Expression and purification of anti-CA125 MAb and scFv
The murine monoclonal IgG1 antibody targeting human CA125 was produced from hybridoma B43.13  that was kindly provided by Quest Pharma Tech Inc., Edmonton, Canada. The hybridoma cell culture supernatant was used to purify the MAb-B43.13 by protein G affinity (P-7700, Sigma-Aldrich, St. Louis, MO, USA) on a BioLogic DuoFlow™ chromatography system (760-0135, Bio-Rad Laboratories, Inc., Hercules, CA, USA). MAb-B43.13-derived anti-CA125 scFv was produced with modifications to previously described constructs ,. Briefly, the DNA sequence encoding scFv-B43.13 was cloned into a pET22b + vector with a modified inter-chain linker, and the protein was expressed in Escherichia coli Rosetta 2 DE3 (Novagen, 71400-3, Merck KGaA, Darmstadt, Germany). The C-terminal hexa-histidine-tagged scFv was purified from the soluble fraction of recombinant cell lysates by immobilized metal affinity chromatography using a TALON® Superflow resin (635507, Clontech Laboratories, Inc., Mountain View, CA, USA).
Cell lines and culture conditions
Ovarian cancer cell lines NIH:OVCAR-3 (ATCC® HTB-161™, ATCC, Manassas, VA, USA) and SKOV3 (ATCC® HTB-77™) were used for in vitro and in vivo studies. Cells were cultured in DMEM-F12 medium supplemented with 10% v/v fetal bovine serum (FBS), 50 IU/mL penicillin, and 50 μg/mL streptomycin (Gibco®, Life Technologies, Carlsbad, CA, USA). NIH:OVCAR-3 cells were additionally supplemented with 7 μg/mL recombinant human insulin (91077C, SAFC Biosciences, Inc., Lenexa, KS, USA). Cells were cultured using sterile techniques and grown in a 37°C incubator providing humidified atmosphere of 5% CO2 in air.
Characterization of CA125-targeting vectors
NIH:OVCAR-3 and SKOV3 cells (7.5 × 106) were lysed with CelLytic™ M (C2978, Sigma-Aldrich). The cell lysates were electrophoresed on a 4% to 15% Mini-PROTEAN® TGX™ precast gel (456-1085, Bio-Rad) and transferred to a Trans-Blot nitrocellulose membrane (162-0115, Bio-Rad). The membranes were probed separately to evaluate MAb versus scFv binding to the target antigen in cell lysates. The blots were blocked for 45 min with 5% non-fat dry milk (Carnation) in PBS having 0.1% Tween-20 (PBST). Anti-CA125 MAb (3 mg/mL), mouse anti-β actin IgG (A1978, Sigma-Aldrich), and anti-CA125 scFv (3 mg/mL) were used as primary antibodies to probe the blots at 1:5,000 dilutions for 1 h at room temperature. Goat anti-mouse HRP conjugate (A4416, Sigma-Aldrich) was used as secondary antibody to probe the blot against anti-CA125 MAb and mouse anti-β actin IgG at 1:5,000 dilution for 1 h at room temperature. 6xHis MAb-HRP conjugate (631210, Clontech) was used as secondary antibody at 1:5,000 dilution to probe against anti-CA125 scFv for 1 h at room temperature. The blots were washed with PBST and developed on Amersham Hyperfilm ECL (28906839, GE Healthcare, Little Chalfont, UK) using Amersham ECL Plus Western Blotting Detection Reagents (RPN2132, GE Healthcare).
Fluorescent labeling of anti-CA125 MAb/scFv
One milligram each of anti-CA125 MAb and scFv at concentrations of 2 mg/mL were fluorescently labeled using the Pierce® fluorescein isothiocyanate (FITC) antibody labeling kit (53027, Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions.
NIH:OVCAR-3 cells (1.5 × 106) were harvested by trypsinization, rinsed twice with fluorescence-activated cell sorting (FACS) buffer (PBS with 0.5% heat-inactivated FBS, 2 mM EDTA, 0.05% sodium azide), and re-suspended by gentle tapping in an approximately 100 μl FACS buffer. Ten micrograms of anti-CA125 MAb or scFv was incubated with the cell suspension for 30 min at room temperature. Cells were rinsed twice in FACS buffer and incubated for 30 min with 4 μg of Alexa Fluor® 488 goat anti-mouse antibody (A-11001, Life Technologies) for the MAb samples and 4 μg of Penta • His Alexa Fluor 488 conjugate (35310, Qiagen, Venlo, the Netherlands) for the scFv samples. Cells were rinsed twice with FACS buffer and analyzed by flow cytometry on a BD FACS Calibur (BD Biosciences, San Jose, CA, USA). A dengue virus targeting IgG1-12A1 and a hexa-histidine-tagged anti-RANK receptor binding scFv were used as isotype controls for the experiments. Negative controls included unstained NIH:OVCAR-3 cells and cells incubated with Alexa Fluor 488 conjugated antibodies alone.
NIH:OVCAR-3 and SKOV3 cells were plated onto glass coverslips in 35-mm tissue culture dishes (100,000 cells/2 mL medium/dish) and incubated at 37°C for 48 h. The cells were rinsed with PBS and fixed in methanol for 30 min at -20°C. The fixed cells were incubated in 5% non-fat dry milk (Carnation) in PBS for 30 min and immunostained separately for 1 h with FITC-labeled versus unlabeled anti-CA125 MAb and scFv. The unlabeled MAb and scFv samples were indirectly stained with corresponding Alexa Fluor 488-labeled secondary antibodies as used for flow cytometry experiments.
Anti-CA125 MAb and scFv (2 mg/mL) were used at 1:250 dilution followed by 1:500 dilutions of secondary antibodies (2 mg/mL) in PBS containing 5% non-fat dry milk. Appropriate blank and control samples were included in the experiments. Antibody incubations were followed by three rinses with PBST for 5 min each. Coverslips were mounted on microscopy slides (Fisherbrand, 12-550-003, Thermo Fisher Scientific) using Mowiol® mounting medium (Calbiochem, 475904, Millipore Co., Billerica, MA, USA) supplemented with DAPI (50 μg/mL). Immunofluorescence was observed through a Zeiss Plan Apochromat 40X/1.3 Oil DIC M27 lens on a confocal laser scanning microscope (Zeiss LSM 710, Carl Zeiss AG, Oberkochen, Germany), and images were analyzed using Zen 2011 software. Separately, 30 μg of FITC-labeled anti-CA125 MAb was added to NIH:OVCAR-3 cells grown on coverslips and allowed to incubate under standard cell culture conditions over a period of 48 h to study uptake in live cells. Coverslips were washed and mounted on glass slides for analysis by confocal microscopy at 0.5-, 1-, 4-, 12-, 24-, and 48-h time points.
Surface plasmon resonance
Kinetic constants for association (ka) and dissociation (kd) and affinity constant KD for anti-CA125 MAb-B43.13 and scFv-B43.13 were determined by surface plasmon resonance on a Biacore 3000 instrument (GE Life Sciences, Piscataway, NJ, USA). Upon activation of a CM5 sensor chip with a 1:1 mixture of N-hydroxysuccinimide/N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride, amine coupling of antigen-grade human ovarian cancer native CA125 protein (MBS318371, MyBioSource, Inc., San Diego, CA, USA) dissolved in 10 mM sodium acetate buffer pH 5.0 was performed to couple approximately 1,200 relative units. The remaining reactive groups in the control and test lanes were inactivated using ethanolamine from the amine coupling kit (GE Life Sciences). The binding of antibody vectors to CA125 immobilized on the CM5 chip was assessed in duplicates across concentration ranges between 18.7 nM and 9.33 μM for MAb-B43.13 and 19.6 nM and 19.6 μM for the scFv-B43.13, respectively. Each sample of MAb/scFv in the aforementioned concentrations in binding buffer (10 mM HEPES, pH 7.0) was injected for 3 min at a flow rate of 30 μL/min to allow binding with the antigen. The binding buffer was allowed to flow over the sensor chip for 15 min at a rate of 30 μL/min to allow for the dissociation of bound MAb/scFv from the antigen on the chip. Next, the regeneration buffer (10 mM HEPES pH 7.0 supplemented with 800 mM KCl) was passed over the chip surface to achieve dissociation of any remaining bound analyte, followed by 2 min for stabilization before the next injection. BIAcore control software 3.2 was used to analyze the data, and the best 1:1 Langmuir binding fit was used to derive kinetic constants.
Preparation of CA125-targeting radioimmunoconjugates
All glassware was rinsed with ultra-pure HCl (Fisherbrand, A508-P500, Thermo Fisher Scientific). Trace metal basis ultra-pure chemicals for buffer preparations were purchased from Sigma-Aldrich. All buffer solutions were treated with biotechnology-grade Chelex 100 (143-2832, Bio-Rad).
p-SCN-Bn-NOTA [S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid] (B-605, Macrocyclics, Dallas, TX, USA) was conjugated to anti-CA125 MAb and scFv to serve as a bifunctional chelator for 64Cu radiolabeling. Briefly, a 6 M excess of p-SCN-Bn-NOTA in DMSO was added to anti-CA125 MAb/scFv in 0.1 M sodium bicarbonate buffer pH 9.0 and allowed to react for 1 h at 37 C. NOTA-functionalized anti-CA125 MAb/scFv was purified from excess unconjugated bifunctional chelator while simultaneously achieving buffer exchange into 0.25 M sodium acetate pH 5.5 by using an Econo-Pac 10DG desalting column (732-2010, Bio-Rad). Protein quantification of the column-eluted fractions was performed using A280 measurements on nanodrop 2000 (Thermo Scientific) and bicinchoninic assay (Pierce BCA Protein Assay kit, 23227) using a Synergy H1 multimode microplate reader for A562 absorbance read-out. The number of bifunctional chelates conjugated per MAb and scFv was determined by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS analysis and according to the method described by Cooper et al. . The purified immunoconjugates were used in radiolabeling experiments.
Cu labeling of NOTA-functionalized MAb/scFv
64Cu was produced via a 64Ni(p, n)64Cu nuclear reaction on a CS-15 biomedical cyclotron at Washington University, St. Louis, USA as previously described  and supplied as high specific activity 64CuCl2 in 0.1 N HCl. Ammonium acetate (0.1 M; pH 5.5) was added to 64CuCl2 to form 64Cu-acetate 64Cu (OAc)2 solution. To 100 μg of NOTA-functionalized MAb/scFv, 85 MBq of 64Cu (OAc)2 was added and allowed to react on a thermomixer at 30°C, 550 rpm for 1 h. EDTA (1 mM) was added to quench the reaction over 10 min. 64Cu-labeled MAb/scFv radioimmunoconjugates were purified on an Econo-Pac 10DG desalting column pre-equilibrated with 0.25 M sodium acetate, pH 5.5 used as the eluant. Elution fractions (350 μL) were collected from the column, and the radioactivity was measured using an Atomlab 400 dose calibrator (Biodex, Shirley, NY, USA). Fifteen microliters of each elution fraction was electrophoresed on a 12% SDS-PAGE gel under non-reducing conditions and evaluated by autoradiography on a BAS-5000 phosphorimager (Fujifilm, Tokyo, Japan). Radiochemical yields and purity were determined by instant thin layer chromatography on ITLC-SG (SGI001, Varian, Inc., Palo Alto, CA, USA), using 10 mM EDTA pH 5.5 as the eluant. Fractions containing high specific activity radioimmunoconjugates were used for in vitro and in vivo radiopharmacological experiments. Monoclonal IgG1 antibody 12A1 was modified into an immunoconjugate and radiolabeled with 64Cu as described above to serve as a non-specific isotype control for in vivo experiments.
Functional characterization of CA125-targeting radioimmunoconjugates
Cell uptake studies
NIH:OVCAR-3 and SKOV3 cells were seeded to obtain 250,000 cells per well in 12-well tissue culture plates. Prior to the uptake experiment, the growth medium was removed and cells were rinsed twice with PBS and incubated in Krebs buffer at 37 C for 1 h. The radioimmunoconjugate (100 KBq) was added to each well except those assigned to measure background activity alone. Cell uptake was terminated at 5, 10, 15, 30, 60, 90, and 120 min by adding ice-cold Krebs buffer and rinsing the wells twice to wash away any unbound radioimmunoconjugates prior to lysing the cells with RIPA buffer.
The cell lysates were transferred to scintillation vials and measured for radioactivity using a γ-counter (Wizard2® 2480 Automatic Gamma Counter, PerkinElmer, Ontario, Canada). Protein levels were quantified using a Pierce™ BCA protein assay kit according to the manufacturer's recommendations. Cell uptake levels were normalized to percentage of the total amount of radioactivity per milligram of protein (% radioactivity/mg protein) and plotted as a function of time. All experiments were performed in triplicates.
Immunoreactivity of the radioimmunoconjugates was assessed by cell binding assays according to the method of Lindmo et al. .
In vivo experiments
All animal experiments were carried out according to guidelines of the Canadian Council on Animal Care (CCAC) and approved by the local animal care committee of the Cross Cancer Institute. Six-week-old BALB/c nude female mice were obtained from Charles River labs (Quebec, Canada). The animals were housed in ventilated cages and provided food and water ad libitum. NIH:OVCAR-3 tumors were induced on the left shoulder by two subcutaneous injections of 15 × 106 and 10 × 106 cells in a 300 μL suspension of 1:1 mixture of PBS and Matrigel (354234, BD Biosciences). The second injection of cells was administered at the same site within 7 to 10 days. NIH:OVCAR-3 tumors grew for 6 to 8 weeks before reaching suitable tumor sizes of 150 to 200 mm3.
SKOV3 tumors were induced on the left shoulder by a single subcutaneous injection of 5 × 106 cells in PBS and were grown for about 2 to 3 weeks before achieving similar tumor sizes.
Small-animal PET experiments were performed on a MicroPET® R4 or INVEON PET scanner (Siemens Preclinical Solutions, Knoxville, TN, USA). Mice were anesthetized by inhalation of isoflurane in 40% oxygen/60% nitrogen, 1 L/min, while maintaining body temperature at 37°C throughout the experiment; 6 to 10 MBq of high specific activity radioimmunoconjugate (approximately 30 to 40 μg) in 150 to 200 μL of 0.25 M sodium acetate (pH 5.5) was administered intravenously via a tail vein catheter. For blocking experiments, 1 mg of unmodified anti-CA125 MAb was administered intraperitoneally 24 h prior to injection of the 64Cu-labeled radioimmunoconjugates. In separate animals, 8 to 10 MBq of 64Cu-labeled 12A1 IgG1 was administered via tail vein injection to evaluate non-specific uptake. Whole-body PET data was acquired by performing static scans for each animal at 24 h post-injection (p.i.) and 48 h p.i. Data acquisition continued for 60 min in 3D list mode. The image files were reconstructed using the maximum a posteriori (MAP) algorithm. The image files were further processed using the ROVER v 2.0.51 software (ABX GmbH, Radeberg, Germany). Masks for defining 3D regions of interest (ROI) were set, and the ROIs were defined by thresholding. Standardized uptake values [SUV = (activity/mL tissue)/(injected activity/body weight), mL g-1] were calculated for each ROI. Additionally, radioactivity uptake was also analyzed as percentage of injected dose per gram tissue (%ID/g). Data are expressed as means ± SEM from n investigated animals.
Ex vivo analyses
An NIH:OVCAR-3 tumor-bearing mouse was injected with 4.55 MBq of 64Cu-labeled anti-CA125 MAb via the tail vein. At 24 h p.i., the animal was euthanized by cervical dislocation. The tumor was submerged into OCT medium and flash frozen using a bath of dry ice-cooled methanol. Seven-micrometer slices were cut on a Leica CM 1850 cryostat (Leica Microsystems, Singapore).
Tissue sections were placed into a BAS-Cassette (2325, Fujifilm) and exposed to a phosphor imaging plate (BAS-MS 2025, Fujifilm) for 15 h at room temperature. The images were developed on a BAS-5000 reader (Fuji Photo Film Co., Ltd.) and analyzed with Adaptive Image Deconvolution Algorithm (AIDA) Image Analyzer v.450 software.
Upon thawing, tissue sections were fixed with formalin for 30 min. The fixed sections were blocked overnight at 4°C using 0.12 mg/mL unconjugated goat anti-mouse Fab fragment (115-003-007, Jackson Immunoresearch, West Grove, PA, USA) in 0.5% fish skin gelatin (G7765, Sigma-Aldrich) pH 7.4, supplemented with 0.1% Triton X-100. The sections were rinsed 3× with Tris-buffered saline having 0.5% Tween 20 (TBST) for 5 min each. MAb-B43.13 was used as primary antibody at a dilution of 1:6,000 in Dako antibody diluent (S0809, Dako, Glostrup, Denmark) and allowed to incubate overnight at 4 C. The sections were rinsed 3× with TBST and incubated with Alexa Fluor® 488 goat anti-mouse antibody (A-11001, Life Technologies) used as secondary antibody at 1:400 dilution in Dako antibody diluent for 2 h at room temperature. After three washes with TBST, the sections were rinsed with water and counterstained using Hoechst H33342 (2 μg/mL) for 5 min. The sections were rinsed and mounted under a coverslip using FluorSave (345789, Calbiochem). The slides were analyzed with a Zeiss Plan Apochromat 10X/0.45 na lens on a confocal laser scanning microscope (Zeiss LSM 710) using Z-stack for image acquisition. The images were registered using Zen 2011 software (Zeiss) and further processed with Adobe Photoshop CS6.
To analyze the samples for immunohistochemistry, the same procedures for treatment, fixation, blocking, and probing of tissue with primary antibody were followed. DakoEnVision™ + HRP-conjugated anti-mouse antibody was used as a secondary antibody, and no counterstain was employed. The sections were observed with a Zeiss Fluar 2.5X/0.12 na lens on an Axioskop 2 Plus microscope. Images were processed and analyzed on AxioVision 4.7 (Zeiss) and further processed with Adobe Photoshop CS6.
All data are expressed as means ± SEM. Graphs were constructed using GraphPad Prism 4.0 (GraphPad Software). Where applicable, statistical differences were tested by unpaired Student's t test and were considered significant for p <0.05.