Pfuse vector

HEK-293 EXPI (Expi293) cells were cultured in Expi293 media (Gibco) at 37 °C and 8% humidity with orbital shaking at 250 r.p.m. ENPP1–Fc or VH–Fc were cloned into a pFUSE vector (InvivoGen) with upstream IL-2 secretion signal. Cells were transfected at 3 M ml⁻¹ density using ExpiFectamine Transfection kit (Gibco), according to manufacturer protocols. To biotinylate the AviTag of ENPP1–Fc, Expi293 cells expressing BirA were used and 0.05 mM biotin was included in the media at the time of transfection. Seventy-two to 120 h after the addition of enhancers, the supernatant was collected (30 min, 4,000g) and filtered using 0.45-micron filters. Proteins were purified with Protein A affinity chromatography or nickel resin and buffer exchanged into PBS using appropriate MW spin filters (Amicon). Protein purity was assessed using SDS–PAGE, and proteins were stored at −80 °C.

The chimeric mAb5E6 (ch5E6) was generated by grafting of PCR amplified variable heavy (V_H) and light (V_L) chain fragments on the human Fc region of IgG1 isotype (Invivogen, USA). Briefly, the cloning was performed using Q5 DNA polymerase (NEB). Primer sequences used for amplification and cloning of heavy and light variable regions from murine mAb5E6 were purchased from Progen (Progen, Germany, Cat. No. F2010)^{52 (link)}. The amplified genes were cloned in pFUSE vectors (Invivogen), and the resulting constructs were co-transfected in Expi-CHO cells (Invitrogen, USA), followed by stable clone generation in the 10-fold excess concentrations of Zeocin and Blatsticidin and limiting dilutions. The culture supernatant was collected for antibody purification through mAbselect Sure Hi-trap Protein A column affinity chromatography (Cytiva). ExpiCHO expression medium (Cat No. A2910001) was used to maintain ExpiCHO cells and antibody production. The amplified V_H, V_L, and full-length antibody constructs were confirmed by DNA sequencing. The primer sequences used for cloning and sequencing are enlisted in Supplementary Table 1.