Biochemical targets

Interleukin 6 (IL6) is a pleiotropic pro-inflammatory cytokine produced by a variety of cell types such as lymphocytes, monocytes, and fibroblasts. The deregulation of IL-6 secretion is involved in several inflammatory processes, e.g. rheumatoid arthritis (RA) and Crohn’s disease. It therefore represents a target of choice in therapeutic antibody development.

Medimmune developed a potent and sensitive antibody by engineering the antibody variable domains using the phage display technology. The description below summarizes the HTRF assay format which they used to screen the ScFv proteins from the phage display selection, based on an immunocompetition.

In the second step, Medimmune ran a random and targeted mutagenesis strategy based on Ribosome display and Phage display technologies respectively, in order to increase the affinity and the potency of their antibodies. They selected the biobetter fragments against a reference antibody generated during the screening phase, using a second HTRF format as illustrated here.

The assay was set up using a mix of FLAG®-tagged human IL-6 and biotinylated reference anti-IL-6 IgG1 antibody. The HTRF® detection was performed by adding an anti-FLAG IgG labeled with cryptate and Steptavidin XLent!. The engineered ScFv fragments were assessed for their capacity to displace the FLAG®-hIL6 / Biotin-Anti-IL-6 Reference Ab interaction. Affinity improved ScFv proteins were identified using this biochemical assay format.

Donna K. Finch & al. Whole-Molecule Antibody Engineering: Generation of a High-affinity Anti-IL6 Antibody with Extended Pharmacokinetics. J.Mol. Biol 2011. 411, 791-807.