Identification of novel single chain antibodies against TNF-α using Phage Display technology

Abstract

Introduction: Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Aims: The aims of this work were to identify anti-TNF-α scFv antibodies and peptides using phage display technology. Methods: Antibody and peptide libraries were used to isolate specific anti-TNF-α binders. Alignment independent 3D-QSAR study was used to generate a predictive model for a series of TACE inhibitors. Results: First, TNF-α was expressed and affinity purified to 95%purity, indicating that the developed method enables successful production of highly purified and fully functional TNF-α. The produced TNF-α was used to identify of anti-TNF-α scFv antibodies using phage display technology. J44 scFv antibody with the highest binding affinity towards TNF-α, was expressed as 6×His-tag fusion protein and purified using affinity column. In ELISA experiment, J44 antibody showed affinity towards TNF-α with Kd value of 0.78 ± 0.16 µM. Secondly, two novel 7-mer TNF-α blocking peptides, P51 and P52, were identified using peptide displaying phage library and showed inhibitory effects (IC50 values of 25.15 ± 2.18 and 7.08 ± 2.24 µM, respectively) on TNF-α induced cytotoxicity on L929 cells. The inhibitory effects of the peptides were attributed to their binding ability at the inter-subunit interfaces leading to subunit dissociation of TNF-α trimer. Molecular docking revealed that the peptides-TNF-α complexes are mostly stabilized through hydrophobic contacts. The identified peptides have the potential to be used as the lead compounds for designing anti-TNF-α peptidomimetics. Finally, a 3D-QSAR analysis was conducted on a set of TACEinhibitors. The analysis led to generate a model with appropriate statistics (q2=0.68, r2 prediction = 0.70) revealing the importance of two main moieties in the potency of the studied compounds: quinoline moietyand hydroxamate moietyinvolved in hydrogen bond acceptor or donor interactions. The result of the current study can be used in designing potent TACE inhibitors where the inhibition of TACE enzyme is required. Conclusions: In the current work, we have used various strategies to inhibit TNF-α activity, and conducted in silico analyses to get insight into the mechanism of action of identified inhibitors and developing models applicable in drug design.