Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: MG1113 binds to KD2 of tissue factor pathway inhibitor (TFPI). (A) TFPI expression vector was transfected into HEK 293 cells. Binding of Kunitz‐2 domain (KD2) of TFPI with MG1113 was then confirmed using immunoprecipitation (IP). A band was observed by western blot (WB) only in cases of TFPI constructs possessing KD2. Mock: human embryonic kidney (HEK) 293 cells were transfected without TFPI expression vector. (B) Two perpendicular views are shown with three polypeptides in different colors. Immunoglobulin (Ig) heavy chain is denoted by VH and CH. Ig kappa light chain is indicated by Vk and Ck. Mapping of epitopes and paratopes, defined as residues within the intersubunit distance of 4.5 Å, on surface representations of KD2 and Fab of MG1113. Left, epitopes on KD2 are shown in three different colors. Orange, yellow, and red indicate putative activated factor X (FXa)‐binding residues, which overlap with Fab of MG1113‐binding residues of KD2. Arg107 (in red) is a key residue in the inhibition of FXa. Right, paratopes on MG1113 are shown in magenta for VH residues and in pink for Vk residues. (C) MG1113 no longer binds to the construct from which the epitope of TFPI is removed. Ab, antibody; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Expressing, Plasmid Preparation, Transfection, Binding Assay, Immunoprecipitation, Western Blot, Construct, Residue, Inhibition

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: Neutralizing effect of MG1113 on the function of tissue factor pathway inhibitor‐α (TFPI‐α). (A) The activity of 1 nM activated factor X (FXa) is reduced by 10 nM TFPI. The activity of FXa is recovered (n = 3) after treatment with MG1113 (0.625‐40 nM). (B) Activation of 10 nM factor X (FX) by extrinsic FXase, comprised of 10.5 pM tissue factor (TF) and 0.5 nM activated factor VII (FVIIa), is inhibited by 9 nM TFPI. Generation and activity of FXa are recovered (n = 4) after treatment with MG1113 (0.13‐800 nM). (C) After factor VIII (FVIII) deficient plasma is treated with MG1113 through thrombin generation assay, with increasing concentration, thrombin generation is also increased (n = 3). (D, E) In a modified prothrombin time (mPT) assay, when FVIII or factor IX (FIX) deficient plasma is treated with MG1113, with increasing concentration, clotting time is further shortened (n = 3). The graph represents mean and standard deviation

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Activity Assay, Activation Assay, Clinical Proteomics, Concentration Assay, Modification, Coagulation, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: MG1113 not only binds to tissue factor pathway inhibitor‐β (TFPI‐β) but also neutralizes it. (A) Binding of human umbilical vein endothelial cells (HUVECs) and MG1113 is confirmed. Differently from human IgG (hIgG), which is a negative control, with increasing concentration of MG1113 (0.003 ‐ 200 nM), increased binding of MG1113 to HUVECs (n = 3) is observed. (B) The factor X (FX) is activated by activated factor VII (FVIIa) and tissue factor, which is expressed on stimulated HUVECs by tumor necrosis factor‐α. With increasing concentration of MG1113 (0.006‐500 nM), generation of activated factor X (FXa) is increased (n = 2). The graph represents mean and standard deviation

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Binding Assay, Negative Control, Concentration Assay, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: MG1113 restores blood loss and clotting time of a hemophilia A (HA)‐induced rabbit. (A) Rabbits received saline or factor VIII (FVIII) neutralizing antibody (Nab) (10 mg/kg) 45 minutes before bleeding induction. Rabbits received saline or MG1113 (0.1, 0.25, 1, 5 mg/kg) 35 min before bleeding induction. Bleeding was observed for 1 hour. Hemoglobin level was reduced by MG1113 treatment in a concentration‐dependent manner. (B) Activated partial thromboplastin time (aPTT) was delayed after treatment with FVIII NAb, but not changed after MG1113 treatment. (C) Modified prothrombin time (mPT) was shortened by MG1113 treatment. (D) Free tissue factor pathway inhibitor (TFPI) level was not changed by FVIII neutralizing antibody (NAb) treatment. However, it was reduced by MG1113 treatment. The graph represents mean and standard deviation. In the test group of 0.25 mg/kg, at which MG1113 started to affect, free TFPI level remained at approximately 20%‐22% of that of the group not treated with MG1113. Each dot represents an entity (n = 8‐15). Outliers were excluded from the data set (Grubb’s test, P < .01). The group not treated with MG1113 and other groups were analyzed using one‐way analysis of variance, assuming a normal distribution. The significance level was set at P < .05. § Two‐stage linear step‐up procedure; † Games‐Howell pairwise comparison; ‡ Dunnett post hoc analysis

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Coagulation, Saline, Concentration Assay, Modification, Standard Deviation, Comparison

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

doi: 10.1002/rth2.12438

Figure Lengend Snippet: Confirmation for the ability of MG1113 in an HA‐induced rabbit through subcutaneous administration. The same concentration of MG1113 was administered intravenously or subcutaneously. Blood loss, activated partial thromboplastin time (aPTT), modified prothrombin time (mPT), and free tissue factor pathway inhibitor (TFPI) levels were then measured. (A) Hemoglobin level, (C) mPT, and (D) the level of free TFPI are reduced by MG1113 treatment through subcutaneous administration. (B) aPTT is delayed by FVIII neutralizing antibody treatment, but not changed by MG1113 treatment. The graph represents mean and standard deviation. Each dot represents an entity (n = 6‐15). The group not treated with MG1113 and other groups were analyzed using one‐way analysis of variance assuming a normal distribution. The significance level was set at P < .05. † Games‐Howell pairwise comparison; ‡ Dunnett post hoc analysis

Article Snippet: MG1113 and recombinant human (rh) TFPI (Creative Biomart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).

Techniques: Concentration Assay, Modification, Standard Deviation, Comparison