Cells and Animals
Expi293F cells (Thermo Fisher Scientific) were cultured in Expi293 expression medium (Thermo Fisher Scientific) on shakers (25 mm shaking diameter) with a shake speed of 120 rpm in a humidified atmosphere of 8% CO2 in air at 37°C. CL1-5 human lung adenocarcinoma cells were a kind gift from Dr. Pan-Chyr Yang (Institute of Biomedical Sciences, Academia Sinica, Taiwan)[61] and were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 6 g L− 1 HEPES, 2 g L− 1 NaHCO3, 10% fetal bovine serum (HyClone), penicillin (100 U mL− 1), and streptomycin (100 mg mL− 1) at 37°C in a humidified atmosphere of 5% CO2 in air. No authentication besides confirming surface expression levels of EphA2 was performed by the authors. Healthy 3- to 5-week-old male SCID mice (C.B17/Icr-Prkdcscid/CrlNarl) were purchased from the National Laboratory Animal Center, Taipei, Taiwan and maintained under specific pathogen-free conditions. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Kaohsiung Medical University and approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (IACUC number: 108225).
Construction and expression of bispecific PEG engagers
The DNA fragment of the anti-mPEG Fab fragment previously cloned from the anti-methoxy PEG Fab (clone h15-2b)[19] or the anti-dansyl (anti-DNS) scFv were placed in frame via a linker sequence (GGGGS)3 with the anti-EphA2 scFv containing an interdomain disulfide bond between residues H44 and L100 to form mPEG×EphA2 or the control PEG engager mPEG×DNS, respectively. The VL-Cκ and VH-CH1-linker-scFv domains were separated with an IRES in the pLNCX retroviral vector (BD Biosciences, San Diego, CA)[62] in the unique Hind III and Cla I restriction enzyme sites to generate pLNCX-mPEG Fab×EphA2 scFv and pLNCX-mPEG Fab×DNS scFv plasmids. Each plasmid was scaled up by transformation into TOP10 E. coli, mixed with 100 mL Luria broth in a 250 mL baffled flask and shaken overnight at 220 rpm. Large-scale plasmid purification was conducted using the PureLink HiPure Plasmid Midiprep Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For protein production, plasmids were transfected into Epxi293F cells using ExpiFectamine and protocols provided by the manufacturer (Thermo Fisher Scientific). Transfected cells were grown at 37°C in an 8% CO2 incubator while shaking at 125 rpm for 5 days. Secreted protein was harvested by centrifugation at 1500 rpm for 5 min. Polyhistidine-tagged BsAbs were passed through 0.22 µm filters before purification with 3 mL CNBr-activated Sepharose™ 4B (GE Healthcare, Little Chalfont, UK) via the ÄKTA™ start protein purification system (GE Healthcare, Little Chalfont, UK)[20].
Characterization of bispecific PEG engagers
Purified mPEG×EphA2 and mPEG×DNS proteins (5 µg) were boiled and separated by electrophoresis on a 10% (wt/vol) SDS-PAGE gel and stained with Coomassie Brilliant Blue under both reducing and non-reducing conditions. Lane M is a PageRuler™ prestained protein ladder. Binding of the bispecific molecules to EphA2 and PEG was determined by incubating 1×106 CL1-5 cells with 2-fold serial dilutions of the proteins for 1 h on ice. Unbound antibodies were removed by extensive washing in cold PBS containing 0.05% (wt/vol) BSA, followed by the addition of 17 nM (200 µL) Lipo-DiD for 1 h on ice. Lipo-DiD (PEGylated DOPC/CHOL liposomes labeled with DiD) which similar to GSL composition were purchased from FormuMax Scientific (Sunnyvale, CA, USA). After removal of unbound Lipo-DiD by extensive washing in cold PBS containing 0.05% (wt/vol) BSA, the surface fluorescence of viable cells was measured on a FACScan flow cytometer (Merck Flow Cytometer, Guava easyCyte System).
Characterize of one-step formulation of GSL with mPEG×EphA2
mPEG×EphA2 and mPEG×DNS were mixed with 9AC-Gw GSL in PBS at 4℃ for 40 min to form αEphA2/GSL and αDNS/GSL, respectively. αEphA2/GSL were prepared with protein: mPEG molar ratios of 4:40, 2:40, 1:40, 0.5:40, 0.25:40. Based on a 100 nm liposome containing ~ 80,000 phospholipid molecules and ~ 4528 mPEG-DSPE molecules (the molar ratio of DSPC: cholesterol: DSPE-PEG2000 = 3:2:0.3), the corresponding number of bispecific molecules per GSL was estimated to be 456, 228, 114, 57, 29, respectively.
EphA2 targeting by αEphA2/GSL
12.5 nM mPEG×EphA2 or control mPEG×DNS were mixed with 10 µg mL− 1 9AC-GW GSL in PBS at 4℃ for 60 min to form αEphA2/GSL and αDNS/GSL, respectively. EphA2 targeting of αEphA2/GSL, αDNS/ GSL and GSL were determined by incubating 5×105 CL1-5 cells for 1 h on ice. Mouse anti human EphA2 antibody (3F7) was a kind gift from Dr. Steve R. Roffler (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan)[63] and was used to detect EphA2 on CL1-5 cells. Unbound liposomes were removed by washing 3 times with cold PBS containing 0.05% (wt/vol) BSA, and 9AC-GW GSL on the surface of CL1-5 cells was determined by staining with 10 µg mL− 1 6.3 anti-PEG antibody[64] for 30 min and 4 µg mL− 1 goat anti-mouse IgG Fcγ-FITC (Jackson ImmunoResearch Laboratories). fiable cells was measured on a FACScan flow cytometer (Merck Flow Cytometer, Guava easyCyte System)
Characterize of αEphA2/GSL stability
For the stability of αEphA2/GSL, mPEG×EphA2 was mixed with 9AC-Gw GSL in PBS at 4℃ for 30 min to form αEphA2/GSL and then incubate under 10% FBS at 37°C water bath for 0, 24, 48, 72, 96, 120 h. 20 ug mL− 1 anti-PEG backbone Ab (clone: AGP4)[65] were coated for 10 min on 37°C incubator and then were removed by extensive washing in filtrated water. 10 µg mL− 1 αEphA2/GSL were stained with 2-fold serial dilutions of the αEphA2/GSL for 30 min on ice. 9AC-GW GSL captured by anti-PEG backbone Ab was measured by staining with 5 µg mL− 1 biotin-conjugated goat anti-human kappa light chain for 30 min and 1 µg mL− 1 horseradish peroxidase (HRP)-conjugated streptavidin (Jackson Immunoresearch Laboratories, West Grove, PA, USA). The plates were washed again and bound peroxidase activity was measured by adding 150 µL/well ABTS solution [0.4 mg mL-1, 2,2′-Azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid] (Sigma-Aldrich, St. Louis, MO, USA), 0.01% (v/v) H2O2, and 100 mM phosphate-citrate, pH4.0] for 1 h at RT. Color development was measured at 405 nm on a microplate reader (Molecular Devices, Menlo Park, CA, USA).
For the anti-EphA2 ability of αEphA2/GSL, CL1-5 cells (1×105/well) were seeded in 96-well plates at 37℃ overnight. 10 µg mL− 1 αEphA2/GSL were stained with 3-fold serial dilutions of the αEphA2/GSL for 30 min on ice. Unbound liposomes were removed by washing 3 times with serum free medium (SFM). 9AC-GW GSL on the surface of CL1-5 cells was determined by staining with 10 µg mL− 1 6.3 anti-PEG antibody for 30 min and 5 µg mL− 1 horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Fcγ (Jackson Immunoresearch Laboratories, West Grove, PA, USA). The plates were washed again and bound peroxidase activity was measured by adding 150 µL/well ABTS solution [0.4 mg mL-1, 2,2′-Azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid] (Sigma-Aldrich, St. Louis, MO, USA), 0.01% (v/v) H2O2, and 100 mM phosphate-citrate, pH4.0] for 1 h at RT. Color development was measured at 405 nm on a microplate reader (Molecular Devices, Menlo Park, CA, USA).
Internalization of αEphA2/GSL
mPEG×EphA2 and mPEG×DNS were mixed with 9AC-Gw GSL in PBS at 4℃ for 40 min to form αEphA2/GSL and αDNS/GSL, respectively. 10 µg mL− 1 Lipo-DiD, αEphA2/Lipo-DiD, or αDNS/Lipo-DiD were incubated with 5×105 CL1-5 cells for 60 min on ice and then unbound liposomes were removed by extensive washing in cold PBS containing 0.05% (wt/vol) BSA. The cells were transferred to fresh culture medium and incubated for 20 or 60 min at 37°C. Control cells were incubated at 4°C for 30 min. Lipo-DiD on the surface of CL1-5 cells was measured by staining with 10 µg mL− 1 6.3 mouse anti-PEG IgG antibody for 30 min and 4 µg mL− 1 goat anti-mouse IgG Fcγ-FITC (Jackson ImmunoResearch Laboratories). After extensive washing with PBS, the fluorescence of cells was measured with a Cytomics FC500 flow cytometer.
The internalization of GSL was determined by incubating 10 µg mL− 1 αEphA2/GSL, αDNS/GSL, or GSL with 5×105 CL1-5 cells for 60 min on ice, removing unbound liposomes by extensive washing in cold PBS containing 0.05% (wt/vol) BSA, and then culturing the cells in fresh medium for 0, 10, 20, 30, or 60 min at 37°C. Control cells were incubated at 4°C. GSL on the surface of CL1-5 cells was determined by staining with 10 µg mL− 1 6.3 anti-PEG antibody for 30 min and 4 µg mL− 1 goat anti-mouse IgG Fcγ-FITC (Jackson ImmunoResearch Laboratories). After extensive washing with PBS, the fluorescence of cells was measured with a Cytomics FC500 flow cytometer.
Internalization imaging of αEphA2/Lipo-DiD by confocal microscopy
CL1-5 cells (2 × 104 cells per well) were seeded on 10 µg mL− 1 poly-L-lysine-coated glass slides in RPMI1640 medium supplemented with 10% FBS (culture medium) at 37°C in a humidified atmosphere of 5% CO2 in air for 24 h. The cells were incubated with 1 nM of LysoTracker Green DND-26 in RPMI (Sigma-Aldrich) to stain lysosomes for 40 minutes at 37°C in an atmosphere of air containing 5% CO2. After washing with fresh culture medium, the cells were incubated with 30 µg mL− 1 of αEphA2/Lipo-DiD or αDNS/Lipo-DiD in fresh culture medium at 37°C for 0, 15 or 30 min. The cells were fixed by 4% paraformaldehyde and covered by DAPI Fluoromount-G® (southern biotech) before fluorescence signals were recorded on an Olympus FluoView 1000 confocal laser scanning microscope.
Drug regeneration of 9AC-G W GSL in vitro and in vivo.
Drug regeneration was examined in a biological system where 5×105 CL1-5 cells were seeded per well in a 24 well-plate in triplicate and incubated overnight. αEphA2/GSL, αDNS/GSL, or unlabeled GSL were added to the cells at 40 µM (9AC-GW concentration) and medium was harvested after 30 min, 8 h, 24 h, and 48 h. An equal volume (500 µL) of 2% Triton X-100 in 20% acetonitrile, 25 mM citric acid pH 2.9 was added to each sample and 50 µL were injected in the HPLC for analysis.
Male SCID mice (C.B17/Icr-Prkdcscid/CrlNarl) between 6 and 8 weeks old were used to assess tumor growth. 5×106 CL1-5 cells in PBS in a total volume of 100 µL were subcutaneously injected into the back of mice. After tumors reached a volume of 100 mm3, mice were randomized into groups of 4 mice with similar mean tumor volumes and dosed intravenously with saline or 2 mg kg− 1 of αEphA2/GSL or αDNS/GSL. Blood was collected in Na-heparinized hematocrit tubes from the tail vein and mice were sacrificed to harvest the tumor tissue at 24 h and 48 h after GSL administration. 100 µL of blood samples were centrifuged at 2500 × g for 3 min and the plasma was collected for analysis. Tumor tissue was homogenized in 3 mL RIPA buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% Na-deoxycholate, 1% Triton X-100). The amount of 9AC was determined in blood and tumor samples by HPLC as described above and normalized to amounts per gram of tissue, according to the relationship 1 mL of whole blood = 1.06 g of weight. Tumor growth measurements were performed twice a week using calipers, and tumor sizes were analyzed by using the equation: volume = (length × width2)/2.
HPLC
Online SPE-HPLC analysis was modified from Prijovich, et al.[66], which was applied for in vitro kinetic and in vivo biodistribution of released-9AC measurement, respectively. Analysis was performed by injecting 100 µL samples into the SPE column (Waters, µbondapak™ C18, 3.9 × 20 mm, 10 µm) and washing away proteins and contaminants with SPE mobile phase containing 10% acetonitrile, 50 mM KH2PO4, pH 2.9 at 2 mL/min with a U.V. detector (Jasco U.V. 975) at 205 nm. The analytic mobile phase (26% acetonitrile, 50 mM KH2PO4, pH 2.9 at constant flow rate) was applied after 2 min washing to elute the captured metabolites to the analytical column (Waters, µbondapak™ C18, 3.9 × 300 mm, 10 µm). A Jasco FP-2020 fluorescence detector with 375 nm excitation and 460 nm emission wavelengths was used to detect 9AC. 9AC concentrations were calculated by comparison with standard curves constructed by recording the peak area versus concentration for three replicate injections (commercial 9AC as standard which was from MedChemExpress, NJ, USA). Data was collected and analyzed on ChromNAV Ver.2 Chromatography software.
In vitro cytotoxicity
CL1-5 cells (5×103/well) were seeded in 96-well plates at 37℃ overnight. The cells were incubated with serial dilutions of 9AC-Gw, αEphA2/GSL, or αDNS/GSL (100 µL /well) at 37℃ for 3 h and then the supernatant was replaced with 100 µL fresh medium. Cell viability was measured with the ATPlite Luminescence Assay System (Perkin-Elmer, Waltham, MA, USA) after 72 h of incubation. Results are expressed as percentage cell viability of luminescence as compared with untreated cells according to the following formula: percentage (%) cell viability = 100 × (treated luminescence/untreated luminescence). Data are presented as means of triplicate determinations.
In vivo antitumor therapy
Male SCID mice (C.B17/Icr-Prkdcscid/CrlNarl) between 6 and 8 weeks old were subcutaneously injected in the back with 5×106 CL1-5 cells. After tumors reached a volume of 150 to 200 mm3, mice were randomized into groups of 5 to 6 mice per group of similar mean tumor volumes and dosed intravenously with saline or 2 mg kg− 1 of GSL, αEphA2/GSL, or αDNS/GSL once a week for 2 weeks, for a total dose of 4 mg kg− 1 9AC-Gw. Tumor growth measurements were performed twice a week using calipers, and tumor sizes were analyzed by using the equation: volume = (length × width2)/2. Mice were weighed once a week to examine treatment toxicity.