Production of a therapeutically active form of the single chain variable fragment derived from the anti-malaria monoclonal antibody 4G2

Abstract

Multi-drug resistance has been observed for Plasmodium falciparum in many malaria-endemic regions. The intention is to develop a last-resort antibody-based therapeutic for patients in which drugs fail. For this, the rat-derived monoclonal antibody (mAB) 4G2 was selected as the primary candidate, as it broadly inhibits growth and invasion of all strains of Plasmodium falciparum. As Fab fragments derived of mAB 4G2 have been shown to have a better inhibition profile compared to the intact antibody, a single chain variable fragments (scFv) of the mAB 4G2 was designed as a polypeptide comprising variable heavy (VH) and light chain domain (VL) of an immunoglobulin (IgG) linked through a polypeptide linker. Attempts to produce the scFv 4G2 in a therapeutically active form have failed so far. The protein is produced in high levels in E. coli but is deposited in insoluble form in inclusion bodies (IBs). In this thesis, three approaches were implemented to obtain a therapeutically active form of the product. The first approach was to use the HEK293-F cells to express the scFv 4G2 protein fused to different secretion factors (CD5, Kappa, SEC, all in pSelect vectors) which should initiate a secretion of the protein into the medium. Although the transfection was successful, and the HEK cells expressed the scFv 4G2, it was and not successfully secreted into the medium, but retained intracellularly.