Preparation of tumor xenograft model
All animal experiments were approved by the Animal Experiments Committee of Gunma University. MDA-MB-231, a triple-negative breast cancer cell line, was purchased from American Type Culture Collection (ATTC; Manassas, VA). Cells were grown in RPMI-1640 medium which contains 10% and 1% fetal bovine serum and antibiotics, respectively. The cell suspension was mixed with Matrigel (Corning Life Sciences; Corning, NY) at a 1:1 (v/v) ratio. Furthermore, the cell suspension (5 × 106 cells/mice) was implanted subcutaneously into the right dorsal flank of a 6-week-old female BALB/c nude mice (Japan SLC, Shizuoka, Japan). The tumor size was measured twice a week using a digital caliper, and the volume was calculated using the 0.5 × (length × width2) formulation. The biodistribution study was performed after the tumor volume reached approximately 200–300 mm3.
Preparation of biotinylated and radiolabeled bevacizumab
BV was purchased from Chugai Pharmaceutical Co. (Tokyo, Japan). Bt-BV was prepared with slight modification according to the previously described procedure [17]. BV was reacted with 6-[6-(biotinylamino)hexanoylamin] hexanoic acid N-hydroxysuccinimide ester (Dojindo Molecular Technologies; Kumamoto, Japan) in 0.1 M borate buffer (pH 8.5) at the molar ratio of 1:6 overnight at 37°C and purified using a Bio-Spin column (Bio-Rad Laboratories; Hercules, CA) eluted with phosphate-buffered saline (PBS). The average number of biotin molecules per BV was determined as 3.6 according to the previous report [15]. To obtain 111In-labeled BV, 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (SCN-Bn-DTPA; Macrocyclics; Dallas, TX) was conjugated to BV and labeled with 111InCl3 (Nihon Medi-Physics; Tokyo, Japan) according to the previously described procedure [17].
Preparation of radiolabeled streptavidin analogs
The SCN-Bn-DTPA in dimethylformamide (DMF) was added to StAv (5 mg/mL in 50 mM borate-buffered saline, pH 8.5) at the molar ratio of 5:1 and incubated at 37°C for 24 h. The purification was performed using a Bio-Spin column eluted with 0.1 M borate buffer (pH 8.5). Then, 50 equivalents of succinic anhydride in DMF (8.3 μL) was added to a 200 μL aliquot of a 5 mg/mL solution of DTPA-StAv. After incubating overnight at room temperature, purification was performed using a Bio-Spin column eluted with PBS. For the preparation of 111In- or 90Y-labeled DTPA-StAv and DTPA-succ-StAv, 30 μl (0.37–1.85 MBq) of 111InCl3 solution or 20–50 μL (74–185 MBq) of 90YCl3 solution (Nucleic; Braunschweig, Germany) was incubated in 0.25 M acetate buffer (pH 5.5, 50 or 100 μL, respectively) for 5 min at room temperature, followed by incubation with DTPA-StAv or DTPA-Succ-StAv (50 or 300 μg in 0.1 M phosphate buffer, respectively) for 1 h at 37°C. To trap unconjugated compounds, 3–5 μL of 100 mM EDTA was added to the reaction mixture. Then, radiolabeled StAv analogs were purified by size-exclusion HPLC using TSKgel SuperSW mAb HR column (7.8 × 300 mm; Tosoh Bioscience, Tokyo, Japan) eluted with 0.1 M phosphate buffer (pH 6.8).
Biodistribution studies
Four types of biodistribution studies were performed in either tumor xenograft mice or normal ddY mice (Japan SLC).
Study 1: Effect of protein dose on biodistribution of 111In-BV in tumor-bearing mice.
To determine the maximum amount of BV binding to the tumor, the biodistribution study of 111In-BVwas performed in tumor-bearing mice. Tumor-bearing mice were injected with 100 μL solution of 111In-BV (20 kBq) with serial amounts of BV (20, 50, 100, 250, and 500 μg/mouse). Mice were sacrificed 24 h after injection, and tissues of interest were excised and weighed. The radioactivity was measured using an automated γ-counter ARC-7001 (Hitachi-Aloka Medical; Tokyo, Japan). The radiotracer uptake was expressed as the percentage of the injected dose per gram of tissue (%ID/g). A reverse sigmoid curve fitting model was used to estimate the nonspecific accumulation of 111In-BV (SigmaPlot software ver. 11.0, Systat Software; San Jose, CA).
Study 2: Comparison between 111In-StAv and 111In-succ-StAv of biodistribution in tumor-bearing mice.
Bt-BV (100 μg/mouse) was injected into tumor-bearing mice, and avidin (44 μg/mouse, a molar equivalent [1 eq]) was injected 24 h later. Three hours after avidin injection, which is 27 h after the Bt-BV injection, 111In-StAv or 111In-succ-StAv (20 kBq/20 μg/mouse) was injected into the tail veins of the tumor-bearing mice. At 1, 3, 6, 24, and 72 h after radioactivity injection, a radiotracer uptake was evaluated as described above.
Study 3: Effect of avidin dose on biodistribution of111In-succ-StAv in normal mice.
Normal mice were injected with 100 μL solution of Bt-BV (100 μg/mouse), and the mice were injected with serial amounts of avidin (1, 5, and 10 eq of BV) 24 h later. Three hours after avidin injection, 111In-succ-StAv (20 kBq/20 μg/mouse) was injected. Radiotracer uptake was evaluated as described above at 1, 3, 6, and 24 h after radioactivity injection.
Study 4: Biodistribution of 111In-succ-StAv with final protocol in tumor-bearing mice.
Finally, the pretargeting 111In-succ-StAv biodistribution study was done with an optimized protocol based on studies 1–3 in tumor-bearing mice. Bt-BV (100 μg/mouse) was injected into the mice, and avidin (10 eq) was injected 24 h later. Three hours after avidin injection, 111In-succ-StAv (20 kBq/20 μg/mouse) was injected into the tail veins of the tumor-bearing mice. Radiotracer uptake was evaluated as described above at 1, 3, 6, 24, 72, and 168 h after radioactivity injection.
Therapeutic Study
When the tumors were fully established, animals were randomly divided into four groups (i.e., PRIT with 11.1 MBq (300 μCi, group 1), 9.25 MBq (250 μCi, group 2), and 7.4 MBq (200 μCi, group 3) of 90Y-succ-StAv and no treatment (group 4)). Bt-BV was injected intravenously into tumor-bearing mice as the PRIT strategy of this study. After 24 h, 10 eq of avidin was administered. Lastly, 3 h after avidin injection, 90Y-succ-StAv was administered. The tumor size was measured twice a week. The observation was terminated when the tumor volume exceeded 2,000 mm3 or the mice were dead.
Statistical Analysis
Data are expressed as mean ± standard deviation (SD). GraphPad Prism software version 6.0 (GraphPad Software; La Jolla, CA) was used for statistical analysis. The Student’s t-test was used for comparing the statistical difference of the two groups, and p < 0.05 was considered significant.