The OX-8 cell line (Health Protection Agency Culture Collections) producing a murine anti-CD8 monoclonal antibody was grown in CD Hybridoma Medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 2 mM l-glutamine and 2.8 mg/L gentamicin (PAA Laboratories GmbH) for 10 to 12 days. The medium was centrifuged, and the supernatant was collected and stored at −20°C. The antibodies were purified utilizing HiTrap™ Protein A HP columns (GE Healthcare Life Sciences, Uppsala, Sweden), and the eluted antibodies were transferred to phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) by repeated centrifugation using an Amicon-15 filter unit (molecular weight cutoff 30,000, Millipore, Billerica, MA, USA). The purity of the antibody solution was analyzed by bioanalyzer and a Protein 230 plus kit (Agilent Technologies, Santa Clara, CA, USA). The protein concentration was determined using the BCA (bicinchoninic acid) standard assay (Sigma-Aldrich, St. Louis, MO, USA). The purified antibody solution was stored at −20°C.
The therapeutic monoclonal antibody
The chimeric (mouse/human) monoclonal IgG1 antibody BR96 (Seattle Genetics Inc., Bothell, WA, USA), which binds to the tumor-associated antigen Lewis Y, was used in this study. The Lewis Y antigen is expressed on the majority of human epithelial tumors, but normal human tissue also contains the BR96-binding antigen, primarily in the epithelial cells of the gastrointestinal tract . The binding affinity between BR96 and the carcinoma cell line used in this study is strong (the dissociation constant being 4 nM) .
Conjugation was performed according to Forrer et al. . Briefly, BR96 was transferred to 0.2 M sodium carbonate buffer, pH 9.5, by repeated centrifugation using the Amicon-15 filter unit. The DOTA-chelate (S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid; 2 mg/mL H2O, Macrocyclics, Dallas, TX, USA) was added to the BR96 antibody (100 mg/mL) at a molar ratio of 3:1 (DOTA:BR96) and incubated for 1 hour at 37°C. The conjugate was purified by repeated centrifugation as described above and transferred to 0.25 M ammonium acetate buffer, pH 5.3. The final concentration was adjusted to 10 mg/mL BR96 by the addition of ammonium acetate buffer. All vials were pretreated with 1% HNO3 and all buffers were pretreated with Chelex-100 (Bio-Rad, Hercules, CA, USA) to remove metals.
MALDI-MS was used to determine the number of DOTA moieties per BR96 molecule, by desalting the sample to 18 MΩ · cm H2O using a centrifugation filter device, and dividing the increase in molecular mass by the molecular mass of the DOTA-chelate (688 u).
Both the 177LuCl3 solution (MDS Nordion, Ottawa, Canada) and the DOTA-BR96 conjugate in 0.25 M ammonium acetate buffer were preheated to 45°C for 10 min. The DOTA-BR96 solution was added to the vial containing the radionuclide and incubated at 45°C for 15 min. The reaction was quenched with an excess of DTPA (diethylene triamine pentaacetic acid) for 5 min. The radiolabeled immunoconjugate was diluted in 1% human serum albumin (HSA, Baxter, Deerfield, IL, USA) to prevent radiolysis from affecting the immunoreactivity. The radiochemical purity was determined by instant thin-layer chromatography (ITLC) using a 1 × 9 cm silica-gel-impregnated fiberglass sheet as the solid phase and 0.1 M EDTA as the mobile phase. To confirm the radiochemical purity and to detect signs of aggregation or fragmentation, separation was performed using size-exclusion chromatography and high-performance liquid chromatography (HPLC) (using a 7.8 × 300 mm molecular sieving column, Phenomenex SEC S3000 (Phenomenex, Torrance, CA, USA), eluted with 0.05 M sodium phosphate at 1.0 mL/min).
Syngeneic animal model
BN7005-H1D2 is a cell line established from a 1,2-dimethylhydrazine-induced rat colon carcinoma in the Brown Norway (BN) rat. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 10 mM HEPES buffer, and 14 mg/L gentamicin (all from PAA Laboratories GmbH) at 37°C, in a humidified environment containing 5% CO2. The cells were washed in PBS and detached by treatment with trypsin (both from PAA Laboratories GmbH). We have previously determined the radiosensitivity of this cell line, expressed as the fraction of survival after exposure to 2 Gy (S2Gy), to be 0.5 (137Cs radiation source, unpublished data). This is similar to the radiosensitivity of human colorectal carcinoma cell lines .
BN rats are immunocompetent and express the BR96 binding antigen in normal tissues, mainly in the epithelium of the gastrointestinal tract , similar to humans. The animals were inoculated with 3 × 105 cells between the peritoneum and the abdominal wall under anesthesia (Isoflurane, Baxter). All experiments were conducted in compliance with European legislation on animal welfare and were approved by the Regional Animal Ethics Committee. The animals were housed under standard conditions and fed with standard pellets and fresh water ad libitum.
Radioimmunotherapy and depletion of CD8-positive cells
The amount of administered anti-CD8 required for efficient depletion, suggested by Holmdahl et al. and Huang et al. [18,19], was confirmed by i.v. injection of 0.5 or 1.0 mg anti-CD8 (in 0.4 mL saline) in 6 tumor-free BN rats. The animals were followed for 25 days p.i., and blood samples were collected twice a week.
On day 0 (13 to 14 days after cell inoculation), 15 tumor-bearing BN rats in the RIT group, were given 400 MBq/kg 177Lu-BR96 (150 μg DOTA-BR96 in 0.4 mL saline with 1% HSA) by intravenous injection. In the anti-CD8 + RIT group, 15 tumor-bearing BN rats were given anti-CD8 on day −3 (0.5 mg in 0.4 mL saline) and day 9 (0.3 mg in 0.4 mL saline), and 400 MBq/kg body weight 177Lu-BR96 day 0. The tumors were measured using a caliper, and the tumor volumes were calculated as (tumor length × tumor width2 × 0.4) . The median tumor volume of all animals on day 0 was 680 mm3 (interquartile range 460 mm3), and the median body weight on day 0 was 272 g (interquartile range 22 g). Body weight and tumor volume were measured twice a week. Blood samples were collected from all animals to monitor myelosuppression and for flow cytometric analyses of CD8-positive cells twice weekly from one week before injection and during the first 4 weeks after injection of the radioimmuno-conjugate, and then once weekly until the end of the study (day 99) or sacrifice. The study was ended 99 days p.i., as in our previous study lasting 180 days all metastatic disease was detectable within 100 days p.i. .
Animals were sacrificed and dissected if the tumor size approached 20 × 20 mm, if the decrease in body weight approached 20% of the normal weight progression, if the general health of the animal was affected during the study, or if metastatic disease was suspected. All remaining animals were sacrificed and dissected at the end of the study (day 99). All dissections were performed by the same person (EE) and the location of metastases was recorded.
Blood samples were used to evaluate the total number of white blood cells, as a measure of myelosuppression, and the CD3+CD4−CD8+ lymphocytes in order to monitor CD8-positive cells. Myelosuppression was determined by counting the white blood cells and platelets with a Vet CA530 Medonic Cell Analyzer (Boule Medical, Spanga, Sweden).
In order to label CD3/CD4/CD8a in blood, EDTA-treated blood samples were incubated with mouse anti-rat CD32 (BD Pharmingen) for 15 min to achieve FcR blockage and stained with FITC-conjugated anti-rat CD3, PE-conjugated anti-rat CD4, and Alexa Fluor 647-conjugated anti-rat CD8a (all from Biolegend, San Diego, CA, USA) for 30 min. Red blood cells were lysed with Erythrolyse Red Blood Cell Lysing Buffer (AbD Serotec, Oxford, UK), and fixed in 0.5% PFA (paraformaldehyde) in PBS/BSA (10 g BSA in 1 L PBS). Labeled cells were analyzed on the same day.
Samples were analyzed with a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA) using CellQuest Pro collection software (BD Bioscience). Corrections were made for spectral overlap. One hundred thousand events per sample were recorded. The collected data were analyzed with Flowing Software 2.5.0 (P. Terho, University of Turku, Finland). CD3+ lymphocytes were gated using forward scatter, side scatter, and FITC channels. CD4−CD8+ cells were counted in both the lymphocyte gate (in the forward scatter and side scatter plot) and CD3+ lymphocyte gate.
The risk of developing metastases was calculated using a log-rank test (Cox-regression) with the STATA software (StataCorp LP, College Station, Texas, USA), while time to complete response of the local tumor (Mantel-Cox test) was calculated using Prism 5.02 (GraphPad Software Inc., La Jolla, CA, USA).