Reagents and antibodies
Mouse IgG1 Tetra-His mAb specific for His-tagged proteins was from Qiagen GmbH (Hilden, Germany). The polyclonal antibodies included: horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, Fc specific, (Sigma-Aldrich, San Louis, MO, USA), and IRDye800-conjugated donkey anti-mouse IgG (H&L) (Rockland Immunochemicals, Gilbertsville, PA, USA). Laminin-111 (LM-111) extracted from the Engelbreth-Holm-Swarm (EHS) mouse tumor was from Invitrogen Life Technologies (Carlsbad, CA, USA), bovine serum albumin (BSA) and isopropyl-1-thio-b-d-galactoside (IPTG) were from Sigma-Aldrich, and human vascular endothelial growth factor 165 (VEGF165) was from Peprotech (London, UK).
Construction of expression vectors
The mammalian expression vector pCR3.1-L36-hNC1encoding the anti-LM-111 L36 scFv-based N-terminal trimerbody, containing a murine TIEXVIII domain, has been previously reported (Blanco-Toribio et al. 2013). To generate the E.coli expression vector pET28a-L36, a 841-bp HindIII/NotI fragment of plasmid pHEN2-L36 (Sanz et al. 2001, 2003), containing the the pectate lyase signal peptide (pelB) of Erwinia carotovora and the L36 scFv sequence, was cloned into the HindIII/NotI digested backbone of plasmid pET28a (Novagen, San Diego, CA, USA). To construct the plasmid pET28a-L36-TIE, a human TIEXVIII domain of was synthesized by GeneartAG (Life Technologies) and subcloned as NotI/BamHI into the vector pET28a-L36. To generate the E.coli expression plasmid pET28a-2H1-TIE, the DNA fragment coding for the anti-VEGF 2H1 scFv (Lamdan et al. 2011) was synthesized by GeneArt AG and subcloned as SfiI/NotI into the vector pET28a-L36-TIE.
Culture conditions and expression in bacteria
E. coli BL21 cells [F-ompT hsdSB (rB−, mB−) gal dcm (DE3)] (Novagen) were transformed with T7 promoter driven expression vectors (pET28a). Bacteria were grown at 37°C in LB-agar plates or in liquid 2xYT medium, supplemented with appropriated antibiotics [ampicillin (Ap), 100 µg/ml; kanamycin (Km), 35 µg/ml]. For periplasm expression, 20 ml 2xYT cultures were grown overnight at 37°C under static culture conditions. The next day, the cultures were inoculated in an appropriate volume of 2xYT and incubated at 37°C and shaking at 250 rpm until OD600 = 0.5, IPTG was added to a final concentration of 0.1 mM and then cultures were incubated for 20 h at 25°C and 180 rpm. Then cultures were centrifuged at 4,000 g for 10 min at 4°C, and the pellets were resuspended in 1/20 of the initial culture volume of precooled periplasmic preparation buffer 30 mM Tris–HCl, pH 8.0, 1 mM EDTA, 20% sucrose. After a 20 min incubation on ice, bacteria were harvested by centrifugation at 6,000g for 10 min at 4°C. The supernatant was stored at 4°C and the pellets were resuspended in 1/20 of initial culture volume of precooled 5 mM MgSO4, incubated for 20 min on ice and centrifuged for 10 min at 4°C. After 20 min incubation on ice, bacteria were harvested by centrifugation at 6,000g for 10 min at 4°C. The two fractions were pooled, cleared by centrifugation at 30,000g for 20 min, and dialyzed (cut-off 10,000 Da) against PBS pH 7.4. Antibody expression was analyzed using ELISA and western blotting.
Culture conditions and expression in mammalian cells
HEK-293 cells (CRL-1573; American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, Walkersville, MD, USA) supplemented with 10% (vol/vol) heat inactivated fetal calf serum (FCS) (Invitrogen Life Technologies), unless otherwise stated. HEK-293 cells were transfected with the appropriate expression vectors using calcium phosphate (Compte et al. 2007). Stable cell lines were generated in HEK 293 cells and selected in DMEM with 0.5 mg/ml G-418 (Sigma-Aldrich), and the proteins were purified from conditioned medium with 0.1% (vol/vol) FCS. Antibody expression was analyzed using ELISA and western blotting.
Harvested conditioned mammalian medium was centrifuged, 0.22 μm filtered (Nalgene, Neerijse, Belgium), concentrated (10×) with a 10.000 MWCO Vivaflow 50 filter (Vivascience GmbH, Hannover, Germany), dialyzed against PBS (pH 7.4) and loaded onto a HisTrap HP 1 ml column using and ÄKTA Prime plus system (GE Healthcare, Uppsala, Sweden). Dialyzed periplasmic preparations were 0.22 μm filtered and loaded onto a HisTrap HP 1 ml column using and ÄKTA Prime plus system. The purified antibodies were dialyzed against PBS, analyzed by SDS-PAGE under reducing conditions and stored at −80°C.
Samples were separated under reducing conditions on 12% Tris–Glycine gels (Bio-Rad Laboratories Ltd., Hercules, CA, USA) and transferred using iBlot system (Invitrogen Life Technologies). After blocking with LI-COR blocking solution (LI-COR, Lincoln, NE, USA), proteins were detected with anti-His mAb, followed by incubation with an IRDye800 conjugated donkey anti-mouse IgG. Images were taken using the Odyssey Infrared Imaging system (LI-COR).
The ability of scFvs and scFv-based N-trimerbodies to bind LM-111 or VEGF165 was studied by ELISA as described (Cuesta et al. 2009). Briefly, Maxisorp plates (NuncA/S, Roskilde, Denmark) were coated with LM-111 (0.5 µg/well) or VEGF165 (0.3 µg/well) and after washing and blocking with 200 µl 5% BSA in PBS, 100 μl with indicated amount of purified protein or periplasmic extract were added for 1 h at room temperature. Antigen titration was performed with serial dilutions of purified L36 scFv-based trimerbodies, and after three washes, 100 µl of HRP-conjugated protein A (1 µg/ml) were added for 1 h at room temperature, after which the plate developed with OPD.
One microgram of each purified scFv-based N-trimerbody was incubated in 60% human serum at 37°C for up to 96 h. Samples were removed for analysis at 3, 24, 48 and 96 h and frozen at −80°C until the entire study was completed. As a control, a second set of serum-exposed samples was frozen immediately to represent a zero time point. Aliquots of the different time-points samples were tested for their capability to bind LM-111 by ELISA.
Size exclusion chromatography-multi-angle laser light scattering (SEC–MALLS)
Static light scattering experiments were performed at room temperature using a Superdex 200 G10/300 GL Size Exclusion Chromatography column (GE HealthCare, Little Chalfont, United Kingdom) attached in-line to a DAWN-HELEOS light scattering detector and an Optilab rEX differential refractive index detector (Wyatt Technology Corporation, Santa Barbara, CA, USA). The column was equilibrated with running buffer (PBS + 0.03% NaN3, 0.1 µm filtered) and the SEC–MALLS system was calibrated with a sample of BSA at 1 mg/ml. Then 100 µl samples of the different antibodies at 0.3 mg/ml in PBS were injected into the column at a flow rate of 0.5 ml/min. Data acquisition and analysis were carried out using ASTRA software (v 126.96.36.199, Wyatt). Based on numerous measurements on BSA samples at 1 mg/ml under the same or similar conditions we estimated that the experimental error in the molar mass is around 5%.