Development and Characterization of an Anti-PD-L1 Immunotoxin for Targeted Cancer Therapy

Background: Immunotoxins (ITs) represent a novel class of therapeutics with bifunctional structures that facilitate their penetration through cell membranes to induce target cell destruction. Programmed cell death ligand-1 (PD-L1), a human cell surface protein, is overexpressed in various cancers. This study aimed to construct a novel IT by genetically fusing an anti-PD-L1 Nanobody (Nb) to a truncated diphtheria toxin (DT).

Methods: The IT construct comprised a 127-amino acid anti-PD-L1 Nb fused to a 380-amino acid fragment of DT, with an N-terminal 6x-His tag. Molecular cloning techniques were employed, followed by transformation and verification through colony-PCR, enzyme digestion, and sequencing. The anti-PD-L1 Nb was expressed in WK6 E. coli cells induced by Isopropyl β-D-1- Thiogalactopyranoside (IPTG) and purified from periplasmic extracts using immobilized Metal Ion Affinity hromatography (IMAC). The IT was similarly expressed, purified, and validated via SDS-PAGE and Western blot analysis.

Results: ELISA confirmed the binding activity of both Nb and IT to immobilized PD-L1 antigen, whereas truncated DT exhibited no binding. MTT assays demonstrated significant cytotoxicity of IT on A-431 cell lines compared to Nb and truncated DT controls. Statistical analyses underscored the significance of these findings.

Conclusion: This study provides a thorough characterization of the constructed IT, highlighting its potential as a therapeutic agent targeting PD-L1-expressing cancer cells. The results support the potential of this IT in cancer immunotherapy, emphasizing the need for further investigation into its efficacy and safety profiles.