Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics

  • Minh Quan Nguyen (Department of Physiology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Do Hyung Kim (Elpis-Biotech) ;
  • Hye Ji Shim (Elpis-Biotech) ;
  • Huynh Kim Khanh Ta (Department of Physiology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Thi Luong Vu (Department of Physiology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Thi Kieu Oanh Nguyen (Department of Physiology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Jung Chae Lim (Fatiabgen Co., Ltd.) ;
  • Han Choe (Department of Physiology, University of Ulsan College of Medicine, Asan Medical Center)
  • Received : 2023.09.21
  • Accepted : 2023.10.17
  • Published : 2023.12.31
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Abstract

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Keywords

Acknowledgement

This study was supported by a grant (KNRF-2019067449) from the National Research Foundation of South Korea and by the South Korean Fund for Regenerative Medicine (KFRM) grant (RS-2023-00215312) funded by the South Korean government (the Ministry of Science and ICT, the Ministry of Health & Welfare).

References

  1. Alewine, C., Hassan, R., and Pastan, I. (2015). Advances in anticancer immunotoxin therapy. Oncologist 20, 176-185. https://doi.org/10.1634/theoncologist.2014-0358
  2. Alewine, C., Xiang, L., Yamori, T., Niederfellner, G., Bosslet, K., and Pastan, I. (2014). Efficacy of RG7787, a next-generation mesothelin-targeted immunotoxin, against triple-negative breast and gastric cancers. Mol. Cancer Ther. 13, 2653-2661. https://doi.org/10.1158/1535-7163.MCT-14-0132
  3. Allahyari, H., Heidari, S., Ghamgosha, M., Saffarian, P., and Amani, J. (2017). Immunotoxin: a new tool for cancer therapy. Tumour Biol. 39, 1010428317692226.
  4. Allured, V.S., Collier, R.J., Carroll, S.F., and McKay, D.B. (1986). Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc. Natl. Acad. Sci. U. S. A. 83, 1320-1324. https://doi.org/10.1073/pnas.83.5.1320
  5. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.M., Lee, T., Pope, S.H., Riordan, G.S., and Whitlow, M. (1988). Single-chain antigen-binding proteins. Science 242, 423-426. https://doi.org/10.1126/science.3140379
  6. Cao, L., Li, Q., Tong, Z., Xing, Y., Xu, K., Wang, J.Y., Li, W., Zhao, J., Zhao, L., and Hong, Z. (2020). HER2-specific immunotoxins constructed based on single-domain antibodies and the improved toxin PE24X7. Int. J. Pharm. 574, 118939.
  7. Cerise, A., Bera, T.K., Liu, X., Wei, J., and Pastan, I. (2019). Anti-mesothelin recombinant immunotoxin therapy for colorectal cancer. Clin. Colorectal Cancer 18, 192-199.e1. https://doi.org/10.1016/j.clcc.2019.06.006
  8. Chang, K. and Pastan, I. (1996). Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc. Natl. Acad. Sci. U. S. A. 93, 136-140. https://doi.org/10.1073/pnas.93.1.136
  9. Chang, K., Pastan, I., and Willingham, M.C. (1992). Isolation and characterization of a monoclonal antibody, K1, reactive with ovarian cancers and normal mesothelium. Int. J. Cancer 50, 373-381. https://doi.org/10.1002/ijc.2910500308
  10. Chaudhary, V.K., Jinno, Y., FitzGerald, D., and Pastan, I. (1990). Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc. Natl. Acad. Sci. U. S. A. 87, 308-312. https://doi.org/10.1073/pnas.87.1.308
  11. Dieffenbach, M. and Pastan, I. (2020). Mechanisms of resistance to immunotoxins containing pseudomonas exotoxin A in cancer therapy. Biomolecules 10, 979.
  12. Do, B.H., Ryu, H.B., Hoang, P., Koo, B.K., and Choe, H. (2014). Soluble prokaryotic overexpression and purification of bioactive human granulocyte colony-stimulating factor by maltose binding protein and protein disulfide isomerase. PLoS One 9, e89906.
  13. Ecker, M., Redpath, G.M.I., Nicovich, P.R., and Rossy, J. (2021). Quantitative visualization of endocytic trafficking through photoactivation of fluorescent proteins. Mol. Biol. Cell 32, 892-902. https://doi.org/10.1091/mbc.E20-10-0669
  14. Ferrer, M., Chernikova, T.N., Timmis, K.N., and Golyshin, P.N. (2004). Expression of a temperature-sensitive esterase in a novel chaperonebased Escherichia coli strain. Appl. Environ. Microbiol. 70, 4499-4504. https://doi.org/10.1128/AEM.70.8.4499-4504.2004
  15. Han, S.H., Joo, M., Kim, H., and Chang, S. (2017). Mesothelin expression in gastric adenocarcinoma and its relation to clinical outcomes. J Pathol. Transl. Med. 51, 122-128. https://doi.org/10.4132/jptm.2016.11.18
  16. Hansen, J.K., Weldon, J.E., Xiang, L., Beers, R., Onda, M., and Pastan, I. (2010). A recombinant immunotoxin targeting CD22 with low immunogenicity, low nonspecific toxicity, and high antitumor activity in mice. J Immunother. 33, 297-304. https://doi.org/10.1097/CJI.0b013e3181cd1164
  17. Hassan, R. and Ho, M. (2008). Mesothelin targeted cancer immunotherapy. Eur. J. Cancer 44, 46-53. https://doi.org/10.1016/j.ejca.2007.08.028
  18. Ho, M., Bera, T.K., Willingham, M.C., Onda, M., Hassan, R., FitzGerald, D., and Pastan, I. (2007). Mesothelin expression in human lung cancer. Clin. Cancer Res. 13, 1571-1575. https://doi.org/10.1158/1078-0432.CCR-06-2161
  19. Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., and Crea, R. (1988). Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 85, 5879-5883. https://doi.org/10.1073/pnas.85.16.5879
  20. Hyun, Y., Baek, Y., Lee, C., Ki, N., Ahn, J., Ryu, S., and Ha, N.C. (2021). Structure and function of the autolysin SagA in the type IV secretion system of Brucella abortus. Mol. Cells 44, 517-528. https://doi.org/10.14348/molcells.2021.0011
  21. Jiang, Q., Ghafoor, A., Mian, I., Rathkey, D., Thomas, A., Alewine, C., Sengupta, M., Ahlman, M.A., Zhang, J., Morrow, B., et al. (2020). Enhanced efficacy of mesothelin-targeted immunotoxin LMB-100 and anti-PD-1 antibody in patients with mesothelioma and mouse tumor models. Sci. Transl. Med. 12, eaaz7252.
  22. Keyaerts, M., Xavier, C., Heemskerk, J., Devoogdt, N., Everaert, H., Ackaert, C., Vanhoeij, M., Duhoux, F.P., Gevaert, T., Simon, P., et al. (2016). Phase I study of 68Ga-HER2-nanobody for PET/CT assessment of HER2 expression in breast carcinoma. J. Nucl. Med. 57, 27-33. https://doi.org/10.2967/jnumed.115.162024
  23. Khaleghi, S., Rahbarizadeh, F., Ahmadvand, D., and Hosseini, H.R.M. (2017). Anti-HER2 VHH targeted magnetoliposome for intelligent magnetic resonance imaging of breast cancer cells. Cell. Mol. Bioeng. 10, 263-272. https://doi.org/10.1007/s12195-017-0481-z
  24. Kim, S., Koh, S., Kang, W., and Yang, J.K. (2022). The crystal structure of L-leucine dehydrogenase from Pseudomonas aeruginosa. Mol. Cells 45, 495-501. https://doi.org/10.14348/molcells.2022.0012
  25. Kreitman, R.J., Hassan, R., Fitzgerald, D.J., and Pastan, I. (2009). Phase I trial of continuous infusion anti-mesothelin recombinant immunotoxin SS1P. Clin. Cancer 15, 5274-5279. https://doi.org/10.1158/1078-0432.CCR-09-0062
  26. Kreitman, R.J. and Pastan, I. (1995). Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor. Biochem. J. 307, 29-37. https://doi.org/10.1042/bj3070029
  27. Lee, S., Park, S., Nguyen, M.T., Lee, E., Kim, J., Baek, S., Kim, C.J., Jang, Y.J., and Choe, H. (2019). A chemical conjugate between HER2-targeting antibody fragment and Pseudomonas exotoxin A fragment demonstrates cytotoxic effects on HER2-expressing breast cancer cells. BMB Rep. 52, 496-501. https://doi.org/10.5483/BMBRep.2019.52.8.250
  28. Liu, W., Onda, M., Lee, B., Kreitman, R.J., Hassan, R., Xiang, L., and Pastan, I. (2012). Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 109, 11782-11787. https://doi.org/10.1073/pnas.1209292109
  29. Nguyen, M.T., Koo, B.K., Thi Vu, T.T., Song, J.A., Chong, S.H., Jeong, B., Ryu, H.B., Moh, S.H., and Choe, H. (2014). Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase. PLoS One 9, e89038.
  30. Park, S., Nguyen, M.Q., Ta, H.K.K., Nguyen, M.T., Lee, G., Kim, C.J., Jang, Y.J., and Choe, H. (2021). Soluble cytoplasmic expression and purification of immunotoxin HER2(scFv)-PE24B as a maltose binding protein fusion. Int. J. Mol. Sci. 22, 6483.
  31. Prantner, A.M., Yin, C., Kamat, K., Sharma, K., Lowenthal, A.C., Madrid, P.B., and Scholler, N. (2018). Molecular imaging of mesothelin-expressing ovarian cancer with a human and mouse cross-reactive nanobody. Mol. Pharm. 15, 1403-1411. https://doi.org/10.1021/acs.molpharmaceut.7b00789
  32. Roovers, R.C., Laeremans, T., Huang, L., De Taeye, S., Verkleij, A.J., Revets, H., de Haard, H.J., and van Bergen en Henegouwen, P.M. (2007). Efficient inhibition of EGFR signaling and of tumour growth by antagonistic antiEFGR Nanobodies. Cancer Immunol. Immunother. 56, 303-317. https://doi.org/10.1007/s00262-006-0180-4
  33. Roovers, R.C., Vosjan, M.J., Laeremans, T., el Khoulati, R., de Bruin, R.C., Ferguson, K.M., Verkleij, A.J., van Dongen, G.A., and van Bergen en Henegouwen, P.M. (2011). A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth. Int. J. Cancer 129, 2013-2024. https://doi.org/10.1002/ijc.26145
  34. Ruiz-Lopez, E. and Schuhmacher, A.J. (2021). Transportation of singledomain antibodies through the blood-brain barrier. Biomolecules 11, 1131.
  35. Rump, A., Morikawa, Y., Tanaka, M., Minami, S., Umesaki, N., Takeuchi, M., and Miyajima, A. (2004). Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion. J. Biol. Chem. 279, 9190-9198. https://doi.org/10.1074/jbc.M312372200
  36. Salema, V. and Fernandez, L.A. (2013). High yield purification of nanobodies from the periplasm of E. coli as fusions with the maltose binding protein. Protein Expr. Purif. 91, 42-48. https://doi.org/10.1016/j.pep.2013.07.001
  37. Sarker, A., Rathore, A.S., and Gupta, R.D. (2019). Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process. Microb. Cell Fact. 18, 5.
  38. Scholler, N., Fu, N., Yang, Y., Ye, Z., Goodman, G.E., Hellstrom, K.E., and Hellstrom, I. (1999). Soluble member(s) of the mesothelin/megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma. Proc. Natl. Acad. Sci. U. S. A. 96, 11531-11536. https://doi.org/10.1073/pnas.96.20.11531
  39. Shahbazi-Gahrouei, D. and Abdolahi, M. (2013). Detection of MUC1- expressing ovarian cancer by C595 monoclonal antibody-conjugated SPIONs using MR imaging. Scientific World Journal 2013, 609151.
  40. Shinoda, H., Shannon, M., and Nagai, T. (2018). Fluorescent proteins for investigating biological events in acidic environments. Int. J. Mol. Sci. 19, 1548.
  41. Shirano, Y. and Shibata, D. (1990). Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level. FEBS Lett. 271, 128-130. https://doi.org/10.1016/0014-5793(90)80388-Y
  42. Siegall, C.B., Chaudhary, V.K., FitzGerald, D.J., and Pastan, I. (1989). Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin. J. Biol. Chem. 264, 14256-14261. https://doi.org/10.1016/S0021-9258(18)71671-2
  43. Song, J.A., Koo, B.K., Chong, S.H., Kwak, J., Ryu, H.B., Nguyen, M.T., Vu, T.T., Jeong, B., Kim, S.W., and Choe, H. (2013). Expression and purification of biologically active human FGF2 containing the b'a' domains of human PDI in Escherichia coli. Appl. Biochem. Biotechnol. 170, 67-80. https://doi.org/10.1007/s12010-013-0140-3
  44. Vera, A., Gonzalez-Montalban, N., Aris, A., and Villaverde, A. (2007). The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol. Bioeng. 96, 1101-1106. https://doi.org/10.1002/bit.21218
  45. Weldon, J.E. and Pastan, I. (2011). A guide to taming a toxin--recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer. FEBS J. 278, 4683-4700. https://doi.org/10.1111/j.1742-4658.2011.08182.x
  46. Weldon, J.E., Xiang, L., Zhang, J., Beers, R., Walker, D.A., Onda, M., Hassan, R., and Pastan, I. (2013). A recombinant immunotoxin against the tumorassociated antigen mesothelin reengineered for high activity, low offtarget toxicity, and reduced antigenicity. Mol. Cancer Ther. 12, 48-57. https://doi.org/10.1158/1535-7163.MCT-12-0336
  47. Wolf, P. and Elsasser-Beile, U. (2009). Pseudomonas exotoxin A: from virulence factor to anti-cancer agent. Int. J. Med. Microbiol. 299, 161-176. https://doi.org/10.1016/j.ijmm.2008.08.003
  48. Wood, L.A., Larocque, G., Clarke, N.I., Sarkar, S., and Royle, S.J. (2017). New tools for "hot-wiring" clathrin-mediated endocytosis with temporal and spatial precision. J. Cell Biol. 216, 4351-4365. https://doi.org/10.1083/jcb.201702188
  49. Wouters, Y., Jaspers, T., De Strooper, B., and Dewilde, M. (2020). Identification and in vivo characterization of a brain-penetrating nanobody. Fluids Barriers CNS 17, 62.
  50. Xiang, Y., Sang, Z., Bitton, L., Xu, J., Liu, Y., Schneidman-Duhovny, D., and Shi, Y. (2021). Integrative proteomics identifies thousands of distinct, multi-epitope, and high-affinity nanobodies. Cell Syst. 12, 220-234.e9. https://doi.org/10.1016/j.cels.2021.01.003
  51. Yi, A.R., Lee, S.R., Jang, M.U., Park, J.M., Eom, H.J., Han, N.S., and Kim, T.J. (2009). Cloning of dextransucrase gene from Leuconostoc citreum HJP4 and its high-level expression in E. coli by low temperature induction. J. Microbiol. Biotechnol. 19, 829-835.
  52. Yu, L., Feng, M., Kim, H., Phung, Y., Kleiner, D.E., Gores, G.J., Qian, M., Wang, X.W., and Ho, M. (2010). Mesothelin as a potential therapeutic target in human cholangiocarcinoma. J. Cancer 1, 141-149. https://doi.org/10.7150/jca.1.141
  53. Zhang, J., Khanna, S., Jiang, Q., Alewine, C., Miettinen, M., Pastan, I., and Hassan, R. (2017). Efficacy of anti-mesothelin immunotoxin RG7787 plus nab-paclitaxel against mesothelioma patient-derived xenografts and mesothelin as a biomarker of tumor response. Clin. Cancer Res. 23, 1564-1574. https://doi.org/10.1158/1078-0432.CCR-16-1667