Production of recombinant human single chain anti-4-1BB molecule antibody in Escherichia coli

Monoclonal antibodies against 4-1BB have been used in the treatment of autoimmune diseases, viral infections, as well as immunotherapy of cancers. Because of their foreign nature and the large size, the murine monoclonal antibodies have restricted efficacy in clinical use. Fully human antibody fragments such as scFv fragments can be an attractive alternative for immunotherapeutic applications. The aim of this study was the production of human scFv antibodies against 4-1BB in order to use it in cancer immunotherapy.

In this study, anti-4-1BB scFv gene was cloned in pET-22(+)and expression in E. coliTOP 10F.The initial analysis of the gene product was performed by western blot and ELISA. Furthermore, its efficacy in stimulating 4-1BB expressingcells was evaluated by assessing the cell surface expression of CD69 and IL-2 mRNA and protein expression.

The results showed that the designed scFvgene was well expressed in E. coliand specifically reacts to the target antigen. The CCRF-CEM cell’ treatment with anti-4-1BB scFv induced IL-2 production and increased CD69 expression on the surface of target cells.

The anti-4-1BB single-chainantibody was successfully expressed in E. coliexpression system and is able to detect target antigen and activate CCRF-CEM cells. This recombinant antibody could be useful for T cell activation in cancer cell therapy approaches.