Tatsuhiko Tachibana

Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization

For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibodys 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.

Reduced elimination of IgG antibodies by engineering the variable region

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.

Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.

Correction of Permeability with Pore Radius of Tight Junctions in Caco-2 Monolayers Improves the Prediction of the Dose Fraction of Hydrophilic Drugs Absorbed by Humans

AbstractPurpose. To improve predictions of fraction dose absorbed (Fa) for hydrophilic drugs, a correction of paracellular permeability using the pore radius of tight junctions (TJs) in Caco-2 monolayers was performed. Methods. The apparent permeability coefficient (Papp) of drugs was measured using the Caco-2 assay and the parallel artificial membrane permeation assay (PAMPA), and values were corrected with the pore radius of TJs. Results. An equation for calculating the pore radius of TJs from the Papp of lucifer yellow was obtained. The optimal pore radius of TJs in Caco-2 monolayers for predicting human Fa was calculated to be 7 Å. The correlation between the actual and predicted Fa was improved by using the Papp corrected with the pore radius of TJs. Permeability in the PAMPA, which was corrected using the pore radius and membrane potential, was well correlated with that in the Caco-2 assay. Most of the hydrophilic drugs tested in this study were absorbed mainly through the paracellular pathway. Conclusions. The results suggest the necessity of optimizing paracellular permeation for the prediction of Fa, and also the importance of the paracellular pathway to the absorption of hydrophilic drugs. This method might contribute to the setting of appropriate dosages and the development of hydrophilic drugs.

Method for predicting the risk of drug–drug interactions involving inhibition of intestinal CYP3A4 and P-glycoprotein

To develop a method to predict the risk of drug–drug interactions involving the inhibition of intestinal CYP3A4 or P-glycoprotein, data from clinical drug–drug interaction studies of CYP3A4 and/or P-glycoprotein substrates were analysed. The ratio of inhibitor dose (Dosei) to inhibition constant (Ki), termed the drug-interaction number, was used to index intestinal drug–drug interaction. From the analysis, it was found that (1) CYP3A4 inhibitors with a drug-interaction number below 2.8 L have a low risk of interacting with substrates which exhibit intestinal first-pass metabolism and those with a drug-interaction number above 9.4 L have a high risk; (2) P-glycoprotein inhibitors with a drug-interaction number below 10.8 L have a low risk of interacting with P-glycoprotein substrates and those with a drug-interaction number above 27.9 L have a high risk; and (3) the drug-interaction number indexes, 2.8 L and 9.4 L for CYP3A4 and 10.8 L and 27.9 L for P-glycoprotein were validated by data from dual CYP3A4/P-glycoprotein substrates. In conclusion, the drug-interaction number is useful for classifying the risk of drug–drug interactions involving the inhibition of intestinal CYP3A4 and P-glycoprotein. This drug-interaction number-based approach is similar to the method that the US Food and Drug Administration (USFDA) recently proposed in the draft guidance for predicting P-glycoprotein-mediated drug–drug interaction.

Model Analysis of the Concentration-Dependent Permeability of P-gp Substrates

PurposeRecently, it was reported that the apparent Michaelis-Menten constant (Km(app)) of a P-glycoprotein (P-gp) substrate, defined for the extracellular substrate concentration, increases as the P-gp expression level in the cell increases. By its nature, the Km value should not depend on the level of P-gp expression. The purpose of this study is to establish a model which can estimate the Km value independent of the P-gp expression level in cells.MethodsThe previously reported concentration-dependent permeability of verapamil, quinidine, and vinblastine in MDR1-MDCKII, P-gp-highly induced Caco-2, P-gp-induced Caco-2, normal Caco-2, and MDR1-knockdown Caco-2 cells data were analyzed using a model in which the Km value was defined for the intracellular substrate concentration.ResultsThe estimated Km values defined for the substrate concentration inside the cells were almost the same among various cells with different levels of P-gp expression. The estimated Vmax values were approximately proportional to the P-gp expression level.ConclusionThe established kinetic model was found to be rational based on the results that the Km values of P-gp substrates were about the same for cells expressing various levels of P-gp, while the Vmax values were proportional to the expression levels of P-gp.

Engineered monoclonal antibody with novel antigen-sweeping activity in vivo.

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.

Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens

Summary Emicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a manner similar to FVIIIa. However, details of the emicizumab–antigen interactions have not been reported so far. In this study, we first showed by surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa with moderate affinities ( K D = 1.58, 1.52, 1.85, and 0.978 μM, respectively). We next showed by immunoblotting analysis that emicizumab recognised the antigens’ epidermal growth factor (EGF)-like domains. We then performed K D -based simulation of equilibrium states in plasma for quantitatively predicting the ways that emicizumab would interact with the antigens. The simulation predicted that only a small part of plasma FIX, FX, and emicizumab would form antigen-bridging FIX–emicizumab–FX ternary complex, of which concentration would form a bell-shaped relationship with emicizumab concentration. The bell-shaped concentration dependency was reproduced by plasma thrombin generation assays, suggesting that the plasma concentration of the ternary complex would correlate with emicizumab’s cofactor activity. The simulation also predicted that at 10.0–100 μg/ml of emicizumab–levels shown in a previous study to be clinically effective–the majority of plasma FIX, FX, and emicizumab would exist as monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar affinities at their EGF-like domains. The K D -based simulation predicted that the antigen-bridging ternary complex formed in circulating plasma would correlate with emicizumab’s cofactor activity, and the majority of FIX and FX would be free and available for other coagulation reactions. Institution where the work was carried out: Research Division, Chugai Pharmaceutical Co., Ltd. Supplementary Material to this article is available online at www.thrombosis-online.com.

Long lasting neutralization of C5 by SKY59, a novel recycling antibody, is a potential therapy for complement-mediated diseases.

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.

Application of human FcRn transgenic mice as a pharmacokinetic screening tool of monoclonal antibody

Abstract 1. For drug discovery, useful screening tools are essential to select superior candidates. Here, we evaluated the applicability of transgenic mice expressing human neonatal Fc receptor (FcRn) (hFcRn Tgm) as a pharmacokinetic screening tool of therapeutic monoclonal antibodies (mAbs) and Fc-fusion proteins that overcomes the species difference in FcRn binding. 2. Marketed 11 mAbs and 2 Fc-fusion proteins were intravenously administered to hFcRn Tgm and WT mice. The half-lives in hFcRn Tgm and WT mice were compared with those in human obtained from literature. The linear half-lives in human and monkey were also calculated by nonlinear pharmacokinetic analysis. For comparison, correlations of half-lives between monkey and human were also evaluated. 3. The half-lives of mAbs and Fc-fusion proteins after intravenous administration ranged from 1.1 to 13.2 days in hFcRn Tgm and from 1.2 to 30.3 days in WT mice. The half-lives in human correlated more closely with those in hFcRn Tgm than in WT mice and monkey. 4. Our results suggest that hFcRn Tgm are a valuable and useful tool for pharmacokinetic screening of mAbs and Fc-fusion proteins in the preclinical stage. Furthermore, we believe that hFcRn Tgm are broadly applicable to preclinical pharmacokinetic screening of mAbs-based therapeutics.