Antibody conjugation and radiolabelling
The murine monoclonal antibody, 11B6, was first described and characterized by Vaisanen et al. [24] and was provided by the University of Turku (Turku, Finland) for this study. Conjugation and radiolabelling was performed as previously described by Tolmachev et al. [25]. Briefly, 2 mg of 11B6 was conjugated with the chelator CHX-A"-DTPA (B-355, Macrocyclics; Dallas, TX, USA) through the isothiocyanate functional group. A solution of 11B6 (4 to 5 mg/mL in PBS) was adjusted to pH 9.2 using 0.07 M sodium borate buffer (Sigma Aldrich; St. Louis, MO, USA). CHX-A"-DTPA was then added to the protein solution at a molar ratio of 3:1 (chelator to antibody) and incubated at 40°C with gentle shaking. The reaction was terminated after 4 h, and CHX-A"-DTPA-11B6, henceforth referred to as DTPA-11B6, was separated from the free chelate by size-exclusion chromatography on a NAP-5 column (GE Healthcare; Uppsala, Sweden) equilibrated with 20 mL of 0.2 M ammonium acetate buffer (Sigma Aldrich), pH 5.5. Conjugated 11B6 was eluted with 1 mL of ammonium acetate buffer, and aliquoted samples were stored at -20°C.
For radiolabelling, approximately 125 μL of DTPA-11B6 (approximately 1 μg/μL in 0.2 M ammonium acetate buffer pH 5.5) was mixed with a predetermined amount (approximately 50 to 100 MBq) of 111InCl3 (Mallinckrodt Medical; Dublin, Ireland), incubated at room temperature for 1.5 to 2 h and then purified on a NAP-5 column (GE Healthcare) equilibrated with PBS (Thermo Scientific; Waltham, MA, USA). Labelling efficiency and kinetics were monitored by instant thin-layer chromatography (ITLC) (Biodex, Shirley, NY, USA) eluted with 0.2 M citric acid (Sigma Aldrich). In this system, the radiolabelled conjugate remains at the origin line, while free 111In and 111In-DTPA migrate with the solvent front. The radioactive distribution was determined using a Cyclone Storage Phosphor System with Optiquant quantification software (Perkin Elmer; Waltham, MA, USA).
Binding kinetics with surface plasmon resonance
The 11B6 binding kinetics were analysed by surface plasmon resonance using a Biacore 2000 (Biacore AB; Uppsala, Sweden). The affinity of 11B6 to hK2 before and after CHX-A"-DTPA conjugation was determined. The hK2 antigen, provided by the University of Turku (Department of Biotechnology; Turku, Finland), was produced and purified as previously described [26]. hK2 antigen (25.9 μg/mL in 10 mM sodium acetate buffer pH 4.0 (Sigma Aldrich)) was immobilized on a CM4 research grade chip (Biacore AB) by amino coupling using N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1 M ethanolamine hydrochloride-NaOH, pH 8.5, in a Biacore 2000 system. Samples were flown over two flow cells, one being a blank reference, in five different concentrations ranging from 0.5 to 100 nM to detect eventual binding. One of the two flow cells contained immobilized hK2, while the other was served as a blank reference. The binding kinetics were studied in a 3-min-long association phase and a 15-min-long dissociation phase with a flow rate of 30 μL/min, followed by regeneration with 25 mM glycine buffer pH 2.7. Kinetic constants were calculated using a 1:1 Langmuir binding model with correction for mass transfer. BIAEvaluation 4.1 software (Biacore AB) was used for calculations.
Stability studies
The stability of 111In-DTPA-11B6 was assessed in triplicate by incubating the compound at 4°C in PBS buffer or at 37°C in murine serum collected from normal NMRI mice. For stability in PBS, 1 μL (n = 3) was taken at 1, 2, 3 and 7 days and analysed by ITLC. For stability in serum, 10 μL of 111In-DTPA-11B6 (corresponding to 3 μg of antibody with 0.8 to 0.9 MBq 111In) was mixed with 100 μL of mouse serum. Approximately 20 μL of each mixture was collected after 2, 3 and 9 days of incubation and analysed by SDS-PAGE on a NuPAGE 4% to 12% Bis-Tris gel (Invitrogen; Carlsbad, CA, USA) in MES buffer (200 V constant, approximately 30 min). 111In-DTPA and free 111In diluted in PBS were run in parallel with the incubated sample as controls. The distribution of the samples along the gel was evaluated using a Cyclone Storage Phosphor System (Perkin Elmer).
Cell lines
LNCaP and DU145 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI 1640 medium (Thermo Scientific) supplemented with 10% foetal bovine serum (Thermo Scientific) with 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Scientific). The cells were maintained at 37°C in a humidified incubator at 5% CO2 and were detached with trypsin-EDTA solution (Thermo Scientific).
Animal models
All animal experiments were conducted in compliance with the national legislation on laboratory animals' protection and with the approval of the Ethics Committee for Animal Research (Lund University, Sweden). Two animal models were used in this study, NMRI-Nu with subcutaneous (s.c.) xenografts and SCID mice with intra-tibial xenografts. NMRI-Nu mice (6-to-8-week-old, Taconic; Ry, Denmark) were inoculated in the right flank by s.c. injection of 5 to 8 × 106 cells in a 200 μL of cell suspension of 1:1 mixture of medium with Matrigel (BD Biosciences; San Jose, CA, USA). Tumours were allowed to develop for 6 to 8 weeks. SCID mice (6-to-8-week-old male, Charles River; Charles River, NJ, USA) were maintained under isoflurane anaesthesia during surgery. For intra-tibial inoculations, the tibia was punctured using a 23-gauge needle, and 1 × 105 LNCaP cells were injected into the tibial cavity. The puncture was closed with bone wax, the incision sutured and the animals received a palliative dose of Temgesic (Buprenorphine, RB Pharmaceuticals; Richmond, VA, USA) once daily for 3 days post-surgery. Intra-tibial tumours were allowed to develop for 8 to 10 weeks. Additionally, a group of normal NMRI mice (n = 4) were used to study the distribution of the tracer in healthy animals. Animals were euthanized by intraperitoneal (i.p.) injection with 20 μL per gram of body weight Ketalar-Rompun solution. (Ketalar, 10 mg/mL; Pfizer; New York, NY, USA, and Rompun, 1 mg/mL; Bayer Animal Health; Monheim, Germany).
Biodistribution studies
Biodistribution studies were conducted to evaluate the uptake of 111In-DTPA-11B6 in human prostate cancer LNCaP xenografts. Mice (n = 3 to 5 per time point) received 111In-DTPA-11B6 (0.4 to 0.6 MBq, 20 μg of mAb, in approximately 100 μL of PBS) through intravenous (i.v.) tail vein injection. Blood and organs (including tumour) were taken at 4, 24, 48, 72 and 168 h post-injection, weighed and measured in a NaI(TI) well counter (Wallac Wizard 1480 Wizard, Perkin Elmer). The activity injected into each animal was measured and used to determine the count rate, in comparison with a standard solution of 111In-DTPA-11B6. Data were corrected for background and physical decay.
Organ-specific uptake values were calculated as percent injected activity per gram of tissue (%IA/g) or percent injected activity (%IA). Among the organs resected were the lateral and ventral prostate, from now on referred to as prostate, and the submandibular glands, from now on called salivary glands.
In vivo binding specificity
In vivo competitive binding studies were performed to investigate the specificity of 111In-DTPA-11B6 to hK2. A 40-fold excess of non-labelled 11B6 was i.v. injected as a co-injection or at 168, 120 and 48 h prior to an i.v. injection of 111In-DTPA-11B6 in hK2-positive LNCaP xenografts (n = 3 to 4 per pre-injection time point). Blood and organs (including tumour) were taken at 48 h post-injection of 111In-DTPA-11B6, weighed and analysed as above. The binding specificity was also evaluated by measuring the uptake of 111In-DTPA-11B6 in hK2-negative DU145 xenografts expressing low levels of hK2 (n = 3) at 48 h post-injection, which are considered to be hK2-negative when compared to other PCa tumours.
Small animal PET/SPECT/CT/MR imaging
Animals were anaesthetized with 2% to 3% isoflurane gas (Baxter; Deerfield, IL, USA) for all imaging purposes. For SPECT/CT imaging, NMRI-nu mice with s.c. LNCaP xenografts (48 h post-injection, n = 4; 72 h post-injection, n = 3; pre-dosed 11B6, n = 4; co-injection 5A10, n = 3) and SCID mice (n = 3) with intra-tibial LNCaP xenografts were i.v. injected with approximately 8 MBq of 111In-DTPA-11B6 (approximately 20 μg of mAb in 150 μL of PBS) and imaged, for 1 h, by using a preclinical SPECT/CT scanner (NanoSPECT/CT Plus, Bioscan; Washington, DC, USA) with the NSP-106 multi-pinhole mouse collimator. SPECT data were reconstructed using HiSPECT software (SciVis; Goettingen, Germany). CT imaging was done before each whole-body SPECT.
Pre-dosed mice were given 0.8 mg of non-labelled mAb 48 h prior to injection of radiolabelled mAb. Co-injections with 111In-DTPA-11B6 and 1.5 mg of fPSA-specific 5A10 were done to evaluate the possible cross-reactivity of 111In-DTPA-11B6 with PSA.
The legs of the SCID mice were resected after imaging, and the radioactivity in the intra-tibial xenografted and the non-xenografted leg were measured in the NaI(Ti) well counter. Radiolabelling for SPECT of intra-tibial xenografts demonstrated 95% radiochemical purity and was injected directly without NAP-5 column purification. Verification of the intra-tibial tumour growth was performed by MR imaging. The legs were imaged in an 11.7 T (500 MHz for protons) vertical bore MR camera (Agilent Technologies; Palo Alto, CA, USA) equipped with Varian 88/55 micro-imaging triple axis gradient coil (1 T/m maximum gradient strength). Samples were placed in the centre of a Millipede imaging probe (Agilent Technologies; Santa Clara, CA, USA), with an inner diameter of 40 mm.
For PET/CT imaging, mice with LNCaP xenografts were i.v. injected with approximately 12 MBq 18F-FDG (n = 4) or approximately 12 MBq18F-Choline (n = 4) and imaged 1 h post-injection using a Bioscan NanoPET/CT Plus preclinical scanner for approximately 15 min. Both SPECT/CT and PET/CT images were analysed using InVivoScope 2.0 software (inviCRO; Boston, MA, USA), and ROIs were drawn using the CT image as anatomical reference.
Autoradiography and staining
After SPECT imaging at 48 and 72 h, s.c. tumours were resected and embedded in Tissue-Tek® O.C.T™ compound (Sakura Finetek; Alphen aan den Rijn, The Netherlands) and frozen on dry ice. The frozen samples were cryosectioned with a thickness of 20 μm for autoradiography analysis on a Cyclone Storage Phosphor System. The tumour sections were stained with Mayer's hematoxylin and chromotrope 2R, Ch2R (both from Histolab; Gothenburg, Sweden), and scanned using a light-microscope slide scanner (Mirax Midi, Carl Zeiss; Oberkochen, Germany). Thresholds for the autoradiograms were set in ImageJ v.1.47.
Statistical analysis
Data was analysed using the unpaired, two-tailed Student's t test (Microsoft Excel or GraphPad Prism v.4). Differences at the 95% confidence level (P < 0.05) were considered to be statistically significant. Figures were produced with GraphPad Prism v.4 (GraphPad Software). All biodistribution data are shown as an average %IA/g of 3 to 5 animals ± SD (standard deviation) unless otherwise stated.