution was used for injection.

The radiochemical purity of ⁶⁴Cu-DOTA-antibodies in PBS (−) was confirmed by reversed phase radio-thin layer chromatography (TLC). This analysis was performed with a TLC aluminum sheet, RP-18 F254 S (Merck Chemicals Co., Ltd.) and methanol∶water∶acetic acid (4∶1∶1) was used as the mobile phase. TLC chromatograms were obtained by autoradiography (FLA-7000IR; GE Healthcare Co., Ltd.). ⁶⁴Cu-DOTA-isotype IgG_2A (⁶⁴Cu-DOTA-Control IgG) was produced in the same way as that for negative control by using rat IgG_2A isotype control (R&D Systems, Inc.).

Assay for CTLA-4 binding activity

The CTLA-4 binding activity of DOTA-anti-CTLA-4 mAb and DOTA-Control IgG was examined by enzyme-linked immunosorbent assay (ELISA) and compared with that of original anti-CTLA-4 mAb and DOTA-Control IgG. Twenty ng of recombinant mouse CTLA-4 (R&D Systems, Inc.) in 50 mM carbonate buffer (pH 9.6) per well was added into a 96-well ELISA plate (R&D Systems, Inc.). After blocking with 3% bovine serum albumin (BSA) and 1% Tween 20 in PBS (−) containing 0.05% Tween 20, 5 ng of the antibodies in PBS (−) containing 1% BSA and 0.05% Tween 20 was added to each well and incubated for one hour. After incubation, each well was treated with 50 uL of HRP-conjugated anti-rat IgG (R&D Systems, Inc.) diluted 1∶6000 with PBS (−) containing 1% BSA and 0.05% Tween 20. Peroxidase activity was visualized with a TMB Microwell Peroxidase Substrate System (Kirkegaard & Perry Laboratories, Inc.) and the absorbance at 450 nm was measured. The absorbance was corrected by performing a blank trial. The corrected absorbance values of DOTA-anti-CTLA-4 mAb and DOTA-Control IgG were respectively divided by the absorbance of anti-CTLA-4 mAb, and relative immunoreactivities were calculated.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis

MALDI-TOF-MS was conducted to determine the extent of DOTA conjugation to antibodies using a method similar to that reported by Lu et al. [22]. MALDI-TOF-MS was performed by using an Ultraflex III MALDI TOF/TOF (Bruker Daltonics Co., Ltd.). Non- and DOTA-conjugated antibodies were desalted with PD Spin Trap G-25 (GE Healthcare Co., Ltd.). Sinapinic acid (Nacalai Tesque, Inc.) at 20 mg/mL in 2∶1 acetonitrile/H₂O with 0.1% trifluoroacetic acid (Wako Pure Chemical Industries, Co., Ltd.) was used as the MALDI matrix.

PET imaging study

⁶⁴Cu-DOTA-anti-CTLA-4 mAb (4 µg, approximately 16 MBq) or ⁶⁴Cu-DOTA-Control IgG (4 µg, approximately 14 MBq) was intravenously administered to CT26-bearing BALB/c mice via the tail vein. Forty-eight hours after administration of the radiolabeled antibodies, probe uptake in the CT26-bearing mice was measured with a small-animal PET scanner (microPET Focus220; Siemens Medical Solutions Inc.). During PET imaging, the mice were anesthetized with 1.5% isoflurane and 1.5% N₂O gas, and placed in the prone position. Emission data were acquired for 60 min. The acquired data were summed into sinograms and three-dimensional images were reconstructed by maximum a posteriori (MAP). Coronal and sagittal images were displayed in 918×760 and 550×760 pixel formats, respectively, with a pixel size of 0.053 mm×0.053 mm. The image intensity was expressed by standardized uptake value (SUV). SUV_max was calculated by ASIPRO software package (Concorde Microsystems, Inc.).

Biodistribution study

Forty-eight hours after administration of ⁶⁴Cu-DOTA-anti-CTLA-4 mAb (4 µg, 1 MBq) or ⁶⁴Cu-DOTA-Control IgG (4 µg, 1 MBq), the animals were immediately sacrificed and the organs and blood were removed. The organs and blood were weighed and radioactivities were counted with a gamma counter (ARC-7001B, ALOKA Co., Ltd.). Decay-corrected uptake was expressed as the percentage of injected dose per gram and calculated as the ratio to blood or muscle for comparison of the accumulation abilities in the CT26 tumor between ⁶⁴Cu-DOTA-anti-CTLA-4 antibody and ⁶⁴Cu-DOTA-Control IgG.

Immunohistological staining

Tumor-bearing BALB/c mice were sacrificed and CT26 tumor tissues including the normal tissues around them were resected and embedded in Optimal Cutting Temperature (O.C.T.) compound (Sakura Finetek Japan Co., Ltd.). Ten-µm-thick frozen tissue sections were prepared and mounted on MAS-coated glass slides (Matsunami Glass Ind., Co., Ltd.). The tissue sections were fixed with 4% paraformaldehyde in PBS (−), blocked with 5% goat serum in PBS (−), and incubated with anti-CTLA-4 antibody (R&D Systems, Inc.). Then, the tissue sections were subjected to endogenous peroxidase inactivation with 0.19% H₂O₂/methanol (Wako Pure Chemical Industries, Ltd.), followed by incubation with horseradish peroxidase conjugated anti-rat IgG antibody (R&D Systems, Inc.). Immunocomplexes were visualized with a DAB substrate kit (Dako Co., Ltd.).

Statistical analysis

SUV_max data are expressed as means ± standard deviation (SD) and other data are expressed as means ± standard error of mean (SEM). Statistical significance was determined using the Student's t-test. The level of significance was taken as p<0.01. The tests were performed using GraphPad Prism software (GraphPad Software, Inc.).

Results

CTLA-4 was strongly expressed in CT26 tumor tissues but not cultured CT26 cells

First, RT-PCR was carried out to examine CTLA-4 expression in CT26 tumor tissues and cultured CT26 cells, and the results were compared to those obtained with normal colon tissues (Fig. 1). CTLA-4 (amplicon length: 920 base pairs (bp)) was strongly expressed in CT26 tumor tissues compared with normal colon tissues.

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Figure 1. RT-PCR in normal colon tissues, CT26 tumor tissues, and cultured CT26 cells.

Expression of CTLA-4, Treg markers, and T cell activation markers in normal colon tissues, CT26 tumor tissues, and cultured CT26 cells.

https://doi.org/10.1371/journal.pone.0109866.g001

On the other hand, CTLA-4 expression was extremely low in cultured CT26 cells. Moreover, Treg markers, such as forkhead box P3 (Foxp3) and folate receptor 4 (FR4) [23], [24], were more strongly expressed in the tumor tissues than the normal colon tissues and the cultured CT26 cells. The expression of CD25 and CD69, which are molecules expressed on regulatory and activated T cells [23]–[25], was also increased in the tumor tissues compared to the normal colon tissues and the cultured CT26 cells. CD154, which is induced on T cells by T cell activation [26], was also more strongly expressed in the tumor tissues than the normal colon tissues and the cultured CT26 cells.

CTLA-4 and T cell marker expression was low in CT26 tumor tissues from tumor-bearing BALB/c nude mice

From the results of Fig. 1, we assumed that T cells were involved in CTLA-4 expression in the CT26 tumor tissues from the tumor-bearing BALB/c mice, and CT26 tumor tissues from the tumor-bearing BALB/c nude mice did not express CTLA-4 due to a marked decrease of T cells in those mice. Thus, we prepared two subcutaneous tumor models by syngeneic subcutaneous transplantation of CT26 into normal BALB/c mice or BALB/c nude mice, and compared CTLA-4 and T cell marker expression in the CT26 tumor tissues from the tumor-bearing BALB/c mice with those from the tumor-bearing BALB/c nude mice by RT-PCR analysis (Fig. 2).

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Figure 2. Gene expression analyses in tissues from tumor-bearing BALB/c and BALB/c nude mice.

CTLA-4 and T cell marker expression in normal colon tissues from normal BALB/c mice, CT26 tumor tissues from tumor-bearing BALB/c mice, and CT26 tumor tissues from tumor-bearing BALB/c nude mice.

https://doi.org/10.1371/journal.pone.0109866.g002

RT-PCR showed that CTLA-4 expression was dramatically decreased in the CT26 tumor tissues from the tumor-bearing BALB/c nude mice, compared with those from the tumor-bearing BALB/c mice. Furthermore, we confirmed that the expression of CD4 and CD8 as well as Foxp3, FR4, CD69, CD154, and CD25 was markedly decreased in the tumor tissues from the tumor-bearing BALB/c nude mice.

DOTA-conjugated antibody probe was synthesized

DOTA chelators were conjugated to each mAb, as shown in Figure 3A. The chromatograms of all the mAbs showed a single peak. In addition, the retention times of anti-CTLA-4 mAb, DOTA-anti-CTLA-4 mAb, Control IgG, and DOTA-Control IgG were 9.42, 9.36, 10.57, and 10.33, respectively (Fig. 3B). The retention times of DOTA-conjugated antibodies were slightly shorter than those of the original antibodies, suggesting that anti-CTLA-4 mAb or Control IgG conjugated to DOTA and was well purified. Furthermore, MALDI-TOF-MS analysis was carried out to measure the average number of DOTA chelators that were conjugated to anti-CTLA-4 mAb or Control IgG (Table 1). The mass differences between anti-CTLA-4 mAb and DOTA-anti-CTLA-4 mAb, and between Control IgG and DOTA-Control IgG were 1634 and 1686, respectively. The mass differences were divided by the mass value of single DOTA conjugation (386 mass units), and the resulting values represented the average number of DOTA chelators that were conjugated to anti-CTLA-4 mAb or Control IgG. From the calculations, 4.2 or 4.4 DOTA chelators on average were conjugated into a single molecule of anti-CTLA-4 mAb or Control IgG.

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Figure 3. Preparation of DOTA-conjugated mAb.

A. Scheme of the synthesis of DOTA-conjugated mAb. B. HPLC analysis of original and DOTA-conjugated mAbs. C. Evaluation of CTLA-4 binding activity of DOTA-anti-CTLA-4 mAb. Data are expressed as means ± SEM.

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Table 1. Average molecular weights of original and DOTA-conjugated antibodies, and estimated numbers of DOTA chelators per unit antibody.

https://doi.org/10.1371/journal.pone.0109866.t001

Then, the binding activity of DOTA-anti-CTLA-4 mAb to CTLA-4 was measured by ELISA (Figure 3C). The binding activity of DOTA-anti-CTLA-4 mAb to CTLA-4 was 86.3±2.8% of that of the original anti-CTLA-4 mAb. The binding activity of DOTA-Control IgG was 0.3±0.1%.

⁶⁴Cu-DOTA-anti-CTLA-4 mAb enabled clear visualization of CTLA-4-positive tumor by PET

⁶⁴Cu-DOTA-anti-CTLA-4 mAb and ⁶⁴Cu-DOTA-Control IgG were obtained in radiochemical yields of 94% and 97%, respectively. The radiochemical purities of both probes were higher than 94%. To evaluate the ⁶⁴Cu-DOTA-anti-CTLA-4 mAb uptake by CTLA-4 positive tumor (CT26), we performed PET and ex-vivo biodistribution analysis. Representative coronal and sagittal images are shown in Figure 4. At 48 hours after administration of the probes, ⁶⁴Cu-DOTA-anti-CTLA-4 mAb clearly visualized the CT26 tumors and ⁶⁴Cu-DOTA-anti-CTLA-4 mAb showed higher accumulation in the tumors than ⁶⁴Cu-DOTA-Control IgG (⁶⁴Cu-DOTA-anti-CTLA-4 mAb: SUVmax = 2.65±0.01, n = 2; ⁶⁴Cu-DOTA-Control IgG: SUVmax = 2.06±0.32, n = 2).

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Figure 4. PET images of ⁶⁴Cu-DOTA-anti-CTLA-4 mAb and ⁶⁴Cu-DOTA-Control IgG.

A. Representative coronal (a) and sagittal (b) PET images of ⁶⁴Cu-DOTA-anti-CTLA-4 mAb in CT26-bearing mice. B. Representative coronal (a) and sagittal (b) PET images of ⁶⁴Cu-DOTA-Control IgG in CT26-bearing mice.

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The results were consistent with those of the ex-vivo biodistribution study (Fig. 5). ⁶⁴Cu-DOTA-anti-CTLA-4 mAb showed significantly higher accumulation in the CT26 tumors than ⁶⁴Cu-DOTA-Control IgG (7.49±0.32%ID/g vs. 5.84±0.38%ID/g, p<0.01). Moreover, ⁶⁴Cu-DOTA-anti-CTLA-4 mAb showed higher tumor-to-blood and tumor-to-muscle ratios than ⁶⁴Cu-DOTA-Control IgG (tumor-to-blood ratio: 0.58±0.03 vs. 0.40±0.02, p<0.001; tumor-to-muscle ratio: 8.48±0.63 and 5.31±0.35, p<0.01).

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Figure 5. Biodistribution analysis of ⁶⁴Cu-labeled antibody probes.

A. %ID/g of ⁶⁴Cu-DOTA-anti-CTLA-4 mAb (n = 7) and ⁶⁴Cu-DOTA-Control IgG (n = 6). B. Tumor-to-blood and tumor-to-muscle ratios of ⁶⁴Cu-DOTA-anti-CTLA-4 mAb (n = 7) and ⁶⁴Cu-DOTA-Control IgG (n = 6). Data are expressed as means ± SEM. Symbols^* and ^** denote p<0.01 and p<0.001 vs. ⁶⁴Cu-DOTA-Control IgG, respectively.

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In addition, CTLA-4 protein expression in the CT26 tumor was confirmed by immunohistochemical staining (Fig. S1A). CTLA-4 was weakly expressed in the normal tissues surrounding the tumor (Fig. S1B).

Discussion

CT26 is a N-nitroso-N-methylurethane-induced, undifferentiated colon carcinoma cell line and recent cancer immunotherapy studies have shown that CTLA-4 blockade reduced CT26 colon tumor size and was effective in CT26 tumor models [27], [28]. Therefore, in this study, we used the CT26 cell line to prepare subcutaneous tumor models for PET imaging.

First, we compared CTLA-4 expression in CT26 tumor tissues, normal colon tissues, and/or cultured CT26 cells by RT-PCR analyses and confirmed that CTLA-4 was strongly expressed in the CT26 tumor tissues compared to the normal colon tissues. There are four functionally different forms of CTLA-4: the full-length form (containing exons 1–4), the soluble form (exons 1, 2, and 4), the ligand-independent form (exons 1, 3, and 4), and the form containing only exons 1 and 4 [29]–[34]. In our experiments, the full-length form of CTLA-4 (amplicon length: 920 bp), which is a representative immunosuppressive form of CTLA-4, was expressed in the CT26 tumor tissues.

On the other hand, we found that CTLA-4 was not expressed in the cultured CT26 cells, although it was strongly expressed in the CT26 tumor tissues. Contardi et al. reported some human tumor cell lines that expressed CTLA-4 [4]. On the other hand, CTLA-4 is expressed also on CD25⁺ (and/or) Foxp3⁺ (and/or) FR4⁺ CD4⁺ Tregs, activated CD4⁺ effector T cells, and activated CD8⁺ effector T cells [2], [3], [24]. In addition, flow cytometry analysis by Valzasina et al. revealed that almost all CD4⁺ T cells in the CT26 tumor tissues expressed CD25 [35]. From those reports, we hypothesized that CTLA-4 expression in the CT26 tumor tissues regulated by T cells. To prove our hypothesis, we examined the expression of several T cell markers in normal colon tissues, CT26 tumor tissues, and cultured CT26 cells. RT-PCR analysis showed that Treg markers and T cell activation markers were strongly expressed in the CT26 tumor tissues but not the normal colon tissues or cultured CT26 cells. Therefore, we assumed that the T cells were responsible for CTLA-4 expression in the CT26 tumor tissues. Then, we used RT-PCR to compare the expression of CTLA-4 and T cell markers in the CT26 tumor tissues of tumor-bearing BALB/c mice with those of tumor-bearing BALB/c nude mice in hopes of elucidating the relationship between CTLA-4 expression in the CT26 tumor tissues and T cells. BALB/c nude mice are thymus-deficient and thus, the number of T cells is greatly reduced in those mice. Interestingly, we found that the expression of CTLA-4 as well as T cell markers was quite low in the CT26 tumor tissues from the BALB/c nude mice. The results indicated that T cells were responsible for the CTLA-4 expression.

Second, we developed a molecular imaging probe that targets CTLA-4 and examined its utility in mice bearing CTLA-4-expressing CT26 tumor. The anti-CTLA-4 mAb for the imaging probe synthesis was made by using recombinant mouse CTLA4 representing an extracellular domain of mouse CTLA-4 (Ala36Phe161) as the immunogen. We selected this mAb for CTLA-4 imaging probe synthesis because clinically used anti-CTLA-4 mAb (ipilimumab) also recognizes the extracellular domain of CTLA-4 and a mAb probe that recognizes the extracellular domain of CTLA-4 is suited for the prediction of the efficacy and drug disposition of ipilimumab.

For CTLA-4 imaging, we conjugated anti-CTLA-4 mAb to DOTA. DOTA-conjugated mAb was prepared by reacting the nucleophilic amino group in the amino acid residue (particularly in lysine) of mAb with the electrophilic DOTA-mono-NHS ester. The binding activity of antibodies to CTLA-4 may be reduced, particularly when DOTA conjugates to lysine residues critical for mAb binding to CTLA-4 [36]. Therefore, it is necessary to check in advance the binding activities of the antibodies. We examined the binding activity of DOTA-conjugated anti-CTLA-4 mAb and compared it with that of control IgG. ELISA confirmed that the binding activity of DOTA-anti-CTLA-4 mAb was preserved for use in CTLA-4 imaging although a slight reduction (86.3±2.8%) was observed relative to the binding activity of the original anti-CTLA-4 mAb. The binding activity of DOTA-Control IgG was extremely low (0.3±0.1%) compared with that of DOTA-anti-CTLA-4 mAb. The results indicate the successful preparation of metal-chelator-conjugated anti-CTLA-4 mAb having CTLA-4 binding activity.

The choice of the positron emitter is an important factor for successful PET imaging. In our study, ⁶⁴Cu was used for labeling mAbs. ⁶⁴Cu decay generates positron emissions applicable to PET and the half-life of ⁶⁴Cu (T_1/2 = 12.7 h) is sufficiently long for imaging up to 24 to 48 h after administration to accommodate the mAb localization time. Therefore, ⁶⁴Cu has been used for the development of mAb-based radiopharmaceuticals [13], [15], [19]. In our preliminary biodistribution analyses, tumor uptake on ⁶⁴Cu-DOTA-anti-CTLA-4 mAb was not significantly higher than that on ⁶⁴Cu-DOTA-Control IgG at 24 h after administration of the probes (data was not shown). Therefore, we chose later time point (48 h) for PET imaging and ex-vivo biodistribution analysis.

The utility of ⁶⁴Cu-DOTA-anti-CTLA-4 mAb was examined by PET and ex-vivo biodistribution analysis. We were able to visualize CTLA-4-positive tumor by PET with ⁶⁴Cu-DOTA-anti-CTLA-4 mAb as the probe. Ex-vivo biodistribution analysis also revealed that ⁶⁴Cu-DOTA-anti-CTLA-4 mAb showed significant accumulation in the CT26 tumor compared to ⁶⁴Cu-DOTA-Control IgG. The results indicated that ⁶⁴Cu-DOTA-anti-CTLA-4 mAb is useful for the noninvasive imaging of CTLA-4 expression in tumor. Furthermore, we quantified PET images and calculated ⁶⁴Cu-DOTA-anti-CTLA-4 mAb/⁶⁴Cu-DOTA-Control IgG ratio in the CT26. The mean ratio of SUV_max values in CT26 was 1.29 in the PET image and that in CT26 was 1.28 in the ex-vivo biodistribution analysis; further, the quantitative value of PET was similar to the ex-vivo biodistribution data. Thus, quantitativity was ensured in our PET experiment. In addition, although further investigation is needed, ⁶⁴Cu-DOTA-anti-CTLA-4 mAb could be used in the diagnosis of other types of tumor invaded by T cells, regardless of CTLA-4 expression in the tumor cells.

In conclusion, we have developed ⁶⁴Cu-DOTA-anti-CTLA-4 mAb and evaluated its potential as a new radiotracer for the noninvasive evaluation of CTLA-4 expression in tumor. Our results demonstrated that ⁶⁴Cu-DOTA-anti-CTLA-4 mAb visualized CTLA-4 expression in CT26 tumor in a noninvasive manner. Therefore, ⁶⁴Cu-DOTA-anti-CTLA-4 mAb is useful for evaluating CTLA-4 expression in the tumor. The evaluation of CTLA-4 expression in tumors using ⁶⁴Cu-DOTA-anti-CTLA-4 mAb would enable selection of patients sensitive to CTLA-4-targeted therapy, thereby eliminating the adverse effects brought about by ineffective therapy and reducing unnecessary financial burden in non-sensitive patients.

Supporting Information

Figure S1.

Immunohistochemically stained images of CTLA-4 in representative CT26 tumor and normal tissue sections. A. CT26 tumor tissue section. B. Normal tissue section surrounding CT26 tumor tissue. Scale bar = 50 ?m.

https://doi.org/10.1371/journal.pone.0109866.s001

(DOC)

Table S1.

List of primer sequences for RT-PCR.

https://doi.org/10.1371/journal.pone.0109866.s002

(DOC)

Acknowledgments

The authors thank Dr. Yasuhiro Wada and Dr. Emi Hayashinaka for technical support in the reconstitution of PET images; Dr. Yousuke Kanayama, Dr. Takanori Sasaki, and Mr. Fumiaki Takenaka for helpful discussions and technical assistance in PET or ex-vivo biodistribution experiments; and Professor Dr. Satoshi Tanaka for relevant suggestions.

Author Contributions

Conceived and designed the experiments: KH KY MH SE. Performed the experiments: KH KY KW. Analyzed the data: KH KY. Contributed reagents/materials/analysis tools: SE. Wrote the paper: KH SK MU MH SE.

References

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32. Araki M, Chung D, Liu S, Rainbow DB, Chamberlain G, et al. (2009) Genetic Evidence That the Differential Expression of the Ligand-Independent Isoform of CTLA-4 Is the Molecular Basis of the Idd5.1 Type 1 Diabetes Region in Nonobese Diabetic Mice. J Immunol 183: 5146–5157.
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Figures

Abstract

Introduction

Materials and Methods

Cell culture

Preparation of subcutaneous tumor model mice

RT-PCR analysis

64Cu-DOTA-anti-CTLA-4 mAb production

Assay for CTLA-4 binding activity

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis

PET imaging study

Biodistribution study

Immunohistological staining

Statistical analysis

Results

CTLA-4 was strongly expressed in CT26 tumor tissues but not cultured CT26 cells

CTLA-4 and T cell marker expression was low in CT26 tumor tissues from tumor-bearing BALB/c nude mice

DOTA-conjugated antibody probe was synthesized

64Cu-DOTA-anti-CTLA-4 mAb enabled clear visualization of CTLA-4-positive tumor by PET

Discussion

Supporting Information

Figure S1.

Table S1.

Acknowledgments

Author Contributions

References

⁶⁴Cu-DOTA-anti-CTLA-4 mAb production

⁶⁴Cu-DOTA-anti-CTLA-4 mAb enabled clear visualization of CTLA-4-positive tumor by PET