Improved immuno-PET imaging of HER2-positive tumors in mice

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Article

Improved immuno-PET imaging of HER2-positive tumors in mice: Urokinase injection-triggered clearance enhancement of Cu-trastuzumab 64

Qin Ren, Kohta Mohri, Shota Warashina, Yasuhiro Wada, Yasuyoshi Watanabe, and Hidefumi Mukai Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01052 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 12, 2019

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Molecular Pharmaceutics

Improved immuno-PET imaging of HER2-positive tumors in mice: Urokinase injection-triggered clearance enhancement of 64Cu-trastuzumab

Qin Ren,†,‡ Kohta Mohri,†,‡ Shota Warashina,†,‡ Yasuhiro Wada,§,║ Yasuyoshi Watanabe,§,║ and Hidefumi Mukai*,†,‡

†Molecular

Network Control Imaging Unit, RIKEN Center for Life Science Technologies,

6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan ‡Laboratory

for Molecular Delivery and Imaging Technology, RIKEN Center for Biosystems

Dynamics Research, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan §Pathophysiological

and Health Science Team, RIKEN Center for Life Science Technologies,

6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan ║Laboratory

for Pathophysiological and Health Science, RIKEN Center for Biosystems

Dynamics Research, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan

*Laboratory for Molecular Delivery and Imaging Technology, RIKEN Center for Biosystems Dynamics Research, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. Tel: +81-78-304-7173. Fax: +81-78-304-7191. E-mail: [email protected]. 1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents/Abstract Graphic

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Abstract Immuno-positron emission tomography (immuno-PET) is expected to improve the specificity of small chemical tracers such as 18F-fluorodeoxyglucose. Whole antibodies significantly accumulate in target molecule-expressing tumors but frequently persist too long in the blood circulation for imaging purposes. We investigated the utility of whole antibodies, 64Cu-labeled

via a urokinase-substrate linker, and their exogenous urokinase-responsive

cleavage to enhance clearance of immuno-PET probes from the blood and shorten the time required to develop adequate imaging contrast. Specifically, we used 64Cu-labeled trastuzumab in human epidermal growth factor receptor 2 (HER2)-positive tumor-bearing mice. 64Cu-labeled trastuzumab with a urokinase-cleavage site (64Cu-CB-TE1A1P-USL-trastuzumab) was synthesized using a bifunctional chelator incorporating a urokinase substrate peptide. Urokinase cleavage was analyzed in vitro by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and radio-gel permeation-high performance liquid chromatography. Improvements in radioisotope clearance and HER2-imaging by urokinase injection were evaluated by PET imaging and ex vivo biodistribution studies in A431 tumor-bearing mice. 64Cu-CB-TE1A1P-USL-trastuzumab

was cleaved into smaller radioactive fragments by

20,000 IU/mL urokinase treatment in vitro at an efficacy of ~95%. The probe targeted HER2 in A431 tumors in mice within 24 h post-injection, and approximately two-thirds of the probe in the blood circulation was eliminated via renal clearance of radioactive fragments after three urokinase injections. Therefore, the tumor/blood ratio increased 3.0-fold. Without urokinase injection, the tumor accumulation of 64Cu-CB-TE1A1P-USL-trastuzumab slowly increased and the blood radioactivity decreased over 72 h. However, the tumor/blood ratios in mice after three urokinase injections were higher at 24 h than those in mice without injections at 72 h. The results indicate that our approach shortened the time required to develop 3 ACS Paragon Plus Environment


adequate imaging contrast of immuno-PET by > 2 days. Therefore, this approach can benefit high-sensitivity imaging under lower radioactive decay conditions and can decrease patient radiation exposure. In addition, it could reduce other adverse effects of radioimmunotherapy.

Keywords: immuno-positron emission tomography, urokinase, trastuzumab, clearance enhancement, imaging contrast, cancer imaging


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Introduction

Immuno-positron emission tomography (immuno-PET), which uses positron emitter-labeled monoclonal antibodies, is expected to improve the specificity of small chemical tracers such as 18F-fluorodeoxyglucose.1,2 To date, it has been used preclinically and clinically to visualize several molecular targets in tumors, including human epidermal growth factor receptor 2 (HER2) and CD20.3-6 Immuno-PET could be a companion diagnostic for molecular target medicines and could become an alternative to biopsy.2,4,6,7 Radiolabeled whole antibodies significantly accumulate in target molecule-expressing tumors but frequently persist in the blood circulation too long for ideal imaging purposes.8 Consequently, time must pass to develop adequate contrast, extending patient radiation exposure unnecessarily. To overcome this problem, several approaches have been investigated. First, antibody fragments and small antibody mimetics were used instead of whole antibodies to shorten retention time in the blood circulation.8,9 Unfortunately, target-site accumulation also dropped, resulting in inadequate imaging contrast in many cases. Second, a pre-targeting approach was proposed.10-12 In this approach, bio-orthogonally conjugatable, positron emitter-labeled small chemicals are injected as PET probes, after whole antibodies with reactive partners have been distributed to the target sites. The PET probes are rapidly cleared from the blood circulation. Therefore, the pre-targeting approach requires shorter times to develop adequate contrast after PET probe injection, which significantly improves imaging contrast. However, it is difficult to optimize the timing of small chemical PET probe injection to obtain the best imaging contrast. Third, triggered clearance enhancement of PET probes in circulation after probe accumulation at the target sites may shorten the time required for adequate imaging contrast. Recently, we reported that intravenous injection of urokinase, a 5 ACS Paragon Plus Environment


clinical thrombolytic drug,13 and its substrate linker was a potential triggered radioisotope clearance enhancement system to improve four-arm polyethylene glycol-conjugated (PEGylated) 64Cu-bombesin analog tetramer imaging.14 This approach is potentially expandable to immuno-PET. In the present study, we investigated the utility of urokinase injection-triggered radioisotope clearance enhancement to improve immuno-PET imaging using the anti-HER2 monoclonal antibody, trastuzumab.4,5,15 We synthesized a urokinase-cleavable bifunctional chelator with 1,4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid) (CB-TE1A1P) for 64Cu-labeling16,17 and dibenzocyclooctyne (DBCO) for the strain-promoting azide-alkyne cycloaddition reaction. Then, the 64Cu-labeled trastuzumab, with an inserted urokinase-cleavage site, was obtained using the bifunctional chelator (Figure 1). After analyzing urokinase-responsive cleavage and radioactive fragment generation in vitro, we evaluated urokinase injection-triggered radioisotope clearance enhancement and HER2-imaging using dynamic PET studies in A431 tumor-bearing mice, which express HER2 in the tumor tissue.18


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Figure 1. Schematic drawing of the synthesis of 64Cu-trastuzumab with a urokinase-cleavage site, 64Cu-CB-TE1A1P-USL-trastuzumab. Abbreviations: EDT, 1,2-ethanedithiol; RT, room temperature; TIS, triisopropylsilane.



Experimental Section

Synthesis of the urokinase-cleavable bifunctional chelator, CB-TE1A1P-urokinase substrate linker (USL)-DBCO. 1,4,8,11-Tetraazabicyclo[6.6.2]hexadecane-4-(methanephosphonic acid di-tert-butyl ester)-11-methanecarboxylic acid (CB-TE1A1P(tBu)2) was synthesized by the NARD Institute, Ltd. (Amagasaki, Japan) using a previously described procedure16 with slight modifications. A urokinase substrate peptide (Gly-Ser-Gly-Arg-Ser-Ala-Gly), with CB-TE1A1P covalently linked through the side chain of a Lys at its N-terminus and Cys at the C-terminus (i.e., CB-TE1A1P-USL-Cys), was synthesized on a NovaSyn® TGR resin (Merck, KGaA, Darmstadt, Germany) by standard solid-phase synthesis based on Fmoc chemistry (Figure 1). After cleavage from the resin and deprotection, the crude product was purified by using a TOYOPAK ODS M (Tosoh Corp., Tokyo, Japan). The CB-TE1A1P-USL-Cys was then reacted with DBCO-PEG4-maleimide (Jena Bioscience GmbH, Jena, Germany) at a molar ratio of 1:1.25 in phosphate-buffered saline (PBS, pH 7.0) at 37 °C for 1 h (Figure 1). The CB-TE1A1P-USL-DBCO was purified by reversed-phase high performance liquid chromatography (RP-HPLC) on a Shimadzu Prominence HPLC system (Shimadzu Corp., Kyoto, Japan). An octadecyl-silica (ODS) column (COSMOSIL C18-AR-II, 10 × 250 mm; Nacalai Tesque, Inc., Kyoto, Japan) was used; the eluents were (A) water with 0.1% trifluoroacetic acid (TFA) and (B) acetonitrile with 0.1% TFA. A linear gradient elution was performed from 5 to 90% B in A over 20 min at 40 °C. The flow rate was 2.5 mL/min. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed using an ultrafleXtreme instrument (Bruker Daltonics, Inc., Billerica, MA, USA) and 2,5-dihydroxybenzoic acid (Bruker Daltonics, Inc.) as a matrix to detect CB-TE1A1P-USL-DBCO (m/z 1897.96 [(M + H)+], calcd. = 1897.90) and 8 ACS Paragon Plus Environment

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CB-TE1A1P-USL-Cys (m/z 1223.60 [(M + H)+], calcd. = 1223.60) (Figure S1). In addition, CB-TE1A1P-DBCO (without USL) was synthesized by the same method as described above.

Conjugation of trastuzumab with CB-TE1A1P-USL-DBCO. Trastuzumab (Herceptin; Chugai Pharmaceutical Co. Ltd., Tokyo, Japan) was reacted with 5 equivalents of 15-azido-4,7,10,13-tetraoxapentadecanoic acid N-succinimidyl ester (N3-PEG4-NHS; Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) in 10 mM PBS (pH 7.4) at room temperature (15– 25 °C) for 3 h, followed by purification using a PD-10 gel filtration column (GE Healthcare UK Ltd., Buckinghamshire, UK). The azide-modified trastuzumab obtained was then reacted with 25 equivalents of CB-TE1A1P-USL-DBCO or CB-TE1A1P-DBCO in PBS (pH 7.4) at 37 °C for 3 h, followed by purification using a PD-10 gel filtration column. The average number of azide and CB-TE1A1P-USL-DBCO substitutions per trastuzumab was determined by MALDI-TOF MS, wherein sinapinic acid (Bruker Daltonics, Inc.) was used as a matrix.

64Cu-labeling. [64Cu]CuCl 2

was obtained as previously described.14 Fifty micrograms of

CB-TE1A1P-USL-trastuzumab or CB-TE1A1P-trastuzumab was reacted with approximately 300 MBq [64Cu]CuCl2 in 0.1 M ammonium acetate buffer (pH 8.2, 250 L) at 37 °C for 1 h. The crude product was filtered three times in 0.2 M glycine buffer (pH 6.5), followed by three times in 10 mM PBS containing 0.05% Tween 20, using an Amicon ultra centrifugal filter (MWCO, 50 kDa; Millipore, Merck KGaA, Darmstadt, Germany). The labeling efficiency and radiochemical purity was were determined by radio-thin-layer chromatography (radio-TLC). A TLC Silica gel 60 RP-18 F254S 20 aluminum sheet (Merck Millipore, Merck KGaA) was used and the mobile phase was 40% methanol containing 100 mM ethylenediaminetetraacetic acid. The labeling efficiency of 64Cu-CB-TE1A1P-USL-trastuzumab

was 64.8  9.5% (n = 3). 9

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Quartz crystal microbalance (QCM) measurements. Recombinant human ErbB2/HER2 Fc chimeric protein (R&D Systems, Inc., Minneapolis, MN, USA) was immobilized by simple adsorption on a sensor chip. After blocking with 5% bovine serum albumin in PBS (pH 7.4), 5 μL aliquots of either trastuzumab or 64Cu-CB-TE1A1P-USL-trastuzumab

were added 5–7 times to the PBS (pH 7.4, 500 μL) in

the reaction vessel; QCM sensorgrams were recorded by a Single-Q 0500 (AS ONE Corporation, Osaka, Japan). The dissociation constants (Kd) were calculated based on the Scatchard method using Q-up Analysis (AS ONE Corporation).

In vitro analyses of urokinase-responsive cleavage. Human urokinase was purchased from Prospec-Tany TechnoGene Ltd. (Ness-Ziona, Israel). The CB-TE1A1P-USL-trastuzumab was treated with 1,000 IU/mL of urokinase in PBS (pH 7.4) at 37 °C for 1 h. The reacted samples and deproteinized supernatants were analyzed by MALDI-TOF MS. 64Cu-CB-TE1A1P-USL-trastuzumab

was treated with 5,000 IU/mL of urokinase in PBS

(pH 7.4) at 37 °C for 1 h. The reacted samples were analyzed by radio-gel permeation HPLC (radio-GP-HPLC) on a Shimadzu Prominence HPLC system with a radio analyzer (RLC-700, Aloka Co., Ltd., Tokyo, Japan). The system used a gel permeation column (COSMOSIL 5Diol-300-II, 7.5 × 300 mm; Nacalai Tesque, Inc.) and 10 mM PBS (pH 7.4) containing 0.3 M NaCl as an eluent at room temperature (15–25 °C) and a flow rate of 1 mL/min. In addition, 64Cu-CB-TE1A1P-USL-trastuzumab was treated with urokinase (0–20,000 IU/mL) in mouse blood at 37 °C for 1 h (test samples). Separately, the probe was completely cleaved by 5,000 IU/mL of urokinase in PBS (pH 7.4) and incubated with mouse blood as a positive control. The reacted samples were mixed with equal volumes of acetonitrile and 10 ACS Paragon Plus Environment

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centrifuged. The radioactivity in the supernatants was then measured. The cleavage rate was calculated using the following formula: cleavage rate (%) = [(radioactivity of test sample)/(radioactivity of positive control)]  100.

Cell line. The A431 human epidermoid carcinoma cell line (RCB0202) was provided by the RIKEN BioResource Center (Tsukuba, Japan) through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. The cells were cultured in RPMI 1640 (Nacalai Tesque, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (South America origin; Life technologies, Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 U/mL; Nacalai Tesque, Inc.), and streptomycin (100 g/mL; Nacalai Tesque, Inc.) at 37 °C in a humidified atmosphere containing 5% CO2.

Experimental animals. Male BALB/c nu/nu mice (4-week-old) and male BALB/c mice (7-week-old) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). A431 tumor-bearing mice were produced via subcutaneous injection of 1 × 106 A431 cells in 100 L Hank’s balanced salt solution (HBSS, Nacalai Tesque, Inc.) into the right axillary region of the BALB/c nu/nu mice. Once the tumors reached diameters of approximately 5–10 mm after ~14 days, the mice were ready for experimentation. All animal experimental protocols were approved by the Ethics Committee on Animal Care and Use of the RIKEN Kobe Institute and were performed in accordance with the Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985).

PET studies and time-activity curve analyses. PET studies were performed as previously described with slight modifications.14 Under anesthesia with a mixture of 1.5% 11 ACS Paragon Plus Environment


isoflurane and nitrous oxide/oxygen (7:3), mice were intravenously injected with 64Cu-CB-TE1A1P-USL-trastuzumab

or 64Cu-CB-TE1A1P-trastuzumab (10–12.5 MBq, 3.0–

3.8 g in 200 µL saline). Conscious mice were subsequently placed in cages. Approximately 24 h later, the mice were anesthetized and placed in a prone position in the center of the PET scanner gantry (microPET Focus 220, Siemens Co., Knoxville, TN, USA). Emission data for 64Cu-CB-TE1A1P-USL-trastuzumab

and 64Cu-CB-TE1A1P-trastuzumab were acquired for

3.5 h and 1.5 h in 3-D list mode, respectively. Urokinase was intravenously injected at a dose of 40,000 IU three times at 0.5, 1.5, and 2.5 h or once at 0.5 h during the PET scan. Acquired emission data were sorted into 42 or 18 dynamic sinograms (42 or 18 × 300 s). For PET imaging without urokinase injection, the 0.5-, 1-, and 2-h emission scans were acquired at 24, 48, and 72 h after probe injection, respectively, and then converted into static sinograms. For the competitive inhibition study, 0.5 mg trastuzumab was intravenously injected 20 min before probe injection. PET images were reconstructed using microPET manager v. 2.4.1.1 (Siemens Co.), as previously described.14 The radioactivity in each pixel was decay-corrected from the time of injection and expressed as the standardized uptake value (SUV), where SUV = [tissue radioactivity concentration (MBq/cm3)]/[injected radioactivity (MBq)/body weight (g)]. The experiments were replicated at least three times for each group and representative images are shown in the figures. Time-activity curve analyses were performed as previously described,14 except that different image-processing software was used (PMOD v. 3.612; PMOD Technologies LLC, Zurich, Switzerland). The average regional radioactivity concentration in the heart, liver, and A431 tumor was expressed as the SUV. The total radioactivity in the bladder was expressed as the percentage of injected dose.

Ex vivo measurements of radioactivity biodistribution. After the PET studies or at the 12 ACS Paragon Plus Environment

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indicated hours after probe injection, blood and tissues were collected from the euthanized mice. Ex vivo measurements of radioactivity biodistribution were performed as previously described,14 except that a different automatic gamma counter was used (2480 WIZARD2; PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). The radioactivity in each tissue was expressed as the percentage injected dose per g of tissue (%ID/g).

Statistical analyses. Each value represents the mean with standard deviation (SD). GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA) and IBM SPSS Statistics v. 24 (IBM Corp., Armonk, NY, USA) were used. Statistical significance was determined using unpaired Student’s t-tests for two groups. One-way or two-way factorial analysis of variance was performed for multiple comparisons among different groups, followed by Tukey’s or Bonferroni’s test. All P values were two-tailed and differences with P < 0.05 were considered statistically significant.



Results

Synthesis of 64Cu-trastuzumab with urokinase-cleavage site, 64Cu-CB-TE1A1P-USL-trastuzumab.

The urokinase-cleavable bifunctional chelator,

CB-TE1A1P-USL-DBCO, was synthesized by a combination of solid-phase peptide synthesis and thiol-maleimide coupling. An average of approximately 2.5 azide groups was introduced into trastuzumab at the amino group of its N-terminus or lysine residues using N3-PEG4-NHS. The azide-modified trastuzumab was then conjugated with CB-TE1A1P-USL-DBCO via a strain-promoting azide-alkyne cycloaddition reaction (Figure 1). The conjugate contained an average of approximately 1.0 chelator per trastuzumab molecule. After radiolabeling, 64Cu-CB-TE1A1P-USL-trastuzumab was produced. Its radiochemical purity and specific activity were 96.7  0.7% (n = 5) and 499  74 MBq/nmol (n = 5), respectively. In addition, the dissociation constant of 64Cu-CB-TE1A1P-USL-trastuzumab

(0.57  0.36 nM; n = 4) did not change from that of the

parent trastuzumab (0.32  0.07 nM; n = 5; P = 0.25).

Urokinase-responsive cleavage of 64Cu-CB-TE1A1P-USL-trastuzumab. The CB-TE1A1P-USL-trastuzumab (not labeled with 64Cu) was examined by MALDI-TOF MS after a 1-h incubation at 37 °C with 1,000 IU/mL urokinase in PBS (Figure S21). In the low-mass region, the major peaks, with m/z values of 906.61 and 928.59, corresponded to those of Ac-Lys(CB-TE1A1P)-Gly-Ser-Gly-Arg-OH (m/z calcd. for [(M + H)+]: 906.51 and calcd. for [(M + Na)+]: 928.91). In the high-mass region, the antibody-related peak shifted by 921 to a smaller m/z value, compared with that of the original CB-TE1A1P-USL-trastuzumab. This shift is consistent with the separation of the peptide fragment detected in the low-mass region described above from 14 ACS Paragon Plus Environment

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CB-TE1A1P-USL-trastuzumab. The 64Cu-CB-TE1A1P-USL-trastuzumab was analyzed by radio-GP-HPLC after a 1-h incubation at 37 °C with 5,000 IU/mL urokinase in PBS (Figure 2A). The 7.8-min peak of the original probe almost completely disappeared. Concurrently, the 11.0-min peak appeared, which corresponded to the retention time of peptide PET probes. The urokinase concentration-dependent cleavage of 64Cu-CB-TE1A1P-USL-trastuzumab in mouse blood was examined based on the recovery of radioactivity in deproteinized supernatants (Figure 2B), because the radioactive peptide fragments generated by urokinase treatment remained soluble after protein precipitation. The cleavage rate increased with increasing urokinase concentration and reached ~95% at 20,000 IU/mL urokinase. Considering that the total blood volume per mouse is approximately 2 mL, the urokinase dose was determined to be 40,000 IU per mouse per injection in the following PET studies.



Figure 2. Urokinase-mediated cleavage of 64Cu-CB-TE1A1P-USL-trastuzumab. (A) Representative radio-GP-HPLC chromatograms of 64Cu-CB-TE1A1P-USL-trastuzumab before and after 1-h incubation with 5,000 IU/mL urokinase at 37 °C. (B) Cleavage rate of the probe reacted with 0–20,000 IU/mL urokinase in mouse blood for 1 h at 37 °C. Each value represents the mean  SD (n = 4).

Urokinase injection-triggered clearance enhancement of 64Cu-CB-TE1A1P-USL-trastuzumab

and HER2-PET imaging improvement in A431

tumor-bearing mice. The impact of multiple intravenous urokinase injections on dynamic radioactivity biodistribution patterns in the PET imaging with 64Cu-CB-TE1A1P-USL-trastuzumab

was investigated (Figure 3). PET images and 16


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time-activity curves of the heart revealed that each urokinase injection enhanced the elimination of radioactivity from the blood circulation within ~15 min (Figures 3B–D). The radioactivity of the heart decreased by 24–31% after each urokinase injection and decreased by 64% after three urokinase injections. In contrast, the cardiac radioactivity did not change in the control group receiving saline. The hepatic radioactivity decreased similarly after urokinase injection (Figure 3D). Specifically, the rate of decrease for each urokinase injection was 11–19% and the total decrease for the triple urokinase injections was 40%. Furthermore, the total bladder radioactivity increased only in the urokinase-injected group and reached 4.3% injected dose after three urokinase injections (Figure 3D). In contrast, the tumor radioactivity barely changed in both groups (Figure 3D). Unlike that of 64Cu-CB-TE1A1P-USL-trastuzumab, 64Cu-CB-TE1A1P-trastuzumab

the radioactivity biodistribution of

was not altered by the urokinase injections (Figure S3).




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Figure 3. Urokinase injection-triggered clearance enhancement of 64Cu-CB-TE1A1P-USL-trastuzumab

and improvement in HER2-positive tumor PET

imaging. (A) Schematic of experimental protocol. (B) and (C) Representative maximum intensity projection PET images over a time course of 3.5 h for A431 tumor-bearing mice receiving 64Cu-CB-TE1A1P-USL-trastuzumab 24 h before the scan. Mice were injected with (B) 40,000 IU urokinase or (C) saline three times over 1-h intervals. Yellow arrows indicate A431 tumor regions. (D) Time-activity curves of the heart, liver, bladder, and tumor for the urokinase-injected (closed circles) and saline-injected control (open circles) groups. Each value represents the mean + or − SD (n = 3–4). Error bars smaller than the symbols are not shown.

The radioactivity biodistribution data measured ex vivo were consistent with those of the PET study (Figure 4A). The radioactivities in the blood and urine of the urokinase-injected group were decreased 3.3× and increased 10×, respectively, compared with those of the control group that received saline, and these differences were significant. Conversely, the tumor accumulation of radioactivity was equivalent between the two groups. In addition, the radioactivities of the lung and heart in the urokinase-treated group were significantly lower than those in the control group receiving saline. Unlike the findings in the PET study, there was no difference in the hepatic radioactivity between the two groups, likely because the tissues were dissected after blood collection. The tumor/blood ratio in the urokinase-injected group, which is a contrast indicator, increased 3.0× compared with that in the control group receiving saline, and this increase was significant (Figure 4B).



Figure 4. Changes in biodistribution pattern of 64Cu-CB-TE1A1P-USL-trastuzumab radioactivity by urokinase injection. (A) Radioactivity biodistribution data measured ex vivo in tissues and (B) tumor/blood ratio of radioactivity at approximately 1 h after PET imaging (approximately 28.5 h after probe injection) for urokinase-injected (black bars) and saline-injected control (white bars) groups. †P < 0.05; †††P < 0.001. Each value represents the mean + SD (n = 3–4). SUV, standardized uptake value.

To validate the HER2 specificity of 64Cu-CB-TE1A1P-USL-trastuzumab for tumor accumulation, a competitive inhibition study was performed. The immediate pre-injection of 0.5 mg trastuzumab reduced tumor accumulation 2.3 times at 24 h and this effect was significant (Figure 5). In addition, when the biodistribution of 64Cu-CB-TE1A1P-USL-trastuzumab

was traced over 72 h, the blood radioactivity decreased

by 32% and the tumor accumulation slowly increased by 63% between 24 and 72 h (Figure 5). Accordingly, the tumor/blood ratio increased and reached 1.83  0.72 at 72 h, which was 20 ACS Paragon Plus Environment

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equivalent to or slightly lower than that of the urokinase-injected group at 24 h (Figures 4B and 5C).

Figure 5. HER2 specificity of 64Cu-CB-TE1A1P-USL-trastuzumab for tumor accumulation and biodistribution over 72 h. (A) Representative maximum intensity projection PET images, (B) radioactivity biodistribution data measured ex vivo in tissues, and (C) tumor/blood radioactivity ratio of A431 tumor-bearing mice receiving 64Cu-CB-TE1A1P-USL-trastuzumab,

approximately 24, 48, and 72 h post-injection. For the 21



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competitive inhibition group, mice received 0.5 mg trastuzumab intravenously 20 min before *

**

probe injection. Yellow arrows indicate A431 tumor regions. P < 0.05; P < 0.01; 0.001;

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P < 0.0001. Each value represents the mean + SD (n = 3–4).


***

P

Improved immuno-PET imaging of HER2-positive tumors in mice

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