Conventional therapeutic immunoglobulin G (IgG) antibodies bind to antigen with high affinity and specificity and have a long serum half-life. The long half-life is due to the pH-dependent protection of IgG from lysosomal degradation, by the neonatal Fc receptor, FcRn. Although capable of neutralising soluble extracellular antigens, a conventional therapeutic IgG may actually lead to antigen persistence, with antigen assuming the long half-life of the bound antibody. A pH-dependent antigen binding antibody can address this limitation by increasing the clearance of antigen via multiple rounds of antigen removal. In contrast to a conventional IgG, a pH-dependent antibody binds antigen with weaker affinity in the acidic endosome than at the neutral pH of the plasma. The weaker affinity in the endosome enables antigen dissociation, with the dissociated antigen trafficked to the lysosome for degradation, while the antibody is salvaged from degradation by FcRn. The salvaged antibody is recycled to the cell surface where it is released back into circulation. However, pH-dependent antibodies still have their limitations, because in order to be internalised they rely on the random sampling of the plasma by fluid-phase endocytosis. A pH-dependent ‘sweeper’ antibody overcomes this limitation by binding to FcRn at both neutral and acidic pH and is therefore internalised by the more efficient receptor-mediated endocytosis. However, a pH-dependent ‘sweeper’ antibody also has limitations; it will block endogenous IgG from binding to FcRn, and is restricted to the endocytosis dynamics of FcRn. To address these limitations, in this thesis a pH-dependent bispecific that does not block FcRn function has been investigated. The bispecific that has been designed is comprised of two Fab’s cross-linked to form a diFab’, where one Fab’ arm binds to antigen in a pH-dependent manner and the other Fab’ arm binds FcRn in a pH-independent manner (see Figure 6.2). To generate pH-dependent binding, histidine mutations have been made in the complementarity-determining regions and flanking regions of an anti-murine IL6 Fab’. The resulting Fab’s have a range of binding affinities with a ~2 log difference in affinity measured between the neutral and acidic pH. The effect of the pH-dependent bispecifics on the mIL6 level was monitored in vitro and the results have shown a statistically significant increase in mIL6 uptake when compared to the pH-independent wild-type bispecific. In addition, to assess the suitability of FcRn as an endocytic receptor to target with the bispecific diFab’, the FcRn trafficking pathways were characterised in a liver cell line expressing endogenous FcRn. The levels of cell surface and intracellular FcRn have been quantified and the rate of internalisation assessed. These data suggest that internalised FcRn mixes with a large internal pool and only returns to cell surface ‘infrequently’. Due to these dynamics, FcRn may not be the most appropriate endocytic receptor for the ‘sweeper’ antigen clearance mechanism. Nevertheless, the FcRn trafficking and endosome acidification of a pH-dependent bispecific is capable of enhancing the clearance of antigen. Thus, this bispecific approach has the potential to reduce antibody dose and/or dosing frequency and target antigens that have previously been seen as ‘undruggable’ due to their high antigen levels.
|Date of Award||7 Feb 2018|
|Supervisor||Paul Whitley (Supervisor), Andrew Chalmers (Supervisor) & Sam Heywood (Supervisor)|