Functional Deimmunization of Interferon Beta-1b by Identifying and Silencing Human T Cells Epitopes
-
Amin Moradi Hasan-Abad
,
Elham Adabi
,
Esmaeil Sadroddiny
,
Mohammad Reza Khorramizadeh
,
Mohammad Ali Mazlomi
,
Saeed Mehravar
,
Gholam Ali Kardar
Abstract
Interferonbeta-1b (IFNβ-1b) developed as therapeutic protein for the treatment of multiple sclerosis (MS). Studies have been shown that Long-term usage of this protein can lead to the development of anti-drug antibodies (ADAs) and this phenomenon cause total loss or reduced efficacy of IFNβ-1b. The aim of this study was to predict and silence IFNβ-1b T-cells epitopes by in silico methods and genetic engineering. Based on bioinformatics studies we identified optimal sets of conservative point mutations for eliminating T-cells epitopes in IFNβ-1b protein. Four synthetic genes with desirable mutation constructed and PET26b+ was used as an expression vector in E. coli. The expression of this proteins confirmed by SDS-PAGE and Western blotting, consequently, IFNβ-1b proteins was purified by His-tag chromatography. To determined activity of mutants’ variants anti-proliferative and anti-viral activity compared to wild form was evaluated using MTT assay in A549 and Vero cells lines respectively. Also the immunogenicity of mutant proteins compared with Betaseron measured in BALB/c mice. The in vitro bioactivity analysis demonstrated that functional activities of all mutant proteins were maintained and is the same as biological activity of Betaseron. Pharmacokinetic studies suggest that, in engineered proteins that contain substitution of Histidine to Glutamic Acid at position 131 (mut 2 and mut 1+2) antibodies response reduced by about 50%, as compared to that for Betaseron. Computational analysis expedites identification and prediction of epitopes in therapeutic protein, therefore, we used immunoinformatic tools for modification of dominant T-cell epitope in IFNβ-1b protein, and this strategy has capacity to create proteins which have naturally reduced immunogenicity.
1.Dhib-Jalbut S, Marks S. Interferon-β mechanisms of action in multiple sclerosis. Neurology. 2010; 74(1 Supplement 1):S17-24.
2.Limmroth V, Putzki N, Kachuck NJ. The interferon beta therapies for treatment of relapsing–remitting multiple sclerosis: are they equally efficacious? A comparative review of open-label studies evaluating the efficacy, safety, or dosing of different interferon beta formulations alone or in combination. Ther Adv Neurol Disord 2011; 4(5):281-96.
3.Runkel L, Meier W, Pepinsky RB, Karpusas M, Whitty A, Kimball K, et al. Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-β (IFN-β). Pharm Res 1998; 15(4):641-9.
4.Kessler M, Goldsmith D, Schellekens H. Immunogenicity of biopharmaceuticals. Nephrology Dialysis Transplantation. 2006; 21(suppl_5):v9-v12.
5.Porter S. Human immune response to recombinant human proteins. J Pharm Sci 2001; 90(1):1-11.
6.Jawa V, Cousens LP, Awwad M, Wakshull E, Kropshofer H, De Groot AS. T-cell dependent immunogenicity of protein therapeutics: preclinical assessment and mitigation. Clin Immunol 2013; 149(3):534-55.
7.Antonelli G. Reflections on the immunogenicity of therapeutic proteins. Clin Microbiol Infect 2008; 14(8):731-3.
8.van Beers MM, Jiskoot W, Schellekens H. On the role of aggregates in the immunogenicity of recombinant human interferon beta in patients with multiple sclerosis. J Interferon Cytokine Res 2010; 30(10):767-75.
9.Bertolotto A, Deisenhammer F, Gallo P, Sørensen PS. Immunogenicity of interferon beta: differences among products. J Neurol 2004; 251(2):ii15-ii24.
10.Mahmoud K. Recombinant protein production: strategic technology and a vital research tool. Research Journal of Cell and Molecular Biology. 2007; 1(1):9-22.
11.Basu A, Yang K, Wang M, Liu S, Chintala R, Palm T, et al. Structure− function engineering of interferon-β-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation. Bioconjug Chem 2006; 17(3):618-30.
12.Schneider-Fresenius C, Otto B, Waschutza G. Recombinant human beta interferon with enhanced solubility. Google Patents; 2003.
13.Bell LD, Smith JC, Boseley PG, Houghton M. Modified (1-28) beta interferons. Google Patents; 1988.
14.Johnson-Jackson D, Furuya K, Zaror I, Larson D. Recombinant interferon-beta with enhanced biological activity. Google Patents; 2009.
15.Kraynov V, Knudsen N, PUTNAM A-MAH, Krawitz D, Pinkstaff J, Myler H. Modified interferon beta polypeptides and their uses. Google Patents; 2012.
16.Song K, Yoon I-S, Kim NA, Kim D-H, Lee J, Lee HJ, et al. Glycoengineering of interferon-β 1a improves its biophysical and pharmacokinetic properties. PloS one 2014; 9(5):e96967.
17.Kay M, Hasan-Abad AM, Hojati Z, Korbekandi H. Targeted mutations in Val101 and Arg27 interferon beta protein increase its transcriptional and translational activities. Cytokine 2016; 78:1-6.
18.Pembroke JT. Bio-molecular modelling utilising RasMol and PDB resources: a tutorial with HEW lysozyme. Biochemistry and Molecular Biology Education 2000; 28(6):297-300.
19.Filpula DR, Basu A. Optimized interferon-beta gene. Google Patents; 2010.
20.Sockolosky JT, Szoka FC. Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expr Purif 2013; 87(2):129-35.
21.Kruger NJ. The Bradford method for protein quantitation. The protein protocols handbook: Springer 2002; 15-21.
22.Kueltzo L, Middaugh C. Ultraviolet absorption spectroscopy. Methods for structural analysis of protein pharmaceuticals 2005; 3:1-25.
23.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature1970; 227(5259):680.
24.Senthilraja P, Kathiresan K. In vitro cytotoxicity MTT assay in Vero, HepG2 and MCF-7 cell lines study of Marine Yeast. J Appl Pharm Sci 2015; 5(3):80-4.
25.Meager A. Biological assays for interferons. J Immunol Methods 2002;261(1-2):21-36.
26.van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays: the MTT assay. Methods Mol Biol 2011; 237-45.
27.Yoon SH, Kim SK, Kim JF. Secretory production of recombinant proteins in Escherichia coli. Recent Pat Biotechnol 2010; 4(1):23-9.
28. van Beers MM, Sauerborn M, Gilli F, Brinks V, Schellekens H, Jiskoot W. Aggregated recombinant human interferon beta induces antibodies but no memory in immune-tolerant transgenic mice. Pharm Res 2010; 27(9):1812-24.
29. Yeung VP, Chang J, Miller J, Barnett C, Stickler M, Harding FA. Elimination of an immunodominant CD4+ T cell epitope in human IFN-β does not result in an in vivo response directed at the subdominant epitope. J Immunol 2004; 172(11):6658-65.
30. Gneiss C, Reindl M, Berger T, Lutterotti A, Ehling R, Egg R, et al. Epitope specificity of neutralizing antibodies against IFN-β. J Interferon Cytokine Res 2004; 24(5):283-90.
31. Runkel L, deDios C, Karpusas M, Betzenhauser M, Muldowney C, Zafari M, et al. Systematic mutational mapping of sites on human interferon-β-1a that are important for receptor binding and functional activity. Biochemistry 2000; 39(10):2538-51.
32. Karpusas M, Whitty A, Runkel L, Hochman P. The structure of human interferon-β: implications for activity. Cell Mol Life Sci 1998; 54(11):1203-16.
33. Liu Z, Williams K, Chang Y-H, Smith J. Single amino acid substitution alters T cell determinant selection during antigen processing of Staphylococcus aureus nuclease. J Immunol 1991; 146(2):438-43.
34. Scheiblhofer S, Laimer J, Machado Y, Weiss R, Thalhamer J. Influence of protein fold stability on immunogenicity and its implications for vaccine design. Expert Rev Vaccines 2017; 16(5):479-89.
35. Lo-Man R, Langeveld JP, Martineau P, Hofnung M, Meloen RH, Leclerc C. Immunodominance does not result from peptide competition for MHC class II presentation. J Immunol 1998; 160(4):1759-66.
36. Finessi V, Nicoli F, Gallerani E, Sforza F, Sicurella M, Cafaro A, et al. Effects of different routes of administration on the immunogenicity of the Tat protein and a Tat-derived peptide. Hum Vaccin Immunother 2015; 11(6):1489-93.
37. Nielsen M, Lund O, Buus S, Lundegaard C. MHC class II epitope predictive algorithms. Immunology 2010; 130(3):319-28.
38. Hall FC, Rabinowitz JD, Busch R, Visconti KC, Belmares M, Patil NS, et al. Relationship between kinetic stability and immunogenicity of HLA‐DR4/peptide complexes. Eur J Immunol 2002; 32(3):662-70.
39. Braciale TJ, Morrison LA, Sweetser MT, Sambrook J, Gething MJ, Braciale VL. Antigen presentation pathways to class I and class II MHC‐restricted T lymphocytes. Immunol Rev 1987; 98(1):95-114.
2.Limmroth V, Putzki N, Kachuck NJ. The interferon beta therapies for treatment of relapsing–remitting multiple sclerosis: are they equally efficacious? A comparative review of open-label studies evaluating the efficacy, safety, or dosing of different interferon beta formulations alone or in combination. Ther Adv Neurol Disord 2011; 4(5):281-96.
3.Runkel L, Meier W, Pepinsky RB, Karpusas M, Whitty A, Kimball K, et al. Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-β (IFN-β). Pharm Res 1998; 15(4):641-9.
4.Kessler M, Goldsmith D, Schellekens H. Immunogenicity of biopharmaceuticals. Nephrology Dialysis Transplantation. 2006; 21(suppl_5):v9-v12.
5.Porter S. Human immune response to recombinant human proteins. J Pharm Sci 2001; 90(1):1-11.
6.Jawa V, Cousens LP, Awwad M, Wakshull E, Kropshofer H, De Groot AS. T-cell dependent immunogenicity of protein therapeutics: preclinical assessment and mitigation. Clin Immunol 2013; 149(3):534-55.
7.Antonelli G. Reflections on the immunogenicity of therapeutic proteins. Clin Microbiol Infect 2008; 14(8):731-3.
8.van Beers MM, Jiskoot W, Schellekens H. On the role of aggregates in the immunogenicity of recombinant human interferon beta in patients with multiple sclerosis. J Interferon Cytokine Res 2010; 30(10):767-75.
9.Bertolotto A, Deisenhammer F, Gallo P, Sørensen PS. Immunogenicity of interferon beta: differences among products. J Neurol 2004; 251(2):ii15-ii24.
10.Mahmoud K. Recombinant protein production: strategic technology and a vital research tool. Research Journal of Cell and Molecular Biology. 2007; 1(1):9-22.
11.Basu A, Yang K, Wang M, Liu S, Chintala R, Palm T, et al. Structure− function engineering of interferon-β-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation. Bioconjug Chem 2006; 17(3):618-30.
12.Schneider-Fresenius C, Otto B, Waschutza G. Recombinant human beta interferon with enhanced solubility. Google Patents; 2003.
13.Bell LD, Smith JC, Boseley PG, Houghton M. Modified (1-28) beta interferons. Google Patents; 1988.
14.Johnson-Jackson D, Furuya K, Zaror I, Larson D. Recombinant interferon-beta with enhanced biological activity. Google Patents; 2009.
15.Kraynov V, Knudsen N, PUTNAM A-MAH, Krawitz D, Pinkstaff J, Myler H. Modified interferon beta polypeptides and their uses. Google Patents; 2012.
16.Song K, Yoon I-S, Kim NA, Kim D-H, Lee J, Lee HJ, et al. Glycoengineering of interferon-β 1a improves its biophysical and pharmacokinetic properties. PloS one 2014; 9(5):e96967.
17.Kay M, Hasan-Abad AM, Hojati Z, Korbekandi H. Targeted mutations in Val101 and Arg27 interferon beta protein increase its transcriptional and translational activities. Cytokine 2016; 78:1-6.
18.Pembroke JT. Bio-molecular modelling utilising RasMol and PDB resources: a tutorial with HEW lysozyme. Biochemistry and Molecular Biology Education 2000; 28(6):297-300.
19.Filpula DR, Basu A. Optimized interferon-beta gene. Google Patents; 2010.
20.Sockolosky JT, Szoka FC. Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expr Purif 2013; 87(2):129-35.
21.Kruger NJ. The Bradford method for protein quantitation. The protein protocols handbook: Springer 2002; 15-21.
22.Kueltzo L, Middaugh C. Ultraviolet absorption spectroscopy. Methods for structural analysis of protein pharmaceuticals 2005; 3:1-25.
23.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature1970; 227(5259):680.
24.Senthilraja P, Kathiresan K. In vitro cytotoxicity MTT assay in Vero, HepG2 and MCF-7 cell lines study of Marine Yeast. J Appl Pharm Sci 2015; 5(3):80-4.
25.Meager A. Biological assays for interferons. J Immunol Methods 2002;261(1-2):21-36.
26.van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays: the MTT assay. Methods Mol Biol 2011; 237-45.
27.Yoon SH, Kim SK, Kim JF. Secretory production of recombinant proteins in Escherichia coli. Recent Pat Biotechnol 2010; 4(1):23-9.
28. van Beers MM, Sauerborn M, Gilli F, Brinks V, Schellekens H, Jiskoot W. Aggregated recombinant human interferon beta induces antibodies but no memory in immune-tolerant transgenic mice. Pharm Res 2010; 27(9):1812-24.
29. Yeung VP, Chang J, Miller J, Barnett C, Stickler M, Harding FA. Elimination of an immunodominant CD4+ T cell epitope in human IFN-β does not result in an in vivo response directed at the subdominant epitope. J Immunol 2004; 172(11):6658-65.
30. Gneiss C, Reindl M, Berger T, Lutterotti A, Ehling R, Egg R, et al. Epitope specificity of neutralizing antibodies against IFN-β. J Interferon Cytokine Res 2004; 24(5):283-90.
31. Runkel L, deDios C, Karpusas M, Betzenhauser M, Muldowney C, Zafari M, et al. Systematic mutational mapping of sites on human interferon-β-1a that are important for receptor binding and functional activity. Biochemistry 2000; 39(10):2538-51.
32. Karpusas M, Whitty A, Runkel L, Hochman P. The structure of human interferon-β: implications for activity. Cell Mol Life Sci 1998; 54(11):1203-16.
33. Liu Z, Williams K, Chang Y-H, Smith J. Single amino acid substitution alters T cell determinant selection during antigen processing of Staphylococcus aureus nuclease. J Immunol 1991; 146(2):438-43.
34. Scheiblhofer S, Laimer J, Machado Y, Weiss R, Thalhamer J. Influence of protein fold stability on immunogenicity and its implications for vaccine design. Expert Rev Vaccines 2017; 16(5):479-89.
35. Lo-Man R, Langeveld JP, Martineau P, Hofnung M, Meloen RH, Leclerc C. Immunodominance does not result from peptide competition for MHC class II presentation. J Immunol 1998; 160(4):1759-66.
36. Finessi V, Nicoli F, Gallerani E, Sforza F, Sicurella M, Cafaro A, et al. Effects of different routes of administration on the immunogenicity of the Tat protein and a Tat-derived peptide. Hum Vaccin Immunother 2015; 11(6):1489-93.
37. Nielsen M, Lund O, Buus S, Lundegaard C. MHC class II epitope predictive algorithms. Immunology 2010; 130(3):319-28.
38. Hall FC, Rabinowitz JD, Busch R, Visconti KC, Belmares M, Patil NS, et al. Relationship between kinetic stability and immunogenicity of HLA‐DR4/peptide complexes. Eur J Immunol 2002; 32(3):662-70.
39. Braciale TJ, Morrison LA, Sweetser MT, Sambrook J, Gething MJ, Braciale VL. Antigen presentation pathways to class I and class II MHC‐restricted T lymphocytes. Immunol Rev 1987; 98(1):95-114.
| Files | ||
| Issue | Vol 18, No 4 (2019) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijaai.v18i4.1421 | |
| PMID | 31522451 | |
| Keywords | ||
| Anti-drug antibodies Computational protein design Deimmunization Interferon-beta-1b T-cell epitope Therapeutic protein | ||
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How to Cite
1.
Moradi Hasan-Abad A, Adabi E, Sadroddiny E, Khorramizadeh MR, Mazlomi MA, Mehravar S, Kardar GA. Functional Deimmunization of Interferon Beta-1b by Identifying and Silencing Human T Cells Epitopes. Iran J Allergy Asthma Immunol. 2019;18(4):427-440.
