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2000
Volume 26, Issue 6
  • ISSN: 1389-2010
  • E-ISSN: 1873-4316

Abstract

Background

Immunotoxins (ITs) represent a novel class of therapeutics with bi-functional 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 over-expressed 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 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 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.

© 2025 Bentham Science Publishers
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Development and Characterization of an Anti-PD-L1 Immunotoxin for Targeted Cancer Therapy
Current Pharmaceutical Biotechnology 26, 854 (2025); https://doi.org/10.2174/0113892010321088240823062243
/content/journals/cpb/10.2174/0113892010321088240823062243
/content/journals/cpb/10.2174/0113892010321088240823062243
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  • Article Type:     Research Article
Keyword(s): anti-PD-L1; Diphtheria toxin; drug delivery; immunotoxin; nanobody; PD-L1; target therapy

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