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Development of expression systems for the production of recombinant human Fas ligand extracellular domain derivatives using Pichia pastoris and preparation of the conjugates by site-specific chemical modifications: A review

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

Human Fas ligand extracellular domain (hFasLECD) is a biomedically important glycoprotein with three potential N-linked carbohydrate-chain attachment sites. hFasLECD can induce an apoptotic cell-death in many malignant cells. Hence, the creation of novel molecular tools exhibiting useful biological activities, based on the exploitation of this protein domain as their components, opens up a great possibility of the advancements in future medical applications. This review mainly focuses on the development of expression systems for obtaining various derivatives of recombinant hFasLECD using Pichia pastoris and the preparation of the conjugates by site-specific chemical modifications of the expressed products. Firstly, a brief introduction of human Fas ligand protein and an overview of the previous works, on the heterologous expression systems for recombinant hFasLECD as well as the associated derivatives aimed at medical applications, were described. Then, the experimental results, obtained during our investigations into the development of the expression systems for the recombinant hFasLECD derivatives using chemically synthesized artificial genes in Pichia pastoris, were summarized. After that, the current state of the methodology for preparation of the hFasLECD conjugates by site-specific chemical modifications, and the functional characterizations of the prepared conjugates, were presented. Finally, conclusions, including a relevant discussion and future perspectives, are provided.
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1. Takahashi T, Tanaka M, Inazawa J, et al. (1994) Human Fas ligand: gene structure, chromosomal location and species specificity. Int Immunol 6: 1567–1574.    

2. Linkermann A, Qian J, Lettau M, et al. (2005) Considering Fas ligand as a target for therapy. Expert Opin Ther Tar 9: 119–134.    

3. Yonehara S, Ishii A, Yonehara M (1989) A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med 169: 1747–1756.    

4. Nagata S (1999) Fas ligand-induced apoptosis. Annu Rev Genet 33: 29–55.    

5. Bodmer JL, Schneider P, Tschopp J (2002) The molecular architecture of the TNF superfamily. Trends Biochem Sci 27: 19–26.    

6. Timmer T, de Vries EG, de Jong S (2002) Fas receptor-mediated apoptosis: a clinical application? J Pathol 196: 125–134.    

7. Peter ME, Hadji A, Murmann AE (2015) The role of CD95 and CD95 ligand in cancer. Cell Death Differ 22: 549–559.    

8. Calmon-Hamaty F, Audo R, Combe B, et al. (2015) Targeting the Fas/FasL system in rheumatoid arthritis therapy: promising or risky? Cytokine 75: 228–233.    

9. Wu J, Wilson J, He J, et al. (1996) Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease. J Clin Invest 98: 1107–1113.    

10. Suda T, Takahashi T, Golstein P, et al. (1993) Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 75: 1169–1178.    

11. Nagata S (1997) Apoptosis by death factor. Cell 88: 355–365.    

12. Wajant H (2006) Fas signaling. New York: Springer Science+Business Media, 1–160.

13. Strasser A, Jost PJ, Nagata S (2009) The many roles of Fas receptor signaling in the immune system. Immunity 30: 180–192.    

14. Bremer E (2013) Targeting of the tumor necrosis factor receptor superfamily for cancer immunotherapy. ISRN Oncol 2013: 371854.

15. Hsieh SL, Lin WW (2017) Decoy receptor 3: an endogenous immunomodulator in cancer growth and inflammatory reactions. J Biomed Sci 24: 39.    

16. Liu W, Ramagopal U, Cheng H, et al. (2016) Crystal structure of the complex of human FasL and its decoy receptor DcR3. Structure 24: 2016–2023.    

17. Muraki M (2012) Heterologous production of death ligands' and death receptors' extracellular domains: structural features and efficient systems. Protein Peptide Lett 19: 867–879.    

18. Tanaka M, Suda T, Yatomi T, et al. (1997) Lethal effect of recombinant human Fas ligand in mice pretreated with Propionibacterium acnes. J Immunol 158: 2303–2309.

19. Muraki M (2006) Secretory expression of synthetic human Fas ligand extracellular domain gene in Pichia pastoris: influences of tag addition and N-glycosylation site deletion, and development of a purification method. Protein Expres Purif 50: 137–146.    

20. Lu Y, Knol JC, Linskens MHK, et al. (2004) Production of the soluble human Fas ligand by Dictyostelium discoideum cultivated on a synthetic medium. J Biotechnol 108: 243–251.    

21. Luo Z, Xu Z, Zhuo S, et al. (2012) Production, purification and cytotoxity of soluble human Fas ligand expressed by Escherichia coli and Dictyostelium discoideum. Biochem Eng J 62: 86–91.    

22. Sun KH, Sun GH, Tsai CY, et al. (2005) Expression, purification, refolding, and characterization of recombinant human soluble-Fas ligand from Escherichia coli. Enzyme Microb Tech 36: 527–534.    

23. Muraki M (2014) Disulfide-bridged proteins with potential for medical applications: therapeutic relevance, sample preparation and structure-function relationships. Integr Mol Med 1: 38–56.

24. Holler N, Tardivel A, Kovacsovicsbankowski M, et al. (2003) Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Mol Cell Biol 23: 1428–1440.    

25. Eisele G, Roth P, Hasenbach K, et al. (2011) APO010, a synthetic hexameric CD95 ligand, induces human glioma cell death in vitro and in vivo. Neuro-Oncology 13: 155–164.    

26. Eisele G, Wolpert F, Decrey G, et al. (2013) APO010, a synthetic hexameric CD95 ligand, induces death of human glioblastoma stem-like cells. Anticancer Res 33: 3563–3571.

27. Oncology venture: APO010, 2012. Available from: http://www.oncologyventure.com/pipeline/apo010/.

28. ClinicalTrials.gov: ID NCT03196947, Safety and pharmacokinetics of rising doses of APO010 in relapsed/refractory multiple myeloma patients selected by DRP (SMR-3184). Available from: https://clinicaltrials.gov/ct2/show/NCT03196947?recrs=ab&cond=APO010&rank=1.

29. Daburon S, Devaud C, Costet P, et al. (2013) Functional characterization of a chimeric soluble Fas ligand polymer with in vivo anti-tumor activity. Plos One 8: e54000.    

30. Samel D, Müller D, Gerspach J, et al. (2003) Generation of a FasL-based proapoptotic fusion protein devoid of systemic toxicity due to cell-surface antigen-resticted activation. J Biol Chem 278: 32077–32082.    

31. Bremer E, Ten CB, Samplonius DF, et al. (2008) Superior activity of fusion protein scFvRit:sFasL over cotreatment with rituximab and Fas agonists. Cancer Res 68: 597–604.    

32. Bremer E, Ten CB, Samplonius DF, et al. (2006) CD7-restricted activation of Fas-mediated apoptosis: a novel therapeutic approach for acute T-cell leukemia. Blood 107: 2863–2870.    

33. Chien MH, Chang WM, Lee WJ (2017) A Fas ligand (FasL)-fused humanized antibody against tumor-associated glycoprotein 72 selectively exhibits the cytotoxic effect against oral cancer cells with a low FasL/Fas ratio. Mol Cancer Ther 16: 1102–1113.    

34. Chan DV, Sharma R, Ju CYA, et al. (2013) A recombinant scFv-FasLext as a targeting cytotoxic agent against human Jurkat-Ras cancer. J Biomed Sci 20: 16.    

35. Hemmerle T, Hess C, Venetz D, et al. (2014) Tumor targeting properties of antibody fusion proteins based on different members of the murine tumor necrosis superfamily. J Biotechnol 172: 73–76.    

36. Orbach A, Rachmilewitz J, Shani N, et al. (2010) CD40.FasL and CTLA-4.FasL fusion proteins induce apoptosis in malignant cell lines by dual signaling. Am J Pathol 177: 3159–3168.

37. Aronin A, Amsili S, Progozhina T, et al. (2014) Highly efficient in-vivo Fas-mediated apoptosis of B-cell lymphoma by hexameric CTLA4-FasL. J Hematol Oncol 7: 64.    

38. Zhang W, Wang B, Wang F, et al. (2012) CTLA4-FasL fusion product suppresses proliferation of fibroblast-like synoviocytes and progression of adjuvant-induced arthritis in rats. Mol Immunol 50: 150–159.    

39. Shi W, Chen M, Xie L (2007) Prolongation of corneal allograft survival by CTLA4-FasL in a murine model. Graef Arch Clin Exp 245: 1691–1697.    

40. Feng YG, Jin YZ, Zhang QY (2005) CTLA4-Fas ligand gene transfer mediated by adenovirus induce long-time survival of murine cardiac allografts. Transpl P 37: 2379–2381.    

41. Kitano H, Mamiya A, Kokubun S, et al. (2012) Efficient nonviral gene therapy with FasL and Del1 fragments in mice. J Gene Med 14: 642–650.    

42. Morello A, Daburon S, Castroviejo M, et al. (2013) Enhancing production and cytotoxic activity of polymeric soluble FasL-based chimeric proteins by concomitant expression of soluble FasL. Plos One 8: e73375.    

43. Franke DDH, Yolcu ES, Alard P, et al. (2007) A novel multimeric form of FasL modulates the ability of diabetogenic T cells to mediate type 1 diabetes in an adoptive transfer model. Mol Immunol 44: 2884–2892.    

44. Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24: 45–66.    

45. Daly R, Hearn MTW (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recogit 18: 119–138.    

46. Ahmad M, Hirtz M, Pichler H, et al. (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biot 98: 5301–5317.    

47. Muraki M (2014) Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications. BMC Biotechnol 14: 19.    

48. Jacobs PP, Geysens S, Vervecken W, et al. (2009) Engineering complex-type N-glycosylation in Pichia pastoris using glycoswitch technology. Nat Protoc 4: 58–70.    

49. Helenius A, Aebi M (2004) Roles of N-linked glycans in endoplasmic reticulum. Annu Rev Biochem 73: 1019–1049.    

50. Pfeffer M, Maurer M, Stadlmann J, et al. (2012) Intracellular interactome of secreted antibody Fab fragment in Pichia pastoris reveals its routes of secretion and degradation. Appl Microbiol Biot 93: 2503–2512.    

51. Muraki M (2008) Improved secretion of human Fas ligand extracellular domain by N-terminal part truncation in Pichia pastoris and preparation of the N-linked carbohydrate chain trimmed derivative. Protein Expres Purif 60: 205–213.

52. Orlinick JR, Elkon KB, Chao MV (1997) Separate domains of the human Fas ligand dictate self-association and receptor binding. J Biol Chem 51: 32221–32229.

53. Gasteiger E, Hoogland C, Gattiker A, et al. (2005) Protein identification and analysis tools on the ExPASy server, In: Walker JM, The Proteomics Protocols Handbook, Totowa: Humana Press, 571–607.

54. Muraki M, Hirota K (2017) Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene-methyltetrazine reactions. BMC Biotechnol 17: 56.    

55. Mahiou J, Abastado JP, Cabanie L, et al. (1998) Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system. Biochem J 330: 1051–1058.    

56. Muraki M, Honda S (2010) Efficient production of human Fas receptor extracellular domain-human IgG1 heavy chain Fc domain fusion protein using baculovirus/silkworm expression system. Protein Expres Purif 73: 209–216.    

57. Muraki M, Honda S (2011) Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus-silkworm expression system. Protein Expres Purif 80: 102–109.    

58. Vogl T, Glieder A (2013) Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnol 30: 385–404.    

59. Allais JJ, Louktibi A, Baratti J (1980) Oxidation of methanol by the yeast, Pichia pastoris. Purification and properties of the alcohol oxidase. Agric Biol Chem 44: 2279–2289.

60. Muraki M (2014) Secretory production of recombinant proteins in methylotrophic yeast Pichia pastoris using a disposable culture-bag. PSSJ Arch 7: e078.

61. Vanamee ÉS, Faustmann DL (2018) Structural principles of tumor necrosis factor superfamily signaling. Sci Signal 11: eaao4910.    

62. Hermanson GT (2013) Chapter 10: Fluorescent Probes, In: Bioconjugate techniques (Third Ed.), London: Academic Press, 395–463.

63. Wu H, Devaraj NK (2016) Inverse electron-demand Diels-Alder biorthogonal reactions. Top Curr Chem 374: 3.    

64. Mayer S, Lang K (2017) Tetrazines in inverse-electron-demand Diels-Alder cycloadditions and their use in biology. Synthesis-Stuttgart 49: 830–848.

65. Muraki M (2016) Preparation of a functional fluorescent human Fas ligand extracellular domain derivative using a three-dimensional structure guided site-specific fluorochrome conjugation. SpringerPlus 5: 997.    

66. Ogasawara J, Watanabe-Fukunaga R, Adachi M, et al. (1993) Lethal effect of the anti-Fas antibody in mice. Nature 364: 806–809.    

67. Wajant H, Gerspach J, Pfizenmaier K (2013) Engineering death receptor ligands for cancer therapy. Cancer Lett 332: 163–174.    

68. Villa-Morales M, Fernández-Piqueras J (2012) Targeting the Fas/FasL signaling pathway in cancer therapy. Expert Opin Ther Tar 16: 85–101.    

69. Oliveira BL, Guo Z, Bernardes GJL (2017) Inverse electron demand Diels-Alder reactions in chemical biology. Chem Soc Rev 46: 4895–4950.    

70. Green NM (1963) Avidin. 4. Stability at extremes of pH and dissociation into sub-units by guanidine hydrochloride. Biochem J 89: 609–620.

71. Paganelli G, Magnani P, Zito F, et al. (1991) Three-step monoclonal antibody tumor targeting in carcinoembryonic antigen positive patients. Cancer Res 51: 5960–5966.

72. Penichet ML, Kang YS, Pardridge WM, et al. (1999) An antibody-avidin fusion protein specific for the transferrin receptor serves as a delivery vehicle for effective brain targeting: initial applications in anti-HIV antisense drug delivery to brain. J Immunol 163: 4421–4426.

73. Schultz J, Lin Y, Sanderson J, et al. (2000) A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy. Cancer Res 60: 6663–6669.

74. Mohsin H, Jia F, Bryan JN, et al. (2011) Comparison of pretargeted and conventional CC49 radioimmunotherapy using 149Pm, 166Ho, and 177Lu. Bioconjugate Chem 22: 2444–2452.    

75. Micheau O, Solary E, Hammann A, et al. (1997) Sensitization of cancer cells treated with cytotoxic drugs to Fas-mediated cytotoxity. J Natl CancerI 89: 783–789.    

76. Yang D, Torres CM, Bardhan K, et al. (2012) Decitabine and vorinostat cooperate to sensitize colon carcinoma cells to Fas ligand-induced apoptosis in vitro and tumor suppression in vivo. J Immunol 188: 4441–4449.    

77. Galenkamp KMO, Carriba P, Urresti J, et al. (2015) TNFα sensitizes neuroblastoma cells to FasL-, cisplatin- and etoposide-induced cell death by NF-ΚB-mediated expression of Fas. Mol Cancer 14: 62.    

78. Xu X, Fu XY, Plate J (1998) IFN-γ induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression. Cancer Res 58: 2832–2837.

79. Horton JK, Siamakpour-Reihani S, Lee CT, et al. (2015) Fas death receptor: a breast cancer subtype-specific radiation response biomarker and potential therapeutic target. Radiat Res 184: 456–469.    

80. Tamakoshi A, Nakachi K, Ito Y, et al. (2008) Soluble Fas level and cancer mortality: findings from a nested case-control study within a large-scale prospective study. Int J Cancer 123: 1913–1916.    

81. Bhatraju PK, Robinson-Cohen C, Mikacenic C, et al. (2017) Circulating levels of soluble Fas (sCD95) are associated with risk for development of a nonresolving acute kidney injury subphenotype. Crit Care 21: 217.    

82. Hamilton SR, Gerngross TU (2007) Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotech 18: 387–392.    

83. Liu L, Stadheim A, Hamuro L, et al. (2011) Pharmacokinetics of IgG1 monoclonal antibodies produced in humanized Pichia pastoris with specific glycoforms: a comparative study with CHO produced materials. Biologicals 39: 205–210.    

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