Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha

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FARAJZADEH DAVOUD; Karimi Gharigh Sadigheh; Jalali Kondori Parisa; DASTMALCHI SIAVOUSH

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Journal Paper

Paper Information

Journal: Iranian Journal of Pharmaceutical Research Year:2019 | Volume:18 | Issue:1 Page(s): 308-319

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Title

Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha

Author(s)

FARAJZADEH DAVOUD | Karimi Gharigh Sadigheh | Jalali Kondori Parisa | DASTMALCHI SIAVOUSH | Issue Writer Certificate

Keywords

TNF-α

Single chain antibody

Affinity chromatography

Pull down

MTT assay

Abstract

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 GSThD2 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.

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APA: Copy

FARAJZADEH, DAVOUD, Karimi Gharigh, Sadigheh, Jalali Kondori, Parisa, & DASTMALCHI, SIAVOUSH. (2019). Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha. IRANIAN JOURNAL OF PHARMACEUTICAL RESEARCH (IJPR), 18(1), 308-319. SID. https://sid.ir/paper/289201/en

Vancouver: Copy

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. IRANIAN JOURNAL OF PHARMACEUTICAL RESEARCH (IJPR)[Internet]. 2019;18(1):308-319. Available from: https://sid.ir/paper/289201/en

IEEE: Copy

DAVOUD FARAJZADEH, Sadigheh Karimi Gharigh, Parisa Jalali Kondori, and SIAVOUSH DASTMALCHI, “Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha,” IRANIAN JOURNAL OF PHARMACEUTICAL RESEARCH (IJPR), vol. 18, no. 1, pp. 308–319, 2019, [Online]. Available: https://sid.ir/paper/289201/en

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