68Ga-labelled NOTA-RGD-GE11 peptide for dual integrin and EGFR-targeted tumour imaging

Accepted Manuscript 68Ga-labelled NOTA-RGD-GE11 peptide for dual integrin and EGFR-targeted tumour imaging

Chien-Jen Chen, Chen-Hsin Chan, Kun-Liang Lin, Jyun-Hong Chen, Chun-Hao Tseng, Ping-Yen Wang, Chuan-Yi Chien, HungMan Yu, Wuu-Jyh Lin PII: DOI: Reference:

S0969-8051(18)30259-2 https://doi.org/10.1016/j.nucmedbio.2018.11.003 NMB 8045

To appear in:

Nuclear Medicine and Biology

Received date: Revised date: Accepted date:

13 August 2018 12 November 2018 22 November 2018

Please cite this article as: Chien-Jen Chen, Chen-Hsin Chan, Kun-Liang Lin, Jyun-Hong Chen, Chun-Hao Tseng, Ping-Yen Wang, Chuan-Yi Chien, Hung-Man Yu, Wuu-Jyh Lin , 68Ga-labelled NOTA-RGD-GE11 peptide for dual integrin and EGFR-targeted tumour imaging. Nmb (2018), https://doi.org/10.1016/j.nucmedbio.2018.11.003

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Ga-labelled NOTA-RGD-GE11 peptide for dual integrin and EGFR-targeted tumour imaging

Chien-Jen Chen, Chen-Hsin Chan, Kun-Liang Lin, Jyun-Hong Chen, Chun-Hao Tseng, Ping-Yen Wang, Chuan-Yi Chien, Hung-Man Yu*, Wuu-Jyh Lin*

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Isotope Application Division, Institute of Nuclear Energy Research, Taoyuan City, Taiwan (ROC)

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*Corresponding authors. Tel.: +886-3-4711400 ext 7177, E-mail address: [email protected] (Hung-Man Yu); Tel.: +886-3-4711400 ext 2761, E-mail address: [email protected] (Wuu-Jyh Lin) Abstract

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Introduction: Multiple peptide receptors are co-rexpressed in many types of cancers. Arg-Gly-Asp (RGD) and GE11 peptides specifically target integrin αVβ3 and EGFR, respectively. Recently, we designed and synthesized a heterodimer peptide NOTA-c(RGDyK)-GE11 (NOTA-RGD-GE11). The aim of this study was to investigate the characteristics of NOTA-RGD-GE11 for dual receptor imaging.

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Methods: NOTA-RGD-GE11 heterodimer was labelled with 68Ga. The dual receptor binding affinity was investigated by antibodies competition binding assay. The in vitro and in vivo characteristics of [68Ga]Ga-NOTA-RGD-GE11 was investigated and compared with that of monomeric peptides [68Ga]Ga-NOTA-RGD and

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[68Ga]Ga-NOTA-GE11. Results: NOTA-RGD-GE11 had binding affinities with both integrin αVβ3 and EGFR. The dual receptor targeting property of [68Ga]Ga-NOTA-RGD-GE11 was validated by blocking studies in a NCI-H292 tumour model. [68Ga]Ga-NOTA-RGD-GE11 showed higher tumour uptake than [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 in biodistribution and PET/CT

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imaging studies. Conclusion: The dual receptor targeting and enhanced tumour uptake of [68Ga]Ga-NOTA-RGD-GE11 warrant its further investigation for dual integrinαVβ3 and EGFR-targeted tumour imaging. Keyword: heterodimer peptide, integrin αVβ3, EGFR, Ga-68, PET imaging 1. Introduction Molecular imaging is a powerful tool to visualize and quantify the in vivo target expression through using positron emission tomography (PET), single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI) in pre-clinical 1

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and clinical cancer research [1,2]. PET is a noninvasive imaging technique and is widely used for diagnosis and monitoring of normal and tumour tissues. In clinical oncology, 18F-fluorodeoxyglucose ([18F]FDG) has been used for tumour imaging due to increased glucose metabolism of tumours compared with normal tissues [3]. In some cancers, [18F]FDG PET remains limited because of nonspecific localization. Tumour receptors as targets for diagnosis and treatment of cancer, are involved in carcinogenesis, growth, and metastasis [4]. Tumour receptor imaging also provides useful information, including the evaluation of the entire tumour burden and heterogeneity of tumour receptor expression [5]. In receptor-targeted cancer therapy, it

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is essential to measure the different expression of tumour receptors for selecting patients [5]. Previous studies have identified many tumour receptor targets, such as the epidermal growth factor receptor (EGFR) [6], integrin αVβ3 [7], gastrin-releasing peptide receptor [8], somatostatin receptors [9], estrogen receptor [10], human epidermal growth factor receptor 2 [11]. Thus, it is important to develop a suitable

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receptor imaging agent to detect the different types of cancer at an early stage. Low-molecular-weight peptide-based probes for targeted molecular imaging have many distinctive advantages compared with macromolecules, such as proteins and antibodies. Small peptides display favorable pharmacokinetic and tissue distribution in

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target tissue and rapid clearance from blood and nontarget tissues in order to obtain high target-to-background ratio for in vivo PET applications. In addition, small peptides are quite flexible in terms of chemical modification and radiolabeling, and they usually have high stability, low nonspecific toxicity and immunogenicity [12,13].The development of heterodimeric peptides, two different specific peptide ligands targeting two types of receptors are covalently linked by either a flexible or rigid linker with adjustable length [14], is based on the fact that many cancer cells co-express multiple peptide receptors [15]. Because expression of some cell-surface

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receptor may be reduced significantly during tumour development, resulting in low binding affinity of monomeric peptides, limiting the quality of the diagnostic imaging compared with heterodimeric peptides for tumour receptors [16]. Arg-Gly-Asp (RGD) and bombesin (BBN) heterodimers peptides targeting integrin αVβ3 and GRPR, which have high tumour-specific binding affinities and high in vivo tumour uptake comparing to monomeric RGD or BBN [17]. The results of these studies indicate that heterodimeric peptides are suitable for early tumour detection and evaluation of cancer progression. The RGD-based radiotracers for imaging of tumour integrin αVβ3 expression were investigated extensively in the last decade. In recent studies, radiolabelled heterodimeric peptides have been developed for dual receptor-targeted imaging, such as RGD-GE11, RGD-A7R and RGD-BBN peptides for integrin Vβ3 and EGFR, 2

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VEGF or GRPR. These peptides were radiolabelled with different isotopes, including gallium-68, fluorine-18, lutetium-177 and copper-64 [17–22]. Gallium-68 generator provides a stable source of positron-emitting isotope without an on-site cyclotron facility [23]. It also simplifies the radiolabeling, kit formulation, and increases precursor availability. Due to the short physical half-life of 68Ga, it matches with the in vivo pharmacokinetics of short peptides [24,25]. RGD peptide sequence has high affinity and selectivity to integrin αVβ3, which is a cell adhesion motif on many plasma proteins and extracellular matrix (ECM). RGD peptide also regulates cell-cell recognition and prevents cell apoptosis, which has been extensively studied in tumour therapy and tissue engineering through recombinant techniques or chemical methods

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[26]. IntegrinαVβ3 play an important role in tumour angiogenesis, invasion and metastasis [27]. Previous studies have shown that the integrinαVβ3 is overexpressed in many different types of tumours, including osteosarcomas, neuroblastomas, glioblastomas, melanomas, lung carcinomas and breast cancer [28]. Based on these

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studies, integrin αVβ3 is a specific biomarker for early detection of tumour, evaluation of disease progression, and monitoring antiangiogenic treatment efficacy [18]. GE11 (Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile) peptide was identified and characterized by phage display screening to have high affinity for EGFR [29]. EGFR

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belongs to the ErbB family of receptor tyrosine kinases (RTKs), which is known to regulate the cell differentiation, migration and proliferation of normal and cancer cells [30]. EGFR are also overexpressed in many types of cancer cells, including esophageal, colorectal, breast, gastric, head and neck, bladder, pancreatic, renal, prostate, ovarian, and nonsmall cell lung cancer [31,32]. These studies indicated that RGD and GE11 peptides are specific molecular probes for the diagnosis and therapy of integrin αVβ3 and EGFR-overexpressing tumours, respectively. In our previous study, we had successfully designed and synthesized a novel [68Ga]Ga-NOTA-c(RGDyK)-GE11

([68Ga]Ga-NOTA-RGD-GE11)

heterodimeric

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peptides (Figure 1) for dual targeting of tumour integrin Vβ3 and EGFR receptors, with high radiochemical purity [21]. The RGD-GE11 heterodimer was linked with 6-aminohexanoic acid (6-Ahx) and cysteine, and was conjugated with 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) to form NOTA-RGD-GE11. In this study, the in vitro and in vivo characteristics of [68Ga]Ga-NOTA-RGD-GE11 was investigated and compared with that of monomeric peptides [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 for evaluation of [68Ga]Ga-NOTA-RGD-GE11 as a dual receptor-targeted PET imaging probe. 2. Materials and methods

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Materials Bifunctional chelators NOTA-NHS ester were obtained from CheMatech (Dijon, France). The Fmoc amino acids and the Wang resin were obtained from AnaSpec, Inc. (Fremont, CA, USA). All other chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA), Acros Organic (Geel, Belgium), Alfa Aesar (Ward Hill, MA, USA), Mallinckrodt Chemicals (St. Louis, MO, USA), or Merck (Darmstadt, Germany) and used without further purification. 68GaCl3 was obtained from a 68Ge/68Ga generator (Eckert & Ziegler, Berlin, Germany) and eluted with 5 mL of 0.1N ultrapure HCl (J.T. Baker, Center Valley, PA, USA). The peptide synthesis

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was performed using a Liberty1™ Automated Microwave Peptide Synthesizer (CEM Microwave Technology Ltd, Buckingham, England, UK). Mass spectrometry was performed on a Thermo LTQ Orbitrap XL® ETD Hybrid Ion Trap-Orbitrap mass spectrometer (San Jose, CA, USA). The peptides were purified by reversed-phase medium-pressure liquid chromatography (RP-MPLC) (CombiFlash® Rf, Teledyne

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Isco, Lincoln Nebraska, USA) using RediSep Rf Gold® C-18 column (15.5 g, 20-40 μm) with an ultraviolet detection wavelength at 214 nm. For quality control of 68 Ga-labelled peptides, a reversed-phase high-performance liquid chromatographic (RP-HPLC) system was used, including a Thermo UltiMate® 3000 pump, a Thermo

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Synthesis of NOTA-RGD-GE11, NOTA-GE11 and NOTA-RGD The NOTA-RGD-GE11, NOTA-GE11 and RGD peptides were synthesized as previously described [21]. For NOTA-c(RGDyK) synthesis, the c(RGDyK) peptide were conjugated with NOTA-NHS ester. c(RGDyK) (20 μmol, 12.4 mg), 25 μL of DIPEA, and 24.0 mg of NOTA-NHS ester (3 equiv) were dissolved in 0.6 mL of DMF. The reaction mixture was stirred at room temperature until all the starting peptide was consumed. The NOTA-RGD was isolated by RP-MPLC. The mobile phase was started, from 80% solvent A (H2O) and 20% solvent B (methanol), to 20% solvent A and 80% solvent B in 12 min at a flow-rate of 18 mL/min. The desired fractions (tR = 4.6 min) were collected and lyophilized to afford the final product. LC-MS: [M+H]+ = 905.18

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UltiMate® 3000 Variable Wavelength ultraviolet/visible detector operating at 214 nm, and a LabLogic Flow-RAM NaI/PMT radioactivity detector. The radioactivity was measured with a dose calibrator (CRC®-25R, Capintec). Distribution of activity on instant thin layer chromatography (iTLC) strip (iTLC-SG, Agilent) was analyzed using a radio-TLC imaging scanner (AR-2000, Bioscan). The activity in organs or tissues was measured with a gamma counter (2470 Wizard, PerkinElmer).

(m/z), calcd 904.44 (C39H60N12O13).

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ACCEPTED MANUSCRIPT Radiolabeling of NOTA-RGD-GE11, NOTA-RGD and NOTA-GE11 peptides with gallium-68 For radiolabeling, 0.5 mL gallium-68 (~185 MBq) and 5 nmol of NOTA-conjugated peptides were added to 0.15 mL 1M HEPES buffer. The final pH of the reaction mixture was 4.1 ~ 4.3, which incubated at room temperature for 15 min. For quality control, the product was analyzed by both radio-HPLC and radio-TLC. For radio-TLC, iTLC with 0.1 M EDTA as the mobile phase was used. In this system, radiolabelled peptides remain at the origin (Rf = 0-0.1) while free gallium-68 migrates with the solvent front (Rf = 0.8-1). For radio-HPLC, A Phenomenex Luna® C-18

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column (5 μm, 100 Å, 250 × 4.6 mm) was used with the mobile phase started from 85% solvent A (0.1% TFA in water) and 15% solvent B (0.1% TFA in acetonitrile) to 100% solvent B in 15 min at a flow-rate of 1 mL/min. The retention times of [68Ga]Ga-NOTA-RGD-GE11, [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 were 9.1 min, 6.2 min and 9.7 min, respectively. Cell culture and tumour xenografts NCI-H292 human lung carcinoma cells and MRC-5 human normal lung cells were cultured in RPMI 1640 medium and in DMEM medium respectively (media

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Western blotting NCI-H292 cells and MRC-5 cells were washed twice with ice-cold PBSV (1 mM Na3VO4). Lysis buffer was the radioimmunoprecipitaion assay (RIPA) buffer containing with 1 mM Na3VO4, 1 mM phenylmethanesulphonylfluoride (PMSF), 10 ng/mL aprotinin and 10 ng/mL leupeptin. The cells were detached by the scraper, and cell suspension was transferred from each dish to a new microcentrifuge tube and then centrifuged at 12000 rpm at 4oC for 10-15 minutes. The supernatants were transferred to new microcentrifuge tubes. The 5X sample buffer (250 mM Tris-HCl pH 6.8, 10%

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contain 10% fetal bovine serum and 1% penicillin/streptomycin), in a humidified atmosphere with 5% CO2 at 37oC. Subcutaneous xenografts of NCI-H292 tumour were produced in BALB/c nude mice (5 ~ 6-weeks old, BioLASCO Taiwan Co., Ltd, Taipei, Taiwan) under anesthesia. Tumour xenografts were produced by subcutaneous injection of 5 x 106 tumour cells in 0.2 mL of PBS at right forefoot. Studies were conducted when the tumours were ~ 0.5 cm in diameter at 3 ~ 4 weeks after inoculation. All experiment protocols were approved by the Institutional Animal Care and Use Committee of the Institute of Nuclear Energy Research (Taoyuan, Taiwan).

SDS, 30% Glycerol, 5% β-mercapitalethanol, 0.02% bromophenol blue) was added into each sample to the final 1X of concentration. Samples were heated at 95oC for 5 minutes. The samples with equal amounts of protein were separated by SDS-PAGE 5

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with 10% separating gel. The gel was then transferred to nitrocellulose (NC) paper at 100V for 100 min (Bio-Rad) in transfer buffer (25 mM Tris base, 190 mM glycine, and 20% (v/v) methanol). Membrane was rinsed two times by Tris-buffered saline/Tween-20 (TBST), and then incubated in blocking buffer (TBST containing 5% skim milk) at room temperature for one hour. After one hour blocking, membrane was incubated with the primary antibody at 4oC, overnight. Afterwards, membrane was washed three times for 15 minutes each with TBST. Subsequently, membrane was incubated with HRP-conjugated secondary antibody at room temperature for two hours and then washed three times for 15 minutes each with TBST. The protein signal was

Immunofluorescence staining NCI-H292 Cells were fixed by 4% paraformaldehyde for 15 mins, washed with

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detected through scanning using ImageQuant LAS-4000 Imaging System (Fujitsu Life Sciences).

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PBS twice and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min, washed three times with PBS and then incubated with blocking buffer of 1% BSA in PBS overnight at 4oC. Fixed cells were incubated with primary antibodies (anti-integrin αV and anti-EGFR, Abcam) for two hours at room temperature, followed

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by Alexa Fluor fluorescent-conjugated secondary antibodies for one hour. DAPI staining was used to mark the location of the nucleus for 15 min at room temperature. Cells were then mounted with ProLong® and the fluorescence images were taken using Olympus fluorescence microscope. Competitive ELISA assay Ninety six-well plates which were pre-seeded with cells (5 x 104/200 mL) were fixed by 4% paraformaldehyde for 15 min, washed with PBS twice. Permeabilized

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with 0.1% Triton X-100 in PBS at room temperature for 10 min, washed three times with PBS and then incubated with blocking buffer of 1% BSA in PBS for 1 h at room temperature. Fixed cells were incubated with increasing NOTA-RGD-GE11 peptide concentration (0 - 10-5 M) for 2 h at room temperature, followed by addition primary antibodies (anti-integrin αV and anti-EGFR) for 2 h. The HRP-conjugated secondary antibodies was added for 1 h, and substrate ABTS was added and incubated for overnight at room temperature. The absorbance of each sample at 450 nm was measured by a multilabel plate reader (Perkin Elmer Victor 2). 2.7

Cell uptake assay Twenty-four hours after seeding in 6-well plates at 1×106 cells/well, the medium were removed and replaced with 2 mL of serum-free medium containing 68Ga-labelled 6

ACCEPTED MANUSCRIPT peptides (74 kBq/mL). The cells were incubated for 30 min - 2 h. The activity in the cells and the supernatants were measured by a γ-counter (2470 Wizard, PerkinElmer). 68 Ga-labelled peptide uptake was calculated as 100% × CPMcells/(CPMcells + CPMsupernatants)/1×106 viable cells. Cytotoxicity Assay NCI-H292 Cells were seeded in a 96-well plate at density of 5000 cells/well in 100 μL culture medium and incubated at 37°C, 5% CO2 for 24 hours. Then 10 μL of different concentrations of NOTA-RGD-GE11 peptide (0 ~ 10-5 M) was added into the

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culture medium and incubated at different time points (0, 0.5, 1, 3 and 5 hours). Finally, 10 μL of CCK-8 solution (Enzo Life Sciences) was added to each well and incubated for 2 h. The absorbance of each sample at 450 nm was measured by a SpectraMax 190 UV/Vis microplate reader (Molecular Devices). In vitro stability test The in vitro stability was evaluated by incubation of 0.1 mL 68 [ Ga]Ga-NOTA-RGD-GE11 (1.11 MBq) with normal saline or mouse serum (0.9 mL) at 37°C at different time points (0, 15, 30, 60 and 120 min). The radiochemical purity

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of peptide was measured by radio-TLC method using iTLC-SG strip with 1.0 M NH4OAc/MeOH (1:1) as eluent. In this system, free gallium-68, insoluble and colloidal products stays at the origin (Rf = 0-0.1) and the radiolabelled peptide with is eluted with the solvent front (Rf = 0.8-1).

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2.10 Biodistribution of [68Ga]Ga-NOTA-RGD-GE11,[68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 peptides in NCI-H292 tumour-bearing nude mice The tumour-bearing mice were injected with 68Ga-labelled peptides via tail vein at

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the dose level of 3.7 q/0.1 mL per mice. The animals were sacrificed at 2 h post-injection (n = 3/group). The major organs and tissues were collected, wet-weighed, and counted in a γ-counter. The percentage injected dose per gram (% ID/g) was determined for each sample. 2.11 PET/CT imaging of [68Ga]Ga-NOTA-RGD-GE11, [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 peptides in NCI-H292 tumour-bearing nude mice When the tumour diameter of about 0.5 cm was reached, PET/CT was performed to evaluate the distribution and tumour targeting of 68Ga-labelled peptides in tumour-bearing mice. PET images and X-ray CT images were acquired using a NanoPET/CT scanner (Bioscan). The mice were anesthetized with 0.1% isofluorane in 7

ACCEPTED MANUSCRIPT oxygen during the imaging. Dynamic PET images were acquired at 0~2 hours after

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intravenous tail injection of 68Ga-labelled peptides (7.4 q/0.1 mL, n = 3/group). For the blocking experiment, 15-min static PET scans of tumour-bearing mice were acquired at 2 h after coinjection with c(RGDyK) at 10 mg/kg + GE11 at 15 mg/kg body weight and 7.4 MBq of [68Ga]Ga-NOTA-RGD-GE11 (n = 3). To estimate the activity concentration, volumes of interest were defined on coregistrated PET/CT images using PMOD software, and the activity in tissues was expressed as %ID/g. 2.12 Statistical analysis

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The statistical significance of comparisons of tissue uptake in animal studies was based on 2-sided, 2-sample Student t tests. Values of P < 0.05 were considered

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statistically significant. Results are reported as mean  SD.

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3. Results

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3.1 Chemistry and Radiolabeling The NOTA conjugates of RGD-GE11, RGD and GE11 (Fig. 1) were analyzed by mass spectroscopy to confirm the molar mass of the products. Gallium-68 eluted from

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a 68Ge/68Ga generator with 0.1N HCl was used directly for the radiolabeling at room temperature. The 68Ga labelled peptides could be obtained within 15 min without further purification, with a molar activity of >35.2 MBq/nmol and a radiochemical purity of >95%. 3.2 Immunostaining and protein level of integrin αVβ3 and EGFR

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The distribution of integrin V3 and EGFR in NCI-H292 cells was shown in Figure 2A, integrin V and integrin3 co-localized in the cytoplasm in NCI-H292 cells.

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The fluorescent signal of integrin V was higher than integrin3. The expression of EGFR was found in the nucleus and cytoplasm (Figure 2B). These data showed that the distribution of integrin V/3 co-localized in the cytoplasm and EGFR localized in nucleus and cytoplasm. NCI-H292 cells have higher expression levels of EGFR than that of integrin V (Figure 2C, 2D). NCI-H292 cells have higher levels of integrin V (1.6 fold) and EGFR (1.8 fold) than that of MRC-5 cells (Figure 2E). 3.3 Competitive ELISA assay The absorbance for anti-integrin V and anti-EGFR antibodies was increased cell concentration (blue line), but this was not for secondary antibody (red line). result indicated that binding of primary antibodies was specific and number-dependent (Figure 3A, 3B). NOTA-RGD-GE11 inhibited the binding of 8

with This cell both

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anti-EGFR antibodies manner (Figure 3C).

to NCI-H292 These results

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NOTA-RGD-GE11 possessed significant both integrin V and EGFR receptor-binding affinities.

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3.4 Cell uptake assay, Cytotoxicity Assay and in vitro stability test The cell uptake of 68Ga-labelled peptides was examined in NCI-H292 cells (Figure 3D). At 15 min after incubation, the cell uptake of the 3 68Ga-labelled peptides had no significant difference (3.92 – 4.86%, P > 0.05). Then [68Ga]Ga-NOTA-GE11 had high

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cell uptake at 60 min (17.91  0.98%) and 120 min (21.35  0.66%) after incubation. [68Ga]Ga-NOTA-RGD-GE11 uptake apparently increased at 120 min after incubation

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(14.11 ± 0.73%). [68Ga]Ga-NOTA-RGD uptake gradually increased from 3.92  0.22% (15 min) to 6.34  0.30% (120 min). In cytotoxicity test, NCI-H292 cells were treated with different concentrations of

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NOTA-RGD-GE11 (0 ~ 10-5 M) for 3 h (Figure 4A), and were treated with peptide (10-5 M) or DMSO (2M) for different time (Figure 4B) by using Cell Counting Kit-8 (CCK-8) assays. With increasing concentration of peptide and incubation time, the cells did not show increased cell death. NOTA-RGD-GE11 (10-5 M) also did not affect

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morphology of the cells compared with DMSO (2M) treatment for 1 day (Figure 4C). The in vitro stability was evaluated by incubation of [68Ga]Ga-NOTA-RGD-GE11 with normal saline or mice serum at 37°C for 0 - 120 min (Figure 4D). [68Ga]Ga-NOTA-RGD-GE11 exhibited good stability in both normal saline and serum for up to 2 h post labeling, and no statistically significant change in radiochemical purity was found between different time points.

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3.5 Biodistribution of [68Ga]Ga-NOTA-RGD-GE11, [68Ga]Ga-NOTA-RGD and

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[68Ga]Ga-NOTA-GE11 peptide in NCI-H292 tumour-bearing nude mice We examined the biodistribution and tumour uptake of 68 68 68 [ Ga]Ga-NOTA-RGD-GE11, [ Ga]Ga-NOTA-RGD and [ Ga]Ga-NOTA-GE11 in NCI-H292 tumour xenograft mouse models (Figure 5). High levels of activity were detected in the liver, kidney and tumour. This result showed that these 68Ga labelled peptides were mainly excreted from the body through hepatic and renal pathways. In addition, the uptake values of other normal organs such as muscle, brain and heart were low. Tumour uptakes of [68Ga]Ga-NOTA-RGD-GE11, [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 were 3.034 ± 0.257, 2.271 ± 0.252 and 2.032 ± 0.350 % ID/g, respectively, at 2 hours after radiopeptides administration. The tumour-to-muscle (T/M) ratios of [68Ga]Ga-NOTA-RGD-GE11, [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 were 4.628 ± 0.926, 3.312 ± 0.637 and 3.092 ± 0.644 9

ACCEPTED MANUSCRIPT respectively. The tumour uptake and T/M ratio of [68Ga]Ga-NOTA-RGD-GE11 was higher than that of [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11. These results suggest that [68Ga]Ga-NOTA-RGD-GE11 showed higher tumour targeting ratio and has better specific binding ability for integrin Vβ3 and EGFR-expressing tumours than monomeric peptides [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11.

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3.6 PET/CT imaging of [68Ga]Ga-NOTA-RGD-GE11, [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 peptides in NCI-H292 tumour-bearing nude mice The tumour-targeting efficacy of [68Ga]Ga-NOTA-RGD-GE11, [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 peptides was evaluated in nude mice bearing NCI-H292 xenograft tumours by PET/CT imaging (Figure 6A). The uptake of tumour and muscle were quantified based on ROI analysis of the nanoPET/CT images by using PMOD

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software. The tumour uptake values and T/M ratios of [68Ga]Ga-NOTA-RGD-GE11, [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 peptides were 3.446 ± 0.548, 2.756

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± 0.483 and 2.408 ± 0.327 % ID/g and 4.397 ± 0.972, 3.114 ± 0.675, 2.876 ± 0.689 respectively at 2 hours after injection (Figure 6B). The T/M ratio of [68Ga]Ga-NOTA-RGD-GE11 was higher than that of [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11. There is no obvious uptake in tumours of blocked mice.

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PET/CT imaging and biodistribution studies demonstrated that the tumour targeting specificity and efficacy of [68Ga]Ga-NOTA-RGD-GE11 heterodimeric peptide were better than that of [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 monomeric peptides.

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4. Discussion In this study, we investigated the in vitro and in vivo characteristics of 68 [ Ga]Ga-NOTA-RGD-GE11 heterodimeric peptide in a dual integrin- and

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EGFR-positive NCI-H292 tumour model. In competitive binding assay, although we used antibodies instead of radiolabelled peptides ([125I]I-c(RGDyK), [125I]I-GE11) as competitive agents, the result did indicate that the NOTA-RGD-GE11 heterodimer can bind both integrin and EGFR in vitro. For verifying the binding specificity of NOTA-RGD-GE11, bombesin peptide was also tested in binding assay and showed no inhibitory effect on binding of both antibodies (data not shown). The limitation of this assay is lack of quantitative estimation (IC50) of binding affinity of NOTA-RGD-GE11 to receptors. In cell uptake study, [68Ga]Ga-NOTA-RGD-GE11 showed lower uptake than [68Ga]Ga-NOTA-GE11, but higher uptake than [68Ga]Ga-NOTA-RGD in NCI-H292 tumour cells. This may be due to the higher numbers of EGFR than integrin of NCI-H292 cells, and the hampered internalization of GE11-EGFR complex in the heterodimer by cell-surface RGD-integrin complex [17]. 10

ACCEPTED MANUSCRIPT In biodistribution and PET imaging studies, the tumour uptake of [ Ga]Ga-NOTA-RGD-GE11 was highest and the two monomeric 68Ga-labelled tracers had comparable tumour uptake. These results seem to conflict with the results of in vitro cell uptake study that [68Ga]Ga-NOTA-GE11 had highest uptake. One of the possible reasons for this discrepancy would be the expression of murine integrin β3 in NCI-H292 tumour vasculature. The increased integrin receptor numbers of tumour could increase the tumour uptake of [68Ga]Ga-NOTA-RGD-GE11 and [68Ga]Ga-NOTA-RGD in vivo. The second reason would be the in vivo synergistic effect of the two binding motifs in the heterodimer. The probability of overall binding

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and rebinding to tumour cells of heterodimer is supposed to be higher than that of monomer. The RGD and GE11 motifs of [68Ga]Ga-NOTA-RGD-GE11 were linked to NOTA through a 6-aminohexanoic acid and a maleimide respectively. It is impossible for the two motifs to bind both integrin and EGFR simultaneously. Another reason may be the results from the improved in vivo pharmacokinetics and circulation time of

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[68Ga]Ga-NOTA-RGD-GE11 over the two monomeric tracers. The in vivo specific dual receptor binding of [68Ga]Ga-NOTA-RGD-GE11 was confirmed by the blocking study. The uptake of [68Ga]Ga-NOTA-RGD-GE11 in the NCI-H292 tumour can be largely inhibited by co-injection with RGD and GE11 monomers. However, the high

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uptake of [68Ga]Ga-NOTA-RGD-GE11 in liver and kidney would be problematic; modification of the ligand structure (especially the linkers) is essential to lead to better pharmacokinetics. In the previous study of Liu et al. [17], The PC-3 tumours were clearly visible with high contrast after injection of [68Ga]Ga-NOTA-RGD-BBN. The tumour uptake of [68Ga]Ga-NOTA-RGD-BBN at 120 min and T/M ratio at 60 min determined from PET images were 4.04 ± 0.28 %ID/g and 4.8 respectively. Another previous study of Ma et al. showed that [18F]F-RGD-A7R heterodimeric peptide exhibited dual integrin and

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VEGF targeting properties both in vitro and in vivo. U-87MG tumours were detected with high contrast after injection of [18F]F-RGD-A7R with T/M ratio of about 4.5 [20]. The tumour uptake level of [68Ga]Ga-NOTA-RGD-GE11 at 2 h p.i. in this report was 3.034 ± 0.257 %ID/g with T/M ratio of 4.628 ± 0.926 and was comparable with that from previous studies. But the liver accumulation of [68Ga]Ga-NOTA-RGD-GE11 was high and has room for improvement by structural modification. Recently, it is encouraging that [68Ga]Ga-BBN-RGD has been translated into the clinic for the first-in-human [33] and early phase [34] studies. These studies indicated heterodimeric peptides would have great value in diagnosis of prostate and breast cancer. It would be interesting and worthy to investigate the effects of different combination of targets, linkers and chelators on the in vivo characteristics of the heterodimeric conjugates in future. 11

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5. Conclusion In this study, we prepared and evaluated three 68Ga-labelled tumour-targeted peptides,

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[68Ga]Ga-NOTA-RGD-GE11, [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11, as molecular probes for PET/CT imaging of a NCI-H292 tumor xenografted mouse model. These studies indicated that [68Ga]Ga-NOTA-RGD-GE11 would have great value in the diagnosis of tumour. The results of this study demonstrated that [68Ga]Ga-NOTA-RGD-GE11 has enhanced accumulation in NCI-H292 tumour in vivo

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when compared with the monomers. These results and other favorable properties of [68Ga]Ga-NOTA-RGD-GE11, such as easy radiolabeling and high molar activity, warrant its further investigation for dual receptor-targeted tumour imaging.

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Acknowledgments

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The authors did not report any conflict of interest. Funding provided by Atomic Energy Council (Taiwan) grant 104-2001-01-05-01.

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