A bispecific antibody NXT007 exerts a hemostatic activity in hemophilia A monkeys enough to keep a nonhemophilic state.

BACKGROUND: Emicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients. OBJECTIVES: To identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation. METHODS: New pairs of light chains for emicizumab's heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A. RESULTS: NXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab's historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well. CONCLUSION: NXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab.

Cynomolgus monkey model of interleukin-31-induced scratching depicts blockade of human interleukin-31 receptor A by a humanized monoclonal antibody.

Scratching is an important factor exacerbating skin lesions through the so-called itch-scratch cycle in atopic dermatitis (AD). In mice, interleukin (IL)-31 and its receptor IL-31 receptor A (IL-31RA) are known to play a critical role in pruritus and the pathogenesis of AD; however, study of their precise roles in primates is hindered by the low sequence homologies between primates and mice and the lack of direct evidence of itch sensation by IL-31 in primates. We showed that administration of cynomolgus IL-31 induces transient scratching behaviour in cynomolgus monkeys and by that were able to establish a monkey model of scratching. We then showed that a single subcutaneous injection of 1 mg/kg nemolizumab, a humanized anti-human IL-31RA monoclonal antibody that also neutralizes cynomolgus IL-31 signalling and shows a good pharmacokinetic profile in cynomolgus monkeys, suppressed the IL-31-induced scratching for about 2 months. These results suggest that the IL-31 axis and IL-31RA axis play as critical a role in the induction of scratching in primates as in mice and that the blockade of IL-31 signalling by an anti-human IL-31RA antibody is a promising therapeutic approach for treatment of AD. Nemolizumab is currently under investigation in clinical trials.

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.

Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody.

Various studies have demonstrated that Fc engineering to enhance neonatal Fc receptor (FcRn) binding is effective for elongating half-life or increasing cellular uptake of IgG. A previous study has shown that a N434H mutation to enhance FcRn binding resulted in increased binding to rheumatoid factor (RF) autoantibody, which is not desirable for therapeutic use in autoimmune disease. In this study, we first showed that all the existing Fc variants with enhanced FcRn binding also show increased RF binding, and then identified specific mutations that could be introduced to those Fc variants to reduce the RF binding. Furthermore, we generated novel Fc variants that do not increase RF binding and show half-lives of 45 d in cynomolgus monkey, which is longer than those of previously reported Fc variants. In addition, we generated novel Fc variants with antigen sweeping activity that do not increase RF binding. We expect that these novel Fc variants will be useful as antibody therapeutics against autoimmune diseases.

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.

A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model.

Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.

Design and synthesis of peptidomimetic factor VIIa inhibitors.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. In previous reports, we described a S3 subsite found in the X-ray crystal structure of compound 2 that bound to FVIIa/soluble tissue factor (sTF). Based on the X-ray crystal structure information and with the aim of improving the inhibition activity for FVIIa/TF and selectivity against other serine proteases, we synthesized derivatives by introducing substituents at position 5 of the indole ring of compound 2. Among them, compound 16 showed high selectivity against other serine proteases. Contrary to our expectations, compound 16 did not occupy the S3-subsite; X-ray structure analysis revealed that compound 16 improved selectivity by forming hydrogen bonds with Gln217, Thr99 and Asn100.

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.

Generation of a humanized monoclonal antibody against human parathyroid hormone-related protein and its efficacy against humoral hypercalcemia of malignancy.

A humanized monoclonal antibody against parathyroid hormone-related protein (PTHrP) was generated from the mouse monoclonal antibody raised against the peptide corresponding to the N-terminal 34 amino acids of the human PTHrP [(PTHrP(1-34)]. The humanized antibody interacted with the PTHrP(1-34) with a kD value of 1.90 x 10(-10) M, and the epitope resides between the amino acids 20 and 30 of the PTHrP. PTHrP(1-34) significantly increased the intracellular cAMP levels in the rat osteosarcoma cells that expressed PTHR1, and the 5 microg/mL or higher concentrations of the humanized antibody almost completely blocked the PTHrP-induced cAMP production even in the presence of 2 microg/mL PTHrP(1-34), demonstrating its ability to fully neutralize PTHrP function. There was no significant difference in the potency of the mouse, chimera, or the humanized antibodies to suppress the PTHrP-induced increase in the intracellular cAMP in ROS cells. Furthermore, at the same doses, the administration of the chimera or the humanized antibody was equally effective in reducing the blood ionized calcium levels of hypercalcemic mice bearing the PAN-7-JCK human pancreatic cancer xenograft or the LC-6-JCK human lung cancer xenograft that secreted PTHrP. Thus, humanized anti-PTHrP may be useful for the treatment of the humoral hypercalcemia of malignancy in humans.