Construction and Periplasmic Expression of a Bispecific Tandem scFv for Dual Targeting of Immune Checkpoints.

Background: Immune checkpoints are molecules that act as regulators of immune system pathways. However, some tumor cells can express the ligands of immune checkpoints to escape from antitumor immune responses. Some agents, such as antibodies, can inhibit these checkpoints that prevent the immune system from targeting and killing cancer cells. The aim of this study was to express a novel bispecific tandem scFv in periplasmic space of Materials and Methods: The bispecific tandem scFv was constructed based on the variable regions gene of anti-PD1 and anti-CTLA-4 antibodies. The optimum codon for expression in Results: Expression of tandem scFv with a molecular weight of 55 kDa was verified by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting analysis. The best condition for soluble periplasmic expression was obtained to be incubation with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 23°C. The protein was successfully purified using affinity chromatography with a final yield of 4.5 mg/L. Binding analysis confirmed the bioactivity of purified the tandem scFv. Conclusion: This bispecific tandem scFv could be a potential candidate to cancer immunotherapy, although more biological activity assessments are still required to be carried out.