A chemical conjugate between HER2-targeting antibody fragment and Pseudomonas exotoxin A fragment demonstrates cytotoxic effects on HER2-expressing breast cancer cells
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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.
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.
참고문헌 (30)
- Mathew M, "Humanized immunotoxins : a new generation of immunotoxins for targeted cancer therapy", Cancer Sci, 100, pp.1359~1365 (2009)
- Yamaizumi M, "One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell", Cell, 15, pp.245~250 (1978)
- Antignani A, "Immunotoxins : the role of the toxin", Toxins, 5, pp.1486~1502 (2013)
- Goldhirsch A, "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, pp.1736~1747 (2011)
- Perou CM, "Molecular portraits of human breast tumours", Nature, 406, pp.747~752 (2000)
- Gutierrez C, "HER2 : biology, detection, and clinical implications", Arch Pathol Lab Med, 135, pp.55~62 (2011)
- Carlomagno C, "c-erb B2overexpression decreases the benefit of adjuvant tamoxifen in early-stage breast cancer without axillary lymph node metastases", J Clin Oncol, 14, pp.2702~2708 (1996)
- Hudis CA, "Trastuzumab--mechanism of action and use in clinical practice", N Engl J Med, 357, pp.39~51 (2007)
- Vu T, "Trastuzumab : updated mechanisms of action and resistance in breast cancer", Front Oncol, 2, pp.62~ (2012)
- Lambert JM, "Antibody-Drug Conjugates for Cancer Treatment", Ann Rev Med, 69, pp.191~207 (2018)
- Wedekind JE, "Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity", J Mol Biol, 314, pp.823~837 (2001)
- Siegall CB, "Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin", J Biol Chem, 264, pp.14256~14261 (1989)
- Kreitman RJ, "Recombinant immunotoxins and other therapies for relapsed", refractory hairy cell leukemia, 2011, pp.52(Suppl 2), 82~86
- Weidle UH, "Prospects of bacterial and plant protein-based immunotoxins for treatment of cancer", Cancer Genomics Proteomics, 11, pp.25~38 (2014)
- Tazzari PL, "Immunotoxins containing recombinant anti-CTLA-4single-chain fragment variable antibodies and saporin : in vitro results and in vivo effects in an acute rejection model", J Immunol, 167, pp.4222~4229 (2001)
- Weldon JE, "A recombinant immunotoxin against the tumor-associated antigen mesothelin reengineered for high activity, low off-target toxicity, and reduced antigenicity", Mol Cancer Ther, 12, pp.48~57 (2013)
- Fuchs H, "Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers", Toxins, 8, pp.200~ (2016)
- Hassan R, "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, pp.5144~5149 (2007)
- Hassan R, "Major cancer regressions in mesothelioma after treatment with an anti-mesothelin immunotoxin and immune suppression", Sci Transl Med, 5 (2013)[DOI]
- Kreitman RJ, "Properties of chimeric toxins with two recognition domains : interleukin 6 and transforming growth factor alpha at different locations in Pseudomonas exotoxin", Bioconjug Chem, 3, pp.63~68 (1992)
- Hassan R, "New Life for Immunotoxin Cancer Therapy", Clin Cancer Res, 22, pp.1055~1058 (2016)
- Mazor R, "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, pp.8571~8576 (2014)
- Liu W, "Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes", Proc Natl Acad Sci U S A, 109, pp.11782~11787 (2012)
- Hu X, "Removal of B-cell epitopes for decreasing immunogenicity in recombinant immunotoxin against B-cell malignancies", J BUON, 21, pp.1374~1378 (2016)
- Mazor R, "Recombinant Immunotoxin with T-cell Epitope Mutations That Greatly Reduce Immunogenicity for Treatment of Mesothelin-Expressing Tumors", Mol Cancer Ther, 14, pp.2789~2796 (2015)
- Mazor R, "Dual B-and T-cell de-immunization of recombinant immunotoxin targeting mesothelin with high cytotoxic activity", Oncotarget, 7, pp.29916~29926 (2016)
- Onda M, "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, pp.5742~5747 (2011)
- Thurber GM, "Antibody tumor penetration : transport opposed by systemic and antigen-mediated clearance", Adv Drug Deliv Rev, 60, pp.1421~1434 (2008)
- Wang L, "New Directions for Half-Life Extension of Protein Therapeutics : The Rise of Antibody Fc Domains and Fragments", Curr Pharm Biotechnol, 17, pp.1348~1352 (2016)
- Vaishya R, "Long-term delivery of protein therapeutics", Expert Opin Drug Deliv, 12, pp.415~440 (2015)