BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

A chemical conjugate between HER2-targeting antibody fragment and Pseudomonas exotoxin A fragment demonstrates cytotoxic effects on HER2-expressing breast cancer cells

  • Lee, Sunju (Department of Physiology, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Park, Sangsu (Department of Physiology, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Nguyen, Minh Tan (Department of Physiology, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Lee, Eunyoung (Department of Physiology, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Kim, Julee (Department of Physiology, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Baek, Sangki (Department of Physiology, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Kim, Chong Jai (Department of Pathology, Asan-Minnesota Institute for Innovating Transplantation, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Jang, Yeon Jin (Department of Physiology, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center) ;
  • Choe, Han (Department of Physiology, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center)
  • Received : 2018.11.05
  • Accepted : 2018.11.28
  • Published : 2019.08.31
Download PDF
⟨ Previous Next ⟩

Abstract

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins.

Keywords

References

  1. Mathew M and Verma RS (2009) Humanized immunotoxins: a new generation of immunotoxins for targeted cancer therapy. Cancer Sci 100, 1359-1365 https://doi.org/10.1111/j.1349-7006.2009.01192.x
  2. Yamaizumi M, Mekada E, Uchida T and Okada Y (1978) One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15, 245-250 https://doi.org/10.1016/0092-8674(78)90099-5
  3. Antignani A and Fitzgerald D (2013) Immunotoxins: the role of the toxin. Toxins 5, 1486-1502 https://doi.org/10.3390/toxins5081486
  4. Goldhirsch A, Wood WC, Coates AS et al (2011) Strategies for subtypes--dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol 22, 1736-1747 https://doi.org/10.1093/annonc/mdr304
  5. Perou CM, Sorlie T, Eisen MB et al (2000) Molecular portraits of human breast tumours. Nature 406, 747-752 https://doi.org/10.1038/35021093
  6. Gutierrez C and Schiff R (2011) HER2: biology, detection, and clinical implications. Arch Pathol Lab Med 135, 55-62 https://doi.org/10.5858/2010-0454-RAR.1
  7. Carlomagno C, Perrone F, Gallo C et al (1996) c-erb B2 overexpression decreases the benefit of adjuvant tamoxifen in early-stage breast cancer without axillary lymph node metastases. J Clin Oncol 14, 2702-2708 https://doi.org/10.1200/JCO.1996.14.10.2702
  8. Hudis CA (2007) Trastuzumab--mechanism of action and use in clinical practice. N Engl J Med 357, 39-51 https://doi.org/10.1056/NEJMra043186
  9. Vu T and Claret FX (2012) Trastuzumab: updated mechanisms of action and resistance in breast cancer. Front Oncol 2, 62 https://doi.org/10.3389/fonc.2012.00062
  10. Lambert JM and Berkenblit A (2018) Antibody-Drug Conjugates for Cancer Treatment. Ann Rev Med 69, 191-207 https://doi.org/10.1146/annurev-med-061516-121357
  11. Wedekind JE, Trame CB, Dorywalska M et al (2001) Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity. J Mol Biol 314, 823-837 https://doi.org/10.1006/jmbi.2001.5195
  12. Siegall CB, Chaudhary VK, FitzGerald DJ 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
  13. Kreitman RJ, Arons E, Stetler-Stevenson M, Fitzgerald DJ, Wilson WH and Pastan I (2011) Recombinant immunotoxins and other therapies for relapsed/refractory hairy cell leukemia. Leuk Lymphoma 52 Suppl 2, 82-86 https://doi.org/10.3109/10428194.2011.565843
  14. Weidle UH, Tiefenthaler G, Schiller C, Weiss EH, Georges G and Brinkmann U (2014) Prospects of bacterial and plant protein-based immunotoxins for treatment of cancer. Cancer Genomics Proteomics 11, 25-38
  15. Tazzari PL, Polito L, Bolognesi A et al (2001) Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model. J Immunol 167, 4222-4229 https://doi.org/10.4049/jimmunol.167.8.4222
  16. Weldon JE, Xiang L, Zhang J et al (2013) A recombinant immunotoxin against the tumor-associated antigen mesothelin reengineered for high activity, low off-target toxicity, and reduced antigenicity. Mol Cancer Ther 12, 48-57 https://doi.org/10.1158/1535-7163.MCT-12-0336
  17. Fuchs H, Weng A and Gilabert-Oriol R (2016) Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers. Toxins 8, 200 https://doi.org/10.3390/toxins8070200
  18. Hassan R, Bullock S, Premkumar A et al (2007) Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers. Clin Cancer Res 13, 5144-5149 https://doi.org/10.1158/1078-0432.CCR-07-0869
  19. Hassan R, Miller AC, Sharon E et al (2013) Major cancer regressions in mesothelioma after treatment with an anti-mesothelin immunotoxin and immune suppression. Sci Transl Med 5, 208ra147
  20. Kreitman RJ, Siegall CB, Chaudhary VK, FitzGerald DJ and Pastan I (1992) Properties of chimeric toxins with two recognition domains: interleukin 6 and transforming growth factor alpha at different locations in Pseudomonas exotoxin. Bioconjug Chem 3, 63-68 https://doi.org/10.1021/bc00013a010
  21. Hassan R, Alewine C and Pastan I (2016) New Life for Immunotoxin Cancer Therapy. Clin Cancer Res 22, 1055-1058 https://doi.org/10.1158/1078-0432.CCR-15-1623
  22. Mazor R, Eberle JA, Hu X et al (2014) Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes. Proc Natl Acad Sci U S A 111, 8571-8576 https://doi.org/10.1073/pnas.1405153111
  23. Liu W, Onda M, Lee B et al (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
  24. Hu X, Zhang M, Zhang C et al (2016) Removal of B-cell epitopes for decreasing immunogenicity in recombinant immunotoxin against B-cell malignancies. J BUON 21, 1374-1378
  25. Mazor R, Zhang J, Xiang L et al (2015) Recombinant Immunotoxin with T-cell Epitope Mutations That Greatly Reduce Immunogenicity for Treatment of Mesothelin- Expressing Tumors. Mol Cancer Ther 14, 2789-2796 https://doi.org/10.1158/1535-7163.MCT-15-0532
  26. Mazor R, Onda M, Park D et al (2016) Dual B- and T-cell de-immunization of recombinant immunotoxin targeting mesothelin with high cytotoxic activity. Oncotarget 7, 29916-29926 https://doi.org/10.18632/oncotarget.9171
  27. Onda M, Beers R, Xiang L et al (2011) Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes. Proc Natl Acad Sci U S A 108, 5742-5747 https://doi.org/10.1073/pnas.1102746108
  28. Thurber GM, Schmidt MM and Wittrup KD (2008) Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearance. Adv Drug Deliv Rev 60, 1421-1434 https://doi.org/10.1016/j.addr.2008.04.012
  29. Wang L and Ying T (2016) New Directions for Half-Life Extension of Protein Therapeutics: The Rise of Antibody Fc Domains and Fragments. Curr Pharm Biotechnol 17, 1348-1352 https://doi.org/10.2174/1389201017666160823144032
  30. Vaishya R, Khurana V, Patel S and Mitra AK (2015) Long-term delivery of protein therapeutics. Expert Opin Drug Deliv 12, 415-440 https://doi.org/10.1517/17425247.2015.961420