Weisong Qin

A novel anti-TNF scFv constructed with human antibody frameworks and antagonistic peptides

The introduction of TNF inhibitors has revolutionized the treatment of some chronic inflammatory diseases, e.g., rheumatoid arthritis and Crohn’s disease. However, immunogenicity is one of the important mechanisms behind treatment failure, and generally, switching to another TNF inhibitor will be the first choice for patients and doctors, which results in unmet need for novel anti-TNF agents. Small antibody molecules with less number of epitope may be valuable in less immunogenicity. In this study, with the help of computer-guided molecular design, single-chain variable fragment (scFv) TSA2 was designed using consensus frameworks of human antibody variable region as scaffold to display anti-TNF antagonistic peptides. TSA2 showed evidently improved bioactivity over TSA1 (anti-TNF scFv explored before) and almost similar activity as S-Remicade (the scFv form of Remicade, anti-TNF antibody approved by FDA), especially in inhibiting TNF-induced cytotoxicity and NF-κB activation. Human antibody consensus frameworks with less immunogenicity have been used in the designing of VH domain antibody, scFv TSA1 and TSA2. A serial of TNF-related works convinced us that the novel design strategy was feasible and could be used to design inhibitors targeting more other molecules than TNF-α. More importantly, these designed inhibitors derived from computer modeling may form a virtual antibody library whose size depends on the number of candidate antagonistic peptides. It will be molecular-targeted virtual antibody library because of the specific antagonistic peptides and the potential antibodies could be determined by virtual screening and then confirmed by biologic experiments.

Overexpression, Effective Renaturation, and Bioactivity of Novel Single‐Chain Antibodies Against TNF‐α

Abstract Neutralization of tumor necrosis factor‐α (TNF‐α) has become an effective therapeutic strategy for TNF‐related autoimmune diseases. Due to the limitations of the large molecular inhibitors in the therapy, development of novel TNF‐α inhibitors is very attractive and useful. In this study, based on the previously designed domain antibody, two novel human anti‐TNF single‐chain antibodies were constructed using modular consensus frameworks of human antibody as scaffold to display the antagonistic peptides. A variety of expression plasmids were used to determine the optimal expression system. The single‐chain antibodies were always overexpressed in E.coli BL21(DE3) host as inclusion bodies. Under the optimized refolding conditions, the inclusion bodies were renatured successfully and the refolded single‐chain antibodies could bind with TNF‐α and block TNF‐induced cytotoxicity on L929 cells. The bioactivity of the single‐chain antibodies was significantly increased over the domain antibody.

Changes of primary sequence and secondary structure proximal to the 5′ end of the stop codon substantially increases the expression of the variable region of an antibody in E. coli

AbstractThe sequence context at the 5′ end of the stop codon may influence the efficiency of termination and translation. To increase the expression of a designed variable region of an antibody (named as VH5) against tumor necrosis factor α(TNFα), two nucleotides (TC) at 25 and 26 nucleotides (nt) upstream of termination codon were substituted with AG, respectively. The free energy of 70 nt (arbitrarily defined from the 32 nt upstream of termination codon to 38 nt downstream) was changed from −13.5 kcal mol-1 to −17.3 kcal mol-1. The expression level was increased from 1 ± 0.3% to 10 ± 1.2% of total cellular protein. Although the precise mechanism of this phenomenon remains to be elucidated, this report provides an alternative means to increase the expression of a foreign gene in E. coli.