Molecular determinants of the immunomodulatory potentials of a novel bifunctional TGF-β1/PD-L1 fusion protein.

Abstract

Autoimmune diseases (AIDs) arise from loss of immune tolerance and are commonly treated with broad immunosuppressive drugs that increase the risk of infection and malignancy. Transforming growth factor-beta 1 (TGF-β1) and programmed death-ligand 1 (PD-L1) are two immunoregulatory proteins whose receptors are expressed on effector and regulatory T cells, and independently modulate immune functions. However, the use of these ligands as a bifunctional regulatory T cell engager (BiTE-like) remains unexplored. This study aimed to design, express, and functionally characterize the development of a TGF-β1/PD-L1 fusion protein. The fusion construct was engineered using overlap-extension PCR and cloned into a mammalian expression vector (pCDNA3.1 +). Recombinant expression in HEK 293 cells resulted in a ~ 70 kDa protein, verified using SDS-PAGE, immunoprecipitation, and western blot. ELISA, co-immunoprecipitation (co-IP) and reverse co-IP experiments confirmed that the TGF-β1 domain in the fusion protein required co-receptors to bind TGFβ-R1, consistent with the canonical receptor binding mechanism of endogenous TGF-β proteins. Parallel immunoassays demonstrated specific binding between the PD-L1 domain and PD-1 receptor. Functional validation in vitro showed differential expression of TGF-β1 and PD-L1 responsive genes, including c-Myc and IFN-γ, upon treatment of leukemic cell model with the fusion protein. SEAP-based reporter assay confirmed the ability of TGF-β1/PD-L1 fusion protein to inhibit NF-κB activation. These results suggested the functional and active binding of TGF-β1 and PD-L1 domains with their cognate receptors. Collectively, this study provided molecular evidence for the structural and functional integrity of the TGF-β1/PD-L1 fusion protein and support its potential development for targeted immunomodulation in AIDs.