The effect of a PD-1/CTLA-4 tandem scFv on T cell cytokine response under immunosuppressive conditions

Background and purpose: Immune checkpoint blockade targeting PD-1 and CTLA-4 has shown promise in cancer immunotherapy; however, limitations such as immune resistance and toxicity from combined full-length antibodies remain. Tandem single-chain variable fragments (scFvs) offer a novel bispecific approach with potentially improved tissue penetration and reduced systemic effects. This study aimed to evaluate the effect of a bispecific tandem scFv targeting PD-1 and CTLA-4 on cytokine secretion of human peripheral blood mononuclear cells (PBMCs) under immunosuppressive conditions. Experimental approach: PBMCs were isolated from healthy donors, activated with concanavalin A (ConA), and treated with various concentrations of the bispecific tandem scFv (0.2-200 nM). Cells were also exposed to soluble CD86, PD-L1, or co-cultured with mitomycin C-treated MDA-MB-231 tumor cells. IFN-γ and IL-2 levels were quantified using ELISA. Findings/Results: The tandem scFv increased IFN-γ and IL-2 secretion from ConA-activated PBMCs, especially at 20 nM. Furthermore, the bispecific tandem scFv significantly enhanced IL-2 secretion in the presence of inhibitory ligands, CD86 and PD-L1. However, the bispecific tandem scFv could reverse the inhibitory effect of CD86 on IFN-γ secretion but not PD-L1. Under co-culture with tumor cells, the modest effect of the bispecific tandem scFv on restoration of IL-2 secretion was observed. Conclusion and implications: Dual CTLA-4/PD-1 blockade using bispecific antibodies such as the bispecific tandem scFv used in the current study could restore the immunosuppressive effect of the tumor microenvironment. However, more in vitro and in vivo experiments are still required to confirm the potential efficacy of PD-1/CTLA-4 tandem scFv for cancer immunotherapy.