Construction and evaluation of a novel Stx2a-based immunotoxin against epidermal growth factor receptor (EGFR)
Negar Akhzari1, Jafar Amani2, Golnaz Tajadod3
1Department of Cell Biology, North Tehran Branch, Islamic Azad University, Tehran, Iran.
Abstract:
Immunotoxins (ITs) are chimeric proteins that combine the targeting specificity of a monoclonal antibody or antibody fragment with the cytotoxic properties of a toxin. They offer a promising strategy for cancer therapy by selectively delivering cytotoxic payloads to tumor cells. The epidermal growth factor receptor (EGFR) is frequently overexpressed in various cancers, making it an attractive target for IT-based therapies. In this study, we designed and constructed a novel IT composed of a single-chain variable fragment (scFv) derived from Panitumumab, a humanized monoclonal antibody targeting EGFR, and the Shiga toxin A subunit 2 (Stx2a), a potent cytotoxic agent. The IT was designed using computational tools to optimize the linker region between the scFv and Stx2a domains. The recombinant IT was expressed in a prokaryotic host and purified to homogeneity. The cytotoxic activity of the IT was evaluated in vitro against human colorectal carcinoma (HCT-116) and human embryonic kidney (HEK293) cells. The IT demonstrated significant cytotoxicity against HCT-116 cells, while exhibiting minimal toxicity to non-target cells. To enhance the delivery and efficacy of the IT, we encapsulated it in chitosan nanoparticles. The nanoparticle-based formulation showed improved cellular uptake and enhanced cytotoxicity compared to the free IT. Our findings suggest that the designed IT has the potential to be a promising therapeutic agent against EGFR-expressing cancers. Further studies are warranted to evaluate its efficacy in vivo and to optimize its formulation for clinical applications.
Insights
We developed a novel immunotoxin targeting EGFR-expressing cancers. Encapsulating this immunotoxin in nanoparticles enhanced its cancer-killing ability and cellular uptake, showing promise for effective cancer therapy.
Area of Science:
- Oncology
- Biotechnology
- Molecular Biology
Background:
- Immunotoxins (ITs) are engineered proteins for targeted cancer therapy.
- Epidermal Growth Factor Receptor (EGFR) is a key target in many cancers.
- Panitumumab-derived scFv and Shiga toxin A subunit 2 (Stx2a) were chosen for their targeting and cytotoxic properties.
Purpose of the Study:
- To design and construct a novel immunotoxin targeting EGFR.
- To evaluate the in vitro cytotoxicity of the immunotoxin and its nanoparticle formulation.
- To assess the potential of this immunotoxin as a cancer therapeutic.
Main Methods:
- Computational design of an immunotoxin linking an anti-EGFR scFv (from Panitumumab) to Stx2a.
- Recombinant expression, purification, and in vitro cytotoxicity assays against HCT-116 and HEK293 cells.
- Encapsulation of the immunotoxin in chitosan nanoparticles to improve delivery and efficacy.
Main Results:
- The novel immunotoxin demonstrated significant, targeted cytotoxicity against EGFR-expressing HCT-116 cancer cells.
- Minimal toxicity was observed in non-target HEK293 cells, indicating specificity.
- Chitosan nanoparticle formulation enhanced cellular uptake and boosted the immunotoxin's cytotoxicity.
Conclusions:
- The designed immunotoxin is a potent and specific agent against EGFR-expressing cancer cells.
- Nanoparticle encapsulation improves immunotoxin delivery and therapeutic efficacy.
- This approach holds promise for developing novel immunotoxin-based cancer therapies.
