Thermo Multiskan Ascent Software 17
Thermo Multiskan Ascent Software 17
Mixed bone marrow cells from 6–8-week-old C57BL/6 mice (5×109 cells) were cultured in 6-well plates (CD11b-enriched cells) or on glass coverslips (pan-monocytes) for 2 days in RMPI media. Non-adherent cells were removed with 5mL RMPI media. Cells were incubated for 15min with cell-trackers (e-fluorosphere kit, Invitrogen, catalogue #65-0866) and washed with Maxpar staining buffer. To block non-specific Ab binding, cells were incubated with anti-mouse CD16/CD32 Abs, normal mouse serum, and rat serum, then incubated with 50L of metal-conjugated Abs (Supplementary Table 1 ). Staining of cells with fluoresce-conjugated primary Abs was followed by metal-conjugated secondary Abs (Supplementary Table 1). Cells were fixed in 2% paraformaldehyde for 15min, washed with Maxpar staining buffer, and then incubated with 125nM Cell-ID Intercalator-Ir (Fluidigm) in Maxpar Fix and Perm Buffer (Fluidigm) overnight. On the following day, cells were washed and filtered through BelArt SP Cell Strainers (Fisher Scientific), and analyzed on a Helios mass cytometer (Fluidigm) with CyTOF6.7 software (Fluidigm). Data were analyzed using Cytobank platform (Cytobank).
After 5-6h culture, CD11b-DCs (CD11b+CD64lowCD24hi) cells were treated with the proteasome inhibitor MG132 (5μM) or DMSO for 2h at 37°C. Cells were then incubated with Cell-ID Cisplatin (Fluidigm) for 20min at room temperature to label dead cells, washed with Maxpar Staining Buffer, and then incubated with anti-mouse CD16/CD32 Abs, normal mouse serum, and rat serum, then incubated with 50L of metal-conjugated Abs (Supplementary Table 1 ). Staining of cells with fluoresce-conjugated primary Abs was followed by metal-conjugated secondary Abs (Supplementary Table 1). Cells were fixed in 2% paraformaldehyde for 15min, washed with Maxpar staining buffer, and then incubated with 125nM Cell-ID Intercalator-Ir (Fluidigm) in Maxpar Fix and Perm Buffer (Fluidigm) overnight. On the following day, cells were washed and filtered through BelArt SP Cell Strainers (Fisher Scientific), and analyzed on a Helios mass cytometer (Fluidigm) with CyTOF 6.7 software (Fluidigm). Data were analyzed using Cytobank platform (Cytobank).
In order to identify conformation-specific antibodies in B-cell culture supernatants, HEK-293 (Thermo Fisher, catalogue number K9000-1) culture supernatants or as purified preparations, a binding ELISA was developed. Nunc maxisorp 384-well ELISA plates (Sigma Aldrich, catalogue number P6366) were coated with 10l per well of 1g/ml human TNFR1-Fc (R&D Systems, catalogue number 372-RI-050) overnight at 4C. Plates were washed and then blocked for 1h with 1% BSA in PBS. After washing plates again, 10l of 100ng/ml apo TNF or TNF pre-incubated with compound at a 50-fold molar excess in 1% DMSO (final concentration) in PBS for 2h at room temperature, were added to each well of the plate and incubated for a further 1h at room temperature. After washing again, mouse IgG or mouse Fab at a range of concentrations diluted in 1% BSA in PBS were added to the plate and incubated for 1h at room temperature. Antibody binding was detected with either an HRP-conjugated goat anti-mouse IgG Fc-specific antibody (Jackson ImmunoResearch, catalogue number 115-036-071) or a goat anti-mouse IgG F(ab)2-specific antibody (Jackson ImmunoResearch, catalogue number 115-036-072) at a 1:5000 dilution in 1% BSA in PBS. Binding was revealed with 3,3,5,5-Tetramethylbenzidine (TMB) Substrate and absorbance at 630nm measured on a synergy 2 microplate reader (BioTek).
To facilitate further characterisation of these small molecule compounds in a variety of contexts, we here generate a monoclonal antibody, known as CA1974, that selectively binds with high affinity to the TNF trimersmall molecule complex. We solve a crystal structure of the conformation-selective antibody in complex with TNF trimer, small molecule inhibitor, and TNFR1 extracellular domain. The structure confirms other studies suggesting that the small molecule inhibitors stabilise a perturbed conformation of the TNF trimer, resulting in a reduction in receptor stoichiometry, which likely leads to the subsequent inhibition of signalling 5, 6 . Furthermore, we show that the antibody, in addition to providing insights into the structure and dynamics of TNF and the mode of action of the small molecules, has potential utility as a tool for measuring target occupancy. This may enable increased understanding of target dynamics and PK-PD effects in preclinical models and when moving into learn-phase clinical studies to confirm proof of mechanism in humans. Collectively, this work highlights the utility of monoclonal antibodies as reagents to support discovery and clinical development of small molecule drug inhibitors of proteinprotein interactions.