For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibody's lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra), an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the interactions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses.

Fc engineering to increase the binding affinity of IgG antibodies to FcRn has been reported to reduce the elimination of IgG antibodies. Herein, we present a novel non-FcRn-dependent approach to reduce the elimination of IgG antibodies. Pharmacokinetic studies conducted in normal mice of various humanized IgG4 antibodies, which had identical constant regions but different variable region sequences, revealed that an antibody with a lower isoelectric point (pI) has a longer half-life. These antibodies exhibited comparable binding affinity to FcRn, and with the antibodies with lower pIs, a longer half-life was also observed in beta2-microglobulin knockout mice, suggesting that differences in the pharmacokinetics were due to a non-FcRn-dependent mechanism. On the basis of our findings, we attempted to engineer the pharmacokinetic properties of a humanized anti-IL6 receptor IgG1 antibody. Selected substitutions in the variable region, without substitution in the Fc region, lowered the pI but did not reduce the biological activity and showed a significant reduction in the clearance of the antibody in cynomolgus monkey. These results suggest that lowering the pI by engineering the variable region could reduce the elimination of IgG antibodies and could provide an alternative to Fc engineering of IgG antibodies.

Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.

Monoclonal antibodies are widely used to target disease-related antigens. However, because conventional antibody binds to the antigen but cannot eliminate the antigen from plasma, and rather increases the plasma antigen concentration by reducing the clearance of the antigen, some clinically important antigens are still difficult to target with monoclonal antibodies because of the huge dosages required. While conventional antibody can only bind to the antigen, some natural endocytic receptors not only bind to the ligands but also continuously eliminate them from plasma by pH-dependent dissociation of the ligands within the acidic endosome and subsequent receptor recycling to the cell surface. Here, we demonstrate that an engineered antibody, named sweeping antibody, having both pH-dependent antigen binding (to mimic the receptor-ligand interaction) and increased binding to cell surface neonatal Fc receptor (FcRn) at neutral pH (to mimic the cell-bound form of the receptor), selectively eliminated the antigen from plasma. With this novel antigen-sweeping activity, antibody without in vitro neutralizing activity exerted in vivo efficacy by directly eliminating the antigen from plasma. Moreover, conversion of conventional antibody with in vitro neutralizing activity into sweeping antibody further potentiated the in vivo efficacy. Depending on the binding affinity to FcRn at neutral pH, sweeping antibody reduced antigen concentration 50- to 1000-fold compared to conventional antibody. Thereby, sweeping antibody antagonized excess amounts of antigen in plasma against which conventional antibody was completely ineffective, and could afford marked reduction of dosage to a level that conventional antibody can never achieve. Thus, the novel mode of action of sweeping antibody provides potential advantages over conventional antibody and may allow access to the target antigens which were previously undruggable by conventional antibody.

Fc engineering can modulate the Fc–FcγR interaction and thus enhance the potency of Abs that target membrane-bound Ags, but it has not been applied to Abs that target soluble Ags. In this study, we revealed a previously unknown function of inhibitory FcγRII in vivo and, using an Ab that binds to Ag pH dependently, demonstrated that the function can be exploited to target soluble Ag. Because pH-dependent Ab dissociates Ag in acidic endosome, its Ag clearance from circulation reflects the cellular uptake rate of Ag/Ab complexes. In vivo studies showed that FcγR but not neonatal FcR contributes to Ag clearance by the pH-dependent Ab, and when Fc binding to mouse FcγRII and III was increased, Ag clearance was markedly accelerated in wild-type mice and FcR γ-chain knockout mice, but the effect was diminished in FcγRII knockout mice. This demonstrates that mouse FcγRII efficiently promotes Ab uptake into the cell and its subsequent recycling back to the cell surface. Furthermore, when a human IgG1 Fc variant with selectively increased binding to human FcγRIIb was tested in human FcγRIIb transgenic mice, Ag clearance was accelerated without compromising the Ab half-life. Taken together, inhibitory FcγRIIb was found to play a prominent role in the cellular uptake of monomeric Ag/Ab immune complexes in vivo, and when the Fc of a pH-dependent Ab was engineered to selectively enhance human FcγRIIb binding, the Ab could accelerate soluble Ag clearance from circulation. We assume such a function would enhance the therapeutic potency of Abs that target soluble Ags.