Antibody formulation

Abstract

In one aspect, the present disclosure provides formulations, pharmaceutical compositions or medicaments containing an antibody, in particular, formulations, pharmaceutical compositions or medicaments containing, as an active ingredient, a bispecific antibody having a blood coagulation factor VIII (FVIII)-like activity higher than that of emicizumab. The formulations, pharmaceutical compositions or medicaments of the present disclosure, which contain the antibody at or above a certain level, can provide stable bioavailability in humans without greatly depending on the drug concentration.

Description

ANTIBODY FORMULATION

[0001] The present disclosure relates to formulations, pharmaceutical compositions or medicaments containing an antibody, in particular, formulations, pharmaceutical compositions or medicaments containing, as an active ingredient, a bispecific antibody having blood coagulation factor VIII (FVIII)-like activity higher than that of emicizumab. In a non-limiting embodiment, the formulations, pharmaceutical compositions or medicaments of the present disclosure are characterized in being administered to a human subject at a certain antibody concentration or more.

[0002] Hemophilia A is a bleeding abnormality caused by a hereditary decrease or deficiency of FVIII function. For bleeding in hemophilia A patients, FVIII formulations are generally administered (on-demand administration). In recent years, FVIII formulations are also administered prophylactically to prevent bleeding events (NPL 1 and NPL 2) (preventive administration). The half-life of FVIII formulations in blood is approximately 12 to 16 hours. Therefore, for continuous prevention, FVIII formulations are administered to patients three times a week (NPL 3 and NPL 4). In on-demand administrations, FVIII formulations are also additionally administered at regular intervals, when necessary, to prevent rebleeding. In addition, the administration of FVIII formulations is done intravenously. Therefore, there has been a strong need for pharmaceutical agents with a lesser burden than FVIII formulations.

[0003] Occasionally, anti-FVIII antibodies (inhibitors) develop in hemophilia patients. Such inhibitors cancel the effects of the FVIII formulations. For bleeding in patients who have developed inhibitors (inhibitor patients), bypass formulations are administered. Their action mechanisms are not dependent on FVIII function, that is, the function of catalyzing the activation of blood coagulation factor X (FX) by activated blood coagulation factor IX (FIXa). Therefore, in some cases, bypass formulations cannot sufficiently stop the bleeding. Accordingly, there has been a strong need for pharmaceutical agents that are not affected by the presence of inhibitors and can substitute for the function of FVIII.

[0004] In recent years, emicizumab, a humanized bispecific monoclonal antibody that binds to FIXa and FX (PTL 1, PTL 2, and PTL 3), was approved for routine supplementation therapy to prevent or reduce the frequency of bleeding episodes in adult and pediatric hemophilia A patients from newborn to elderly (congenital FVIII deficiency) with or without FVIII inhibitors.

[0005] Routine supplementation therapy with emicizumab resulted in a significant reduction in bleeds that required treatment, in comparison with no routine supplementation therapy in patients with hemophilia A with / without inhibitors and in intra-patient comparisons with routine supplementation therapy with FVIII or routine infusion with bypass formulations (NPL 5 and NPL 6). Since emicizumab can be injected subcutaneously, it is easy to administer. The t1 / 2of emicizumab is estimated to be about 4 to 5 weeks (NPL 7), which is remarkably longer compared with the bypass formulations and the FVIII concentrates. Thus, its usefulness is also promising from the viewpoint of convenience. On the other hand, it is estimated that the FVIII function-substituting activity for the approved dose of emicizumab is approximately 15 IU / dL, which is comparable to the mild hemophilia level (NPL 8 and NPL 9). Emicizumab did not completely prevent bleeds, as 30% to 40% of patients experienced at least one bleed in the clinical studies (NPL 5 and NPL 6). Therefore, it is necessary to administer hemostatic agents intravenously at the time of bleeding, and the risk of developing inhibitors by the FVIII on-demand hemostatic therapy remains.

[0006] In conclusion, although FVIII supplementation therapy is essential for the management of hemophilia A, it requires a great burden to administer, and there is still room for improvement to obtain a healthy and less burdensome life, although it is presumed that emicizumab will be a new standard-of-care in hemophilia A treatment.

[0007] Recently, bispecific antibodies with higher FVIII cofactor function-substituting activity than emicizumab were found from human antibody libraries (PTL 4). NXT007 (rINN：zemocimig), one of such antibodies, is a humanized IgG4 bispecific monoclonal antibody that binds to FIXa and FX to substitute for the function of FVIII as emicizumab (NPL 10). The results of nonclinical studies showed that NXT007 has higher FVIII function-substituting activity than emicizumab (NPL 11). However, the safety, tolerability, PK, PD, and efficacy of NXT007 in humans have not been known and developability of formulations containing NXT007 was unknown.

[0008] In recent years, various antibody formulations have been developed and put into practical use, but many antibody formulations are used as formulations for intravenous injection. Meanwhile, due to needs in actual medical practice, there is high demand for developing antibody-containing formulations as formulations for subcutaneous injection which can be self-injected. Especially, due to its convenience, there is high demand for developing solution formulations enclosed in a pre-filled syringe.

[0009] When designing an antibody-containing formulation for subcutaneous injection, the concentration of antibodies in the liquid to be administered must be made high, because while the amount of antibody to be administered per dose is high (about 80 to 200 mg), the volume of fluid to be injected is generally restricted in subcutaneous injection. However, in the absence of experimental studies, it was unknown whether high concentration NXT007 formulations could be administered to a patient in order to increase blood concentration in a dose-dependent manner.

[0010] Forming of particles in an aqueous solution is a problem in an antibody-containing formulation. The particles formed are aggregates that are larger than multimers such as dimers and trimers, and known particles include sub-visible particles (SVPs) which are microparticles with a particle size of 1.5 micrometers to less than 50 micrometers that are generally difficult to see by eyes, and visible particles (VPs, larger than 100 micrometers) which are visually detectable under standard illuminance (approximately 2,000 to 3,000 lx). Visual detection rate of visible particles in a medicinal formulation varies greatly depending on the person performing detection, and under standard illuminance prescribed in the pharmacopoeia (approximately 2,000 to 3,000 lx), it is reported that detection sensitivity of particles with a particle size of 100 micrometers is about 40%, detection sensitivity of particles with a particle size of 150 micrometers is about 70%, and detection sensitivity of particles with a particle size of 200 micrometers is almost 100% (NPL 12). Actually, particles with smaller particle size of a minimum of about 40 micrometers may be visually detected by increasing illuminance for observing a medicinal formulation or by longer observation times. In the present specification, such particles of 40 micrometers or more to 100 micrometers are particularly referred to as particles visually detectable only under high illuminance. Further, particles having a particle size of 40 micrometers or more are particles visually detectable under high illuminance and are referred to as visually detectable particles.

[0011] [PTL 1] WO 2012 / 067176 [PTL 2] WO 2015 / 194233 [PTL 3] WO 2018 / 047813 [PTL 4] WO 2019 / 065795

[0012] [NPL1] Blood 58, 1-13 (1981) [NPL2] Nature 312, 330-337 (1984) [NPL3] Nature 312, 337-342 (1984) [NPL4] Biochim.Biophys.Acta 871, 268-278 (1986) [NPL5] N Eng J Med. 2017;377(9):809-18. [NPL6] N Eng J Med. 2018;379(9):811-22. [NPL7] Blood. 2016;127(13):1633-41. [NPL8] Thromb Haemost. 2017;117(7):1348-57. [NPL9] N Engl J Med. 2016;374(21):2044-53. [NPL10] Blood (2022) 140 (Supplement 1): 11295-11296. [NPL11] J Thromb Haemost. 2024;22:430-440 [NPL12] James A. Melchore, AAPS PharmSciTech; 2011; 12(1): 215-221

[0013] The present invention was achieved in view of the above circumstances. An objective of the present invention is to provide formulations, pharmaceutical compositions or medicaments containing NXT007 (rINN：zemocimig) that can be administered to human subjects with different body weights, preferably formulations, pharmaceutical compositions or medicaments containing high concentrations of NXT007 that can be administered subcutaneously to achieve sufficient bioavailability. Another objective of the present invention is to provide formulations, pharmaceutical compositions or medicaments containing (preferably high concentrations of) NXT007 that are stable, e.g., have reduced particle formation during storage.

[0014] To achieve the above objective, the present inventors have conducted Phase I / II clinical study (NXT001JG study, also referred to as NXTAGE study) to evaluate the safety, tolerability, PK, PD, and efficacy of NXT007 in healthy adults and hemophilia A patients. During Part A of this study where healthy adults received a single SC administration of NXT007, an issue of nonlinearity (i.e., blood level did not increase in proportion to the dose when the dose of NXT007 was increased) was observed. Due to this issue, it was concerned that the target blood level could not be achieved even if the dose was increased. However, since the dose was increased by increasing the antibody concentration in the NXT007 formulation (also referred to as “drug concentration” in this specification) under a fixed dosing volume (also referred to as “drug volume” in this specification) in the part involving heathy adults, it was unclear whether the increase in dose affected bioavailability or the increase in drug concentration affected bioavailability. Thus, Part D to confirm the effect of drug concentration and Part E to investigate the effect provided when a predetermined dose was divided and administered to multiple sites were additionally examined, and it was found that bioavailability of NXT007 is affected by drug concentration rather than dose. It was also found that if dose is increased in proportion to drug volume while keeping drug concentration constant, blood level increases in proportion to the dose. It was also found that for the drug concentration at or above a certain level, bioavailability does not change and blood level increases in proportion to the dose, thus nonlinearity issue does not occur. It was also found that there is no difference in blood level between a case when the same dosing amount is divided and administered to multiple sites and when it is administered to a single site, and blood level increases in proportion to the total dose irrespective of whether or not the dose is divided, under the same drug concentration.

[0015] As stated above, in Part A of the NXT001JG study, the inventors observed a non-linearity PK issue, in that NXT007 displayed sub-proportional increases in blood levels relative to the doses administered. This non-linearity PK issue was specific to NXT007and was not observed with other antibodies.. Because the concentrations of NXT007 in the drug formulation increased with dose in Part A of the study, the inventors recognized an NXT007-specific problem--that sufficient blood levels of NXT007 may not be achieved with high concentrations of NXT007 in the drug formulation.

[0016] By conducting subsequent experiments in Parts D and E, the inventors found that PK non-linearity was dependent on the concentration of NXT007 in the formulation (also referred to herein as “NXT007 concentration” or “NXT007 formulation concentration” or “drug concentration”) rather than dependent on dose. Furthermore, the inventors found a surprising technical effect: the PK non-linearity occurs only within a certain NXT007 concentration window. For NXT007 concentrations at or above a specific threshold (the “plateau zone”), the non-linearity issue resolves, such that bioavailability stabilizes and remains substantially constant at sufficient levels. This discovery allowed the inventors to design high-concentration NXT007 pharmaceutical formulations characterized by a linear PK profile and stable bioavailability. Accordingly the NXT007 formulations of the present invention are characterized by a stable bioavailability at high concentrations - a property that could not have been predicted from the sub-proportionality PK profile initially observed at lower concentrations.

[0017] The present invention has been made based on such findings, and specifically encompasses the embodiments exemplarily provided below: [1] A formulation or pharmaceutical composition comprising an antibody, wherein the antibody is a bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the first antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the second antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, wherein the concentration of the antibody in the formulation or pharmaceutical composition is 9.94 mg / ml or more (e.g., 10.7 mg / ml or more). [2] The formulation or pharmaceutical composition according to [1], wherein the antibody is a bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 13, and the first antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 14; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 15, and the second antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 16. [3] The formulation or pharmaceutical composition according to [1], wherein the antibody is a bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 21, and the first antibody light chain comprises the amino acid sequence of SEQ ID NO: 22; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 23, and the second antibody light chain comprises the amino acid sequence of SEQ ID NO: 24. [4] The formulation or pharmaceutical composition according to any of [1] to [3], comprising: 9.94 mg / mL or more (e.g., 10.7 mg / ml or more) of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 5 to 80 mmol / L; 100 to 300 mmol / L (preferably, 150 to 225 mmol / L) of L-arginine; 0 to 100 mmol / L (preferably, 20 to 100 mmol / L) of L-methionine; and 0.5 to 5.0 mg / mL of Polysorbate 80, Polysorbate 20, or Poloxamer 188 (preferably, Polysorbate 80) wherein the pH of the formulation or pharmaceutical composition is 5.5 to 7.0. [5] The formulation or pharmaceutical composition according to any of [1] to [3], comprising: 9.94 mg / mL or more (e.g., 10.7 mg / ml or more) of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 18 to 22 mmol / L; 135 to 165 mmol / L of L-arginine; 18 to 22 mmol / L of L-methionine; and 0.45 to 0.55 mg / mL of Polysorbate 80 wherein the pH of the formulation or pharmaceutical composition is 5.4 to 6.6. [6] The formulation or pharmaceutical composition according to any of [1] to [3], comprising: 9.94 mg / mL or more (e.g., 10.7 mg / ml or more) of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 19 to 21 mmol / L; 142.5 to 157.5 mmol / L of L-arginine; 19 to 21 mmol / L of L-methionine; and 0.475 to 0.525 mg / mL of Polysorbate 80 wherein the pH of the formulation or pharmaceutical composition is 5.7 to 6.3. [7] The formulation or pharmaceutical composition according to any of [1] to [3], comprising: 9.94 mg / mL or more (e.g., 10.7 mg / ml or more) of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Polysorbate 80, wherein the pH of the formulation or pharmaceutical composition is 6.0. [8] The formulation or pharmaceutical composition according to any of [1] to [7], wherein the concentration of the antibody is 9.94 to 235 mg / mL (e.g., 10.7 to 235 mg / mL). [9] The formulation or pharmaceutical composition according to any of [1] to [7], wherein the concentration of the antibody is 9.94 to 150 mg / mL (e.g., 10.7 to 150 mg / mL).

[0010] The formulation or pharmaceutical composition according to any of [1] to [7], wherein the concentration of the antibody is 26.67 mg / mL or more.

[0011] The formulation or pharmaceutical composition according to any of [1] to [5], wherein the concentration of the antibody is 26.67 to 150 mg / mL. [12-1] The formulation or pharmaceutical composition according to any of [1] to

[0011] , comprising: 80 mg / mL or 150 mg / ml of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Polysorbate 80, wherein the pH of the formulation or pharmaceutical composition is 6.0. [12-2] The formulation or pharmaceutical composition according to any of [1] to

[0011] , comprising: 150 mg / ml of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Polysorbate 80, wherein the pH of the formulation or pharmaceutical composition is 6.0. [12-3] A formulation or pharmaceutical composition comprising: 150 mg / ml of zemocimig; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Polysorbate 80, wherein the pH of the formulation or pharmaceutical composition is 6.0. [13-1] The formulation or pharmaceutical composition according to any of [1] to

[0011] , comprising: 80 mg / mL or 150 mg / ml of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Polysorbate 20, wherein the pH of the formulation or pharmaceutical composition is 6.0. [13-2] The formulation or pharmaceutical composition according to any of [1] to

[0011] , comprising: 150 mg / ml of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Polysorbate 20, wherein the pH of the formulation or pharmaceutical composition is 6.0. [13-3] A formulation or pharmaceutical composition comprising: 150 mg / ml of zemocimig; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Polysorbate 20, wherein the pH of the formulation or pharmaceutical composition is 6.0. [14-1] The formulation or pharmaceutical composition according to any of [1] to

[0011] , comprising: 80 mg / mL or 150 mg / ml of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Poloxamer 188, wherein the pH of the formulation or pharmaceutical composition is 6.0. [14-2] The formulation or pharmaceutical composition according to any of [1] to

[0011] , comprising: 150 mg / ml of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Poloxamer 188, wherein the pH of the formulation or pharmaceutical composition is 6.0. [14-3] A formulation or pharmaceutical composition comprising: 150 mg / ml of zemocimig; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Poloxamer 188, wherein the pH of the formulation or pharmaceutical composition is 6.0.

[0015] The formulation or pharmaceutical composition according to any of [1] to [9], which is used such that the antibody is administered at a concentration of 9.94 mg / mL or more (e.g., 10.7 mg / ml or more).

[0016] The formulation or pharmaceutical composition according to any of [1] to [8], which is used such that the antibody is administered at a concentration of 9.94 to 235 mg / mL (e.g., 10.7 to 235 mg / mL).

[0017] The formulation or pharmaceutical composition according to any of [1] to [9], which is used such that the antibody is administered at a concentration of 9.94 to 150 mg / mL (e.g., 10.7 to 150 mg / mL).

[0018] The formulation or pharmaceutical composition according to any of [1] to

[0014] , which is used such that the antibody is administered at a concentration of 26.67 mg / mL or more.

[0019] The formulation or pharmaceutical composition according to any of [1] to

[0014] , which is used such that the antibody is administered a concentration of 26.67 to 150mg / mL.

[0020] The formulation or pharmaceutical composition according to any of [1] to

[0019] , which is for administration to a human.

[0021] The formulation or pharmaceutical composition according to any of [1] to

[0020] , which is for use in subcutaneous administration.

[0022] The formulation or pharmaceutical composition according to any of [1] to

[0021] , which is for use in preventing or treating hemophilia A or von Willebrand's disease.

[0023] The formulation or pharmaceutical composition according to

[0022] , which is for use in preventing or treating congenital hemophilia A or acquired hemophilia A.

[0024] The formulation or pharmaceutical composition according to

[0023] , which is for use in preventing or treating severe or moderate congenital hemophilia A without Factor VIII inhibitors.

[0025] The formulation or pharmaceutical composition according to

[0023] , which is for use in preventing or treating severe, moderate or mild congenital hemophilia A with Factor VIII inhibitors.

[0026] The formulation or pharmaceutical composition according to any of [4] to [7] or

[0012] to

[0014] , characterized by having reduced formation of insoluble particles as compared to a reference formulation or pharmaceutical composition having the same component composition except for a lower concentration of L-arginine (i.e., containing the same concentrations of antibody and other components, but with a lower concentration of L-arginine than the formulation or pharmaceutical composition).

[0027] The formulation or pharmaceutical composition according to

[0026] , wherein the insoluble particles are those in the formulation or pharmaceutical composition after stored at 5 to 40 °C.

[0028] The formulation or pharmaceutical composition according to

[0026] , wherein the insoluble particles are those in the formulation or pharmaceutical composition after stored at 40 °C for at most 6 months.

[0029] The formulation or pharmaceutical composition according to any of

[0026] to

[0028] , wherein the insoluble particles are sub-visible particles, and the number of the particles in the formulation or pharmaceutical composition are measured by a micro flow imaging (MFI) method.

[0030] The formulation or pharmaceutical composition according to any of

[0026] to

[0028] , wherein the insoluble particles are visible particles, and the number of the particles in the formulation or pharmaceutical composition are measured by visual inspection, optionally under conditions of an EP method or an EVI method.

[0031] The formulation or pharmaceutical composition according to any of [4] to [7] or

[0012] to

[0014] , characterized by having reduced formation of insoluble particles as compared to a reference formulation or pharmaceutical composition having the same component composition except for absence of surfactant (i.e., containing the same concentrations of antibody and other components, but without surfactants).

[0032] The formulation or pharmaceutical composition according to

[0031] , wherein the surfactant is Polysorbate 80, Polysorbate 20, or Poloxamer 188.

[0033] The formulation or pharmaceutical composition according to any of

[0031] or

[0032] , wherein the insoluble particles are those after a shipping stress test and / or a drop / vibration stress test is conducted.

[0034] The formulation or pharmaceutical composition according to

[0033] , wherein the drop / vibration stress test is conducted in accordance with ASTM D4169

[0035] The formulation or pharmaceutical composition according to any of [4] to [7] or

[0012] to

[0014] , characterized by having reduced formation of antibody size variants as compared to a reference formulation or pharmaceutical composition having the same component composition except for absence of methionine (i.e., containing the same concentrations of antibody and other components, but without methionine).

[0036] The formulation or pharmaceutical composition according to any of [4] to [7] or

[0012] to

[0014] , characterized by having reduced formation of antibody size variants as compared to a reference formulation or pharmaceutical composition having the same component composition except that the buffer is phosphate buffer (i.e., containing the same concentrations of antibody and other components, but the buffer is phosphate buffer).

[0037] The formulation or pharmaceutical composition according to any of [4] to [7] or

[0012] to

[0014] , characterized by having reduced formation of antibody size variants as compared to a reference formulation or pharmaceutical composition having the same component composition except for absence of aspartic acid (i.e., containing the same concentrations of antibody and other components, but without aspartic acid).

[0038] The formulation or pharmaceutical composition according to any of

[0035] to

[0037] , wherein the antibody size variants are HMW forms and / or LMW forms.

[0039] The formulation or pharmaceutical composition according to any of

[0035] to

[0038] , wherein content of the antibody size variants in the formulation or pharmaceutical composition is evaluated by the area ratio [area%] of each peak in size exclusion chromatography (SE-HPLC) or the area ratio [CPA%] of each peak by capillary electrophoresis (CE-SDS (NR: Non-reduction))

[0040] The formulation or pharmaceutical composition according to any of [4] to [7] or

[0012] to

[0014] , characterized by having reduced formation of antibody charge variants as compared to a reference formulation or pharmaceutical composition having the same component composition except that the pH is less than 5.5 (i.e., containing the same concentrations of antibody and other components, but the pH is less than 5.5).

[0041] The formulation or pharmaceutical composition according to

[0040] , wherein the antibody charge variants are acidic components and / or basic components.

[0042] The formulation or pharmaceutical composition according to

[0040] or

[0041] , wherein content of the antibody charge variants is evaluated by the percentage [%] of the peaks of each component by imaged capillary isoelectric focusing (icIEF).

[0043] The formulation or pharmaceutical composition according to any of [4] to [7] or

[0012] to

[0014] , characterized by having reduced viscosity as compared to a reference formulation or pharmaceutical composition having the same component composition except that hydrochloric acid is used to adjust the pH (i.e., containing the same concentrations of antibody and other components, but the pH is adjusted using hydrochloric acid rather than aspartic acid).

[0044] The formulation or pharmaceutical composition according to

[0043] , wherein the viscosity is measured by an EMS viscometer.

[0045] The formulation or pharmaceutical composition according to any of [4] to [7] or

[0012] to

[0014] , characterized by having higher colloidal stability as compared to a reference formulation or pharmaceutical composition having the same component composition except that L-arginine concentration is less than 100 mmol / L (i.e., containing the same concentrations of antibody and other components, but L-arginine concentration is less than 100 mmol / L).

[0046] The formulation or pharmaceutical composition according to

[0045] , wherein the colloidal stability is evaluated by measuring the diffusion coefficient (D) by a dynamic scattering (DLS) method and calculating the kD value by plotting the D value against the antibody concentration.

[0047] The formulation or pharmaceutical composition according to any of [1] to

[0025] , characterized by absence of visible particles in the formulation or pharmaceutical composition after stored at 5 °C for at most 36 months.

[0048] The formulation or pharmaceutical composition according to

[0047] , wherein the absence of visible particles is determined by EVI method.

[0049] The formulation or pharmaceutical composition according to any of [1] to

[0025] , characterized by absence of visible particles in the formulation or pharmaceutical composition following at most 4 months of a shipping stress test and / or drop / vibration test.

[0050] The formulation or pharmaceutical composition according to

[0049] , wherein the absence of visible particles is determined by EP method.

[0051] A medicament comprising a bispecific antibody for use in treatment of hemophilia A with or without an inhibitory substance, wherein the bispecific antibody comprises: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the first antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the second antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, wherein the concentration of the bispecific antibody in the medicament is 9.94 mg / ml or more (e.g., 10.7 mg / ml or more), and wherein the bispecific antibody is administered subcutaneously to a human patient.

[0052] A medicament comprising a bispecific antibody for use in treatment of hemophilia A with or without an inhibitory substance, wherein the bispecific antibody comprises: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the first antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the second antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, wherein the concentration of the bispecific antibody in the medicament is 26.67 mg / ml or more, and wherein the bispecific antibody is administered subcutaneously to a human patient.

[0053] The medicament according to

[0051] or

[0052] , wherein the bispecific antibody comprises: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 13, and the first antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 14; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 15, and the second antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 16.

[0054] The medicament according to

[0051] or

[0052] , wherein the bispecific antibody comprises: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 21, and the first antibody light chain comprises the amino acid sequence of SEQ ID NO: 22; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 23, and the second antibody light chain comprises the amino acid sequence of SEQ ID NO: 24.

[0055] The formulation or pharmaceutical composition according to any of [1] to

[0050] or the medicament according to any of

[0051] to

[0054] , further comprising a recombinant human hyaluronidase PH20 (rHuPH20).

[0018] Fig. 1 depicts a linear chart (A) and a single logarithmic chart (B) showing time-dependent changes of NXT007 plasma concentration (mean + / - SD) in healthy adults who did not develop anti-drug antibodies against NXT007 after administration of the NXT007 formulation of various doses, in NXT001JG study (also referred to as NXTAGE study), Part A. Numerical values in legends indicate the doses of the administered NXT007 formulation.See, the description of Fig. 1A.Fig. 2 depicts linear charts (A)(B) and single logarithmic charts (C)(D) showing time-dependent changes of NXT007 plasma concentration (mean + / - SD) in healthy adults who did not develop anti-drug antibodies against NXT007 after single dose administration of the NXT007 formulation (nominal dose: 0.054 mg / kg) of various concentrations, in NXT001JG study, Part A and Part D. For Cohort A-3 of Part A, which has a nominal dose of 0.018 mg / kg, mean and SD of the plasma NXT007 level were triplicated to indicate as the data of nominal dose of 0.054 mg / kg. (A) and (C) show the time-dependent change up to 42 days after administration, and (B) and (D) show the time-dependent change up to the final observation time point. Numerical values shown along with cohort names in legends indicate the drug concentrations (mg / mL) of the administered NXT007 formulation.See, the description of Fig. 2A.See, the description of Fig. 2A.See, the description of Fig. 2A.Fig. 3 depicts linear charts (A)(B) and single logarithmic charts (C)(D) showing time-dependent changes of NXT007 plasma concentration (mean + / - SD) in healthy adults who did not develop anti-drug antibodies against NXT007 after single dose administration of the NXT007 formulation (nominal dose 0.18 mg / kg) of various concentrations, in NXT001JG study, Part A and Part D. For Cohort A-4 of Part A, which has a nominal dose of 0.054 mg / kg, mean and SD of the plasma NXT007 level were multiplied by 3.33 to indicate as the data of nominal dose of 0.18 mg / kg. (A) and (C) show the time-dependent change up to 42 days after administration, and (B) and (D) show the time-dependent change up to the final observation time point. Numerical values shown along with cohort names in legends indicate the drug concentrations (mg / mL) of the administered NXT007 formulation.See, the description of Fig. 3A.See, the description of Fig. 3A.See, the description of Fig. 3A.Fig. 4 depicts linear charts (A)(B) and single logarithmic charts (C)(D) showing time-dependent changes of NXT007 plasma concentration (mean + / - SD) in healthy adults who did not develop anti-drug antibodies against NXT007 after the NXT007 formulation of a nominal dose of 0.054 mg / kg were administered as a single dose to a single site or divided and administered as single doses to three sites, in NXT001JG study, Part A and Part E. For Cohort A-3 of Part A, which has a nominal dose of 0.018 mg / kg, mean and SD of the plasma NXT007 level were triplicated to indicate as the data of nominal dose of 0.054 mg / kg. (A) and (C) show the time-dependent change up to 42 days after administration, and (B) and (D) show the time-dependent change up to the final observation time point. Numerical values shown along with cohort names in legends indicate the drug concentrations (mg / mL) of the administered NXT007 formulation.See, the description of Fig. 4A.See, the description of Fig. 4A.See, the description of Fig. 4A.Fig. 5 depicts linear charts (A)(B) and single logarithmic charts (C)(D) showing time-dependent changes of NXT007 plasma concentration (mean + / - SD) in healthy adults who did not develop anti-drug antibodies against NXT007 after the NXT007 formulation of a nominal dose of 0.162 mg / kg were administered as a single dose to a single site or divided and administered as single doses to three sites, in NXT001JG study, Part A and Part E. For Cohort A-4 of Part A, which has a nominal dose of 0.054 mg / kg, mean and SD of the plasma NXT007 level were triplicated to indicate as the data of nominal dose of 0.162 mg / kg. For Cohort A-5 of Part A, which has a nominal dose of 0.18 mg / kg, mean and SD of the plasma NXT007 level were multiplied by 0.9 to indicate as the data of nominal dose of 0.162 mg / kg. (A) and (C) show the time-dependent change up to 42 days after the administration, and (B) and (D) show the time-dependent change up to the final observation time point. Numerical values shown along with cohort names in legends indicate the drug concentrations (mg / mL) of the administered NXT007 formulation.See, the description of Fig. 5A.See, the description of Fig. 5A.See, the description of Fig. 5A.Fig. 6 depicts single logarithmic charts showing dose-normalized Cmax(A) and dose-normalized AUCinf(B) of NXT007 in healthy adults who did not develop anti-drug antibodies against NXT007 after single dose administration of the NXT007 formulation of various drug concentrations, in NXT001JG study, Part A, Part D, and Part E. Cmaxrefers to maximum plasma concentration, and AUCinfrefers to area under the plasma concentration-time curve extrapolated to infinity. The solid line curves show predictive values of PK parameters when the relationship between dose-normalized Cmax(A) and dose-normalized AUCinfis applied to an inhibitory sigmoid maximum effect model. Numerical values shown along with cohort names in legends indicate the doses (mg / kg) of the administered NXT007 formulation.See, the description of Fig. 6A.Fig. 7A depicts a graph showing the results of the measurement of the percentage [area%] of HMW Forms (HMWS) by size exclusion chromatography (SE-HPLC), when the samples with each of the component compositions shown in Table 16 were stored under each condition.Fig. 7B depicts a graph showing the results of the measurement of the percentage [CPA%] of LMW Forms (LMWS) by capillary electrophoresis (CE-SDS (non-reduction (NR)), when the samples with each of the component compositions shown in Table 16 were stored under each condition.Fig. 8 depicts graphs showing the results of the measurement of the percentages [area%] of HMW Forms (upper graph) and LMW Forms (lower graph) by SE-HPLC, when the samples with each of the component compositions shown in Table 20 were stored under each condition.Fig. 9 depicts a graph showing the results of the measurement of the percentage [area%] of HMW Forms by SE-HPLC, when the samples with each of the component compositions shown in Table 23 were stored under each condition.Fig. 10 depicts graphs showing the results of the measurement of the percentages [%] of acidic peaks (upper graph) and basic peaks (lower graph) by image capillary isoelectric focusing (icIEF), when the samples with each of the component compositions shown in Table 23 were stored under each condition.Fig. 11 depicts graphs showing the results of the measurement of the percentages [%] of acidic components (upper graph) and basis components (lower graph) by icIEF, when the samples with each of the component compositions shown in Table 27 were stored under each condition.Fig. 12 depicts a graph showing the results of the measurement of the viscosity of the samples with each of the component compositions shown in Table 30 by EMS viscometer.Fig. 13 depicts a graph showing the results of the measurement of the percentage [area%] of HMW forms in the samples with each of the component compositions shown in Table 33 by SE-HPLC.Fig. 14 depicts a graph showing the results of the measurement of kD values [mL / g] of the samples with each of the component compositions shown in Table 36 by a dynamic scattering (DLS) method.Fig. 15 depicts a graph showing the results of the measurement of the number of insoluble particles (the number of sub-visible particles with each particle size per mL) by a micro flow imaging (MFI) method, when the samples with each of the component compositions shown in Table 39 were stored under each condition.

[0019] (Detailed Description) 0 Abbreviations and Definitions of terms

[0020] 1. Hemophilia A 1.1 Definition and Epidemiology of Hemophilia A Hemophilia A is a life-long hemorrhagic disease attributed to a congenital absence or dysfunction of blood coagulation factor VIII (FVIII). The gene that encodes FVIII is located on the X chromosome, and therefore the disease is in an X-linked recessive mode of inheritance, and more than 99% of hemophilia patients are males. The incidence of hemophilia A is approximately one in 5,000 live-born males[1-4]. No ethnic differences have been reported, and the numbers of patients with hemophilia A registered in 2017 on the World Federation of Hemophilia (WFH) report were 5,450 in Japan, 13,639 in the United State (U.S.), 6,713 in the United Kingdom (UK), 6,126 in France, 6,342 in Russia, 12,533 in China, and 10,395 in Brazil. The numbers of patients with hemophilia A with inhibitors were 839 in the U.S., 212 in the UK, 153 in France, 187 in Germany, 200 in Russia, and 384 in Brazil[5].

[0021] 1.2 Diagnosis and Clinical Manifestation of Hemophilia A In patients with hemophilia A, although clotting time, platelet counts, and prothrombin time (PT) are normal, activated partial thromboplastin time (APTT) is prolonged. A test result of less than 40 international units (IU) / dL of FVIII activity is diagnosed as hemophilia A. However, the diagnosis must be confirmed by normal level of von Willebrand factor antigen, because FVIII is also decreased in von Willebrand disease[6]. The severity of hemophilia A is classified according to the patient's residual endogenous FVIII activity (severe: <1 IU / dL, moderate: 1-5 IU / dL, and mild: >5-40 IU / dL)[2,4,7]. In severe hemophilia A, recurrent spontaneous bleeds, typically into joints or muscles, are observed. In moderate hemophilia A, spontaneous bleeds are less frequent than severe, but minor trauma can lead to bleeding. Mild hemophilia A leads to hemostasis difficulties after tooth extraction, surgery, and trauma, but spontaneous bleeds are rare[6]. Repeated intra-articular bleeding is the main reason leading to reduced QoL in hemophilia A, because the patients develop hemophilic arthropathy, which is associated with joint damage and difficulty in walking, and may ultimately require surgical intervention, including joint replacement. Patients with hemophilia A can also develop subcutaneous, oral, gastrointestinal, and intranasal bleeding, and intracranial bleeding can be fatal.

[0022] 1.3 Current Treatment Option and Unmet Medical Need The WFH has issued “Guidelines for the management of hemophilia”[8]. As described in the Guidelines, the standard therapy for patients with hemophilia A is FVIII supplementation therapy using recombinant FVIII (rFVIII) formulations or plasma derived FVIII formulations. FVIII supplementation therapies can be broadly categorized into 2 types: on-demand hemostasis therapy, in which FVIII formulations are administered whenever bleeding symptoms occur, and routine supplementation (prophylaxis) therapy, in which FVIII formulations are administered regularly to prevent the onset of bleeding symptoms. Routine supplementation therapy maintains blood FVIII activity at 1 IU / dL or higher at trough, and effectively prevents the onset or progression of hemophilic arthropathy by inhibiting bleeding symptoms (particularly repeated bleeding into joints)[7,9,10]. The rate of routine supplementation therapy has recently been increasing in developed countries in recent days due to an expanded supply of rFVIII concentrates, in addition to the therapeutic advantages described above

[0011] . Although intra-articular bleeding in patients with severe disease is seen from about one year after birth, the proportion of patients with no arthropathy is markedly higher among patients who started routine supplementation therapy before the age of 2, than in patients who started the therapy at a later age

[0012] . Hence, to forestall development of arthropathy, the recommended treatment in pediatric patients with severe hemophilia A is “primary routine supplementation therapy” - that is, routine supplementation therapy initiated before the age of 2, or after the first episode of intra-articular bleeding[7,12,13,14].

[0023] Meanwhile, unfortunately, it has recently been reported that progressive joint disability was observed by magnetic resonance imaging (MRI) examination in up to two thirds of patients with hemophilia A receiving sufficient primary routine supplementation therapy[15,16]. The symptoms were observed before the age of 10, and also appeared in joints where clinically no bleeding was observed. In addition, it was reported that Hemophilia Joint Health Score of 10 or more was observed in 11% to 46% of patients, and in another report, the existence of target joints

[0017] , a decrease in walking ability, or chronic pain were observed in 40% of patients

[0018] . These findings suggest that progression of hemophilic arthropathy occurs in some patients because of synovitis due to asymptomatic micro bleeding

[0019] . Thus, it suggests that routine supplementation therapy does not completely prevent the occurrence of musculoskeletal disorders in the long-term

[0020] .

[0024] Generally, to introduce routine supplementation therapy (especially primary routine supplementation therapy), home injection therapy is required. It is difficult for patients and caregivers to inject at home twice or 3 times a week at the beginning. In particular, insertion of a central venous access device is sometimes necessary because of the difficulty of securing a blood vessel in the case of an infant. However, insertion of a central venous access device carries the risk of causing pneumothorax or hemothorax when inserting the catheter into the subclavian vein or the risk of infection via the central venous access device. In addition, although it is rare, thrombosis and breakdown of the central venous access device may occur[7]. Thus, there are various problems in the introduction of routine supplementation therapy (especially primary routine supplementation therapy).

[0025] In addition, patients with hemophilia A can develop alloantibodies (inhibitors) that neutralize FVIII after repetitive exposure to FVIII concentrates. The inhibitors are likely to develop within 50 days of treatment initiation (if they occur)[7,21]and the risk of developing inhibitors is the highest before the age of 5

[0022] . It is reported that the prevalence of inhibitors in congenital hemophilia A is 20% to 30%[23-26]. The inhibitors markedly reduce the hemostatic effect of FVIII concentrates, so the development of inhibitors is a serious problem. In the therapeutic guidelines of the WFH, inhibitors are classified as low-titer (<5 BU / mL) or high-titer (5 BU / mL or more) based on immune response to FVIII concentrates[5]. In most patients with low-titer inhibitors, FVIII concentrates at a higher dose to neutralize the inhibitors could be a first choice. For patients with inhibitors at high titers, the first choice for treatment is “bypass formulations” such as recombinant activated blood coagulation factor VII (rFVIIa), activated prothrombin complex formulation (aPCC), or plasma-derived activated blood coagulation factor VII and blood coagulation factor X (pdFVIIa / FX). However, bypass formulations present difficulties in the management of hemostasis. Because the hemostatic effect of bypass formulations is unstable in comparison with that of FVIII concentrates, rFVIIa and pdFVIIa / FX require frequent administration due to short blood half-life (t1 / 2) [2.3 hours for rFVIIa

[0027] and 2.79 hours for FVIIa of pdFVIIa / FX

[0028] ], and the injection time of aPCC is long (25 to 50 minutes per injection

[0029] ).

[0026] In recent years, routine infusion of rFVIIa and aPCC showed results suggesting efficacy[30,31]. However, the hemostatic effect of routine bypass formulation infusion in patients with inhibitors is not sufficient: patients who take routine aPCC infusion achieve an annualized bleeding rate (ABR) of 7.9

[0032] , and patients who take routine rFVIIa infusion experience 2-3 bleeds / month

[0031] . In addition, the short blood t1 / 2of bypass formulations requires frequent injection. Furthermore, since aPCC is a plasma derived formulation, it is theoretically impossible to completely eliminate the risk of infection derived from blood donors.

[0027] Patients with inhibitors can also be treated with immune tolerance induction (ITI) therapy to eliminate the inhibitors, and 60 to 80% of the patients are possible to return to FVIII routine supplementation therapy[33,34]. However, patients to whom ITI can be applied are limited in terms of their inhibitor titer, and because it requires daily or several times weekly use of high-dose FVIII formulations over an extended period of time, the physical burden on patients is substantial. Especially, pediatric patients who are being treated with ITI have a risk of complication such as infections, because the treatment requires placement of central venous access devices. Patients who failed ITI may have persistent inhibitors.

[0028] In recent years, emicizumab, a humanized bispecific monoclonal antibody that binds to activated blood coagulation factor IX (FIXa) and FX, was approved for routine supplementation therapy to prevent or reduce the frequency of bleeding episodes in adult and pediatric hemophilia A patients from newborn to elderly (congenital FVIII deficiency) with or without FVIII inhibitors.

[0029] Routine supplementation therapy with emicizumab resulted in a significant reduction in bleeds that required treatment, in comparison with no routine supplementation therapy in patients with hemophilia A with / without inhibitors, and in intra-patient comparisons with routine supplementation therapy with FVIII or routine infusion with bypass formulations[35,36]. Since emicizumab can be injected subcutaneously, it is easy to administer. The t1 / 2of emicizumab is estimated to be about 4 to 5 weeks

[0037] , which is remarkably longer compared with the bypass formulations and the FVIII concentrates. Thus, its usefulness is also promising from the viewpoint of convenience. On the other hand, it is estimated that the FVIII function-substituting activity for the approved dose of emicizumab is approximately 15 IU / dL, which is comparable to the mild hemophilia level[38,39]. Emicizumab did not completely prevent bleeds, as 30% to 40% of patients experienced at least one bleed in the clinical studies[35,36]. Therefore, it is necessary to administer hemostatic agents intravenously at the time of bleeding, and the risk of developing inhibitors by the FVIII on-demand hemostatic therapy remains. In addition, there is no evidence for prevention of hemophilic arthropathy.

[0030] In conclusion, although FVIII supplementation therapy is essential for the management of hemophilia A, it requires a great burden to administer, and there is still room for improvement to obtain a healthy and less burdensome life, although it is presumed that emicizumab will be a new standard-of-care in hemophilia A treatment.

[0031] 2 NXT007 NXT007 is a humanized monoclonal modified immunoglobulin G4 (IgG4) antibody with a bispecific antibody structure that binds to FIXa and FX to substituted for the function of FVIII, which was invented by Chugai pharmaceutical Co. Ltd. NXT007 has the same mode of action that binds to FIXa and FX as emicizumab, which has already been approved in the U.S., the EU, and Japan. NXT007 can accelerate FX activation by FIXa in patients with hemophilia A who have loss of function of FVIII and / or reduced FVIII, and thus expected to promote coagulation reaction and promote effective hemostasis. From the results of nonclinical studies, NXT007 showed higher FVIII function-substituting activity than emicizumab. It is expected that NXT007 increases FVIII activity to a non-hemophilic level, at least 40 IU / dL. Since NXT007 shares no sequence homology with FVIII, it is suggested that NXT007 can exert the FVIII function-substituting activity in patients with hemophilia A irrespective of the presence of FVIII inhibitors, and does not induce the development of FVIII inhibitors.

[0032] 2.1 Molecule and Nonclinical Data 2.1.1 Physical, Chemical, and Pharmaceutical Properties and Clinical Formulation NXT007 is a humanized monoclonal modified IgG4 antibody with a bispecific antibody structure that binds to FIXa and FX as FVIII does. NXT007 has two different heavy chains (H chains) (447 and 444 amino acid residues, respectively) and two different light chains (L chains) (214 and 213 amino acid residues, respectively), including 4 inter-chain disulfide bonds and 12 intra-chain disulfide bonds.

[0033] 2.1.2 Nonclinical Pharmacology NXT007 is a bispecific antibody composed of two different heavy chains and two different light chains. The two heavy chains were generated by modifying those of emicizumab, while the two light chains were newly generated. The FVIII function-substituting activity and characteristics of NXT007 were investigated by in vitro and in vivo pharmacological tests and reported (Non-patent Document 11). In in vitro evaluation, a thrombin generation assay using hemophilia A plasma was conducted. In vivo hemostatic activity was evaluated with non-human primate models of acquired hemophilia A. The results demonstrated that NXT007 showed in vitro thrombin generation activity (when triggered by tissue factor) comparable to the international standard activity (100 IU / dL) for FVIII, which is far above that of emicizumab. NXT007 also showed potent in vivo hemostatic activity at the plasma concentration of about 30-fold lower than that of emicizumab in previous data. Regarding the dose shift of NXT007 and emicizumab, the in vivo result was consistent with the in vitro result. Regarding PK, NXT007 showed lower in vivo clearance than common monoclonal antibodies, which suggests that Fc engineering to enhance FcRn binding successfully functioned.

[0034] (Description of Embodiments) In a non-limiting aspect, the present disclosure provides formulations, pharmaceutical compositions, or medicaments (herein, these may be comprehensively referred to as “the formulations of the present disclosure”) containing at least 9.94 mg / mL (e.g., at least 10.7 mg / mL) of NXT007 (rINN：zemocimig), a bispecific antibody that binds to both FIX and / or FIXa and FX to substitute for the function of FVIII (i.e., the concentration of the active ingredient in the formulation, pharmaceutical composition or medicament is at least 9.94 mg / mL, e.g., at least 10.7 mg / mL). In some embodiments, the formulation of the present disclosure is a stable antibody-containing formulation, characterized by having reduced formation of insoluble particles, antibody size variants, and / or antibody charge variants during storage; reduced viscosity; and / or higher colloidal stability.

[0035] In the present specification, the phrases “substitute for the function of FVIII” and “substitute for the function of FVIIIa” can be used interchangeably, and mean recognizing FIX or FIXa, and FX, and promoting FX activation by FIXa (promoting FXa production by FIXa). FXa production-promoting activity can be evaluated using, for example, a measurement system comprising FXIa, FX, synthetic substrate S-2222 (synthetic substrate of FXa), and phospholipids. Such a measurement system shows a correlation with the disease severity and clinical symptoms in hemophilia A cases (Rosen S, Andersson M, Blomba¨ck M et al. Clinical applications of a chromogenic substrate method for determination of FVIII activity. Thromb Haemost 1985; 54: 811-23).

[0036] NXT007, a bispecific antibody contained in the formulations of the present disclosure as an active ingredient, is specifically a bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively (QH06-QC3 chain), and the first antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively (QL32-CL2 chain); and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively (JH07-JC3 chain), and the second antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively (JL07-CL4 chain).

[0037] More specifically, NXT007 is a bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 13 (QH06-QC3 chain), and the first antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 14 (QL32-CL2 chain); and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 15 (JH07-JC3 chain), and the second antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 16 (JL07-CL4 chain). More specifically, NXT007 comprises: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises a heavy chain constant region which comprises the amino acid sequence of SEQ ID NO: 17 (QH06-QC3 chain), and the first antibody light chain comprises a light chain constant region which comprises the amino acid sequence of SEQ ID NO: 18 (QL32-CL2 chain); and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises a heavy chain constant region which comprises the amino acid sequence of SEQ ID NO: 19 (JH07-JC3 chain), and the second antibody light chain comprises a light chain constant region which comprises the amino acid sequence of SEQ ID NO: 20 (JL07-CL4 chain).

[0038] More specifically, NXT007 is a bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 21 (QH06-QC3 chain), and the first antibody light chain comprises the amino acid sequence of SEQ ID NO: 22 (QL32-CL2 chain); and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 23 (JH07-JC3), and the second antibody light chain comprises the amino acid sequence of SEQ ID NO: 24 (JL07-CL4).

[0039] The amino acids contained in the amino acid sequences disclosed in the specification may be modified after translation (for example, modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation is well known for those skilled in the art). Those having amino acids with such post-translational modifications are of course included in the antibodies used in the formulations of the present disclosure.

[0040] In the present invention, polypeptides generally refer to peptides and proteins having a length of approximately ten amino acids or longer. They are usually biologically derived polypeptides, but are not particularly limited thereto, and may be, for example, polypeptides comprising an artificially designed sequence. Furthermore, they may be any of naturally-occurring polypeptides, synthetic polypeptides, recombinant polypeptides, and such. Additionally, fragments of the above-mentioned polypeptides are also included in the polypeptides of the present invention.

[0041] The term “antibody” is used in the broadest sense, and can be monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (such as bispecific antibodies), antibody derivatives, and modified antibodies (Miller K et al. J Immunol. 2003, 170(9), 4854-61) so long as they show a desired biological activity. The antibodies may be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or those derived from another species or artificially synthesized. The antibodies disclosed herein can be of any type (for example, IgG, IgE, IgM, IgD, and IgA), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules. The immunoglobulins can be derived from any species (for example, human, mouse, or rabbit). The terms “antibody”, “immune globulin” and “immunoglobulin” are used interchangeably in a broad sense.

[0042] “Bispecific” antibodies refer to antibodies having two variable regions that each recognize different epitopes, where the variable regions are present in the same antibody molecule. Bispecific antibodies may be antibodies that recognize two or more different antigens, or antibodies that recognize two or more different epitopes on the same antigen. Bispecific antibodies may include not only whole antibodies but antibody derivatives.

[0043] Recombinant antibodies produced by using genetic engineering techniques can be used as the antibodies. A recombinant antibody can be obtained by cloning a DNA encoding the antibody from hybridomas or antibody-producing cells such as sensitized lymphocytes that produce antibodies; incorporating this into a vector; and then introducing it into hosts (host cells) to produce the antibody.

[0044] Bispecific antibodies are not limited to those of the IgG type; for example, IgG-type bispecific antibodies can be secreted from a hybrid hybridoma (quadroma) produced by fusing two types of hybridomas that produce IgG antibodies (Milstein C. et al., Nature 1983, 305: 537-540). They can also be secreted by introducing into cells the L-chain and H-chain genes constituting the two kinds of IgGs of interest, i.e., a total of four kinds of genes, to co-express the genes.

[0045] Antibodies of the present invention can be produced by methods known to those skilled in the art. Specifically, a DNA encoding the antibody of interest is inserted into an expression vector. The insertion into the expression vector is carried out such that the expression will take place under the control of expression regulatory regions such as an enhancer and a promoter. Next, host cells are transformed using this expression vector to express the antibody. When doing so, appropriate combinations of a host and an expression vector can be used.

[0046] The antibodies of the present invention thus obtained can be isolated from the inside of host cells or the outside of the cells (medium, etc.), and purified to be substantially pure, homogeneous antibodies. The antibodies can be separated and purified by methods ordinarily used for separating and purifying antibodies, and the methods are not limited in any way. For example, the antibodies can be separated and purified by appropriately selecting and combining column chromatography, filtration, ultrafiltration, salting-out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectrofocusing, dialysis, recrystallization, and such.

[0047] In a non-limiting embodiment, the formulation of the present disclosure is an NXT007 antibody-containing formulation that provides excellent bioavailability in a human. The concentration of the antibody contained in the formulation of the present disclosure is, for example, 9.94 mg / mL or more, 10.7 mg / mL or more, 26.67 mg / mL or more, 50 mg / mL or more, 80 mg / mL or more, or 150 mg / mL or more. The concentration of the antibody contained in the formulation of the present disclosure is, for example, 5 to 235 mg / mL, 10 to 235 mg / mL, 5 to 220 mg / mL, 10 to 220 mg / mL, 5 to 150 mg / mL, or 10 to 150 mg / mL, preferably 9.94 to 235 mg / mL, 10.7 to 235 mg / mL, 26.67 to 235 mg / mL, 50 to 235 mg / mL, 80 to 235 mg / mL, 9.94 to 220 mg / mL, 10.7 to 220 mg / mL, 26.67 to 220 mg / mL, 50 to 220 mg / mL, 80 to 220 mg / mL, 9.94 to 150 mg / mL, 10.7 to 150 mg / mL, 26.67 to 150 mg / mL, 50 to 150 mg / mL, or 80 to 150 mg / mL, such as 9.94 mg / mL, 10.7 mg / mL, 26.67 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 125 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 180 mg / mL, 200 mg / mL, or 220 mg / mL. The upper limit of the concentration of the antibody contained in the formulation of the present disclosure is, for example, 150 mg / mL, 220 mg / mL, or 235 mg / mL. The formulation of the present disclosure is appropriately diluted to be administered to a subject (in particular, human subject). In some embodiments, the formulation of the present disclosure is used such that it is administered to the subject (in particular, human subject) at the antibody concentration of 0.0994 mg / mL or more, 0.107 mg / mL or more, 0.2667 mg / mL or more, 0.8 mg / mL or more, 0.994 mg / mL or more, 1.07 mg / mL or more, 2.667 mg / mL or more, 8 mg / mL or more, 9.94 mg / mL or more, 10.7 mg / mL or more, 26.67 mg / mL or more, 50 mg / mL or more, 80 mg / mL or more, or 150 mg / mL or more, such as 9.94 to 235 mg / mL, 10.7 to 235 mg / mL, 26.67 to 235 mg / mL, 9.94 to 150 mg / mL, 10.7 to 150 mg / mL, 26.67 to 150 mg / mL, or 80 to 150 mg / mL, such as 9.94 mg / mL, 10.7 mg / mL, 26.67 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, or 180 mg / mL. In preferred embodiments the antibody concentration in the formulation is 150 mg / ml.

[0048] In some embodiments, the formulation of the present disclosure is a stable antibody-containing formulation which can be administered to a human. In the present invention, the term “stable antibody-containing formulation” refers to a formulation in which aggregates (herein also be referred to as size variants) and / or components with charge heterogeneity (herein also be referred to as charge variants) from proteins such as antibodies are less likely to be generated therein, i.e., the formulations in which deterioration reactions, including generation of insoluble aggregates, soluble aggregates, or components with charge heterogeneity, are less likely to occur. “Components with charge heterogeneity” refer to components having protein surface charges that are different from those of the major component due to deamidation, oxidation, hydrolysis, and such. Stability of antibody-containing formulations during storage can be evaluated by freeze-thaw test, thermal acceleration test, long term storage test, cryopreservation test, and such.

[0049] In some embodiments, the formulation of the present disclosure comprises a histidine buffer, a citrate buffer, or a Tris buffer, preferably a histidine buffer, as a buffer. The concentration (amount) of the buffer in the formulation of the present disclosure is 5 to 80 mmol / L, such as 5 mmol / L, 10 mmol / L, 20 mmol / L, 40 mmol / L, or 80 mmol / L, preferably 10 to 40 mmol / L, and particularly preferably 20 mmol / L. The histidine contained in the formulation of the present disclosure is preferably L-histidine. In the formulation of the present disclosure, histidine may be included as a salt, and examples of such salt include histidine-hydrochloride, histidine-aspartate, and histidine-glutamate.

[0050] In some embodiments, the formulation of the present disclosure includes a histidine-aspartate buffer containing, in addition to histidine, aspartic acid, malic acid, citric acid, succinic acid, tartaric acid, malonic acid, or alpha-ketoglutaric acid, preferably aspartic acid, which is an acidic amino acid, as a counterion species thereof. A preferable embodiment of the histidine-aspartate buffer in the formulation of the present invention is a buffer prepared by titrating a solution such as an aqueous solution supplemented with histidine as a free amino acid, with a liquid such as an aqueous solution containing aspartic acid as a free amino acid. Alternatively, the buffer can be prepared by adding the amino acids in the reverse order, or by direct titration with powders.

[0051] The concentration (amount) of the histidine-aspartate buffer in the formulation of the present invention is 5 to 80 mmol / L, such as 5 mmol / L, 10 mmol / L, 20 mmol / L, 40 mmol / L, or 80 mmol / L, preferably 10 to 40 mmol / L, and particularly preferably 20 mmol / L. These concentrations represent histidine concentrations in the histidine-aspartate buffer in the formulation of the present invention, and a skilled artisan can readily recognize the concentration (amount) of aspartic acid, which is added for pH control as the counterion species of histidine, from the pH of the formulation and the histidine concentration.

[0052] In one embodiment, the formulation of the present disclosure is a solution formulation. In another embodiment, the formulation of the present disclosure is a lyophilized formulation. The pH in a solution state (pH of the solution formulation, or pH of a solution after the lyophilized formulation is redissolved in water) of the formulation of the present disclosure is preferably 5.5 to 7.0, more preferably 5.5 to 6.5, and further preferably 5.5 to 6.0, particularly preferably 6.0.

[0053] In some embodiments, the formulation of the present disclosure comprises arginine as a stabilizer and / or isotonizing agent. The arginine is preferably L-arginine. In the formulation of the present disclosure, arginine can be included as a single substance, or a derivative or salt thereof, and examples of such salt include arginine-hydrochloride, arginine-aspartate, and arginine-glutamate. The concentration (amount) of arginine in the formulation of the present disclosure is 100 to 300 mmol / L, or 150 to 300 mmol / L, preferably 150 to 225 mmol / L, such as 100 mmol / L, 150 mmol / L, 175 mmol / L, 200 mmol / L, or 225 mmol / L, particularly preferably 150 mmol / L.

[0054] In some embodiments, the formulation of the present disclosure comprises methionine as an antioxidant. The methionine is preferably L-methionine. The concentration (amount) of methionine in the formulation of the present disclosure is 20 to 100 mmol / L, such as 20 mmol / L, 40 mmol / L, 60 mmol / L, or 100 mmol / L, preferably 20 mmol / L.

[0055] Surfactants contained in the formulation of the present invention are, for example, Polysorbate 20 (or PS20), Polysorbate 80 (or PS80), Poloxamer 188 (also referred to as Pluronic F-68; polyethylene (160) polyoxypropylene (30) glycol), and Polysorbate 80 is particularly preferred. The amount of Polysorbate 80 added relative to the formulation of the present invention is preferably 0.5 mg / mL to 5.0 mg / mL, such as 0.5 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, and 5.0 mg / mL, and particularly preferably 0.5 mg / mL.

[0056] The formulation of the present invention can further contain amino acids. Preferred amino acids for use in the present invention are natural amino acids or amino acid derivatives, and particularly preferred amino acids are L-methionine and L-proline. The formulation of the present invention can further contain sugars. Preferred sugars used in the present invention are sucrose, trehalose, meglumine, and sorbitol. The amount of amino acid or sugar added to the formulation of the present invention is generally 1 mM to 1000 mM, preferably 5 mM to 500 mM, and more preferably 10 mM to 300 mM.

[0057] The formulation of the present invention can further contain inorganic salts. The preferred inorganic salts used in the present invention are magnesium salts and calcium salts.

[0058] If needed, cryoprotectants, suspending agents, solubilizing agents, isotonizing agents, preservatives, adsorption inhibitors, diluents, excipients, pH adjustors, analgesics, sulfur-containing reducing agents, antioxidants, and such can be appropriately added to the formulation of the present invention.

[0059] Cryoprotectants include sugars such as trehalose, sucrose, and sorbitol.

[0060] Solubilizing agents include polyoxyethylene hardened castor oil, Polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl ester.

[0061] Isotonizing agents include sodium chloride, potassium chloride, and calcium chloride.

[0062] Preservatives include methyl-p-hydroxybenzoate, ethyl-p-hydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

[0063] Adsorption inhibitors include human serum albumin, lecithin, dextran, ethylene oxide / propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hardened castor oil, and polyethylene glycol.

[0064] Sulfur-containing reducing agents include those containing sulfhydryl groups such as N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanol amine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and thioalkanoic acids having one to seven carbon atoms.

[0065] Antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, alpha-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as disodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0066] In one aspect of the present invention, the container to be filled with the formulation of the present disclosure includes a syringe and cartridge. In one embodiment, the container to be filled with the formulation of the present disclosure is a syringe, and preferably a pre-filled syringe. In an embodiment, the pre-filled syringe is housed in an autoinjector.

[0067] In one aspect of the present invention, “pre-filled syringe” implies a syringe wherein the syringe as a container is filled with a liquid composition (in particular, the formulation, pharmaceutical composition or medicament of the present disclosure). In one embodiment, a pre-filled syringe includes a syringe filled with a composition for administration to a patient. Herein, the syringe may be covered with a syringe closure such as for example a stopper, but is not limited thereto. In one embodiment, the composition is filled into the syringe in a filling facility for production. In one embodiment, the syringe is sterilized before filling the composition into the syringe. In one embodiment, the pre-filled syringe may be stored before administering the composition to a patient for a period of one day, or at least 7 days, or at least 14 days, or at least 1 month, or at least 6 months, or at least 1 year, or at least 2 years. In one embodiment, the pre-filled syringe is exposed to storage and / or shipping conditions.

[0068] In some embodiments, the formulation of the present disclosure comprises: 9.94 mg / mL or more (e.g., 10.7 mg / mL or more) of a bispecific antibody, the bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises heavy chain CDRs 1, 2, and 3, which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the first antibody light chain comprises light chain CDRs 1, 2, and 3, which comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises heavy chain CDRs 1, 2, and 3, which comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the second antibody light chain comprises light chain CDRs 1, 2, and 3, which comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 5 to 80 mmol / L; 100 to 300 mmol / L (preferably, 150 to 225 mmol / L) of L-arginine; 0 to 100 mmol / L (preferably, 20 to 100 mmol / L) of L-methionine; and 0.5 to 5.0 mg / mL Polysorbate 80, Polysorbate 20, or Poloxamer 188 (preferably, Polysorbate 80) wherein the pH of the formulation is 5.5 to 7.0.

[0069] In the specific preferable embodiments, the formulation of the present disclosure is a formulation comprising: 9.94 mg / mL or more (e.g., 10.7 mg / mL or more) of a bispecific antibody NXT007; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL Polysorbate 80, wherein the pH of the formulation is 6.0. In the particularly preferable embodiments, the formulation of the present disclosure is a formulation comprising: 150 mg / mL of a bispecific antibody NXT007; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL Polysorbate 80, wherein the pH of the formulation is 6.0.

[0070] In other embodiments, the formulation of the present disclosure is an aqueous formulation comprising (i) an antibody of the present invention, (ii) histidine buffer at a concentration of 10 mM to 50 mM, (iii) sucrose at a concentration of 200 mM to 300 mM, (iv) Polysorbate 80 at a concentration of 0.01% to 0.1% (w / v), wherein the pH of the formulation is in the range of 5.0 to 6.0. Optionally, this formulation may further comprise 0.5 mM to 5 mM L-methionine and / or 0.1 mM to 0.5 mM disodium edetate. In still other embodiments, the formulation of the present disclosure comprises a buffer to maintain the pH in the range of 5.0 to 6.0, a stabilizer, and a surfactant. Here, the buffer is selected from the group consisting of histidine, citric acid, and acetic acid, preferably a histidine buffer, particularly preferably a histidine-aspartate buffer; the stabilizer is selected from the group consisting of sucrose, trehalose, sorbitol, and mannitol, preferably sucrose; and the surfactant is selected from the group consisting of Polysorbate 80, Polysorbate 20 and Poloxamer 188, preferably Polysorbate 80. In the specific embodiments, the formulation of the present disclosure further comprises a recombinant human hyaluronidase PH20 (rHuPH20). In the other specific embodiments, the formulation of the present disclosure does not comprise a recombinant human hyaluronidase PH20 (rHuPH20).

[0071] The formulation of the present invention is preferably a formulation for administration to a human (formulation for human administration), and in some embodiments, formulation for use in prevention or treatment of hemophilia A in a human patient. As used herein, the terms “treating”, “treatment” and the like are understood as affecting a subject to reach a desired pharmacological and / or physiological effect in terms of a partial or complete cure of a disease or condition or associated symptoms. The terms “preventing”, “prevention” and the like are understood as prophylactic treatment of the subject in terms of completely or partially preventing the occurrence or frequency, arresting the development, delaying the onset or reducing the severity of a disease or condition or symptoms associated therewith. In the context of the invention, “preventing” or “treating” preferably refers to reducing the incidence of bleeding or excessive bleeding or reducing the likelihood of bleeding or excessive bleeding in a subject (e.g., by increasing blood clotting activity). Most preferably “preventing” or “treating” means routine prophylaxis to prevent or reduce the frequency of bleeding episodes in a subject. Bleeding or excessive bleeding in such subject is preferably caused by a decrease or deficiency in the activity of FVIII and / or FVIIIa. The formulation of the present invention can be administered to a patient via any suitable route, for example, as a bolus or by continuous infusion for a certain period of time, via intravenous, intramuscular, or subcutaneous route. Intravenous administration or subcutaneous administration is preferred. Particularly preferably, the formulation of the present invention is a formulation for subcutaneous administration. Preferably, the antibody-containing formulation for subcutaneous administration contains high concentration of antibodies, because the dosing volume is generally limited in subcutaneous administration.

[0072] The dosing amount of NXT007 is, for example, 0.001 to 1000 mg / kg, and the interval of administration is at least one day or longer.

[0073] The formulation of the present invention can be used for diseases that develop and / or progress due to the reduction or deficiency in the activity of FVIII and / or activated blood coagulation factor VIII (FVIIIa). For example, it can be used for hemophilia A or von Willebrand's disease, preferably congenital hemophilia A or acquired hemophilia A, more preferably severe, moderate or mild congenital hemophilia A in which inhibitors against FVIII / FVIIIa have appeared (i.e., with Factor VIII inhibitors), or severe or moderate congenital hemophilia A in which inhibitors against FVIII / FVIIIa have not appeared (i.e., without Factor VIII inhibitors) without being particularly limited thereto.

[0074] Stability of formulation (1) Risk characterization for formation of insoluble particles In some embodiments, the formulation of the present disclosure is characterized by having reduced formation of insoluble particles (sub-visible particles or visually detectable particles). In one embodiment, the formulation of the present disclosure is characterized by having reduced formation of sub-visible particles and / or visible particles as compared to a reference formulation having a different component composition from the formulation of the present disclosure (e.g., containing the same concentrations of antibody and other components, but with a lower concentration of L-arginine than the formulation of the present disclosure). In one embodiment, the formulation of the present disclosure is characterized by having reduced formation of visible particles as compared to a reference formulation having a different component composition from the formulation of the present disclosure (e.g., containing the same concentrations of antibody and other components, without surfactants). In preferable embodiments, the formulation of the present disclosure is characterized by absence of visible particles in the formulation or pharmaceutical composition over a certain period of time at certain storage conditions (e.g., 6 to 36 months of static storage at 5 °C, 4 months of a shipping stress test and / or drop / vibration test, etc.). Herein, a visually detectable particle includes particles that are visually detectable under high illuminance and have a particle size of 40 micrometers or more. Among them, particles that are visually detectable under standard illuminance as prescribed in the pharmacopoeia (approximately 2,000 to 3,000 lx) are referred to as “visible particles” or “insoluble visible particles”. Generally, visible particles have a particle size greater than 100 micrometers (NPL 12). Particles that are smaller in size than visible particles which cannot be seen by eye with standard illuminance as prescribed in the pharmacopoeia (approximately 2,000 to 3,000 lx) but which can be visually detected by increasing illuminance or by longer observation time are referred to as “particles visually detectable only under high illuminance” and have a particle size of 40 micrometers to 100 micrometers. Visible particles can be confirmed by visual inspection with the naked eye for 5 seconds or more under illumination at standard illuminance (approximately 2,000 to 3,000 lx), by slowly rotating or inverting the container in front of a black background or a white background. Particles visually detectable only under high illuminance can be confirmed by visual inspection with the naked eye for 30 seconds or more under illumination at high illuminance (6,000 lx or more), by slowly rotating or inverting the container in front of a black background. Visible particles can also be confirmed with inspection under high illuminance. Particles except those generated from protein molecules in the solution are not considered as “visually detectable particles” regardless of size. Whether the visually detectable particles are derived from protein molecules can be confirmed by Raman microspectroscopic measurement. The only protein contained in the solution is the active pharmaceutical ingredient (API), and visually detectable particles are generated from API. Thus, as used herein, “visually detectable particles,” “visible particles,” and “insoluble visible particles” in the formulations, pharmaceutical compositions or medicaments of the present disclosure are aggregates of proteins (particularly antibodies that are APIs of the formulations, pharmaceutical compositions or medicaments of the present disclosure). The particle size and number of visually detectable particles may be determined by light obscuration particle count method, microscopic particle count method, flow cytometric particle image analysis method, visual inspection, and infrared spectroscopy (IR) or Raman microspectroscopic measurement after isolation of particles, and preferably by a combination of visual inspection and infrared spectroscopy or Raman microspectroscopic measurement. As used herein, sub-visible particles are particles detectable by a micro flow imaging (MFI) method, with a particle size of 2 micrometers or more, 5 micrometers or more, 10 micrometers or more, 25 micrometers or more, or 50 micrometers or more.

[0075] (2) Reducing particle formation In one aspect of the present invention “reducing formation of particles” refers to adjusting the component composition so that insoluble particles are not formed or the number of formed particles is reduced in a solution of a formulation (in particular, a pharmaceutical formulation), a pharmaceutical composition, or a medicament wherein insoluble particles are formed under a given condition. Reduction of formation of insoluble particles can be confirmed by counting the number of particles in the formulations having different component compositions. The size and number of particles may be determined by MFI method, light obscuration particle count method, microscopic particle count method, flow cytometric particle image analysis method, visual inspection (e.g., EP method or EVI method), and infrared spectroscopy (IR) or Raman microspectroscopy after isolation of particles, and preferably by a combination of visual inspection and infrared spectroscopy or Raman microspectroscopy.

[0076] (3) Evaluation of formation of various variants, viscosity, and colloidal stability In some embodiments, the formulation of the present disclosure is characterized by having reduced formation of antibody size variants and / or antibody charge variants; reduced viscosity; and / or higher colloidal stability. In one embodiment, the formulation of the present disclosure is characterized by having reduced formation of antibody size variants as compared to a reference formulation having a different component composition from the formulation of the present disclosure (e.g., containing the same concentrations of antibody and other components, but without methionine). In one embodiment, the formulation of the present disclosure is characterized by having reduced formation of antibody size variants as compared to a reference formulation having a different component composition from the formulation of the present disclosure (e.g., containing the same concentrations of antibody and other components, but the buffer is phosphate buffer). In one embodiment, the formulation of the present disclosure is characterized by having reduced formation of antibody size variants as compared to a reference formulation having a different component composition from the formulation of the present disclosure (e.g., containing the same concentrations of antibody and other components, but without aspartic acid). The content of the antibody size variants (HMW Forms (high molecular weight forms), which may also be referred to as HMWS (high molecular weight species) and correspond to post-peaks, and LMW Forms (low molecular weight forms), which may also be referred to as LMWS (low molecular weight species) and correspond to pre-peaks) in the formulation can be evaluated by the area ratio [area%] in size exclusion chromatography (SE-HPLC) or the area ratio [CPA%] of each peak by capillary electrophoresis (CE-SDS (NR: Non-reduction)). In one embodiment, the formulation of the present disclosure is characterized by having reduced formation of antibody charge variants as compared to a reference formulation having a different component composition from the formulation of the present disclosure (e.g., containing the same concentrations of antibody and other components, but the pH is less than 5.5). The content of the antibody charge variants (acidic components and basic components) in the formulation can be evaluated by the percentage [%] of the peaks of each component by imaged capillary isoelectric focusing (icIEF). In one embodiment, the formulation of the present disclosure is characterized by having reduced viscosity as compared to a reference formulation having a different component composition from the formulation of the present disclosure (e.g., containing the same concentrations of antibody and other components, but the pH is adjusted using hydrochloric acid rather than aspartic acid). The viscosity of the formulation can be measured, for example, by an EMS viscometer. In one embodiment, the formulation of the present disclosure is characterized by having higher colloidal stability as compared to a reference formulation having a different component composition from the formulation of the present disclosure (e.g., containing the same concentrations of antibody and other components, but L-arginine concentration is less than 100 mmol / L). The colloidal stability of the formulation can be evaluated by measuring the diffusion coefficient (D) by a dynamic scattering (DLS) method and calculating the kD value by plotting the D value against the antibody concentration. These measurement methods are well known and can be performed as appropriate by those skilled in the art.

[0077] All cited documents in the present specification are incorporated herein by reference. The present invention is further illustrated in the following Examples, however, it is not limited thereto.

[0078] A PHASE I / II CLINICAL STUDY TO EVALUATE THE SAFETY, TOLERABILITY, PHARMACOKINETICS, PHARMACODYNAMICS, AND EFFICACY OF NXT007 IN HEALTHY ADULTS AND PATIENTS WITH HEMOPHILIA A1. STUDY RATIONALE AND BENEFIT-RISK ASSESSMENT Hemophilia A is an X-linked recessive bleeding disorder that occurs in approximately 1 in 5,000 live male births. Patients with hemophilia A have a deficiency or absence of FVIII, an essential component of the intrinsic pathway in the coagulation cascade. The goal of treatment for patients with hemophilia A is continuous restoration of normal FVIII activity (40 IU / dL or more). Indeed, effective routine supplementation with FVIII formulations is adopted as a desired approach. Although FVIII supplementation therapy is the gold standard, it requires frequent intravenous (IV) infusion (2 to 3 times / week) in order to obtain effective prophylaxis of bleeding, which poses a significant challenge for patients, caregivers, and healthcare providers. In addition, FVIII supplementation therapy also has a problem of the development of FVIII inhibitors (neutralizing antibodies).

[0079] Emicizumab overcame these challenges associated with FVIII supplementation therapy. Emicizumab can be administered by subcutaneous (SC) injection at a rate of 1 to 4 times / 4 weeks and is effective regardless of the existence of FVIII inhibitors. An annualized bleeding rate (ABR) in adult and adolescent patients with hemophilia A treated with emicizumab was significantly lower than in those received current FVIII supplementation therapy. Although it is a great step forward, there are some limitations. In clinical studies for adult and adolescent patients with hemophilia A with / without FVIII inhibitors, about 40% of patients still experienced one or more bleeding that required treatment, within the first 24 weeks from the start of the treatment. Therefore, no current existent therapies have been able to reach the goal of complete treatment of hemophilia.

[0080] NXT007 bridges FIXa and FX to substitute for the function of missing FVIIIa that is needed for effective hemostasis, in the same way as emicizumab. NXT007 has potential clinical usefulness as follows due to improved drug effect than emicizumab and features of an IgG molecule.

[0081] (1) Less frequent administration due to the longer blood half-life (t1 / 2) NXT007 is expected to demonstrate efficacy as long as the t1 / 2of IgG (2 to 4 weeks); this is longer than FVIII concentrates (t1 / 2; 8 to 19 hours)[4,40], rFVIIa (t1 / 2; 2.9 hours), and aPCC (t1 / 2; 4 to 8 hours). The t1 / 2of emicizumab is 4 to 5 weeks, and thus NXT007 is also expected to be administered less frequently.

[0082] (2) Enables SC administration It would be possible to develop NXT007 as SC injection formulations, since bioavailability of IgG in SC administration is high.

[0083] (3) Efficacious irrespective of the presence of FVIII inhibitors NXT007 is structurally unrelated to FVIII, and therefore the possibility that FVIII inhibitors, neutralizing antibodies to FVIII, recognize NXT007 is extremely low. Thus, NXT007 is expected to show efficacy not only in patients without inhibitors, but also in patients with inhibitors.

[0084] (4) Will not induce FVIII inhibitors In the case that anti-NXT007 antibodies develop, the possibility that the anti-NXT007 antibody would cross-react with FVIII to inhibit the activity of FVIII is extremely low.

[0085] (5) Possess higher FVIII function-substituting activity than emicizumab The bleeding rate for the approved dose of emicizumab suggested that it corresponded to approximately 15 IU / dL of FVIII activity

[0041] . It was reported that 30 to 40% of patients still experienced at least 1 bleed in routine supplementation therapy by emicizumab, according to the result of clinical studies of emicizumab[35,36]. On the other hand, the results of nonclinical studies showed that NXT007 have higher FVIII function-substituting activity than emicizumab. It is expected that NXT007 provides high hemostatic activity by achieving the normal level of FVIII activity (> 40 IU / dL). NXT007 may result in fewer patients who experience bleeding, the suppression of progression of hemophilic arthropathy, and the reduction of psychological stress in patients and caregivers.

[0086] 2. OBJECTIVES AND ENDPOINTS This study was designed with the aim of evaluating the safety, tolerability, PK, PD, and efficacy of NXT007 in healthy adults and patients with hemophilia A. Objectives and corresponding endpoints for the study regarding the part involving healthy adults (Part A, Part D, and Part E) are outlined below.

[0087] 2.1 Objectives 2.1.1 Primary Objectives -- To evaluate the safety and tolerability of a single dose of NXT007 in healthy adults -- To evaluate the PK of a single dose of NXT007 in healthy adults -- To evaluate the PD of a single dose of NXT007 in healthy adults

[0088] 2.1.2 Secondary Objectives -- To evaluate the immunogenicity of a single dose of NXT007 in healthy adults

[0089] 2.2 Study Endpoints 2.2.1 Primary Endpoints -- Safety and tolerability endpoints - Incidence and severity of adverse events including severe adverse events (SAEs), and their relationship to NXT007 - Incidence of abnormal laboratory values - Incidence of abnormal vital signs and ECG parameters - Cytokines

[0090] -- PK endpoints - Plasma NXT007 level

[0091] -- PD endpoints - APTT - Thrombin generation - Plasma FIX concentration - Plasma FX concentration

[0092] 2.2.2 Secondary Endpoints -- Immunogenicity endpoints - Incidence of anti-drug antibodies (ADAs) to NXT007

[0093] 3. OVERVIEW OF STUDY DESIGN The present study is adaptive, and was designed to continuously assess safety, tolerability, PK, and PD data of NXT007, and to proceed to the examination of the next dose and cohort based on the results. This study was originally designed as a first-in-human, proof-of-concept study consisting of 3 sequential parts: Part A, a Single-Ascending Dose (SAD) study in healthy adults; and Part B and Part C, Multiple Ascending Dose (MAD) studies in patients with hemophilia A to be conducted with doses determined based on the results of Part A. However, since unexpected problems were found from the results of Part A, two parts for healthy adults to examine the problems (Part D and Part E) were newly added. Details of the study design in each parts are described below.

[0094] 4. TARGET POPULATION (Inclusion Criteria) Healthy adults who met all of the following criteria were included in Part A, Part D, and Part E studies: -- Signed informed consent form (ICF) -- Able to comply with the study protocol, in the primary / sub investigators' judgment -- Male sex -- For men who are not surgically sterile: agreement to remain abstinent (refrain from heterosexual intercourse) or use condoms, and agreement to refrain from donating sperm, as defined below: - With female partners of childbearing potential or pregnant female partners, men must remain abstinent or use a condom during the treatment period and for at least 5 half-lives or 12 months after the last dose of NXT007, whichever is longer, to avoid exposing the embryo. Men must refrain from donating sperm during this same period. - The reliability of sexual abstinence should be evaluated in relation to the duration of the clinical trial and the preferred and usual lifestyle of the healthy adult / patient. Periodic abstinence (e.g., calendar, ovulation, symptothermal, or post-ovulation methods) and withdrawal are not acceptable methods of contraception. -- Japanese race / ethnicity -- Age greater than or equal to 20 and less than 45 years -- BMI at screening: (Body weight [kg] / height [m]2) greater than or equal to 18.5 to less than 25.0 kg / m2 -- Absence of evidence of any active or chronic disease following a detailed medical and surgical history and complete physical examination, vital signs, 12-lead ECG, and laboratory tests -- Weight greater than or equal to 60 and less than 88 kg at Day 1 (Pre-dose) (Cohorts D-1, D-2, and D-3 only)

[0095] (Exclusion Criteria) Healthy adults who met any of the following criteria were excluded from Part A, Part D, and Part E studies: -- Previous or current history of drug- or alcohol-dependence -- Previous or concomitant thromboembolic disease such as deep vein thrombosis (DVT), or signs of thromboembolic disease, or thrombotic microangiopathy (TMA) -- Protein C activity (chromogenic assay), protein S free antigen, or antithrombin III activity levels below the lower limit of the reference range at screening -- History of clinically significant allergy (anaphylactic shock or anaphylactoid symptoms) -- Previous or concomitant autoimmune or connective tissue disease -- Previous or concomitant malignancy or leukemia -- History of hypersensitivity associated with globulin preparations -- Received or planned to receive a live vaccine within 4 weeks before the start of the study drug administration -- Received or planned to receive an inactivated vaccine within 1 week before the start of the study drug administration -- Planned surgery (including tooth extraction) during the study period -- Participated in another clinical study within 4 weeks before screening, or within 5 half-lives of the investigational product, whichever is longer. -- Clinically significant ECG abnormalities at screening such as the following: - The average QT interval with Fridericia's correction of triplicate-measurements in supine position at 10-minute rest >450 msec - Bradycardia at rest (average heart rate <40 beats per minute [bpm]) - Tachycardia at rest (average heart rate >100 bpm) - Other clinically significant abnormalities in ECG -- Any family history of congenital long QT syndrome or known congenital arrhythmia -- Any other reason that, in the judgment of the primary / sub investigators, would render the subject unsuitable for study participation -- Clinically significant respiratory, circulatory, endocrine, hematological, gastrointestinal, immune, psychological / nervous system, renal, hepatic, or allergic disorder -- Family history of thromboembolic disorder such as serious DVT -- Use or planned use of other drugs (i.e., prescription or over-the-counter drugs) within 2 weeks before the start of study drug administration (provided, however, that non-systemic topical antiseptics and ophthalmic solutions not affecting the PK or safety of the study drug will be permitted) -- Blood draw of 200 mL within 4 weeks or 400 mL within 12 weeks prior to screening (e.g., blood donations), annual total blood draw volume (including blood drawn for this study) exceeding 1200 mL, or blood component donation within 2 weeks prior to screening -- FVIII activity of 120 IU / dL or more at screening -- Positive screening result for human immunodeficiency virus (HIV) antigen / antibodies, hepatitis B surface (HBs) antigen, or hepatitis C virus (HCV) antibodies. Healthy adults who showed HBs antigen positive due to vaccination against hepatitis B are eligible. -- Evidence of infection at screening -- Abnormal clinical findings at screening -- Prior treatment with antibody preparations (commercially available or investigational)

[0096] 5. STUDY DRUG (Test drug) NXT007 1.0 mL (in a 3.0 mL vial, 80 mg / mL) for SC administration; or NXT007 1.0 mL (in a 3.0 mL vial, 150 mg / mL) for SC administration (only for Cohort D-4). These test drugs were provided in a 3.0 mL vial filled with 1.0 mL of solution containing 80 mg or 150 mg (only for Cohort D-4) of NXT007, and stored protected from light at 2°C to 8°C until use.

[0097] NXT007 is a bispecific antibody disclosed in a patent document, WO2019 / 065795 (according to WO2019 / 065795, the bispecific antibody comprises: a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 124 (SEQ ID NO: 21 in the present specification) and the first antibody light chain comprises the amino acid sequence of SEQ ID NO: 136 (SEQ ID NO: 22 in the present specification); and a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 144 (SEQ ID NO: 23 in the present specification) and the second antibody light chain comprises the amino acid sequence of SEQ ID NO: 155 (SEQ ID NO: 24 in the present specification)).

[0098] NXT007 formulation for SC administration prescribed as shown in Table 1 was suitably diluted and used in the study. A formulation that includes the components in Table 1 except for NXT007 (placebo) was used to dilute the test drug.

[0099]

[0100] (Control drug) In Part A, placebo 1.0 mL (in 3.0 mL vial) for SC administration was used as a control drug. The placebo was subcutaneously administered to placebo groups with the same dosing volume and the same method as in corresponding NXT007 administration groups in each cohort.

[0101] 6. OBSERVATION AND ASSESSMENT ITEMS 6.1 Assessment Items 6.1.1 Assessments conducted at Investigational Sites The following assessments were conducted at investigational sites: -- Vital signs -- Weight -- Height -- Complete physical examination -- 12-Lead ECG 1: To detect ECG abnormalities. If subjects discontinue out of treatment period, ECG evaluation should be performed as 12-lead ECG 1. -- Hematology 1: Includes WBC count, RBC count, hemoglobin, hematocrit, platelet count, differential WBC count (neutrophils, eosinophils, basophils, monocytes, lymphocytes, other cells), MCV, MCH, and MCHC. -- Blood chemistry: Includes Na, K, Cl, glucose, BUN, creatinine, total protein, albumin, P, Ca, total and direct bilirubin, alkaline phosphatase, ALT, AST, uric acid, LDH, CRP, gamma-GTP, CK, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and A / G ratio. For 8 hours post-dose on Day 1 and 24 hours post-dose on Day 2 of Parts A, D, and E, includes only CRP. -- Coagulation 1: Includes APTT, PT, PT-INR, and D-dimer. -- Urinalysis: Includes dipstick (glucose, protein, blood, and urobilinogen).

[0102] 6.1.2 Assessments Measured at Central Laboratories The following assessments are measured at central laboratories: -- Coagulation 2: Includes PT, PT-INR, fibrinogen, D-dimer, prothrombin fragment 1+2. For all time points of Parts A, D, and E except for screening, includes only fibrinogen and prothrombin fragment 1+2. -- Coagulation 3: Includes APTT, protein C activity (chromogenic assay), protein S free antigen, antithrombin III activity, FVIII activity, FVIII inhibitors. -- Immunology: Includes HBs antigen, HCV antibody, and HIV antigen and antibodies. -- Special test (Cytokines): Includes IL-2, IL-6, IL-8, TNF-alpha, and IFN-gamma. -- Special test (PK 1): Includes plasma NXT007 level by ELISA. -- Special test (PD 1): Includes plasma FIX concentration and plasma FX concentration. -- Special test (PD 2): Includes APTT and thrombin generation. -- Special test (PD 3): Includes APTT and thrombin generation on FVIII neutralization. -- Special test (ADA): Includes anti-NXT007 antibodies. -- CCSR (Optional) -- Optional assessment of FVIII activity

[0103] 6.2 Informed Consent and Screening Log Written informed consent for participation in the study was obtained before performing any study-related procedures (including screening evaluations). Signed informed consent forms (ICFs / IAFs) for enrolled or non-enrolled subjects were maintained at the investigational sites. All screening tests were completed before enrolment and reviewed to confirm if subjects meet the inclusion and exclusion criteria. Some of these tests or processes were performed as a part of the patient's regular medical care and done even if he / she did not take part in the study. If patients had had some of the results recently, no test or evaluation was required to be repeated. The primary / sub investigators reviewed details of all subjects screened and eligibility or reasons for screening failure of subjects and recorded them in source materials.

[0104] 6.3 Medical History, Concomitant Medication, and Subject Background Data Medical history included clinically significant diseases, surgeries, cancer history (including prior cancer therapies and procedures) within the past 5 years, and these were recorded at baseline. Reproductive status, smoking history, and use of alcohol and drugs of abuse within the past 5 years were also recorded. In addition, all medications (e.g., prescription drugs, over-the-counter drugs, vaccines, herbal drugs, homeopathic remedies, nutritional supplements) used by the subject before observation period were recorded. At the time of each follow-up physical examination, an interval medical history and any changes in medications and hypersensitivities were recorded. Subject background data included age (date of birth), sex, and self-reported race / ethnicity. Background data for Part A, D, and E includes medical history, concurrent diseases, and drug hypersensitivities.

[0105] 6.4 Physical Examinations A complete physical examination included an evaluation of the head, eyes, ears, nose, throat, neck, lymph nodes, and the cardiovascular, dermatological, musculoskeletal, respiratory, gastrointestinal, genitourinary, and neurological systems. Additional systemic examinations were performed in case of evocative symptoms at the primary / sub investigators' discretion. The primary / sub investigators paid special attention to clinical signs related to previous serious illnesses. Height was recorded at screening in all Parts and body weight was recorded at the time point specified in the schedule of observation and evaluation. The BMI was calculated from the measurements at screening. Any abnormality identified at baseline was recorded as general medical history and baseline conditions on eCRF. At subsequent visits, physical examinations directed to specific symptoms were performed at specified post-baseline visits and as clinically indicated. Changes from baseline abnormalities were recorded in source materials. New or worsened clinically significant abnormalities were recorded as adverse events on eCRF. -- Physical Examination Items: Interview, visual inspection, auscultation, percussion, palpation

[0106] 6.5 Vital Signs Vital signs included heart rate, systolic and diastolic blood pressure, and body temperature. Blood pressure and heart rate were measured with a completely automated device. Manual measurements were performed only if an automated device was not available. When possible, the same arm was used for all blood pressure measurements. These measurements were taken before blood collection and performed in a seated position after 10 minutes rest. Abnormalities observed at baseline were recorded as general medical history and baseline conditions on eCRF. At subsequent visits, new or worsened clinically significant abnormalities were recorded as adverse events on eCRF.

[0107] 6.6 Electrocardiography 12-lead ECG measurements were performed to detect abnormalities in Parts A, D, and E. Triplicate ECG measurements were performed at screening, and single ECG measurements were performed at specified time points except screening and at unscheduled time points. ECG measurements during administration period were performed using a standard high-quality, high-fidelity digital electrocardiograph machine equipped with computer-based interval measurements. Lead placement was as consistent as possible. ECGs were measured after the patient had been resting in a supine position for at least 10 minutes. All ECGs were measured prior to other assessments / procedures scheduled at that same time (e.g., vital sign measurements, blood draws) other than urinalysis and were not measured within 2 hours after any meal. Factors that may induce changes in heart rate (e.g., television, radio, conversation) were avoided during the pre-ECG resting period and during ECG recording. The conditions during the ECG measurements were required to be as close as possible to those at pre-dose time-points. The conditions specifically included food intake, activity level, stress, and room temperature, but other conditions were also preferably made to be as close as possible. To confirm safety, the primary / sub investigators reviewed, signed, and dated all ECG records. Paper or electronic copies of ECG tracings for each subject were kept as part of the patient's study file at the investigational site. The following were recorded in the corresponding eCRF of 12-lead ECG: heart rate, RR interval, QRS interval, PR interval, uncorrected QT interval, and QTcF interval, read by machine from the individual ECG records. Any morphologic waveform changes or other ECG abnormalities were recorded on the eCRF. If considered appropriate by the Sponsor, ECGs were analyzed retrospectively at a central laboratory. If the mean QTcF interval was shown to be > 500 ms and / or 60 ms longer than the baseline value, another triplicate ECGs were measured (ideally within 5 minutes), and triplicate ECG measurements were continued until QTcF value stabilized on 2 successive measurements. The necessity of measuring another triplicate ECGs was allowed to be determined by the data on the immediate reports, not by the data on the reports from the central laboratories. When such findings were observed, a report was sent to a medical monitor. Standard-of-care treatment was permitted per the discretion of the primary / sub investigators. If PK sampling was not scheduled for that timepoint, an unscheduled PK sample for PK1 assessment was obtained.

[0108] 6.7 Patient-Reported Outcomes In Part A, patient-reported outcome (PRO) data was collected via Bleed / Medication Questionnaires, distributed to the subjects. The questionnaires included start date and time, end date and time, reason, type, location and associated symptoms of bleeds, as well as start date and time, reason, type, and dose of bleeding medications if used.

[0109] (1) Standardized Criteria for Bleed The definition of “bleed” used for this study was adapted from standard criteria defined by FVIII / FIX subcommittee of the International Society of Thrombosis and Hemostasis. -- An event was considered a bleed if coagulation factor formulations were administered to treat signs or symptoms of bleeding (pain, swelling, etc.). -- Bleeds during the period starting from the first sign of bleed and ending 72 hours after the last treatment for the bleed were considered the same bleed if the symptoms of them occurred at the same location or within 72 hours after injections. -- Any injection to treat the bleed, which had passed > 72 hours after the preceding injection, was considered the first injection to treat a new bleed at the same location. -- Any bleed at a different location was considered a separate bleed regardless of time passed from last injection.

[0110] (2) Definitions of Bleed Sites -- Target joints: defined as a major joint (e.g., hip, elbow, wrist, shoulder, knee, and ankle) in which repeated bleeds occur (frequency of 3 bleeds or more at the same joint over the last 24 weeks prior to study entry) -- Joint bleeds (other joint except target joints) defined as an unusual sensation (“aura”) in the joint, in combination with any of the following: - Increasing swelling or warmth of the skin over the joint - Increasing pain -- Progressive loss of range of motion or difficulty in using the limb as compared with baseline -- Muscle bleeds (Bleed Questionnaire) -- Bruise / hematoma (Bleed Questionnaire) -- Other (Bleed Questionnaire)

[0111] (3) Definitions of Bleed Types The assessment of a bleed was made to separate it into spontaneous bleeds, traumatic bleeds, and bleeds related to procedure / surgery. Data of both spontaneous bleeds (i.e., hemorrhage where neither the patient nor a caregiver can identify a reason) and traumatic bleeds (i.e., hemorrhage occurred secondary to an event such as trauma, “strenuous” activity, or “severe” activity) were collected. -- Spontaneous bleeds: Bleeds should be classified as spontaneous bleeds if a patient recorded a bleed when there was no known contributing factor, such as definite trauma, antecedent “strenuous” activity or “severe” activity. The determination of what constitutes “strenuous” or “severe” activity would be at the discretion of the patient. For example, light jogging may be considered “non-strenuous” while sprinting may be considered “strenuous,” lifting of weights for a short period of time may be considered “moderate” while repetitive weightlifting may be considered “severe”. -- Traumatic bleeds: Bleeds should be classified as traumatic if a patient records a bleed when there is a known reason for the bleed. For example, if a patient were to exercise “strenuously” and then have a bleed in the absence of any obvious injury, the bleed would be recorded as a traumatic bleed (because, although no injury occurred, there was antecedent “strenuous” activity). Bleed subsequent to injuries would certainly be classified as traumatic. -- Bleeds related to procedure / surgery: Bleeds resulting from any surgeries or invasive procedures (e.g., tooth extractions, venipuncture, or SC drug administrations) or invasive diagnostic procedures (e.g., lumbar puncture, arterial blood gas determination, or any endoscopy with biopsy, etc.) would not be counted as bleeds. Bleeds related to procedure / surgery are not associated with any trauma, except procedure / surgery-induced trauma.

[0112] 7. STATISTICAL CONSIDERATIONS AND ANALYSIS PLAN The safety, tolerability, PK, PD, and efficacy were analyzed for the following analysis population.

[0113] 7.1 Determination of the Number of Subjects The purpose of this study is to evaluate the safety, tolerability, PK, PD, and efficacy of NXT007 in healthy adults or patients. The minimum sample sizes required to evaluate the safety and tolerability are at least 8 healthy adults (6 for the test drug and 2 for a placebo) per cohort for Part A. The purpose of Parts D and E is to investigate the PK and PD of NXT007 in healthy adults. The minimum sample sizes required to evaluate the PK and PD are at least 6 healthy adults per cohort for Part D (except for Cohort D-4) and Part E. In Part A, the number of subjects in each dose cohort was determined as 8 (6 for the test drug and 2 for the placebo), as an adequate number of subjects (6 being the minimum) for investigating the tolerability, safety, PK, and PD in healthy adults receiving the test drug. In Parts D and E (except for Cohort D-4), the number of subjects in each dose cohort was determined as 6, as an adequate number of subjects (6 being the minimum) for investigating the PK and PD in healthy adults. For Cohort D-4, the number of subjects was determined as 8.

[0114] 7.3 Safety Analyses The safety analysis population consisted of all subjects who received at least one administration of the study drug. In this analysis, the subjects were grouped according to the drug received. Adverse events were read according to the ICH Medical Dictionary for Regulatory Activities (MedDRA) code, and the severity was graded according to the WHO Toxicity Grading Scale. For adverse events, numbers of subjects with adverse events and numbers of adverse events were summarized by event as classified by System Organ Class and Preferred Term, and by severity grade.

[0115] 7.4 PK Analyses The PK analysis population consisted of all subjects who received at least one administration of NXT007 and had at least one post-administration measurement of PK endpoints. In this analysis, the subjects were grouped according to the drug received. Summary statistics were calculated for the plasma NXT007 levels by treatment group. The PK parameters of NXT007 (Cmax, Tmax, AUC, t1 / 2, CL / F, and Vd / F, etc.) were calculated, and the pharmacokinetic linearity were evaluated from the relationship between the dosing amount of NXT007 and the PK parameters. The effects of the drug concentration of NXT007 and multiple site injections on PK were evaluated in Parts D and E, respectively, in conjunction with the results of Part A.

[0116] 7.5 PD Analyses The PD analysis population consisted of all subjects who received at least one administration of the study drug and had at least one post-administration measurement of PD endpoints. In this analysis, the subjects were grouped according to the drug received. Summary statistics were calculated for APTT, thrombin generation, plasma FIX concentration, and plasma FX concentration, by treatment group.

[0117] 7.6 Immunogenicity Analyses The immunogenicity analysis population consisted of all subjects who received at least one administration of NXT007 and had at least one pre- or post-administration measurement of immunogenicity endpoints. In this analysis, the subjects were grouped according to the drug received. The numbers and proportions of ADA-positive subjects and ADA-negative subjects at the baseline and after the baseline (baseline prevalence and postbaseline incidence) were summarized by treatment group. When determining the postbaseline incidence, subjects were considered to be ADA positive, if they were ADA negative or had missing data at the baseline but developed an ADA response following study drug administration (study drug-induced ADA response), or if they were ADA positive from the baseline and the ADA titer of one or more postbaseline samples was at least 4-fold increase of the baseline titer (study drug-boosted ADA response). Meanwhile, subjects were considered to be ADA negative, if they were ADA negative or had missing data at the baseline and all postbaseline samples showed negative, or if they were ADA positive from the baseline but did not have any postbaseline samples with the ADA titer that is at least 4-fold increase of the baseline titer (study drug-unaffected ADA response). The relationship between ADA occurrence status and safety, efficacy, PK, and PD were analyzed.

[0118] 8. DETAILS OF METHODS AND RESULTS8.1 Part A: Single-Ascending Dose in Healthy Adults Part A is a placebo-controlled, randomized, double-blind, inter-individual, single- ascending dose, single center study in healthy Japanese adult males. The aim of Part A was to evaluate the safety, tolerability, PK, and PD of a single dose administration of NXT007 in healthy adults. Subjects received a single SC injection of NXT007 at a single site on abdomen, with the dose escalating in the order of Cohorts A-1, A-2, A-3, A-4, and A-5.

[0119] Forty healthy adults were enrolled in Part A. At each cohort, 8 healthy adults were randomized, of which 6 received the test drug and 2 received the placebo by single SC administration. The first 2 healthy adults were included in a sentinel group and dosed first (1 for the test drug and 1 for the placebo). After confirming the laboratory test values, cytokines, vital signs, 12-lead ECG results, and adverse events up to Day 22 in the sentinel group by the primary investigator, the remaining 6 healthy adults were administered with the study drug sequentially (5 for the test drug and 1 for the placebo) at least 10 minutes apart under body condition monitoring. When repeating the same dose level, no sentinel subject was needed. All healthy adults were admitted to the hospital (investigational site) on Day -1 (minus 1) and were required to remain at the site until Day 22. The healthy adults underwent all tests on Day -1, and after an overnight fasting (at least 10 hours), they were examined in the morning of Day 1 and administered with the study drug. On Day 22, if the primary investigator judged that the healthy adult had no health problems based on all available test results, then the healthy adult was released from the hospital.

[0120] Cohort transition was proceeded in the order of Cohorts A-1, A-2, A-3, A-4, and A-5. Before proceeding the cohort transition, both of the following items in a prior cohort were evaluated: -- PK and PD up to at least Day 29 -- Laboratory test values, cytokines, vital signs, 12-lead ECG results, and adverse events up to at least Day 43

[0121] Determination of Dose and Dosage Regimen in Part A (1) Determination of Minimum Dose As this study is the first clinical study to administer NXT007-containing formulations (hereinafter referred to as the present drug) to humans, the investigation was started from single SC administration in healthy adults to confirm its safety and tolerability. The minimum dose was determined as 0.0018 mg / kg, with reference to the maximum dose in healthy adults described later, so that inter-individual dose escalation is performed in steps with a dose escalation ratio of approximately 3-fold. With this dose, the FVIII function-substituting activity (equivalent FVIII activity) at the mean Cmaxof the present drug in humans, which was predicted based on the results of a single-dose PK study in cynomolgus monkeys, was predicted to be no greater than 1 / 10 of the equivalent FVIII activity (2.96 IU / dL

[0038] ) of emicizumab, which has the same mechanism of action as the present drug, at its mean Cmax(7.56 microgram / mL

[0042] ) after a single SC administration of 1 mg / kg, which is the maximum dose at which safety has been confirmed in the clinical studies of emicizumab in healthy adults. The predicted mean Cmaxafter a single SC administration of 0.0018 mg / kg of the present drug was 0.0120 microgram / mL, and the equivalent FVIII activity was predicted to be 0.0751 IU / dL, based on the amount of FIX-present drug-FX complex formed which was calculated from the dissociation constant KDof the present drug for human FIX and FX by a method

[0038] similar to that used for emicizumab.

[0122] In FVIII-deficient human plasma, the present drug shortened APTT in a concentration-dependent manner from 0.0000300 microgram / mL. For emicizumab, which has the same mechanism of action, the minimum effective concentration based on APTT was 0.01 microgram / mL

[0043] and the safety of emicizumab has been confirmed up to a mean Cmaxof 7.56 microgram / mL in healthy adults

[0042] , and thus, an up to 756-fold safety margin relative to the minimum effective concentration has been ensured. The predicted mean Cmaxof a single SC administration of 0.0018 mg / kg of the present drug was 400 times the minimum effective concentration of the present drug based on APTT, and since this value is within the range of the safety margin relative to the minimum effective concentration which has been experienced with emicizumab, it was considered as appropriate to select 0.0018 mg / kg as the starting dose for the part of the study involving healthy adults.

[0123] Of note, a 43400- to 44600-fold safety margin relative to the NOAEL for the 3-month GLP study in cynomolgus monkeys (20 mg / kg SC Q2W), and a 84100- to 90800-fold safety margin relative to the NOAEL for the 6-month GLP study in cynomolgus monkeys (20 mg / kg SC Q2W) have been ensured for the minimum dose for Part A.

[0124] (2) Determination of Maximum Dose In healthy adults with a normal level of FVIII activity, in order to minimize the potential risk of hypercoagulation due to the administration of the present drug, the maximum dose was determined as 0.18 mg / kg, so that the sum of the endogenous FVIII activity and the FVIII activity from administration of the present drug would not exceed the upper limit of the normal range, 150 IU / dL

[0045] .

[0125] In Part A, subjects with FVIII activity of 120 IU / dL or more at screening were excluded. For a single SC administration of 0.18 mg / kg of the present drug, the predicted mean Cmaxwas 1.20 microgram / mL and the equivalent FVIII activity was predicted to be 7.37 IU / dL, and therefore, even taking into account inter-individual variability in PK, it was considered that the sum of the endogenous FVIII activity and the FVIII activity from administration of the present drug would not exceed 150 IU / dL.

[0126] Since the present drug is a bispecific antibody that binds to FIX, FIXa, FX, and FXa, it was considered to potentially inhibit the coagulation reactions related to FIX or FX. However, as it was predicted that neither of the unbound fractions of FIX and FX at the maximum dose in Part A would not fall below the minimum unbound fractions experienced in the clinical studies of emicizumab in hemophilia A patients, it was considered unlikely that the present drug would cause clinically significant inhibition of FIX and FX coagulation activity.

[0127] Of note, the maximum dose for Part A has a 434- to 446-fold safety margin relative to the NOAEL for the 3-month GLP study in cynomolgus monkeys (20 mg / kg SC Q2W) and a 841- to 908-fold safety margin relative to the NOAEL for the 6-month GLP study in cynomolgus monkeys (20 mg / kg SC Q2W).

[0128] (3) Determination of Dose Ascending Steps The doses and numbers of subjects planned for Part A, based on the above rationales for the determination of minimum and maximum doses, are shown in Table 2. In Part A, in Japanese healthy adults, a single SC administration was started from the minimum dose of 0.0018 mg / kg and escalated at an equal ratio of approximately 3-fold to test safety, tolerability, and PK of up to 0.18 mg / kg.

[0129]

[0130] Safety and tolerability of NXT007 were confirmed for all cohorts in Part A. Meanwhile, the pharmacokinetics (PK) of NXT007 showed linearity in the dose range of Cohort A-1 to A-3, but nonlinearity was observed in the dose range higher than that. For each cohort in Part A, PK data observed in the subjects who did not develop anti-drug antibodies against NXT007 were shown in FIG. 1 and Table 3.

[0131]

[0132] For the subjects who did not develop anti-drug antibodies against NXT007, Cmaxand AUCinfincreased dose-proportionally, and dose-normalized values (Cmax / Dose and AUCinf / Dose) were comparable, in the dose range of Cohorts A-1 to A-3. However, in the dose range higher than that, the increase in Cmaxand AUCinffell below the dose proportional, and Cmax / Dose and AUCinf / Dose decreased. Tmaxand t1 / 2were comparable irrespective of dose, and the elimination phase of plasma NXT007 level was monophasic. It was considered from these results that the PK characteristics observed over the entire dose range in Part A were nonlinear, whereas the elimination process was linear. Thus, it was assumed that the cause of nonlinearity was in the absorption process, and bioavailability of NXT007 possibly decreased dose-dependently. It followed from this assumption that the development of high dose NXT007 formulations might be challenging and the target blood level might not be achieved simply by increasing dose, and thus, frequent low-dose administration, larger dosing volume per administration site, or multiple site administration might be required. Moreover, in order to achieve the target efficacy in patients, it would be necessary to administer higher dose than in healthy adults to achieve higher blood level; however, it was considered as difficult or impossible (at least in a realistic dosage regimen) if bioavailability decreases dose-dependently.

[0133] In Part A, however, NXT007 was administered with adjusted doses by changing the drug concentration by suitably diluting the NXT007 formulation for SC administration prescribed as shown in Table 1, while keeping the dosing volume constant in each cohort (Table 4). Thus, it was unclear whether the decrease in bioavailability suggested in Part A was dependent on the dose of NXT007 administered or dependent on the drug concentration in the formulation. In addition, to determine the dose for patients, which was assumed to be higher than the dose for healthy adults, it was considered necessary to reveal the degree of effects on bioavailability that the dose, drug concentration, and dosing volume have.

[0134] * The following excipients and excipient concentrations were kept constant for all of formulations administered to Cohorts A-1 to A-5: 150 mmol / L of L-arginine, 20 mmol / L of L-histidine, 20 mmol / L of L-methionine, 0.5 mg / mL of Polysorbate 80, and pH 6.0.

[0135] 8.2 Part D: Single Administration at Various Drug Concentrations in Healthy Adults Part D was newly added to further clarify the PK characteristics that showed nonlinearity. Part D was an open-label, randomized (except for newly-added Cohort D-4), inter-individual, single center study in healthy Japanese adult males. The aim of Part D was to investigate the effect of drug concentration on PK and PD of NXT007 in healthy adults. Subjects received a single SC injection of NXT007 at a single site on abdomen, as in Part A. The dosing regimen for Part D are outlined in Table 5.

[0136] * The following excipients and excipient concentrations were kept constant for all of formulations administered to Cohorts D-1 to D-4: 150 mmol / L of L-arginine, 20 mmol / L of L-histidine, 20 mmol / L of L-methionine, 0.5 mg / mL of Polysorbate 80, and pH 6.0.

[0137] Cohort D-1 (N=6) In Cohort D-1, the same dose of NXT007 as for Cohort A-4 (i.e., 0.054 mg / kg; 3-fold relative to Cohort A-3), which was confirmed to exhibit nonlinear PK characteristics, was evaluated to explore how the exposure would be when the dose was given with a 1 / 3-fold drug concentration relative to Cohort A-4 (i.e., 2.667 mg / mL, the same drug concentration as for Cohort A-3, which was confirmed to exhibit linear PK characteristics) and a 3-fold dosing volume relative to Cohort A-4.

[0138] Cohort D-2 (N=6) In Cohort D-2, the same dose of NXT007 as for Cohort A-5 (i.e., 0.18 mg / kg; 3.33-fold relative to Cohort A-4), which was confirmed to exhibit nonlinear PK characteristics, was evaluated to explore how the exposure would be when the dose was given with a 1 / 3.33-fold drug concentration relative to Cohort A-5 (i.e., 8 mg / mL, the same drug concentration as for Cohort A-4, which was confirmed to exhibit nonlinear PK characteristics) and a 3.33-fold dosing volume relative to Cohort A-5.

[0139] Cohort D-3 (N=6) In Cohort D-3, the same dose of NXT007 as for Cohort A-5 (i.e., 0.18 mg / kg) was evaluated to explore how the exposure would be when the dose was given with a 3-fold drug concentration relative to Cohort A-5 (i.e., 80 mg / mL with no dilution, which corresponds to the maximum available concentration in Part A) and a 1 / 3-fold dosing volume relative to Cohort A-5.

[0140] Cohort D-4 (N=8) In Cohort D-4 (drug concentration: 150 mg / mL), the same dose of NXT007 (0.18 mg / kg) as for Cohort D-2 (8 mg / mL), Cohort A-5 (26.67 mg / mL), and Cohort D-3 (80 mg / mL) was administered to healthy adults as a single SC administration to explore how the NXT007 exposure would be at higher concentration and lower dosing volume relative to Cohort D-2, Cohort A-5, and Cohort D-3.

[0141] Exposures in Cohorts D-1 to D-3 were predicted based on the blind analysis results of interim data from Part A. Given no useful prior information or certain quantitative anticipation on the effect of drug concentration, the most conservative approach to predict the exposure in Part D from a safety perspective was to assume dose-proportional increase under the same drug concentration. With this approach, the mean Cmaxfor Cohorts D-1 and D-2 were predicted respectively to be 0.699 and 1.81 microgram / mL (i.e., 3-folds relative to Cohort A-3 and 3.33-folds relative to Cohort A-4, respectively). Mean Cmaxfor Cohort D-3 was unpredictable in this context due to the lack of PK data with 80 mg / mL in humans; however, assuming that the PK nonlinearity could be caused by increased drug concentration, Cohort D-3 was predicted to result in a lower mean Cmaxthan that observed in Cohort A-5 or that predicted for Cohort D-2 under the same dosing amount. The selected dose for Cohort D-2, which was anticipated to result in the highest mean Cmaxamong the cohorts in Part D, has a 558- to 602-fold safety margin relative to the NOAEL for the 6-month GLP study in cynomolgus monkeys (20 mg / kg SC Q2W) and a 162- to 183-fold safety margin relative to the NOAEL for the single-dose GLP study in cynomolgus monkeys (15 mg / kg IV).

[0142] Table 6 shows comparison results of the PK parameters of NXT007, which were observed in the subjects who did not develop anti-drug antibodies against NXT007 in all cohorts in Part A and Cohort D-1, Cohort D-2, Cohort D-3, and Cohort D-4 (for Cohort D-4, values in parentheses indicate interim data). Tmaxand t1 / 2were comparable between each cohort, and the dose and the drug concentration of NXT007 were considered as not affecting the elimination process of NXT007. When comparing Cohort A-3 (dose = 0.018 mg / kg, NXT007 conc. = 2.667 mg / ml, dosing volume = 6.750 microliter / kg) and Cohort D-1 (dose = 0.054 mg / kg, NXT007 conc. = 2.667 mg / ml, dosing volume = 20.250 microliter / kg), Cmax / Dose and AUCinf / Dose were comparable when the NXT007 concentration was kept constant at 2.667 mg / ml despite a 3-fold difference in dose and dosing volume, suggesting that bioavailability was also comparable. Furthermore, when comparing Cohort A-4 (dose = 0.054 mg / kg, NXT007 conc. = 8 mg / ml, dosing volume = 6.750 microliter / kg) and Cohort D-2 (dose = 0.18 mg / kg, NXT007 conc. = 8 mg / ml, dosing volume = 22.500 microliter / kg) of the same drug concentration, Cmax / Dose and AUCinf / Dose were comparable when the NXT007 concentration was kept constant at 8 mg / ml despite a 3.33-fold difference in dose and dosing volume, suggesting that bioavailability was also comparable. These results suggest that dose and dosing volume do not contribute to the PK non-linearity issue. Meanwhile, when comparing Cohort D-1 and Cohort A-4 or Cohort D-2 and Cohort A-5 of the same dose, Cmax / Dose and AUCinf / Dose decreased at higher drug concentration, suggesting that bioavailability of NXT007 decreased at higher drug concentration. These results suggested that the primary factor of the PK nonlinearity of NXT007 is drug concentration rather than dose. Furthermore, results indicate that when dose is increased in proportion to dosing volume while keeping drug concentration constant, blood level increases in proportion to the dose, suggesting that nonlinearity issue is not dose-dependent. When Cmax / Dose and AUCinf / Dose of Cohorts A-1 to A-5 and D-1 to D-4 were compared, they were comparable when the drug concentration was 2.667 mg / mL or less, decreased when the drug concentration was > 2.667 mg / mL and < 26.67 mg / mL, and then became stable again when the drug concentration was 26.67 mg / mL or more. These results suggest that non-linearity was dependent on drug concentration up to a certain threshold; for drug concentrations at or above the threshold, bioavailability does not change and blood level increases in proportion to the dose, thus nonlinearity issue does not occur.

[0143]

[0144] FIGs. 2A to D show the PK profiles (time-dependent changes in plasma concentration) of NXT007, which were observed in the subjects who did not develop anti-drug antibodies against NXT007 in Cohort A-3, Cohort A-4, and Cohort D-1. As shown in FIG. 2A and FIG. 2C, in 42 days after administration, the plasma NXT007 level in Cohort D-1, in which a dose of 0.054 mg / kg was administered at 2.667 mg / mL, was equivalent to 3-fold of the plasma NXT007 level in Cohort A-3, in which a dose of 0.018 mg / kg was administered at the same drug concentration. Meanwhile, the plasma NXT007 level when a dose of 0.054 mg / kg was administered at the drug concentration of 8 mg / mL (Cohort A-4) was lower than that when the same dose was administered at the lower drug concentration of 2.667 mg / mL (Cohort D-1). In addition, as shown in FIG. 2B and FIG. 2D, the PK profile curves in the elimination process up to the final observation time point showed consistent form at the drug concentrations between 2.667 and 8 mg / mL. These results suggest that the drug concentration in the formulation does not affect the elimination process but affects the degree of bioavailability.

[0145] FIGs. 3A to D show the PK profiles (time-dependent changes in plasma concentration) of NXT007, which was observed in the subjects who did not develop anti-drug antibodies against NXT007 in Cohort A-4, Cohort A-5, Cohort D-2, Cohort D-3, and Cohort D-4. As shown in FIG. 3A and FIG. 3C, in 42 days after administration, the plasma NXT007 level in Cohort D-2, in which a dose of 0.18 mg / kg was administered at a drug concentration of 8 mg / mL, was equivalent to 3.33-fold of the plasma NXT007 level in Cohort A-4, in which a dose of 0.054 mg / kg was administered at the same drug concentration of 8 mg / ml. Meanwhile, the plasma NXT007 levels when a dose of 0.18 mg / kg was administered at the drug concentration of 26.67 mg / mL (Cohort A-5), 80 mg / mL (Cohort D-3), and 150 mg / mL (Cohort D-4) were comparable, and all of them were lower than that when the same 0.18 mg / kg dose was administered at the lower drug concentration of 8 mg / mL (Cohort D-2). In addition, as shown in FIG. 3B and FIG. 3D, the PK profile curves in the elimination process showed consistent form at the drug concentrations between 8 and 150 mg / mL. These results suggest that the drug concentration does not affect the elimination process but affects the degree of bioavailability, such that the higher NXT007 concentrations (8 mg / ml and above) results in reduced bioavailability as compared to the lower NXT007 concentration (2.667 mg / ml).

[0146] 8.3 Part E: Multiple Sites Administration in Healthy Adults Part E was newly added to further clarify the PK characteristics of NXT007 that showed nonlinearity. Part E was an open-label, randomized, inter-individual, single center study in healthy Japanese male adults. The aim of Part E was to investigate the effect of multiple sites administration on PK and PD of NXT007 in healthy adults, and unlike Part A, subjects received a single SC injection of NXT007 at each of three different sites on abdomen. The dosing regimen for Part E is outlined in Table 7.

[0147]

[0148] Cohort E-1 (N=6) In Cohort E-1, the same dose as for Cohort A-4 (drug concentration: 8 mg / mL) (i.e., 0.054 mg / kg; 3-fold relative to Cohort A-3) was tested to explore how the exposure would be when a dose, which was confirmed to exhibit linear PK characteristics in Cohort A-3 (0.018 mg / kg), was given to three sites per administration. In Cohort E-1, each of the three sites received the same drug concentration (2.667 mg / mL) and dosing volume per site as Cohort A-3.

[0149] Cohort E-2 (N=6) In Cohort E-2, the comparable (0.9-fold) dose as for Cohort A-5 (drug concentration: 26.67 mg / mL) (i.e., 0.162 mg / kg; 3-fold relative to Cohort A-4) was tested to explore how the exposure would be when a dose, which was confirmed to exhibit nonlinear PK characteristics in Cohort A-4 (0.054 mg / kg), was given to three sites per administration. In Cohort E-2, each of the three sites received the same drug concentration (8 mg / mL) and dosing volume per site as Cohort A-4.

[0150] Exposures in Part E were predicted based on the blind analysis results of interim data from Part A. Given no useful prior information or certain quantitative anticipation on the effect of drug concentration, the most conservative approach to predict the exposure in Part D from a safety perspective was to assume dose-proportional increase under the same drug concentration. For Part E, exposure was expected to increase in proportion to the number of injection sites per administration, by which the mean Cmaxfor Cohorts E-1 and E-2 were predicted respectively to be 0.699 and 1.63 microgram / mL (i.e., 3-fold relative to Cohorts A-3 and A-4, respectively).

[0151] Table 8 shows comparison results of the PK parameters of NXT007, which were observed in the subjects who did not develop anti-drug antibodies against NXT007 in all cohorts in Part A and Cohort E-1 and Cohort E-2. Tmaxand t1 / 2were comparable between each cohort, and the dosing amount of NXT007 per site, the total dose, and the drug concentration were considered as not affecting the elimination process of NXT007. When comparing Cohort A-3 and Cohort E-1 of the same drug concentration of 2.667 mg / ml and the same dosing volume per site (or Cohort A-4 and Cohort E-2 of the same drug concentration of 8 mg / ml and the same dosing volume per site), although the total dosing volume and the total dose in both cohorts were different, Cmax / Dose and AUCinf / Dose were comparable, suggesting that bioavailability was also comparable. In other words, the comparisons between Cohort A-3 to E-1, and the comparison between Cohort A-4 to E-2 revealed that exposure increased proportionally to the total dose. This confirms that dose escalation itself does not trigger non-linearity if the concentration is held constant. Meanwhile, when comparing Cohort E-1 (drug concentration = 2.667 mg / ml) and Cohort A-4 (drug concentration = 8 mg / ml), in which the same total dose of 0.054 mg / kg was administered to both cohorts, Cmax / Dose and AUCinf / Dose decreased in the higher drug concentration cohort, even though the total dose was kept constant. The same trend was observed when comparing Cohort E-2 (drug concentration = 8 mg / ml, total dose = 0.162 mg / kg divided across 3 sites) and Cohort A-5 (drug concentration = 26.67 mg / ml, total dose = 0.18 mg / kg administered at 1 site. Cmax / Dose and AUCinf / Dose decreased in the higher drug concentration cohort despite the slightly higher dose, suggesting that bioavailability of NXT007 decreased at higher drug concentration. In other words, both comparisons revealed that dose-normalized exposure decreases with higher drug concentrations. These results reinforce the notion that, at least in comparisons between these cohorts, the primary factor of PK nonlinearity of NXT007 is drug concentration rather than dosing amount per site or total dose.

[0152]

[0153] FIGs. 4A to D show the PK profiles (time-dependent changes in plasma levels) of NXT007, which were observed in the subjects who did not develop anti-drug antibodies against NXT007 in Cohort A-3, Cohort A-4, and Cohort E-1. As shown in FIG. 4A and FIG. 4C, in 42 days after administration, the plasma NXT007 level in Cohort E-1, in which a dose of 0.018 mg / kg was administered at the drug concentration of 2.667 mg / mL at each of three sites (i.e., the total dosing volume and the total dose of Cohort E-1 were 3-fold relative to Cohort A-3, respectively), was equivalent to 3-fold of the plasma NXT007 level in Cohort A-3, in which a dose of 0.018 mg / kg was administered at the same drug concentration at one site. Meanwhile, the plasma NXT007 level when a dose of 0.054 mg / kg was administered at the drug concentration of 8 mg / mL at one site (Cohort A-4) was lower than that when the same dose was administered at the drug concentration of 2.667 mg / mL at each of three injection sites (Cohort E-1). This observation suggested that when the administered doses were kept constant, NXT007 blood levels were decreased in Cohort A-4 (the higher concentration formulation) as compared to Cohort E-1. These results taken together further reinforce the notion that PK non-linearity issue was dictated by NXT007 drug concentration and not the dose or number of injection sites. In addition, as shown in FIG. 4B and FIG. 4D, the PK profile curves in the elimination process showed consistent form at the drug concentrations between 2.667 and 8 mg / mL. These results revealed that the time-course changes in plasma NXT007 level were comparable between a case when the same dose was divided and administered to three sites and when it was administered to a single site, and that the dosing amount per site does not affect PK nonlinearity of NXT007. In addition, when the same drug concentration was used but dose amounts and dosing volumes are increased, the increase in blood level in proportion to the total dose was observed, irrespective of whether or not the dose was divided. These results highlight that the PK non-linearity issue is not dictated by dose or number of injection sites.

[0154] FIGs. 5A to D show the PK profiles (time-dependent changes in plasma concentration) of NXT007, which were observed in the subjects who did not develop anti-drug antibodies against NXT007 in Cohort A-4, Cohort A-5, and Cohort E-2. As shown in FIG. 5A and FIG. 5C, in 42 days after administration, the plasma NXT007 level in Cohort E-2, in which a dose of 0.054 mg / kg was administered at 8 mg / mL at each of three sites, was equivalent to 3-fold of the plasma NXT007 level in Cohort A-4, in which a dose of 0.054 mg / kg was administered at the same drug concentration. Meanwhile, 0.9-fold of the plasma NXT007 level when a dose of 0.18 mg / kg was administered at the drug concentration of 26.67 mg / mL (Cohort A-5) was lower than the plasma NXT007 level when a 0.162 mg / kg, i.e., 0.9-fold of 0.18 mg / kg in Cohort A-5, was administered at 8 mg / mL (Cohort E-2). In addition, as shown in FIG. 5B and FIG. 5D, the PK profile curves in the elimination process showed consistent form at the drug concentrations between 8 and 26.67 mg / mL. These results revealed that the time-course changes in plasma NXT007 level were comparable between a case when the same dosing amount was divided and administered to three sites and when it was administered to a single site, and that the dosing amount per site does not affect PK nonlinearity of NXT007. In addition, when the same drug concentration was used, the increase in blood level in proportion to the total dose was observed, irrespective of whether or not the dose was divided. These results taken together further reinforce the notion that PK non-linearity issue was dictated by NXT007 drug concentration and not the dose or number of injection sites.

[0155] FIG. 6 shows the graphs plotted with dose-normalized Cmax(A) and dose-normalized AUCinf(B) of NXT007 on the Y-axis and drug concentration on the X-axis, which were observed in the subjects who did not develop anti-drug antibodies against NXT007 in all cohorts in Part A, Part D, and Part E. Both dose-normalized Cmaxand dose-normalized AUCinfof NXT007 decreased as drug concentration increased and reached a plateau at about 50 % decrease at maximum. The predicted values of PK parameters (“Predicted”) are also depicted in the graphs by solid line curves, which values were obtained by applying the relationship between dose-normalized Cmaxor dose-normalized AUCinf(“Observed”) and drug concentration to an inhibitory sigmoid maximum effect model. Parameter estimates in the inhibitory sigmoid maximum effect model are summarized in Table 9 (the values in parentheses indicate the values when interim data were used for cohorts D-4).

[0156] C: Drug Concentration; E: PK parameter value; E0: PK parameter value when the drug concentration is 0; IC50: Drug concentration that achieves 50% of maximum effect; Imax: Maximum effect of drug concentration; γ: Hill's coefficient

[0157] The drug concentrations at which dose-normalized Cmaxand dose-normalized AUCinf(which decrease dependent on NXT007 drug concentration) reach the plateau were investigated by using the inhibitory sigmoid maximum effect model with parameters shown in Table 9. Specifically, the minimum values of PK parameters, i.e., values of PK parameters when the drug concentration is infinite, were calculated first. Next, the drug concentrations that give values of 1.25-fold relative to the minimum values of PK parameters were calculated. The 1.25-fold criterion was determined based on the acceptable upper limits for bioequivalence set forth in the Guideline for Bioequivalence Studies51), 52), 53)issued by the drug regulatory authority. Thus, it was calculated that the values of 1.25-fold relative to the minimum values were achieved at the drug concentration of 9.94 mg / mL (9.71 mg / mL when interim data were used for cohorts D-4) for dose-normalized Cmaxand at 8.92 mg / mL (10.7 mg / mL when interim data were used for cohorts D-4) for dose-normalized AUCinf. It was considered from the above results that the bioavailability of NXT007 at the drug concentration of 9.94 mg / mL or more (e.g., 10.7 mg / mL or more) fall within the range of 80% to 125% relative to the bioavailability at infinite drug concentration based on any of dose-normalized Cmaxand dose-normalized AUCinf, and reach the plateau.

[0158] It was found from the above results that bioavailability of NXT007 is affected by drug concentration rather than dose; that if dose is increased in proportion to drug volume under a constant drug concentration, blood level increases in proportion to the dose, thus nonlinearity issue does not occur; that for the drug concentration at or above 9.94 mg / mL (e.g., 10.7 mg / mL or more), bioavailability does not change significantly with further drug concentration increases, and blood level increases in proportion to the dose, thus nonlinearity issue does not occur; and that there is no change in blood level between a case when the same dosing amount is divided and administered to multiple sites and when it is administered to a single site, and blood level increases in proportion to the total dose irrespective of whether or not the dose is divided, under the same drug concentration. For patients, higher plasma NXT007 level than that in healthy adults must be reached to achieve the target efficacy. For the drug concentration at or above 9.94 mg / mL (e.g., 10.7 mg / mL or more), bioavailability does not change and blood level increases in proportion to the dose, thus the target plasma NXT007 level can be achieved. In summary, the inventors found that PK non-linearity was dependent on the concentration of NXT007 in the formulation rather than dependent on dose. The inventors also found a surprising technical effect: the PK non-linearity occurs only within a certain NXT007 concentration window. For NXT007 concentrations at or above 9.94 mg / ml, e.g., at 10.7 mg / ml or more, the non-linearity issue resolves, such that bioavailability stabilizes and remains substantially constant at sufficient levels. This discovery allowed for the design of high-concentration NXT007 pharmaceutical formulations characterized by a linear PK profile and stable bioavailability.

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Factor VIII-Mimetic Function of Humanized Bispecific Antibody in Hemophilia A. N Engl J Med. 2016;374(21):2044-53. 40) Lambert T, Benson G, Dolan G, Hermans C, Jimenez-Yuste V, Ljung R, et al. Practical aspects of extended half-life products for the treatment of haemophilia. Ther Adv Hematol. 2018;9(9):295-308. 41) Yoneyama K, Schmitt C, Kotani N, Levy GG, Kasai R, Iida S, et al. A Pharmacometric Approach to Substitute for a Conventional Dose-Finding Study in Rare Diseases: Example of Phase III Dose Selection for Emicizumab in Hemophilia A. Clin Pharmacokinet. 2018 Sep ;57(9):1123-34. 42) Kotani N, Yoneyama K, Kawakami N, Shimuta T, Fukase H, Kawanishi T. Relative and Absolute Bioavailability Study of Emicizumab to Bridge Drug Products and Subcutaneous Injection Sites in Healthy Volunteers. Clin Pharmacol Drug Dev. 2018;19.doi:10:1002 / cpdd.617. 43) Common Technical Document 2.6.2.2.4 on Hemlibra 30 mg for SC-chu / Hemlibra 60mg for SC-chu / Hemlibra 90mg for SC-chu / Hemlibra 105mg for SC-chu / Hemlibra 150mg for SC-chu. Chugai Pharmaceutical Company. 23 Mar 2018. 44) International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline, Addendum to ICH S6: Preclinical Safety Evaluation of Biotechnology-derived Pharmaceuticals, S6(R1). [Accessed January 31, 2023] https: / / database.ich.org / sites / default / files / S6_R1_Guideline_0.pdf. Published 2011. 45) World Federation of Hemophilia. Severity of hemophilia [(Accessed Dec 26, 2018]. Available from: https: / / www.wfh.org / en / page.aspx?pid=643 46) European Medicines Agency EMA Committee for Medicinal Products for Human Use (CHMP) Guideline on the clinical investigation of recombinant and human factor VIII products. 26 July 2018 EMA / CHMP / BPWP / 144533 / 2009 rev. 2 47) Blanchette VS, Key NS, Ljung LR, Manco-Johnson MJ, van den Berg HM, Srivastara A. Definitions in hemophilia: communication from the SSC of the ISTH. J Thromb Haemost. 2014;12:1935-9. 48) EU Ethical considerations for clinical trials on medicinal products conducted with the pediatric population 2017. 49) Stephen RH. Blood sample volumes in child health research: review of safe limits. Bull World Health Organ 2011:89:46-53. 50) Hemlibra(R)(emicizumab-kxwh) Prescribing Information; US Package Insert, Genentech, Inc. [Accessed Sep 7 2021] https: / / www.gene.com / download / pdf / hemlibra_prescribing.pdf 51) Bioavailability Studies Submitted in NDAs or INDs - General Considerations, Center for Drug Evaluation and Research, April 2022. https: / / www.fda.gov / media / 121311 / download 52) Guideline on the Investigation of Bioequivalence, Committee for Human Medicinal Products (CHMP), January 2010. https: / / www.ema.europa.eu / en / documents / scientific-guideline / guideline-investigation-bioequivalence-rev1_en.pdf 53) Guideline for Bioequivalence Studies of Generic Products, PSEHB / PED Notification No. 0319-1, March 19, 2020. https: / / www.nihs.go.jp / drug / be-guide(e) / 2020 / GL1_BE_2020.pdf

[0160] EVALUATION OF OCCURRENCE RATE OF VP IN MAB FORMULATION BY ADDITION OF PS80 In this Example, the effect of different concentrations of Polysorbate 80 (PS80) in a monoclonal antibody (mAb) formulation on the occurrence rate of visible particles (VPs) in the formulation was evaluated. In this and subsequent examples, a formulation containing NXT007 (rINN: zemocimig) as the mAb was used.

[0161] (Evaluation methods) Each component shown in Table 11 was dissolved in purified water and the pH was adjusted to 6.0 using aspartic acid. Then the solution was filtered through a 0.22 micrometer filter using a Terumo syringe and filled into a syringe. For each syringe, a shipping stress test and a drop / vibration stress test (test condition: ASTM D4169) were conducted in the following order (1) through (5), and the occurrence of VPs was evaluated by visual inspection when (3), (4), and (5) were conducted: (1) actual shipping → (2) ASTM → (3) actual shipping (= 2nd shipping) → (4) ASTM (= 2nd ASTM) → (5) stored at 5°C for 1 month (1M). Samples without VPs were determined as non-defective samples and samples with VPs were determined as defective samples, and the number of non-defective / defective samples were counted at each evaluation point.

[0162]

[0163]

[0164] (Evaluation results) The results of the visual inspection are shown in Tables 12 to 14. The results show that at each evaluation point, defective samples containing VPs were observed when PS80 was not added, while no VPs were observed when PS80 was added at any concentration (0.5 to 5.0 mg / mL), showing an improvement in the occurrence rate of VPs.

[0165]

[0166]

[0167]

[0168] EVALUATION OF THE EFFECT OF SURFACTANT TYPE ON THE STABILITY OF MAB FORMULATION In this Example, the effect of different surfactant types in a monoclonal antibody (mAb) formulation on the stability of the formulation was evaluated.

[0169] (Evaluation methods) Each component shown in Table 16 was dissolved in purified water and the pH was adjusted to 6.0 using L-aspartic acid. The prepared solution was sterilized by filtration using a syringe and a 0.22 micrometer filter, and 0.5 mL of the solution was filled into a glass vial. The vials were then stored in an inverted position at 5°C, 25°C, and 40°C for 1, 2, 3, 4.5, 5, and 6 months. At each time point, the presence or absence of insoluble particles (visible particles (VP)) formation was confirmed by visual inspection. In addition, samples stored at 25°C and 40°C for 5 months were rotated at room temperature, 18 rpm for 3 days and 7 days, and then visual inspection was performed. Samples without VPs were determined as non-defective samples and samples with VPs were determined as defective samples, and the number of non-defective / defective samples were counted at each evaluation point.

[0170]

[0171]

[0172] (Evaluation results) Table 17 shows the results of the visual inspection performed after storage at each temperature condition. The results show that no VPs were observed for any sample or storage condition. Furthermore, Table 18 shows the results of the visual inspections performed after 7 days of rotation at room temperature. The results show that no VPs were observed in any samples due to the rotation. These results indicate that there is no significant difference in the VP formation in the mAb formulation regardless of the surfactant used.

[0173] Furthermore, when these surfactants were used, no difference was observed in terms of size variant, charge variant, potency, and binding after a rotation stress test and a storage stability test were conducted.

[0174]

[0175]

[0176] EVALUATION OF MAB FORMULATION STABILITY BY ADDITION OF METHIONINE In this Example, the effect of different methionine (Met) concentrations in a monoclonal antibody (mAb) formulation on the stability of the formulation was evaluated.

[0177] (Evaluation methods) Each component shown in Table 20 was dissolved in purified water and the pH was adjusted to 6.0 using aspartic acid. Then the solution was filtered through a 0.22 micrometer filter using a Terumo syringe, filled into a syringe, and stored at 5°C, 25°C, or 40°C for 0.5 to 24 months (M). Furthermore, some samples were exposed to light under a white fluorescent lamp at an illuminance of 1,000 lux at 25°C for 7 days to evaluate the effect of light exposure stress. For each formula, area ratios [area%] of the antibody size variants were evaluated with size exclusion chromatography (SE-HPLC), the size variants being a monomer (corresponding to main peak), HMW Forms (HMWS: corresponding to post-peaks), and LMW Forms (LMWS: corresponding to pre-peaks). In addition, area ratios [CPA%] of pre-peaks, post-peaks, and a main peak were evaluated by capillary electrophoresis (CE-SDS (NR: Non-reduction)).

[0178]

[0179]

[0180] (Evaluation results) The results of the SE-HPLC measurement are shown in Table 21 and FIG. 7A. The results show that the peak area ratio of HMW Forms (post-peak) decreased in most of the storage temperatures and durations, when Met concentration was increased from 0 mM to 20 mM. No further decrease in area ratio was observed when the Met concentration was increased to 40 mM, 60 mM, and 100 mM. The results of the CE-SDS (NR) measurement are shown in Table 22 and FIG. 7B. The results show that the area ratio of LMW Forms (pre-peaks) decreased in most of the storage temperatures and durations, when Met concentration was increased from 0 mM to 20 mM. No further decrease in area ratio was observed when Met concentration was increased to 40 mM, 60 mM, and 100 mM. Repeating this experiment with 150 mg / ml instead of 180 mg / ml NXT007 (INN: zemocimig) will demonstrate the same effect of Met concentration.

[0181] Image capillary isoelectric focusing (icIEF) was conducted for similar formulas, and no improvement in charge variants by the addition of Met was observed.

[0182]

[0183]

[0184] EVALUATION OF THE EFFECT OF BUFFER TYPE ON THE STABILITY OF MAB FORMULATION In this Example, the effect of different buffer types in a monoclonal antibody (mAb) formulation on the stability of the formulation was evaluated.

[0185] (Evaluation methods) Each component shown in Table 24 was dissolved in purified water and the pH was adjusted to 6.0 using NaOH or HCl. Then the solution was filtered through a 0.22 micrometer filter using a Terumo syringe and filled into a microtube. SE-HPLC measurement was conducted for a sample immediately after preparation (Initial) and for samples stored at 25°C or 40°C for 1, 2, or 4 weeks (W), and area ratios [area%] of the antibody size variants were evaluated, the size variants being a monomer (main peak), HMW Forms (high molecular weight forms), and LMW Forms (low molecular weight forms).

[0186]

[0187] *Tris: 2-Amino-2-Hydroxymethyl-1,3-propanediol

[0188] (Evaluation results) The results of the SE-HPLC measurement are shown in Table 25 and FIG. 8. In all samples, the amount of HMW Forms increased with increasing storage temperature and long-term storage. Little difference was observed among buffer types under the Initial and 25°C storage conditions, however, under the 40°C storage conditions, the rate of increase in the amount of HMW Forms varied depending on the buffer type, increasing in the order of N20H < N20C < N20T < N20P (L-histidine < citric acid < Tris < phosphate buffer). The increase in the amount of HMW Forms was particularly significant in N20P, increasing to 1.9% at 40°C4W. On the other hand, in N20H, which showed the slowest increase in the amount of HMW Forms, it remained at 1.0%. The amount of LMW forms in N20P increased up to 7.5% (25°C4W) after storage. However, other samples showed almost no increase even after storage at 40°C4W. These results indicate that the mAb is most stable when L-histidine is used as the buffer type.

[0189]

[0190] EVALUATION OF THE EFFECT OF PH ON THE STABILITY OF MAB FORMULATION In this Example, the effect of different buffer pHs in a monoclonal antibody (mAb) formulation on the stability of the formulation was evaluated.

[0191] (Evaluation methods) Each component shown in Table 27 was dissolved in purified water and the pH was adjusted to each value shown in Table 27 using NaOH or HCl. Then the solution was filtered through a 0.22 micrometer filter using a Terumo syringe and filled into a microtube. SE-HPLC measurement was conducted for a sample immediately after preparation (Initial) and for samples stored at 25°C or 40°C for 1 or 2 weeks (W), and area ratios [area%] of the antibody size variants were evaluated, the size variants being a monomer (main peak), HMW Forms (high molecular weight forms), and LMW Forms (low molecular weight forms). In addition, icIEF measurement was conducted for the same samples, and content ratios [%] of the antibody charge variants were evaluated, the charge variants being a main component (main peak), acidic components (Acidic Region), and basic components (Basic Region).

[0192]

[0193]

[0194] (Evaluation results: SE-HPLC) The results of the SE-HPLC measurement are shown in Table 28 and FIG. 9. Under all storage conditions, N20H5.0 showed the highest amount of HMW Forms (Max. 1.8%) and the differences among other samples were relatively small (Max. 0.7-1.0%). Furthermore, under all storage conditions, the amount of HMW Forms increased in the following order: The amount of LMW forms was comparable under all buffer pH conditions (0.0-0.1%). These results demonstrate that the aggregation stability of the mAb formulation is high in a pH 6.0 buffer (N20H6.0).

[0195] (Evaluation results: icIEF) The results of the icIEF measurement are shown in Table 29 and FIG. 10. In all samples, the content ratio of the main peak decreased with increasing storage temperature and long-term storage. With increasing pH, the content ratio of the main peak decreased (40°C2W, N20H5.0: 74.8% → N20H7.0: 62.7%) and the content ratio of the acidic components increased (40°C2W, N20H5.0: 21.8% → N20H7.0: 35.1%). Meanwhile, the content ratio of the basic components was highest at pH 5.0 (N20H5.0) (40°C2W, 3.4%) and comparable at other pH (2.1-2.2%). These results demonstrate that at the intermediate buffer pH, i.e., pH 6.0 (N20H6.0), the increase of both the acidic components and basic components after storage were relatively small and the mAb was considered to be in a stable charge condition.

[0196] The results of the above SE-HPLC and icIEF measurements indicated that the pH of the mAb formulation was optimal at pH 6.0 in terms of both aggregation stability and charge stability.

[0197]

[0198]

[0199] EVALUATION OF THE EFFECT OF BUFFER (L-HISTIDINE) CONCENTRATION ON THE STABILITY OF MAB FORMULATION In this Example, the effect of different buffer (L-histidine) concentrations in a monoclonal antibody (mAb) formulation on the stability of the formulation was evaluated.

[0200] (Evaluation methods) Each component shown in Table 31 was dissolved in purified water and the pH was adjusted to 6.0 using NaOH or HCl. Then the solution was filtered through a 0.22 micrometer filter using a Terumo syringe and filled into a microtube. icIEF measurement was conducted for a sample immediately after preparation (Initial) and for samples stored at 25°C or 40°C for 1 or 2 weeks (W), and content ratios [%] of the antibody charge variants were evaluated, the charge variants being a monomer (main peak), HMW Forms (high molecular weight forms), and LMW Forms (low molecular weight forms).

[0201]

[0202]

[0203] (Evaluation results) The results of the icIEF measurement are shown in Table 32 and FIG. 11. In all samples, the content ratio of the main peak decreased with increasing storage temperature and long-term storage. With increasing L-histidine concentration, the content ratio of the main peak slightly decreased (40°C2W, N5H: 73.7% → N80H: 70.9%) and the content ratio of the acidic components increased (40°C2W, N5H: 24.3% → N80H: 27.5%). However, the change is small and the basic components showed little difference. These results demonstrate that buffer (L-histidine) concentration does not have a large impact on mAb stability.

[0204]

[0205] EVALUATION OF THE EFFECT OF THE TYPE OF COUNTERION ON THE STABILITY OF MAB FORMULATION In this Example, the effect of different types of counterion in a monoclonal antibody (mAb) formulation on the stability of the formulation was evaluated.

[0206] 1. Evaluation of viscosity (Evaluation methods) Each component shown in Table 34 was dissolved in purified water and the pH was adjusted to 6.0 using each counterion. Then the solution was filtered through a 0.22 micrometer filter using a Terumo syringe. For each formula, viscosity was measured by an EMS viscometer under the condition of 25°C

[0207]

[0208] QS: sufficient quantity to adjust to pH 6.0

[0209] (Evaluation results) The results of the viscosity measurement are shown in Table 35and FIG. 12. In particular, under the conditions where mAb concentrations were 150 mg / mL or higher, the viscosity of the formulation was reduced when aspartic acid, malic acid, citric acid, succinic acid, tartaric acid, malonic acid, and alpha-ketoglutaric acid were used to adjust the pH, compared to when hydrochloric acid was used.

[0210]

[0211] 2. Evaluation of size variants (SEC) (Evaluation methods) Each component shown in Table 37 was dissolved in purified water and the pH was adjusted to 6.0 using each counterion. Then the solution was filtered through a 0.22 micrometer filter using a Terumo syringe and filled into a syringe. Size exclusion chromatography (SE-HPLC, SEC) was conducted for a sample immediately after preparation (Initial) and samples stored at 40°C temperature and 75% humidity for 2 or 4 weeks (W) to evaluate area ratio [area%] of the antibody size variants: HMW Forms (high molecular weight forms).

[0212]

[0213]

[0214] (Evaluation results) The results of the SEC measurement are shown in Table 38 and FIG. 13. At each measurement time point, the HMW value was suppressed when aspartic acid was used for pH adjustment, compared to when other pH adjusting agents were used. In addition, similar results were obtained regarding the rate of increase of HMW.

[0215]

[0216] EVALUATION OF THE EFFECT OF ARGININE CONCENTRATION ON THE STABILITY OF MAB FORMULATION In this Example, the effect of different arginine (Arg) concentrations in a monoclonal antibody (mAb) formulation on the stability of the formulation was evaluated.

[0217] 1. Evaluation of colloidal stability (DLS measurement) (Evaluation methods) Each component shown in Table 40 was dissolved in purified water and the pH was adjusted to 6.0 using aspartic acid. Then the solution was filtered through a 0.22 micrometer filter using a Terumo syringe and filled into a syringe. For each formula, diffusion coefficient (D) was measured by dynamic scattering (DLS) method. The D values were plotted against antibody concentrations to calculate kD values.

[0218]

[0219]

[0220] (Evaluation results) The results of the DLS measurement are shown in Table 41 and FIG. 14. The results showed that the kD was -8.98 mL / g at an Arg concentration of 80 mM, whereas increasing the Arg concentration to 150 mM resulted in a kD of -4.63 mL / g, indicating an improvement in colloidal stability. Increasing the Arg concentration to 225 mM or 300 mM did not result in any further improvement in colloidal stability.

[0221]

[0222] 2. Evaluation of insoluble particles during storage (MFI measurement, visual inspection) (Evaluation methods) Each component shown in Table 43 was dissolved in purified water and the pH was adjusted to 6.0 using aspartic acid. Then the solution was filtered through a 0.22 micrometer filter using a Terumo syringe and filled into a syringe. Samples were taken out immediately after preparation (Initial) and after storage at the 40°C temperature and the 75% humidity for 1, 3, or 6 months (M), and then the number of microparticles was measured by MFI and the number of insoluble microparticles was measured by visual inspection. Visual inspection was performed under the conditions of an EP method (illumination 2,700-3,300 lux, white and black backgrounds for 5 seconds each) and an EVI method (illumination 10,000 lux or more, black background for 30 seconds).

[0223]

[0224]

[0225] (Evaluation results) The results of the MFI measurement are shown in Table 44 and FIG. 15. In particular, in the ranges of 10 micrometers or more and 5 micrometers or more, the number of microparticles was suppressed more at Arg concentrations of 100 mM or higher than at 50 mM. Increasing the Arg concentration to 150 mM or 225 mM did not result in a further suppression of the number of microparticles. The results of the visual inspection are shown in Table 45. Both the EP method and the EVI method showed that the number of insoluble particles was suppressed in an Arg concentration-dependent manner in the range of Arg concentrations from 50 mM to 150 mM. Increasing the Arg concentration to 225 mM did not result in a further suppression of the number of insoluble particles.

[0226]

[0227]

[0228] EVALUATION OF THE STABILITY OF 150 MG / ML MAB FORMULATION UNDER 5°C STATIC STORAGE CONDITIONS In this Example, the stability of the monoclonal antibody (mAb) formulation under 5°c static storage conditions was evaluated. Each component shown in Table 47 was dissolved in purified water and the pH was adjusted to 6.0 using aspartic acid. Samples were taken out after storage at 5°C for a certain period, and then the presence or absence of insoluble particles was confirmed by visual inspection. Visual inspection was performed under the conditions of an EVI method (illumination of 10,000 lux or more, black background for 30 seconds).

[0229]

[0230]

[0231] (Evaluation results) The results of the visual inspection are shown in Table 48. No insoluble particles were detected in the EVI method over a storage period of 6 months (6M) to 36 months (36M).

[0232]

[0233] EVALUATION OF INSOLUBLE PARTICLE FORMATION IN MAB FORMULATIONS FOLLOWING ACTUAL SHIPPING (VISUAL INSPECTION) In this Example, the effect of actual shipping (air and truck shipping) of the monoclonal antibody (mAb) formulation on insoluble particle formation was evaluated. Each component shown in Table 50 was dissolved in purified water and the pH was adjusted to 6.0 using aspartic acid. After preparation (Initial), the number of insoluble microparticles was measured by visual inspection. Visual inspection was performed under the conditions of an EP method (illumination 2,700-3,300 lux, white and black backgrounds for 5 seconds each). After the visual inspection, the syringes were transported back and forth between Japan and Switzerland by air and truck, and then another visual inspection was performed in the same manner.

[0234]

[0235]

[0236] (Evaluation results) The results of the visual inspection are shown in Table 51. The results of the EP method test showed that no insoluble particles were detected in the syringe formulation even after actual shipment by air and truck.

[0237]

[0238] The present disclosure revealed for the first time the relationship between the concentration of NXT007 in a formulation to be administered to a human subject and blood level in the human. By administering the formulation of the present disclosure containing NXT007 at or above a certain level to a human subject, stable bioavailability can be provided in the human without greatly depending on the drug concentration, and the formulation can be used in the prevention and treatment of hemophilia.

Claims

1. A formulation comprising an antibody, wherein the antibody is a bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the first antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the second antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, wherein the concentration of the antibody in the formulation is 10.7 mg / ml or more.

2. The formulation according to claim 1, wherein the antibody is a bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 13, and the first antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 14; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 15, and the second antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 16.

3. The formulation according to claim 1, wherein the antibody is a bispecific antibody comprising: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 21, and the first antibody light chain comprises the amino acid sequence of SEQ ID NO: 22; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 23, and the second antibody light chain comprises the amino acid sequence of SEQ ID NO: 24.

4. The formulation according to any one of claims 1 to 3, comprising: 10.7 mg / mL or more of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 5 to 80 mmol / L; 100 to 300 mmol / L of L-arginine; 20 to 100 mmol / L of L-methionine; and 0.5 to 5.0 mg / mL of Polysorbate 80, Polysorbate 20, or Poloxamer 188 wherein the pH of the formulation is 5.5 to 7.0.

5. The formulation according to any one of claims 1 to 3, comprising: 10.7 mg / mL or more of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 18 to 22 mmol / L; 135 to 165 mmol / L of L-arginine; 18 to 22 mmol / L of L-methionine; and 0.45 to 0.55 mg / mL of Polysorbate 80 wherein the pH of the formulation is 5.4 to 6.6.

6. The formulation according to any one of claims 1 to 3, comprising: 10.7 mg / mL or more of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 19 to 21 mmol / L; 142.5 to 157.5 mmol / L of L-arginine; 19 to 21 mmol / L of L-methionine; and 0.475 to 0.525 mg / mL of Polysorbate 80 wherein the pH of the formulation is 5.7 to 6.3.

7. The formulation according to any one of claims 1 to 3, comprising: 10.7 mg / mL or more of the antibody; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Polysorbate 80, wherein the pH of the formulation is 6.0.

8. The formulation according to any one of claims 1 to 7, wherein the concentration of the antibody is 10.7 to 235 mg / mL.

9. The formulation according to any one of claims 1 to 7, wherein the concentration of the antibody is 10.7 to 150 mg / mL.

10. The formulation according to any one of claims 1 to 7, wherein the concentration of the antibody is 26.67 mg / mL or more.

11. The formulation according to any one of claims 1 to 7, wherein the concentration of the antibody is 26.67 to 150 mg / mL.

12. A formulation comprising: 150 mg / mL of zemocimig; histidine-aspartate buffer, wherein the concentration of histidine in the buffer is 20 mmol / L; 150 mmol / L of L-arginine; 20 mmol / L of L-methionine; and 0.5 mg / mL of Polysorbate 80, wherein the pH of the formulation is 6.0.

13. The formulation according to any one of claims 1 to 12, which is for administration to a human.

14. The formulation according to any one of claims 1 to 13, which is for use in subcutaneous administration.

15. The formulation according to any one of claims 1 to 14, which is for use in preventing or treating hemophilia A.

16. The formulation according to any one of claims 4 to 7 or 12, characterized by having reduced viscosity as compared to a reference formulation having the same component composition except that hydrochloric acid is used to adjust the pH.

17. The formulation according to claim 16, wherein the viscosity is measured by an EMS viscometer.

18. A medicament comprising a bispecific antibody for use in treatment of hemophilia A with or without an inhibitory substance, wherein the bispecific antibody comprises: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the first antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the second antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, wherein the concentration of the bispecific antibody in the medicament is 10.7 mg / ml or more, and wherein the bispecific antibody is administered subcutaneously to a human patient.

19. A medicament comprising a bispecific antibody for use in treatment of hemophilia A with or without an inhibitory substance, wherein the bispecific antibody comprises: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the first antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises heavy chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the second antibody light chain comprises light chain CDRs 1, 2, and 3 which comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, wherein the concentration of the bispecific antibody in the medicament is 26.67 mg / ml or more, and wherein the bispecific antibody is administered subcutaneously to a human patient.

20. The medicament according to claim 18 or 19, wherein the bispecific antibody comprises: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 13, and the first antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 14; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises a heavy chain variable region which comprises the amino acid sequence of SEQ ID NO: 15, and the second antibody light chain comprises a light chain variable region which comprises the amino acid sequence of SEQ ID NO: 16.

21. The medicament according to claim 18 or 19, wherein the bispecific antibody comprises: (1) a first antibody heavy chain and a first antibody light chain that bind to blood coagulation factor IX and / or activated blood coagulation factor IX, wherein the first antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 21, and the first antibody light chain comprises the amino acid sequence of SEQ ID NO: 22; and (2) a second antibody heavy chain and a second antibody light chain that bind to blood coagulation factor X, wherein the second antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 23, and the second antibody light chain comprises the amino acid sequence of SEQ ID NO: 24.

22. The medicament according to any one of claims 18 to 21, characterized in that in said concentration range of the antibody in the medicament, the concentration of the antibody in the blood of the human patient administered the medicament increases linearly with the increase in the concentration of the antibody in the medicament.

23. The medicament according to any one of claims 18 to 21, characterized in that the dose of the antibody is set by increasing or decreasing the dosing volume, while keeping the antibody concentration in the medicament constant, and the concentration of the antibody in the blood of the human patient administered the medicament increases linearly with the increase in the set dose of the antibody.

24. A method of reducing viscosity in an antibody formulation comprising zemocimig, comprising adding at least one of aspartic acid, malic acid, citric acid, succinic acid, tartaric acid, malonic acid, and alpha-ketoglutaric acid to a starting antibody formulation comprising 150 mg / ml zemocimig , in an amount sufficient to adjust the antibody formulation to pH 6.0, wherein the viscosity in the resulting formulation is reduced as compared to a corresponding antibody formulation prepared by adding hydrochloric acid to the starting antibody formulation in an amount sufficient to adjust the corresponding antibody formulation to pH 6.0.

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