Expressão heteróloga e avaliação da capacidade de ligação da região variável do anticorpo monoclonal Ofatumumabe

Abstract

Ofatumumab is a human anti-CD20 monoclonal antibody used in the treatment of diseases in which B cell depletion might be beneficial, such as Chronic Lymphoid Leukemia, Non-Hodgkin's Lymphoma and Multiple Sclerosis. The single-chain fragment variable (scFv) is a construct in which the light and heavy chain variable domains of an antibody are joined by a linker. Some advantages are associated with the scFv format, mainly due to its reduced size when compared to whole antibodies and its ability to be expressed in prokaryotic system. The present study aimed to express, purify and assess the binding capacity of the scFv from Ofatumumab. In future studies, it is intended to apply this scFv as a reference in biological assays associated with the determination of crucial Ofatumumab residues involved in its binding capacity to the CD20 molecule, as well as in the context of the production of mutants with improved properties. For expression, pET-SUMO was used as the vector and the Escherichia Coli SHuffle was the strain chosen. Purification was performed by nickel-containing column affinity chromatography. Purification was evaluated by SDS-PAGE, and immunoassays were performed to assess binding capacity (Western blot and ELISA). There was an average yield of 4.3 mg/L of culture from expression and purification of the soluble fraction. By electrophoresis and Western blot, evidence of scFv expression was obtained. The immunoassays performed could not confirm retention of scFv binding capacity to CD20+ cells applied in these assays. It was concluded that the recombinant protein was expressed partially soluble. Future studies of process optimization will still be necessary to achieve higher yield in scFv production. The search for experimental strategies to properly assess scFv binding capacity remains a challenge. The restrictions associated with the use of cells that must keep the target antigen available on their membranes so that they adequately expose the double epitope region for interaction of the analyzed fragment throughout the assay, as well as the possible limitations of the fragment type itself for application in these tests – in addition to probable structural changes in scFv – may underlie the difficulty of detecting the binding capacity of the scFv obtained.