Expi293 system

Transient expression of IgGs was carried out using the Expi293™ system (Thermo Fisher, Waltham, MA, USA) according to protocols provided by the supplier. Cells were transfected with two plasmids for mAb production of with four plasmids for bsAb production, using a 1:2 heavy-chain: light chain plasmid DNA ratio. IgGs were purified from conditioned media harvested 4–6 days post-transfection. The conditioned medium was filtered using a 0.45 µm filter and the IgG was purified by protein-A (or MabSelect, GE Healthcare, Chicago, IL, USA) affinity chromatography or by sequential Kappa-select–Lambda-select (GE Healthcare, Pittsburgh, PA, USA) affinity chromatography according to the supplier’s recommendations. Purified IgGs were stored in Phosphate-buffered saline (PBS) at −80 °C.

For B cell receptor reconstruction from the scRNAseq data, we used BraCeR^{18 (link)} assigning the sequences to the IMGT (Immunogenetics) database. The output tables were filtered based on completeness and expression levels of the paired V(D)J transcripts using a customized R script. Combined heavy and light chain sequences were aligned using Clustal Omega. The alignment was visualized using FigTree. Selected immunoglobulin heavy and light chain pairs were cloned into expression vectors^{19 (link)}. Recombinant monoclonal antibodies were expressed in the Expi293 system (Thermo Fisher Scientific). Purified antibody concentration was determined by ELISA.

Soluble FcγRs and poIgGs were expressed in human embryonic kidney 293F (HEK293F) suspension cells cultured in shaker flasks (120 rpm, 37°C, 5% CO₂, 85% humidity) using F17 expression medium supplemented with Pluronic and GlutaMAX (Gibco). Plasmids coding for FcγRs were transfected alone and poIgG heavy chains were co-transfected in equimolar ratio with plasmids coding for poIg-kappa light chain. Transient transfection was performed using 293free (Merck Millipore) premixed with OptiMEM (Gibco) and expression was enhanced by feeding and addition of valproic acid. The fed-batch culture was harvested by centrifugation 7 days after transfection and the supernatant was cleared by filtration.
Full length FcγRs were transiently expressed using the Expi293 system (Thermofisher). Suspension cells were seeded in 6 well-plates (120 rpm, 37°C, 5% CO₂, 85% humidity) and co-transfected with porcine or human FcγRs together with the related FcR-γ chain in an equimolar ratio. The transfected cells were used 48 h post transfection.

PTH1R ECD-TEV-human Fc (residues M1-G188, Uniprot: Q03431) was expressed using the pMH vector (UCB proprietary) using the Expi293 system (Thermo Fisher). The ECD was captured from the supernatant using Protein A affinity resin (MabSelect SuRE, GE Healthcare) in batch. The resin was washed with 20 CVs of Tris-buffered saline (TBS) and incubated overnight with 200 µg of TEV-6His protease, at 4 °C. The resin was sedimented and the supernatant containing TEV protease and PTH1R-ECD was recovered. Ni-NTA beads were used to remove TEV-6His and the PTH1R- ECD was buffer exchanged into TBS pH 8.0 using 50 kDa molecular weight cut-off filters (Amicon, Millipore), concentrated to 11 mg/ml, flash-frozen and stored at −80 °C for future use. The protein was assessed for purity by SDS-PAGE and Western blot analysis. PTH (1–34) binding function of the purified ECD was confirmed using isothermal titration calorimetry (ITC).