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

Area of Science:

• Immunology
• Biotechnology
• Cancer Therapy

Background:

• Monoclonal antibodies targeting PD-1 have transformed cancer treatment but present challenges like adverse events and poor tumor penetration.
• Single-chain variable fragments (scFvs) offer potential advantages, including better tissue penetration and cost-effective production.

Purpose of the Study:

• To design and express a novel anti-PD-1 single-chain variable fragment (scFv) with optimized framework regions.
• To evaluate the binding affinity, specificity, and anti-tumor efficacy of the engineered scFv in vitro.

Main Methods:

• An anti-PD-1 scFv (L2H5) was engineered based on the Opdivo sequence with framework region mutations.
• The scFv gene was codon-optimized for E. coli expression and purified from inclusion bodies.
• Bioactivity was assessed using immunoblotting, cytotoxicity assays, immunofluorescence, and a tumor cell killing assay.

Main Results:

• Successfully expressed and purified L2H5 anti-PD-1 scFv at 4 mg/L.
• Demonstrated specific binding to PD-1 via immunoblotting and immunofluorescence.
• Showed no significant cytotoxicity to normal cells and enhanced T-cell mediated tumor cell killing in vitro.

Conclusions:

• Engineered L2H5 anti-PD-1 scFv exhibits specific PD-1 binding and potent anti-tumor activity.
• Prokaryotic expression of the scFv is feasible, yielding a functional protein with no observed toxicity to normal cells.
• This scFv represents a promising candidate for next-generation cancer immunotherapies.