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

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.