Reagents
All oligonucleotide primers and plasmids used in this study are described in Supplementary Tables 1 and 2. Restriction enzymes, Phusion® High-Fidelity DNA polymerase, and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA, USA). Oligonucleotide primers and VCSM13 helper phage stock were obtained from Integrated DNA Technologies (Coralville, IA, USA) and Agilent Technologies (Santa Clara, CA, USA), respectively. 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC), an anti-M13 antibody conjugated to horseradish peroxidase (HRP), and 1-StepTM Ultra 3,3′,5,5′-tetramethylbenzidine (TMB) substrates were purchased from Avanti Polar Lipids (Alabaster, AL, USA), Bethyl Laboratories (Montgomery, TX, USA), and Thermo Fisher Scientific (Waltham, MA, USA), respectively. All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise.
Construction of plasmids
The mouse ETA (mETA) gene (NCBI Gene ID: 13617) was synthesized by GenScript (Piscataway, NJ, USA). The pP9-mETA plasmid was constructed by Gibson assembly19 of the mETA DNA fragments amplified by polymerase chain reaction (PCR) using primers (MSJ#01 and MSJ#02) and the pP9 plasmid20 digested with the SmaI restriction enzyme. The gene encoding membrane scaffold protein (MSP-1), derived from the apolipoprotein A-I gene (NCBI Gene ID: 335), was assembled by PCR using primers (MSJ#03–MSJ#08) and was subcloned into pET28a(+) (Novagen, Burlington, MA, USA) at the NdeI/BamHI restriction endonuclease sites to generate pET28-MSP-1. To construct plasmids encoding the heavy and light chains of full-length IgG for AG8, each VH and VL gene was PCR amplified using a phagemid (pEL3X-AG8) isolated from the phage library screen and the primer pairs MSJ#42/MSJ#44 for VH and MSJ#46/MSJ#48 for VL. Then, the DNA fragments encoding the IgG constant region (CH1-CH2-CH3) of trastuzumab, which were prepared by PCR amplification using a primer pair (MSJ#43/MSJ#45) and a template (pMAZ-IgH-GlycoT)21, were assembled with the VH DNA fragments using a primer pair (MSJ#42/MSJ#45). A primer pair (MSJ#46/MSJ#49) was used to assemble the DNA fragments for the VL gene, and the human Ck DNA fragments were amplified using primers (MSJ#47/MSJ#49) and a template (pMAZ-IgL-GlycoT)21. pMAZ-AG8H and pMAZ-AG8L were constructed by ligation of the resulting heavy and light-chain DNA of AG8 IgG, respectively, into the pMAZ-IgL-GlycoT plasmid at the BssHII and XbaI sites.
Expression and purification of human ETA, mouse ETA, and membrane scaffold protein-1
Human ETA (hETA), mouse ETA (mETA), and membrane scaffold protein-1 (MSP-1) proteins were expressed and purified as described in the literature20,22. E. coli BL21(DE3) harboring pP9-hETA20 or pP9-mETA (for pP9-derived plasmids), or pET28a-MSP-1 (for pET28a-derived plasmids) was inoculated in Luria-Bertani (LB) medium supplemented with 100 μg/ml ampicillin (Millipore Sigma, Burlington, MA, USA) and 50 μg/ml kanamycin (Millipore Sigma, Burlington, MA, USA) and cultivated for 16 h at 37 °C and 250 rpm. Then, 100-fold dilutions of overnight-grown cells were inoculated in LB medium supplemented as needed with the same antibiotics and incubated at 37 °C until the absorbance of the culture broth at 600 nm (OD600) reached 0.6. After the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) (0.5 mM for hETA and mETA, 1 mM for MSP-1) and incubation under specific culture conditions (25 °C for 16 h for hETA and mETA, 30 °C for 4 h for MSP-1) to induce protein expression, cells were harvested by centrifugation at 8000×g and disrupted using a microfluidizer (Microfluidics, Westwood, MA, USA). To prepare endothelin receptors (hETA and mETA), the resulting lysates were centrifuged at 12,000×g for 20 min, and the supernatants were ultracentrifuged at 100,000×g for 1.5 h to recover the membrane fractions from the pellets. After the membrane fractions were dissolved in 0.5% sarkosyl and centrifuged at 30,000×g for 30 min to remove insoluble aggregates, the recovered supernatants were bound to Ni-NTA agarose (Qiagen, Germantown, MD, USA) equilibrated with Buffer A (25 mM Tris–HCl and 1 mM phenylmethylsulfonylfluoride (pH 7.8)). After the resin was washed with 20 column volumes (CV) of Buffer A supplemented with 20 mM imidazole, the resin-bound proteins were eluted using 5 CV of Buffer A supplemented with 300 mM imidazole. Then, the eluents were loaded onto a PD-10 desalting column (Cytiva, Marlborough, MA, USA) to remove excess imidazole, and the buffer was exchanged with 25 mM Tris–HCl (pH 7.8) containing 10% glycerol. The purified endothelin receptors (hETA and mETA) were stored at −80 °C before use. To prepare MSP-1 proteins, cell lysates were centrifuged at 12,000×g, and the resulting supernatants were loaded onto a Ni-NTA column equilibrated with 10 ml of 50 mM Tris-Cl and 1% Triton X-100 (pH 7.4). After adding 10 ml of 50 mM Tris-Cl and 50 mM imidazole (pH 7.4) for washing and 10 ml of 50 mM Tris-Cl and 300 mM imidazole (pH 7.4) for elution, the eluent buffer was exchanged with 1 × phosphate-buffered saline (PBS, pH 7.4) containing 10% glycerol using a PD-10 desalting column.
Preparation of reconstituted hETA nanodiscs
Purified hETA and MSP-1 were mixed with POPC dissolved in 100 mM sodium cholate at a hETA:MSP-1:POPC molar ratio of 1:30:60. After the addition of 200 mg/ml Bio-BeadsTM SM-2 (Bio-Rad, Hercules, CA, USA), the resuspended solution was incubated at 4 °C for 16 h with mixing by rotation at 100 rpm and centrifuged at 12,000×g for 5 min to remove detergents. Then, the supernatants were dialyzed in 1× PBS (pH 7.4) and concentrated using Amicon Ultra®4 spin columns (Merck Millipore; 30 kDa cutoff). The concentrated supernatants were loaded onto a Superdex 200 gel filtration chromatography column (Cytiva, Marlborough, MA, USA) for development in 35 ml of 1× PBS (pH 7.4), and the fractions showing both hETA and MSP-1 protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) analysis were recovered.
Construction of a human naive immune scFv library
VH and VL genes of human immunoglobulins, which were prepared from peripheral blood mononuclear cells (PBMCs) of anonymous donors as described in the literature23, were PCR amplified using 200 μM dNTPs, 1 μM mixed oligonucleotides (MSJ#07–MSJ#16 for VH and MSJ#17–MSJ#37 for VL), 2.5 units of Phusion® High-Fidelity DNA polymerase, and 100 ng of cDNA as a template. Then, the VH and VL genes were assembled by PCR with two primers (MSJ#38/MSJ#39) to connect the resulting VH and VL genes with a flexible glycine–serine linker (GGGSSGGGGSGGGGSGGGGS), and the resulting PCR products encoding the single-chain variable fragments (scFvs) were digested with SfiI and ligated into the pEL3X phagemid, which is a derivative of pComb3X24 with modified SfiI sequences (GGCCCAGCCGGCC/GGCCTCGGGGGCC). Then, the ligation products were transformed into E. coli ER2738 (F´proA+B+ lacIq Δ(lacZ)M15 zzf::Tn10(TetR)/fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5) to generate the human naive scFv antibody library.
Preparation of phage particles from the scFv library
E. coli ER2738 cells harboring naïve immune scFv library plasmids were inoculated and grown for 1 h in 10 ml of Super Broth (SB) medium (Becton Dickinson Diagnostic Systems, DifcoTM, USA) supplemented with 100 μg/ml carbenicillin. The culture broth was diluted 1:100 in 1 L of SB medium containing the same antibiotic and incubated at 37 °C with shaking at 250 rpm until the absorbance of the culture broth at 600 nm reached 0.8–1.0. Then, 1 ml of VCSM13 helper phage (1 × 1012 pfu) and 70 μg/ml kanamycin were added, and the infected cells were incubated for 16 h at 37 °C with shaking at 250 rpm to induce the production of scFv-displaying phage particles. The culture broth was centrifuged at 10,000×g, and the supernatants were mixed with polyethylene glycol (PEG)/NaCl solution containing 4% (w/v) PEG 8000 and 3% (w/v) NaCl. The pellets were resuspended in 1× PBS and 3% bovine serum albumin (pH 7.4), and the recovered phage particles were stored at 4 °C prior to use.
Library panning and screening
In total, 50 μl of 4 μg/ml Gαi3 protein purified as described previously25 was coated onto a 96-well plate (Corning, Corning, NY, USA) at 4 °C for 16 h. After extensive washing of the wells, 50 μl of hETA reconstituted nanodiscs (4 μg/ml) was added, and the plate was incubated at room temperature for 2 h. Before loading the library phage particles into the wells of the plate immobilized with hETA nanodiscs, a negative selection procedure was conducted. The library phage particles were incubated in wells immobilized with empty nanodiscs consisting of only MSP-1 and a lipid that did not contain hETA. Next, 50 μl of the resulting supernatants were added to the wells preimmobilized with hETA nanodiscs. After the plate was washed with 1× PBS (pH 7.4), bound phage particles were eluted in 100 μl of glycine-HCl buffer (pH 2.2) and neutralized by the addition of 20 μl of 2 M Tris (pH 8.0). Then, 120 μl of the resulting neutralized, recovered phages and 1 ml of VCSM13 helper phage particles were added to infect E. coli ER2738, and the amplified phages were used for the next round of biopanning. The number of washing cycles was increased in each subsequent round of biopanning to enrich high-affinity binders. After five rounds of biopanning, E. coli ER2738 cells were infected with eluted phages, and 400 individual clones were cultured in 1 ml of SB medium at 37 °C with shaking at 250 rpm until the OD600 reached 0.6. Then, 50 μl of VCSM13 helper phages and 70 μg/ml kanamycin were added to the infected E. coli ER2738 cells. After overnight cultivation, the supernatant was used for phage enzyme-linked immunosorbent assay (ELISA).
Phage ELISA
To isolate phage particles displaying specific anti-hETA antibodies, 50 μl of 4 μg/ml purified human Gαi3 protein diluted in 0.05 M Na2CO3 (pH 9.6) was added to each well of a 96-well plate (Corning, Corning, NY, USA) and incubated at 4 °C for 16 h. After blocking with 150 μl of 4% skim milk in 1× PBS (pH 7.4) and washing four times with 150 μl of PBS (pH 7.4) containing 0.02% n-dodecyl-β-d-maltoside (DDM), 50 μl of 5 μg/ml hETA reconstituted nanodiscs was added to each well of the plate. Then, the plate was incubated at 25 °C for 1 h, washed with 150 μl of 1× PBS (pH 7.4) containing 0.02% DDM, and treated with 50 μl of rescued phage particles displaying scFvs. After incubating at 25 °C for 1 h and washing four times, 50 μl of anti-M13-HRP conjugates diluted 4000-fold in 1× PBS (pH 7.4) containing 0.02% DDM was added to the plate. After incubation for 1 h at 25 °C and four washes in 150 μl of 1× PBS containing 0.02% DDM (pH 7.4), 50 μl of 1-Step™ Ultra TMB was added to each well, and the plate was incubated for 20 min to develop the signal. After quenching the signal by the addition of 50 μl of 4 N H2SO4, the ELISA-binding signal was detected by measuring the absorbance at 450 nm in an Epoch plate reader (BioTek, Winooski, VT, USA).
Luciferase assay
A luciferase assay was performed using a dual-luciferase reporter assay system (Promega, Madison, WA, USA) according to the manufacturer’s instructions. Poly-l-lysine was coated onto 96-well plates (Corning, Corning, NY, USA) by incubation at 37 °C for 1 h, and cells were then seeded at a density of 5 × 103 cells/well. The luciferase reporter plasmids were cotransfected with the control plasmid encoding Renilla luciferase into the cells in the plate, and AG8 phage supernatants were added after 24 h. Then, a mixture of dye reagent was added after 48 h, and luciferase activity was measured using a VICTOR Light luminometer (PerkinElmer, Inc., Waltham, MA, USA). The transfection efficiency was evaluated by normalization to Renilla luciferase activity as a control.
Mammalian cell culture
CHO-K1 cells expressing human ETA were maintained as monolayer cultures on 100-mm cell culture dishes in Ham’s F12 medium supplemented with 10% fetal bovine serum (FBS) and 1 × antibiotic–antimycotic solution at 37 °C in a humidified atmosphere containing 5% CO2. The established human colorectal cancer cell lines HT-29 and HCT-116 were purchased from the Korean Cell Line Bank (Seoul, Korea) and maintained in HyClone RPMI-1640 medium (Cytiva, Marlborough, MA, USA) supplemented with 10% HyClone FBS (Cytiva, Marlborough, MA, USA), 1% penicillin-streptomycin, and 1% sodium pyruvate at 37 °C in a humidified atmosphere of 5% CO2.
Expression and purification of AG8 IgG
The pMAZ-AG8H and pMAZ-AG8L plasmids, which encode the heavy and light chains of AG8 IgG, respectively, were constructed using an eCube Plasmid DNA Mini Kit (PhileKorea, Seoul, Korea) and transfected into Expi293 cells using polyethyleneimine, as described in the literature26. After resuspension of the cells in 300 ml of GIBCO FreeStyleTM medium (Thermo Fisher Scientific, Waltham, MA, USA), incubation at 37 °C with shaking at 125 rpm under 8% CO2 for 6 days, and centrifugation at 4000×g, the supernatants were mixed with 40 ml of 25 × PBS (pH 7.4) and 1 ml of a slurry of Protein A agarose resin (GenScript, Piscataway, NJ, USA). The resuspension was incubated at 4 °C for 16 h and passed through a polypropylene column (Thermo Fisher Scientific, Waltham, MA, USA) to recover the resin. Next, 100 ml of 1× PBS (pH 7.4) was added to the column to wash the resin, and 3 ml of 100 mM glycine-HCl buffer (pH 2.5) was loaded onto the column for elution. The eluents were immediately neutralized by the addition of 1 ml of Tris-Cl (pH 8.0). After buffer exchange with 1× PBS (pH 7.4) using Amicon Ultra®4 spin columns (Merck Millipore; 3-kDa cutoff), the concentration and purity of AG8 IgG were analyzed by measuring the absorbance at 280 nm and by 4–15% SDS–PAGE.
Physicochemical analysis of AG8 IgG
Antibody aggregation was measured with a Waters Alliance 2695 system (Milford, MA, USA) and a Waters BioSuite high-resolution size-exclusion chromatography (SEC) column (7.5 mm × 300 mm, 10-μm particle size). Samples (10 μl, 1 mg/ml) were injected, and separation was conducted using isocratic elution with 1 × PBS (pH 7.4) at a flow rate of 1 ml/min. The purity was analyzed with reversed-phase high-performance liquid chromatography (RP-HPLC) using an Agilent 1260 Infinity system (Santa Clara, CA, USA). A Waters XBridge BEH 300 C4 (4.6 mm × 150 mm, 3.5-μm particle size) column was used to separate analytes at a flow rate of 1.44 ml/min. The mobile phase was 0.1% trifluoroacetic acid (TFA) in water (Eluent A) and 0.1% TFA in acetonitrile (Eluent B) applied in gradient mode: 0–18 min, a linear increase from 20 to 80% Eluent B; 18–30 min, washing, and re-equilibration. The injection concentration and volume were the same as those used for SEC. The intact masses of the antibody were determined with RP-HPLC using a Waters Acquity I class UPLC system. Separation was performed on a Thermo MabPacTM RP column (2.1 mm × 50 mm, 4-μm particle size) at a flow rate of 0.2 ml/min. The mobile phase was prepared by mixing 0.1% formic acid in water (Eluent A) and 0.1% formic acid in acetonitrile (Eluent B). After linear gradient elution for 2 min with an increase in the ratio of Eluent B to 25% followed by isocratic elution with 25% Eluent B, the sample was separated by linear gradient elution (25–45% Eluent B). The effluent was analyzed with a Thermo Fisher LTQ Orbitrap mass spectrometer (Thousand Oaks, CA, USA) using Fourier transform (FT) mode. The resolution and mass range of the FT-based mass spectrometer were 120,000 and m/z 400–4000, respectively. The injection concentration and volume were 0.1 mg/ml and 5 μl, respectively. Glycan profiling was performed with a Rapi-Fluor labeling kit (Waters, Milford, MA, USA), and all procedures were performed as described previously27,28.
ELISA
For coating, 50 μl of Gαi3 (4 μg/ml, diluted in 0.05 M Na2CO3 (pH 9.6)) was added to a 96-well polystyrene plate, and the plate was incubated at 4 °C for 16 h. After the addition of 150 μl of 4% skim milk in 1× PBS (pH 7.4) and incubation for 2 h for blocking, 50 μl of 4 μg/ml hETA/mETA reconstituted in 0.5% sarkosyl was added to the plate. Then, the plate was washed four times with 150 μl of 1× PBS containing 0.05% Tween 20 (PBST, pH 7.4), and 50 μl of AG8 IgG serially diluted in 1× PBS (pH 7.4) was added. After the plate was washed with 150 μl of PBST, 50 μl of a goat anti-human IgG (H + L) antibody-HRP conjugate (5000-fold dilution; Thermo Fisher Scientific, Waltham, MA, USA) was added. After the plate was washed with 150 μl of PBST, 50 μl of 1-Step™ Ultra TMB was added, the plate was incubated for 20 min, and 50 μl of 4 N H2SO4 was added to the wells to quench the ELISA signal. The absorbance at 450 nm was analyzed in an Epoch plate reader (BioTek, Winooski, VT, USA).
Calcium flux assay
Changes in the cytosolic Ca2+ concentration upon hETA binding to the ET-1 ligand were analyzed as described in the literature29. After incubation of 1 × 105 hETA-overexpressing CHO-K1 cells or HT-29 colorectal cancer cells with 5 μM fura-2-acetoxymethyl ester (Fura-2-AM) dye at 25 °C for 1 h, serially diluted scAb AG8 was added. After incubation at 25 °C for 1 h, 10 nM ET-1 was added, and the resulting fluorescence emission at 510 nm, with separate excitation at 380 and 340 nm, was monitored using a FluoroMate FS-2 fluorescence spectrometer (Scinco, Seoul, Korea) to evaluate the Ca2+ concentration changes upon intracellular endothelin signaling.
Proliferation assay
Cancer cell proliferation was analyzed using a CyQUANTTM NF cell proliferation kit (Thermo Fisher Scientific, Waltham, MA, USA). Cells were seeded in 96-well plates at a density of 2–3 × 103 cells/well. After 24 h of incubation, ET-1 and the anti-ETA antibody were mixed at a 1:1 ratio in 2% FBS medium, and the medium was replaced with RPMI-1640 medium. After 24 h, CyQUANT® NF dye reagent was added, and the cells were incubated at 37 °C for 30 min. Then, the fluorescence intensity was measured as the ratio of the fluorescence at 530 nm to that at 485 nm using an Infinite M200 Pro microplate reader (TECAN, Männedorf, Switzerland).
Western blot analysis
Whole-cell protein lysates were prepared using RIPA buffer (iNtRON Biotechnology, Seongnam, Korea) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland), and total protein samples were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). After separation of equal amounts of the protein lysates on 10% Bis–Tris protein gels (Thermo Fisher Scientific, Waltham, MA, USA), transfer to PVDF membranes (Merck Millipore, USA), and blocking with 5% skim milk, the membranes were incubated with HRP-conjugated anti-β-actin, anti-phospho-ERK1/2, anti-total-ERK1/2, anti-phospho-AKT (S473), or anti-total-AKT antibodies (Cell Signaling Technology, Danvers, MA, USA). Then, the membranes were washed in 0.05% Tween 20 in Tris-buffered saline and incubated with a 1:5000 dilution of anti-rabbit IgG -HRP conjugate (Bio-Rad, USA) as the secondary antibody. Specific bands were detected using a WEST-ZOL plus Western Blot Detection System (iNtRON Biotechnology, Seongnam, Korea).
RNA extraction and quantitative real-time PCR (qRT–PCR) analysis
The total RNA was isolated using an RNeasy Mini Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol. Reverse transcription was conducted using 1 μg of total RNA as a template and SuperScriptTM III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). qRT–PCR was performed in triplicate in LightCycler® 480 system with SYBR Green I Master Mix (Roche, Mannheim, Germany) and the appropriate primers (MSJ#50/MSJ#51), and the target gene expression levels were normalized to the β-actin level. The values from independent experiments were averaged, and are presented as the means ± standard deviations.
Mouse xenograft model
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Cancer Center Research Institute (NCCRI). The NCCRI is an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International)-accredited facility and abides by the Institute of Laboratory Resources (ILAR) guidelines. Five-week-old female nude mice (BALB/c nude) were purchased from OrientBio (Seongnam, Korea). After 1 week, colorectal cancer cells (2 × 106) resuspended in 100 μl of 1× PBS (pH 7.4) were subcutaneously injected using a 31-gauge needle. The tumor-bearing mice were randomized into the control and treatment groups (n = 4 mice per group) after 7 days. Then, AG8 IgG (1.125 mg/kg) was injected intratumorally into each mouse at 2-day intervals, and 1 × PBS (pH 7.4, 50 µl/mouse) was injected as the negative control. After tumor volumes and body weights were measured prior to antibody injection, the tumors were measured using a caliper, and the volumes were calculated as follows: [W(width)2 × L(length)] × 1/2. The mice were sacrificed 27 days after cancer cell injection.
Statistical analysis
Statistical analyses were performed with Student’s t test, and P < 0.05 was considered statistically significant.
In silico modeling to predict the AG8 binding site in ETA
Structural modeling of the single-chain variable fragment antibody (scFv) was carried out using the AG8 sequence and the antibody modeling tool of the Discovery Studio 2019 program (Biovia, San Diego, CA, USA). The crystal structures for ETB from the Protein Data Bank (PDB IDs: 5GLI and 5GLH for ligand-free hETB and ET-1-bound ETB, respectively) were used for modeling and docking analysis. The potential binding site in AG8 was limited to the extracellular region of the ETB structure, and the most stable binding site was determined using the “ZDOCK” function in Discovery Studio 201930.
