Cancer cell lines
The human cancer cell lines (Colo205, LoVo, BxPC-3, Panc-1 and Aspc-1) were purchased from American Type Culture Collection (ATCC, Rockville, MD). The colon carcinoma cell lines, Colo205 and LoVo, were cultured in RPMI 1640 medium (Sigma Aldrich, UK) and F-12K medium (Invitrogen, UK) respectively, supplemented with 10% foetal calf serum (FCS) (PAA, Laboratories, Somerset, UK) and L-glutamine (10 nmol/L) (Invitrogen, UK). BxPC-3, Aspc-1 and Panc-1 (pancreatic carcinoma) were cultured in RPMI 1640 medium (Invitrogen, UK) supplemented with 10% foetal calf serum (FCS) (PAA, Laboratories, Somerset, UK) and L-glutamine (10 nmol/L) (Invitrogen, UK). All cultures were maintained in a humidified environment at 37°C with 5% CO2.
Immunohistochemistry (IHC) staining
Tissue microarray sections, containing biopsy cores taken from colorectal cancer patient specimens, were stained for cathepsin S as previously described  using an automated IHC platform (Bond Max™, Leica Microsystems, Newcastle, UK). Mouse anti-cathepsin S antibody Fsn0503 (Fusion Antibodies Ltd.) was used at 4 ug/mL. A polymer-based detection system (Refine cat#DS9800) was used with 3',3-Diaminobenzidine (DAB) as the chromogen.
Western blotting and Flow cytometry
To prepare whole cell lysates of Colo205, LoVo, BxPC-3, Aspc-1 and Panc-1, cell pellets were washed in PBS and lysed using standard RIPA buffer supplemented with a Calbiochem® protease inhibitor cocktail set III (1:50 dilution of lysis buffer) (Merck Chemicals Ltd., UK) as previously described . Samples were incubated for 30 minutes on ice prior to a 20 minute centrifugation at 13,000 rpm. Protein concentration was determined by BCA assay (Thermo Scientific, UK). Denatured samples were analysed by SDS-PAGE on 12% (w/v) polyacrylamide gels. Gels were transferred by semi-dry blotting onto nitrocellulose membrane, blocked with 3% dried milk before probing with 0.4 μg mouse anti-Cathepsin S antibody in 15 mL PBS overnight 4°C. Following washes in phosphate-buffered saline tween (PBS-T), membranes were probed with goat anti-mouse-HRP (1:3000) (Bio-Rad Laboratories Ltd., UK) for 1 hour at room temperature. After a series of further washes with PBS-T, membranes were developed using Chemiluminescent Substrate (West Pico Chemiluminescent Substrate, Pierce) for 5 minutes.
The surface expression of cathepsin S on Colo205, LoVo, BxPC-3, Aspc-1 and Panc-1 cell lines was examined and quantified by flow cytometry. Briefly, cells (0.5 × 106) were washed with cold Cell Wash (BD Biosciences, UK) and incubated with 0.5μg of polyclonal goat anti-cathepsin S antibody (R&D Systems, UK) in PBS for 1.5 hour on ice. The cells were then washed with cold Cell Wash and incubated with R-PE-conjugated donkey anti-goat antibody (1:50) (Abcam, UK) in PBS for 1 hour on ice. The cells were washed with cold PBS and stored at 4°C in the dark prior to analysis on the FACSCanto (BD Biosciences, UK). Target quantification was performed using QuantiBRITE PE beads (BD Biosciences, UK). Briefly, QuantiBRITE PE beads were run on the BD FACSCanto using the same parameters for fluorescence as used for the analysis of the cell lines. Geometric means were displayed in the statistic view (BD FACS Diva Software v6.1) and the calculation of Antibody Bound per Cell (ABC) values was carried out according to the manufacturer's protocol.
To assess localized cathepsin S expression Colo205, LoVo, BxPC-3, Aspc-1 and Panc-1 cells were grown on coverslips at 20,0000 cells/well in a humidified environment at 37°C with 5% CO2 overnight. The cells were then rinsed with Cell Wash (BD Biosciences, UK) and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After fixing, the cells were washed with PBS and non-specific sites were quenched with a blocking buffer (PBS containing 10% goat serum) for 2 hours. The cells were then incubated with 32 μg of mouse anti-cathepsin S antibody Fsn0503 or mouse isotype antibody control (Fusion Antibodies Ltd., UK) for 1 hour at 37°C. After extensive rinsing with PBS, the cells were incubated with 10 μg/mL goat anti-mouse AlexFlour 488-conjugated secondary antibody (Invitrogen, UK) in blocking buffer for 1 hour in the dark. Labelling of actin filaments was performed with Molecular Probes® rhodamine phalloidin (1:150) (Invitrogen, UK). After extensive washing, the coverslips were mounted upside-down on glass microscope slides using Vectashield® media (Vector Laboratories Inc., Burlingame, CA, USA) and viewed on laser confocal microscope.
Humanization of the murine antibody
mRNA was extracted from 106 cells of Fsn0503 using STAT60 reagent. RT-PCR was performed using an oligo(dT) primer and reverse transcriptase, followed by PCR with murine specific IgG degenerate primers. The PCR products were cloned into the pCR2.1 vector (Invitrogen) for sequencing analysis. The vector was transformed into TOP10 cells and positive clones identified by PCR. The plasmid clones were mini-prepped and sequenced by the dideoxy-chain termination method on an ABI 3130xl genetic analyser. A consensus sequence for the light chain and heavy chain variable domains was determined from a minimum of 5 clones.
A Composite Human Antibody™ version of the Fsn0503 antibody, Fsn0503h, was designed from multiple human antibody sequences, with CDRs (complementarity Determining Regions) identified using Kabat and Chothia definitions (REF). The composite sequence was screened for T cell epitopes binding to human MHC Class II alleles (Antitope). The Fsn0503h VH and VL sequences were synthetically constructed and cloned into an expression vector containing human IgG1 isotype constant domains. Human IgG1 isotype was selected as the optimum isotype for ADCC activity in humans. NS01 cells were stably transfected via electroporation with both heavy chain and light chain expression vectors. Electroporated cells were distributed into 5 × 96 well plates and selected with 200 nM MTX (methotrexate). Wells containing methotrexate resistant colonies were sampled and tested for IgG expression levels, and the best expressing line was selected and frozen down (Antitope). Fsn0503h was then further selected by two rounds of limiting dilution in 96 well plates in the presence of 200 nM MTX. Fsn0503h cells were cultured in Dulbeccos Minimum Essential medium (Invitrogen, Paisley, UK) supplemented with 10% Ultra Low IgG serum (Invitrogen, Paisley, UK), 1% Penicillin Streptomycin (PAA laboratories, Austria), and 200 nM methotrexate (Sigma, Poole, UK). Cells were cultured in T-flasks (Greiner, GmbH). Cells were routinely passaged twice a week at ratios between 1:3 and 1:6 according to the cell density. To express large quantities of antibodycells were seeded in T-175 flasks at a seeding density of 3.0-5.0 × 104 cells/cm2 in 30 mL of complete DMEM. Cells were incubated at 37°C 6% CO2 for 11 days prior to harvesting of culture medium by centrifugation at 5,000 g for 40 minutes. Antibody was purified on a protein A column (GE Healthcare, UK) using AKTA prime chromatography system (GE Healthcare, UK).
A PK study was performed to assess the stability and half-life of Fsn0503h in serum. All animal experiments were carried out in accordance with the Animal (Scientific Procedures) Act 1986 and conformed to current UK Co-ordinating Committee on Cancer Research guidelines. Eight week old male and female Sprague-Dawley (SD) rats (three male; three female), were housed in a temperature and humidity-controlled room for the duration of the study. Blood samples (pre-bleed) were taken from each animal before treatment with Fsn0503h. Each animal was administrated with 10 mg/kg Fsn0503h by intravenous (i.v.) tail injection. Blood samples (0.5 - 1 mL) were collected in heparinised tubes at selected time points following administration of Fsn0503h: 1, 4, 24, 48 hours, then once a week (7, 21, 28, 35, 42 days). Samples were centrifuged following collection, and serum was stored at -20°C until analysis by ELISA. Briefly, recombinant human cathepsin S was coated onto a 96-well plate (40 nM) and incubated with varying dilutions of serum or drug standards for 1 hour at room temperature. After rinsing with PBS-T secondary antibody (rat anti-human horseradish peroxidase-conjugated antibody) was added to each well. Plates were incubated for 1 hour at room temperature, washed with PBS-T and then incubated with TMB for 5 minutes at room temperature. The reaction was stopped by the addition of 500 mmol/L HCL, and absorbance was read at 450 nm.
ADCC was determined by conducting a standard CytoTox 96® Non-Radioactive Cytotoxicity LDH-Release Assay purchased from Promega. The LoVo colorectal cancer cell line and BxPC3 pancreatic cancer cell line were selected for analysis. The LoVo and BxPC-3 cells were seeded at 7000 cells/well. Human peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were used as effectors cells following IL-2 stimulation (100 U/mL). Both the target (T) and effector (E) cells were resuspended in 5% FCS RPMI medium with 15 mM HEPES and incubated in triplicate on 96-well microtiter plates with various concentrations of Fsn0503h (from 6670 to 833.75 nM) with or without CD16 neutralizing antibody (6670 nM) at an E:T cell ratio of 40:1.
Statistical analysis was performed on experimental results using the student t test of variance. All in vitro/ex vivo experiments were repeated a minimum of three times and datais expressed as means ± SD.