Iranian Journal of Medical Sciences

Document Type : Original Article(s)

Authors

Department of Medical Biotechnology, School of Medicine, Fasa University of Medical Sciences, Fasa, Iran

10.30476/ijms.2021.90916.2199

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the prevalent cancers in the world with a high recurrence rate. In recent years, different researches have focused on designing efficient multi-epitope peptide vaccines against HCC. In designing these vaccines, over-expressed antigens in HCC patients, such as α- fetoprotein (AFP) and Glypican-3 (GPC-3), have been considered. In our previous study, a multi-epitope peptide vaccine for HCC was designed by in silico methods. The designed vaccine construct included the AFP, GPC-3, and Aspartyl-β-hydroxylase (ASPH) as CytoLoxic T cell Lymphocytes (CTL), one epitope from Tetanus Toxin Fragment C (TTFrC) as Helper T cell Lymphocytes (HTL), and a segment of microbial heat shock protein (HSP70) peptide 407-426 as an adjuvant. All the mentioned parts were connected by appropriate linkers. The aim of this study is the production of the designed vaccine.
Methods: This research is experimental and was carried out in Fasa, Iran, in 2017. The designed vaccine construct was transformed to the E.coli BL21 (DE3) strain and expressed in different Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations (0.6 and 1 mM), times (4, 6, 8, 16 hours), and temperatures (25 and 37 °C). Then, the expressed protein was analyzed by Sodium Dodecyl Sulfate-polyacrylamide (SDS) gel and the Western blot method.
Results: The best conditions for protein expression were obtained in the Super Optimal Broth (SOB) medium at 37 °C after the induction of expression by 1 mM IPTG for six hour.
Conclusion: The recombinant HCC vaccine was produced with a proper concentration.

Keywords

  1. Shimada S, Mogushi K, Akiyama Y, Furuyama T, Watanabe S, Ogura T, et al. Comprehensive molecular and immunological characterization of hepatocellular carcinoma. EBioMedicine. 2019;40:457-70. doi: 10.1016/j.ebiom.2018.12.058. PubMed PMID: 30598371; PubMed Central PMCID: PMCPMC6412165.
  2. Tabrizian P, Jibara G, Shrager B, Schwartz M, Roayaie S. Recurrence of hepatocellular cancer after resection: patterns, treatments, and prognosis. Ann Surg. 2015;261:947-55. doi: 10.1097/SLA.0000000000000710. PubMed PMID: 25010665.
  3. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008;359:378-90. doi: 10.1056/NEJMoa0708857. PubMed PMID: 18650514.
  4. Johnston MP, Khakoo SI. Immunotherapy for hepatocellular carcinoma: Current and future. World J Gastroenterol. 2019;25:2977-89. doi: 10.3748/wjg.v25.i24.2977. PubMed PMID: 31293335; PubMed Central PMCID: PMCPMC6603808.
  5. Ghahremanifard P, Afzali F, Rostami A, Nayeri Z, Bambai B, Minuchehr Z. Designing a novel multi-epitope T vaccine for “targeting protein for Xklp-2”(TPX2) in hepatocellular carcinoma based on immunoinformatics approach. International Journal of Peptide Research and Therapeutics. 2020;26:1127-36. doi: 10.1007/s10989-019-09915-2.
  6. Nakagawa H, Mizukoshi E, Kobayashi E, Tamai T, Hamana H, Ozawa T, et al. Association Between High-Avidity T-Cell Receptors, Induced by alpha-Fetoprotein-Derived Peptides, and Anti-Tumor Effects in Patients With Hepatocellular Carcinoma. Gastroenterology. 2017;152:1395-406. doi: 10.1053/j.gastro.2017.02.001. PubMed PMID: 28188748.
  7. Tomimaru Y, Mishra S, Safran H, Charpentier KP, Martin W, De Groot AS, et al. Aspartate-beta-hydroxylase induces epitope-specific T cell responses in hepatocellular carcinoma. Vaccine. 2015;33:1256-66. doi: 10.1016/j.vaccine.2015.01.037. PubMed PMID: 25629522; PubMed Central PMCID: PMCPMC4331251.
  8. Butterfield LH, Economou JS, Gamblin TC, Geller DA. Alpha fetoprotein DNA prime and adenovirus boost immunization of two hepatocellular cancer patients. J Transl Med. 2014;12:86. doi: 10.1186/1479-5876-12-86. PubMed PMID: 24708667; PubMed Central PMCID: PMCPMC4021640.
  9. Yamauchi N, Watanabe A, Hishinuma M, Ohashi K, Midorikawa Y, Morishita Y, et al. The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol. 2005;18:1591-8. doi: 10.1038/modpathol.3800436. PubMed PMID: 15920546.
  10. Tsuchiya N, Hosono A, Yoshikawa T, Shoda K, Nosaka K, Shimomura M, et al. Phase I study of glypican-3-derived peptide vaccine therapy for patients with refractory pediatric solid tumors. Oncoimmunology. 2017;7:e1377872. doi: 10.1080/2162402X.2017.1377872. PubMed PMID: 29296538; PubMed Central PMCID: PMCPMC5739579.
  11. Jia S, VanDusen WJ, Diehl RE, Kohl NE, Dixon RA, Elliston KO, et al. cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase. J Biol Chem. 1992;267:14322-7. PubMed PMID: 1378441.
  12. Aihara A, Huang CK, Olsen MJ, Lin Q, Chung W, Tang Q, et al. A cell-surface beta-hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma. Hepatology. 2014;60:1302-13. doi: 10.1002/hep.27275. PubMed PMID: 24954865; PubMed Central PMCID: PMCPMC4176525.
  13. McKee AS, Munks MW, Marrack P. How do adjuvants work? Important considerations for new generation adjuvants. Immunity. 2007;27:687-90. doi: 10.1016/j.immuni.2007.11.003. PubMed PMID: 18031690.
  14. Baek KH, Zhang H, Lee BR, Kwon YG, Ha SJ, Shin I. A small molecule inhibitor for ATPase activity of Hsp70 and Hsc70 enhances the immune response to protein antigens. Sci Rep. 2015;5:17642. doi: 10.1038/srep17642. PubMed PMID: 26631605; PubMed Central PMCID: PMCPMC4668564.
  15. Kelly M, McNeel D, Fisch P, Malkovsky M. Immunological considerations underlying heat shock protein-mediated cancer vaccine strategies. Immunol Lett. 2018;193:1-10. doi: 10.1016/j.imlet.2017.11.001. PubMed PMID: 29129721.
  16. Srivastava P. Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu Rev Immunol. 2002;20:395-425. doi: 10.1146/annurev.immunol.20.100301.064801. PubMed PMID: 11861608.
  17. Walker KB, Keeble J, Colaco C. Mycobacterial heat shock proteins as vaccines - a model of facilitated antigen presentation. Curr Mol Med. 2007;7:339-50. doi: 10.2174/156652407780831575. PubMed PMID: 17584074.
  18. Xu M, Zhou L, Zhang Y, Xie Z, Zhang J, Guo L, et al. A Fixed Human Umbilical Vein Endothelial Cell Vaccine With 2 Tandem Repeats of Microbial HSP70 Peptide Epitope 407-426 As Adjuvant for Therapy of Hepatoma in Mice. J Immunother. 2015;38:276-84. doi: 10.1097/CJI.0000000000000091. PubMed PMID: 26261891.
  19. Li J, Xing Y, Zhou Z, Yao W, Cao R, Li T, et al. Microbial HSP70 peptide epitope 407-426 as adjuvant in tumor-derived autophagosome vaccine therapy of mouse lung cancer. Tumour Biol. 2016;37:15097-105. doi: 10.1007/s13277-016-5309-2. PubMed PMID: 27662838.
  20. Dehbarez FM, Nezafat N, Mahmoodi S. In Silico Design of a Novel Multi-Epitope Peptide Vaccine Against Hepatocellular Carcinoma. Letters in Drug Design and Discovery. 2020;17:1164-76. doi: 10.2174/1570180817999200502030038.
  21. Chang AY, Chau V, Landas JA, Pang Y. Preparation of calcium competent Escherichia coli and heat-shock transformation. JEMI methods. 2017;1:22-5.
  22. Gravel P, Golaz O. Protein blotting by the semidry method. The protein protocols handbook. New York: Springer; 1996. p. 621-9.
  23. Kim RD, Sarker D, Meyer T, Yau T, Macarulla T, Park JW, et al. First-in-Human Phase I Study of Fisogatinib (BLU-554) Validates Aberrant FGF19 Signaling as a Driver Event in Hepatocellular Carcinoma. Cancer Discov. 2019;9:1696-707. doi: 10.1158/2159-8290.CD-19-0555. PubMed PMID: 31575541.
  24. Villanueva A, Hernandez-Gea V, Llovet JM. Medical therapies for hepatocellular carcinoma: a critical view of the evidence. Nat Rev Gastroenterol Hepatol. 2013;10:34-42. doi: 10.1038/nrgastro.2012.199. PubMed PMID: 23147664.
  25. Gnoni A, Santini D, Scartozzi M, Russo A, Licchetta A, Palmieri V, et al. Hepatocellular carcinoma treatment over sorafenib: epigenetics, microRNAs and microenvironment. Is there a light at the end of the tunnel? Expert Opin Ther Targets. 2015;19:1623-35. doi: 10.1517/14728222.2015.1071354. PubMed PMID: 26212068.
  26. Herzer K, Hofmann TG, Teufel A, Schimanski CC, Moehler M, Kanzler S, et al. IFN-alpha-induced apoptosis in hepatocellular carcinoma involves promyelocytic leukemia protein and TRAIL independently of p53. Cancer Res. 2009;69:855-62. doi: 10.1158/0008-5472.CAN-08-2831. PubMed PMID: 19141642.
  27. Palmer DH, Midgley RS, Mirza N, Torr EE, Ahmed F, Steele JC, et al. A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma. Hepatology. 2009;49:124-32. doi: 10.1002/hep.22626. PubMed PMID: 18980227.
  28. Liu YK, Yang KZ, Wu YD, Gang YQ, Zhu DN. Treatment of advanced primary hepatocellular carcinoma by 131-I-anti-AFP. Lancet. 1983;1:531-2. doi: 10.1016/s0140-6736(83)92220-1. PubMed PMID: 6186874.
  29. Sawada Y, Yoshikawa T, Ofuji K, Yoshimura M, Tsuchiya N, Takahashi M, et al. Phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for hepatocellular carcinoma patients. Oncoimmunology. 2016;5:e1129483. doi: 10.1080/2162402X.2015.1129483. PubMed PMID: 27467945; PubMed Central PMCID: PMCPMC4910752.
  30. Negahdaripour M, Nezafat N, Eslami M, Ghoshoon MB, Shoolian E, Najafipour S, et al. Structural vaccinology considerations for in silico designing of a multi-epitope vaccine. Infect Genet Evol. 2018;58:96-109. doi: 10.1016/j.meegid.2017.12.008. PubMed PMID: 29253673.
  31. Zhang L. Multi-epitope vaccines: a promising strategy against tumors and viral infections. Cell Mol Immunol. 2018;15:182-4. doi: 10.1038/cmi.2017.92. PubMed PMID: 28890542; PubMed Central PMCID: PMCPMC5811687.
  32. Gilboa E. The promise of cancer vaccines. Nat Rev Cancer. 2004;4:401-11. doi: 10.1038/nrc1359. PubMed PMID: 15122211.
  33. Greten TF, Manns MP, Korangy F. Immunotherapy of HCC. Rev Recent Clin Trials. 2008;3:31-9. doi: 10.2174/157488708783330549. PubMed PMID: 18474013.
  34. Pardee AD, Yano H, Weinstein AM, Ponce AA, Ethridge AD, Normolle DP, et al. Route of antigen delivery impacts the immunostimulatory activity of dendritic cell-based vaccines for hepatocellular carcinoma. J Immunother Cancer. 2015;3:32. doi: 10.1186/s40425-015-0077-x. PubMed PMID: 26199728; PubMed Central PMCID: PMCPMC4509479.
  35. Zhou Y, Li C, Shi G, Xu X, Luo X, Zhang Y, et al. Dendritic cell-based vaccine targeting aspartate-beta-hydroxylas represents a promising therapeutic strategy for HCC. Immunotherapy. 2019;11:1399-407. doi: 10.2217/imt-2019-0081. PubMed PMID: 31608722.
  36. Shokouhi H, Farahmand B, Ghaemi A, Mazaheri V, Fotouhi F. Vaccination with three tandem repeats of M2 extracellular domain fused to Leismania major HSP70 protects mice against influenza A virus challenge. Virus Res. 2018;251:40-6. doi: 10.1016/j.virusres.2018.05.003. PubMed PMID: 29730305.