Iranian Journal of Pathology

Targeted Therapy with a Novel Superantigen-based Fusion Protein Against Interleukin-13 Receptor α2-overexpressing Tumor Cells: An in-Silico Study

Document Type : Original Research

Authors

Zahra Gholipour 1
Abbas Ali Imani Fooladi 2
Kazem Parivar 1
Raheleh Halabian 2

1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

https://doi.org/10.30699/ijp.2024.2014231.3200
Abstract
Background & Objective: Superantigens are bacterial toxins that induce a massive immune response in the host. Superantigen staphylococcal enterotoxin B (SEB) can form a ternary complex with its receptors, MHC class II (MHCII) and TCR, and can be used in tumor-targeting therapy, particularly when cooperating with a specific vector. In this study, SEB was fused to interleukin-13 (IL13), which forms a complex with IL13 receptor α2 (IL13Rα2) overexpressed in glioblastoma multiforme (GBM) cells for therapeutic goals.
Methods We designed four fusion proteins based on the arrangement of SEB (N- or C-terminal domain) and provided a flexible inter-domain linker (no or yes), resulting in the formation of SEB-IL13, SEB-L-IL13, IL13-SEB, and IL13-L-SEB, respectively. These fusion proteins were then evaluated for their various physicochemical properties and structural characteristics. Bioinformatics tools were employed to predict, refine, and validate the three-dimensional structure of the fusion proteins. In addition, the fusion proteins were docked with IL13Rα2, MHCII, and TCR receptors through the HADDOCK 2.4 server. The candidate fusion protein was subjected to molecular dynamics simulation.
Results: There were differences among the designed fusion proteins. The model with the N-terminal domain of IL13 and containing an inter-domain linker (IL13-L-SEB) was stable and had a long half-life. The docking analysis revealed that the IL13-L-SEB fusion protein had a higher binding affinity to the IL13Rα2, MHCII, and TCR receptors. Finally, using molecular dynamics simulation through iMODS, acceptable results were obtained for the IL13-L-SEB docked complexes.
Conclusion: The results suggest IL13-L-SEB is a promising novel fusion protein for cancer therapeutic application.

Keywords

Fusion protein
Glioblastoma multiforme
Interleukin-13
Molecular docking
Staphylococcal enterotoxin B

Subjects

  1. Ostrom QT, Gittleman H, Truitt G, Boscia A, Kruchko C, Barnholtz-Sloan JS. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2011–2015. Neuro Oncol. 2018;20(suppl_4):iv1-86. [DOI:10.1093/neuonc/noy131] [PMID]
  2. Xu S, Tang L, Li X, Fan F, Liu Z. Immunotherapy for glioma: current management and future application. Cancer Lett. 2020 Apr 28;476:1-2. [DOI:10.1016/j.canlet.2020.02.002] [PMID]
  3. Debinski W, Gibo DM, Hulet SW, Connor JR, Gillespie GY. The receptor for interleukin 13 is a marker and therapeutic target for human high-grade gliomas. Clin Cancer Res. 1999;5(5):985-90.
  4. Mintz A, Gibo DM, Slagle-Webb B, Christensent ND, Debinski W. IL-13Rα2 is a glioma-restricted receptor for interleukin-13. Neoplasia. 2002;4(5):388-99. [DOI:10.1038/sj.neo.7900234] [PMID]
  5. Husain SR, Puri RK. Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside. Neuro Oncol. 2003;65:37-48. [DOI:10.1023/A:1026242432647] [PMID]
  6. Lupardus PJ, Birnbaum ME, Garcia KC. Molecular basis for shared cytokine recognition revealed in the structure of an unusually high affinity complex between IL-13 and IL-13Rα2. Structure. 2010;18(3):332-42. [DOI:10.1016/j.str.2010.01.003] [PMID]
  7. Jaén M, Martín-Regalado Á, Bartolomé RA, Robles J, Casal JI. Interleukin 13 receptor alpha 2 (IL13Rα2): Expression, signaling pathways and therapeutic applications in cancer. Biochim. Biophys Acta - Rev Cancer. 2022:188802. [DOI:10.1016/j.bbcan.2022.188802] [PMID]
  8. Knudson KM, Hwang S, McCann MS, Joshi BH, Husain SR, Puri RK. Recent advances in IL-13rα2-directed cancer immunotherapy. Front. Immunol. 2022;13:878365. [DOI:10.3389/fimmu.2022.878365] [PMID]
  9. Debinski W, Obiri NI, Pastan I, Puri RK. A novel chimeric protein composed of interleukin 13 and pseudomonas exotoxin is highly cytotoxic to human carcinoma cells expressing receptors for interleukin 13 and interleukin 4 (∗). J Biol Chem. 1995;270(28):16775-80. [DOI:10.1074/jbc.270.28.16775] [PMID]
  10. Rustamzadeh E, Vallera DA, Todhunter DA, Low WC, Panoskaltsis-Mortari A, Hall WA. Immunotoxin pharmacokinetics: a comparison of the anti-glioblastoma bi-specific fusion protein (DTAT13) to DTAT and DTIL13. Neuro Oncol. 2006;77:257-66. [DOI:10.1007/s11060-005-9051-7] [PMID]
  11. Lichan-Wang, Huajie-Zhang, Shumin-Zhang, Mosong-Yu, Xiaoming-Yang. Construction and characterization of a novel superantigen fusion protein: bFGF/SEB. Cancer Investig. 2009;27(4):376-83. [DOI:10.1080/07357900802487228] [PMID]
  12. White J, Herman A, Pullen AM, Kubo R, Kappler JW, Marrack P. The Vβ-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell. 1989;56(1):27-35. [DOI:10.1016/0092-8674(89)90980-X] [PMID]
  13. Truong K, Khorchid A, Ikura M. A fluorescent cassette-based strategy for engineering multiple domain fusion proteins. BMC Biotechnol. 2003;3(1):1-8. [DOI:10.1186/1472-6750-3-8] [PMID]
  14. Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013;65(10):1357-69. [DOI:10.1016/j.addr.2012.09.039] [PMID]
  15. Bhaskara RM, de Brevern AG, Srinivasan N. Understanding the role of domain–domain linkers in the spatial orientation of domains in multi-domain proteins. J Biomol. Struct. Dyn. 2013;31(12):1467-80. [DOI:10.1080/07391102.2012.743438] [PMID]
  16. Glockshuber R, Malia M, Pfitzinger I, Plueckthun A. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry. 1990;29(6):1362-7. [DOI:10.1021/bi00458a002] [PMID]
  17. Klein JS, Jiang S, Galimidi RP, Keeffe JR, Bjorkman PJ. Design and characterization of structured protein linkers with differing flexibilities. Protein Eng Des Sel. 2014;27(10):325-30. [DOI:10.1093/protein/gzu043] [PMID]
  18. Tong Q, Liu K, Lu XM, Shu XG, Wang GB. Construction and characterization of a novel fusion protein MG7-scFv/SEB against gastric cancer. Biomed Res Int. 2010;2010. [DOI:10.1155/2010/121094] [PMID]
  19. Boehr DD, Wright PE. How do proteins interact?. Science. 2008;320(5882):1429-30. [DOI:10.1126/science.1158818] [PMID]
  20. Gao W, Mahajan SP, Sulam J, Gray JJ. Deep learning in protein structural modeling and design. Patterns. 2020;1(9). [DOI:10.1016/j.patter.2020.100142] [PMID]
  21. Berman H, Henrick K, Nakamura H. Announcing the worldwide protein data bank. Nat Struct. Biol 2003;10(12):980-. [DOI:10.1038/nsb1203-980] [PMID]
  22. Daga PR, Patel RY, Doerksen RJ. Template-based protein modeling: recent methodological advances. Curr Med Top Chem. 2010;10(1):84-94. [DOI:10.2174/156802610790232314] [PMID]
  23. Wollacott AM, Zanghellini A, Murphy P, Baker D. Prediction of structures of multidomain proteins from structures of the individual domains. Protein Sci. 2007;16(2):165-75. [DOI:10.1110/ps.062270707] [PMID]
  24. Almsned F, Gogovi G, Bracci N, Kehn-Hall K, Blaisten-Barojas E, Shehu A. Modeling the tertiary structure of a multi-domain protein: structure prediction of multi-domain proteins. In Proceedings of the 2018 ACM International Conference on Bioinformatics, Computational Biology, and Health Informatics 2018 (pp. 615-620). [DOI:10.1145/3233547.3233702]
  25. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015;10(6):845-58. [DOI:10.1038/nprot.2015.053] [PMID] []
  26. Gasteiger E, Hoogland C, Gattiker A, Duvaud SE, Wilkins MR, Appel RD, Bairoch A. Protein identification and analysis tools on the ExPASy server. Humana press; 2005. [DOI:10.1385/1-59259-890-0:571]
  27. Kelley LA, Sternberg MJ. Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc. 2009;4(3):363-71. [DOI:10.1038/nprot.2009.2] [PMID]
  28. Jefferys BR, Kelley LA, Sternberg MJ. Protein folding requires crowd control in a simulated cell. J Mol Biol. 2010;397(5):1329-38. [DOI:10.1016/j.jmb.2010.01.074] [PMID]
  29. Ko J, Park H, Heo L, Seok C. GalaxyWEB server for protein structure prediction and refinement. Nucleic Acids Res. 2012;40(W1):W294-7. [DOI:10.1093/nar/gks493] [PMID]
  30. DeLano WL. The PyMOL molecular graphics system. http://www. pymol. org/. 2002.
  31. Meiler J, Baker D. Coupled prediction of protein secondary and tertiary structure. Proc Natl Acad Sci. 2003;100(21):12105-10. [DOI:10.1073/pnas.1831973100] [PMID]
  32. Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW, Arendall III WB, Snoeyink J, Richardson JS, Richardson DC. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 2007;35(suppl_2):W375-83. [DOI:10.1093/nar/gkm216] [PMID]
  33. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr. 1993;26(2):283-91. [DOI:10.1107/S0021889892009944]
  34. Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res. 2007;35(suppl_2):W407-10. [DOI:10.1093/nar/gkm290] [PMID]
  35. Van Zundert GC, Rodrigues JP, Trellet M, Schmitz C, Kastritis PL, Karaca E, Melquiond AS, van Dijk M, De Vries SJ, Bonvin AM. The HADDOCK2. 2 web server: user-friendly integrative modeling of biomolecular complexes. J Mol Biol. 2016;428(4):720-5. [DOI:10.1016/j.jmb.2015.09.014] [PMID]
  36. Honorato RV, Koukos PI, Jiménez-García B, Tsaregorodtsev A, Verlato M, Giachetti A, Rosato A, Bonvin AM. Structural biology in the clouds: the WeNMR-EOSC ecosystem. Front Mol Biosci. 2021;8:729513. [DOI:10.3389/fmolb.2021.729513] [PMID]
  37. Rödström KE, Elbing K, Lindkvist-Petersson K. Structure of the superantigen Staphylococcal Enterotoxin B in complex with TCR and peptide–MHC demonstrates absence of TCR–peptide contacts. J Immunol. 2014;193(4):1998-2004. [DOI:10.4049/jimmunol.1401268] [PMID]
  38. Laskowski RA, Swindells MB. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. [DOI:10.1021/ci200227u] [PMID]
  39. López-Blanco JR, Aliaga JI, Quintana-Ortí ES, Chacón P. iMODS: internal coordinates normal mode analysis server. Nucleic Acids Res. 2014;42(W1):W271-6. [DOI:10.1093/nar/gku339] [PMID]
  40. Lopéz-Blanco JR, Garzón JI, Chacón P. iMod: multipurpose normal mode analysis in internal coordinates. Bioinformatics. 2011;27(20):2843-50. [DOI:10.1093/bioinformatics/btr497] [PMID]
  41. Moy FJ, Diblasio E, Wilhelm J, Powers R. Solution structure of human IL-13 and implication for receptor binding. J Mol Biol. 2001;310(1):219-30. [DOI:10.1006/jmbi.2001.4764] [PMID]
  42. Bolin DR, Swain AL, Sarabu R, Berthel SJ, Gillespie P, Huby NJ, Makofske R, Orzechowski L, Perrotta A, Toth K, Cooper JP. Peptide and peptide mimetic inhibitors of antigen presentation by HLA-DR class II MHC molecules. Design, structure− activity relationships, and x-ray crystal structures. J Med Chem. 2000;43(11):2135-48. [DOI:10.1021/jm000034h] [PMID]
  43. Okamoto H, Yoshimatsu Y, Tomizawa T, Kunita A, Takayama R, Morikawa T, Komura D, Takahashi K, Oshima T, Sato M, Komai M. Interleukin-13 receptor α2 is a novel marker and potential therapeutic target for human melanoma. Sci Rep. 2019;9(1):1281. [DOI:10.1038/s41598-019-39018-3] [PMID]
  44. Gholipour Z, Fooladi AA, Parivar K, Halabian R. Targeting glioblastoma multiforme using a novel fusion protein comprising interleukin-13 and staphylococcal enterotoxin B in vitro. Toxicology in Vitro. 2023;92:105651. [DOI:10.1016/j.tiv.2023.105651] [PMID]
  45. Kalland T, Hedlund G, Dohlsten M, Lando PA. Staphylococcal enterotoxin-dependent cell-mediated cytotoxicity. Superantigens. 1991:81-92. [DOI:10.1007/978-3-642-50998-8_6] [PMID]
  46. Mondal TK, Bhatta D, Biswas S, Pal P. Superantigen-induced apoptotic death of tumor cells is mediated by cytotoxic lymphocytes, cytokines, and nitric oxide. Biochem Biophys Res Commun. 2002;290(4):1336-42. [DOI:10.1006/bbrc.2002.6359] [PMID]
  47. Kong Y, Tong Y, Gao M, Chen C, Gao X, Yao W. Linker engineering for fusion protein construction: improvement and characterization of a GLP-1 fusion protein. Enzyme Microb Technol 2016;82:105-9. [DOI:10.1016/j.enzmictec.2015.09.001] [PMID]
  48. Rahmani F, Imani Fooladi AA, Ajoudanifar H, Soleimani NA. In silico and experimental methods for designing a potent anticancer arazyme-herceptin fusion protein in HER2-positive breast cancer. J Mol Model. 2023;29(5):160. [DOI:10.1007/s00894-023-05562-z] [PMID]
  49. Imani-Fooladi AA, Yousefi F, Mousavi SF, Amani J. In silico design and analysis of TGFαl3-seb fusion protein as “a new antitumor agent” candidate by ligand-targeted superantigens technique. Iran J Cancer Prev. 2014;7(3):152.
  50. Mehrab R, Sedighian H, Sotoodehnejadnematalahi F, Halabian R, Fooladi AA. A comparative study of the arazyme-based fusion proteins with various ligands for more effective targeting cancer therapy: an in-silico analysis. Res Pharm Sci. 2023;18(2):159. [DOI:10.4103/1735-5362.367795] [PMID]
  51. Hu W, Xie Q, Xiang H. Improved scFv Anti-LOX-1 binding activity by fusion with LOX-1-binding peptides. Biomed Res Int. 2017;2017. [DOI:10.1155/2017/8946935] [PMID]
  52. Validi M, Karkhah A, Prajapati VK, Nouri HR. Immuno-informatics based approaches to design a novel multi epitope-based vaccine for immune response reinforcement against Leptospirosis. Mol Immunol. 2018;104:128-38. [DOI:10.1016/j.molimm.2018.11.005] [PMID]
  53. Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng. 2001;14(8):529-32. [DOI:10.1093/protein/14.8.529] [PMID]
  54. Guo H, Yang Y, Xue F, Zhang H, Huang T, Liu W, Liu H, Zhang F, Yang M, Liu C, Lu H. Effect of flexible linker length on the activity of fusion protein 4-coumaroyl-CoA ligase:: stilbene synthase. Mol Biosyst. 2017;13(3):598-606. [DOI:10.1039/C6MB00563B] [PMID]
  55. Milano T, Angelaccio S, Tramonti A, Di Salvo ML, Contestabile R, Pascarella S. Structural properties of the linkers connecting the N-and C-terminal domains in the MocR bacterial transcriptional regulators. Biochim Open. 2016;3:8-18. [DOI:10.1016/j.biopen.2016.07.002] [PMID]
Iranian Journal of Pathology
Volume 19, Issue 2
Spring 2024
Pages 193-204

  • Receive Date 26 October 2023
  • Revise Date 26 December 2023
  • Accept Date 26 December 2023

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