The [111In]In-labeled hu3F8 and Rituximab were prepared as previously described [22, 23]. Briefly, the antibody was conjugated to p-SCN-Bn-DTPA (molar ratio: 1:7–9 (hu3F8), 1:1 (Rituximab)) at 37°C for 1 hour, and the conjugate was purified by size-exclusion centrifugation. The resulting antibody conjugate was then added to 40.7-129.5 MBq of [111In]In-InCl3, 0.5 mL of 0.2 M HCl, and 0.06 mL of 3 M NH4OAc, pH 4. The resulting mixture was incubated at 37°C for 30–60 minutes and then purified by a PD-10 column. The radiochemical purity was determined by radio-thin layer chromatography and the protein concentration was determined by spectrophotometry (Nanodrop, Wilmington, DE, USA).
In vitro studies
Immunocytochemical and immunohistochemical Staining: The cross-species reactivity of hu3F8 with human (U2OS) and canine (OSCA78) OS cells and spontaneously occurring canine OS tumor tissue was first evaluated by immunocytochemistry (ICC) and immunohistochemistry (IHC), respectively. Cells grown in 8-well chambers were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature. After washing with PBS, cells were blocked for 30 min by serum-free antigen blocking solution (Dako Agilent, Carpinteria, CA, USA), followed by overnight incubation with hu3F8 (1:200) at 4°C. Cells were then washed and incubated with FITC-labeled anti-human Fc secondary antibody (1:1000, ThermoFisher Scientific, Waltham, MA, USA) for 1 hour. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA). For IHC of canine OS tissue, six micron-thick sections were deparaffinized. Endogenous peroxidase was quenched by 0.3% H2O2. The samples were incubated in an antigen retrieval solution (Dako) and pressure cooked for 3 minutes. The samples were blocked and incubated with hu3F8 (1:200) for overnight. After washing with PBS, OS samples were incubated with a biotinylated anti-human Fc secondary antibody (1:800 dilution, ThermoFisher Scientific), followed by incubation with streptavidin-horseradish peroxidase complex and developed with 3,3’-diaminobenzidine peroxidase substrate kit (Vector Laboratories). The OS sections were then counterstained with hematoxylin. Human IMR32 cells known to express GD2 were used as a positive control, and SK-N-SH cells and dog spleen tissue were used as GD2 negative controls. Images were captured at 200× magnification using an upright microscope (Eclipse Ti, Nikon Instruments Inc., Melville, NY, USA).
Human IMR32, U2OS, canine OSCA78 cells, and canine white blood cells (negative control) (1 x 106) were collected and fixed with 4% PFA. The cells were then incubated with hu3F8 (5 µg/mL) for 30 min at 4°C. After washing with PBS, cells were incubated with Alexa Fluor 647-labeled goat anti-human IgG (Southern Biotech, Birmingham, AL, USA) for 30 min, washed with PBS, and analyzed on a FACS flow cytometer (BD Biosciences).
Receptor binding assay
The binding affinity of [111In]In-DTPA-hu3F8 was determined in IMR32 and OSCA78 cells as described previously with modifications [23, 24]. In brief, cells were seeded in 24-well plates at 1.5 x 105 cells/well 24 hours prior to the experiment. After 24 hours, the cells were washed twice with PBS and treated with a serial dilution of [111In]In-DTPA-hu3F8 (0.05–100 nmol/L) for 4 hours at 4°C. Non-specific binding was assessed by pretreating cells with 10 µg/well of unlabeled hu3F8 for 30 minutes at 4 ℃ followed by [111In]In-DTPA-hu3F8 incubation. Each assay was conducted in triplicate. After 4 hours of incubation, cells were washed twice with ice cold PBS, dissolved in 0.5% sodium dodecyl sulfate solution and counted on a γ-well counter (PerkinElmer 2470 WIZARD2®). The protein concentration of the cell lysates was determined with a BCA protein assay kit (Pierce). The measured activity was normalized to the number of cells (sites/cell). The Michaelis-Menten equation was fitted to the binding curve to determine the dissociation constant (Kd) and maximum number of binding sites (Bmax) using Prism 9 (GraphPad, La Jolla, CA, USA).
The immunoreactivity of [111In]In-labeled antibodies was determined as previously described  by antibody adherence to IMR32 and OSCA78 cells. Two sets of tubes (1 x 107 cells/tube) were prepared for each cell line and 0.1µg of [111In]In-DTPA-hu3F8 or [111In]In-DTPA-Rituximab was used for immunoreactivity assessment. Immunoreactivity (%) = (Bound activity in cells / Total activity) x 100.
In vivo mouse studies
All animal studies were approved by the Animal Care and Use Committee of the Johns Hopkins University, School of Medicine. Six- to 8-week-old healthy female Nu/Nu mice were obtained from Charles River Laboratories (Wilmington, MA, USA). Mice received subcutaneous injections of either 1 × 107 IMR32 or 3 x 106 of OSCA78 in Matrigel in the right flank for biodistribution studies or both flanks for SPECT imaging.
Biodistribution studies were carried out as previously described . Following a growth period of approximately 8 weeks, IMR32 and OSCA78 tumor-bearing mice (n = 3–4) were injected intravenously with 100 µL of [111In]In-DTPA-hu3F8 (3.03 MBq/10 µg). Mice were euthanized with isoflurane at either 24 or 72 hours after injection. The major organs, including blood, heart, lungs, liver, kidneys, spleen, stomach (with content), intestine (with content), femur, muscle, and tumors were harvested, weighed and measured in an automatic γ- well counter. The percentage of injected activity per gram (%IA/g) was calculated by comparison with a weighed, diluted standard. In addition, to examine the specific accumulation of radiolabeled hu3F8 in OS tumors, additional biodistribution studies were performed in OSCA78 tumor-bearing mice using an [111In]In-labeled irrelevant antibody, [111In]In-DTPA-Rituximab (1.74 MBq/10 µg) (n = 3). These mice were sacrificed at 72 hours after injection and their organs were harvested and processed, as described above. After counting, fresh OSCA78 tumors were embedded in OCT compound and sectioned for IHC staining to detect radiolabeled hu3F8 using an anti-human Fc antibody.
Mice with established OSCA78 tumors in both flanks were injected intravenously with either [111In]In-DTPA-hu3F8 (3.03 MBq, 10 µg) or [111In]In-DTPA-Rituximab (1.74 MBq, 10 µg). IMR32 xenografted mice received intravenous injection of [111In]In-DTPA-hu3F8 (3.03 MBq, 10 µg). At 24-, 48-, and 96-hours post-injection, SPECT/CT images were acquired with a NanoSPECT/CT system (Bioscan Inc., Washington, DC, USA) using multiplexed multipinhole gamma detectors and high-resolution collimators. Images were acquired for 60 min at each time point and were reconstructed at voxel size of 0.6 mm3 isotropic using the vendor-supplied iterative algorithm. SPECT images were co-registered with CT images for an anatomic reference using PMOD, version 3.7(PMOD Technologies LLC, Zurich Switzerland), and the voxel intensity was calibrated using images of a standard with known activity and volume.
In Vivo dog studies
Tumor-free dogs: Three healthy research dogs (13.6–17.0 kg) received intravenous injection of either the unlabeled hu3F8 (100 µg; n = 2) or [111In]In-DTPA-hu3F8 (17.0 MBq/100 µg; n = 1) to assess the tolerance of the antibody. Fifteen minutes prior to injection, the dogs were fasted and received intravenous analgesia and antihistamine prophylaxis with 0.05 mg/kg hydromorphane and 1 mg/kg of diphenhydramine, respectively. The dog that received [111In]In-DTPA-hu3F8 was sedated with 0.005 mg/kg fentanyl and induced with 0.25 mg/kg midazolam and 4 mg/kg propofol, intubated, and placed under isoflurane anesthesia with mechanical ventilation. Whole body SPECT/CT imaging with two bed positions (SPECT: voxel size = 2.4 mm3, final matrix = 256 x 256 x 337; CT: matrix size = 512 x 512, pixel size = 0.9766 x 0.9766 mm2, slice thickness = 1.0 mm, tube voltage = 130 kVp, tube current = 26 mA) was performed at 4 and 24 hours after injection on the Symbia T16 Series scanner (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) using a medium energy collimator. SPECT images were reconstructed using vendor’s Flash3D OSEM algorithm with compensation for attenuation and resolution. The attenuation map was generated from CT images. Volume of interests (VOIs) were drawn for the liver, spleen, kidneys, and heart on the first 4-hour CT images, and were then transposed to the 24-hour time point after deformable registration of the scans had been performed using Velocity software (version 3.1, Varian Medical Systems, Palo Alto, CA, USA). One syringe containing 18.35 MBq [111In]In was imaged for 5 min to determine the calibration factor (MBq/CPM) of the system. VOI for urinary bladder was draw on both 4- and 24-hour CT images due to the change of the bladder size with time.
Spontaneous OS Dogs: Two companion dogs (A: 30.0 kg and B: 32.1 kg) were diagnosed with spontaneously occurring OS. Metastatic recurrence of OS after amputation and chemotherapy was confirmed by fine needle aspirate at 5 weeks in dog A and 18F-fluorodeoxyglucose-positron emission tomography ([18F]F-FDG-PET) at 12 months in dog B. Dog A received 100 MBq of [111In]In-DTPA-hu3F8 (0.35 mg) intravenously while dog B received 36 MBq of [111In]In-DTPA-hu3F8. Whole body SPECT/CT imaging was performed 48 hours post-injection using similar parameters to those for tumor-free dogs. Dog A was euthanized after pain palliation failed, four months after [111In]In-DTPA-hu3F8 injection. Necropsy was performed by a board-certified pathologist. The shoulder and lung metastatic tissues were obtained and processed for histological analysis as described above.