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Long circulating XTEN864-HGV-Apoptin fusion protein for selective cancer therapy
Liu Yang1, Akvile Haeckel1, Nicola Beindorff2
1Charité - Universitätsmedizin Berlin, Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Radiology, Charitéplatz 1, 10117 Berlin, Germany.
Abstract:
The virus protein CAV-Apoptin and its homologue HGV-Apoptin selectively kill cancer cells but are not suitable for systemic treatment. The aim was to develop Apoptin-based fusion proteins for intravenous application in cancer therapy, which also contain the hydrophilic polypeptide XTEN, a cleavage site for MMP-2/9, and a TAT peptide for cell penetration. Expression of XTEN864-HGV-Apoptin in E. coli and purification using XTEN as a tag yielded 100 mg protein/L tissue culture. The expression of XTEN864-CAV-Apoptin did not generate a sufficient yield. Cytotoxic effects were assessed using MTT and Annexin A5 assays, whereas cellular uptake was visualized using Cy3.5-XTEN864-HGV-Apoptin. Blood half-life and biodistribution were evaluated with 99mTc-XTEN864-HGV-Apoptin using SPECT-CT and gamma counting. The fusion protein significantly reduced cancer cell growth and induced apoptosis with minimal effects on non-cancerous cells. It accumulates in the nucleus and associates with F-actin. In mice, the protein showed a blood half-life of 0.68 h (fast phase) and 17 h (slow phase), with a tumor/muscle ratio of 9.36 ± 6.22 (SD). In a 4T1 mouse tumor model, it effectively inhibited tumor growth. The cancer-specific cytotoxicity and prolonged circulation of XTEN864-HGV-Apoptin suggest its potential for systemically applicable, biodegradable, and E. coli-producible antitumor drugs.
Insights
Researchers developed a novel Apoptin-based fusion protein, XTEN864-HGV-Apoptin, for effective intravenous cancer therapy. This engineered protein demonstrates potent cancer cell killing and prolonged circulation in vivo.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Virus proteins CAV-Apoptin and HGV-Apoptin show selective cancer cell killing but are unsuitable for systemic therapy.
- Development of Apoptin-based fusion proteins is needed for intravenous cancer treatment.
Purpose of the Study:
- To engineer and evaluate Apoptin-based fusion proteins for systemic cancer therapy.
- To incorporate XTEN, MMP-2/9 cleavage site, and TAT peptide for enhanced delivery and efficacy.
Main Methods:
- Expression and purification of XTEN864-HGV-Apoptin in E. coli.
- Assessment of cytotoxicity using MTT and Annexin A5 assays.
- Evaluation of cellular uptake, blood half-life, biodistribution (SPECT-CT), and in vivo tumor growth inhibition (4T1 model).
Main Results:
- XTEN864-HGV-Apoptin was successfully produced (100 mg/L) and purified.
- The fusion protein exhibited significant cancer cell apoptosis and reduced growth with minimal toxicity to normal cells.
- In vivo studies showed prolonged blood circulation (17 h slow phase) and effective tumor growth inhibition in mice.
Conclusions:
- XTEN864-HGV-Apoptin demonstrates potent, cancer-specific cytotoxicity and favorable pharmacokinetic properties.
- The fusion protein is a promising candidate for developing systemically applicable, biodegradable, and E. coli-producible antitumor drugs.
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