Abstract
The purpose of this study was to develop 64Cu-labeled trastuzumab with improved pharmacokinetics for human epidermal growth factor receptor 2 (HER2). Methods: Trastuzumab was conjugated with SCN-Bn-NOTA and radiolabeled with 64Cu. Serum stability and immunoreactivity of 64Cu-NOTA-trastuzumab were tested. Small-animal PET imaging and biodistribution studies were performed in a HER2-positive breast cancer xenograft model (BT-474). The internal dosimetry for experimental animals was determined using the image-based approach with the Monte Carlo N-particle code. Results: 64Cu-NOTA-trastuzumab was prepared with high radiolabeling yield and radiochemical purity (>98%) and showed high stability in serum and good immunoreactivity. Uptake of 64Cu-NOTA-trastuzumab was highest at 48 h after injection as determined by PET imaging and biodistribution results in BT-474 tumors. The blood radioactivity concentrations of 64Cu-NOTA-trastuzumab decreased biexponentially with time in both mice with and mice without BT-474 tumor xenografts. The calculated absorbed dose of 64Cu-NOTA-trastuzumab was 0.048 mGy/MBq for the heart, 0.079 mGy/MBq for the liver, and 0.047 mGy/MBq for the spleen. Conclusion: 64Cu-NOTA-trastuzumab was effectively targeted to the HER2-expressing tumor in vitro and in vivo, and it exhibited a relatively low absorbed dose due to a short residence time. Therefore, 64Cu-NOTA-trastuzumab could be applied to select the right patients and right timing for HER2 therapy, to monitor the treatment response after HER2-targeted therapy, and to detect distal or metastatic spread.
See an invited perspective on this article on page 23.
Human epidermal growth factor receptor 2 (HER2), a transmembrane receptor tyrosine kinase 2, is overexpressed in gastric cancer, ovary cancer, prostate cancer, and lung cancer, as well as breast cancer. HER2 is a proven therapeutic target for breast and gastric cancer (1,2); it is highly expressed in 15%–20% of breast cancers, and HER2-positive characteristics are associated with more aggressive growth, worse prognosis, greater possibility of recurrence, and shorter survival than for HER2-negative breast cancer (3). Several anti-HER2 agents have been successfully developed and applied to HER2-positive breast cancer, including trastuzumab and pertuzumab as antibody therapeutics and lapatinib and neratinib as HER2 tyrosine kinase inhibitors (4).
Trastuzumab is a humanized monoclonal antibody that targets the extracellular portion of HER2 and is the first HER2-targeted agent approved by the U.S. Food and Drug Administration for treating both early-stage and metastatic HER2-overexpressing breast cancer (5). For effective treatment with HER2-targeting agents, it is important to validate HER2 expression in the primary tumor and metastatic sites. Although there is a routine examination of HER2 expression using immunohistochemistry or fluorescence in situ hybridization, technical problems can arise when lesions are not easily accessible by core-needle biopsy, including whole-body metastasis. In addition, HER2 expression can vary during the course of the disease and even among tumor lesions in the same patient. Thus, a PET imaging probe using radiolabeled antibodies based on Food and Drug Administration–approved trastuzumab has been studied for the noninvasive evaluation of HER2 expression (6). Additionally, a molecular imaging technique with radiolabeled trastuzumab can be applied to select the right timing for HER2 therapy, to monitor the treatment response after HER2-targeted therapy, and to detect distal or metastatic spread. For this purpose, several PET or SPECT agents with trastuzumab using 64Cu, 124I, 111In, and 89Zr were developed (7–10). Especially, 64Cu-DOTA-trastuzumab using DOTA as a bifunctional chelator was developed and applied to clinical studies for individualizing treatment of HER2-positive metastatic tumors and HER2-positive breast cancer (11–15).
In addition, a properly designed antibody–chelate immunoconjugate could serve as a therapeutic agent or imaging probe by introducing a radioisotope. 90Y-labeled ibritumomab tiuxetan (Zevalin; Spectrum Pharmaceuticals) is a radioimmunotherapy pharmaceutical for recurrent and resistant forms of low-grade follicular B-cell non-Hodgkin lymphoma. SPECT imaging with 111In-ibritumomab tiuxetan was performed to select the right patients for 90Y-ibritumomab tiuxetan treatment (16).
Because antibody molecules are labile to heat, pH, and agents such as salt and detergents, forming antibody–chelator conjugates before introducing the radioisotope is a proper strategy for preparation and quality control of radiopharmaceuticals with antibody molecules. In particular, an appropriately structured antibody–chelator conjugate could be applicable for therapeutic or imaging purposes by introducing a metallic therapeutic or imaging radioisotope such as 90Y-labeled ibritumomab tiuxetan or 111In-labeled ibritumomab tiuxetan. 64Cu, with a half-life of 12.7 h, is the most widely studied PET radioisotope. It can be produced via the 64Ni(p,n)64Cu reaction using a cyclotron in a carrier-free state (17). The optimal structure of the antibody–chelator conjugate for 64Cu could be used for therapeutic β-emitter 67Cu (half-life, 2.58 d) as a pair of radioisotopes of 64Cu (18). Because 64Cu-radiolabeled complexes with improved stability have been reported with NOTA derivatives (19), we introduced NOTA as a chelator for 64Cu labeling into trastuzumab antibody to provide the more favorable pharmacokinetic of 64Cu-labeled trastuzumab than that of DOTA as a chelator. Moreover, we can predict the internal absorbed radiation dose of 67Cu-labeled antibody using 64Cu-labeled antibody (20,21), which is a powerful advantage for 64Cu/67Cu-labeled antibody development as a PET probe or therapeutic agent (Supplemental Fig. 1; supplemental materials are available at http://jnm.snmjournals.org).
The purpose of this study was to develop 64Cu-labeled trastuzumab using NOTA as a chelator with improved pharmacokinetics for HER2 and to evaluate the pharmacokinetic characteristics and image-based absorbed dose of 64Cu-NOTA-trastuzumab for human study.
MATERIALS AND METHODS
Preparation of 64Cu-NOTA-Trastuzumab
Trastuzumab (Herceptin; Roche) was buffer-exchanged and concentrated to 10 mg/mL in 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, pH 8.5, using Vivaspin-20 centrifugal concentrators (Sartorius). A 20-fold molar excess of p-SCN-Bn-NOTA (Macrocyclics) in 100% ethanol was added to the antibody in 0.1 M HEPES buffer. After incubation at 4°C overnight for conjugation, the reaction mixture was purified and concentrated to 5 mg/mL with 0.1 M ammonium acetate buffer, pH 6, using Vivaspin-20. Mass spectrometry was performed to determine the number of chelates per antibody.
The immunoconjugate (NOTA-trastuzumab) was radiolabeled with 64Cu produced at KIRAMS by 50-MeV cyclotron irradiation (22). 64CuCl2 (74 MBq) was added to 1 mg of NOTA-trastuzumab. The reaction mixtures were incubated at room temperature for 1 h with constant agitation followed by testing of the radiolabeling yield and purity by instant thin-layer chromatography using silica gel paper (Agilent Technologies) as the stationary phase and 0.1 M ammonium acetate buffer, pH 6, with 50 mM ethylenediaminetetraacetic acid as the mobile phase.
Stability Testing of 64Cu-NOTA-Trastuzumab
We tested the in vitro serum stability of the radiolabeled antibodies using instant thin-layer chromatography as described above by incubating 100 μL (7.4 MBq) of radiolabeled antibodies in 100 μL of fresh human serum, mouse serum, and phosphate-buffered saline at 37°C for 1, 4, 6, 12, 24, and 48 h. To determine the shelf life at 4°C, we measured the radiochemical purity of 64Cu-NOTA-trastuzumab immediately and 24 h after labeling.
Cell Culture and Animals
The HER2-positive cell line, BT-474, was obtained from the American Type Culture Collection and was grown in RPMI-1640 medium (Corning) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and antibiotics (penicillin G, 100 units/mL, and streptomycin, 10 μg/mL; Gibco) at 37°C in a humidified 5% CO2 atmosphere.
All animal experiments were carried out under a protocol approved by the KIRAMS Institutional Animal Care and Use Committee (kirams2016-0097). Six-week-old female BALB/c nude mice were purchased from NARA Biotech. Ectopic or orthotopic xenografts were produced by implanting 1 × 107 BT-474 cells in right flank skin or mammary fat pad, respectively.
Immunoreactivity
In vitro immunoreactivity of the 64Cu-NOTA-trastuzumab samples was determined using specific radioactive cell-binding assays following procedures modified from Lindmo et al. (23). Briefly, 7 different serial dilutions of BT-474 cells starting at 6 × 106 cells/500 μL were prepared in phosphate-buffered saline (pH 7.4) supplemented with 1% bovine serum albumin. 64Cu-NOTA-trastuzumab (200 ng) was added to each tube (n = 3) and incubated for 3 h at 4°C. Cells were collected by centrifugation, resuspended, and washed twice with cold phosphate-buffered saline before the 64Cu-activity associated with the cell pellet was counted. Competitive inhibition (blocking) studies were conducted using the same procedure but with the addition of nonradiolabeled trastuzumab (A >1,000-fold excess of monoclonal antibody; 210 μg) to the 64Cu-NOTA-trastuzumab solutions. After incubation, the radioactivity of tubes was read using a 1480 Wizard γ-counter (PerkinElmer). The immunoreactive fraction was determined by performing a linear regression analysis on a double inverse plot of (total/bound) activity versus normalized cell concentration. The immunoreactive fraction was obtained from the inverse of the intercept on the plot.
Autoradiography and Immunohistochemistry
Immediately after PET scanning, the mice were killed and tumor tissues were removed and frozen in FSC22 tissue-freezing medium (Leica Biosystems). After the tissues had decayed for 48 h, we obtained sections with a thickness of 20 μm using a CM1800 cryostat microtome (Leica Instruments) and exposed them on imaging plates for 24 h. We scanned the plates with BAS-5000 (Fujifilm). We quantified image intensity in the region of interest as units of photostimulated luminescence per square millimeter using Fujifilm Multi Gauge software, version 3.0 (Fujifilm).
Frozen tumor tissues were sectioned at 5 μm, incubated overnight at 4°C with anti-HER2 antibody (1:50 dilution; Cell Signaling Technology), and incubated for 30 min at room temperature with goat antirabbit IgG-horseradish peroxidase (1:200 dilution; Santa Cruz Biotechnology). We used a 3,3′-diaminobenzidine kit (DAKO) for color development and subsequently counterstained the slides with hematoxylin.
Biodistribution Study
Biodistribution studies were performed to evaluate the uptake of 64Cu-NOTA-trastuzumab in normal and subcutaneous BT-474 tumor–bearing mice. At 2, 6, 24, 48, and 72 h after the intravenous administration of 3.7 MBq of 64Cu-NOTA-trastuzumab, the mice were sacrificed, their organs were removed and weighed, and radioactivity was measured. Results are expressed as percentage injected dose (%ID) per gram of tissue (n = 4).
Pharmacokinetic Study of 64Cu-NOTA-Trastuzumab in Nude Mice With or Without HER2-Positive Tumor
64Cu-NOTA-trastuzumab was injected into nude mice with or without BT-474 tumor xenografts via their tail veins at 5.5–6.7 MBq (150–180 μCi)/100 μg/head (n = 6 each). Blood samples were collected from the retroorbital plexus into a 75-mm sodium-heparinized capillary tube at 0, 0.167, 0.5, 1, 2, 6, 24, 48, and 72 h after administration. Dosing solution (2 μL) and blood samples (30 μL) were transferred to polyethylene tubes. Radioactivity was counted by a 1480 Wizard γ-counter. The radioactivity concentration of the blood sample was expressed as the %ID per milliliter of blood.
Pharmacokinetic parameters were estimated from the blood radioactivity concentration versus time by a noncompartmental method using the nonlinear least-squares regression program WinNonlin, version 2.0 (Pharsight).
S Value and Absorbed Dose Calculation
We used tumor and organ tissue density to determine S value. CT density and PET radioactivity were used as input data in the Monte Carlo simulation. S value and a dose map of organs and tumor were calculated using Geant4 Monte Carlo N-particle code. The S value equation is
where yi is the number of energy, Ei is the energy per radiation, and m is the mass of the target region.
The absorbed dose(
) was calculated according to the formula
where 
is the initial injected radioactivity, 
is the radiotracer residence time of a source organ 
, and 
is the dose deposited in target 
per unit of cumulated activity in source 
. Each organ and tumor absorbed dose includes contributions from the self-dose and the cross-dose from other segmented regions (24).
Small-Animal PET/CT Imaging
At 1, 2, and 6 h, and 1, 2, and 3 d after the intravenous injection of 7.4 MBq of 64Cu-NOTA-trastuzumab per animal, the mice were placed in a spread prone position under inhalation anesthesia (isoflurane, 2%) and imaged for 20 min with an Inveon dedicated small-animal PET/CT scanner (Siemens Healthcare; n = 3). The counting rates in the reconstructed images were converted to activity concentrations (%ID per gram of tissue). To define each organ and tumor in the mice, we acquired contrast CT data using ExiTron nano 12000 (Miltenyi Biotec). Blocking studies (n = 2) were performed to evaluate the HER2 specificity of 64Cu-NOTA-trastuzumab in vivo, where each mouse in a group of two was injected with 1 mg of nonlabeled trastuzumab within 1 h before 64Cu-NOTA-trastuzumab administration.
Analysis of Time–Activity Curve and Residence Time
Mouse images were acquired at 6 time points after the radiotracer administration. PET image data and CT image data were colocalized, and the PET image data were segmented for each organ and tumor to calculate time–activity curve and S value. Individual mouse CT data were segmented by referral to the contrast CT image. Tumors and organs were segmented by mean-based region-growing 3-dimensional segmentation. The time–activity curve was expressed as %ID/g. Residence times were calculated via time–activity curves of acquired region-segmented PET data.
RESULTS
Quantification of 64Cu-NOTA-Trastuzumab in HER2-Positive Breast Tumor PET Images
To evaluate the potential of 64Cu-NOTA-trastuzumab as a PET imaging agent for HER2 expression, we performed PET imaging in ectopic BT-474 tumor models. BT-474 tumors were clearly visible on PET images after 24 h (Fig. 1). Quantitative data obtained from region-of-interest analysis of the PET results in the nonblocking group are shown in Figure 2A. Liver uptake for 64Cu-NOTA-trastuzumab was 14.64 ± 2.23, 13.23 ± 1.34, and 9.50 ± 0.32 %ID/g at 24, 48, and 72 h, respectively (n = 3). Radioactivity in the blood was 19.23 ± 4.43, 15.00 ± 2.45, and 13.03 ± 3.71 %ID/g at 24, 48, and 72 h, respectively (n = 3). Importantly, tumor uptake of 64Cu-NOTA-trastuzumab accumulated and was clearly visible at 24 h, peaked at 48 h, and remained prominent over time (24.82 ± 11.16, 29.24 ± 16.45, and 28.34 ± 15.89 %ID/g at 24, 48, and 72 h, respectively; n = 3).
64Cu-NOTA-trastuzumab PET images in HER2-positive tumor model. Right 2 columns display PET images after cold trastuzumab pretreatment (n = 2), and left 3 show nonblocking images (n = 3). Arrows indicate BT-474 tumors.
64Cu-NOTA-trastuzumab PET image quantification of nonblocking group (A) and cold trastuzumab–pretreated group (B) in HER2-positive tumor model. 64Cu-NOTA-trastuzumab specificity was suggested by blocking experiments with excess of unlabeled trastuzumab.
Administering a blocking dose of trastuzumab 1 h before 64Cu-NOTA-trastuzumab injection significantly reduced tumor uptake to 6.80 ± 0.77, 6.85 ± 1.73, and 6.70 ± 0.49 %ID/g at 24, 48, and 72 h, respectively (n = 2; Fig. 2B), demonstrating that 64Cu-NOTA-trastuzumab maintained the HER2 specificity of its parent antibody in vivo. Liver uptake of 64Cu-NOTA-trastuzumab in the blocking group was similar to that in mice injected with 64Cu-NOTA-trastuzumab alone: 17.72 ± 2.53, 15.67 ± 0.27, and 12.30 ± 1.66 %ID/g at 24, 48, and 72 h, respectively (n = 2).
Moreover, orthotopic BT-474 tumor uptake of 64Cu-NOTA-trastuzumab accumulated and was clearly visible at 6 h, peaked at 51 h, and remained prominent over time (2.48 ± 1.69, 3.08 ± 2.47, 3.53 ± 2.99, and 2.92 ± 3.92 %ID/g at 6, 28, 51, and 122 h, respectively). An autoradiogram of the frozen section prepared from the removed HER2-positive tumor revealed high accumulation in the areas where HER2-positive cells were detected by immunohistochemistry (Fig. 3).
64Cu-NOTA-trastuzumab PET and immunohistochemistry in orthotopic HER2-positive BT-474 breast tumor model. Tumor uptake of 64Cu-NOTA-trastuzumab was clearly visible at 6 h and peaked at 51 h, and autoradiogram of frozen section prepared from removed tumor revealed high accumulation in area where HER2-positive cells were detected by immunohistochemistry. IHC = immunohistochemistry.
Biodistribution of 64Cu-NOTA-Trastuzumab in HER2-Positive Breast Tumor Model and Normal Mice
Biodistribution data of 64Cu-NOTA-trastuzumab in BT-474 HER2–positive tumor models were compared and are summarized in Figure 4. The 64Cu-NOTA-trastuzumab uptake of major organs and tissues in tumor-bearing mice was similar to that in normal mice. The radioactivity in blood and spleen was high at 2 h but gradually decreased over time. The uptake of 64Cu-NOTA-trastuzumab in BT-474 HER2–positive tumors steadily increased and peaked at 48 h, at 64.44 ± 31.11 %ID/g.
Biodistribution of 64Cu-NOTA-trastuzumab in normal mice (A) and HER2-positive tumor–bearing mice (B). At 2, 6, 24, 48, and 72 h after injection of 64Cu-NOTA-trastuzumab, both mice were euthanized, and radioactivity in organs was measured (n = 4).
Pharmacokinetics of 64Cu-NOTA-Trastuzumab in Nude Mice With or Without HER2-Positive Tumors
The blood radioactivity concentration–time profiles after a single intravenous bolus injection of 64Cu-NOTA-trastuzumab in nude mice with or without BT-474 tumor xenografts are shown in Figure 5. In both groups of mice, blood radioactivity concentrations decreased biexponentially with time. The concentration–time profiles in tumor-bearing nude mice were similar to those in the nude mice without tumors. Table 1 summarizes the pharmacokinetic parameters obtained by noncompartmental analysis. The blood concentration extrapolated to time zero was 46.8 ± 7.52 and 48.7 ± 4.64 %ID/mL; the terminal elimination half-life was 135 ± 41.1 and 190 ± 40.2 h; the area under the blood concentration–time curve from 0 to 72 h was 1,354 ± 166 and 1,480 ± 124 %ID·h/mL; the volume of distribution at the terminal phase was 4.74 ± 0.786 and 4.69 ± 0.461 mL; the systemic clearance was 0.0254 ± 0.00538 and 0.0176 ± 0.00289 mL/h; and the mean retention time was 31.0 ± 0.512 and 31.6 ± 0.390 h, respectively, after intravenous injection of 64Cu-NOTA-trastuzumab in nude mice with and without tumors.
Average blood radioactivity concentration vs. time after intravenous injection of 64Cu-NOTA-trastuzumab in nude mice with or without BT-474 tumor xenografts. Data are mean ± SD.
Blood Radioactivity Parameters After 64Cu-NOTA-Trastuzumab* in Mice With and Without BT-474 Tumors
Residence Time Calculation
To clearly define each organ region, we acquired CT images using a contrast agent. As a result, we clearly defined the region of interest in the PET image data. The segmented tumor and major organs are shown in Figure 6A. The time–activity curve was calculated at each time point (1, 2, and 6 h, and 1, 2, and 3 d) using the defined specific region in the PET image (Fig. 6B). The highest mean residence time was in the tumor, followed by the kidney, heart, liver, lung, urinary bladder, spleen, and stomach; the lowest residence time was in the brain (Fig. 6C). The mean residence time in the tumors was 7.42 ± 3.3 MBq-h/MBq. The residence time in the heart was 3.33 ± 0.8 MBq-h/MBq. The residence time in the lung was 2.28 ± 0.7 MBq-h/MBq, and that in the liver was 2.66 ± 0.8 MBq-h/MBq. The residence time in the stomach was 1.31 ± 0.2 MBq-h/MBq, and that in the spleen was 2.19 ± 0.6 MBq-h/MBq.
(A) Acquired CT and PET images. (B) Time–activity curve evaluated by segmented PET image data at 6 time points. (C) Calculated residence time in each organ and tumor. (D) Energy map generated from Monte Carlo simulation.
Absorbed Dose Calculation
S value was calculated with both 64Cu and 67Cu using Monte Carlo simulation with the CT images. As shown in Tables 2 and 3, S value was higher with the self-irradiation than with the cross-irradiation and was highest in the HER2-positive tumors, followed by the spleen, brain, urinary bladder, heart, kidney, and liver. We calculated the absorbed dose of 64Cu-NOTA-trastuzumab using an image-based approach. The absorbed dose of 64Cu-NOTA-trastuzumab was 0.048 ± 0.012 for heart, 0.079 ± 0.004 for liver, 0.047 ± 0.010 for spleen, and 2.43 ± 1.09 mGy/MBq for tumor. According to a study by Tamura et al. (12), the absorbed dose of 64Cu-DOTA-trastuzumab was 0.34 ± 0.046 for heart, 0.24 ± 0.117 for liver, and 0.14 ± 0.04 mGy/MBq for spleen (Fig. 7). Consequently, the absorbed dose of 64Cu-NOTA-trastuzumab was lower than that of 64Cu-DOTA-trastuzumab in the heart, liver, and spleen. We also calculated the absorbed dose of 67Cu-NOTA-trastuzumab using Monte Carlo simulation to develop 67Cu-NOTA-trastuzumab as a therapeutic agent for HER2-positive breast cancer based on 64Cu-NOTA-trastuzumab. The absorbed doses of 67Cu-NOTA-trastuzumab and 67Cu-DOTA-trastuzumab in liver were 0.042 and 0.934 mGy/MBq, respectively, calculated using the biodistribution results published by Paudyal et al. (25). Therefore, 67Cu-NOTA-trastuzumab might be safer than 67Cu-DOTA-trastuzumab because of the lower absorbed dose in major organs such as liver, heart, and spleen.
64Cu S Values Calculated Using Monte Carlo Simulation with CT Image
67Cu S Values Calculated Using Monte Carlo Simulation with CT Image
64Cu-DOTA-trastuzumab exhibited higher absorbed dose than 64Cu-NOTA-trastuzumab in heart, liver, and spleen (12).
DISCUSSION
Breast cancer is the most common malignancy. It is a heterogeneous disease that can be classified by microscopic appearance and molecular profiles that include the expression of estrogen receptor and overexpression of HER2 (3). Overexpression of HER2 portends a poor prognosis with an increased risk for disease progression and decreased overall survival (26).
The discovery of many novel molecular targets for anticancer treatment has led to the development of therapeutic antibodies. Overexpression of HER2 enables constitutive activation of growth factor signaling pathways and thereby serves as an oncogenic driver in breast cancer. Through both genetic and pharmacologic approaches, it was determined that HER2 is both necessary and sufficient for tumor formation and maintenance in models of HER2-positive breast cancer (27). Targeting HER2 with monoclonal antibody, such as trastuzumab and pertuzumab, is a well-established therapeutic strategy for HER2-positive breast cancer in neoadjuvant (28), adjuvant (29,30), and metastatic settings (31,32). Especially trastuzumab, a humanized recombinant monoclonal antibody against HER2, is widely used as a standard treatment for HER2-expressing breast cancer. Recently, a need has been recognized for repetitive visualization of HER2 expression due to the success of trastuzumab–emtansine as an antibody–drug conjugate to minimize toxicity against major organs and enhance therapeutic efficacy. Detection of HER2-positive metastatic tumors using radiolabeled HER2 antibodies would be valuable to provide safety, treatment economy, and other therapeutic options to HER2-negative tumor–bearing patients. Conversely, Ulaner et al. reported that 89Zr-labeled trastuzumab PET/CT could be applied to detect unsuspected HER2-positive metastases in patients with HER2-negative primary breast cancer as a proof-of-concept clinical study (32). Thus, it is important to evaluate HER2 expression in metastatic and primary tumors to determine whether anti-HER2 therapy is indicated.
Although HER2 expression is routinely determined using histologic analysis (33), technical problems can arise when lesions cannot be easily accessed by core-needle biopsy, and the analysis lacks specificity and sensitivity (34). In addition, HER2 expression can vary during the course of the disease and even across tumor lesions within the same patient. To overcome these problems, novel techniques such as PET and SPECT have been studied for evaluating HER2 expression. Molecular imaging using radiolabeled antibodies can provide real-time information and noninvasively assess the presence of specific targets throughout the body (35). PET imaging depends on the delivery of a targeting ligand containing a positron-emitting radionuclide to a tissue or organ of interest. 64Cu (half-life, 12.7 h) is an attractive radionuclide for PET imaging that can be used for both diagnostic imaging and radionuclide therapy because of its dual decay characteristics (36). PET images acquired by 64Cu-labeled trastuzumab may potentially achieve good contrast with high resolution and low radiation exposure because of the shorter half-life of the radioisotope (12). However, the application of 64Cu as a radioisotope for antibody molecules has been hindered by the lack of an optimal chelator to form a stable conjugate complex in vivo. The high uptake and retention of copper-containing compounds in the blood and liver are well known (37,38). Therefore, an optimal chelator for copper metal ion for a more stable conjugate is required for in vivo studies using 64Cu. NOTA is an intensively investigated macrocyclic, multidentate chelator used for complexation of a broad variety of bi- and trivalent metal ions (39–41). Bifunctional NOTA forms an exceedingly stable complex with Ga3+ ions, and PET agents can feature its use. NOTA conjugates labeled with 67Ga, 68Ga, 64Cu, 67Cu, and 111In are suitable for diagnostic and therapeutic approaches (42,43). According to a report of Paudyal et al. (25), uptake of 64Cu-DOTA-trastuzumab in liver was 26.9 ± 7.4 %ID/g at 24 h. In contrast, uptake of 64Cu-NOTA-trastuzumab in liver was 5.44 ± 1.84 %ID/g at 24 h (Fig. 4). This result suggests that release of 64Cu from 64Cu-NOTA-trastuzumab is less than that from 64Cu-DOTA-trastuzumab and, therefore, that 64Cu-NOTA-trastuzumab is more stable than 64Cu-DOTA-trastuzumab.
According to the pharmacokinetic results of 64Cu-NOTA-trastuzumab in HER2-positive tumor–bearing and non–tumor-bearing nude mice, the systemic clearance and terminal half-life were significantly different from each other (P < 0.05; Fig. 5), whereas other parameters were quite similar to each other. The systemic clearance in nude mice with HER2-positive tumors was higher than that in nude mice without tumors. This result suggests that the elimination rate of 64Cu-NOTA-trastuzumab in nude mice with HER2-positive tumors is faster than that in nude mice without tumors and that this might be because 64Cu-NOTA-trastuzumab is targeted, trapped to HER2-positive tumors, and removed from mouse circulation.
The image-based methods basically include Monte Carlo simulation and entail manually drawing regions of interest to calculate the individual specific dosimetry based on the patient’s anatomic image. Monte Carlo simulation can be used for acquiring the S values of different radionuclides. If the same pharmacokinetic or pharmacodynamic characteristics exist between a diagnostic radiopharmaceutical, such as emitting γ-rays, and a therapeutic radiopharmaceutical, such as emitting α- or β-particles, it may be possible to calculate the subject-specific internal dosimetry using the diagnostic radiopharmaceutical (44). To develop 67Cu-NOTA-trastuzumab as a therapeutic agent based on 64Cu-NOTA-trastuzumab, we calculated the absorbed dose of 67Cu-NOTA-trastuzumab in this study using Monte Carlo simulation. The lower absorbed dose of 67Cu-NOTA-trastuzumab in major organs showed it to have greater safety than 67Cu-DOTA-trastuzumab. Therefore, NOTA-conjugated trastuzumab might be suitable for both therapeutic and PET-diagnostic applications in HER2-positive breast cancer.
CONCLUSION
Trastuzumab as a HER2-targeting antibody can be efficiently radiolabeled with 64Cu using NOTA as a chelator with high labeling yield and excellent stability. The calculated absorbed dose of 64Cu-NOTA-trastuzumab was lower than that of 64Cu-DOTA-trastuzumab in heart, liver, and spleen because of a short residence time. Furthermore, 64Cu-NOTA-trastuzumab was efficiently targeted to the HER2-expressing tumors in vivo and in vitro. Therefore, 64Cu-NOTA-trastuzumab might be applicable in human studies for selecting patients with the right timing for HER2 therapy and for monitoring the response after HER2-targeted therapy.
DISCLOSURE
This work was supported by National Research Foundation of Korea (NRF) grants funded by the Ministry of Science and ICT (NRF-2012M2A2A7013480, NRF-2015R1C1A1A02036885, and NRF-2017M2A2A02070985). No other potential conflict of interest relevant to this article was reported.
Footnotes
Published online May 18, 2018.
- © 2019 by the Society of Nuclear Medicine and Molecular Imaging.
REFERENCES
- Received for publication February 21, 2018.
- Accepted for publication May 14, 2018.
