Molecular Determinants of the Immunomodulatory Potentials of a Novel Bifunctional TGF-β1/PD-L1 Fusion Protein

Purpose Autoimmune diseases (AIDs) are inflammatory conditions characterized by dysregulated immune tolerance. Conventional immunosuppressive therapies for AIDs may reduce overall immune activity, which pose risks of infection and malignancy. Transforming growth actor-beta 1 (TGF-β1) and programmed death-ligand 1 (PD-L1) are two immunoregulatory proteins known to independently modulate immune cell function. Their combined potential in a single therapeutic entity, however, remains unexplored. This study aimed to design, express, and functionally characterize a bifunctional fusion protein that composed of TGF-β1 and PD-L1 domains. Methods and Results The fusion construct was engineered using overlap-extension PCR and cloned into a mammalian expression vector (pCDNA3.1+). Recombinant expression in HEK293 cells resulted in a ~70 kDa protein, verified using SDS-PAGE, immunoprecipitation, and western blot. 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 assays 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. This result suggested the functional and active binding of TGF-β1 and PD-L1 domains with their cognate receptors. Conclusion Collectively, this study provided molecular evidence for the structural and functional integrity of the TGF-β1/PD-L1 fusion protein and support its potential as a platform for targeted immunomodulation in autoimmune conditions.