Targeting Oncolytic Adenoviruses to Cancer Cells Using a Designed Ankyrin Repeat Protein Lipocalin-2 Fusion Protein

Area of Science:

• Oncolytic virotherapy
• Molecular targeting strategies
• Adenovirus engineering

Background:

• Oncolytic viruses offer a promising approach for cancer treatment by selectively targeting cancer cells and stimulating anti-tumor immunity.
• The Lipocalin-2 receptor (LCN2R) is frequently overexpressed on various cancer cells, presenting a potential target for selective viral delivery.
• Adenoviruses (Ads) are commonly used vectors for oncolytic virotherapy due to their safety profile and genetic payload capacity.

Purpose of the Study:

• To engineer and characterize a novel LCN2-based targeting system for oncolytic adenoviruses (Ads) to enhance cancer cell specificity and uptake.
• To evaluate the efficacy of the LCN2-targeted Ads in vitro using cancer cell lines and 3D models.
• To investigate the mechanism underlying the enhanced viral uptake mediated by the LCN2 targeting system.

Main Methods:

• Fusion of a Designed Ankyrin Repeat Protein (DARPin) adapter binding to Ad type 5 knob (knob5) with LCN2 to retarget Ads to LCN2R.
• In vitro testing using Chinese Hamster Ovary (CHO) cells expressing LCN2R and 20 cancer cell lines (CCLs) infected with Ad5 vectors encoding luciferase and green fluorescent protein.
• Quantification of viral infection via luciferase assays, viral uptake via flow cytometry and hexon immunostaining, and virus spread in 3D cell culture models.

Main Results:

• The LCN2 adapter (LA) mediated a 10-fold higher infection rate compared to a blocking adapter (BA) in LCN2R-expressing cells.
• Most tested CCLs exhibited increased viral uptake with LA-bound Ads compared to BA-bound Ads; five CCLs showed uptake comparable to unmodified Ad5.
• Enhanced viral spread was observed in nine CCLs using LA-bound Ads in 3D models, with increased and earlier fluorescence signals.
• Mechanistically, enhanced uptake was dependent on the absence of LCN2's ligand, Enterobactin (Ent), and independent of iron.

Conclusions:

• A novel DARPin-based LCN2 targeting system was successfully developed for oncolytic adenoviruses.
• This system significantly enhances viral uptake and spread in various cancer cell lines, demonstrating potential for improved oncolytic virotherapy.
• The targeting efficacy is modulated by the presence of Enterobactin, suggesting context-dependent applications.