Quantitative prediction of CYP3A-mediated drug-drug interactions by correctly estimating fraction metabolized using human liver chimeric mice.

BACKGROUND AND PURPOSE: Fraction metabolized (fm ) and fraction transported (ft ) are important for understanding drug-drug interactions (DDIs) in drug discovery and development. However, current in vitro systems cannot accurately estimate in vivo fm due to inability to reflect the ft by efflux transporters (ft,efflux ). This study demonstrates how CYP3A-mediated DDI for CYP3A/P-gp substrates can be predicted using Hu-PXB mice as human liver chimeric mice. EXPERIMENTAL APPROACH: For estimating human in vitro fm by CYP3A enzyme (fm,CYP3A,in vitro ), six drugs, including CYP3A/P-gp substrates (alprazolam, cyclosporine, docetaxel, midazolam, prednisolone, and theophylline) and human hepatocytes were incubated with or without ketoconazole as a CYP3A inhibitor. We calculated fm,CYP3A,in vitro based on hepatic intrinsic clearance. To estimate human in vivo fm,CYP3A (fm,CYP3A,in vivo ), we collected information on clinical DDI caused by ketoconazole for these six drugs. We calculated fm,CYP3A,in vivo using the change of total clearance (CLtotal ). For evaluating the human DDI predictability, the six drugs were administered intravenously to Hu-PXB and SCID mice with or without ketoconazole. We calculated the change of CLtotal caused by ketoconazole. We compared the CLtotal change in humans with that in Hu-PXB and SCID mice. KEY RESULTS: The fm,CYP3A,in vitro was overestimated compared to the fm,CYP3A,in vivo . Hu-PXB mice showed much better correlation in the change of CLtotal with humans (R2 = 0.95) compared to SCID mice (R2 = 0.0058). CONCLUSIONS AND IMPLICATIONS: CYP3A-mediated DDI can be predicted by correctly estimating human fm,CYP3A,in vivo using Hu-PXB mice. These mice could be useful predicting hepatic fm and ft,efflux .

Physiologically based pharmacokinetic (PBPK) model that describes enhanced FcRn-dependent distribution of monoclonal antibodies (mAbs) by pI-engineering in mice.

We previously reported that monoclonal antibodies (mAbs) with a high isoelectric point (pI) value tended to exhibit fast plasma clearance (CL) and large steady-state volume of distribution (Vdss) in mice. However, the positive correlation between pI, CL, and Vdss cannot be described by the reported physiologically based pharmacokinetic (PBPK) models, in which FcRn-mediated transcytosis of mAbs is set to be minimal compared to convection-mediated transport. To address this issue, physiological parameters (lymph flow rate, reflection coefficient, endothelial uptake clearance, and FcRn concentration) were optimized based on the pharmacokinetic profiles of mAbs with various pI values in wild type and FcRn-deficient (beta-2-microglobulin knockout [KO]) mice. Simulations using the PBPK model developed in this study showed a positive correlation between pI, CL and Vdss observed in wild-type mice. Therefore, this model successfully characterized our hypothetical mechanism that an electrostatic positive interaction between mAbs and the endothelial membrane enhances FcRn-mediated transcytosis of mAbs, resulting in large Vdss. We sought to determine the right contribution of the two pathways of antibody distribution to the interstitial space and established a new model that could effectively capture the effect of pI on FcRn-mediated distribution of mAbs in the body.

Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N-alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

Quantitative Prediction of OATP-Mediated Disposition and Biliary Clearance Using Human Liver Chimeric Mice.

Drug biliary clearance (CLbile) in vivo is among the most difficult pharmacokinetic parameters to predict accurately and quantitatively because biliary excretion is influenced by metabolic enzymes, transporters, and passive diffusion across hepatocyte membranes. The purpose of this study is to demonstrate the use of Hu-FRG mice [Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice transplanted with human-derived hepatocytes] to quantitatively predict human organic anion transporting polypeptide (OATP)-mediated drug disposition and CLbile To predict OATP-mediated disposition, six OATP substrates (atorvastatin, fexofenadine, glibenclamide, pitavastatin, pravastatin, and rosuvastatin) were administered intravenously to Hu-FRG and Mu-FRG mice (FRG mice transplanted with mouse hepatocytes) with or without rifampicin as an OATP inhibitor. We calculated the hepatic intrinsic clearance (CLh,int) and the change of hepatic clearance (CLh) caused by rifampicin (CLh ratio). We compared the CLh,int of humans with that of Hu-FRG mice and the CLh ratio of humans with that of Hu-FRG and Mu-FRG mice. For predicting CLbile, 20 compounds (two cassette doses of 10 compounds) were administered intravenously to gallbladder-cannulated Hu-FRG and Mu-FRG mice. We evaluated the CLbile and investigated the correlation of human CLbile with that of Hu-FRG and Mu-FRG mice. We found good correlations between humans and Hu-FRG mice in CLh,int (100% within threefold) and CLh ratio (R2 = 0.94). Moreover, we observed a much better relationship between humans and Hu-FRG mice in CLbile (75% within threefold). Our results suggest that OATP-mediated disposition and CLbile can be predicted using Hu-FRG mice, making them a useful in vivo drug discovery tool for quantitatively predicting human liver disposition. SIGNIFICANCE STATEMENT: OATP-mediated disposition and biliary clearance of drugs are likely quantitatively predictable using Hu-FRG mice. The findings can enable the selection of better drug candidates and the development of more effective strategies for managing OATP-mediated DDIs in clinical studies.

Translational Approach for Predicting Human Pharmacokinetics of Engineered Therapeutic Monoclonal Antibodies with Increased FcRn-Binding Mutations.

INTRODUCTION: Recently, increasing FcRn binding by Fc engineering has become a promising approach for prolonging the half-life of therapeutic monoclonal antibodies (mAbs). This study is the first to investigate the optimization of an allometric scaling approach for engineered mAbs based on cynomolgus monkey data to predict human pharmacokinetics. METHODS: Linear two-compartmental model parameters (clearance [CL]; volume of distribution in the central compartment [Vc]; inter-compartmental clearance [Q]; volume of distribution in the peripheral compartment [Vp]) after the intravenous (IV) injection of engineered mAbs (M252Y/S254T/T256E or M428L/N434S mutations) in cynomolgus monkeys and humans were collected from published data. We explored the optimal exponent for allometric scaling to predict parameters in humans based on cynomolgus monkey data. Moreover, the plasma concentration-time profile of engineered mAbs after IV injection in humans was predicted using parameters estimated based on an optimized exponent. RESULTS: For engineered mAbs, a significant positive correlation between cynomolgus monkeys and humans was observed for CL, but not for other parameters. Whereas conventional exponents (CL: 0.8, Q: 0.75, Vc: 1.0, Vp: 0.95) previously established for normal mAbs showed poor prediction accuracy for CL and Q of engineered mAbs, the newly optimized exponents (CL: 0.55, Q: 0.6, Vc: 0.95, Vp: 0.95) achieved superior predictability for engineered mAbs. Moreover, the optimized exponents accurately predicted plasma mAb concentration-time profiles after IV injection of engineered mAbs in humans. CONCLUSIONS: We found that engineered mAbs require specially optimized exponents to accurately predict pharmacokinetic parameters and plasma concentration-time profiles after IV injections in humans based on cynomolgus monkey data. This optimized approach can contribute to a more accurate prediction of human pharmacokinetics in the development of engineered mAbs.

Recent Advances in Translational Pharmacokinetics and Pharmacodynamics Prediction of Therapeutic Antibodies Using Modeling and Simulation.

Therapeutic monoclonal antibodies (mAbs) have been a promising therapeutic approach for several diseases and a wide variety of mAbs are being evaluated in clinical trials. To accelerate clinical development and improve the probability of success, pharmacokinetics and pharmacodynamics (PKPD) in humans must be predicted before clinical trials can begin. Traditionally, empirical-approach-based PKPD prediction has been applied for a long time. Recently, modeling and simulation (M&S) methods have also become valuable for quantitatively predicting PKPD in humans. Although several models (e.g., the compartment model, Michaelis-Menten model, target-mediated drug disposition model, and physiologically based pharmacokinetic model) have been established and used to predict the PKPD of mAbs in humans, more complex mechanistic models, such as the quantitative systemics pharmacology model, have been recently developed. This review summarizes the recent advances and future direction of M&S-based approaches to the quantitative prediction of human PKPD for mAbs.

Elimination of plasma soluble antigen in cynomolgus monkeys by combining pH-dependent antigen binding and novel Fc engineering.

A conventional antibody targeting a soluble antigen in circulation typically requires a huge dosage and frequent intravenous administration to neutralize the antigen. This is because antigen degradation is reduced by the formation of antigen-antibody immune complexes, which escape from lysosomal degradation using neonatal Fc receptor (FcRn)-mediated recycling. To address this, we developed an antigen-sweeping antibody that combines pH-dependent antigen binding and Fc engineering to enhance Fc receptor binding. The sweeping antibody actively eliminates the plasma antigens by increasing the cellular uptake of the immune complex and dissociating the antigens in the acidic endosome for degradation. Strong antigen sweeping can reduce the dosage, potentially achieve higher efficacy, and expand the scope of antigen space available for targeting by antibodies. In this study, to further improve the sweeping efficacy, we developed a novel antibody Fc variant by enhancing Fcγ receptor IIb (FcγRIIb) binding and modulating charge characteristics for increased cellular uptake of the immune complex, together with enhancing FcRn binding for efficient salvage of the antigen-free antibodies. Our Fc variant achieved strong antigen sweeping in cynomolgus monkeys with antibody pharmacokinetics comparable to a wild-type human IgG1 antibody. The positive-charge substitutions enhanced uptake of the immune complex by FcγRIIb-expressing cells in vitro, which was completely inhibited by an anti-FcγRIIb antibody. This suggests that the strong in vivo sweeping efficacy improved by the charge engineering is more likely achieved by FcγRIIb-dependent uptake of the immune complex rather than nonspecific uptake. We expect this novel Fc engineering can maximize the antigen sweeping efficacy even in humans and create novel therapeutic antibodies that meet unmet medical needs for patients.

Quantitative prediction of P-glycoprotein-mediated drug-drug interactions and intestinal absorption using humanized mice.

BACKGROUND AND PURPOSE: P-glycoprotein (P-gp) exhibits a broad substrate specificity and affects pharmacokinetics, especially intestinal absorption. However, prediction, in vivo, of P-gp-mediated drug-drug interaction (DDI) and non-linear absorption at the preclinical stage, is challenging. Here we evaluate the use of human MDR1 mouse artificial chromosome (hMDR1-MAC) mice carrying human P-gp and lacking their own murine P-gp to quantitatively predict human P-gp-mediated DDI and non-linear absorption. EXPERIMENTAL APPROACH: The P-gp substrates (aliskiren, betrixaban, celiprolol, digoxin, fexofenadine and talinolol) were administered orally to wild-type, Mdr1a/b-knockout (KO) and hMDR1-MAC mice, and their plasma concentrations were measured. We calculated the ratio of area under the curve (AUCR) in mice (AUCMdr1a/b-KO /AUCwild-type or AUCMdr1a/b-KO /AUChMDR1-MAC ) estimated as attributable to complete P-gp inhibition and the human AUCR with and without P-gp inhibitor administration. The correlations of AUCRhuman with AUCRwild-type and AUCRhMDR1-MAC were investigated. For aliskiren, betrixaban and celiprolol, the Km and Vmax values for P-gp in hMDR1-MAC mice and humans were optimized from different dosing studies using GastroPlus. The correlations of Km and Vmax for P-gp between human and hMDR1-MAC mice were investigated. KEY RESULTS: A better correlation between AUCRhuman and AUCRhMDR1-MAC (R2 = 0.88) was observed. Moreover, good relationships of Km (R2 = 1.00) and Vmax (R2 = 0.98) for P-gp between humans and hMDR1-MAC mice were observed. CONCLUSIONS AND IMPLICATIONS: These results suggest that P-gp-mediated DDI and non-linear absorption can be predicted using hMDR1-MAC mice. These mice are a useful in vivo tool for quantitatively predicting P-gp-mediated disposition in drug discovery and development.

Analysis of Non-linear Pharmacokinetics of P-Glycoprotein Substrates in a Microfluidic Device Using a Mathematical Model that Includes an Unstirred Water Layer (UWL) Compartment.

PURPOSE: The purpose of this research is to analyze non-linear pharmacokinetics of P-glycoprotein (P-gp) substrates in a cell based assay of a microfluidic device, which might be affected by hydrodynamic barrier (unstirred water layer, UWL). RESULTS: Apparent permeability (Papp) were obtained using non-P-gp substrates (propranolol, metoprolol, and atenolol) and P-gp substrates (quinidine and talinolol) in a commercially available microfluidic device, organoplate ® of Caco-2 cell based assay. The previous UWL resistance model was well fitted to Papp of static and flow condition by assuming UWL including and negligible condition, while P-gp substrates of higher passive permeability (quinidine) was apart from the fitting curve. The concentration dependent non-linear kinetics of P-gp substrates, quinidine and talinolol, was more analyzed in detail, and apparent Vmax discrepancy between static and flow assay condition in the quinidine assay was observed, while that was not observed in talinolol, the lower permeable substrate. Based on the experimental results, a mathematical model for P-gp substrates including UWL compartment on the previous 3-compartment model was developed, and it indicated that the apparent Vmax was variable along with the ratio between passive permeability and UWL permeability. CONCLUSIONS: The mathematical model adding UWL compartment well explained non-linear pharmacokinetics of apparent permeability of P-gp substrate in the microfluidic device. The model also has a potential to be applied to P-gp substrate permeability analysis in vivo.

Estimation of Clearance and Bioavailability of Therapeutic Monoclonal Antibodies from Only Subcutaneous Injection Data in Humans Based on Comprehensive Analysis of Clinical Data.

INTRODUCTION: Theoretically, the separate estimation of clearance (CL) and bioavailability (F) requires both intravenous and extravascular injection data. This study investigated whether CL and subcutaneous F of therapeutic monoclonal antibodies (mAbs) in humans can be separately estimated from subcutaneous injection data only. METHODS: First, the geometric mean of linear pharmacokinetic parameters (CL, intercompartmental CL [Q], volume of distribution in the central compartment [Vc], and volume of distribution in the peripheral compartment [Vp]) after intravenous injection for mAbs in humans that have been reported in public data sources was estimated from 103 mAbs with linear pharmacokinetics and 44 mAbs with nonlinear pharmacokinetics. Next, we estimated the CL and F of 25 mAbs with linear pharmacokinetics from plasma/serum mAb concentration-time profiles after subcutaneous injection in humans by fixing the geometric mean of Q, Vc, and Vp based on the public data. Moreover, the plasma/serum concentration-time profile of 25 mAbs after intravenous injection was simulated using the estimated CL and the geometric mean of Q, Vc, and Vp. RESULTS: There were no significant differences in parameters among subclasses (immunoglobulin [Ig] G1, 2, and 4) or in linearity (derivation from linear and nonlinear pharmacokinetics). Using only subcutaneous injection data, we successfully estimated the CL of 23/25 mAbs (92%) and F of all 25 mAbs (100%) within 1.5-fold of the observed value. Moreover, overall, the simulated concentration-time profiles were largely consistent with observed data (90.8% within 1.5-fold of the observed values). CONCLUSIONS: This approach does not require intravenous injection data to separately estimate CL and F after subcutaneous injection in humans and can therefore accelerate the clinical development of mAbs.

A Novel Total Drug Assay for Quantification of Anti-C5 Therapeutic Monoclonal Antibody in the Presence of Abundant Target.

SKY59 or RO7112689 is a humanized monoclonal antibody against complement protein C5 with pH-dependent C5-binding and neonatal Fc receptor-mediated recycling capabilities, which result in long-lasting neutralization of C5. We developed and validated a novel total drug assay for quantification of target-binding competent SKY59 in the presence of endogenous C5 in cynomolgus monkey plasma. The target-binding competent SKY59 was determined after complex formation by the addition of recombinant monkey C5 using goat anti-human IgG-heavy chain monkey-adsorbed polyclonal antibody as a capture antibody and rabbit anti-C5 monoclonal antibody (mAb) non-competing with SKY59 for detection. The total SKY59 assay was shown to be accurate and precise over the range of 0.05-3.2 µg/mL as well as be tolerant to more than 400 µg/mL of C5 (~ 3000-fold molar excess of target). We also developed and validated a total C5 assay, confirmed selectivity and parallelism, and verified the utility of recombinant monkey C5 for the total C5 assay as well as the total SKY59 assay. Furthermore, we used these validated methods to measure SKY59 and C5 concentrations in cynomolgus monkey plasma samples in a toxicology study. This total drug assay can be applied not only to other antibody therapeutics against shed/soluble targets when a non-competing reagent mAb is available but also for clinical studies when a reagent mAb specific for engineered Fc region on a therapeutic mAb is available.

Simple Approach to Accurately Predict Pharmacokinetics of Therapeutic Monoclonal Antibodies after Subcutaneous Injection in Humans.

BACKGROUND AND OBJECTIVE: The subcutaneous injection of therapeutic monoclonal antibodies is increasingly used in the treatment of several diseases because of its convenience. Thus, a simple and accurate method of predicting the pharmacokinetics of monoclonal antibodies after a subcutaneous injection in humans would be a valuable tool for preclinical/clinical development. In this study, we investigated whether the pharmacokinetics of monoclonal antibodies after a subcutaneous injection in humans can be predicted using only pharmacokinetic data after a subcutaneous injection in cynomolgus monkeys. METHODS: First, we compared the accuracy of three approaches to predict the apparent clearance (CL/F) and apparent volume of distribution (Vd/F) for 15 monoclonal antibodies in humans (1) allometric scaling from cynomolgus monkeys; (2) geometric mean of reported values in humans; (3) estimation from a regression line based on CL/F in humans [only Vd/F]). Then, using the predicted CL/F and Vd/F, and the geometric mean of reported absorption rate constant of mAbs the plasma concentration-time profiles of 13 monoclonal antibodies after subcutaneous injections in humans were simulated. RESULTS: In a comparison of approaches, the first approach showed the best prediction accuracy for CL/F with an exponent of 0.9 (100% and 73% prediction accuracy within 2- and 1.5-fold of the observed value),and the third approach was the best for Vd/F (100% prediction accuracy within 1.5-fold of the observed value). Next, using the first approach for CL/F and the third approach for Vd/F, we accurately predicted the plasma concentration-time profiles of 13 monoclonal antibodies after subcutaneous injections in humans. CONCLUSION: This simple approach can be applied in preclinical and clinical settings to predict the pharmacokinetics of monoclonal antibodies after subcutaneous injections in humans. Further, this approach requires only CL/F after a subcutaneous injection in cynomolgus monkeys, contributing to animal welfare and reducing costs.

Antibody to CD137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.

Improvement of pharmacokinetic properties of therapeutic antibodies by antibody engineering.

Monoclonal antibodies (mAbs) have become an important therapeutic option for several diseases. Since several mAbs have shown promising efficacy in clinic, the competition to develop mAbs has become severe. In efforts to gain a competitive advantage over other mAbs and provide significant benefits to patients, innovations in antibody engineering have aimed at improving the pharmacokinetic properties of mAbs. Because engineering can provide therapeutics that are more convenient, safer, and more efficacious for patients in several disease areas, it is an attractive approach to provide significant benefits to patients. Further advances in engineering mAbs to modulate their pharmacokinetics were driven by the increase of total soluble target antigen concentration that is often observed after injecting a mAb, which then requires a high dosage to antagonize. To decrease the required dosage, several antibody engineering techniques have been invented that reduce the total concentration of soluble target antigen. Here, we review the various ways that antibody engineering can improve the pharmacokinetic properties of mAbs.

Antibody engineering to generate SKY59, a long-acting anti-C5 recycling antibody.

Modulating the complement system is a promising strategy in drug discovery for disorders with uncontrolled complement activation. Although some of these disorders can be effectively treated with an antibody that inhibits complement C5, the high plasma concentration of C5 requires a huge dosage and frequent intravenous administration. Moreover, a conventional anti-C5 antibody can cause C5 to accumulate in plasma by reducing C5 clearance when C5 forms an immune complex (IC) with the antibody, which can be salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling. In order to neutralize the increased C5, an even higher dosage of the antibody would be required. This antigen accumulation can be suppressed by giving the antibody a pH-dependent C5-binding property so that C5 is released from the antibody in the acidic endosome and then trafficked to the lysosome for degradation, while the C5-free antibody returns back to plasma. We recently demonstrated that a pH-dependent C5-binding antibody, SKY59, exhibited long-lasting neutralization of C5 in cynomolgus monkeys, showing potential for subcutaneous delivery or less frequent administration. Here we report the details of the antibody engineering involved in generating SKY59, from humanizing a rabbit antibody to improving the C5-binding property. Moreover, because the pH-dependent C5-binding antibodies that we first generated still accumulated C5, we hypothesized that the surface charges of the ICs partially contributed to a slow uptake rate of the C5-antibody ICs. This idea motivated us to engineer the surface charges of the antibody. Our surface-charge engineered antibody consequently exhibited a high capacity to sweep C5 and suppressed the C5 accumulation in vivo by accelerating the cycle of sweeping: uptake of ICs into cells, release of C5 from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, our engineered anti-C5 antibody, SKY59, is expected to provide significant benefits for patients with complement-mediated disorders.

Quantitative prediction of therapeutic antibody pharmacokinetics after intravenous and subcutaneous injection in human.

Prediction of the plasma/serum mAb concentration-time profile in human is important to determine the required dose regime. This study proposes an approach for predicting the plasma/serum mAb concentration-time profile after intravenous and subcutaneous injection in human based on comprehensive analysis of reported pharmacokinetic data. Optimal scaling exponents from cynomolgus monkey to human for CL, Q, Vc, and Vp were estimated as 0.8, 0.75, 1.0, and 0.95, respectively. The estimated exponents were used to predict plasma/serum mAb concentration-time profile in human from pharmacokinetic data in cynomolgus monkey, and the results had reasonable accuracy with symmetric variability of prediction. Then, data reported for pharmacokinetics in human were used to estimate optimal ka and F after subcutaneous injection. The geometric mean of ka was suitable to predict Tmax, and F which was estimated from CL was suitable to predict Cmax. Our approach is useful for predicting the plasma/serum mAb concentration-time profile after intravenous and subcutaneous injection in human. Moreover, the study also investigated the possibility of predicting pharmacokinetic parameters of mAbs with increased FcRn binding mutations in human and found that our approach of prediction based on reported pharmacokinetic data may also be applicable to mAbs with these mutations.

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.

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 (KD = 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 KD-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 KD-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.

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.