Advertisement

Co-Solvents Effects on the Stability of Recombinant Immunotoxin Denileukin Diftitox: Structure and Function Assessment

  • Sh. Bayat
  • M. ZeinoddiniEmail author
  • A. Azizi
  • M. A. Nasiri Khalili
Research Paper
  • 2 Downloads
Part of the following topical collections:
  1. Biology
  2. Biology

Abstract

Denileukin diftitox (brand name: Ontak, DAB389IL2) is the first recombinant immunotoxin approved by FDA for treatment of CTCL (cutaneous T cell lymphoma). This smart toxin is contains truncated diphtheria toxin (DT387), genetically fused to human interleukin 2 (IL2). Like other pharmaceutical productions, there is an aggregation problem in this fusion protein production and achieving a stable protein structure is mainly recommended for clinical use. Herein, we have investigated the effects of co-solvents on the structure and function of denileukin diftitox. In this work, the histidine-tagged DAB389IL2 was expressed in Escherichia coli and after solubilizing and refolding, the inclusion body was purified using nickel chelate affinity chromatography. Then, the structural alterations of the refolded protein in the presence of different co-solvents (sorbitol, trehalose, and sucrose) were investigated using fluorescence and circular dichroism (CD) techniques. Moreover, the nuclease activity of the refolded protein was traced to confirm the refolding process,. Finally, the biological activity of produced DAB389IL2 was carried out using MTT assay. The results revealed that, DAB389IL2 is more stable in PBS including sorbitol (30 mM) than other stabilizers. The tryptophan residues flexibility—exposure of DAB389IL2 to the solvent, in the presence of sorbitol, with respect to fluorescence quenching by acrylamide, indicated the best characterization. Also, in the presence of 2 ng/ml of protease, DAB389IL2 was completely undigested and so it indicated the highly stability of the fusion protein to proteases. Furthermore, the refolding and function of the produced fusion protein is verified by DNase activity and bioassay. Therefore, the data of this research could give further insights into the protein refolding mechanism and formulation of the protein in the presence of co-solvents.

Keywords

Denileukin diftitox Immunotoxin Expression Stability Function 

Notes

Acknowledgements

This study has been supported by the Malek-Ashtar University of Technology.

References

  1. Arakawa T, Prestrelski SJ, Kenney WC, Carpenter JF (2001) Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev 46:307–326CrossRefGoogle Scholar
  2. Chi EY, Krishnan S, Randolph TW, Carpenter JF (2003) Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res 20:1325–1336CrossRefGoogle Scholar
  3. D’Amico S, Marx JC, Gerday C, Feller G (2003) Activity-stability relationships in extremophilic enzymes. J Biol Chem 278:7891–7896CrossRefGoogle Scholar
  4. Duvic M, Talpur R (2008) Optimizing denileukin diftitox (ontak) therapy. Future Oncol 4:457–469CrossRefGoogle Scholar
  5. Ebrahimi Bagha M, Zeinoddini M, Saeedinia AR, Xodadadi N (2018) Overexpression and purification of diphtheria fusion toxin: DAB389IL-2. J Bionanosci 12:688–693Google Scholar
  6. Eklund JW, Kuzel TM (2005) Denileukin diftitox: a concise clinical review. Expert Rev Anticancer Ther 5:33–38CrossRefGoogle Scholar
  7. Figgitt DP, Lamb HM, Goa KL (2000) Denileukin diftitox. Am J Clin Dermatol 1:67–72CrossRefGoogle Scholar
  8. Foss FM (2000) DAB(389)IL-2 (denileukin diftitox, ONTAK): a new fusion protein technology. Clin Lymphoma 1:S27–S31CrossRefGoogle Scholar
  9. Frankel AE, Kreitman RJ, Sausville EA (2000) Targeted toxins. Clin Can Res 6:326–334Google Scholar
  10. Frokjaer S, Otzen DE (2005) Protein drug stability: a formulation challenge. Nat Rev Drug Discov 4:298–306CrossRefGoogle Scholar
  11. Howman RA, Prince HM (2011) New drug therapies in peripheral T-cell lymphoma. Expert Rev Anticancer Ther 11:457–472CrossRefGoogle Scholar
  12. Iyer PV, Ananthanarayan L (2008) Enzyme stability and stabilization: aqueous and non-aqueous environment. Process Biochem 43:1019–1032CrossRefGoogle Scholar
  13. Kaminetzky D, Hymes KB (2008) Denileukin diftitox for the treatment of cutaneous T-cell lymphoma. Biol Targ Thera 2:717–724Google Scholar
  14. Kreitman RJ (2006) Immunotoxins for targeted cancer therapy. AAPS J 8:532–551CrossRefGoogle Scholar
  15. Lansigan F, Stearns DM, Foss F (2010) Role of denileukin diftitox in the treatment of persistent or recurrent cutaneous T-cell lymphoma. Cancer Manag Res 2:53–59CrossRefGoogle Scholar
  16. Li YM, Hall WA (2010) Targeted toxins in brain tumor therapy. Toxins 2:2645–2662CrossRefGoogle Scholar
  17. Manoukian G, Hagemeister F (2009) Denileukin diftitox: a novel immunotoxin. Expert Opin Biol Ther 9:1445–1451CrossRefGoogle Scholar
  18. Moghaddas M, Zeinoddini M, Saeedinia AR, Bayat S (2018) Structural and functional assessment of diphtheria fusion toxin: DT389GCSF. J Bionanosci 12(2):240–244CrossRefGoogle Scholar
  19. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxcity assays. J Immunol Methods 65(1–2):55–63CrossRefGoogle Scholar
  20. Murakami S, Kinoshita M (2016) Effects of monohydric alcohols and polyols on the thermal stability of a protein. J Chem Phys 144:125105CrossRefGoogle Scholar
  21. Oshima H, Kinoshita M (2013) Effects of sugars on the thermal stability of a protein. J Chem Phys 138:245101CrossRefGoogle Scholar
  22. Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ (2006) Immunotoxin therapy of cancer. Nat Rev Cancer 6:559–565CrossRefGoogle Scholar
  23. Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ (2007) Immunotoxin treatment of cancer. Annu Rev Med 58:221–237CrossRefGoogle Scholar
  24. Sek DC (2008) Protein formulations containing sorbitol. US20080200655 A1Google Scholar
  25. Shire SJ (2009) Formulation and manufacturability of biologics. Curr Opin Biotechnol 20:708–714CrossRefGoogle Scholar
  26. Stevenson CL (2000) Characterization of protein and peptide stability and solubility in non-aqueous solvents. Curr Pharm Biotechnol 1:165–182CrossRefGoogle Scholar
  27. Talpur R, Jones DM, Alencar AJ, Apisarnthanarax N, Herne KL, Yang Y, Duvic M (2006) CD25 expression is correlated with histological grade and response to denileukin diftitox in cutaneous T-cell lymphoma. J Invest Dermatol 126:575–583CrossRefGoogle Scholar
  28. Wang W (2005) Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm 289:1–3CrossRefGoogle Scholar
  29. Weiss WF, Young TM, Roberts CJ (2009) Principles, approaches, and challenges for predicting aggregation rates and shelf life. J Pharm Sci 98(4):1246–1277CrossRefGoogle Scholar
  30. Zeinoddini M, Khajeh K, Hosseinkhani S, Saeedinia AR, Robatjazi SM (2013) Stabilisation of recombinant aequorin by polyols: activity, thermostability and limited proteolysis. Appl Biochem Biotechnol 170:273–280CrossRefGoogle Scholar

Copyright information

© Shiraz University 2019

Authors and Affiliations

  1. 1.Department of Bioscience and BiotechnologyMalek Ashtar University of TechnologyTehranIran

Personalised recommendations