Influence of FcRn binding properties on the gastrointestinal absorption and exposure profile of Fc molecules.

The neonatal Fc receptor (FcRn) represents a transport system with the potential to facilitate absorption of biologics across the gastrointestinal barrier. How biologics interact with FcRn to enable their gastrointestinal absorption, and how these interactions might be optimized in a biological therapeutic are not well understood. Thus, we studied the absorption of Fc molecules from the intestine using three IgG4-derived Fc variants with different, pH-dependent FcRn binding and release profiles. Using several different intestinal models, we consistently observed that FcRn binding affinity correlated with transcytosis. Our findings support targeting FcRn to enable intestinal absorption of biologics and highlight additional strategic considerations for future work.

Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics.

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.

Comparison of the kinetics and extent of muscarinic M1-M5 receptor internalization, recycling and downregulation in Chinese hamster ovary cells.

We characterized agonist-induced internalization, recycling and downregulation of each muscarinic receptor subtype (M(1)-M(5)) stably expressed in Chinese hamster ovary (CHO) cells. The radioligands [(3)H]QNB and [(3)H]NMS were used to measure the total and plasma membrane populations of muscarinic receptors, respectively. Following carbachol treatment (1 mM), the rank orders for the rate of carbachol-induced internalization of the muscarinic subtypes were M(2)>M(4)=M(5)>M(3)=M(1), respectively. Unlike the M(2) receptor, M(1), M(3), M(4) and M(5) receptors recycled back to the plasma membrane after 1 h carbachol treatment. The receptor downregulation elicited to 24h carbachol treatment was similar for M(2), M(3), M(4) and M(5) receptors, whereas that for the M(1) receptor was greater. Our results indicate that there are subtype-specific differences in the rate and extent of agonist-induced muscarinic receptor internalization, recycling and downregulation in CHO cells.