Characterization of a High-Affinity Fully Human IgG2 Antibody Against Tissue Factor Pathway Inhibitor As a Bypass Agent for the Treatment of Hemophilia

Abstract The tissue factor initiated coagulation pathway is intact in patients with hemophilia; however, this pathway is normally inhibited by tissue factor pathway inhibitor (TFPI), resulting in insufficient thrombin generation to stop bleeding and prevent recurrence of bleeding. Antagonizing this natural inhibition through anti-TFPI antibodies is a potential mechanism to effectively restore hemostasis in patients with hemophilia. A human phage-displayed antibody library was used to identify optimal anti-TFPI antibodies. From the panning campaign, unique antibodies were identified that bound to human TFPI and were cross-reactive to murine TFPI. In vitro characterization demonstrated that 6 of the antibodies could block TFPI activity, partially or completely restoring factor Xa activity. These 6 antibodies also shortened clotting time of hemophilic human (Hem A) plasma in a diluted prothrombin assay. When the ROTEM assay (Tem International GmbH, Basel, Switzerland) was used to measure clotting in human Hem A plasma from congenitally deficient individuals (factor VIII [FVIII] levels <0.38%), anti-TFPI antibodies significantly shortened clotting time, alone or in combination with FVIII or activated factor VII. In addition, the antibodies were effective in shortening clotting time in human blood containing anti-FVIII antibodies, indicating that anti-TFPI antibodies can be applied to inhibitor patients. One of the identified anti-TFPI antibodies was tested in a hemophilia A mice tail vein transection model and improved survival rate to 60%, significantly higher than the 10% survival rate of mice treated with an isotype control antibody. Additionally, combined with low-dose FVIII, this antibody could further increase the survival rate in hemophilic mice, and it was selected for further optimization. Optimization covered 2 different aspects: (1) affinity maturation to increase its affinity to human and murine TFPI; and (2) sequence optimization to reduce sequence deviation from germ-line sequences and to remove critical residues prone to undergo unwanted chemical modifications during production and/or storage. Both processes consisted of 2 rounds of sequence optimization and screening. Following affinity maturation, target affinity was increased by >200-fold on human and >500-fold on murine TFPI. Sequence optimization was performed on the backbone of the final affinity-matured variant. BAY 1093884, the final optimized variant, is a fully human IgG2 antibody with <10 pM binding affinity to human and murine TFPI. Disclosures Bauzon: Bayer: Employment. Liu:Bayer: Employment. Grudzinska-Goebel:Bayer: Employment. Tebbe:Bayer: Employment. Pan:Bayer: Employment. Cifrese:Bayer: Employment. Wang:Bayer: Employment. Sim:Bayer HealthCare LLC: Employment.

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The Significance of TFPI in Clotting Assays – Gomparison and Combination with other Anticoagulants

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649603 ◽

1993 ◽

Vol 70 (03) ◽

pp. 448-453 ◽

Cited By ~ 12

Author(s):

Ole Nordfang ◽

Hanne I Kristensen ◽

Sanne Valentin ◽

Per Østergaard ◽

Johnny Wadt

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Antithrombin Iii ◽

Anticoagulant Activity ◽

Assay System ◽

Thrombin Inhibitor ◽

Clotting Time ◽

Normal Plasma ◽

Tissue Factor Pathway ◽

Plasma Heparin

SummaryThe anticoagulant activities of Tissue Factor Pathway Inhibitor (TFPI), heparin and hirudin were compared in intrinsic (APTT) and extrinsic (PT) activated clotting assays. In contrast to the thrombin inhibitor hirudin, heparin was 10 fold more potent in the APTT assay than in the PT assay, indicating that inhibition of intrinsic activation is important for the anticoagulant activity of heparin as measured in an APTT assay. TFPI was most potent in the PT assay and the effect of TFPI was most pronounced in the presence of other anticoagulants (heparin and hirudin). The activities of the two natural anticoagulants antithrombin III (ATIII) and TFPI were compared in a PT assay with very dilute tissue factor. In this assay system TFPI in normal plasma affected the clotting time more than ATIII in the plasma. However, when heparin was added ATIII was the major anticoagulant, but profound Prolongation of the clotting time was only seen when TFPI was also added. In an ATIII deficient plasma heparin did not augment the effect of TFPI, showing that the increased effect of TFPI in the presence of heparin is dependent on the anticoagulant activity of ATIII/heparin. The effect of TFPI at prolonged clotting times was also illustrated by the significant effect of blocking TFPI in the plasma from warfarin-treated patients. Thus TFPI is a major anticoagulant in normal plasma and the effect of TFPI is especially seen at prolonged clotting times.

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Daily and Circadian Rhythms of Tissue Factor Pathway Inhibitor and Factor VII Activity

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/01.atv.0000153140.13148.e0 ◽

2005 ◽

Vol 25 (3) ◽

pp. 646-649 ◽

Cited By ~ 56

Author(s):

Mirko Pinotti ◽

Cristiano Bertolucci ◽

Francesco Portaluppi ◽

Ilaria Colognesi ◽

Elena Frigato ◽

...

Keyword(s):

Circadian Rhythms ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Vii ◽

Tissue Factor Pathway

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Effects of different progestin regimens in hormone replacement therapy on blood coagulation factor VII and tissue factor pathway inhibitor

Human Reproduction ◽

10.1093/humrep/17.12.3235 ◽

2002 ◽

Vol 17 (12) ◽

pp. 3235-3241 ◽

Cited By ~ 31

Author(s):

E.M. Bladbjerg

Keyword(s):

Hormone Replacement Therapy ◽

Replacement Therapy ◽

Tissue Factor ◽

Blood Coagulation ◽

Tissue Factor Pathway Inhibitor ◽

Hormone Replacement ◽

Coagulation Factor ◽

Factor Vii ◽

Coagulation Factor Vii ◽

Tissue Factor Pathway

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Peroxisome Proliferator-Activated Receptor γ Induces the Expression of Tissue Factor Pathway Inhibitor-1 (TFPI-1) in Human Macrophages

PPAR Research ◽

10.1155/2016/2756781 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

G. Chinetti-Gbaguidi ◽

C. Copin ◽

B. Derudas ◽

N. Marx ◽

J. Eechkoute ◽

...

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Mononuclear Cells ◽

Coagulation Cascade ◽

Factor Vii ◽

Peroxisome Proliferator ◽

Peripheral Blood Mononuclear ◽

Peroxisome Proliferator Activated Receptor ◽

Human Macrophages ◽

Tissue Factor Pathway

Tissue factor (TF) is the initiator of the blood coagulation cascade after interaction with the activated factor VII (FVIIa). Moreover, the TF/FVIIa complex also activates intracellular signalling pathways leading to the production of inflammatory cytokines. The TF/FVIIa complex is inhibited by the tissue factor pathway inhibitor-1 (TFPI-1). Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that, together with PPARα and PPARβ/δ, controls macrophage functions. However, whether PPARγ activation modulates the expression of TFP1-1 in human macrophages is not known. Here we report that PPARγ activation increases the expression of TFPI-1 in human macrophages in vitro as well as in vivo in circulating peripheral blood mononuclear cells. The induction of TFPI-1 expression by PPARγ ligands, an effect shared by the activation of PPARα and PPARβ/δ, occurs also in proinflammatory M1 and in anti-inflammatory M2 polarized macrophages. As a functional consequence, treatment with PPARγ ligands significantly reduces the inflammatory response induced by FVIIa, as measured by variations in the IL-8, MMP-2, and MCP-1 expression. These data identify a novel role for PPARγ in the control of TF the pathway.

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Factor VII, Tissue Factor Pathway Inhibitor, and Monocyte Tissue Factor in Diabetes Mellitus: Influence of Type of Diabetes, Obesity Index, and Age

Thrombosis Research ◽

10.1016/s0049-3848(00)00424-2 ◽

2001 ◽

Vol 101 (5) ◽

pp. 367-375 ◽

Cited By ~ 29

Author(s):

Anne Vambergue ◽

Lucia Rugeri ◽

Valérie Gaveriaux ◽

Patrick Devos ◽

Annie Martin ◽

...

Keyword(s):

Diabetes Mellitus ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Vii ◽

Obesity Index ◽

Tissue Factor Pathway

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Role of tissue factor in ischemic reperfusion injury: iv) the effect of tissue factor pathway inhibitor on the survival rate in vivo

Transplantation Proceedings ◽

10.1016/s0041-1345(98)00630-7 ◽

1998 ◽

Vol 30 (5) ◽

pp. 2300-2301 ◽

Cited By ~ 4

Author(s):

N Yoshimura ◽

Y Kobayashi ◽

K Nakamura ◽

R Yoshimura ◽

H Yamagishi ◽

...

Keyword(s):

Survival Rate ◽

Reperfusion Injury ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Ischemic Reperfusion Injury ◽

Ischemic Reperfusion ◽

Tissue Factor Pathway

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Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Endothelial Tissue Factor Pathway Inhibitor.

Blood ◽

10.1182/blood.v104.11.803.803 ◽

2004 ◽

Vol 104 (11) ◽

pp. 803-803

Author(s):

Haiwang Tang ◽

Lacramioara Ivanciu ◽

Todd Hamm ◽

Fletcher B. Taylor ◽

Cristina Lupu ◽

...

Keyword(s):

Electron Microscopy ◽

Endothelial Cells ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Vii ◽

Sublethal Dose ◽

Saline Control ◽

E Coli ◽

Activity Measurements ◽

Tissue Factor Pathway

Abstract Activation of tissue factor (TF)-dependent coagulation is an early event in the pathogenesis of sepsis, responsible for microvascular thrombosis and consequent organ injury. We hypothesized that sepsis-induced TF procoagulant activity may be paralleled by a decreased expression or function of its natural inhibitor, tissue factor pathway inhibitor (TFPI). To test this hypothesis we used a non-human primate sepsis model in which adult baboons were infused with live E. coli at 109 CFU/kg (sublethal dose), 1010 CFU/kg (lethal dose) or saline (control group) and the animals were sacrified after 2, 8, or 24 hours. Lung tissue was snap frozen for protein and mRNA extraction or fixed and processed for light and electron microscopy. Tissue cryosections were immunostained using double/multiple fluorescence labeling approaches for TF, TFPI, factor VII/FVIIa and fibrin, in conjunction with cell markers for endothelial cells (CD31 or von Willebrand factor), leukocytes (PSGL-1), macrophages (CD68), PMN (myeloperoxidase) and platelets (gpIIb/IIIa). Large amounts of TF were detected in leukocytes, endothelial cells and platelet-rich microthrombi, starting from 2 hours and throughout the examined period. Concomitantly, confocal and electron microscopy analysis revealed increased leukocyte infiltration, platelet aggregates and fibrin deposition in the intravascular and interstitial compartments. In addition, TF induction was documented by semiquantitative RT-PCR, ELISA, western blot and factor Xa activation assays. Whereas TFPI mRNA showed only a modest increase, tissue-associated TFPI protein was found considerably decreased, especially during the first eight hours post E. coli infusion. Moreover, TFPI inhibitory activity of lung extracts from septic animals was 6–8 fold lower comparing to controls. TF activity measurements in the presence of inhibitory anti TFPI antibodies showed that only a very small fraction of endogenous TF was inhibited by tissue-associated TFPI, suggesting that most of the active TFPI available in the vascular compartment was depleted. The decrease of TFPI inhibitory potency cannot be exclusively explained by its proteolytic degradation, as we did not find significant amounts of truncated TFPI on western blots. In conclusion, our studies demonstrate that the exposure to septic and inflammatory stimuli lead to a decrease of TFPI-dependent endothelial anticoagulant potential, simultaneous with a strong TF-dependent procoagulant response. Activation of TF-dependent coagulation pathway not adequately countered by TFPI may have important roles in the pathogenesis of sepsis-associated disseminated intravascular coagulation. Strategies aimed to restore the physiological anticoagulant function of TFPI may help preventing sepsis-induced multiple organ dysfunction syndrome and death.

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