A hemophilia A mouse model for the in vivo assessment of emicizumab function

Abstract The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip–bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab.

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Prevention of Premature Fibrinolysis and Reduction of Bleeding in Vivo in Hemophilia with Inhibitors By a Stabilized TAFI Variant

Blood ◽

10.1182/blood.v124.21.694.694 ◽

2014 ◽

Vol 124 (21) ◽

pp. 694-694 ◽

Cited By ~ 2

Author(s):

Maarten L.V. Hendrickx ◽

Vikas Bhat ◽

Ann Gills ◽

Paul J. Declerck ◽

Annette von Drygalski ◽

...

Keyword(s):

Half Life ◽

Hemophilia A ◽

High Titer ◽

Bleeding Complications ◽

Clot Lysis ◽

Saline Control ◽

Normal Plasma ◽

Deficient Mice ◽

Tail Clip

Abstract Introduction: Approximately 20-30% of patients with hemophilia A and ~5% of patients with hemophilia B develop inhibitors against FVIII or FIX, leaving patients unresponsive to standard treatments, and vulnerable to joint bleeding with the consequence of debilitating arthropathy. With the increasing life span of hemophilia patients, there is a growing need for optimization of treatment strategies for inhibitor patients. Most approaches focus on FVIIa-based bypassing agents to achieve hemostasis and rescue bleeding, but in addition to impaired coagulation, the bleeding complications in hemophilia are also exacerbated by premature break down of the clot by the fibrinolytic system. Thrombin activatable fibrinolysis inhibitor (TAFI) integrates the coagulation and fibrinolytic systems, and its activation is severely defective in hemophilia. Moreover, the enzymatic activity of activated TAFI (TAFIa) is short-lived (~8 min at 37oC) requiring continuous generation of TAFIa to attenuate fibrinolysis. Here we investigate the efficacy of a stabilized TAFI mutant with a 180-fold increased enzymatic half-life to normalize the premature lysis and rescue bleeding in hemophilia with inhibitors. Materials and Methods: The effects of the stabilized S305C-T325I-T329I-H333Y-H335Q -TAFI mutant (stTAFI; T½ ~19 h) and plasma-derived TAFI (pTAFI) on clot lysis were determined in hemophilia A, B, and normal plasma with or without high titer inhibitors. In vivo efficacy of stTAFI and pTAFI for bleed reduction in hemophilia was tested using the tail clip model in FVIII-deficient mice and wt-BalbC mice injected i.v. with inhibitory anti-FVIII antibodies. Results: Titrations of stTAFI and pTAFI indicated that a ~7-fold lower concentration of stTAFI (0.75 µg/ml) compared to pTAFI (5 µg/ml) was required to normalize the clot lysis time (CLT) in FVIII deficient plasma to that obtained in the presence of FVIII (2 U/ml). The generation of TAFIa activity in FVIII deficient plasma during clot formation was severely diminished, but returned to normal levels upon addition of stTAFI (1 µg/ml) or pTAFI (7.5 µg/ml). In normal plasma, the addition of an inhibitory anti-FVIII antibody (GMA-8015; 10 µg/ml) decreased the CLT from 95 ± 9 to 62 ± 7 min, which was restored more efficiently to normal by stTAFI (1.25 µg/ml) than by pTAFI (7.5 µg/ml). Similarly, stTAFI normalized the CLT in FIX deficient plasma or normal plasma with inhibitory anti-FIX antibodies (GMA-001; 10 µg/ml) at a 10-fold lower concentration compared to pTAFI. Also in 3 individual plasma samples of hemophilia A patients with inhibitors (49-178 BU/ml), CLT were very short (~50 min) and stTAFI normalized the CLT (~110 min) more efficiently than pTAFI. To determine whether correction of clot lysis in vitro translates to reduction of bleeding in vivo, the effects of stTAFI and pTAFI on bleeding were determined in hemophilia A mice after tail clip. StTAFI (0.1 mg/kg) reduced bleeding significantly after 10 min (5.1 ± 2.4 µl/g compared to saline control 10.5 ± 1.8 µl/g; p < 0.05) and 20 min (15.9 ± 5.5 µl/g vs saline 24.9 ± 2.9 µl/g; p < 0.05). Reduction of blood loss was similar to that achieved by a therapeutic dose of murine FVIIa (90 µg/kg) after 10 min (2.2 ± 1.0 µl/g) and 20 min (13.5 ± 4 µl/g). Neither human pTAFI (0.1 mg/kg) nor murine wt-TAFI (0.1 mg/kg) reduced bleeding. Compared to stTAFI, a 10-fold higher dose of pTAFI (1 mg/kg) was required to reduce bleeding significantly. Next, reduction of bleeding by stTAFI was determined in hemophilia A with inhibitors. Wt-mice received an inhibitory anti-FVIII antibody (GMA-8015; 0.25 mg/kg) that increased bleeding after 10 min (10.5 ± 3.2 µl/g vs no inhibitor 1.7 ± 0.6 µl/g; p < 0.02) and 20 min (20.9 ± 4.1 µl/g vs no inhibitor 1.7 ± 0.6 µl/g; p < 0.0001). Administration of stTAFI (0.1 mg/kg) reduced the bleeding to 4.0 ± 1.5 µl/g after 10 min (p < 0.05) and 9.1 ± 3.3 µl/g after 20 min (p < 0.05). Conclusions: The stTAFI mutant with a 180-fold increased enzymatic half-life provided effective protection against premature fibrinolysis in hemophilia A and B plasmas with inhibitors. In vivo, stTAFI reduced bleeding in FVIII-deficient mice and wt-mice with a high titer FVIII inhibitor. These results provide proof-of-principle that clot protection by TAFI can reduce bleeding in hemophilia and warrant additional studies investigating stabilized TAFI mutants as a novel strategy to diminish the risk of bleeding in hemophilia patients with inhibitors. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v120.21.42.42 ◽

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.

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Novel Severe Hemophilia a Mouse Model with Factor VIII Intron 22 Inversion

Biology ◽

10.3390/biology10080704 ◽

2021 ◽

Vol 10 (8) ◽

pp. 704

Author(s):

Jeong Pil Han ◽

Dong Woo Song ◽

Jeong Hyeon Lee ◽

Geon Seong Lee ◽

Su Cheong Yeom

Keyword(s):

Mouse Model ◽

Hemophilia A ◽

Clinical Symptoms ◽

Structural Similarity ◽

Coagulation Disorder ◽

Severe Hemophilia ◽

Spontaneous Bleeding ◽

Intron 22 Inversion ◽

Fviii Activity

Hemophilia A (HA) is an X-linked recessive blood coagulation disorder, and approximately 50% of severe HA patients are caused by F8 intron 22 inversion (F8I22I). However, the F8I22I mouse model has not been developed despite being a necessary model to challenge pre-clinical study. A mouse model similar to human F8I22I was developed through consequent inversion by CRISPR/Cas9-based dual double-stranded breakage (DSB) formation, and clinical symptoms of severe hemophilia were confirmed. The F8I22I mouse showed inversion of a 391 kb segment and truncation of mRNA transcription at the F8 gene. Furthermore, the F8I22I mouse showed a deficiency of FVIII activity (10.9 vs. 0 ng/mL in WT and F8I22I, p < 0.0001) and severe coagulation disorder phenotype in the activated partial thromboplastin time (38 vs. 480 s, p < 0.0001), in vivo bleeding test (blood loss/body weight; 0.4 vs. 2.1%, p < 0.0001), and calibrated automated thrombogram assays (Thrombin generation peak, 183 vs. 21.5 nM, p = 0.0012). Moreover, histological changes related to spontaneous bleeding were observed in the liver, spleen, and lungs. We present a novel HA mouse model mimicking human F8I22I. With a structural similarity with human F8I22I, the F8I22I mouse model will be applicable to the evaluation of general hemophilia drugs and the development of gene-editing-based therapy research.

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152 IN VIVO ANALYSIS OF THE ADENOVIRUS-SPECIFIC CYTOTOXIC T-LYMPHOCYTE RESPONSE IN CD28-, FASL-, AND PERFORIN-DEFICIENT MICE.

Journal of Investigative Medicine ◽

10.2310/6650.2005.x0008.151 ◽

2006 ◽

Vol 54 (1) ◽

pp. S283.1-S283

Author(s):

J. Chen ◽

H. -C. Hsu ◽

A. Zajac ◽

Q. Wu ◽

P. -A. Yang ◽

...

Keyword(s):

T Lymphocyte ◽

Cytotoxic T Lymphocyte ◽

Lymphocyte Response ◽

In Vivo Analysis ◽

Deficient Mice ◽

Cytotoxic T Lymphocyte Response

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In Vivo Assessment of Airway Function in the Mouse Model

Human Respiratory Syncytial Virus - Methods in Molecular Biology ◽

10.1007/978-1-4939-3687-8_16 ◽

2016 ◽

pp. 219-230

Author(s):

Azzeddine Dakhama ◽

Erwin W. Gelfand

Keyword(s):

Mouse Model ◽

Airway Function ◽

In Vivo Assessment

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Abstract 4169: Systematic in vivo analysis of PI3K pathway aberrations in a mouse model of invasive lobular carcinoma

10.1158/1538-7445.am2016-4169 ◽

2016 ◽

Author(s):

Martine Van Miltenburg

Keyword(s):

Mouse Model ◽

Invasive Lobular Carcinoma ◽

Lobular Carcinoma ◽

Pi3k Pathway ◽

In Vivo Analysis

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Humanized CD22 transgenic mouse model for in vivo analysis of anti‐CD22‐based immunotherapy

European Journal of Immunology ◽

10.1002/eji.202048636 ◽

2020 ◽

Vol 50 (11) ◽

pp. 1834-1837

Author(s):

Carolin Brandl ◽

Sieglinde Angermüller ◽

Lars Nitschke

Keyword(s):

Mouse Model ◽

Transgenic Mouse ◽

Transgenic Mouse Model ◽

In Vivo Analysis

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Feiba And Autoplex: A Comparison Of These Two Activated Prothrombin Complex Concentrates

10.1055/s-0038-1652326 ◽

1981 ◽

Author(s):

E Lechler ◽

B Eggeling ◽

D Meyer-Börnecke ◽

H Stoy

Keyword(s):

Hemophilia A ◽

Gel Filtration ◽

Activated Partial Thromboplastin Time ◽

Factor Ix ◽

Prothrombin Complex Concentrates ◽

Minor Degree ◽

Activated Prothrombin Complex Concentrates ◽

A Minor

These activated concentrates are used for the treatment of patients with factor VIII inhibitors . Both shorten the activated and non-activated partial thromboplastin time of inhibitor plasma and hemophilia A plasma in vitro. They do not or only to a minor degree improve the prothrombin consumption of hemophilia A plasma in vitro. In gel filtration of AUTOPLEX the activity which shortens the PTT of hemophilia A plasma eluted in a volume higher than that of the nonactivated factors of the prothrombin complex and contains activated factor IX. The activity of FEIBA elutes at a lower filtration volume in a rather broad peak together with the factors of the the non-activated prothrombin complex. BaSO4- adsorbed plasma and purified antithrombin (Behring) abolish the activity of AUTOPLEX more readily than of FEIBA. Both concentrates have only a low amidolytic effect (S 2222) and are not inhibited with SBTI and PMSF. In the crossed two-dimensional immunelectrophoresis with heparin in the agarose of the first dimension and anti-antithrombin (Behring) in the agarose of the second dimension (method of Sas), a mixture of AUTOPLEX and antithrombin results into a two peak precipitation of antithrombin, whereas with FEIBA a broadened intermediate peak develops. In vivo both concentrates do not improve the prothrombin consumption and AUTOPLEX shortens the PTT for at least 90 minutes. In summary, these two concentrates differ considerably.

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