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Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha
The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of diseases such as Crohn’s disease, psoriasis, psoriatic arthritis, and rheumatoid arthritis, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Shaheed Beheshti University of Medical Sciences
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487432/ https://www.ncbi.nlm.nih.gov/pubmed/31089365 |
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author | Farajzadeh, Davoud Karimi-Gharigh, Sadigheh Jalali-Kondori, Parisa Dastmalchi, Siavoush |
author_facet | Farajzadeh, Davoud Karimi-Gharigh, Sadigheh Jalali-Kondori, Parisa Dastmalchi, Siavoush |
author_sort | Farajzadeh, Davoud |
collection | PubMed |
description | The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of diseases such as Crohn’s disease, psoriasis, psoriatic arthritis, and rheumatoid arthritis, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to the corresponding receptor has mainly served as a therapeutic strategy against some inflammatory diseases. This study aimed to investigate the production of a humanized single chain antibody (scFv) against TNF-α. Therefore, a murine monoclonal antibody, D2 mAb, was selected for humanizing by the complementarity determining region (CDR)-grafting method. Briefly, the replacement of the CDRs from D2 mAb with the specific human single chain scaffold led to the production of a novel humanized single chain fragment variable mAb against human TNF-α (hD2). The subsequent cloning of hD2 into a suitable expression vector, pGEX-6P-1, resulted in the expression of a 52-kDa GST-fusion protein in E. coli, mostly in the form of inclusion bodies. The solubilization and refolding of GST-hD2 inclusion bodies was achieved with the addition of 4 M urea and subsequent dialysis to recover the fusion protein in soluble form. Then the soluble GST-hD2 was purified by affinity chromatography through immobilized glutathione. The GST pull-down experiment showed a positive interaction between GST-hD2 and TNF-α protein. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity (Kd of 1.03 nM) and hence hD2 has the potential to be developed into a therapeutic agent. However, more investigation is needed to elucidate the potential of in-vivo TNF-α neutralizing activity of hD2 in comparison to other anti-TNF-α antibodies. |
format | Online Article Text |
id | pubmed-6487432 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Shaheed Beheshti University of Medical Sciences |
record_format | MEDLINE/PubMed |
spelling | pubmed-64874322019-05-14 Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha Farajzadeh, Davoud Karimi-Gharigh, Sadigheh Jalali-Kondori, Parisa Dastmalchi, Siavoush Iran J Pharm Res Original Article The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of diseases such as Crohn’s disease, psoriasis, psoriatic arthritis, and rheumatoid arthritis, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to the corresponding receptor has mainly served as a therapeutic strategy against some inflammatory diseases. This study aimed to investigate the production of a humanized single chain antibody (scFv) against TNF-α. Therefore, a murine monoclonal antibody, D2 mAb, was selected for humanizing by the complementarity determining region (CDR)-grafting method. Briefly, the replacement of the CDRs from D2 mAb with the specific human single chain scaffold led to the production of a novel humanized single chain fragment variable mAb against human TNF-α (hD2). The subsequent cloning of hD2 into a suitable expression vector, pGEX-6P-1, resulted in the expression of a 52-kDa GST-fusion protein in E. coli, mostly in the form of inclusion bodies. The solubilization and refolding of GST-hD2 inclusion bodies was achieved with the addition of 4 M urea and subsequent dialysis to recover the fusion protein in soluble form. Then the soluble GST-hD2 was purified by affinity chromatography through immobilized glutathione. The GST pull-down experiment showed a positive interaction between GST-hD2 and TNF-α protein. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity (Kd of 1.03 nM) and hence hD2 has the potential to be developed into a therapeutic agent. However, more investigation is needed to elucidate the potential of in-vivo TNF-α neutralizing activity of hD2 in comparison to other anti-TNF-α antibodies. Shaheed Beheshti University of Medical Sciences 2019 /pmc/articles/PMC6487432/ /pubmed/31089365 Text en This is an Open Access article distributed under the terms of the Creative Commons Attribution License, (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Article Farajzadeh, Davoud Karimi-Gharigh, Sadigheh Jalali-Kondori, Parisa Dastmalchi, Siavoush Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha |
title | Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha |
title_full | Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha |
title_fullStr | Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha |
title_full_unstemmed | Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha |
title_short | Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha |
title_sort | design and construction of a novel humanized single-chain variable-fragment antibody against the tumor necrosis factor alpha |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487432/ https://www.ncbi.nlm.nih.gov/pubmed/31089365 |
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