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Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo
Published on: July 20, 2019
A mutated human tumor necrosis factor-alpha improves the therapeutic index in vitro and in vivo
1Biotechnology Center, School of Pharmacy of Fourth Military Medical University, Shaanxi, People's Republic of China.
Background:
Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that has cytotoxic, cytostatic and immunomodulatory effects on malignant tumors. However, clinical trials have revealed high systemic toxicity and this has hampered its utilization as an anti-cancer agent. In this study, a human TNF-alpha mutant was created and tested for its anti-tumor effects.
Methods:
The TNF mutant (recombinant mutated human TNF; rmhTNF) was prepared by protein engineering in which amino acids Pro, Ser and Asp at positions 8, 9 and 10 of TNF-alpha were substituted by Arg, Lys and Arg, and C terminal Leu157 was substituted by Phe, along with deletion of the first seven N-terminal amino acids. Prokaryotic expression recombinant vector pBV-mhTNF containing the PLPR promotor was constructed and transformed into E. coli DH5alpha. The rmhTNF was expressed in a partially soluble form in DH5alpha, purified from the supernatant of cell lysate by ammonia sulfate precipitation and two sequential chromatographic steps.
Results:
The purified rmhTNF was >95% pure by SDS-PAGE stained with silver and high-pressure size exclusion chromatography (SEC-HPLC). Its yield was about 1.22 mg/g wet cell paste. The mutant rmhTNF exhibited an approximately 50-fold increase in cytotoxicity relative to the wild-type rhTNF on the mouse fibroblast cell line L929 in a standard cytotoxicity test, and at least and at least 50 times higher LD50 as wild type rhTNF in mice. In vivo biological activity studies carried out on tumor cell transplanted mice and nude mice also showed a more effective cytotoxicity of rmhTNF than rhTNF.
Discussion:
These results suggest that rmhTNF has potential for developing an effective anti-tumor reagent for some tumors.
Insights
A novel mutant of tumor necrosis factor-alpha (TNF-alpha) shows significantly enhanced anti-tumor effects and reduced toxicity. This engineered TNF-alpha mutant holds promise as a potent anti-cancer therapeutic agent.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- Tumor necrosis factor-alpha (TNF-alpha) is a cytokine with anti-cancer properties.
- High systemic toxicity limits clinical use of wild-type TNF-alpha.
- A modified TNF-alpha was engineered to improve anti-tumor efficacy and safety.
Purpose of the Study:
- To create and evaluate a novel mutant of human TNF-alpha (rmhTNF) for anti-tumor activity.
- To assess the cytotoxicity and in vivo efficacy of rmhTNF compared to wild-type TNF-alpha.
- To determine the potential of rmhTNF as an anti-cancer therapeutic.
Main Methods:
- Protein engineering was used to create rmhTNF by specific amino acid substitutions and N-terminal deletion.
- Recombinant rmhTNF was expressed in E. coli and purified using chromatography.
- Cytotoxicity was assessed using L929 mouse fibroblast cell line and LD50 determination in mice.
Main Results:
- Purified rmhTNF achieved >95% purity with a yield of 1.22 mg/g wet cell paste.
- rmhTNF demonstrated approximately 50-fold higher cytotoxicity than wild-type rhTNF in vitro.
- In vivo studies showed rmhTNF to be more effective than rhTNF in reducing tumor burden in mice.
Conclusions:
- The engineered rmhTNF exhibits significantly enhanced anti-tumor activity and reduced toxicity.
- rmhTNF shows potential as a promising anti-cancer reagent for specific tumor types.
- Further development of rmhTNF could lead to a more effective cancer therapy.
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