scholarly journals FVIIIa-mimetic bispecific antibody (Mim8) ameliorates bleeding upon severe vascular challenge in hemophilia A mice

Blood ◽  
2021 ◽  
Author(s):  
Henrik Østergaard ◽  
Jacob Lund ◽  
Per Jr Greisen ◽  
Stine Kjellev ◽  
Anette Henriksen ◽  
...  
Keyword(s):  
Bispecific Antibody ◽  
Hemophilia A ◽  
Factor Ix ◽  
Factor X ◽  
Equilibrium Dissociation Constant ◽  
Biophysical Properties ◽  
Kd Values ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd ◽  
Apparent Equilibrium

Hemophilia A (HA) is a bleeding disorder resulting from deficient Factor VIII (FVIII), which normally functions as a cofactor to activated Factor IX (FIXa) that facilitates activation of Factor X (FX). To mimic this property in a bispecific antibody (biAb) format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting biAb (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant (KD) of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with KD-values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by four orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation with potencies 13 and 18 times higher than a sequence-identical analog of emicizumab, respectively. A similar potency difference was observed in a tail-vein transection model in hemophilia A mice, while reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetics of Mim8 were investigated and a half-life of 14 days demonstrated in cynomolgus monkey. In conclusion, Mim8 is a FVIIIa-mimetic with a potent and efficacious hemostatic effect based on preclinical data.

Generation of a Novel Bispecific Antibody (ACE910) Against Activated Factor IX and Factor X Mimicking the Function of Factor VIII Cofactor Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1126-1126
Author(s):  
Tomoyuki Igawa ◽  
Zenjiro Sampei ◽  
Tetsuhiro Soeda ◽  
Yukiko Okuyama-Nishida ◽  
Chifumi Moriyama ◽  
...  
Keyword(s):  
Large Scale ◽  
Bispecific Antibody ◽  
Hemophilia A ◽  
Therapeutic Potential ◽  
Factor Ix ◽  
Factor X ◽  
Liquid Formulation ◽  
Igg Antibody ◽  
Fviii Activity ◽  
Routine Prophylaxis

Abstract Abstract 1126 Exogenous factor VIII (FVIII) is used to reduce bleeding complications in patients with severe hemophilia A. However, there are two drawbacks of current routine prophylaxis by FVIII. One is the requirement of frequent intravenous administration due to its short half-life and low subcutaneous bioavailability of FVIII. Second is the development of anti-FVIII antibodies (inhibitors) in approximately 30% of the severe patients which deprives the patients from routine prophylaxis by FVIII. To overcome these drawbacks, bispecific IgG antibody against activated factor IX (FIXa) and factor X (FX), which mimics the cofactor function of FVIII by placing these two factors into spatially appropriate positions, was screened from approximately 40,000 bispecific antibodies recognizing FIXa by the one arm and FX by the other arm. The therapeutic potential of the bispecific antibody identified from the screening was marginal due to insufficient FVIII-mimetic activity and poor pharmacokinetics, and moreover, large scale purification of recombinant bispecific IgG antibody was challenging. Therefore, the lead bispecific antibody, after humanization, was subjected to multidimensional optimization process in order to improve both the therapeutic potential and the manufacturability of the bispecific antibody. FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulty of manufacturing bispecific antibody was overcome by identifying common light chain for anti-FIXa and FX heavy chain through framework/complementarity determining region shuffling, and by isoelectric point engineering of the two heavy chain variable regions to facilitate ion exchange chromatography purification of the bispecific antibody. Engineering to overcome low solubility and deamidation was also performed to enable stable high concentration liquid formulation for clinical use. ACE910, multidimensionally optimized bispecific antibody, exhibited potent FVIII-mimetic activity in human FVIII deficient plasma (more than 10% of FVIII activity at 300 nM in thrombin generation assay), and half-life of approximately 3 weeks with high subcutaneous bioavailability in cynomolgus monkey, enabling effective prophylaxis by subcutaneous administration with long dosing interval. In silico immunogenicity prediction analysis suggested that ACE910 was minimally immunogenic in human, in contrast to high immunogenicity of FVIII in human. Importantly, the activity of ACE910 was not affected by the presence of inhibitors, while polyclonal anti-ACE910 antibody did not inhibit FVIII activity, allowing the use of ACE910 without considering the development or presence of inhibitors. Furthermore, ACE910 could be purified in a large scale manufacturing, and formulated into patient-friendly subcutaneously injectable liquid formulation for clinical use. We believe that ACE910, with its multidimensionally optimized profile, would significantly improve the quality of life of hemophilia A patients by reducing not only bleeding but also the burden on the patients themselves, their parents, and all medical staff. Disclosures: Igawa: Chugai Pharmaceutical Co.,Ltd: Employment. Sampei:Chugai Pharmaceutical Co., Ltd.: Employment. Soeda:Chugai Pharmaceutical Co.,Ltd: Employment. Okuyama-Nishida:Chugai Pharmaceutical Co., Ltd.: Employment. Moriyama:Chugai Pharmaceutical Co.,Ltd: Employment. Wakabayashi:Chugai Pharmaceutical Co.,Ltd: Employment. Tanaka:Chugai Pharmaceutical Co.,Ltd: Employment. Muto:Chugai Pharmaceutical Co., Ltd.: Employment. Kojima:Chugai Pharmaceutical Co.,Ltd: Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment. Yoshihashi:Chugai Pharmaceutical Co.,Ltd: Employment. Harada:Chugai Pharmaceutical Co.,Ltd: Employment. Funaki:Chugai Pharmaceutical Co.,Ltd: Employment. Haraya:Chugai Pharmaceutical Co.,Ltd: Employment. Tatsuhiko:Chugai Pharmaceutical Co.,Ltd: Employment. Suzuki:Chugai Pharmaceutical Co.,Ltd: Employment. Esaki:Chugai Pharmaceutical Co.,Ltd: Employment. Nabuchi:Chugai Pharmaceutical Co.,Ltd: Employment. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment.

Hemostatic Effect of a Novel Bispecific Antibody (ACE910) Against Activated Factor IX and Factor X in an Acquired Hemophilia A Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 42-42
Author(s):  
Atsushi Muto ◽  
Takehisa Kitazawa ◽  
Kazutaka Yoshihasi ◽  
Minako Takeda ◽  
Tetsuhiro Soeda ◽  
...  
Keyword(s):  
Single Dose ◽  
Bispecific Antibody ◽  
Hemophilia A ◽  
Factor Ix ◽  
Factor X ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Repeated Doses ◽  
Cofactor Activity

Abstract Abstract 42 Background: Hemophilia A is treated by intravenous replacement therapy with factor VIII (FVIII), either on demand to resolve bleeding or as a prophylactic to prevent bleeding. Recently, routine prophylactic treatment is recommended to effectively prevent bleeding and to reduce bleeding-related chronic joint damage. However, the need for frequent intravenous injections of FVIII negatively affects patients' quality of life and their adherence to the routine prophylactic regimen. More importantly, approximately 30% of severe hemophilia A patients develop inhibitory antibodies toward the injected FVIII, rendering the replacement therapy ineffective. To overcome these drawbacks, we generated a bispecific antibody (termed ACE910) against activated factor IX (FIXa) and factor X (FX), which mimics the cofactor function of FVIII. Objectives: The aims of the present study were to examine the FVIII-mimetic cofactor activity of ACE910 in vitro and its hemostatic activity in vivo. Methods: The FVIII-mimetic cofactor activity of ACE910 was evaluated by a thrombin generation assay in human FVIII-deficient plasma as well as by an enzymatic assay using purified coagulation factors. For in vivo studies, an acquired hemophilia A model was established in cynomolgus monkeys by a single intravenous injection of mouse monoclonal anti-FVIII neutralizing antibody, which was cross-reactive to cynomolgus monkey FVIII but not to porcine FVIII. After artificial bleeding had been induced, ACE910 or porcine FVIII was intravenously administered in a single dose or in twice-daily repeated doses, respectively. Bleeding symptoms, including anemia and skin bruising, were monitored for three days. A pharmacokinetic study of ACE910 was also performed with a single intravenous or subcutaneous administration to cynomolgus monkeys. Results: ACE910 concentration-dependently showed FVIII-mimetic cofactor activity in the enzymatic assay and improved thrombin generation parameters in human FVIII-deficient plasma. Intravenous administration of ACE910 (a single dose of 3 mg/kg) significantly reduced the bleeding symptoms in the acquired hemophilia A model of a non-human primate. This hemostatic effect was comparable to twice-daily intravenous administration of porcine FVIII (repeated doses of 10 U/kg). The half-life of ACE910 was approximately three weeks for both single intravenous and subcutaneous administrations. The subcutaneous bioavailability of ACE910 was nearly 100%. Conclusion: The bispecific antibody against FIXa and FX, ACE910, exerted FVIII-mimetic cofactor activity in vitro. Furthermore, a single dose of ACE910 demonstrated hemostatic activity comparable to twice-daily repeated doses of 10 U/kg porcine FVIII in vivo. Moreover, ACE910 exhibited high subcutaneous bioavailability and approximately three-week half-life in a non-human primate. Our bispecific antibody against FIXa and FX is a subcutaneously injectable, long-acting agent that removes the need to consider the induction or presence of FVIII inhibitors and may establish a novel principle for the prophylactic treatment of hemophilia A patients. Disclosures: Muto: Chugai Pharmaceutical Co., Ltd.: Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment. Yoshihasi:Chugai Pharmaceutical Co., Ltd.: Employment. Takeda:Chugai Pharmaceutical Co., Ltd.: Employment. Soeda:Chugai Pharmaceutical Co., Ltd.: Employment. Igawa:Chugai Pharmaceutical Co., Ltd.: Employment. Sampei:Chugai Pharmaceutical Co., Ltd.: Employment. Sakamoto:Chugai Pharmaceutical Co., Ltd.: Employment. Okuyama-Nishida:Chugai Pharmaceutical Co., Ltd.: Employment. Saito:Chugai Pharmaceutical Co., Ltd.: Employment. Kawabe:Chugai Pharmaceutical Co., Ltd.: Employment. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor X ◽  
Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IXa in Activated Prothrombin Complex Concentrates Is the Dominant Contributor to Enhanced Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
State Level ◽  
Peak Level ◽  
Coagulation Factors ◽  
Major Effect ◽  
Factor Ix ◽  
Factor X ◽  
Factor Ixa ◽  
Activated Prothrombin Complex Concentrates

Background. Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa and factor X has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to treating this bleeding in hemophilia A patients with inhibitors is to give an activated prothrombin complex concentrate (APCC; FEIBA) (disfavored in NHF MASAC #255). APCC contains a number of coaguation factors including prothrombin, factor X (FX), and factor IX (FIX). APCC also contains activated factor X (FXa) and factor IX (FIXa). Previous work has shown that when APCCs are added to hemophilia A plasma containing emicizumab there is a significant increase in thrombin generation [J Thromb Haemost 2018;16:1580-1591]. The goal of this work was to study thrombin generation in hemophilia A plasma with emicizumab and to examine the role of the zymogen and activated components of an APCC in the increased thrombin generation. Methods. In hemophilia A plasma, thrombin generation assays were done using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). The components of APCC were studied at concentrations that should mimic the levels seen in the plasma of a patient given a dose of 50 U/kg: prothrombin 1800 nM; FX 130 nM; FIX 90 nM; and FIXa 0.4 nM. Results. When initiated with low TF, hemophilia A plasma alone had essentially no thrombin generation. As expected, adding emicizumab enhanced thrombin generation. The addition of zymogen coagulation factors, prothrombin, FIX, and FX, separately or together gave a small increase in thrombin generation. However, addition of FIXa to emicizumab gave a large increase in peak thrombin. In hemophilia A plasma with emicizumab and FIXa, addition of prothrombin further increased thrombin generation and specifically increased the peak level of thrombin. Further addition of FX or FIX, separately or together, only minimally increased thrombin generation. Discussion. The strong contribution of factor IXa to the effects of APCCs on thrombin generation in hemophilia A plasma depends on the presence of emicizumab. In the absence of emicizumab, a study of the individual components of APCC showed that a combination of FXa and prothrombin at levels found in APCCs had the major effect on thrombin generation [Haemophilia. 2016;22:615-24]. That study found that FIXa did not increase thrombin generation. However, in the presence of emicizumab, despite the weak solution phase affinity of the bifunctional antibody for FIXa, small amounts of FIXa were the most significant contributor to thrombin generation. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

Ontogeny of gastric mucosal muscarinic receptor and sensitivity to carbachol

1984 ◽  
Vol 246 (5) ◽  
pp. G550-G555
Author(s):  
E. R. Seidel ◽  
L. R. Johnson
Keyword(s):  
Dissociation Constant ◽  
Cholinergic Receptor ◽  
Equilibrium Dissociation Constant ◽  
Muscarinic Cholinergic Receptor ◽  
Fetal Rat ◽  
Gastric Lumen ◽  
Basal Acid ◽  
Muscarinic Cholinergic ◽  
Equilibrium Dissociation ◽  
Apparent Equilibrium

Development of the muscarinic cholinergic receptor and sensitivity of oxyntic gland mucosa to a muscarinic agonist were studied in rats of various ages. The gastric lumen of the fetal rat at the 20th day of gestation contained a statistically significant amount of basal pepsin, which increased log linearly over the first 30 days of life. Carbachol was effective in stimulating the secretion of pepsin as early as 12 h after birth. Basal acid could be measured in the gastric lumen 12 h after birth. The secretion of basal acid increased log linearly over the first 30 days of life. Carbachol was an effective secretagogue even in the fetal rat. The density of the muscarinic cholinergic receptor of the adult rat oxyntic gland mucosa was 99.3 fmol/mg protein with an apparent equilibrium dissociation constant for quinuclidinyl benzilate of 0.40 nM. The receptor was well developed even in the fetal rat, which bound 79.6 fmol/mg protein with an apparent equilibrium dissociation constant of 0.26 nM. Except for immediately after birth, receptor density was maintained between 70 and 90% of the adult level over the first 30 days of life. These results suggest that cholinergic regulation of gastric acid and pepsin secretion is probably functional by either late gestation or at least immediately after birth.

scholarly journals Novel Insights and New Developments Regarding Coagulation Revealed by Studies of the Anti-Factor IXa (Activated Factor IX)/Factor X Bispecific Antibody, Emicizumab

2020 ◽  
Vol 40 (5) ◽  
pp. 1148-1154
Author(s):  
Koji Yada ◽  
Keiji Nogami
Keyword(s):  
Hemophilia A ◽  
Activated Protein C ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
New Developments ◽  
Activated Factor Vii ◽  
Factor Ixa ◽  
Thrombin Activation ◽  
No Activation

Emicizumab is a humanized anti-FIXa/FX (factor IXa/X) bispecific monoclonal antibody that mimics FVIIIa (activated factor VIII) cofactor function. The hemostatic efficacy of emicizumab has been confirmed in clinical studies of patients with hemophilia A, irrespective of the presence of FVIII inhibitors. Emicizumab differs in some properties from FVIIIa molecule. Emicizumab requires no activation by thrombin and is not inactivated by activated protein C, but emicizumab-mediated coagulation is regulatable and maintains hemostasis. A small amount of FIXa (activated factor IX) is required to initiate emicizumab-mediated hemostasis, whereas tissue factor/FVIIa (activated factor VII)-mediated FXa (activated factor X) and thrombin activation initiates FVIIIa-mediated hemostasis. Fibrin formation, followed by fibrinolysis, appears to be similar between emicizumab- and FVIIIa-mediated hemostasis. These results suggest possible future uses of emicizumab for treating hemorrhagic diseases other than hemophilia A and reveal previously unobservable behaviors of procoagulation and anticoagulation factors in conventional hemostasis. Here, we have reviewed novel insights and new developments regarding coagulation highlighted by emicizumab.

scholarly journals Specific binding of 1,25-dihydroxycholecalciferol in human medullary thyroid carcinoma

1982 ◽  
Vol 206 (1) ◽  
pp. 181-184 ◽  
Author(s):  
H C Freake ◽  
I MacIntyre
Keyword(s):  
Specific Binding ◽  
Binding Protein ◽  
Sedimentation Coefficient ◽  
C Cells ◽  
Equilibrium Dissociation Constant ◽  
Normal Thyroid ◽  
Medullary Thyroid ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd ◽  
Medullary Thyroid Carcinomas

A specific 1,25-dihydroxycholecalciferol-binding protein has been detected in high-salt cytosols prepared from human medullary thyroid carcinomas. The binding protein had the same equilibrium dissociation constant (Kd = 0.17 +/- 0.05 nM; n = 4) and sedimentation coefficient on sucrose gradients (3.7S) as than seen in established vitamin D target tissues. This protein was not detected in normal thyroid cytosols, which may reflect the low proportion of C-cells within the gland.

scholarly journals Bridging the Missing Link with Emicizumab: A Bispecific Antibody for Treatment of Hemophilia A

2020 ◽  
Vol 120 (10) ◽  
pp. 1357-1370
Author(s):  
Georg Gelbenegger ◽  
Christian Schoergenhofer ◽  
Paul Knoebl ◽  
Bernd Jilma
Keyword(s):  
Clinical Trials ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Current Standard ◽  
Bleeding Events ◽  
Recombinant Activated Factor Vii

AbstractHemophilia A, characterized by absent or ineffective coagulation factor VIII (FVIII), is a serious bleeding disorder that entails severe and potentially life-threatening bleeding events. Current standard therapy still involves replacement of FVIII, but is often complicated by the occurrence of neutralizing alloantibodies (inhibitors). Management of patients with inhibitors is challenging and necessitates immune tolerance induction for inhibitor eradication and the use of bypassing agents (activated prothrombin complex concentrates or recombinant activated factor VII), which are expensive and not always effective. Emicizumab is the first humanized bispecific monoclonal therapeutic antibody designed to replace the hemostatic function of activated FVIII by bridging activated factor IX and factor X (FX) to activate FX and allow the coagulation cascade to continue. In the majority of hemophilic patients with and without inhibitors, emicizumab reduced the annualized bleeding rate to almost zero in several clinical trials and demonstrated a good safety profile. However, the concurrent use of emicizumab and activated prothrombin complex concentrate imposes a high risk of thrombotic microangiopathy and thromboembolic events on patients and should be avoided. Yet, the management of breakthrough bleeds and surgery remains challenging with only limited evidence-based recommendations being available. This review summarizes published clinical trials and preliminary reports of emicizumab and discusses the clinical implications of emicizumab in treatment of hemophilia A.

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