Biacore 3000, Biacore T100, research grade CM5 chips, amine coupling reagents, 10× HBS-EP buffer, and rabbit anti-mouse IgG were purchased from Biacore (GE Healthcare). Human BMP2, BMP2/7, BMP3, BMP3b, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8b, BMP9, BMP10, BMP15, GDF11, Nodal, Artemin, Neurturin, Persephin, GDF3, GDF15, MIS, glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, Activin A, Activin B, Activin AC, Activin AB, TGFβ3, and GDF9b were purchased from R&D Systems, and GDF8 was purchased from Fitzgerald Industries Int. Human TGFβ1, TGFβ2, GDF5, and GDF6 were purchased from PeproTech. mEngECD-hFc 27–581 (ECD of mouse endoglin fused to human Fc), hEngECD 26–586 (ECD of human endoglin), and type I (hALK1-hFc, hALK2-hFc, hALK3-hFc, hALK4-hFc, mALK5-hFc, hALK6-hFc, rALK7-hFc) and type II (hTGFβRII-hFc, hBMPRII-hFc, hMISRII-hFc) receptors containing the ECD of the respective receptors fused to human Fc were purchased from R&D Systems and reconstituted as suggested by the manufacturer. hActRIIA-hFc, hActRIIB-hFc, mActRIIB-mFc, mALK1-mFc, and hALK1-His were produced and purified in-house. Goat anti-human Fc-specific IgG antibody was purchased either from Biacore (GE Healthcare), The Jackson ImmunoResearch Laboratories, or Thermo Fisher Scientific. Novex 4–12% gels, Native PAGE gels, and other electrophoresis reagents were from Invitrogen.
Cloning, Expression, and Purification of mEngECD-mFc 27–581, hEngECD-hFc 26–586, and Truncation Variants Thereof from HEK 293 Cells
Mouse (27–581) and human (26–586, 26–437, 26–378, 26–359, 26–332, 26–329, 360–586, and 438–586) endoglin extracellular domain constructs were obtained by PCR amplification of the gene sequence obtained from Open Biosystems (Huntsville, AL). Purified PCR fragments were ligated into pAID4mFc or pAIDhFc vectors to create the pAID4-endoglin expression constructs. The sequences of the endoglin constructs were confirmed by DNA sequencing. mEngECD-mFc 27–581 and hEngECD-hFc 26–586 constructs and truncation variants were transiently transfected into human embryonic kidney (HEK) 293 cells (Invitrogen). Cells were seeded at a density of 0.5 × 106 cells/ml into 500 ml of FreeStyle medium (Invitrogen) in a 3-liter spinner flask. Cells were grown at 37 °C overnight, and a complex of DNA/polyethyleneimine (1 μg/ml) was added to the cells. After 5–8 h, an additional 500 ml of FreeStyle medium was added. After 1 week of growth at 37 °C, the conditioned media was harvested and concentrated for purification.
The filtered conditioned media material containing mEngECD-mFc 27–581, hEngECD-hFc 26–586, or truncated variants of human endoglin was purified by affinity chromatography using mAb Select SuRe protein A chromatography. The protein mEngECD-mFc 27–581 was subsequently purified by Q-Sepharose column chromatography. hEngECD-hFc 26–437 was further purified by flow through a ceramic hydroxyapatite (CHT-II, Bio-Rad) column in 5 mm sodium phosphate, pH 7.0, to remove high and low molecular weight impurities. hEngECD-hFc 26–359 was purified by MabSelect SuRe and ceramic hydroxyapatite columns as described above for hEngECD-hFc 26–437 followed by a Q-Sepharose chromatography as for mEngECD-mFc 27–581. The purified proteins were dialyzed into 10 mm Tris, 137 mm NaCl, 2.7 mm KCl, pH 7.14 (modified TBS). The purity of the protein preparations was analyzed by SDS-PAGE under reducing and non-reducing conditions and visualized by SilverSNAP Stain for mass spectrometry (Pierce). Analytical size-exclusion chromatography (SEC) was performed on Agilent 1100 Series HPLC with a Tosoh column TSK gel G3000 SWxl, 7.88 (ID) × 30 cm (length), 5-μm particle size. The purity of the protein preparations was greater than 95%. The protein concentration was measured at 280 nm with NanoDrop 1000 (Thermo) using the extinction coefficient and the molecular weight calculated from the theoretical protein sequence. The N terminus of each construct was verified by N-terminal sequencing using Procise 494 Sequencer (Applied Biosystems).
Cloning, Expression, and Purification of mEngECD-mFc 27–581, hEngECD-hFc 26–586, and hEngECD-hFc 26–359 from CHO Cells
The mEngECD-mFc 27–581, hEngECD-hFc 26–586, and hEngECD-hFc 26–359 constructs were subcloned into a UCOE vector (Millipore). 25 μg of linearized UCOE construct was transfected into CHO cells using TransIT transfection reagent (Mirus); cells were incubated at 37 °C with 5% CO2 in alpha media (Sigma) supplemented with 10% dialyzed FBS (Invitrogen) and 10 μg/ml ADT mixture (Sigma) with one media change after 16 h. On the 2nd day post-transfection, the cells were split 1:6 into selection media containing methotrexate (5 and 10 nm) and grown for 5 days before the media were changed with the appropriate methotrexate concentration. Fourteen days post-transfection, pools were selected by anti-Fc ELISA or Western blotting. Selected pools were then adapted to serum-free conditions in CHO P3 media (Irvine Scientific). The protein was purified from the filtered conditioned media in two steps (affinity and ion-exchange chromatography) as described above for mEngECD-mFc 27–581 from HEK293 cells.
Receptor-ligand binding affinities were determined by SPR using Biacore 3000 and T100 instruments (GE Healthcare).
Kinetic Assay in a Capture Format
Goat anti-human Fc-specific IgG or rabbit anti-mouse IgG was immobilized onto research grade CM5 chip using standard amine coupling chemistry following the manufacturer's protocol. hEngECD-hFc 26–586 or mEngECD-mFc 27–581 and truncated variants thereof were captured on the experimental flow cell. Another flow cell was used as a reference (control) to subtract for nonspecific binding, drift, and bulk refractive index. The endoglin-antibody complex was stable over the time course of each ligand binding cycle. A concentration series of BMP9 and BMP10 (0.00977–1.25 nm, with 2-fold serial dilutions) was injected over experimental and control flow cells at a flow rate of 70 μl/min at 20 °C and 37 °C. Duplicate injections of each ligand concentration were performed, with buffer blanks injected periodically for double referencing. The antibody surface was regenerated between binding cycles by injection of 3 m MgCl2 (for Biacore antibody) or 10 mm glycine, pH 1.7 (for The Jackson ImmunoResearch Laboratories or Thermo Fisher Scientific antibodies). Buffer containing 0.01 m HEPES, 0.5 m NaCl, 3 mm EDTA, 0.005% v/v Surfactant P20, pH 7.4, supplemented with 0.5 mg/ml BSA was used as the running buffer. All sensorgrams were processed by double referencing (subtraction of the responses from the reference surface and from an average of blank buffer injections). To obtain kinetic rate constants, the corrected data were fit to a 1:1 interaction model that includes a term for mass transport using BIAevaluation software (GE Healthcare). The equilibrium dissociation constant KD was determined by the ratio of binding rate constants kd/ka.
Solution Inhibition Assay
hEngECD-hFc 26–359 (20 μg/ml) was injected at 5 μl/min over an anti-hFc IgG CM5 chip on the Biacore 3000 for 3 min followed by an injection of BMP9 (0.625 nm) premixed with mActRIIB-mFc (0, 1, or 2.5 nm) or mALK1-mFc (0, 2.5, or 50 nm) at 50 μl/min for 5 min followed by a dissociation of 6 min.
Epitope Exclusion Assay
hEngECD-hFc 26–359 (20 μg/ml) was injected at 5 μl/min over an anti-hFc IgG CM5 chip on the Biacore 3000 for 3 min followed by subsequent injections of BMP9 (0.625 nm, 5 min) and mActRIIB-mFc, mALK1-mFc, or hALK1-His (100 nm, 5 min) at 50 μl/min. The results were analyzed with the BIAevaluation software.
Reporter Gene Assay in A204 Cells
A reporter gene assay in A204 cells was used to evaluate the effects of hEngECD
-hFc and mEngECD
-mFc fusion proteins on signaling by BMP9 and BMP10. This assay is based on human rhabdomyosarcoma cell line A204 transfected with a pGL3 BRE-luciferase reporter plasmid (
- David L.
- Mallet C.
- Mazerbourg S.
- Feige J.J.
- Bailly S.
- Korchynskyi O.
- ten Dijke P.
) as well as a Renilla reporter plasmid (pRLCMV-luciferase) to control for transfection efficiency. The BRE motifs are present in BMP-responsive genes (Id1 promoter), so this vector is of general use for factors signaling through Smad1 and/or Smad5. In the absence of hEngECD
-hFc and mEngECD
-mFc fusion proteins, BMP9 and BMP10 stimulate signaling in A204 cells in a dose-dependent manner.
A204 cells (ATCC HTB-82) were distributed in 48-well plates at 105 cells per well. The following day a solution containing 12 μg of pGL3 BRE-luciferase, 0.1 μg of pRLCMV-luciferase, 30 μl of FuGENE 6 (Roche Diagnostics), and 970 μl of Opti-MEM (Invitrogen) was preincubated for 30 min at room temperature before adding to 24 ml of assay buffer (McCoy's medium supplemented with 0.1% BSA). This mixture was applied to the plated cells (500 μl/well) for incubation overnight at 37 °C. On the third day, medium was removed and replaced with test substances (250 μl/well) diluted in assay buffer, which included BMP9 and BMP10 at a final concentration of 5 ng/ml. After an overnight incubation at 37 °C, the cells were rinsed and lysed with passive lysis buffer (Promega) and frozen at −70 °C. Before the assay, the plates were warmed to room temperature with gentle shaking. Cell lysates were transferred in duplicate to a chemiluminescence plate (96-well) and analyzed in a luminometer with reagents from a Dual-Luciferase Reporter Assay system (Promega) to determine normalized luciferase activity.
Directed in Vivo Angiogenesis Assay
Directed in vivo angiogenesis assay (Trevigen, Gaithersburg, MD) was used as a quantitative method for assaying angiogenesis. Implant-grade silicone cylinders were filled with 20 μl of basement membrane extract premixed with or without a combination of vascular endothelial growth factor (VEGF, 600 ng) and fibroblast growth factor (FGF-2, 1.8 μg). The silicone angioreactors were implanted subcutaneously in the dorsal flank of athymic nude mice, 2 angioreactors on each side of the animal. Mice were dosed subcutaneously daily with either vehicle (modified TBS) or mEngECD-mFc 27–581 (10 mg/kg). After 11 days, animals received an intravenous injection of FITC-dextran (20 mg/kg) and were euthanized 20 min later by CO2 inhalation. Angioreactors were immediately excised from the animal, photographed, and solubilized. The amount of FITC-dextran in the angioreactor was quantified at 520-nm emission and 485-nm excitation using a fluorescence plate reader (Infinite m200, Tecan, Mannedorf, Switzerland). The intensity of the signal is proportional to the amount of blood vessels in each angioreactor.
Chick Chorioallantoic Membrane (CAM) Assay
Nine-day-old fertilized chick embryos were maintained in a 48-place tabletop egg incubator at 37 °C and specific humidity (60%). Prominent blood vessels were visualized through the eggshell with the aid of an egg lamp. The area of the outer eggshell, where the prominent blood vessels are located, was swabbed with 70% alcohol, a small hole was made, and the air sack was displaced causing a “blister” to form between the shell membrane and the CAM; finally, a small window was cut through the eggshell with a hobby grinding wheel (Dremel Emerson Electric Co., Racine, WI). Small filter disks infused with the angiogenic substance VEGF (50 ng) either in the presence or absence of mEngECD-hFc 27–581 (3 doses of 14 μg) in Biacore running buffer, pH 7.4, was then placed at the opening. For hEngECD-hFc 26–359, 3 doses of 20 μg were used in the assay. Each group (8 eggs) was treated daily for 3 days, and CAM results were analyzed on the fourth day. Quantification of the number of resulting vessels per disk post-treatment was made visually with the help of an egg lamp. Statistical analysis of the results was performed with Excel and SigmaStat software.
Colon-26 Cancer Model
Five- to six-week-old BALB/c mice were obtained from Harlan Laboratories. They were supplied autoclaved food and water ad libitum. Colon 26 (ATCC# CRL-CL25) cells were grown in T-175 flasks at 37 °C + 5% CO2 in RPMI 1640 medium (ATCC) + 10% FBS (Invitrogen). Cells were trypsinized with TrypLE Select (Invitrogen) and counted using a CEDEX cell counter. Cells were washed once with PBS and resuspended in sterile water at a concentration of 10 × 106 cells/ml. 100 μl of the cell suspension was injected subcutaneously in the dorsal right flank. After 3 days, subcutaneous tumors became palpable, and animals were subcutaneously administered daily either mEngECD-mFc 27–581 (10 mg/kg) or modified TBS. Tumor volumes were measured manually using digital calipers (VWR International), and tumor volume was estimated using the formula (0.5 × length × width2). At the conclusion of the study, animals were euthanized by CO2 inhalation, and tumors were excised and frozen in liquid nitrogen-cooled isopentane.