Characterization of the novel anti-TNF-α single-chain fragment antibodies using experimental and computational approaches

Abstract

Single-chain fragment variable (scFv) antibodies are antibody fragments consist of variable domains of full antibodies known to retain antigen binding properties while having much lower molecular weights granting some beneficial properties to them. In our previous study, three phage particles each displaying an individual scFv antibody (i.e. J43, J44, and J48) were identified as tumor necrosis factor alpha (TNF-α) binders. The current study aimed to produce previously identified anti-TNF-α scFv antibodies and to investigate their abilities to bind and inhibit TNF-α biological effect. The estimated free energy of folding determined using spectrofluorimetry method for the prepared scFv proteins was in the range of 6.35–12.45 kJ mol⁻¹ indicating their proper folding in the solution. In ELISA experiment, the produced scFvs showed an appropriate affinity towards TNF-α with K_d values in the range of 0.5–2.18 µM. They also inhibited the TNF-α-induced cytotoxicity on L929 cells with sub-micromolar IC₅₀ values (0.12 and 0.73 μM for J44 and J48, respectively). Molecular docking studies showed that J44 could mimic adalimumab interactions with TNF-α, confirming its relatively high TNF-α inhibitory effect compared to J43 and J48. It seems that the findings in the current study can be useful for designing more potent anti-TNF-α antibodies.

KEYWORDS:

• Cell cytotoxicity
• molecular docking
• phage display
• protein folding
• scFvs
• TNF-α

Acknowledgments

We would like to thank Doctor Haleh Hamedifar, CEO at CinnaGen Company, for a kind gift of adalimumab and the Research Office and Biotechnology Research Center of Tabriz University of Medical Sciences for providing financial support.

Additional information

Funding

This study was financially supported by Research Office and Biotechnology Research Center of Tabriz University of Medical Sciences.