In vitro characterization of a bispecific anti-(PD-1/CTLA-4) single-chain diabody for cancer immunotherapy.

Immunotherapy with a combination of anti-CTLA-4 and anti-PD-1 antibodies has exhibited promising results for the treatment of many kinds of cancers in clinical practice. In the present study, we developed a novel bispecific daibody for simultaneous targeting of PD-1 and CTLA-4 to induce antitumor immunity. The bispecific single-chain diabody was designed on the basis of the amino acid sequences of variable regions of anti-PD1 and anti-CTLA4 antibodies. After expression in the bacterial system and affinity purification, its binding properties alone or in the presence of PD-1 and CTLA-4 ligands were evaluated by flow cytometry. The expected molecular weight of the full-length His-tagged protein was approximately 55 kDa, which was confirmed by SDS‒PAGE analysis and western blotting analysis. The bispecific diabody could selectively bind to cells expressing PD-1 and CTLA-4 and inhibit PD-1/PD-L1 and CD86/CTLA-4 interactions. This novel protein significantly increased IL-2 and IFN-γ secretion in a dose-dependent manner in T-cell activation experiments. Furthermore, it reversed the suppressive effect of PD-L1 on ConA-stimulated PBMCs. The diabody also led to a significant increase in T-cell proliferation and cytokine release in the MLR assay. This bispecific diabody can be considered a promising checkpoint inhibitor candidate, although more in vitro and in vivo biological function evaluations still need to be performed.