In fact, as shown here, affinity maturation would tend to impair the antibody potency

In fact, as shown here, affinity maturation would tend to impair the antibody potency. an example, a recent evaluate1highlights that three competing theories, namely receptor aggregation, conformational change and segregation of receptors from intracellular inhibitors, are all supported by experimental data and may all be partly responsible for transmission transduction. An important group of agonistic receptors is the tumour necrosis factor (TNF) receptor superfamily, users of which are known to form trimeric signalling complexes upon ligation with their cognate trimeric ligands.2As many TNF receptor superfamily users have significant functions in the progression of human disease,3a better understanding of their signalling mechanisms could enable the design of potent agonist and antagonist molecules as candidate drugs for clinical development. Monoclonal antibodies, with their high affinity, target selectivity and long serum half-lives, are well suited as receptor agonist drug molecules. Accordingly, you will find agonistic antibodies in preclinical and clinical development against several TNF receptor family members, such as TNF-related apoptosis inducing ligand (TRAIL) receptor 1,4TRAIL receptor 2,5glucocorticoid-induced tumor necrosis factor receptor (GITR),6CD1377and CD40.8 The TNF receptor superfamily member Fas (CD95, APO-1, TNFRSF6) is a receptor, which participates in transmission transduction to trigger cellular apoptosis after binding to Fas ligand (FasL).9Because of its expression on many tumours and its ability to trigger apoptosis,10Fas could be considered an interesting target for therapeutic intervention. However, the finding that an agonistic Fas antibody administered to mice caused severe liver damage and rapid death has led to the conclusion that Fas is not IDO-IN-4 a valid therapeutic target.11Despite the possible limitations of Fas in the therapeutic setting it can still IDO-IN-4 be considered a relevant model system to explore the nature of agonistic antibody signalling in the TNF receptor superfamily. Agonistic antibodies, as opposed to the more frequently isolated antagonistic or neutralising antibodies, have rarely been analyzed from a structurefunction perspective and little is known about the importance of epitope and affinity in the biological potency of such antibodies. Whereas neutralising antibodies are expected to exhibit a direct positive IDO-IN-4 correlation between affinity and potency, agonist antibodies are potentially more delicate and difficult to understand and as a consequence it could be more challenging to enhance their potency. In this study, phage-display technology was used to identify a human anti-Fas antibody, which could efficiently trigger apoptosis and therefore demonstrate potentin vitrotumour cell-killing activity. This model agonistic antibody was then used as a starting point for mutational and crystallographic studies to explore the binding interface and better understand the agonistic activity. This systematic analysis of an agonistic antibody interacting with its receptor, in particular the exploration of the relationship between affinity and potency, has led to some amazing conclusions about the nature of agonistic antibody signalling. == Results == == Isolation of agonistic anti-Fas antibody E09 and comparison with other agonists == Antibodies to human Fas receptor were isolated by performing phage-display selections12on the recombinant extracellular domain name (ECD) of Fas. Antibodies specific for Fas ECD were IDO-IN-4 detected by phage ELISA and a total of 264 unique scFv were sequenced. Of the 264 different scFv antibodies screened for agonism in a cell-viability assay, Rabbit Polyclonal to TCEAL4 only one was identified as having anti-proliferative activity. This scFv, E09, was converted to human IgG1 antibody format for further characterisation. To confirm the agonistic activity towards human Fas receptor, assays were performed on Jurkat cells to measure caspase 3/7 activation and DNA fragmentation, which, respectively, are early and late readouts for apoptosis. The E09 antibody was compared with the natural ligand FasL in recombinant form and two agonistic anti-Fas antibodies, the mouse monoclonal antibodies DX2 and SM1.1.10,13All agonists were able to induce IDO-IN-4 caspase 3/7 activity and DNA fragmentation, as shown inFigure 1, but to differing extents. E09 was as potent as the natural ligand FasL at triggering caspase 3/7 activity and even more potent than FasL at inducing DNA fragmentation with an EC50of 0.7 and 2.8 nM for E09.