Production and characterization of a functional anti-mouse PD-1 single-chain variable fragment (scFv) expressed from...

Bifidobacterium species preferentially accumulate in solid tumors and have emerged as a promising platform for localized production of therapeutic molecules within the tumor microenvironment. In this study, we constructed a recombinant B. longum strain capable of secreting an anti-mouse programmed cell death-1 (PD-1) single-chain variable fragment (scFv) and evaluated its expression, antigen-binding activity, blocking activity, and binding kinetics. Purified anti-mouse PD-1 scFv was detected as a ∼27 kDa protein by SDS-PAGE and confirmed by Western blot analysis. The antigen-binding activity of the purified scFv was evaluated by ELISA using recombinant mouse PD-1, yielding an apparent dissociation constant (Kd) of 0.053 μg/mL (1.9 nM). Furthermore, an in vitro PD-1/PD-L1 blocking assay demonstrated that preincubation of immobilized PD-1 with the scFv resulted in a concentration-dependent reduction in PD-L1 binding at PD-L1 concentrations of 0.03, 0.1, and 0.3 μg/mL, with relative IC₅₀ values of 0.58, 0.96, and 1.59 μg/mL, respectively. Although PD-L1 binding was inhibited in a concentration-dependent manner, residual binding remained even at the highest scFv concentration tested. Biolayer interferometry further showed concentration-dependent binding of the purified scFv to mouse PD-1. Collectively, these findings demonstrate that B. longum can produce an anti-mouse PD-1 scFv with in vitro binding and blocking activities and provide a foundation for the future development of bacteria-mediated localized immune checkpoint blockade for solid tumors.