A comparative evaluation of the chelators H₄octapa and CHX-A″-DTPA with the therapeutic radiometal ⁹⁰Y

Abstract

Objectives

To compare the radiolabeling performance, stability, and practical efficacy of the chelators CHX-A″-DTPA and H₄octapa with the therapeutic radiometal ⁹⁰Y.

Methods

The bifunctional chelators p-SCN-Bn-H₄octapa and p-SCN-Bn-CHX-A″-DTPA were conjugated to the HER2-targeting antibody trastuzumab. The resulting immunoconjugates were radiolabeled with ⁹⁰Y to compare radiolabeling efficiency, in vitro and in vivo stability, and in vivo performance in a murine model of ovarian cancer.

Results

High radiochemical yields (>95%) were obtained with ⁹⁰Y-CHX-A′-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab after 15 min at room temperature. Both ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab exhibited excellent in vitro and in vivo stability. Furthermore, the radioimmunoconjugates displayed high tumoral uptake values (42.3 ± 4.0%ID/g for ⁹⁰Y-CHX-A″-DTPA-trastuzumab and 30.1 ± 7.4%ID/g for ⁹⁰Y-octapa-trastuzumab at 72 h post-injection) in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. Finally, ⁹⁰Y radioimmunotherapy studies performed in tumor-bearing mice demonstrated that ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab are equally effective therapeutic agents, as treatment with both radioimmunoconjugates yielded substantially decreased tumor growth compared to controls.

Conclusions

Ultimately, this work demonstrates that the acyclic chelators CHX-A″-DTPA and H₄octapa have comparable radiolabeling, stability, and in vivo performance, making them both suitable choices for applications requiring ⁹⁰Y.

1. Introduction

The β⁻-emitting radiometal ⁹⁰Y is commonly used for peptide receptor radionuclide therapy (PRRT, e.g., ⁹⁰Y-DOTATOC) and radioimmunotherapy (e.g., ⁹⁰Y-DOTA-trastuzumab) to deliver a therapeutic dose of radiation to tumor cells [1,2]. One advantage of ⁹⁰Y (t_1/2=64.1 h, β⁻=72%, $E_{\beta }^{-}=2.288$ MeV) compared to other therapeutic isotopes is that it can treat relatively large and poorly vascularized tumors due to its long in vivo β⁻ range of ~12 mm [3–5]. This relatively long particle range means that tumor cells up to ~550 cell diameters away from the radiopharmaceutical will also receive therapeutic irradiation, a phenomenon known as the “crossfire effect” [3–5]. While the crossfire effect is generally regarded as positive due to the ability to treat tumors of larger sizes, an inevitable disadvantage of the long β⁻ range is the potential irradiation of healthy tissue near the tumor.

Critically, the use of ⁹⁰Y for PRRT and radioimmunotherapy necessitates the use of a pre-therapy scout imaging scan to perform dosimetry, as ⁹⁰Y itself emits no particles or gamma rays suitable for imaging [6,7]. In this type of scout imaging, ⁹⁰Y is replaced with an isotope appropriate for imaging: for example, ¹¹¹In-DOTATOC will be used as an imaging counterpart to ⁹⁰Y-DOTATOC [6,7]. A number of isotopes – including ¹¹¹In, ⁶⁴Cu, ⁸⁹Zr, and ⁶⁸Ga – have been used for dosimetry imaging scans prior ⁹⁰Y-based radiotherapy. However, these approaches leave much to be desired, as labeling a targeting vector (e.g., DOTATOC) with different radiometals can drastically change its in vivo properties [6–8]. Indeed, changes in the coordination environment, charge, or stability a radiometal–chelator complex [e.g., ⁹⁰Y(DOTA)¹⁻ vs. ⁶⁴Cu(DOTA)²⁻] can cause significant discrepancies in tumor uptake and organ distribution [9].

For this reason, the use of the positron-emitting radiometal ⁸⁶Y (⁸⁶Y, t_1/2 = 14.7 h, β⁺ = 33%, $E_{\beta }^{+}=640$ keV) for PET imaging prior to ⁹⁰Y-based therapy would appear to be ideal, as the ⁸⁶Y- and ⁹⁰Y-labeled radiopharmaceuticals would be chemically identical isotopologues [10]. Imaging with ⁸⁶Y is a more attractive option than imaging with the commonly-used surrogate isotope ¹¹¹In due to the former's suitability for PET (rather than SPECT) and its identical aqueous coordination chemistry to ⁹⁰Y³⁺. However, the use of ⁸⁶Y³⁺ nonetheless has its fair share of drawbacks. First, when compared to ⁹⁰Y (t_1/2 ~ 64.1 h), ⁸⁶Y possesses a shorter half-life (14.7 h), which can preclude imaging studies at later time points. In addition, ⁸⁶Y emits a relatively high-energy β⁺ (E_mean = 640 keV), meaning that the positrons have a long mean-free path length (R_mean = 2.5 mm) compared to other popular positron-emitting radiometals such as ⁶⁴Cu (E_mean = 278.2 keV, R_mean = 0.56 mm) and ⁸⁹Zr (E_mean=402.7 keV, R_mean=1.27 mm) [11]. As a result of this higher β⁺ energy and an abundance of gamma emissions (1077, 1153, 1854, 1921 keV), the image quality of ⁸⁶Y-PET scans is reported as being sub-optimal. However, the subtraction of some of these prompt gamma coincidences can improve image quality and useful images can be obtained from ⁸⁶Y, despite the shortcomings described above [12]. Another concern that arises from this abundance of high-energy gamma emissions from ⁸⁶Y is the radiation dose to anyone handling it. As a result, substantial radioactive shielding is required during radiolabeling, and the quantity of ⁸⁶Y that can be transported in one container is limited [5]. For these reasons – as well as its limited availability in North America and the low purity of the available ⁸⁶Y – experiments with ⁸⁶Y were not included in this study. Although ⁸⁶Y has previously been used successfully, the issues encountered with the available ⁸⁶Y during this study prevented its inclusion [6,10,13–22].

Regardless of the identity of the metal, any radiopharmaceutical labeled with a metallic isotope must have a suitable chelator attached to the targeting vector (e.g., peptide, antibody) to facilitate the stable coordination of the radiometal ion [9,23–25]. The acyclic chelating ligand H₄octapa (Fig. 1) has been previously synthesized and studied with the radiometals ¹¹¹In (t_1/2 ~ 2.8 days) and ¹⁷⁷Lu (t_1/2 ~ 6.6 days) and was observed to possess rapid radiolabeling kinetics and excellent in vitro and in vivo stability with both radiometals [26–29]. A popular class of targeting vectors to use with longer half-life radiometals such as ¹¹¹In, ¹⁷⁷Lu, and ⁹⁰Y are antibodies, proteins that possess exquisite selectivity and binding affinity to their target antigens but are also sensitive to elevated temperatures (>40 °C). As a result, a chelator must be used that can coordinate radiometals at relatively low temperatures (e.g., 25–40 °C) so that the structural integrity of the antibody is not compromised. The acyclic chelator H₄octapa was demonstrated to radiolabel with ¹¹¹In and ¹⁷⁷Lu in less than 15 min at room temperature. This suggests that radiolabeling performance with the chemically similar tricationic radiometals ⁸⁶Y³⁺ and ⁹⁰Y³⁺ should also be favorable.

Fig. 1.

The bifunctional chelators discussed in this work: p-SCN-Bn-H₄octapa, p-SCN-Bn-CHX-A″-DTPA, and p-SCN-Bn-DOTA (C-DOTA).

One of the more common chelators used for ⁸⁶Y/⁹⁰Y is the macrocycle 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA; Fig. 1), which typically requires elevated temperatures of 60–90 °C over a period of 30–60 min for radiometalation [26–29]. The high temperatures required for radiolabeling make DOTA a very poor choice for use with antibodies. A newer and more suitable chelator for this radiometal pair is CHX-A″-DTPA, which – in contrast to DOTA – can be effectively radiolabeled at ambient temperatures within 15–30 min [26–31]. Although DOTA and CHX-A″-DTPA are the most established Y³⁺ chelators, a new NOTA derivative 3p-C-NETA has also been used with ¹⁷⁷Lu, ⁸⁶Y, and ⁹⁰Y and gives high radiolabeling yields in only 5 min at room temperature [32–34].

H₄octapa was previously studied with non-radioactive yttrium, with which it formed a complex with thermodynamic stability and solution chemistry comparable to the H₄octapa complexes with Lu³⁺ and In³⁺[26,28,29]. These results with non-radioactive yttrium suggested that testing H₄octapa with ⁹⁰Y should yield fast radiolabeling kinetics and robust stability, as was found with ¹¹¹In and ¹⁷⁷Lu. To determine the suitability of H₄octapa for use with isotopes of Y³⁺, the bifunctional chelators p-SCN-Bn-H₄octapa and p-SCN-Bn-CHX-A″-DTPA were conjugated to the antibody trastuzumab and radiolabeled with ⁹⁰Y, and the resulting radioimmunoconjugates were evaluated in vitro and in vivo. The stability of the ⁹⁰Y chelate complexes were evaluated in vitro by serum stability and in vivo by measuring the uptake of ⁹⁰Y in organs in which “free” ⁹⁰Y³⁺ is known to accumulate (e.g., bone, liver, kidneys).

2. Materials and methods

2.1. General materials

All solvents and reagents were purchased from commercial suppliers (Sigma Aldrich, St. Louis, MO; TCI America, Portland, OR; Fisher Scientific, Waltham, MA) and were used as received unless otherwise indicated. DMSO used for chelator stock solutions was of molecular biology grade (>99.9%: Sigma, D8418). The bifunctional chelator p-SCN-Bn-H₄octapa was synthesized as previously reported [28], and [(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (p-SCN-Bn-CHX-A″-DTPA) and 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine] thiourea (p-SCN-Bn-DFO) were purchased from Macrocyclics, Inc. (Dallas, TX). UV–Vis measurements for determining antibody stock solution concentrations were taken on a Thermo Scientific Nanodrop 2000 spectrophotometer (Wilmington, DE), using the full-sized antibody (150 kDa) molar extinction coefficient of 210,000 L mol⁻¹. ⁹⁰YCl₃ (0.5 M HCl) was purchased from PerkinElmer. ⁸⁹Zr was produced at Memorial Sloan Kettering Cancer Center using an EBCO TR19/9 variable-beam energy cyclotron (Ebco Industries Inc., British Columbia, Canada) via the ⁸⁹Y(p,n)⁸⁹Zr reaction. ⁸⁹Zr was purified in accordance with previously reported methods to create ⁸⁹Zr with a specific activity of 5.3–13.4 mCi/μg (195–497 MBq/μg) [35]. Labeling reactions were monitored using silica–gel impregnated glass-microfiber instant thin layer chromatography paper (iTLC-SG, iTLC-SA, Varian, Lake Forest, CA) and analyzed on a Bioscan AR-2000 radio-TLC plate reader using Winscan Radio-TLC software (Bioscan Inc., Washington, DC). All radiolabeling chemistries were performed with ultra-pure water (>18.2 MΩ cm⁻¹ at 25 °C, Milli-Q, Millipore, Billerica, MA) that had been stirred with Chelex-100 resin (1.2 g/L, 24 h) (BioRad Laboratories, Hercules, CA). Radioactivity in samples was measured using a Capintec CRC-15R dose calibrator (Capintec, Ramsey, NJ). For ⁸⁹Zr biodistribution studies, a PerkinElmer (Waltham, MA) Automated Wizard Gamma Counter was used for counting radioactivity in organ samples and creating calibration curves for ⁸⁹Zr. For ⁹⁰Y biodistribution studies, a PerkinElmer (Waltham, MA) Automated Tri-Carb® 2910 TR liquid scintillation counter was used with samples that had been treated with Solvable™ (PerkinElmer) and Ultima Gold™ high flash-point universal liquid scintillation-cocktail (PerkinElmer).

2.2. Antibody modification

Trastuzumab (purchased commercially as Herceptin; Genentech, San Francisco, CA) was purified using PD10 size-exclusion columns [with phosphate buffered saline (PBS, pH 7.4), 3 times], followed by centrifugal filter units with a 50,000 molecular weight cut off (Amicon® ultra centrifuge filters, Ultracel®-50: regenerated cellulose, Millipore Corp., Billerica, MA) (PBS, pH 7.4) to remove α-α-trehalose dihydrate, L-histidine, and polysorbate 20 additives. After purification, the antibody was taken up in PBS pH 7.4 and kept in the fridge at 4 °C as a stock solution (12.8 mg/mL). Subsequently, 150 μL of antibody solution (~1.92 mg antibody) was combined with 850 μL PBS (pH 7.4), the pH of the resulting solution was adjusted to 8.8–9.0 with 0.1 M Na₂CO₃ (~30 μL), and 6 equivalents of p-SCN-Bn-H₄octapa or p-SCN-Bn-CHX-A″-DTPA were added in 10–15 μL DMSO. The reactions were incubated at 37 °C for 1 h, followed by PD10 purification (PD10, Sephadex G-25 M, PD10 column, GE Healthcare; dead volume = 2.5 mL, product eluted with 2.0 mL fractions of sterile saline), and centrifugal filtration (Amicon 50 kDa) to purify the resultant antibody conjugate. The final immunoconjugate stock solutions were stored in PBS (pH 7.4) at 4 °C.

2.3. ⁹⁰Y-radiolabeling of H₄octapa- and CHX-A″-DTPA-trastuzumab

Aliquots of chelate-modified antibody (200 μg for ⁹⁰Y) bearing either H₄octapa or CHX-A″-DTPA were transferred to 2 mL microcentrifuge tubes and made up to 0.5 mL with ammonium acetate buffer (pH 5.5, 200 mM, treated with Chelex-100 resin at 1.2 g/L overnight before use). Aliquots of ⁹⁰Y (~0.35 mCi, ~12.9 MBq) were added. The radiolabeling mixtures were allowed to react at 37 °C (500 rpm, thermomixer) for 60 min and then analyzed via iTLC-SA (acidic) with an eluent of 50 mM DTPA (pH 2.5). Crude radiochemical yields for ⁹⁰Y-CHX-A″-DTPA-trastuzumab were ~96% and for ⁹⁰Y-octapa-trastuzumab were ~99%. After 60 min, EDTA solution was added to quench the reactions (20 μL EDTA, 50 mM, pH 5.5), and were then purified using size-exclusion chromatography (PD10), followed by centrifugal filtration (Amicon® ultra 50 kDa, rinsed 3 times with saline). The radiochemical purity of the final purified radiolabeled bioconjugate was assayed by radio-iTLC-SA (DTPA mobile phase, 50 mM, pH 2.5). For ⁹⁰Y reactions, crude radiochemical yields of both compounds were >95%, and purified yields were all >99%. In the iTLC experiments, ⁹⁰Y-octapa-trastuzumab and ⁹⁰Y-CHX-A″-DTPA-trastuzumab remained at the baseline, while ⁹⁰Y³⁺ ions eluted with or near the solvent front (~100–150 mm) with the DTPA mobile phase (50 mM, pH 2.5).

2.4. ⁸⁹Zr-DFO-trastuzumab radiolabeling

⁸⁹Zr-DFO-trastuzumab was prepared as previously published, and p-SCN-Bn-DFO was conjugated to trastuzumab using the same method as described above for p-SCN-Bn-H₄octapa and p-SCN-Bn-CHX-A″-DTPA.[36,37] Briefly, aliquots of ⁸⁹Zr oxalate were neutralized to ~pH 7.0–7.4 using sodium carbonate (1 M), and subsequently mixed with DFO-trastuzumab (~500 μg, 0.5 mL PBS, pH 7.4) and reacted for 60 min at 37 °C. Radiochemical yields obtained were >99% after 1 h, and ⁸⁹Zr-DFO-trastuzumab was purified as described above for the ⁹⁰Y-labeled radioimmunoconjugates.

2.5. MALDI-TOF MS/MS analysis to determine the number of chelates per antibody

The number of benzylthiourea-linked H₄octapa or CHX-A″-DTPA chelates conjugated to trastuzumab was determined using MALDI-TOF MS/MS (Alberta Proteomics and Mass Spectrometry Facility, University of Alberta, Canada). All experiments were performed in triplicate, and all samples were run along with standard samples of unmodified trastuzumab (run on the same day). 1 μL of each sample (1 mg/mL) was mixed with 1 μL of sinapic acid (10 mg/mL in 50% acetonitrile:water and 0.1% trifluoroacetic acid). 1 μL of the sample/matrix solution was then spotted onto a stainless steel target plate and allowed to air dry. All mass spectra were obtained using a Bruker Ultraflex MALDI-TOF/TOF (Bruker Daltonic GmbH). Ions were analyzed in positive mode, and external calibration was performed using of a standard protein mixture (bovine serum albumin). The mass signals (M⁺²/2) at half the molecular weight of the antibody were taken from each chromatogram and averaged (n = 3), and the molecular weight of the unmodified trastuzumab was subtracted from the molecular weight of the modified antibody to determine the mass contributed by the conjugated chelators. The mass difference was divided by the molecular weight of the attached bifunctional chelator (p-SCN-Bn-H₄octapa, p-SCN-Bn-CHX-A″-DTPA), and the error in each triplicate set of measurements (unmodified trastuzumab vs. modified trastuzumab) was propagated to the final value of the number of chelates per antibody. The MALDI-TOF spectra are available in the supporting information Figs. S1–S9.

2.6. Determination of immunoreactivity

The immunoreactivity of ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab was determined using specific radioactive cellular-binding assays in which an excess of cells was used (~10 million per microcentrifuge tube). This quantity of cells was assumed to contain effectively “infinite antigen” relative to the quantity of antibody added to each (~ 6 ng of radiolabeled antibody). To this end, 10 million SKOV3 cells were suspended in microcentrifuge tubes in ~0.2 mL of media in microcentrifuge tubes. Aliquots of the bioconjugates – ⁹⁰Y-octapa-trastuzumab and ⁹⁰Y-CHX-A″-DTPA-trastuzumab (20 μL of a stock solution containing ~10–20 μCi, ~0.37–0.73 MBq, ~3 μg of radioimmunoconjugate, in 10 mL of 1% bovine serum albumin in PBS pH 7.4) – were added to each tube and mixed by pipette (n = 4 each immunoconjugate; final volume: ~220 μL) The samples were then left to sit on ice for 60 min. Subsequently, the treated cells were pelleted via centrifugation (600 g, 4 °C, for 2 min), and the media was transferred by pipette to clean microcentrifuge tubes. The cell pellets were not re-suspended and were washed three times with ice-cold PBS (1 mL) and centrifuged (600g, 4 °C, 2 min) between each wash. Each wash was collected into an empty microcentrifuge tube (1 mL). Each tube (cell pellet, media, and 3 PBS washes) was measured in a scintillation counter (other isotopes can be counted using an automated gamma counter). Each tube was rinsed with 0.5 mL of PBS and transferred to scintillation vials (5 mL), 4.5 mL of Ultima gold scintillation fluid was added, samples were vortex mixed for 30 s, and then measured on an automated scintillation counter (PerkinElmer). The activity data were background-corrected, and the amount of activity (antibody) bound to the cells was compared to the total amount of activity present in the media and washes. The immunoreactivity was determined by dividing the amount of radioactivity bound to the cell pellet by the total amount of radioactivity present in the cell pellet, the media, and the washes.

2.7. ⁹⁰Y-octapa-trastuzumab and ⁹⁰Y-CHX-A″-DTPA-trastuzumab blood serum competition experiments

Frozen human blood serum was thawed for 30 min, and 500 μL aliquots were transferred to 2.0 mL Corning microcentrifuge vials. A portion of radiolabeled immunoconjugate (~150 μCi, ~50–75 μg, 500 μL, purified) was transferred to the blood serum (n=3 for each chelator). Serum competition samples were then incubated at 37 °C with gentle agitation (500 rpm, Eppendorf thermomixer) and analyzed via iTLC-SA elution with a DTPA eluent (50 mM, pH 2.5) followed by scanning (Bioscan AR-2000) at time points of 0, 1, 24, 48, 72, and 96 h.

2.8. Cell culture

Human ovarian cancer cell line SKOV3 was obtained from the American Tissue Culture Collection (ATCC, Bethesda, MD) and maintained in a 1:1 mixture of Dulbecco's modified Eagle medium: F-12 medium, supplemented with 10% heat-inactivated fetal calf serum (Omega Scientific, Tarzana, CA), 2.0 mM glutamine, nonessential amino acids, 100 units/mL penicillin, and 100 units/mL streptomycin in a 37 °C environment containing 5% CO₂. Cell lines were harvested and passaged weekly using a formulation of 0.25% trypsin/0.53 mM EDTA in Hank's Buffered Salt Solution without calcium and magnesium.

2.9. Subcutaneous SKOV3 xenografts

All experiments were performed under an Institutional Animal Care and Use Committee-approved (IACUC) protocol, and the experiments followed institutional guidelines for the proper and humane use of animals in research. Six- to eight-week-old athymic nu/nu female mice (NCRNUM) were obtained from Charles River Laboratories (Kingston, NY). Animals were housed in ventilated cages, were given food and water ad libitum, and were allowed to acclimatize for approximately 1 week prior to treatment. SKOV3 tumors were induced on the left shoulder by a subcutaneous injection of 5.0 × 10⁶ cells in a 100 μL cell suspension of a 1:1 mixture of fresh media/BD Matrigel (BD Biosciences, Bedford, Ma). Experiments were performed 3–4 weeks following the injection of the cancer cells.

2.10. ⁹⁰Y-octapa-trastuzumab and ⁹⁰Y-CHX-A″-DTPA-trastuzumab biodistribution studies

The purified ⁹⁰Y immunoconjugates (radiolabeled and purified as described above) were suspended in sterile saline (0.9% NaCl) to a concentration of ~20–25 μCi (1.1 MBq, ~ 15–20 μg) in 200 μL. The specific activity of both ⁹⁰Y-octapa-trastuzumab and ⁹⁰Y-CHX-A″-DTPA-trastuzumab was ~1.8 mCi/mg. Biodistribution experiments could not be performed using a gamma counter for ⁹⁰Y, as the strictly β⁻-emitting isotope requires organ digestion, bleaching, and scintillation counting. After doses were drawn in syringes, they were weighed before and after injection to obtain the total mass of injected antibody solution. This weight was then used with a set of standards to determine the total injected dose in each mouse in scintillation counts (in counts per mg of injected dose; see below).

Each mouse was injected with ⁹⁰Y-trastuzumab via the tail vein and euthanized by CO₂ (g) asphyxiation at time points of 24 and 72 h after the administration of the radioimmunoconjugate (n = 4 per time point). After sacrifice, the blood, tumor, heart, lungs, liver, spleen, pancreas, kidneys, large intestine, small intestine, muscle, bone (femur), and skin (ear) were collected. All organs were rinsed in water after removal and air-dried for 5 min. The organs were collected into pre-weighed glass scintillation vials (20 mL), and samples of well-vascularized tissues with higher blood content – including the blood, spleen, and liver – were obtained in small quantities (50–100 mg) to minimize color quenching in the final digested organ solutions. Subsequently, tissues were weighed and then digested for scintillation counting. Full tables of biodistribution data for ⁹⁰Y (%ID/g values), and experimental details for organ digestion are available in the supporting information Tables S1–S4.

A control biodistribution experiment using unbound ⁹⁰YCl₃ was performed as well. In this experiment, an aliquot of pure ⁹⁰YCl₃ (~100 μCi) was transferred to sterile saline (1.0 mL), and doses were drawn (~20 μCi in 200 μL) for injection (n = 3). In this case, the biodistribution experiment was performed only a 72 h post-injection. The organ samples were weighed and measured by scintillation counting as described above.

Standards for the ⁹⁰Y biodistribution experiments were prepared by transferring a small volume of the same injected ⁹⁰Y-antibody or ⁹⁰YCl₃ doses in saline (in the same formulations as were injected into mice, ~20 μL, ~2 μCi) into pre-weighed glass scintillation vials (20 mL vials, same as used for biodistributions, n = 5) and then weighing the mass of the 20 μL transferred mixture. These standards were then processed in the same manner as described above for organ samples and counted on the scintillation counter at the same time as the organ samples. The average number of scintillation counts per milligram of standard injected dose was calculated from these standards (counts per milligram), and the mass of injected dose for each biodistribution (obtained by subtracting weight of empty syringes from full syringes after injection) was then multiplied by this standard factor to obtain a value for the total injected dose for each mouse in scintillation counts.

2.11. ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab preliminary radioimmunotherapy pilot study

Female nude mice (n = 24) were xenografted with SKOV3 cells (subcutaneous, left shoulder) as described above. Tumor growth and volume were monitored by caliper measurements, in which the length (L) was taken as the longest dimension, and the width (W) as the shortest. Tumor volume (V) was calculated using the formula [V = L × (W/2)²]. Once the tumors grew to 50–100 mm³, the mice with the most consistent tumors were arranged in cages of 4 with approximately equal average tumor volumes amongst all mice. Mice were arranged into 3 groups (n = 4 each) using ear tags and ear punching as methods of sorting and identification. Tumors in several mice did not take, and some tumor sizes were very inconsistent, so smaller groups of n = 4 were used for this preliminary radioimmunotherapy pilot study (n = 5 for controls). Groups were injected with ⁹⁰Y-octapa-trastuzumab (n = 4, ~100 μCi, 200 μL saline, ~30 μg antibody, ~3.3 mCi/mg SA), ⁹⁰Y-CHX-A″-DTPA-trastuzumab (n = 4, ~100 μCi, 200 μL saline, ~30 μg antibody, ~3.3 mCi/mg), and controls (n = 5, 200 μL saline, ~30 μg trastuzumab antibody). New batches of CHX-A″-DTPA-trastuzumab and octapa-trastuzumab were synthesized to preclude aggregation and abnormal spleen uptake similar to that observed in the biodistribution of ⁹⁰Y-octapa-trastuzumab. Mice were assessed via tumor caliper measurements and bodyweight twice per week for the duration of the experiment, which was terminated when tumors approached the size limit of 1000 mm³. At the termination of the ⁹⁰Y therapy study (day 36), mice were injected with ⁸⁹Zr-DFO-trastuzumab (~130 μCi, ~30 μg) to allow for PET imaging, biodistribution, and autoradiography/histology experiments. These experiments could not be performed with the strictly β⁻-emitting isotope ⁹⁰Y (due both to the 36 day therapy study and ⁹⁰Y's lack of analytically useful gamma or positron emissions).

2.12. Cerenkov luminescence imaging (CLI)

Optical images of Cerenkov radiation (CR) were obtained using an IVIS 200 (Caliper Life Sciences) optical imaging machine. This system uses a cryo-cooled charge-coupled device for high-sensitivity detection of low-intensity sources. The mice that had been injected with ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab for the radioimmunotherapy experiments (n = 4 per group; ~100 μCi/mouse) were imaged using two-minute exposures 72 h after the administration of the therapeutic radioimmunoconjugates.

2.13. ⁸⁹Zr-DFO-trastuzumab PET imaging and biodistribution studies

Mice from the ⁹⁰Y therapy study (36 days post-injection of ⁹⁰Y or control) were imaged via PET with ⁸⁹Zr-DFO-trastuzumab (n = 4 from each therapy group). To this end, ⁸⁹Zr-DFO-trastuzumab was radiolabeled, purified, and prepared as described above. PET imaging was performed using a micro-PET R4 rodent scanner (Focus 120, Concord Microsystems). Mice were administered radiolabeled antibody (~30 μg, ~130 μCi, ~4.8 MBq) in 200 μL sterile saline (0.9% NaCl) via intravenous tail vein injection. Approximately 5 min prior to PET image acquisition, mice were anesthetized via inhalation of 2% isoflurane/oxygen gas mixture (Baxter Healthcare, Deerfield, IL) and placed on the scanner bed where anesthesia was maintained. PET images were acquired at 72 h, with PET data being recorded via static scans with a minimum of 15 million coincident events (~30 min). An energy window of 350–700 keV and a coincidence timing window of 6 ns was used. Data were sorted into 2D histograms by Fourier rebinning, and transverse images were reconstructed by filtered back-projection (FBP) into a 128 × 128 × 63 (0.72 × 0.72 × 1.3 mm³) matrix. The image data were normalized to correct for non-uniformity of response of the PET, dead-time count losses, positron branching ratio, and physical decay to the time of injection, but no attenuation, scatter, or partial-volume averaging correction was applied. The counting rates in the reconstructed images were converted to activity concentrations (percentage injected dose [%ID] per gram of tissue) by use of a system calibration factor derived from the imaging of a mouse-sized water-equivalent phantom containing ⁸⁹Zr. Images were analyzed using ASIPro VM software (Concorde Microsystems).

These ⁸⁹Zr-DFO-trastuzumab-treated mice were euthanized for the biodistribution experiments immediately following the single 72 h PET imaging time point. Biodistribution experiments with ⁸⁹Zr-DFO-trastuzumab did not require organ digestion or scintillation counting, as the gamma emissions from ⁸⁹Zr could be quantified using an automated gamma counter. The organs from ⁸⁹Zr biodistribution experiments were placed in pre-weighed test tubes, weighed, and then the amount of radioactivity present was determined using automated gamma counting (PerkinElmer). The counts were background- and decay-corrected from the time of injection and then converted to the percentage of injected dose (%ID) per gram of organ tissue (%ID/g). The radioactivity counts measured in each organ were converted to activity (μCi) using a calibration curve created from known standards of ⁸⁹Zr (serial dilution from ~3 μCi ⁸⁹Zr). Biodistribution data from mice injected with ⁸⁹Zr-DFO-trastuzumab can be found in the supporting information (Tables S5–S7).

2.14. Autoradiography and histology of ⁸⁹Zr-DFO-trastuzumab in ⁹⁰Y treated tumors

Following PET imaging, tumors were excised and embedded in optimal-cutting-temperature mounting medium (OCT, Sakura Finetek) and frozen on dry ice. Series of 10 μm frozen sections were then cut. To determine radiotracer distribution, digital autoradiography was performed by placing tissue sections in a film cassette against a phosphor imaging plate (Fujifilm BAS-MS2325; Fuji Photo Film) for an appropriate exposure period at −20 °C. Phosphor imaging plates were read at a pixel resolution of 25 μm with a Typhoon 7000 IP plate reader (GE Healthcare). After autoradiographic exposure, the same frozen sections were then used for immunohistochemical (IHC) staining and microscopy. The immunohistochemistry detection of Androgen Receptor was performed using a Discovery XT processor (Ventana Medical Systems). A rabbit polyclonal C-erbB2 antibody (Enzo, cat.# ALX-810-227) was used at 10 μg/mL concentration. The tissue sections were fixed in 4% paraformaldehyde and blocked for 30 min in 10% normal goat serum, 2% BSA in PBS. The incubation with the primary antibody was done for 5 h, followed by 16 min incubation with biotinylated goat anti-rabbit IgG (Vector labs, cat#:PK6101) in 1:200 dilution. Blocker D, streptavidin–HRP and DAB detection kit (Ventana Medical Systems) were used according to the manufacturer instructions. Sequential sections were stained with H&E. Whole mount images were acquired at ×100 magnification using a BX60 microscope (Olympus America, Inc.) equipped with a motorized stage (Prior Scientific Instruments Ltd.) and DP80 camera (Olympus). Whole-tumor montage images were obtained by acquiring multiple fields at ×40 magnification, followed by alignment using MicroSuite Biologic Suite (version 2.7; Olympus). IHC and autoradiographic images were registered using Adobe Photoshop (CS6) as previously described [38].

3. Results and discussion

3.1. H₄octapa coordination chemistry

Solution nuclear magnetic resonance (NMR) data and density functional theory (DFT) calculations from a previous study exploring the solution chemistry of H₄octapa with Y³⁺ suggested that this metal–ligand pair forms complexes similar to those of H₄octapa with In³⁺ and Lu³⁺[26]. More specifically, the structures of [Y(octapa)]⁻ and [Lu(octapa)]⁻ were predicted for both 8-coordinate (ligand only) and 9-coordinate (ligand and one H₂O) architectures and showed striking similarities. Furthermore, the molecular electrostatic potentials (MEP) of the [Y(octapa)]⁻ and [Lu(octapa)]⁻ complexes were mapped onto the surface of the DFT-calculated structures, suggesting very similar charge distributions between the two complexes [26,28]. Potentiometric titrations determined H₄octapa to have a formation constant (log K_ML) of 18.3 ± 0.1 with Y³⁺ and 20.08 ± 0.9 with Lu³⁺, revealing high thermodynamic stability [26,28]. Another previous study compared the in vivo performance of H₄octapa-trastuzumab and DOTA-trastuzumab with the radiometals ¹¹¹In and ¹⁷⁷Lu, highlighting the improved radiolabeling properties of H₄octapa compared to DOTA [28]. This preliminary in vivo work demonstrated that H₄octapa is a strong chelator for the radiometals ¹¹¹In and ¹⁷⁷Lu, and the similarities in non-radioactive characterization between the [Y(octapa)]⁻ and [Lu(octapa)]⁻ complexes suggest that H₄octapa may be an excellent ligand for radiopharmaceutical applications with ⁸⁶Y and ⁹⁰Y. The chelator H₄octapa presents an alternative to the “gold standard” chelator CHX-A″-DTPA for radiometals such as ¹¹¹In, ¹⁷⁷Lu, and ⁸⁶Y/⁹⁰Y.

3.2. Bioconjugation, radiolabeling, and in vitro characterization

The purpose of this study was to evaluate the in vitro and in vivo performance of the chelator H₄octapa with the radiometal ⁹⁰Y and compare it with the existing `gold standard' yttrium chelator CHX-A″-DTPA. Although DOTA forms superbly stable complexes with radiometals such as ¹¹¹In, ¹⁷⁷Lu, and ⁸⁶Y/⁹⁰Y, its radiolabeling kinetics are very slow, and it the high temperature conditions that it requires for radiolabeling are not compatible with antibodies. To begin, trastuzumab (HER2-targeting antibody, ~150 kDa) was incubated under slightly basic conditions (pH 8.5–9.0) with 6 equivalents of p-SCN-Bn-H₄octapa or p-SCN-Bn-CHX-A″-DTPA and purified via size exclusion chromatography (2 × PD10) and spin filtration (2 × Amicon Ultra 50 kDa). After purification, aliquots of trastuzumab immunoconjugates (10 μL, 1 μg/μL, n = 3) were frozen and sent for MALDI-TOF analysis. Mass spectrometry results indicated that these modifications yielded 1.5 ± 0.1 chelates per antibody in the case of p-SCN-Bn-H₄octapa and 1.0 ± 0.6 chelates per antibody in the case of p-SCN-Bn-CHX-A″-DTPA.

H₄octapa-trastuzumab and CHX-A″-DTPA-trastuzumab were radiolabeled with ⁹⁰Y³⁺ in NH₄OAc buffer (pH 5.5, 200 mM, treated with Chelex-100 resin) for 60 min at 37 °C. Radiolabeling yields were evaluated using iTLC-SA (acidic) silica-embedded paper strips, and a mobile phase of DTPA (pH 2.5, 50 mM) was used to resolve “free” ⁹⁰Y³⁺ from antibody-bound radiometal (Fig. 2). High radiochemical yields were easily achieved with ⁹⁰Y³⁺ (>95% radiochemical yield crude, >99% purity after purification). Both H₄octapa-trastuzumab and CHX-A″-DTPA-trastuzumab (50 μg) were also radiolabeled with ⁹⁰Y at room temperature to compare their kinetics. In these experiments, the radiochemical yields were evaluated after 15 min, with both immunoconjugates obtaining crude radiochemical yields of >95% (by iTLC), revealing equally facile radiolabeling kinetics with ⁹⁰Y.

Fig. 2.

Radiolabeling yields with ⁹⁰Y as determined by iTLC (iTLC-SA acidic strips, mobile phase 50 mM DTPA, pH 2.5). Un-coordinated ⁹⁰Y³⁺ elutes with the solvent front of the iTLC-SA strip (~100–125 mm), while the ⁹⁰Y-labeled radioimmunoconjugate remains at the baseline (~25 mm).

The radiolabeled immunoconjugates were evaluated by a cell-binding assay to determine their immunoreactivity (Table 1, % of native binding affinity). This in vitro assay used the same SKOV3 cancer cells used for in vivo experiments. Briefly, the antigen over-expressing cells (10 million SKOV3 per microcentrifuge tube [39]) were mixed with a small quantity of radioimmunoconjugate (~6 ng), and the percent radioimmunoconjugate bound to cells after washing was determined as the % immunoreactivity. The immunoreactivity values for ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab were 93.9 ± 0.9% and 91.2 ± 0.8%, respectively. These values indicate that chelator conjugation and radiolabeling yielded the final radioimmunoconjugates with minimal antibody degradation or radiolysis, and did not disrupt their ability to bind their target antigen. In order to test the stability of the radioimmunoconjugates under biologically relevant conditions, both constructs were incubated in human serum for 3 days at 37 °C. At the end of the experiment (96 h), the stability of the ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab conjugates were found to be excellent: 87.1 ± 0.9% and 94.8 ± 0.6%, respectively (Table 1, Fig. 3). In summary, the radiolabeling yields, radiochemical purity, immunoreactivity, and serum stability results were excellent for both ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab. Despite the problems encountered with ⁸⁶Y that precluded its inclusion in this study, ⁸⁶Y remains an attractive and potentially useful isotope for PET imaging applications.

Table 1.

Chemical and in vitro biological characterization data for ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab radioimmunoconjugates.

Chelator	Isotope	Radiolabeling yields (37 °C, 60 min)	Chelates/mAba	Specific activity (mCi/mg)	Immunoreactive fraction (%)b	Serum stability 96 h (%)c
H4octapa	90Y	Crude = 99%, Pure = 99%	1.5 ± 0.1	1.8	91.2 ± 0.8	94.8 ± 0.6
CHX-A″-DTPA	90Y	Crude = 96%, Pure = 99%	1.0 ± 0.6	1.8	93.9 ± 0.9	87.1 ± 0.9

^aMALDI-TOF MS/MS mass spectrometry (n = 3) spectra in Figs. S1–S9.

^bDetermined by saturation binding cell assay (10 × 10⁶ SKOV3 cells per vial, n = 4).

^cDetermined by incubation in human serum (37 °C for 96 h; n = 3).

Fig. 3.

Stability comparison of ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab in human blood serum from 0 to 96 h (37 °C, 500 rpm mixing), showing excellent stability for both radioimmunoconjugates (87.1 ± 0.9% and 94.6 ± 0.3% at 96 h, respectively).

3.3. Biodistribution studies of ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab

To expand further our comparison of the new chelator H₄octapa to the incumbent CHX-A″-DTPA, the in vivo performance of the ⁹⁰Y-labeled immunoconjugates was assessed via biodistribution experiments in a model system using HER2-expressing SKOV3 ovarian cancer cells. Each of the radiolabeled antibodies – ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab – was injected via tail vein into female nude athymic mice bearing subcutaneous SKOV3 ovarian cancer xenografts on the left shoulder (~20–30 μCi, 0.74–1.1 MBq, ~15–20 μg, in 200–250 μL of sterile saline; tumor volume ~ 100–150 mm³). After 24 and 72 h (n = 4 per time point), the mice were euthanized via CO₂ (g) asphyxiation, and 14 organs including the SKOV3 tumors were removed, weighed, and assayed for radioactivity content on a scintillation counter (digested and bleached organ samples). A control biodistribution experiment was also performed with ⁹⁰YCl₃, which illustrated that “free” ⁹⁰Y³⁺ accumulates primarily in the lungs, liver, spleen, kidneys, and bones.

The biodistribution profiles of both ⁹⁰Y-labeled immunoconjugates were nearly identical with the exception of higher uptake of ⁹⁰Y-octapa-trastuzumab in the spleen and slightly higher tumor uptake with ⁹⁰Y-CHX-A″-DTPA-trastuzumab. Higher spleen uptake is typically a result of antibody aggregation, which can occur during the storage, radiolabeling, purification, or formulation of radiolabeled antibodies. The slightly lower tumor uptake observed for ⁹⁰Y-octapa-trastuzumab could be because part of the injected dose was trapped in the spleen (possibly due to aggregation) instead of being free to accumulate in the tumor. The uptake of both radioimmunoconjugates was identical (within error) in organs that are known to accumulate free ⁹⁰Y³⁺ (e.g., kidneys, liver, bone), which suggests that the higher spleen uptake observed for ⁹⁰Y-octapa-trastuzumab was not a result of chelate instability. The control group of ⁹⁰YCl₃ clearly demonstrates which organs “free” ⁹⁰Y accumulates in, suggesting that both chelators offer equivalent and impressive in vivo stability (Fig. 4). Importantly, subsequent radioimmunotherapy experiments performed with ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab (vide infra) showed no signs of abnormal spleen uptake for either tracer. Antibody aggregation sometimes occurs during radiolabeling, purification, and handling, and it appears that it did not occur for this ⁹⁰Y therapy study. This comparison suggests that both H₄octapa and CHX-A″-DTPA are highly stable and appropriate choices for ⁹⁰Y chelation for in vivo use.

Fig. 4.

Biodistribution data showing a comparison of the stability and performance of ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab (S.I.=small intestine, L.I.=large intestine), including a control group inject with ⁹⁰YCl₃ used to demonstrate the primary organs in which “free” ⁹⁰Y³⁺ accumulates.

3.4. ⁹⁰Y Radioimmunotherapy and Cerenkov luminescence imaging using ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab in mice bearing subcutaneous SKOV3 xenografts

After the successful radiolabeling and biodistribution results with ⁹⁰Y described above, it was deemed prudent to test these two radioimmunoconjugates for their practical ability to deliver therapeutic doses of ⁹⁰Y to target tissue. To this end, therapeutic doses (100 μCi, 3.7 MBq) of both ⁹⁰Y-labeled radioimmunoconjugates were injected into cohorts of mice bearing subcutaneous SKOV3 xenografts. More specifically, three cohorts of mice were prepared (n = 4 each treatment, n = 5 control): ⁹⁰Y-CHX-A″-DTPA-trastuzumab (30 μg, 100 μCi, 3.7 MBq), ⁹⁰Y-octapa-trastuzumab (30 μg, 100 μCi, 3.7 MBq), and controls (30 μg non-radiolabeled trastuzumab). Cerenkov luminescence imaging (CLI) was performed 3 days after the injection of the radioimmunoconjugates, which provided visual confirmation of tumor uptake that could not be obtained by PET or SPECT imaging with ⁹⁰Y (Fig. 5). To ensure that no abnormal uptake in the spleen was observed for either antibody (typically the result of antibody aggregation during radiolabeling and the formulation of doses), the mice were also imaged by CLI on the left side (the side opposite the subcutaneous SKOV3 xenografts). The spleen sits close to the skin on the left side of mice, and therefore high spleen uptake should be observable by CLI. No accumulation in the spleen could be observed by CLI in this experiment, suggesting no abnormal uptake in the spleen.

Fig. 5.

Cerenkov luminescence imaging of ⁹⁰Y therapy mice 72 h after the administration of ⁹⁰Y-CHX-A″-DTPA-trastuzumab or ⁹⁰Y-octapa-trastuzumab (100 μCi per mouse, ~30 μg trastuzumab, 2-min imaging time), anesthetized with isoflurane, imaged for 2 min, showing substantial accumulation in the HER2-expressing SKOV3 tumors.

The tumor volumes of the ⁹⁰Y-treated mice were monitored twice per week using caliper measurements, and the general health of the mice was observed by appearance and body weight. This preliminary radioimmunotherapy pilot study was started with larger cohorts of mice bearing SKOV3 xenografts (n=8 per group) to account for tumors that did not take and for tumors that were much larger or smaller than average. This approach yielded cohorts of n=4 with similarly sized tumors for each therapy group and n=5 for the control group. By day 36, the control tumors were approaching the 1000 mm³ limit of the study, so this time was chosen as the end point for the study. A plot of the normalized tumor growth vs. time is displayed in Fig. 6 and clearly shows nearly identical treatment efficacy for the 90Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab groups, with substantial reductions in tumor growth rate being compared to the control group. This demonstrates that the serum stability and biodistribution experiments – which suggested comparable stability and in vivo performance of ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab – did indeed translate to equivalent radioimmunotherapeutic efficacy as measured by tumor growth response. The slightly lower tumor uptake observed in the biodistribution study of ⁹⁰Y-octapa-trastuzumab could have been a result of higher spleen uptake due to a minor amount of aggregation of the antibody during radiolabeling, which was not observed during the ⁹⁰Y therapy study. These results confirm that the chelators H₄octapa and CHX-A″-DTPA are effective choices for ⁹⁰Y in terms of radiolabeling kinetics, stability, and in vivo performance.

Fig. 6.

Normalized tumor growth during the 36 day therapy experiment, showing substantial reductions in tumor growth in the cohorts treated with ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab compared to the control group that was treated with trastuzumab only.

3.5. Post-radioimmunotherapy ⁸⁹Zr-DFO-trastuzumab PET imaging, biodistribution, and autoradiography in mice following radioimmunotherapy

PET scans are commonly used in the clinic to monitor the response of cancer patients to therapy. However, in the case of this radioimmunotherapy study, the therapeutic isotope (⁹⁰Y) does not emit any particles suitable for imaging. Furthermore, 36 days after the administration of the ⁹⁰Y-labeled radioimmunoconjugates in this study, too little radioactive ⁹⁰Y was left to perform autoradiography experiments. At the conclusion of the therapy study, the treated mice were injected with ⁸⁹Zr-DFO-trastuzumab to facilitate PET imaging and autoradiography of tumor sections. To this end, at the end of the ⁹⁰Y radioimmunotherapy study (day 36), the mice from each group (n = 4) were injected with ⁸⁹Zr-DFO-trastuzumab (~130 μCi, ~4.8 MBq, ~30–35 μg) and imaged after 72 h. The PET images in Fig. 7 illustrate substantial uptake of ⁸⁹Zr-DFO-trastuzumab in the SKOV tumors of the mice from the ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab radioimmunotherapy therapy cohorts. Not surprisingly, these images provide a telling picture of the substantially larger tumors of the animals in the control group. Furthermore, lower activity concentrations of ⁸⁹Zr-DFO-trastuzumab were observed in tumors of the control group, most likely due to the large size of the tumors (~1000 mm³) and the presence of poorly-perfused necrotic regions.

Fig. 7.

⁸⁹Zr-DFO-trastuzumab PET imaging of representative mice from the ⁹⁰Y radioimmunotherapy experiment. The mice were injected with ⁸⁹Zr-DFO-trastuzumab (~30 μg, ~130 μCi, ~4.8 MBq) on day 36 of the radioimmunotherapy study and imaged 72 h later. The images clearly show higher uptake of ⁸⁹Zr-DFO-trastuzumab in the tumors of the mice that had been treated with ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab and lower uptake in the much larger tumors of the mice from the control group.

The localization of ⁸⁹Zr-DFO-trastuzumab in the mice from the therapy study was evaluated via biodistribution, which showed similar tumor uptake and tissue distribution in both therapy groups. Tumor uptake in control mice was lower than therapy groups as a result of the substantially larger tumors (expressed as %ID/g). During the biodistribution necropsy (Fig. 8), pieces of SKOV3 tumor were taken from the ⁹⁰Y treated and control mice that had been injected with ⁸⁹Zr-DFO-trastuzumab. Autoradiography and histology (hematoxylin and eosin as well as HER2 staining) were performed on sections of tumors from each therapy group (Fig. 9). Results confirm high expression of HER2 in SKOV3 tumors, with the uptake of ⁸⁹Zr-DFO-trastuzumab mimicking the distribution that would be expected from the ⁹⁰Y-labeled radioimmunoconjugates. Interestingly, the distribution of ⁸⁹Zr-DFO-trastuzumab in the tumors of the ⁹⁰Y-octapa-trastuzumab therapy group (Fig. 9, row 2) shows high concentrations of ⁸⁹Zr in a region with low HER2 expression. This is most likely a result of fluid pooling in a necrotic region of the tumor, which likely formed as a result of radioimmunotherapy. ⁸⁹Zr-DFO-trastuzumab showed similar uptake in both ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab groups (Figs. 7 and 8).

Fig. 8.

Biodistribution of ⁸⁹Zr-DFO-trastuzumab in the mice from the ⁹⁰Y radioimmunotherapy experiment. The mice were injected with ⁸⁹Zr-DFO-trastuzumab on day 36 of the radioimmunotherapy trial and euthanized for biodistribution 72 h after the administration of the imaging agent.

Fig. 9.

Sections of tumors from the ⁸⁹Zr-trastuzumab PET/biodistribution obtained at the conclusion of the ⁹⁰Y radioimmunotherapy trial, showing representative SKOV3 tumors from the (1) ⁹⁰Y-CHX-A″-DTPA-trastuzumab group, (2) ⁹⁰Y-octapa-trastuzumab group, and (3) control (trastuzumab only) group. Panels display (A) hematoxylin- and eosin (H&E)-stained tumor sections outlining the architecture of the tumor; (B) immunofluorescence staining of HER2 expression; (C) digital autoradiography demonstrating the distribution of ⁸⁹Zr-DFO-trastuzumab within the tumors; and (D) overlay of the digital autoradiograph with the H&E and HER2 staining.

4. Conclusions

The principal benefit of acyclic chelators such as H₄octapa and CHX-A″-DTPA over macrocyclic chelators such as DOTA lies in the ability of the former to be radiolabeled with rapid kinetics without significant heating. Radiolabeling experiments revealed that both CHX-A″-DTPA-trastuzumab and H₄octapa-trastuzumab could be labeled with ⁹⁰Y³⁺ in very high radiochemical purity after only 15 min at room temperature. Both ⁹⁰Y-CHX-A″-DTPA-trastuzumab and ⁹⁰Y-octapa-trastuzumab exhibited excellent in vitro and in vivo stability, high immunoreactivity, and comparable biodistribution profiles in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. Furthermore, both ⁹⁰Y-labeled radioimmunoconjugates displayed identical therapeutic efficacy in radioimmunotherapy studies in mice bearing SKOV3 tumors. In these experiments, 36 days after the administration of the radioimmunoconjugates, the average tumor volume in both treatment groups was ~300 mm³, a remarkable shift compared to the average volume of ~1000 mm³ for the tumors of the control group. In the end, these data clearly suggest that H₄octapa and CHX-A″-DTPA are both suitable chelators for use with ⁹⁰Y.