Human ovarian epithelial cancer (HOEC), ovary surface epithelial (HOSE), human artery endothelial (HAE) cells
Human epithelial growth factor receptor positive (HER-2+) human ovarian epithelial cancer cell lines—OV-90 (CRL-11732) and TOV-112D (CRL-11731) were from the American Type Culture Collection (ATCC, Rockville, MD, USA) [17, 18]. They were derived from the ascites of the advanced, metastatic ovarian adenocarcinoma grade 3, stage IIIc. It is her2/neu+ and p53 mutated. It is cultured in the base medium: 1:1 mix of MCDB105 and 199 with the final concentration of sodium bicarbonate 2.2 g/l. It is supplemented with 15% of human serum. It contains 100 units/ml penicillin, 200 mg/ml streptomycin, in the cell culture 75 cm2 flask (Corning) (catalog #430641) in incubators set at 5% CO2 at 37 °C. The medium is replaced every 3 days. To split, the cultures are briefly rinsed with 0.25% (w/v) Trypsin, 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor and thereafter treated with that solution. After dispensing into new flasks, they are grown in the same conditions.
Human epithelial growth factor receptor positive (HER-2+) human ovarian epithelial cancer cell lines—SK-OV-3 (HTB-77) and OVCAR-3 (HTB-161) were from ATCC [19, 20]. They were derived from the ascites of the advanced ovarian adenocarcinoma. They were grown in McCoy’s Modified Medium supplemented with 10% human serum. The cells were grown in the cell culture 75 cm2 flask (corning) (catalog #430641) in incubators with 5% CO2 at 37 °C. The cultures were split as described above.
Human ovary surface epithelial (HOSE) cells were derived from ovaries removed by prophylactic oophorectomies [21]. The cells were transfected with SV40 to extend their life span. They carried BRCA 1, 2 mutations. They were cultured in the base medium: 1:1 mix of MCDB105 and 199 supplemented 15% of human serum, 0.25 U/ml of insulin, 2 mM of l-glutamine. The media also contained 100 U/ml penicillin, 200 mg/ml streptomycin. The cells were grown in the cell culture 75 cm2 flask (Corning) (catalog #430641) in the CO2 incubators at 37 °C. The cultures were propagated as described above.
Biotechnology of anti-HER-2 and anti-HBsAg antibodies and biosimilars
Biotechnology of anti-HER-2 synthetic antibodies was pursued by adaptation of that originally described, either as new antibodies or as biosimilars to the FDA approved: trastuzumab and pertuzumab as the positive controls [8, 21–23]. For verification, the DNA plasmid constructs for the anti-HER-2 antibodies variable fragments were imported from the International ImMunoGeneTics (IMGT, Paris, F, EU) antibody sequences’ bank [8, 21–23].
Briefly, in the first technology, the B cells were isolated from the blood of patients suffering from the cancers. White blood cells (WBC) were isolated using Ficoll-Hypaque technique. The total mRNA was isolated using Trizol reagent (Molecular Research Center, Inc. Cincinnati, OH). The cDNA was generated using random hexamers (Intergrated DNA Technologies, Coralville, IA) and reverse transcriptase (Promega, Madison, WI) in reactions involving denaturing RNA at 70 °C followed by reverse transcription carried at 42 °C for 15 min. The cDNA quality was tested by the polymerase chain reaction (PCR) of beta actin and GAPDH as reference genes with the commercially available primers (ABI, Foster City, CA). For amplification of coding sequences of the variable fragments, the primers' sets were designed using the Kabat database. They were synthesized on 380A DNA Synthesizer (ABI, Foster City, CA). The variable fragments were amplified with polymerase chain reaction using the mix of the generated cDNA, the synthesized primers, dNTPs, and Taq DNA polymerase (Hoffmann–La Roche, Basel, Switzerland) on the Robocycler (Stratagene, San Diego, CA) or Mastercycler (Eppendorf, New York, NY). The blunt ended amplicons were inserted into the plasmid coding for the constant regions of the human antibodies with sequences imported from the Gene Bank. The DNA plasmid constructs also contained metal binding domains capable of chelating superparamagnetic and fluorescent metals as detailed [8]. After electroporation of plasmids into fresh B cells or cultured human myelomas, they were propagated and expressed.
For selection and in vitro evolution, the HER-2 receptors were extracted from the human HER-2+ ovarian cancer cells by immunoprecipitation of rapidly frozen, crushed, thawed, and lyophilized. Alternatively, the mimotopes of HER-2 were manufactured. Both served as the baits and references for validation of antibodies.
Alternatively, biotechnology of trastuzumab and pertuzumab biosimilars was crafted on such a way that the coding sequences for the anti-HER-2 antibodies’ variable fragments were imported from the IMGT. These sequences were synthesized, cloned, expressed, and modified on the same way as the newly developed anti-HER-2 antibodies as described above.
For generating of anti-HBsAg antibodies the B cell were acquired from the patients suffering from the Acute hepatitis B. The protocol was identical to that published [27]. Dane particles, isolated from the patients’ blood by PEG gradients precipitation or from liver biopsies by CsCl gradient centrifugations, were rapidly frozen, lyophilized and stored. Alternatively, HBsAg were produced in transfected with the plasmid DNA human hepatoma cells. Prior to selection, during in vitro evolution, they were reconstituted with buffer and served as the baits. They also served as the negative controls for anti-HER-2 antibodies.
The metal binding domains of the antibodies were saturated with Gd, Tb, Ru, Ni, Co, or Eu or linked with Au coated Fe3O4 (Au(Fe3O4)) nanoparticles. The specificity and sensitivity were determined based upon elemental spectra acquired with EDXS (Noran, Middleton, WI, USA), EELS (Zeiss, Oberkochen, D, EU), or TRXFS (Bruker AXS, Fitchburg, WI, USA). The fluorescent properties were measured with the RF-5301PC spectrofluorometer (Shimadzu, Tokyo, Japan). The magnetic relaxivities were measured on the DMX 400 WB or AVANCE II NMR spectrometers (Bruker Optics, Dallas, TX, USA).
Biotechnology of HBsAg
HBsAg was isolated from the patients suffering from acute hepatitis B: either from the blood by PEG fractionations or from the liver biopsies by CsCl gradient centrifugation.
To assure exact immunogenic compatibility with the immunity induced by vaccinations with the FDA approved HBsAg, which were produced in yeast, the HBsAg in this project were also generated in yeast as originally described [8, 21–23]. Biotechnology of the recombinant HBsAg was pursued based upon the published DNA coding sequence [24, 25]. Hepatitis B virus like particles (VLP) were initially synthesized in yeast—Saccharomyces cerevisiae as originally described. In particular, the expression plasmid pHBS-16 included the HBsAg surface antigen (HBsAg) controlled by the yeast alcohol dehydrogenase (ADHI) promoter through introduced by EcoRI restriction sites into the DNA construct of the pBR322 plasmid. That followed by yeast replication origin, yeast trp1 gene. This biotechnology was later modified to be pursued in Pichia pastoris [24]. Briefly, yeast cultures of Pichia pastoris were grown at 30 °C in rich medium (YPD; 1% yeast extract, 2% bactopeptone, 2% glucose) initially and shifted either to synthetic media (YNM, 0.67% yeast nitrogen base supplemented with 0.5% (v/v) methanol) for immunoprecipitation and immunofluorescence experiments, or to mineral media (MMOT, 0.2% (v/v) oleate and 0.02% (v/v) Tween-40) for fractionation studies.
All the protocols’ products—HBsAg VLPs were referenced and validated to the FDA approved and the CDC recommended Engerix B and Recombivax and the anti-HBV antibody titer assays [15, 16].
Biotechnology of fluorescent and superparamagnetic mimotopes
Design of HER-2 cyclic mimotopes was initiated by importing the DNA from the GenBank and in vitro translation into amino acid sequences or direct amino acid sequences from SwissProt into the Peptide 3D or LaserGene software. That followed by determination of surface displayed domains. Further, molecular computer aided modeling led to selection of the most likely immunogenic domains. The 12–40 amino acids long sequences were selected. The amino acid sequences were exported. The selected sequences were altered by introducing glycine linkers with terminal cysteines at both amino and carboxyl terminus of the peptide designs. The designed peptides were synthesized as linear on the peptide synthesizer. After detachment from the cartridges, the peptides were converted into cyclics by means of the cysteines. The synthetic products—HER-2 mimotopes were selected and validated on the high pressure liquid chromatography columns. The specificity of the mimotopes was validated by binding to trastuzumab and ant-HER-2 biosimilars with the aid of MACS or FACS.
Biotechnology of anti-HER-2 × HBsAg biomolecular clusters
The synthetic anti-HER-2 antibodies and synthetic HBsAg VLPs were linked with heterospecific, bifunctional linker–sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide ester (SMBS) after adapting the protocol [27]. Briefly, the anti-HER-2 antibody was dialyzed against 0.15 M sodium chloride, 0.1 M sodium phosphate, at pH 7.2. Sulfo-MBS stock in DMSO was added to this solution up to the final 2% w/v concentration to assure at least 80× molar excess. After 1 h at room temperature, the reaction solution was rapidly applied to desalting columns. Performing chromatography with the 0.15 M sodium chloride, 0.1 M sodium phosphate, at pH 7.2 carrier solution was followed by pooling the activated anti-HER-2 antibodies’ 1 ml fractions. To this solution, the synthetic HBsAg diluted in the same carrier solution was promptly added to assure 1:1 ratio. The reaction continued for 1 h at room temperature. The effective anti-HER-2 × HBsAg clusters were isolated by chromatography.
The specificity of the anti-HER-2 × HBsAg to label HER-2 receptors was validated by FCM, NMR and XRFS on cells and mimotopes. The specificity of the anti-HER-2 × HBsAg to attract immune response was validated by labeling with anti-HBsAg antibodies rendered fluorescent for FCM or superparamagnetic for NMR or element specific by XRFS [8, 21].
Immunoblotting and immunoprecipitation
The cells and tissues were either frozen crushed in the rapid controlled rate freezer (the NSF grant support to MM).or native disintegrated with ultrasonicator (Branson Ultrasonic, Danbury, CT, USA). After being homogenized within the sample buffer they were either stored in liquid nitrogen or lyophilized. They were electrophoresed in the native buffer (Invitrogen, Carlsbad, CA, USA). They were vacuum- or electro-transferred onto the PVDF membranes (Amersham, Buckinghamshire, UK, EU). The membranes carrying the transferred proteins were first soaked within human serum and thereafter labeled with the bioengineered, biosimilar, and referenced anti-HER-2 antibodies. The anti-HBsAg isotype antibodies served as the controls. The images of the blots were acquired and quantified with Fluoroimager (Molecular Dynamics, Sunnyvale, CA, USA) or Storm 840 (Amersham, Buckinghamshire, UK, EU).
The anti-HER-2 and anti-HBsAg antibodies were rendered magnetic or fluorescent by conjugating Au coated Fe3O4 nanoparticles. The sera and liver biopsies’ homogenates were mixed with these superparamagnetic antibodies. The targeted molecules rendered superparamagnetic were pulled out by the means of 1.5T magnet. The intensity of fluorescence was measured on the spectrofluorometer to determine the concentration of HER-2 mimotopes of HBsAg VLPs.
Fluorescent antibodies
Fluorescent, activated cell sorting
Flow cytometry
Multiphoton fluorescence spectroscopy
Ovarian cancer cells were labeled with the fluorescent antibodies. They were sorted on the Calibur, Vantage SE, or Aria (Becton–Dickinson, Franklin Lakes, NJ, USA). The antibodies were dissolved and all washing steps carried in phenol-free, Ca+/Mg+—free, PIPES buffered saline solution, supplemented with 20 mM glucose, 10% human serum. Sorting was performed on Aria, Calibur, Vantage SE (Becton–Dickinson, Franklin Lakes, NJ, USA) with the sheath pressure set at 20 lb per square inch and low count rate. The sorted batches were analyzed on Calibur or Aria using FACS Diva software or on the FC500 (Beckman-Coulter, Brea, CA, USA). For the measurement of the fluorescently labeled cells, these settings were tuned at the maximum emission for the Eu chelated antibody at 500 V with references to isotype antibodies and non-labeled cells. This assured the comparisons between populations of cells labeled with multiple antibodies without changing the settings on PMTs.
The fluorescently labeled cells or tissues were imaged with the Axiovert (Zeiss, Oberkochen, D, EU) equipped with the Enterprise argon ion (457, 488, 529 nm lines) and ultraviolet (UV) (364 nm line) lasers; Odyssey XL digital high-sensitivity with instant deconvolution confocal laser scanning imaging system operated up to 240 frames/s (Noran, Madison, WI, USA), and the Diaphot (Nikon, Tokyo, Japan) equipped with the Microlase diode-pumped Nd:YLF solid state laser (1048 nm line).
Superparamagnetic antibodies
Nuclear magnetic resonance spectroscopy
Magnetic activated cell sorting
Ovarian cancer cells were labeled with the superparamagnetic anti-HER-2 and anti-phosphatidylserine (anti-PS) antibodies [8]. The antibodies were dissolved and all washing steps carried in phenol-free, Ca+/Mg+—free, PIPES buffered saline solution, supplemented with 20 mM glucose, 10% human serum. The aliquots were dispensed into the magnetism-free NMR tubes (Shigemi, Tokyo, Japan). The relaxation times T1 were measured in resonance to the applied pulse sequences on the NMR spectrometers: DMX 400 WB or AVANCE II NMR (Bruker, Billerica, MA) or the Signa clinical scanners (GE, Milwaukee, WI, USA). The superparamagnetic antibodies were also used to isolate the labeled cells from the solution. The cells labeled with the superparamagnetic antibodies were isolated on the magnetic, activated cell sorter operated at 1.5 T (the NSF grant support to MM).
Elemental-tags modified antibodies
Energy dispersive x-ray spectroscopy
x-ray reflection fluorescence spectroscopy
The samples, which were cryo-immobilized, presented the life-like antigenicity and supramolecular organization. Elemental analyses were pursued by EDXS and XRFS as described (40). The field emission, scanning transmission, electron microscope FESTEM HB501 (Vacuum Generators, Kirkland, WA, USA) was equipped with the energy dispersive x-ray spectrometer (EDXS) (Noran, Middleton, WI, USA) and post-column electron energy loss spectrometer (EELS) (Gatan, Pleasanton, CA). The cryo-energy filtering transmission electron microscope 912 Omega was equipped with the in-column, electron energy loss spectrometer (EELS) and the energy dispersive x-ray spectrometer (EDXS) (Zeiss, Oberkochen, D, EU). The cryo-energy filtering transmission electron microscopes 410 and 430 Phillips were equipped with the post-column, electron energy loss spectrometers (EELS) and the energy dispersive x-ray spectrometer (EDXS) (Noran, Middleton, WI, USA). The field emission, scanning electron microscope SEM1530 (Zeiss, Oberkochen, D, EU) was equipped with the energy dispersive x-ray spectrometer (EDXS) (Noran, Middleton, WI, USA). The field emission, scanning electron microscope 3400 was equipped with the energy dispersive x-ray spectrometer (EDXS) (Hitachi, Tokyo, Japan). The S2 Picofox XRFS spectrometer was equipped with a molybdenum (Mo) x-ray target and the Peltier cooled Xflash Silicon Drift Detector (Bruker AXS, Fitchburg, WI, USA). Scan times ranged upto 1000 s. The ICP standard of 1000 mg/l of mono-element Gallium or Gadolinium (CPI International, Denver, CO, USA) was added to 500 microL of each sample to the final concentration of 10 mg/l. Instrument control, data collection, and analysis were under the SPECTRA 7 software (Bruker AXS, Fitchburg, WI, USA).
Antibody-vaccine engineered construct-induced toxicity (AVECIT)
To study collective killing effects of the anti-HER-2 and anti-HER-2 × HBsAg upon the ovarian cancer cells, the patients’ cellular and serum fractions described below were pooled making erythrocytes-free blood (EFB). Anti-HER-2 and anti-HER-2 × HBsAg were added to the EFB. Similarly, anti-HBsAg, anti-HPV, and anti-EGFR1 antibodies were added as the controls. The incubation with the antibodies continued at the 37 °C incubators. The labeling continued for 1–24 h. It was terminated by washing with the cold buffer.
To quantify by flow cytometry (FCM) and fluorescent activated cell sorting (FACS) the numbers of killed cells, the samples were stained with propidium iodide (PI) (Sigma-Aldrich, Milwaukee, WI, USA) at 50 µg/ml. To determine the numbers of apoptotic cells, they were labeled with anti-phosphatidylserine antibodies or annexin.
Antibody dependent cell cytotoxicity (ADCC)
To study toxicity to the cancer cells caused by the patients’ cytotoxic cells—the effectors triggered by the anti-HER-2 antibodies and AVEC, the peripheral blood mononuclear cells were separated from the blood on Ficoll-Hypaque density gradients. The cells were washed by three cycles of spinning down and suspending in the PBS at pH 7.3. They were rendered fluorescent by adding the stock solution of the DiI membrane dye (Molecular Probes, Inc., Eugene, OR, USA) in DMSO for 10 min at 26 °C. Small aliquots were washed with the buffer and the cells quantified on FCM as the way to determine the effector to target cells’ ratios (ETR). These ratios varied: 10:1, 50:1, and 100:1. Incubations lasted 1–24 h in a 37 °C, 5% CO2 incubator.
The numbers of killed cells were determined due to staining with the PI at 50 µg/ml or anti-dsDNA, apoptotic with anti-PS or annexin, and of surviving cells from the DiO staining cell counts.
Complement dependent cytotoxicity (CDC)
To study toxicity to the cancer cells caused by the patients’ complement system—the effector triggered by the anti-HER-2 antibodies and AVEC, the serum was separated by gentle centrifugation from the freshly drawn blood. It was supplemented with the anti-HER-2 and anti-HER-2 × HBsAg. Incubations lasted 1–24 h in a 37 °C, 5% CO2 incubator. The numbers of killed cells were determined due to staining with the PI at 50 µg/ml apoptotic with anti-PS or annexin, and of surviving cells from the DiO staining counts.
Statistical analysis
All the measurements were run in triplicates for each sample from six patients. The numbers were analyzed and displayed using GraphPad software (GraphPad Software, Inc, La Jolla, CA). Data were presented as mean of standard error of the mean (SEM). Statistical significance was calculated by t test for two groups (trial vs control).
