1E10-AF
Hybridoma cell line was cultured in DMEM/F-12 medium supplemented with 5% of fetal calf serum (Invitrogen, USA), 5 mM of glutamine (Invitrogen, USA), 2.546 g/L of HEPES (Sigma, USA), 2.18 g/L of sodium bicarbonate (Sigma, USA) and 1 mL/L of 2-mercaptoethanol (Sigma, USA). Mouse ascites fluid from BALB/c mice was sterilized by depth filtration and 1E10 mAb was purified using the following chromatographic sequence: ion exchange by DEAE Sepharose, affinity chromatography using Protein A Sepharose, and Sepharose G-25 gel filtration chromatography (all from GE Heathcare, USA).
1E10-ST
Supernatant of 1E10-ST was obtained from the stirred tank bioreactors hybridoma cell culture using a proprietary protein free medium in perfusion mode. Clarified 1E10 supernatant was purified by protein A affinity chromatography using ProSep vA ultra matrix (Millipore, USA, followed by cation exchange chromatography (SP Sepharose FF, GE Healthcare, USA) and Sepharose G-25 size exclusion chromatography (GE Healthcare, USA).
Aluminum hydroxide-precipitated 1E10-AF and 1E10-ST mAb formulations
Purified mAb-1E10 molecules were aseptically mixed at a final concentration of 1 mg/ml with 5 mg/ml of aluminum hydroxide as adjuvant (Superfos Biosector, Frederikssund, Denmark). The mixture was gently stirred for 3 h at room temperature. The aluminum hydroxide-precipitated mAb was stored in aliquots at 4°C until use.
Amino acid sequence and posttranslational modifications (PTMs) analysis
Lyophilized mAbs (50 μg) obtained from each process were dissolved in 50 μL of 25 mM of Tris-HCl buffer, pH 8.5 with 8 M Urea. After addition of DTT to a final concentration of 50 mM, the mixture was flushed with N2 and incubated for 3 h at 37°C. Following this step the sample was cooled to room temperature, acrylamide was added to a final concentration of 100 mM and the mixture incubated for 20 min in the dark at room temperature. The samples were diluted 8 times to a final concentration of 1 M Urea. After addition of 1 μg trypsin, the mixture was incubated at 37°C for 18 h. The reaction was stopped by addition of 2% (v/v) TFA in water. The peptide mixture was analyzed directly by MALDI-MS and ESI-MS.
Analyses of peptides by MALDI-TOF2 MS were performed on an Axima Performance mass spectrometer (Shimadzu Biotech, Japan) equipped with a 337 nitrogen laser and a collision-induced dissociation (CID) chamber with helium gas. Analyses were carried out in reflector mode (Mr < 5000) with delayed extraction. The instrument was calibrated externally using ProteoMass Peptide Calibration kit (Sigma), low mass gate value of 700 was selected. Data analyses were performed using Shimadzu Biotech Launchpad software (Shimadzu Biotech, Japan)
Digested peptides were purified using CleanUp Pippet Tips C18 (Agilent, USA) as recommended by the manufacturer. Sample was deposited on the MALDI plate by the dried-droplet sample preparation method [44] using α-cyano-4-hydroxycinnamic acid (CHCA), 10 μg uL-1, in TFA-water-acetonitrile (0.1:30:70, v/v).
ESI-MS and MS2 were performed in the positive ion mode on a Micromass Q-TOF II mass spectrometer (Manchester, UK) equipped with a Z-spray sample introduction system. Acquisition and analysis of the data was performed using MassLynx software v 4.0 (Micromass, Manchester, UK). For calibration, a sodium and cesium iodide mixture solution was used.
Aggregation analysis
High performance-size exclusion chromatography (HPSEC) of 100 μl sample was performed as standard using a 7.8 × 300 mm TSKgel G2000SWXL column protected by a 6 × 40 mm guard column (Tosoh Bioscience, Stuttgart, Germany) on a HP1100 analytical chromatography system (Hewlett-Packard, San Jose, CA, USA) at 0.5 ml/min in 100 mM sodium phosphate pH 6.6, 150 mM NaCl, 0.05% NaN3. Using a combination of the built-in UV detector measuring at 280 nm and a sequential refractive index (RI), intrinsic viscosity (IV), and right-angle light scattering detection (TDA 302, Viscotek Corp., Houston, TX, USA) the MW and hydrodynamic radius of eluting peaks were determined. Detector calibration was performed using bovine serum albumin from Sigma (A7638) weighed out at 1.0 mg/ml.
Thermal stability of mAb-1E10 by FTIR
Infrared spectra were recorded using a Tensor 27 spectrometer (Bruker Optics, Ettlingen, Germany). Protein samples were prepared in a BioATR cell connected to a thermostat. For each spectrum, a 120 scan interferogram was collected in single beam mode at 4 cm-1 resolution. Reference spectra were recorded under identical conditions with only the reference buffer in the cell. To determine the melting temperature (TmFTIR), temperature-dependent spectra were acquired every 5°C in a ranging from 25 to 90°C. Recorded infrared spectra were analyzed by the Protein Dynamics software for Opus 4.2 (Bruker Optics) and displayed as vector-normalized second-derivative amide I spectra. Melting temperature was calculated by plotting the 1639 cm-1 to 1625 cm-1 signal ratio vs. temperature.
Analysis of charge heterogeneity
1E10-ST and 1E10-AF (approximately 100 μg each) were diluted five-fold with buffer A (10 mM sodium acetate pH 5) loaded onto a ProPac10 WCX column (Dionex, Houston, USA) and eluted with buffer B (10 mM sodium acetate, 1 M NaCl pH 5). The gradient was performed in two steps, from 8 to 13% B in 5 min and 13 to 20% B in 20 min. Protein elution was monitored by absorbance at 280 nm.
The mAb solutions (1.5 mg/ml in 15 mM sodium phosphate pH 7) were treated with carboxypeptidase B (Boehringer, Germany) at an enzyme-to-substrate ratio of 1:5000 (w/w) at 25°C. Reaction was stopped by adding 1 μl of acetic acid. Digested samples were analyzed by WCX HPLC as described above.
N-Glycosylation analysis
N-glycans were released by digestion with peptide-N4-(N-acetyl-β-d-glucosaminyl) asparagine amidase F (PNGase F, BioLabs, Beverly, MA, USA), using the method described by the manufacturer. Briefly, mAbs-1E10 were denatured at 100°C for 10 min in 0.1% SDS, 5% β-mercaptoethanol. Nonidet P-40 (NP-40) was added to a final concentration of 1% before enzyme addition. The digestion was carried out at a ratio of 5 U of PNGase F per milligram of glycoprotein at 37°C for 2 h. The protein was precipitated by adding three volumes of cold ethanol and the mixture was kept at -20°C for 30 min. The oligosaccharides were concentrated under vacuum and subjected to 2-aminobenzamide (2AB) labeling.
Oligosaccharides were fluorescently labeled with 2AB by reductive amination [45]. Briefly, oligosaccharides were dissolved in 5 μL DMSO-acetic acid (7:3) containing 2AB (0.35 M) and 1 M NaCNBH3 and incubated at 65°C for 2 h. Excess of fluorophore was removed by 2 h vertical chromatography on Whatman 3 MM paper using acetonitrile. The paper bearing the oligosaccharide signal (application point) was cut. The oligosaccharides were eluted from the paper by adding double-distilled water (2 × 500 μL). The eluate was filtrated in a syringe through a 0.45 μm PTFE filter (Millex-LCR, Millipore) and then concentrated under vacuum.
Normal-phase HPLC (NP-HPLC) of the labeled portion was performed using a TSK-Gel Amide-80 4.6 × 250 mm column (Tosoh BioSep, Japan) on a separation module (Merck-Hitachi, Japan) equipped with a fluorescence detector. Labeled N-glycans were separated by a linear gradient of 20-58% of 50 mM ammonium formiate pH 4.4 against acetonitrile over 152 min at a flow rate of 0.4 mL/min. Samples were injected in 80% acetonitrile. The fluorescence detection was carried out using an excitation wavelength of 330 nm and an emission wavelength of 420 nm [46]. The elution positions of the N-glycans were determined in glucose units (GU) by comparison with a standard dextran hydrolysate 2AB labeled (dextran ladder) [47].
Animals
Female BALB/c/cenp mice (ten weeks old) and Leghorn chicken (10 weeks old) were purchased from the Center for Laboratory Animal Breeding (CENPALAB, Havana, Cuba). Animals were housed under conventional conditions with free access to water and food and maintained in accordance with the guidelines stipulated by the Animal Subject Committee Review Board of CENPALAB. Animal studies were performed with approval from CENPALAB's and CIM's Institutional Animal Care and Use Committees.
Antitumor experiments
The sarcomatoid mammary carcinoma cell line F3II is a highly invasive and metastatic variant established from a clone of a spontaneous BALB/c mouse mammary tumor. F3II cells grow as spindle-cell carcinoma tumors with a high local invasiveness and a 90-100% incidence of lung metastases [48]. F3II cells were maintained in minimal essential medium (MEM 41500, Gibco, BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 80 mg/ml Gentamycin, and 20 mg/ml tetracycline in monolayer culture.
All mice were injected subcutaneously with 2 × 105 F3II cells in the right flank. Seven days after tumor inoculation, the animals were separated in different groups and injected as follows: subcutaneously with normal saline; subcutaneously with 100 μg of 1E10-ST/Alum; intravenously with 150 mg/m2 of Cyclophosphamide (Cy-150); intravenously with 150 mg/m2 of Cyclophosphamide and subcutaneously with 100 μg of 1E10-AF/Alum; intravenously with 150 mg/m2 of Cyclophosphamide and subcutaneously with 100 μg of 1E10-ST/Alum or intravenously with 60 mg/m2 of Doxorubicin (Dox) and 600 mg/m2 of Cyclophosphamide (Cy-600). The time of appearance of local tumors was monitored by palpation and further confirmed by histopathology. In all cases, tumors were diagnosed as spindle-cell carcinomas. Tumor size was measured with a caliper twice a week.
Induction of anti-anti-idiotype antibody (Ab3) response
Chicken were immunized subcutaneously with 100 μg of 1E10 vaccine in the days 0, 7 and 21. Blood extractions were done before and one week after the last immunization (day 28).
The presence of anti-1E10 antibodies in chicken sera was determined in a solid-phase ELISA Polystyrene Maxisorp microtiter plates (Nunc, Roskilde, Denmark) were coated with 50 μl of a solution of 10 μg/ml of mAb-1E10 in carbonate buffer pH 9.6 and incubated overnight at 4°C. After washing with PBS containing 0.05% Tween 20, the plates were blocked for 1 h at room temperature with PBS containing 1% BSA. Then, diluted serum samples were added to each well and the plates were incubated for 2 h at 37°C. Chicken antibodies were detected with an AP-conjugated rabbit anti-chicken IgY (Sigma). The plates were washed four times and the reaction was developed with a substrate solution consisting of 1 mg/ml p-nitrophenylphosphate (Sigma) in diethanolamine buffer, pH 9.8. Absorbance was measured at 405 nm in an ELISA reader (Tekan, Salzburg, Austria).
The highest serum dilution giving OD values ≥0.2 and being at least three times the value corresponding to the preimmune serum at the same dilution was considered as titer. Assays were performed in triplicate for each sample and the coefficient of variation (CV) was less than 15%. The ODs of the blanks were less than 0.1.