Optimization, Expression, Purification, and Activity Identification of Anti-PD-1 scFv

Introduction: Anti-PD-1 monoclonal antibodies have revolutionized cancer therapy but face challenges, including immune-related adverse events, poor tumor penetration due to their large size, and high production costs. Single-chain variable fragments offer a promising alternative, with enhanced tissue penetration and the potential for cost-effective prokaryotic expression. Methods: Based on the Opdivo sequence, an anti-PD-1 scFv (L2H5) was designed using a framework region-frequency-guided optimization approach. Two sites in the light chain and five in the heavy chain FR were mutated to high-frequency residues. The gene was codon-optimized for E. coli and cloned into the pET-28a vector. The protein was expressed in E. coli BL21(DE3), purified via Ni-NTA chromatography from inclusion bodies, and refolded by dialysis. Its bioactivity was assessed by immunoblotting, cytotoxicity assay, immunofluorescence, and a luciferase-based tumor cell killing assay. Results: The codon-optimized L2H5 scFv was successfully expressed, yielding 4 mg/L after purification. Immunoblotting confirmed its specific binding to PD-1. The CCK-8 assay demonstrated no significant cytotoxicity towards normal 293T cells. Immunofluorescence revealed specific binding to PD-1 on A549 cell surfaces. In a co-culture system with tumor cells and lymphocytes, the L2H5 scFv significantly enhanced T cell-mediated tumor cell killing in a dose- and time-dependent manner. Discussion: This study connected the VH and VL region sequences of the PD-1 monoclonal antibody drug Opdivo through a linker to form a PD-1 single-chain antibody and tried to mutate and optimize the protein sequence of the antibody framework region. The activity and binding of L2H5 PD-1 scFv to PD-1 protein were verified by in vitro experiments, and the binding ability of L2H5 PD-1 scFv to PD-1 protein was verified by immunoblotting and immunofluorescence. In terms of binding ability, the results of immunoblotting showed that the corresponding bands were detected at the size position of PD-1 protein, and the results of immunofluorescence intuitively showed the binding of L2H5 PD-1 scFv protein on cells. In terms of biological activity, the results of CCK-8 experiments showed that L2H5 PD-1 scFv had no obvious toxicity to normal cells and did not affect the growth and proliferation of normal cells. Conclusion: The L2H5 anti-PD-1 scFv protein was successfully expressed by a prokaryotic vector. The expressed protein had no obvious toxicity to normal cells and retained PD-1-binding activity.