CT26 (CRL-2638) murine colorectal adenocarcinoma cells were purchased from the American Type Culture Collection (ATCC) and grown in McCoy's 5A Medium (Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS, HyClone) at 37°C with 5% CO2. GL261 glioma cells were kindly provided by Dr. Michael Lim (Johns Hopkins University, Baltimore) and maintained in DMEM media (ATCC) supplemented with 10% FBS.
Bolton-Hunter reagent (BH, N-succinimidyl-3-(4-hydoxyphenyl)-propionate) and TNF-α (mouse, recombinant) were purchased from Sigma-Aldrich. Radioiodines (Sodium 125- or 131-iodide) were purchased from MP Biomedicals and Nordion, respectively. IODO-GEN was purchased from Pierce. Mouse monoclonal IgG1 isotype control antibody (ab18447) and CD20 antibody (ab8237) were purchased from Abcam. PEGylated liposomal doxorubicin (DOXIL®) was purchased from Tibotec Therapeutics. Hydrogenated Chicken Egg L-α-Phosphatidylcholine (HEPC), 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000] (DSPE-PEG2000) and Cholesterol (Chol) were purchased from Avanti Polar Lipids. C. novyi-NT
spores were prepared as previously described [11
All animal experiments were overseen and approved by the Animal Welfare Committee of Johns Hopkins University, and were in compliance with the University standards. For the subcutaneous tumor model, female, six to eight week-old BALB/c mice (Harlan Breeders, ~20 g in weight) were used. Five million CT26 cells were injected subcutaneously into the right flank of each mouse and allowed to grow for ~10 days before randomization, group assignment, and treatment. C. novyi-NT spores were administered by a bolus tail vein injection of 300 million spores suspended in 0.2 mL of phosphate buffered saline, pH 7.5 (PBS). Cytotoxic anticancer agents were administered 16 hours later via the same route. TNF-α was reconstituted freshly before administration in doubly-distilled H2O at 100 μg/mL and diluted into 0.1% (w/v) BSA in PBS at a final concentration of 10 μg/mL. Cytotoxic agents were injected within a few minutes thereafter. Tumor volume was calculated as length × width2 × 0.5. For the orthotopic brain tumor model, female C57BL6 mice, 5-6 weeks of age, were purchased from the NCI-Frederick. Mice were anesthetized via intraperitoneal injection of 60 μL of a stock solution containing ketamine hydrochloride (75 mg/kg, Abbot Laboratories), xylazine (Xyla-ject®, 7.5 mg/kg, Phoenix Pharmaceutical), and ethanol (14.25%) in a sterile 0.9% NaCl solution. Following a 1-cm midline scalp incision, a 1-mm burr hole was placed over the right frontal bone, with its center 2 mm lateral to the sagittal suture and 1 mm anterior to the coronal suture. On a stereotactic frame, a sterile needle loaded with 20,000 GL261 cells was placed at a depth of 3 mm below the dura and the cells were injected slowly at a rate of 1 μL/minute. Afterwards, the animal was removed from the frame and the scalp incision closed with surgical staples. On day 12 post implantation of the tumor cells, a significant tumor was formed and 1 μg of mouse recombinant TNF-α or 100 μL of Doxil at 20 mg/kg, or both, were administered intravenously through the tail vein. Animals were monitored for potential side effects following drug administration. Animals were observed daily for any signs of deterioration, neurotoxicity, or movement disorders. They were inspected for signs of pain and distress, as per the Johns Hopkins Animal Care and Use Guidelines. If the symptoms persisted and resulted in debilitation, the moribund animals were euthanized. The brain and other organs were dissected and placed in formalin for further pathological studies. A single dose was administered for all therapeutic agents described above.
Three peptides (JHU009A: CNVDLQQKLIEN; JHU009B: CYPEWGTKDENGNIRK; JHU009C: CDMAQMLRNLPVTE) were used to immunize the mice for generating antibodies against C. novyi-NT
liposomase (A&G Pharmaceutical). After screening ~500 hybridoma clones by ELISA, one clone (JHU009-5F5) specific to the JHU009C peptide was eventually selected for the imaging study. The affinity and specificity of the JHU009-5F5 mAb were also confirmed by both ELISA and western blot analyses against purified liposomase protein [12
Radioiodination of Antibodies
Typically, 20 μg of purified antibody in 100 μL of PBS was added to an iodogen-coated vial. Sodium 125- or 131-iodide was then added to the vial at 2 to 5 mCi in 2 to 5 μL of 0.1 M NaOH, pH 10. The reaction was then incubated for 10 minutes at room temperature before purification on a PBS-equilibrated Sephadex G-25 desalting column (Amersham Biosciences) to remove unincorporated radioiodine. The radiochemical yield was typically 30% to 40%. The radiochemical purity was at least 95% as determined by thin-layer chromatography. Antibodies were labeled within 24 hours of use and stored in PBS at 4°C after labeling and purification.
Preparation of Liposomes
A mixture of HEPC:Chol:DSPE-PEG2000 at a molar ratio of 50:45:5 was solubilized in chloroform, followed by drying to a thin film under rotary evaporation and then under vacuum for 2 hours. The film was hydrated with arginine solution (80 mmol/L) in 4-(2-hydroxyethyl)-piperazine-1-sulphonic acid (HEPES, 80 mmol/L, pH 8.0) and submerged in a 65°C sonication bath (Bransonic) to form Large Multilamellar Vesicles (MLVs). This lipid suspension was extruded 10 times through a double stack of 0.1 μm Nuclepore filters (Whatman) using a Lipex Thermobarrel Extruder (Northern Lipids). The resulting colloidal suspension of Single Unilamellar Vesicles (SUV) was dialyzed against 150 mmol/L phosphate buffer (pH 5.6) at 4°C to exchange the external milieu of the liposomes and then filter-sterilized. The mean size of the SUVs was ~100 nm in diameter and polydispersity index ~0.1 as determined by quasi-elastic light scattering using a Malvern Zetasizer 3000 (Malvern).
Radioiodination of Bolton-Hunter reagent
Bolton-Hunter reagent (BH, N-hydroxysuccinimide (NHS) ester of HPPA) was labeled with sodium 125- or 131-iodide by the chloramine-T method and purified by solvent extraction. Briefly, 50 μL of chloramine T (4 mg/mL in phosphate buffer) and 3.7 to 37 MBq (0.1–1.0 mCi) of 125I-NaI or 131I-NaI were added to 2 μL of BH freshly solubilized in anhydrous dioxin (0.5 mg/mL). Iodination was achieved by incubation at room temperature for approximately 15 sec and then 400 μL of 100 mmol/L phosphate buffer (pH 7.4) was added. The radiolabeled BH was immediately extracted with 500 μL of toluene and the organic phase was removed and transferred to a glass tube. For the encapsulation of the reagent into liposomes, the organic solvent was evaporated using a dry nitrogen stream before adding the liposome suspension.
Encapsulation of the Iodinated Reagents into the Liposomes
For the chemical entrapment of the iodinated BH, arginine-containing liposomes were incubated for 30 min at 37°C with 125
I-BH. The labeling efficiency was determined by counting the liposome suspension before and after chromatography on a PD-10 column (GE Healthcare)[18
]. The radiochemical yield was typically 50% to 70%.
CT26-bearing BALB/c mice were injected via the tail vein with 50 μCi of 125I-liposomes or 125I-IgG1. Three to four mice in each experimental arm were sacrificed by cervical dislocation at 48 hours post injection. The liver, spleen, kidneys, muscle, and tumor were quickly removed as was ~0.1 mL of blood. The organs and blood were weighed and their radioactivity was measured with an automated gamma counter (1282 Compugamma CS, Pharmacia/LKB Nuclear). The percent injected dose per gram of tissue (ID%/g) was calculated by comparison with samples of a standard dilution of the initial dose. All measurements were corrected for decay.
BALB/c mice bearing subcutaneous CT26 tumor or C57BL6 mice bearing orthotopic GL261 brain tumor were injected intravenously with 37.5 MBq (1 mCi) of either 125
I-IgG1 or 125
I-SSLs in saline. The mice were positioned on the X-SPECT (Gamma Medica-Ideas) gantry and scanned using two low-energy, high resolution pinhole collimators (Gamma Medica-Ideas) rotating through 360° in 6° increments for 40 seconds per increment. Immediately following SPECT acquisition, the mice were scanned by CT (X-SPECT) over a 4.6 cm field of view using a 600 mA, 50 kV beam. Data were reconstructed using the ordered subsets-expectation maximization algorithm. The SPECT and CT data were then coregistered using the instrument supplied software and displayed using AMIDE (http://amide.sourceforge.net/
) or Amira software (Visage Imaging).
The statistical significance of percent survival between different experimental arms was determined by Long-rank analysis.