Periplasmic Expression of a Novel Diabody for Co-Targeting Two Immune Checkpoints, PD-1 and CTLA-4

Clinical studies have shown that the simultaneous use of two different immune checkpoint inhibitors, nivolumab and ipilimumab, has significantly improved the safety and efficacy compared to using each one alone. Diabodies are engineered antibodies with dual specificity that simultaneously target two different antigens and can be produced in bacterial expression systems. The present study aimed to produce a novel diabody to simultaneously target two immune checkpoints in the periplasmic space of Escherichia coli.

The amino acid sequence of diabody was designed based on the variable regions of nivolumab and ipilimumab antibodies; its genetic code was optimized for expression in the bacterial system and after chemical synthesis, the gene was ligated into pET-22b plasmid. Protein expression was performed in E. coli BL21 (DE3) at different temperatures and with different inducer concentrations. After periplasmic protein extraction, the diabody purification was carried out using nickel affinity chromatography.

SDS-PAGE results confirmed a protein's expression with a molecular weight of approximately 55 kilodaltons. The highest amount of soluble protein was obtained with 1 mM IPTG (the inducer) and at 23 °C. The expressed soluble protein was successfully purified by affinity chromatography with a purity of about 90%.

Our results indicated that optimization of culture conditions can lead to improvement of soluble expression of similar proteins in the periplasmic space.